CROSS REFERENCE TO RELATED APPLICATIONS This application is a division of U.S. application Ser. No. 14/998,939, filed Mar. 8, 2016, which application is a continuation of U.S. application Ser. No. 13/914,701, filed Jun. 11, 2013, which application is a continuation of U.S. application Ser. No. 13/372,542, filed Feb. 14, 2012 (now abandoned), which application is a continuation of U.S. application Ser. No. 12/001,025, filed Dec. 6, 2007 (now abandoned), which application claims benefit of priority under 35 USC § 119(e) of U.S. provisional application Ser. No. 60/873,247, filed Dec. 6, 2006, herein incorporated by reference in their entireties.
INCORPORATION OF SEQUENCE LISTING Two copies of the sequence listing (Copy 1 Sep. 12, 2018 and Copy 2 Sep. 12, 2018) and a computer readable form (CRF Sep. 12, 2018) of the sequence listing, all on CD-Rs, each containing the file named 3126005US5.TXT, which is 67,123,200 bytes (measured in MS-WINDOWS) and was created on Sep. 12, 2018, are incorporated herein by reference in their entirety.
INCORPORATION OF COMPUTER PROGRAM LISTING Two copies of Computer Program Listing (Copy 1 Sep. 12, 2018 and Copy 2 Sep. 12, 2018) containing folders “hmmer-2.3.2” and “161pfamDir” are provided on separate CD-ROMs and are incorporated herein by reference in their entirety. Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis. Folder 161pfamDir contains 161 profile Hidden Markov Models. Both folders were created on the disk on Sep. 12, 2018 having a total size of 15,534,080 bytes when measured in MS-WINDOWS® operating system.
FIELD OF THE INVENTION Disclosed herein are transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds
BACKGROUND OF THE INVENTION Transgenic plants with enhanced 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 useful recombinant DNA for production of transformed plants with improved properties, e.g. by actually selecting a transgenic plant from a screen for such improved property. An object of this invention is to provide transgenic plant cell nuclei, plant cells, plants and seeds by screening transgenic crop plants for one of more enhanced agronomic traits where the nucleus in cells of the plant or seed has recombinant DNA that was identified as imparting an improved trait in a model plant, e.g. Arabidopsis thaliana. In some cases the model plant may exhibit an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like. In other cases the model plant may exhibit an improved trait that is a surrogate to an enhanced agronomic trait, e.g. salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield. A further object of the invention is to provide screening methods requiring routine experimentation by which such transgenic plant cell nuclei, cells, plants and seeds can be identified by making a reasonable number of transgenic events and engaging in screening identified in this specification and illustrated in the examples.
SUMMARY OF THE INVENTION This invention provides plant cell nuclei with recombinant DNA that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells. Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17. In more specific embodiments of the invention plant cells are provided which express a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table 2. Amino acid sequences of homologs are SEQ ID NO: 395 through 19,938. In even more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1 by annotation to a related protein in Genbank and alternatively identified in Table 16 by identification of protein domain family.
Other aspects of the invention are specifically directed to transgenic plant cells, and transgenic plants comprising a plurality of plant cells with such nuclei, progeny transgenic seed, embryo and transgenic pollen from such plants. Such plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus transformed with recombinant DNA by screening the transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA in their nucleus, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance to salt exposure, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil. In some aspects of the invention the recombinant DNA expresses a protein that imparts the enhanced trait; in other aspects of the invention the recombinant DNA expresses RNA for suppressing the level of an endogenous protein. In yet another aspect of the invention the nucleus of plant cells in plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention. In aspects of the invention the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.
Yet other aspects of the invention provide nuclei in cells of transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants.
In other embodiments for practice of various aspects of the invention in Argentina the recombinant DNA in the nucleus is provided in plant cells derived from corn lines that are and maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or to both.
This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA in the nucleus of the plant cells. In some aspects of the invention the recombinant DNA can express a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 17; in other aspects the recombinant DNA suppresses the level of such a protein. More specifically the method comprises (a) screening a population of plants for an enhanced trait and recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA; (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants; (c) verifying that the recombinant DNA is stably integrated in said selected plants; (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO: 1-197; and (e) collecting seed from a selected plant. In one aspect of the invention the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and where the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed selected as having at least one of the enhanced traits described above.
Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has a nucleus of this invention with stably-integrated, recombinant DNA The method further comprises producing corn plants from said hybrid corn seed, where a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.
Another aspect of the invention provides a method of selecting a plant comprising a nucleus of this invention in its plant cells by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a nucleus of this invention with unique recombinant DNA.
Aspects of the invention relating to nucleus in plant cells having recombinant DNA for suppressing the expression of a protein are identified in Table 1 and Table 16. More specific aspects of the invention provide plant cells having recombinant DNA for suppressing the expression of a protein having the function in a plant of the protein with amino acid sequence of SEQ ID NO: 200, 201, 205, 207, 211 and 394 or the corresponding Pfam identified in Table 16, i.e. SNF5, LMBR1, TFIIS_M, TFIIS_C, Glyco_transf_8, respectively. Such suppression can be effected by any of a number of ways known in the art, e.g. anti-sense suppression, RNAi or mutation knockout and the like.
Another aspect of this invention relates to growing transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency. Alternatively methods comprise applying reduced amount of nitrogen input as compared to the conventional input during the production of a corn crop.
The various aspects of this invention are especially useful for transgenic plant cells in seeds and transgenic plants having any of the above-described enhanced traits in crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
The invention also provides recombinant DNA constructs comprising the DNA useful in the nuclei in plant cells for imparting enhanced traits in plants having those cells.
BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1AA illustrate a consensus amino acid sequence of SEQ ID NO: 227 and its homologs.
FIGS. 2, 3 and 4 illustrate plasmid maps.
DETAILED DESCRIPTION OF THE INVENTION In the attached sequence listing:
SEQ ID NO: 1-197 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;
SEQ ID NO: 198-394 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-197;
SEQ ID NO: 395-19938 are amino acid sequences of homologous proteins;
SEQ ID NO: 19939 is a consensus amino acid sequence.
SEQ ID NO: 19940 is a nucleotide sequence of a plasmid base vector useful for corn transformation; and
SEQ ID NO: 19941 is a DNA sequence of a plasmid base vector useful for soybean or canola transformation.
SEQ ID NO: 19942 is a DNA sequence of a plasmid base vector useful for cotton transformation.
The nuclei of this invention are identified by screening transgenic plants for one or more traits including 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.
“Gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequences involved in the regulation of expression. In aspects of the invention where an improved trait is provided by expression of a protein, “gene” refers at least to coding nucleotide sequence for a protein or a functional polypeptide fragment of a protein that imparts the trait. In aspects of the invention where an improved trait is provided by suppression of expression of an endogenous protein, “gene” refers to any part of the gene that can be a target for suppression.
“Transgenic seed” means a plant seed whose nucleus has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein. The term “transgenic plant” is used to refer to the 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 a nucleus with the recombinant DNA in its genome.
“Recombinant DNA” means a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous 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. A recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or 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, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as 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.
A “control plant” is a plant without trait-improving recombinant DNA in its nucleus. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. A suitable control plant may be a nontransgenic plant of the parental line used to generate a transgenic plant herein. Alternatively, a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement. A control plant may also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, 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. As use herein a “reference” is 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, e.g., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.
“Trait enhancement” 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 enhancement can be 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 enhancement is only measured qualitatively. It is known that there can be a natural variation in a trait. Therefore, the trait enhancement 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 enhancement includes, but is 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 enhanced plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
“Yield-limiting environment” means the 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 affect 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” used herein preferably refers to the sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) can be used as a physiological measure of plant water deficit. It measures the effect of osmotic adjustment in plant water status, when a plant is under stress conditions. Conditions that may result in water deficit stress include heat, drought, high salinity and PEG induced osmotic stress.
“Cold stress” means the exposure of a plant to a temperature below (two or more degrees Celsius below) those normal for a particular species or particular strain of plant.
“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. The term ammonium as used herein 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.
“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. For example, a low nitrogen condition can refer to a growth condition with 50% or less of the conventional nitrogen inputs. “Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives optimal 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. One skilled in the art would recognize what constitute such soil, media and fertilizer inputs for most plant species.
“Shade stress” means a growth condition that has 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 may become exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few 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 (Cheikh, e.g., (2003) U.S. Patent Application No. 20030101479).
“Increased yield” of a transgenic plant of the present 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, maize yield can be 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 can result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved tolerance to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA can also be 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 may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology. Expression also refers to production of encoded protein from mRNA.
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 Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions. As used herein, “antisense orientation” includes reference to a polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
As used herein, “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
A “consensus sequence” refers to an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.
Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes may be generated by the event of speciation (see ortholog) or by the event of genetic duplication (see paralog). “Orthologs” refer to a set of homologous genes in different species that evolved from a common ancestral gene by specification. Normally, orthologs retain the same function in the course of evolution; and “paralogs” refer to a set of homologous genes in the same species that have diverged from each other as a consequence of genetic duplication. Thus, homologous genes can be from the same or a different organism. As used herein, “homolog” means a protein that performs the same biological function as a second protein including those identified by sequence identity search.
Percent identity refers to 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 a test sequence and a reference sequence is the number of identical components which are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “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.
“Pfam” refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 protein families and is based on the Swissprot 47.0 and SP-TREMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam is currently maintained and updated by a Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the Pfam alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low. Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins in the same protein family are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Model which characterizes the Pfam domain using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the gathering cutoff for the alignment of a particular Pfam are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein in a common Pfam for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. The HMMER software and Pfam databases are version 18.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 198 through SEQ ID NO: 394. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 17 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfams for use in this invention, as more specifically disclosed below, are L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RrnaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1_30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE 1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1_49 kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS_1, Thioredoxin, DUF1005, LEA_3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN_2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding_1, Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT_2, GDC-P, zf-CCHC, NPH3, TPR_1, TFIIA, DHBP_synthase, UQ_con, TPR_2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf_11, CBM_20, Methyltransf_12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf_8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch_1, PGAM, Aminotran_1_2, Kelch_2, UPF0261, CoA_binding, DUF868, Peptidase_S10, Lung_7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM_1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran_3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin_2, LRR_1, Cupin_3, zf-CSL, FAR1, HD, FH2, APC8, PTR2, MannoseP_isomer, Rib_5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp, Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH_11, Tim 17, and PBD.
Recombinant DNA Constructs The present invention provides recombinant DNA constructs comprising one or more polynucleotides disclosed herein for imparting one or more improved traits to transgenic plant when incorporated into the nucleus of the plant cells. Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells. Other construct components may include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides. Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.
In a preferred embodiment, a polynucleotide of the present invention is operatively linked in a recombinant DNA construct to a promoter functional in a plant to provide for expression of the polynucleotide in the sense orientation such that a desired protein or polypeptide fragment of a protein is produced. Also provided are embodiments wherein a polynucleotide is operatively linked to a promoter functional in a plant to provide for expression of gene suppression RNA to suppress the level of an endogenous protein.
Recombinant constructs prepared in accordance with the present invention also generally include a 3′ untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3′ UTRs include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens.
Constructs and vectors may 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. Nos. 5,188,642 and 5,728,925, incorporated herein by reference.
Table 1 provides a list of genes that provided recombinant DNA that was expressed in a model plant and identified from screening as imparting an improved trait. When the stated orientation is “sense”, the expression of the gene or a homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. When the stated orientation is “antisense”, the suppression of the native homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. In some cases the expression/suppression in the model plant exhibited an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like. In other cases the expression/suppression in the model plant exhibited an improved trait that is a surrogate to an enhance agronomic trait, e.g. salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield. Even when expression of a transgene or suppression of a native gene imparts an improved trait in a model plant, not every crop plant expressing the same transgene or suppressing the same native gene will necessarily demonstrate an indicated enhanced agronomic trait. For instance, it is well known that multiple transgenic events are required to identify a transgenic plant that can exhibit an enhanced agronomic trait. However, by with routine experimentation a transgenic plant cell nuclei, cell, plant or seed of this invention can be identified by making a reasonable number of transgenic events and engaging in screening process identified in this specification and illustrated in the examples. An understanding of Table 1 is facilitated by the following description of the headings:
“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 ID” 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.
TABLE 1
NUC PEP
Seq SEQ
ID ID Construct
No. No Gene ID ID Orientation
1 198 CGPG106 11029 SENSE
2 199 CGPG1133 12223 SENSE
3 200 CGPG117 10422 ANTI-SENSE
4 201 CGPG1226 13485 ANTI-SENSE
5 202 CGPG1288 13235 SENSE
6 203 CGPG1301 13411 SENSE
7 204 CGPG1458 73944 SENSE
8 205 CGPG1542 13846 ANTI-SENSE
9 206 CGPG170 12602 SENSE
10 207 CGPG1828 74065 ANTI-SENSE
11 208 CGPG2206 72783 SENSE
12 209 CGPG2217 17210 SENSE
13 210 CGPG2292 72724 SENSE
14 211 CGPG2457 17805 ANTI-SENSE
15 212 CGPG2499 16610 SENSE
16 213 CGPG2653 76602 SENSE
17 214 CGPG2813 18456 SENSE
18 215 CGPG3002 18414 SENSE
19 216 CGPG3154 71538 SENSE
20 217 CGPG3235 76532 SENSE
21 218 CGPG3274 18231 SENSE
22 219 CGPG3275 18232 SENSE
23 220 CGPG3363 18256 SENSE
24 221 CGPG3367 18258 SENSE
25 222 CGPG3375 19193 SENSE
26 223 CGPG3528 71301 SENSE
27 224 CGPG3534 18354 SENSE
28 225 CGPG3638 77334 SENSE
29 226 CGPG3918 19767 SENSE
30 227 CGPG3920 19774 SENSE
31 228 CGPG3962 70992 SENSE
32 229 CGPG3972 19956 SENSE
33 230 CGPG3990 70948 SENSE
34 231 CGPG3994 70201 SENSE
35 232 CGPG4026 19973 SENSE
36 233 CGPG4048 70987 SENSE
37 234 CGPG4052 70950 SENSE
38 235 CGPG4057 70962 SENSE
39 236 CGPG4058 70915 SENSE
40 237 CGPG4069 19947 SENSE
41 238 CGPG4087 70969 SENSE
42 239 CGPG4088 70985 SENSE
43 240 CGPG4102 70971 SENSE
44 241 CGPG4121 70963 SENSE
45 242 CGPG4122 70994 SENSE
46 243 CGPG4140 70956 SENSE
47 244 CGPG4154 70995 SENSE
48 245 CGPG4311 73306 SENSE
49 246 CGPG4363 70657 SENSE
50 247 CGPG4369 70660 SENSE
51 248 CGPG442 74536 SENSE
52 249 CGPG4454 71328 SENSE
53 250 CGPG4456 71329 SENSE
54 251 CGPG4473 70755 SENSE
55 252 CGPG4588 70684 SENSE
56 253 CGPG4765 73330 SENSE
57 254 CGPG4788 76202 SENSE
58 255 CGPG4912 72807 SENSE
59 256 CGPG4926 72811 SENSE
60 257 CGPG4967 73235 SENSE
61 258 CGPG4977 72813 SENSE
62 259 CGPG5001 72825 SENSE
63 260 CGPG5025 73628 SENSE
64 261 CGPG5041 76105 SENSE
65 262 CGPG5116 73242 SENSE
66 263 CGPG5144 74217 SENSE
67 264 CGPG5171 73735 SENSE
68 265 CGPG5194 73256 SENSE
69 266 CGPG5200 73260 SENSE
70 267 CGPG5210 75822 SENSE
71 268 CGPG5221 72001 SENSE
72 269 CGPG5269 72056 SENSE
73 270 CGPG5404 77308 SENSE
74 271 CGPG5432 73766 SENSE
75 272 CGPG5518 72774 SENSE
76 273 CGPG5535 72788 SENSE
77 274 CGPG5540 72753 SENSE
78 275 CGPG5568 72709 SENSE
79 276 CGPG5577 73954 SENSE
80 277 CGPG5587 73137 SENSE
81 278 CGPG5594 73161 SENSE
82 279 CGPG5633 73057 SENSE
83 280 CGPG5640 73127 SENSE
84 281 CGPG5646 73033 SENSE
85 282 CGPG5656 73105 SENSE
86 283 CGPG5659 73141 SENSE
87 284 CGPG5661 73165 SENSE
88 285 CGPG5684 73155 SENSE
89 286 CGPG5694 73026 SENSE
90 287 CGPG5704 73120 SENSE
91 288 CGPG5714 73133 SENSE
92 289 CGPG5721 73134 SENSE
93 290 CGPG5728 73123 SENSE
94 291 CGPG5757 73981 SENSE
95 292 CGPG5764 73136 SENSE
96 293 CGPG5783 73172 SENSE
97 294 CGPG5791 73020 SENSE
98 295 CGPG5799 72946 SENSE
99 296 CGPG5856 74746 SENSE
100 297 CGPG5927 77312 SENSE
101 298 CGPG5941 75237 SENSE
102 299 CGPG5957 75240 SENSE
103 300 CGPG5967 74349 SENSE
104 301 CGPG6040 76422 SENSE
105 302 CGPG607 70812 SENSE
106 303 CGPG6178 77322 SENSE
107 304 CGPG6185 74662 SENSE
108 305 CGPG6306 76527 SENSE
109 306 CGPG6318 77020 SENSE
110 307 CGPG6326 77609 SENSE
111 308 CGPG6370 73485 SENSE
112 309 CGPG6429 73433 SENSE
113 310 CGPG6440 73411 SENSE
114 311 CGPG6516 73568 SENSE
115 312 CGPG6653 74688 SENSE
116 313 CGPG6712 74420 SENSE
117 314 CGPG6737 74435 SENSE
118 315 CGPG6747 74460 SENSE
119 316 CGPG6796 74566 SENSE
120 317 CGPG6805 77610 SENSE
121 318 CGPG6810 77618 SENSE
122 319 CGPG6952 77517 SENSE
123 320 CGPG6953 77518 SENSE
124 321 CGPG7121 76460 SENSE
125 322 CGPG7163 77069 SENSE
126 323 CGPG7168 76161 SENSE
127 324 CGPG7206 76171 SENSE
128 325 CGPG7225 76178 SENSE
129 326 CGPG7267 76467 SENSE
130 327 CGPG7272 77536 SENSE
131 328 CGPG7281 76576 SENSE
132 329 CGPG7308 74862 SENSE
133 330 CGPG7316 74863 SENSE
134 331 CGPG7371 74858 SENSE
135 332 CGPG7457 74933 SENSE
136 333 CGPG7520 75379 SENSE
137 334 CGPG7529 77816 SENSE
138 335 CGPG7636 75434 SENSE
139 336 CGPG7737 77821 SENSE
140 337 CGPG7767 75685 SENSE
141 338 CGPG7804 75654 SENSE
142 339 CGPG7823 75692 SENSE
143 340 CGPG7828 75657 SENSE
144 341 CGPG7833 75622 SENSE
145 342 CGPG7933 77549 SENSE
146 343 CGPG7986 77917 SENSE
147 344 CGPG8012 77568 SENSE
148 345 CGPG8015 77570 SENSE
149 346 CGPG8055 77338 SENSE
150 347 CGPG8062 77580 SENSE
151 348 CGPG8082 77928 SENSE
152 349 CGPG8083 77349 SENSE
153 350 CGPG8106 77357 SENSE
154 351 CGPG8107 77587 SENSE
155 352 CGPG8136 77933 SENSE
156 353 CGPG8152 77619 SENSE
157 354 CGPG8166 77621 SENSE
158 355 CGPG8377 77629 SENSE
159 356 CGPG8976 77832 SENSE
160 357 CGPG8987 76802 SENSE
161 358 CGPG9013 76829 SENSE
162 359 CGPG9080 76961 SENSE
163 360 CGPG9081 76973 SENSE
164 361 CGPG9130 77150 SENSE
165 362 CGPG9133 77186 SENSE
166 363 CGPG9134 77103 SENSE
167 364 CGPG9137 77139 SENSE
168 365 CGPG9141 77187 SENSE
169 366 CGPG9145 77140 SENSE
170 367 CGPG9147 77164 SENSE
171 368 CGPG9148 77176 SENSE
172 369 CGPG9155 77165 SENSE
173 370 CGPG9163 77166 SENSE
174 371 CGPG9170 77155 SENSE
175 372 CGPG9180 77180 SENSE
176 373 CGPG9183 77121 SENSE
177 374 CGPG9186 77157 SENSE
178 375 CGPG9205 77195 SENSE
179 376 CGPG9207 77124 SENSE
180 377 CGPG9219 77261 SENSE
181 378 CGPG9220 77273 SENSE
182 379 CGPG9230 77203 SENSE
183 380 CGPG9236 77275 SENSE
184 381 CGPG9238 77204 SENSE
185 382 CGPG9259 77266 SENSE
186 383 CGPG9271 77220 SENSE
187 384 CGPG9275 77268 SENSE
188 385 CGPG9278 77209 SENSE
189 386 CGPG9283 77269 SENSE
190 387 CGPG9309 77451 SENSE
191 388 CGPG9311 77452 SENSE
192 389 CGPG9322 77430 SENSE
193 390 CGPG9335 77432 SENSE
194 391 CGPG9341 77433 SENSE
195 392 CGPG9344 77444 SENSE
196 393 CGPG9345 77409 SENSE
197 394 CGPG976 12313 ANTI-SENSE
10 207 CGPG1828 16322 SENSE
Recombinant DNA DNA for use in the present invention to improve traits in plants have a nucleotide sequence of SEQ ID NO:1 through SEQ ID NO:197, as well as the homologs of such DNA molecules. A subset of the DNA for gene suppression aspects of the invention includes fragments of the disclosed full polynucleotides consisting of oligonucleotides of 21 or more consecutive nucleotides. Oligonucleotides the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO: 197 are useful as probes and primers for detection of the polynucleotides used in the invention. Also useful in this invention are variants of the DNA. Such variants may be naturally occurring, including DNA from homologous genes from the same or a different species, or may be 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 base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a DNA useful in the present invention may 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 genes providing DNA demonstrated as useful in improving traits in model plants disclosed herein will generally have significant identity with the DNA disclosed herein. DNA is substantially identical to a reference DNA if, when the sequences of the polynucleotides are optimally aligned there is about 60% nucleotide equivalence; more preferably 70%; more preferably 800% equivalence; more preferably 85% equivalence; more preferably 90%; more preferably 95%; and/or more preferably 98% or 99% equivalence over a comparison window. A comparison window is preferably at least 50-100 nucleotides, and more preferably is the entire length of the polynucleotide provided herein. Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably by computerized implementations of these algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference polynucleotide may be a full-length molecule or a portion of a longer molecule. Preferentially, the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
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: 198 through SEQ ID NO: 394, as well as homologs of such proteins.
Homologs of the proteins useful in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources, 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. As used herein, a homolog is a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared. An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog. Other complicating factors include alternatively spliced transcripts from the same gene, limited gene identification, redundant copies of the same gene with different sequence lengths or corrected sequence. A local sequence alignment program, e.g., BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) 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 in the present invention 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 describe 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: 395 to SEQ ID NO: 19,938 to the proteins with amino acid sequences of SEQ ID NO: to 198 to SEQ ID NO: 394 are found in the listing of Table 2.
Other functional homolog proteins differ in one or more amino acids from those of a trait-improving protein disclosed herein 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 can be 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 can be 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 comprises proteins that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.
Homologs of the trait-improving proteins provided herein will generally demonstrate significant sequence identity. Of particular interest are proteins having at least 50% sequence identity, more preferably at least about 70% sequence identity or higher, e.g., at least about 80% sequence identity with an amino acid sequence of SEQ ID NO:198 through SEQ ID NO: 394. Of course useful proteins also include those with higher identity, e.g., 90% to 99% identity. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence 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: 198 through SEQ ID NO: 394.
Genes that are homologous to each other can be grouped into families and included in multiple sequence alignments. Then a consensus sequence for each group can 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. The consensus sequence can be used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship. Thus, the present 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 sequences.
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 polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.
Furthermore, the promoters can include multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 and rice actin 2 genes. Examples of other enhancers that can be used in accordance with the invention 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 to result in the production of an effective amount of a polypeptide 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. application Ser. No. 10/739,565, incorporated herein by reference.
In some 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 some 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 suppression includes any of the well-known methods for suppressing transcription of a gene or the accumulation of the mRNA corresponding to that gene thereby preventing translation of the transcript into protein. Posttranscriptional gene suppression is mediated by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that can 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 can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.
Gene Stacking The present invention also contemplates that the trait-improving recombinant DNA provided herein can be used in combination with other recombinant DNA to create plants with multiple desired traits or a further enhanced trait. The combinations generated can include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations can be created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.
In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the proteins disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goal. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include using glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified antifungal protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×107 to 2×108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Spend virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).
Fusion procedures usually produce viable hybrids at low frequencies, about 1×10−6 to 1×10−8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azasenne blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
Transformation Methods Numerous methods for producing plant cell nuclei with recombinant DNA are known in the art and may be used in the present 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 will 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 may be 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-lox 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” refers to 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 may be regenerated is useful as a recipient cell. Callus may be 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. patent application Ser. No. 09/757,089, which are incorporated herein by reference.
In practice DNA is introduced into only a small percentage of target cell nuclei in any one experiment. Marker genes are used to provide an efficient system for identification of those cells with nuclei 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 with a nucleus of the invention are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA in the nucleus. 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 can also be 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, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets can be 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° C. After regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
Progeny may be 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 nuclei with recombinant DNA that confer improved traits to plants, Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an improved trait.
Arabidopsis thaliana is used a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, e.g., Methods in Arabidopsis Research e.g., (1992), World Scientific, New Jersey, New Jersey, in “Preface”).
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 3 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.
In particular, Table 3 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.
“% id” 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” identify by two letter 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 3
PEP Annotation
SEQ Construct e %
ID ID value id description trait
198 11029 1.00E−170 85 gb|AAD02882.1|gamma-tocopherol DS LN
methyltransferase [Arabidopsis
thaliana]
199 12223 / / / CK PEG
200 10422 1.00E−133 100 gb|AAB47766.1|SNF5 homolog BSH LN
[Arabidopsis thaliana]
201 113485 4.00E−39 55 emb|CAB79347.1|hypothetical LN
protein [Arabidopsis thaliana]
202 13235 7.00E−76 100 einb|CAB78153.1|putative protein LN
[Arabidopsis thaliana]
203 13411 3.00E−29 100 ref|NP_179638.1|unknown protein LN CK
[Arabidopsis thaliana]
204 73944 0 95 ref|NP_189578.1|phosphorylase] CS
transferase, transferring glycosyl
groups [Arabidopsis thaliana]
205 13846 0 91 gb|AAN-60291.1| unknown LN
[Arabidopsis thaliana]
206 12602 0 100 ref|NP_565974.1|PP2.A-4; hydrolase/ LN
protein phosphatase type 2A/protein
serine/threonine phosphatase
[Arabidopsis thaliana]
207 74065 0 94 ref|NP_181390.1|DNA binding/ CS DS
transcription factor [Arabidopsis
thaliana] gb|AAN13033.1| putative
elongation
208 72783 0 83 ref|NP_564690.1|unknown protein CK PEG HS
[Arabidopsis thaliana]
209 17210 1.00E−92 100 ref|NP_563622.1|unknown protein CS CK PEG
[Arabidopsis thaliana]
210 72724 1.00E−128 90 ref|NP_190525.1| protein translocase/ LL
protein transporter [Arabidopsis
thaliana
211 17805 1.00E−172 80 ref|NP_563761.1|unknown protein LN
[Arabidopsis thaliana]
212 16610 0 100 gb|AAL44550.1|fructose bisphosphate LN HS
aldolase [Agrobacterium tumefaciens
str. C58]
213 76602 0 78 ref|NP_566515.1|signal transducer CS HS
[Arabidopsis thaliana]
214 18456 1.00E−135 81 ref|NP_176820.1|DNA binding/ CK SS PEG
transcription factor [Arabidopsis
thaliana]
215 18414 1.00E−169 70 ref|NP_195583.1| translation initiation CK
factor [Arabidopsis thaliana]
216 71538 1.00E−137 90 emb|CAE85115.1|synaptotagmin SP
[Arabidopsis thaliana]
217 76532 0 99 ref|NP_564775.1|ATP binding/ CK CS
carbohydrate binding/kinase/protein
kinase/protein serine/threonine
kinase/protein-tyrosine kinase/sugar
binding [Arabidopsis thaliana]
218 18231 1.00E−142 100 emb|CAC83762.1| allene oxide CK LN
cyclase [Arabidopsis thaliana]
219 18232 6.00E−85 89 ref|NP_191628.1|unknown protein CS
[Arabidopsis thaliana]
220 18256 1.00E−102 100 dbj|BAD44508.1| putative zinc finger SS CK HS
protein (PMZ) [Arabidopsis thaliana]
221 18258 1.00E−175 83 emb|CAA11525.1|transcription factor SP
IIA large subunit [Arabidopsis
thaliana]
222 19193 1.00E−103 100 ref|NP_564682.1|lipid binding SP HS
[Arabidopsis thaliana]
dbj|BAE73268.1|xylogen like protein
12 [Arabidopsis thaliana]
223 71301 0 84 ref|NP_568393.1|nucleic acid binding LN
[Arabidopsis thaliana]
224 18354 0 96 ref|NP_567568.1|ATP binding/ LN
kinase/protein kinase/protein
serine/threonine kinase/protein-
tyrosine kinase [Arabidopsis thaliana]
225 77334 1.00E−117 100 ref|NP_178399.1|RNS1 LL
(RIBONUCLEASE 1);
endoribonuclease [Arabidopsis
thaliana]
226 19767 1.00E−180 95 gb|AAO69665.1|serine threonine CS DS
protein phosphatase [Phaseolus
acutifolius]
227 19774 1.00E−100 57 ref|NP_974271.1|unknown protein CS PP
[Arabidopsis thaliana]
228 70992 1.00E−108 65 gb|ABE87200.1|hypothetical protein CS PEG
MtrDRAFT_AC151.668g4v1
Medicago truncatula]
229 19956 1.00E−146 100 gb|AAF64040.1|14-3-3-like protein PP
[Glycine max]
230 70948 3.00E−59 45 ref|NP_201297.1|CIP8 (COP1- PP HS
INTERACTING PROTEIN 8);
protein binding/zinc ion binding
[Arabidopsis thaliana]
231 70201 0 68 ref|NP_567072.1|ATP binding/ CS SS
kinase/protein kinase/protein
serine/threonine kinase protein-
tyrosine kinase [Arabidopsis thaliana]
232 19973 0 96 gb|AAB04057.1|S-adenosyl-L- PP SS
methionine delta24-sterol-C-
methyltransferase
233 70987 0 93 gb|AAM83095.1|SOS2-like protein CK CS HS SS PEG
kinase [Glycine max]
234 70950 1.00E−116 77 dbj|BAD38167.1|putative leucine PP PEG
zipper protein [Oryza sativa (japonica
cultivar-group)]
235 70962 1.00E−179 78 gb|ABE93200.1|conserved PP HS
hypothetical protein [Medicago
truncatula]
236 70915 0 76 dbj|BAE711210.1|hypothetical protein CS PP
[Trifolium pratense] dbj|BAE71208.1|
hypothetical protein [Trifolium
pratense]
237 19947 1.00E−116 65 gb|AAS38575.1|short-chain CS DS SS
dehydrogenase Tic32 [Pisum
sativum]
238 70969 1.00E−144 76 ref|NP_174321.2|unknown protein SS
[Arabidopsis thaliana]
239 70985 0 69 dbj|BAB08296.1|unnamed protein CS HS PEG
product [Arabidopsis thaliana]
sp|Q9FMT4|Y541.7_ARATH Protein
At5g14170
240 70971 1.00E−92 63 gb|ABE80664.1|GNS1/SUR4 SS
membrane protein [Medicago
truncatula] gb|ABE78502.1|
GNSI/SUR4 membrane protein
[Medicago truncatula]
241 70963 1.00E−96 56 ref|NP_177178.1|CYCD1; I; cyclin- CS PP HS
dependent protein kinase regulator
[Arabidopsis thaliana]
242 70994 2.00E−33 44 ref|NP_172962.1|RHA2A; protein PP DS HS SS
binding/ubiquitin-protein ligase/zinc
ion binding [Arabidopsis thaliana]
243 70956 0 85 gb|AAO23063.1|ent-kaurenoic acid CS SS HS
oxidase [Pisum sativum]
244 70995 1.00E−132 66 dbj|BAD73789.1|putative CK CS PP LL PEG
uncharacterized hypothalamus protein
HT010 [Oryza sativa (japonica
cultivar-group)]
245 73306 0 93 ref|NP_201243.1|phosphoric ester SS PEG
hydrolase [Arabidopsis thaliana]
246 70657 9.00E−31 73 emb|CAB78150.1|probable wound- SP LL
induced protein [Arabidopsis
thaliana]
247 70660 2.00E−32 79 ref|NP_56821.7|transcription SP
regulator [Arabidopsis thaliana]
248 74536 0 91 ref|NP_001031061.1|PGDH; CS
phosphoglycerate dehydrogenase
[Arabidopsis thaliana]
249 71328 0 99 gb|AAD46022.1|Strong simlarity to PP
gb|286426 F10M6.190 cytochrome
p450 homolog from Arabidopsis
thaliana BAC
250 71329 0 94 ref|NP_177477.1|heme binding/iron LN
ion binding/monooxygenase/oxygen
binding [Arabidopsis thaliana]
251 70755 1.00E−142 100 ref|NP_188925.1|unknown protein LL
[Arabidopsis thaliana]
252 70684 3.00E−53 100 emb|CAB80194.1|putative protein CS LN HS PP
[Arabidopsis thaliana]
253 73330 1.00E−158 72 emb|CAB96855.1|putative protein CK CS SS
[Arabidopsis thaliana]
254 76202 3.00E−83 67 emb|CAA16586.1|putative protein LL
[Arabidopsis thaliana]
255 72807 0 100 ref|NP_173478.2|nucleotide binding PEG CK HS SS
[Arabidopsis thaliana]
256 72811 1.00E−176 100 ref|NP_565782.1|nucleotide binding CK SS PEG
[Arabidopsis thaliana]
257 73235 0 89 ref|NP_564867.1|pepsin A S HS
[Arabidopsis thaliana]
258 72813 2.00E−79 93 ref|NP_850506.1|unknown protein LL LN
[Arabidopsis thaliana]
259 72825 1.00E−154 95 ref|NP_563635.1|oxidorecluctase CK PEG HS
[Arabidopsis thaliana]
260 7 3628 1.00E−163 92 ref|NP_849428.1|oxidoreductase CS DS LL HS PEG
[Arabidopsis thaliana]
261 76105 0 93 emb|CAB79123.1|receptor kinase-like DS
protein [Arabidopsis thaliana]
262 73242 0 100 ref|NP_567292.1|unknown protein SP PEG
[Arabidopsis thaliana]
263 74217 0 100 emb|CAB93726.1|cytochrome P450- DS CK
like protein [Arabidopsis thaliana]
264 73735 0 97 gb|AAU94404.1|At3g48520 CK
[Arabidopsis thaliana]
265 7 3256 1.00E−114 77 ref|NP_566329.1|unknown protein CS PP PEG
[Arabidopsis thaliana]
gb|AAL06975.1|
266 73260 4.00E−37 100 ref|NP_566488.1|unknown protein CS
[Arabidopsis thaliana]
267 75822 1.00E−90 88 emb|CAB63015.1|putative protein LL
[Arabidopsis thaliana]
268 72001 0 96 ref|NP_200045.1|CYP96A4; heme CS PP HS
binding/iron ion binding/
monooxygenase/oxygen binding
[Arabidopsis thaliana]
269 72056 0 92 ref|NP_010186.1|Essentia1, non- SP
ATPase regulatory subunit of the 26S
proteasoine lid required for the
assembly and activity of the 26S
proteasome;
270 77308 0 100 ref|NP_178642.1|SCPL38; serine PEG
carboxypeptidase [Arabidopsis
thaliana]
271 73766 1.00E−163 100 emb|CAB62363.1|MTN3-like protein LL
[Arabidopsis thaliana]
272 72774 1.00E−139 95 ref|NP_011052.1|Constituent of 66S LL CK HS
pre-ribosomal particles, involved in
60S ribosomal subunit biogenesis;
273 72788 0 81 ref|NP_010007.1|General repressor of CK CS SS
transcription, forms complex with
Cyc8p, involved in the establishment
of repressive chromatin structure
through interactions with histones H3
and H4, appears to enhance
expression of some genes
274 72753 1.00E−105 85 ref|NP_013125.1|One of four subunits CS PP HS PEG
of the endosomal sorting complex
required for transport III (ESCRT-
III);
275 72709 0 94 ref|NP_011827.1|Low affinity LL
methionine pennease, similar to
Mup1p;
276 73954 0 98 ref|NP_200954.1|MIM; ATP binding HS
[Arabidopsis thaliana]
277 73137 1.00E−110 94 gb|AAD32800.1|putative thioredoxin CS
H [Arabidopsis thaliana]
278 73161 1.00E−36 65 emb|CAB85993.1|putative protein CS PEG
[Arabidopsis thaliana]
279 73057 1.00E−133 95 ref|NP_012770.1|Tetrameric SP
phosphoglycerate mutase, mediates
the conversion of 3-phosphoglycerate
to 2-phosphoglycerate during
gtycolysis and the reverse reaction
during gluconeogenesis;
280 73127 0 99 dbj|BAB05414.1|aspartate SS HS
aminotransferase [Bacillus halodurans
C-125] ref|NP_242561.1| aspartate
aminotransferase [Bacillus halodurans
C-125]
281 73033 0 96 dbj|BAB07276.1|2,3- CK
bisphosphoglycerate-independent
phosphoglycerate mutase [Bacillus
halodurans C-125]
282 73105 0 99 emb|CAB13630.|glutamine PP PEG
synthetase [Bacillus subtilis subsp.
subtilis str. 168]
283 73141 0 95 ref|NP_414696.1|glutamate-1- CS
semialdehyde aminotransferase
[Escherichia coli K12]
284 73165 0 99 ref|NP_288110.1|pyruvate kinase DS PEG
[Escherichia coli O157:H7EDL933]
285 73155 1.00E−173 92 emb|CAC47346.1|PROBABLE CS
FRUCTOSE-BISPHOSPHATE
ALDOLASE CLASS I PROTEIN
[Sinorhizobium meliloti]]
286 73026 1.00E−116 87 emb|CAG77169.1|triophosphate PEG
isomerase [Erwinia carotovora subsp.
atroseptica SCRI1043]
287 73120 8.00E−82 100 ref|NP_441918.1|nucleoside CS SS HS
diphosphate kinase [Synechocystis sp.
