CROSS REFERENCE TO RELATED APPLICATIONS This application claims benefit under 35USC § 119(e) of U.S. provisional application Ser. No. 60/592,978, filed Jul. 31, 2004, herein incorporated by reference.
INCORPORATION OF SEQUENCE LISTING Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-ROMs, each containing the file named “pa01184—53452B.rpt”, which is 67,108,864 bytes (measured in MS-WINDOWS, MEDIUM TYPE: CD-ROM (ASCII TEXT) COMPUTER: IBM PC/XT/AT,IBM PS/20R COMPATIBLES. OPERATING SYSTEM: DOS/WINDOWS 2000/NT) and was created on Jul. 18, 2005, are herein incorporated by reference.
FIELD OF THE INVENTION Disclosed herein are inventions in the field of plant genetics and developmental biology. More specifically, this invention provides transgenic seeds for crops, wherein the genome of said seed comprises recombinant DNA, the expression of which results in the production of transgenic plants that have improved trait(s).
BACKGROUND OF THE INVENTION Transgenic plants with improved traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of genes that are useful in recombinant DNA constructs for production of transformed plants with improved properties.
SUMMARY OF THE INVENTION This invention provides transgenic seeds, transgenic plants and DNA constructs with trait-improving recombinant DNA from a gene for a protein having an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group consisting of SEQ ID NO: 270 and its homologs through SEQ ID NO: 538, where the respective homolog proteins have amino acid sequences SEQ ID NO: 539 through SEQ ID NO: 22568, as indicated in Table 17. In some cases of trait improvement, the recombinant DNA encodes a protein; in other cases, the recombinant DNA suppresses endogenous protein expression. In a broad aspect this invention provides transgenic seeds for growing crop plants with improved traits, such crop plants with improved traits and the plant parts including transgenic seed produced by such crop plants. The improved traits provided by the recombinant DNA in the transgenic crop plant of this invention are identified by comparison to a control plant, i.e., a plant without the trait-improving recombinant DNA. In one aspect of the invention, transgenic crop plant grown from the transgenic seed has improved yield, as compared to the yield of a control plant, e.g., a plant without the recombinant DNA that produces the increased yield. Some plants of this invention exhibit increased yield by producing a yield increase under non-stress conditions. Other plants of this invention exhibit increased yield by producing a yield increase under one or more environmental stress conditions including, but not limited to, water deficit stress, cold stress, heat stress, high salinity stress, shade stress, and low nitrogen availability stress. Still other plants of this invention have other improved phenotypes, such as improved plant development, plant morphology, plant physiology or seed composition as compared to a corresponding trait of a control plant. The various aspects of this invention are especially useful for transgenic seed and transgenic plants having improved traits in corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
The invention also comprises recombinant DNA constructs. In one aspect, such recombinant DNA constructs useful for the transgenic seed and transgenic plants of this invention comprise a promoter functional in a plant cell operably linked to a DNA segment for expressing a protein associated with a trait in a model plant or a homologue. In another aspect the recombinant DNA constructs useful for the transgenic seed and transgenic plants of this invention comprise a promoter functional in a plant cell operably linked to a DNA segment for suppressing the level of an endogenous plant protein which is a homologue to a model-plant protein, the suppression of which is associated with an improved trait. Suppression can be effected by any of a variety of methods known in the art, e.g. post transcriptional suppression by anti-sense, sense, dsRNA and the like or by transcriptional suppression.
This invention also provides a method of producing a transgenic crop plant having at least one improved trait, wherein the method comprises providing to a grower of transgenic seeds comprising recombinant DNA for expression or suppression of a trait-improving gene provided herein, and growing transgenic plant from said transgenic seed. Such methods are used to generate transgenic crop plants having at least one improved trait under one or more environmental stress conditions including, but not limited to, water deficit stress, cold stress, heat stress, high salinity stress, shade stress, and low nitrogen availability stress. In another aspect, such methods are used to generate transgenic crop plants having improved plant development, plant morphology, plant physiology or seed component phenotype as compared to a corresponding phenotype of a control plant. Of particular interest are uses of such methods to generate transgenic crop plants having increased yield under non-stress condition, or under one or more stress conditions.
DETAILED DESCRIPTION OF THE INVENTION This invention provides transgenic plant seed having in its genome trait-improving recombinant DNA and transgenic plants grown from such seed which exhibit an improved trait as compared a control plant. In one aspect, the invention provides transgenic plants where the improved trait is one or more of improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity. Particular transgenic plants grown from transgenic seeds of this invention exhibit increased seed yield. Recombinant DNA constructs used in this invention comprise recombinant DNA disclosed herein which produces mRNA to modulate gene expression imparting improved traits to plants.
“Gene” means all or part of the DNA that encodes a protein or mRNA, e.g., chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA, and includes DNA regions flanking the coding sequences, e.g., introns, 5′UTR, 3′UTR, promoters and other DNA involved in the regulation of expression.
“Transgenic seed” means plant seed having a genome altered by the incorporation of recombinant DNA, e.g., by transformation. “Transgenic plant” means a plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains the recombinant DNA in its genome. “Recombinant DNA” means a DNA molecule having a genetically engineered modification introduced through a combination of endogenous and/or exogenous DNA elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. Recombinant DNA can exist outside of a cell, e.g., as a PCR fragment or in a plasmid, or can be integrated into a genome such as a plant genome.
“Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances the characteristic is visible to the human eye, e.g., seed or plant size, or can be measured by biochemical techniques, e.g., detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
“Control plant” is a plant without trait-improving recombinant DNA. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. One suitable control plant is a non-transgenic plant of the parental line that was used to generate a transgenic plant. Another suitable control plant is a transgenic plant that comprises recombinant DNA without the specific trait producing DNA, e.g., simply a marker gene. Another suitable control plant is a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, e.g., in field conditions, the use of a limited number of control plants can cause a wide variation in the control dataset. To minimize the effect of the variation within the control dataset, a “reference” is used, i.e., a trimmed mean of all data from both transgenic and control plants grown under the same conditions and at the same developmental stage. The trimmed mean is calculated by eliminating a specific percentage, i.e., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.
“Trait improvement” means a detectable and desirable difference in a characteristic in a transgenic plant relative to a control plant or a reference. In some cases, the trait improvement is measured quantitatively. For example, the trait improvement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference. In other cases, the trait improvement is only measured qualitatively. It is known that there are natural variations in a trait. Therefore, the trait improvement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein. Trait improvement includes, but not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Also of interest is the generation of transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include improved plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
“Yield-limiting environment” means a condition under which a plant would have the limitation on yield including environmental stress conditions.
“Stress condition” means a condition unfavorable for a plant, which adversely affects plant metabolism, growth and/or development. A plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield. Specifically, “water deficit stress” means sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) is one physiological measure of plant water deficit. RWC measures the effect of osmotic adjustment in plant water status, when a plant is under stressed conditions. RWC can result from heat, drought, high salinity and induced osmotic stress.
“Cold stress” means exposure of a plant to temperatures below, e.g., at least two or more degrees Celsius below, those temperatures that are normal for a particular species or particular strain of plant.
“Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives enough amounts of nitrogen nutrient to sustain a healthy plant growth and/or for a plant to reach its typical yield for a particular plant species or a particular strain. “Nitrogen nutrient” means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate. “Ammonium” means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc. Those skilled in the art know what constitutes such soil, media and fertilizer inputs for most plant species. “Low nitrogen availability stress” means a plant growth condition that does not contain sufficient nitrogen nutrient to maintain a healthy plant growth and/or for a plant to reach its typical yield under a sufficient nitrogen growth condition; a useful low nitrogen availability stress is a growth condition with 50% or less of the conventional nitrogen inputs.
“Shade stress” means a limited light availability that triggers the shade avoidance response in plant. Plants are subject to shade stress when localized at lower part of the canopy, or in close proximity of neighboring vegetation. Shade stress is exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few other examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre.
“Increased yield” of a transgenic plant of this invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare. For example, corn yield is measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield is often achieved from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA is used to provide transgenic plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.
“Expression” means transcription of DNA to produce RNA. The resulting RNA includes mRNA encoding a protein, antisense RNA that is complementary to an mRNA encoding a protein, or an RNA transcript comprising a combination of sense and antisense gene regions, such as for use in RNAi gene suppression. Expression also means production of encoded protein from mRNA.
“Promoter” means a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. “Tissue preferred” promoters preferentially regulate expression in certain tissues, such as leaves, roots, or seeds. “Tissue specific” promoters predominately regulate expression only in certain tissues. “Cell type” specific promoter primarily regulate expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. “Inducible” and “repressible” promoters regulate expression under environmental influences, under the effect of anaerobic conditions, certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute a class of “non-constitutive” promoters. “Constitutive” promoters are promoters which are active under most conditions. “Anti-sense orientation” refers to a DNA sequence that is operably linked to a promoter in an orientation where the anti-sense strand is transcribed. “Operably linked” refers to an association of two or more DNA elements in a single construct so that the function of one is affected by the other. For example, a promoter is operably linked with transcribable DNA when it is capable of affecting the expression of that DNA; that is, the coding DNA is under the transcriptional control of the promoter.
“Consensus sequence” means an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTAL W alignment of amino acid sequence of homolog proteins.
“Homologs” means genes that produce functionally similar proteins, e.g., in the same organism or in different organisms. A gene can be related to a homolog gene by descent from a common ancestral DNA. Homologs include genes where the relationship is by speciation, e.g., often called orthologs, or by genetic duplication, e.g., often called paralogs. More specifically, “orthologs” include homologs in different species that evolved from a common ancestral gene by specification. Normally orthologs retain the same function in the course of evolution. “Paralogs” include homologs in the same species that have diverged from each other as a consequence of genetic duplication.
“Percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g. nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of sequences is the number of identical components which are shared divided by the total number of sequence components in the segment used as a reference over a window of alignment which is the smaller of the sequences. “Percent identity” (“% identity”) is the identity fraction times 100. “% identity” to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.
“Arabidopsis” means plants of Arabidopsis thaliana.
Recombinant DNA Constructs
This invention provides recombinant DNA constructs comprising DNA elements for imparting one or more improved traits to transgenic plant. Such constructs typically comprise a promoter operatively linked to DNA to provide for expression of a protein or RNA for gene suppression in a target plant. Recombinant DNA constructs can also include additional regulatory elements, such as 5′ or 3′ untranslated regions (UTRs) such as polyadenylation sites, introns, and transit or signal peptides. Such recombinant DNA constructs are assembled using methods known to those of ordinary skill in the art.
In certain embodiments, recombinant DNA constructs comprise sense-oriented, trait-imparting DNA operably linked to a promoter that is functional in a plant to provide for expression of the trait-imparting DNA in the sense orientation such that a desired protein is produced. In other embodiments at least a part of the trait-imparting DNA is in an anti-sense orientation for gene suppression activity.
Recombinant DNA constructs, especially for expressing proteins are typically prepared with a 3′ UTR that a polyadenylation site and signal. Recombinant DNA constructs can also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference.
Table 1 provides a list of genes that can provide trait-imparting DNA for recombinant DNA constructs. DNA from each gene was used in a model plant (Arabidopsis) to discover associations with improved traits. The DNA was also used to identify homologs from which a consensus amino acid sequence is defined for characterizing the aspects of the invention where recombinant DNA is incorporated in the transgenic seeds, transgenic plants, DNA constructs and methods of this invention. With reference to Table 1:
- “NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing.
- “PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA
- “construct_id” refers to an arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA.
- “gene” refers to an arbitrary name used to identify the particular DNA.
- “orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter.
“species” refers to the organism from which the particular DNA was derived. TABLE 1
Nuc SEQ ID Pep SEQ ID construct_id Gene orientation Species
1 270 14324 CGPG1560 SENSE Arabidopsis thaliana
2 271 17484 CGPG2630 SENSE Arabidopsis thaliana
3 272 19109 CGPG1381 ANTI-SENSE Arabidopsis thaliana
4 273 70423 CGPG3165 SENSE Arabidopsis thaliana
5 274 70424 CGPG3180 SENSE Arabidopsis thaliana
6 275 70480 CGPG3833 SENSE Arabidopsis thaliana
7 276 70509 CGPG2420 SENSE Arabidopsis thaliana
8 277 70647 CGPG4334 SENSE Arabidopsis thaliana
9 278 70675 CGPG4519 SENSE Arabidopsis thaliana
10 279 70829 CGPG518 SENSE Arabidopsis thaliana
11 280 70849 CGPG596 SENSE Arabidopsis thaliana
12 281 71627 CGPG1270 SENSE Arabidopsis thaliana
13 282 71934 CGPG2294 SENSE Arabidopsis thaliana
14 283 72615 CGPG4829 SENSE Arabidopsis thaliana
15 284 72927 CGPG1477 SENSE Arabidopsis thaliana
16 285 73014 CGPG5692 SENSE Xenorhabdus nematophilus 85816
17 286 73559 CGPG6535 SENSE Bacillus subtilis 168
18 287 74251 CGPG5489 SENSE Arabidopsis thaliana
19 288 19631 CGPG3627 SENSE Arabidopsis thaliana
20 289 70121 CGPG2380 SENSE Saccharomyces cerevisiae
21 290 70654 CGPG4352 SENSE Arabidopsis thaliana
22 291 70696 CGPG4590 SENSE Arabidopsis thaliana
23 292 70713 CGPG1462 ANTI-SENSE Arabidopsis thaliana
24 293 70740 CGPG3700 SENSE Arabidopsis thaliana
25 294 71321 CGPG4418 SENSE Arabidopsis thaliana
26 295 71835 CGPG4634 SENSE Arabidopsis thaliana
27 296 72934 CGPG5798 SENSE Saccharomyces cerevisiae
28 297 72945 CGPG5787 SENSE Saccharomyces cerevisiae
29 298 72980 CGPG5773 SENSE Saccharomyces cerevisiae
30 299 73504 CGPG6480 SENSE Synechocystis sp. PCC 6803
31 300 73507 CGPG6504 SENSE Bacillus subtilis 168
32 301 73573 CGPG6462 SENSE Agrobacterium tumefacians C58
33 302 73586 CGPG6471 SENSE Bacillus subtilis 168
34 303 73770 CGPG5435 SENSE Arabidopsis thaliana
35 304 74105 CGPG6574 SENSE Xenorhabdus nematophilus 86068
36 305 74111 CGPG6622 SENSE Escherichia coli K-12
37 306 74136 CGPG6632 SENSE Synechocystis
38 307 74139 CGPG6561 SENSE Escherichia coli K-12
39 308 74267 CGPG5364 SENSE Arabidopsis thaliana
40 309 74291 CGPG5363 SENSE Arabidopsis thaliana
41 310 74318 CGPG5826 SENSE Arabidopsis thaliana
42 311 74319 CGPG5831 SENSE Arabidopsis thaliana
43 312 74324 CGPG5885 SENSE Arabidopsis thaliana
44 313 74512 CGPG32 SENSE Arabidopsis thaliana
45 314 74583 CGPG6649 SENSE Ralstonia metallidurans CH34
46 315 70427 CGPG3067 SENSE Arabidopsis thaliana
47 316 71811 CGPG4426 SENSE Arabidopsis thaliana
48 317 73463 CGPG6384 SENSE Ralstonia metallidurans CH34
49 318 72081 CGPG5279 SENSE Glycine max
50 319 10139 CGPG101 ANTI-SENSE Arabidopsis thaliana
51 320 11410 CGPG103 SENSE Arabidopsis thaliana
52 321 11604 CGPG48 ANTI-SENSE Arabidopsis thaliana
53 322 12368 CGPG1006 SENSE Arabidopsis thaliana
54 323 13502 CGPG1354 SENSE Arabidopsis thaliana
55 324 13745 CGPG1576 ANTI-SENSE Arabidopsis thaliana
56 325 13821 CGPG1569 SENSE Arabidopsis thaliana
57 326 14240 CGPG1697 SENSE Arabidopsis thaliana
58 327 14718 CGPG1082 SENSE Arabidopsis thaliana
59 328 17022 CGPG1774 SENSE Arabidopsis thaliana
60 329 17924 CGPG2882 SENSE Arabidopsis thaliana
61 330 18259 CGPG3368 SENSE Arabidopsis thaliana
62 331 19171 CGPG2952 SENSE Saccharomyces cerevisiae
63 332 19201 CGPG2332 SENSE Arabidopsis thaliana
64 333 19317 CGPG3662 SENSE Xanthomonas
65 334 70417 CGPG3427 SENSE Arabidopsis thaliana
66 335 70467 CGPG3785 SENSE Arabidopsis thaliana
67 336 70806 CGPG712 SENSE Arabidopsis thaliana
68 337 70818 CGPG479 SENSE Arabidopsis thaliana
69 338 70820 CGPG655 SENSE Arabidopsis thaliana
70 339 70919 CGPG4029 SENSE Glycine max
71 340 71623 CGPG4696 SENSE Arabidopsis thaliana
72 341 71662 CGPG4679 SENSE Glycine max
73 342 71693 CGPG4652 SENSE Glycine max
74 343 72384 CGPG4639 SENSE Saccharomyces cerevisiae
75 344 72439 CGPG5075 SENSE Arabidopsis thaliana
76 345 72619 CGPG4835 SENSE Arabidopsis thaliana
77 346 72624 CGPG4842 SENSE Arabidopsis thaliana
78 347 72715 CGPG5521 SENSE Saccharomyces cerevisiae
79 348 72754 CGPG5548 SENSE Saccharomyces cerevisiae
80 349 72819 CGPG4989 SENSE Arabidopsis thaliana
81 350 75516 CGPG7689 SENSE Glycine max
82 351 75701 CGPG7856 SENSE Glycine max
83 352 73515 CGPG6473 SENSE Bacillus subtilis 168
84 353 74684 CGPG6360 SENSE Arabidopsis thaliana
85 354 19542 CGPG3069 SENSE Arabidopsis thaliana
86 355 19618 CGPG3574 SENSE Arabidopsis thaliana
87 356 19649 CGPG3140 SENSE Arabidopsis thaliana
88 357 19745 CGPG3973 SENSE Glycine max
89 358 19768 CGPG4096 SENSE Glycine max
90 359 19772 CGPG3939 SENSE Glycine max
91 360 19779 CGPG4113 SENSE Glycine max
92 361 19833 CGPG4074 SENSE Glycine max
93 362 19862 CGPG3961 SENSE Glycine max
94 363 19879 CGPG4009 SENSE Glycine max
95 364 70445 CGPG3728 SENSE Arabidopsis thaliana
96 365 70738 CGPG3195 SENSE Arabidopsis thaliana
97 366 71437 CGPG4043 SENSE Glycine max
98 367 71572 CGPG4520 SENSE Arabidopsis thaliana
99 368 71617 CGPG1227 SENSE Arabidopsis thaliana
100 369 72532 CGPG4780 SENSE Arabidopsis thaliana
101 370 72757 CGPG5572 SENSE Arabidopsis thaliana
102 371 73412 CGPG6448 SENSE Pseudomonas syringae var tomato DC3000
103 372 74102 CGPG6550 SENSE Bacillus halodurans C-125
104 373 72633 CGPG4853 SENSE Arabidopsis thaliana
105 374 72456 CGPG4745 SENSE Arabidopsis thaliana
106 375 72963 CGPG1746 SENSE Arabidopsis thaliana
107 376 70426 CGPG3199 SENSE Arabidopsis thaliana
108 377 70772 CGPG4627 SENSE Arabidopsis thaliana
109 378 71137 CGPG125 SENSE Arabidopsis thaliana
110 379 71529 CGPG2808 SENSE Arabidopsis thaliana
111 380 71601 CGPG1858 SENSE Arabidopsis thaliana
112 381 72362 CGPG983 SENSE Arabidopsis thaliana
113 382 72466 CGPG4767 SENSE Arabidopsis thaliana
114 383 72524 CGPG4770 SENSE Arabidopsis thaliana
115 384 73085 CGPG5689 SENSE Synechocystis sp. PCC 6803
116 385 74241 CGPG5457 SENSE Arabidopsis thaliana
117 386 74247 CGPG5475 SENSE Arabidopsis thaliana
118 387 74284 CGPG5413 SENSE Arabidopsis thaliana
119 388 74652 CGPG6168 SENSE Arabidopsis thaliana
120 389 70437 CGPG3706 SENSE Arabidopsis thaliana
121 390 71633 CGPG857 SENSE Arabidopsis thaliana
122 391 72948 CGPG5617 SENSE Arabidopsis thaliana
123 392 72519 CGPG4749 SENSE Arabidopsis thaliana
124 393 10475 CGPG399 SENSE Arabidopsis thaliana
125 394 11120 CGPG459 ANTI-SENSE Arabidopsis thaliana
126 395 19736 CGPG4129 SENSE Glycine max
127 396 71606 CGPG4715 SENSE Arabidopsis thaliana
128 397 71840 CGPG4353 SENSE Arabidopsis thaliana
129 398 74240 CGPG5454 SENSE Arabidopsis thaliana
130 399 74331 CGPG5834 SENSE Arabidopsis thaliana
131 400 74610 CGPG6048 SENSE Arabidopsis thaliana
132 401 75527 CGPG7682 SENSE Glycine max
133 402 70681 CGPG4584 SENSE Arabidopsis thaliana
134 403 71663 CGPG4638 SENSE Xanthomonas
135 404 72769 CGPG5573 SENSE Arabidopsis thaliana
136 405 71508 CGPG1541 SENSE Arabidopsis thaliana
137 406 74248 CGPG5476 SENSE Arabidopsis thaliana
138 407 72771 CGPG2166 SENSE Arabidopsis thaliana
139 408 72085 CGPG5228 SENSE Arabidopsis thaliana
140 409 72744 CGPG5563 SENSE Saccharomyces cerevisiae
141 410 73039 CGPG810 SENSE Arabidopsis thaliana
142 411 73054 CGPG5754 SENSE Saccharomyces cerevisiae
143 412 73501 CGPG6456 SENSE Agrobacterium tumefacians C58
144 413 19707 CGPG4179 SENSE Glycine max
145 414 19951 CGPG3941 SENSE Glycine max
146 415 19967 CGPG4032 SENSE Glycine max
147 416 70543 CGPG3815 SENSE Arabidopsis thaliana
148 417 70707 CGPG1273 ANTI-SENSE Arabidopsis thaliana
149 418 70719 CGPG1712 ANTI-SENSE Arabidopsis thaliana
150 419 71134 CGPG817 SENSE Arabidopsis thaliana
151 420 71146 CGPG2928 SENSE Arabidopsis thaliana
152 421 71660 CGPG4690 SENSE Arabidopsis thaliana
153 422 72086 CGPG5236 SENSE Arabidopsis thaliana
154 423 72632 CGPG4852 SENSE Arabidopsis thaliana
155 424 72716 CGPG5529 SENSE Saccharomyces cerevisiae
156 425 72723 CGPG1848 SENSE Arabidopsis thaliana
157 426 72987 CGPG1787 SENSE Arabidopsis thaliana
158 427 74109 CGPG6606 SENSE Xenorhabdus nematophilus 86068
159 428 74140 CGPG6569 SENSE Bacillus halodurans C-125
160 429 74191 CGPG6597 SENSE Rhodobacter sphaeroides 2.4.1
161 430 74265 CGPG5356 SENSE Arabidopsis thaliana
162 431 74369 CGPG6076 SENSE Arabidopsis thaliana
163 432 70217 CGPG6 SENSE Arabidopsis thaliana
164 433 72711 CGPG1846 SENSE Arabidopsis thaliana
165 434 70932 CGPG4089 SENSE Glycine max
166 435 73518 CGPG6497 SENSE Pseudomonas fluorescens PfO-1
167 436 19771 CGPG4011 SENSE Glycine max
168 437 73549 CGPG6460 SENSE Xenorhabdus nematophilus 85816
169 438 72994 CGPG5803 SENSE Saccharomyces cerevisiae
170 439 71928 CGPG1617 SENSE Arabidopsis thaliana
171 440 72903 CGPG5584 SENSE Arabidopsis thaliana
172 441 73017 CGPG5733 SENSE Saccharomyces cerevisiae
173 442 74587 CGPG6774 SENSE Agrobacterium tumefacians C58
174 443 72453 CGPG4735 SENSE Arabidopsis thaliana
175 444 72967 CGPG5742 SENSE Saccharomyces cerevisiae
176 445 72961 CGPG5591 SENSE Arabidopsis thaliana
177 446 73070 CGPG5627 SENSE Arabidopsis thaliana
178 447 73475 CGPG6385 SENSE Rhodopseudomonas palustris CGA009
179 448 72916 CGPG1814 SENSE Arabidopsis thaliana
180 449 72969 CGPG5789 SENSE Saccharomyces cerevisiae
181 450 74449 CGPG6659 SENSE Agrobacterium tumefaciens
182 451 16615 CGPG2539 SENSE Agrobacterium
183 452 19187 CGPG3310 SENSE Arabidopsis thaliana
184 453 19648 CGPG3134 SENSE Arabidopsis thaliana
185 454 70354 CGPG3995 SENSE Glycine max
186 455 70421 CGPG2942 SENSE Arabidopsis thaliana
187 456 70459 CGPG3758 SENSE Arabidopsis thaliana
188 457 70465 CGPG3775 SENSE Arabidopsis thaliana
189 458 70683 CGPG4587 SENSE Arabidopsis thaliana
190 459 70725 CGPG2097 ANTI-SENSE Arabidopsis thaliana
191 460 70852 CGPG1465 SENSE Arabidopsis thaliana
192 461 71112 CGPG934 SENSE Arabidopsis thaliana
193 462 71127 CGPG945 SENSE Arabidopsis thaliana
194 463 71132 CGPG1561 SENSE Arabidopsis thaliana
195 464 71217 CGPG95 SENSE Arabidopsis thaliana
196 465 71645 CGPG4688 SENSE Arabidopsis thaliana
197 466 71726 CGPG3894 SENSE Arabidopsis thaliana
198 467 72432 CGPG4562 SENSE Arabidopsis thaliana
199 468 72450 CGPG4732 SENSE Arabidopsis thaliana
200 469 72455 CGPG4742 SENSE Arabidopsis thaliana
201 470 72727 CGPG5522 SENSE Saccharomyces cerevisiae
202 471 72817 CGPG4987 SENSE Arabidopsis thaliana
203 472 72992 CGPG5777 SENSE Saccharomyces cerevisiae
204 473 73007 CGPG5760 SENSE Saccharomyces cerevisiae
205 474 73073 CGPG5688 SENSE Synechocystis sp. PCC 6803
206 475 73506 CGPG6496 SENSE Pseudomonas fluorescens PfO-1
207 476 74107 CGPG6590 SENSE Sinorhizobium meliloti 1021
208 477 74117 CGPG6575 SENSE Xenorhabdus nematophilus 86068
209 478 74131 CGPG6592 SENSE Synechocystis sp. PCC 6803
210 479 74344 CGPG5929 SENSE Arabidopsis thaliana
211 480 14320 CGPG1229 SENSE Arabidopsis thaliana
212 481 16756 CGPG2117 SENSE Arabidopsis thaliana
213 482 17448 CGPG2673 SENSE Arabidopsis thaliana
214 483 17633 CGPG2839 SENSE Arabidopsis thaliana
215 484 18876 CGPG3096 SENSE Arabidopsis thaliana
216 485 19120 CGPG1976 ANTI-SENSE Arabidopsis thaliana
217 486 19221 CGPG2958 SENSE Arabidopsis thaliana
218 487 70206 CGPG4116 SENSE Glycine max
219 488 70223 CGPG53 SENSE Arabidopsis thaliana
220 489 70347 CGPG3147 SENSE Arabidopsis thaliana
221 490 70406 CGPG1687 SENSE Arabidopsis thaliana
222 491 70469 CGPG3791 SENSE Arabidopsis thaliana
223 492 70564 CGPG1864 SENSE Arabidopsis thaliana
224 493 70601 CGPG2917 SENSE Arabidopsis thaliana
225 494 70612 CGPG3721 SENSE Arabidopsis thaliana
226 495 70720 CGPG1358 ANTI-SENSE Arabidopsis thaliana
227 496 70735 CGPG2661 SENSE Arabidopsis thaliana
228 497 70846 CGPG377 SENSE Arabidopsis thaliana
229 498 70923 CGPG4020 SENSE Glycine max
230 499 71149 CGPG3457 SENSE Arabidopsis thaliana
231 500 71608 CGPG4687 SENSE Arabidopsis thaliana
232 501 71739 CGPG4345 SENSE Arabidopsis thaliana
233 502 72014 CGPG5230 SENSE Arabidopsis thaliana
234 503 72051 CGPG5241 SENSE Arabidopsis thaliana
235 504 74259 CGPG5343 SENSE Arabidopsis thaliana
236 505 72463 CGPG4760 SENSE Arabidopsis thaliana
237 506 72902 CGPG5597 SENSE Arabidopsis thaliana
238 507 74572 CGPG6640 SENSE Synechocystis
239 508 73055 CGPG5768 SENSE Saccharomyces cerevisiae
240 509 74103 CGPG6558 SENSE Escherichia coli K-12
241 510 72921 CGPG5781 SENSE Saccharomyces cerevisiae
242 511 72968 CGPG5772 SENSE Saccharomyces cerevisiae
243 512 19703 CGPG4172 SENSE Glycine max
244 513 19946 CGPG4097 SENSE Glycine max
245 514 19980 CGPG3914 SENSE Glycine max
246 515 70435 CGPG3701 SENSE Arabidopsis thaliana
247 516 71114 CGPG1657 SENSE Arabidopsis thaliana
248 517 72451 CGPG4733 SENSE Arabidopsis thaliana
249 518 72947 CGPG5607 SENSE Glycine max
250 519 73012 CGPG5786 SENSE Saccharomyces cerevisiae
251 520 73022 CGPG5622 SENSE Arabidopsis thaliana
252 521 73488 CGPG6394 SENSE Bacillus subtilis 168
253 522 73901 CGPG5237 SENSE Arabidopsis thaliana
254 523 73964 CGPG5804 SENSE Saccharomyces cerevisiae
255 524 74019 CGPG5706 SENSE Bacillus subtilis 168
256 525 74022 CGPG5724 SENSE Arabidopsis thaliana
257 526 74114 CGPG6551 SENSE Agrobacterium tumefacians C58
258 527 74262 CGPG5353 SENSE Arabidopsis thaliana
259 528 74292 CGPG5367 SENSE Arabidopsis thaliana
260 529 74302 CGPG5384 SENSE Arabidopsis thaliana
261 530 74325 CGPG5898 SENSE Arabidopsis thaliana
262 531 74429 CGPG6689 SENSE Bacillus subtilis 168
263 532 74440 CGPG6682 SENSE Bacillus halodurans C-125
264 533 74462 CGPG6668 SENSE Synechocystis
265 534 74465 CGPG6692 SENSE Bacillus subtilis 168
266 535 74474 CGPG6669 SENSE Synechocystis
267 536 74505 CGPG6783 SENSE Escherichia coli K-12
268 537 74507 CGPG6799 SENSE Xenorhabdus nematophilus 85816
269 538 74562 CGPG6764 SENSE Bacillus subtilis 168
Recombinant DNA
Trait-imparting DNA for use in this invention for improved traits in plants is disclosed herein as having a DNA sequence of SEQ ID NO:1 through SEQ ID NO:269 and any of the respective homologs. A subset of the trait-imparting DNA includes fragments with less than the full DNA sequence, e.g., consisting of oligonucleotides of at least about 15 to 20 or more consecutive nucleotides from one of the disclosed sequences. Such oligonucleotides are fragments of the larger molecules having a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 269, and find use, for example as probes and primers for detection of the polynucleotides of the invention or for cloning DNA for use in this invention.
Useful DNA includes variants of the disclosed DNA. Such variants include naturally occurring, including homologous DNA from genes of the same or a different species, or non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one nucleotide of a disclosed DNA without causing the amino acid sequence of the protein produced to be changed. Hence, useful DNA can have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.
Homologs of the trait-imparting DNA generally demonstrate significant identity with the DNA provided herein. Homologous DNA is substantially identical to a trait-imparting DNA if, when the nucleotide sequences are optimally aligned there is at least about 60% nucleotide identity, or higher, e.g., at least 70% or 80% or 85% or even 90% identity or higher, such as 95% or 98% identity over a comparison window of at least 50 to 100 nucleotides, and up to the entire length of the trait-imparting DNA. Optimal alignment of sequences for aligning a comparison window can be conducted by algorithms including computerized implementations of the algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference DNA sequence can represent a full-length coding sequence or a portion.
Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 270 through SEQ ID NO: 538, as well as homologs of such proteins.
One method to identify homologs of the proteins useful in this invention is by comparison of the amino acid sequence of the trait-imparting protein to amino acid sequences of proteins from the same or different organisms, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. In one method a local sequence alignment program, e.g., BLAST, is used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) is used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. Thus, homolog is used herein to described proteins that are assumed to have functional similarity by inference from sequence base similarity. The relationship of homologs with amino acid sequences of SEQ ID NO: 539 through SEQ ID NO: 22568 to the proteins with amino acid sequences of SEQ ID NO: 270 through SEQ ID NO: 538 is found is found in Table 17.
Aspects of the invention also use DNA encoding functional homolog proteins which differ in one or more amino acids from those of protein encoded by disclosed trait-imparting DNA as the result of one or more of the well-known conservative amino acid substitutions, e.g., valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence are selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence are selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention uses DNA encoding proteins that differ in one or more amino acids from those of protein encoded from a described trait-imparting DNA as the result of deletion or insertion of one or more amino acids in a native sequence.
Homologs of the proteins encoded by disclosed trait-improving DNA will generally demonstrate significant sequence identity, e.g., at least 50% amino acid sequence identity or higher such as at least 70% identity or at least 80% or at least 90% identity with an amino acid sequence of SEQ ID NO:270 through SEQ ID NO:538. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence of a protein encoded by a disclosed trait-imparting DNA and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. The window of comparison for determining identity can be the entire amino acid sequence disclosed herein, e.g., the full sequence of any of SEQ ID NO:270 through SEQ ID NO:538.
Genes that are homologs to each other can be grouped into families and included in multiple sequence alignments to allow a consensus sequence to be derived. This analysis enables the derivation of conserved and class- (family) specific residues or motifs that are functionally important. These conserved residues and motifs can be further validated with 3D protein structure if available. A consensus sequence is used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship and the corresponding trait-imparting DNA. Thus, this invention contemplates that protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence.
Promoters
Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus or figwort mosaic virus promoters. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters, U.S. Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/078,972 which discloses a coixin promoter, U.S. patent application Ser. No. 09/757,089 which discloses a maize chloroplast aldolase promoter, and U.S. patent application Ser. No. 10/739,565 which discloses water-deficit inducible promoters, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant DNA to provide for expression of desired genes in transgenic plant cells.
It is well known in the art that promoters are usefully altered to contain multiple “enhancer sequences” to assist in elevating gene expression. By including an enhancer sequence with such constructs, expression is generally enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, and can also be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, 5′ enhancing elements are introns. Particularly useful enhancers are the 5′ introns of the rice actin 1 gene and the rice actin 2 gene. Other enhancers include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.
In some aspects of the invention it is preferred that the promoter element in the DNA construct be capable of causing sufficient expression in water deficit conditions. Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions. Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize. Such water-deficit-inducible promoters are disclosed in U.S. 2004-0123347 A1, incorporated herein by reference.
In other aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell, et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger, et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Perl) (Stacy, et al., (1996) Plant Mol. Biol. 31(6): 1205-1216).
In still other aspects of the invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol. Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).
“Gene overexpression” means expression, e.g., of a gene at a level in its native host that exceeds levels of expression in a non-transgenic host. In many embodiments of the invention, a recombinant DNA construct provides gene overexpression, e.g., as identified in Table 1.
Gene Suppression
Gene suppression includes any of the well-known methods for suppressing expression, typically indicated by reduced levels of protein. Posttranscriptional gene suppression is mediated by transcription of integrated recombinant DNA to form double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. This formation of dsRNA most commonly results from transcription of an integrated inverted repeat of an element of a target gene, and is a common feature of gene suppression methods known as anti-sense suppression, co-suppression and RNA interference (RNAi). Transcriptional suppression can be mediated by a transcribed dsRNA having homology to a promoter DNA sequence to effect what is called promoter trans suppression.
More particularly, posttranscriptional gene suppression by inserting a recombinant DNA construct with one or more copies of anti-sense oriented DNA to regulate gene expression in plant cells is disclosed in U.S. Pat. No. 5,107,065 (Shewmaker, et al.,) and U.S. Pat. No. 5,759,829 (Shewmaker, et al.,). Transgenic plants transformed using such anti-sense oriented DNA constructs for gene suppression can comprise integrated DNA arranged as an inverted repeats that result from insertion of the DNA construct into plants by Agrobacterium-mediated transformation, as disclosed by Redenbaugh, et al., in “Safety Assessment of Genetically Engineered Flavr Savrm Tomato, CRC Press, Inc. (1992). Inverted repeat insertions can comprises a part or all of the T-DNA construct, e.g., an inverted repeat of a complete transcription unit or an inverted repeat of transcription terminator sequence. Screening for inserted DNA comprising inverted repeat elements can improve the efficiency of identifying transformation events effective for gene silencing whether the transformation construct is a simple anti-sense DNA construct which must be inserted in multiple copies or a complex inverted repeat DNA construct (e.g., an RNAi construct) which can be inserted as a single copy.
Posttranscriptional gene suppression by inserting a recombinant DNA construct with sense-oriented DNA to regulate gene expression in plants is disclosed in U.S. Pat. No. 5,283,184 (Jorgensen, et al.) and U.S. Pat. No. 5,231,020 (Jorgensen, et al.). Inserted T-DNA providing gene suppression in plants transformed with such sense constructs by Agrobacterium is organized predominately in inverted repeat structures, as disclosed by Jorgensen, et al., Mol. Gen. Genet., 207:471-477 (1987). See also Stam, et al., The Plant Journal, 12(1), 63-82 (1997) who used segregation studies to support Jorgensen's finding that gene silencing is mediated by multimeric transgene T-DNA loci in which the T-DNAs are arranged in inverted repeats. Screening for inserted DNA comprising inverted repeat elements can improve the gene silencing efficiency when transforming with simple sense-orientated DNA constructs. Gene silencing efficiency can also be improved by screening for single insertion events when transforming with an RNAi construct containing inverted repeat elements
As disclosed by Redenbaugh, et al., gene suppression can be achieved by inserting into a plant genome recombinant DNA that transcribes dsRNA. Such a DNA insert can be transcribed to an RNA element having the 3′ region as a double stranded RNA. RNAi constructs are also disclosed in EP 0426195 A1 (Goldbach, et al.,—1991) where recombinant DNA constructs for transcription into hairpin dsRNA for providing transgenic plants with resistance to tobacco spotted wilt virus. Double-stranded RNAs were also disclosed in WO 94/01550 (Agrawal, et al.,) where anti-sense RNA was stabilized with a self-complementary 3′ segment. Agrawal, et al., referred to U.S. Pat. No. 5,107,065 for using such self-stabilized anti-sense RNAs for regulating gene expression in plant cells; see International Publication No. 94/01550. Other double-stranded hairpin-forming elements in transcribed RNA are disclosed in International Publication No. 98/05770 (Werner, et al.,) where the anti-sense RNA is stabilized by hairpin forming repeats of poly(CG) nucleotides. See also U.S. Patent Application Publication No. 2003/0175965 A1 (Lowe, et al.,) which discloses gene suppression using and RNAi construct comprising a gene coding sequence preceded by inverted repeats of 5′UTR. See also U.S. Patent Application Publication No. 2002/0048814 A1 (Oeller) where RNAi constructs are transcribed to sense or anti-sense RNA which is stabilized by a poly(T)-poly(A) tail. See also U.S. Patent Application Publication No. 2003/0018993 A1 (Gutterson, et al.,) where sense or anti-sense RNA is stabilized by an inverted repeat of the 3′ untranslated region of the NOS gene. See also U.S. Patent Application Publication No. 2003/0036197 A1 (Glassman, et al.,) where RNA having homology to a target is stabilized by two complementary RNA regions.
Gene silencing can also be affected by transcribing RNA from both a sense and an anti-sense oriented DNA, e.g., as disclosed by Shewmaker, et al., in U.S. Pat. No. 5,107,065 where in Example 1 a binary vector was prepared with both sense and anti-sense aroA genes. See also U.S. Pat. No. 6,326,193 where gene targeted DNA is operably linked to opposing promoters.
Gene silencing can also be affected by transcribing from contiguous sense and anti-sense DNA. In this regard see Sijen, et al., The Plant Cell, Vol. 8, 2277-2294 (1996) discloses the use of constructs carrying inverted repeats of a cowpea mosaic virus gene in transgenic plants to mediate virus resistance. Such constructs for posttranscriptional gene suppression in plants by double-stranded RNA are also disclosed in International Publication No. WO 99/53050 (Waterhouse, et al.,), International Publication No. WO 99/49029 (Graham, et al.), U.S. 2004-0029283 A1 (Fillatti), U.S. Pat. No. 6,506,559 (Fire, et al.,). See also U.S. 2004-0006792 A1 (Shewmaker, et al.,) that discloses constructs and methods for simultaneously expressing one or more recombinant genes while simultaneously suppressing one or more native genes in a transgenic plant. See also U.S. Pat. No. 6,448,473 (Mitsky, et al.,) that discloses multi-gene suppression vectors for use in plants. All of the above-described patents, applications and international publications disclosing materials and methods for posttranscriptional gene suppression in plants are incorporated herein by reference. Transcriptional suppression such as promoter trans suppression can be affected by a expressing a DNA construct comprising a promoter operably linked to inverted repeats of promoter DNA for a target gene. Constructs useful for such gene suppression mediated by promoter trans suppression are disclosed by Mette, et al., The EMBO Journal, Vol. 18, No. 1, pp. 241-148, 1999 and by Mette, et al., The EMBO Journal, Vol. 19, No. 19, pp. 5194-5201-148, 2000, both of which are incorporated herein by reference.
Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformations with the T-DNA of Agrobacterium are readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that are efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest are identified using the polynucleotides of this invention. For example, a large population of mutated plants are screened to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.
Gene Stacking
This invention also contemplates that the trait-improving recombinant DNA is used in combination with other recombinant DNA to create plants with a multiple desired traits. The combinations generated include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations are created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.
Plant Transformation Methods
Numerous methods for transforming plant cells with recombinant DNA are known in the art and are useful in producing the transgenic seeds of this invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
In general it is preferred to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it is useful to target heterologous DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-10× as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” means any of the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant is regenerated is useful as a recipient cell. Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. 2004-0216189 A1, which are incorporated herein by reference.
In practice DNA is introduced into only a small percentage of target cells in any one experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells are tested further to confirm stable integration of the exogenous DNA. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants are also often employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.
Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in regeneration media and allowed to mature into plants. Developing plantlets are transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO2, and 25-250 microeinsteins m−2 s−1 of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28 degrees C. After regenerating plants have reached the stage of shoot and root development, they are transferred to a greenhouse for further growth and testing. Plants are pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
Progeny are recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Discovery of Trait-Improving Recombinant DNA
To identify recombinant DNA that confer improved traits to plants, Arabidopsis plants were transformed with a large population of recombinant DNA constructs for expressing a large variety of distinct DNA. Transgenic plants were produced and screened to identify those plants having recombinant DNA constructs expressing trait-imparting DNA. A two-step screening process was employed which comprised two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait. The following Table 2 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.
In particular, Table 2 reports
- “PEP SEQ ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.
- “construct_id” is an arbitrary name for the recombinant DNA describe more particularly in Table 1.
- “annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.
- “description” refers to the description of the top BLAST hit.
- “e-value” provides the expectation value for the BLAST hit.
- “identity” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.
- “traits” identified by two letters codes the confirmed improvement in a transgenic plant provided by the recombinant DNA. The codes for improved traits are:
- “CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen;
- “CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen;
- “DS” which indicates drought tolerance improvement identified by a soil drought stress tolerance screen;
- “PEG” which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen;
- “HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen;
- “SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen;
- “LN” which indicates nitrogen use efficiency improvement identified by a limited nitrogen tolerance screen;
- “LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition;
- “PP” which indicates improved growth and development at early stages identified by an early plant growth and development screen;
“SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein. TABLE 2
Pep annotation
SEQ construct e %
Id id gene value identity ncbi id description traits
270 14324 CGPG1560 1.00E − 127 86 gi|15232185| ref|NP_191546.1|expressed CK SS
protein [Arabidopsis
thaliana]]
271 17484 CGPG2630 0 93 gi|15220912| ref|NP_173239.1|zinc finger CK
(C3HC4-type RING finger)
family protein [Arabidopsis
thaliana]
272 19109 CGPG1381 9.00E − 31 81 gi|18404521| ref|NP_565870.1|expressed CK
protein [Arabidopsis
thaliana]
273 70423 CGPG3165 1.00E − 134 96 gi|30688808| ref|NP_850953.1|MADS- CK CS CK HS PP
box protein (AGL9)
[Arabidopsis thaliana]
gb|AAM65812.1| putative
floral homeotic protein,
AGL9 [Arabidopsis
thaliana]
274 70424 CGPG3180 1.00E − 142 81 gi|25405039| pir||H96827protein CK
F20B17.12 [imported]-
Arabidopsis thaliana
gb|AAF68121.1|F20B17.12
[Arabidopsis thaliana]
275 70480 CGPG3833 1.00E − 122 99 gi|18411867| ref|NP_565174.1|14-3-3 CK
protein GF14 pi (GRF13)
[Arabidopsis thaliana]
276 70509 CGPG2420 1.00E − 113 82 gi|15225186| ref|NP_180770.1|ovate CK
protein-related [Arabidopsis
thaliana]
277 70647 CGPG4334 1.00E − 171 94 gi|15237269| ref|NP_200093.1|ornithine CK
cyclodeaminase/mu-
crystallin family protein
[Arabidopsis thaliana]
dbj|BAB10429.1|
278 70675 CGPG4519 0 100 gi|15224730| ref|NP_180115.1|2- CK
oxoglutarate-dependent
dioxygenase, putative
[Arabidopsis thaliana]
pir||E84648 probable
dioxygenase]
279 70829 CGPG518 0 92 gi|15232841| ref|NP_186854.1|potassium CK
transporter (KUP3)
[Arabidopsis thaliana]
280 70849 CGPG596 1.00E − 166 96 gi|15224801| ref|NP_179547.1|cytidine CK
deaminase (CDD)/cytidine
aminohydrolase
[Arabidopsis thaliana]
281 71627 CGPG1270 0 99 gi|18398032| ref|NP_566315.1|ABC1 CK
family protein [Arabidopsis
thaliana]
282 71934 CGPG2294 1.00E − 154 79 gi|15233973| ref|NP_195575.1|26S CK
proteasome regulatory
subunit S5A (RPN10)
[Arabidopsis thaliana]
sp|P55034|PSD4_ARATH
26S proteasome non-
ATPase regulatory subunit
4 (26S proteasome
regulatory
283 72615 CGPG4829 2.00E − 49 88 gi|18422886| ref|NP_568693.1|expressed CK
protein [Arabidopsis
thaliana]
284 72927 CGPG1477 1.00E − 114 81 gi|15234815| ref|NP_194797.1|MA3 CK
domain-containing protein
[Arabidopsis thaliana]
pir||A85359 translation
initiation factor-like protein
285 73014 CGPG5692 1.00E − 180 93 gi|37528369| ref|NP_931714.1|Fructose- CK PP
1,6-bisphosphatase (D-
fructose-1,6-bisphosphate
1-phosphohydrolase)
(FBPase) [Photorhabdus
luminescens subsp.
laumondii TTO1]
286 73559 CGPG6535 0 93 gi|16078422| ref|NP_389241.1|similar to CK
aspartate aminotransferase
[Bacillus subtilis]
sp|O31665|MTNE_BACSU
Transaminase mtnE
pir||F69863 probable
transaminase (EC 2.6.1.-)
ykrV
287 74251 CGPG5489 1.00E − 171 87 gi|15238437| ref|NP_200760.1|zinc CK SP
transporter (ZIP2)
[Arabidopsis thaliana]
sp|Q9LTH9|ZIP2_ARATH
Zinc transporter 2 precursor
(ZRT/IRT-like protein 2)
288 19631 CGPG3627 1.00E − 94 90 gi|18410249| ref|NP_565053.1|SNF7 CS
family protein [Arabidopsis
thaliana] pir||G96755
developmental protein
homolog DG1118
[imported] - [Arabidopsis
thaliana]
289 70121 CGPG2380 1.00E − 111 100 gi|6323765| ref|NP_013836.1|Hypothetical CS
ORF; Ymr118cp
[Saccharomyces
cerevisiae]
sp|Q04487|YM07_YEAST
Putative succinate
dehydrogenase cytochrome
B subunit, mitochondrial
precursor
290 70654 CGPG4352 2.00E − 63 88 gi|18400941| ref|NP_566531.1|expressed CS
protein [Arabidopsis
thaliana]
291 70696 CGPG4590 8.00E − 92 87 gi|25408379| pir||E84768hypothetical CS PP
protein At2g35430
292 70713 CGPG1462 0 95 gi|30678679| ref|NP_191966.2|malate CS
oxidoreductase, putative
293 70740 CGPG3700 0 94 gi|18402759| ref|NP_566667.1|transcription CS LL PP
factor jumonji (jmjC)
domain-containing protein
294 71321 CGPG4418 0 91 gi|13878402| sp|Q9STL0|C71N_ARATH CS
Cytochrome P450 71A23
pir||T06712 probable
cytochrome P450
T29H11.180
295 71835 CGPG4634 0 100 gi|15234361| ref|NP_192100.1|DC1 CS SP
domain-containing protein
[Arabidopsis thaliana]
pir||E85024 probable CHP-
rich zinc finger protein
296 72934 CGPG5798 0 98 gi|6323512| ref|NP_013583.1|High- CS
affinity inorganic phosphate
(Pi) transporter and low-
affinity manganese
transporter; regulated by
Pho4p and Spt7p; mutation
confers resistance to
arsenate; exit from the ER
during maturation requires
Pho86p; Pho84p
[Saccharomyces
cerevisiae]
297 72945 CGPG5787 0 94 gi|6319991| ref|NP_010071.1|GABA- CS
specific transport protein;
Uga4p [Saccharomyces
cerevisiae]
sp|P32837|UGA4_YEAST
GABA-specific permease
(GABA-specific transport
protein)
298 72980 CGPG5773 0 89 gi|63219690| ref|NP_012036.1|Subunit of CS
the anaphase-promoting
complex/cyclosome
(APC/C), which is a
ubiquitin-protein ligase
required for degradation of
anaphase inhibitors,
including mitotic cyclins,
during the
metaphase/anaphase
transition; Cdc23p
299 73504 CGPG6480 1.00E − 178 100 gi|16330153| ref|NP_440881.1|fructokinase CS PP
[Synechocystis sp. PCC
6803] pir||S77227
fructokinase (EC 2.7.1.4)-
Synechocystis sp. (strain
PCC 6803)
300 73507 CGPG6504 1.00E − 173 100 gi|16078547| ref|NP_389366.1|similar to CS LL PP PEG
glutaminase [Bacillus
subtilis]
301 73573 CGPG6462 0 100 gi|15890038| ref|NP_355719.1|AGR_C_5 CS PP
067p [Agrobacterium
tumefaciens str. C58]
ref|NP_533456.1| 3-
isopropylmalate
dehydrogenase
302 73586 CGPG6471 1.00E − 177 100 gi|16077684| ref|NP_388498.1|similar to CS DS PP
fructokinase [Bacillus
subtilis]
303 73770 CGPG5435 2.00E − 34 68 gi|15235771| ref|NP_193383.1|cysteine CS PP
protease inhibitor family
protein/cystatin family
protein [Arabidopsis
thaliana]
304 74105 CGPG6574 0 73 gi|23059330| ref|ZP_00084307.1|COG1012: CS
NAD-dependent
aldehyde dehydrogenases
[Pseudomonas fluorescens
PfO-1]
305 74111 CGPG6622 0 99 gi|16131442] ref|NP_418028.1|alpha- CS
amylase [Escherichia coli
K12]
306 74136 CGPG6632 9.00E − 81 99 gi|16330993| ref|NP_441721.1|unknown CS CK HS
protein [Synechocystis sp.
PCC 6803]
307 74139 CGPG6561 1.00E − 180 95 gi|24112825| ref|NP_707335.1|glyceraldehyde- CS LL
3-phosphate
dehydrogenase A [Shigella
flexneri 2a str. 301]
308 74267 CGPG5364 0 97 gi|18399375| ref|NP_566402.1|U-box CS
domain-containing protein
[Arabidopsis thaliana]
309 74291 CGPG5363 0 94 gi|18401867| ref|NP_565676.1|armadillo/ CS
beta-catenin repeat family
protein/U-box domain-
containing protein
[Arabidopsis thaliana]
310 74318 CGPG5826 0 100 gi|15219730| ref|NP_176847.1|cell CS HS
division protein kinase,
putative [Arabidopsis
thaliana]
311 74319 CGPG5831 0 96 gi|15224359| ref|NP_181907.1|mitogen- CS
activated protein kinase,
putative/MAPK, putative
(MPK6) [Arabidopsis
thaliana]
312 74324 CGPG5885 1.00E − 174 95 gi|42569304| ref|NP_180094.2|protein CS
kinase family protein
[Arabidopsis thaliana]
313 74512 CGPG32 0 96 gi|15217945| ref|NP_176132.1|amino CS HS SP
acid permease I (AAP1)
[Arabidopsis thaliana]
314 74583 CGPG6649 1.00E − 151 83 gi|22978283| ref|ZP_00024043.1|COG0252: CS PP
L-asparaginase/archaeal Glu-
tRNAGln amidotransferase
subunit D [Ralstonia
metallidurans]
315 70427 CGPG3067 0 100 gi|42572771| ref|NP_974481.1|kelch CS DS
repeat-containing F-box
family protein [Arabidopsis
thaliana]
316 71811 CGPG4426 0 97 gi|15223341| ref|NP_171627.1|cytochrome CS DS LL LN
P450, putative
[Arabidopsis thaliana]
317 73463 CGPG6384 0 100 gi|22977164| ref|ZP_00022985.1|COG0538: DS
Isocitrate
dehydrogenases [Ralstonia
metallidurans]
318 72081 CGPG5279 7.00E − 66 77 gi|42570373| ref|NP_850277.2|CCAAT- DS PEG
box binding transcription
factor, putative [Arabidopsis
thaliana]
319 10139 CGPG101 0 89 gi|15229877| ref|NP_187154.1|sodium DS
proton exchanger, putative
(NHX2) [Arabidopsis
thaliana]
320 11410 CGPG103 0 82 gi|15236418| ref|NP_192555.1|homeobox DS
protein knotted-1 like 1
(KNAT1) [Arabidopsis
thaliana]
321 11604 CGPG48 0 92 gi|15233457| ref|NP_194642.1|hexokinase DS
1 (HXK1) [Arabidopsis
thaliana]
322 12368 CGPG1006 1.00E − 146 85 gi|15231451| ref|NP_190238.1|epsin N- DS
terminal homology (ENTH)
domain-containing protein/
clathrin assembly protein-
related [Arabidopsis
thaliana]
323 13502 CGPG1354 0 95 gi|15224557| ref|NP_180632.1|serine/ DS PP
threonine protein kinase,
putative [Arabidopsis
thaliana]
324 13745 CGPG1576 1.00E − 112 84 gi|15222987| ref|NP_177749.1|hypothetical DS
protein [Arabidopsis
thaliana] gb[AAF17642.1|
T23E18.15 [Arabidopsis
thaliana]
325 13821 CGPG1569 1.00E − 155 85 gi|18416499| ref|NP_567716.1|expressed DS
protein [Arabidopsis
thaliana]
326 14240 CGPG1697 0 94 gi|15241302| ref|NP_197527.1|expressed DS
protein [Arabidopsis
thaliana]
327 14718 CGPG1082 0 86 gi|18407200| ref|NP_566090.1|expressed DS
protein [Arabidopsis
thaliana]
328 17022 CGPG1774 1.00E − 159 100 gi|15237803| ref|NP_197755.1|nodulin DS
MtN3 family protein
[Arabidopsis thaliana]
329 17924 CGPG2882 3.00E − 92 100 gi|15233350| ref|NP_192875.1|zinc finger DS
(C3HC4-type RING finger)
family protein (RHA1b)
[Arabidopsis thaliana]
330 18259 CGPG3368 2.00E − 94 88 gi|30685085| ref|NP_849549.1|zinc finger DS PP
protein (LSD1) [Arabidopsis
thaliana]
331 19171 CGPG2952 0 91 gi|6320063| ref|NP_010143.1|plasma DS
membrane glucose sensor;
Rgt2p [Saccharomyces
cerevisiae]
332 19201 CGPG2332 0 96 gi|15233948| ref|NP_194205.1|protein DS
kinase (AFC2) [Arabidopsis
thaliana]
sp|P51567|AFC2_ARATH
Protein kinase
333 19317 CGPG3662 1.00E − 151 91 gi|21232858| ref|NP_638775.1|conserved DS
hypothetical protein
[Xanthomonas campestris
pv. campestris str. ATCC
33913]
334 70417 CGPG3427 0 81 gi|18396278| ref|NP_566180.1|integral DS PP SP
membrane family protein
[Arabidopsis thaliana]
335 70467 CGPG3785 0 100 gi|15241416| ref|NP_196953.1|no apical DS
meristem (NAM) family
protein [Arabidopsis
thaliana]
336 70806 CGPG712 0 100 gi|15218225| ref|NP_173010.1|cyclin, DS
putative [Arabidopsis
thaliana]
337 70818 CGPG479 1.00E − 157 92 gi|30691978| ref|NP_568508.2|bZIP DS
transcription factor family
protein [Arabidopsis
thaliana]
338 70820 CGPG655 0 93 gi|15224342| ref|NP_181899.1|acyl-[acyl- DS
carrier-protein] desaturase/
stearoyl-ACP desaturase
(SSI2) [Arabidopsis
thaliana]
339 70919 CGPG4029 1.00E − 169 73 gi|6996560| emb|CAB75429.1|oligouridylate DS
binding protein
[Nicotiana plumbaginifolia]
340 71623 CGPG4696 1.00E − 150 95 gi|15236511| ref|NP_192588.1|mitogen- DS
activated protein kinase,
putative [Arabidopsis
thaliana] pir||T01835
serine/threonine-specific
protein kinase ARA.KIN
homolog T15F16.3-
Arabidopsis thaliana
341 71662 CGPG4679 1.00E − 173 91 gi|5929964| gb|AAD56659.1|malate DS
dehydrogenase [Glycine
max]
342 71693 CGPG4652 6.00E − 86 56 gi|21553460| gb|AAM62553.1|snap25a DS
[Arabidopsis thaliana]
343 72384 CGPG4639 0 98 gi|1169548| sp|P38604|ERG7_YEAST DS
Lanosterol synthase
(Oxidosqualene-lanosterol
cyclase) (2,3-
epoxysqualene-lanosterol
cyclase) (OSC)
gb|AAA64377.1|2,3-
oxidosqualene-lanosterol
cyclase
344 72439 CGPG5075 8.00E − 57 90 gi|22331337| ref|NP_683594.1|NPR1/NI DS
M1-interacting protein 2
(NIMIN-2)
345 72619 CGPG4835 6.00E − 83 88 gi|15237317| ref|NP_200108.1|expressed DS
protein [Arabidopsis
thaliana]
346 72624 CGPG4842 0 100 gi|15242148| ref|NP_200558.1|expressed DS
protein [Arabidopsis
thaliana]
347 72715 CGPG5521 0 91 gi|6323933| ref|NP_014004.1|Carboxy- DS SS
terminal domain (CTD)
phosphatase, essential for
dephosphorylation of the
repeated C-terminal
domain of the RNA
polymerase II large subunit
(Rpo21p); Fcp1p
[Saccharomyces
cerevisiae]
348 72754 CGPG5548 1.00E − 169 100 gi|728961| sp|Q00618|BET4_YEAST DS
Geranylgeranyl transferase
type II alpha subunit (Type
II protein geranyl-
349 72819 CGPG4989 0 100 gi|18417026| ref|NP_567780.1|pfkB-type DS
carbohydrate kinase family
protein [Arabidopsis
thaliana]
350 75516 CGPG7689 1.00E − 138 70 gi|42568081| ref|NP_197938.2|zinc finger DS
(C3HC4-type RING finger)
family protein [Arabidopsis
thaliana]
351 75701 CGPG7856 8.00E − 41 37 gi|15225413| ref|NP_182037.1|zinc finger DS LN
(C2H2 type) family protein
[Arabidopsis thaliana]
352 73515 CGPG6473 1.00E − 162 100 gi|16079626| ref|NP_390450.1|similar to HS CS PEG
6-phosphogluconate
dehydrogenase (pentose
phosphate) [Bacillus
subtilis]
353 74684 CGPG6360 1.00E − 64 100 gi|18390735| ref|NP_563782.1|expressed CS HS
protein [Arabidopsis
thaliana]
354 19542 CGPG3069 0 90 gi|18403574| ref|NP_564592.1|F-box HS
family protein [Arabidopsis
thaliana]
355 19618 CGPG3574 1.00E − 121 100 gi|15218423| ref|NP_177373.1|trypsin HS
and protease inhibitor
family protein/Kunitz
family protein [Arabidopsis
thaliana] pir||F96746
probable drought induced
protein
356 19649 CGPG3140 1.00E − 141 87 gi|18412787| ref|NP_567287.1|vesicle- HS
associated membrane
family protein/VAMP
family protein
357 19745 CGPG3973 2.00E − 60 46 gi|15239303| ref|NP_201424.1|expressed HS
protein [Arabidopsis
thaliana]
358 19768 CGPG4096 1.00E − 179 81 gi|25052804| gb|AAN65180.1|mitogen- HS SS
activated protein kinase 4
[Petroselinum crispum]
359 19772 CGPG3939 3.00E − 82 79 gi|7488744| pir||T09700MADS-box HS
protein - alfalfa (fragment)
gb|AAB51377.1|MADS-box
protein [Medicago sativa]
360 19779 CGPG4113 1.00E − 153 89 gi|30681126| ref|NP_196201.2|phosphate CS HS
translocator-related
[Arabidopsis thaliana]
361 19833 CGPG4074 1.00E − 107 79 gi|6683777| gb|AAF23363.1|CAGL2 CS HS PP
[Cucumis sativus]
362 19862 CGPG3961 2.00E − 89 56 gi|15229637| ref|NP_188469.1|no apical HS
meristem (NAM) family
protein [Arabidopsis
thaliana] dbj|BAB01106.1|
unnamed protein product
[Arabidopsis thaliana]
363 19879 CGPG4009 0 75 gi|18401703| ref|NP_564504.1|protein HS CS SS
phosphatase 2C-related/
PP2C-related [Arabidopsis
thaliana]
364 70445 CGPG3728 2.00E − 51 88 gi|30696602| ref|NP_200357.2|protease HS
inhibitor/seed storage/lipid
transfer protein (LTP)
family protein [Arabidopsis
thaliana]
365 70738 CGPG3195 1.00E − 96 100 gi|15234797| ref|NP_194791.1|expressed HS PP
protein [Arabidopsis
thaliana]
366 71437 CGPG4043 1.00E − 164 81 gi|15241535| ref|NP_196433.1|serine/ HS
threonine protein kinase,
putative [Arabidopsis
thaliana]
367 71572 CGPG4520 3.00E − 83 92 gi|18403850| ref|NP_565804.1|expressed HS
protein [Arabidopsis
thaliana]
368 71617 CGPG1227 0 100 gi|15236219| ref|NP_195218.1|1- HS CK
phosphatidylinositol
phosphodiesterase-related
[Arabidopsis thaliana]
369 72532 CGPG4780 1.00E − 118 90 gi|15236659| ref|NP_194120.1|expressed HS
protein [Arabidopsis
thaliana]
370 72757 CGPG5572 0 89 gi|15242402| ref|NP_197088.1|zinc finger HS LL PEG
protein CONSTANS (CO)
[Arabidopsis thaliana]
371 73412 CGPG6448 0 99 gi|28867589| ref|NP_790208.1|glutamine HS
synthetase, type I
[Pseudomonas syringae pv.
tomato str. DC3000]
372 74102 CGPG6550 1.00E − 167 94 gi|15614187| ref|NP_242490.1|L- HS
asparaginase [Bacillus
halodurans C-125]
373 72633 CGPG4853 1.00E − 145 86 gi|15238013| ref|NP_199519.1|casein CS LL PEG
kinase II beta chain,
putative [Arabidopsis
thaliana]
374 72456 CGPG4745 1.00E − 75 92 gi|15239846| ref|NP_196763.1|17.6 kDa DS LL
class II heat shock protein
(HSP17.6-CII) [Arabidopsis
thaliana]
375 72963 CGPG1746 1.00E − 151 87 gi|15222239| ref|NP_172174.1|ovate LL LN
family protein [Arabidopsis
thaliana]
376 70426 CGPG3199 8.00E − 54 88 gi|18397268| ref|NP_564336.1|double- LL
stranded DNA-binding
family protein [Arabidopsis
thaliana]
377 70772 CGPG4627 1.00E − 92 80 gi|15220084| ref|NP_173175.1|MADS- LL
box protein (AGL100)
[Arabidopsis thaliana] P
378 71137 CGPG125 1.00E − 111 90 gi|15218957| ref|NP_176202.1|two- LL
component responsive
regulator/response
regulator 3 (ARR3)
[Arabidopsis thaliana]
379 71529 CGPG2808 1.00E − 131 72 gi|42562375| ref|NP_174152.3|Dof-type LL
zinc finger domain-
containing protein
[Arabidopsis thaliana]
380 71601 CGPG1858 1.00E − 168 92 gi|15231425| ref|NP_187378.1|transcriptional LL
activator, putative
[Arabidopsis thaliana]
381 72362 CGPG983 1.00E − 163 95 gi|15242779| ref|NP_200562.1|xyloglucan: LL
xyloglucosyl transferase,
putative/xyloglucan
endotransglycosylase,
putative/endo-xyloglucan
transferase, putative
[Arabidopsis thaliana]
382 72466 CGPG4767 3.00E − 60 83 gi|15234046| ref|NP_195030.1|glutaredoxin CK LL PEG
family protein
[Arabidopsis thaliana]
383 72524 CGPG4770 1.00E − 134 91 gi|18412649| ref|NP_567140.1|expressed CK LL
protein [Arabidopsis
thaliana]
384 73085 CGPG5689 1.00E − 134 100 gi|16331347| ref|NP_442075.1|triosephos- LL
phate isomerase
[Synechocystis sp. PCC
6803]
385 74241 CGPG5457 0 89 gi|444790| prf||1908224Anucleotide LL
translocator
386 74247 CGPG5475 1.00E − 159 100 gi|18411863| ref|NP_565172.1|protein LL
phosphatase 2C, putative/
PP2C, putative
[Arabidopsis thaliana]
387 74284 CGPG5413 0 97 gi|15230577| ref|NP_190087.1|serine LL
carboxypeptidase III,
putative [Arabidopsis
thaliana]
388 74652 CGPG6168 1.00E − 90 80 gi|15235970| ref|NP_194879.1|expressed LL DS
protein [Arabidopsis
thaliana]
389 70437 CGPG3706 1.00E − 172 100 gi|30678824| ref|NP_186983.2|short- CK LN
chain
dehydrogenase/reductase
(SDR) family protein
[Arabidopsis thaliana]
390 71633 CGPG857 1.00E − 100 86 gi|6690274| gb|AAF24061.1|v-SNARE DS LN
AtVTI1a [Arabidopsis
thaliana]
391 72948 CGPG5617 0 94 gi|15225456| ref|NP_182059.1|leucine- LN PEG
rich repeat transmembrane
protein kinase, putative
[Arabidopsis thaliana]
392 72519 CGPG4749 LN SS
393 10475 CGPG399 1.00E − 164 96 gi|15240972| ref|NP_195761.1|stress- LN
responsive protein, putative
[Arabidopsis thaliana]
394 11120 CGPG459 0 100 gi|15227169| ref|NP_179812.1|inositol-3- LN
phosphate synthase
isozyme 2/myo-inositol-1-
phosphate synthase 2/MI-
1-P synthase 2/IPS 2
[Arabidopsis thaliana]
395 19736 CGPG4129 2.00E − 94 67 gi|13346194| gb|AAK19619.1|GHMYB9 LN
[Gossypium hirsutum]
396 71606 CGPG4715 0 91 gi|15218674| ref|NP_171800.1|phototropic- LN
responsive NPH3 family
protein [Arabidopsis
thaliana]
397 71840 CGPG4353 0 96 gi|18401087| ref|NP_566542.1|mitotic DS LL LN
phosphoprotein N′ end
(MPPN) family protein
[Arabidopsis thaliana]
398 74240 CGPG5454 1.00E − 155 90 gi|15233884| ref|NP_194188.1|mitochondrial CK LN
substrate carrier family
protein [Arabidopsis
thaliana] pir||T05577
uncoupling protein homolog
F22K18.230 - Arabidopsis
thaliana
399 74331 CGPG5834 0 94 gi|15220416| ref|NP_172003.1|protein LN
kinase family protein
[Arabidopsis thaliana]
400 74610 CGPG6048 1.00E − 117 100 gi|15217568| ref|NP_172434.1|Ras- LL LN
related GTP-binding
protein, putative
[Arabidopsis thaliana]
sp|O04486|RB1C_ARATH
Ras-related protein
Rab11C
401 75527 CGPG7682 1.00E − 55 65 gi|15240946| ref|NP_195750.1|phosphatidyl- LN
ethanolamine-binding
family protein [Arabidopsis
thaliana]
402 70681 CGPG4584 9.00E − 64 93 gi|18411465| ref|NP_567196.1|auxin- CK PEG
responsive family protein
[Arabidopsis thaliana]
403 71663 CGPG4638 0 93 gi|21230153| ref|NP_636070.1|conserved CK PEG
hypothetical protein
[Xanthomonas campestris
pv. campestris str. ATCC
33913]
404 72769 CGPG5573 0 100 gi|15225499| ref|NP_182075.1|cytochrome CK PEG
P450, putative
[Arabidopsis thaliana]
405 71508 CGPG1541 2.00E − 24 100 gi|15241504| sp|Q9SD80|OM05_ARATH PEG CS SP PEG
Mitochondrial import
receptor subunit TOM5
homolog (Translocase of
outer membrane 5 kDa
subunit homolog)
406 74248 CGPG5476 0 100 gi|15226152| ref|NP_180926.1|protein PEG CS
phosphatase 2C, putative/
PP2C, putative
[Arabidopsis thaliana] p
407 72771 CGPG2166 5.00E − 44 100 gi|18395032| ref|NP_564151.1|expressed PEG CK HS SS
protein [Arabidopsis
thaliana]
408 72085 CGPG5228 0 94 gi|15241541| ref|NP_199275.1|cytochrome PEG HS
P450 family protein
[Arabidopsis thaliana]
dbj|BAA98115.1|flavonoid
3′,5′-hydroxylase-like;
cytochrome P450
[Arabidopsis thaliana]
409 72744 CGPG5563 1.00E − 136 96 gi|6321574| ref|NP_011651.1|20S HS PEG CK
proteasome beta-type
subunit; the only
nonessential 20S subunit;
Pre9p [Saccharomyces
cerevisiae]
410 73039 CGPG810 0 96 gi|15242124| ref|NP_197599.1|molybdopterin HS PEG
biosynthesis CNX1
protein/molybdenum
cofactor biosynthesis
enzyme CNX1 (CNX1)
[Arabidopsis thaliana]
411 73054 CGPG5754 2.00E − 98 100 gi|6324827| ref|NP_014896.1|Nat5p PEG HS SS PEG
[Saccharomyces
cerevisiae] pir||S67150
hypothetical protein
YOR253w - yeast
(Saccharomyces
cerevisiae)
412 73501 CGPG6456 0 97 gi|15888752| ref|NP_354433.1|AGR_C_2631p HS PEG
[Agrobacterium
tumefaciens str. C58]
sp|Q8UFH1|ENO_AGRT5
Enolase (2-
phosphoglycerate
dehydratase) (2-phospho-
D-glycerate hydro-lyase)
413 19707 CGPG4179 1.00E − 86 49 gi|15236282| ref|NP_195242.1|O- PEG CS
methyltransferase family 2
protein [Arabidopsis
thaliana]
414 19951 CGPG3941 5.00E − 91 54 gi|15221582| ref|NP_177064.1|basic PEG CK
helix-loop-helix (bHLH)
family protein [Arabidopsis
thaliana]
415 19967 CGPG4032 1.00E − 127 67 gi|4760710| dbj|BAA77395.1|SLL2-S9- PEG
protein [Brassica rapa]
416 70543 CGPG3815 0 95 gi|15220994| ref|NP_175222.1|E2F PEG CK
transcription factor-2
(E2F2)/transcription factor
E2Fc (E2Fc) [Arabidopsis
thaliana]
417 70707 CGPG1273 1.00E − 108 100 gi|15219558| ref|NP_177523.1|Ssu72- PEG
like family protein
[Arabidopsis thaliana]
pir||F96765 unknown
protein F
418 70719 CGPG1712 0 86 gi|18394560| ref|NP_564043.1|expressed PEG
protein [Arabidopsis
thaliana]
419 71134 CGPG817 8.00E − 55 100 gi|15240471| ref|NP_200327.1|small PEG HS PP
ubiquitin-like modifier 2
(SUMO) [Arabidopsis
thaliana]
420 71146 CGPG2928 1.00E − 86 92 gi|29165403| gb|AAO65311.1|MADS PEG
affecting flowering 3 variant
II [Arabidopsis thaliana]
421 71660 CGPG4690 3.00E − 80 100 gi|18415773| ref|NP_567637.1|methionine PEG
sulfoxide reductase
domain-containing protein/
SelR domain-containing
protein [Arabidopsis
thaliana]
422 72086 CGPG5236 0 100 gi|15232215| ref|NP_191556.1|methylenetetra- PEG PP PEG
hydrofolate reductase
1 (MTHFR1) [Arabidopsis
thaliana] -
423 72632 CGPG4852 4.00E − 99 90 gi|18425032| ref|NP_569028.1|expressed PEG
protein [Arabidopsis
thaliana]
424 72716 CGPG5529 2.00E − 85 89 gi|6320196| ref|NP_010276.1|subunit of PEG
the Anaphase Promoting
Complex; all known APC
subunits co-
immunoprecipitate with
epitope-tagged Apc11p;
Apc11p [Saccharomyces
cerevisiae]
425 72723 CGPG1848 0 97 gi|15237500| ref|NP_199487.1|human PEG
Rev interacting-like family
protein/hRIP family protein
[Arabidopsis thaliana]
dbj|BAB08919.1|zinc finger
protein Glo3-like
[Arabidopsis thaliana]
426 72987 CGPG1787 5.00E − 81 77 gi|15231568| ref|NP_189282.1|octicosapep- PEG SP
tide/Phox/Bem1p (PB1)
domain-containing protein
[Arabidopsis thaliana]
427 74109 CGPG6606 0 77 gi|37524479| ref|NP_927823.1|maltodextrin PEG
phosphorylase
[Photorhabdus luminescens
subsp. laumondii TTO1]
428 74140 CGPG6569 0 99 gi|15613102| ref|NP_241405.1|NADP- PEG PP PEG
dependent aldehyde
dehydrogenase [Bacillus
halodurans C-125]
429 74191 CGPG6597 0 96 gi|22960294| ref|ZP_00007935.1|COG1850: PEG
Ribulose 1,5-
bisphosphate carboxylase,
large subunit [Rhodobacter
sphaeroides]
430 74265 CGPG5356 1.00E − 117 100 gi|15237288| ref|NP_197727.1|GRAM PEG PP PEG
domain-containing protein/
ABA-responsive protein-
related [Arabidopsis
thaliana]
431 74369 CGPG6076 2.00E − 86 96 gi|18409647| ref|NP_564994.1|ubiquitin- PEG CK PP PEG
conjugating enzyme family
protein [Arabidopsis
thaliana]
432 70217 CGPG6 0 97 gi|15231536| ref|NP_189259.1|cytochrome CK PP SP
P450 family protein
[Arabidopsis thaliana]
433 72711 CGPG1846 4.00E − 75 79 gi|15221048| ref|NP_175816.1|transcription CK PP SP
initiation factor IID
(TFIID) 31 kDa subunit
(TAFII-31) family protein
[Arabidopsis thaliana]
434 70932 CGPG4089 1.00E − 129 56 gi|15223134| ref|NP_177792.1|expressed CS HS PP
protein [Arabidopsis
thaliana]
435 73518 CGPG6497 1.00E − 177 63 gi|22981996| ref|ZP_00027327.1|COG1012: CS CK PP
NAD-dependent
aldehyde dehydrogenases
[Burkholderia fungorum]
436 19771 CGPG4011 3.00E − 90 80 gi|18418200| ref|NP_568342.1|rubredoxin PP HS SS
family protein
[Arabidopsis thaliana]
dbj|BAB10504.1|
gene_id:MKP11.2˜unknown
protein [Arabidopsis
thaliana] g
437 73549 CGPG6460 0 90 gi|37524978| ref|NP_928322.1|5- HS DS PP
carboxymethyl-2-
hydroxymuconate
semialdehyde
dehydrogenase
[Photorhabdus luminescens
subsp. laumondii TTO1]
438 72994 CGPG5803 0 83 gi|6322702| ref|NP_012776.1|Vacuolar CK PEG CS PP PEG
transporter, exports large
neutral amino acids from
the vacuole; member of a
family of seven S.
cerevisiae genes (AVT1-7)
related to vesicular GABA-
glycine transporters; Avt3p
[Saccharomyces
cerevisiae]
439 71928 CGPG1617 0 100 gi|18394888| ref|NP_564120.1|catalase 3 CS PEG CK PP PEG
(SEN2) [Arabidopsis
thaliana]
440 72903 CGPG5584 0 91 gi|6322293| ref|NP_012367.1|Histone PEG PP SS
methyltransferase with a
role in transcriptional
elongation, methylates a
lysine residue of histone
H3; associates with the C-
terminal domain of Rpo21p;
histone methylation activity
is regulated by
phosphorylation status of
Rpo21p; Set2p
[Saccharomyces
cerevisiae]
sp|P46995|SET2_YEAST
SET domain protein 2
441 73017 CGPG5733 0 94 gi|6325368| ref|NP_015436.1|kinase PEG PP PEG
required for late nuclear
division; Dbf20p
[Saccharomyces
cerevisiae]
442 74587 CGPG6774 0 94 gi|17938451| ref|NP_535240.1|succinate PEG DS HS PP SS
semialdehyde
dehydrogenase
[Agrobacterium
tumefaciens str. C58]
443 72453 CGPG4735 6.00E − 67 91 gi|15218924| ref|NP_174236.1|auxin- CK PP SP SS
responsive family protein
[Arabidopsis thaliana]
pir||A86417 probable auxin-
induced protein, 45653-
45228
444 72967 CGPG5742 0 99 gi|6321525| ref|NP_011602.1|Cytosolic CS CK HS LL PP SS
catalase T, has a role in
protection from oxidative
damage by hydrogen
peroxide; Ctt1p
[Saccharomyces
cerevisiae]
445 72961 CGPG5591 0 95 gi|15228498| ref|NP_186975.1|UTP- PEG SS HS PP
glucose-1-phosphate
uridylyltransferase, putative/
UDP-glucose
pyrophosphorylase,
putative/UGPase, putative
[Arabidopsis thaliana]
446 73070 CGPG5627 0 90 gi|15225044| ref|NP_181451.1|protein PEG PP SS
kinase family protein
[Arabidopsis thaliana]
447 73475 CGPG6385 0 100 gi|39934021| ref|NP_946297.1|glyceraldehyde- PEG PP SS
3-phosphate
dehydrogenase(GAPDH)
[Rhodopseudomonas
palustris CGA009]
448 72916 CGPG1814 0 97 gi|15228871| ref|NP_188303.1|protein PP SS
phosphatase 2C, putative/
PP2C, putative
[Arabidopsis thaliana]
449 72969 CGPG5789 0 94 gi|6321886| ref|NP_011962.1|Low- PP SP SS
affinity glucose transporter
of the major facilitator
superfamily, expression is
induced by Hxk2p in the
presence of glucose and
repressed by Rgt1p when
glucose is limiting; Hxt1p
[Saccharomyces
cerevisiae]
450 74449 CGPG6659 0 96 gi|15890426| ref|NP_356098.1|AGR_L_619p PP SS
[Agrobacterium
tumefaciens str. C58]
pir||A98170 hypothetical
protein AGR_L_619
[imported] - Agrobacterium
451 16615 CGPG2539 0 98 gi|15890896| ref|NP_356568.1|AGR_L_ PP
1560p [Agrobacterium
tumefaciens str. C58]
ref|NP_534561.1|glucose-
1-phosphate
adenylyltransferase
[Agrobacterium
tumefaciens str. C58]
452 19187 CGPG3310 0 91 gi|18423163| ref|NP_568731.1|squamosa PP
promoter-binding protein,
putative [Arabidopsis
thaliana]
453 19648 CGPG3134 1.00E − 179 96 gi|18413950| ref|NP_568102.1|short- PP
chain
dehydrogenase/reductase
(SDR) family protein
[Arabidopsis thaliana]
454 70354 CGPG3995 0 64 gi|15241312| ref|NP_196916.1|nodulin DS PP SP
family protein [Arabidopsis
thaliana]
455 70421 CGPG2942 0 88 gi|30677977| ref|NP_178317.2|zinc finger PP
(C2H2 type) family protein
[Arabidopsis thaliana]
456 70459 CGPG3758 0 95 gi|15233315| ref|NP_188242.1|F-box PP
family protein [Arabidopsis
thaliana] dbj|BAB01261.1|
unnamed protein product
[Arabidopsis thaliana]
457 70465 CGPG3775 1.00E − 155 90 gi|15236937| ref|NP_195254.1|zinc finger PP SP
(C2H2 type) family protein
[Arabidopsis thaliana]
458 70683 CGPG4587 3.00E − 65 64 gi|18423239| ref|NP_568751.1|polyadenylate- PP
binding protein,
putative/PABP, putative
[Arabidopsis thaliana]
459 70725 CGPG2097 0 91 gi|18420505| ref|NP_568066.1|expressed CS PP
protein [Arabidopsis
thaliana]
460 70852 CGPG1465 0 93 gi|15237075| ref|NP_195290.1|isocitrate PP SP
dehydrogenase, putative/
NAD+ isocitrate
dehydrogenase, putative
[Arabidopsis thaliana]
461 71112 CGPG934 1.00E − 130 94 gi|15218701| ref|NP_171806.1|expressed CS PP
protein [Arabidopsis
thaliana] pir||E86161
F1003.11 protein-
[Arabidopsis thaliana]
gb|AAD25802.1|Belongs to
the PF|01027
Uncharacterized protein
family UPF0005 with 7
transmembrane domains.
[Arabidopsis thaliana]
462 71127 CGPG945 0 97 gi|15225307| ref|NP_179604.1|26S PP
protease regulatory
complex subunit 4, putative
[Arabidopsis thaliana]
pir||E84585 26S
proteasome subunit 4
[imported]- Arabidopsis
thaliana
463 71132 CGPG1561 0 98 gi|15232209| ref|NP_191550.1|expressed PP
protein [Arabidopsis
thaliana]
464 71217 CGPG95 0 100 gi|15221476| ref|NP_172127.1|shaggy- PP
related protein kinase iota/
ASK-iota (ASK9) (GSK1)
[Arabidopsis thaliana]
(EC 2.7.1.-)
465 71645 CGPG4688 3.00E − 69 100 gi|18401105| ref|NP_566544.1|phosphotransfer PP
family protein
[Arabidopsis thaliana]
466 71726 CGPG3894 0 93 gi|15217677| ref|NP_171725.1|no apical HS PP
meristem (NAM) family
protein [Arabidopsis
thaliana]
467 72432 CGPG4562 1.00E − 144 92 gi|20152540| emb|CAD29662.1|putative PP SP
auxin response factor 23
[Arabidopsis thaliana]
468 72450 CGPG4732 1.00E − 170 100 gi|15238890| ref|NP_197366.1|zinc finger LL PP
(C3HC4-type RING finger)
family protein [Arabidopsis
thaliana]
469 72455 CGPG4742 1.00E − 144 93 gi|15242893| ref|NP_200597.1|anthranilate PP PEG
synthase beta subunit,
putative [Arabidopsis
thaliana]
470 72727 CGPG5522 1.00E − 118 100 gi|6324107| ref|NP_014177.1|functionally PP
related to TFIIB, affects
start site selection in vivo;
Ssu72p [Saccharomyces
cerevisiae]
471 72817 CGPG4987 0 96 gi|30679158| ref|NP_567238.2|AAA-type PP
ATPase family protein
[Arabidopsis
472 72992 CGPG5777 0 90 gi|6324981| ref|NP_015049.1|S- PP PEG
adenosylMethionine
Permease; Sam3p
[Saccharomyces
cerevisiae]
473 73007 CGPG5760 0 93 gi|6320865| ref|NP_010944.1|One of PP
three possible beta-
subunits of the Snf1 kinase
complex, allows nuclear
localization of the Snf1
kinase complex in the
presence of a
nonfermentable carbon
source; contains glycogen-
binding domain; Gal83p
[Saccharomyces
cerevisiae]
474 73073 CGPG5688 0 95 gi|16331010| ref|NP_441738.1|fructose PP
1,6-bisphosphatase
[Synechocystis sp. PCC
6803]
475 73506 CGPG6496 0 96 gi|23062569| ref|ZP_00087347.1|COG1012: PP
NAD-dependent
aldehyde dehydrogenases
[Pseudomonas fluorescens
PfO-1]
476 74107 CGPG6590 0 95 gi|15965198| ref|NP_385551.1|PYRUVATE PP SS
DEHYDROGENASE
ALPHA2 SUBUNIT
PROTEIN [Sinorhizobium
meliloti 1021]
477 74117 CGPG6575 0 81 gi|37528116| ref|NP_931461.1|Phenylacetalde- CS PP
hyde dehydrogenase
(PAD) [Photorhabdus
luminescens subsp.
laumondii TTO1]
478 74131 CGPG6592 0 96 gi|16329404| ref|NP_440132.1|transaldolase PP SS
[Synechocystis sp.
PCC 6803] B -
479 74344 CGPG5929 1.00E − 111 100 gi|15236410| ref|NP_193147.1|COP9 HS PP
signalosome subunit,
putative/CSN subunit,
putative (CSN8)
[Arabidopsis thaliana]
480 14320 CGPG1229 0 100 gi|18418018| ref|NP_567894.1|expressed SP
protein [Arabidopsis
thaliana]
481 16756 CGPG2117 1.00E − 142 85 gi|18391249| ref|NP_563885.1|expressed SP
protein [Arabidopsis
thaliana]
482 17448 CGPG2673 1.00E − 102 72 gi|15239624| ref|NP_197993.1|PHD SP
finger family protein
[Arabidopsis thaliana]
gb|AAM64729.1|nucleic
acid binding protein-like
[Arabidopsis thaliana]
483 17633 CGPG2839 1.00E − 145 85 gi|18395124| ref|NP_564171.1|basic SP
helix-loop-helix (bHLH)
family protein [Arabidopsis
thaliana]
484 18876 CGPG3096 1.00E − 172 89 gi|18394949| ref|NP_564133.1|transporter- SP
related [Arabidopsis
thaliana] pir||G86343
hypothetical protein
T22I11.10
485 19120 CGPG1976 0 100 gi|15232345| ref|NP_188710.1|fertilization- SP
dependent endosperm
protein (FIE) [Arabidopsis
thaliana]
486 19221 CGPG2958 1.00E − 159 78 gi|30690446| ref|NP_182182.2|Dof zinc SP
finger protein DAG2/Dof
affecting germination 2
(DAG2) [Arabidopsis
thaliana]
487 70206 CGPG4116 1.00E − 139 64 gi|18412918| ref|NP_565249.1|phospholipid/ SP
glycerol acyltransferase
family protein [Arabidopsis
488 70223 CGPG53 0 93 gi|15240313| ref|NP_198006.1|hexose SP
transporter, putative
[Arabidopsis thaliana]
489 70347 CGPG3147 1.00E − 121 66 gi|18416267| ref|NP_567693.1|Dof-type SP
zinc finger domain-
containing protein
[Arabidopsis thaliana]
490 70406 CGPG1687 0 93 gi|18397470| ref|NP_564354.1|early- SP
responsive to dehydration
stress protein (ERD4)
[Arabidopsis thaliana]
491 70469 CGPG3791 1.00E − 171 89 gi|15237581| ref|NP_198936.1|MADS- SP HS
box family protein
[Arabidopsis thaliana]
492 70564 CGPG1864 0 89 gi|15219067| ref|NP_173589.1|SWIRM SP
domain-containing protein/
DNA-binding family protein
gb|AAD41423.1|Contains
similarity to gb|AF033823
moira protein from
Drosophila melanogaster
and contains a PF|00249
Myb-like DNA-binding
domain.
493 70601 CGPG2917 0 91 gi|15235140| ref|NP_193702.1|zinc finger SP PP
(C3HC4-type RING finger)
family protein [Arabidopsis
thaliana] pir||T04748
hypothetical protein
T16H5.30 - Arabidopsis
thaliana
494 70612 CGPG3721 0 96 gi|18416732| ref|NP_568256.1|conserved SP
oligomeric Golgi complex
component-related/COG
complex component-related
[Arabidopsis thaliana]
495 70720 CGPG1358 0 93 gi|15238483| ref|NP_198387.1|lectin SP
protein kinase family
protein [Arabidopsis
thaliana]
496 70735 CGPG2661 1.00E − 109 100 gi|15231241| ref|NP_187953.1|transcription SP
initiation factor IID-1
(TFIID-1)/TATA-box factor
1/TATA sequence-binding
protein 1 (TBP1)
[Arabidopsis thaliana]
497 70846 CGPG377 1.00E − 151 100 gi|15221223| ref|NP_177577.1|zinc finger SP
(C3HC4-type RING finger)
family protein [Arabidopsis
thaliana] pir||D96772
probable RING zinc finger
protein
498 70923 CGPG4020 0 87 gi|8132347| gb|AAF73257.1|MAP SP
kinase PsMAPK2 [Pisum
sativum]
499 71149 CGPG3457 0 83 gi|20141566| sp|P48001|HKL4_ARATH-
Homeobox protein knotted-1
like 4 (KNAT4) pir||T51795
HOMEOBOX PROTEIN
KNOTTED-1 LIKE 4
(KNAT4) -
500 71608 CGPG4687 0 100 gi|15220438| ref|NP_172008.1|ent- SP
kaurenoic acid hydroxylase
(KAO1)/cytochrome P450
88A3, putative (CYP88A3)
[Arabidopsis thaliana]
501 71739 CGPG4345 7.00E − 89 80 gi|18406944| ref|NP_566061.1|expressed SP
protein [Arabidopsis
thaliana]
502 72014 CGPG5230 0 100 gi|25410898| pir||D84423probable WD- SP
40-repeat protein [imported] -
Arabidopsis thaliana
gb|AAD14533.1|putative
stress protein [Arabidopsis
thaliana]
503 72051 CGPG5241 0 93 gi|18401606| ref|NP_566585.1|cyclic SP
nucleotide-binding
transporter 1/CNBT1
(CNGC20) [Arabidopsis
thaliana]
sp|Q9LD37|CG20_ARATH
Probable cyclic nucleotide-
gated ion channel 20,
chloroplast precursor
(Cyclic nucleotide-binding
transporter 1)
504 74259 CGPG5343 0 96 gi|15222882| ref|NP_175431.1|branched- CS HS SS
chain amino acid
aminotransferase 6/
branched-chain amino acid
transaminase 6 (BCAT6)
[Arabidopsis thaliana] s
505 72463 CGPG4760 8.00E − 48 100 gi|15236351| ref|NP_193115.1|auxin- CS SS HS LN PP
responsive protein, putative
[Arabidopsis thaliana]
506 72902 CGPG5597 0 88 gi|15240576| ref|NP_199800.1|chloride SS CS DS
channel protein (CLC-c)
[Arabidopsis thaliana]
sp|Q96282|CLCC_ARATH
Chloride channel protein
CLC-c (AtCLC-c)
507 74572 CGPG6640 1.00E − 109 93 gi|16331001| ref|NP_441729.1|unknown CS PP SS
protein [Synechocystis sp.
PCC 6803]
508 73055 CGPG5768 0 97 gi|6321588| ref|NP_011665.1|Hypothetical SS CS HS
ORF; Ygr149wp
[Saccharomyces
cerevisiae]
509 74103 CGPG6558 0 99 gi|15833050| ref|NP_311823.1|fructose- HS PP SS
bisphosphate aldolase
class II [Escherichia coli
O157:H7] r
510 72921 CGPG5781 0 93 gi|6322892| ref|NP_012965.1|general CK PEG SS
amino acid permease;
Gap1p [Saccharomyces
cerevisiae]
511 72968 CGPG5772 0 99 gi|6321546| ref|NP_011623.1|role in PEG LL SS
DNA replication during S
phase; Clb6p
[Saccharomyces
cerevisiae]
512 19703 CGPG4172 0 83 gi|7488676| pir||T07150G-box binding HS SS
factor 2A - soybean
(fragment) gb|AAB00097.1|
G-box binding factor
513 19946 CGPG4097 1.00E − 46 34 gi|15219099| ref|NP_175691.1|2- SS
oxoglutarate-dependent
dioxygenase, putative
[Arabidopsis thaliana]
514 19980 CGPG3914 2.00E − 63 49 gi|28629811| gb|AAO45179.1|transcription CS SS
factor Myb1 [Malus
xiaojinensis]
515 70435 CGPG3701 1.00E − 150 91 gi|15236597| ref|NP_193499.1|casein SS PP SP
kinase II beta chain,
putative [Arabidopsis
thaliana]
516 71114 CGPG1657 0 88 gi|30680729| ref|NP_849990.1|K+ efflux SS
antiporter, putative (KEA4)
[Arabidopsis thaliana]
517 72451 CGPG4733 0 94 gi|15239622| ref|NP_197992.1|mitochondrial SS
substrate carrier family
protein [Arabidopsis
thaliana]
518 72947 CGPG5607 3.00E − 62 53 gi|1483230| emb|CAA67968.1|MADS4 SS
protein [Betula pendula]
519 73012 CGPG5786 0 97 gi|6324187| ref|NP_014257.1|belongs SS
to a ubiquitous family of
cytoplasmic membrane
proteins that transport only
ammonium (NH(4)(+) +
NH(3)).; Mep2p
[Saccharomyces
cerevisiae]
520 73022 CGPG5622 0 86 gi|15225518| ref|NP_182083.1|protein SS
kinase family protein
[Arabidopsis thaliana]
521 73488 CGPG6394 1.00E − 154 94 gi|16080620| ref|NP_391447.1|UTP- SS CS PP
glucose-1-phosphate
uridylyltransferase [Bacillus
subtilis]
522 73901 CGPG5237 0 92 gi|18400284| ref|NP_565553.1|extra- SS
large guanine nucleotide
binding protein/G-protein
(XLG)
523 73964 CGPG5804 0 88 gi|6319773| ref|NP_009855.1|Na+/Pi SS
cotransporter, active in
early growth phase; similar
to phosphate transporters
of Neurospora crassa;
transcription regulated by
inorganic phosphate
concentrations and Pho4p;
Pho89p [
524 74019 CGPG5706 2.00E − 92 100 gi|16079815| ref|NP_390639.1|adenine SS
phosphoribosyltransferase
[Bacillus subtilis]
525 74022 CGPG5724 0 97 gi|18378991| ref|NP_563659.1|glycosyl SS SP
hydrolase family 3 protein
[Arabidopsis thaliana]
526 74114 CGPG6551 0 99 gi|15888903| ref|NP_354584.1|AGR_C_ SS
2921p [Agrobacterium
tumefaciens str. C58]
pir||H97551 probable
aminotransferase aatc
527 74262 CGPG5353 0 100 gi|18416245| ref|NP_568226.1|histidinol- SS PP
phosphate
aminotransferase, putative
[Arabidopsis thaliana]
528 74292 CGPG5367 0 96 gi|15239204| ref|NP_201393.1|U-box SS
domain-containing protein
[Arabidopsis thaliana]
529 74302 CGPG5384 1.00E − 59 82 gi|25313155| pir||A96787protein F10A5.6 PP SS
[imported] - Arabidopsis
thaliana
530 74325 CGPG5898 1.00E − 174 86 gi|15230382| ref|NP_188576.1|cinnamyl- SS
alcohol dehydrogenase
(CAD) [Arabidopsis
thaliana]
531 74429 CGPG6689 0 96 gi|16077873| ref|NP_388687.1|acetoin SS
dehydrogenase E1
component (TPP-
dependent alpha subunit)
[Bacillus subtilis]
532 74440 CGPG6682 6.00E − 90 82 gi|15613838| ref|NP_242141.1|uridine SS
kinase [Bacillus halodurans
C-125]
533 74462 CGPG6668 5.00E − 67 99 gi|16332127| ref|NP_442855.1|unknown HS SS
protein [Synechocystis sp.
PCC 6803]
534 74465 CGPG6692 1.00E − 119 99 gi|16078642| ref|NP_389461.1|similar to PP SS
ribulose-5-phosphate 3-
epimerase [Bacillus subtilis]
535 74474 CGPG6669 2.00E − 85 82 gi|16331209| ref|NP_441937.1|unknown LL SS
protein [Synechocystis sp.
PCC 6803]
536 74505 CGPG6783 0 100 gi|16129426| ref|NP_415984.1|cryptic SS
nitrate reductase 2 beta
subunit [Escherichia coli
K12]
537 74507 CGPG6799 0 82 gi|27479656| gb|AAO17183.1|Orf17 SP SS
[Photorhabdus
luminescens]
538 74562 CGPG6764 0 95 gi|16077501| ref|NP_388315.1|similar to SS
pyruvate oxidase [Bacillus
subtilis]
Please note that this file doesn't have Table 3.
Screens for Identifying Trait Improving Genes
DS—Improvement of drought tolerance identified by a soil drought stress tolerance screen: Drought is a water deficit condition that imposes osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage. The drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting. The drought tolerance-imparting DNA defined for this invention are used in recombinant DNA constructs that improve plant survival rate under drought conditions. Exemplary recombinant DNA which has been identified for conferring such drought tolerance is identified as such in Table 2. Such identified recombinant DNA is useful in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle. As demonstrated from the model plant screen, in some embodiments of transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.
PEG-Improvement of drought tolerance identified by PEG induced osmotic stress tolerance screen: Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits et al., (1995) Plant Physiol. 107:125-130). Several physiological characteristics have been reported as being reliable indications for selection of plants possessing drought tolerance. These characteristics include the rate of seed germination and seedling growth. The traits can be assayed relatively easily by measuring the growth rate of seedling in PEG solution. Thus, a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen. Certain embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen survive drought conditions providing a higher yield potential as compared to control plants.
SS-Improvement of drought tolerance identified by high salinity stress tolerance screen: Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought. Exemplary recombinant DNA which has been identified to help plants maintain biomass, root growth and/or plant development in high salinity conditions are identified as such in Table 2. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, embodiments of trait-improving recombinant DNA identified in a high salinity stress tolerance screen also provide transgenic crops with improved drought tolerance. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen survive drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.
HS-Improvement of drought tolerance identified by heat stress tolerance screen: Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified as heat stress tolerance conferring genes may also impart improved drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.
CK and CS-Improvement of tolerance to cold stress: Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential. Two screening conditions, i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS), were set up to look for transgenic plants that display visual growth advantage at lower temperature. In cold germination tolerance screen, the transgenic Arabidopsis plants were exposed to a constant temperature of 8 degrees C. from planting until day 28 post planting. The trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants. In cold shock tolerance screen, the transgenic plants were first grown under the normal growth temperature of 22 degrees C. until day 8 post planting, and subsequently were placed under 8 degrees C. until day 28 post planting. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen survive cold conditions providing a higher yield potential as compared to control plants.
Improvement of tolerance to multiple stresses: Different kinds of stresses often lead to identical or similar reaction in the plants. Genes that are activated or inactivated as a reaction to stress can either act directly in a way the genetic product reduces a specific stress, or they can act indirectly by activating other specific stress genes. By manipulating the activity of such regulatory genes, i.e., multiple stress tolerance genes, plants are enabled to react to different kinds of stresses. For examples, DNA for expressing proteins of SEQ ID NO:352 and SEQ ID NO:353 is useful to improve both heat stress tolerance and cold stress tolerance in plants. Plants transformed with DNa for expressing protein of SEQ ID NO:508 resist heat stress, salt stress and cold stress. Thus, the disclosed stress tolerance conferring genes are useful in combinations to generate transgenic plants that resist multiple stress conditions.
PP-Improvement of early plant growth and development: It is known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy vigorous plants. This means avoiding seed and seedling diseases, leading to increased nutrient uptake and increased yield potential. Traditionally early planting and applying fertilizer are the methods used for promoting early seedling vigor. In early development stage, plant embryos establish only the basic root-shoot axis, a cotyledon storage organ(s), and stem cell populations, called the root and shoot apical meristems, that continuously generate new organs throughout post-embryonic development. “Early growth and development” encompasses the stages of seed imbibition through the early vegetative phase. Certain DNA is identified as useful to produce transgenic plants that have advantages in one or more processes including, but not limited to, germination, seedling vigor, root growth and root morphology under non-stressed conditions. The transgenic plants starting from a more robust seedling are less susceptible to the fungal and bacterial pathogens that attach germinating seeds and seedling. Furthermore, seedlings with advantage in root growth are more resistant to drought stress due to extensive and deeper root architecture. Therefore, genes conferring the growth advantage in early stages to plants are used to generate transgenic plants that are more resistant to various stress conditions due to improved early plant development. Exemplary recombinant DNA that confers both stress tolerance and growth advantages to plants, is identified as such in Table 2, e.g., DNA encoding a protein of SEQ ID NO:444 can improve the plant early growth and development, and impart heat and cold tolerance to plants. Embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.
SP-Improvement of late plant growth and development: “Late growth and development” encompasses the stages of leaf development, flower production, and seed maturity. Transgenic plants with late growth and development advantages express DNA that is identified as such in Table 2. Such plants exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length. For example, the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant. Seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield. Embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
LL-Improvement of tolerance to shade stress identified in a low light screen: The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased. As the result, a plant under low light condition increases significantly its stem length at the expanse of leaf, seed or fruit and storage organ development, thereby adversely affecting of yield. Recombinant DNA that enables plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently provides embodiments of those plants with higher yield as compared to the wild type plants. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by this invention are suitable for a higher density planting, thereby resulting increased yield per unit area.
LN-Improvement of Tolerance to Low Nitrogen Availability Stress
Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves. All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.
Recombinant DNA that imparts enhanced nitrogen use efficiency in transgenic plants is identified in Table 2. Such plants exhibit one or more desirable traits including, but not limited to, increased seedling weight, increased number of green leaves, increased number of rosette leaves, altered root length and advanced flower bud formation. Such plants can also have altered amino acid or protein compositions, increased yield and/or better seed quality. Embodiments of such transgenic plants are productively cultivated under nitrogen nutrient deficient conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs that cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless under such conditions. The transgenic plants also are advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.
Stacked Traits: This invention also provides transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a glyphosate resistance trait, or insect resistance, such as using a gene from Bacillus thuringiensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides. To illustrate that the production of transgenic plants with herbicide resistance is a capability of those of ordinary skill in the art, reference is made to U.S. 2003-0106096 A1 and 2002-0112260 A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which are incorporated herein by reference. To illustrate that the production of transgenic plants with pest resistance is a capability of those of ordinary skill in the art reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plants expressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses control of invertebrates which feed on transgenic plants which express dsRNA for suppressing a target gene in the invertebrate, to U.S. Pat. No. 5,986,175 which discloses the control of viral pests by transgenic plants which express viral replicase, and to U.S. Patent Application Publication 2003/0150017 A1 which discloses control of pests by a transgenic plant which express a dsRNA targeted to suppressing a gene in the pest, all of which are incorporated herein by reference.
Once one recombinant DNA has been identified as conferring an improved trait of interest in transgenic Arabidopsis plants, several methods are available for using the sequence of that recombinant trait-imparting DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant and different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, this invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO:1 through SEQ ID NO:269, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO:270 through SEQ ID NO:538. In another aspect, this invention provides a consensus amino acid sequence for respective homologs for each of SEQ ID NO:270 through SEQ ID NO:538. In yet another aspect, this invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence disclosed herein.
The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by this invention are not limited to any particular plant species. Indeed, the plants of this invention encompass many species of monocots and dicots and include agriculturally useful plants which are cultivated for purposes of food production or industrial applications, e.g., corn and soybean plants and cotton plants. Recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are disclosed in the following examples. Other plants of this invention include canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
Thus, embodiments of this invention include the use of both DNA identified in Table 3 and homologs in recombinant DNA for transgenic crop plants with improved traits. Transgenic crop plants with improved traits are identified from populations of plants grown from transgenic events by screening to segregate the plants of this invention from plants without the improved traits. Preferred screens for transgenic crop plants identify plants with improved responses to stress conditions, e.g., assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures are made on greenhouse or field grown plants and include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.
Trait data on morphological changes is collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil. Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other enhanced traits are identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barrenness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain are confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
To confirm hybrid yield in transgenic corn plants expressing trait-imparting DNA of this invention, it is useful to test hybrid plants over multiple years at multiple locations in a geographical location where corn is conventionally grown, e.g., in Iowa, Illinois and Kansas, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.
Transgenic crop plants are used to provide other aspects of this invention such as transgenic seeds of crop plants. Seeds of transgenic plants are used to propagate more progeny plants which contain the trait-improving recombinant DNA constructs of this invention. These progeny plants are within the scope of this invention when they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.
Screening Methods for Crop Transgenic Plants with Enhanced Agronomic Trait
Due to variability in transformation many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. Thus, screening is necessary to identify the transgenic events that produce the transgenic plants and seeds of this invention. Transgenic crop plants having enhanced traits are identified from populations of plants transformed as described herein by evaluating the trait in a variety of assays to detect an enhanced agronomic trait. Useful assays include analyses to detect changes in the chemical composition, biomass, physiological properties and morphology of the plant. Changes in chemical compositions such as nutritional composition of grain are detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics are detected in greenhouse or field grown plants and include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties are identified by evaluating responses to stress conditions, e.g., assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology are measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other screening properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barrenness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain are evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
Seeds for transgenic crop plants with enhanced agronomic traits of this invention are corn, soybean and cotton seeds, as well as seeds for canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
A. Screening for Nitrogen Use Efficiency
Many transgenic crop plants of this invention exhibit enhanced nitrogen use efficiency as compared to control plants. Higher nitrogen soil applications increase seed protein and starch accumulation, and lead to larger seed weight and larger kernel number per ear. Recent improvements in elite high yielding corn hybrid genotypes include the ability to utilize nitrogen efficiently. DNA causing the enhanced nitrogen use efficiency in crop plants are especially useful, e.g., for improving yield. Enhanced nitrogen use efficiency is assessed by measuring changes in plant growth such as leaf area production, shoot biomass, chlorophyll content in plants grown in nitrogen limiting conditions and/or nitrogen sufficient conditions. It is useful to conduct a first screen in nitrogen limiting conditions and confirm replicate transgenic events in both nitrogen limiting and nitrogen sufficient conditions. Table 4 shows an amount of nutrients in the nutrient solution for nitrogen limiting conditions (low N) and nitrogen sufficient conditions (high N) which are useful for nitrogen use efficiency screening. For example in a greenhouse screen pots of transgenic plants and control plants are treated with 100 ml of nutrient solution three times a week on alternate days starting at 8 and 10 days after planting for high N and low N screening, respectively. TABLE 4
2 mM NH4NO3 20 mM NH4NO3
Nutrient stock Low nitrogen High nitrogen
1 M NH4N03 2 mL/L 20 mL/L
1 M KH2PO4 0.5 0.5
1 M MgSO4.7H2O 2 2
1 M CaCl2 2.5 2.5
1 M K2SO4 1 1
Note:
Adjust pH to 5.6 with HCl or KOH
After 28 days of plant growth for low N screening and 23 days for high N screening, measurements are taken for total shoot fresh mass, leaf chlorophyll, leaf area, leaf fresh mass and leaf dry mass.
B. Screening for Increased Yield
Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from a variety or other traits such as enhanced seed sink potential, e.g., the number and size of endosperm cells or kernels, and/or enhanced sink strength, e.g., the rate of starch biosynthesis. Sink potential is established very early during kernel development, as endosperm cell number and cell size are determined within the first few days after pollination.
Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.
Effective yield screening of transgenic corn uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Useful screening in multiple and diverse geographic locations, e.g., up to 16 or more locations, over one or more planting seasons, e.g., at least two planting seasons, is useful to statistically distinguish yield improvement from natural environmental effects. Useful hybrid screening includes planting multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, e.g., 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Plants from separate transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre.
Surrogate indicators for screening for yield improvement include source capacity (biomass), source output (sucrose and photosynthesis), sink components (kernel size, ear size, starch in the seed), development (light response, height, density tolerance), maturity, early flowering trait and physiological responses to high density planting, e.g., at 45,000 plants per acre.
When screening for yield improvement a useful statistical measurement approach comprises three components, i.e., modeling spatial autocorrelation of the test field separately for each location, adjusting traits of recombinant DNA events for spatial dependence for each location, and conducting an across location analysis.