PCC 6803]
288 73133 2.00E−63 90 gb|ABA76342.1|Nucleoside LL
diphosphate kinase [Pseudomonas
fluorescens PfO-1]
289 73134 9.00E−98 81 emb|CAE15979.1|ribose 5-phosphate SS CK
isomerase A (phosphoriboisomerase
A) [Photorhabdus luminescens subsp.
laumondii TTO1]
290 73123 0 95 ref|NP_180412.2|nucleic acid binding CS PEG
[Arabidopsis thaliana]
291 73981 0 93 ref|NP_014797.1|hypothetical protein; DS PEG
SlpIp [Saccharomyces cerevisiae]
292 73136 0 91 ref|NP_012454.1|Nuclear actin- DS PP
related protein involved in chromatin
remodeling
293 73172 0 100 ref|NP_014952.1|Protein involved in CK PEG HS
ER-to-Golgi transport; Sly41p
[Saccharomyces cerevisiae]
294 7 3020 0 93 ref|NP_014993.1[Proline permease, LL
required for high-affinity transport of
proline
295 72946 0 95 ref|NP_012534.1|Vacuolar PEG LL
transporter, imports large neutral
amino acids into the vacuole
296 74746 0 93 ref|NP_566298.1|ATP binding/ CK
kinasel protein kinase/protein
serine/threonine kinase/protein-
tyrosine kinase [Arabidopsis thaliana]
297 77312 0 90 ref|NP_178463.1|CDC4813; ATP PP SS HS
binding/ATP-dependent peptidase/
ATPase/nucleoside-triphosphatase/
nucleotide binding/serine-type
endopeptidase [Arabidopsis thaliana]
298 75237 0 100 ref|NP_173390.1|kinase [Arabidopsis LN
thaliana] gb|AAF98405.1| Unknown
protein [Arabidopsis thaliana]
299 75240 0 100 emb|CAB69854.1|putative protein CK
[Arabidopsis thaliana]
300 74349 1.00E−148 79 ref|NP_565187.1|transcription LN
regulator [Arabidopsis thaliana]
301 76422 5.00E−44 100 ref|NP_171781.1|unknown protein LN
[Arabidopsis thaliana]
302 70812 1.00E−152 92 ref|NP_850182.1|PUR ALPHA-1; PP PEG
nucleic acid binding [Arabidopsis
thaliana]
303 77322 1.00E−172 100 gb|AAW38983.1|At5g10750 LN LL
[Arabidopsis thaliana]
304 74662 4.00E−89 100 ref|NP_001031939.1|ubiquitin PP HS
conjugating enzyme/ubiquitin-like
activating enzyme [Arabidopsis
thaliana]
305 76527 0 96 emb|CAB51062.1|cell division cycle CS
protein 23 homolog [Arabidopsis
thaliana]
306 77020 0 98 emb|CAB75781.1|putative transporter CS
protein [Arabidopsis thaliana]
ref|NP_190154.1| transporter
[Arabidopsis thaliana]
307 77609 0 96 gb|AAU95452.1|At5g04420 LL SS
[Arabidopsis thaliana]
308 73485 0 100 emb|CAD84238.1|Glyceraldehyde 3- PEG
phosphate dehydrogenase
309 73433 1.00E−132 65 ref|ZP_0081.9153.1|putative alcohol SP LN PP
dehydrogenase [Marinobacter
aquaeolei VT8]
310 73411 0 99 emb|CAB14878.1|pyruvate kinase CS HS PP PEG
[Bacillus subtilis subsp. subtilis str.
168]
311 73568 0 97 emb|CAC41401.1|PROBABLE SP
SUCCINAIE-SEMIALDEHYDE
DEHYDROGENASE [NADP+]
PROTEIN [Sinorhizobium meliloti]
312 74688 0 98 emb|CAA16688.1|receptor protein CS PP SS PEG
kinase-like protein [Arabidopsis
thaliana]
313 74420 1.00E−142 99 emb|CAD85691.1|Phosphoglycerate PP LN SS
mutase family [Nitrosomonas
europaea ATCC 19718]
314 74435 1.00E−117 100 emb|CAC41549.1|PROBABLE LL
PHOSPHOGLYCERATE MUTASE
1 PROTEIN [Sinorhizobium meliloti]
315 74460 0 95 ref|NP_441738.1|fructose-1,6- CK
bisphosphatase [Synechocystis sp.
PCC 6803]
316 74566 0 93 emb|CAC48499.1|putative trehalose SP PP SS PEG HS
synthase protein [Sinorhizobium
meliloti 1021]
317 77610 0 94 gb|AAC23406.1|hypothetical protein CS HS
[Arabidopsis thaliana]
318 77618 0 100 ref|NP_850453.1|JAR1 PP HS
(JASMONAIE RESISTANT 1)
[Arabidopsis thaliana]
319 77517 7.00E−77 100 emb|CAB75802.1|putative protein CS LL
[Arabidopsis thaliana]
320 77518 1.00E−49 100 ref|NP199600.1|oxidoreductase, S'S LL HS
acting on NADH or NADPH, quinone
or similar compound as acceptor
[Arabidopsis thaliana]
321 76460 / / / CS PEG
322 77069 0 85 ref|NP_201196.1|unknown protein PP
[Arabidopsis thaliana]
323 76161 1.00E−161 100 ref|NP_851282.1|unknown protein CK
[Arabidopsis thaliana]
324 76171 1.00E−178 95 ref|NP_176204.1|oxidoreductase LL
[Arabidopsis thaliana]
325 76178 5.00E−60 86 ref|NP_565029.1|unknown protein CS PP HS LL PEG
[Arabidopsis thaliana]
326 76467 6.00E−86 89 ref|NP_565671.1|unknown protein LL
[Arabidopsis thaliana]
327 77536 1.00E−119 83 gb|AAS99692.1|At1g10020 SP CS DS PP LN
[Arabidopsis thaliana]
328 76576 1.00E−145 100 ref|NP_181023.1|FAH1 (FATTY DS
ACID HYDROXYLASE 1); catalytic
[Arabidopsis thaliana]
329 74862 1.00E−145 100 gb|ABA77057.1|Delta 1-pyrroline-5- PP
carboxylate reductase [Pseudomonas
fluorescens PfO-1]
330 74863 1.00E−93 100 ref|NP_012420.1|Nucleosome LL PEG CK SS
assembly factor, involved in
chromatin assembly after DNA
replication]
331 74858 5.00E−50 68 gb|AAK14395.1|response regulator LL
protein [Dianthus caryophyllus]
332 74933 1.00E−177 95 gb|AAK85899.1|AGR_C_118p LL
[Agrobacterium tumefaciens str. C58]
333 75379 1.00E−105 76 dbj|BAD73205.1|unknown protein LN
[Oryza sativa (japonica caltivar-
group)]
334 77816 4.00E−41 89 gb|ABA98984.1|expressed protein SP PP HS
[Oryza sativa (japonica cultivar-
group)]
335 75434 3.00E−52 79 ref|XP_472650.1|OSJNBa0027P08.15 LN
[Oryza sativa (japonica cultivar-
group)]
336 77821 0 71 gb|ABE84883.1|conserved PP SS HS PEG
hypothetical protein [Medicago
truncatula]
337 75685 0 82 ref|XP_475937.1|unknown protein LL
[Oryza sativa (japonica cultivar-
group)]
338 75654 0 100 ref|NP_563865.1|unknown protein SP CK HS
[Arabidopsis thaliana]
339 75692 1.00E−119 87 dbj|BAD30296.1|peptidyl-prolyl cis- CK LN
trans isomerase-like protein [Oryza
sativa (japonica cultivar-group)]
340 75657 6.00E−57 67 dbj|BAD38392.1|DNAJ heat shock N- CS LL HS
terminal domain-containing protein-
like [Oryza sativa (japonica cultivar-
group)]
341 75622 1.00E−30 78 dbj|BAD45825.1|unknown protein LN
[Oryza sativa (japonica cultivar-
gyoup)]
342 77549 1.00E−132 89 emb|CAB80891.1|AT400820 PP HS
[Arabidopsis thaliana]]
343 77917 0 91 ref|NP_197917.1|EBF2 (EIN3- PP PEG
BINDING F BOX PROTEIN 2)
[Arabidopsis thaliana]
344 77568 9.00E−99 76 ref|NP_849792.1|nucleic acid binding PP
[Arabidopsis thaliana]
345 77570 7.00E−54 100 gb|AAS75309.1|multidomin CK SS
cyclophilin type peptidyl-prolyl cis-
trans isomerase [Arabidopsis
thaliana]]
346 77338 4.00E−24 65 gb|AAM61454.1|unknown PEG
[Arabidopsis thaliana]
347 77580 3.00E−45 100 dbj|BAB01457.1|unnamed protein PEG CS HS SS
product [Arabidopsis thaliana]
348 77928 4.00E−38 100 ref|NP_196244.1|unknown protein HS
[Arabidopsis thaliana]
349 77349 1.00E−81 93 emb|CAC05463.1|putative lipid LL SS
transfer protein [Arabidopsis thaliana]
350 77357 1.00E−155 95 ref|NP_175357.1|unknown protein PEG
[Arabidopsis thaliana]
351 77587 / / / CS PP HS SS
352 77933 / / / SS
353 77619 3.00E−40 98 db|BAB09403.1|unnamed protein CK SS PEG HS
product [Arabidopsis thaliana]
354 77621 4.00E−58 74 ref|NP_197632.1|Rac GTPase CK
activator [Arabidopsis thaliana]
[Arabidopsis thaliana]
355 77629 5.00E−36 100 ref|NP_196372.1|GRP19 [Arabidopsis SS HS
thaliana]
356 77832 0 83 ref|NP_917762.1|p050IG0I.24 CS SS PEG HS LN
[Oryza sativa (japonica cultivar-
group)]
357 76802 0 76 ref|NP_181908.1|actin binding CK
[Arabidopsis thaliana]
gb|AAB64026.1| unknown protein
[Arabidopsis thaliana]
358 76829 0 89 ref|NP_909912.1|ferredoxin-NADP+ CK
reductase [Oryza sativa]
359 76961 0 65 ref|XP_473189.1|OSJNBa0073E02.11 LL LN
[Oryza sativa (japonica cultivar-
group)]
360 76973 1.00E−151 76 ref|XP_480055.1|unknown protein LL
[Oryza sativa (japonica cultivar-
group)]
361 77150 0 76 dbj|BAD61385.1|putative SP HS PEG
nucleostemin [Oryza sativa (japonica
cultivar-group)]
362 77186 0 85 dbj|BAD27898.1|putative PP
pentatricopeptide (PPR) repeat-
containing protein [Oryza sativa
(japonica cultivar-group)]
363 77103 1.00E−174 66 ref|NP_568580.1|catalytic LL DS
[Arabidopsis thaliana]
364 77139 2.00E−93 58 ref|NP_171690.1|PFC1 (PALEFACE CS SS
I) [Arabidopsis thaliana]
365 77187 1.00E−160 83 ref|NP_913437.1|3-methyl-2 - CS PP LL PEG
oxobutanoate hydroxy-me hyl-
transfe rase-like protein [Oryza sativa
(japonica cultivar-group)]
366 77140 0 77 dbj|BAD36145.1|membrane protein CK PEG SS
PTM1-like [Oryza sativa (japonica
cultivar-group)]
367 77164 0 91 gb|ABF93778.1|DNA polymerase PP HS
delta small subunit, putative,
expressed [Oryza sativa (japonica
cultivar-group)]
368 77176 0 76 ref|NP_914476.1|putative LL
phytochrome P450 [Oryza sativa
(japonica cultivar-group)]
369 77165 1.00E−139 75 ref|NP_914949.1|serine/threonine CK LL LN
protein kinase-like protein [Oryza
sativa (japonica cultivar-group)]
370 77166 0 85 dbj|BAB75233.1|all3534 [Nostoc sp. CK PP
PCC 7120]
371 77155 0 97 emb|CAA35550.1|hycE [Escherichia PP
coli]
372 77180 2.00E−62 61 gb|ABA99663.1|expressed protein CS PP HS PEG
[Oryza sativa (japonica cultivar-
group)]
373 77121 1.00E−166 93 dbj|BAB05404.1|transcriptional CK
repressor of the biotin operon
[Bacillus halodurans C-125]
374 77157 0 100 ref|NP_53I453.1|3,4-dihydroxy-2- CK LL
butanone-4-phoshate synthase/GTP
cyclohydrolase II [Agrobacterium
tumefaciens str. C58]
375 77195 0 94 gb|AAM71555.1|mannose-6- PP SS PEG LL
phosphate isomerase/mannose-1-
phosphate guanylyl transferase
[Chlorobium tepidum TLS]
376 77124 1.00E−180 100 ref|NP_418404.1|biotin--protein PP SS PEG
ligase [Escherichia coli K12]
377 77261 0 100 ref|NP_416982.1|hydrogenase 4, PEG
subunit [Escherichia coli K12]
378 77273 0 93 dbj|BAB06534.1|glycine PP PEG
dehydrogenase subunit 1 [Bacillus
halodurans C-125]
379 77203 1.00E−176 60 dbj|BAD33942.1|putative serine PEG CK
carboxypeptidase precursor [Oryza
sativa (japonica cultivar-group)]
380 77275 4.00E−37 55 gb|ABA91490.1|expressed protein SS HS PEG
[Oryza sativa (japonica cultivar-
group)]
381 77204 2.00E−27 44 ref|XP_469963.1|putative protease CK CS
inhibitor [Oryza sativa (japonica
cultivar-group)]
382 77266 7.00E−25 73 gb|AAT93978.1|unknown protein CS PP PEG
[Oryza sativa (japonica cultivar-
group)]
383 77220 1.00E−169 78 dbj|BAD52854.1|putative non- LL
phototropic hypocotyl 3 [Oryza sativa
(japonica cultivar-group)]
384 77268 4.00E−71 85 gb|ABF95596.1|ETC complex I PP SS
subunit conserved region family
protein, expressed [Oryza sativa
(japonica cultivar-group)]
385 77209 9.00E−86 85 ref|XP_479456.1|putative 60S PP LL
ribosome subunit biogenesis protein
[Oryza sativa (japonica cultivar-
group)]
386 77269 2.00E−56 70 dbj|BAD32031.1|unknown protein SS
[Oryza sativa (japonica cultivar-
group)]
387 77451 0 71 ref|XP_475231.1|putative PP SS
microtubule-associated protein [Oryza
sativa (japonica cultivar-group)]
388 77452 1.00E−106 69 dbj|BAD69045.1|unknown protein PP
[Oryza sativa (japonica cultivar-
group)]
389 77430 1.00E−160 83 dbj|BAD33328.1|putative protein CS PP SS
serine/threonine kinase [Oryza sativa
(japonica cultivar-group)]
390 77432 0 86 ref|NP_849565.1|carbohydrate PP HS PEG
transporter/nucleoside transporter/
sugar porter [Arabidopsis thaliana]
gb|AAM19835.1|
AT4g35300/F23E12_140
[Arabidopsis thaliana]
391 77433 0 100 gb|AAK59487.1| putative cleavage CK PP
and polyadenylation specificity factor
[Arabidopsis thaliana]
392 77444 0 96 ref|NP_199947.1|unknown protein CS PP
[Arabidopsis thaliana]
393 77409 0 91 ref|NP_001032163.1|unknown protein PP
[Arabidopsis thaliana]
394 12313 0 94 ref|NP_189150.1|QUA1 SP
(QUASIMODO1); transferase,
transferring glycosyl groups/
transferase, transferring hexosyl
groups [Arabidopsis thaliana]
Trait Improvement Screens DS—Improvement of Drought Tolerance Identified by a Soil Drought Stress Tolerance Screen:
Drought or water deficit conditions impose mainly 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 present invention provides recombinant DNA that can improve the plant survival rate under such sustained drought condition. Exemplary recombinant DNA for conferring such drought tolerance are identified as such in Table 3. Such recombinant DNA may find particular use 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 can 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 e.g., (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. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen can survive better 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, and (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. The present invention provides genes that help plants maintain biomass, root growth, and/or plant development in high salinity conditions, which are identified as such in Table 3. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, trait-improving recombinant DNA identified in a high salinity stress tolerance screen can also provide transgenic crops with improved drought tolerance. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen can survive better 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 by the present invention 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. In the present invention, 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° C. from planting until day 28 post plating. 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° C. until day 8 post plating, and subsequently were placed under 8° C. until day 28 post plating. As demonstrated from the model plant screen, 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 can survive better 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, the plant can be enabled to react to different kinds of stresses. For examples, PEP SEQ ID NO: 231 can be used to improve both salt stress tolerance and cold stress tolerance in plants. Of particular interest, plants transformed with PEP SEQ ID NO: 233 can resist heat stress, salt stress and cold stress. In addition to these multiple stress tolerance genes, the stress tolerance conferring genes provided by the present invention may be used in combinations to generate transgenic plants that can resist multiple stress conditions.
PP-Improvement of Early Plant Growth and Development:
It has been known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy and 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” used herein encompasses the stages of seed imbibition through the early vegetative phase. The present invention provides genes that are 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, it can be recognized by those skilled in the art that genes conferring the growth advantage in early stages to plants may also be used to generate transgenic plants that are more resistant to various stress conditions due to improved early plant development. The present invention provides such exemplary recombinant DNA that confer both the stress tolerance and growth advantages to plants, identified as such in Table 3, e.g., PEP SEQ ID NO: 268 which can improve the plant early growth and development, and impart heat and cold tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen can 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” used herein encompasses the stages of leaf development, flower production, and seed maturity. In certain embodiments, transgenic plants produced using genes that confer growth advantages to plants provided by the present invention, identified as such in Table 3, 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. On one hand, 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. On the other hand, the 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. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen can 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. The present invention provides recombinant DNA that enable plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently, resulting higher yield as compared to the wild type plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen can have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by the present invention may be 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.
According to the present invention, transgenic plants generated using the recombinant nucleotides, which confer enhanced nitrogen use efficiency, identified as such in Table 3, exhibit one or more desirable traits including, but not limited to, increased seedling weight, greener leaves, increased number of rosette leaves, increased or decreased root length. One skilled in the art may recognize that the transgenic plants provided by the present invention with enhanced nitrogen use efficiency may also have altered amino acid or protein compositions, increased yield and/or better seed quality. The transgenic plants of the present invention may be productively cultivated under low nitrogen growth conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs, which would cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless. The transgenic plants also may be 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:
The present invention also encompasses 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 RoundUp Ready® trait, or insect resistance, such as using a gene from Bacillus thuringensis 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. patent application publications 2003/0106096A1 and 2002/0112260A1 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 DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant or different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, the invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO: 1 through SEQ ID NO: 197, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 198 through SEQ ID NO: 394. In another aspect, the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 395 through SEQ ID NO: 19938. In yet another aspect, the present invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence provided herein.
The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by the present invention are not limited to any particular plant species. Indeed, the plants according to the present invention may be of any plant species, i.e., may be monocotyledonous or dicotyledonous. Preferably, they will be agricultural useful plants, i.e., plants cultivated by man for purposes of food production or technical, particularly industrial applications. Of particular interest in the present invention are corn and soybean plants. The recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are provided by the present invention. Other plants of interest in the present invention for production of transgenic plants having improved traits include, without limitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
In certain embodiments, the present invention contemplates to use an orthologous gene in generating the transgenic plants with similarly improved traits as the transgenic Arabidopsis counterpart. Improved physiological properties in transgenic plants of the present invention may be confirmed in responses to stress conditions, for example in 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 may be made on greenhouse or field grown plants and may 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 may be 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 may be 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, barreness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain may be 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 genes of the present invention, it may be desirable to test hybrids over multiple years at multiple locations in a geographical location where maize is conventionally grown, e.g., in Iowa, Illinois or other locations in the midwestern United States, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.
Transgenic plants can be used to provide plant parts according to the invention for regeneration or tissue culture of cells or tissues containing the constructs described herein. Plant parts for these purposes can include leaves, stems, roots, flowers, tissues, epicotyl, meristems, hypocotyls, cotyledons, pollen, ovaries, cells and protoplasts, or any other portion of the plant which can be used to regenerate additional transgenic plants, cells, protoplasts or tissue culture. Seeds of transgenic plants are provided by this invention can be used to propagate more plants containing the trait-improving recombinant DNA constructs of this invention. These descendants are intended to be included in the scope of this invention if 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.
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.
Examples Example 1. Identification of Recombinant DNA that Confers Improved Trait(s) to Plants A. Plant Expression Constructs for Arabidopsis 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, e.g., PNAS 95:376-381, 1998; Guyer, e.g., 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, e.g., 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-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). A list of recombinant DNA constructs which improve drought tolerance in transgenic plants is illustrated in Table 4.
TABLE 4
PEP Drought Time to wilting
SEQ score Seed yield Risk
ID Construct Nomination Delta P- Delta P- score P-
NO ID ID Orientation mean value mean value mean value
198 11029 CGPG106 SENSE 0.109 0.381 0.072 0.717 0.119 1.000
292 73136 CGPG5764 SENSE 0.030 0.090 0.136 0.067 0.104 1.000
284 73165 CGPG5661 SENSE −0.031 0.504 0.526 0.018 −0.075 1.000
260 73628 CGPG5025 SENSE 0.342 0.038 −0.474 0.022 0.208 1.000
291 73981 CGPG5757 SENSE 0.517 0.026 −0.096 0.000 0.573 1.000
207 74065 CGPG1828 ANTI- −0.078 0.164 0.646 0.027 0.048 1.000
SENSE
263 74217 CGPG5144 SENSE −0.026 0.414 0.418 0.032 0.071 1.000
261 76105 CGPG5041 SENSE 0.288 0.029 −1.780 0.027 0.173 1.000
328 76576 CGPG7281 SENSE 0.241 0.032 −0.555 0.442 0.212 1.000
327 77536 CGPG7272 SENSE 0.089 0.194 1.059 0.001 0.042 1.000
226 19767 CGPG3918 SENSE 0.117 0.038 0.164 0.296 / /
237 19947 CGPG4069 SENSE −0.009 0.852 −0.104 0.504 / /
242 70994 CGPG4122 SENSE 0.040 0.021 −0.131 0.447 / /
363 77103 CGPG9134 SENSE 0.145 0.023 −0.499 0.096 / /
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (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) If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 226, 237, 242, or 363 showed improved drought tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
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, e.g., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve heat tolerance in transgenic plants illustrated in Table 5.
TABLE 5
Root length Growth stage Seedling weight
PEP at day 14 at day 14 at day 14
SEQ Construct Nomination Delta Risk score Delta
ID ID ID Orientation mean P-value mean P-value mean P-value
239 70985 CGPG4088 SENSE 0.480 0.036 1.176 0.196 1.333 0.018
233 70987 CGPG4048 SENSE 0.228 0.021 −0.036 0.670 1.214 0.002
268 72001 CGPG5221 SENSE 0.273 0.004 0.210 0.330 1.393 0.009
274 72753 CGPG5540 SENSE 0.434 0.006 0.414 0.258 1.100 0.006
276 73954 CGPG5577 SENSE 0.406 0.002 0.455 0.282 1.274 0.000
325 76178 CGPG7225 SENSE 0.714 0.001 1.093 0.145 1.537 0.000
361 77150 CGPG9130 SENSE 0.219 0.032 0.178 0.237 1.159 0.002
367 77164 CGPG9147 SENSE 0.317 0.026 0.651 0.099 1.152 0.002
390 77432 CGPG9335 SENSE 0.581 0.001 0.457 0.124 1.551 0.000
351 77587 CGPG8107 SENSE 0.488 0.006 0.628 0.072 1.328 0.001
348 77928 CGPG8082 SENSE 0.462 0.032 0.191 0.301 1.498 0.004
212 16610 CGPG2499 SENSE 0.055 0.640 0.109 0.609 0.983 0.020
220 18256 CGPG3363 SENSE 0.070 0.138 0.071 0.541 1.021 0.004
222 19193 CGPG3375 SENSE 0.255 0.094 0.267 0.368 1.023 0.003
252 70684 CGPG4588 SENSE 0.010 0.781 0.003 0.979 1.002 0.045
230 70948 CGPG3990 SENSE 0.282 0.095 0.351 0.335 1.045 0.023
243 70956 CGPG4140 SENSE 0.155 0.313 0.091 0.320 1.129 0.018
235 70962 CGPG4057 SENSE 0.469 0.085 0.267 0.508 1.410 0.007
241 70963 CGPG4121 SENSE −0.002 0.979 −0.131 0.311 0.976 0.005
242 70994 CGPG4122 SENSE 0.140 0.574 1.204 0.096 1.207 0.030
244 70995 CGPG4154 SENSE 0.374 0.112 1.640 0.171 1.328 0.013
208 72783 CGPG2206 SENSE 0.387 0.057 0.053 0.509 1.013 0.019
255 72807 CGPG4912 SENSE 0.357 0.071 0.443 0.020 1.099 0.009
259 72825 CGPG5001 SENSE 0.171 0.349 0.213 0.234 1.118 0.012
287 73120 CGPG5704 SENSE 0.096 0.490 0.193 0.364 1.080 0.033
280 73127 CGPG5640 SENSE −0.220 0.077 −0.194 0.030 0.643 0.014
293 73172 CGPG5783 SENSE 0.283 0.197 0.887 0.162 1.154 0.007
310 73411 CGPG6440 SENSE 0.198 0.389 0.631 0.345 1.320 0.003
260 73628 CGPG5025 SENSE 0.406 0.149 1.475 0.365 1.169 0.016
316 74566 CGPG6796 SENSE 0.627 0.092 0.777 0.466 1.714 0.014
338 75654 CGPG7804 SENSE 0.292 0.237 0.804 0.315 0.940 0.017
340 75657 CGPG7828 SENSE 0.390 0.067 1.222 0.307 1.521 0.011
213 76602 CGPG2653 SENSE 0.036 0.748 0.092 0.670 0.752 0.025
372 77180 CGPG9180 SENSE 0.068 0.555 −0.045 0.177 0.871 0.022
380 77275 CGPG9236 SENSE 0.282 0.125 0.971 0.285 1.027 0.013
297 77312 CGPG5927 SENSE 0.201 0.344 0.710 0.372 1.039 0.032
320 77518 CGPG6953 SENSE 0.238 0.344 0.069 0.655 1.188 0.015
342 77549 CGPG7933 SENSE 0.184 0.180 −0.012 / 0.978 0.007
347 77580 CGPG8062 SENSE 0.157 0.143 −0.074 / 0.891 0.049
317 77610 CGPG6805 SENSE 0.023 0.937 −0.063 / 1.207 0.029
318 77618 CGPG6810 SENSE 0.002 0.942 −0.074 / 1.015 0.006
353 77619 CGPG8152 SENSE 0.213 0.316 −0.076 0.457 0.956 0.019
355 77629 CGPG8377 SENSE 0.095 0.124 −0.063 / 1.133 0.005
334 77816 CGPG7529 SENSE 0.033 0.705 0.856 0.385 0.611 0.038
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 307, 313, 327, 330, 349, 366, or 387 showed improved heat stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
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 umol/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. A list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants illustrated in Table 6.
TABLE 6
Root Root Growth Seedling
length at length stage at weight at
PEP day 11 at day 14 day 14 day 14
SEQ Construct Delta P- Delta P- Delta P- Delta P-
ID ID Orientation mean value mean value mean value mean value
220 18256 SENSE 0.192 0.232 0.236 0.080 1.291 0.151 0.598 0.039
214 18456 SENSE 0.213 0.089 0.209 0.197 2.062 0.197 0.476 0.046
232 19973 SENSE 0.289 0.012 0.299 0.002 0.976 0.054 1.002 0.013
231 70201 SENSE 0.363 0.030 0.403 0.052 2.083 0.229 1.057 0.014
243 70956 SENSE 0.006 0.923 0.128 0.016 −0.045 NA −0.364 0.371
238 70969 SENSE 0.435 0.091 0.471 0.027 0.653 0.473 0.994 0.058
240 70971 SENSE 0.337 0.063 0.257 0.041 1.418 0.226 0.593 0.030
233 70987 SENSE 0.363 0.030 0.284 0.044 1.434 0.380 0.865 0.012
273 72788 SENSE 0.484 0.019 0.456 0.016 1.813 0.314 0.929 0.008
256 72811 SENSE 0.385 0.024 0.409 0.002 2.968 0.031 0.805 0.004
287 73120 SENSE 0.341 0.001 0.244 0.019 0.613 0.103 0.755 0.015
280 73127 SENSE 0.501 0.028 0.395 0.055 0.212 0.175 0.739 0.023
289 73134 SENSE 0.226 0.134 0.266 0.045 0.152 0.487 0.378 0.056
257 73235 SENSE 0.431 0.020 0.360 0.018 1.618 0.327 0.650 0.009
245 73306 SENSE 0.228 0.046 0.219 0.018 0.249 0.217 0.708 0.008
253 73330 SENSE 0.196 0.225 0.186 0.029 0.000 NA 0.543 0.045
316 74566 SENSE 0.331 0.001 0.076 0.422 0.597 0.031 0.716 0.003
312 74688 SENSE 0.480 0.076 0.415 0.031 0.000 NA 0.808 0.062
376 77124 SENSE 0.340 0.034 0.322 0.015 2.306 0.137 0.411 0.168
364 77139 SENSE 0.339 0.096 0.287 0.041 1.683 0.294 0.602 0.039
375 77195 SENSE 0.445 0.009 0.345 0.010 0.367 0.185 0.582 0.022
384 77268 SENSE 0.495 0.047 0.468 0.102 1.906 0.079 0.992 0.042
386 77269 SENSE 0.454 0.038 0.426 0.004 1.996 0.065 1.136 0.001
380 77275 SENSE 0.457 0.008 0.487 0.040 2.119 0.154 1.070 0.010
297 77312 SENSE 0.335 0.086 0.361 0.013 0.960 0.208 0.723 0.063
389 77430 SENSE 0.303 0.030 0.360 0.004 0.131 0.495 0.894 0.022
320 77518 SENSE 0.171 0.017 0.185 0.026 −0.134 0.443 0.339 0.109
345 77570 SENSE 0.326 0.091 0.241 0.134 1.396 0.090 0.862 0.026
351 77587 SENSE 0.178 0.083 0.096 0.665 0.995 0.426 0.632 0.036
353 77619 SENSE 0.389 0.058 0.398 0.011 1.913 0.257 0.857 0.040
355 77629 SENSE 0.404 0.043 0.389 0.019 2.523 0.083 0.826 0.040
336 77821 SENSE 0.393 0.063 0.444 0.033 0.196 0.228 0.619 0.015
356 77832 SENSE 0.134 0.238 0.304 0.011 0.914 0.156 0.413 0.050
352 77933 SENSE 0.112 0.044 0.165 0.009 0.749 0.482 0.517 0.171
347 77580 SENSE −0.019 0.814 0.087 0.018 −0.158 0.151 0.124 0.204
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 242, 255, 307, 313, 327, 330, 349, 366, or 387 showed improved salt stress tolerance evidenced by the second criteria as illustrated in Example 1L.
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. A list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants illustrated in Table 7.
TABLE 7
Root Root Growth Seedling
length at length at stage at weight at
PEP day 11 day 14 day 14 day 14
SEQ Construct Delta P- Delta P- Delta P- Delta P-
ID ID Orientation mean value mean value mean value mean value
199 12223 SENSE / / / / / / 0.353 0.024
214 18456 SENSE 0.320 0.018 0.274 0.033 3.109 0.073 0.553 0.006
302 70812 SENSE 0.195 0.078 0.078 0.296 4 0.000 0.363 0.016
234 70950 SENSE 0.322 0.056 0.427 0.016 4 0.000 0.381 0.127
239 70985 SENSE 0.270 0.066 0.221 0.005 4 0.000 0.372 0.007
233 70987 SENSE 0.227 0.205 0.128 0.470 4 0.000 0.495 0.000
228 70992 SENSE 0.369 0.002 0.370 0.013 4 0.000 0.574 0.027
244 70995 SENSE 0.379 0.049 0.320 0.065 4 0.000 0.491 0.003
208 72783 SENSE 0.154 0.084 0.043 0.553 4 0.000 0.264 0.044
255 72807 SENSE 0.165 0.080 0.158 0.150 4 0.000 0.333 0.047
256 72811 SENSE 0.489 0.052 0.491 0.050 4 0.000 0.784 0.016
259 72825 SENSE 0.423 0.055 0.388 0.080 2.741 0.161 0.608 0.020
295 72946 SENSE 0.345 0.053 0.473 0.050 0.898 0.632 0.415 0.181
286 73026 SENSE 0.636 0.021 0.619 0.028 2.741 0.161 0.752 0.045
290 73123 SENSE 0.460 0.048 0.445 0.007 NA NA 0.609 0.069
278 73161 SENSE 0.510 0.024 0.506 0.006 2.624 0.246 0.626 0.073
284 73165 SENSE 0.304 0.101 0.285 0.044 2.499 0.270 0.643 0.077
293 73172 SENSE 0.197 0.056 0.137 0.204 2.374 0.293 0.660 0.081
262 73242 SENSE 0.305 0.029 0.332 0.022 2.249 0.317 0.677 0.085
265 73256 SENSE 0.325 0.084 0.182 0.178 2.124 0.341 0.694 0.089
308 73485 SENSE 0.133 0.070 0.039 0.583 1.999 0.365 0.711 0.093
291 73981 SENSE 0.239 0.147 0.238 0.281 1.874 0.389 0.728 0.096
316 74566 SENSE 0.451 0.004 0.248 0.118 1.749 0.412 0.745 0.100
312 74688 SENSE 0.190 0.191 0.102 0.520 1.624 0.436 0.762 0.104
330 74863 SENSE 0.174 0.164 0.216 0.109 1.499 0.460 0.779 0.108
366 77140 SENSE 0.644 0.005 0.572 0.015 1.374 0.484 0.796 0.112
361 77150 SENSE 0.313 0.047 0.359 0.044 1.249 0.508 0.813 0.116
365 77187 SENSE 0.273 0.084 0.032 0.763 1.124 0.532 0.830 0.120
375 77195 SENSE 0.319 0.021 0.256 0.041 NA NA 0.847 0.124
379 77203 SENSE 0.113 0.485 0.019 0.829 0.999 0.555 0.864 0.127
377 77261 SENSE 0.115 0.230 0.085 0.315 0.874 0.579 0.881 0.131
270 77308 SENSE 0.174 0.321 0.074 0.700 0.749 0.603 0.898 0.135
346 77338 SENSE 0.227 0.158 0.196 0.272 0.624 0.627 0.915 0.139
350 77357 SENSE 0.169 0.300 0.259 0.066 2.715 0.169 0.303 0.020
390 77432 SENSE 0.306 0.043 0.122 0.270 2.57 0.214 0.734 0.019
347 77580 SENSE 0.300 0.107 0.240 0.009 2.288 0.119 0.500 0.179
353 77619 SENSE 0.142 0.216 0.139 0.002 4 0.000 0.129 0.582
356 77832 SENSE 0.236 0.006 0.266 0.001 1.333 0.435 0.286 0.037
343 77917 SENSE 0.174 0.163 0.160 0.006 4 0.000 0.330 0.009
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference.
If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 209, 245, 260, 274, 282, 310, 321, 325, 336, 372, 376, 378, 380, or 382 showed improved osmotic stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
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. A list of recombinant nucleotides that improve cold shock stress tolerance in plants illustrated in Table 8.
TABLE 8
Rosette area Rosette area Rosette area
at day 8 at day 28 difference
PEP Construct Nomination Delta P- Risk score P- Delta P-
SEQ ID ID ID Orientation mean value mean value mean value
199 12223 CGPG1133 SENSE 0.112 0.617 0.334 0.014 0.273 0.143
218 18231 CGPG3274 SENSE −0.131 0.269 1.144 0.002 1.179 0.003
215 18414 CGPG3002 SENSE 0.997 0.034 1.665 0.001 1.895 0.001
214 18456 CGPG2813 SENSE 0.099 0.757 0.799 0.001 0.684 0.001
233 70987 CGPG4048 SENSE 0.864 0.136 1.213 0.010 1.146 0.002
244 70995 CGPG4154 SENSE 1.088 0.008 1.262 0.023 1.407 0.025
208 72783 CGPG2206 SENSE 0.814 0.023 0.957 0.018 0.899 0.052
273 72788 CGPG5535 SENSE 0.262 0.058 1.090 0.016 1.212 0.021
256 72811 CGPG4926 SENSE 0.880 0.058 0.669 0.002 0.715 0.018
259 72825 CGPG5001 SENSE 0.032 0.887 0.338 0.071 0.357 0.027
281 73033 CGPG5646 SENSE 0.322 0.394 1.311 0.012 1.480 0.017
293 73172 CGPG5783 SENSE 0.395 0.209 1.065 0.030 1.099 0.074
253 73330 CGPG4765 SENSE 0.347 0.515 0.835 0.065 1.043 0.007
264 73735 CGPG5171 SENSE 0.624 0.076 0.655 0.001 0.718 0.005
315 74460 CGPG6747 SENSE 0.452 0.005 0.542 0.043 0.455 0.154
296 74746 CGPG5856 SENSE 0.304 0.346 0.978 0.001 1.061 0.007
299 75240 CGPG5957 SENSE 0.479 0.153 0.652 0.143 0.875 0.039
338 75654 CGPG7804 SENSE 0.531 0.140 1.712 0.010 1.979 0.015
339 75692 CGPG7823 SENSE 0.526 0.063 2.027 0.001 2.246 0.001
323 76161 CGPG7168 SENSE 0.768 0.000 1.810 0.004 2.146 0.003
217 76532 CGPG3235 SENSE 0.921 0.008 1.216 0.001 1.324 0.002
357 76802 CGPG8987 SENSE 0.468 0.097 1.578 0.000 1.872 0.000
358 76829 CGPG9013 SENSE −0.434 0.329 0.709 0.010 0.314 0.118
373 77121 CGPG9183 SENSE 0.081 0.385 0.757 0.023 0.698 0.014
366 77140 CGPG9145 SENSE −0.221 0.165 0.896 0.007 1.043 0.005
374 77157 CGPG9186 SENSE 0.190 0.566 1.110 0.016 1.176 0.015
369 77165 CGPG9155 SENSE 1.573 0.043 0.683 0.038 0.699 0.022
370 77166 CGPG9163 SENSE 0.777 0.011 1.432 0.016 1.570 0.025
381 77204 CGPG9238 SENSE 0.545 0.139 1.313 0.003 1.436 0.006
391 77433 CGPG9341 SENSE 1.054 0.103 0.895 0.027 0.659 0.084
345 77570 CGPG8015 SENSE 0.538 0.215 0.866 0.025 0.898 0.023
353 77619 CGPG8152 SENSE −0.589 0.322 0.503 0.068 0.558 0.049
354 77621 CGPG8166 SENSE 0.053 0.863 0.716 0.044 0.792 0.040
203 13411 CGPG1301 SENSE 0.183 0.427 0.577 0.033 0.573 0.031
209 17210 CGPG2217 SENSE −0.244 0.685 0.162 0.038 0.156 0.104
220 18256 CGPG3363 SENSE 0.272 0.400 0.639 0.008 0.719 0.020
289 73134 CGPG5721 SENSE 0.014 0.947 1.053 0.000 1.062 0.025
263 74217 CGPG5144 SENSE 0.168 0.390 0.261 0.043 0.206 0.075
379 77203 CGPG9230 SENSE 0.606 0.051 0.620 0.032 0.778 0.039
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (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) If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 255, 272, or 330 showed improved cold stress tolerance evidenced by the second criterial as illustrated in Example 1L.
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 umol/m2/s. At 28 days post plating, 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. A list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants illustrated in Table 9.
TABLE 9
Root length Growth stage at
at day 28 day 28
PEP Construct Nomination Delta Delta
SEQ ID ID ID Orientation mean P-value mean P-value
209 17210 CGPG2217 SENSE 0.164 0.089 4.000 0.000
219 18232 CGPG3275 SENSE −0.081 0.613 4.000 0.000
226 19767 CGPG3918 SENSE 0.212 0.007 4.000 0.000
227 19774 CGPG3920 SENSE 0.140 0.011 4.000 0.000
237 19947 CGPG4069 SENSE / / 4.000 0.000
231 70201 CGPG3994 SENSE 0.311 0.010 4.000 0.000
252 70684 CGPG4588 SENSE 0.335 0.209 4.000 0.000
236 70915 CGPG4058 SENSE 0.231 0.258 4.000 0.000
243 70956 CGPG4140 SENSE 0.113 0.525 4.000 0.000
241 70963 CGPG4121 SENSE 0.210 0.252 4.000 0.000
239 70985 CGPG4088 SENSE 0.283 0.063 4.000 0.000
233 70987 CGPG4048 SENSE 0.392 0.202 4.000 0.000
228 70992 CGPG3962 SENSE 0.008 0.959 4.000 0.000
244 70995 CGPG4154 SENSE 0.426 0.002 4.000 0.000
268 72001 CGPG5221 SENSE 0.070 0.728 4.000 0.000
274 72753 CGPG5540 SENSE 0.189 0.010 2.889 0.121
273 72788 CGPG5535 SENSE 0.548 0.009 4.000 0.000
287 73120 CGPG5704 SENSE 0.401 0.146 4.000 0.000
290 73123 CGPG5728 SENSE 0.171 0.166 4.000 0.000
277 73137 CGPG5587 SENSE 0.382 0.019 4.000 0.000
283 73141 CGPG5659 SENSE 0.348 0.029 1.891 0.219
285 73155 CGPG5684 SENSE 0.194 0.105 4.000 0.000
278 73161 CGPG5594 SENSE 0.327 0.018 2.889 0.121
265 73256 CGPG5194 SENSE 0.176 0.473 4.000 0.000
266 73260 CGPG5200 SENSE 0.256 0.047 4.000 0.000
253 73330 CGPG4765 SENSE 0.229 0.016 4.000 0.000
310 73411 CGPG6440 SENSE 0.232 0.213 4.000 0.000
260 73628 CGPG5025 SENSE 0.304 0.040 1.330 0.467
204 73944 CGPG1458 SENSE 0.426 0.026 4.000 0.000
207 74065 CGPG1828 ANTI-SENSE 0.215 0.026 2.599 0.205
248 74536 CGPG442 SENSE −0.054 0.431 4.000 0.000
312 74688 CGPG6653 SENSE 0.645 0.006 4.000 0.000
340 75657 CGPG7828 SENSE 0.394 0.017 2.680 0.180
325 76178 CGPG7225 SENSE 0.334 0.107 4.000 0.000
321 76460 CGPG7121 SENSE 0.136 0.381 4.000 0.000
305 76527 CGPG6306 SENSE 0.238 0.001 0.335 0.421
217 76532 CGPG3235 SENSE 0.250 0.331 4.000 0.000
213 76602 CGPG2653 SENSE 0.283 0.387 4.000 0.000
306 77020 CGPG6318 SENSE 0.507 0.038 0.000 0.000
364 77139 CGPG9137 SENSE 0.445 0.005 0.000 0.000
372 77180 CGPG9180 SENSE 0.370 0.046 4.000 0.000
365 77187 CGPG9141 SENSE 0.456 0.036 4.000 0.000
381 77204 CGPG9238 SENSE 0.314 0.002 4.000 0.000
382 77266 CGPG9259 SENSE 0.017 0.948 4.000 0.000
389 77430 CGPG9322 SENSE 0.095 0.233 4.000 0.000
392 77444 CGPG9344 SENSE 0.171 0.292 4.000 0.000
319 77517 CGPG6952 SENSE 0.132 0.001 2.507 0.235
327 77536 CGPG7272 SENSE −0.084 0.675 4.000 0.000
351 77587 CGPG8107 SENSE 0.415 0.112 4.000 0.000
317 77610 CGPG6805 SENSE 0.267 0.180 4.000 0.000
356 77832 CGPG8976 SENSE 0.153 0.579 4.000 0.000
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 347 showed improved cold stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.
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 may be 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.
A list of recombinant DNA constructs that improve shade tolerance in plants illustrated in Table 10.
TABLE 10
Leaf Seedling Petiole
angle at weight at length at
day 23 day 23 day 23
PEP Construct Nomination RS P- Delta P- Delta P-
SEQ ID ID ID Orientation mean value mean value mean value
251 70755 CGPG4473 SENSE NA NA −0.751 0.017 −0.307 0.059
244 70995 CGPG4154 SENSE NA NA 0.251 0.070 0.008 0.944
275 72709 CGPG5568 SENSE NA NA −0.903 0.070 −0.608 0.019
210 72724 CGPG2292 SENSE NA NA −0.707 0.098 −0.638 0.020
272 72774 CGPG5518 SENSE NA NA −0.268 0.110 −0.195 0.087
258 72813 CGPG4977 SENSE NA NA −0.469 0.092 −0.186 0.054
294 73020 CGPG5791 SENSE NA NA −1.443 0.010 −1.265 0.010
288 73133 CGPG5714 SENSE NA NA −0.852 0.009 −0.896 0.012
260 73628 CGPG5025 SENSE NA NA 0.311 0.011 0.317 0.146
271 73766 CGPG5432 SENSE NA NA −0.988 0.048 −0.671 0.088
314 74435 CGPG6737 SENSE NA NA −0.735 0.042 −0.755 0.049
331 74858 CGPG7371 SENSE NA NA −0.667 0.010 −0.808 0.008
330 74863 CGPG7316 SENSE NA NA 0.176 0.060 0.168 0.515
332 74933 CGPG7457 SENSE NA NA −0.100 0.801 −0.533 0.094
340 75657 CGPG7828 SENSE NA NA 0.193 0.059 0.244 0.085
337 75685 CGPG7767 SENSE NA NA −0.917 0.075 −0.974 0.049
267 75822 CGPG5210 SENSE NA NA −0.267 0.194 −0.297 0.090
324 76171 CGPG7206 SENSE NA NA −0.350 0.065 −0.570 0.049
254 76202 CGPG4788 SENSE NA NA −1.153 0.009 −1.713 0.019
326 76467 CGPG7267 SENSE NA NA −0.484 0.177 −0.743 0.088
359 76961 CGPG9080 SENSE NA NA −0.425 0.098 −0.265 0.051
360 76973 CGPG9081 SENSE NA NA 0.333 0.052 0.248 0.003
363 77103 CGPG9134 SENSE NA NA 0.363 0.093 0.198 0.072
374 77157 CGPG9186 SENSE NA NA 0.287 0.044 0.107 0.086
369 77165 CGPG9155 SENSE NA NA −0.081 0.413 −0.572 0.012
368 77176 CGPG9148 SENSE NA NA −0.577 0.025 −0.651 0.085
365 77187 CGPG9141 SENSE NA NA 0.335 0.002 0.263 0.046
385 77209 CGPG9278 SENSE NA NA 0.671 0.027 0.451 0.011
383 77220 CGPG9271 SENSE NA NA 0.463 0.008 0.231 0.228
225 77334 CGPG3638 SENSE NA NA 0.531 0.067 0.336 0.031
349 77349 CGPG8083 SENSE NA NA 0.133 0.001 0.035 0.781
319 77517 CGPG6952 SENSE NA NA −0.843 0.142 −1.458 0.062
320 77518 CGPG6953 SENSE NA NA 0.354 0.007 0.380 0.010
307 77609 CGPG6326 SENSE NA NA 0.252 0.087 0.211 0.145
For “seeding weight” and “leaf angle”, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2.
For “petiole length”, if p<0.05 and delta <0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta <0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in 1 to SEQ ID NO: 246, 295, 303, 325, or 375 showed enhanced shade tolerance by the second criteria as illustrated in Example 1L and 1M.
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 uE/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.
A list recombinant DNA constructs that improve early plant growth and development illustrated in Table 11.
TABLE 11
Root Root Seedling
length at length at weight at
day 10 day 14 day 14
PEP Construct Nomination Delta P- Delta P- Delta P-
SEQ ID ID ID Orientation mean value mean value mean value
227 19774 CGPG3920 SENSE 0.488 0.097 0.307 0.100 0.550 0.163
229 19956 CGPG3972 SENSE 0.248 0.062 0.154 0.214 0.284 0.001
232 19973 CGPG4026 SENSE 0.326 0.047 0.066 0.577 0.444 0.012
302 70812 CGPG607 SENSE 0.704 0.037 0.546 0.031 0.740 0.033
236 70915 CGPG4058 SENSE 0.292 0.005 0.278 0.008 0.331 0.114
230 70948 CGPG3990 SENSE 0.255 0.269 0.093 0.229 0.375 0.026
234 70950 CGPG4052 SENSE 0.106 0.577 0.124 0.374 0.239 0.041
235 70962 CGPG4057 SENSE 0.171 0.075 0.144 0.226 0.283 0.015
241 70963 CGPG4121 SENSE 0.149 0.057 0.112 0.074 0.062 0.513
242 70994 CGPG4122 SENSE 0.198 0.063 0.121 0.114 0.240 0.124
244 70995 CGPG4154 SENSE 0.132 0.052 0.101 0.006 0.198 0.045
249 71328 CGPG4454 SENSE 0.209 0.136 0.251 0.122 0.445 0.093
268 72001 CGPG5221 SENSE 0.183 0.060 0.142 0.042 0.195 0.149
274 72753 CGPG5540 SENSE 0.113 0.045 0.053 0.348 0.081 0.730
282 73105 CGPG5656 SENSE 0.046 0.577 0.138 0.057 0.298 0.142
292 73136 CGPG5764 SENSE 0.212 0.077 0.133 0.027 0.330 0.037
265 73256 CGPG5194 SENSE 0.123 0.503 0.371 0.135 0.623 0.069
313 74420 CGPG6712 SENSE 0.249 0.085 0.093 0.358 0.363 0.042
316 74566 CGPG6796 SENSE 0.169 0.003 0.147 0.114 0.173 0.171
304 74662 CGPG6185 SENSE −0.205 0.337 0.009 0.878 −0.009 0.958
312 74688 CGPG6653 SENSE 0.221 0.083 0.059 0.358 0.295 0.163
329 74862 CGPG7308 SENSE 0.201 0.122 0.044 0.515 0.383 0.006
325 76178 CGPG7225 SENSE 0.217 0.018 0.093 0.364 0.240 0.234
322 77069 CGPG7163 SENSE / / / / 0.641 0.074
376 77124 CGPG9207 SENSE 0.337 0.121 0.256 0.045 0.762 0.034
371 77155 CGPG9170 SENSE 0.181 0.262 0.265 0.035 0.419 0.000
367 77164 CGPG9147 SENSE 0.589 0.068 0.369 0.066 0.899 0.051
370 77166 CGPG9163 SENSE 0.383 0.025 0.391 0.006 0.787 0.019
372 77180 CGPG9180 SENSE 0.442 0.058 0.267 0.031 0.402 0.339
362 77186 CGPG9133 SENSE 0.448 0.022 0.383 0.079 0.654 0.057
365 77187 CGPG9141 SENSE 0.453 0.043 0.227 0.127 0.660 0.013
375 77195 CGPG9205 SENSE 0.221 0.114 0.188 0.160 0.259 0.064
385 77209 CGPG9278 SENSE 0.360 0.092 0.181 0.203 0.594 0.047
382 77266 CGPG9259 SENSE / / / / 0.470 0.020
384 77268 CGPG9275 SENSE 0.353 0.111 0.170 0.244 0.386 0.041
378 77273 CGPG9220 SENSE 0.255 0.070 0.023 0.586 0.510 0.141
297 77312 CGPG5927 SENSE / / / / 1.006 0.065
393 77409 CGPG9345 SENSE 0.410 0.027 0.396 0.011 0.461 0.040
389 77430 CGPG9322 SENSE 0.209 0.183 0.211 0.247 0.399 0.092
390 77432 CGPG9335 SENSE 0.342 0.113 0.285 0.122 0.425 0.055
391 77433 CGPG9341 SENSE 0.204 0.120 0.222 0.041 0.382 0.085
392 77444 CGPG9344 SENSE 0.185 0.068 0.227 0.003 0.276 0.002
387 77451 CGPG9309 SENSE 0.266 0.097 0.208 0.042 0.014 0.981
388 77452 CGPG9311 SENSE 0.286 0.021 0.182 0.123 0.284 0.201
327 77536 CGPG7272 SENSE 0.121 0.389 0.147 0.026 0.384 0.027
342 77549 CGPG7933 SENSE 0.349 0.006 0.207 0.032 0.383 0.022
344 77568 CGPG8012 SENSE 0.312 0.020 0.157 0.015 0.359 0.071
351 77587 CGPG8107 SENSE 0.151 0.128 0.223 0.014 0.370 0.030
318 77618 CGPG6810 SENSE 0.179 0.018 0.104 0.276 0.154 0.388
334 77816 CGPG7529 SENSE 0.145 0.400 0.171 0.142 0.312 0.181
336 77821 CGPG7737 SENSE 0.114 0.406 0.126 0.208 0.311 0.079
343 77917 CGPG7986 SENSE 0.136 0.083 0.162 0.011 −0.242 0.458
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 252, 309, or 310 showed improved early plant growth and development evidenced by the second criteria as illustrated in Example 1L and 1M.
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, Ohio) consisting of 30-40/o 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.
A list of recombinant DNA constructs that improve late plant growth and development illustrated in Table 12.
TABLE 12
Rosette dry Rosette Seed net dry Silique dry Silique
weight radius at weight weight length at
PEP at day 53 day 25 at day 62 at day 53 day 40
SEQ Construct Delta P- Delta P- Delta P- Delta P- Delta P-
ID ID mean value mean value mean value mean value mean value
394 12313 −0.075 0.489 0.654 0.000 0.157 0.174 −0.024 0.275
221 18258 −0.494 0.017 −0.183 0.083 0.533 0.006 −0.269 0.234 0.056 0.009
246 70657 0.373 0.040 0.187 0.037 0.355 0.081 −0.187 0.222 −0.003 0.972
247 70660 −0.010 0.936 −0.072 0.431 0.445 0.026 0.116 0.369 0.145 0.045
216 71538 −0.167 0.168 0.017 0.686 0.521 0.005 −0.196 0.113 0.023 0.557
269 72056 0.563 0.008 NA NA −0.251 0.159 −0.289 0.314 −0.046 0.314
279 73057 −0.266 0.035 0.167 0.114 0.396 0.034 −0.304 0.085 0.023 0.543
311 73568 −0.140 0.115 −0.085 0.340 0.553 0.019 0.344 0.010 0.040 0.458
222 19193 −0.296 0.110 0.081 0.385 1.076 0.011 NA NA 0.073 0.001
262 73242 0.159 0.132 0.133 0.050 1.074 0.009 0.652 0.002 0.045 0.048
309 73433 0.477 0.026 0.113 0.120 1.140 0.006 0.552 0.016 −0.009 0.872
316 74566 −0.387 0.023 0.104 0.047 1.213 0.003 −0.169 0.130 0.012 0.598
338 75654 0.604 0.012 −0.006 0.899 −1.193 0.004 −0.258 0.057 −0.004 0.962
361 77150 0.611 0.003 −0.098 0.115 −0.363 0.011 −0.398 0.203 0.001 0.990
327 77536 0.043 0.692 0.089 0.040 0.725 0.015 0.419 0.016 0.075 0.105
334 77816 −0.324 0.021 0.005 0.954 1.207 0.000 −0.038 0.679 0.039 0.378
If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.
K. Low 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 BASTA resistance, 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. The leaf color raw data were collected on each plant as the percentages of five color elements (Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using a computer imaging system. A statistical logistic regression model was developed to predict an overall value based on five colors for each plant.
A list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants illustrated in Table 13.
TABLE 13
Root length Leaf color Rosette weight
PEP Construct Nomination Delta Risk score P- Delta P-
SEQ ID ID ID Orientation mean P-value mean value mean value
200 10422 CGPG117 ANTI- −0.435 0.008 1.311 0.021 0.050 0.396
SENSE
206 12602 CGPG170 SENSE −0.004 0.941 −0.052 0.895 0.115 0.048
202 13235 CGPG1288 SENSE −0.047 0.267 −0.085 0.864 0.128 0.068
203 13411 CGPG1301 SENSE 0.323 0.202 −3.233 0.126 0.174 0.044
201 13485 CGPG1226 ANTI- 0.311 0.096 1.162 0.038 0.241 0.019
SENSE
205 13846 CGPG1542 ANTI- −0.267 0.190 1.506 0.004 0.176 0.281
SENSE
212 16610 CGPG2499 SENSE −0.037 0.677 1.325 0.004 −0.004 0.918
211 17805 CGPG2457 ANTI- −0.348 0.016 3.523 0.067 −0.105 0.116
SENSE
218 18231 CGPG3274 SENSE −0.207 0.117 0.925 0.062 −0.093 0.094
224 18354 CGPG3534 SENSE 0.104 0.070 −0.407 0.124 0.189 0.048
252 70684 CGPG4588 SENSE 0.379 0.001 −3.676 0.003 0.328 0.030
223 71301 CGPG3528 SENSE −0.394 0.027 1.269 0.042 −0.128 0.026
250 71329 CGPG4456 SENSE −0.169 0.230 0.475 0.074 −0.365 0.337
258 72813 CGPG4977 SENSE −0.361 0.193 0.521 0.076 0.013 0.120
309 73433 CGPG6429 SENSE 0.129 0.201 −0.091 0.794 0.186 0.002
300 74349 CGPG5967 SENSE −0.139 0.019 0.592 0.080 0.045 0.080
313 74420 CGPG6712 SENSE 0.612 0.043 −3.086 0.063 0.173 0.002
298 75237 CGPG5941 SENSE −0.097 0.237 0.948 0.031 0.074 0.136
333 75379 CGPG7520 SENSE 0.000 0.998 0.529 0.048 −0.017 0.755
335 75434 CGPG7636 SENSE −0.336 0.039 1.208 0.076 −0.109 0.107
341 75622 CGPG7833 SENSE −0.503 0.001 1.776 0.007 0.096 0.497
339 75692 CGPG7823 SENSE −0.055 0.165 −0.377 0.328 0.132 0.083
301 76422 CGPG6040 SENSE 0.197 0.178 −0.263 0.734 0.162 0.094
359 76961 CGPG9080 SENSE −0.361 0.136 1.472 0.013 −0.190 0.066
369 77165 CGPG9155 SENSE 0.366 0.041 −1.982 0.143 0.253 0.099
303 77322 CGPG6178 SENSE / / 1.691 0.060 −0.185 0.052
207 16322 CGPG1828 SENSE / / −0.021 0.982 0.116 0.043
356 77832 CGPG8976 SENSE / / 2.472 0.011 −0.028 0.445
For leaf color and rosette weight, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2. For root length, if p<0.05, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2, the transgenic plants showed a trend of trait improvement as compared to the reference.
Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 198 or 327 showed improved tolerance to low nitrogen condition evidenced by the second criteria as illustrated in Example 1L and 1M.
L. Statistic Analysis for Qualitative Responses A list of responses that were analyzed as qualitative responses illustrated in Table 14.
TABLE 14
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 Table 14. 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. Two criteria were used to determine a transgenic with enhanced trait(s). Transgenic plants comprising recombinant DNA disclosed herein showed trait enhancement according to either or both of the two criteria.
For the first criteria, the risk scores from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., 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. If p<0.05 and risk score mean >0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference.
For the second criteria, the RS from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The RS with a value greater than 0 indicates that the transgenic plants from this events perform better than the reference. The RS with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and risk score mean >0, the transgenic plants from this event showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed improvement in the same response, the transgene was deemed to show trait enhancement.
M. Statistic Analysis for Quantitative Responses A list of responses that were analyzed as quantitative responses illustrated in Table 15.
TABLE 15
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 from cold shock tolerance screen
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 day14 Early plant growth and development
screen
Rosette dry weight at day 53 Late plant growth and development screen
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). Two criteria were used to determine trait enhancement. A transgene of interest could show trait enhancement according to either or both of the two criteria. The measurements (M) of each plant were transformed by log2 calculation. The Delta was calculated as log2M(transgenic)-log2M(reference). If the measured response was Petiole Length for the Low Light assay, Delta was subsequently multiplied by −1, to account for the fact that a shorter petiole length is considered an indication of trait enhancement.
For the first criteria, the Deltas from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. 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. If p<0.05 and risk score mean >0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference.
For the second criteria, the delta from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The Delta with a value greater than 0 indicates that the transgenic plants from this event performs better than the reference. The Delta with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and delta mean >0, the transgenic plants from this event showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed enhancement in the same response, the transgene was deemed to show trait improvement.
Example 2 Identification of Homologs A BLAST searchable “All Protein Database” is 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” is 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 is queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 198 through SEQ ID NO: 394 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 is 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 is queried using amino acid sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST searchable database is 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 is 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: 395 to SEQ ID NO: 19938. These orthologs are reported in Tables 2 as homologs to the proteins cognate to genes used in trait-improving recombinant DNA.