A first step in modeling spatial autocorrelation is estimating the covariance parameters of the semivariogram. A spherical covariance model is assumed to model the spatial autocorrelation. Because of the size and nature of the trial, it is likely that the spatial autocorrelation may change. Therefore, anisotropy is also assumed along with spherical covariance structure. The following set of equations describes the statistical form of the anisotropic spherical covariance model.
where I(•) is the indicator function, h=√{square root over ({dot over (x)}2+{dot over (y)}2, and
{dot over (x)}=[cos(ρπ/180)(x1−x2)−sin(ρπ/180)(y1−y2)]/ωx
{dot over (y)}=[sin(ρπ/180)(x1−x2)+cos(ρπ/180)(y1−y2)]/ωy
where s1=(x1, y1) are the spatial coordinates of one location and s2=(x2, y2) are the spatial coordinates of the second location. There are 5 covariance parameters, θ=(v, σ2, ρ, ωn, ωj), where v is the nugget effect, σ2 is the partial sill, ρ is a rotation in degrees clockwise from north, ωn is a scaling parameter for the minor axis and ωj is a scaling parameter for the major axis of an anisotropical ellipse of equal covariance. The five covariance parameters that define the spatial trend will then be estimated by using data from heavily replicated pollinator plots via restricted maximum likelihood approach. In a multi-location field trial, spatial trend are modeled separately for each location.
After obtaining the variance parameters of the model, a variance-covariance structure is generated for the data set to be analyzed. This variance-covariance structure contains spatial information required to adjust yield data for spatial dependence. In this case, a nested model that best represents the treatment and experimental design of the study is used along with the variance-covariance structure to adjust the yield data. During this process the nursery or the seed batch effects can also be modeled and estimated to adjust the yields for any yield parity caused by seed batch differences.
After spatially adjusted data from different locations are generated, all adjusted data is combined and analyzed assuming locations as replications. In this analysis, intra and inter-location variances are combined to estimate the standard error of yield from transgenic plants and control plants. Relative mean comparisons are used to indicate statistically significant yield improvements.
C. Screening for Water Use Efficiency
Many transgenic crop plants of this invention exhibit improved yield resulting from improved water use efficiency and/or drought tolerance.
A greenhouse screen for transgenic corn plants for water use efficiency measures changes in plant growth rate, e.g., at least a 10% improvement, in height and biomass during a vegetative drought treatment, as compared to control plants. The hydration status of the shoot tissues following the drought is also measured. Shoot Initial Height (SIH) is plant height after 3 weeks of growth under optimum conditions. Shoot Wilt Height (SWH) is plant height at the end of a 6 day drought. Time course experiments have shown that at about 3 days of drought, wild type plants basically stop growing and begin to wilt. Thus a transgenic plant with improved water use efficiency will continue to grow (probably to a lesser extent than with water) and thereby end up as a significantly taller plant at the end of a drought experiment. Shoot Wilt Mass (SWM) is the amount of wet and dry matter in the shoot (plant separated from root ball at the soil line) at the end of the drought; SDM is measure after 2 to 3 weeks in a drying chamber. Shoot Turgid mass (STM) is the SWM plus the mass of the water that is transported into plant tissues in 3 days of soaking in 40 degree C. water in the dark. Experiments show that most of the water is pulled up in 24 hours but it takes 2 more days before additional increase becomes insignificant. STM-SWM is indicative of water use efficiency in plants where recovery from stress is more important than stress tolerance per se. Relative water content (RWC) is a measurement of how much (%) of the plant is water at harvest. RWC=(SWM−SDM)/(STM−SDM)*100. Fully watered corn plants are about 98% RWC. Typically, in a wilt screen the plants are about 60% RWC. Plants with higher RWC at the end of a drought are considered to be healthier plants and more fit for post-drought recovery and growth.
Relative Growth Rate (RGR) is calculated for each shoot using the formula RGR=(SWH−SIH)/((SWH+SIH)/2)*100
D. Screening for Growth Under Cold Stress
Many transgenic crop plants of this invention exhibit improved growth under cold stress, e.g., in a cold germination assay, in a cold shock assay, in an early seedling growth assay and in root-shoot biomass assay.
In a cold germination assay transgenic seeds from transgenic plants, e.g., R2 inbred seeds or F1 hybrid seeds, seeds of two types of control plants, e.g., negative segregants from the transgenic event or wild type, non-transgenic seeds of the transformed genotype, are treated with fungicide. A useful fungicide such as Captan fungicide (available from Arvesta Corp as MAESTRO® 80DF Fungicide) is applied at the rate of 0.43 mL Captan per 45 g of corn seeds which are dried to provide fungicide-coated seeds.
In a useful cold screen for transgenic corn seeds ten seeds per transgenic event are placed on filter paper (e.g., Whatman No. 1) in the lid of a Petri dish with 5 ml of water. A closed Petri dish is placed in a growth chamber set at 11 degrees C. for inbred corn seed or 9.5 degrees C. for hybrid corn seed. 2 ml of water is added on day 3 and day 10. Seeds are considered germinated if the emerged radicle size is 1 cm. Cold seeds are scored every 2 days from day 10 up to day 30. Tissue samples are collected at random on the last day of the experiment for confirmation of RNA expression. A germination index (GI) is calculated as
GI=(Σ([T+1−ni]*[Pi−Pi−1]))/T
where “T” is the number of days for the experiment, “n” is the number of days after start, “i” is number of times germination is counted including the current day, “P” is the percentage of seed germinated during any given rating. Statistical differences are calculated between positive and wild type control.
In a cold shock assay, seeds are planted in potting media and placed in a growth chamber set at 23 degrees C., relative humidity of 65% with 12 hour day and night photoperiod (300 uE/m2-min). Planted seeds are watered for 20 minute every other day by sub-irrigation and flats are rotated every third day. On day 10 after planting the transgenic positive and wild type control plants are positioned in flats in an alternating pattern. Chlorophyll fluorescence of plants is measured on the tenth day during the dark period of growth by using a Walz PAM-2000 portable fluorometer following manufacturer's instructions. After chlorophyll measurements, leaf samples from each event are collected for confirming the expression of recombinant DNA. The plants are then exposed to temperatures of 5 degrees C. for 4 days. On the fourth day chlorophyll fluorescence is measured and plants are restored to a 23 degrees C. environment for recovery over 3 days. During the recovery period the length of the V3 leaf is measured on the first and third days. After two days of recovery V2 leaf damage is determined visually by estimating percent of green V2 leaf. Statistical differences in V3 leaf growth, V2 leaf necrosis and fluorescence during pre-shock and cold shock can be used for estimation of cold shock damage on corn plants.
In an early seedling growth assay three sets of seeds are assayed. The first set is a group of transgenic seeds from transgenic plants; the second set is negative segregants of the transgenic seed; and the third seed set is seed from two cold tolerant and two cold sensitive wild-type controls. All seeds are treated with a fungicide as indicated above. Seeds are grown in germination paper (12 inch×18 inch pieces of Anchor Paper #SD7606), wetted in a solution of 0.5% KNO3 and 0.1% Thyram. For each paper fifteen seeds are placed on the line evenly spaced such that the radicles will grow toward the same edge. The wet paper is rolled up evenly and tight enough to hold the seeds in place. The roll is secured into place with two large paper clips, one at the top and one at the bottom. The rolls are incubated in a growth chamber at 23 degrees C. for three days in a randomized complete block design within an appropriate container. The chamber is set for 65% humidity with no light cycle. For the cold stress treatment the rolls are then incubated in a growth chamber at 12 degrees C. for fourteen days. The chamber is set for 65% humidity with no light cycle. For the warm treatment the rolls are incubated at 23 degrees C. for an additional two days. After the treatment the germination papers are unrolled and the seeds that did not germinate are discarded. The lengths of the radicle and coleoptile for each seed are measured. A coleoptile sample is collected from six individual kernels of each entry for confirming the expression of recombinant DNA. Statistical differences in the length of radicle and shoot during pre-shock and cold shock are used for an estimation of the effect of the cold treatment on corn plants. The analysis is conducted independently for the warm and cold treatments.
In a root-shoot biomass assay two sets of seeds are used. The first set is transgenic seeds with recombinant DNA, e.g., R2 inbred seeds or F1 hybrid seeds; the second seed set is non-transgenic, wild type negative control made from the same genotypes as the transgenic seeds. All seeds are treated with a fungicide as indicated above. The seeds are planted in potting media in pots arranged in a randomized complete block design with 6 replications. Pots are watered as and when needed by filling water up to the brim of the pot. Plants are grown in a greenhouse to a V6 stage or approximately for 28 days. Greenhouse lights are turned on after emergence of seedlings with 14 hours of light 10 hours of dark. Plants are fertilized twice each week with water-soluble fertilizer containing 200-ppm nitrogen. For measurement of root and shoot dry weight, two pots are separated carefully to remove adhering sand by washing with water. Washed roots are cut at the first node. The roots are placed in a paper bag after squeezing excess water, folded once and stapled. The shoots are then folded up to a convenient size (approximately 15 cm), placed in a paper bag. Bags are placed over a wire shelve to facilitate drying in a ventilated room maintained at 120 degrees F. to a moisture content of about 13% then weighed to determine dried root and shoot biomass.
E. Screen for Enhanced Oil, Starch, or Protein Levels in Plant Seeds
Oil concentrations are determined in kernels by Near Infrared Transmittance (NIT) from inbred and from hybrid lines. Data are also obtained for protein and starch content from this measurement.
Inbred Kernel Oil Screen
The primary transformants are selfed to produce R1 seed which is planted to segregating seed. An untransformed control line is planted every sixth row. All plants are self-pollinated. A molecular assay is conducted to determine zygosity of the transgene in each plant. Ears are harvested at maturity, and well-filled ears are chosen for proximate analysis. Proximate analysis is conducted on up to 5 homozygous ears. If 5 good homozygous ears are not available, then hemizygous ears will be used to obtain 5 good transgene-positive ears. Statistical analysis is conducted to determine whether proximate values for transgenic events are different from controls. Events with an increase in oil with a p-value of less than or equal to 0.1 are termed “putative leads.” Kernel composition is confirmed in an inbred confirmation nursery which is conducted with selected events, and is run with a design similar to that of the Gen2 nursery. A “confirmed lead event” demonstrates an increase in oil with a p-value of less than or equal to 0.1 in two nurseries.
Hybrid Kernel Oil Screen
Grain samples from the multilocation hybrid yield trials are collected at the time of harvest and are analyzed by NIT. Controls are negative segregants, untransformed controls, or pollinators. Data from 3 to 12 locations are pooled for the statistical analysis. Putative leads have increased oil with a p-value of less than or equal to 0.1.
The various aspects of the invention are illustrated by means of the following examples which are in no way intended to limit the full breath and scope of claims.
Example 1 Identification of Recombinant DNA that Confers Improved Trait(s) to Plants A. Expression Constructs for Arabidopsis Plant Transformation
Each gene of interest was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, et al., PNAS 95:376-381, 1998; Guyer, et al., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577). The transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide. The transformation of Arabidopsis plants was carried out using the vacuum infiltration method known in the art (Bethtold, et al., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds, were subsequently grown in a glufosinate-containing selective medium to select for plants which were actually transformed and which produced T2 transgenic seed.
B. Soil Drought Tolerance Screen
This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions
T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA). Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 μmol/m2/s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.
To identify drought tolerant plants, plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.
At the end of this assay, seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.
Two approaches were used for statistical analysis on the wilting response. First, the risk score was analyzed for wilting phenotype and treated as a qualitative response according to the example 1L. Alternatively, the survival analysis was carried out in which the proportions of wilted and non-transgenic wilted transgenic and control plants were compared over each of the six days under scoring and an overall log rank test was performed to compare the two survival curves using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). Table 5 provides a list of recombinant DNA constructs that improve drought tolerance in transgenic plants. TABLE 5
Wilt Response Risk Seed Weight/ Survival Analysis of
Pep score plant wilt response
SEQ RS p- p- diff time p-
ID Construct_id Gene Orientation mean value c delta value c to wilting value c
319 10139 CGPG101 ANTI-SENSE 0.115 0.024 S −0.123 0.804 / −0.01 0.469 /
320 11410 CGPG103 SENSE 0.226 0.003 S −0.366 0.926 / 0 1 /
321 11604 CGPG48 ANTI-SENSE 0.25 0.034 S 0.257 0.01 S −0.24 0.38 /
322 12368 CGPG1006 SENSE 0.148 0.044 S 0.359 0.02 S −0.06 0.764 /
323 13502 CGPG1354 SENSE 0.431 0 S 0.624 0 S 1.34 0.366 /
324 13745 CGPG1576 ANTI-SENSE −0.021 0.711 / 0.664 0 S −0.29 0.453 /
325 13821 CGPG1569 SENSE 0.135 0.025 S 0.128 0.402 / 0.24 0.972 /
326 14240 CGPG1697 SENSE 0.377 0.002 S −1.305 0.991 / 0 1 /
327 14718 CGPG1082 SENSE 0.168 0.001 S 0.135 0.351 / 0.25 0.208 /
328 17022 CGPG1774 SENSE 0.06 0.124 T 0.563 0.043 S 0 0.961 /
329 17924 CGPG2882 SENSE −0.093 0.914 / 0.288 0.021 S 0.09 0.935 /
330 18259 CGPG3368 SENSE 0.07 0.28 / 0.391 0.058 T −0.27 0.591 /
331 19171 CGPG2952 SENSE 0.227 0.005 S 0.846 0.001 S 0.12 0.543 /
332 19201 CGPG2332 SENSE 0.124 0.027 S −0.435 0.785 / 0.35 0.256 /
333 19317 CGPG3662 SENSE 0.338 0 S −0.071 0.61 / 0.63 0.106 T
334 70417 CGPG3427 SENSE 0.253 0.016 S −1.424 0.984 / 0 1 /
315 70427 CGPG3067 SENSE −0.033 0.818 / 1.004 0.002 S 0.01 0.977 /
335 70467 CGPG3785 SENSE 0.127 0.023 S −0.448 0.946 / 0.71 0.046 S
336 70806 CGPG712 SENSE 0.246 0.046 S 0.174 0.276 / 0.14 0.07 T
337 70818 CGPG479 SENSE 0.07 0.115 T 0.558 0.009 S 0.61 0.283 /
338 70820 CGPG655 SENSE 0.172 0.048 S 0.036 0.441 / 0.26 0.554 /
339 70919 CGPG4029 SENSE 0.167 0.009 S −0.565 0.904 / 0.31 0.508 /
340 71623 CGPG4696 SENSE 0.158 0.047 S 0.421 0.04 S 0 1 /
390 71633 CGPG857 SENSE 0.121 0.017 S −0.823 0.967 / 0.45 0.139 T
341 71662 CGPG4679 SENSE 0.063 0.013 S −0.037 0.631 / 0.16 0.957 /
342 71693 CGPG4652 SENSE 0.074 0.042 S 0.246 0.06 T 0.34 0.616 /
316 71811 CGPG4426 SENSE 0.359 0.015 S −0.729 0.903 / 0.15 0.822 /
318 72081 CGPG5279 SENSE 0.269 0.005 S −0.372 0.987 / 0.17 0.404 /
343 72384 CGPG4639 SENSE 0.133 0.018 S 0.62 0.002 S 0.17 0.359 /
344 72439 CGPG5075 SENSE 0.175 0.013 S −0.035 0.604 / 0.23 0.244 /
374 72456 CGPG4745 SENSE 0.53 0.01 S −0.737 0.979 / −0.08 0.823 /
345 72619 CGPG4835 SENSE 0.178 0.039 S 0.219 0.072 T 0.96 0.691 /
346 72624 CGPG4842 SENSE 0.163 0.021 S 0.356 0.051 T 0.3 0.375 /
347 72715 CGPG5521 SENSE 0.082 0.1 T 0.767 0.002 S 0.12 0.628 /
348 72754 CGPG5548 SENSE 0.131 0.031 S −0.365 0.974 / 0 0.923 /
349 72819 CGPG4989 SENSE 0.094 0.026 S 0.362 0.069 T 0.03 0.83 /
350 75516 CGPG7689 SENSE 0.067 0.066 T 0.965 0.001 S 0.24 0.464 /
351 75701 CGPG7856 SENSE 0.147 0.006 S 0.986 0.015 S 0.15 0.448 /
317 73463 CGPG6384 SENSE 0.174 0.048 S −1.359 0.959 / 0.09 0.984 /
454 70354 CGPG3995 SENSE −0.005 0.563 / 0.444 0.002 S 10.29 0.9 /
397 71840 CGPG4353 SENSE 0.142 0.007 S −0.212 0.859 / 9.29 0.99 /
506 72902 CGPG5597 SENSE 0.009 0.162 T 0.034 0.2 T 5 1 /
437 73549 CGPG6460 SENSE 0.119 0.037 S −0.774 0.949 / 6.26 0.25 /
302 73586 CGPG6471 SENSE 0.003 0.451 / 0.588 0.002 S 6.34 0.723 /
442 74587 CGPG6774 SENSE 0.262 0.001 S −0.117 0.574 / 7.49 0.041 S
388 74652 CGPG6168 SENSE 0.475 0 S −0.766 0.92 / 7.48 0 S
S: represents that the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05, p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance)
T: represents that the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2
/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset.
C. Heat Stress Tolerance Screen
Under high temperatures, Arabidopsis seedlings become chlorotic and root growth is inhibited. This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature.
T2 seeds were plated on ½× MS salts, 1% phytagel, with 10 μg/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was ˜140 μmol/m2/s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, et al., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1L. Table 6 provides a list of recombinant DNA constructs that improve heat tolerance in transgenic plants. TABLE 6
Pep Growth stage Root Length Seedling Weight
SEQ RS p- p- p-
ID Construct_id Gene Orientation mean value c delta value c delta value c
354 19542 CGPG3069 SENSE 0.842 0.021 S 0.296 0.023 S 1.616 0 S
355 19618 CGPG3574 SENSE 1.072 0.005 S 0.328 0.006 S 1.569 0 S
356 19649 CGPG3140 SENSE 0.72 0.042 S 0.243 0.054 T 1.665 0 S
357 19745 CGPG3973 SENSE 0.65 0.016 S 0.19 0.021 S 1.505 0 S
358 19768 CGPG4096 SENSE 1.06 0.035 S 0.17 0.034 S 1.171 0 S
436 19771 CGPG4011 SENSE 0.822 0.023 S 0.201 0.001 S 1.43 0 S
359 19772 CGPG3939 SENSE 0.247 0.014 S 0.151 0.014 S 1.344 0 S
360 19779 CGPG4113 SENSE 0.605 0.051 T 0.181 0.003 S 1.436 0 S
361 19833 CGPG4074 SENSE 0.965 0.026 S 0.266 0.007 S 1.01 0 S
362 19862 CGPG3961 SENSE 0.341 0.119 T 0.132 0.007 S 1.22 0 S
363 19879 CGPG4009 SENSE 0.734 0.002 S 0.22 0.001 S 1.499 0 S
364 70445 CGPG3728 SENSE 0.413 0.062 T 0.148 0.114 T 1.249 0 S
365 70738 CGPG3195 SENSE 0.687 0.055 T 0.205 0.041 S 1.261 0 S
366 71437 CGPG4043 SENSE 0.094 0.198 T 0.092 0.064 T 1.301 0 S
367 71572 CGPG4520 SENSE 0.938 0.052 T 0.441 0 S 1.633 0 S
368 71617 CGPG1227 SENSE 0.809 0.012 S 0.143 0.029 S 1.05 0.003 S
408 72085 CGPG5228 SENSE 1.234 0.02 S 0.192 0.043 S 1.162 0 S
369 72532 CGPG4780 SENSE 1.028 0.022 S 0.198 0.052 T 1.043 0.001 S
409 72744 CGPG5563 SENSE 0.17 0.146 T 0.04 0.359 / 0.827 0.004 S
370 72757 CGPG5572 SENSE 1.82 0.004 S 0.14 0.091 T 1.121 0 S
407 72771 CGPG2166 SENSE 1.776 0.001 S 0.36 0 S 1.428 0 S
444 72967 CGPG5742 SENSE 0.273 0.063 T 0.147 0.102 T 1.03 0 S
410 73039 CGPG810 SENSE −0.048 0.774 / −0.135 0.957 / 0.59 0.022 S
411 73054 CGPG5754 SENSE 0.055 0.312 / 0.236 0.001 S 1.434 0 S
508 73055 CGPG5768 SENSE 0.154 0.123 T 0.269 0 S 1.524 0 S
371 73412 CGPG6448 SENSE 0.187 0.118 T 0.134 0.06 T 1.181 0 S
412 73501 CGPG6456 SENSE 1.6 0.003 S 0.081 0.136 T 1.119 0 S
352 73515 CGPG6473 SENSE −0.037 0.758 / −0.024 0.604 / 0.694 0.008 S
437 73549 CGPG6460 SENSE 2.612 0 S 0.199 0.017 S 1.432 0 S
372 74102 CGPG6550 SENSE 0.34 0.035 S 0.268 0.002 S 1.355 0 S
509 74103 CGPG6558 SENSE −0.013 1 / −0.021 0.608 / 0.86 0 S
353 74684 CGPG6360 SENSE 0.44 0.018 S 0.254 0.002 S 1.383 0 S
512 19703 CGPG4172 SENSE 0.211 0.079 T 0.059 0.301 / 1.262 0 S
273 70423 CGPG3165 SENSE 1.456 0 S 0.418 0 S 1.912 0 S
491 70469 CGPG3791 SENSE 0.143 0.28 / 0.028 0.349 / 1.001 0.001 S
434 70932 CGPG4089 SENSE 0.354 0.1 T 0.03 0.419 / 1.254 0 S
419 71134 CGPG817 SENSE 0.522 0.106 T 0.077 0.248 / 1.225 0 S
466 71726 CGPG3894 SENSE 0.196 0.225 / 0.082 0.2 T 1.243 0 S
505 72463 CGPG4760 SENSE 1.182 0.003 S 0.202 0.026 / 1.702 0 S
445 72961 CGPG5591 SENSE 1.195 0.013 S 0.106 0.105 T 1.084 0 S
306 74136 CGPG6632 SENSE 0.886 0.012 S 0.306 0.009 S 1.42 0 S
504 74259 CGPG5343 SENSE 1.044 0.023 S 0.081 0.187 T 1.274 0 S
310 74318 CGPG5826 SENSE 0.407 0.075 T 0.116 0.052 T 1.118 0 S
479 74344 CGPG5929 SENSE 1.256 0.018 S 0.118 0.125 T 1.275 0 S
533 74462 CGPG6668 SENSE 0.846 0.022 S 0.26 0.011 S 1.425 0 S
313 74512 CGPG32 SENSE 0.241 0.118 T 0.146 0.044 S 1.062 0 S
442 74587 CGPG6774 SENSE 0 / / 0.084 0.054 T 1.046 0 S
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2
/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset
D. Salt Stress Tolerance Screen
This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.
T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the experiment were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 mmol/m2. On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth stage data was analyzed as a qualitative response according to example 1L. Table 7 provides a list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants TABLE 7
Seedling
Root Length Root Length Weight
Pep Growth Stage at day 11 at day 14 at day 14
SEQ RS p- p- p- p-
ID Construct id Gene Orientation mean value c delta value c delta value c delta value c
512 19703 CGPG4172 SENSE 1.124 0.139 T 0.198 0.021 S 0.072 0.116 T 0.582 0.023 S
513 19946 CGPG4097 SENSE 1.201 0.072 T 0.02 0.89 / 0.069 0.573 / 0.443 0.266 /
514 19980 CGPG3914 SENSE 0.904 0.146 T 0.101 0.259 / 0.144 0.058 T 0.706 0.016 S
515 70435 CGPG3701 SENSE 1.363 0.031 S −0.118 0.228 / 0.161 0.038 S 0.053 0.697 /
516 71114 CGPG1657 SENSE 0.138 0.399 / 0.245 0.009 S 0.187 0.025 S 0.472 0.069 T
517 72451 CGPG4733 SENSE 2.226 0.02 S 0.186 0.006 S 0.069 0.23 / 0.466 0.011 S
443 72453 CGPG4735 SENSE 1.539 0.031 S 0.232 0.002 S 0.216 0 S 0.737 0.001 S
505 72463 CGPG4760 SENSE 3.026 0.002 S 0.119 0.269 / 0.202 0.073 T 1.26 0 S
392 72519 CGPG4749 SENSE 0.598 0.041 S 0.126 0.055 T 0.172 0.003 S 0.635 0.008 S
506 72902 CGPG5597 SENSE 1.418 0.039 S 0.114 0.286 / 0.226 0.078 T 0.426 0.091 T
448 72916 CGPG1814 SENSE 0.682 0.181 T 0.07 0.532 / 0.201 0.046 S 0.387 0.079 T
510 72921 CGPG5781 SENSE 1.977 0.029 S 0.163 0.176 T 0.211 0.073 T 0.663 0.006 S
518 72947 CGPG5607 SENSE 1.505 0.028 S 0.024 0.899 / 0.204 0.022 S 0.466 0.216 /
445 72961 CGPG5591 SENSE 1.879 0.007 S 0.228 0.122 T 0.229 0.003 S 0.817 0.04 S
444 72967 CGPG5742 SENSE 2.427 0.004 S 0.386 0.009 S 0.369 0.001 S 1.254 0 S
511 72968 CGPG5772 SENSE 1.531 0.055 T 0.302 0.06 T 0.209 0.073 T 0.761 0.003 S
449 72969 CGPG5789 SENSE 0.67 0.078 T 0.029 0.789 / 0.239 0.008 S 0.603 0.013 S
519 73012 CGPG5786 SENSE 2.371 0.001 S 0.366 0 S 0.342 0 S 1.08 0 S
520 73022 CGPG5622 SENSE 1.408 0.036 S 0.22 0.056 T 0.347 0 S 0.492 0.03 S
508 73055 CGPG5768 SENSE 3.291 0.001 S 0.369 0.087 T 0.417 0.005 S 1.096 0.005 S
446 73070 CGPG5627 SENSE 2.755 0.005 S 0.188 0.392 / 0.275 0.011 S 0.685 0.025 S
447 73475 CGPG6385 SENSE 1.11 0.059 T 0.127 0.361 / 0.216 0.001 S 0.474 0.06 T
521 73488 CGPG6394 SENSE 2.373 0.013 S 0.314 0.006 S 0.262 0.002 S 1.126 0.005 S
522 73901 CGPG5237 SENSE 1.141 0.078 T 0.207 0.197 T 0.202 0.097 T 0.639 0.03 SW
523 73964 CGPG5804 SENSE 1.235 0.043 S 0.428 0.008 S 0.317 0.022 S 0.955 0.002 S
524 74019 CGPG5706 SENSE 0.105 0.168 T 0.074 0.649 / 0.171 0.106 T 0.773 0.011 S
525 74022 CGPG5724 SENSE 0.033 0.327 / −0.065 0.616 / 0.172 0.032 S 0.484 0.068 T
509 74103 CGPG6558 SENSE 1.225 0.074 T 0.26 0.042 S 0.267 0.004 S 0.543 0.042 S
526 74114 CGPG6551 SENSE 3.627 0 S 0.265 0.119 T 0.26 0 S 0.561 0.063 T
504 74259 CGPG5343 SENSE 2.802 0.003 S 0.249 0.098 T 0.256 0.037 S 0.995 0 S
527 74262 CGPG5353 SENSE 0.225 0.319 / 0.238 0.062 T 0.247 0 S 0.629 0.006 S
528 74292 CGPGS367 SENSE 0.327 0.199 T 0.16 0.067 T 0.105 0.166 T 0.565 0.013 S
529 74302 CGPG5384 SENSE 1.246 0.016 S 0.296 0.004 S 0.25 0 S 0.705 0.004 S
530 74325 CGPG5898 SENSE 1.596 0.021 S −0.035 0.76 / 0.094 0.106 T 0.685 0.004 S
531 74429 CGPG6689 SENSE 1.796 0.008 S 0.298 0.037 S 0.207 0.006 S 0.496 0.029 S
532 74440 CGPG6682 SENSE 0.223 0.334 / 0.43 0.007 S 0.272 0.01 S 0.744 0.017 S
450 74449 CGPG6659 SENSE 0.693 0.19 T 0.204 0.104 T 0.205 0.022 S 0.451 0.095 T
533 74462 CGPG6668 SENSE 2.14 0.028 S 0.244 0.038 S 0.239 0.001 S 0.64 0.013 S
534 74465 CGPG6692 SENSE 1.245 0.016 S 0.35 0.01 S 0.215 0.001 S 0.575 0.043 S
535 74474 CGPG6669 SENSE 3.312 0.002 S 0.233 0.083 T 0.338 0.003 S 0.589 0.044 S
536 74505 CGPG6783 SENSE 1.731 0.043 S 0.272 0.007 S 0.208 0.01 S 0.493 0.009 S
537 74507 CGPG6799 SENSE 2.32 0.009 S 0.056 0.567 / 0.227 0.035 S 0.476 0.126 T
538 74562 CGPG6764 SENSE 1.405 0.038 S −0.052 0.776 / 0.215 0.014 S 0.104 0.766 /
507 74572 CGPG6640 SENSE 1.425 0.025 S 0.201 0.009 S 0.267 0.001 S 1.184 0 S
270 14324 CGPG1560 SENSE 1.708 0.017 S 0.307 0.01 S 0.408 0 S 0.995 0.002 S
358 19768 CGPG4096 SENSE 1.496 0.061 T 0.163 0.187 T 0.129 0.013 S 0.66 0.002 S
436 19771 CGPG4011 SENSE 1.666 0.051 T 0.319 0.005 S 0.201 0.028 S 0.68 0.031 S
363 19879 CGPG4009 SENSE 2.117 0.029 S 0.16 0.139 T 0.091 0.202 / 0.545 0.028 S
347 72715 CGPG5521 SENSE 1.078 0.002 S 0.207 0.141 T 0.176 0.014 S 0.519 0.015 S
407 72771 CGPG2166 SENSE 0.564 0.256 / −0.019 0.857 / 0.206 0.013 S 0.306 0.05 S
440 72903 CGPG5584 SENSE 0.645 0.196 T −0.031 0.569 / 0.097 0.389 / 0.482 0.015 S
411 73054 CGPG5754 SENSE 1.52 0.035 S 0.198 0.089 T 0.098 0.187 T 0.696 0.004 S
476 74107 CGPG6590 SENSE 0.772 0.036 S 0.411 0 S 0.431 0.001 S 1.579 0 S
478 74131 CGPG6592 SENSE 1.747 0.044 S 0.316 0.004 S 0.115 0.01 S 0.663 0.001 S
442 74587 CGPG6774 SENSE 1.754 0.01 S 0.121 0.243 / 0.194 0.001 S 0.681 0 S
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2
/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset
E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen
There are numerous factors, which can influence seed germination and subsequent seedling growth, one being the availability of water. Genes, which can directly affect the success rate of germination and early seedling growth, are potentially useful agronomic traits for improving the germination and growth of crop plants under drought stress. In this assay, PEG was used to induce osmotic stress on germinating transgenic lines of Arabidopsis thaliana seeds in order to screen for osmotically resistant seed lines.
T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, ½× MS salts, 1% phytagel, and 10 μg/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.
Seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm. The growth stage data was analyzed as a qualitative response according to example 1L. Table 8 provides a list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants. TABLE 8
Seedling
Root Length Root Length Weight
Pep Growth Stage at day 11 at day 14 at day 14
SEQ RS p- p- p- p-
ID Gene Construct_id Orientation mean value c delta value c delta value c delta value c
413 19707 CGPG4179 SENSE 2.653 0.019 S 0.074 0.58 T −0.031 0.81 / 0.427 0.063 T
414 19951 CGPG3941 SENSE 1.432 0.134 T 0.017 0.864 T −0.08 0.28 / 0.476 0.054 T
415 19967 CGPG4032 SENSE 2.691 0.014 S 0.01 0.934 T 0.19 0.039 S 0.537 0.056 T
416 70543 CGPG3815 SENSE 1.735 0.077 T 0.084 0.561 T 0.323 0.007 S 0.676 0.006 S
402 70681 CGPG4584 SENSE 2.528 0.006 S −0.065 0.682 / −0.179 0.05 / 0.374 0.146 T
417 70707 CGPG1273 ANTI- 1.42 0.095 T 0.248 0.006 S 0.3 0.002 S 0.331 0.007 S
SENSE
418 70719 CGPG1712 ANTI- 1.43 0.106 T −0.007 0.968 / 0.101 0.255 T 0.155 0.349 T
SENSE
419 71134 CGPG817 SENSE 1.478 0.13 T 0.119 0.15 T 0.035 0.684 T 0.198 0.277 T
420 71146 CGPG2928 SENSE 2.624 0.016 S 0.227 0.101 T 0.242 0.1 T 0.278 0.296 T
405 71508 CGPG1541 SENSE 3.153 0.001 S 0.446 0.054 T 0.384 0.048 S 0.782 0.003 S
421 71660 CGPG4690 SENSE 2.893 0.005 S −0.024 0.795 / 0.225 0.074 T 0.165 0.329 T
403 71663 CGPG4638 SENSE 0.116 0.444 T −0.061 0.536 / 0.358 0.041 S 0.058 0.702 T
439 71928 CGPG1617 SENSE 2.076 0.041 S 0.353 0.013 S 0.289 0.035 S 0.531 0.035 S
318 72081 CGPG5279 SENSE 1.262 0.138 T 0.174 0.243 T 0.2 0.197 T −0.105 0.714 /
408 72085 CGPG5228 SENSE 4 0 S 0.22 0.046 S 0.371 0.004 S 0.929 0 S
422 72086 CGPG5236 SENSE 2.589 0.022 S 0.281 0.033 S 0.136 0.314 T 0.32 0.043 S
423 72632 CGPG4852 SENSE 1.663 0.053 T 0.254 0.069 T 0.094 0.548 T 0.471 0.015 S
424 72716 CGPG5529 SENSE 2.914 0.004 S 0.146 0.058 T 0.007 0.925 T 0.582 0.016 S
425 72723 CGPG1848 SENSE 2.138 0.066 T 0.043 0.728 T 0.2 0.068 T −0.326 0.225 /
409 72744 CGPG5563 SENSE 1.636 0.05 / 0.195 0.151 T 0.059 0.751 T 0.539 0.005 S
404 72769 CGPG5573 SENSE 2.207 0.055 T 0.086 0.464 T −0.134 0.162 / 0.389 0.086 T
407 72771 CGPG2166 SENSE 2.569 0.021 S 0.169 0.221 T 0.192 0.038 S 0.56 0.018 S
440 72903 CGPG5584 SENSE 2.161 0.061 T 0.035 0.856 T 0.245 0.162 T 0.06 0.871 T
510 72921 CGPG5781 SENSE 2.249 0.025 S 0.034 0.788 T 0.225 0.077 T 0.306 0.28 T
391 72948 CGPG5617 SENSE 2.267 0.005 S 0.054 0.495 T 0.117 0.019 S 0.289 0.077 T
445 72961 CGPG5591 SENSE 1.19 0.14 T 0.037 0.76 T 0.136 0.339 T 0.588 0.027 S
511 72968 CGPG5772 SENSE 3.142 0.007 S 0.178 0.072 T −0.031 0.781 / 0.698 0.016 S
426 72987 CGPG1787 SENSE 2.055 0.078 T −0.058 0.64 / 0.097 0.144 T 0.325 0.054 T
438 72994 CGPG5803 SENSE 2.674 0.013 S 0.245 0.124 T 0.07 0.657 T 0.599 0.035 S
441 73017 CGPG5733 SENSE 4 0 S 0.343 0.09 T 0.462 0.002 S 0.996 0 S
410 73039 CGPG810 SENSE 4 0 S 0.319 0.019 S 0.237 0.05 / 0.426 0.011 S
411 73054 CGPG5754 SENSE 3.048 0.002 S 0.556 0.003 S 0.26 0.063 T 1.12 0.002 S
446 73070 CGPG5627 SENSE 3.439 0.001 S 0.139 0.49 T 0.19 0.135 T 0.24 0.365 T
447 73475 CGPG6385 SENSE 1.933 0.033 S 0.04 0.476 T 0.009 0.897 T 0.459 0.073 T
412 73501 CGPG6456 SENSE 1.29 0.121 T 0.143 0.118 T 0.043 0.564 T 0.433 0.013 S
427 74109 CGPG6606 SENSE 3.517 0 S 0.159 0.136 T 0.249 0.004 S 0.333 0.025 S
428 74140 CGPG6569 SENSE 2.05 0.039 S 0.168 0.138 T 0.196 0.086 T 0.71 0.012 S
429 74191 CGPG6597 SENSE 2.565 0.019 S 0.336 0.092 T 0.199 0.112 T 0.54 0.02 S
406 74248 CGPG5476 SENSE 3.158 0.007 S 0.14 0.192 T 0.204 0.051 T 0.377 0.037 S
430 74265 CGPG5356 SENSE 2.208 0.023 S 0.317 0.034 S 0.419 0.006 S 0.577 0.008 S
431 74369 CGPG6076 SENSE 3.522 0 S 0.347 0.045 S 0.272 0.107 T 0.624 0.02 S
442 74587 CGPG6774 SENSE 3.325 0.002 S 0.073 0.468 T 0.414 0.002 S 0.577 0.016 S
405 71508 CGPG1541 SENSE 3.153 0.001 S 0.446 0.054 T 0.384 0.048 S 0.782 0.003 S
439 71928 CGPG1617 SENSE 2.076 0.041 S 0.353 0.013 S 0.289 0.035 S 0.531 0.035 S
422 72086 CGPG5236 SENSE 2.589 0.022 S 0.281 0.033 S 0.136 0.314 / 0.32 0.043 S
469 72455 CGPG4742 SENSE 1.367 0.15 T 0.026 0.764 / 0.024 0.786 / 0.255 0.04 S
382 72466 CGPG4767 SENSE 0.735 0.101 T 0.068 0.333 / 0.249 0.006 S −0.341 0.278 /
373 72633 CGPG4853 SENSE 1.179 0.122 T 0.227 0.009 S 0.097 0.065 T 0.442 0.013 S
370 72757 CGPG5572 SENSE 2.272 0.017 S 0.125 0.361 / 0.11 0.317 / 0.425 0.047 S
472 72992 CGPG5777 SENSE 2.233 0.056 T 0.176 0.261 / 0.116 0.34 / 0.511 0.019 S
438 72994 CGPG5803 SENSE 2.674 0.013 S 0.245 0.124 T 0.07 0.657 / 0.599 0.035 S
441 73017 CGPG5733 SENSE 4 0 S 0.343 0.09 T 0.462 0.002 S 0.996 0 S
411 73054 CGPG5754 SENSE 3.048 0.002 S 0.556 0.003 S 0.26 0.063 T 1.12 0.002 S
300 73507 CGPG6504 SENSE 0.343 0.334 / 0.347 0.007 S 0.261 0.032 S 0.3 0.122 T
352 73515 CGPG6473 SENSE 3.336 0.002 S 0.279 0.009 S 0.241 0.003 S 0.328 0.05 S
428 74140 CGPG6569 SENSE 2.05 0.039 S 0.168 0.138 T 0.196 0.086 T 0.71 0.012 S
430 74265 CGPG5356 SENSE 2.208 0.023 S 0.317 0.034 S 0.419 0.006 S 0.577 0.008 S
431 74369 CGPG6076 SENSE 3.522 0 S 0.347 0.045 S 0.272 0.107 T 0.624 0.02 S
422 74587 CGPG6774 SENSE 3.325 0.002 S 0.073 0.468 / 0.414 0.002 S 0.577 0.016 S
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2
/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset
F. Cold Shock Tolerance Screen
This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.
Eleven seedlings from T2 seeds of each transgenic line plus one control line were plated together on a plate containing ½× Gamborg Salts with 0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then oriented horizontally and stratified for three days at 4° C. At day three, plates were removed from stratification and exposed to standard conditions (16 hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At day eight, plates were removed from standard conditions and exposed to cold shock conditions (24 hr photoperiod, 8° C. at both day and night) until the final day of the assay, i.e., day 28. Rosette areas were measured at day 8 and day 28, which were analyzed as quantitative responses according to example 1M. Table 9 provides a list of recombinant nucleotides that improve cold shock stress tolerance in plants. TABLE 9
difference in
rosette area
rosette area rosette area between day 28
Pep at day 8 at day 28 and day 8
SEQ p- p- p-
ID Construct_id Gene Orientation delta value c delta value c delta value c
270 14324 CGPG1560 SENSE 0.054 0.429 / 0.258 0.017 S −0.071 0.631 /
271 17484 CGPG2630 SENSE −0.189 0.759 / 0.544 0.025 S 0.275 0.121 T
272 19109 CGPG1381 ANTI- −0.016 0.523 / 0.541 0.008 S 0.818 0.014 S
SENSE
273 70423 CGPG3165 SENSE 0.316 0.012 S 0.521 0.022 S 0.89 0.018 S
274 70424 CGPG3180 SENSE 0.474 0.003 S 0.695 0.003 S 0.693 0.043 S
275 70480 CGPG3833 SENSE −0.066 0.591 / 0.175 0.159 T 0.474 0.059 T
276 70509 CGPG2420 SENSE 0.023 0.438 / 0.117 0.216 / 0.609 0.032 S
277 70647 CGPG4334 SENSE −0.508 0.894 / 0.604 0.049 S 0.895 0.047 S
278 70675 CGPG4519 SENSE 0.2 0.2 / 0.303 0.153 T 0.507 0.034 S
279 70829 CGPG518 SENSE −0.319 0.823 / 0.804 0.002 S 1.082 0.002 S
280 70849 CGPG596 SENSE −0.039 0.564 / 0.698 0.001 S 0.707 0.001 S
281 71627 CGPG1270 SENSE −0.146 0.748 / 0.349 0.05 T 0.3 0.12 T
282 71934 CGPG2294 SENSE −0.068 0.796 / 0.757 0 S 0.922 0 S
283 72615 CGPG4829 SENSE 0.477 0.007 S 0.834 0 S 0.979 0.001 S
286 73559 CGPG6535 SENSE 0.143 0.093 T −0.265 0.878 / −0.344 0.821 /
287 74251 CGPG5489 SENSE 0.377 0.021 S 0.439 0.045 S 0.45 0.07 T
389 70437 CGPG3706 SENSE −0.273 0.916 / 0.147 0.165 T 0.682 0.034 S
402 70681 CGPG4584 SENSE 0.352 0.155 T 0.252 0.261 / 0.269 0.328 /
403 71663 CGPG4638 SENSE 0.358 0.013 S 0.032 0.423 / −0.031 0.585 /
404 72769 CGPG5573 SENSE 0.381 0.049 S 0.881 0.006 S 1.102 0.005 S
407 72771 CGPG2166 SENSE 0.993 0 S 1.381 0.003 S 1.536 0.003 S
432 70217 CGPG6 SENSE 0.275 0.067 T 0.126 0.289 / 0.362 0.215 /
433 72711 CGPG1846 SENSE 0.774 0.001 S 0.579 0.004 S 0.429 0.038 S
438 72994 CGPG5803 SENSE 0.116 0.381 / 0.708 0.068 T 0.744 0.069 T
510 72921 CGPG5781 SENSE 0.265 0.057 T 0.31 0.162 T 0.367 0.11 T
414 19951 CGPG3941 SENSE 0.729 0.006 S 0.473 0.017 S 0.846 0.006 S
273 70423 CGPG3165 SENSE 0.316 0.012 S 0.521 0.022 S 0.89 0.018 S
416 70543 CGPG3815 SENSE 1.584 0 S 0.86 0 S 0.82 0.002 S
368 71617 CGPG1227 SENSE 0.204 0.136 T 0.408 0.025 S 0.458 0.057 T
439 71928 CGPG1617 SENSE 0.104 0.265 / 0.786 0 S 0.836 0.001 S
382 72466 CGPG4767 SENSE 0.497 0.017 S 0.565 0.017 S 0.963 0.002 S
383 72524 CGPG4770 SENSE 0.438 0.02 S 0.377 0.025 S 0.385 0.043 S
409 72744 CGPG5563 SENSE 0.52 0.058 T 0.859 0.026 S 0.454 0.189 T
444 72967 CGPG5742 SENSE 0.955 0 S 0.629 0.009 S 0.403 0.189 T
435 73518 CGPG6497 SENSE 0.114 0.278 / 0.319 0.01 S 0.195 0.114 T
306 74136 CGPG6632 SENSE 0.606 0.007 S 0.523 0.036 S 0.598 0.025 S
398 74240 CGPG5454 SENSE −0.099 0.644 / 1.277 0.003 S 1.498 0.006 S
431 74369 CGPG6076 SENSE 0.623 0.002 S 0.62 0.04 S 0.737 0.096 T
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2
/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset.
G. Cold Germination Tolerance Screen
This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions.
T2 seeds were plated and all seedlings used in the experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of ½× Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH. Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 mmol/m2/s. At 28 days post planting, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.
The root length at day 28 was analyzed as a quantitative response according to example 1M. The growth stage at day 7 was analyzed as a qualitative response according to example 1L. Table 10 provides a list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants. TABLE 10
Growth stage Root Length
Pep at day 28 at day 28
SEQ RS p- p-
ID Construct_id Gene Orientation mean value c delta value c
288 19631 CGPG3627 SENSE 2.229 0.052 T 0.094 0.252 /
289 70121 CGPG2380 SENSE 2.732 0.042 S 0.126 0.238 /
290 70654 CGPG4352 SENSE 2.474 0.026 S 0.263 0.019 S
291 70696 CGPG4590 SENSE 3.092 0.01 S 0.086 0.145 T
292 70713 CGPG1462 ANTI- 4 0 S 0.268 0.014 S
SENSE
293 70740 CGPG3700 SENSE 2.485 0.024 S 0.244 0.012 S
294 71321 CGPG4418 SENSE 1.837 0.126 T 0.014 0.474 /
295 71835 CGPG4634 SENSE 3.349 0.002 S 0.353 0.036 S
296 72934 CGPG5798 SENSE 2.222 0.023 S 0.243 0.101 T
297 72945 CGPG5787 SENSE 3.478 0.001 S 0.236 0.011 S
298 72980 CGPG5773 SENSE 3.265 0.003 S 0.239 0.006 S
299 73504 CGPG6480 SENSE 4 0 S 0.521 0 S
300 73507 CGPG6504 SENSE 4 0 S 0.404 0.001 S
301 73573 CGPG6462 SENSE 4 0 S 0.268 0.004 S
302 73586 CGPG6471 SENSE 4 0 S 0.314 0.064 T
303 73770 CGPG5435 SENSE 3.091 0.01 S 0.303 0.085 T
304 74105 CGPG6574 SENSE 3.329 0.002 S 0.109 0.116 T
305 74111 CGPG6622 SENSE 2.226 0.021 S 0.445 0.006 S
306 74136 CGPG6632 SENSE 3.192 0.005 S 0.328 0.002 S
307 74139 CGPG6561 SENSE 4 0 S 0.254 0.092 T
308 74267 CGPG5364 SENSE 3.054 0.002 S 0.3 0 S
309 74291 CGPG5363 SENSE 4 0 S 0.142 0.142 T
310 74318 CGPG5826 SENSE 4 0 S 0.272 0.008 S
311 74319 CGPG5831 SENSE 3.207 0.005 S 0.201 0.002 S
312 74324 CGPG5885 SENSE 3.144 0.007 S 0.232 0.017 S
313 74512 CGPG32 SENSE 4 0 S 0.332 0.011 S
314 74583 CGPG6649 SENSE 3.249 0.004 S 0.28 0.001 S
315 70427 CGPG3067 SENSE 1.567 0.108 T 0.222 0.044 S
352 73515 CGPG6473 SENSE 4 0 S 0.324 0.003 S
353 74684 CGPG6360 SENSE 2.927 0.004 S 0.426 0.003 S
373 72633 CGPG4853 SENSE 2.121 0.027 S 0.289 0.048 S
405 71508 CGPG1541 SENSE 1.99 0.039 S 0.263 0.033 S
406 74248 CGPG5476 SENSE 2.385 0.011 S 0.217 0.017 S
434 70932 CGPG4089 SENSE 3.268 0.003 S 0.146 0.067 T
435 73518 CGPG6497 SENSE 3.373 0.002 S 0.352 0.032 S
439 71928 CGPG1617 SENSE 3.062 0.011 S 0.18 0.002 S
504 74259 CGPG5343 SENSE 3.511 0 S 0.308 0.024 S
505 72463 CGPG4760 SENSE 2.736 0.01 S 0.032 0.386 /
506 72902 CGPG5597 SENSE 3.105 0.009 S 0.278 0.037 S
507 74572 CGPG6640 SENSE 4 0 S 0.125 0.155 T
508 73055 CGPG5768 SENSE 3.173 0.006 S 0.407 0.004 S
413 19707 CGPG4179 SENSE 1.829 0.061 T 0.169 0.018 S
360 19779 CGPG4113 SENSE 4 0 S 0.213 0.017 S
361 19833 CGPG4074 SENSE / / / 0.292 0.022 S
363 19879 CGPG4009 SENSE 4 0 S 0.34 0.001 S
514 19980 CGPG3914 SENSE 0.798 0.122 T 0.278 0.011 S
273 70423 CGPG3165 SENSE 2.906 0.004 S 0.105 0.114 T
459 70725 CGPG2097 ANTI- 1.949 0.044 S 0.122 0.148 T
SENSE
461 71112 CGPG934 SENSE 2.579 0.018 S 0.185 0.17 T
444 72967 CGPG5742 SENSE 4 0 S 0.287 0.007 S
438 72994 CGPG5803 SENSE 1.072 0.098 T 0.161 0.04 S
521 73488 CGPG6394 SENSE 4 0 S 0.211 0.012 S
477 74117 CGPG6575 SENSE 2.567 0.02 S 0.123 0.04 S
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2
/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined compared to the reference in the current dataset
H. Shade Tolerance Screen
Plants undergo a characteristic morphological response in shade that includes the elongation of the petiole, a change in the leaf angle, and a reduction in chlorophyll content. While these changes may confer a competitive advantage to individuals, in a monoculture the shade avoidance response is thought to reduce the overall biomass of the population. Thus, genetic alterations that prevent the shade avoidance response are associated with higher yields. Genes that favor growth under low light conditions may also promote yield, as inadequate light levels frequently limit yield. This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.
T2 seeds were plated on glufosinate selection plates with ½ MS medium. Seeds were sown on ½× MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m2/s, far/red ratio (655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1L.
Table 11 provides a list of recombinant DNA constructs that improve shade tolerance in plants TABLE 11
flowerbud Petiole Number of seedling
formation Leaf Angle length rosette leaves weight
Pep at day 23 at day 23 at day 23 at day 23 at day 23
SEQ RS p- RS p- RS p- RS p- p-
ID Construct_id Orientation mean value c mean value c mean value c mean value c delta value c
376 70426 SENSE 0.719 0.09 T 0.163 0.244 / −0.206 0.185 T −0.225 0.984 / 0.484 0.003 S
377 70772 SENSE −0.501 0.858 / −0.066 0.724 / −0.692 0.004 S −0.626 0.983 / −0.693 0.023 /
378 71137 SENSE 0.26 0.384 / 0.11 0.168 T −0.869 0.029 S 1.064 0.073 T −0.453 0.288 /
379 71529 SENSE 0.89 0.108 T −0.014 1 / −0.44 0.006 S 1.817 0.024 S −0.022 0.914 /
380 71601 SENSE 1.79 0.066 T 0.195 0.172 T −0.057 0.59 / −0.483 0.961 / 0.264 0.308 /
381 72362 SENSE 1.763 0.072 T 0.218 0.262 / −0.109 0.242 / 0.003 0.484 / 0.311 0.119 T
374 72456 SENSE 0.563 0.313 / 0.751 0.186 T −0.848 0.001 S −0.624 0.999 / −0.85 0.025 /
382 72466 SENSE −1.382 1 / −0.092 0.645 / −0.894 0 S 1.212 0.122 T −0.879 0.027 /
383 72524 SENSE −0.153 1 / 0.152 0.09 T −0.952 0.002 S −0.57 1 / −1.269 0.01 /
373 72633 SENSE 3.513 0 S −0.431 0.977 / −0.426 0.001 S −0.735 0.994 / 0.269 0.129 T
375 72963 SENSE −0.911 0.988 / 0.236 0.396 / −0.753 0.003 S −0.083 0.665 / −0.485 0.015 /
384 73085 SENSE −0.073 0.756 / 1.212 0.122 T 0.175 0.097 / 4 0 S 0.51 0.027 S
385 74241 SENSE −0.195 0.935 / −0.098 0.64 / −1.077 0.01 S −0.821 0.999 / −1.024 0.002 /
386 74247 SENSE 0.43 0.16 T 0.203 0.095 T −0.22 0.197 T 1.166 0.07 T 0.483 0.018 S
387 74284 SENSE 0.062 0.22 / 2.077 0.038 S −0.014 0.943 / 2.348 0.04 S 0.03 0.938 /
388 74652 SENSE 0.015 0.442 / 0.093 0.455 / −0.073 0.621 / 0.967 0.184 T 0.173 0.569 /
363 19879 SENSE 0.585 / / 0 / / 0.152 / / 0 / / 0.708 / /
293 70740 SENSE 0.44 0.137 T 0.609 0.103 T 0.161 0.186 / / / / 0.45 0.001 S
316 71811 SENSE −0.025 0.637 / 0.725 0.113 T −0.205 0.01 S / / / −0.517 0.037 /
397 71840 SENSE 1.498 0.12 T 0.712 0.178 T −0.379 0.001 S −0.188 0.765 / −0.95 0.124 /
468 72450 SENSE 0.068 0.349 / −0.042 1 / 0.166 0.016 / 1.62 0.049 S 0.501 0.038 S
370 72757 SENSE 3.595 0 S 0.872 0.079 T 0.064 0.138 / −0.767 0.997 / 0.546 0.001 S
444 72967 SENSE 1.829 0.063 T 1.123 0.065 T 0.133 0.196 / −0.18 0.794 / 0.509 0.008 S
511 72968 SENSE 0.185 0.005 S 0.48 0.179 T 0.22 0.162 / 1.698 0.085 T 0.382 0.031 S
300 73507 SENSE 0.248 0.086 T 0.307 0.287 / −0.304 0.031 S / / / 0.05 0.86 /
307 74139 SENSE −0.011 0.556 / −0.071 1 / −0.124 0.068 T −0.511 1 / −0.413 0.124 /
535 74474 SENSE 0.755 0.154 T −0.145 0.857 / 0.142 0.396 / / / / 0.765 0.005 S
400 74610 SENSE 0.572 0.222 / 0.177 0.22 / −0.18 0.071 T / / / −0.341 0.173 /
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2
/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined compared to the reference in the current dataset.
I. Early Plant Growth and Development Screen
This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth. In this screen, we were looking for genes that confer advantages in the processes of germination, seedling vigor, root growth and root morphology under non-stressed growth conditions to plants. The transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.
T2 seeds were plated on glufosinate selection plates and grown under standard conditions (˜100 □E/m2/s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.
Table 12 provides a list recombinant DNA constructs that improve early plant growth and development. TABLE 12
Pep Root Length Seedling Weight
SEQ ID Construct_id gene Orientation delta p-value c delta p-value c
432 70217 CGPG6 SENSE 0.038 0.469 / 0.375 0.047 S
433 72711 CGPG1846 SENSE 0.132 0.021 S 0.601 0.001 S
434 70932 CGPG4089 SENSE 0.328 0.005 S 0.473 0.017 S
435 73518 CGPG6497 SENSE 0.287 0 S 0.634 0.036 S
436 19771 CGPG4011 SENSE 0.218 0.076 T 0.581 0.018 S
437 73549 CGPG6460 SENSE 0.139 0.003 S 0.349 0.03 S
438 72994 CGPG5803 SENSE 0.44 0.004 S 0.791 0.001 S
439 71928 CGPG1617 SENSE 0.073 0.427 / 0.494 0.005 S
440 72903 CGPG5584 SENSE 0.298 0.002 S 0.399 0.044 S
441 73017 CGPG5733 SENSE 0.284 0.004 S 0.199 0.488 /
442 74587 CGPG6774 SENSE 0.111 0.075 T 0.538 0.006 S
443 72453 CGPG4735 SENSE 0.215 0.005 S 0.416 0.069 T
444 72967 CGPG5742 SENSE 0.103 0.212 / 0.568 0.008 S
445 72961 CGPG5591 SENSE 0.177 0.046 S 0.548 0.006 S
446 73070 CGPG5627 SENSE 0.221 0.007 S 0.652 0.005 S
447 73475 CGPG6385 SENSE 0.084 0.014 S 0.336 0.014 S
448 72916 CGPG1814 SENSE 0.182 0.067 T 0.353 0.012 S
449 72965 CGPG5789 SENSE 0.133 0.245 / 0.45 0.069 T
450 74449 CGPG6659 SENSE 0.314 0.002 S 0.579 0.027 S
451 16615 CGPG2539 SENSE 0.223 0.023 S 0.571 0.009 S
452 19187 CGPG3310 SENSE 0.264 0.001 S 0.51 0.08 T
453 19648 CGPG3134 SENSE 0.218 0.013 S 0.27 0.106 T
454 70354 CGPG3995 SENSE 0.152 0.046 S 0.406 0.029 S
455 70421 CGPG2942 SENSE 0.179 0.225 / 0.581 0.013 S
456 70459 CGPG3758 SENSE 0.187 0.036 S −0.034 0.884 /
457 70465 CGPG3775 SENSE −0.009 0.899 / 0.188 0.196 T
458 70683 CGPG4587 SENSE 0.133 0.088 T 0.402 0.014 S
459 70725 CGPG2097 ANTI- 0.326 0.001 S 0.116 0.548 /
SENSE
460 70852 CGPG1465 SENSE 0.237 0 S 0.297 0.127 T
461 71112 CGPG934 SENSE 0.199 0.013 S 0.316 0.034 S
462 71127 CGPG945 SENSE 0.097 0.02 S 0.4 0.054 T
463 71132 CGPG1561 SENSE 0.195 0.02 S 0.08 0.524 /
464 71217 CGPG95 SENSE 0.234 0 S 0.566 0.036 S
465 71645 CGPG4688 SENSE 0.475 0.003 S 0.361 0.133 T
466 71726 CGPG3894 SENSE 0.223 0.056 T 0.458 0.033 S
467 72432 CGPG4562 SENSE 0.209 0 S 0.581 0.041 S
468 72450 CGPG4732 SENSE 0.335 0 S 0.79 0 S
469 72455 CGPG4742 SENSE 0.278 0.019 S 0.482 0.051 T
470 72727 CGPG5522 SENSE 0.123 0.002 S 0.315 0.004 S
471 72817 CGPG4987 SENSE 0.254 0.023 S 0.485 0 S
472 72992 CGPG5777 SENSE 0.219 0.023 S 0.664 0.015 S
473 73007 CGPG5760 SENSE 0.139 0.093 T 0.462 0.008 S
474 73073 CGPG5688 SENSE 0.164 0.022 S 0.285 0.247 /
475 73506 CGPG6496 SENSE 0.512 0 S 0.986 0 S
476 74107 CGPG6590 SENSE 0.282 0.002 S 0.538 0.057 T
477 74117 CGPG6575 SENSE 0.211 0.002 S 0.449 0.005 S
478 74131 CGPG6592 SENSE 0.142 0.047 S 0.586 0.003 S
479 74344 CGPG5929 SENSE 0.27 0.01 S 0.474 0.105 T
323 13502 CGPG1354 SENSE 0.57 0.002 S 0.625 0 S
330 18259 CGPG3368 SENSE 0.226 0.026 S 0.551 0.021 S
361 19833 CGPG4074 SENSE 0.259 0 S 0.472 0.016 S
334 70417 CGPG3427 SENSE 0.187 0.056 T 0.113 0.65 /
273 70423 CGPG3165 SENSE 0.03 0.747 / 0.185 0.007 S
515 70435 CGPG3701 SENSE 0.131 0.051 T 0.394 0.014 S
493 70601 CGPG2917 SENSE 0.177 0 S 0.365 0.063 T
291 70696 CGPG4590 SENSE 0.079 0.205 / 0.331 0.024 S
365 70738 CGPG3195 SENSE 0.012 0.864 / 0.472 0.003 S
293 70740 CGPG3700 SENSE 0.103 0.082 T 0.387 0.021 S
419 71134 CGPG817 SENSE 0.063 0.548 / 0.279 0.066 T
422 72086 CGPG5236 SENSE 0.083 0.234 / 0.398 0.05 S
505 72463 CGPG4760 SENSE −0.131 0.416 / 0.422 0.003 S
285 73014 CGPG5692 SENSE 0.11 0.391 / 0.373 0.096 T
521 73488 CGPG6394 SENSE 0.018 0.793 / 0.398 0.1 T
299 73504 CGPG6480 SENSE −0.152 0.479 / 0.538 0.012 S
300 73507 CGPG6504 SENSE 0.15 0.002 S 0.053 0.854 /
301 73573 CGPG6462 SENSE 0.112 0.07 T 0.375 0.006 S
302 73586 CGPG6471 SENSE 0.309 0 S 0.611 0 S
303 73770 CGPG5435 SENSE 0.21 0.059 T 0.281 0.484 /
509 74103 CGPG6558 SENSE 0.374 0 S 0.561 0.024 S
428 74140 CGPG6569 SENSE 0.16 0.017 S 0.376 0.066 T
527 74262 CGPG5353 SENSE 0.203 0.012 S 0.375 0.045 S
430 74265 CGPG5356 SENSE 0.107 0.259 T 0.432 0.067 T
529 74302 CGPG5384 SENSE 0.115 0.101 T 0.269 0.056 T
431 74369 CGPG6076 SENSE 0.138 0.03 S 0.195 0.21 /
534 74465 CGPG6692 SENSE 0.2 0.02 S 0.688 0.042 S
507 74572 CGPG6640 SENSE 0.162 0.023 S 0.617 0 S
314 74583 CGPG6649 SENSE 0.144 0.023 S 0.403 0.008 S
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2
/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset
J. Late Plant Growth and Development Screen
This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants.
Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, OH) consisting of 30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat moss 10-20% processed bark ash, 1-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft3. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of 5 per 2½″ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat. Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of ˜200 μmoles/m2/s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.
Application of the herbicide glufosinate was performed to select T2 individuals containing the target transgene. A single application of glufosinate was applied when the first true leaves were visible. Each pot was thinned to leave a single glufosinate-resistant seedling ˜3 days after the selection was applied.
The rosette radius was measured at day 25. The silique length was measured at day 40. The plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53. The seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.
Table 13 provides a list of recombinant DNA constructs that improve late plant growth and development. TABLE 13
Rosette Dry Rosette Seed Dry Silique Dry Silique
Pep Weight Radius Weight Weight Length
SEQ p- p- p- p- p-
ID Construct_id Orientation delta value c delta value c delta value c delta value c delta value c
480 14320 SENSE −0.145 0.94 / 0.137 0.038 S −0.702 1 / 0.477 0.002 S 0.016 0.276 /
481 16756 SENSE 0.485 0.016 S 0.223 0.025 S 0.148 0.042 S 0.481 0.002 S 0.147 0.013 S
482 17448 SENSE −0.288 0.991 / −0.054 0.829 / 0.376 0.008 S 0.185 0.034 S −0.064 0.981 /
483 17633 SENSE 0.018 0.359 / 0.13 0.106 T 0.416 0.055 T 0.399 0.044 S 0.116 0.08 T
484 18876 SENSE 0.257 0.016 S 0.026 0.448 / −0.213 0.786 / 0.388 0 S −0.015 0.705 /
485 19120 ANTI- −0.252 0.936 / 0.022 0.006 S −1.042 0.95 / 0.165 0.076 T 0.046 0.021 S
SENSE
486 19221 SENSE −0.316 0.986 / 0.153 0.097 T −0.35 0.903 / 0.394 0.068 T 0.183 0.028 S
487 70206 SENSE 0.125 0 S 0.074 0.18 T 0.712 0.022 S 0.12 0.026 S 0.019 0.403 /
488 70223 SENSE 0.197 0.026 S 0.23 0.018 S −0.781 0.998 / 0.134 0.039 S −0.205 0.91S /
489 70347 SENSE 0.156 0.038 S −0.082 0.868 / −0.153 0.752 / 0.405 0.006 S 0.056 0.068 T
490 70406 SENSE −0.275 0.948 / −0.245 0.992 / 0.759 0.025 S −0.282 0.949 / −0.121 0.939 /
491 70469 SENSE 0.032 0.392 / 0.348 0.004 S −0.733 0.996 / 0.325 0.059 T −0.141 0.922 /
492 70564 SENSE 0.17 0.037 S 0.051 0.234 / 0.772 0 S −0.381 0.977 / −0.015 0.655 /
493 70601 SENSE 0.231 0.086 T 0.247 0.004 S −0.257 0.959 / 0.323 0.024 S 0.082 0.03 S
494 70612 SENSE 0.053 0.112 T 0.082 0.096 T 1.049 0.011 S −0.212 0.992 / 0.07 0.004 S
495 70720 ANTI- −0.16 0.898 / 0.028 0.384 / 1.312 0.009 S 0.219 0.121 T 0.128 0.018 S
SENSE
496 70735 SENSE −0.058 0.672 / 0.156 0.087 T 0.421 0.036 S 0.532 0.003 S 0.033 0.038 S
497 70846 SENSE 0.086 0.255 / 0.134 0.063 T −0.33 0.918 / 0.142 0.151 T 0.011 0.439 /
498 70923 SENSE 0.484 0.011 S 0.108 0.081 T −0.4 0.844 / 0.091 0.198 T 0.099 0.001 S
499 71149 SENSE −1.085 0.993 / −0.043 0.681 / 0.346 0.017 S 0.136 0.133 T 0.066 0.077 T
500 71608 SENSE −0.849 0.907 / −0.132 0.976 / 0.816 0.006 S 0.279 0.004 S 0.038 0.238 /
501 71739 SENSE −0.275 0.937 / −0.107 0.955 / 0.334 0 S −0.075 0.815 / −0.119 0.874 /
502 72014 SENSE −0.038 0.94 / 0.07 0.278 / 0.732 0.06 T −0.026 0.584 / −0.013 0.596 /
503 72051 SENSE 0.026 0.28 / 0.311 0.003 S 0.222 0.236 / 0.453 0.009 S 0.052 0.168 T
432 70217 SENSE −0.202 0.893 / 0.203 0.024 S −0.079 0.743 / 0.27 0.07 T −0.063 0.856 /
454 70354 SENSE 0.134 0.147 T −0.119 0.684 / 0.48 0.02 S −0.119 0.736 / 0.014 0.3 /
334 70417 SENSE −0.35 0.988 / −0.041 0.637 / 0.69 0.012 S −0.136 0.978 / 0.004 0.411 /
515 70435 SENSE 0.664 0.014 S 0.146 0.036 S 0.138 0.097 T 0.33 0.038 S 0.039 0.16 T
457 70465 SENSE −0.106 0.79 / / / / 0.883 0.001 S −0.27 0.945 / −0.097 0.818 /
460 70852 SENSE 0.178 0.031 S 0.145 0.034 S −0.525 0.904 / −0.315 0.861 / −0.059 0.774 /
405 71508 SENSE 0.195 0.162 T 0.251 0.017 S −0.515 0.929 / −0.322 0.997 / −0.196 0.982 /
295 71835 SENSE 0.139 0.055 T 0.163 0.049 S 0.538 0.019 S 0.27 0.068 T 0 0.496 /
467 72432 SENSE 0.146 0.021 S 0.139 0.02 S 0.325 0.012 S 0.074 0.149 T −0.106 0.879 /
443 72453 SENSE 0.204 0.037 S 0.116 0.046 S −2.198 0.995 / 0.016 0.448 / −0.013 0.534 /
433 72711 SENSE 0.292 0.058 T 0.143 0.024 S −0.114 0.76 / −0.04 0.667 / −0.093 0.946 /
449 72969 SENSE 0.046 0.072 T −0.158 0.887 / 0.39 0.031 S 0.477 0.001 S 0.095 0.054 T
426 72987 SENSE 0.385 0.006 S / / / 0.098 0.104 T 0.153 0.016 S 0.057 0.061 T
525 74022 SENSE 0.11 0.226 / −0.069 0.844 / 0.71 0.009 S −0.05 0.613 / −0.004 0.544 /
287 74251 SENSE −0.56 0.961 / −0.184 0.916 / 0.611 0.001 S 0.174 0.229 / −0.137 0.923 /
537 74507 SENSE 0.255 0.017 S 0.178 0.03 S 0.318 0.032 S 0.188 0.011 S 0.013 0.078 T
313 74512 SENSE −0.247 0.953 / 0.107 0.015 S −0.073 0.86 / 0.113 0.034 S 0.05 0.175 T
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents data points not determined or the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2
/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset
K. Limited Nitrogen Tolerance Screen
Under low nitrogen conditions, Arabidopsis seedlings become chlorotic and have less biomass. This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.
T2 seeds were plated on glufosinate selection plates containing 0.5× N-Free Hoagland's T 0.1 mM NH4NO3 T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i.e., viable or non-viable) and root length. After 21 days of growth, plants were scored for visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1L.
Table 14 provides a list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants. TABLE 14
Flowerbud Number of Number of Seedling
Pep formation green leaves Root Length rosette leaves Weight
SEQ RS p- RS p- p- RS p- p-
ID Construct_id Orientation mean value c mean value c delta value c mean value c delta value c
375 72963 SENSE 1.1 0.004 S 0.293 0.021 S −0.446 0.002 S −0.246 0.786 / 0.137 0.001 S
389 70437 SENSE −0.28 0.982 / −0.08 0.769 / 0.259 0.006 S 0.5 0.005 S 0.133 0.003 S
390 71633 SENSE 0.26 0.26 / 0.254 0.114 T −0.1 0.539 / 0.647 0.06 T 0.106 0.023 S
391 72948 SENSE 0.587 0.06 T 0.539 0.029 S −0.237 0.003 S 0.479 0.1 T 0.078 0.121 T
392 72519 SENSE 0.749 0.033 S 0.209 0.104 T −0.09 0.274 / 0.276 0.264 / 0.116 0.006 S
393 10475 SENSE 1.256 0.026 S 0.588 0.005 S −0.378 0.002 S 0.081 0.319 / 0.018 0.75 /
394 11120 ANTI- 0.795 0.033 S 0.608 0.015 S −0.45 0.001 S 0.287 0.106 T −0.041 0.387 /
SENSE
395 19736 SENSE −0.24 0.907 / 0.355 0.033 S 0.014 0.864 / 0.64 0.006 S −0.075 0.005 /
396 71606 SENSE 0.605 0.088 T 0.176 0.11 T −0.033 0.708 / 1.239 0.005 S 0.133 0.002 S
397 71840 SENSE 0.408 0.235 / 0.879 0.006 S −0.137 0.248 / 0.524 0.032 S 0.066 0.198 T
398 74240 SENSE −0.06 0.602 / 0.13 0.076 T −0.107 0.306 / 0.714 0.034 S 0.108 0.002 S
399 74331 SENSE −0.44 1 / 0.054 0.203 / 0.132 0.055 T 1.045 0.021 S 0.134 0.003 S
400 74610 SENSE −0.59 1 / −0.08 0.922 / 0.289 0 S 1.241 0.017 S 0.137 0.001 S
401 75527 SENSE 0.242 0.228 / 0.376 0.028 S −0.183 0.045 / 0.352 0.083 T −0.005 0.8 /
316 71811 SENSE −0.45 0.91 / / / / −0.112 0.202 / 0.438 0.014 S −0.054 0.161 /
505 72463 SENSE −0.16 0.976 / / / / 0.112 0.109 T 0.366 0.024 S 0.13 0.006 S
351 75701 SENSE 0.736 0.048 S 0.07 0.861 / −0.4 0.018 S / / / −0.109 0.193 /
S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)
T: represents the transgenic plants showed a trend of trait improvement compared than the reference with p < 0.2
/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset
L. Statistic Analysis for Qualitative Responses
Table 15 provides a list of responses that were analyzed as qualitative responses TABLE 15
response Screen categories (success vs. failure)
wilting response Risk Soil drought tolerance screen non-wilted vs. wilted
Score
growth stage at day 14 heat stress tolerance screen 50% of plants reach stage1.03 vs. not
growth stage at day 14 salt stress tolerance screen 50% of plants reach stage1.03 vs. not
growth stage at day 14 PEG induced osmotic stress tolerance 50% of plants reach stage1.03 vs. not
screen
growth stage at day 7 cold germination tolerance screen 50% of plants reach stage 0.5 vs. not
number of rosette leaves Shade tolerance screen 5 leaves appeared vs. not
at day 23
flower bud formation at Shade tolerance screen flower buds appear vs. not
day 23
leaf angle at day 23 Shade tolerance screen >60 degree vs. <60 degree
number of green leaves at limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not
day 21
number of rosette leaves limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not
at day 21
Flower bud formation at limited nitrogen tolerance screen flower buds appear vs. not
day 21
Plants were grouped into transgenic and reference groups and were scored as success or failure according to criteria in Table 15. First, the risk (R) was calculated, which is the proportion of plants that were scored as of failure plants within the group. Then the relative risk (RR) was calculated as the ratio of R (transgenic) to R (reference). Risk score (RS) was calculated as −log2RR. Subsequently the risk scores from multiple events for each transgene of interest were evaluated for statistical significance by t-test using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). RS with a value greater than 0 indicates that the transgenic plants perform better than the reference. RS with a value less than 0 indicates that the transgenic plants perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.
M. Statistic Analysis for Quantitative Responses
Table 16 provides a list of responses that were analyzed as quantitative responses. TABLE 16
response screen
seed yield Soil drought stress tolerance screen
seedling weight at day 14 heat stress tolerance screen
root length at day 14 heat stress tolerance screen
seedling weight at day 14 salt stress tolerance screen
root length at day 14 salt stress tolerance screen
root length at day 11 salt stress tolerance screen
seedling weight at day 14 PEG induced osmotic stress tolerance screen
root length at day 11 PEG induced osmotic stress tolerance screen
root length at day 14 PEG induced osmotic stress tolerance screen
rosette area at day 8 cold shock tolerance screen
rosette area at day28 cold shock tolerance screen
difference in rosette area cold shock tolerance screen
from day 8 to day 28
root length at day 28 cold germination tolerance screen
seedling weight at day 23 Shade tolerance screen
petiole length at day 23 Shade tolerance screen
root length at day 14 Early plant growth and development screen
Seedling weight at day 14 Early plant growth and development screen
Rosette dry weight Late plant growth and development screen
at day 53
rosette radius at day 25 Late plant growth and development screen
seed dry weight at day 58 Late plant growth and development screen
silique dry weight at day 53 Late plant growth and development screen
silique length at day 40 Late plant growth and development screen
Seedling weight at day 21 Limited nitrogen tolerance screen
Root length at day 21 Limited nitrogen tolerance screen
The measurements (M) of each plant were transformed by log2 calculation. The Delta was calculated as log2M(transgenic)-log2M(reference). Subsequently the mean delta from multiple events of the transgene of interest was evaluated for statistical significance by t-test using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). The Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. The Delta with a value less than 0 indicates that the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.
EXAMPLE 2 Identification of Homologs A BLAST searchable “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database was queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 240 through SEQ ID NO: 478 using “blastp” with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.
The Organism Protein Database was queried using amino acid sequences of SEQ ID NO: 270 through SEQ ID NO: 538 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Likely orthologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 539 to SEQ ID NO: 22568. The relationship of the homologs to the identified trait-improving genes on an amino acid sequence basis is found in Table 17 where the amino acid sequence of a protein encoded by a trait-improving DNA, e.g., SEQ ID NO:270, is followed by the amino acid sequences of protein encoded by homologous genes, e.g., SEQ ID NO:19844, 4248, 2761, 15944, etc. The source organism of each homolog is reported in the Sequence Listing. TABLE 17
Sequence IDs for homolog proteins
Seq ID NO: homolog Seq ID NOs
270: 19844 4248 2761 15944 11776 16144 10470 9742 6776 1010 2285 16333
9154 20620 16454 20025 8388 10646 1208 6001 1706 2448 14768 10226
12626 19846 9302 17295 17794 6354 5098 1789 6430 17749 821 10109
7542 17855 15562 17462
271: 1715 5418 7208 19338 7440 711 8113 17151 17592 18879 4807 8671
9936 11315 10681 3177 10519 6830 13563 12162 15155 1860 18072 20945
6715 15032 5192 10928
272: 0
273: 13771 8553 4219 10043 10800 8345 17501 13569 954 17197 6188 3760
13267 16169 8132 2667 11216 15637 4652 2270 10309 5708 18374 9446
12844 7790 7569 4786 9725 14187 12859 16948 18626 13741 5525 7877
4550 15544 9706 7616 14358 15163 13182 14560 16722 1129 1472 4261
10693 20144 6437 21413 17893 17984 17116 9925 19953 20648 983 2837
5663 2943 10465 1841 12497 6435 14763 13495 12676 7513 8363 16389
8162 7945 14956 15029 12433 22241 16071 13003 16940 18847 12354 7732
14013 5735 11505 8833 17658 16048 17609 575 6641 6331 3738 10842
18927 21518 20097 14117 5309 13744 15880 19484 9648 22509 1221 15515
6785 852 6466 17423 14164 658 8704 16710 19375 3306 14050 16883
16322 1722 15481 13636 10680 6347 3552 8885 21794 17703 22557 14777
21189 13711 3601 3968 3692 4003 20044 12943 19749 1865 6355 14902
6137 22370 19468 10410 21460 10451 17175 20965 12916 1206 6796 11329
9139 11008 10569 9058 7988 19743 20088 14111 8231 4522 18497 11952
2866 15466 3609 2403 16796 13539 14806 5364 12620 20699 12940 15426
4409 12452 5296 9156 629 12665 20947 3649 7530
274: 16167 5178 2412 10455 20036 11246 19666 6400 5573 22539 8547 21845
2413 20290 4036 19351 5886 6071 17184 9738
275: 14665 16694 12678 14928 21489 7918 1571 3959 2490 2517 14615 3788
10022 16096 21248 13293 8541 13446 6120 4360 3812 15574 18938 19203
2284 2215 10054 14052 9653 10183 17752 20776 4240 22343 8270 9192
17217 7374 12141 20657 7674 6445 2522
276: 12496 3460 13599 7043 9150 1664
277: 16949 9640 8150 2014 12188 5779 17876 14612 18293 11053 15958 15263
18370 20984 13094 18734 7380 10318 21641 12737 13028 20561 7087 10686
9894 7528 12573 16043 14846 20513 2802 8897 14716 10257 16407 2727
6151 1484 6831 16916 10146 17756 13193 7670 15946 7750 9397 20046
12547 5399 18644 11883 12531 12530 17188 2130 3805 17493 16821 10181
3639 3934 1419 780
278: 16531 9228 5799 19821 10980 17656 3449 19982 13335 20959 11238 13084
10281 17610 19623 17614 9736 21375 19978 5859 4943 12390 18806 1349
8759 21741 20400 13707 9170 15899 11361 4333 10631 12909 2136 12776
20323 7676 5847 9065 19902 4545 6768 546 9246 14134 4442 20731
9931 6700 8677 19305 4828 3655 17550 7579 21629 9044 19475 637
4209 3519 3873 15884 12247 10177 1407 10312 15957 16955 17469 20241
7267 21862 16864 22192 19599 6365 12324 10985 6424 6449 14564 3115
14885 10603 911 18609 3359 7059 2851 5801 9613 8391 18695 7987
14964 1081 15171 1312 14747 3060 13390 22115 5060 16536 19729 13468
11109 21989 2230 9462 9096 18775 10721 11999 8340 16607 22199 8687
3652 8147 22073 4090 3491 20506 7835 10890 6781 7839 14478 16371
579 6442 9721 5423 2566 7876 17580 2023 13164 9424 14096 8115
15304 14818 7282 6422 4841 10106 1075 12995 9297 21280 2309 895
12373 5366 22159 4499 737 11369 4308 13974 2794 12771 16584 9250
6930 8792 10185 17718 13148 5054 22383 9674 9868 21546 15881 20000
19029 4938 14024 15026 15535 16386 15402 21036 12653 9490 4170 11551
3711 4179 18619 16370 14161 11294 9094 2689 21881
279: 13577 10344 17371 5840 13904 12689 11795 21912 17226 14519 8854 14229
7998 8326 1400 16163 11785 10117 21511 21043 18205 2933 2043 22181
21628 20090 18018 20578 19081 11011 4768 2834 4635 13475 6306 13119
1671 21348 16926 20801 6361 4269 4366 15615 941 7046 3166 16526
6250 4790 825 16541 14270 3574 8127 18015 13147 17950 15243 6671
20662 3487 11787 6762 5488 7117 9417 15702 4859 20527 19240 17200
7115 19523 12823 9115 11770 20530 4791 20334 11019 20645 14383 1914
8945 15358 9347 2496 3098 11604 16441 19352 18451 12451 8877 15655
791 12609 12588 19785 18328 8143 22185 1488 10212 14002 16826 11779
1890 3879 20130 21633 20930 17222 2691 18852 7120 7853 644 21495
14630 21041 5676 8447 16750 10436 4523 2686 8237 2948 13559 19008
3686 18465 3895 7416 10170 18867 14574 1159
280: 16310 3796 10205 19486 13581 11022 21960 15598 12604 9662 20210 21764
8626 17323 11666 21316 6121 7438 982 2135 17499 12340 20711 6553
10296 17874 914 17716 3723
281: 20551 21096 13717 4006 19762 10017 14425 17785 6291 3855 15232 11917
21856 18564 19010 3910 13621 19087 670 10539 851 5698 9112 12694
14218 11947 18104 21227 22348 14178 1862 9829 9325 14533 13149 8679
17127 6507 10375 2782 3357 594 7548 9316 5728 7981 8022 13688
1294 13817 21893 10781 5263 11292 5492 9542 8000 16102 6328 11687
13695 15807 4068 3478 6486 13660 22165 17881 19166 3613 7013 6393
15983 17688 5124 4243 19684 17008 12366 7161 20062 12194 15870 14385
9124 9865 6211 628 6448 700 8869 17941 10697 21134 5586 4469
21167 855 20538 9251 1036 10678 1977 17337 4575 19974 3520 10195
14572 3870 21293 19011 12921 20120 11647 15054 13976 21163 20362 12988
15636 2345 4740 3205 17504 1953 15208 16834 7654 15907 8961
282: 19261 3860 7076 12616 1790 4886 9735 12611 20478 4501 18874 7032
18024 7225 4544 11443 2127 19283 7367 1338 4482 15213 20554 3826
14978 21769 11755 4250 15506 20020 6593 1286 20750 18985 16069 4571
22536 3773 11152 9745 13196 2190 8120 7914 16863 11987 16172 15399
14422 12490 8076 17180 19067 14493 13105 16459 18285 15863 14085 18130
11566 17352 20003 2995 5386 8757 19103 15685 20563 18739 20815 19454
7820 20771 7972
283: 0
284: 10750 5276 3894 3486 12240 18158 12170 15393 9765 11266 5031 2792
9334 20684 1144 13799 10858 16622 20849 22001 6897 17710 15401 18589
9550 1757 10249 21993 2001 19689 15058 4297 19990 643 11414 18208
19995
285: 17916 15231 15741 15829 4645 21977 10291 1806 21573 474 6018 2663
8036 9618 16693 3960 15864 14578 17125 15924 21826 13440 17249 8650
20159 1986 15742 19706 22092 8766 6813 17830 10853 21281 13394 5285
8139 21004 14220 17563 2086 2488 1597 4698 13233 4654 1250 15737
2907 1469 9957 13288 6516 22526 16496 14873 10471 18290 3086 11953
18592 3185 9418 17135 8081 9593 19180 4673 7979 16544 13933 1300
16782 15551 8460 15960 3405 13997 1566 21046 8636 17134 6512 6596
13346 15639 14396 9252 12093 21591 15042 6953 18637 16784 22523 6262
16933 22448 4612 19863 6076 4133 19601 3344 12192 16828 17089 19303
6118 15088 14986 21070 771 3291 2153 21234 18173 11970 21215 10644
20638 4377 21183 9519 13810 10948 17764 3781 21029 16613 18091 6526
5846 22213 22003 20765 3801 21866 21771 14860 861 6743 5007 5529
14267 14880 21391 10210 5693 5970 3793 15855 1007 13001 6878 9875
16912 19329 13614 10333 13714 6903 21112 8204 1133 21262 16852 15703
21338 6248 21547 15242 13567 16788 11020 18655 10528 19496 17440 22414
17480 8142 7760 20388 2829 16249 12914 2569 14595 7096 6689 12534
6105 16041 9242 9145 1552 10313 1379 9596 11771 5820 593 15445
3268 14744 18410 6984 10872 10053 9713 8837 1383 4305 10421 2944
20363 19120 7463 16753 20969 18430 12905 10227 11066 6057 13677 18640
4083 1527 19285 5385 17557 20851 15693 17304 1683 14391 15965 19854
8425 14916
286: 16099 8075 8242 429 13036 10217 18174 507 11062 15313 16399 14594
3820 5216 5717 10394 20464 15374 15526 18461 17556 14810 4502 12391
11462 14924 1652 18690 9504 13188 8137 3327 12056 2537 4030 1064
15390 6367 19001 8581 6859 7602 14778 7681 18172 18107 12112 7417
13896 2465 12130 19906 4949 17359 16220 13607 526 20229 16469 10705
15889 16978 8246 16554 18467 9002 9780 17882 21655 20242 21051 3977
19627 9063 10810 15051 5705 9204 11991 9915 15840 16148 14589 6171
6614 20389 6007 6226 5373 19830 9030 14898 11912 16971 18279 19602
13078 18533 1840 4491 7812 8977 13027 6371 15170 21310 6443 9755
12535 2819 14876 3052 12267 8377 3804 7983 22450 22167 22369 21674
891 13549 7113 14532 11064 9354 7858 8394 2489 17243 2044 9345
6836 2341 19148 17763 13516 14240 9032 15489 8304 7822 17083 9993
8188 5481 11010 1093 7789 21699 21306 11113 8956 18128 7880 3416
2857 17664 21589 11961 19154 11542 4812 7486 21609 5307 21506 3533
6663 4677 18511 4730 696 9569 17797 18687 7097 20259 15252 13221
3425 3261 450 6778 14692 22502 14915 21157 20371 3563 12591 16308
7626 4212 19798 16130 21296 2832 830 14553 14135 6434 18295 17956
20230 476 10392 12043 3289 20779 1281 17745 21065 5487 3754 8587
2882 12578 11548 12705 15830 16586 7955 15449 988 20859 1768 11129
6547 22518 5552 9854 11376 6669 5359 9132 10964 22087 1879 17298
13137 3333 20301 13752 19370 2425 6946 19538 14273 8003 15340 6427
17165 15603 12419 10809 13658 14064 4221 7387 3386 13284 20981 2316
3116 8418 3578 6508 18217 992 22249 14855 13442 3471 19949 5277
19885 2816 10917 21954 9436 18538 18659 12816 12308 20364 15943 2649
2515 14701 9329 8193 17579 22076 8890 21073 8376 15177 10882 859
5990 9814 16053 12078 3144 5209 13848 22428 19076 9604 5454 3141
17056 14918 953 4088 6615 15729 11270 5837 14568 12544 17630 2371
4694 12396 15369 16597 3697 10741 13396 21621 16662 8841 11120 14081
13456 21472 7487 12898 12787 7866 11748 8692 9484 2336 15204 6565
14320 2978 9294 7482 9493 1277 4576 15513 21001 3242 4952 7813
7306 12296 12021 6645 21060 9286 22489 6264 13745 16653 19069 7780
15219 16969 9271 18873 14762 10802 10479 16663 3678 16713 7751 13703
3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482
14526 7151 2644 835 10655 12264 17372 788 15452 14361 11678 2754
15289 14415 9468 1673 2117 17672 3643 6809 9280 22095 18903 3706
15256 18593 5764 11115 12583 11568 622 13613 2331 5136 8073 15998
5630 10889 9673 9014 7950 6748 9771 11716 18929 14842 6789 1773
981 18575 19878 21485 14269 9584 12794 21399 20485 9218 18691 3350
18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 2376
15699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848
13211 22530 15360 12863 9975 6398 14067 16683 21170 1924 890 8155
15866 10131 7187 4332 1235 20330 12927 7088 5099 2302 12424 8303
17466 14322 11383 2282 14043 15956 1818 3414 12982 18548 15665 10961
21084 10824 18440 16819 3730 13940 18821 14864 19607 7969 6546 16771
5441 8459 18266 5000 15749 13014 14274 8444 4707 13097 15930 6281
18662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143 6097
786 6709 13766 5954 9650 17926 9083 20839 17473 5462 22351 12788
5453 13277 11817 9672 8255 6463 15113 9053 15528 1319 926 17317
6368 7704 18694 3644 12154 3794 21257 21638 18386 18111 21498 10731
13776 5539 14530 20282 7762 9529 17675 15191 12380 14865 15825 2818
16442 10262 10965 15558 5973 22476 19408 5773 5016 20819 18982 19997
4572 640 19168 17905 2970 2132 20312 4655 16568 21093 13882 5730
7976 7079 13035 3505 2800 12522 8939 11219 2155 15905 3206 2017
15587 6277 1297 1423 9549 6119 18728 12545 2037 11704 9571 21379
13089 11197 13107 22367 2420 17943 7599 5997 1801 21374 16528 8722
16672
287: 8532 19537 5344 9149 21995 6924 15909 4097 11901 8844 7649 2133
2202 5183 18294 19005 11338 19082 14317
288: 19787 18784 5642 15617 18773 2850 21740 18774 8659 17768 4774 15786
13450 5349 3435 11594 3337 19500 18011 5026 20455 17036 22287 17395
9621 6514 1386 11394 7035 21823 15347 11387 15362 4210 5017 10279
19441 22361 7943 7642 8141 16188 15181 3603 7428 2749 21346 17018
10086
289: 652 22063 8339 21393 2555 8373 21778 11080 15895 10755 10659 16023
19651 17328 17996 10829
290: 7684 9445 17343 1187 6793 10461 6245 10467 20367 12284
291: 19558 4465 12410 21267 12416
292: 13619 7334 20532 21542 12253 16189 8764 2507 21921 6586 19095 9485
5381 5599 12372 14529 13331 15295 19004 1397 21520 14556 20510 22236
2986 7080 8521 6732 13272 16558 4313 20499 22483 14588 9523 7470
18752 12181 20429 12348 695 10907 14321 14033 18144 3919 8802 20993
8165 3312 17714 14662 16218 6883 15696 5826 8318 13408 11637 4264
1882 4226 19194 12589 20866 1376 21972 8106 9328 11225 6391 8315
20980 16513 6488 21387 10825 871 22553 7860 20986 5123 7178 8148
19174 5633 3500 7737 17944 19517 19101 2424 1678 1758 16439 22408
11623 7905 18353 18641 17321 18471 6775 8307 11843 9722 15205 20080
16600 6416 10161 8449 8858 14419 19903 14156 15652 13343 15817 3739
12654 14241 7430 8851 6919 22017 6531 1962 3371 18557 2125 6815
15392 19947 6480 15023 1479 1138 21122 20521 21201 1677 17815 9610
8319 5949 7617 10353 9342 4915 6495 1682 14875 17940 1204 13538
16452 19603 8029 2886 14466 19507 12125 6822 10351 4123 9222 9605
18244 18276 17153 17212 12619 14496 11337 8683 15935 10084 14862 5228
20909 9138 702 17034 5542 16281 16905 2247 17007 15480 13602 16745
3223 6163 1245 10228 21595 18522 6698 5750 8543 21981 15735 3961
16417
293: 10002 18611 6406 14073 5127 8351 1241 12550 10077 11772 10606 3231
10112 15857 13002 6686 8210 8327 4270 16125 8696 5110 12311 7006
19199 6994 12597 5342 12722 21575 5723 2670 6046 12012
294: 9532 10331 15934 2046 6293 19503 22316 15147 14129 5699 7047 13130
2151 1110 20162 9143 9176 4233 8431 6088 7434 18942 5356 18099
10361 3212 21551 20873 2419 8077 3140 13009 21068 6829 20236 14326
6208 847 2330 15785 1052 14929 4869 4567 16757 19546 10070 1727
6317 8558 3880 18261 6409 6931 17666 21501 12516 16760 16947 3896
3358 6554 14909 1974 4835 20560 11275 2659 12987 18209 16896 15952
20752 15130 15569 7406 13923 1051 11602 3912 10042 4592 22204 2430
1382 18088 15862 20314 9057 10845 5041 7582 21742 12835 17242 15658
8522 22493 7831 1594 13654 7414 12151 12808 14165 15368 17979 10215
8018 22247 22220 7254 654 20380 2845 5061 7274 5018 9568 9818
1494 3725 11235 6804 8938 12700 17001 19302 11190 4028 3173
295: 21192
296: 10114 6052 18680 17170 11936 20081 9148 3938 18585 12840 14672 14951
1544 20775 4870 8723 3360 19138 1416 1901 18744 2625 951 6936
16543 3550 9203 11070 9360 21434 3920 1693 10869 11881 12895 1003
5405 14684 21089 14531 21626 20145 17875 7549 2428 2613 18036 1421
18003 6973 20577 12790 22102 3375 13691 15302 6589 15430 18154 16596
19645 21006 17187 14709 19975 1854 17428 11844 10033 1792 934 845
5533 21419 6582 2314 19307 8902 8278 20861 7030 10654 9636 16002
2969 15306 5928 1894 592 13534 4985 5623 5150 2342 22174 1686
2688 10718 20383 19440 19860 618 22308 10355 12565 8585 15993 12725
22563 5578 14369 13712 9428 20205 10767 1546 5387 8364 18905 18948
2545 17156 20131 22338 2223 7037 15860 5111 16844 14702 1634 13698
16173 17860 16802 21238 20479 18313 18815 16556 12453 18481 19778 12153
19470 20701 1477 19657 2220 5033 6117 12286 12326 6566 16110 13469
17077 20231 4058 9834 3780 19094 6308 9548 1549 21687 13080 7340
18746 2266 11139 2005 14735 5543 4941 2921 8468 19501 3139 16192
10723 6333 11355 3845 11452 19385 14092 1395 5545 2216 10682 21311
15661 21100 2597 14697 9008 17498 17514 9946 20269 5669 5062 15447
2775 6975 12793 12639 8185 19766 8932 4278 14889 4981 22471 5952
6818 16336 21140 14098 21953 4979 13968 16410 18878 9696 4978 16146
2609 22097 16986 20921 22527 16920 18126 16034 5362 933 15779 16770
1772 9517 6866 16982 19232 19937
297: 9912 18441 5938 12696 19188 22233 2840 10426 16052 9508 12778 15536
19286 9563 19034 20209 15518 6498 21608 11026 14044 10165 12621 12028
16923 21297 5646 16170 12477 16660 1378 2624 3809 12760 16057 7256
15500 18959 10317 10326 5977 4830 1143 1499 10983 2571 15543 13839
12370 9041 1331 8508 16831 13832 4303 18599 21584 9787 1548 8292
1560 18466 11683 17762 17468 9815 4374 18826 20637 18709 16467 7307
16512 904 18899 17113 10700 5006 22084 5463 20856 18792 1535 13608
11312 8634 19381 11435 870 7232 5079 6299 13314 10487 15382 14552
14878 20590 14820 7911 8924 12070 7581 2122 18063 8338 12227 14479
3229 17500 9776 16213 12767 17455 5438 1839 9982 4649
298: 18555 7838 6652 1982 15254 2218 6543 13709 5154 14481 4355 8406
21382 21663 19102
299: 19421 10037 3803 5108 3343 20336 11002 8946 12661 9168 15378 18020
16075 5607 19118 22336 19724 8555 2295 21579 1259 8494 18083 960
13560 20787 22477 19264 6401 8583 9620 5248 701 10759 22290 2593
21024 17543 1417 15845 17793 18019 6222 10575 1775 8903 15288 2885
7993 20494 10001 13641 11853 614 12502 13330 17952 13069 9420 1771
17636 13434 5588 2165 19494 4998 1751 16904 10306 3123 12413 16515
21706 9144 19364 2394 2677 15207 13710 9045 11258 1042 11230 13140
18738 15056 11058 930 22417 8107 16349 7322 16450 15822 16451 6646
18926 2197 302 8630 17924 14996 19753 4515 10245 7300 6710 21723
12990 8111 14791 13977 19581 12911 22090 603 15053 14851 21092 14284
20327 13846 9587 21947 7441 12133 14923 3660 13939 6002 5590 11909
22216 4929 14884 9941 9040 12896 7903 21683 11508 22006 11550 5658
2777 9977 15523 5791 21963 16141 18240 5322 2499 19702 9292 20325
5274 9028 10531 1688 20146 5153 7485 18445 4165 5614 10277 16781
4563 3161 6533 5957 7089 2687 7105 21351 21515 22453 19592 7173
1968 19358 11469 8780 17266 7586 12142 3066 7281 14577 9430 3270
13924 12341 15761 12637 10962 2998 19035 4877 659 9821 20906 12168
1736 20717 2543 16421 1821 18042 14352 15050 8399 19775 21325 7525
17680 18001 19979 18487 6598 5555 10444 21216 21128 8987 1493 17533
16118 1639 12595 68552 20981 8912 3290 5745 6378 9285 11287 14670
11840 9243 9669 14995 13742 20118 9371 11429 12747 19245 1590 13573
3695 20005 2712 20008 9988 1732 20129 19987
300: 16422 16652 2702 1006 429 10217 533 507 306 535 478 10780
18650 18461 13998 11343 7459 5240 10390 15192 6367 19001 17659 17306
18166 3899 19411 17674 6453 14183 9624 20799 11585 14626 21314 12049
7812 18212 3745 13797 8320 4940 22404 21496 12527 18023 6674 2559
15926 17879 9075 14547 21758 1689 16481 15621 18121 13519 12731 5774
10662 11761 10902 17148 12381 2857 17664 21589 11961 19154 6201 21437
11195 20933 21506 3533 4677 18511 4730 696 9569 17797 18687 7097
20259 15252 13221 450 11335 20777 12713 22502 14915 21157 20371 3563
12591 16308 7626 4212 19798 19458 5691 18292 16130 21296 2832 830
14553 6434 18295 17956 829 12043 20779 1281 17745 1768 6547 16586
7955 12705 11376 15449 988 20859 11129 22518 5552 9854 6669 5359
9132 10964 1879 22087 13137 17298 3333 13752 20301 19370 2425 6946
19538 14273 17165 15340 6427 15603 12419 10809 8003 13658 14064 4221
7387 13284 20981 2316 3116 8418 3578 3386 6508 18217 22249 992
13442 3471 14855 19949 5277 19885 2816 10917 9436 18538 18659 12816
12308 15943 2515 20364 14701 8193 17579 22076 8890 2649 9329 21073
8376 15177 10882 859 5990 9814 16053 5209 3144 13848 22428 19076
5454 17056 14918 4088 11270 5837 9604 22455 19840 801 12234 21301
1149 953 9960 1656 3755 12278 22119 21098 1005 10264 13037 11702
20954 5581 5063 1118 6454 20632 1696 19474 14911 9381 4111 13374
2932 3071 18551 22362 1431 12923 6509 19229 14012 5372 12362 17380
20272 16391 13395 5132 901 9540 19228 14568 17630 2371 4694 12396
15369 16597 3697 10741 16309 4927 13396 21621 8841 16662 11120 14081
8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813
7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969
14762 18330 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472
10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644
835 10655 12264 9315 2786 16253 9488 5634 17372 788 9280 22095
18903 3706 15256 18593 5764 11115 12583 11568 13613 2331 5136 8073
15998 5630 11304 19137 5817 5580 18341 8588 12540 2454 4970 17445
2401 11869 6193 21516 10889 5190 13207 16465 9673 9771 11716 18575
9584 12794 21399 20485 9218 18691 3350 18263 4846 544 19667 9933
4140 1318 20418 11128 20105 16734 2376 15699 7061 4232 15357 14036
5339 7107 19030 7165 21370 12103 4848 13211 22530 15360 12863 9975
6398 14067 16683 21170 1924 890 8155 15866 10131 7187 4332 1235
20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 2282 15956
3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 13940 18821
14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 15749 13014
14274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 10837
21928 13800 5188 19614 16117 4719 19714 1833 13499 3107 21492 12503
21929 7921 1429 14398 22189 6439 12993 12307 6650 18994 747 5932
18630 6683 1921 15651 5594 6958 13597 19763 10097 14124 14687 1094
5780 7770 7688 15110 5797 7907 21169 3329 12627 4065 19882 22067
19211 2061 7038 8909 16914 13129 19881 13235 16317 1551 14888 1201
20264 7800 7526 2209 5887 10798 15317 10223 11351 10105 10661 15014
2908 16412 22277 19851 12003 19616 11003 7768 6166 4620 13850 16231
7016 20541 3458 1240 15787 20099 9282 11480 4994 6281 18662 7992
9661 19875 11156 3619 14086 20948 16946 3456 6143 786 6709 5954
17926 20839 22351 13277 15113 6368 7704 18694 3644 12154 3794 21257
21638 18386 18111 21498 10731 13776 5539 14530 20282 7762 9529 17675
15191 12380 14865 15825 2818 16442 5901 8220 18578 13297 2495 21913
4526 16085 10965 15558 18982 2984 11245 17095 7536 5117 2132 20312
4655 16568 21093 13882 5730 7976 7079 13035 6801 2800 5619 10126
12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 11197 13107
16006 21848 20337
301: 19000 5175 17102 15893 7025 2120 12938 12299 15442 11496 10074 5733
14018 4497 12860 17771 17375 8161 11868 22009 7849 15996 13313 6451
9249 12173 13520 22245 9579 19128 12786 1276 5812 2509 15030 5404
7335 18498 6363 16355 19266 14084 8908 19966 8863 19169 539 3459
13467 15720 2898 9093 9837 17258 1976 20125 1460 8614 10175 5871
15351 6426 7149 12032 11271 8224 11194 11907 4519 3493 18304 22188
18791 10516 18657 11078 6908 20626 14734 4564 18762 9992 1434 6230
9846 4840 12934 18658 3437 11327 16354 4241 1568 14609 10156 19085
969 13387 20299 14003 18420 8635 5706 21190 1432 6124 1526 18029
16093 11179 1324 22474 8016 22059 5181 15454 1309 4573 20643 18651
6220 9683 15336 12171 3835 20676 15459 11187 6374 19257 8887 12249
15479 8309 2813 3769 17901 16142 2063 6092 19535 21118 1210 8401
7375 9531 10590 7738 17854 1657 9700 15494 7894 15287 1226 6551
13070 19534 13610 1802 22458 17237 8915 14991 3591 9885 9545 21263
14344 19754 11799 10504 11873 18078 16699 19633 15816 12846 4878 20466
21081 22381 17475 18403 21905 22085 18453 5736 17957 4021 7834 14499
2154 13058 11572 3583 10692 7183 16133 15670 12332 1756 16507 18601
11306 18332 20634 7994 12919 9064 5045 14968 9551 2414 1646 4753
1805 1502 18175 18991 19442 17821 1888 18587 10549 18855 4858 7028
14908 22205 5964 4933 4340 5271 927 8374 3316 9426 12837 20014
16546 20576 5008 9914 10822 13163 7197 6338 17347 21212 3445 14444
561 1401 13989 15266 20426 12281 7477 7453 766 14757 19524 4093
17235 17808 16762 10080 9217 14800 9794 18834 2718 6390 20406 7926
924 20355 13005 19727 8981 4683 7215 4131 6381 3407 11741 12599
16185 18844 16344 19195 11057 8355 8958 11977 19693 20196 6610 14503
22133 4744
302: 8946 9168 14628 299 6214 4182 16075 11899 18083 13560 20787 22477
16789 19264 6401 9620 5248 701 22290 21024 4074 1417 17543 11432
17793 6222 18019 21867 3019 20723 20357 10575 3819 6741 17742 1775
8903 15288 14774 2885 20494 20292 10001 11853 13330 17952 16001 17214
1751 16904 10306 20788 12413 1843 16515 21706 2394 15207 18738 15056
9630 930 15269 6646 18926 4515 7300 14791 6976 603 21092 2483
21947 7441 14923 3660 12133 22216 4929 5590 11909 14884 9941 6002
9040 13939 12896 7903 14465 21683 11508 8978 22006 5322 21963 2499
2777 18240 5791 16141 9977 5658 15523 9292 11550 19702 1688 20325
5274 10531 9028 20146 5614 7485 10844 2674 18445 4072 7836 5153
4165 15169 10277 16781 7814 3161 5957 6533 4563 7089 2687 21351
21515 22453 19592 7105 7173 1968 19358 10994 20056 7763 9851 13457
8780 11469 7586 17266 3066 12142 9430 14577 13924 7281 3270 19033
15761 12341 2998 12637 2543 10962 19035 20906 4877 659 9821 12168
20717 14352 1821 18042 16421 1736 15050 8399 19775 17680 18001 21325
6598 18487 15537 14525 21216 7525 19979 5555 10444 11512 21128 1493
8987 17533 22098 6855 12595 18912 15079 5745 6378 9285 11287 14670
11840 14995 13573 3695 19886 12402 20005 2712 9988 1732 1437
303: 11613 15600 900 21525 7534 8911 20350 17575 7154 10397 5219 15446
9261 1171 16015
304: 14477 14507 15421 17494 18062 2648 7991 15716 18037 11158 2783 2025
16965 22007 22121 19530 11719 9318 11530 17738 1299 21214 12334 21809
4864 10621 8796 1194 4280 19732 18120 15953 2967 3266 19426 6571
6888 17781 1697 10045 1251 7276 7126 12814 4960 7248 3598 17583
17847 7140 8316 18820 21056 7036 15143 13926 18581 5501 1142 3253
7339 4338 10287 9635 11127 20511 19012 13060 4347 12691 12055 20545
21425 3704 15743 19675 16542 16559 21229 11524 9274 11075 8804 17224
3133 3607 10123 13418 2973 11620 15872 11732 19109 740 1359 17419
6843 1680 16067 3406 4022 6395 9511 2595 20688 4623 22346 2369
17333 5395 11866 17305 10611 7631 15733 18432 1959 6814 6323 13183
9078 3032 21942 3286 13158 20759 22152 18157 1337 3062 16447 2772
10847 14538 4797 21087 7452 19143 16659 16530 16620 11651 17070 1585
22147 10166 6601 22431 15463 10652 13842 11862 1897 19136 9220 17946
12697 22065 3138 16162 17146 7667 19062 9071 11793 11610 7968 7594
7221 10376 5672 11711 3151 4084 14756 8726 18769 3480 10028 2838
20595 20275 1292 17594 12254 4853 20642 8186 16379 11759 11502 8591
4288 11992 15333 9201 14724 3846 15765 5294 9934 3608 4038 2297
1916 7144 15894 19202 4614 3319 11943 21007 5703 7304 9147 4298
11434 11341 10440 4734 13768 7039 7695 14607 13737 10493 13662 16005
5151 2008 20772 4271 19954 15488 16991 11303 17894 5027 7608 5609
16228 18306 2556 13676 8198 5829 17178 6626 16081 16487 20616 939
20854 11693 16083 22378 12968 16437 7624 6039 10274 13230 16495 10978
13806 5631 6912 22114 2955 21137 8847 12918 16985 22400 13954 16430
1905 19874 9524 6234 15225 4942 7297 20802 5035 10180 21875 20321
12454 9019 4029 11248 16017 3758 13953 2462 4887 8042 7962 11126
15081 22027 16352 2791 1889 15457 8559 17058 7622 4393 1128 8437
21033 5138 12634 1952 5842 21247 15955 3808 10420 14719 17939 4794
21353 19950 18656 13160 3900 11932 16466 13358 19797 5206 2289 9733
10875 12426 15784 10942 12064 7723 14341 5120 18048 8171 11041 17196
16314 20434 21075 20432 4222 8716 4904 18464 630 8403 6184 18819
17228 16302 10762 9202 16022 17350 15237 12703 11159 17792 4597 7291
15512 1072 6812 15612 4183 5466 17341 9980 4892 1108 1406 22046
1630 11923 17859 5617 18198 5043 6967 20318 22123 1264 12469 12218
10452 13091 8047 6109 14712 428 22157 7745 16695 22271 6934 7635
18516 6231 1574
305: 8099 12755 17111 978 1006 1542 15083 18937 12887 2035 10217 535
533 507 306 478 8894 18461 12108 1501 12486 21909 7034 7130
11072 14307 10425 15925 19332 1367 13146 7730 1937 9628 18825 609
18335 21615 4370 7330 8575 15629 12213 5331 3191 19367 4875 5240
11492 21592 7018 6367 19001 20564 13807 16754 21328 19708 4343 9940
1066 2399 12207 1629 18478 7812 13667 3746 5720 16976 18312 3470
5618 19921 17739 4546 14856 18520 3007 531 7912 8917 11529 6283
8622 8288 17072 4299 7840 5830 6060 19606 3409 7000 8429 19346
2857 17664 21589 11961 19154 18239 18127 11549 16493 21437 11195 20933
21506 1079 9281 838 3533 4677 18511 4730 696 9569 17797 18687
7097 20259 15252 13221 450 11335 20777 12713 22502 14915 21157 20371
12591 3563 16308 7626 4212 19798 19458 5691 18292 16130 2832 21296
830 14553 6434 18295 17956 829 476 12043 20779 1281 17745 7955
9854 988 20859 6669 5359 9132 16586 12705 15449 1768 11129 6547
22518 5552 11376 10964 1879 22087 13137 17298 19370 3333 13752 20301
2425 6946 19538 14273 15340 6427 17165 15603 12419 8003 10809 13658
14064 4221 7387 13284 20981 2316 3116 8418 3386 6508 19949 18217
3578 22249 992 18538 13442 3471 14855 5277 19885 2816 10917 9436
18659 12816 12308 15943 2515 20364 14701 8193 17579 22076 8890 2649
9329 21073 8376 15177 10882 859 5990 9814 16053 5454 5209 3144
9604 13848 22428 19076 17056 14918 4088 9960 11270 5837 22455 19840
12234 21301 1149 801 953 1656 20632 3755 12278 22119 21098 1005
10264 13037 11702 20954 5581 5063 1118 14568 17630 2371 4694 12396
15369 16597 3697 10741 16309 4927 13396 21621 8841 16662 11120 14081