TABLE 2
SEQ ID NO: homolog SEQ ID NOs
198: 3331 2914 8408 6836 10845 14799 13562 8848 2509 13003 875 10945
4176 7318 4820 19156 19137 14942 17166 13809 3506 3410 1343 8019
7882 6490 3550 4999 3130 12635 8774 10864 17857 18089 16263 7295
10882 18252 3728 14833 11485 15463 18826 2924 18723 1446 2812 7847
3395 4209 15768 10679 18379 14040 3974 1087 11574 6329 5482 5609
6441 5537 17042 4716 4516 12643 11384 12736 8137 1344 19473 8452
4094 2460 18071 16891 5620 2364 7018 5200 11131 18133 12170 2731
1687 5014 18511 9343 19283 6682 8123 8124 10597 10622 10608 10600
16221 4280 11494 4867 9035 9507 7969 19159 19425 5248 17367 8135
8773 1287 2671 9365 18729 13828 8754 7170 14529 16604 8341 1135
6224 9739 11212 12962 4331 13555 14356 14357 14783 7509 2032 4692
5243
199: 15320
200: 7710 14519 16336 7019 7872 7719 18299 10453 4650 15008 10089 699
15645 10533 11751 12924 10526 5313 17532 8825 6941 7743 4155 14541
9797 16905 8304 12080 19803 3087 1397 7534 5547 2423
201: 11777 6444 1416 4353 15071 3413 14526
202: 19073 6651 15740 7399 15791 2977 7789 9248 14697 15923 12131 5470
965 11100 11069 12624 12892 3580 5270 18663 2916 1684 8107 4240
19240 18582 19054 15775 7301 17416 10566 16351 13846 16386 18431 4439
4056 8159 8292 12484 7316 3763 4783 18427 15501 16164 7762 4015
4556 18955 12061 8636 9665 9713 10212 17555 5330 12850 16436 19045
16034 16692 2571 11676 12666 11853 5348 8771 12218 3157 10749
203: 5608 2382 17993 16598 7505 6976
204: 11369 13527 10897 6893 6962 11592 837 5116 19669 1655 7244 2623
2591 16504 17797 4636 15133 4141 7112 7939 6898 15965 16706 16979
11784 16115 9620 15750 14652 3008 7838 14787 4563 1873 15811 7271
4317 16603 1715 14288 11341 6188 1546 5683 5225 3033 6069 11268
15981 15490 4181 8633 6058 7993 6763 9671 14892 17296 990 8184
2935 8536 11232 12598 12638 5112 10787 1107 13858 5864 16398 16380
17071 16520 1973 5455 13852 19228 1695 17402 2865 17120 2507 5601
14952 1462 6616 6973 3092 1382 7755 3870 9765 5711 1257 6119
15310 3792 15970 16032 14584 7282 15140 7273 3013 15249 13253 13241
2265 17700 18976 4009 17354 2223 17559 3151 6320 17010 8939 11258
734 573 15015 3050 16344 3931 11238 7691 11499 1112 4825 8344
13519 11669 5640 13588 5473 15108 7210 17617 5994 5980 16627 17906
5876 6079 18894 6956 12887 14694 10968 1061 2778 6096 10570 680
974 3120 16300 5885 7626 13881 4244 4233 15039 4486 3103 3115
7654 11890 12359 10800 9409 12657 8977 9182 17818 2942 9780 9899
18496 4118 18188 10437 13907 17312 15730 13906 2603 12770 4837 12953
16663 9237 9546 16293 18238 6022 13908 10266 9180 5538 15536 10370
15958 5217 8154 13095 15972 14841 15262 12702 10766 15960 18726 19395
10345 5964 13064 3946 17225 17262 17220 17266 17224 17252 5465 5495
12800 12814 12758 3021 12400 12492 16461 13638 5616 17449 8397 17258
5669 5675 6852 9796 17601 17569 9775 17664
205: 14023 6117 12539 6696 18601 1463 19893 6621 9577 9166 17890 17889
528 5413 18313 2784 8102 3988 890 19106 571 6791 2402 912
206: 17912 8799 6566 12137 4356 5803 5253 6078 9123 17523 12392 17806
6917 19039 7350 17767 11311 17082 15535 19392 11233 470 8318 14963
16736 8867 14135 5246 4674 2124 9901 13302 15459 18997 2954 1026
667 7561 16841 8092 9084 4065 1836 13713 13697 17640 7422 19580
4565 15193 526 13108 7065 11703 11362 3633 19430 6221 17433 15616
10082 10189 3089 15238 6633 8106 16180 15317 16364
207: 14575 8831 11211 14985 17619 18829 2521 5219 15413 15835 1204 6569
4661 4457 6066 14578 932 13694 7541 4813 15707 19563 1326 16107
5009 7518 14825 13777 6175 15925 13617 5765 14838 9091 10776 15679
5909 19373 756
208: 19581 18655 16310 7497 6382 8261 936 7358 8883 4866 2146 8601
2932 1626 10354 15192 11509 9909 13503 488 17477 14483 16941 2824
1072 4989 14179 4883 13751
209: 3493 16588 7031 5155 11150 17294 5241 10621 5786 5591 16509 2021
1324 12454 15061 1670 15143 16506 17002 8741 18091 4013 5959 19370
4239 12885 12174 3709 3431 9596 3378 1018 5963 13212 3150 2952
1619 19861 19114 15446 19582 12663 19603
210: 17777 843 11020 1337 18495 1493 6396 15336 14087 2568 4517 1157
10741 4553 14999 425 4258 17388 5327 748 11691 10174 17805 16628
10117 7980 8902
211: 9637 17261 13232 7387 10506 19067 12021 872 11620 13603 841 1450
2848 16911
212: 14398 1558 18339 13428 13431 4182 16260 10619 6127 12005 7704 5064
13072 4395 9032 19907 9588 478 12782 9917 13100 5202 10562 2044
4452 10775 3543 14840 14071 14020 10406 14218 1797 16955 3879 10312
12432 14824 7082 14554 15744 1508 15866 16232 6797 7077 5816 3597
3596 3311 15022 881 2638 906 12037 12039 3458 2270 8812 3217
9466 18102 610 9042 2431 18349 16650 14418 2251 4235 6826 1244
16651 13004 7852 14435 9553 9262 15603 4340 9235 1413 10538 8296
17223 16048 16071 2120 19847 10420 11239 18968 2995 7551 15854 12345
5685 18180 2381 4814 2159 16862 8677 13466 11435 6649 6015 8097
11230 16025 9440 1353 16151 16249 15930 11226 9398 2870 12373 15043
11765 10145 11071 285 9270 9537 9095 2350 13362 5496 7924 11756
2624 7967 6715 15074 17068 9275 10177 11007 14857 8951 12182 11643
13590 7684 1740 929 910 9093 10018 13344 16978 17544 6305 11378
1393 14108 15937 16631 11803 13126 11969 18524 9644 8425 8421 8420
10755 10757 16696 15994 1143 3549 5868 5126 7506 12547 12669 19032
4707 4710 4708 4732 4736 4733 8772 4730 4711 3385 1831 2528
2304 15098 13223 15197 11673 4386 11884 2336 14693 9118 9633 14548
12725 2399 18359 11011 14692 5652 5434 9755 9513 2681 4364 12903
19777 13525 4326 6859 6088 11611 3068 13940 3578 14440 8582 9826
19618 18012 17233 10057 18222 15021 9371 5985 14888 14001 1279 7826
14232 18172 7425 11502 15565 17431 8317 10294 17742 5564 5531 18144
7400 10835 14389 13461 13857 7113 16444 4722 2115 7276 18254 1237
5256 13951 2920 16719 1045 15371 19022 684 8004 2781 19811 19766
3279 18712 4940 14312 6426 10076 15772 7906 19836 11219 18985 2673
3933 14263 11860 1907 4672 15599 13737 4944 2252 15415 11543 3433
7746 17114 4115 4881 9445 14696 9525 14484 16112 5392 10428 1400
9349 7616 18930 2533 12585 10497 10236 18415 4874 19793 4633 12544
1995 17255 6130 10439 15261 6768 15985 17752 8504 12863 7487 14875
12020 2036 18127 9264 3628 5019 7498 15865 1448 9636 2840 3161
6207 2644 1059 2065 10031 2171 16927 4747 18701 19271 4174 1389
9893 12527 9018 10035 5039 10698 13943 7894 12671 5651 19586 12810
1254 3519 16114 4286 13948 14493 8294 14606 2586 14069 7712 16988
737 16797 16826 18966 5145 17610 6304 8895 7744 9280 9284 10647
16558
213: 12662 5295 19093 10971 15359 1163 18513 889 2151 3536 7105 3292
8466 18205 3579 1504 19486 13768 18047 6871 7174 11029 11505 17782
5697 18822 4892 12656 1284 3062 4697 5993 18343 18790 3057 7014
12265 10981 15204 11631 17704 17941 10633 19776 933 10363 8446 17800
9822 2953 13693 19862 14551 15692 17673 6081 5049 12581 6910
214: 15345 19484 4871 3871 6695 3993 11459 9199 15899 13821 14206 19489
13765 712 19072 11649
215: 1607 15669 13168 19044 790 9515 4511 13776 11573 13000
216: 4929 17143 10058 17506 15064 3332 5268 6968 16252 15949 6180 3000
14937 18454 13071
217: 6807 19716 480 14021 4179 12236 6337 12237 6338 6336 12234 4815
2261 18555 4706 7836 9176 18851 3443 9382 9439 8870 16691 16646
11516 11500 11515 11498 16670 16660 16683 11513 11496 16668 16664 11519
16686 11479 11481 7146 16643 16659 4850 17951 7144 4824 7163 4851
11234 4860 17956 7160 17977 4827 4826 4859 7147 11475 7165 17928
17958 4853 11473 7143 11477 5129 18893 18914 17526 17529 17548 17547
17549 18917 13686 19089 13683 4938 16999 13666 4494 12074 17097 12093
16874 12095 15878 17159 17154 17156 17153 5681 15126 10052 19712 4288
15399 742 11607 15654 7006 8041 715 2713 11522 7695 3581 7865
3776 15738 19821 17259 3948 13388 17427 15523 14216 8711 15423 19060
16814 14298 18720 7885 3967 9710 3568 3573 3575 8503 15486 12932
7870 989 3595 17756 9202 520 11205 14771 3574 2443 8036 19537
14641 6739 15375 2400 6753 3336
218: 13099 6217 4454 4699 11292 4702 1554 6467 15339 15722 11035 12964
16317 13914 17486 18809 14651 4189 9581 14957 2871 1561 17618 3835
14955 3868 9158 13663 10042 7575 19824 6200 10522 3600 4064 14590
12731 7201 19572 9060 4035 6255 3457 11320 7986 8818
219: 19325 10450 4487 12312 2564 10096 5641 1947 4367 6964 18031 1012
11041 15078 12255 5761 5061 9832 16834 18348
220: 8238 19207 18657 1358 2734 9320 13526 12631 17660 16548 15528 18501
18049 15274 19470
221: 2724 17089 9639 9208 17722 6817 11848 18904 13673 2347 11585 3054
7486 6056 1290 14932 14930 12636 14230 1572 15708 18912 18177 1699
10460 2090 5583 18802 606 5929 5041 19148 14224 9144 2643 17144
14142 14674 16275
222: 14820 18626 11591 649 3005 3037 5453 5430 14248 2371 4626 11661
9784 10560 16219 11699
223: 17739 13288 4139 15466 19773 753 1277 14333 4211 14510 662 7930
18703 3268 19400 17533 2302 9078
224: 13859 17816 12692 10417 7051 5444 12975 1611 7050 17105 5023 3397
13149 14751 10564 7331 7329 11868 9210 9211 18860 18879 14568 17920
14497 12723 19352 10587
225: 11265 17061 11261 17058 11267 17064 8014 8033 8018 508 8035 8038
8013 8017 3554 13411 12545 12528 11997 12566 11922 12520 11958 12525
12522 12553 11961 11979 5030 3464 3910 18901 14601 14600 840 15664
15668 15121 15142 15119 15686 15690 15657 15688 15146 15164 15716 15148
15656 11191 13623 12732 5050 5029 5011 5055 5035 5036 5032 5081
5058 5013 5078 5026 5059 5077 5008 5053 5076 11939 11976 12541
2898 17207 5179 5115 13674 13423 16078 9689 11174 5252 17279 17277
11177 1867 1865 1823 1844 1842 1793 1824 1820 10671 3139 1123
5805 12934 12755 7504 1091 17904 17909 17926 17902 17938 17907 17929
10598 17935 17933 5298 17980 10601 5383 14050 6585 11379 16583 17978
17814 4838 17812 2633 3257 3189 774 12008 9988 14268 18392 12567
14920 12980 11959 7682 7685 13235 4127 3740 3766 3770 3769 3795
14966 457 8521 19464 4847 13386 5024 13387 13401 5957 6571 7932
8369 10496 10492 10490 10495 10517 7943 4958 4956 4923 4986 4961
4951 5007 4937 4934 4984 4933 4924 4921 4936 4979 4932 4981
4988 18200 18196 7176 4739 5479 8351 7177 12886 9521 6464 17681
14504 907 16793 1869 14509 8882 19727 8798 1197 3419 14168 12846
15739 1115 14165 19715 492 8873 1119 497 1194 3251 1116 8876
13982 12860 14430 451 14969 6102 4358 2650 2649 11790 12925 5882
19696 9551 3867 19796 18933 18929 833 4908 14217 4852 4858 16064
14208 10736 4886 14214 16062 14172 10735 14210 10720 1885 14187 14190
14184 14207 14703 14707 14709 14706 14390 7658 7659 7661 2582 13794
5204 17305 16541 8914 16959 17283 9043 3924 2696 8656 6648 2675
18504 17311 6052 9425 9406 9410 9411 6051 9452 6033 15964 6267
6031 8489 8463 8865 9427 6268 8490 8493 15602 15621 14616 15649
15652 15606 11723 17247 9423 15595 15624 16148 2240 7428 11722 11363
2225 2196 5480 15579 15626 16142 7413 5483 11741 16145 16162 11725
5481 18072 10973 12451 12340 15511 12185 4348 12754 8722 14544 6171
10732 9492 19795 12165 15082 6617 9613 739 19520 2497 14005 11128
18795 14331 14329 14334 16439 12070 4053 3942 2387 2391 2394 755
754 2950 752 2377 19819 17119 7118 1360 2273 11458 13758 11215
11274 11229 13178 5110 16239 12884 18765 16637 19693 16831 17824 10810
15395 15420 17470 10005 16397 16394 14711 14728 15503 4401 801 12244
12292 12308 12219 9033 16763 14392 16804 16785 9566 16806 14409 14391
16762 9027 9568 9029 16788 9009 16780 14410 16781 16782 16802 3330
3357 3326 9001 3358 5789 14387 14412 3360 3356 1643 1805 14061
15787 5632
226: 19514 17912 10095 10091 10252 15927 5360 18385 12313 19719 12823 5182
1491 8631 4666 2562 9358 10461 3952 19610 9255 7782 16516 5161
6343 4894 5577 16734 1381 16378 14552 12707 11907 7651 10404 17767
2417 11311 17082 17741 11253 11254 7473 13718 6269 3242 7085 5224
8787 16268 19692 10769 8867 1434 16082 19530 14789 13615 18997 2954
9175 8092 9082 1813 1815 1817 639 3883 15427 2035 4435 3601
414 7005 8000 1812 1131 1455 6221 11231 10817 15601 16270 6118
2193 17500 14186 9141 3449 3089 13886 15786 15238 2001 3945 14973
16180 15317 18083 18695
227: 17372 8093 5527 2439 13784 14768 10702 11535 7750 761 18735 5946
17426 17988 10652 2928 18398 2009 9559 5094 12948 8522 1305 17785
5852
228: 2790 5299 15433 5501 18128 16466 14960 6564 8782 4397 19662 16008
10829 19118 18086 3428 17943 10935 15806 8101 1184 4968 6059 10388
229: 7539 3499 12437 19471 14158 11575 6399 15966 17187 13512 7688 2131
10858 4169 19567 7834 7585 14073 3954 1991 12940 7756 8167 18994
9449 15859 4341 17025 14373 12191 12982 983 13764 19774 16247 12665
12664 2619 18875 7515 11785 2849 10913 10717 4653 11887 905 1641
3563 6183 16765 17773 4166 4694 14328 11986 12084 1625 12695 9681
14082 17341 15047 17498 3530 3531 7241 7972 4790 15643 14365
230: 12430 9914 10470 14976 6139 18895 8072 18544 7257 6437 15066
231: 7185 13859 12150 12692 5555 6319 14735 16754 3121 2208 14915 18531
2991 7950 2419 4378 18922 4377 18705 17105 5023 3397 2203 5350
895 18953 5090 9210 9211 14568 19723 12433 3877 17892 19476 11685
9653 9066 16392 7989 5056 6545 19418
232: 10845 14019 10546 14942 7302 8774 13992 10864 7129 12564 10723 2440
19878 3172 18252 12532 9942 2129 18826 18723 17324 19791 8580 3395
5915 9604 7572 16359 8047 3408 5482 12915 9969 16513 1344 16044
19210 18748 11940 14402 19311 5620 2364 19283 5249 3646 17251 17844
14459 5315 9268 16190 19143 15124 418 3076 16204 17414 18692 13164
8191 10636 13894 3423 2842 1173 7663 13250 18208 14090 19680 15815
4805 10584 1287 15495 9256 2229 10166 19534 11163 2292 2687 19082
9365 2465 5381 18660 3073 9545 17750
233: 19344 12031 1229 15123 14123 19501 7767 5944 11173 4596 11614 10841
14129 2393 7360 621 14382 11612 8136 14668 7577 1652 2491 3392
2160 4767 17696 6615 12377 3471 2985 17194 17177 10988 5084 9695
16519 11445 3213 10846 16453 19346 18274 17478 17798 15663 6823 5131
14272 3777 19911 19724 6132 19722 14803 17148 1605 11787 14359 15796
645 10159 13264 12931 6357 4621 16774 18913 5746 7161
234: 11849 7119 1272 13625 6244 7780 7229 640 15779 14967 5388 14480
1349 17525 12768 10327 8976 5113 5895 15006 4428 16619 4613 3393
18954 4451 3799 17712 12488 15863 18697 18482 7844
235: 8664 9584 8904 18497 19265 8237 2617 7729 17728 8659 13842 11016
9197 10827 5351 3224 7379
236: 12797 7459 12082 1618 7740 16856 7623 8994 7602 19549 12596 7644
18396 13883 7266 18758 3328 18998 1660 12434 2139 19899 12252 5883
3266 19587 11474 5870 5443
237: 14746 16460 7754 9978 16450 18053 18050 18466 8866 10337 8245 1604
8568 13606 19627 18283 15611 10648 13871 9910 10297 3025 18486 8908
14474 11654 2349 10867 14488 13901 7336 11511 14836 1906 13400 5568
19527 4351 18319 6689 10519 15977 15760 7605 16652 14204 14665 727
9225 12804 2776 11108 14258 1882 4357 1972 8324 6435 18239 6728
18983 9555 13551 10797 18458 4622 14064 2771 1818 18410 13553 15912
10854 9475 10873 15530 14816 6283 703 8105 16216 7038 957 11908
13853 10718 11237 5623 10262 12248 6541 7053 4480 14011 17142 6691
7795 17981 2822 1473 3561 4545 13347 1348 11256 6511 11356 13955
6699 6555 6559 3185 16166 17894 9307 6011 10635 1391 13177 16458
702 18335 18740 16787 4175 10950 3412 4193 951 13870 19183 18644
947 16280 6184 13111 3667 10338 4125 14782 18479 18004 19084 12471
16645 866 1895 10049 11122 12521 7132 2660 12759 14534 18429 15615
17611 9347 12704 1525 7492 3451 18373 17125 5972 9777 16039 12350
4073 8962 17221 1961 1958 11156 12899 3908 7429 10977 15040 16234
17004 17293 13355 4977 260 10645 14133 9251 12220 12633 7087 9535
8894 11452 2346 6125 2062 6710 13299 12947 12943 4422 5817 13502
14378 13504 14379 4770 1451 19815 12842 4405 13837 9184 10051 10999
9565 12305 11330 16546 17073 1402 5263 7189 6160 2372 6567 1248
3858 14455 1766 2516 1656 14550 16864 14989 14368 15573 9312 7786
13516 2969 1952 2541 11207 10625 9198 19674 10334 18065 3495 8519
12201 3921 13249 18592 857 17545 11757 15455 2164 789 4350 3117
8649 11708 11463
238: 9324 12427 663 6064 9836 19556 9348 1900 17219 13657 7086 7381
6006 7357 10469 6466 8472 10588 17047 609 1637 10699 6450 14299
15101 10536 18759 2378 18013 9269 3343 12489 11305 18178 2269 16890
10629 18665 15962 9375 9437 16312 6501 11737 6339 10847 5290 4667
5522 18388 1804 11566 2369 15872 12465 7687 6209 11354 15667 15938
18971 18258 3774
239: 13622 9907 10802 1635 19607 8218 11862 18989 19820 1430 15998 4855
18040 3455 2471 6506 7587 11932 17440 16155 13643 12011 13012 11289
14270 17959 10271 5763 19020 5904 7809
240: 19167 14809 6116 11171 10424 1445 13848 1387 18366 9887 3732 10953
2175
241: 939 3338 2461 7962 16158 15297 19138 7398 3035 1767 9983 16494
10256 11165 13953 18376 4113 4116 10457 11995 19241 2063 19340 15435
14028 14611 14366 18488 9171 5494 10438 18123 11822 13093 15526 9516
18346 4375 8716
242: 12077 11851 16904 8919 18979 6443 17768 3571 5105
243: 12862 6311 17138 19420 12939 8147 18538 16023 6580 8709 16080 4700
2940 9554 5556 503 12303 7072 8226 11973 19415 12551 9852 13704
7982 1030 14887 12346 1435 1437 6242 16606 2041 5356 12706 9139
2872 10869 9028 7957 7190 19892 16505 9383 6589 11472 8394 8396
3626 16568 19220 18648 14425 15305 13545 8443 8427 1247 17078 14914
5103 19922 14358 18892 8972 16582 12124 19870 9538 19845 19867 19195
19221 19191 19323 19242 19215 19307 19275 19273 19356 19192 19173 19350
19279 19216 19852 19223 19250 19326 19848 19330 19214 19869 19351 19332
19276 19247 19177 19163 19305 19328 19851 19353 19197 19850 19358 19175
11034 19871 6578 17445 3832 3831 6233 7090 9456 6970 13917 13920
4539 4537 4542 4028
244: 1245 4324 12202 4379 6967 9036 9880 10540 13263 13138 18122 19047
2008 19483 2468 7439 5787 10979 15035 10982 3699 11097 14482 17829
11866 4198 14130 17239 6111 14956 17492 11385 4004 10448 9464 9894
15398 1231 12878 12829 3833 2080
245: 11361 5661 8686 17264 8328 8330 7550 10232 18956 315 8720 1696
5922 16632 14340 11996 8012 4522 16791 10038 5066 4969 4634 17213
14401 7284 2099 6718 8030 14321 1903 15554 15028 12204 3060 19178
11653 9316 17858 18132 18589 9667 17297 5418 17508 15450 6147 5012
18588 18020 862 4920 15050 16525 3064 2884 5607 12297 11396 18052
2912 17060 1697 17604 18870 1732 5968 18761 4044 5088 10593 5242
4527 13682 8054 16503 1962 19380 1623 10664 4597 15319 3478 16945
18584 3108 12724 15546 15764 15754 18354 18416 4890 19678 19130 15532
5213 11374 9753 14702 15217 15921 5855 18752 1904 15721 18203 18156
18864 8700 7560 16924 636 6811 16379 19046 11063 14929 16207 15425
17368 11052 14975 6574 9560 10349 3830 8250 2015 18785 8196 7713
11586 14490 18301 18792 15378 7699 18803 19816 16104 14468 17612 11556
1765 5525 2247 5751 11815 17200 10136 6205 16567 10487 18141 6202
8537 8518 7037 8133 6764 827 4941 818 8356 19365 7588 3017
6400 5637 8416 12444 510 12026 11634 9859 1460 18194 11314 2685
4226 984 19513 8932 12905 4112 18873
246: 13549 2112 6794 8833 12086 2984 5421 7868 9325 19092 9699 19026
1631 4555 804 8588 19422 8685 6218 17573 17039 12147 4754 10543
6834 6733 864 9209 4190 9301 16571 2360 4996 8081 11140 13006
10637 5281 17394 4637 9663 1828 12569 1215 14407 13300 6644 11688
17465 631 16880 3874 16141 975 8011 16154 18389 13579 15094 8944
3735 8021 2288 15334 16083 15801 12760 10618 13208 19889 18999 4870
13710 12132 15259 9267 11470 13144 17588 628 17272 14844 14000 19900
15096 12603 13869 6532 16013 5431 1192 10395
247: 14941 2202 18124 13094 7945 3968 8551 2458 18176 15639 12789 5427
16829 9519 1674 19789 16819 4712 18517 13990 16185 6271 15089 5575
1227 1331 10634 4978 6366 14209 9402 12738 589 6685 16243 2070
14026 12494
248: 4050 14993 16855 19797 5528 9097 18611 19631 18510 8376 8956 10289
11370 13805 18078 19606 8202 15826 16478 19041 5700 6765 8134 14325
10101 16816 4765 16287 5377 18675 7230 7964 876 9597 19802 11403
4833 17530 18265 13417 12779 8885 11923 18564 3790 19147 1406 7066
10573 7108 3783 4727 19785 1458 16375 8415 9522 10729 6071 9047
9113 3248 6378 1799 14779 3630 4294 7608 13707 16321 15368 12743
11299 2413 1584 11214 4012 2710 4268 19546 17625 6940 1273 5755
413 5952 3098 15675 11098 3286 8068 2690 1222 13381 15257 4756
4725 9351 7586 5770 10868 3420 12012 775 7742 8422 6103 15004
16010 12445 13845 17033 16212 3842 19401 4100 8417 13255 1233 5307
7664 11820 4960 4282 2333 17315 12161 4217 10485 8151 16203 1002
11057 5823 9298 1567 411 5686 2787 6556 17045 5742 3090 12019
12930 14231 15548 11694 8266 16248 1579 4806 2747 11349 6186 8303
4499 14141 8094 16001 12109 10916 10761 2142 4592 5947 1880 5294
16111 2594 9869 6575 13083 17382 14364 11324 2341 14545 1596 15428
18963 1929 10976 3736 16709 5662 4501 19083 17865 14066 8045 18782
2866 19741 12401 690 3507 2118 8721 10292 3096 7017 5861 9569
15479 6800 14522 15996 3926 12508 9266 7508 3882 1770 18129 2452
16753 7303 5038 17638 14421 11636 19188 2092 5721 781 700 15887
8872 5320 5466 10259 7441 13041 10874 7249 4514 9725 18158 17609
18140 18293 17059 18509 4888 9306 5484 7091 1826 19360 3124 1056
2563 3177 16370 10946 6177 11856 17835 15766 9377 14858 9666 2881
4713 18553 10081 3260 6030 16206 11892 10319 18672 3859 2663 15443
5089 10992 2411 12439 17434 6053 18213 1552 16896 16303 2170 4062
9729 5506 4195 2496 9435 18250 3643 10322 8871 11520 8307 10814
4172 3616 467 2220 2765 10994 7181 9294 9019 14302 16474 5797
4407 12612 9186 18928 4415 11982 14037 3959 1335 507 14948 9483
17387 6806 8933 14886 13678 506 12462 6785 2667 19322 5005 14242
11045 12403 2390 19573 11726 9949 5099 9853 16159 15920 13256 615
3439 10834 8240 12330 19303 17872 12750 7494 2539 12602 5373 16910
3341 11873 10613 7321 18101 12158 14798 8096 5837 1986 13521 5384
7186 4164 12183 5907 11172 15553 726 4059 6816 6374 16761 7732
6913 1868 15780 18634 16779 6640 3707 4959 10722 8632 18898
249: 8837 9354 459 481 15638 15637 10951 1014 1011 10934 460 456
1750 19841 3504 7770 7490 2370 12273 8627 10651 13584 13749 13618
14339 12485 7233 18749 12207 7057 408 14300 5215 19033 14163 1226
1228 5775 5424 8505 18275 18314 10203 8674 11707 15070 19005 8386
2693 9051 4549 8477 13649 15215 13783 10149 13027 13294 15862 674
9926 18724 12578 8169 1419 6285 15077 2147 14574 6516 1177 13510
9181 2520 4618 7286 7290 7135 6455 7288 9787 11002 551 2679
8942 1701 8928 8930 1097 17269 12778 15439 17593 12347 1453 10948
15909 19108 8061 16885 8964 5642 15311 18841 8881 9067 2902 10703
8544 18226 18698 18197 7766 17357 6778 13002 10083 15189 7944 1067
13701 18247 5067 8858 2375 10780 12562 6618 1888 368 13569 16443
14088 17735 6201 1162 9317 18221 17786 9563 9562 2386 3903 5156
18001 547 12480 13049 2495 8339 18977 12208 19466 10488 15681 11974
4385 19012 8291 16077 16024 5878 19014 8302 17408 16026 16018 16022
4841 9829 9828 17831 4690 2646 9830 13874 14131 9842 9827 9849
8403 9844 9847 9865 9851 8406 8404 6995 7012 8405 4019 10234
7448 17662 1677 19490 2475 6216 13658 5135 4005 982 15959 17990
3508 17774 17778 15478 7855 9687 17808 10782 7592 268 18627 12803
17776 12927
250: 12939 8147 18538 6832 13653 16376 2940 8025 9554 5556 503 12303
11973 12551 9852 1030 10923 12346 1435 1437 2658 15612 16349 9028
13226 19568 6589 11472 4670 17175 1302 15214 13545 992 5103 19327
18189 7434 13340 9873 7090 16545 6970 15069 19166 16647
251: 6603 10030 13559 6740 11390 7765 3614 11615 15027 11616 14213 11329
19168 2567 13201 10961 3308 11492 2325 6423 11527 3741 11560 8630
7463 1786 14408 10585 15696 14167
252: 17378 17392 17375 19787 819 9037 18778 15271 2631 2615 2616 2606
15288 2607 15269 15283 15286 15282 15287 15285 2609 2629 2613 2604
1472 3730 6707 4595
253: 16186 14031 2169 19019 14212 16029 16170 16973 2492 10956 13426
254: 9072 16169 1130 12933 1592 17856 19381 14964 19031 3682
255: 15936 5121 14535 14860 1527 1041 997 4031 18021 12960 12659 6385
17513 15910 6705 15441 15200 3272 16975 18974 12341 5796
256: 15904 2266 1296 6085 10365 4818 7630 6936 18652 4715 9134 15714
2506 5391 14801 12410 18804 2831 19264 7856 15547 14814 17301 17179
18891 11410 1475 11170 7067 17441 12196
257: 1879 1595 12797 7459 8363 12502 12756 5854 465 1425 1893 12362
12596 7644 16328 15689 4599 17970 5443 17245 10708 17790
258: 3828 1761 11325 17218 18307 14003 12997 4456 6236 19270 8499 7402
5087 10067 3938 3953 3939 17913 19475 5664 3918 12870 8678 9700
259: 12266 4307 12661 6842 6841 6413 17238 16962 15778 516 7796 7046
7155 8841 7260 9891 13142 2868 8814 9110 9530 14169 17860 3971
6657 13557 14715 9995 14954 9514 14428 5521 11000 8008 6231 6796
6894 12969 15894 13220 469 19780 17022 2260 17210 1668 1624 17554
9991 2888 10752 8717 1792 7940 17358 7238 580 582 583 2828
4970 4995 18273 1838 4998 4967 8488 14536 1477 13690 10891 14229
7730 15048 9774 18163 9481 10644 2992 6652 4943 7469 6637 19611
6346 6577 5487 15216 8436 7531 19737 9498 7963 8585 17924 10815
10055 9630 14139 8171 9921 12928 12473 13070 793 5190 2500 9328
1480 13432 15272 14189 13728
260: 14746 16460 18053 7754 9978 16450 18050 18466 8866 10337 10391 8245
1604 8568 8843 13606 19627 18283 15825 15808 15611 13871 2627 2601
19077 1485 5898 5916 8835 2570 9910 10297 3025 18486 8908 14474
11654 2349 10867 14488 9940 13901 7336 11511 19746 14836 1906 13400
19527 5568 4351 18319 6689 10519 15977 15760 7605 16652 14204 14665
2776 9225 1972 12804 727 14258 18708 1882 6435 11108 8324 18239
3913 10328 4357 12240 6728 18983 18808 5969 9555 13551 18458 4622
13553 10797 1818 14064 18410 6283 10854 2771 9475 10873 15912 15530
8946 16216 7038 957 13243 13853 11908 12331 10718 11237 5623 10262
12248 6541 17142 7053 14011 4480 2822 17981 7795 6691 3561 1473
17141 4545 13347 1348 11256 6511 11356 8651 13955 6699 6555 16166
6559 3185 17894 9307 6011 10635 1391 13177 702 16458 18740 16787
4175 10950 3412 4193 951 13870 19183 18644 947 18835 6184 13111
3667 10338 4125 14782 18479 18004 19084 12471 16645 866 1895 10049
11122 12521 7132 14534 2660 12759 18429 17611 15615 12704 1525 9347
7492 3451 18373 17125 5972 9777 16039 12350 4073 8962 17221 1961
1958 11156 12899 18769 11606 3225 3930 12985 3908 7429 18285 10977
15040 16234 17004 17293 13355 15499 2062 11463 11416 11708 6710 4422
5817 13502 14378 14379 13504 1451 19815 12842 4405 10999 9565 11330
5263 7189 6160 2372 3858 1248 6567 14455 1766 14550 2516 1656
14989 16864 9312 19300 19408 13516 2556 2969 1952 2541 11207 10625
19674 9198 10334 18065 3495 8519 12201 3921 13249 18592 857 17545
11757 15455 2164 789 4350 3117 14133 12220 7087 11452 6125 4977
261: 6807 480 6337 12236 4678 12917 9439 9382 11513 11500 16670 16646
16691 16660 11516 11515 11496 11498 16668 16683 16686 16664 11519 16659
16643 14322 2737 1932 12955 12317 19334 12435 12976 5809 18730 11183
705 12278 16937 13026 12999 1928 15878 8321 12130 11053 9846 17306
15324 10531 11328 12167 19474 12203 2827 878 9720 4228 5762 4288
13228 10010 14908 1017 679 14784 677 1487 10919 18296 10669 11200
9055 18733 17838 16389 728 715 1375 1396 6901 3743 12599
262: 11852 1409 16572 9074 6600 4427 824 15705 6709 10791 14587 9823
18325 515 12450 18527 604 6043 12455 2071 10310 18162 3176 2577
15203 12952 14467 6645 6698 12807 6113 4709 8824 6274 8311 3411
18810 17940 19805 17162 665 12693 1309 15412 18084 5235 8314 13722
15858 15969 18135 2305 13611 4030 14864 5363 4470 8271 5178 12458
263: 8837 481 15637 10951 10934 19841 460 3504 15638 1014 1011 459
1750 456 12273 4264 8627 13584 13749 19375 12485 18749 7057 408
7233 12207 13068 5215 5515 14163 8781 1228 1226 5775 5424 8505
18275 18314 10203 8674 2693 19005 14053 9051 8386 8477 4549 6597
15215 10149 13783 1372 1370 13027 13294 4287 12578 1419 15077 2147
2905 14574 17504 6516 6455 9181 2520 7135 7288 7290 2679 4618
7286 551 9787 11002 1701 8942 8928 8930 1097 17269 12778 13750
18947 15439 17593 1453 19108 8061 17556 5642 16885 8964 10019 2902
1548 5257 1728 7766 17357 5647 10631 15019 1888 18777 18221 17786
9562 9563 5156 2386 3903 18001 547 12480 13049 8197 2495 8339
19466 12208 18977 11974 4385 19014 8302 19012 17408 8291 16077 5878
16024 16026 16016 16018 16022 4841 9828 9829 9830 9827 14131 13874
9849 9847 4690 2646 9842 17831 8403 9851 9865 8405 8404 6995
9844 7012 4019 15785 10234 7448 17896 1513 13878 7287
264: 10139 8837 1318 481 15637 10951 10934 19841 460 3504 15638 1014
1011 459 456 1750 17473 5931 11424 7490 1243 2370 12273 4264
8627 13584 13749 14339 12485 18749 7233 12207 408 7057 19033 6702
13068 5215 5515 14163 8781 5775 1228 5424 1226 8505 8674 18275
18314 10203 11707 15070 2693 15215 13884 10149 13783 13027 13294 15807
4287 9926 12578 2905 17504 6516 7290 9181 6455 7135 2679 2520
7286 4618 551 7288 9787 11002 1701 8928 8930 8942 1097 17269
15267 13750 18947 15439 17593 9816 11871 13684 1453 19108 8061 5642
10443 12994 4925 6825 16676 7766 13011 12993 13010 17357 9582 4071
15183 1888 11394 1145 18221 17786 9563 9562 18001 3903 2495 17171
19146 18977 12208 8339 19466 10488 15681 4385 17408 19012 8291 5878
16077 19014 8302 16018 16024 16026 16016 16022 4841 9842 9830 9828
8403 4690 2646 9827 9829 13874 14131 17831 9847 9849 9844 9851
8405 9865 8404 6995 7012 4019 7448 538 4587 4576 14863 2709
10770
265: 10339 11051 4273 6693 11042 16475 7274 1052 18146 6194 6195 18166
10865 4396 638 10021 15834 6518
266: 12371 1790 10658 8747 10474 4583
267: 19068 13736 19730 2398 4156 8189 14176 3642 16457 13680 12542
268: 16601 2370 12273 10651 18749 7233 7057 12207 408 14163 1226 1228
5775 5424 8505 18314 10203 18275 8674 11707 14053 19005 8477 8386
7370 6597 2693 9051 4549 13649 15215 10149 13027 9926 8169 1419
6285 2147 15077 2905 17504 13510 7290 7286 7135 7288 2520 6455
9181 9787 2679 4618 551 11002 1701 8930 8942 8928 1097 12778
13750 15439 18947 17593 13684 19108 8061 16885 5642 8964 8881 18841
15311 10703 8544 18226 18698 7766 13011 12993 17357 6778 15189 13002
10083 1067 7944 13701 18247 8858 2375 10780 12562 6618 1888 13569
368 16443 17735 14088 6201 18221 17786 9563 9562 5156 3903 2386
547 12480 18001 13049 8197 2495 8339 12208 19466 18977 10488 11974
4385 8302 19014 8291 16077 19012 16024 5878 16026 17408 16018 16022
16016 4841 4690 2646 17831 9830 9842 9828 9829 13874 14131 9849
7012 9847 9827 8403 9851 9865 8405 9844 8406 4019 10234 7448
538 12803 17808 10782 7592 17774 7855 9687 15478 3508 2475 17776
12927 17662 982 4005 17990 6216 13658
269: 14096 14101 3422 9935 9907 11404 3149 1688 10616 10023 13889 4400
13959 6604 8205 13382 8564 13019 14460 15364 2532 8729 14099 19644
19650 19653 11186 19315 2228 13473 8252 19754 7420 12978 18550 13931
11080 8842 17395 1271 18960 9745 4617 1713 12914 5411 11902 15306
3958 14094 14091 14074 7042 19574 7859 17118 9165 9458 1459 14832
13165 14520 10512 17335 15352 14170 13097 5335 9461 13601 12995 8362
2662 13567 14785 3435 11646 11805 18390 4017 8969 6711 4962 8794
9291 7926 18526 14047 14393 7407 3866 5766 11775 19021 15525 1840
13661 14072 18260
270: 16324 10027 16399 19626 19600 18167 7908 9950 15476 16909 13757 17032
10125 16306 5372 19301 1764 9641 11812 14642 18106 18647 13221 9050
13225 2608 858 9561 5437 14594 5439 6002 12210 12290 5485 6003
13981 16840 17398 15782 13385 4464 894 19838 9505 11810 18088 18145
16187 1329 14353 14351 14349 10819 8846 9429 16618 17714 16634 13050
10112 19589 3140 14486 3444 16943 12766 19496 9070
271: 2051 2869 19263 12625 10041 11778 3745 15945 19372 16735 5498 12285
7206 9395 16559 5318 10659 1955 8048 19028 5197 13490 4306 15568
14829 8639
272: 16396 2024 2104 12134 16012 12790 10384 16093 7594 16424 3265 5193
1401 2076 7270 19843 9331 4976 4079 18320 7860 6861 14631 5218
2559 6330 4039 736 7978 11712 7058 13306 9913 17623 5261 3627
1734 12108 18532 10878 3240 10925 8343 13484 10007 17652 6295 15677
4058 5272
273: 12662 15327 19093 953 5512 10971 2513 18064 17932 1221 19584 4462
3536 19638 12776 5617 18924 7810 19428 13350 11809 13768 18047 11036
10750 3825 17782 18804 2831 18060 18535 9083 17800 9822 14551 11830
6598 8161 6081 5049 6923 6951 12249
274: 4873 8617 18355 6309 6356 19579 11946 18342 7376 17347 13047 4927
14649 12482 15068 14383 10603 3075 5557 16054 5542 5206 17331 19720
11637 5366 16537 14444 11823 2345 18756 7228 11287 3136 3972 2054
13176 4279 12726 19609 19002 3887 18393 12190 2873 4093 15745 19571
15950 5128
275: 3391 5673 17748 12970 2049 2756 10716 780 5570 15625 17600 2057
17079 14041 6494 10663 7787 10440 10550 12319 2852 13899 6288 6170
14404 7408 2857 4218 2808 18604 6362 17551 11818 511 5580 16146
1803 12576 13505 7130 9614 2287 6383 500 13389 19443 13855 8834
1779 5989 19035 7368 11731 19702 17630 783 15741 15774 15442 17674
2205 5474 2153 18915 13290 9728 17044 8267 18171 1334 15222 15195
7733 5047 8922 18281 12929 8992 12922 5696 12619 2026 6208 13543
17275 13703 15244 18936 16496 17942 12986 13118 16224 18419 10277 4864
9332 2640 16067 11844 6736 15403 4550 3500 4202
276: 9040 9153 13748 18710 9288 19447 1096 7103 8455 3624 18607 16311
18304 14306 9854 17310 16393 9187 17043 17574 17689 8273 13937 14386
4911 19063 18212 18817 18819 3310 3329 18815 3312 18959 8476 1386
2263 8742 15253 7396 3159 5367 18632 11510 4953 18618 495 19426
10261 849 13607 1099 15505 16314 14154 11065 14523 19079 3856
277: 6298 11547 9455 3560 3298 1224 6573 17886 3688 17535 6514 11001
16832
278: 9342 1148 8880 13995 9598 18620 16354 596 3713 12096 7035 13429
19465 13583 8623 4778 4775 4758 6813 14722 2970 16245 18993 16658
3106 6966 18030 8468 3622 3111 5706 1317 7832 18473 17421 13587
13609 3484 2614 11857 11213 7994 14581 14105 8667 8513 779 14862
6228 14830 2526 13752 2961 4199 676 14238 15588 17472 5776 1150
15882 3232 11072 7812 12851 3715 13664 13123 9048 17974 449 2155
3081 9548 12652 8280 15483 16721 8031 13645 18580 7580 3619 3479
6971 18732 15594 9508 10074 14377 14469 1702 11710 13599 19866 8968
12053 12047 4245 4259
279: 19107 3731 10544 1365 18558 3521 315 15072 9222 995 13810 10579
12184 1169 10820 8682 17063 17115 11426 7610 8335 6680 19590 669
12808 2095 3210 7981 8975 13797 19673 10108 12140 14891 4849 9589
10013 11489 5848 8508 16961 14635 3335 5341 9626 18112 14644 12785
9168 9484 8109 9837 3294 15636 3494 2017 19052 19769 16972 7811
19903 14579 7377 4121 7557 13806 6626 9547 10641 12326 19729 7235
1411 3644 10157 10154 2648 7794 10100 12628 15633 12168 7589 15853
19745 14933 10255 1291 17736 2414 7188 16138 9089 9415 11728 12818
6609 8114 10065 12609 4638 16498 2517 12709 17410 15136 17048 16713
18469 19832 12689 1479 15584 12811 17562 13267 13892 10949 9327 472
5713 11273 9716 15698 5082 17282 1612 7565 10794 11434 8216 10240
6588 15316 15700 6461 7231 10826 9945 1966 767 9017 8428 9216
1791 12429 5223 4910 19009 13274 12648 16308 4798 9611 5747 13303
2839 10405 4049 4046 969 5871 13542 17764 7597 9272 18722 14315
13433 12396 17102 19120 3267 2911 2934 2480 15593 1566 15118 17099
13495 18798 16391 13630 12936 4072 17390 11981 14556 12247 14559 12268
14560 14577 14558 12267 14576 12226 9387 332 2109 7841 18828 9615
11137 19936 14986 3553 9388 17887 11929 4554 18866 5440 3007 845
18184 4097 2805 16099 12066 16033 3934 3003 1594 10929 12343 16214
17420 5606 4643 10415 14917 6601 15702 10449 19711 13552 1507 19835
1698 9321 11696 2863 14802 17698 1498 17572 14405 13598 12043 2262
567 1583 2618 3734 18793 19614 14449 16036 8175 4878 19636 13279
17286 5486 17542 9087 15980 2290 11904 428 10747 9550 13537 3804
8971 5843 3250 1514 18175 14056 11021 12535 9436 8113 10697 15175
13365 7725 5679 15598 19442 16718 6306 9803 17134 10762 2355 13282
9344 18414 962 10707 13732 15247 16140 14944 8665 3901 12556 13238
3850 16213 15720 5407 7133 17222 15571 4342 952 16118 708 14879
19403 2997 14894 498 14617 11730 18476 3794 6451 3609 16585 3917
3066 16371 13698 6380 5627 5614 3881 18493 6416 12573 15178 11348
7043 8200 17880 11947 15915 7621 7041 8209 18926 10681 14294 3702
8567 18263 9236 1978 3932 19800 9976 13414 8046 11624 1429 3768
17319 17779 7609 16512 3551 630 3171 7227 9996 16499 5172 8855
13654 9117 3812 16292 19434 10320 1982 3126 14582 9645 1807 19478
7519 12941 17861 14481 4909 1899 3459 1922 19333 4083 1541 12260
11304 16432 3209 9163 2838 17263 2745 9173 17072 5826 7351 9534
19739 19536 7374 17185 14542 17759 5670 4857 6297 13448 7546 11655
18214 11988 3497 12119 18421 2238 9138 8088 14015 6428 10563 6419
14637 4955 9470 1521 11999 10694 19034 314 18961 1819 15545 10660
10122 1038 15537 16448 8891 10556 10553 13996 16085 16717 16720 16629
3416 6712 18028 18027 18046 18045 18018 2046 2280 11438 7044 432
10580 10575 17329 2438 2435 12466 13227 13331 3373 4354 1137 14182
14180 17288 17285 17287 17309 18279 17281 5134 13133 13134 7591 7579
7578 7590 7622 7593 7619 8710 11257 10155 17393 5497 7611 7613
3368 3371 3345 3347 3346 11146 18562 15875 3327 11270 617 12175
16942 6398 14919 6019 6427 16524 15844 4568 13742 8545 8646 17237
10441 15117 4903 16684 7548 4237 4891 11339 18131 14205 916 15666
812 8359 2239 6482 16408 10426 1946 16250 7313 7296 10169 13861
16153 7466 6375 11598 1049 14326 7059 9757 6237 2331 4295 3739
1600 4339 9125 15701 18725 10147 17364 5245 694 5869 7830 14712
3680 8784 12459 4338 15013 3889 4760 2323 2136 16586 3470 11223
13993 8676 4320 6154 958 7689 4997 6877 12357 17840 823 1007
2433 5629 7080 8534 2047 12554 18572 15080 13812 14010 15955 19762
16570 11714 12363 13476 19136 3340 8548 16230 13481 15749 6845 2819
2803 2874 2798 2851 2774 2772 2847 2769 11161 2801 14362 3590
1712 8501 2235 2242 2245 2243 18869 17430 17096 16063 16061 4419
4399 7500 7499 7479 7507 7501 7478 2530 9228 852 5988 15329
19925 15542 16445 15520 16918 16920 16451 14285 16922 15560 3473 2729
2751 2748 2730 1449 313 6008 10997 19320 14355 13633
280: 15402 17754 15436 17629 9518 8079 14324 10849 9618 16771 13444 11169
12000 16733 11507 10938 18192 18292 19813 13096 7912 8166 14940 10205
3198 12339 17093 6121 18940 9368 16294 13760 16750 10258 19864 717
17453 996 3004 19619 15600 2042 7099 14437 10711 13380 19640 16532
2201 1802 2309 6647 9806 6879 9457 8208 12113 10109 11945 9605
12639 15237 13105 11307 15891 3950 15781 412 17950 8118 5602 1129
18116 15989 10069 5174 1902 2069 8549 11084 3460 2119 3544 5463
19760 6025 2277 7245 14680 12142 11315 10103 11686 7410 5674 18381
3114 6700 7140 6390 1801 5927 9743 14343 16776 14417 6038 14869
8795 12626 14704 15315 19906 16747 15411 2945 6780 4297 8158 3758
5621 3569 4034 12467 19872 13169 1081 8095 1736 18688 9902 11293
1423 19566 17541 1444 2075 19482 17202 17020 15746 15846 16117 2321
9835 4123 7173 5998 4409 19765 10646 9885 10056 18669 12506 11744
14243 2630 5877 7111 5671 1529 1675 7444 6146 3023 3898 18187
1165 11993 15839 10330 6061 5491 4227 3781 9058 17595 11079 11202
7362 10144 11840 8333 13318 3170 6878 6916 16201 15777 19884 5750
12644 18885 13200 8731 18368 8181 14701 7566 13576 7966 16096 3440
4942 4573 2833 10605 7925 14032 3404 4390 11831 17429 15424 19414
16495 16622 11298 10131 6694 18316 8214 4497 14461 11010 15850 4991
15394 15401 5554 7553 7642 6491 5072 16712 18970 17407 1013 5799
15429 13670 4021 17706 15034 10501 15109 5127 9817 12262 12837 1717
18387 17051 1748 3020 8327 5847 16045 1172 5230 13057 10892 8743
3975 4171 5329 579 17409 16177 5306 5472 11154 9931 4136 5374
2437 15963 2773 17124 17810 18806 13785 2757 10243 16360 5612 16322
12186 2514 18051 19245 15150 17624 1481 7727 15029 17521 3890 2359
5655 15000 19595 10596 9889 9655 9987 11358 19111 11956 4272 6625
9408 15892 4615 17792 13508 980 2199 5653 15516 15373 15610 13373
9133 4362 10774 17361 10744 477 554 11553 14415 17110 12358 14292
13332 11804 11811 9941 13978 11248 18909 19512 16233 3315 13090 18834
11850 7862 19388 5804 19237 19460 4704 13932 6772 6661 6659 2180
12650 18850 12384 12309 430 13903 13110 18218 9954 6627 8550 4388
18332 16577 10907 816 14925 18100 1185 19304 652 9511 10006 6151
12106 6150 12493 7145 15562 2025 18478 7025 7026 4485 14496 7121
15494 13310 16051 8638 5236 11905 7716 16724 17428 13977 12065 14350
8192 14225 12919 19150 3456 17883 4875 7007 6429 17497 12548 9527
12164 398 13983 6190 3241 2352 5757 2425 19500 3188 13614 16480
1042 2584 18280 17452 1855 15112 8640 9646 8269 17863 2113 19671
4472 8213 10111 7758 11259 15393 17215 10615 14115 18114 19469 2426
8337 9601 19412 1557 13838 5949 16547 12016 6009 17996 16020 13608
18118 4335 2510 2358 6156 10954 3031 10072 16042 9333 971 1426
6023 16744 18147 18700 17680 17679 4200 2825 14313 17189 9300 3978
3376 2704 12344 17083 7635 8941 10128 19694 13357 12141 8860 8119
8265 15908 3690 8287 5054 16229 19707 8187 12767 8492 1917 743
3160 17877 8353 12737 8570 6610 16241 17568 17643 18099 7570 6986
13046 14790 11141 18103 19929 4945 4863 7823 1957 13295 8615 7666
19503 18182 17400 18683 13167 474 4854 8015 15919 17049 6539 8967
18743 18229 1022 7970 16839 19367 17290 18980 13370 3941 6507 16184
8973 2098 2078 3254 17794 10311 17483 2883 18643 13517 3729 1887
1368 14447 2804 10407 12834 17444 18805 15242 15905 18413 11332 15147
5790 12206 12866 11142 2298 19828 19319 18179 10150 5563 17462 11283
10674 17994 7653 11921 8670 18362 11531 10803 14348 18130 4425 7792
3742 4069 13020 8929 12909 5891 3491 11742 10960 1707 7447 11965
14102 16993 19751 4688 6075 5409 928 14103 17862 5849 11486 3424
13098 6417 1263 14274 3937 4584 19652 3860 4922 7959 16775
281: 8694 12679 18529 10066 2678 7179 6485 2766 9063 12300 13395 10238
7333 12286 11528 7686 10627 19423 16297 3477 14307 14332 4683 6386
14764 18946 11842 6708 4206 7511 14161 4948 18236 16555 19706 16935
9276 1705 8251 17729 14148 10222 1114 14562 7443 7171 12646 525
3814 3651 11471 13348 17253 3355 5958 18560 9986 5060 4869 4769
4656 9642 648 1410 2931 18886 8791 11423 16482 11075 17009 10151
4552 4158 14670 5141 19526 19457 6070 18446 10680 4669 10087 10957
4753 6048 16338 807 6752 7620 2892 903 10046 10520 10397 14613
1203 19013 13180 13847 9021 8726 11936 9469 19630 17914 1539 16852
2684 10230 15144 7414 15169 16341 18719 4705 14987 1054 1301 8183
10260 12916 14291 9101 5800 9224 19194 2786 14896 17070 13453 10805
8590 1236 3657 17563 5588 19064 6364 15198 2518 14895 12534 11548
11147 5143 4729 18858 19784 5715 6997 19753 10859 13393 5535 514
6792 15326 7576 4957 14042 1374 8856 9698 10313 12942 14998 18662
4919 12571 16319 920 17169 6335 5044 6370 18927 5610 2268 12436
6087 3030 2039 16323 8447 18750 16277 2412 18682 18358 12549 18406
1390 8657 15832 8609 6974 6281 8591 1357 5973 16208 12678 10423
18094 17711 4111 10235 10848 1743 3109 9476 6992 16202 18853 17948
2213 12570 4347 4954 1510 15130 3018 12888 12264 16727 16884 19498
10768 14695 16944 12594 14452 18055 12881 19189 10784 2720 13143 5352
7662 15194 18003 5874 16708 14822 14749 15941 2031 15715 10075 12830
6407 9819 6114 8177 6890 5432 15138 19857 18139 1174 8978 8120
9900 6781 18900
282: 9488 13616 15590 2297 2284 4876 8155 4503 12483 1657 3791 899
884 18026 18515 18521 18025 17955 559 18537 557 16157 10429 13191
7467 11321 7432 14150 2723 12764 12504 16859 19196 19198 19204 18436
19288 19227 19280 19200 19248 19885 18438 19282 18463 19259 19224 19904
19285 18461 19231 19909 19908 19292 19887 19310 19924 19920 19912 19255
19201 19905 18443 19860 19222 18441 18425 19252 19253 19233 16129 15836
3701 13372 6579 3072 10251 10921 11241 1852 14936 13471 2795 13570
13592 5773 13546 3179 4658 5425 19564 6136 5441 19623 527 19056
16702 16685 2755 4581 1965 4319 3694 3892 6668 6060 18290 1483
12475 6925 2154 2749 11421 12831 15432 10442 12991 12491 11934 17139
10324 10227 2579 15389 7706 18794 1036 10124 11030 10073 15797 12617
3046 10666 11189 6165 6563 8600 2237 11718 1494 17677 1968 11225
3759 19617 1963 1230 9162 14979 2576 10989 16101 1141 17522 11388
19518 14314 9391 12490 9259 9858 9861 9878 3027 8297 6634 2580
564 536 513 13257 12751 2800 4947 6596 6727 12672 13807 4898
6067 7002 9776 9305 5205 17859 10706 9919 5631 3026 14137 17947
13952 10969 17270 11134 11135 1464 1994 7337 4302 19858 14835 3132
18541 18685 15115 13477 7242 5034 4232 4229 7849 3936 9400 7074
1711 13593 1267 7020 6203 5752 11017 19211 16500 5464 16481 16479
11262 9474 2657 9912 17012 17455 16989 2421 11405 4134 4135 13791
3760 4746 7250 11301 16165 19238 6369 8863 6260 10640 806 1373
1942 9798 10528 1672 6827 6035 18378 2844 12503 12481 18439 9335
6502 16929 6137 3738 14085 11558 7893 670 2543 714 7694 12393
8619 7916 5416 4052 6540 18074 16465 12699 15044 7738 6074 5428
10870 18932 19505 13905 15650 18814 14236 16809 16695 16811 6358 6377
9099 13233 15830 15855 16971 9952 13720 19312 2882 17849 13346 11894
13927 10068 15728 15724 1669 10842 5211 5573 11524 15993 10964 11561
8701 8076 2340 9030 12177 8173 15874 13078 13080 15578 16552 11954
17694 13066 11930 9502 8839 10015 14690 15514 14413 543 5118 19602
14943 17995 1533 15898 17708 13695 8703 7375 12243 12246 10408 1134
7047 7069 1384 664 14725 13989 14727 12311 550 588 2405 3105
1325 3085 4328 12896 10416 5716 5734 5080 10206 14083 5736 12511
7935 9350 11129 2367 15487 18863 6310 16136 16616 17976 3605 11762
6296 7735 3664 15502 16857 13747 16858 1704 8970 8242 3966 18776
2037 13001 18364 8423 12792 14596 1694 2481 2503 17868 531 18007
7674 7678 2470 5590 15264 15917 13152 11243 11441 18206 19145 19302
5018 2605 15951 15519 11689 3205 8293 4752 4648 2589 3319 3377
6240 2569 2542 4651 4796 4774 4647 4644 4101 4693 4777 4022
4018 4020 4714 3995 4133 4060 4668 4197 1988 16865 1441 16612
758 17827 17825 6420 6905 12143 5753 17242 18540 553 5233 15097
3540 2308 14280 13379 15914 9672 9677 653 5107 10689 462 16974
6046 7495 7028 14188 17597 18006 10434 5334 13292 9733 15419 1170
1187 16770 17137 16828 16680 5597 11937 16404 17267 5157 13849 8607
18671 4234 14051 1528 11389 17864 17893 17962 561 12269 12414 14893
13196 10758 18547 563 558 18543 532 5357 13969 17925 3015 560
3362 5820 7763 960 17879 10047 537 3873 8224 535 555 556
585 17895 562 2019 11745 11746 8692 18545 533 19830 13796 13716
3761 15630 15770 10070 7827 6148 2018 7825 7828 8464 8157 7805
3409 3407 3366 3430 3387 3429 7806 7768 7804 7797 7772 7799
7771 7776 7822 7820 5838 18949 16551 2875 14511 15973 10383 9041
11443 17876 17871 1216 3604 18867 11364 7984 7564 5619 16508 9249
19817 11408 4489 7775 16600 11204 11343 6760 16109 12509 11456 1471
12963 1159 14149 17898 4525 8069 5516 3522 17718 17147 2073 10352
9958 12355 4590 8758 17922 17979 15581 2550 4547 11843 1492 9863
9732 12824 4458 4446 2432 8850 10795 2573 12282 1601 2523 1313
4569 6686 7903 18104 7660 11455 7569 10308 14385 17379 16758
283: 15531 6256 3677 12101 13037 10190 10248 11043 822 16817 7460 8374
602 15713 1872 6858 11835 10862 12518 10351 1380 17170 18399 10269
9599 10039 5643 1398 5309 2941 13830 626 17116 19140 11467 18598
13371 8055 11493 2929 12146 10801 14962 19804 16176 17439 2811 496
2695 19371 12254 9322 16426 17763 3303 7474 8440 6587 7356 10022
7182 7542 19708 4269 14632 12375 12229 13513 5894 17174 11372 14162
15843 13669 2318 4478 4374 2133 1853 5420 9504 9838 8996 1878
5702 13717 1634 18754 3402 15053 14759 16550 3082 4918 14755 14416
9281 8312 16172 11469 9564 18095 19075 13665 14192 9419 14729 2368
10551 14620 6612 19450 454 8874 19239 7854 8878 17842 15819 18002
13934 12440 12425 7219 13659 3489 18080 14572 6789 15128 8759 17772
11836 13231 12637 8764 17762 17882 18242 8340 4916 1839 12027 518
6044 6520 14817 9982 15655 16991 6478 8575 17011 7819 19454 11008
3852 16238 12816 18889 19856 3049 6004 4420 14200 19451 3399 3287
757 4411 3141 3803 3354 2485 600 11184 11333 15248 4728 19407
13987 16072 13120 493 7888 811 14946 2583 8687 9939 14742 4000
19160 16329 17352 7798 9075 11087 16289 13321 3576 5304 6605 16610
12700 5370 15030 7544 2014 8691 16557 17396 12352 3485 16266 13480
19873 8847 11387 13745 2392 8223 5726 12517 14311 13416 14639 10763
14203 18402 3129 12616 9840 18774 18715 15765 15812 8125 18204 4445
799 8531 4265 17783 16560 14953 11093 3886 5539 6863 15521 13084
19629 18230 15314 6456 5130 7907 9874 4068 9185 2982 8263 8178
11555 12456 2286 17432 6568 1183 1923 13056 400 18905 11046 17586
15182 10135 12752 9213 14279 7205 650 6593 6212 18450 15704 9964
7123 5422 14997 16892 14156 16838 19853 13287 1951 6717 14456 16168
7220 6525 16069 12673 11280 9572 7263 16613 1000 3100 1497 17231
9541 2750 6439 3980 10290 15824 17100 4572 9670 8215 6984 12801
10455 3696 17104 11037 10557 7817 7141 11642 18768 18492 12742 7415
8653 14608 13015 13405 13121 8857 17133 16228 14222 5302 7237 7488
5999 3641 566 2958 11175 2130 2764 16189 18380 6548 9673 14038
14035 7139 15190 5426 19562 18437 17501 644 2919 7071 7261 14062
13482 15191 11743 19472 19437 6920 13727 15618 15631 19891 11927 10932
8010 16223 14499 6723 19485 6581 14854 14856 7405 12499 14495 885
6550 15800 14507 17436 16225 11331 3216 8486 4438 7918 5382 661
9656 5184 1208 3516 15538 15408 11613 11439 5451 15131 18608 4901
4791 17602 8576 6930 9997 16304 16968
284: 14546 4993 1029 7514 2917 10152 11335 9594 14726 17937 6935 14151
10273 5997 7000 10002 6010 16908 7529 2137 5389 15290 8071 6027
4413 15840 17540 6072 10477 13780 16667 4735 851 17780 8426 4344
3194 6508 4023 12288 16472 15236 18423 2712 6253 7180 8745 16575
12645 11798 15025 14872 12821 12055 5069 8763 5400 5829 7669 2817
5344 1590 8606 18818 16682 18975 16411 12120 1632 2145 10107 3618
17320 7159 19374 8892 12001 19521 2499 7464 11752 2395 19468 2094
17945 13685 3703 7073 8473 15278 11750 6376 722 1788 12276 2177
3659 2253 13161 10545 826 3249 6401 10373 5991 4410 9993 9994
4785 9814 10962 18365 854 5075 4178 13328 18181 19525 14890 1603
6161 1186 310 587 2986 3592 12649 3080 15617 11601 13501 13181
6206 12788 7897 2732 11732 7914 4586 18225 17758 4002 14369 2043
9570 3991 19823 19209 11521 18784 14970 6131 8658 15987 11465 8315
19681 17975 1559 5862 3880 11271 2108 14500 11285 16316 19436 10432
7652 11110 19523 2843 17746 1118 7774 19578 18424 4835 14580 15727
5582 2332 2821 17034 16853 10210 12320 9478 7636 16940 5379 13944
8103 651 3131 7232 11772 19236 15809 4006 529 19806 7702 4689
17509 5782 10158 6488 7040 13260 10093 686 10592 15289 19347 9038
13009 13925 7052 14143 8739 14688 12107 15485 11400 6474 3104 3107
14423 1690 5872 1941 9668 3685 11900 10682 15943 14081 8352 10943
8232 18499 14024 18737 9701 5604 13245 3263 5326 14254 14253 14255
14251 19565 4990 2198 4887 16593 4192 623 16711 18391 10980 7532
10316 16098 911 19278 8228 17323 16757 2097 18771 1562 8768 11770
13930 13210 9302 4394 18666 1679 19230 14127 8256 3565 3486 8305
17338 12200 14095 16106 16554 5101 9627 11323 13709 19928 15448 7178
6851 9856 17693 5450 14758 6440 4840 16948 10481 18098 12809 9820
8612 5560 2328 16487 4316 9793 9587 6024 4610 3518 14472 8379
17788 16283 940 18073 12073 12239 12641 1517 4520 4253 19655 19157
7457 14257 13738 9744 6631 11117 7100 10958 1663 19658 830 850
981 19109 1871 7127 18552 12232 3002 16895 13330 7142 5045 8449
15114 15111 19687 19859 2450 5262 5468 1622 15404 10115 3091 1432
7857 9421 10367 16178 16767 18032 14517 10922 4845 5596 17422 17656
14974 16264 17181 461 10535 13155 18603 4629 7200 12897 12379 19551
3158 16835 2030 2535 10521 8602 5328 6340 5196 12908 4510 6303
5493 710 15944 18673 17709 16365 17701 3274 14621 2173 3229 12063
8399 9226 19709 3984 17274 5355 11541 16698 15860 19429 17598 1573
5354 3074 10753 6512 4856 14767 3042 14876 3811 856 14273 14271
2338 13640 5456 5459 8719 10281 6810 7883 15726 15508 17683 3047
16285 12882 13574 3723 18442 11447 14287 18277 8323 8320 19125 8734
18846 8429 8735 6351 18016 7745 18581 16456 13515 4296 16800 3915
7965 11716 12382 5285 552 18741 19308 12228 713 1275 3193 601
4292 18067 7027 15113
285: 14398 1558 18339 13431 13428 4182 16260 10619 6127 12005 7704 5064
13072 4395 9032 19907 9588 478 12782 9917 13100 5202 10562 2044
4452 10775 3543 14840 14071 14020 10406 14218 1797 16955 3879 10312
12432 14824 7082 14554 15744 1508 15866 16232 6797 7077 5816 3596
3597 3311 15022 881 2638 906 212 12037 12039 3458 9042 8812
3217 9466 2270 18102 610 2431 16650 14418 2251 1244 4235 6826
16651 14435 13004 7852 18349 9553 9262 4340 9235 15603 1413 17223
8296 10538 16071 16048 2120 19847 11239 10420 18968 12345 2995 2159
2549 3987 7551 19585 5685 4861 16862 8677 13466 11435 11230 6015
6649 8097 16025 9440 1353 16249 16151 9398 15930 11226 2870 12373
15043 11765 10145 11071 9537 9095 5496 2350 11756 7967 13362 2624
7924 6715 15074 17068 9275 10177 11007 14857 8951 12182 11643 13590
1740 7684 910 929 9093 10018 13344 16978 6305 11378 17544 11969
1393 15937 11803 16631 13126 14108 18524 9644 8425 8421 8420 10755
16696 15994 10757 1143 3549 5868 5126 7506 19032 12669 12547 4707
4710 4708 4732 4736 4733 8772 4730 4711 3385 1831 2528 2304
13223 15197 15098 11673 4386 11884 2336 9633 9118 14693 2399 12725
14548 11011 18359 14692 5652 5434 9755 9513 2681 4364 19777 13525
12903 6859 4326 6088 11611 3068 3578 13940 8582 14440 19618 9826
13275 18012 9371 10057 5985 18222 15021 11502 14888 14001 1279 17431
18172 7826 14232 8317 10294 7425 15565 5531 17742 5564 18144 14389
13857 7113 16444 7400 10835 13461 4722 2115 13951 19022 5256 18254
7276 16719 1045 2920 3279 19766 684 19811 1237 15371 10076 8004
18712 2781 4940 11219 2673 19836 18985 14312 15772 3933 6426 7906
15599 11860 1907 18897 14263 16522 4672 13737 574 16112 15415 11543
3433 7746 4115 9525 5392 9445 2252 14696 17114 1400 9349 4881
14484 18930 2533 10428 7616 18415 10497 12585 10236 4874 19793 4633
12544 1995 17255 6130 10439 8504 7487 15261 6768 12863 3628 15985
17752 5019 2036 2840 18127 12020 9264 14875 7498 1448 1059 9636
2644 15865 6207 9893 3161 10031 16927 2065 7894 2171 4174 1254
18701 10035 19271 1389 12810 4747 12671 19586 10698 5651 9018 1931
4286 7712 3519 12527 13943 6181 5039 13948 16797 16826 18966 5145
17610 6304 8895 7744 9280 9284 10647 16558
286: 9313 1653 17351 14049 14657 1383 1015 18068 4392 10779 16383 9920
487 9934 2830 3247 2351 18430 10228 17404 17658 5336 5337 17655
1350 17686 17699 17685 5353 17632 5339 17682 1645 1642 1640 1620
1616 8916 8212 9895 2832 2783 4293 6486 10048 4251 5819 5822
5393 7543 8453 7971 5457 12283 11526 4620 4594 4593 12447 12446
12443 12421 12418 12419 4579 6639 4625 4624 4598 8851 4628 4580
4631 14489 13819 14308 18593 5807 15337 2081 12213 5581 14765 7934
18948 16079 6446 10336 6213 8673 10129 12010 19004 19003 13184 10392
11721 7649 9054 15693 14867 14868 7482 1784 9674 1270 19176 1199
8500 19667 4212 4448 3986 12515 15430 7679 18854 16043 4242 10959
13366 2878 11272 5100 3297 7169 19051 3475 13999 17136 17946 7102
9260 9177 5942 18576 8622 9353 18500 14477 16377 4784 16796 16425
17653 13145 16135 7307 4655 9544 12992 17516 2232 1408 2230 1599
11103 9661 8918 17531 7259 10941 16332 19728 2216 11076 2670 18069
18160 18744 1206 2891 5478 5142 4382 2683 19169 7606 11398 12629
18745 3786 17936 5887 3425 5214 8401 14931 18934 15251 6533 4671
6531 17015 1412 5092 8805 5545 6049 12372 578 17463 10263 1161
2814 5758 10686 7449 1330 10508 15984 8027 1288 5176 19668 1710
19355 3454 13918 14445 2162 14905 2727 17196 7122 9749 10816 13053
15837 11705 1700 2315 17710 12014 19134 14514 14744 2761 1547 12385
7818 16211 18823 9872 2759 2806 12795 9882 6415 11514 9287 8821
10229 14451 7419 9477 11295 15793 15660 11783 701 2955 17659 2504
17348 16342 15186 6406 8247 5380 13891 1315 1256 19915 1724 2012
14065 10299 8282 616 13375 9102 16056 11991 9014 9223 19193 5659
12275 17960 6092 13451 8592 8527 7016 15734 1394 3654 2224 9086
6674 15201 5282 3178 18367 19531 17843 8903 4152 2519 4236 14485
8563 18395 2904 12192 17438 7558 8708 17132 14463 2926 3606 14880
4126 13192 11148 18831 19343 3854 3197 1662 15209 1164 6993 19768
5846 5825 13392 3964 18837 3676 6815 1563 14197 4983 16851 1080
10309 13336 6679 6681 19226 1139 18217 6677 11476 10458 11376 11534
15281 15284 4323 17519 4170 18686 3737 12407 9314 18763 18235 2522
424 12163 6909 13949 13705 2221 17613 6620 10223 18017 9192 4434
7858 4011 19099 15849 2703 7837 16579 10683 5630 13463 4008 5853
15102 13390 15974 3534 9370 7120 13281 10677 6277 7442 19758 3296
12002 5857 6669 12717 15152 973 15151 5971 2689 2692 14737 15184
19235 11966 9246 1051 16723 7949 2967 13928 1691 17376 8438 6021
5801 5705 16980 13383 18063 14100 12568 5238 12085 17418 17386 7929
19078 16133 15135 4577 8414 19149 3621 10044 6007 16987 3208 3567
14540 9431 6837 9742 14981 18407 10889 5572 15933 2966 14048 10199
19043 8146 11747 2107 1220 19179 13356 3167 14446 7973 13997 10221
14201 10975 11488 8663 6381 2578 2575 14911 6552 16339 768 1468