8692 9484 15514 10745 15204 6565 14320 2978 9294 7482 9493 1277
4952 7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780
15219 16969 14762 18330 10280 10802 10479 16663 3678 7751 16713 13703
3630 4691 9472 10709 8542 7060 6112 22457 21974 5278 4682 10694
19198 20476 7333 6482 14526 7151 2644 10655 12264 835 9315 2786
16253 9488 5634 17372 788 15256 22095 18903 3706 9280 18593 5764
12583 11568 13613 2331 5136 8073 5630 11115 11304 15998 19137 5817
5580 18341 2454 8588 12540 4970 17445 2401 11869 7142 4488 19396
10889 5190 13207 16465 9673 9771 11716 16290 14313 9218 15665 15699
5441 20485 18548 18575 18821 3350 18263 20418 11128 16734 19030 21370
13211 22530 15360 6398 14067 21170 15866 10131 4332 1235 12927 7088
5099 2302 12424 9584 8303 11383 2282 8459 3414 15749 18691 12982
12794 10961 10824 18440 16819 9975 21399 8444 4707 13097 15930 11872
2621 7158 14864 6942 19607 7969 6546 5408 18266 10837 21928 13800
21492 5339 20330 1833 4846 544 19667 9933 4140 1318 20105 2376
7061 4232 15357 14036 7107 7165 12103 4848 12863 16683 1924 3730
890 8155 7187 17466 14322 14398 22189 15956 21084 16117 13940 1818
19714 16771 6683 1921 5000 13014 14274 18502 6650 13499 19763 10097
5188 19614 4719 3107 5780 12503 21929 7921 1429 5797 6439 12993
12307 15651 18994 747 5932 18630 7907 5594 6958 3329 4065 19882
13597 14124 14687 7770 1094 7688 15110 21169 12627 22067 19211 16914
2061 1201 19616 7038 8909 13129 19881 16317 1551 13235 14888 20264
7800 7526 2209 11351 5887 10798 10105 15317 10223 10661 15014 2908
16412 15519 22277 19851 12003 6166 7768 11003 20541 3458 1240 15787
20099 9282 11480 4994 6281 18662 3619 7992 9661 19875 14086 11156
20948 16946 3456 6143 786 6709 5954 17926 20839 22351 13277 13192
15113 6368 18694 3644 12154 3794 21257 21638 7704 21498 14865 18386
18111 10731 13776 5539 14530 20282 7762 9529 17675 15191 12380 16442
15825 2818 8220 5901 18578 13297 2495 21913 4526 16085 10965 15558
21784 775 7504 10887 3135 11204 15835 17679 3002 18189 7516 6483
9518 14034 11913 9964 2126 10118 3160 10150 10347 14405 15486 7545
12799 13960 1340 3431 18982 2984 15366 2275 6738 11245 5295 2132
20312 4655 16568 21093 13882 5730 7976 7079 13035 18086 2800 4062
8280 13990 6955 16633 12655 12545 2037 11704 9571 21379 3712 7685
4483 22341 18456 11197 13107 16006 9320 19090
306: 14420 1006 429 10217 10190 2439 478 314 18461 5240 6367 19001
3122 4403 526 11601 7812 6192 17664 2857 21589 11961 19154 21437
11195 20933 21506 3533 4677 18511 4730 696 9569 17797 18687 7097
20259 15252 13221 450 11335 20777 12713 22502 14915 21157 20371 3563
12591 16308 7626 4212 19798 19458 5691 18292 5019 17572 15704 16130
21296 2832 830 14553 6434 18295 17956 829 476 12043 20779 1281
17745 16586 7955 12705 15449 988 20859 1768 11129 6547 22518 5552
9854 11376 6669 5359 9132 10964 1879 14844 22087 13137 17298 3333
13752 20301 19370 2425 6946 19538 14273 15340 6427 17165 15603 12419
18044 10809 8003 13658 14064 4221 7387 13284 20981 19949 2316 3116
8418 3386 6508 18217 3578 22249 992 14855 13442 3471 5277 19885
8739 2816 10917 15943 9436 18538 1973 18659 12816 12308 2515 20364
14701 8193 17579 22076 8890 2649 9329 21073 8376 15177 10882 859
5990 9814 16053 5209 3144 13848 22428 19076 14918 5454 17056 4088
11270 5837 9604 953 22455 19840 12234 1149 801 21301 9960 1656
3755 12278 22119 21098 1005 10264 13037 11702 20954 5581 5063 1118
6454 20632 1696 19474 14911 9381 22362 4111 13374 2932 3071 18551
1431 12923 6509 19229 14012 5372 12362 17380 20272 16391 901 13395
5132 9540 19228 6169 11589 13306 3179 14568 17630 2371 4694 12396
15369 11784 16597 3697 10741 16309 4927 13396 21621 16662 8841 11120
14081 8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952
7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219
16969 14762 18330 10280 10802 10479 16663 3678 16713 7751 13703 3630
4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526
7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 788
18247 9280 22095 18903 3706 15256 18593 5764 11115 12583 11568 13613
2331 5136 8073 15998 5630 11304 19137 5817 5580 18341 8588 12540
2454 3016 4970 17445 2401 11869 6193 21516 2447 10889 5190 13207
16465 9673 9771 11716 13123 18575 9584 12794 21399 20485 9218 18691
3350 18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734
2376 15699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103
4848 13211 22530 15360 12863 9975 6398 14067 16683 21170 1924 890
8155 15866 10131 7187 4332 1235 20330 12927 7088 5099 2302 12424
8303 17466 14322 11383 2282 14274 15956 3414 12982 18548 15665 10961
21084 10824 18440 16819 3730 13940 18821 14864 1818 19607 7969 6546
16771 5441 8459 18266 5000 15749 13014 8444 4707 13097 15930 11872
2621 7158 6942 18502 5408 10837 21928 13800 5188 19614 16117 4719
1833 13499 3107 21492 12503 21929 7921 1429 14398 22189 6439 12993
12307 19714 6650 18994 747 5932 18630 6683 1921 15651 19882 5594
6958 13597 19763 10097 14124 14687 1094 5780 7770 7688 15110 5797
7907 21169 3329 4065 12627 22067 19211 2061 13235 7038 8909 16914
13129 16317 19881 1551 14888 1201 20264 7800 7526 2209 5887 10798
15317 10223 11351 10105 10661 15014 2908 16412 12003 22277 19851 19616
11003 7768 6166 13850 4620 16231 7016 2717 20886 14522 5254 15519
20541 3458 1240 15787 20099 9282 11480 4994 6281 18662 7992 9661
19875 11156 3619 14086 20948 16946 3456 6143 786 6709 5954 17926
20839 22351 4726 13277 15113 6368 7704 18694 3644 12154 3794 21257
21638 18386 18111 21498 10731 13776 5539 14530 20282 7762 9529 17675
15191 12380 14865 15825 2818 16442 5901 8220 18578 13297 2495 21913
4526 16085 10965 15558 18982 2984 11245 11137 12977 2132 20312 4655
16568 21093 13882 5730 7976 7079 13035 2800 7101 509 307 305
12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 11197 13107
14927 16006 532 428 372
307: 3349 11861 16061 5336 12949 18887 6597 3983 22099 2324 447 4512
11806 9383 17441 7957 16578 12877 12285 6745 8158 22143 14055 16201
1754 1561 18005 11717 9710 6672 1451 10276 2243 21572 5576 13128
10885 6027 17037 15496 10454 6068 15308 2899 11018 14627 20697 3974
7859 15270 15938 5811 2698 16879 8859 21272 14729 18033 840 5497
13669 21714 19513 5502 16413 15747 1844 4651 13831 22094 8195 21288
3065 16776 4822 18118 19091 5553 14834 21349 12572 16815 8955 13075
12733 4648 22049 5403 1029 1877 22486 14277 19152 1410 21143 7372
21858 17690 6467 9703 12831 4606 4506 19173 2942 1265 17141 18407
8705 18889 4746 3811 9106 17506 7669 11911 20449 10409 22212 9181
21792 22305 9003 8407 9234 9976 16656 2745 18946 4081 1944 12668
7458 13728 15650 11471 11697 10966 5324 9173 19342 21172 9379 17192
21538 8949 22434 3555 19594 13734 9786 21821 21772 21456 21196 9187
10524 11998 20944 6938 7779 8979 13537 5360 18851 21252 15297 11734
1781 17886 3931 16508 9825 5806 12904 4987 1438 8935 16959 16382
1164 22141 2007 17210 14488 6399 14781 6373 22109 599 9398 9473
5195 4504 5963 14256 6939 6345 1162 18406 17784 1442 4331 6935
5879 18326 2240 20428 19414 11272 14731 3444 1809 21833 14869 12121
5793 8845 20490 14861 12563 4528 17811 1567 9570 733 11892 13985
11612 1512 1070 2951 19948 9341 9353 1718 16198 8546 15048 9919
20674 3909 10843 16432 10218 15654 4043 14847 6714 8707 9886 20997
22088 13038 8037 9178 5352 3851 6484 16180 20444 13259 20733 21480
17651 22149 13792 13689 6933 21822 7825 13935 22010 15175 7093 21104
21571 14166 13059 1320 19388 12387 18647 10615 2572 16738 17277 10671
13485 4638 2352 7397 5048 13275 7941 11316 9127 6631 17029 14783
5081 2879 3446 12504 11878 2839 3228 19752 1088 1402 6179 16798
22525 16859 10813 2895 20050 10689 9920 13359 6382 16804 22048 15372
17024 19135 2131 20708 19186 7679 13583 7611 8668 2471 18443 8080
14254 18267 2356 17724 10645 21850 7740 18043 7128 21146 15408 8078
2828 15730 8513 18968 650 14046 3085 13243 11222 15011 1105 3976
1507 15891 10269 17937 6229 13505 13897 17262 1497 9774 5505 5939
7480 3041 12199 18054 15132 17133 9987 21835 578 22529 20727 13110
17553 15284 5186 15066 12304 21091 9863 4969 14128 13927 2253 22173
20557 7177 21588 7134 20606 17839 15095 21594 19310 4399 21404 1167
2570 8102 16155 14831 9194 9119 2558 21715 11856 11165 7573 10855
15158 22322 8996 12383 18252 20458 20237 9891 4099 16104 8881 12231
2561 647 22387 19765 4615 21305 15139 4964 20281 11812 22153 16216
18833 21797 7671 15097 11028 2172 17562 12574 2664 9816 12353 2954
21428 12735 2392 5226 8680 17177 18591 20343 15667 21341 11930 10157
19825 5210 7091 22444 3844 717 18902 14808 11659 8670 1785 8233
21693 8835 7167 12659 3869 22226 15589 13405 5280 13156 10456 10253
16881 14660 15728 6461 7132 8140 20493 14049 12322 15683 846 12989
4867 5866 20584 15020 1964 20885 21697 12785 2196 12335 15689 9927
763 1531 20138 11358 1132 19079 14162 5361 7543 18763 17112 21617
7915 20412 1643 20533 8020 9158 2930 19107 21064 3201 11077 14726
20135 9411 15348 9402 12669 15995 8168 7755 3112 12219 551 21933
8916 13876 14513 6877 8597 18116 8268 9085 9769 11890 21961 11709
10758 12800 8640 1212 10509 21023 11267 21751 1557 18735 18051 21254
15200 5771 17912 3381 18702 19564 12819 17593 8712 8128 1909 6496
14338 16091 948 17565 16614 18595 11540 18075 9306 5464 18421 10608
12167 17453 9268 11122 12880 13525 21522 5843 7885 10162 18002 13496
9474 11489 14139 5078 13260 10989 5480 3428 22424 15138 4252 2826
5882 20175 15811 16116 7879 12274 19326 5318 8325 1069 22306 6156
14294 21302 22394 4521 10893 5702 4766 568 15271 1077 17778 9726
14452 21497 5648 10532 15330 3647 8350 19227 17520 19834 16427 12852
9016 11208 9994 5906 20197 1115 15244 22452 15164 4258 6471 14545
6910 6417 21796 17471 17991 14998 15383 3215 10382 2580 8289 8452
12605 11475 6041 3868 2991 13102 15010 21365 21059 6894 21208 11757
308: 13659 8503 5417 2636 18255 14258 18186 14480 2997 12520 20393 16958
14070 11296 3421 20754 17033 17928
309: 22368 4230 16648 2237 2516 16152 10415 5473 14703 19022 18480 3997
17101 4156 10514 13955 12976 4059
310: 4051 3527 13345 2678 8550 12739 5968 11252 1249 13757 11908 13883
642 18741 863 15128 20888 14323 4672 8571 16644 10920 8004 22023
10377 10056 4837 19713 7234 1632 16113 13478 12526 19784 19698 2701
14658 15220 8365 18917 19359 21121 8529 17053 2009 2989 6906 5789
4862 8968 21524 13803 21337 5536 3377 936 20910 2445 21144 20276
15766 10716 15947 17616 7904 2437 12791 8028 5119 21686 20353 6765
13032 12566 2929 19589 6025 12404 5688 7908 7436 18460 14549 1975
9403 18910 15871 5414 21648 22373 15001 8960 8512 14839 13626 2150
18521 4687 4104 15162 19653
311: 7246 7633 13566 4922 7189 6648 3075 2678 8937 18998 20791 2442
4593 2102 13757 8484 11908 21901 9697 3719 21432 10971 8004 22023
10056 4837 6659 9021 8001 9591 4169 18319 12526 20405 19739 12836
12342 14209 15108 19698 21200 14093 4820 21147 16711 686 18917 21915
21121 8529 17053 2009 2989 6906 12853 7577 6127 14095 21524 6916
13803 7336 19598 11276 21713 14811 21337 20910 936 2445 11616 15947
17616 8331 11034 19471 5916 17862 12115 14492 18721 8414 19179 5371
9277 5119 17025 20374 3996 5057 1399 16384 9470 18719 18399 3401
8557 2158 3928 19925 15780 1172 19003 4754 6752 7557 21304 14324
15694 2929 15268 11094 19589 8713 6025 14222 14987 11178 3467 7373
1975 9403 2856 8286 16534 8056 9369 2719 15871 5414 22373 21648
1955 5457 18895 17041 22161 13812 8512 14839 13626 18521 21678 2002
4687 18560 4104 7854 18098 13647 15831 11038 19653 8205
312: 12932 12556 10612 8324 20661 21261 10886 21109 3162 13645 12519 14154
4627 9952 7890 12288 13254 22516 21541 11332 12510 6556 17293 7951
6797 12554 2010
313: 15796 4326 10345 13029 13317 20973 19570 14613 13103 3759 11764 3247
11747 9513 3029 20696 20193 9454 5495 18809 16012 8066 7668 15744
1836 6499 12779 2481 14822
314: 7479 533 507 535 306 12575 15642 18461 5073 9804 18997 8666
3659 6320 6367 19001 6157 11916 2258 21356 9857 2163 2584 3331
20086 6423 4662 3838 14933 8860 19820 15915 7403 4850 4714 9730
12137 14919 12016 19643 3913 17315 7812 21939 13951 1478 7199 10536
9491 3917 15376 4608 20333 7216 21640 8527 16880 19870 10959 4725
15069 5944 17598 6786 14232 4110 10546 16887 4811 15616 10586 10067
11756 20433 13391 16270 10468 7655 21719 5755 2857 17664 21589 11961
18800 19154 879 12644 16878 2030 4833 5428 3533 8904 4677 18511
4730 696 9569 17797 18687 7097 20259 15252 13221 450 22502 14915
21157 20371 3563 12591 16308 7626 4212 19798 19458 5691 18292 16130
21296 2832 830 14553 6434 18295 476 12043 20779 1281 17745 16586
7955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854 11376
6669 5359 9132 10964 1879 22087 13137 17298 3333 13752 20301 19370
2425 6946 19538 14273 15340 6427 17165 15603 12419 10809 8003 13658
14064 4221 7387 13284 20981 2316 3116 8418 3386 6508 18217 3578
22249 992 13442 3471 14855 19949 5277 19885 2816 10917 9436 18538
18659 12816 12308 15943 2515 20364 14701 8193 17579 22076 8890 2649
9329 21073 8376 15177 10882 859 5990 9814 16053 5209 3144 13848
22428 19076 14568 4694 12396 15369 16597 3697 10741 13396 21621 8841
16662 11120 14081 15204 6565 14320 2978 9294 7482 9493 1277 7813
7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969
14762 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709
8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835
10655 12264 788 9673 9771 11716 18575 9584 12794 21399 6281 18662
7992 9661 19875 11156 3619 14086 786 20948 16946 3456 6143 6709
5954 17926 20839 22351 13277 10965 15558 8891 10152 15189 13161 3256
13226 1748 21271 22258 10136 18982 20726 10154 17115 12652 1426 12623
4475 16706 17850 21904 545 2132 20312 4655 16568 21093 13882 5730
7976 7079 13035 7273 14619 2800 12728 15228 19263 10088 305 16894
6203 22106 19049 10427 11733 8133 372 12866 13782
315: 3342 12667 2100 12617 1136 6158 17567 4891 9212 13652 2310 14039
8257 4890 20474 16825 8834 4127 1369 14688 17548
316: 10331 15934 2046 6293 19503 17105 13130 7604 20972 611 2151 19670
1110 20162 9143 9176 19023 19084 18099 5356 15439 3212 10361 21551
20873 18300 6915 11130 19131 17963 7910 4920 4372 18216 8334 5551
11009 21068 13009 6829 20236 2330 15785 1052 14929 4869 4064 14251
6409 4771 21722 3477 22283 4071 13419 2771 12516 2855 6554 5053
15301 13629 22091 9959 8014 18394 7184 5437 10014 15912 7764 9611
13654 12151 12808 1755 10215 21979 12492 8018 7254 20380 2845 654
5061 7274 9568 5018 11235 6804 8938 12700 17001
317: 7147 17102 22335 21107 13529 17232 12299 15442 10074 12860 8161 6451
9249 12173 13520 2381 7179 2397 4681 8032 10026 17837 1679 750
13467 22304 9093 12920 8746 12849 8975 8614 11049 15351 6414 11194
18365 22000 10060 950 10516 16860 18657 11078 20626 15349 9992 11321
16068 13071 20575 10629 4241 12058 21534 18040 574 21837 6124 1526
12392 2919 22474 21550 1309 19435 13170 2632 14407 19257 15479 17487
7797 19535 13125 7697 9338 11284 15648 2144 9885 3591 21263 9545
14344 6947 19431 21279 4756 13704 4125 11670 6044 22381 21905 15904
14499 10692 12332 1756 4806 18332 7994 9064 18991 17391 20098 22081
5015 11957 13991 17712 22029 21507 3779 6619 4824 1847 11561 2927
7497 1618 16567 1625 1011 14504 1983 2353 13906 16938 11796 2922
13819 606 3676 2458 20599 20531 13948 7520 12498 13705 8646 9042
9750 14969 563 1092 8562 6341 3562 11565 15599 16895 12974 1939
16577 1711 21985 5754 19800 15748 4371 12415 2463 4827 5656 6520
14881 1513 19711 6790 20528 13821 9526 8611 6058 17821 1888 18587
4858 18855 7028 14908 22205 5964 4340 5271 17773 927 8374 3316
2666 20014 21527 9914 10822 7197 21212 13163 13989 17347 15266 1401
6338 14444 3445 561 9349 20565 2095 7453 18711 19524 4093 8735
14800 9217 16762 10080 19195 11057 11977 21372 20196 6610 14503 22133
19181
318: 4562 9606 1444 14309 10107 21463 18996 14333 1334 13632 22339 19891
15309 3134 19973 17693 15433 13252 20233 7289 15373 8208 21182 21283
14523 6862 8920 22389 7427 19075 14622 18190 16360 12132 1586 14883
3538 22299 4909 6348 9295 11703 6913 16728 19809 21668 18897 20235
616 10625 12272 10812 4295 2449 11240 20715 10788 12331
319: 11506 8025 19504 17096 9236 5967 13265 13150 20345 2575 15093 720
9238 20846 7110 18462 16689 3666 13238 20249 22504 5681 15821 11308
7263 2587 3204 8488 1135 3170 19880 7687 4736 5783 20141 8454
11622 3641 10121 15474 15016 3635 12476 2148 6474 12754 10278 4251
2157 17396 18150 5472 2435 9967 3440 12802 21724 16131 7711 10389
5452 18194 15428 5298 16650 13697 3148 15338 9355 4542 7607 12624
9835 6871 1450 17202 4346 18156 2256 5469 11562 11593 8125 13465
21018 14122 6978 1534 5284 19934 13992 9438 9860 16616 13159 22237
9239 12122 7442 20605 16637 625 10834 2546 3875 5692 13941
320: 5584 14500 1413 1303 11425 12432 20553 5678 19728 1702 21840 16132
13978 8053 11638 17005 7048 12602 12897 18092 1532 16921 11347 8929
2022 22014 21806 20100 17247 5940 13718 10507 20917 13044 3334 11629
3850 19276 8082 15982 2091 8247 2827 4647 21352 16763 13875 2729
11068 14988 22366 6081 6500 17279 7348 9062 12873 13199 7794 6832
16726 7795 12202 12467 5788 10596 12756 21948 11263 10951 13316 8432
13127 9984 13493 8019 12038 1826 14472 9125 4955 19548 16684 5905
16785 3169 20313 12232 10211 6536 20084 4322 19294
321: 11679 7712 4765 2778 10428 15613 13759 8748 15529 4789 4143 22492
10520 5899 5524 1150 15997 6728 7250 19339 3882 2486 16390 13804
4361 20834 20825 586 2032 2861 14363 17505 15033 6284 9806 19295
8378 20979 6962 22356 6126 18081 16902 16016 11797 14905 12294 1932
4974 560 4872 19916 7886 18272 11004 21567 6122 3929 10292 20964
14636 4316 16294 16670 6202 3283 3979 708 10683 3722 9024 10315
7707 21597 12187 21031 11517 17433 1323 3512 20375 12464 21941 10366
13212 15089 3993 3918 17676 18119 2709 17107 15788
322: 10548 4897 5425 7818 3878 10642 3310 16365 9020 12789 17219 2142
13865 19002 946 18211
323: 12932 12556 10612 8471 9836 7200 3244 4931 6155 11441 5925 9952
1283 20609 2129 3224 8050 2693 11630 8765 8258 10271 17801 10768
7008 3010 16997 6501 20840 3978 2010
324: 10963 8235 5742 21966 17140
325: 15756 6758 15758 5287 19343 4588 17303 13336 21407 16436 6718
326: 16712 1166 15865 2733 17246 15303 1381 13492 6572 7728 13605 12382
9290 21152 7868 21359 6654 2544
327: 12913 16464 16273 20905 3701 8623 4539 11237 7785 12606 12734 1726
4555 7538 13748 14436 18787 11199 15234 14408 15929 7578 1919 8057
11210
328: 14125 20217 16758 5776 15086 18339 9344 13845 5197 20795 5897 9137
9973 704 18058 11958 7252 2114 7827 5752 4689 7592 15102 22013
12505 15413 5397 16345 11067 11445 21494 14366 3450 8189 13758 10214
21577 10815 1385 19042
329: 3497 2714 8101 4216 8074 1106 1600
330: 11223 1913 6535 17340 17586 15949 10628 5279 16989 13504 22441 20437
16847 20368 18727 20627 677 8442 2847 12004 5478
331: 13633 5900 8539 14672 12233 964 10478 20775 5875 5805 1804 1598
8760 4906 10137 12276 20894 3902 1838 1416 13999 8927 20239 21067
2846 1536 951 9346 9547 21436 4596 12781 4010 11786 8601 10761
6187 19637 20291 12933 1404 16680 16262 4172 6985 11107 10370 17700
13722 7971 3488 17731 14386 6750 13862 2765 17564 11582 6358 16691
21558 16323 14350 16234 10634 8984 7275 21757 12586 15643 921 10884
20529 8362 4086 15778 8213 645 8641 14759 21903 9293 2872 7350
12282 7277 21911 19806 10064 12113 15339 20238 1645 15286 17911 15808
9799 12685 4914 19213 10728 2852 4839 20536 7995 7379 15240 12971
591 20690 5666 5145 10145 1126 13494 13052 13798 22187 6567 2224
22255 22276 19919 8963 4880 11857 15344 20168 20601 21712 7009 15363
10724 8126 7044 2098 12067 13898 7690 17967 22554
332: 4051 7246 14199 4757 2211 19939 7119 18627 18219 10672 19725 20509
7686 2989 10561 7284 936 20910 3464 21563 5884 11537 21063 1972
3103 11411 17085 21614 20041 6920 896 19271 21101 6679 18617 19003
7557 2937 8512
333: 11864 1840 17916 16388 781 8033 2364 22015 9748 2475 765 14146
13993 16445 7070 19406 13139 11260 21048 22437 16423 12388 15277 9405
13452 21701 15472 21519 18268 18140 17189 10852 15461 8741 9641 20297
21384 9522 16319 4702 6436 9574 18726 3628 16593 18634 13820 16639
4189 17966 3524 13210 12214 13114 18483 11648 5974 15580 4570 17225
19309 11814 7466 15434 16431 13455 9128 17770 17597 11744 5668 18077
20332 12252 6487 5722 19526 13902 14620 19013 12349 14056 3612 8715
15923 4913 11247 1777 16114 9983 8124 7153 20006 10025 1074 21879
4977 19631 8517 5456 9877 4139 21162 4364 7696 9702 1769 9365
5341 7159 5323 19419 19967 19719 15541 14191 10391 612 17071 22520
2596 16797 826 17040 18360 6069 16301 22129 12523 19596 3074 6712
12666 21205 18284 21965
334: 22427 862 20385 6265 12399 22070 17740 7828 4067 1153 11326 2935
9841 19586 16621 4285 4474 5816 1468 12636 2870 12256 15396 20978
13936 20481 9196 15850 17759 14083 17877 11918 14099 22052 4350 14967
3030 4936 6573 20255 16179 17938 6661 14748 18841 8544 17559 16186
13096 10817 20956 15131 16981 20307 6896 9262 16287 14784 4658 18310
6506 1358 8689 18061 6280 1214 4418 13166 8421 2972 19253 6332
1033 16181 10378 16100 12772 1621 974 9705 11370 21857 9111 13663
6502 15769 18274 20182 18579 16062 18202 10290 15049 10207 10284 15057
16286 790 10968 5513 1193 17665 4712 12568 13764 17032 778 11095
19866 6900 21331 931 14040 5675 18493 5449 2779 10498 12780 10602
10664 9704 2890 2696 4265 20376 6349 5782 11580 15846 4001 15619
21400 16911 11079 4939 15709 14567 17601 14494 2988 2987 3279 14867
3013 15708
335: 15578 12136 19850 745 14354 11893 10400 16221 5102 12842 19938 8836
6356
336: 1655 8875 17969 16217 19832 4954 10256 13907 2280 7705 2896 5571
6016 2423 14230 7769 16575 6956 11570 638 22347 12730 3084 7136
21153 979 18133 14470 4762 13775 13517 18643 14115 20989 4195 14211
17841 21732 2378 6456 2604 16482 9054 9876 6696 20958 9729 13852
22311 12997 5664 17434 511 11309 2192 7514 21788 10558 17846 13447
19287 9827 17314 19480 8685 19289 9913 16783 7798 15355 21362
337: 16000 2106 21931 7956 15630 12717 10346 15324 20316 3593 13203 3167
2799 12590 13815 4459 3056 18383 12953 14071 22160 2913 15509 16846
14685 8814 5931 19964 20780 14871 13615 6779 997
338: 20669 3012 4857 6538 14648 21791 21544 15892 20656 17483 5020 19379
16363 12470 5494 9395 17737 5910 3081 8480 20365 7510 1412 19099
8801 14807 16161 2393 10298 8123 1239 10701 8769 20670 18966 739
17909 2578 9830 13785 17449 6339 13378 17851 10673 17975 18270 18869
20842 16312 5144 8392 13794 20142 22008 4642 7811 9505 7022 21431
7054 13460 4715 13880 15813 16187 18556 12071 5168 4445 8109 6238
19089 10095 2361 20340 16200 16747 6028 22386 10599 2796 3476 14418
7731 5135 20971 13422 21726
339: 2298 2436 19901 14207 19971 5786 15380 16523 17629 1543 2607 3939
13893 13623 19506 1858 2400 20267 18864 3159 17867 6744 4520 5184
1289 5953 16468 8962 5695 6006 21230 5243 4876 2057 22012 13975
18334
340: 5094 13921 9689 10104 19369 17978 21082 2690 17645 3853 21213 17435
20373 10839 8089 4282 11406 17050 9439 11816 3709 8068 13476 14133
17945 20398 2090 4164 2229 4799 13949 13578 8589 10573 17122 18689
4604 7491 11399 9657 1351 17934 21685 9266 20395 18832 4616 13018
17935 782 21358 4923 2048 7355 19368 10540 3418 15391 6352 1454
584 6023 7278 13416 7550 12889 8632 13053 6040 1583 16498 7544
2590 9046 601 4785 7174 3594 18195 11021 9022 8275 1685 3624
341: 13668 15794 6342 14448 9278 20512 8200 16696 13521 1647 7170 15662
12079 14698 21329 1853 5038 11976 2478 11937 22112 21927 3832 2707
4810 16842 15999 3541 7952 9937 1893 9025 19963 10155 18805 2652
3473 21709 10794 17386 16395 9463 14603 17255 17919 4407 21734 1453
17376 5269 14505 22325 7898 5201 11700 7509 3737 5907 11800 14887
18221 3408 1391 6603 945 2741 11668 22559 13979 16492 19717 12052
13678 12316 20893 10627 18110 9396 17457 12447 17706 4188 12736 10213
5657 1361 17750 2164 20757 10342 5557 13427 1459 5874 11035 14177
18713 8770 13138 12686 12216 4228 12420 12044 4116 20177 7717 20582
6544 20287 13392 5213 21383 20420 12277 610 7242 13209 21266 15605
17695 1713 6386 7399 3771 15478 14510 14171 22266 6037 14656 20179
7964 7223 20189 1994 21418 7850 8756 18014 14079 726 12260 21444
10406 2633 8511 11121 10904 3092 2853 17906 16524 8187 17998 20778
2286 20199 3729 6145 21008 2565 12962 1257 15354 10610 7157 1796
6315 17300 22423 14435 16471 16628 6594 21171 9956 9842 18573 17327
8572 9431 16077 13255 10967 18428 7421 19940 17552 22215 6311 22128
15501 21897 19865 6983 7587 17139 17241 18872 10791 18810 8812 5978
20019 19994 6664 13673 2650 8721 16038 14722 18896 21111 2762 17606
10806 18027 9130 18559 12287 11513 6131 3172 1990 4027 5292 3287
13481 11191 19301 11297 10021 9171 14682 17190 14540 1140 8118
342: 2631 10657 18699 2536 6440 2600 16629 20719 4729 7634 14681 5022
8936 10883 18494 6327 9575 8157 8096 14112 2076 9896 11151 16731
2204 9035
343: 2755 21932 4777 6313 21861 12723 19508 1200 15989 16994 5687 6685
14644 15427 18561 20955 5319 10512 18861 4752 4558 9237 14495 2731
18748 20853 21624 4158 6682 15121 13328 11123 17204 3588 8417 12524
14426 12139 5914 22397 13082 3579 4217 4229 10653 20451 3376 17804
6004 10303 2139 15227 11831 18631 19845 12159 11935 18000 18066 13477
5998 16773 6555 6113 18981 769 10905 22461 18369 7501 12698 17209
22284 15974 3000 1328 15319 13157 21846 21899 1244 17408 7057 674
11714 14760 3856 22403 13952 5958 18866 16803 5945 7999 10446 17518
21309 12969 5105 10656 10777 18972 14634 7566 9182 10453 6623 6029
3857 1041 17997 10387 10643 1035 3246 9229 18431 9219 13687 4600
14823 1650 1911 1506 20935 12204 6588 14467 12894 12964 21745 4773
4206 8608 11403 21291 15104 9577 22470 10395 7106 18085 1045 18438
20164 5627 6989 3125 20664 9899 17477 20384 8914 13339 21568 14346
15753 18139 2610 22040 12680 1930 10147 11672 21762 14042 4281 8445
18322 7765 11544 8117 10562 17367 2534 14153 16918 7771 16968 14118
14857 20095 4419 15545 5134 11331 9802 4911 9938 3150 22321 8071
13544 4128 7168 8967 22547 3271 5965 9073 16529 16953 7176 17918
1026 16418 11579 13510 11163 21202 11509 15037 10348 12555 7467 9767
15877 20472 13410 15361 12759 10511 8273 5946 3774 8121
344: 0
345: 11342 13022 13189 13375 19355 4603 9001 693 10423 19460 11989 3379
20654 19018 22196
346: 4152 1714 20305 10236 21335 10203 4070 20587 13194 15640 10242 11539
16862 1124 10029 12355 19807 3466 21983 17696 21768 12283 20617 17748
17513 7821 7744 10704 11320 3178 16909 21864 9099 11919
347: 15403 7222 17930 22425 8943 8767 5431 9412 9840 7098 10706 20039
19399 18679 3063 13922 12891 19177 14765 8888 3766 15264 15112 8346
19249 9496 8467 12097 7066 13354 10438 19683 1867 13713 11392 20463
8419 9530 13590 19828 17789 3181 19673 20308 20048
348: 22289 699 7239 11768 21811 2946 15818 18586 10674 14308 3720 10934
14107 19528 13730 3925 20977 22047 4398 22391 16849 20631
349: 6607 11545 4182 16789 10863 11170 7754 13889 17668 6741 14774 8116
16721 683 13808 10075 2147 13320 7970 20540 8978 11576 16025 12292
10319 5002 13246 7075 22235 11091 9999 20403 14955 7021 7400 15424
1403 1576 15201 1547 21389 2191 20891 18977 17393 11512 10958 14007
6534 12539 20887 4792
350: 21637 9766 13473 10430 2373 10229 22472 2533 8573 9183
351: 21639 6558 15717 6583 1489 21115 14590 22491 22170 13729 14708 11897
9169 16003 16657 18303 2503 7619 9667 19462
352: 14832 4680 730 8944 19323 9756 8807 16549 6800 8830 14997 16942
1293 1086 19167 624 11497 15185 11336 10574 6438 21022 10877 3498
1533 10113 8352 18565 10340 12363 3314 18700 8755 21000 5888 14051
11660 10255 20975 16898 1028 4899 9807 15549 1039 16373 21500 3757
4390 13334 5158 7861 19157 5010 21692 13142 13526 12155 3025 15524
4634 18132 20879 11836 6517 5390 6944 17642 10919 12777 22312 12374
12089 2107 8665 10063 15827 10534 19377 14693 4078 16983 7351 21180
353: 9803 5872 20880 7736 9394 19214 20066 4813 3867 8129 6810 8821
14360 9758 10208 22542 16794 8724 2461 5260 11314 17374 20064 22105
20232 17121 17288 16874 14516 1119 2781 15198 20079 14792 3439 22298
12105 21450
354: 6321 5225 13406 4040 4448 20067 13787 8883 20377 876 9126 21945
4701 11644
355: 8510 12886 3986 1767 22183 9676 17318 13827 6552 22440 15136 22229
15255 8556
356: 20329 7584 18151 12782 19780 869 1234 4543 4443 6003 10879 6195
14766 2261 5303 18455 14900 13423 4770 7064 4996
357: 1090 22239 4778 3892 10096 3089 20675 8225 3300 3785 10016 16285
358: 7246 7633 13566 4922 7189 8550 21432 6659 8001 9021 4169 9591
18319 13478 12836 12342 14209 15108 19698 21556 16875 20650 5851 17486
6249 13780 15776 11726 21200 14093 18917 21915 21121 8529 17053 2009
2989 6906 7928 14192 7577 6127 6916 13803 19598 11276 7336 21713
21337 2445 11616 17616 21069 3827 12208 819 16095 11103 17030 6075
5459 17025 20374 5057 1399 3996 16384 3401 8557 9470 18719 18399
2158 3928 19925 15780 1172 19003 4754 6752 7557 15694 2929 15268
8286 16534 8056 2719 1955 18895 5457 17041 22161 8512 21678 7854
13647 18098 15831 11038 15162 8205
359: 11518 14223 17501 6542 13569 4821 16169 8132 15637 2270 10309 5708
18374 9446 7790 12784 7569 14187 5525 7188 21691 15258 4550 7616
5261 15163 14560 20144 10693 12244 21413 6135 7464 11142 13491 17655
15676 14174 18333 13472 20648 6435 14763 13495 12676 18906 12036 16066
21593 21667 14370 16048 17609 19658 5604 575 21518 5309 13744 15880
19484 6704 658 8704 16710 12907 19375 3306 19468 11042 3984 17175
20965 1206 9155 20970 6796 17057 11008 18605 3353 4650 9058 7988
19743 8369 3654 18497 11952 2866 16796 8504 20034 19086 4409 12452
12908 7530
360: 13327 3981 5070 16490 20204 5909 15321 10310 12746 5760 6045 1730
1018 15233 13486 17387 20991 5406 13208 21716 9902 21763 22315 6472
824 1364 18250 18767 12499 21700 3665 8788 19915 12114 17927 19363
361: 13771 10043 10800 17501 13569 6854 4637 16169 8132 2667 15637 2270
10309 18374 5708 9446 9291 12844 7790 12784 10720 9725 13741 5525
15920 7877 7188 21691 15258 6705 4550 14008 15544 9706 7616 14358
13182 14560 16722 1472 4000 1388 4261 10984 9823 21413 4976 9514
16806 9972 9224 13061 15247 17984 9925 19953 10247 2676 11680 9751
7643 12752 14983 15727 20648 3200 6435 14763 13495 13003 16940 7214
15976 5735 6270 22352 12931 14053 16048 17609 575 18286 21834 10842
18927 21518 20097 5309 13744 15880 19484 658 8704 16710 19375 3306
6347 19468 10410 10451 11618 20965 12916 1206 20970 11008 1237 7988
19743 20088 8231 4522 18497 11952 2866 15466 3609 2403 16796 13539
14806 5364 12620 20699 15426 4409 12452 5296 3102 20918 9156 12665
629 20947 3649 7530
362: 1670 8427 4513 13628 17835 1981 20663 1254 19692 11385 11148 18372
18636 1663 18409 21940 17744 17838 14190 16086 18411 682
363: 2344 7205 10404 18090 3530 22282 2138 1522 5878 7220 18971 21529
18900 21077 6911 22318 1047 9881 13404 7614 13515 13092 13300 20784
3818 8910 19715 892 11279 3262
364: 7108 14914 4339 11633 1095 18100 17413 19557 4087 5710 14536 8563
365: 16767 7229 7241 19193 14780 8455 1669 2113 16394 13341 4965 20987
21669 16094 13151 2242 9452 6246 4432 1828 6419 11839 6713 3448
5129 7881 21433 18954 18710 15718 12146 16149 12186 2849 17256 2464
11803 21181 15484 8463
366: 6504 7524 14750 15585 13755 6170 12273 16565 1049 12336 18307 21260
18677 18863 18346 15597 19755 3751 19920 2278 12086 1347 858 13286
14325 1020 15133 10620 2444 3050 16139 16876 1573
367: 16254 15810 10496
368: 4014 5426 1644 4368 10252 8347 14623 6336 3143 17492 2883 8191
13701 14972 3068 11055 13245 907 1811 17743 3222 22379 5180 8300
10258 19899 11656 9989
369: 12917 22561 8545 4583 16643 6177 17097 3916 4541 14252 13870 6493
5606 20188 10820 20279 8970 21193
370: 4823 10748 15527 15435 796 14678 9764 10076 11244 9175 7609 5115
21377 11924 10595 20985 18928 3164 9788 11682 14840 9374 7368 12999
6258 1876 19771 13726 657 7929 12344 4713 4417 19488 11641 3883
6787 11345 16897 10763 19559 1322
371: 7873 16841 5024 667 14126 19489 20718 20114 6307 13749 4296 12328
20713 14817 9449 17512 12753 3363 11373 19490 20600 14723 1330 10565
10981 22124 10789 16941 4863 19070 12635 18547 1307 13095 22242 5955
10082 21555 19391 1056 17810 15497 12220 6532 15511 7786 2790 6035
18667 7196 17474 2121 6729 19563 5810 3048 746 3566 16497 12050
8774 22138 1519 13512 22303 2143 10708 12991 12651 3454 16182 10091
18474 18979 19291 18720 9390 8536 17155 6630 10710 15964 7870 4033
10458 1637 16790 15145 11065 1445 18055 8688 19251 19006 13222 19365
2339 7595 4454 13908 19220 15364 18914 10062 2726 22250 16571 5841
19972 14541 2092 17011 19981 20191 13421 15044 15913 3884 17551 14501
21005 6049 4670 18512 20116 22469 12494 12091 21526 21355 8895 11631
16944 11810 10807 21779 906 756 15963 10502 12400 1579 15470 8436
8930 2675 20539 7266 19290 13829 1855 2679 8784 9380 16462 6530
22546 22416 17960 15631 1720 21887 5290 18327 18553 4590 7391 7917
14645 15614 13247 14642 12300 11299 19418 1740 14311 18764 7547 14528
635 9762 6404 17436 12422 16799 6528 15127 16603 13586 8483 3332
2647 22390 2094 17405 13174 3330 5025 17812 3810 19126 12711 9225
18885 11043 21479 7774 21789 17915 1800 13040 16606 13023 2103 2956
11730 6062 11525 15548 21598 14233 4710 17481 6481 20502 21631 14591
703 7865 21847 18542 17238 15120 19632 2874 12903 20473 16809 19278
15968 14351 21898 19284 8790 7247 6870 19794 764 16079 17554 10015
14157 5749 18287 4764 3645 13045 14959 2521 18913 21473 9257 12508
4304 1449 12138 6240 11499 7386 10591 20874 4447 5379 5272 10120
8267 6643 6108 22137 17643 4910 15972 4782 15186 2068 5528 12337
11658 13756 3142 15763 12030 13236 17079 5380 18622 5896 4307 3158
17067 12487 14339 613 6726 15106 15675 11379 7371 5597 12017 17274
14824 18417 20721 11498 16295 15954 12677 9580 12641 17409 19499 9907
10576 21600 1559 1247 18152 17273 13554 16429 16780 20611 10103 3507
20153 6861 13556 3496 16183 11289 3492 3006 5273 3374 9357 22451
22004 19394 18283 11166 15318 8768 18770 9309 16332 16630 17527 9479
8561 16576 10695 18749 17951 10930 9616 3752 10469 2422 4538 18515
20865 7082 1739 12250 6190 16535 9858 7630 15712 2642 22497 1808
8526 18181 7296 21659 16761 14816 21998 13413 14852 10402 11131 6668
16521 11910 2019 5412 11257 11346 4369 17646 5667 12585 1224 11092
5532 10200 16673 17220 17006 6359 17169 14019 20289 17599 5659 9944
9538 3683 3521 19933 16463 13655 5686 21698 20758 784 11603 2553
8439 21559 7563 14314 13522 15633 1741 1518 15412 5891 1375 2785
14686 15611 21250 11653 21545 17884 18298 2764 3864 12813 11218 5410
16378 1570 7383 9592 15460 16787 1050 10032 6964 19685 13788 11507
3504 17418 3664 20387 12935 8777 20035 14635 11626 3618 6519 743
1923 12738 13872 12436 5348 10557 14330 1015 17903 15405 4174 2069
16438 1021 19206 4166 13172 10851 2780 1326 13679 7285 11220 4129
8228 10294 19721 22543 1447 5468 4193 6618 3849 14474 3736 3595
18582
372: 1006 429 14179 7479 10217 535 533 507 306 478 12575 314
2254 5697 5950 15642 18461 5073 9804 18997 8666 5240 3659 6320
6367 19001 6157 11916 21356 2258 11071 9857 2163 2584 3331 526
15153 20086 6423 4662 3838 14933 8860 19820 15915 7403 13646 4850
4714 9730 12137 14919 12016 19643 3913 17315 7812 21939 13951 1478
7199 10536 9491 3917 18676 15376 4608 16936 20333 1999 7216 1589
17342 21640 8527 5069 16880 19870 10959 4725 15069 13329 20496 5944
17598 6786 7612 14232 18318 534 531 286 4110 10546 16887 4811
15616 10586 10067 4095 692 11756 20433 13391 9954 10468 16270 7655
21719 5755 4146 2857 17664 21589 11961 18800 19154 879 12644 16878
2030 4833 21437 5428 11195 20933 21506 3533 8904 13072 4677 18511
4730 696 9569 17797 18687 7097 20259 15252 13221 450 11335 20777
12713 22502 14915 21157 20371 3563 12591 16308 7626 4212 19798 19458
5691 18292 16130 21296 2832 830 14553 6434 18295 17956 829 476
12043 20779 1281 17745 16586 7955 12705 15449 988 20859 1768 11129
6547 22518 5552 9854 11376 6669 5359 9132 10964 1879 22087 13137
17298 3333 13752 20301 19370 2425 6946 19538 14273 15340 6427 17165
15603 12419 10809 8003 13658 4221 14064 22249 7387 13284 20981 2316
3116 8418 3386 6508 18217 3578 992 13442 3471 14855 19949 5277
19885 2816 10917 9436 18538 18659 12816 12308 15943 2515 20364 14701
8193 17579 22076 8890 2649 9329 21073 8376 15177 10882 8591 6053
5990 9814 14918 5209 3144 13848 22428 19076 5454 17056 4088 11270
5837 9604 22455 19840 1656 12234 1149 801 953 9960 21301 5581
3755 12278 22119 21098 1005 10264 13037 11702 20954 5063 1118 6454
20632 1696 19474 14911 9381 4111 13374 2932 3071 18551 22362 12923
1431 6509 19229 14012 5372 12362 17380 20272 16391 9540 13395 5132
901 19228 14568 17630 2371 4694 12396 15369 16597 3697 10741 16309
4927 13396 21621 8841 16662 11120 14081 8692 9484 15204 6565 14320
2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645 9286
22489 13745 16653 19069 7780 15219 16969 14762 18330 10802 10479 16663
3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457
21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786
16253 9488 5634 17372 788 9280 22095 18903 3706 15256 18593 5764
11115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817
5580 18341 8588 12540 2454 4970 17445 2401 11869 6193 21516 10889
5190 13207 16465 9673 9771 11716 18575 9584 12794 21399 20485 9218
18691 14036 3350 18263 4846 544 19667 9933 4140 1318 20418 11128
20105 16734 2376 15699 7061 4232 15357 5339 7107 19030 7165 -21370
12103 4848 13211 22530 15360 12863 9975 6398 14067 16683 21170 1924
890 8155 15866 10131 7187 4332 1235 20330 12927 7088 5099 2302
12424 8303 17466 14322 11383 2282 15956 3414 12982 18548 15665 10961
21084 10824 18440 16819 3730 13940 188211 4864 1818 19607 7969 6546
16771 5441 8459 18266 5000 15749 13014 14274 8444 47071 3097 15930
11872 2621 7158 6942 18502 5408 10837 21928 13800 5188 19614 16117
4719 1833 13499 3107 21492 12503 21929 7921 1429 14398 22189 6439
12993 12307 19714 6650 18994 747 5932 18630 6683 1921 15651 5594
6958 13597 19763 10097 19882 14124 14687 1094 5780 7770 7688 15110
5797 7907 21169 3329 12627 4065 22067 19211 2061 7038 8909 16914
13129 19881 16317 1551 13235 14888 1201 20264 7800 7526 2209 5887
10798 15317 10223 11351 10105 10661 15014 2908 16412 22277 19851 12003
19616 11003 7768 6166 4620 13850 16231 7016 20541 3458 1240 15787
20099 9282 11480 4994 6281 18662 7992 9661 19875 11156 3619 14086
20948 16946 3456 6143 786 6709 5954 17926 20839 22351 13277 15113
6368 7704 18694 3644 12154 3794 21257 21638 18386 18111 21498 10731
13776 5539 14530 20282 7762 9529 17675 15191 12380 14865 15825 2818
16442 5901 8220 18578 13297 2495 21913 4526 16085 10965 15558 8891
13161 15189 10152 3256 22258 1748 21271 13226 10136 22036 22190 18982
10154 20726 17115 12652 2984 12623 1426 4475 16706 17850 21904 11245
427 304 477 545 2132 20312 4655 16568 21093 13882 5730 7976
7079 13035 14619 7273 2800 12728 15228 8972 19263 509 305 16894
12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 11197 13107
22106 6203 19049 10427 16006 11733 8133 2264 12866 13782
373: 21829 10688 1896 2695 15238 6923 13321 678 19909 6620 21886 4554
4937 9312 12000 1954 4679 2233 18331 21411
374: 6020 13479 5421 1087 1989 14406 16303 744 882 12075 4026 22268
4422 9416 14897 9425 12945 10738 8798 854 13950 9451 21908 8474
22358 10772 3605 17605 21264 20769 15607 2985 11015 4905 7656 19436
14257 8375 8062 4389 12310 14819 10371 1341 10899 5014 18145 16619
22100 13463 2860 1177 15365 14952 17870 14006 9642 5832 15041 1884
15546 4286 20157 10385 20872 9791 8771 2815 22262 21484 5747 8299
21256 22118 17091 16759 13043 22140 18776 20913 940 1270 18159 1798
2743 20782 13248 7678 22155 12200 20855 14226 12099 17769 16419 12580
3054 20908 14508 1936 4781 6302 8832 7652 17179 5548 11581 15473
20439 4751 15772 5864 2710 20430 12577 14048 13591 22217 16257 11511
9747 18047 5923 8265 7628 7942 7892 18958 3784 7603 8156 22139
12033 735
375: 17388 7507 6853 5756 19170 605 19514 774 18117 6385 7365 5325
15832 13388 6982 805 9639 15679 21883
376: 6489 1778 6165 13947 15105 6175 12001 16671 13761 6928 4769 20271
21491 10938 7900 12795 10505 14143 12592 21354 13777 3510 5052 16591
12548 849 3088 6774 12148 9161 12039 2189 17027 1969 2627 11841
9768 10979 13916 11981 6032 1926 3963 5427 18199 9453 4199 2366
9495 7328
377: 15533 9191 10043 2753 19146 7790 4261 10984 17984 14174 13472 11804
22074 3317 10460 14013 11181 20629 17263 5966 14272 21460 17175 8369
11952 21759 11479 16405 2498 10527
378: 22075 3099 19424 9211 2977 18514 18017 15584 2234 15782 4438 4584
3716 9235 16307 16304 1846 2682 20821 956 5704 7923 18167 11822
6140 3047 10275 21133 18224 5583 15900 13725 19275 17302 20181 18021
3015 5962 12164 9319 20992 4622 8799 22151 20338 14742 22201 10555
17777 10102 7163 15444 9363 21307 8269 10110 4429 6632 20691 9638
14736 14020 10138 20244 11555 5116 14225 2312 21414 10821 2249 22550
13699 938 20457 5430 12992 13398
379: 2406 15415 587 9691 5895 21357 17711 19549 17251 13715
380: 10085 21760 4289 13995 7446 9558 14863 11606 20045 17971 9039 18616
9373 14535 4825 12951 3787 17677 5214 18052 6875 18135 21630 5232
20890 1770 10991 8146 11813 4697 17003 7055
381: 21131 21645 7243 20820 5674 11731 6667 10151 4817 4113 1832 17312
4832 4163 2924 20194 9682 19740 4054 16537 8531 19852 9450 8277
12513 18904 5869 17443 18723 22349 1228 589 14210 7986 10598 6036
822 5873 20860 8502 1122 16499 3922 18794 17929 19216 19270 21895
2758 12314 2468 11214 6717 21474 11827 3203 6754 13557 17019 1712
19477 13019 5645 10923 3817 6215 7503 816 4760 3589 12238 12773
10459 10744 9856 10749 18103 19048 3457 21457 19189 16910 16867 19415
16265 3682 16610 16470 16356 21013 5777 8013 18006
382: 19957 3226 10525 8530 7982 3708 16506 20265 19422 17319 17973 16769
6887 12979 5654 18163
383: 8694 8251 3871 9924 7519 16224 22555 14009
384: 12702 17415 4664 4449 4325 19760 21746 3267 10368 3093 11350 3531
16380 21838 19608 11587 8367 20351 2902 19733 18161 18210 22500 20226
13535 5126 18613 16364 11894 11183 4057 11226 17119 9098 3211 2185
20288 20489 21647 4103 11973 11885 10260 12393 7832 4511 20170 1928
13833 21900 14890 1027 7922 6811 5790 14015 17065 6010 13795 9245
9566 11876 8337 15174 2263 19217 5530 9598 9388 1659 10101 17925
9717 14228 12740 14255 18590 12269 14674 17257 22064 8551 1059 11112
5338 15072 18204 15279 6969 14944 11583 18391 14690 6503 2317 785
6692 22080 20243 22421 4743 11968 5398 1750 12965 11391 12878 4721
9384 20075 5074 2118 14173 6821 5012 9798 11794 9811 10818 7495
4569 11830 12957 2497 1617 4507 10667 12465 5796 18993 12481 3573
13319 20523 21343 19968 8041 15867 18712 11232 18921 4109 16995 2484
14332 5833 21284 1405 18264 14069 17528 20295 17890 20822 18580 1268
2953 21324 21132 13085 21661 13618 878 13234 5382 11592 6749 16229
5848 4012 12378 1929 19687 9013 10182 7228 2028 14882 10560 6987
11978 10065 16975 15791 15013 5286 17958 17087 8422 21303 2200 3761
1089 18218 15951 21747 17124 2300 8857 8486 8383 19233 3427 1197
12998 7171 8654 10108 13462 17054 18871 15851 8291 18960 4494 15966
22488 18543 7773 12235 12178 22041 19129 20411 2027 19172 15806 6469
6370 22096 4105 13426 4809 19640 4659 12156 13332 9311 588 10476
22560 15961 14413 21548 7803 12072 17825 16719 17270 21888 3990 11256
4092 19664 21590 20602 20735 3685 16999 11453 18129 8997 1186 3952
7692 11037 17899 10950 13564 21682 15755 6178 12242 16297 14421 11436
3560 21531 1486 13983 14970 9231 6305 2459 19412 13256 16829 9649
11045 1317 16681 21053 19661 21038 7395 6841 1114 9000 14705 10494
9108 17796 20404 8446 10945 13307 15299 1073 16289 6701 11667 17949
18645 17044 20488 19663 20206 14400 4114 15577 20198 18423 6253 6078
16870 15568 10782 8621 17031 19360 1692 11227 9123 16836
385: 2321 2906 21872 10635 21078 7065 15156 1425 16772 13825 20591 13813
12899 1192 19892 18999 8889 11465 20504 3946 12048 20666 9727 18484
4435 1280 12630 17995 1807 1366 12411 2542 17186 15294 18206 15878
13956 14389 4053 13057 11138 9564 13365 14654 2060 6116 1599 22293
3117 16686 18549 738 1017 18097 20366 10906 7412 972 3303 9331
22033 22401 5340 18297 5439 11324 19746 22005 10666 2640 21017 10814
15503 15916 4700 10384 8762 17479 12301 18760 11416 17765
386: 7205 16239 22108 14974 15094 21071 5878 17987 14219 8882 17848 8491
10730 10766 11279 3262 17261
387: 11850 8199 14384 16243 558 13030 19215 3479 17782 5827 19788 4416
9270 4145 3077 7366 3904 3442 6662 14375 15690 11650 11821 1949
1649 4587 11211 6236 21165 7837 18707 13824 13754 3121 9516 1966
16645 15795 15859 18232 16579 16020 15502 22210 11865 21270 5088 2239
6314 11933 1605 15281 22051 13665 9116 6153 21677 5334 21914 20518
6808 1927 14427 10241 5400 1667 20796 14866 19395 5422 7965 11136
5440 20442 14551 4855 21855 2066 15805 15888 3582 2334 3800 4016
7072 16877 16230 17326 7806 2071 7990 18865 641 16590 21955 17280
17308 2396 13681 3877 6148 11519
388: 3596 7515 17959 3549 15024 13576 15991 5407 13215 8370 14374 9790
11193 17162 12026 4861 2328 9153 3383 2598 20929 5662 2982 14262
15841 5775 18375
389: 1348 15757 11966 16108 15222 19104 21321 4489 18184 6470 8478 20190
12628 857 9167 18660 16328 21136 12320 10268 9965 9361 754 20342
17110 8283 11192 16702 18986 16654 9350 12474 2031 22089 14245 18814
2961 13231 21209 11352 13736 3385 5559 5208 19587 21576 13363 21166
21877 19184 16154 13751 7235 18147 10895 7829 1343 5632 11790 12936
5995 4717 22228 14767 9074 21907 10830 11760 8348 21924 13987 8023
20096 10747 11879 2097 22475 2738 9421 8592 11715 20682 10679 9928
390: 11664 19373 14010 12174 19349 11769 4498 2274 21231 2630 10572 10953
8931 12005 7156 11098 17092 4856 20031 3585 18340 15441 19792 8259
5870 21654 18857 1161 9343 17931 22246 9364
391: 10049 13487 22261 19856 3119 7342 21599 5351 21514 16751 9423 5727
5042 8667 12431 7989 2965 4617 5682 19205 18518 12013 6053 9576
5713 11119 9862 16190
392: 18732 10751 14514 17429 18757 4995 12463 17063 16460 10638 21916 17344
6287 10622 9084 7641 13773 14486 3055 1922 8058 12612 3059 4605
19053 5881 16145 10408 6114 13634 14741 604 18842 18868 21777 10350
10325 1510 2222 7494 12825 2470 17446 15874 22549 13588 17843 20862
393: 14137 6491 1356 15653 9544 17800 4657 6563 18761 19273 9502 12445
4359 12437 16199 18995 2176 8009 10587 4063 4005 20824 17620 19815
19234 17093 19511 10545 5626 9305 16800 12265 9897 6943 20358 12184
3707 16211 21688 10582 3368 2742 2365 10339 3898 8612 3028 11721
15331 5828 13918 3634 19884 16538 1111 1554 14399 14371 5104 1829
8104 17482 13063 16292 21720 7729 11408 5629 16271 21292 11860 3424
9033 21268 21695 15296 18354 4934 13381 16156 13739 13034 4472 20876
17313 16924 12087 18839 13963 22413 3248 944 6344 11007 8619 11418
9828 5268 14031 21703 8516 10388 866 2809 7449 17438 17555 16805
1636 15432 1152 10419 17275 12821 6115 15623 5603 15002 12960
394: 3202 16855 14393 16047 12109 20443 2004 17625 4349 19221 15436 5819
17066 19649 12670 1965 14910 21969 9893 3600 12536 3822 12850 4180
6132 13648 21798 18884 15981 21322 10336 20424 7005 13279 15017 2477
13011 10124 4373 14416 7377 13006 8088 4485 10568 6181 12408 4013
2391 10416 1220 14803 9678 2564 13861 16954 3199 4561 21312 6627
14292 4410 5823 16748 12403 14414 8669 12884 734 17278 562 6518
1881 4518 21380 6494 15852 17368 13840 12084 19239 3091 7517 5984
14268 4629 5191 19861 16641 17406 4144 18973 21415 21258 6950 14555
7041 13342 18344 19371 17541 1834 8664 16235 11478 14116 6540 10401
15100 22060 16196 11176 13397 19092 19747 10990 13435 9728 7131 8313
8349 9269 11964 1483 16810 1500 2320 14402 11184 3721 1256 10000
920 14238 8919 5685 20117 4235 9693 6412 5084 10715 7364 9817
5924 6015 15928 18253 21113 17516 623 7726 7224 14801 7682 8966
18692 11054 14527
395: 15084 19366 8811 1190 6727 4321 12649 19433 12507 3995 12015 20152
21429 17657 22463 5534 8079 16111 6899 3734 13762 19928 19272 17507
6452 12037 16857 11523 12569 6792 18238 5763 14939 12699 21275 3930
16987 2452 2193 21219 4796 14984 6885 7888 6130 13135 14954 1935
2387 6161 8499 19730 6609 2287 21882 2634 15588 11487 4376 17509
21610 2093 18802 7506 11993 4406 20544 8974 1016 16510 12662 20736
14836 15691 17920 7734 11742 21957 21727 7743 12157
396: 14699 6724 12500 6101
397: 4537 6708 17900 6960 21012 6873 21604 2141 7919 10956 4205 10702
6611 13911 7162 10797 21569 16752 6012 6869
398: 16076 2321 4451 19562 5982 1411 2156 12558 8727 16772 14543 9800
22191 22531 13050 12479 8829 9890 18225 2623 2538 1627 16157 3656
21344 15134 15798 9715 5142 2700 1902 22110 11093 7017 1409 20559
10443 9498 11500 15719
399: 5094 13921 11673 21082 2690 17779 21361 3853 21213 10839 20373 8089
10464 18576 8718 15705 2229 4135 17410 12062 1263 10684 4844 14376
13578 8589 10573 17122 19674 11088 5570 7753 17311 3616 11735 4466
20847 13877 22288 22274 8170 8179 8272 5234 21652 20395 3764 17831
14489 8729 13018 2082 3516 719 14264 7662 13451 8424 15025 6352
2590 8632 13053 6040 1583 16498 5182 601 17476 6142 7174 3594
4785 18195 11021 9022 3624 8275 1685
400: 20186 17002 688 20774 5087 2884 14965 22528 18781 1690 4912 5577
11728 16448 16807 10976 1638 15429 8957 580 4493 5637 5091 16774
7160 16922 15404 15627 18041 10475 11189 17861 6458 12796 2292 15539
10144 3302 20470 15550 17536 14443 18302 11535 18222 3339 19330 20436
12330 4213 11378 13719 1446 18705 18359 16604 7847 4055 1915 4452
7369
401: 8043 17358 11675 2116 16697 17985 22056 14520 11577 10169 8676 13033
5205 2318 6687 11694 2175 13809 11141 20423 6267 21105 10537 11944
20585 17725 18065 11722 4293 11413 1336 17250 5565 20625 13141 8797
11621 2433 14925 19876 20907 16241 14430 8061 5447 21832 6570 13938
7924 2863 13858 15223 21239 17416 17732
402: 12881 6207 13353 11751 15876 1666 22177 13826 8030 20136 19068 10849
6185 15941 3633 6034 14463 19984 9404 13322
403: 6523 1729 4595 16330 20515 15902 19588 3043 18919 11708 20497 2327
1441 669 18540 19877 6224 20250 22038 16853 15290 17729 14201 6522
6639 958 6067 9352 10585 13076 22253 20296 15055 16741 856 13376
4656 741 7600 3292 2041 12712 9536 7469 18070 17534 11560 1612
12461 21026 10734 16245 11563 6734 14728 2065 12210 7856 6660 21451
19555 3908 11196 7863 3537 4208 19879 1538 19512 18260 12688 20261
4516 10861 13969 17758 753 6479 4375 12255 14160 16212 16720 16343
8787 1275 20369 8862 14259 6209 11213 8389 15695 8160 17885 1848
22162 17147 13696 14312 4009 8381 15581 21090 8703 15075 12930 5299
2236 11851 22418 6073 6225 7353 12257 3365 8254 20943 7181 15879
9322 10783 967 9671 5003 19304 21876
404: 20121 4845 16755 15934 12656 20703 22316 10903 1987 15141 15570 565
19322 15992 21094 9068 15986 19707 21551 8077 3154 5118 3193 22498
2768 10551 21158 3298 8643 5107 10916 16268 21127 14373 13081 4348
2952 5053 3842 20761 1985 10588 14743 18380 9448 6324 8451 11229
12521 5321 13117 13654 3189 9466 973 16177 20685 18436 6742 8800
8329 17207 10301 16839 21523 20549 8368 16906 21295 16634 14165 16440
18858 11979 19688 11752 20704 8049 5101 12140 6150 18457 17776 10823
1055 10714 2685 17863 16832 18771 9387 18448 11154 16274 12172 16594
19936 10508 11531 11286 3525 16791 20273 17444 20714 13542 11356 22016
9568 15624 2045 8938 12700 17001
405: 15903 14278 19149 8514 3419 8145 3547 17612 11032 20571 12663
406: 17617 22319 7205 6865 16239 9494 6282 3807 1614 20370 12460 4224
10012 14713 14403 11097 4784 11736 5165 8086 18191 18915 19519 8491
10730 11279 3262
407: 2891 3958 11588 15385 3069 2735 20029 11448 11546
408: 9532 10331 19503 14129 5699 15147 7047 5185 7604 2151 19670 9143
1141 10189 18230 4233 9176 19023 18942 18099 5356 3212 10361 20873
12438 9248 1233 17267 19131 21068 13009 6829 20236 14326 6208 21773
15785 1052 14929 4869 4397 13900 6409 3254 21501 12516 1974 20560
11275 2659 5053 9051 18988 22150 21521 17242 12835 5328 12151 12808
14165 1755 10215 22220 22247 7254 20380 5061 2845 654 5018 7274
9568 11235 6804 8938 3173 12045 19302 4028 11190 14318
409: 17878 16387 12478 9552 20335 20588 14328 20566 9985 11470 9847 3078
10774 13004 16036 17980 12297 19736 12682 20732 15994 18840 14761 7615
3382 13301 14704 1253 8175 1203 14879 16511 18949 17910 4951 12305
12551 984 5460 6545 17611 15062 3611 21636 4337 22142 17887 5729
13561 7326 7817 4775 12683 18165 11442
410: 19444 20278 7007 15467 10031 19083 20001 8653 7474 9943 2052 11515
1185 15759 16848 7867 18893 18562 4415 7309 16700 646 22398 21783
11681 20677 13180 18105 19115 9525 21934 4239 9535 3136 7961 4042
17090 13917 13550 6807 15815 5083 14397 15406 5715 18413 1545 1181
18323 18109 4215 21611 15464 20382 18532 10295 3243 557 7871 21405
21910 742 17206 11102 8305 8131 8342 13021 21027 6288 21249 8657
3604 2514 17634 15990 20228 10314 8380 7680 4394 11472 9447 8886
14484 732 21164 5885 1582 18950 4256 13943 10878 4921 18817 9457
13643 5491 10600 6106 7932 12303 8523 7259 15802 15381 10690 5244
2547 9595 13530 3278 7558 787 14423 1602 7317 8244 875 22340
2864 14957 7393 18265 8733 9103 19853 11368 18367 16756 15487 19985
15688 15114 9775 5561 11959 3943 9409 7846 14829 11027 10403 18628
18693 10737 1258 16202 22195 7356 22522 660 14611 1131 8690 1624
19314 19584 2161 13946 12833 2228 22353 20610 5071 22372 14247 6017
7666 12648 14029 21735 14114 20694 13015 18234 681 10081 11147 18808
19321 6914 2616 12379 10311 15491 18766 3328 17588 18807 17753 12618
11319 20579 10231 2975 15216 2793 16097 1795 18974 14473 20268 21825
19495 18545 14979 5483 4788 9820 13325 2386 8747 14646 4173 8861
11690 17653 8169 5892 19300 15684 9216 6651 6990 4535 11520 8301
5772 10373 13686 13843 9665 19610 10235 17272 17334 22154 18621 865
3789 1076 18122 12966 12081 14558 1943 10356 2299 11243 18708 10234
14621 11538 20401 7508 14752 11476 8779 13046 15566 2029 5433 2096
1102 12085 13461 16044 8180
411: 9901 16263
412: 2162 18233 20049 12022 11201 15202 8052 7238 12584 4327 7460 9562
19525 21286 14793 5936 10320 1414 6411 4718 9260 8095 16930 16698
18606 2323 7175 13143 20113 14775 19888 9560 2539 9038 9327 18125
11362 1014 9813 17983 8842 7807 2893 11880 14342 9378 3540 3096
19574 14434 16029 13644 4168 6269 3249 12029 19054 8470 14290 17635
13553 18138 15398 9069 18935 2699 5624 1368 7752 20623 8011 16598
2959 7392 16359 2033 13502 14870 6425 15173 5707 18089 13368 20148
5202 12293 7625 10367 22375 9160 16642 1146 8644 18822 6890 4761
16943 2968 15397 10530 4187 21770 6917 9741 21455 11557 12319 13100
16735 11024 6580 16704 17268 12608 10727 17252 2824 12882 11405 20525
6174 16284 14249 11514 4780 19770 4323 7269 18976 6189 4008 10736
14087 19567 22011 14971 14023 17424 2305 22078 3592 16956 7560 1738
12942 21285 20899 11460 19580 21557 17786 17161 3227 5470 5918 1584
6167 21290 18980 5133 22328 17322 3423 15190 20764 18753 17010 16011
3336 4391 7094 15896 7833 16618 16299 7848 21863 6217 20647 1230
22513 18373 7471 12642 20594 15221 18254 7661 18439 4382 21156 10463
3841 9623 18957 1044 10437 20165 17833 1374 15273 7844 19571 16820
11643 1575 1920 1695 11209 7958 10668 13971 5416 7100 13690 4695
12664 13185 8604 18811 916 3631 20925 6998 12127 1325 18046 17290
2206 18785 10243 13121 8060 8498 7423 7638 19073 2589 3866 17454
20816 16178 20033 1467 16026 8181 1941 8720 17669 4852 1609 20526
4534 20245 18223 21627 17568 18939 13054 14168 5856 18898 11014 19510
9810 12845 4047 20300 8236 15824 1098 16088 13424 14404 7164 14512
21944 14356 18936 12143 10894 5086 16950 18427 2265 12701 10676 14700
636 7556 1255 21562 20004 4018 14365 7792 21836 22515 4626 7954
2981 3277 20156 14224 17076 11402 8579 1038 19802 8390 4147 20742
19929 17647 3352 12885 17628 22490 3315 8629 6094 6966 14651 5256
8279 11688 6703 11108 1457 14601 9351 8986 2075 8490 2577 15644
14025 2586 2646 3999 9581 14960 19190 15504 18393 7405 2505 7583
10066 9356 8520 21298 7640 20817 21774 15316 7810 6357 19520 17972
21917 15777 13859 21642 3703 5853 1354 21366 12726 4471 1978 4480
2810 5335 21970 14779 22384 3393 5141 2928 5815 5890 8839 5761
19941 2036 7211 760 9097 1184 5514 15671 4895 2808 12236 11124
11100 16580 8441 6767 10567 19813 7882 20630 8524 3825 22209 13732
18168 5582 4896 6403 15710 3400 12466 7217 21299 649 918 4342
3021 10027 21150 13145 6798 20339 2003 8395 18183 5409 11431 19105
15632 1529 11293 12867 2841 12448 8092 20698 17823 17746 9681 10359
16092 22422
413: 22034 8322 12008 13273 812 11765 21273 20302 7255 570 20174 13723
6666 22086 18541 9801 12646 17511 3238 4002 10019 21775 19200 1284
18777 16126 10941 18730 16545 2655 10677 1823 3378 22507 22487 2105
21401 16277 16134 17411 11463 10282 1387 17316 814 12211 10675 5919
2531 8234 15950 13303 20240 15142 6343 17332 698 8256 18799 16961
7465 15483 15126 11265 1248 13144 4991 910 12748 14534 20200 1113
9537 549 10854 14460 6577 14432 1556 6735 19416 16300 21874 14903
5716 1511 13958 10119 16522 6561 5477 12441 6266 14367 2568 7559
19472 19810 11611 12517 13165 12364 21402 12768 9589 5941 15456 1565
5435 14242 21793 4776 7231 1961 19032 1380 19110 4711 18525 20467
16120 1422 22131 18435 17158 21488 4267 21287 8136 21470 13657 7855
7462 20952 6100 21395 1091 7935 12221 4220 4275 22272 11605 1109
6087 13735 21333 11724 18084 1628 1346 8072 15856 19515 2451 14016
9102 7499 20103 6129 8681 14265 22363 10518 18395 20658 15572 20900
17981 16009 11950 11074 707 8846 9190 18652 12428 10283 19835 9740
19502
414: 6834 6684 1593 14718 639 18288 5930 12385 11749 15764 10482 14872
20500 17253 17922 10034 9808 1699 3559 8697 16121 8791 12687 718
18476 13223 1062 17385 11440
415: 13988 14945 6635 15700 20023 11536 17004 12834 5317 19374 10116 7960
5032 17692 21336 1492 19786 6860 14502 20681 5259 9724 20051 567
3863 6677 19709 7411 16573
416: 5198 4396 18094 11888 17143 5694 21019 8021 21392 3175 21815 18035
12729 6083 4640 17336 18325 1938 5227 15019 16822 2110 2441 1780
11073 2067
417: 4094 4249 6447 4266 21799 11386 7114 19998 8507 4118 4930 21755
7422 470 10048 18342 16272
418: 12195 18355 21055 18016 20402 12963 12102 17702 3106 18678 18424 22227
19413 1979 17990 22510 17357 1360 17490 9376 2639 8398 14303 1563
9512 20310 20534 2405 3798 12350 16708 19648 22564 959 8091 13262
18778
419: 8007 9185 12493 17152 21173 10594 13523 16563 10079 2431 10753 19487
20147 9647 14147 9929 1830 1654 17021 19317 3147 1112 10779 8538
3237 6408 13120 3049 22221 899 15039 8907 16324 4879 3957 783
19624 12762 8261 15668 12832 19114 10457 3297 5741 17704 16175 15159
6999 11766 8413 11239 8954 8566 11458 11367 8923 11782 3384 18544
8298 18012 21849 14689 1883 11942 728 1701 15090 13910 20183 12952
1872 3776 5981 21830 14524 4742 9043 15217 19665 12092 13500 7051
5615 9310 10058 672 11082 16588 21831
420: 11518 6542 21976 16169 2667 11216 15637 2270 12844 12784 7569 16948
13741 15920 7877 4550 7616 14358 13182 14560 4000 4261 10693 20144
8084 21413 5610 16074 7572 9778 1745 15641 21565 16376 10773 18848
1071 17984 9925 22043 15210 10247 20648 3200 983 20360 6435 14763
13495 12676 12528 8206 20705 3157 4565 9878 16048 17609 21518 13885
7244 5309 13744 15880 19484 658 8704 16710 12907 7984 6137 22370
19468 10410 9826 17175 20965 6796 9139 11329 17057 3353 18605 9058
8231 2866 8504 16796 20034 12940 6134 15426 4409 917 9156 12665
629 12908 7530
421: 15638 19372 18500 2446 1148 7739 10757 13616 14797 4533 4130 21278
9633 2203 20379 16812 11738 4453 7875 17989 11775 6848 21289 15564
17637 18961 2109 17883 5751 4470 13318 19225 8496 999 19409 7694
4874 3523 21721 18661 5643 6235 15475 4993 8534 2734 16838 841
8087 7343 11926 5112 4748 2697 17805 22050 13444 20480 12313 19959
1236 14235 21517 18071 19134 12869 2268 16339 6198 11473 8372 13642
19849 19983 19150 7473 17895 14624 12838 18434 8867 10445 17390 4706
4050 4847 16631 13356 6505 18534 1687 1794
422: 5236 21619 6397 2990 20047 22291 6084 2760 5267 5082 12765 16136
9918 13383 21449 6268 20961 21232 5855 7138 1995 14576 1013 12134
10069 15073 6212 15451 8899 18412 4849 4386 4988 16988 4076 12237
5275 16208 20027 947 6757 10538 21003 4705 12576 20998 19553 17590
14359 11013 19927 6707 17088 18975 18429 14985 13721 17584 21718 20322
1390 19701 8055 6576 18610 20621 22331 12128 6286 7906 19843 18623
20065 11146 18095 21935 18101 573 11564 9180 14490 7031 11030 15407
20068 22044 7315 18849 17381 20667 5814 8717 2914 14041 6827 11083
22344 21424 1648 14123 11982 18740 20583 4741 19612 1957 6206 19071
15555 14347 15150 22534 1958 16979 5465 17615 13167 9714 3221 17417
1787 20324 4795 20150 11040 19333 5877 2311 2374 14089 20555 1581
853 11842 5451 3675 12229 8405
423: 7073 8473 19204 13490 15419 10302 3067 4727 8682 22082 19922 8178
18347 11384 16646 9838
424: 21999 10083 17047 19988 17430 10414 4089 1514 16232 9556 12818 19256
18022 1716 9583 12552 17581 13890 9464 2246 15706 18577 15783 9368
425: 8789 9460 12638 14787 661 13830 20570 19781 2510 17126 17660 1608
2111 19080 6968 16599 5238 20160 4150 9643 16795 2152 20203 17640
20673 20994 13131 12812 8991 987 22355 19043 18108 15045 14625 18638
2854 600 13108 14921 11424 11994 19459 3294 9066 11477 13026 15931
6981 9953 20837 1046 8701 4413 3741 4408
426: 6366 17652 5554 20635 17360 4793 14236 21110 8866 5732 11691 19410
427: 1006 14786 20756 8495 10217 5541 16973 535 3260 11264 12854 314
18315 18461 5544 18823 5240 2104 12358 16966 17291 6367 19001 15260
3597 5549 20568 10778 12440 1988 13743 11485 1783 18629 526 17425
9565 20151 9300 11185 5246 15752 2084 18185 2918 8400 3763 3837
5746 20378 7812 13638 17369 4080 10663 17802 2070 4989 7198 21781
14194 4437 8103 12106 16298 13285 19237 5066 19712 16970 17382 14671
9686 5137 13854 11867 2123 3011 15148 11900 22193 17908 9773 16561
14739 22164 2058 837 20811 16636 19327 18664 13237 18684 19318 3797
6139 2857 17664 21589 11961 19154 21437 16338 11195 20933 21506 3533
9255 4677 18511 4730 696 9569 17797 18687 7097 20259 4425 3003
22502 14915 21157 1139 2541 4460 16130 21296 2832 830 14553 6434
18295 17956 829 476 12043 3677 7659 5486 9761 5540 8443 15590
15367 14935 4602 18472 9627 7490 20779 1281 14568 17630 2371 4694
12396 15369 16597 3697 10741 16309 4927 13396 2277 21198 5308 21621
8841 16662 11120 14081 7321 10424 15204 6565 14320 2978 9294 7482
9493 1277 4952 7813 7306 12296 12021 6645 9286 22489 15165 9809
16089 15681 13745 16653 19069 7780 12587 19236 8110 4594 14762 10802
10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060
6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264
9315 2786 16253 9488 5634 17372 10399 10547 16039 14345 5365 10379
14566 788 5943 10889 5190 13207 16465 10488 17136 6257 8306 2432
11948 16340 15326 5415 9673 15586 20108 20541 3458 1240 15787 20099
9282 11480 4994 9189 12928 10743 7111 16707 19389 2889 19161 9422
21510 10965 22018 15558 4315 21926 12010 3187 21634 19556 2333 13869
18982 20495 2984 2763 5448 20219 12749 2389 7476 15067 3515 5375
1465 12770 18281 1243 13198 9265 1763 1615 5056 11143 12570 11245
2293 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 16007
2800 3186 7053 15790 7591 22480 20744 22093 6055 15875 305 12545
2037 11704 9571 21379 3712 7685 4483 18456 22341 10909 11197 13107
16635 16006 372 18722 18324
428: 14507 15421 14477 17494 566 7991 15716 19769 6346 11158 2783 18037
18920 14200 1991 21228 2025 16965 22007 22121 19530 17216 16560 11719
9318 17738 1299 21214 12334 12769 13914 10771 21809 10996 18789 3482
8428 15625 18025 21461 4486 4864 18259 1869 18894 5076 10621 8796
15953 1037 18120 21047 8656 2967 13154 6569 12338 19732 3033 10633
15359 15028 16103 12423 19646 3561 2797 6835 16222 3266 19426 6571
21319 4126 22537 6888 17781 1697 10045 1251 7003 7276 19243 14694
15687 11467 11444 12814 7126 7580 4960 7248 3598 14355 7140 17847
8316 17583 16488 4421 19550 8999 1180 18026 20573 8479 18820 21056
7036 15143 13926 18737 18581 16569 13403 1142 3253 7394 4463 11832
14382 7339 16547 15305 10604 4338 16236 16768 4138 18754 442 18246
10039 12847 6541 21176 20831 13811 12065 10287 11127 9635 20511 19012
13060 12724 898 4347 8702 12055 21425 20545 19336 8006 14571 5579
15743 3704 19675 16542 16559 21229 11524 9274 11075 8804 4132 17224
3133 3607 12684 13418 10123 13007 14891 18308 10517 2973 11620 15395
10374 3168 3051 15872 11732 9301 740 19109 1359 17419 10051 6843
1680 5509 10865 11707 16067 22296 1000 3406 18908 8719 9844 20469
12031 11698 9632 4041 4022 2595 20688 6395 9511 16615 4623 22346
2369 17333 14975 19292 12095 14281 11866 5395 16733 3840 17783 15322
17305 10611 1428 7631 15733 5563 18432 3777 1959 18192 1274 9859
16608 6676 5894 4159 21902 6323 6814 7759 17670 10954 17330 3090
8154 15122 14026 9078 475 20224 15939 13183 2171 20320 12959 18620
6196 16678 13224 21618 98701 7904 435 11481 4738 13205 11111 6886
4385 10224 21396 16032 22294 3286 13158 7249 8239 1725 17508 13407
19123 1165 2912 20759 22152 18157 16447 2772 12222 10847 14538 17604
14596 20901 4797 3679 21087 19143 7452 9348 17993 21440 14394 16530
16659 10434 15793 9088 17239 4831 6340 2684 689 15731 9528 16620
17353 14180 11651 17070 2073 1585 8281 12298 22147 10166 22431 15463
6601 8700 16540 2993 4260 9664 10652 15975 13842 14457 15948 11412
5572 7389 11862 1897 19136 11526 10222 1819 9220 12697 22065 20558
17946 2983 18633 13258 13996 5429 20450 10529 15553 3208 12002 14696
12270 16162 9071 7594 17146 7667 11793 2416 11610 19062 19931 6868
7221 5047 7968 10376 5672 2355 11711 4084 8726 14756 9731 18769
5680 10960 10209 17713 4926 14217 6624 9314 3151 6850 4560 18134
17898 12386 5636 3480 10028 2838 20595 1292 20275 12254 17594 4853
20642 8186 16379 10383 4591 11759 11502 8591 4288 11992 17721 15333
9201 12553 14724 3846 22481 1723 15765 17537 17720 5294 9934 3608
16724 3395 2370 22184 6889 3455 7902 13093 4038 2297 7376 1916
7144 16736 15894 19202 4614 10304 12063 3319 11943 21007 5703 6099
3369 4162 18750 20614 3762 6621 7015 7304 4298 9147 11434 11341
10440 4734 13768 14607 7695 13737 10493 7039 16005 5151 2008 20772
4271 19954 7608 5609 5027 11303 8198 15488 16991 16228 17894 18306
2556 6626 20616 16083 12968 22378 16437 7624 19737 2476 20184 8948
13230 16495 6039 10274 10978 13806 5631 6912 22114 2955 21137 13954
22400 1905 19874 16430 12918 16985 8847 4942 18505 6234 9524 15225
3274 6369 1408 7297 20802 5035 20321 12454 13377 9019 4029 11248
3758 16017 6780 2467 8343 727 18768 2737 1889 15457 2791 8559
7622 17058 4393 1128 13953 2462 12634 4887 5138 7962 11126 15081
22027 8042 16352 21033 8437 1952 10420 21247 20139 11118 19561 15955
3808 17939 13239 4794 21353 13160 11932 16466 19950 18656 9789 3900
11330 1626 21761 16275 839 16058 1199 5355 10804 10251 19141 19986
19797 13358 5206 9701 1661 11677 2289 10875 9733 12426 10942 7723
15784 12064 14341 5120 6223 5983 18048 8171 11041 10670 17196 16314
20434 21075 20432 1709 8716 4904 18464 630 8403 6184 18819 13412
17228 16302 10762 4918 9392 16256 4056 5523 9202 16022 17350 304
477 437 537 12703 15237 17792 11159 4597 7291 15512 1072 6812
15476 15612 4183 5466 17341 12261 10729 21044 4892 1108 1406 1630
5617 11923 17859 18198 6967 1264 20318 12469 22123 10452 5701 13091
15516 2225 1997 712 14602 8047 16361 6109 14712 22157 7745 16695
22271 11050 6934 6231 7635 1574 21574 18516 4245
429: 4085 2527 1065 15525 14455 18955 8686 14616 12315 16572 15135 18501
11081 5251 21737 20520 691 6761 19795 13031 3691 6753 3599 17478
19773 21114 6014 3843 12406 18706 4803 19871 5593 19334 19269 16267
7830 3347 21925 18962 16676 22334 12165 16013 9753 7457 5446 17495
20783 3532 19992 4641 17715 4676 10300 11655 22264 21804 2482 3288
11375 10949 13571 5392 22506 4117 12147 9177 12485 12082 14102 16550
8227 7887 9151 10009 2194 8035 3263 2456 10489 4324 17182 18539
10448 18491 18289 3508 9164 17132 9114 3276 7050 5077 679 14103
7303 20808 18812 5767 9247 20679 11242 19578 10187 6168 13518 22465
10647 6086 8411 19641 21890 15074 12875 17324 6537 5971 5621 4007
3671 5808 7390 2901 10617 17896 3947 21371 18983 19065 5064 8637
18056 8308 7938 11745 19805 2000 1635 14446 18226 8264 6511 6429
20857 18717 10669 18388 21752 2576 14131 18309 6243 2219 5023 15274
22359 8379 1196 17230 21410 4530 10035 18136 20678 8008 3975 22206
7027 14101 9482 8164 18390 11397 4582 20372 17760 16433 21946 13871
7967 9095 17117 9950 6587 14695 5445 3749 17681 19020 11758 8793
9612 10927 2881 20172 17530 3965 10328 18563 5640 17022 14773 3998
19518 6413 13513 21860 10059 4735 8090 17595 22329 18064 11254 4553
5196 7698 6952 13389 11495 4755 6529 14647 755 3688 17603 11692
15799 7721 11607 1907 5972 12763 20832 5479 6622 14733 843 8870
17397 3483 11493 19618 5858 18527 4566 20709 6716 19382 5368 13548
7884 20519 2015 18102 11949 19869 7193 21204 8094 22071 1179 8989
687 3082 1061 9091 4149 3083 19222 3949 20094 5822 13984 6059
18698 5004 9193 7842 2013 15901 10055 17023 9839 17902 6839 14943
21717 9782 8458 1749 6940 4589 14640 12339 16830 2964 18200 9685
7385 3184 18909 12110 11069 17845 2560 4950 3891 6893 12525 5770
4468 20897 13934 19823 2551 11904 3681 22519 2294 4619 581 12706
20766 3718 18030 8652 13568 7092 5748 13595 21125 11323 6294 7245
16341 18013 21906 7756 20468 12807 8952 4854 12892 20932 17233 15059
14597 10337 3390 2083 16457 2550 6863 18213 15149 18389 13296 2910
16845 800 9561 7305 5114 20251 14853 21958 20953 13079 4928 12827
18923 12922 12166 4134 8385 16411 6965 15063 1198 16204 2303 10341
9006 17038 3101 4223 12243 5951 19417 14082 8354 18716 5876 1912
10613 2494 17074 6579 21936 20024 9076 15771 8144 18836 21381 752
15962 2074 7618 7564 6180 14316 20422 20988 5743 1333 16210 5635
6971 21853 13791 11654 848 13920 16054 21997 16890 8874 3252 14854
18045 15844 1948 2167 19790 7872 15068 14737 9874 19597 19839 10412
18454 3700 11597 3064 19734 6549 14176 16329 18535 22267 1097 4574
8296 12640 20748 9752 18291 15724 1817 20274 2199 11298 12660 14557
21233 19952 16014 18187 20646 4479 15036 19259 10006 11951 14208 807
17472 22405 14906 11172 3156 4287 8159 7673 17535 1537 14931 1539
22567 5737 13603 19699 9407 1651 6379 10581 19260 3165 14667 7864
9195 1596 13152 16816 7742 6806 1616 17544 17707 20454 6937 3411
18482 20603 12839 21210 2018 11104 7947 15160 8893 5218 15123 19207
2443 10521 17888 19288 11689 13671 1060 7139 4723 16589 12750 20823
21620 13574 8321 3982 8782 19710 7192 18007 8616 10833 19376 1910
15804 21373 14004 10937 1232 14661 15988 3876 11632 4668 17814 12594
19491 10832 9955 715 15438 10841 3640 9007 971 6389 21095 16446
17276 14461 2963 15035 11615 3420 8456 9895 20407 21560 16473 17496
7402 5937 16861 18408 18177 14755 15184 9227 12946 18987 11729 4477
14072 12541 20612 4203 11063 18943 22467 12279 17726 13181 1130 19124
12801 22244 16404 4962 17046 2469 2626 10801 569 7725 2704 8430
18169 17621 14239 22342 20154 12018 6110 3490 20465 12047 553 5605
2628 16250 2059 9087 3888 731 1827 21528 6858 5758 9971 9055
19121 12596 9582 4484 11624 14798 6138 15283 7826 6756 11874 17055
9759 17852 4798 22395 20864 12972 13464 710 16153 11807 13928 12179
4667 18624 20452 2395 16837 8335 12798 16367 2473 10503 15101 11372
17828 16195 20015 2994 16716 1892 19350 3636 17068 17570 11645 1327
7475 20149 10068 20414 14198 6292 22295 12872 21980 3940 12542 18299
9655 2119 18038 8871 8658 17301 11886 2039 3626 2453 8245 11903
20419 2748 18668 6038 20076 11931 18793 3924 4613 6432 14061 11825
10785 4319 20931 20293 12868 20904 13240 9080 5315 18550 5568 3790
19960 18123 4607 21807 11927 7809 10838 3410 7219 3828 9117 4802
17766 7596 18685 14850 2868 2410 17525 18227 5587 748 9922 6817
13414 4122 14886 6795 20797 1302 13981 13919 12280 20227 13694 3432
3581 17689 19868 22144 2368 4292 15152 803 7632 8409 12829 22148
18235 17947 2085 4436 22035 19521 12094 1279 20990 7209 22178 9906
5312 962 12098 7610 20672 14380 1096 4197 11149 11974 5660 10939
21240 21535 6515 17591 13604 20996 6136 21870 19319 4234 6030 1908
3858 15291 7352 15060 2897 10563 1195 7862 18600 13786 13041 4898
2026 19040 19171 16313 14963 5034 1080 22252 3765 15468 11773 5638
12937 5393 13796 9419 21859 15071 8173 759 8785 10233 12251 10936
13190 19896 6977 14271 14981 14593 7058 1904 16109 1104 9497 9142
17439 2429 3251 1743 15595 776 11599 957 18835 15453 11273 11954
11554 9554 14263 13171 11110 17426 11221 20644 11573 20653 13888 8606
21124 21083 881 7084 6656 7589 1372 21363 12603 1875 14364 12080
923 17921 6852 4045 16278 13047 5535 17042 3118 9872 3494 11980
20390 19044 13195 11777 10230 14014 9456 5040 5358 12359 10174 7896
15061 19132 17294 9949 7651 19331 16609 5096 11833 5926 12692 793
7709 17994 10740 21923 4044 18803 7948 12708 4621 7049 15789 3183
19757 20773 18237 13118 11870 7824 12271 20914 19595 2904 7799 5575
11905 17160 7767 15836 1947 9981 9284 16661 19158 10592 7940 12764
14604 1540 8753 2822 16765 15532 1631 9675 17723 5039 9590 12994
21045
430: 10036 20702 8519 2198 16727 19676 5461 9855 22269 16042 7071 17892
17842 9521 21420 10703 799 9651 7127 11809 20361 1641 6056 19650
2134 9375 17502 21016 902 22224 8402 19626 7011 22503 19144 15490
2388 16766 6922 16601 9905
431: 10556 12984 17708 9241 19697 1495 14030 9734 14027 5836 7290 3964
13438 20803 17948 4643 8743 19246 8822 22019 15937 17296 18828 14438
21756 1588 19060 15098 4646 10462
432: 9532 10331 15934 2046 6293 19503 15147 5699 7047 14129 17105 9079
5185 13130 2151 9143 1141 10189 18230 19023 8431 18942 18099 5356
3212 10361 22388 21551 20873 5482 17963 7910 21068 13009 6829 14326
20236 6208 21773 15785 14929 1052 4869 16502 3926 4567 14892 13401
6433 9709 18261 6409 22223 21368 11301 6931 15940 17666 21501 12516
3358 6554 1974 4835 20560 11275 2659 5053 16358 7498 14297 15569
19582 20331 6857 8522 22493 2638 19738 13482 13654 7414 12151 12808
14165 1755 10215 8018 22220 22247 7254 20380 2845 654 7274 5061
5018 9568 9818 1494 3725 11235 10859 8938 12700 17001 19302 11190
4028 12045 3173 14318
433: 540 20745 4392 9539 7190 4968 12434 2894 9916 18824 15969 2549
13484 14142 5455 8922 14475 9503 12306 9461 18965 943 1591 20055
3802 8501 6629 7934 6828 21142
434: 18584 20810 16027 15628 21406 7354 15327 14802 20743 2149 9585 6022
1273 9506 9718 22409 3023
435: 20813 11290 7991 15677 22007 11719 11530 9318 1246 18259 1869 18894
5076 8796 1194 21241 4280 6152 3266 19426 6888 17781 10045 1251
15687 11467 7270 18820 4999 20923 20417 3253 11348 11482 14368 12055
19675 16542 16559 11524 9274 4132 12684 10517 11459 11732 9301 1359
17419 6843 11707 16067 9844 20469 16615 1849 2369 12095 14281 7631
725 21902 14026 3032 7802 1337 3062 21087 17626 17682 15601 15731
689 16620 17353 12317 12298 15292 10166 14717 13048 9220 15209 1054
10376 2355 11711 10028 2838 4853 11759 11502 9201 22481 15765 5294
9934 19837 18485 1903 16398 7376 3319 11943 7304 11341 13768 5151
2008 20772 16083 22378 12968 19737 8948 3528 5985 21137 7297 17939
4794 21353 18048 8171 10670 8716 12196 630 6184 9202 16022 537
15237 12703 1072 5466 12261 21044 4892 1108 11923 17859 5617 712
6109 7745 16695 11050
436: 16500 8982 2601 9214 5987 9273 2383 3132 16871 4889 1471
437: 14507 14477 15421 18062 17494 2654 566 7991 19769 6346 18037 2783
11158 1991 2025 16965 22007 22121 19530 17738 1299 10771 4864 1869
18259 5076 18894 10621 8796 1194 21241 4280 16103 3266 19426 6888
1697 10045 1251 7276 7003 19243 7580 12814 7126 16291 7248 3598
18820 18737 12671 18581 16569 13403 4999 20923 1142 14188 5501 20417
21625 3253 10604 7339 7394 4463 10287 9635 12724 898 4347 12691
8702 12055 20545 15743 3704 10510 16542 16559 21229 11524 9274 11075
8804 17224 3133 3607 10123 13418 15872 11732 740 19109 1359 17419
10051 6843 5509 1680 11707 10865 22296 16067 1000 18908 3406 9844
20469 8719 4041 6395 20688 22346 4623 17333 14975 11866 3840 16733
5395 17783 17305 10611 7631 1274 3777 16608 21902 3032 10224 13407
20759 22152 18157 1337 16447 12222 2772 10847 17604 4797 20901 21087
19143 7452 8263 17993 16659 22147 10166 2993 14457 11862 9220 12697
16162 17146 7667 9071 7594 19062 11610 5047 11793 10376 5672 2355
3480 10028 20595 20275 1292 17594 12254 20642 4853 8186 16379 11759
11502 8591 4288 15333 9201 12553 3846 15765 17537 5294 9934 16724
3395 2370 4038 2297 1916 7144 12063 3319 11943 4162 7304 4298
9147 11434 11341 10440 4734 13768 5151 2008 20772 4271 19954 22378
12968 22114 2955 21137 20802 5035 10180 17939 4794 16466 19950 18656
13358 19797 9733 18048 8171 11041 10670 17196 16314 21075 20434 20432
4222 8716 4904 18464 630 8403 6184 18819 5523 6647 14751 16022
17350 12703 4597 17792 11159 7291 15512 1072 15476 6812 15612 4183
17341 12261 10729 21044 9980 1108 4892 1406 22046 17859 11923 5617
18198 6967 5043 1264 22123 20318 12469 10452 12218 13091 15516 5701
8047 16361 6109 14712 21918 22157 7745 16695 22271 11050 6934 7635
6231
438: 3768 9465 20939 10052 11543 2583 2455 10554 14401 2398 18197 15151
12975 22406 10665 9509 15663 5927 13360 6164 16012 7099 21660 8046
5207 4255 5194 15420 1613 12275 14710 16908 3629 20166 7194 4457
5992 7677 13929 12101 15458 11274 20283 7521 14721
439: 14966 4405 7118 6146 8600 7777 22037 13873 8489 21468 8655 19547
10433 17970 9362 20687 16031 887 7010 20077 4253 21851 14561 6562
15040 11590 1611 13370 21623 10658 13700 6147 16428 6376 14491 7527
12266 22380 14286 8408 4247 10369 7295 19540 12488 11421 18418 20410
4446 3422 5527 5306 1906 20728 7396 20941 2529 6606 4873 7218
19654 13098 12189 18969 15325 4309 13598 20706 9719 8328 20122 16518
4685 8609 21086 1946 9979 2746 15560 7370 17671 15455 6005 3018
4686 4101 22066 4236 21839 3651 4990 11914 15738 6771 3325 20546
14826 19572 9615 13650 13899 13584 8742 3543 17869 19591 17545 22430
2960 4200 2077 8177 7719 2622 6263 9849 1470 5989 1311 21426
17824 7804 768 15927 14310 9131 19750 19247 21469 14334 14266 14771
6820 17114 15394 15812 13253 2599 12929 5170 19859 15229 15346 19250
1984 14570 11409 5996 20660 21736 20018 22326 19741 21896 19744 14409
13692 3399 3194 17618 14100 18368 2518 690 20346 14843 6521 4745
19127 22103 15018 18736 20115 1684 21656 3590 20651 2319 16368 7672
3740 21690 1173 6846 5212 19894 8599 13173 8940 12978 16135 15091
18853 20615 1491 18437 15682 13879 16777 19476 17619 1577 7748 13631
13915 10365 12457 15298 8901 8625 5241 7358 14305 18568 18524 14410
11871 21891 15823 20498 6200 9264 16240 19297 18458 2821 14785 3126
14169 5798 9737 21776 19585 3198 14858 22300 7338 8554 11016 18990
9617 18528 8024 16993 22122 4137 8570 22146 12226 9200 11674 21612
14895 9476 16664 3321 16193 18447 16049 5313 18953 8533 11938 13683
5508 2740 18932 19696 7262 8603 9275 18376 10691 20818 7714 1458
12356 7720 7819 17020 17844 13115 5156 10998 6550 16814 12176 777
14663 18952 3941 6256 10163 1231 14652 12351 20934 5346 3674 3417
3413 20689 13348 3105 5424 8660 8772 19183 22278 21459 16873 20072
17128 16743 19690 4218 3991 11096 10955 5602 886 19026 21015 19125
13706 10726 19356 12958 17574 18510 16516 22357 8610 3240 7095 3522
21988 5616 7937 3174 1747 3775 10024 6702 4908 6372 2579 18940
21345 5519 15356 13540 14244 5795 14821 14518 20309 12120 2485 17587
14150 15819 9978 12910 11595 16932 1960 694 14412 11488 18681 8778
12679 17521 8810 5092 21175 2974 13020 10931 21585 2789 4153 18786
18329 1083 19178 2493 21206 9256 15592 13324 16856 12883 15416 22022
685 13962 7186 2183 1824 7815 9340 13838 4490 22377 18686 9359
16226 1355 9864 4384 15337 14450 4671 8942 10589 3962 9323 19836
12509 15215 5193 8404 6144 21817 7541 10871 1525 21274 13622 9110
8628 2177 7874 14877 8824 18554 3163 18907 15713 2835 16426 3153
20949 2770 7004 14833 12848 4690 18093 18783 15352 8130 7593 18583
3501 4178 14120 14899 3694 21340 1642 6921 16503 11048 5058 2817
9969 13347 19955 11925 20475 21679 16040 21148 2936 11490 9879 3668
15510 9259 5266 11317 15129 15828 17404 18074 18271 12632 22568 6154
19613 4529 14306 9441 17517 20968 2859 19635 2252 15293 3989 7716
16280 13326 11344 11586 14234 9625 5215 13361 1156 16246 444 20628
5316 13449 3080 16715 10685 19801 5171 1815 3509 16084 17427 995
9018 10526 18574 10099 13402 17849 6159 17431 3824 555 9659 14148
9887 22551 16739 20950 2267 11455 5169 15137 17589 4048 18400 8026
1816 14675 4107 22512 7319 16725 20176 1188 3250 10266 3394 15649
6275 16233 11157 7931 10836 20212 6842 21580 18201 18602 7675 17976
14515 12856 12375 20806 1835 10204 928 877 18843 14059 6197 22517
12239 14250 21061 548 2492 19311 18449 5757 16595 21416 19162 10349
18182 5204 19779 22446 2313 11619 20729 18442 6595 12083 6350
440: 13290 13287 12491 9089 17221 9777 9141 4749 6525 10696 2418
441: 5159 5787 3587 20298 1786 13310 1389 10380 15437 20220 15554 14498
1082 9121 21871 18718 1610 17142 16098 20306 17432 20695 8310 15692
2042 20770 15746 11972 20448 7362 5959 17865 8865 4630 3037 6655
4037 16478 11125 6160 10003 14982 9652 7150 5560 21884 7298 3602
9622 20738 13828 4091 21632 1885 10098 5784 19579 11915 1619 4973
4953 15722 18588 9009 3874 3245 15203 22499 1147 19826 15124 8880
21552 2999 14657 21323 6418 1393 5831 1681 17953 9370 9708 13283
7660 9230 20542 6634 21649 6803 7555 12376 14548 16219 9559 21885
10474 5522 13930 9391 12268 15620 22031 14151 12757 13437 1509 3933
21317 8728 11995 13090 16778 17402 11699 16105 1278 2276 10571 834
14094 1564 4767 5915 1665 19312 9166 2756 14136 20976 11340 16028
13738 1229
442: 12225 16626 7991 19769 20707 21228 18920 15677 22007 1246 1299 4864
1869 18259 5076 18894 8796 1194 21241 3266 19426 6888 17781 10045
1251 11444 7126 7580 7248 17460 18820 16569 4999 20923 5501 20417
12724 12691 8702 12055 8006 14571 19675 16542 11524 14891 11459 15395
15872 9301 740 19109 15861 1359 17419 10051 6843 5509 16067 22296
18908 12031 4041 3840 17305 10611 1428 10769 7631 21902 14896 20759
22152 18157 1337 10847 14596 3679 21087 17626 10092 3743 21220 22333
4831 12317 17353 2073 22147 9664 11526 9220 2983 5429 13258 10529
12270 3138 6868 10376 2355 11711 9314 6624 17713 17898 12386 10028
20595 10383 4591 11502 11759 11992 15333 9201 12553 15765 5294 4038
7144 1916 10304 3319 11943 7304 9147 4298 11434 11341 9677 12289
5151 20772 19954 22175 19748 3020 16083 19737 21137 3274 6780 14060
10848 7124 17939 21761 1661 9701 6223 5983 18048 10670 1709 8716
12196 630 8582 9202 16022 17350 7291 21223 15476 12261 21044 1108
4892 1406 22046 11923 18198 6967 22123 20318 12469 10452 8047 6109
14712 7745 11374
443: 620 12442 20304 1418 14021 9656 19898 1753 11261 6048 9339 20785
14195 2548 20087 4601 12996 2180 21054 8933 8763 20471 14140 10491
10186 6478 12177 17437 1521 8613 20482 15739 2169 12190 15573 15908
1717 6770 16901 15379 7349 10141 17028 12150 9400 11457 15837 15021
17531 12650 16974 10219 14993
444: 7118 14966 4405 6146 8600 7777 13873 22037 8489 21468 8655 10433
19547 9362 20687 16031 20077 4253 21851 14561 6562 15040 11590 1611
13370 21623 10658 6147 16428 19540 10369 13700 14491 7527 4247 14286
12266 12488 6376 22380 18418 8408 11421 7295 20410 4446 3422 5527
5306 1906 20728 7396 2529 20941 4873 7218 19654 13098 12189 18969
15325 4309 13598 20706 8328 9719 20122 2746 15560 1946 9979 21086
17671 6005 7370 15455 3018 4101 4686 22066 4236 21839 3651 4990
11914 15738 3325 6771 20546 14826 19572 13650 9615 13899 13584 3543
17869 19591 17545 22430 2960 2077 4200 8177 7719 2622 6263 9849
1470 5989 1311 17824 21426 7804 768 15927 14310 9131 19750 19247
21469 14771 14334 14266 15812 15394 17114 6820 2599 19859 12929 5170
15229 15346 19250 1984 14570 11409 5996 21736 20018 22326 20660 19741
21896 19744 14409 13692 3194 17618 3399 17236 14100 18368 2518 690
20346 14843 15018 1684 18736 20115 21656 3590 20651 1173 5212 6846
8599 19894 13173 12978 8940 16135 15091 18853 20615 1491 15682 13879
16777 19476 17619 1577 7748 13631 13915 10365 15298 12457 8901 8625
14305 18568 18524 14410 11871 21891 6326 13311 6747 13835 5310 2038
11820 1116 18682 439 21539 20042 6396 15465 17351 4069 12600 18504
4527 20683 15823 12851 18338 6200 20498 9264 16240 12538 14814 15115
18458 19297 9737 21776 14169 5798 3126 14785 19585 3198 14858 22300
21664 7338 8554 18990 21738 9617 20013 11016 8024 16993 22122 4137
8570 22146 12226 9200 11674 21612 9476 3321 16049 16193 18447 11938
8533 5508 13683 2740 18932 19696 7262 8603 10691 18376 20818 7714
1970 8639 12180 20962 1458 12356 17844 7720 17020 7819 13115 5156
10998 6550 16814 12176 14663 777 18952 6256 3941 3417 3413 3105
5424 8660 20689 13348 8772 19183 11096 16873 22278 886 21459 3991
19690 4218 5602 3174 1747 6702 4908 10024 2579 6372 18940 5795
3775 14821 5519 15356 13540 14518 21345 14244 20309 12120 2485 9978
17587 11595 12910 15819 14150 694 16932 8778 1960 5092 17521 2770
2183 685 22022 12883 15416 13962 6144 1824 7815 19836 4384 4671
16226 8942 1355 9864 10589 14450 7186 13838 9359 9340 18686 9323
3962 4490 22377 15337 15215 7874 5193 8404 21817 7541 12509 3501
10871 1525 9110 13622 8628 14833 3163 18554 15713 21274 8824 16426
20949 2177 14877 7004 3153 18907 12848 18783 14120 4690 11925 18093
14899 15352 6921 18583 19635 16503 3694 21340 9969 7593 11048 11344
11586 14234 13449 3080 16715 19801 3509 17427 16084 10099 17849 13402
6159 17431 14148 3824 555 9659 22551 9887 2267 16739 20950 11455
5169 15137 16725 1188 20176 3250 10266 3394 15649 6275 16233 11157
7931 10836 20212 6842 17976 21580 18201 7675 18602 14515 12856 12375
20806 1835 928 877 14059 18843 6197 22517 12239 14250 21061 548
2492 19311 18449 5757 16595 10349 18182 5204 19779 22446 2313 11619
20729 18442 6595 12083 6350
445: 21014 8217 3568 15315 16602 22197 20633 10719 22273 7927 12557 3214
5391 17035 16717 18176 3239 21949 541 17286 20722 21074 17292 19789
4688 6904 4467 1219 19816 19041 17181 21332 18859 22211 8218 2257
6585 7606 16882 11061 6736 15760 18569 22238 10997 3586 4444 11325
16288 5712 12397 13355 14617 8607 12841 4510 17747 8828 10495 7078
11921 20794 5507 14664 17923 2708 13572 15775 2513 11167 14105 3111
11389 3370 20781 7301 10605 20844 14047 14130 22186 16977 10134 11180
19900 10713 1464 7899 2349 6473 6149 17373 2862 15797 14327 10299
17623 20126 6074 2179 9070 18653 19230 14764 18671 15774 11854 9254
7597 9029 4935 1891 7776 14288 7040 20201 10153 16227 4268 19425
11328 17458 2842 14940 6228 15942 6091 22429 723 13304 7975 9105
16127 19403 11783 823 6252 21376 13980 4302 22514 6008 19617 15008
5641 9634 543 14573 2873 11280 18674 12111 5653 8223 7146 12775
12377 16801 7816 4017 16666 19793 7002
446: 10867 18970 4967 22194 5235 18316 19541 18967 16679 12435 17897 14632
9836 7200 3244 4931 6155 724 767 11956 2291 15034 1158 4627
22468 2941 13822 6431 20840 3978 3058 2820 4352 17463 13357 17354
2351 1025 3451 8214 2208
447: 3349 11861 16061 20185 18887 5336 6597 12949 13433 7265 3983 19704
21765 4512 9383 17441 11806 19145 7957 1660 16578 8361 12877 12285
6745 14055 16201 22143 8158 1754 1561 9710 18005 11717 18079 2243
21572 10276 6672 1451 5576 13128 10885 6027 6068 15496 10454 833
15308 2899 11018 20697 14627 3974 7859 15270 16400 15938 15725 15423
5811 19619 2698 19513 5502 3862 14005 6242 14353 16413 5739 5131
1844 15747 8386 4651 22360 19139 7085 13831 22094 8195 21288 3065
16776 4822 18118 19091 5553 12572 14834 21349 16815 8955 13075 12733
4648 21653 22049 5403 1029 1877 1485 22486 14277 19152 7372 4606
21858 17690 9703 12831 6467 10808 13527 19173 11175 11657 2942 16325
1265 17141 11133 17532 18407 20507 14912 8705 18889 792 12024 4746
6954 5377 14106 3811 9106 7669 17506 18207 11911 10409 20449 9181
18488 21792 22305 9003 12668 7458 11471 11697 19342 10966 5324 17192
9173 21172 21538 9379 8949 22434 3555 19594 13734 5195 4504 5963
14256 6939 6345 1162 18406 2357 17784 1442 4331 6935 5879 18326
2240 20428 19414 11272 3444 14731 8631 1809 21833 14869 9442 3632
12121 8845 20490 19745 13155 12792 12563 14861 14746 14813 4528 17811
1567 2911 10316 11855 21730 13905 22042 3872 20386 9570 6304 9122
6011 10819 2612 733 11892 20852 15230 16137 13985 13024 11612 8752
1512 3948 22301 14196 1070 2951 8433 842 12944 19948 22053 9341
9353 1718 16198 8546 15048 9919 20674 21812 3909 10843 16432 15654
4043 6714 14847 8707 9886 20997 22088 13038 8037 9178 2878 21388
5352 3851 6484 13259 20733 16180 20444 5834 17651 21480 13792 22149
13049 13689 6933 5650 21822 22010 13935 7093 15175 21104 21571 13059
14166 1320 14676 19388 12387 10615 2572 18647 16738 21225 7210 15278
11859 4902 21097 13308 18378 12809 17277 1707 10671 4638 13485 7397
2352 5048 7941 13275 5252 11316 3053 9127 6631 5081 2879 17029
14783 12504 3446 5574 11878 2839 3228 16798 6179 1088 1402 19752
22525 21445 16859 10813 2895 8708 2051 20050 10689 13359 6382 9920
16804 3557 13937 22048 15372 19186 2356 21850 10645 17724 7740 18043
18443 14254 8080 7679 18267 13583 7611 8668 2471 7128 21146 15408
1163 8078 5556 13897 17262 5961 22314 15730 18968 650 8513 11528
7068 3085 14046 13243 11222 15011 3976 1105 15891 1507 6229 10269
17937 9774 5505 5939 7480 1497 3041 15132 9987 18054 121992 1835
22529 20727 7448 2979 5186 13110 17553 15284 15066 21091 12304 13927
2253 22173 14128 9863 4969 20557 7177 21588 7134 20606 17839 15095
4399 2214 21594 19310 3008 2570 8102 16155 4099 18252 20237 9891
20458 14831 9119 9194 7573 11856 2558 21715 10855 5766 15158 3389
1154 11165 21797 3985 15097 8881 12231 16104 647 19765 22387 4155
2561 4615 21305 15139 4964 16030 20281 8285 15689 12785 2196 1531
12335 20138 9927 1132 4098 6925 14275 10776 22136 15697 16176 15977
2664 9816 17562 11028 11930 10157 8670 12353 12574 2954 21428 12735
2392 5226 8680 17177 8835 7167 15667 18591 21341 2172 3869 15589
20343 19825 5210 7091 22444 3844 717 18902 14808 11659 1785 13405
22226 8233 21693 10456 13156 14660 12659 6461 10253 7132 5280 16881
15728 12322 846 14049 12744 8140 20493 15683 11033 12989 5866 18113
1964 14109 15020 20584 17016 18813 5845 7915 21617 20412 1643 20533
8020 9158 19107 2930 21064 15660 21586 18572 10523 4610 13545 7585
1761 2480 16383 16486 1541 8462 2592 3201 11077 14726 20135 9411
9402 12669 15995 15348 7755 8168 551 8916 3112 13876 21933 12219
14513 6877 8898 8597 18116 8268 9085 9769 11890 21961 11709 10758
12800 8640 1212 10509 21023 21751 11267 1557 18735 18051 21254 15200
5771 17912 3381 18702 19564 12819 15172 17593 8712 8128 1909 6496
14338 16091 948 17565 16614 18595 11540 18075 9306 10608 12167 9268
17453 12880 21522 13525 10162 5843 7885 18002 13496 9474 11489 13260