18066 12089 17874 13394 9319 919 12747 7881 17584 17587 6226 19910
7627 18249 9111 10610 1083 6509 17564 5851 15935 14750 1415 7919
8561 13034 1757 7298 10482 17985 2674 18011 18159 6510 8905 4745
13968 5850 6365 18717 6735 7063 6097 7741 6692 16850 9791 1250
1976
287: 14033 3069 10887 17423 15804 10725 1039 8823 13656 1864 18512 3164
11788 9149 8808 5438 15880 3992 800 12872 18261 8766 14438 3187
10085 4070 901 13535 2677 8285 10764 19705 18563 18309 4608 19842
15814 13706 2652 1108 2600 11436 8221 18056 9465 14769 5688 5397
3900 1598 6393 5004 17760 15162 12125 12647 7151 11629 14327 14070
16315 15318 15939 1024 17173 7378 11794 17214 14034 2307 3146 11136
745 14629 1219 8349 9131 14106 2574 6164 13786 12212 288 9930
5003 15379 17113 11587 10350 16113 1863 10534 16794 10418 3427 14528
9660 19113 14120 18931 693 10649 15876 620 11764 4473 14614 15160
2527 7562 15931 15942 9412 10452 1278 9417 14663 6750 3059 18739
5729 11588 8690 1571 1240 12230 10197 3943 9396 19704 5587 3849
6395 2512 1364 4248 9916 17140 19660 9460 2383 2545 10421 10451
7528 17188 5982 10710 4743 17869 13867 15895 18341 13127 9124 11340
15482 4846 15991 3001 8402 17663 13283 11123 13596 1151 10214 18536
11869 2959 17628 2207 1422 5187 12698 16068 18921 3969 18923 15856
13136 15313 11878 7997 15604 14266 18654 16893 14982 19298 8644 8268
18840 18022 3726 3772 13530 12761 10851 9602 19087 12685 5286 4627
16074 10376 1178 17450 2560 825 10966 6542 5902 17622 13520 16539
494 18321 9756 4078 13338 11983 15481 17256 1850 10700 6895 19331
5603 16542 8629 14464 8541 11955 15582 1615 9393 11799 9705 10217
3894 5124 17122 10037 19916 3503 17417 13729 5423 16076 1843 12859
17649 1593 15088 11536 19105 6404 17208 9567 4208 3180 3864 9156
12512 15134 8390 16883 10879 8150 9719 12593 14926 14282 17273 6293
6538 13207 13438 1179 13854 1534 12605 12235 11858 6290 13936 15363
13652 15361 19700 3899 1614 9390 19686 12223 19528 1763 6129 2975
18334 4649 2415 18042 5063 4001 2807 5550 868 2976 14624 3321
10604 2149 11085 14039 5615 8502 14424 9369 14136 4541 3019 11951
18036 14808 13308 6822 19066 15323 12540 9754 11217 12299 6562 19219
2211 1980 409 1970 8961 14118 1424 14183 13802 3223 12957 11050
12155 17192 14643 15307 6911 12324 7373 11544 13247 17437 12217 11827
14360 17578 12753 6662 19268 1665 7212 10672 7843 1289 5216 4585
19139 7937 16633 5644 16320 11433 9472 12965 12360 3202 11429 11427
19689 1587 773 8864 14046 5266 9201 17227 10510 7291 10904 10655
4965 17040 2183 16534 15799 9967 13632 6850 19691 8788 16409 4673
17193 14098 11916 2451 4834 18460 18183 14691 19318 1489 12987 16441
17707 16821 18525 6452 5795 10306 808 5051 9434 4528 13359 1168
13904 3700 15002 12152 6475 2944 15440 18551 1414 4231 14124 8358
6492 16565 17743 5954 16843 954 5541 5859 18096 12827 15357 11338
14884 7974 10246 5834 3697 450 16197 8091 11058 8780 10822 12036
3632 1407 632 5735 2700 8621 4284 18605 5544 3482 7892 18630
6701 13975 18530 11623 10209 10341 12178 19162 4832 13434 19639 7936
1306 11386 4376 19409 7887 13961 19229 11753 10931 1751 18762 698
7891 10920 8887 5093 14119 434 10475 8949 9491 15868 17642 9386
8713 17515 11664 12640 4630 9414 7285 9296 14419 9418 14870 5136
2943 8767 9080 9232 9233 9981 8666 11032 6927 1544 17476 16917
19548 12188 2244 15586 11795 14909 4751 13962 9106 429 18945 4616
5199 10178 18224 7863
288: 14033 3069 10887 17423 15804 10725 1039 8823 287 9557 13656 1864
18512 3164 11788 9149 8808 5438 15880 3992 800 12872 18261 8766
14438 3187 10085 4070 901 13535 2677 8285 10764 19705 18563 18309
4608 19842 15814 13706 2652 1108 2600 11436 8221 18056 9465 14769
5688 5397 3900 1598 6393 5004 17760 15162 12125 12647 7151 11629
14327 14070 16315 15318 15939 1024 17173 7378 11794 17214 14034 2307
3146 11136 745 14629 1219 8349 9131 14106 2574 6164 13786 12212
5003 15379 17113 11587 10350 16113 1863 10534 16794 10418 3427 14528
9660 19113 14120 18931 693 10649 15876 620 11764 4473 14614 15160
2527 7562 9412 15931 15942 10452 1278 9417 14663 6750 3059 18739
5729 11588 8690 1571 1240 12230 10197 3943 9396 19704 5587 3849
6395 2512 1364 4248 9916 17140 19660 5811 9460 2383 2545 10421
10451 7528 17188 5982 10710 4743 17869 13867 15895 18341 13127 9124
11340 15482 4846 15991 3001 8402 17663 13283 11123 13596 1151 10214
18536 11869 2959 17628 2207 1422 5187 12698 16068 18923 3969 18921
15856 13136 15313 11878 7997 15604 14266 18654 16893 14982 19298 8644
8268 18840 18022 3726 3772 13530 12761 10851 9602 19087 12685 5286
4627 16074 10376 1178 17450 2560 825 10966 6542 5902 17622 13520
16539 494 18321 9756 4078 13338 11983 15481 17256 1850 10700 6895
19331 5603 16542 8629 14464 8541 11955 15582 1615 9393 11799 9705
10217 3894 5124 17122 10037 19916 3503 17417 13729 5423 16076 1843
12859 17649 1593 15088 11536 19105 6404 17208 9567 4208 3180 3864
9156 12512 15134 8390 16883 10879 8150 9719 12593 14926 14282 17273
6293 6538 13207 13438 1179 13854 1534 12605 12235 11858 6290 13936
15363 15361 13652 19700 3899 1614 9390 19686 12223 19528 1763 2975
6129 14424 9369 868 4001 2807 5550 18042 4649 2415 5063 14136
18334 4541 2976 3321 2149 14624 10604 14865 4260 2050 11085 14039
5615 11951 13308 18036 12540 14808 6822 19066 15323 9754 11217 6562
19219 2211 1980 409 1970 1424 14183 3223 14118 17192 13802 12957
12155 11050 12753 8961 6911 12324 7373 14643 15307 11544 6662 13247
17578 14360 17437 1289 5216 7937 4585 19139 16633 5644 16320 11433
9472 12965 12360 3202 11429 11427 19689 1587 773 8864 14046 5266
9201 10510 17227 16534 7291 9967 10904 10655 19691 8788 16409 4673
14098 2451 17193 11916 14691 4834 18460 12987 18183 2944 1489 19318
17707 10306 5795 6452 13359 16821 18525 16441 808 5051 4528 9434
1168 13904 954 5541 5859 17743 14884 5954 16843 18096 12827 15357
6492 16565 11338 19639 5834 10246 3697 7974 4376 19409 11058 16197
8091 1751 18762 450 1407 3632 8780 2700 8621 12036 18605 5735
632 10822 5544 6701 7892 10341 13975 4284 3482 5093 14119 434
10475 8949 9491 15868 17642 9386 8713 17515 11664 12640 4630 9414
7285 9296 14419 9418 14870 5136 2943 8767 9080 9232 9233 9981
8666 11032 6927 1544 16917 19548 12188 2244 15586 11795 14909 4751
13962 9106 429 18945 4616 5199 10178 18224 7863
289: 5523 4313 17448 17841 12777 10355 19605 19897 13150 13788 8075 5624
13087 8533 15156 16282 4418 5362 17314 16470 9691 6830 3539 8947
11303 15847 12513 12958 12763 11550 1760 7062 5192 9953 3474 3481
8116 13424 12304 5690 13692 18678 7503 12740 12452 4939 13248 3994
5264 3961 4726 4657 19545 17062 16253 1040 7214 5231 11985 5953
3077 10399 11073 3556 17461 15325 9938 4761 10626 12944 12889 11220
6105 15005 17775 7340 16006 1428 19624 11919 12601 642 3843 3912
4096 12974 13254 2664 16161 8861 13261 13575 9768 14949 19126 4880
5824 9299 14874 5687 2782 17505 17726 6275 3175 13677 4398 4025
5376 4677 18843 7388 15255 14128 2596 1938 14569 13062 4742 13091
16870 9122 11112 12762 3052 11166 2182 15922 13893 6314 10549 17230
10781 6932 11582 16011 6946 9000 6251 9407 11924 9877 6838 15556
15794 11808 19431 2295 1090 12514 7454 18403 2529 18862 19419 12173
3773 11501 1975 14174 16124 8277 11572 2186 4994 16607 12215 7089
1851 16812 1689 3238 11697 1993 14319 8424 1113 3997 2880 18417
15224 9191 8757 16205 13252 3135 18125 1294 2016 407 744 18246
19479 15883 18721 4036 14388 15680 19029 5232 12705 8217 16267 8222
11264 961 12449 16916 13761 3615 7172 14831 14301 16476 14882 7390
14607 14441 5660 11599 11359 4092 17343 13568 581 5071 859 5934
10571 12380 8016 725 9697 6714 8705 17074 1644 11944 12826 11957
4502 5499 5549 14898 1200 7101 18165 6291 6133 13712 11143 13419
3259 14157 11554 16748 6359 2357 941 11901 18451 1984 931 4926
6943 18727 6551 17793 4278 4905 3893 11593 10509 15596 4274 3302
3510 8231 18704 13258 4695 1347 8641 4776 16869 9696 16749 12938
4466 5831 2176 13023 7184 5489 8886 12920 6529 871 15574 8579
17493 7527 18633 2515 3708 13536 7991 3379 16897
290: 13013 2572 15390 1939 14078 15522 9968 9379 18412 18308 9944 6870
12684 3058 2845 13741 4261 19592 4701 17626 13030 8728 11077 4091
4089 17112 14261 13039 2448 19588 19632 10171 3875 16215 6248 6904
17027 14757 14672 18336 6783 16361 13415 7175 19494 2544 3982 12302
12865 16091 6469 16191 15776 6721 2879 4779 2699 14780 16535 10778
10809 10295 3281 8912 10017 11633 16868 2259 19421 16353 2746 14045
11766 14772 5191 8574 17749 1617 17592 15384
291: 17667 17054 19440 15213 7334 2141 3727 9845 11081 9526 12337 15918
15597 3255 10276 970 16639 19309 2222 15291 19583 14945 7813 9966
1286 1910 10491 19293 16030 19104 463 11758 9810 14676 9957 4241
14669
292: 9221 9220 6193 3253 14842 13721 4160 4741 7322 10303 16057 13497
13494 13514 3142 3155 3203 3201 3204 3231 3227 3230 3228 5987
3206 17127 17830 17527 19410 19413 12404 13866 13360 13864 13860 1092
18317 16638 3344 11672 1048 5311 11768 18957 9486 5520 4119 3271
14470 11906 13464 5650 8589 1858 2994 2964 2947 2923 14662 12783
5730 10701 3718 10132 8593 12855 6873 15376 15374 19023 14646 14645
9650 10687 844 17322 17326 6887 4652 3798 14561 15275 12003 17738
3837 3822 3819 3846 15647 8778 10880 17001 8637 3841 8776 18718
14843 3863 3817 13443 13421 13442 13498 3923 13787 3280 3276 18190
4216 12478 8586 7030 8614
293: 13730 7076 16121 2661 2089 15753 13834 1549 18755 5880 14792 946
10952 15392 19376 15572 11083 1945 12301
294: 19341 4724 19896 3258 5111 599 6431 13589 6942 6192 6252 13509
19882 4075 1265 10657 10589 5296 15003 18223 18248 2327 1261 9965
4027 15223 16926 14122 18287 13182 19642 13929 3304 730 11185 4607
2628 9008 18151 2691 11457 19048 6876 6857 15174 15158 17254 6991
11670 4110 4109 12772 10184 4872 12819 12694 6264 7048 9607 9648
12614 8990 7650 19393 5863 3904 10368 7157 16438 13128 17560 7535
1575 10105 10134 568 13578 4304 9818 7538 9257 18048 15120 18444
17647 18149 3239 2052 591 16792 10643 9825 9839 18609 3056 3720
12370 9361 1519 17206 2733 2404 2274 10638 1810 1681 794 5509
8149 6109 16956 4055 7526 9906 3806 4482 3406 8669 11941 8434
17353 1718 16055 16094 11109 13291 14317 16938 8688 12395 11729 19065
14012 4384 7554 19069 18471 16995 10788 16414 3527 4972 4041 7910
6178 2598 12998 9686 12367 8259 9712 9092 15589 17494 13102 3555
4223 2428 5186 13868 17026 19357 3670 16415 4465 5164 14396 8679
11667 4509 11014 4526 10211 3983 11652 13984 11918 3989 1532 19637
1516 8361 9956 2740 19492 19752 7125 504 18859 1252 13539 6656
17606 8695 18363 828 4343 4885 16387 12180 16021 6095 15293 7951
15342 16946 16808 5255 15684 11760 19481 5992 13140 3529 12315 13875
7700 1035 5477 5476 17832 8331 8176 7166 12559 4721 7461 1735
1862 4162 19901 18566 16648 17875 1044 17901 16027 3656 5177 4493
8389 7751 16700 19251 7033 8539 8515 6547 17753 6527 7218 4305
18907 2441 16894 8368 15641 7365 8148 6445 5873 7736 9634 9685
9688 5086 9147 19697 6980 13974 14084 14057 14234 16981 7596 2200
9879 6560 7032 4346 9104 12902 9061 16803 18628 5254 6524 19495
6553 1235 13979 4589 5122 8755 5028 18168 8279 14683 17291 3546
19846 17369 19927 12627 14146 16105 17234 15988 10866 12145 8926 6958
8927 4271 16119 10894 10871 14052 4521 4546 19411 17524 542 18878
6595 2294 11153 9855 9127 6844 8065 9659 15206 10237 10286 5228
7708 16193 16173 1816 2487 7234 18474 15871 18498 18386 9860 16437
13117 11881 10754 18738 9129 5234 17737 539 13246 11876 5966 6301
18615 2022 1249 15906 6134 1486 1737 17536 16641 16126 2117 18070
1727 19256 772 771 16084 13449 415 1588 4731 11912 13036 12904
10167 10170 10181 11796 7931 4436 19552 15340 9069 6065 9576 14476
4033 6144 6355 9580 11373 3845 10555 6026 1538 14252 11149 12524
19249 7977 8112 14058 7344 7310 12622 12584 4642 6874 16745 440
1628 829 15558 18297 1682 2486 4188 5990 15852 3101 17511 647
6316 8897 18910 1311 5918 17076 9692 4085 13054 13063 16969 6300
295: 10020 1439 9915 16163 18062 10972 18887 18356 4431 1884 10727 13800
12798 2858 7343 14738 2791 14434 9539 17180 8662 5767 8275 2125
6101 2121 7866 9540 2780 5578 19289 18868 12356 8526 2380 13296
3693 16875 2379 13074 19810 949 14344 1531 16649 14889 14044 4090
3535 14795 14076 11583 15037 3570 4564 3572 16352 16416 1362 5033
6120 979 439 15995 19886 17585 14283 1830 6945 9194 11025 1647
6731 8953 4154 14591 490 17151 13499 11392 12621 8859 12715 16405
19405 18614 9282 12579 6899 1876 7114 15543 19677 5323 11508 12990
19006 18475 3862 7010 19748 1860 15260 4534
296: 4540 6354 1755 1754 17371 3891 9752 4896 3390 13183 12597 8675
19057 6229 4548 17590 5708 4363 6198 17103 9094 2389 11801 14837
6762
297: 4370 7533 2597 5919 4081 1338 1069 10498 19778 13926 7098 7281
7968 12746 5461 13420 11565 11748 7477 1523 7224 17897 18082 14625
17717 1367 15055 3466 18152 3922 5925 14810 16421 9929 17561 14752
18760 19598 12786 11882 9247 5551 4562 3594 11415 17495 11692 12861
16921 838 16714 10602 15187 4256 6989 12555 10731 13076 7618 12876
10208 869 12690 7839 12981 17982 15557 6454 5181 4327 12171 8233
18590 17657
298: 16642 4906 6546 8807 15527 1974 3476 14666 13472 17605 12833 9706
9679 4373 19761 15703 11680 18191 19790 19596 6939 14155 3044 7880
10088 3490
299: 13175 3648 1095 19091 6471 18453 11391 11863 12390 14399 8543 2686
18964 17580 17565
300: 15769 8388 13307 16886 9617 9239 11198 2854 892 2335 11948 5159
11294 8727 8073 11276 15356 19272 11054
301: 10572 5247 19764 5097 11353 18884 8479 9341 11247 19532 7752 2918
6108 14397 6315
302: 17017 11635 5436 17789 15563 8900 12793 3944 11383 6001 6054 7021
17088 19664 6262 2642 18990 4151 14818 7272 8345 5258 16301 1266
4095 18986 14878 10156
303: 9378 2246 16919 2665 10325 6537 6515 8525 17336 2540
304: 3039 5198 11864 16540 18772 6379 16878 17787 1436 12983 13229 16103
16127 8943 12794 16102 16100 4759 19321 8190 2913 15179 3181 5096
12048 6045 692 3816 5920 473 489 14828 10454 1781 15219 3647
12472 10767 4582 2698 3361 17036 3796 8573 19690 16677 8338 13554
16002 17464 6225 3244 7491 16139 13938 11138 14770 18781 12979 6776
12028 7778 6586
305: 6411 7134 8906 16488 13440 2289 8244 2283 7468 6684 10347 4285
18467 13646 6223 12937 4443 18455 1856 14107 6321 2537 3970 3246
12156 16596 16256 12890 15409 19151 4718 5175 11879 18126 18711 9204
13563 9579 16836
306: 12951 10398 11346 19186 15452 7802 7096 13491 13681 11482 14807 3733
17000 671 17496 8110 12773 17405 2034 7911 17108 17121 17123 10527
13104 9923 15163 15161 15672 1133 7107 15159 16049 2789 5414 19524
9684
307: 17075 12342 704 19539 6820 19480 17050 17008 19402 15902 18440 5435
2820 13268 13272 6889 3099 441 18043 8140 7516 17639 18575 12426
10811 14604 7842 4222 5559 17265 12910 16743 603 5928 7036
308: 9770 17349 9595 3084 14563 12042 12040 1361 7692 7690 7693 11111
13156 9807 11464 16693 16672 16674 16694 16675 11027 16798 4507 7680
7462 15210 11446 7522 12111 10983 13675 15116 10650 17747 18142 6554
18344 14846 3610 1916 1918 1920 10514 10515 5814 16298 16963 8419
5633 10896 11971 11972 11360 9381 15416 8935 9451 16130 7252 12038
12592 11897 11899 9157 12294 15591 3634 3640 6918 1028 1025 1010
7954 6924 6937 3533 5048 13534 13474 10893 902 10939 9585 2197
2948 10393 11720 14453 7240 11026 11092 11088 13756 13185 13798 13762
13187 13799 16822 9450 14147 11126 10984 11917 14454 6394 8064 8050
1033 4682 5085 13556 3724 3725 3721 3722 3710 18855 16128 4243
13813 13676 13159 17397 9345 13367 5940 18157 464 11157 3264 3704
3705 16760 646 11952 7153 3598 8442 9161 5867 7339 1404 15165
12587 12670 19102 11078 3824 18746 9973 5144 1144 7739 4130 12607
1009 1004 1006 11992 1285 16777 7909 4484 8987 406 3215 13397
1047 5188 16783 5519 18583 11625 11004 11056 11059 6050 3963 13604
8117 14971 18716 17730 12013 7877 17204 5195 19070 4915 13843 19554
4417 15788 11190 1693 2846 16330 11950 7243 10375 3691 3684 3687
17915 16005 1671 19540 10499 10225 15761 1933 15767 2505 8246 16343
19493 18295 12056 10278 16556 16134 13206 13209 16090 16088 16110 9103
10581 9834 3635 13203 4829 5828 1395 2655 597 19923 5896 16913
5772 1466 3658 3683 821 19840 795 820 15202 10661 15913 19756
19772 405 19504 5314 3672 6313 9762 9767 14249 2904 2899 2900
8635 18813 7724 8042 16951 14555 9160 2926 14241 2897 2907 6915
6922 6897 9718 18623 16845 656 654 659 16108 18227 12270 12018
10063 10061 13077 8431 11119 11118 16150 16966 13427 3374 18426 7386
11478 3612 3613 3636 6900 6903 16373 19217 6757 19030 10360 13103
6818 14125 6759 13921 13923 1196 13924 4819 16656 1658 8471 1659
11962 14022 10872 13157 16640 7948 18681 18699 9529 8931 12442 1512
3143 16501 19154 6032 6908 18549 9542 12945 3916 18656 12708 15592
14240 9740 9737 14661 1110 13204 886 14017 9925 2939 15031 19131
1242 483 2701 9085 19779 2445 3183 8143 14002 9189 19747 2742
4298 18369 5914 14761 2103 10374 8783 8779 8777 17066 2087 8596
4717 10877 14774 8813 8334 16562 10853 8024 16960 13916 13919 15016
15751 12115 10855 2038 746 14851 19743 15226 16549 2267 6875 7927
1005 9649 9959 7485 19025 14411 14374 9783 13960 2711 9164 3716
2547 17424 18941 14075 13323 5543 14815 2165 1756 13839 16367 15644
11187 13216 6388 12024 17755 13911 9905 17809 19499 10196 18637 7216
7392 11656 13835 19161 16251 11503 6720 2060 15709 5305 7634 17852
18207 16818 3165 3167 12327 9373 14446 11406 9769 2446 11776 15103
15104 9805 5938 19634 3749 1241 11031 13445 16440 11033 3821 17474
17796 16654 11605 11600 17919 4230 11602 17229 15737 7785 3844 3847
519 688 4144 11023 1132 10390 15833 11665 4077 12918 16563 15661
7525 9509 17053 887 778 9741 9759 13398 9612 8193 17328 1726
10300 10279 9459 4992 8844 9571 11468 16657 10900 10059 4404 16431
11209 1678 3452 10909 10062 4426 1897 4429 8284 16799 18573 16149
16964 6742 6744 5361 16614 5359 12651 15347 15877 15350 15344 4440
3577 3488 4442 10910 10911 14004 10912 10807 15901 13213 4453 4455
4477 4149 10914 4145 4506 4504 4500 4481 577 17702 16492 16427
16446 11291 16449 16422 11428 16623 11430 19757 14384 11120 19257 11484
6191 11608 846 12045 12044 14753 16772 3629 13129 8555 3650 3653
10024 12068 12069 1079 9792 9323 9336 9318 12730 11003 10356 10985
6919 18233 18757 9875 9790 9802 11466 5912 9363 883 11049 11994
14457 3872 11895 9736 10974 19097 11659 19098 11658 9788
309: 16801 13132 15648 16447 8652 17833 5712 8074 10690 3442 8077 16017
4336 4334 18570 13315 10785 16462 16900 17538 1921 19152 17383 12486
10936 16527 2668 17086 13286 16325 15106 13376 6034 10789 18519 10507
14473 19441 13022 10831 1501 10537 763 17615 18865 8762 11975 17249
1355 1352 1731 3928 897 5821 9053 17271 7633 3483 11371 720
6786 10425 4104 19770 14043 16580 16413 15086 13278 2632 19416 11567
16915 397 7115 6155 10323 4935 10287 7095 9624 14788 15196 8456
15783 6496 1418 747 8160 15292 7226 422 8460 777 12388 7677
4483 18613 12189 5109 11791 10558 6465 10804 9227 17539 7309 3145
8845 16327 2894 1639 629 7456 18787 8517 4205 13792 7154 8540
19386 12121 8153 18037 14847 2324 11604 5469 9801 18468 17366 14140
12739 8760 7404 15266 14677 8556 8459 8628 6999 13739 10675 6389
19049 14756 908 978 13391 15622 2116 3418 6687 7203 11089 7349
10890 12729 12757 12691 8258 15154 5394 2810 6594 13059 13162 13166
13276 13782 13550 9631 11740 13230 18487 12114 13130 765 19329 16985
16984 5165 16244 3083 4150 10606 8313 3214 11734 4026 14277 12261
15383 15691 14747 10250 7726 5530 9870 13909 4204 5398 7793 3405
7816 1214 655 5288 9771 18267 18824 18445 5386 6029 17183 14760
17340 14748 14228 707 15468 7547 7657 4329 6352 16661 19814 2314
3041 8145 7582 19453 4067 11236 9815 11632 10505 5399 5401 5701
3262 7403 12127 7921 3166 6436 1351 6654 13970 6833 12197 8822
8554 1854 17475 5955 3220 10389 18522 18779 11151 13163 16644 6673
19122 8310 11845 11773 14281 8380 9076 16982 13329 19679 12412 10783
16471 12667 4679 11260 17650 666 16258 15997 18585 8921 10213 7197
5699 9843 6432 2595 1776 740 11551 15041 10033 14055 10738 16517
16515 13985 6821 4687 3337 14627 7093 14433 13024 16428 7275 3380
4043 18350 608 14968 2172 16860 13317 8029 18848 11693 11576 15045
11780 19931 1062 11038 10639 11224 11221 2227 3034 6350 5148 7749
12030 8913 7568 3467 5635 3053 4469 12468 7698 10930 8896 10567
5095 10895 11854 16996 7668 15453 7431 7583 8611 5945 3403 2901
7239 8207 19926 4267 5634 2493 15967 18640 17304 14193 13803 13885
12365 19133 15544 17454 12090 5312 11677 14113 8439 6438 1093 1935
8879 6523 2410 4492 1778 17725 1981 17098 9203 11167 16434 12877
12668 8547 1722 11460 9512 9552 5897 11717 12745 12744 4809 4793
17479 11627 8724 14508 4543 10264 4971 8893 12051 5043 4355 1461
19129 13714 13778 4337 1055 12988 17873 9876 17972 770 14159 4321
9397 9625 4982 12453 12399 15388 13950 19243 9355 1752 1060 16605
8940 17507 5292 4698 6678 1898 8775 4290 2447 16769 3911 11807
5120 6985 4114 3219 17056 14361 17019 4749 8281 1577 17419 16965
17327 5163 19684 6013 18109 15816 18702 17013 15234 16355 14223 9490
1545 18506 18978 18616 9311 10823 8457 8366 18318 6280 1223 3810
19685 10902 7638 4578 7075 3196 14114 9172 3199 14132 12017 11925
1300 1299 18838 3719 12015 11644 5046 3195 19721 18624 15377 19934
6278 1999 1378 12351 5552 13478 7204 16346 1190 12660 10133 10832
11577 11662 11896 13158 19477 7317 4352 1153 5239 13031 8108 17846
9200 7760 2474 1105 8336 10576 16823 13973 16952 16876 12397 479
1881 1602 6077 2802 12398 4605 2551 8786 2140 2855 2834 9841
17023 18871 17373 17344 16728 10808 18676 16746 8129 2385 8180 3562
5933 4787 2996 10676 15651 12169 19544 7801 7900 2278 855 7521
18383 2546 1234 12032 19919 3652 721 8963 1814 10032 8605 10275
18902 12424 12526
310: 4993 14546 1029 7514 2917 10152 11335 9594 14726 1405 17937 6935
14151 10273 685 5997 7222 14658 11749 10121 7000 10002 6010 16908
7529 2137 5389 15290 8071 6027 4413 15840 17540 6072 5406 10477
13780 15060 14826 18210 10116 10120 10142 16667 851 4735 17780 8426
10161 10163 18631 4975 4344 3194 6508 3557 11735 16259 4023 12288
16472 15236 18423 2712 6253 7180 8745 16575 12645 11798 15025 14872
12821 12055 5069 8763 5400 5829 7705 7669 2817 5344 1590 8606
18818 5065 13075 16682 18975 16411 12120 1632 2145 10107 6360 3618
17320 18641 7159 19374 8892 1683 10110 12001 19521 2499 7464 11752
11792 2395 3322 19468 1246 2094 17945 13685 3703 7073 8473 15278
11750 6376 722 8952 1788 12276 2177 8655 3659 2253 15358 9315
2534 8070 15012 13161 10545 826 3249 6401 10373 13341 5991 4410
9993 9994 4785 3349 11306 9814 13902 10962 18365 854 5075 13311
4178 13328 18181 19894 19525 14890 1603 6161 14478 9243 587 2986
3592 12649 3080 15617 11601 13501 13181 6206 12788 7897 11732 2732
7914 3063 8862 19937 4586 2634 18225 17758 1058 4002 4561 14369
2043 9570 3991 19823 19209 11521 18784 14970 6473 6131 8658 7280
15987 11465 8315 15256 19681 16402 17975 1559 5862 3880 11271 16235
13872 2108 14500 11285 16316 19436 10432 7652 11110 16345 19523 2843
17746 1118 7774 19578 18424 4835 14580 15727 5582 2332 15462 2821
17034 16853 10210 605 14264 12320 9478 13425 7636 16940 15239 5379
13944 8103 651 15792 12912 15640 3131 7232 11772 19236 15809 4006
529 19806 7702 4689 17509 3421 19306 5782 5183 10158 6488 7040
13260 10093 13627 8732 686 10592 15289 19347 9038 13009 13925 7052
14143 12780 12107 8739 14688 11400 7874 6474 3104 3107 14423 1690
5872 1941 9668 3685 11900 10682 15943 14081 19665 8352 10943 8232
4088 18499 14024 14211 18737 9701 5604 13245 3263 5326 14254 14253
14255 14251 19565 4990 2198 4887 7549 16593 10140 4192 623 1967
10165 10137 16711 3289 18400 18391 18244 10980 7532 10316 16098 911
19278 11727 8228 17323 16757 8819 2097 7207 18771 1562 7815 9992
16477 8768 11770 13930 10114 10138 10097 10099 13210 9302 4394 5658
18666 1679 11104 1773 19230 14524 6799 14127 8979 14095 16554 5101
8256 3565 16106 3486 17338 8305 12200 9627 7178 11323 13709 19928
6851 15448 5450 14758 6440 4840 16948 9856 17693 10481 18098 12809
9820 8612 5560 2328 16487 4312 4316 9793 17788 16283 4610 8379
6024 3518 14472 940 18073 4253 1517 12073 19157 19655 12239 12641
4520 7457 14257 13738 6631 9744 7100 10958 1663 19658 19657 850
830 981 19109 1871 7127 18552 12232 3002 16895 13330 7142 5045
8449 15114 15111 19687 19859 2450 5262 5468 1622 15404 3091 1432
7857 10115 9421 16767 16178 10367 18032 14517 10922 5596 4845 17422
17656 15742 14974 16264 17181 461 10535 7200 13155 18603 12379 4629
12897 19551 3158 16835 2030 2535 10521 5328 8602 6303 12908 6340
16365 17709 15944 18673 710 17701 14621 2173 12063 3274 3229 8399
3984 9226 19429 19709 17274 5355 16698 11541 15860 3074 17598 1573
5354 6512 10753 4856 3042 14767 3811 14876 856 14273 14271 2338
13640 5456 5459 8719 10281 6810 7883 15726 15508 17683 3047 16285
12882 13574 3723 18442 11447 18277 8323 8320 19125 8734 18846 8429
8735 6351 18016 7745 18581 16456 13515 4296 16800 3915 7965 11716
9558 12382 5285 893 552 10143 18741 19308 284 3200 12228 1654
713 1275 3193 1053 601 4292 7027 15113 11082 1186
311: 13531 15014 19184 4117 8168 10792 10850 19284 5526 10160 888 19833
19024 13115 9575 3212 14092 11196 16286 14686 5558 17090 17081 16907
15798 5743 12604 593 8040 15926 19225 11909 5613 8553 2850 19417
15940 5913 17199 13547 15258 14739 402 14718 10577 2249 2275 7788
11681 6460 1636 8604 12864 2293 8498 7265 8520 7952 12354 16688
12128 1747 6881 10130 17339 3222 18097 865 18477 16994 5237 19366
395 5648 7676 8241 14849 16511 1950 3133 15141 12703 6805 4165
17466 13619 14951 10472 5901 7341 11090 7199 6418 19254 13897 13967
6098 8683 6978 11297 10739 18490 11417 13326 15170 8985 4283 10620
10050 2430 5975 15405 7764 12075 11938 5271 18351 2785 12328 14687
15954 10173 10759 17131 7683 6392 9694 19508 11227 14431 10991 10955
5333 17596 14716 12198 17690 13772 11828 9888 1787 3935 15629 403
11568 12658 9592 16152 16381 18209 19448 16752 14848 2937 5447 7889
8469 9447 13876 12634 18731 15627 12984 10207 3116 8826 6318 8934
3762 3827 1708 7049 9274 12720 9444 9420 7354 14664 6570 4045
1225 17512 4782 7308 1264 7406 3352 15122 18219 1822 2876 14134
2291 3261 16739 2348 11942 11875 9500 6270 9871 6173 5722 16368
13322 16507 13154 9751 11960 1438 3048 4276 19604 2680 11597 11557
8211 1536 11015 10386 6448 17614 15609 11101 10314 7156 6322 4619
11816 14775 1023 3639 18377 18621 18433 10468 17302 1949 17435 9669
4544 1990 7409 11278 9214 16595 11711 18807 4491 14566 15498 15400
17691 19615 14984 2401 18622 13403 8647 3919 733 12287 9690 2714
4040 7221 18345 19682 16469 10317 16625 3496 2388 12510 10301 19547
15032 8347 1998 2048 14532 10402 4795 13067 6630 12135 4047 7346
15387 1896 11660 3389 19879 2061 6988 19738 13900 4474 2621 17457
11124 8595 8920 19461 11829 16912 9638 10219 14338 4225 12172 9374
10078 11281 3024 15023 11963 14871 13655 10963 11216 16949 17195 6722
17870 4862 12097 5511 1927 6433 11365 12620 12118 8817 4187 18833
16333 4734 2143 19881 4183 19538 447 7211 18329 5917 9943 14372
8806 16953
312: 9795 6261 5149 5170 5168 12820 7573 12233 9643 9693 2068 11819
18352 16729 2793 17847 7225 12946 8557 1832 444 10898 1956 4691
7777 13647 19902 13284 13061 12536 5153 5151 16950 2990 2989 2988
3487 2993 9433 3848 1198 1195 12880 1340 18680 17799 7617 13361
9528 3585 6230 9821 3940 6243 18903 7878 9794 11915 6414 8130
3463 15900 17558 2602
313: 19107 3731 10544 1365 315 15072 9222 995 13810 10579 12184 1169
10820 8682 17063 17115 11426 7610 8335 6680 19590 669 12808 2095
3210 7981 8975 13797 19673 10108 12140 14891 4849 9589 10013 11489
5848 8508 16961 14635 3335 5341 9626 18112 14644 12785 9168 9484
8109 9837 3294 15636 3494 2017 19052 19769 16972 7811 19903 14579
7377 4121 7557 13806 6626 9547 10641 12326 19729 7235 1411 3644
10154 10157 2648 7794 10100 12628 15633 12168 7589 15853 4368 19745
14933 10255 1291 17736 2414 7188 16138 9089 9415 11728 12818 6609
8114 10065 12609 4638 16498 2517 12709 17410 17048 15136 16713 18469
19832 12689 1479 15584 12811 17562 13267 13892 10949 9327 472 5713
11273 9716 15698 5082 17282 1612 7565 10794 11434 8216 10240 6588
15316 15700 6461 7231 10826 9945 1966 767 9017 8428 9216 1791
12429 5223 4910 19009 13274 12648 16308 10486 4798 9611 5747 13303
1346 2839 10642 10405 4049 4046 969 5871 13542 17764 7597 9272
18722 14315 13433 12396 17102 19120 3267 2911 2934 2480 15593 1566