14139 10989 5078 5480 16165 3428 2826 5882 20175 15811 16116 7879
5318 8325 1069 22306 21302 6156 22394 14294 5702 4766 568 15271
1077 16409 17778 9726 14452 21497 5648 10532 3647 15330 8350 18444
19227 19834 3890 21780 12302 13191 21151 13104 12185 20101 10857 11960
7382 18469 4948 11036 12852 11208 9994 14197 5906 16060 20197 3576
1115 6471 4258 14545 21796 17471 17991 14998 3215 307 10382 6929
2580 6731 8289 9480 9118 11333 12900 8452 12605 11475 19577 6041
2991 13102 15010 21059 21365 6894 11757 21208
448: 17617 22319 7205 5436 22282 1508 1614 12460 8290 21670 8595 18790
19872 21481 5282 5245 2363 9283 17848 10766 892 11279 3262
449: 13633 5900 8539 14672 20311 964 12233 10478 5805 8760 4906 10137
3902 1838 13999 21067 4596 12781 4010 10761 8601 11786 6187 19637
20291 12933 1404 16680 16262 4172 11107 2757 4381 11802 14386 6750
3881 20169 3124 11582 4779 14837 16691 21558 16323 14350 7275 16234
10634 8984 12657 3036 20253 12586 15643 8362 20529 4086 15778 15634
10975 7311 15107 8618 8213 4431 2716 12985 488 2869 18696 12025
7468 6477 4900 14759 645 8641 3783 17104 16899 21903 9293 2872
7277 12282 7350 15064 2602 14650 13112 15517 7433 889 15267 21911
19806 10064 15339 1645 20835 16525 12947 16705 22317 19397 18277 11661
16082 8992 9337 17911 12718 11845 12581 12948 1001 4151 15609 5506
19445 5163 11501 20741 15240 10944 3217 5255 11017 20435 15575 13834
12427 2375 8287 6080 20581 16884 20690 12613 4380 15312 5145 10145
1126 13494 15530 15431 22255 22276 19919 8963 4880 20168 20601 7009
15363 10724 8126 7044 20981 3898 22554 17967
450: 1006 429 10217 535 507 533 306 478 314 12870 18461 5240
6367 19001 17394 8913 526 17484 7812 286 534 531 300 21030
2857 17664 21589 11961 19154 21437 11195 20933 21506 3533 4677 18511
4730 696 9569 17797 18687 7097 20259 15252 13221 22502 14915 21157
22126 18059 20371 3563 12591 16308 7626 4212 20262 19798 19458 5691
18292 11428 18877 16130 21296 2832 830 14553 6434 18295 17956 829
476 12043 9077 20779 1281 17745 16586 7955 12705 15449 988 20859
1768 11129 6547 22518 5552 9854 11376 6669 5359 9132 10964 1879
22087 13137 17298 3333 13752 20301 19370 2425 6946 19538 14273 15340
6427 17165 15603 12419 10809 8003 13658 18296 14064 4221 14568 17630
2371 4694 12396 15369 16597 3697 10741 16309 4927 13396 9785 21621
8841 16662 11120 14081 15204 6565 14320 2978 9294 7482 9493 1277
4952 7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780
15219 16969 14762 18330 10802 10479 16663 3678 16713 7751 13703 3630
4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526
7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 16548
13727 19096 10973 788 10889 5190 13207 16465 13178 9673 5374 9771
11716 10164 20541 3458 1240 15787 20099 9282 11480 4994 4865 1120
6281 18662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143
786 6709 5954 17926 20839 22351 13277 10965 15558 18982 2984 11245
2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800 305
307 509 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341
3094 11197 13107 16006 532
451: 3345 751 19630 15890 21334 2502 16406 10364 15556 10330 10995 14378
13458 16021 7590 20173 3502 572 19897 3673 12693 3848 6462 21994
20804 17582 4276 16244 8615 17497 11060 9796 2390 21178 7166 2759
19905 12817 14186 14243 5490 6784 5792 13470 1101 12561 6617 11241
6402 9027 20751 1813 16709 11278 10008 3046 7793 13443 5592 8736
3403 14205 19027 2511 14794 10004 16347 3927 13109 8732 21235 10480
19155 2047 21601 2804 11826 19016 20937 21978 13295 3255 17452 6272
19822 8274 14395 20792 19448 1967 4644 19536 3230 8051 21408 16552
17526 4434 6675 10332 16527 11695 11834 4106 21315 20826 18978 17269
22168 21255 19151 14377 2774 19782 12983 9784 9962 16564 850 18492
20740 21582 7445 1779 10977 17687 11634 18743 18450 11001 19140 11439
11743 13008 6064 17602 13592 13552 15418 11134 16033 20106 17363 12458
12874 6513 15906 16223 1784 874 1125 9165 17183 4284 8998 583
17287 963 5591 19466 4320 9188 20258 21605 17648 19691 3335 6816
19609 22222 3733 6330 8114 19196 6605 22558 3373 5980 4992 10542
21937 2186 4838 2923 20053 20960 4335 13352 2747 5898 8059 18499
19914 7292 2255 21251 19565 10896 14138 1474 1803 22420 2054 18604
4980 12543 19544 7953 17754 18426 21889 21464 7125 21785 14725 10578
7357 2460 2281 4747 13666 6881 21446 19660 14216 1004 17442 21195
9433 21820 7213 8549 21471 8344 17607 6026 13716 12371 18944 19208
20867 13887 15334 8749 16682 14433 12986 7761 12475 15833 15505 8725
627 20270 7644 15253 15959 13593 13483 12343 18196 3966 1640 20730
3351 10803 1315 1603 19482 6421 12053 1137 8850 12068 12197 8734
19465 2535 2409 22297 7627 19726 1553 13474 15450 3620 5719 11963
3815 19768 758 13039 16269 11639 7621 5670 7361 8209 17309 14298
22309 19799 6584 3893 10041 3814 2858 7122 20012 8232 12230 820
11268 7920 3024 942 16980 14141 2812 19328 19907 16962 16151 12559
3658 994 16669 2146 14691 14451 2080 3146 18837 7883 935 4402
21465 15552 7472 6823 18275 16984 17026 7620 5223 8813 21417 1708
22055 15118 13508 19764 22214 16335 1874 6227 19122 5567 20812 16276
19961 11837 11145 14544 15166 9889 16056 20845 14946 6840 722 13349
14215 14913 12175 2181 19628 13753 5595 13445 8711
452: 21725 4108 17790 10816 10072 11215 7845 21938 14000 12290 9072 2188
13197
453: 17069 19235 13531 17697 11206 10541 21126 13101 21378 21802 1329 11808
2880 22230 1218 9626 19047 3040 17741 3001 15767 17052 3724 7152
16952 20547 5794 17379 9932 18642 18433 2347 8182 3667 10010 9258
4330 16872 13931 10125 15345 15750 14295 6095 14904 11788 22535 2662
5270 2833 2917 19197 11031 8241 18241 12357 932 9546 3545 5011
5922 15762 21458 11169 19093 2950 4496 8786 17325 12345 8819 14838
11101 7574 21973 11052 5177 10577 9162 18537 19268 11571 9690 8586
17826 10897 21454 12803 2767 20158 11415 6066 16018 13857 10790 8706
12567 13232 21790 2221 16327 22364 9930 11940 14381 12027 5625 20421
6213 18883 15885 991 14231 1117 8576 18603 5865 1558 1668 7347
20793 5291 16647 5611
454: 10637 2260 13315 5883 11574 14796 9991 4552 7533 6653 8525 1420
15386 21880 6261 12607 8500 22030 1956
455: 7653 1606 10840 3715 10418 14035 4843 2101 19615 10922 3554 1735
3155 13503 6907 7784 20054 21733 1169 1452 5818 22385
456: 0
457: 3034 22132 9120 13729 6791 11897 5149 22382 7782 1951
458: 21696 15323 6711 794 10202 21441 6649 17320 15012 6988 16064 7702
2108 7337 13972 18351 2384 18596 11446 5769 20712 818 9873 11398
20352 19777 18363 16843 5030 6560 11056 12761 9908 4279 12906 8596
16833 6392 2335 20071 16868 4580 21828 17448 14949 21452 5673 10050
5093 22411 1705 11285 10329 5612
459: 655 11975 8069 20491 17684 1394 19340 17701 5908 684 14348 21852
5434 14730 2715 13227 21854 8282 16106 10308 21982 7567 4049 15967
16174 14809 13372 20868 798 20016 19024 1342 3575 14227
460: 19142 17102 15893 22335 12299 15442 11496 10074 5733 14018 4497 12860
17771 17375 8161 11868 317 11262 6451 9249 12173 13520 2381 4681
6996 6490 9579 1276 5404 8032 6363 10026 7316 14084 8908 17837
8863 19169 750 18053 13467 9093 12920 8746 301 4625 12849 8975
1460 8614 20285 11049 15351 15199 22494 14045 10173 6414 13682 21841
1623 17211 18365 4519 21203 2011 22000 10060 950 10516 18657 11078
6908 20626 18762 9992 11321 16068 13071 20575 9335 10629 4241 17420
13570 10156 14609 12058 7665 21534 18040 5706 574 11168 21837 3354
20317 6124 1526 12392 16093 22474 21550 2099 15300 14848 1309 13170
19435 2632 6374 14407 14649 12249 8309 550 7797 3769 17901 16142
2063 6092 19535 13070 9812 1776 3443 17767 15918 12414 2144 9885
3591 9545 21263 14344 19754 19431 11670 6760 19223 12846 21920 21081
22381 17475 18403 21905 12709 15904 5736 14499 13058 11572 10692 7183
16133 15670 12332 1756 16939 22104 4806 15031 18332 20634 7994 12919
9064 5045 14968 9551 2414 1646 4753 1805 18175 18991 9750 1618
17712 22081 22029 14969 2458 1011 14504 3779 21507 6619 4824 5015
20098 2353 13906 16938 1847 13819 11957 12498 17391 1983 2927 13991
20531 20599 1625 606 8646 7497 11561 11796 8562 3676 16567 11565
15599 13705 12415 13948 1711 2922 7520 21985 563 3562 1939 5754
19800 6341 9042 15748 16577 12974 4827 4371 1092 14881 16895 20528
1513 6790 19711 13821 5656 6520 2463 9526 17821 1888 18587 10549
18855 4858 7028 14908 22205 5964 4933 4340 5271 927 8374 3316
20014 21527 5008 9914 10822 13989 15266 13163 17347 7197 21212 6338
1401 3445 561 14444 20426 12281 9349 20565 7453 19524 4093 17235
8735 14800 9217 16762 10080 19195 11057 11977 8958 21372 19693 20196
6610 14503 22133 19181
461: 21226 10722 9090 9917 17608 3307 17248 19659 12888 16315 15869 13541
11628 14989 5547 13844 21236 9910 11202 10862 3786 7783 20043 16159
21123 21149 1353 5976 20875 12245 2040 1496 18804 8260 2945 3653
21684 4427 20639 22232 20700 626 868 17999 20858 2479 14206 10988
22292 12471 7299 12462 21503 15092 2732 4462 16160 13957 17698 19014
462: 10915 15022 4450 11366 20814 8548 7447 2554 10246 14947 16967 18188
8578 3396 12439 21412 21085 10559 15980 13299 6199 5121 3610 8985
5265 4663 6420 19456 9646 7090 9324 14666 21020 13611 3580 3309
3503 10431 15493 1658 4414 10484 19347 18475 3196 10873 7062 5347
14932 697 8959 9970 13746 17123 19066 3791 6974 21237 4075 21728
13369 5804 15214 3905 7104 15534 15425 15645 14841 13863 17523 14583
13266 16008 19678 809 17661
463: 15610 19505 3188 13558 22207 789 18180 4508 22145 6608 12144 14753
3301 17632 6183 18032 8172 21197 9377 13065 15119 10305 14673 12954
12468 8469
464: 7246 2525 8937 1249 20791 2442 13757 2102 8484 20391 22436 8284
6769 10971 8004 22023 4837 6659 8039 542 11456 2562 13478 12526
20405 19784 19698 17561 14237 21200 20356 7320 10248 1737 18419 11847
10624 18917 21915 21121 18416 17053 8529 2009 1335 2989 17827 5789
7577 14095 21524 13803 19598 11276 12421 21337 2916 21144 20276 15766
10716 15947 19883 13961 9267 13637 16960 15005 18795 2343 10609 15000
4509 3285 1871 22443 9056 14293 11451 17137 11684 5432 21943 5119
17025 20216 6625 22445 4609 21217 19722 18262 17059 19393 8393 18989
14155 18379 19003 4754 6752 7557 21304 14324 2929 15268 11094 6025
8713 14222 2187 1975 2856 8286 16534 8056 9369 15871 22373 5414
5457 8512 14839 13626 13511 18521 18560 2002 4104 11038 15162 19653
9129
465: 6459 6394 20221 2332 21782 13055 10580 2279 21367 19818 15567 13294
11815 16237 17819 16570 1622 15858 19159 18654 9082 10360 8884
466: 17835 13628 20663
467: 20037 7346 1314 12511 16115 17491 18854 2329
468: 17297 12034 9186 19531 5960 3320 6107 989 17365 1448 5740 10535
13789 18153 3130 2668 16090 6274 11423 19113 19718
469: 8693 16592 5005 19306 6604 16732 9754 13116 8230 21139 4079 5166
18517 6465 2795 16685 16688 21959 6043 13767 6173 22310 3259 13685
20252 7455 5044 3534 9389 10900 11649 8773 10993 11006 9330 11087
5753 19387 21478 15236 21689 1435 21991 913 7484 14285 18519 20798
16122 2721 770 15723 20967 12158 21702 18984 12751 12455 5999 11116
11824 9843 12418 7936 7869 5363 4568 3972 16205 22566 18031 20763
17285 18882 7781 5471 19353 4312 4716 18663 9603 15043 11217 14246
16326 9289 1155 13216 13122 13400 3648 8238 13201 897 19164 9520
12035 8212 12560 22169 3281 12562 11339 17667 12309 8806 22280 18666
22125 9307 6640 20092 2145 19241 15914 1213 13453 5332 61332 1968
9060 20524 10171 20916 20091 19282 10648 21566 4242 19533 11739 14917
21058 17627 7451 13855 20246 872 19965 10868 11173 2843 8856 6802
6353 5160 6251 13175 12961 12248 21646 18733 1820 15077 6061 2722
19163 13367 10128 22117 17264 16640 22485 19560 8034 1100 17788 13010
7758 19682 1515 12783 21145 7575 3152 16168 2508 13380 8293 1473
9684 17719 8565 4032 17866 18951 14592 14121 19857 5090 19600 22439
21996 16150 952 14805 7722 18382 14204 12365 18343 18356 17329 19831
6021 4198 17799 3839 721 9477 11433 18772 8387 1287 8906 17051
17649 19824 20739 20085 3542 5975 20850 9850 9401 8873 15065 7512
19446 6993 10543 8853 18552 12124 18137 16070 1895 21102 9226 7191
4365 12533 4665 5929 15384 20652 15768 3859
470: 4094 4249 6447 417 9699 4266 21799 19998 8507 4118 4930 21755
7422 16272
471: 5778 13042 20693 6337 16080 1704 20548 2346 20737 5152 6633 17650
2524 19716 3465 3548 16517 9459 12501 10090 1759 3273 12472 21412
14456 8135 18797 3137 17061 3402 9770 1269 2404 4759 7648 8568
8662 21076 11318 18004 13312 14804 6000 10393 14666 21987 3309 18931
14379 6524 14185 6945 8382 7629 16509 1933 4728 21025 17218 17638
1752 16078 3430 16885 19841 13228 577 3035 15647 12625 14586 21237
4075 7439 13720 15834 12529 21705 3905 17339 10133 5868 20567 18269
22275 13814 5994 10239 18618 20112 19325 13266 2962 2905 3571
472: 1296 2585 13077 15698 8567 10322 16480 13708 15389 17974 17451 15792
19280 10142 3469 11718 8642 15847 3638 2801 9326 3742 10221 13816
4884 3280 9923 15873 20214 10492 13528 8475 3713 15933 21666 11740
1633 11990 9134 17965 1788 6764 20492 17131 19212 13533 16793 11371
8594 20347 3728 17461 13823 8134 22260 7419 19185 22026 3272 1884
8730 8990 19977 1859 10999 4586 16348 19281 19991 11567 1008 20028
7658 20830 19756 20878 3207 20070 2980 18632 2087 6612 5122 7489
19025 20408 5867 8896 19056 19348 18818 3699 779 22456 11541 15161
11135 20058 2201 6739 16612 14992 19392 15341 8872 4204 21922 22540
19457 14075 4186 15353 17073 5661 13025 14487 6849 20671 6103 10343
4142 17366 14159 18164 2380 8153 15839 19958 6670 2506 8947 15249
9712 1377 18798 8620 19625 10515 9429 5493 7939 18930 8714 19761
3572 20073 19642 9602 11161 7180 10987 2971 8438 4675 8709 2938
4636 3485 9435 18162 15411 20078 14563 15262 14349 19829 9555 8928
16342 19361 1362 12060 11259 1940 18358 7454 21475 16972 20517 14458
5311 4246 3026 9206 21394 17932 5647 18756 632 3100 13204 18856
14390 13351 12707 15711 10265 18751 14958 5221 17138 17198 808 10940
11642 19473 3009 13298 15678 3551 14990 9133 19864 6205 1024 9670
22172 21447 19672 2996 6377 6123 21676 2672 19700 21984 15674 9974
14920 2725 11780 7537 15508 9998 18115 3670 16824 20178 4972 18635
8472 13466 21265 2831 8951 14511 7701 18486 7554 17622 11381 6309
22198 20143 15854 21658 12398 12681 11609 16625 12161 6162 3022 5109
7314 14439 8366 13945 12198 11746 13801 5301 15197 3210 11365 15206
17546 19669 2720 10926 1742 6963 11828 6972 1267 14550 8450 11591
19644 11212 14447 15125 18385 14497 17159 5903 832 17245 7432 3663
3061 9406 7511 4860 10188 13056 4196 11132 3684 3623 8675 8119
10193 16296 17100 11852 16004 17566 10552
473: 2421 20836 18507 17807 18922 14668 9172 2056 648 16255 7978 2322
11200
474: 17916 15231 15741 4645 21977 15829 10291 21573 1806 2663 8036 9618
16693 3960 15864 14578 17125 15924 21826 13440 17249 8650 20159 15742
1986 19706 22092 8766 6813 17830 10853 21281 13394 5285 8139 21004
14220 17563 2086 2488 1597 4698 13233 4654 1250 15737 2907 1469
9957 13288 6516 22526 16496 14873 10471 18290 3086 11953 18592 3185
9418 17135 8081 9593 19180 4673 7979 1300 13933 16544 16782 15551
8460 15960 13997 3405 1566 21046 8636 17134 6596 6512 13346 15639
21591 15042 12093 14396 9252 18637 22523 6953 16784 6262 16933 22448
4612 19863 6076 19303 12192 16828 17089 4133 19601 6118 3344 15088
14986 21070 2153 771 3291 10644 20638 4377 21183 9519 21234 11970
21215 18173 18301 17764 13810 10948 3781 21029 11162 16613 18091 22003
3801 21866 22213 6526 5846 20765 21771 14860 5007 861 6743 5529
14267 14880 21391 10210 5693 5970 3793 15855 1007 13001 6878 9875
16912 19329 13614 10333 13714 8204 21112 6903 1133 21262 21547 15703
21338 6248 15242 13567 16788 11020 18655 19496 10528 22414 8142 17440
20388 7760 17480 18357 14595 12914 2829 16249 2569 7096 6689 12534
6105 16041 9145 9242 1552 1379 10313 9596 10872 11771 3268 15445
593 5820 18410 6984 14744 9713 10053 1383 8837 676 4305 10421
2944 20363 19120 7463 16753 20969 18430 10227 12905 11066 6057 13677
18640 4083 1527 285 19285 5385 17557 20851 15693 17304 1683 14391
15965 19854 8425 14916
475: 17494 14507 18062 7991 15677 2025 16965 15154 22007 1299 4864 1869
18259 5076 18894 10621 1194 21241 4280 3561 13154 8656 12338 18120
3266 19426 6888 17781 1697 1251 15687 7276 7126 4960 7248 3598
8316 21056 7036 15143 13926 18581 16569 4999 20923 5501 4338 10604
10287 9635 11127 8702 12055 20545 19675 16542 11075 17224 15872 11732
6843 1680 5509 10865 16067 3406 18908 8719 4022 6395 4623 22346
12095 17333 5395 17305 10611 7631 18432 21902 7759 3032 10224 17508
1337 3062 10847 4797 20901 3679 21087 9348 17993 3743 10092 14394
9088 16620 9528 17353 12317 17070 22147 22431 10652 9220 12697 20558
11610 11793 17146 2355 4084 3480 10028 2838 20595 20642 8186 16379
10383 4591 4288 8591 15333 9201 15765 5294 3608 4038 2297 7144
1916 16736 3319 11341 10440 4734 20772 4271 19954 7624 2955 17939
21353 11932 19797 2289 9733 10875 12426 18048 20434 18464 630 8403
6184 18819 5523 17228 16302 10762 9202 16022 17350 11159 4597 17792
15612 4183 5466 17341 10729 21044 9980 4892 1108 1406 18198 1264
712 13091 8047 14712 22157 7745 6934 7635
476: 3284 22524 15910 13589 1006 11575 20349 10911 18523 22545 13067 2372
10217 9609 535 306 507 533 5233 478 4966 9015 21390 314
1764 12941 18461 9866 21665 7323 5049 9997 7294 5240 7571 6367
19001 13429 2814 21443 20459 1880 1396 3615 13867 7787 22538 13411
13344 8482 1205 18398 13083 12473 14941 17485 1550 757 11281 11029
21967 827 1873 21174 14091 5281 885 7332 8229 10933 12672 21103
4400 1012 15883 7812 7121 22179 16424 1339 1866 3474 11617 19918
16996 14296 1352 17699 9135 12367 19265 15111 3388 20896 20167 6697
6564 10047 19989 9358 22350 21749 15375 4618 531 16283 300 534
286 6575 7081 11468 2641 13543 15224 3097 15540 19380 19031 14287
6260 5051 12104 13901 11086 12459 12824 919 13271 15196 6303 4194
14638 1182 15803 1842 19299 7287 2857 17664 21589 11961 19154 12506
21814 4986 5343 21106 6334 21437 20936 11195 21506 2582 22069 5476
3533 7949 19583 22113 4677 18511 4730 696 9569 17797 18687 7097
20259 15252 13221 13305 450 22502 14915 21157 19855 11089 1744 10324
19917 6730 2195 2037 13563 12591 16308 7626 4212 19798 19458 5691
18292 8005 20912 21422 6721 10358 13601 6637 1272 17775 16130 21296
2832 830 14553 6434 18295 17956 829 12043 2457 5731 4395 10908
3209 13959 12384 5220 21843 19341 16539 19751 20779 1281 13763 17745
16586 7955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854
11376 6669 5359 9132 10964 14922 1879 22087 13137 17298 3333 13752
20301 19370 2425 14659 6946 19538 14273 15340 6427 17165 15603 7257
12419 10809 8003 13658 14064 4221 7387 13284 20981 2316 3116 8418
3386 6508 18217 3578 22249 992 13442 3471 14855 19949 5277 19885
2816 10917 9436 18538 18659 12816 12308 15943 2515 20364 14701 8193
17579 22076 8890 2649 9329 21073 8376 15177 10882 859 5990 9814
16053 5209 7718 3144 13848 22428 19076 5454 17056 14918 4088 11270
5837 9604 22455 19840 12234 1149 801 953 9960 21301 1656 3755
12278 22119 13793 21098 1005 10264 13037 11702 20954 5581 5063 1118
6454 20632 1696 6301 9792 5396 19474 14911 20315 9381 4111 11600
7691 13374 2932 3071 18551 22362 1431 8710 12923 6509 19229 14012
5372 12362 17380 14108 20272 16391 19694 13395 5132 901 14568 17630
2371 4694 12396 15369 13051 16597 3697 10741 16309 4927 13396 21621
8841 16662 11120 14081 21259 19449 8692 9484 15204 6565 18317 14304
2769 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 21386
12021 6645 16505 9286 22489 3622 13745 16653 19069 7780 15219 16969
14762 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709
8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835
10655 12264 9315 2786 16253 9488 5634 17372 3836 18362 11559 7637
4946 18143 12059 6995 17942 3772 3625 788 20809 16318 18862 7143
13134 9280 22095 18903 3706 15256 18593 5764 11115 12583 11568 13613
2331 5136 8073 15998 5630 11304 19137 5817 5580 18341 8588 12540
2454 4970 17445 2401 11869 6193 2434 3951 10889 5190 13207 16465
5825 17346 3323 13177 5354 2210 9333 13864 21502 19147 15973 9673
11863 9771 9608 11716 16811 18575 9584 12794 21399 20485 9218 18691
3350 18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734
2376 15699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103
4848 13211 22530 15360 12863 9975 6398 14067 16683 21170 1924 890
8155 15866 10131 7187 4332 1235 20330 12927 7088 5099 2302 12424
8303 17466 14322 11383 2282 15956 3414 12982 18548 15665 10961 21084
10824 18440 16819 3730 13940 18821 14864 1818 19607 7969 6546 16771
5441 8459 18266 5000 15749 13014 14274 8444 4707 13097 15930 11872
2621 7158 6942 18502 5408 10837 21928 13800 5188 19614 16117 4719
1833 13499 3107 21492 12503 21929 7921 1429 14398 22189 6439 12993
12307 19714 6650 21398 18994 747 5932 18630 6683 1921 15651 5594
6958 13597 19763 10097 14124 14687 1094 5780 7770 7688 15110 5797
7907 21169 3329 12627 4065 19882 22067 19211 2061 7038 8909 16914
13129 19881 16317 1551 13235 14888 1201 20264 7800 7526 2209 7384
5887 10798 15317 10223 11351 10105 10661 15014 2908 16412 22277 19851
12003 19616 11003 7768 6166 4620 8674 20541 3458 1240 15787 20099
9282 11480 4994 19545 802 17144 13420 13439 14301 1357 14011 7026
1145 6281 5258 6013 2805 18662 7992 9661 19875 11156 3619 14086
20948 16946 3456 6143 786 6709 5954 17926 20839 22351 21072 13277
15307 11701 15113 22057 6368 7704 18694 3644 12154 3794 21257 21638
18386 18111 21498 10731 13776 5539 14530 20282 7762 9529 17675 15191
12380 14865 15825 2818 5956 16442 6255 5901 8220 6918 18578 17465
13297 2495 21913 4526 16085 10965 15558 22565 22251 810 14127 7570
2692 13168 10912 15666 20574 4894 2526 10115 18982 19796 4020 2984
11522 11245 21570 8695 427 16749 17392 7325 10184 2132 20312 4655
16568 21093 13882 5730 7976 7079 13035 2800 16658 18348 16408 4157
5264 8112 509 307 305 12545 2037 11704 9571 21379 3712 7685
4483 18456 22341 3397 11197 13107 675 16006 532 20604 18536
477: 14477 7991 15716 18037 2783 11158 22007 11719 9318 11530 1246 21214
10621 8796 4280 19646 1037 3266 19426 6571 6888 17781 1697 10045
1251 14694 7276 7126 12814 14355 17583 1180 17847 8316 20573 7140
18820 7036 21056 15143 13926 18581 4999 20923 5501 1142 3253 7339
4338 16547 15305 10287 11127 20511 19012 13060 4347 12691 12055 3704
15743 19675 16542 21229 11524 9274 11075 8804 17224 3133 3607 10123
11459 11620 2973 15872 11732 19109 740 1359 17419 10051 6843 1680
16067 3406 4041 4022 9511 2595 4623 2369 19292 17333 5395 17305
10611 1428 7631 15733 18432 1959 21902 6814 13183 20320 12959 3032
4738 13205 22294 3286 19123 13158 20759 22152 18157 16447 10847 14538
4797 21087 7452 19143 16659 16620 11651 17070 10166 6601 15463 22431
10652 13842 11862 19136 1897 9220 12697 22065 20558 17946 13996 16162
7667 10376 2355 4084 14756 8726 10960 18769 3480 2838 20595 20275
1292 4853 20642 11502 11759 11992 9201 12553 14724 15765 17537 5294
9934 3608 15894 19202 4614 3319 11943 7304 4298 9147 11434 11341
10440 4734 13768 7695 7039 10493 13737 14607 16005 5151 2008 20772
4271 19954 4883 11303 15488 5027 17894 16991 7608 5609 16228 8198
18306 2556 6626 5829 13676 20616 12968 22378 16437 7624 20184 6039
10274 13230 16495 10978 13806 5631 6912 2955 21137 12918 16985 16430
8847 22400 13954 1905 19874 18505 9524 7297 20802 5035 20321 12454
9019 13953 2462 4887 16352 1889 17058 15457 8559 7962 11126 15081
22027 2791 7622 8042 4393 8437 1128 12634 21033 21247 5138 1952
10420 4794 21353 11932 13160 19797 13358 2289 10875 9733 12426 14341
7723 5120 12064 10942 15784 8171 17196 20434 21075 20432 4222 8716
18464 12196 630 8403 8582 6184 18819 9392 16256 4056 17228 16302
10762 13412 17350 12703 17792 4597 11159 7291 1072 4183 5466 17341
4892 1108 22046 1630 17859 11923 5617 18198 20318 1264 22123 2225
1997 13091 5701 8047 6109 22157 7745 16695 6934 1574 18516
478: 19887 10135 21002 14580 12123 15919 12579 5844 2744 4525 1601 15195
13066 547 20326 18377 314 8202 4733 7841 16690 17977 2012 6367
10918 3637 18964 16866 17780 7398 19262 8152 16337 16385 15522 10127
11277 18934 3901 22130 7812 16808 7324 19680 2408 19428 11882 19522
16434 9215 10784 19210 9199 9487 12925 15096 6578 16140 12862 976
11288 12658 20505 12973 11420 19873 8358 17414 300 8602 17310 6259
15328 15736 16123 1022 4666 6077 8815 10323 18236 1980 4532 17009
7605 19668 1127 2857 17664 21589 11961 19154 6093 3017 11359 3533
21439 16651 4677 18511 4730 696 9569 17797 18687 7097 20259 450
9399 14915 21157 20002 20371 3563 12591 16308 15626 7639 2807 13480
8017 8740 16746 716 21161 1662 7626 4212 19798 19458 5691 18292
16130 21296 2832 830 14553 6434 18295 22345 20848 19451 4148 6755
20779 1281 17745 16586 7955 12705 15449 988 20859 1768 11129 6547
22518 5552 9854 11376 6669 5359 9132 10964 1879 22087 13137 17298
3333 13752 20301 19370 2425 6946 14568 4694 21621 8841 16662 11120
14081 7280 15204 6565 1345 14320 2978 9294 7482 9493 1277 7813
7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 16663 20476
7333 6482 14526 7151 14429 6992 788 4653 2925 17755 9673 7253
9771 3362 11716 21088 4458 18575 9584 12794 21399 14605 15265 10699
6281 18662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143
16455 786 6709 5954 17926 20839 22351 19605 13277 16667 9507 1392
9772 15558 21992 22438 5600 18371 10363 19255 11144 11875 3318 21951
4300 19052 16019 10566 17447 7523 21878 18982 5589 4804 15183 17109
19009 17084 20892 22180 11635 13124 4102 12865 2079 3506 509 6351
4815 532 372 6844 9299 5904 14428
479: 21743 1760 3495 6194 14521 17761 15144 21403 16214 6891 10614 7713
1057 3816 17150 12489 10225 5157 8783
480: 21754 6794 12450 9614 4412 5824 9276 21813 1463 11059 4808 13206
9578 13428 3072 15596 4328 3690 8876 7086 15462 6444 12263 8993
5420 6407 12246 1242 4404 4259 22020 4611 9408 14680 3680 10238
9797 6568 8934 7500
481: 17081 17400 17733 17377 12323 9631 5203 18639 16889 16035 17467 19493
3726 14585 14637
482: 6638 7562 10477 1852 4661 1068 13778 18473 9233 12209 5474 831
3220 16416 19485 21657 17736 2773 6559 19274 11791 2362 1290
483: 15521 9926 9372 3535 14815 17378 15342 16992 22270 6694 8651 10442
9501 20248 14291 19051 3324 8926 19254 5893 16393 10149 19420 11920
8831 8248 4517 12007 9386 15721 21801 17774 10932 11965 11946
484: 3750 6233 2713 11380 12023 5512 8397 9963 4401 17914 21042 20938
15212 2563 21028 4455 18141 21707 15335 2976 12163
485: 12915 8353 10649 15559 21532 1766 2706 8964 20052 20655 17361 14417
5510 14469 5001 8855 14600 20649 14614 15591 20286 2606 9471 1305
12051 13261 12077 17015 15608 2231 9209 13625 14471 16566 18336 20911
10270 15117 22496 11939 2250 12212 10593 15979 5988
486: 21080 8598 20902 564 3747 14584 4202 15248 1415 21487 2776 8440
6441 6856 4639 13249 14411 6300 7237 7568 8497 20720 5330 2288
3218 10413 16585 19827 2466 13415 19277 19479 4274 5139 8341 13715
487: 6455 20477 1176 8864 15945 3915 15571 16266 6450 1211 18350 6737
21409 1653 14641 22433 16158 5655 11725
488: 13633 11464 17818 4487 22079 19811 14672 20311 5875 1804 5805 8760
3617 12276 17631 6644 1838 1416 13999 2846 1536 21067 951 9346
9547 21436 4596 13884 12781 4010 10761 12933 16680 14715 12582 16262
4172 17700 6985 11107 17731 21246 3488 4816 11798 4381 9680 2173
8661 11802 13722 4211 7971 6750 14655 2765 2588 17564 9533 14335
13862 22447 12417 16557 9483 20169 6497 2050 3881 20747 3124 21130
11454 11582 14837 2614 4779 6358 16323 14350 7275 16234 10634 8984
18673 3036 12657 921 5211 10884 8362 20529 8627 7103 8149 15778
6773 11819 1672 12073 7102 20447 2872 7433 19593 19806 21911 5516
15286 11661 8992 14562 3219 17911 15808 5506 20741 21530 15240 20344
18214 15575 12427 3217 20435 7796 2375 11017 10944 13834 12971 20581
6080 16884 1900 7505 3937 11188 811 9207 2830 20690 12613 5666
4380 15312 18349 17639 10145 13494 664 6567 13052 6979 13432 17364
2224 22187 6961 13798 7944 4112 9995 15431 15530 10764 1676 8678
7407 22255 22276 8963 4880 11857 15344 7420 20168 20601 21712 7009
15363 8126 3045 7044 2098 12067 13898 7690
489: 21080 8598 20111 19932 20211 6441 6856 13249 4639 14411 6300 15103
8497 20720 5330 2288 19827 16585 13715
490: 18170 1316 6082 19848 9272 9871 15911 5917 19405 7429 21327 20596
8045 10178 4720 16346 7401 11417 5176 9427 22532 19611 6616 10826
2235 7529 22399 668 22163 10888 10947 17345 20889 1019 13964 12858
12745 21803 19731 15370 19455
491: 14629
492: 9026 18203 9034 17176 19316 18425 10191 3057 4291 19993 19117 633
19344 14181 6581 17129 12690 10130 10179 8823 14022 7424 4363
493: 11676 10500 20010 22061 13770 5046 17871 10828 15052 1942 11251 10057
14213
494: 2867 20983 17787 12926 13702 2680 4336 1898 7033 4692 1931 11547
4004 17355 7966 21453 6141 17522 20009 20155 3861 20641 13805 9004
5518 9100 6706 6460 9101 19945 14203 18615 8818 2078 21644 13350
9869 8593
495: 10049 5921 9432 4046 3753 21873 9833 14362 8333 13672 19390 1216
3131 18992 9486 11253
496: 3127 1607 15003 2751 9086 18256 7201 3526 20063 8184 14575 14132
3886 15754 11997 10334 8002 19176 6325 12719 15443 14454 10921 14581
14745 9990 9911 1762 10972 16402 1520 16638 1856 10874 3967 20026
4426 20686 9163 20462 20942 17529 11988 17803 8476 6783 21168 19191
2426 12096 15664 7561 11891 12826 17289 3387 19858 12743 9853 19481
615 8569 14610 1310 14283 3903 16792 22556 12950 6759 5113 5803
20040 13866 7531 12876 9534 7024 13251 663 2377 20537 22479 20413
17045 11984 22225 3341 1812 7226 19298 20768 6766 10544 12090 22407
18361 8222 5162 22240 11887 8295 15781 14468 2137 9880 12861 16260
21244 21191 22495
497: 4536 12720 13909 16764 14068 5500 2174 17809 20213
498: 7246 13566 4922 7189 11908 21432 19713 9021 8001 4169 9591 18319
12836 14209 15108 20572 4881 2021 2629 13362 21200 14093 18917 5199
21121 8529 17053 2009 2989 6906 19492 5789 6127 7577 22482 6916
4185 8063 10716 15947 17616 22376 714 9711 8995 19804 16484 10432
16415 18397 17120 5711 20348 11410 22426 5459 17025 20374 5057 3996
1399 16384 8557 3401 18399 9470 18719 2158 3928 19925 15780 1172
19003 4754 6752 7557 15694 2929 15268 8286 16534 8056 2719 1955
18895 5457 22161 17041 8512 21678 7854 18098 13647 15831 11038 15162
8205
499: 14500 1413 1282 1702 21840 19911 17917 16929 17820 4161 16375 12532
18092 1532 5902 19438 13718 6024 19735 13044 2787 19276 8082 9213
13875 2729 19946 19636 13441 11906 5009 21052 8166 12467 5788 10596
21948 8953 11461 18890 14782 22256 22200 7502 22419 22028 9688 19279
20234 20394 3662 13624 5037 17822 19294
500: 17384 19962 11922 12514 975 11652 22218 7145 9489 16374 595 10439
11174 6276 2673 2273 5345 20790 14336 21369 16362 13555 12890 20920
16827 21155 6409 16703 4160 18249 5053 1371 16010 3265 21504 10158
7203 22449 12631 15261 19335 9198 7318 16504 7195 4503 21207 10641
8201 15388 10167 17571 2611 3434 12571 13501 9308 8098 13986 4294
6690 17338 11438 16124 14788 9510 20668 22077 11494 16723 5320 3714
18672 19620 11228 1183 17223 1503 20710 7345 17503 10327 18402 18415
3756 1040 19384 13970 1810 20021 12766 10244 7980 16489 3481 1266
2213 5652 5230 6678 19037 22307 5389 18546 3014 22259
501: 16649 18243 16414 4207 20589 9444 16449
502: 22464 13448 15971 16611 16582 2337 16931 10640 13818 19924 15868 13017
5333 7271 18106 6613 20999 5327 19529 19566 15187 19703 13925 6927
17013 11388 20074 15701 3553 10267 14357
503: 10199 16886 2241 18245 1886 9716 7985 8744 20562 11084 13088 19819
22302 17049 990 2306 15235 2417 1433 2115 19923 19064 1002 16264
8492 2272 709 13399 12601 4956 19182 6191 7426 9434 14994 11105
6247 10698 8645
504: 8253 556 21865 11532 13731 17389 13431 10078 1271 12610 10733 21490
10485 18060 14669 11177 20124 7109 12716 1462 14936 5986 8097 5080
6289 3372 19867 2939 10924 773 11608 16138 4495 6375 38291 0354
13565 9492 7772 17813 19072 19386 6691 804 12615 3475 9031 16143
1291 12201 12820 1857 14937 19224 20789 10619 4585 1580 8663 12262
20460 1321 17853 6723 5237 11503 749 5651 8384 14280 17145 5419
15332 13013 5297 12732 19655 7901 7020 16059 19539 4387 1475 21505
2671 5714 1191 2608 21672 3296 15495 21919 1209 12295 15820 14193
14631 6254 7207 20109 13220 15146
505: 15140 1261 13853 2474 14749 2848 6699 13353 5174 10742 4290 12981
5167 19956 3782 14080 3433
506: 4704 7409 3027 1782 17403 2728 12116 22243 18039 19464 20608 21816
20161 9586 9832 1168 20257 8215 2055 2915 21159 8994 19097 4800
10831 3258 3511 5759 8010 5683 18509 3517 13649 8892 8848 13892
15978 11205 19106 17806 10914 8163 22263 5036 9958 17199 17195 12100
14343 7851 2637 16675 11823
507: 1698 1006 429 10217 7363 6232 18461 5240 6367 19001 18925 7778
526 3438 7812 20057 286 2857 17664 21589 11961 19154 21437 11195
20933 21506 3533 4677 18511 4730 696 9569 17797 18687 7097 20259
15252 13221 450 11335 20777 12713 22502 14915 21157 20371 3563 12591
16308 7626 4212 19798 19458 5691 18292 17864 4456 16130 21296 2832
830 14553 6434 18295 17956 829 476 12043 20779 1281 17745 16586
7955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854 11376
6669 5359 9132 10964 1879 22087 13137 884 17298 3333 13752 20301
19370 2425 6946 19538 14273 15340 6427 17165 15603 12419 10809 8003
13658 14064 4221 7387 13284 20981 2316 3116 8418 3386 6508 18217
3578 22249 992 13442 3471 14855 19949 5277 19885 2816 10917 9436
18538 18659 12816 12308 15943 2515 20364 14701 8193 17579 22076 8890
2649 9329 21073 8376 15177 10882 859 5990 9814 16053 5209 3144
13848 22428 19076 5454 17056 14918 4088 11270 5837 9604 22455 19840
12234 1149 801 953 9960 21301 1656 3755 12278 22119 21098 1005
10264 13037 11702 20954 5581 5063 1118 6454 20632 1696 19474 14911
9381 4111 13374 2932 3071 18551 22362 1431 12923 6509 19229 14012
5372 12362 17380 20272 16391 13395 5132 901 9540 19228 14568 17630
2371 4694 12396 15369 16597 3697 10741 16309 4927 13396 21621 8841
16662 11120 14081 21135 8692 9484 15204 6565 14320 2978 9294 7482
9493 1277 4952 7813 7306 12296 12021 6645 9286 22489 13745 16653
19069 7780 15219 16969 14762 18330 10802 10479 16663 3678 16713 7751
13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333
6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634
17372 788 11307 11198 9280 22095 18903 3706 15256 18593 5764 11115
12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817 5580
14145 18341 8588 12540 2454 4970 17445 2401 11869 6193 21516 10889
5190 13207 16465 9673 9771 11716 22279 18575 9584 12794 21399 20485
9218 18691 3350 18263 4846 544 19667 9933 4140 1318 20418 11128
20105 16734 2376 15699 7061 4232 15357 14036 5339 7107 19030 7165
21370 12103 4848 13211 22530 15360 12863 9975 6398 14067 16683 21170
1924 890 8155 15866 10131 7187 4332 1235 20330 12927 7088 5099
2302 12424 8303 17466 14322 11383 2282 15956 3414 12982 18548 15665
10961 21084 10824 18440 16819 3730 13940 18821 14864 1818 19607 7969
6546 16771 5441 8459 18266 5000 15749 13014 14274 8444 4707 13097
15930 11872 2621 7158 6942 18502 5408 10837 21928 13800 5188 19614
16117 4719 1833 13499 3107 21492 12503 21929 7921 1429 14398 22189
6439 12993 12307 19714 6650 18994 747 5932 18630 6683 1921 15651
5594 6958 13597 19763 10097 14124 14687 1094 5780 7770 7688 15110
5797 7907 21169 3329 4065 19882 12627 22067 19211 2061 7038 8909
16914 13129 19881 16317 1551 13235 14888 1201 20264 7800 7526 2209
5887 10798 15317 10223 11351 10105 10661 15014 2908 16412 22277 19851
12003 19616 11003 7768 6166 4620 13850 16231 7016 2717 20541 3458
1240 15787 20099 9282 11480 4994 6281 18662 7992 9661 19875 11156
3619 14086 20948 16946 3456 6143 786 6709 5954 17926 20839 22351
13277 21057 15113 6368 7704 18694 3644 12154 3794 21257 21638 18386
18111 21498 10731 13776 5539 14530 20282 7762 9529 17675 15191 12380
14865 15825 2818 16442 5901 8220 18578 13297 2495 21913 4526 16085
10965 15558 18982 2984 11245 20659 477 304 427 12456 2132 20312
4655 16568 21093 13882 5730 7976 7079 13035 2800 19890 12545 2037
11704 9571 21379 3712 7685 4483 18456 22341 11197 13107 12449 16006
532
508: 19543 4932 20425 19108 19812 10140 21350 7766 14738 13289 13966 559
10501 18670 21326 12614 16425 9945 1996 21651 12806 13430 17362 21330
509: 5942 14707 1006 429 20069 10217 6051 533 535 478 10352 18305
21211 21561 20755 17549 15547 13386 8817 2523 18461 15773 19497 5240
22508 3614 6367 19001 3539 12446 15801 15469 13944 13012 20409 13620
15377 4732 4983 11962 10129 873 20622 7812 18242 8809 14606 18228
2931 3322 9023 6902 3558 3304 13684 21120 3950 12291 4958 21675
7657 4473 17017 4318 22410 19999 2415 22330 534 6329 4819 10093
8054 966 16945 14639 6901 14032 21397 2354 2669 10194 5075 2367
11025 6751 20059 21308 14038 2857 17664 21589 11961 19154 2340 7408
6244 17569 13074 21437 14633 11195 20933 21506 4167 3533 18759 4677
18511 4730 696 9569 17797 18687 7097 20259 15252 13221 450 10725
22502 14915 21157 18816 20371 3563 12591 16308 7626 4212 19798 19458
5691 18292 16130 21296 2832 830 14553 6434 18295 17956 829 476
12043 2658 7437 19427 14568 17630 2371 4694 12396 15369 16597 3697
10741 16309 4927 13396 21621 8841 16662 11120 14081 8692 9484 15204
6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021
6645 9286 22489 13745 16653 19069 7780 15219 16969 14762 18330 10802
10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060
6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264
9315 2786 16253 9488 5634 17372 21009 788 5630 10889 5190 13207
16465 3748 4100 8138 14088 9673 20541 3458 1240 15787 20099 9282
11480 4994 5496 4783 20946 10965 15558 12797 4801 18251 14926 14677
6310 18248 15246 7925 15740 18982 2984 5765 4060 11245 20786 4379
2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2360 2800
16617 17577 19315 17164 15726 12545 2037 11704 9571 21379 3712 7685
4483 18456 22341 11197 13107 2227 16006 17470 10632 372 16331 4975
18567 13264
510: 2585 13077 1296 15698 1085 3129 8567 7012 16480 1063 15389 13708
17974 17451 15792 19280 10142 3469 4227 11718 15847 3638 2801 9326
10221 3742 13816 3280 4884 9923 11360 17722 15873 1490 20214 10492
13528 3713 8475 15933 21666 11740 9134 1633 11990 17965 20492 19212
13533 17131 16793 11371 3728 8594 17461 20347 8134 13823 22260 9279
7419 22025 5521 19185 8730 10999 4586 16348 19281 11567 19991 17098
20028 1067 6210 7658 19756 20830 10712 16037 2865 20878 3207 18632
20070 2980 6612 2087 5122 7489 19025 20408 5867 9692 13244 17130
19056 19348 18818 779 3699 15218 22456 11541 11135 2201 20058 15161
16612 6739 10651 14992 19392 15341 4204 21922 22540 19457 14075 17880
4186 15353 17073 5661 13025 14487 20671 6849 10343 6103 4142 17366
14159 2380 18164 13366 8153 19958 15839 6670 8947 2506 15249 9712
22234 1377 18798 8620 19625 10515 15887 9429 5493 7939 18930 19761
8714 3572 20073 19642 9602 11161 19552 7180 10987 2971 8438 4675
2938 8709 9900 4636 9435 3485 18162 15411 20078 15262 14563 3415
19829 9555 8928 16342 19361 1362 1940 12060 11259 18358 21475 7454
16972 20517 14458 5311 9206 3026 4246 21394 11663 5647 21680 18756
632 3100 18856 13204 14390 13351 12707 15711 10265 5221 14958 18751
17138 17198 2504 8197 10940 11642 7410 19473 13298 3551 3009 15678
14990 9133 18124 6205 1024 9670 14097 21447 2996 6377 19672 22172
21975 4551 21676 6123 2672 21984 2725 19700 14920 15674 7537 9974
11780 5242 13895 9998 3670 18115 16824 20178 18010 7493 13466 21265
8951 14511 18486 7701 6098 7554 17622 11381 6309 22198 19059 21658
15854 16625 12681 12161 11609 12398 6162 3022 5109 7314 14439 8366
13945 12198 11746 19463 8918 13801 5301 15197 3210 11365 15206 19669
17546 2720 10926 1742 6963 11828 6972 8450 14550 1267 11591 19644
11212 14447 15125 21242 13309 14497 17159 832 17245 5903 7432 18321
3663 3061 4860 9406 7511 10188 13056 4196 11132 3684 3623 8675
14569 21430 19252 10739 16443 8119 10193 17100 16296 16004 11852 17566
10552
511: 1655 3326 8875 4957 8311 19832 4954 10256 13907 20915 2896 5571
12394 6016 4944 12546 7522 2423 2681 8552 10982 7116 5314 12119
21099 18607 12329 4674 13459 21805 3969 14769 20266 22203 3114 336
12480 7283 9746 8207 7204 14337 11300 20397 20746 1530 5147 14828
21034 3906 2359 21177 8537 10139 16166 2594 13784 16485 20871 14720
1825 16147 17234 8065 17167 16501 11902 18924 5861 14539 14230 2788
2072 18571 2501 4192 13524 4842 3095 7769 10089 16575 6956 16293
11570 10846 19759 20187 21448 19904 10891 15038 1868 12675 6957 653
16915 12203 12409 8067 4947 19061 2619 13364 4888 5566 10650 17254
18049 16316 7313 11076 14440 1765 10770 13783 5839 16261 12061 9852
10237 10732 19814 4893 6882 6805 4262 4557 3971 6381 1527 13153
22347 12730 10796 30841 8028 9061 7136 21153 925 8750 18082 12774
8584 13536 12518 6788 979 8900 18133 5696 15970 16063 14470 16701
5862 12183 3705 6658 16350 9557 8971 3717 1724 22354 16718 3854
3536 4599 16869 11895 16555 16258 4214 20552 21819 7135 16259 15280
4762 13775 4531 3367 13517 21549 8105 18643 4066 13282 14115 5130
10429 5920 4306 3192 5443 21499 6557 11929 20598 20903 4176 9050
15849 2723 15314 5993 12041 21869 14163 7930 9081 20569 9795 20396
11150 12443 4358 9475 10811 2552 11877 17685 21729 20974 12598 14790
20989 15004 17240 6237 17208 21930 8580 11818 5442 17259 20256 16046
5065 2308 2128 12182 17118 2251 7155 2892 13229 9845 20431 4311
16583 21037 18490 9763 6826 11713 15211 21554 16813 7056 18009 3039
19296 6673 11484 17841 21732 6456 2378 9054 19996 15355 21362 7381
512: 11789 20550 12924 14464 4011 19063 17858 14825 17933 18215 18506 8574
11311 14714 10870 5173 18503 12871 11792 6319 5991 16072 5679 7664
7997 7724 13724 15843 5179 21486 13257 20843 5253 15239 8745 4317
11829 5762 17201
513: 5799 17656 10980 3449 13335 20959 11238 9736 21154 8759 1349 18282
16665 17907 1107 9170 6492 20516 7727 5847 9065 19902 9931 8677
21320 19055 9044 19475 21629 4209 3873 10407 3831 21694 7261 11569
1480 1407 5128 14542 607 2784 18463 12009 8535 4917 22501 18918
12152 19362 6874 3988 10827 20140 18080 14167 1710 4124 3650 20877
6031 11044 12822 11665 16321 4669 19156 10011 15957 10312 16955 17469
9415 12205 21650 12324 10985 4763 17086 19498 18609 9205 19889 13760
11234 3128 3060 17868 7045 10721 18775 8340 16607 22199 8687 3652
8147 19720 4090 22058 13973 15250 5813 3005 6442 5423 4181 11396
20254 11686 18648 22257 3514 5054 17718 19944 22383 13148 10185 9304
9674 13580 20000 5106 14024 15026 15535 16386 22442 21036 12653 8647
894 4170 18619 16370 14161 13064 13337 2689
514: 19430 2217 13653 7909 3447 18614 2248 3672 3914 9037 2661 14057
515: 21829 10688 1896 2695 15238 6923 13321 678 19909 21886 6620 4554
4937 9312 1954 4679 2233 18331 21411
516: 12014 16420 2750 15538 18414 1466 5013 18876 17795 12046 9951 6600
5934 11750 5835 2034 4693 13740 10307 909 4231 1436 13779 16112
13219 4984 13214 18231 18179 2811 4814 18531 554 6050 18257 2903
3484 19943 17271 7129 1721 20263 20319 16055 20881 9942 13994 18838
11636 7029 16119 5257 1043 19478 20619 13417 11422 10160 20613 5029
8196 19429 9805 1861 17540 5288 14683 10272 7443 2656 16627 2315
772 9104 9410 4096 4548 9824 8485 17962 16475 22107 21011 15087
8840 15594 20392 9440 6125 9757 11164 17673 20924 4254 8176 16401
14437 18646 12057 12830 10159 17174 8040
517: 13062 19774 2825 8829 8332 18558 21964 19050 1627 16157 11534 19742
19647 13280 1517 9695 19320 10835 10293 13202 9174 14643 15719
518: 3195 21607 10760 14223 22323 21616 11305 21976 16169 8132 2667 11216
15637 4652 2270 8808 10309 18374 9291 7790 12784 7569 1295 5525
20767 17464 15920 7877 6705 4550 14008 15544 7616 1791 14358 15686
13182 14560 16722 8357 1472 4000 4261 10693 20144 4624 13338 17893
7464 11142 13491 15676 17655 17116 17984 9925 18333 10247 16818 4579
2385 20648 983 3200 6435 14763 13495 12036 16066 6740 14948 2307
14063 6851 10240 1728 16533 12629 10196 17078 16048 17609 11181 20629
575 18286 21518 5309 13744 15880 19484 16840 4959 15622 1288 18892
15176 815 7329 1175 20218 8827 7588 658 8704 16710 12907 17705
21364 16998 4945 15440 14372 12879 20119 17194 19468 10410 10451 11618
12006 12191 17175 20965 12916 6796 9139 17057 11008 18605 3353 4650
12741 1237 7988 20088 8231 4522 18497 11952 2866 15466 10449 5850
8504 22473 14806 5364 12620 20699 20034 4409 917 9156 629 12665
12908 7530
519: 11427 19017 6602 1864 10522 13385 3923 15070 3821 6415 1572 5050
15882 10935 5721 20535 12645 22402 8448 1350 7112 8649 5148 21245
11236 6476 14907 12714 20807 15311 9469 4739 13575 9732 8564 14799
12721 13373 4868 12893 19038 13333 8973 17817 15414 18146 11390 22248
13609 10100 15109 10007 5125 12633 6880 5388 20060 21032 8509 17168
19622 20280 21442 12066 17299 20123 12902 2062 656 6457 19453 20032
10850 8420 3813 21339 19516 16171 21635 2450 21079 19226 22202 17856
19847 1215 16964 11846 9253 18683 16927 3145 2877 4684 8416 4696
4925 14172 18178 10122 20134 11437 19509 20038 17265 10957 11114 11985
13382 4424 21277 6642 11395 19354 3216 17000 10584 4383 608 11099
3606 4061 6926 11774 3799 5172 3696 17840 9961 17641 22548 13507
6182 1731 2207 5353 5367 13860 8912 20922 11967 3241 16963 17734
10073 13903 22521 12149 4120 617 13506 13612 8031 6763 7546 6218
16311 10707 1223 21269 9414 8360 4885 3852 955 17284 19833 11354
2803 14827 1301 19175 10717 10910 12224 11516 13340 5262 5684 2178
13200 5913 9157 7959 17542 20440 18229 4805 8038 13587 16238 15602