15118 17099 13495 18798 16391 13630 12936 4072 17390 11981 12247 14556
14559 12268 14558 14560 14577 12267 14576 12226 9387 332 2109 7841
18828 9615 9388 17887 3007 11137 19936 14986 3553 4554 11929 16099
5440 845 18184 2805 4097 18866 12066 14917 16033 3934 3003 1594
10929 12343 16214 17420 5606 4643 10415 6601 15702 10449 19711 13552
1507 19835 1698 9321 11696 2863 14802 17698 1498 17572 14405 13598
12043 2262 567 1583 2618 3734 18793 19614 14449 16036 8175 19636
13279 17286 5486 17542 9087 15980 2290 4878 11904 428 10747 9550
13537 3804 8971 5843 3250 1514 18175 14056 11021 12535 9436 8113
10697 15175 7725 13365 5679 15598 19442 16718 6306 9803 17134 10762
2355 13282 9344 18414 962 10707 13732 15247 16140 14944 8665 3901
12556 13238 3850 16213 15720 5407 7133 17222 15571 4342 952 16118
708 14879 2997 19403 14894 498 14617 11730 18476 3794 6451 3609
16585 3917 3066 11348 16371 13698 6380 5627 5614 3881 18493 6416
12573 15178 7043 8200 17880 11947 15915 7621 7041 8209 18926 10681
14294 3702 8567 18263 9236 1978 3932 19800 9976 13414 8046 11624
1429 3768 17319 17779 7609 16512 630 3171 7227 3551 9996 16499
8855 13654 9117 3812 16292 19434 10320 5172 1982 3126 14582 9645
1807 19478 7519 12941 17861 14481 4909 1899 3459 1922 19333 4083
1541 12260 11304 16432 3209 9163 2838 17263 2745 9173 17072 5826
7351 9534 19739 19536 7374 17185 14542 17759 5670 4857 6297 13448
7546 11655 18214 11988 3497 12119 18421 2238 9138 8088 14015 6428
10563 6419 14637 4955 9470 1521 11999 10694 314 18961 1819 15545
10660 10122 4646 13358 18428 15805 12921 13439 13112 10688 18791 3218
10153 5875 15881 14320 2938 13237 12193 16805 2072 13687 15276 12110
9603 12516 3748 11719 18595 2719 7437 2981 5840 16220 2127 19699
11867 10298 3040 4738 17635 17582 9714 2444 8326 10594 16463 11355
16573 18034 10940 2168 14219 13085 7255 8220 15653 19511 18649 7840
8684 2272 9676 5395 14310 13304 17528 4645 2741 19834 12291 3607
4917 15188 1038 15537 16448 8891 10556 10553 13996 16085 16717 16720
16629 3416 6712 18027 18028 18046 18045 18018 2046 2280 11438 7044
432 17329 10580 2438 10575 2435 12466 13227 13331 3373 4354 1137
14182 14180 17288 17285 17287 17309 18279 17281 5134 13133 13134 7591
7579 7578 7590 7622 7593 7619 8710 11257 10155 17393 5497 7611
7613 3368 3371 3345 3347 11146 3346 15875 3327 11270 617 12175
15844 16942 6398 16524 14919 6019 6427 4568 13742 8545 8646 17237
10441 15117 18131 812 16684 7548 4903 4891 11339 14205 916 13861
4237 5869 14326 7059 9757 15666 8359 2239 6482 16408 10426 1600
4339 9125 15701 10147 6375 17364 694 11598 14712 1946 16250 7313
3680 8784 12459 4338 15013 3889 4760 2323 2136 16586 3470 11223
13993 8676 4320 6154 958 7689 4997 6877 12357 17840 14977 10169
1152 12474 11366 1049 7296 3453 823 1007 2433 5629 7080 8534
2047 12554 18572 15080 13812 14010 15955 19762 16570 11714 12363 13476
11640 13481 6845 19136 3340 15749 8548 16230 2819 2803 2874 2851
2798 2774 2772 2847 2769 2801 11161 14362 3590 279 19320 14355
13633 12071 4122 10856 1712 8501 2235 2242 2243 2245 18869 17430
17096 16063 16061 4419 4399 7499 7500 7479 7507 7501 7478 2530
9228 852 5988 15329 19925 15542 16445 15520 16918 16920 16451 14285
15560 16922 3473 2729 2751 2748 2730 1449 6008 10997
314: 19107 3731 10544 1365 3521 18558 315 15072 9222 995 13810 10579
12184 1169 10820 8682 17063 17115 11426 7610 8335 6680 19590 669
12808 2095 3210 7981 8975 13797 19673 10108 12140 14891 4849 9589
10013 11489 5848 8508 16961 14635 3335 5341 9626 18112 14644 12785
9168 9484 8109 9837 3294 15636 3494 2017 19052 19769 16972 7811
19903 14579 7377 4121 7557 13806 6626 9547 7168 10641 12326 19729
7235 1411 3644 10157 10154 2648 7794 10100 12628 15633 12168 7589
15853 4368 19745 14933 10255 1291 17736 2414 7188 16138 9089 9415
11728 12818 6609 8114 10065 12609 4638 16498 2517 12709 17410 17048
15136 16713 18469 19832 12689 1479 15584 12811 17562 13267 13892 10949
9327 472 5713 11273 9716 11114 15698 5082 17282 1612 7565 10794
11434 8216 10240 6588 15316 15700 6461 7231 10826 17284 9945 1966
767 9017 8428 9216 1791 12429 5223 4910 19009 13274 12648 16308
4798 9611 5747 13303 2839 10405 4049 4046 969 5871 13542 17764
7597 9272 18722 14315 13433 12396 17102 19120 3267 2911 2934 2480
15593 16755 1566 15118 17099 13495 18798 16391 13630 12936 4072 17390
11981 14556 12247 14559 12268 14560 14577 14558 12267 14576 12226 9387
332 2109 7841 18828 9615 17887 9388 3007 16099 12066 845 18184
4097 18866 11137 19936 14986 3553 4554 11929 5440 2805 16033 3934
3003 1594 10929 12343 16214 17420 5606 4643 10415 14917 6601 15702
10449 19711 13552 1507 19835 1698 9321 11696 2863 14802 17698 1498
17572 14405 13598 12043 2262 567 1583 2618 3734 18793 19614 14449
16036 8175 19636 13279 17286 5486 17542 9087 15980 2290 4878 11904
428 10747 9550 13537 3804 8971 5843 3250 1514 18175 14056 11021
12535 9436 8113 10697 15175 7725 13365 5679 15598 19442 16718 6306
9803 17134 10762 2355 13282 9344 18414 962 10707 3609 13732 15247
16140 14944 8665 3901 12556 13238 3850 16213 15720 5407 7133 17222
15571 4342 952 16118 708 14879 19403 2997 14894 498 14617 11730
18476 3794 6451 16585 3917 3066 16371 13698 6380 5627 5614 3881
18493 6416 12573 15178 11348 7043 8200 17880 11947 15915 7621 7041
8209 18926 10681 14294 3702 8567 18263 9236 1978 3932 19800 9976
13414 8046 11624 1429 3768 17319 17779 7609 16512 630 3171 7227
3551 9996 16499 8855 13654 9117 3812 16292 19434 10320 5172 1982
3126 14582 9645 1807 19478 7519 12941 17861 14481 4909 1899 3459
1922 19333 4083 1541 12260 11304 16432 3209 9163 2838 17263 2745
9173 17072 5826 7351 9534 19739 19536 7374 17185 14542 17759 5670
4857 6297 13448 7546 11655 18214 11988 3497 12119 18421 2238 9138
8088 14015 6428 10563 6419 14637 4955 9470 6734 1521 11999 10694
1819 15545 10660 10122 4646 13358 12921 18428 15805 10688 13439 13112
10153 5875 3218 18791 2938 12193 15881 16805 14320 13237 2072 13687
15276 12110 9603 12516 3748 11719 18595 2719 7437 2981 5840 16220
19699 10298 2127 3040 11867 4738 17635 17582 9714 2444 10594 8326
16463 11355 16573 10940 2168 14219 13085 7255 18034 15653 19511 18649
7840 8220 2272 9676 5395 14310 8684 4645 2741 19834 12291 4917
15188 3607 13304 1038 15537 16448 8891 10556 10553 13996 16085 16717
16720 16629 3416 6712 18028 18027 18046 18045 18018 2046 2280 11438
7044 432 17329 10580 2438 10575 2435 12466 13227 13331 3373 4354
1137 14182 14180 17288 17285 17287 17309 18279 17281 5134 13133 13134
7591 7579 7578 7590 7622 7593 7619 8710 11257 10155 17393 5497
7611 7613 3368 3371 3345 3347 11146 3346 3327 11270 15875 617
12175 15844 16942 6398 16524 14919 6019 6427 4568 13742 8545 17237
8646 812 10441 15117 16684 7548 11339 18131 4903 4891 14205 916
13861 14326 4760 4997 7059 9757 15666 8359 2239 6482 16408 10426
1600 4339 9125 15701 10147 6375 17364 694 5869 11598 14712 1946
16250 7313 4800 3680 8784 12459 4338 15013 3889 2323 2136 16586
3470 11223 13993 8676 4320 6154 958 7689 6877 12357 17840 14977
10169 1152 12474 11366 1049 7296 3453 16153 10586 1007 2433 5629
7080 8534 2047 12554 18572 15080 13812 14010 15955 823 19762 16570
11714 12363 13476 13481 6845 19136 3340 15749 8548 16230 2803 2874
2819 2798 2851 2774 2772 2847 2769 2801 11161 14362 3590 279
19320 14355 13633 12071 1712 8501 2235 2242 2245 2243 18869 17430
17096 16063 16061 4419 4399 7500 7499 7479 7507 7501 7478 2530
9228 852 5988 15329 19925 15542 15520 16445 16918 14285 16922 15560
3473 2729 2751 2748 2730 1449 313 6008 10997 18961
315: 2185 11361 5661 8686 17264 8328 8330 7550 10232 18956 1696 5922
16632 14340 10579 4523 1169 11996 8012 4522 16791 8335 10038 5066
4969 4634 17213 14401 7284 2099 15619 17243 6718 8030 14321 1903
15554 15028 12204 3060 19178 8109 11653 9316 17858 18132 18589 9667
17297 5418 17508 15450 6147 5012 18588 18020 862 4920 15050 16525
3064 2884 5607 12297 11396 18052 2912 17060 1697 17604 18870 8264
17736 1732 5968 5593 15864 18761 4044 5088 17149 10593 5242 4527
13682 8054 16503 1962 19380 1623 10664 4597 15319 3478 16945 18584
3108 12724 15546 15764 15754 18354 18416 4890 19678 19130 15532 5213
11374 9753 14702 15217 17235 15921 5855 18752 1904 15721 18203 18156
18864 8700 7560 16924 636 6811 16379 19046 11063 10405 4049 4046
969 14929 16207 7597 9272 15425 17368 11052 14975 6574 9560 10349
3830 8250 2911 2934 2015 18785 8645 8196 7713 11586 14490 18301
18792 15378 7699 18803 19816 16104 14468 17390 11981 14556 12247 14559
12268 14560 14577 14558 12267 14576 15277 12222 16710 12224 12226 9387
332 17612 2109 7841 18828 11556 1969 9578 7424 5369 245 18873
16602 11137 19936 14986 3553 4554 11929 5440 2805 12674 16033 3934
3003 1594 10929 12343 16214 17420 5606 4643 10415 14917 6601 15702
10449 19711 13552 2863 1507 19835 1698 9321 11696 14802 17698 1498
17572 14405 13598 12043 2262 567 1583 2618 3734 18793 19614 14449
16036 8175 19636 13279 17286 5486 17542 9087 15980 2290 4878 11904
428 10747 9550 13537 3804 8971 5843 3250 1514 18175 14056 11021
12535 9436 8113 10697 15175 7725 13365 5679 15598 19442 16718 6306
9803 17134 10762 2355 13282 9344 18414 962 10707 13732 15247 16140
14944 8665 3901 12556 13238 3850 16213 15720 5407 7133 17222 15571
4342 952 16118 708 14879 19403 2997 14894 13269 498 14617 11730
18476 3794 6451 4601 7256 19396 2011 16585 3917 3066 16371 13698
6380 5627 5614 3881 18493 6416 12573 15178 8209 11348 7043 8200
17880 11947 15915 7621 7041 18926 10681 14294 3702 8567 18263 9236
1978 3932 19800 9976 13414 8046 11624 1429 3768 17319 17779 7609
16512 630 3171 7227 3551 9996 16499 8855 13654 9117 3812 16292
19434 10320 5172 1982 3126 14582 9645 1807 19478 7519 12941 17861
14481 4909 1899 3459 1922 19333 4083 1541 11695 12260 11304 16432
6090 18117 19599 18305 3209 9163 2838 17263 2745 9173 17072 5826
7351 9534 19739 19536 7374 17185 14542 17759 5670 4857 6297 13448
7546 11655 18214 11988 3497 12119 18421 2238 15982 9138 8088 14015
6428 10563 6419 14637 4955 15467 9470 1521 11999 10694 314 1765
1819 4359 18984 14947 13368 6080 8594 12041 17552 5525 3665 12817
1783 2890 18428 15805 9454 10688 13439 13112 10153 5875 3218 18791
2938 12193 15881 16805 18770 14320 13237 2072 10668 13687 15276 12110
9603 12516 3748 11719 18595 2719 7437 2981 5840 16220 19699 10298
2127 3040 11867 4738 17635 17582 9714 2444 10594 8326 16463 16175
11355 16573 10940 2168 14219 13085 7255 18034 15653 19511 18649 7840
8220 2272 9676 5395 14310 8684 4645 2741 19834 12291 1038 15537
16448 8891 10556 10553 13996 16085 8752 6235 10378 16031 9773 9748
8634 1983 5010 16717 16720 16629 3416 6712 18028 18027 18046 18045
2247 17307 5751 13958 2046 2280 11815 11438 7044 432 17329 10580
2438 10575 2435 12466 13227 13331 3373 4354 1137 14182 14180 1685
6522 17288 17285 17287 17309 18279 17281 5134 13133 13134 7591 7579
7578 7590 7622 7593 7619 8710 11257 10136 10155 17393 5497 7611
7613 3368 3371 3345 3347 11146 3346 3342 14996 9789 7217 2775
6179 6205 617 5167 7673 18690 12874 2313 15844 16942 6398 16524
14919 6019 6427 13742 5524 12408 15818 13998 17952 15058 2676 443
16567 8545 9337 11440 6373 12996 16653 18303 8646 8910 18008 14978
10441 2409 16827 2374 16684 7548 18131 4903 4891 11339 14205 916
13861 14326 7296 2136 7059 9757 15666 8359 2239 6482 1101 8053
8470 8156 10487 1094 11152 5629 7080 6211 8534 2047 12554 18572
15080 13812 14010 15955 9662 19762 16570 11714 2862 11347 12363 2885
13476 19136 3340 13481 6845 8548 16230 15749 18141 2803 2874 2819
2798 2851 2774 2772 2847 2769 2801 11161 14362 6202 8537 8518
7037 8133 6764 827 279 4941 1712 818 19365 8356 7588 3017
2235 2242 2245 2243 18869 6400 5637 915 8416 12444 510 12026
11634 16063 16061 4419 4399 7500 7499 7479 7507 7501 7478 2530
9859 1460 18194 11314 2685 4226 15542 19925 15520 16922 15560 5221
8058 14961 984 19513 2729 2751 2748 2730 8932 313 12905 6008
4112 8720
316: 5462 8702 15896 14144 15464 19725 9380 4042 545 16095 1526 8816
16014 8322 3022 1553 2635 18516 10285 6421 5546 6489 8482 8478
5585 16227 2190 10760 1465 2255 11395 7990 14793 10172 16192 7992
12560 3462 14906 12272 16970 2134 2867 2758 7489 6672 10233 12166
15802 1638 918 18996 2473 4032 8620 3162 3756 12149 13374 1308
1307 11769 13333 10034 11874 12574 4659 9026 13327 2753 14426 2429
11931 6246 16533 2250 3513 4318 4974 3869 15232 3820 4665 16608
13406 16766 3671 4310 8986 18992 8995 19933 19932 8988 7545 3888
1293 17163 11580 4168 4167 16335 4007 13342 13297 2864 18382 15220
6521 15457 12531 12431 4213 7363 7483 7328 2936 8542 8532 1997
2463 1996 7728 9652 15509 7851 12529 2192 8974 9761 13146 8836
9376 9401 2717 11802 11806 2558 2553 18119 3929 423 19081 8210
9326 5275 14503 3110 14226 5291 5571 4513 5138 6994 5137 17365
1785 5345 11683 6635 7064 18861 13492 12133 805 10146 9632 15755
16143 8100 13038 16967 19575 19594 3996 16196 1721 16725 14719 12580
11240 5778 618 8966 8955 19713 9948 9946 3468 7884 16898 1191
18670 7152 18629 2566 12157 3148 14714 9023 16483 14008 17781 13594
7412 4148 15789 2191 19158 8063 16731 853 6697 3043 4675 3548
5951 5342 3186 6258 12561 3976 16231 7192 3154 16854 9100 11130
3448 9146 12098 14067 15841 18357 879 3661 7236 1239 14934 1964
8301 13913 18880 2792 2960 8392 12600 19792 13251 1556 8938 11739
11738 17186 14126 8704 6167 15076 7923 10995 8699 4137 19459 2768
11490 2285 468 3256 3962 3526 6749 785 4270 18693 14414 12394
15009 15406 7292 12653 1253 9468 10332 19444 7625 16015 5343 16491
5692 14537 13107 10267 5745 11145 6063 13844 12413 7513 1555 19155
8411 14705 6809 12611 16041 16179 16590 16403 14059 5845 15694 17067
9812 2968 9811 11761 5358 7512 10371 10369 6536 5488 6872 5893
5937 18324 3446 15225 17313 18360 16566 17334 12104 16784 4519 11690
1160 2536 14367 18839 10104 11824 2933 10654 19865 12293 12274 12271
7670 10692 7530 2086 7632 7453 5948 6608 10756 17250 3010 4463
3520 2896 3517 10296 1847 11132 11288 6317 16569 19515 4801 16530
3438 12214 12211 3078 7475 6795 4591 15447
317: 2231 3383 14866 16544 7418 9107 4315 9446 19269
318: 4830 2716 9062 17385 658 13025 18942 1913 7312 19788 1716 3538
4143 17321 815 18938 9848 2344 5906 7890 446 6885 11380 759
13452 4140 13032 458 17467 1500 8433 6737 15173 681 2424 16493
320: 1136 7051 5444 7050 15227 18825 10186 7331 7329 11868 7003 6249
4750 4038 15916 19050 15491 14585 17723 15235 17636 18713 18024 13564
3655 7523 2416
321: 15725 14852 5781 6187 9364 6675 12873 12050 2178 18054 18447 870
12686 19055 13293 6047 3502 15480 6688 6643 16132 12103 18489 19742
17678 2836 12364 8309 14776 7846 17350 17117 11028 7278 1841
322: 19529 18972 10678 6073 19135 18539 10466 12487 17080 14595 7367 8165
4913 16318 11781
323: 9079 9955 19786 2490 2106 6931 4177 11411 14494 401 16768 10346
324: 11512 8262 6323 11449 12376 15280 6345 6342 6758 18459 5739 3469
6326 6372 4249 12966 5720 5704 5725 5724 5756 5810 5785 5764
5783 5788 5723 5727 11451 8412 19543 15504 16837 1442 3587 7395
11317 13364 750 15231 7715 3807 7915 8643 10574 16564 2736 16086
7850 13459 10318 9779 731 10396 10077 6470 798 7137 6756 6754
14009 17356 13319 2236 18981 17018 6664 803 19647 1274 15145 18856
9265 1809 1806 1811 9896 17998 8581 15642 5684 17295 7254 7960
5636 8730 9734 9831 19625 7753 8179 7998 5300 6623 12543 16137
18696 5777 1167 4524 1077 11970 3765 4567 14316 9593 16226 5513
12102 3788 19164 15971 3631 12022 13285 9219 5808 2023 12867 12139
14800 9148 9151 5703 19445 3505 1417 9937 12900 5835 8793 17627
9980 10119 15928 3285 12749 534 19027 7116 10086 3784 18420 9372
3809 16983 19749 4147 2561 14685 19010 5508 15932 1043 8298 15444
13412 8608 15947 13976 8067 3584 19918 10353 11595 11284 14370 8853
4973 8385 5589 1798 1207 509 7947 11095 15540 3437 7615 7612
10821 10244 18075 7438 19651 8598 9205 7814 14515 14553 7875 9013
4551 17654 6535 4686 15510 9928 14939 9130 13820 10857 11701 13734
9606 633 7757 10685 964 7436 502 18371 10090 18370 5553 15365
16452 3436 13450 1356 18559 15301 2361 18638 10471 17333 9747 13715
12322 6368 6349 6775 6779 6773 6777 7269 6774 7262 6328 17232
17226 17209 17212 17211 5741 6344 6348 6476 12159 4157 7574 8332
6766 5759 5760 12701 2721 10839 17534 8451 14104 12537 13141 14902
14903 4314 14916 4309 13007 10518 3401 7876 1104 8383 14730 14720
12059 1106 6982 1031 15090 15093 6907 17634 8009 484 7723 1281
5926 776 6614 7009 15697 12348 13028 2330 12655 15081 6495 13560
18571 14791 19199 6442 7955 9241 7078 6798 10503 15218 8382 19172
938 11115 6504 5016 5836 18241 7022 17216 3591 6384 2085 8286
9708 14861 18111 4952 13581 14185 2853 14506 14516 6690 10730 1934
2612 10612 11767 14612 3998 3009 16097 19296 15380 18005 12479 8182
4663 14202 1064 1476 5622 11833 13437 12832 3282 17447 4447 5740
1103 6324 5794 13746 19613 13540 3119
325: 16615 14754 4057 8715 5965 14877 1866 8104 17176 4366 14518 9167
9065 10806 584 14195 17198 8201 4762 17268 2697 6174 2254 8981
13193 12420 6622 8325 5839 16464 6347 11709 18411 6294 10583
326: 11367 14736 13964 6308 3036 11437 10175 5974 9622 2625 2363 10917
11431 9207 5827 1582 5507
327: 10691 6846 18734 10944 7446 14465 1433 5628 16454 18586 10494 17172
7904 9524 7196 17084 9159 445 5749
328: 1385 15829 19449 11943 5936 18464 6928 7520 1074 12457 797 5586
11159 6704 13511 5517 4980 15756 19404 11162 11144 11682 8750 6782
14571 796 7484 18881 2581 19645 16081 10523 17997 6738 18661 512
13506 959 14965 17458 6459 1020 16188 3545 7761 17240 19190 3663
4263 17715 14220
329: 8714 3717 5566 10565 1859 668 9746 12025 1427 7417 530 14121
7988 11832 3990 15583 10329 7800 2906 9120 14177 14111 16467 2762
4786 19733 3949 3152 8034 16589 3235 4766 6719 8060 10844 12009
12714 15999 13202 19675 9263 15341 11412 11538 1175 13971 9989 3509
7314 9081 5349 6882 4703 8235 2796 5908 10247 12378 16419 16844
12799 14375 5098 6430 9862 12923 12774 18038 9506 4345 14983 1075
17160 6513 3472 1454 13829 6880 6497 17891 11910 6204 10379 14721
14990 10513 5022 5978 10881 5325 4900 11308 9150 18664 5881 12552
6583 6062 13051 8792 15886 882 6896 2502 9724 4391 16217 12349
5780 2148 13289 1827 6149 7304 5083 12091 2946 7643 17480 13408
14394 7079 11009 5514 9020 19826 17411 8276 9974 16420 12687 6975
522 5006 10709 7584 18456 15757 4215 10986 2166 5442 6286 9293
9482 944 7759 18574 18169 6499 4685 7873 8584 17543 9884 7382
7352 7061 17128 14603 3906 11674 10746 1503 13826 14262 12721 17380
11192 13270 15052 9833 12894 19206 19502 19182 12719 6650 17024 11885
12688 5510 1496 14710 18472 12216 4163 6189 13422 1597 8924 12843
10721 10028 1759 14239 14549 4604 10040 7920 1403 10667 14918 7430
13189 11618 14583 3524 4966 17459 839 13522 3834 6998 2999 6865
7167 13043 1901 11888 1209 6472 14813 788 3885 3956 7158 18767
16576 15007 18092 16591 6954 6172 3857 2555 2459 2669 18256 10830
6666 8480 11442 12577 9271 14060 9245 14655 15710 17030 7711 7709
3351 6487 4533 2082 17184 8698 14994 13896 7835 8142 814 14855
3617 935 10433 15296 18619 17848 3434 4185 18019 521 13339 14025
5754 12949 10467 9496 5158 19096 2135 576 993 12906 11309 11504
5691 9623 12589 3192 8950 6480 10293 11074 16003 19115 966 1849
14063 14904 657 1848 16007 14717 4142 16489 12381 2754 8869 14112
8121 19313 10861 19088 3712 6558 7559 10653 9717 16715 18087 10084
17845 19383 16584 14634 8560 18766 7631 18659 1540 9731 6883 17687
15181 15549 6519 11663 486 13767 15635 13224 15907 5070 10188 9295
9297 19656 13831 9250 18896 6462 10456 4816 6498 11564 14166 10726
13462 2420 6005 5935 11870 8991 1892 3542 12006 14673 1339 15348
11771 7607 19801 18944 7773 6641 18882 7567 18085 12565
330: 904 12835 3861 1482 5979 9709 13954 6123 6143 16986 17855 18668
1909 5240 12004 9616 16459 11407 4107 15822 4812 9206 13190 986
738 8516 7209 9218 9155 15166 1046 9972 9234 14160 10926 9007
12402 13404 14565 1314
331: 14505 19187 14237 2004 13667 15330 8712 2983 10833 6141 8832 10908
14924 5518 1621 10016 549 17499 11318 10215 16348 19205 15747 5139
1782 19628 11628 15831 15263 14491 13634 7647 10268 16730 6410 1484
1366 16382 14778 1874
332: 625 19038 18542 16881 3521 18558 315 11296 10579 14173 1169 11180
14471 696 8335 11062 1469 7394 18110 7821 19076 5860 4906 11277
2138 9025 2195 14684 8109 11176 9010 14713 5710 3301 16679 1176
8811 8290 4792 18465 8807 17003 8983 9258 15527 15529 14912 17091
17736 13621 7248 8229 8007 10799 19406 11113 13933 9338 9443 15873
16790 5467 4303 11067 15230 15245 17641 9012 19616 6000 14666 2725
14734 17332 12078 13496 13912 13472 10405 4049 4046 969 16147 12289
7597 9272 835 17757 15353 2123 17605 2911 2934 13460 10176 18009
1003 13239 12374 3372 17390 11981 14556 12247 14559 12268 14560 14577
14558 12267 14576 12226 9387 2109 7841 18828 11137 19936 14986 3553
4554 11929 5440 2805 16033 3934 3003 1594 10929 12343 16214 17420
5606 4643 10415 14917 6601 15702 10449 19711 13552 1507 19835 1698
9321 11696 2863 14802 17698 1498 17572 14405 13598 12043 2262 567
1583 2618 3734 18793 19614 14449 16036 8175 19636 13279 17286 5486
17542 9087 15980 2290 4878 11904 428 10747 9550 13537 3804 8971
5843 3250 1514 10411 13008 5960 18175 14056 11021 12535 9436 8113
10697 15175 7725 16718 13365 5679 15598 19442 6306 9803 17134 10762
2355 13282 9344 18414 962 10707 13732 15247 16140 14944 8665 3901
12556 13238 3850 16213 15720 5407 7133 17222 15571 4342 952 16118
708 14879 298 7886 19403 2997 14894 498 14617 11730 18476 3794
6451 16585 3917 3066 16371 13698 6380 5627 5614 3881 18493 6416
12573 15178 11348 7043 8200 17880 11947 15915 7621 7041 8209 18926
10681 10164 14294 3702 8567 18263 9236 1978 3932 19800 9976 13414
8046 11624 1429 3768 17319 17779 7609 16512 630 3171 7227 3551
9996 16499 19516 8855 13654 9117 3812 16292 19434 10320 5172 1982
3126 14582 9645 1807 19478 2739 7519 5561 12941 2436 17861 14481
4909 1899 3459 15199 19324 1922 19333 4083 1541 12260 11304 16432
8512 4387 10025 6653 9022 6289 7641 9339 2859 11610 9145 8381
8830 8028 17745 3541 482 2045 8753 3209 9163 2838 17263 2745
9173 17072 5826 7351 9534 19739 19536 7374 17185 14542 17759 5670
4857 6297 13448 7546 11655 18214 11988 3497 12119 18421 2238 9138
8088 14015 6428 10563 6419 14637 4955 9470 6734 18494 1521 11999
10694 19034 6803 18967 314 1819 14284 18428 15805 9706 10688 13439
13112 10153 5875 3218 18791 2938 12193 15881 16805 14320 13237 2072
12833 13687 15276 12110 9603 12516 3748 11719 10927 18595 13468 2719
7437 2981 5840 16220 19699 10298 2127 3040 11867 4738 17635 17582
9714 2444 10594 8326 16463 2150 4184 2007 11355 16573 10940 9501
2168 14219 13085 7255 18034 15653 19511 18649 7840 8220 2272 4799
9676 5395 14310 16236 8684 4645 2741 19834 12291 4917 15188 3607
13304 10053 19863 17528 10883 622 943 12590 14403 18836 9723 10918
19559 18677 1038 15537 16448 8891 10556 10553 922 3334 13996 16085
16717 16720 16629 13354 3416 6712 18027 18028 18046 18045 15020 9679
4373 2046 2280 16254 15252 11438 7044 432 17329 10580 2438 10575
2435 14080 12466 13227 13331 3373 4354 1137 9112 9115 8626 9114
14182 14180 17288 17285 17287 17309 18279 17281 19761 5134 13133 13134
7591 7579 7578 7590 7622 7593 7619 8710 11257 10155 17393 5497
7611 7613 3368 3371 3345 3347 11146 3346 19018 18562 4131 617
15703 18059 11680 15149 6854 10204 14371 15844 16942 6398 16524 14919
6019 6427 4568 2096 17412 15137 6371 442 13742 19059 13740 18548
8545 8646 10441 19790 9654 2971 11164 12791 16684 7548 18131 4903
4891 11339 14205 916 13861 14326 7296 7059 9757 15666 8359 2239
6482 16408 10426 1600 4339 9125 15701 10147 6375 17364 694 5869
11598 14712 1946 16250 7313 3680 8784 12459 4338 15013 3889 4760
2323 2136 16586 3470 11223 13993 8676 4320 6154 958 7689 4997
6877 12357 17840 14977 10169 1152 12474 11366 1049 3453 16153 10586
7466 18725 19446 12713 6237 2331 1068 5956 14116 4295 5532 19458
9119 2957 3739 1926 5245 13988 1520 18033 18302 19042 17337 4538
4879 14267 1733 12961 6254 3681 7830 2074 16366 1543 11786 9016
14318 19058 3564 8810 13044 12256 15026 4210 6152 6276 7015 19314
14293 8809 10942 9006 4237 5540 4066 15205 2922 7718 9533 18546
10937 14422 11800 15473 1522 19100 8937 2303 11886 7722 16621 18323
896 9132 14097 11222 15658 13479 11450 7853 10321 18832 7922 19744
6867 15240 11684 18174 1205 4611 5375 16992 2152 19112 4946 19596
16620 19036 18565 19016 18568 18507 18505 18503 18502 5629 7080 13753
1126 8534 2047 12554 18939 18572 15080 13812 14010 15955 6939 14155
19762 16570 11714 12363 13544 7253 10903 17939 19714 11964 15759 1212
3044 7880 13476 18635 9531 13481 6845 19136 3340 1470 15749 8548
16230 16655 5529 5322 6804 2189 2803 2874 2819 2798 2851 2774
2772 2847 2769 2801 11161 14362 279 18610 1712 3375 2235 2242
2245 2243 18869 9808 16261 14527 6784 8618 13941 16063 16061 4419
4399 7500 7499 7479 7507 7501 7478 5967 5140 7332 2530 9726
5117 1829 3095 15542 19925 15520 16445 16918 16920 16451 15541 16209
16922 15560 15559 11352 19040 2729 2751 2748 2730 6517 313 6884
6008
333: 417 6257 8790 13789 16313 14699 10733 16574 8828 5654 11626 2210
11562 3826 12856 1109
334: 8128 4221 17278 17046 18251 12696 6743 3779 8868 11244 10704 12642
6965 16337 9523
335: 18958 12883 14235 19663 17014 1182 2397 1912 8514 1875 540 4252
8725 6215 16237 4063 729 2593 724 8219 17866 6912 6447 18691
2763 5446 2066 15270 17205 19591
336: 12057 12728 5689 13469 14260 4138 1692 19601 15628 9152 17930 16195
2028 8172 17751 16934 16401
337: 3127 12366 13856 16497 1925 19818 4950 15674 5793 15496 15493 2188
2206 5492 1835 6671 11106 1524 9890 1111
338: 5950 18742 15397 6802 19876 12654 8206 17804 1971 16998 18602 18337
4016 5119 2979 4844 10291 19710 18136 5748 10284 7029 5412 5278
11920 1908 6210 762 9715 12314 6273 6084 19740 8804 15410 16699
8756 13731 3128 3144 10582 15564
339: 11402 410 12775 2861 5260 5387 19121 1001 11194 18533 19260 466
4275 18650 7675 1919 4821 7323 16863 9487 12895 16977 15299 2339
8707 2334 9658 18289 19688 13814 2715 19839 17620 11898 1578 12967
4603 13211 930 19517 19938 15569 15063 8098 19557 8485 18333 16609
14853 9045 18679 9143 17460 7537 13585 18153 18578 1495 6307 4430
14054 6332 15889 17603 11064 948 2013 18684 6751 1586 17167 17443
8350 2105 8487 9467 13324 1745 1746 5173 4505 11300 7665 13801
6730 2418 12477 12495 19560 8467 6040 6166 5180 14885 7681 18736
6363 10978
340: 6122 1518 18485 14501 10446 5892 19266 13779 16587 8559 9244 8689
11841 16626 17021
341: 10333 3897 11487 9936 11989 10885 8445 11048 9005 17316 16689 13965
11249 5452 8186 2457 10098
342: 3006 14845 848 12847 17916 1499 5698 15349 8481 2524 12033 2479
19698 8954 6422 13377 17520
343: 6892 17589 9778 16830 14586 3829 7946 7552 7372 2949 6234 6869
12368 9735 13309 2554 7769 4764 8126 18134 2656 2408 9463 16486
19750 2406 15386 7481 14227 7803 18842 18844 4369 15153 18161 19455
3782 7008 2599 4449 1837 2204 6481 10484 18534 15820 3300 18577
14013 8365 13671 11914 3706
344: 4518 2816 7393 8749 3714 18587 12336 13565 834 14466 2212 18658
3417 2925
345: 471 17836 1008 8569 18625 14950 11789 8572 1102 10998 17791
346: 16406 9039 13935 10414 14191 11070
347: 6068 14653 2218 9394
348: 8023 17381 14654 13733 18394 10569 4912 9979 1032 937 13915 10361
1651 5040 10191 12530
349: 1648 10364 2132 11847 18514 11591 2053
350: 13558 19061 6182 985 13642 11733 7195 10366 10180 4120 16484 13042
18195 12100 8507
353: 4575 1730 16597 3415 11245 13804 9868 8538 15670 7953 11687 14656
1891
354: 19676 7455 15869 13573 16262 19202 4246 19397 2728 12796 9583 7861
5152 12632 18294 12083 15810
355: 2962 6855 10045 10561 12389 12391 2422 18108 12563 12416 4416 11255
8450 16833 11444 14745 12519 14732 12470 14479 1758
356: 8236 1667 6732 4010 6333 999 12332 2454 12092 2743 7397 3753
12781 16704 6391 18035 3785 1321 8174 19017 8597 16697 12677 9178
12405 17672 12838 19432 2856 7305 5899 5645 1905 926 4508 6831
11594 13483 19577 13571 17403 19874
357: 18266 7571 19462 4882 3947 18198 19775 19771 19267 9864 15678 7599
19000 3750 3851 12623 17165 17571 14922 2910 16737 8164 5433 14442
13244 5939 1262 17766 9254 2064
358: 4219 1193 4372 8802 10387 9975 16842 12280 12281 12279 17094 11872
2301 709 13862 19439 1073 17661 15857 5981 5996 10915 2469 18211