18860 3544 7650 12674 17624 7074 576 17060 7413 22374 15047 21681
651 17560 6085 12160 12805 10775 4709 11401 6838 18364 17014 10752
17012 8980 17510 10711 8219 16107 16692 19401 17191 18405 15984 5384
19201 4362 883 6527 6410 867 12321 17889 17964 11941 14152 6591
4578 7461 4633 14845 14113 9478 16306 10792 5394 8423 16472 5538
5726 15272 22541 13274 19969 1344 17982 3299 6241 17488 6970 16632
2724 14874 2212 22392 12804 12135 15618 11858 552 17686 5585 19912
21050 1222 14554 13068 15732 8905 5854
520: 12932 18316 8070 15251 8216 15182 21583 18345 17227 19404 5489 17421
19679 6636 1134 14441 20247 13276 19767 5072
521: 14618 7172 4577 10111 4971 9921 11310 2304 19058 4919 2407 21318
6019 13841 12405 6079 22166 21222 11447 3834 2957 2876 3847 10030
3657 8737 2532 19313 13162 19695 11283 21477 13270 11090 20277 5569
11224 1524 7689 5785 1443 1174 5378 3463 7893 2512 20456 11012
5357 17062 17203 17936 14894 10285 19219 19980 15015 19783 22068 1918
19153 5369 14302 9654 16742 15180 4073 14772 14901 3954 13470 8012
15898 12241 11155 18311 11712 3109 18530 17791 1700 6065 18830 8838
7741 20399 7063 8515 13562 2024 8969 7647 12054 19078 2184 16655
8540 7852 16491 13169 20514 20202 7535 18320 11710 7747 15987 17730
7302 1993 2766 4031 5562 21493 10046 1799 4731 18131 8100 10005
7123 2520 10396 22156 9867 7052 11207 4505 21049 11504 21868 11614
5889 12118 13000 22505 2411 10176 6296 9619 18566 20017 11928 3767
11046 7551 2528 11302 21596 6991 7083 2350 736 10023 5402 21313
14934 14110 14248 11521 15498 3429 17829 15576 19218 22393 10506 19723
20354 6720 17694 8466 4273 4916 22208 20427 9107 15921 4439 10197
2338 16397 13942 15715 20725 18142 3311 2926 10795 3889 17244 10288
19160 8477 12011 3565 21062 9658 16357 6172 2617 1734 15245 22324
7974 4678 3698 3992 10876 20303 19258 17048 4272 20359 11598 8412
6693 19116 5690 8276 912 4024 10472 12647 6733 5738 15800 21800
3295 8590 20011 17717 4177 7227 20102 5504 13132 11762 15477 18479
9793 17968 11404 21844 17913 17281 14938 1225 7663 13582 11349 16247
19951 3778 8781 7268 10618 18594 14849 15471 22337 22454 4787 4244
12088 1992 9332 15917 17106 13790 19542 19573 2089 1592 7878 12361
19383 14253 17356 4238 9481 1439
522: 14431 6239 11377 22466 13606 11763 4871 19242 19036 9779 970 4882
15417 18050 2958 13661 18314 19970 8044 8672 8262 4077 12369 12815
15194 7431 19112 585 5289 17307 6089 5537 5935 21766 1304 15168
4356 6485 15400 19439 706 14453 1031 5411 11160 12131 10020 11249
8415 21643 14449 14017 1963 10261 1703 1878 4023 12040 9720 17260
21578 15343 11117 16514 14299 20222 12107 18114 13664 4184 14261 16823
13551 22313 3921 1998 666 4628 20928 18508 9588 8794 5200 619
4115 20171 1555 3070 5376 7843 10040 19791 19447 20093 16957 19231
523: 5718 10805 6298 2166 20556 21748 9437 8027 3472 10338 7212 20501
11491 4341 11669 9303 16917 8868 14506 2160 21476 17596 18688 9966
15557 11767 2124 18956 7553 18747 18697 17873 7757 16320 22134 13292
7913 9948 6980 17735 634 12444 16435 5350 17955 19423 15565 11353
20128 13016 5239 860 22552 2379 6033 16520 21986 17573 5222 14074
2603 19569 20484 2875 1313 5161 7288 21179 5370 19621 980 10087
20381 3366 6986 21533 16164 20883 3426 1157 16729 6898 20593 17576
9698 12169 10986 8048 17173 5809 21767 582 9317 13750 3110 18945
19532 13594 14859 19926 14149 16203 18916 2694 2259 3885 16893 6833
6096 16248 7576 1925 6278 10533 5649 20618 13912 17331 9831 9223
1851 20107 18714
524: 5146 21711 9443 19434 17798 8271 15157 6680 1121 22072 17654 19028
13218 6405 9244 5217 7137 8194 21221 12019 13471 17578 11153 9140
11727 17633 3462 17166 6295 21753 828 13617 9059 2402 16458 7601
13675 21744 12228 3556 9011 10297 8699 1058 17727 3732 888 19098
19007 20328 22435 9743 8151 9884 5912 13874 1793 20963 14276 11662
13913 18489 16461 9898 8731 15745 3973 880 2296 9092 18765 17370
18888 13547 17231 13680 4367 14754 17832 2159 8925 6054 4836 21950
16483 17585 20898 15188 9783 19045 2088 7805 5781 7540 21423 10626
3232 3412 16951 12593 7133 7230 17422 10172 19629 20061 15226 16396
13106 16858 12395 8064 18608 10913 18148 14559 11640 22231 949 3806
922 6047 14424 20294 21512 2844 21750 10411 1285 598 9413 13278
5979 15078 9781 11382 15932 16934 17171 3994 21427 3887 3113 10974
15635 18526 5503 9904 16377 17644 12020 15507 18396 5802 16207 16206
6951 13409 14980 12042 9197 20762 11969 14942 19111 1373 9467 797
8518 2947 13489 6590 10901 7185 7646 6204 6548 8825 7264 13425
13837 10201 11983 19803 21421 11553 9049 3356 19634 16334 3944 7749
20163 662 8190 6651 20724 22285 8240 12389 5598 11233 14315 15485
11282 20624 12145 12327 13242 17891 1217 8203 12727 22111 8826 9668
20863 11483 21587
525: 6387 20127 15922 6468 22478 5164 14961 7042 16305 2182 16677 7733
8221 13250 4225 10787 11039 2823 13281 6380 6824 6360 20082 1398
14175 14977 21194 5933 9986 10799 2049 7444 4924 15193 2703 12215
13851 836 5401 12967 18193 18801 1103 3308 20749 18446 18404 15606
19308 7496 14462 10044 21187 16900 8754 16101 22544 1746 5089 13111
9288 15179 4423 14999 11584 15673 5028 18076 21294 12622 21553 14776
12325 6837 10013 20597 20543 11805 18067 18468 4428 3795 8921 3661
8624 4476 13323 11313 7963 22062 20636 18513 1030 8805 22083 5807
3830 19568 5055 4025 10497 21824 10898 19772 590 10473 8226 20982
6273 21435 16209 9607 18881 10143 5700 11596 10735 19808 17961 19021
1837 21662 20927 1365 7598 21243 15848 17757 8577 12564 9707 7488
9892 1675 13967 1620 3282 2358 18278 15275 15531 18220 21540 22120
14812 13263 6574 14679 2574 16215 1945 10259 15499 11627 12347 2605
6895 7286 10483 2487 10289 3346 7293 9601 10220 7996 4547 4237
7808 7341 5250 14483
526: 8075 16099 8242 429 13036 10217 18174 535 533 507 306 11062
15313 16399 14594 314 3820 5717 20464 15374 18461 18690 9504 8137
11945 3327 4030 1064 15390 4708 6367 19001 8581 6859 18172 3305
18107 12112 13896 2465 12130 19906 4949 17359 16220 13607 10705 15889
16978 8246 16554 18467 9002 9780 17882 21655 20242 9063 5705 9204
9915 11991 16148 15840 14589 6171 20389 6007 14898 1840 4491 7812
8977 13027 6371 15170 21310 12535 9755 2819 3052 12267 8377 3804
7983 22167 21674 22450 891 13549 7113 14532 9354 7858 8394 2044
9345 2341 6836 19148 17763 286 18665 2645 14240 13516 9032 15489
8304 7822 9993 17083 7310 5481 11010 1093 7789 21306 21699 11113
8956 18128 7880 3416 6279 2857 11961 19154 10481 11542 21609 21506
3533 6663 3391 4677 18511 4730 696 9569 17797 18687 7097 20259
15252 13221 3261 14692 22502 14915 21157 20371 3563 12591 16308 7626
16130 21296 2832 830 14553 6434 18295 17956 20230 10392 12043 21065
20779 1281 17745 3289 3754 5487 8587 11548 12578 2882 5552 16586
7955 1768 22518 12705 15449 988 20859 11129 6547 9854 11376 6669
5359 9132 10964 19776 1879 22087 13137 13752 17298 3333 20301 19370
15830 2425 6946 19538 14273 15340 6427 17165 15603 12419 10809 8003
13658 14568 12544 17630 2371 4694 12396 15369 16597 3697 10741 13396
21621 16662 8841 11120 14081 13456 21472 7487 12898 12787 8692 9484
15204 6565 11203 14320 2978 9294 7482 9493 1277 4952 7813 7306
12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969 19357
14762 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709
8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835
10655 12264 17372 10889 6281 10965 15558 5973 1482 22476 19408 9637
5773 5016 20819 18982 19997 4572 640 19168 8691 17905 2970 2132
20312 4655 16568 21093 13882 5730 7976 7079 13035 3505 2800 12522
8939 2155 3206 2017 15587 6277 1297 1423 18728 305 509 12545
2037 11704 9571 21379 13089 11197 13107 22367 17943 2420 7599 5997
372 1801 21374 16528 8722 16672
527: 3642 15482 8461 18796 11646 4433 18911 12512 19895 14727 7891 5229
8941 8192 671 3621 19293 20133 7775 16444 5601 9047 7456 1332
15241 20870 3570 13126 6335 14830 17816 4052 1048 11934 7415 16817
11466 15006 17149 16740 13546 20225 18675 12360 11085 4464 12955 7636
18831 17524 21731 2244 8988 9012 18649 8617 6285 18612 1053 1455
4829 16779 15889 18827 14953 12980 10447 1034 15656 2581 22412 4354
10756 21138 3468 6872 6990 15371 16865 14170 14485 16553 7703 21199
6847 729 14001 18779 7182 2836 8312 3038 18069 16737 4357 10422
3567 6722 22039 3264 5085 20829 6719 2934 10564 14973 13027 2205
9393 19576 7148 2665 6959 16605 4997 17039 7857 4310 11556 19590
12715 6725 8377 3489 3770 22024 3546 20586 5189 20926 2245 1604
7700 18947 5485 22116 21537 11706 18715 21536 17691 17398 2949 3079
6290 961 6042 4750 8302 20882 5852 13136 16392 2643 14077 9240
11996 3702 14090 19402 7233 5546 10148 20680 8638 3269 15282 9663
15009 15604 8648 14392 18782 10570 6318 19862 13514 13217 3980 5231
11578 4812 18337 3004 3073 8249 18941 9036 3233 864 15080 19930
6746 1476 10866 20483 19942 7450 762 21276 3235 15320 22462 11955
1562 13099 7710 9136 7251 16129 19467 10892 10630 13600 12857 14732
4581 18096 3518 12368 1009 17283 4039 6864 5773 6063 15842 16851
18149 7977 14608 4572 10553 4514 11848 18068 17157 12223 18597 15178
10550 11835 16242 11778 20884 16907 14445 5068 7404 1516 6277 17663
17662 21513 5526 14789 7565 21347 1238 10660 18496 16381 20827 19639
528: 22368 2262 4500 18087 11171 3257 5531 17538 11849 9017 9287 9644
19677 20195 3911 3945 21129 6446
529: 18280 2326 4826 11407 11186 16051 1917 13113 10636 10206 19238 18366
6941 11986 7683 16587 14340 18850 6892 3404 17954 20223 11450 10435
19074 13384 7518 15985 8336 5383 3907 13379 12855 14509 12217 3398
530: 1719 15257 12828 16494 8849 18495 10061 1831 11005 20446 21892 17075
9052 1023 10386 2348 6364 14482 15814 7746 9263 3529 9935 10786
20919 12407 11705 8493 5095 8481 6104 3104 14770 19267 1032 17399
8085 17728 7344 673 15714 3361 5103 21218 15350 7539 761 19758
16925 17515 8371 9594 13769 16744 17772 4420 8605 6819 6628 9541
19100 13982 7360 20665 9903 5639 8487 13488 9666 5511 11106 17709
5021 19088 20007 12811 14758 15329 7823 21543 20508 1178 5558 16935
1252 7312 20192 18963 5305 19575 17193 1262 3577 20951 2711 18829
9159 6009 6176 17751 9694 17992 12429 15448 4598 18452 7077 10597
16197 705 8820 11971 19192 1370 996 1578 21971 6312 17349 2736
3936 10254 10398 22265 20083 15853 14066 5564 9113 3087 1078 7435
14028 16372 9321 18704 8983 21564 9660 11558 17282 12695 19057 13847
17094 11400 7613 2232 17213 17103 19378 6072 14868 8879 6825 12193
1427 3275 9458 15007 6271 4411 4190 12430 16366 20438 15680 10929
4035 20030 4556 17229 4660 597 1587 15707 4334 22484 21035 8211
16251 13133 11295 2966 14058 5665 6592 8738 6102 13087 3436 12074
14546 13772 20692 10970 19345 13509 5329 19187 16282 9968 21671 9048
8976 7492 21462 19662 8457 8317 18846 12864 10746 4141 8673 21010
10417 4329 7895 998 10132 8852 14795 5620 6695 13781 19165 7258
20895 4154 7359 12259 11737 12704 21606 3865 5520 6932 12901 20940
15027 3236 4907 20734 15492 10607 2472 15046 8506 18724 11671 15259
15936 7889 5596 2053 6905 8323 16351 10263 15826 21160 19461 17456
3953 3584 4314 64281 4221 4758 5800 22365 17678 16252 14893 17154
19437 16453 21224 9572 16225 9367 1899 8751 8426 12483 4441 20592
6867 2168 16928 13225 22460 20208 12970 2591 5304 13498 21581 4699
21894 1461 5860 21622 18703 2500 7014 15116 596 14962 5517 13693
21739 19681 11533 3338 3956 11364 17988
531: 15910 22524 13589 1006 11575 20349 18523 10911 13067 10217 9609 5233
4966 9015 21390 18461 9866 21665 7323 5049 9997 5240 6367 19001
2814 21443 1880 1396 3615 13867 7787 22538 13411 13344 1205 18398
13083 17485 1550 757 11281 11029 21967 827 1873 21174 14091 5281
885 7332 8229 21103 4400 1012 15883 7812 7121 16424 1339 1866
3474 11617 19918 14296 1352 17699 12367 3388 20896 20167 6697 19989
9358 15375 4618 11468 13543 3097 15540 19380 14287 5051 11086 12824
13271 15196 6303 4194 1182 15803 1842 19299 2857 17664 21589 11961
19154 12506 21814 6334 21437 20936 11195 20933 21506 22069 5476 3533
19583 7949 4677 18511 4730 696 9569 17797 18687 7097 20259 15252
13221 13305 450 22502 14915 21157 6730 2195 19855 10324 11089 1744
19917 16130 21296 2832 830 14553 6434 18295 17956 829 476 12043
2457 13959 12384 5220 3209 19341 5731 10908 4395 21843 14568 17630
2371 4694 12396 15369 13051 16597 3697 10741 16309 4927 13396 21621
8841 16662 11120 14081 19449 21259 15204 6565 18317 14304 4952 7813
7306 12296 12021 6645 21386 16505 9286 22489 3622 14762 18330 10802
10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060
6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264
9315 2786 16253 9488 5634 17372 3836 7637 4946 18143 11559 18362
12059 10889 5190 13207 16465 17346 3323 5354 5825 2210 19147 9333
13177 20541 3458 1240 15787 20099 9282 11480 4994 14011 1145 19545
802 17144 13420 13439 14301 1357 10965 15558 22251 22565 810 7570
2692 15666 13168 10912 20574 4894 2526 18982 4020 19796 2984 11522
11245 21570 8695 16749 17392 2132 20312 4655 16568 21093 13882 5730
7976 7079 13035 2800 16658 18348 16408 4157 8112 5264 305 12545
2037 11704 9571 21379 3712 7685 4483 18456 22341 3397 11197 13107
675 16006 20604 18536
532: 15542 3190 1006 429 7236 21108 10856 10217 1694 1170 507 478
7946 4963 10952 8465 18461 9739 1850 5240 2382 6367 19001 19407
14319 21808 15520 1498 14212 14282 19450 526 3987 18034 12939 14517
22533 14740 7812 18725 15422 12515 6665 8294 20640 9749 7933 18742
10601 1528 14184 8297 11430 21253 7897 4901 7308 16574 19527 9882
8330 6186 16730 3076 15579 16073 19400 17335 2857 17664 21589 11961
19154 6997 3197 21437 4440 11195 20933 21506 3533 4677 18511 4730
696 9569 17797 18687 7097 20259 2920 15252 13221 3452 14930 450
11335 20777 12713 22502 14915 21157 14054 10864 5059 21184 8816 9146
14653 14579 20371 3563 12591 16308 7626 4212 19798 19458 5691 18292
7706 5100 4540 16130 21296 2832 830 14553 6434 18295 17956 829
476 12043 13241 11023 4301 9298 2269 1306 13291 17064 20779 5613
1281 17745 11486 9723 16586 7955 12705 15449 988 20859 1768 11129
6547 22518 5552 9854 11376 6669 5359 9132 10964 1879 22087 13137
17298 3333 13752 20301 19370 2425 20461 6946 19538 14273 15340 6427
17165 15603 12419 10809 8003 13658 14064 14568 17630 2371 4694 12396
15369 16597 3697 10741 12482 16309 4927 13396 21621 8841 16662 11120
14081 15751 4903 21508 18755 14599 6384 8692 9484 15204 6565 14320
2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645 9286
22489 13745 16653 19069 7780 15219 16969 14762 18330 10802 10479 16663
3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457
21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786
16253 9488 5634 17372 1733 20453 21787 16624 7001 20828 13302 10357
4549 788 10490 19133 12318 5515 9280 22095 18903 3706 15256 18593
5764 11115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137
5817 5580 3044 18341 8588 5857 12540 2454 4970 16919 17445 2401
11869 6193 21516 10889 5190 13207 16465 9673 11625 9771 2283 11716
19554 905 7481 12401 19686 13656 22135 9553 18575 9584 12794 5644
21399 20485 9218 18691 3350 18263 4846 544 19667 9933 4140 1318
20418 11128 20105 16734 2376 15699 7061 4232 15357 14036 5339 7107
19030 7165 21370 12103 4848 13211 22530 15360 12863 9975 6398 14067
16683 21170 1924 890 8155 15866 10131 7187 4332 1235 20330 12927
7088 5099 2302 12424 8303 17466 14322 11383 2282 15956 3414 12982
18548 15665 10961 21084 10824 18440 16819 3730 13940 18821 14864 1818
19607 7969 6546 16771 5441 8459 18266 5000 15749 13014 14274 8444
4707 13097 15930 11872 2621 7158 6942 18502 5408 10837 21928 13800
5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 7921 1429
14398 22189 6439 12993 12307 19714 6650 18994 747 5932 18630 6683
1921 15651 5594 6958 13597 19763 10097 14124 14687 1094 5780 7770
7688 15110 5797 7907 21169 3329 12627 4065 19882 22067 19211 2061
7038 8909 16914 13129 19881 11720 16317 1551 13235 14888 1201 20264
7800 7526 2209 5887 15285 10798 15317 10223 11351 10105 10661 15014
2908 16412 22277 19851 12003 19616 11003 7768 6166 21613 4620 13850
16231 7016 2717 20541 3458 1240 15787 20099 9282 11480 4994 937
16775 18459 14476 9221 17450 6281 18662 7992 9661 19875 11156 3619
14086 20948 16946 3456 6143 786 6709 5954 17926 20839 22351 13277
11255 5140 4283 9455 4818 14459 5187 16668 10965 15558 17215 14289
11685 4388 14119 18982 2984 11245 304 477 427 18112 965 2900
2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 5498 2800
19337 9600 307 12545 2037 11704 9571 21379 3712 7685 4483 18456
22341 11197 13107 16006 7623
533: 6090 1006 429 10217 10250 19910 18933 314 18461 5240 6367 19001
16913 19119 526 3693 7812 13179 534 300 531 2857 17664 21589
11961 19154 21437 11195 20933 21506 3533 4677 18511 4730 696 9569
17797 18687 7097 20259 15252 13221 450 11335 20777 12713 22502 14915
21157 20371 3563 12591 16308 7626 4212 19798 19458 5691 18292 16130
21296 2832 830 14553 6434 18295 17956 829 476 12043 20779 1281
17745 16586 7955 12705 15449 988 20859 1768 11129 6547 22518 5552
9854 11376 6669 5359 9132 10964 1879 22087 13137 17298 3333 13752
20301 19370 2425 6946 19538 14273 15603 15340 6427 17165 12419 10809
8003 13658 14064 4221 7387 13284 20981 2316 3116 8418 3386 6508
18217 3578 22249 992 13442 3471 14855 19949 5277 19885 2816 10917
9436 18538 18659 12816 12308 15943 2515 20364 14701 8193 17579 22076
8890 2649 9329 21073 8376 15177 10882 859 5990 9814 16053 5209
3144 13848 22428 19076 5454 17056 14918 4088 11270 5837 9604 22455
19840 12234 1149 801 953 9960 21301 1656 3755 12278 22119 21098
1005 10264 13037 11702 20954 5581 5063 1118 6454 20632 1696 19474
14911 9381 4111 13374 2932 3071 18551 22362 1431 12923 6509 19229
14012 5372 12362 17380 20272 16391 13395 5132 901 9540 19228 11589
13306 3179 14568 17630 2371 4694 12396 15369 11784 16597 3697 10741
16309 4927 13396 21621 8841 16662 11120 14081 8692 9484 15204 6565
14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645
9286 22489 13745 16653 19069 7780 15219 16969 14762 18330 10280 10802
10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060
6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264
9315 2786 16253 9488 5634 17372 788 9280 22095 18903 3706 15256
18593 5764 11115 12583 11568 13613 2331 5136 8073 15998 5630 11304
19137 5817 5580 18341 8588 12540 2454 4970 17445 2401 11869 6193
21516 2447 10889 5190 13207 16465 9673 9771 11716 18575 9584 12794
21399 20485 9218 18691 3350 18263 4846 544 19667 9933 4140 1318
20418 11128 20105 16734 2376 15699 7061 4232 15357 14036 5339 7107
19030 7165 21370 12103 4848 13211 22530 15360 12863 9975 6398 14067
16683 21170 1924 890 8155 15866 10131 7187 4332 1235 20330 12927
7088 5099 2302 12424 8303 17466 14322 11383 2282 15956 3414 12982
18548 15665 10961 21084 10824 18440 16819 3730 13940 18821 14864 1818
19607 7969 6546 16771 5441 8459 18266 5000 15749 13014 14274 8444
4707 13097 15930 11872 2621 7158 6942 18502 5408 10837 21928 13800
5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 7921 1429
14398 22189 747 6439 12993 12307 19714 6650 18994 5932 18630 6683
1921 15651 5594 6958 13597 19763 10097 4065 14124 14687 1094 5780
7770 7688 15110 5797 7907 21169 3329 12627 19882 22067 19211 2061
7038 8909 16914 13129 19881 16317 1551 13235 14888 1201 20264 7800
7526 2209 5887 10223 10798 15317 11351 10105 10661 15014 2908 16412
22277 19851 12003 19616 11003 7768 6166 4620 16231 13850 7016 2717
20886 14522 5254 15519 20541 3458 1240 15787 20099 9282 11480 4994
6281 18662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143
786 6709 5954 17926 20839 22351 13277 15113 6368 7704 18694 3644
12154 3794 21257 21638 18386 18111 21498 10731 13776 5539 14530 20282
7762 9529 17675 15191 12380 14865 15825 2818 16442 5901 8220 18578
13297 2495 21913 4526 16085 10965 15558 18982 2984 11245 21040 477
11140 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800
15563 509 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341
11197 13107 13802 16006 428
534: 6383 10450 1006 8266 17986 13269 9010 9822 18387 3735 10217 533
535 1774 16191 18891 13849 14706 571 8356 3340 18461 20284 20753
10192 13836 5240 2653 20833 3564 1870 11250 6367 19001 20137 15672
13176 20207 8314 4015 1887 8795 21466 7023 18171 6876 8560 16477
13630 10793 4136 5249 17099 9152 2730 4724 8684 5821 3710 16562
22562 9500 21952 12129 1151 5863 2635 13579 5947 9543 6219 4851
7812 10216 19130 12258 1950 8843 1084 2519 12495 7141 1845 6128
9067 2618 1308 1202 16045 2887 3970 22176 2992 14189 2020 3687
18669 17082 7279 10946 10754 10583 4481 915 16903 18701 14442 11182
12710 8108 4737 8174 21990 14565 20445 20022 9861 2888 17080 6216
3441 9629 4034 4019 17401 1207 22219 1481 5143 2857 17664 21589
11961 19154 12425 21437 17489 11195 20933 21506 3533 12843 4677 18511
4730 696 9569 17797 18687 7097 20259 20966 15252 13221 11335 20777
12713 22502 14915 21157 13596 7735 6111 14062 20371 3563 12591 16308
7626 21483 4212 19798 19458 5691 18292 16130 21296 2832 830 14553
4632 6434 18295 17956 829 12043 1189 3932 18731 6845 21360 6772
19039 21673 20779 1281 16128 14568 17630 2371 4694 12396 15369 14260
13396 21621 8841 16662 11120 14081 6657 8692 9484 14065 2540 15204
6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021
6645 9286 22489 19908 893 14762 18330 10802 10479 16663 3678 16713
7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476
7333 6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488
5634 17372 2081 12643 15099 7425 15838 10094 788 3348 9280 22095
18903 3706 15256 18593 5764 11115 12583 5630 11568 13613 2331 5136
8073 15998 11304 19137 5817 4430 5580 8588 18341 12540 2454 4970
17445 2401 10889 5190 13207 16465 11291 16479 21467 4263 2705 9673
20541 3458 1240 15787 20099 9282 11480 4994 2798 5475 1814 2683
8435 4345 3627 15897 9366 6281 20716 18662 7992 9661 19875 11156
21818 3619 14086 20948 16946 3456 6143 786 6709 5954 17926 20839
13891 10965 15558 15276 3513 17600 11426 1123 16786 5326 17407 18982
2984 19817 11245 5838 21039 15310 2132 20312 4655 16568 21093 13882
5730 7976 7079 13035 2800 16353 4982 22032 3731 7388 13436 8698
509 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 15886
11197 13107 5444 16006 9573 20805 372 19913 9499 18057 7973 22459
21704 8464
535: 9382 1006 429 10217 2438 9883 314 18461 5240 6367 19001 16674
12484 526 11896 7812 9819 2857 17664 21589 11961 19154 21437 11195
20933 21506 3533 4677 18511 4730 696 9569 17797 18687 7097 20259
15252 13221 450 11335 20777 12713 22502 14915 21157 20371 3563 12591
16308 7626 4212 19798 19458 5691 18292 16130 21296 2832 830 14553
6434 18295 17956 829 476 12043 20779 1281 17745 16586 7955 12705
15449 988 20859 1768 11129 6547 22518 5552 9854 11376 6669 5359
9132 10964 1879 22087 13137 17298 3333 13752 20301 19370 2425 6946
19538 14273 15340 6427 17165 15603 12419 10809 8003 13658 14064 4221
7387 13284 20981 2316 3116 8418 3386 6508 18217 3578 22249 992
14855 13442 3471 19949 5277 19885 2816 10917 9436 18538 18659 12816
12308 15943 2515 20364 14701 8193 17579 22076 8890 2649 9329 21073
8376 15177 10882 859 5990 9814 16053 5209 3144 13848 22428 19076
5454 17056 14918 4088 11270 5837 9604 22455 19840 12234 1149 801
953 9960 21301 1656 3755 12278 22119 21098 1005 10264 13037 11702
20954 5581 5063 1118 6454 20632 1696 19474 14911 9381 4111 13374
2932 3071 18551 22362 1431 12923 6509 19229 14012 5372 12362 17380
20272 16391 13395 5132 901 9540 19228 14568 17630 2371 4694 12396
15369 16597 3697 10741 16309 4927 13396 21621 8841 16662 11120 14081
8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813
7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969
14762 18330 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472
10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644
835 10655 12264 9315 2786 16253 9488 5634 17372 788 9280 22095
18903 3706 15256 18593 5764 11115 12583 11568 13613 2331 5136 8073
15998 5630 11304 19137 5817 5580 8588 18341 12540 2454 4970 17445
2401 11869 6193 21516 10889 5190 13207 16465 9673 9771 11716 18575
9584 12794 21399 20485 9218 18691 3350 18263 4846 544 19667 9933
4140 1318 20418 11128 20105 16734 2376 15699 7061 4232 15357 14036
5339 7107 19030 7165 21370 12103 4848 13211 22530 15360 12863 9975
6398 14067 16683 21170 1924 890 8155 15866 10131 7187 4332 1235
20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 2282 15956
3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 13940 18821
14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 15749 13014
14274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 10837
21928 13800 5188 19614 16117 4719 1833 13499 3107 21492 12503 21929
7921 1429 14398 22189 6439 12993 12307 19714 6650 18994 747 5932
18630 6683 1921 15651 5594 6958 13597 19763 10097 14124 14687 1094
5780 7770 7688 15110 5797 7907 21169 3329 19882 12627 4065 22067
19211 2061 7038 8909 16914 13129 19881 16317 1551 13235 14888 1201
20264 7800 7526 2209 5887 10798 15317 10223 11351 10105 10661 15014
2908 16412 22277 19851 12003 19616 11003 7768 6166 4620 13850 16231
7016 2717 20541 3458 1240 15787 20099 9282 11480 4994 6281 18662
7992 9661 19875 11156 3619 14086 20948 16946 3456 6143 786 6709
5954 17926 20839 22351 13277 15113 6368 7704 18694 3644 12154 3794
21257 21638 18386 18111 21498 10731 13776 5539 14530 20282 7762 9529
17675 15191 12380 14865 15825 2818 16442 5901 8220 18578 13297 2495
21913 4526 16085 10965 15558 18982 2984 11245 16474 304 477 427
5067 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800
20416 509 307 305 12545 2037 11704 9571 21379 3712 7685 4483
18456 22341 11197 13107 13268 16006 428
536: 8250 5293 17519 10969 8775 16532 10286 11811 5689 2871 680 20415
14104 16888 17043 10273 6777 22396 602 4866 12333 9909 22327 4344
9005 16476 2573 10616 19638 15657 8950 4378 2657 3364 21708 985
6316 8776 2238 13856 1099 10466 19656 6879 9313 19604 12069 8122
21385 16087 20487 1424 14078 2427 4175 7418 9385 16892 15809 3499
16714 5768 6322 13627 19469 8252 19551 14976 12742 10499 11322 9597
3569 9336 20132 5283 22127 10381 3149 7067 13886 7478 3897 20869
19398 13086 4191 3669 20800 6799 8453 17539 21956 5724 13878 16687
1797
537: 12225 7991 19769 20707 6346 21228 18920 15677 22007 17216 1246 1299
3482 8428 4864 1869 18259 5076 18894 10621 1194 21241 4280 10372
9939 3461 22101 14388 9687 1934 3266 19426 6888 17781 1697 1251
15687 11467 11444 7248 4960 3598 18026 17460 7270 18737 20522 18581
16569 13403 4999 20923 3253 12847 6541 12065 13811 442 18246 10039
21176 20831 13532 19077 10287 9635 12724 898 43471 1482 12691 8702
12055 8006 14571 19675 16542 21229 14891 11459 15395 15872 9301 10051
6843 5509 11707 10865 18908 12031 9844 20469 4041 4623 22346 1849
12095 14281 3840 16733 15322 21786 17305 10611 1428 7631 22045 6676
5894 8015 21902 14896 17330 3090 15122 7759 6196 16678 475 20224
3032 21119 21396 1165 20759 22152 18157 1337 3062 12222 10847 14596
3679 21087 17626 3743 10092 21220 22333 4831 6340 2684 14394 17353
12317 2073 22147 10166 9664 11412 21185 7389 11862 11526 9220 2983
5429 13258 10529 12270 6868 5672 2355 11711 9314 17713 6624 12386
17898 3480 10028 20275 1292 12254 17594 20642 4853 10383 4591 8186
16379 15333 9201 15765 17720 5294 9934 7376 4038 2297 1916 7144
16736 10304 3319 3762 20614 11341 10440 4734 9677 12289 20772 4271
19954 22175 3020 19748 16083 8948 19737 22114 2955 21137 3274 1408
6780 10848 7124 14060 11449 17939 4794 16058 21761 10251 4961 19141
1661 9701 6223 5983 18048 10670 17196 1709 4904 12196 630 8403
8582 6184 18819 3380 9202 16022 17350 12703 15237 17792 4597 11159
1072 20441 15476 15612 17341 12261 21044 4892 1108 1406 22046 18198
6967 20318 12469 22123 7645 10452 1264 712 14602 6109 14712 22157
7745 16695 11050 22271 21574
538: 8633 21066 20486 15659 19893 20341 18625 7202 9210 15561 8243 19452
10486 9599 20104 5911 17836 3744 3313 22371 13393 8083 14598 8878
4121 11696 16024 6949 9760 18073 21021 1691 3955 13639 14076 5677
2739 10860 20215 14300 10639 13585 11393 3392 7331 2940 4631 3646
14037 5709 8396 7169 3234 9515 13640 21141 9848 13213 16854 10018
17683 4171 20607 15041 2557 19483 17558 19443 20760 13774 16065 5097
13747 713 8505 18788 18352 817 5302 8167 10335 22171 13497 18845
11474 8093 22332 17185 18392 15387 7019 17613 13932 22281 9232 21810
19046 14214 6221 10362 21188 12117 2909 9179 16519 21842 14950 5628
5467 18729 7240 8434 11000 2806 17205 1863 9645 18886 6782 20838
7708 6070 7788 22286 19842 16891 9527 18008 7791 11889 5849 2006
3823 12758 17857 15410 11552 11047 5550 18384 903 18870 12346 18758
1298 11723 11231 13187 11334 10943 11898 11754 4492 6688 19935 13184
13651 1487 20841 6884 6464 8803 16050 6362 1430 1363 10992 6297
3031 2620 18570 22021 21438 13674 22415 1440 6599 17348 16403 2064
5725 621 12956 13371 3689 813 8410 17547 5880 7069 7272 13965
9296 18273 16456 7801 2112 17834 8758 7378 19838 6388 10579 19671
16279 806 14835 2530 4277 795 12076 5622 13454 3935 4559 18529
18155 19209 15646 5247 6539 16369 5499 21300 14158 12126 17163 16184
993 21342 16623 17172 10687 11363 1456 21186 21282 21603 10623 6475
17459 18745 21602 9996 3042 16551 1505 5224 5300 3833 4119 10925
11838 21509 15409 13868 11753 16835 13881 7206 18880 6510 4201 3942
968 5155 13073 10765 5458 12673 3180 3108 15770 908 21482 5413
18381 14279 4351 15582 22158 16937 12412 13635 14711 5969 9888 21962
11801 16194 10513 4703 11781 15593 17383 18258 9184 7699 9109 1971
18780 3171 11419 10405 631 15583 21710 7483 5744 22254 12810 13894
20089 6909 5948 3120 15669 17412 19244 2016 16850 22002 7260 14202
3293 4461 2271 14582 18598 17872 1569 18470 5671 8965 4082 7327
22511 15167 1160 15734 5450 19432 14387 14587 3176 8359 18160 11357
15085 6948 5337 2567 10321 22182 10881 10168 18477 12537 19015 20260
2170 18422 2440 3225 5484 1260 1822 7532 19248 7693 4353 10441
2660 5734 10038 19976 3453 19019 1674 12312 16581 10232 3727 1523
2140 22432 20580 9947 19324 20180 1384 2325 18901 10198 20110 9744
9208 3182 14144 18875 14329 929 22054 2752 4524 986 977 21117
9567 9679 12352 20957 4772 12912 19652 11510 5608 21827 13765 8183
14331 13670 20503 12206 21116 22320 844 2226 21795 13733 11051 20995
2615 7715 4478 2290 12549 15082 2491 2301 14182 15076 1227 10071
3792 13186 4722 1595 10880 11269 8528 3213 14537 4257 8761 17108
4834 2651 19705 3355 7552 6681
Example 3 Consensus Sequence Build ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 379 and 10 homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. Attached are the sequences of SEQ ID NO: 379, its homologs and the consensus sequence SEQ ID NO: 22569 at the end. The symbols for consensus sequence are (1) uppercase letters for 100% identity in all positions of multiple sequence alignment output; (2) lowercase letters for >=70% identity; symbol; (3) “X” indicated <70% identity; (4) dashes “-” meaning that gaps were in >=70% sequences.
SEQ ID NO
2406 --------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQQQQE-
15414 --------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQHPQE-
587 ------------------------------------MMGRVMEKPSQDLLQQQQQ-----
9696 ------------------------------------MMGRVMEKPSQDLLQQQQQ-----
5895 ------------------------------------MMGRVMEKPSQDLLQQQQQ-----
17251 MFGNGNCDVDNEKTIITSSKWTQSEIDDHKVSMASSTGNRVMEKPGQELLQQQQQ-----
19549 -----------------------------------------MEKQGQELLQQHHQQQQQQ
379 -----------------------------------MQSKNMIVASSHQQQQQQQPQQPQP
21357 --------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTQPNSE
17711 --------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTQPNSE
13715 -----------------------------------LTLTKCCMQRGSHFRSRSGSQEARS
consensus --------------------------xxxxxxxxxxxxxxxxxxxxqxxxqqqqxxxxxx
22569
APKCPRCDSSNTKFCYYNNYSLSQPRHECKACKRYWTRGGTLRNVPVGGGCRRNKRVKRP
APKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRRNKRVERP
ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP
ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP
ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP
ALRCPRCDSSNTKFCYYNNYSLTQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRLKRP
ALKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGYRRNNKRSTS
QLKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGSYRKNKRVKRP
SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVKKS
SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVRKS
GSSMSRCNSMDTKFCYYNNYNVNQPRHFCKNCQRYWTAGGSMRNVPVGAGRRKNKHTGSV
xlkcpRCxSsnTKFCYYNNYslsQPRHFCkaCkRyWTrGGtlRNVPVGggXRkNkrvxxx
LITTNPSSAAIDTAASNNSSN-SSSAPLQPPIDTASTS--------------NHINPLFY
TTSPCSAAIDTASNSSNSSSAPTAAASLQPQIDTASTS--------------NHTNPLFY
TNHGDSSSSAANSPSSSNSNPPSQPHLDNIIASSSTTN------------HINNISPFFY
TNHGDSSSSAANSPSSSNSNPPSQPHTDNIIASSSTTN------------HINNTSPFFY
TNHGDSSSSAANSPSSSNSNPPSQPHIDNIIASSSTTN------------HINNISPFFY
TYPCSNNNNIDFSASPSSSTPSSVVANPNPPSQSQQQQQQQQHHSFDIAATSNHINTMLY
SSNGPTSTTTLIKRPISTIETATTSNSSSPSSTHSSTS--------------NHMNPMEY
------STATTTTASTVSTTNSSSPNNPHQISHFSSMN----------------HHPLFY
TTPTTTSSTTTTPITTATSTCTATVTTSIGNNNNNMDAMLG----------CYSHMTIQT
ITTPITTSSTTTHQSQPPLQLALPQSQPQLATTTTTWMLCW----------VVTAT----
YRHTVITPDSLASLQVDGPDLVDHKPLSPEKVNGTILKFG--------------PDAPLC
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx----------xxxxxxxxxy
GLPSSS-SDVNLPLFSRFGSRISSS----GFDLQLNNALGLGFSSGVLSNEASDNNGYR-
GLPSSS-SDVNLPLFSRFGSRISSS----GFDLQLNNALGLGFSSRVLSNEASDNNRYR-
GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN--
GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN--
GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN--
GGNSCH-DVMNFPFSTRFNSTTRVSNPASGYDNLPQNGLGLGFSSGILMSAAGGEVNLNH
GLSSTNNPCDPNLPFSRENITSRLSTSSGYDLQPQMNFFGLGFSSGFENNGYTNGFNTS-
GLSDHMSSCNNNLPMIPSRFSDSSK------------TCSSSGLESEFLSSGFSSLSALG
PLADDQKNMSSSLYQALIRPPPLLLQQQNLLNTRELEGKDFGIGIGNGNNGTFPSSTLAL
------------------------------------------------------------
ESMASILNLGEQNLSSQLDFTAGAE-----------NREETSCSSACKPVKKKDITQHN-
gxxxxxxxxxxxxxxxxxxxxxxxx----xxxxxxxxxxxxgxsxxxxxxxxxxxxxxx-
-------------NWFGSNNTLLSSYTSTTSTTTPAMSSLLSSSLLQQKFMTDGVD----
-------------SGEGSNNMLLSSYTSTT-TTTPAMSSLLLQQKFISGGLKNDAD----
------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSSTLLQQNLMGGGG---GG
------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSCTLLQQNFMGGGG---GG
------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSSTLLQQKLMSGGG---GG
HHHHHHDEGSYRNGFSTSNNNNYSSIFGSSSTTTPVMASLLSSTLLQQKFMGTGGGIKGG
----------------NNNYDSIFSSSTSASNNTSVMPSVLSSTLLQHKFFDDGLK----
--------LGLPHQMSHDHTINGSFINNSTTNKPFLLSGLFGSSMSSSSTLLQHP-----
P-------------IPHQSQSLLFPFSASSRSFDTNPCSVVSTSLRSSNVYNYGED----
------------------------------------------------------------
------------------------------------------------------------
------------xxxxxxxxxxxxxxxxxxxxxxxxxxsxxxxxxxxxxxxxxxxx--xx
--------STNTFQHGLGLTPLEQLQMASDHSSEAGMVALKDVKVELGQNNRLEWNNGAA
--------SSNTPQHGLSLTSLEQLQIASDHSSEAGMVALKDVKVELGQNNNRLEWNGGA
VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRPEWSNNNNNNNNNGGG
VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRFEWSNNNNNNNNNGGG
EGEEVVIMIKVATLSMAWHRYKGCKWKGTIIIVTILAQKK--------------------
GGGGGGDDDPFHHHQEMDSKEVKLGEGLQNRLDQWNMNNLNGNGGAVFQNQMENMGLSDN
----------YGSDAGSNGAFQDLQFGSKMQNQMEHIGGFYDPASSIYLNATSSSAIGVW
----------HKPMNNGGDMLGQSHLQTLASLQDLHVGGNNEDMKYKEGKLDQISGNING
---------QFKAIEEPTTNSTTATIVPSTGGTNNTHHPWEIAAATSGVGLGTSSNSNYW
------------------------------------------------------------
------------------------------------------------------------
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
CQSQIQHVGLYDPSLYWNNSAATALGVWNDQAANIGSSVTSLI
FQSQIQHVGLYDPLLYWNN-SATALGVWNDQAANIGSSVTSLI
GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPNNIGPSVTSLI
GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPN-IGPSVTSLI
-------------------------------------------
NASLYWNNNHNNSNNNTSA-TATGLSSVWSTDQPGSNSVSSLI
NDQGANNTGSSVTSLI---------------------------
FMSSSSSLDPSNYNNMWNNASVVNGAWLDPTNNNVGSSLTSLI
NWEDFDSLVSTDLKDPWDDSDIKP-------------------
-------------------------------------------
-------------------------------------------
xxxxxxxxxxxxxxxxxxx-xxxxxxxxxxxxxxxxxxxxxxx
Example 4 Corn Transformation Construct GATEWAY™ destination vectors (available from Invitrogen Life Technologies, Carlsbad, Calif.) are constructed for insertion of trait-improving DNA for corn transformation. The elements of each destination vector are summarized in Table 18 below and include a selectable marker transcription region and a DNA insertion transcription region. The selectable marker transcription region comprises a Cauliflower Mosaic Virus 35S promoter operably linked to a gene encoding neomycin phosphotransferase II (nptII) followed by both the 3′ region of the Agrobacterium tumefaciens nopaline synthase gene (nos) and the 3′ region of the potato proteinase inhibitor II (pinII) gene. The DNA insertion transcription region comprises a rice actin 1 promoter, a rice actin 1 exon 1 intron1 enhancer, an att-flanked insertion site and the 3′ region of the potato pinII gene. Following standard procedures provided by Invitrogen the att-flanked insertion region is replaced by recombination with trait-improving DNA, in a sense orientation for expression of a trait-improving protein and in a gene suppression orientation (i.e., either anti-sense orientation or in a sense- and anti-sense orientation) for a trait-improving suppression of a protein. Although the vector with trait-improving DNA inserted at the att-flanked insertion region is useful for plant transformation by direct DNA delivery, such as microprojectile bombardment, it is preferable to bombard target plant tissue with tandem transcription units that have been cut from the vector. For Agrobacterium-mediated transformation of plants the vector also comprises T-DNA borders from Agrobacterium flanking the transcription units.
Vectors for Agrobacterium-mediated transformation are prepared with each of the trait-improving genes having a sequence of SEQ ID NO:1 through SEQ ID NO:269 with the DNA solely in sense orientation for expression of the encoded, cognate trait-improving protein and in a gene suppression orientation for suppression of the cognate protein. Each vector is transformed into corn callus which is propagated into a plant that is grown to produce transgenic seed for each transgenic event. Progeny plants are self-pollinated to produce seed which is selected for homozygous seed. Homozygous seed is used for producing inbred plants, for introgressing the trait into elite lines, and for crossing to make hybrid seed. The progeny transgenic plants comprising the trait-improving DNA with a sequence of SEQ ID NO: 1 through SEQ ID NO: 269 have one or more improved traits identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic corn including inbred and hybrids are also produced with DNA from each of the identified homologs of DNA of SEQ ID NO: 1 through SEQ ID NO: 269 to provide transgenic seeds and plants which are identified from total transgenic events by screening for the improved agronomic trait. Transgenic corn plants are also produced where the trait-improving DNA is transcribed by each of the promoters from the group selected from, a maize globulin 1 promoter, a maize oleosin promoter, a glutelin 1 promoter, an aldolase promoter, a zein Z27 promoter, a pyruvate orthophosphate dikinase (PPDK) promoter, a soybean 7S alpha promoter, a peroxiredoxin antioxidant (Perl) promoter and a CaMV 35S promoter.
Seed produced by the plants is provided to growers to enable production of corn crops with improved traits associated with the trait-improving DNA. TABLE 18
FUNCTION ELEMENT REFERENCE
DNA insertion Rice actin 1 promoter U.S. Pat. No. 5,641,876
transcription region Rice actin 1 exon 1, intron 1 U.S. Pat. No. 5,641,876
enhancer
DNA insertion AttR1 GATEWAY ™ Cloning Technology
transcription region Instruction Manual
(att -flanked insertin CmR gene GATEWAY ™ Cloning Technology
region) Instruction Manual
ccdA, ccdB genes GATEWAY ™ Cloning Technology
Instruction Manual
attR2 GATEWAY ™ Cloning Technology
Instruction Manual
DNA insertion Potato pinII 3′ region An et al., (1989) Plant Cell 1: 115-122
transcription region
selectable marker CaMV 35S promoter U.S. Pat. No. 5,858,742
transcription region nptII selectable marker U.S. Pat. No. 5,858,742
nos 3region U.S. Pat. No. 5,858,742
PinII 3′ region An et al., (1989) Plant Cell 1: 115-122
E. coli maintenance ColE1 origin of replication
region F1 origin of replication
Bla ampicillin resistance
Example 5 Soybean Transformation Construct Constructs for use in transformation of soybean are prepared by restriction enzyme based cloning into a common expression vector. Elements of an exemplary common expression vector are shown in Table 19 below and include a selectable marker expression cassette and a gene of interest expression cassette. The selectable marker expression cassette comprises Arabidopsis act 7 gene (AtAct7) promoter with intron and 5′UTR, the transit peptide of Arabidopsis EPSPS, the synthetic CP4 coding region with dicot preferred codon usage and a 3′ UTR of the nopaline synthase gene.
The gene of interest expression cassette comprises a Cauliflower Mosaic Virus 35S promoter operably linked to a trait-improving gene in a sense orientation for expression of a trait-improving protein and in a gene suppression orientation (i.e., either anti-sense orientation or in a sense- and anti-sense orientation for a trait-improving suppression of a protein.
Vectors similar to that described above are constructed for use in Agrobacterium mediated soybean transformation systems, with each of the trait-improving DNA having a sequence of SEQ ID NO:1 though SEQ ID NO:269 and the respective identified homologs with the DNA in sense orientation for expression of the encoded, cognate protein and in a gene suppression arrangement for suppression of the cognate protein. Each vector is transformed into soybean embryo tissue to produce transgenic events which are grown into plants that produce progeny transgenic plants and seed for screening to identify the transgenic soybean plants of this invention that exhibit the enhanced agronomic trait imparted by DNA with a sequence of SEQ ID NO:1 through SEQ ID NO:269 or a respective homolog. The transgenic soybean plants of this invention are identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic soybean plants are also produced where the trait-improving DNA is transcribed by a napin promoter and Arabidopsis SSU promoter.
Seed produced by the plants is provided to growers to enable production of soybean crops with improved traits associated with the trait-improving DNA. TABLE 19
Function Element Reference
Agro transformation B-ARGtu.right border Depicker, A. et al (1982) Mol
Appl Genet 1: 561-573
Antibiotic resistance CR-Ec.aadA-SPC/STR
Repressor of primers from the ColE1 plasmid CR-Ec.rop
Origin of replication OR-Ec.oriV-RK2
Agro transformation B-ARGtu.left border Barker, R. F. et al (1983) Plant
Mol Biol 2: 335-350
Plant selectable marker expression cassette Arabidopsis act 7 gene McDowell et al., (1996) Plant
(AtAct7) promoter with Physiol. 111: 699-711.
intron and 5′UTR
5′ UTR of Arabidopsis act 7 gene
Intron in 5′UTR of AtAct7
Transit peptide region of Klee, H. J. et at (1987) MGG
Arabidopsis EPSPS 210: 437-442
Synthetic CP4 coding region with
dicot preferred codon usage
A 3′ UTR of the nopaline synthase U.S. Pat. No. 5,858,742
gene of Agrobacterium
tumefaciens Ti plasmid
Plant gene of interest expression cassette Promoter for 35S RNA from U.S. Pat. No. 5,322,938
CaMV containing a duplication of
the −90 to −350 region
Gene of interest insertion site
Cotton E6 3′ end GenBank accession U30508
Example 6 Cotton Transformation Vectors similar to that described above for soybean transformation are constructed for use in Agrobacterium mediated cotton transformation systems, with each of the trait-improving DNA having a sequence of SEQ ID NO:1 though SEQ ID NO:269 and the respective identified homologs with the DNA in sense orientation for expression of the encoded, cognate protein and in a gene suppression arrangement for suppression of the cognate protein. Each vector is transformed into cotton embryo tissue to produce transgenic events which are grown into plants that produce progeny transgenic plants and seed for screening to identify the transgenic soybean plants of this invention that exhibit the enhanced agronomic trait imparted by DNA with a sequence of SEQ ID NO:1 through SEQ ID NO:269 or a respective homolog. The transgenic cotton plants of this invention are identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic cotton plants are also produced where the trait-improving DNA is transcribed by a napin promoter and Arabidopsis SSU promoter.
Seed produced by the plants is provided to growers to enable production of cotton crops with improved traits associated with the trait-improving DNA.