13345 7869 8911 18950 8909 18952 8225 2963 8049 6934 2376 6638
15632 8089 9727 17744 4332 3625 2818 3137 17921 3318 16305 764
13749 12825 10113 546 11893 19759 13294 15366 11250 13771 8642 15051
6891 15845 12578 4987 6516 18800 4333 18374 4360 14269 17370 9277
18408 4808 4029 12225 19338 787 15279 9722 10304 1332 9721 3088
11139 6037 16019 8571 5449 4424 5365 2467 3432 7502 1283 2362
17765 12194 5682 9922 14289 9064 13109 2442 5695 13898 1251 6239
13774 860 2056 6839 6840 612 11105 11286 2407 12769 13122 9044
13743 3836 3447 8243 6953
359: 12802 5201 10302 17468 16751 7450 9413 19290 5280 19612 15469 15474
13637 6949 3348 2194 4865 3207 16722 3441 14427 7198 3775 3802
12181
360: 19570 9059 8115 3359 3370 16218 4471 2588 3190 1342 1327 7790
7330 15451 1506 12550 3293 13170 13832 8062 11326 2156 5293 5104
3243 672 10504 12238 7614 14171 3051 10249 4843 15762 12428
361: 3065 950 8348 12335 19427 14109 13455 16581 18561 19522 5471 7987
13435 14619 9480 6424 5284 10400 10362 7024 1443 7277 15946 6856
5638 955 2157 1720 9881 18969 15813 7701 12321 3920 8603 9215
3907 11529 14014 6824 2329 8668 10607 9238 2565 4098 9193 16372
18237 17803 19666 2639 7110 12353 660 13242 17052 10200 12815 11310
17724 3805 1392 19435 12595 5074 10118 5073 8413 6434 8087 3649
6611 4839 1944 2006 14636 15167 19849 3029 5269 18965 9850 11641
4433 12460 3711 13980 19654 16418 4220 14462 14907 12618 7928 11377
1861 16288 3333 15079 11178 5930 14221 15099 7106 13591 11342 6453
9428 3138 4529 18919 10578 11967 3123 15132 7246 1399 19641 1877
15059 10489 18120 12869 12088 11647 7054 2316 4238 4224 17713 13334
18328 6713 10427 5017 1537 13171 3450 18694 3746 11425 11266 11206
14036 8884 13754 3840 5694 12680 14821 15322 12298 15719 13691 13441
2511 17203 3588 3365 11160 4803 13723 19123 12875 17986 9764 4606
4403 4531 10348 11668 16400 16295 8960 3752 5598 15851 14513 7149
15712 16423 6990 14475 12448 4402 11453 9183 3764 10933 3278 7656
10372 17668 18799 2893 12854 5169 17911 17413 11409 13265 438 10127
17867 12591 2654 6083 11279 17487 15623 19297 15212 13033 4207 10444
16705 11619 8797 17292 14740 6849 19452 2484 14138 14897 9108 16156
4383 5020 6543 6549 11168 3290 6961 16871 3270 15512 2895 14733
15488 2322 14667 15731 18753 18312 12557 13197 3823 7435 13672 2033
5162 8163 7655 6409 8511 14850 8444 8899 15067
362: 15752 17695 9990 5932 15211 8418 6238 13628 14659 485 972 7628
19124 17248 10742 14839 7306 6162 2258 9285
363: 15587 7536 11706 19935 8746 17583 13021 11251 695 6660 14178 15828
10282 7088 4536 3957 2909 10712 8289 1371 15458 19277 18642 4003
15273 4301 13644 7311 11813 1680 735 15095 1457 1128 3122 9711
3793 1155 18202 5222 16291 5429 8099 7324 11246 909 2980 6952
4744 9471 15817 19001 6196 6665 2449 16000 14068 9442 4512 15890
11197 18150 14278 19695 6747 10772 7524 8625 11005 5911 9704 1140
3532 6565 12151 16037 13454 9169 3324 9517 19274 3692 4037 3896
1082 8078 12333 17688 6926 9760 17178 8565 15303 18874 5274 3608
11418 15790 13582 9809 13016 12263 17135 18105 2163 12533 9985 14786
6012 15171 3012 7976 18090 4450 16120 10148 9586 1280 9405 9430
10773 8152 17676 19007 13766 7476 16279 18231 12787 12805 1089 16681
3955 2488 673 12058 8888 7696 12741 2767 7629
364: 18935 11480 10102 2128 611 16123 4822 16369 3396 5943 18617 12251
9404 19062 5185 6458 17236 4412 6457 4432 11891 4444 17731 5068
13351 18269 1188 18262 18243 18264 18245 15524 18268 7045 10216 15254
15585 9962 6016 6018 5983 10695 4247 11334 2233 11399 14429 17303
18326 5114 4325 13602 3559 12081 18081 5579 15294 19095 8364 12907
14773 15870 10476 3925 8957 18029 8308 2927 19174 3169 6014 4048
6676 17157 11935 4361 4103 7348 3589 16742 2320 8945 12971 1753
2476 9137 14089 16990 18646 2973 10662 10632 13827 11754 1568 5396
13363 13991 6017 6232 13824 9366 1808 7556 18911 16773 9136 4823
14910 396 19794 17197 7423 9283 517 4437 17158 6128 6636 12989
7092 2462 14623 6769 11837 19463 3305 13186 5079 2059 2342 5833
5569 1515 5189 675 4488 17733 8803 17720 1127 7371 13624 3909
4748 1217 8255 9253 16038 5961 6590 16131 19015 11099 19890 2161
12546 10713 925 11622 813 16009 13081 15471 17703 8306 13620 1120
7320 18557 7452 7470 13172 15551 3325 1780 2126 16004 10743 14145
11018 15477 18982 10765 13029 15065 4084 8672 12316 10611 3273 11834
6279 6327 17101 19914 19661 12771 7385 9866 8494 1768 15903 6534
13088 18689 5562 9170 7829 3895 17666 16326 15100 13815 9286 8993
6944 16914 7289 8901 17669 12277 14660 16867 11040 1316 3645 2585
2770 4895 1589 399 6220 18600 18397 917 5160 14743 19767 11532
1354 13972 3182 2310 15434 2040 5147 8430 14935 13335 16362 10812
11006 12064 11533 16872 18830 15354 1960 11327 15506 19294 14823 1796
7366 15566 5646 12469 10813 15208 13430 17355 4773 1940 14330 14354
12329 4797 2889 595 9772 14913 17811 14573 7131 12710 6929 13194
15743 17161 14900 19555 4173 17031 5984 7342 16617 8400 3525 3512
3611 3679 3669 3674 10419 9782 9140 17719 6888 2353 8032 12136
15475 934 10480 19362 17217 16756 19071 18554 12338 17276 2282 13612
1421 5210 19246 14938 4414 10326 861 19438 16936 5565 10516 7011
365: 13631 2299 17761 900 706 18107 11275 3061 17510 586 1894 15867
13092 19868 17581 6902 13125 3191 17566 7355 11590 2877 15662 10745
10009 17671 18076 2217 2055 11552 10714 19648 16707 9924 9503 19883
12972 8648 6287 14196 10126 7645 11125 1084 2706 1420 1739 11228
2641 9918 13470 15676 15827 15328 968 809 8295 15038 16125 2326
13641 2592 15180 7187 19519 8254 7001 15948 419 12496 7126 5605
18193 15842 6741 17608 8770 10875 17317 16846 9813 6726 15155 11019
16116 16028 16531 1857 17795 11282 12582 8230 14029 9438 10288 9360
9453 5792 14648 3353 782 13500 19798 3960 2396 13841 6828 19659
4186 5277 570 7389 8319 1775 5548 697 6933 4532 9927 7345
5347 17289 12608 7808 2718 9932 14265 15975 13060 14443 6331 18432
13873 12306 4371 15241 12122 15732 5458 13523 8923 17770 1063 17401
17362 14436 7068 13174 12734 11814 1336 10705 3094 7480 832 4811
15561 12242 19576 792 9707 13586 7094 13475 8199 5714 15555 18801
1992 5212 10193 18728 19345 15911 1076 1019 2978 11483 2478 14400
8170 1943 17567 15417 5317 11195 14991 18347 7672 19335 19822 11797
13910 8435 16903 15659 6408 13305 6028 491 12784 19379 8454 3118
1686 10876 13040 17491 11393 2077 3299 874 13487 11838 9664 10552
2610 8066 13005 12361 6853 3757 2887 15577 10529 14247 16087 18789
15976 16374 10771 4277 14521 12901 18215 11495 3853 4255 1629 12007
2466 9883 11432 13572 8080 5273 3638 18449 18786 16514 12076 1511
6812 17300 5015 8838 9304 10185 7905 11345 15952 766 13089 2956
613 8059 1979 12501 8535 16930 3363 10502 9217 16331 12258 3097
9416 10257 11596 2590 11883 5567 18847 4461 15605 10990 10192 17106
6082 15250 19258 19683 6624 17570 5102 9195 8650 11158
366: 7895 6135 5404 5150 6602 19424 2002 11648 2102 11953 8198 18899
10410 16171 8925 5672 16257 7833 4196 7416 8915 14363 1706 15695
9308 7164 13079
367: 17228 9424 6076 17145 3914 10656 6091 14671 7138 1890 732 16240
11984 11978 17029 18186 17451 1323 5667 18155 19103 8458 10719 1530
1664 4884 11793 11316 1027 569 6663 18298 6906 1989 11679 8546
17917 7183 4074 5267 8898 17813
368: 10139 8837 7747 7598 13605 1318 9532 7490 2370 12273 4264 14618
8627 10651 13584 18749 7057 7233 408 12207 5515 14163 1226 1228
5775 5424 18314 18275 10203 8674 1800 2693 19005 8477 14053 9051
10777 4549 6597 9738 7370 13649 5856 10149 13783 12613 17815 11337
9362 13027 15862 4287 18724 8169 1419 6285 15077 2147 2905 14574
1177 13510 9181 7135 7290 2520 6455 4618 7286 7288 2679 551
9787 11002 1701 8930 8928 8942 1097 13702 15267 12778 15439 18947
17593 12250 12347 19108 15478 7855 9687 17776 17774 12927 17808 3508
7592 10782 2475 17662 17990 268 18627 12803 4005 6216 13658 8061
16885 5642 8881 8964 10019 2902 8544 10703 1548 4925 12994 10443
7766 13011 12993 13010 17357 15189 1888 18221 17786 9563 9562 2386
5156 3903 547 18001 12480 13049 8197 2495 17171 18977 12208 19466
8339 10488 4385 19012 16077 8291 17408 5878 19014 8302 16024 16026
16018 16022 4841 9827 9828 9829 8403 9847 9830 9842 4690 2646
13874 9849 14131 17831 9865 8405 9851 9844 7012 8406 4019 499
9951 11218 13124 538 13569
369: 6354 13600 13597 13402 8495 11462 4678 17651 2271 18508 9403 8227
19337 18039 11414 13507 8796 16388 13135 10409 13151 13116 13153 14811
13648 16385 17968 17992 18014 17987 17989 17964 17984 17963 18015 17961
17934 17931 18010 17966 17884 17850 17851 17885 17905 17839 17878 17900
17881 17854 17903 17360 12858 19170 13662 7781 19569 19153 13650 13595
5154 17820 2688 17837 17834 17821 2707 2705 17822 17363 3754 3771
3775 3751 3778 3747 17028
370: 13195 17260 15550 15298 16070 13528 18906 6667 3884 19731 16485 15332
11024 4720 7335 10724 9903 16073 9800 2005 2226 17918 14439 11382
1930 15821 6790 3307 2184 3678 6020 2483 16932 11759 18061 8407
17553 9683 10479 3498 7938 11193 10141 10064 5340 13160 1065 1050
1066 8697 5057 1948 19456 10401 4381 5626 3147 4807 15513 12411
1085 17692 15803 9678 13378 594 3808 6353 1729 12195 14567 9675
19783 10272 1789 7097 4737 17591 9334 11855 9329 14859 6302 14086
8083 15671 12954 13947 17819 5460 14531 15105 16035 18528 15990 19316
10591 3086 18057 7458 4476 12898 14681 5385 6706 12606 19781 4024
18079 12117 14093 15233 18591 7496 19829 4740 7646 6599 7671 19185
12054 10751 9600 19620 8234 8875 8090 5475 9292 16255 13529 6245
17823 2300 18651 19369 988 5505 16473 15879 14117 9448 8299 7128
1260 12844 6801 11913 12153 3878 16199 3426 6628 5890 3275 9071
19553 19622 5677 2987 5813 8587 5410 7917 10123 11678 19390 5338
10026 16740 6793 1954 9499 1886 18780 8529 17665 18483 19399 13823
10599 10014 11713 13942 16536 6829 13457 12144 4468 15992 10473 13518
2219 10524 420 421 14605 14570 6724 1269 15608 11671 5125 6124
12849 7013 12911 427 17957 7104 18943 14199 10798 14689 13399 9608
5287 17769 9135 11977 16390 8020 11559 16849 475 16412 9947 18271
8288 16395 11055 10283 8052 3695 4102 6848 1576 5301 7717 994
2477 9188 4330 6788 6219 12712 8475 8239 14804 13446 8249 3014
11497 719 15957 17377 11889 19701 16334 9730 14899 17716 13795 14007
15861 9886 16290 1777 16807 13147 5390 5656 16417 11861 16848 8738
10094 18845 16925 17514 13485 17384 15049 13629 16050 15795 15733 10818
17446 13946 5886 1298 18331 2672 1478 4124 15983 15934 3583 13711
1676 19213 15367 19827 17298 12179 5594 17065 6157 13890 4408 9574
11208 1762 15748 10092 2000 6115 8274 11506 15771 13770 17670 13724
4250 18579 18707 18706 18338 11926 18667 19212 9804 19854 14597 7979
5533 5584 7353 4291 17318 16669 687 880 11774 18653 11461 14110
6979 18340 8989 12588 16340 12839 4132 12893 15176 12464 5250 13418
19208 6505 2317 5276 12735 5595 3237 2548 769 11313 4897 4054
13148 18282 8680 17055 9088 13069 7258 15620 4755 2281 4479 14873
14276 18888 1613 2084 2083 2525 15057 8026 1769 6771 18311 14286
18220 3288 3011 1673 18154 842 9128 10218 12711 4076 14589 13986
9857 976 14336 10790 16144 19128 6185 10202 19394 10344 5962 15092
13101 13349 1772 5842 17973 9011 7194 12615 6592 16662 6996 4557
2794 9462 13636 1591 641 17482 711 1379 4654 19782 16899 4214
17607 4609 14972 12409 6334 8398 2027 15897 1125 2319 5865 18872
5227 19597 6259 16933 6862 12848 17576 9766 5970 13699 9543 10511
17456
371: 8393 9015 13413 2587 10796 17631 11121 433 4963 9556 575 5319
15391 10530 9031 4635 9004 9073 3306 11645 3981 19101 19141 19812
10554 17490 13822 14492 17246 1376 15351 19391 12231 4365 5194 2111
5408 17546 18797 5903 6042 1218 4848 19643 4899 6748 1341 2965
13343 10436 3965 12507 16052 10080 14622 15172 1156 15431 19244 5027
7279 4985 13179 18612 9230 6746 8566 5265 5208 3815 11698 18164
13048 16879 4441 8300 11252 16442 505 453 12406 1723 6094 17550
19037 5884 14923 4421 18481 6530 12498 17991 3134 8890 3350 7985
7864 10001 10340 991 5289 19317 10465 7319 2659 5841 13312 1585
8483 2167 16410 18310 17967 11530 8907 847 867 8377 12438 12935
1630 8528 11657 16778 524 942 13396 13561 12813 15308 14883 14448
5321 8706 4558 4612 4061 16274 14724 2777 3112 607 6814 11242
4393 19491 1312 1238 437 18920 2620 5133 8260 9619 13014 8506
10965 2373 14342 18448 19165 17471 5405 6607 1741 14959 15075 16265
13994 1258 14304 8744 10343 19837 12926 7213 3174 10993 14602 10617
7779 10187 9485 15515 19398 18257 19349 1202 15472 7901 12094 13651
1646 14731 1180 12154 16047 18288 2248 8051 19670 7996 7995 3855
1551 14194 18409 5592 11704 13409 18199 12052 7391 8999 15302 4964
15580 16350 15735 14708 15312 6145 15699 6159 15221 18322 10493 501
6886 13963 12295 8982 13436 16066 11980 4664 9057 9034 16666 15687
2427 11094 13668 3320 14079 9090 11181 8003 4257 11638 6544 6957
3999 12296 10381 2653 4772 4810 8751 10568 10162 5600 5649 15893
14376 16307 17697 9785 10884 16276 6169 6176 1037 9196 8367 7783
14533 9056 15139 19917 12676 3038 10673 5503 7023 15682 10198 898
1297 5858 14777 6955 17092 945 3032 10231 16741 4106 11060 10054
18270 12505 13314 1821 7784 11537 6572 3603 13635 5021 9052 7601
6284 8815 10431 18883 18773 17616 11724 3501 16356 11013 14805 9024
14557 7384 17109 7703 18849 14347 5364 6468 8936 3979 19281 10693
13957 3977 13486 7380 17077
372: 4087 6477 5737 5639 1608 6099
373: 11821 15570 15362 17684 12417 16687 6089 15073 1359 9786 19736 8877
10254 5707 15924 3173 5784 19735 9392 2552 15018 11302 10241 18422
14812 5768 9824 19506 6981 10715 19649 3163 9998 2058 6405 6584
7848 14615 15185 2020 16636 15445 11375 18820 10331 1794 6619 8395
6787 15426 6658 7293 10195 2815 19672 5744 14420 15534 15370 14303
15177 12323 14958 14797 11491 452 5665 6655 9310 16947 3284 15338
17037 6312 3537 1320 3839 1070 13922 11570 9105 5536 15484 14763
14487 9068 12682 9682 10905 17502 4201 4406 8085 7056 8194 19718
4289 19086 2788 10609 1276 11826 13369 15331 5251 8761 16059 6055
8829 15454 5769 404 6808 15129 1725 10525 13488 7737 8203 2464
17537 3566 8056 4191 10305 16861 7136 8740 1328 6582 7383 9441
7191 15884 17130 5403 5900 19080 12583 10011 3547 16174 11235 17910
9121 5888 18291 17740 7117 12383 15552 8043 8998 8613 9367 10541
17801 8616 10220 1388 8599 4600 15321 13524 873 3602 1369 11422
7039 17579 16732 1166 9497 14498 11817 691 12683 11133 10036 10168
9240 7540 16302 2752 10828 18058 9911 15729 17807 1719 19117 1924
3905 6227 4868 10358 11763 18113 5209 8329 10740 6938 9635 14512
17399 18121 12733 13966 5279 1953 1959 15567 18234 3818 5220 2744
18925 13352 15418 7193 635 9142 8342 1452 16526 8583 14626 10595
15517 3675 15449 18995 1550 12913 1574 10748 15207 689 10793 16997
12087 3593 9495 12369 16764 13313 16678 1034 9702 12049 15046 15685
1505 4817 6591 10004 17280 1057 2209 16242 6770 11621 11066 19348
13114 5818 18606 11096 2735 11091 16599 13119 10928 11061 18170 18353
3156 18093 8497 15157 14638 11736 1201 11263 18000 17999 11949 914
4681 8852 14381 624 7004 5368 718 17732 11589 12259 1569 3028
5910 8948 4159 8737 11933 15646 4723 2738 4789 376 8578 16888
18857 6948 802 19507 5226 6977 6646 17557 2557
374: 16075 17346 1488 14198 4254 1650 2972 17330 12199 963 19510 17391
5693 10970 15087 18216 17575 18987 17965 11155 16523 17488 9549 11549
10008 17518 8820 12423 1502 16789 5728 716 10447 10464 16976 5052
6412 5108 3314 6292 8733 16269 15372 10825 12586 10413 10380 12879
10280 19593 3339 9629 3902 1154 10253 8577 7055 2366 3283 13877
9422 4904 10226 1456 18827 6632 11525 14700 16759 2841 3514 3755
17006 17415 7347 3067 13384 3558 2974 1846 3323 6629 3184 19561
19703 15961 8827 2835 9303 1071 10179 14834 435 12500 4768 8437
7517 2760 14250 12828 10786 1268 13055 13052 12497 16738 9520 19359
10335 2636 2886 9647 6341 6642 7315 12387 19717 11540 13887 5657
16407 18284 16813 16160 19921 18470 14030 12523 13879 8162 18330 4380
12845 8696 15369 16716 14346 16690 4719 3277 5678 5680 10403 3623
5774 2508 1744 19808 4467 13035 11579 7401 2215 4902 11210 11968
15518 8984 3055 3245 18372 7421 8387 11179 7162 12630 5371 6163
5316 14827 17771 590 15838 18775 7975 11865 15784 2067 15507 17191
10843 14530 15228 9977 13811 16873 2501 8524 6493 9867 12072 8122
14458 2666 15333 9399 4828 15607 12871 15613 1771 16671 7034 11542
15229 10987 6960 14341 14525 7247 4763 19509 5283 18405 8785 16222
14297 18434 14175 18636 10459 13065 17257 16561 1889 14928 6483 19913
2456 1117 3838 6107 6860 7721 6402 1845 12187 10183 3985 7714
6914 17425 9651 13259 791 15634 15360 10630 8039 9174 19497 1774
12575 11290 11928 14723 17308 16624 18115 9049 7707 4914 15036 6484
13266 7824 18137 7109 4623 13337 19831 7899 18876 14992 15343 4660
14819 3465 1564 18556 19844 4349 19085 9971 13851 16931 2174 8283
19354 11651 4757 15968 6959 10967 11188 17645 8006 6247 8959 10307
13566 11039 13744 17853 863 11617 7364 10342 548 17035 13825 1142
13781 9657 5941 1581 17069 12968 15470 16299 4281 15614 1937 4559
14610 4696 13538 7697 3637 7603 11413 2179 7933 19467 8355 1987
19608 967 15718 3480 8769 12950 16296 15497 18747 16455 2726 19646
1232 9750 12105 12718 12852 5921 12836 3388 7411 16468 8997 11368
1122 7595 14296 9278 19558 12461 14633 16726 17802 7427 18821 8484
8671 7208 18639 13316 13956 4535 18240 4560 14766 13755 10837 5042
14741 10899 6606 836 5977 3221 7913 9252 12441 5332 6266 4788
11839 7268 17954 2356 3394 7563 11666 4588 13840 956 15465 19389
17489 4423 4266 13131 10548
375: 16510 13467 14806 18916 13489 19287 6963 11609 12123 6168 14647 17406
19726 10559 16928 16901 7555 12727 15736 13018 2908 998 11571 14927
11630 8370 8372 13218 10996 17016 7958 9384 13045 13447 8144 1147
15001 2276 2823 913 14592 3364 18232 14988 977 13106 11518 14539
12112 17129 2915 10863 1580 4515 5798 8253 8082 416 19385 19171
1100 16820 6199 4571 18783 1742 10182 13173 16635 17155 13139 6972
5995 17971 7440 13865 9190 7070 17784 2010 723 15385 11201 10394
4498 13493 13320 14352 678 13273 448 5709 11312 18597 11116 7510
15024 12386 4804 16278 7734 1363 7445 19533 12959 1292 16198 16824
12162 2158 10445 8558 12176 1883 5802 17888 7202 9357 9212 9289
15706 15848 18962 11998 16594 7471 9904 6557 5534 7297 14395 12716
14630 1709 8057 5106 13882 13240 3414 8391 13833 18908 11344 5879
12334 16882 8131 13298 15056 11047 2998 9898 12476 14335 16430 17095
2214 1606 8257 16665 13613 6921 7748 14323 15979 5733 12116 6947
8474 13708 15492 18185 6299 13610 18148 14215 18714 2930 12129 15295
15268 14650 18384 18143 3511 6197 6361 17944 17442 5229 2611 565
16358 16357 15084 16089 1181 17594 9098 2241 19299 12023 7283 10377
6819 4949 8736 13205 2694 19763 13769 19621 13407 9290 16521 1825
10029 12822 2903 15953 15986 11911 13793 11546 2234 14881 9908 11523
15533 17577 11068 17517 17721 17374 10696 12227 19181 13058 8185 15758
4153 877 8465 19011 4495 6613 16284 8138 18523 4389 15763 3113
17826 2647 1098 10483 10463 19364 11397 11401 11381 6868 15127 1474
16902 5599 19363 17325 9309 19361 5037 4928 3787 16923 4146 16939
19535 15773 19339 15539 15125 18484 4203 18687 9493 10628 16954 18751
5417 19261 19262 19234 1714 19898 19336 19855 19291 19286 19880 19877
19295 19895 19875 1295 10357 6086 10734 11846 6683 12722 18816 18796
7791 4566 18951 15711 11715 3398 18259 16246 1255 3813 1431 15683
7433 3767 5490 5324 10201 9680 13775 10924 11448 5618 3801 6703
2837 11877 2626 19090 4080 8371 9573 3492 13188 3045 16273 5297
2403 2365 6847 7359 12126 16429 13888
376: 11821 15570 15362 17684 12417 16687 6089 1359 9786 19736 8877 10254
5707 15924 3173 5784 19735 9392 2552 11302 15018 18422 14812 5768
9824 19506 10715 19649 3163 9998 2058 6584 7848 14615 15185 2020
15445 16636 11375 18820 10331 1794 6619 8395 15426 6787 6658 7293
10195 2815 19672 5744 14420 15534 15370 14303 15177 12323 14958 14797
11491 452 5665 6655 9310 16947 3284 15338 17037 6312 3537 1320
3839 8693 1070 13922 11570 9105 5536 5454 15484 14763 14487 9068
12682 9682 10905 17502 4201 4406 8085 8086 7056 8194 4289 2788
19086 10609 17621 1276 11826 13369 15331 5251 8761 8829 15454 5769
6808 15129 1725 10525 13488 7737 8203 2464 17537 3566 8056 4191
16861 7136 8740 1328 6582 7383 9441 7191 15884 17130 5403 5900
19080 10011 3547 16174 11235 17910 5888 18291 17740 7117 12383 15552
8043 8613 9367 10541 17801 13465 8616 10220 1388 8599 4600 15321
13524 873 3602 1369 11422 7039 16732 1166 9497 14498 11817 691
12683 11133 10036 10168 9240 7540 16302 2752 10828 18058 9911 15729
17807 1719 19117 1924 3905 6227 4868 10358 11763 18113 5209 8329
10740 6938 14512 17399 18121 12733 13966 5279 1953 15567 3818 2744
18925 13352 15418 7193 635 9142 8342 1452 16526 8583 14626 10595
15517 3675 15449 18995 1550 12913 1574 10748 15207 689 10793 12087
9495 12369 16764 13313 16678 1034 9702 12049 15046 9046 15685 1505
4817 6591 10004 17280 18300 1057 2209 13541 16242 6770 17146 11621
11066 2735 11091 11096 19348 10928 13114 5818 18353 3156 18093 15157
11263 11949 914 4681 7472 8852 624 7004 5368 718 17732 12259
3028 1569 5910 8948 4159 5738 8737 15646 4723 4789 12067 16888
18857 373 6948 802 5226 6977 6646 17557 2557
377: 8393 9015 13413 2587 10796 17631 11121 634 433 4963 9556 575
5319 15391 10530 431 9031 4635 9004 9073 3306 11645 3981 19141
19101 19812 10554 16183 13822 14492 17246 1376 15351 19391 12231 4365
5194 2111 5408 17546 18797 3789 5903 1218 4848 19643 6748 4899
1341 2965 13343 10436 3965 12507 16052 10080 14622 1156 15431 5027
7279 4985 13179 3070 3071 18612 2494 9230 6746 8566 5265 5208
3815 11698 12148 18164 13048 16879 8300 4441 9892 11252 16442 505
453 12406 1723 6094 17550 19037 5884 16347 17111 14923 4421 18481
6530 12498 17991 3134 8890 3350 7985 7864 10001 10340 991 5289
19317 10465 7319 2659 5841 13312 1585 8483 2167 16410 18310 17967
11530 8907 847 867 8377 12935 12438 1630 8528 11657 18567 17817
16778 524 942 2110 13396 13561 12813 15308 14883 14448 5321 8706
4558 4612 4061 16274 14724 2777 607 3112 6814 11242 4393 19491
1312 1238 437 18920 2620 5133 9619 8260 13014 8506 10965 3168
2373 14342 18448 19165 17471 5405 6607 1741 14959 15075 16265 13994
1258 14304 8744 10343 19837 12926 7213 3174 10993 14602 10617 7779
10187 9485 15515 19398 18257 19349 1202 15472 7901 12094 13651 1646
14731 1180 12154 16047 18288 7639 2248 8051 7996 7995 3855 1551
14194 18409 5592 11704 8432 13409 18199 12052 7391 8999 15302 4964
15580 16350 15735 14708 15312 6145 15699 6159 15221 18322 10493 501
6886 13963 12295 8982 13436 16066 11980 4664 9057 9034 16666 15687
2427 11094 13668 3320 14079 9090 11181 8003 4257 11638 6544 6957
3999 12296 10381 2653 4772 4810 8751 10568 10162 5600 5649 15893
14376 16307 17697 9785 10884 16276 6169 6176 1037 9196 8367 7783
14533 9056 15139 19917 12676 3038 10673 5503 7023 15682 10198 898
1297 5858 14777 6955 17092 945 3032 16741 4106 11060 10054 18270
12505 13314 1821 7784 11537 6572 3603 13635 5021 9052 7601 6284
8815 10431 18883 18773 17616 11724 3501 16356 11013 14805 14557 7384
17109 7703 14347 18849 5364 8936 3979 19281 10693 13957 3977 7380
13486 3384
378: 13086 19930 5574 17164 15382 2809 1565 9389 817 831 5123 14538
9536 1834 1833 13082 18812 19542 12765 13660 1610 1609 15422 15421
1627 13773 5445 16272 10838 19127 19142 9628 10532 7251 8084 9179
10060 3233 3234 10382 15456 12062 12060 8373 598 11569 18041 16210
3876 5308 5310 4105 19053 17469 5866 18520 18518 17299 16592 13763
1649 2826 1088 5244 17633 8002 6325 8510 8509 17342 10265 8523
11182 2722 2708 14181 17828 10012 8552 6241 10590 5676 10274 8661
3295 6576 8917 12079 16538 11322 11319 15823 16815 16795 13353 14432
2003 1985 7807 7667 1870 12245 15011 15010 7081 7327 7326 3951
11419 11420 5668 5402 2091 2088 10840 16194 9002 9003 8127 8141
1440 6106 6104 17126 17005 4082 9591 9590 1915 6725 10684 7300
3317 3316 9330 19487 19488 14152 14153 2122 1914 1911 10665 14295
14309 2813 2489 17345 11755 18173 14628 16281 4570 426 12610 17201
17057 16060 16058 9609 987 9610 15673 921 1086 18306 11336 2498
17087 17085 12034 12035 14678 14675 1138 544 16271 12318 12812 4308
19378 14598 14599 7369 18594 7648 17481 19008 14345 5166 7493 13726
13725 8384 3400 16200 7902 8410 8409 13137 16958 16957 15723 12257
12253 15978 12973 4460 4459 13017 1570 1078 2531 15107 15091 8965
13199 13198 19203 19218 2645 523 5806 17244 17241 9229 13301 12697
16182 14543 17648 7956 541 7604 12160 14921 4194 19094 17637 18276
18272 14246 14245 6265 11350 6263 11351 1447 9984 5779 16703 16701
8272 8270 15033 637 11702 3382 13215 6987 17734 17485 17484 8448
11578 16065 14275 12748 19807 2279 9116 2296 2779 13325 13222 5611
2538 12956 15346 10824 1158 4300 18255 6983 15168 4680 19799 12284
6950 16092 3445 2799 10239 3660 12422 2797 18788 11987 9273 10359
2829 13626 19734 592 14588 3599 6112 6093 6110 572 5844 11779
4108 3093 11517 11539 455 10947 11127 13214 3927 11199 12891 15461
15460 7465 5719 5718 3367 13458 13456 1149 14244 6969 12138 9126
1661 15396 5031 16825 1467 3689 6716 6528 6526 8496 18877
379: 16324 10027 16399 19626 19600 476 10737 6503 2637 5207 7908 18645
13757 8204 17032 1213 10125 9070 5372 15085 10478 7338 18023 9641
11812 14642 18106 9561 2651 1666 5437 14594 5439 12210 6002 12290
5485 6003 14353 14349 14351 10819 8846 9429 2453 10112 16543 3140
1146 1124 1121 15438 2354
380: 15042 683 16630 18404 5663 2482 10670 11782 18811 18709 11700 19144
6755 9799 13818 6126 2455 8789
381: 17923 18890 5500 4771 6745 5378 3236 1210 11603 19433 19342 15300
17953 11454 9261 12841 11591 5415 5453 5430 8278 14564 6761 17927
18569 14781 10539 6214 3620 3528 17899 8748 12221 5504 5502 9489
6425 8958
382: 19132 10614 8139 10836 16040 11545 7879 2100 1703 14547
383: 1189 16611 3586 4641 19633 17705 17150 8849 3552 7294 2029 6153
18596
384: 741 1633 5923 8765 16673 14609 3698 6449 2951 2093 11563 614
14593 14762 6397 7731 14290 17038 4311 14640
385: 11357 13113 2101 9359 2343 5986 9703 12857 8854 17190 4889 13808
12572 3386 7845 17983 4794
386: 1016 16046 10106 6272 16847 10542 15062 5576 4893 5448 15407 7831
16578 18278 2702 7215 9961 6250 3252 18599 16433 3226 17007
387: 7223 8111 7867 1322 9479 3291 18674 5132 12538 3515 18852 16518
14682 5419 9781
388: 13217 19387 1319 13219 1333 2384 4322 7600 13280 5924 13688 2181
16877 16122 5091 927 6100 3686
389: 11269 4678 13073 1755 1754 8654 3662 17371 3891 3973 2187 15246
18462 4802 3390 619 643 13183 4014 1542 10000 19057 6229 1262
14305 14256 17590 5708 4877 3775 8462
390: 17182 3673 6158 5731 8195 15335 7264 7267 5346 10728 11675 2337
19110 7961 8491 12046 10224 6729 18918 18228 3153 8723 3309 3313
6222 15956 4632 12325 6835 6387 2622 4496 9897 10860 8044 10003
13262 13236 6403 5331 4640 1303 4602 1304 18435 16053 18480 17646
17644 17675 16363 4781 3269 8610 10852 3582 5717 9242 9340 9346
13271 1345 3780 11086 14006 2264 5062 5146 16786 891 1282 11639
2472 9621 19732 15665 10462 16384 18138 9510 19888 19119 4931 18991
13945 18937 13580 18401 13863 1936 4639 10245 760 13679 14995 17389
5259 10385 9763 15381 10901 10886 10888 12415 15437 8840 7871 4684
12209 17107 11203 16435 8624 13410 8530 10422 11650 15110 13639 12675
13689 19825 4662 13277 5625 9279 3369 5976 1377 2144 4086 18044
16889 6767 18418 11859 8188 6500 16529 16528 13234 14259 18077 11012
10071 11825 16181 6479 3800 9154 19377 924 923 7361 4574 14380
13895 17168 14901 17969 13735 17599 10270 4099 18973 3523 14337 8378
4490 18286 5203 2114 13850 9109 9096 7060 4161 6561 1211 9640
13790 19180 14796 5771 15355 14027 6282 12681 16502 4842 4262 4930
15500 16490 14450 18361 15929 1171 7624 13836 15083 19541 1490 8022
9426 19755 13700 10435 8346 13719 8681 19368 10906 19116 1509 3865
12853 3125 5889 8316 18315 9077 436 1977 8354 5002 5025 16553
5171 17152 9352 17908 9960 9963 4907 2079 14164 3744 3102 18375
4128 4129 14794
391: 5830 10547 4780 6670 1560 15309 12977 11581 15243 10500 7299 19550
4614 11584 18988 17503 13880 18201 7124 6036 17359 15414 19074 13696
4180 810 12806 13939 1259 2434 12029 5666 8718 751 18491 15717
1795 4475 1738 627 5732 18253 8660 11107 16309 12868 4299 14698
14679 14502 8248 11990 9758 16167 9385 10043 10430 16906 12099 5791
16810 13759 8562 2921 8889 10315 14077 2682 18457 14406 12241 10194
5832 13548 15304 4676
392: 18452 9432 5303 14233 9970 7637 14980 15265 7581 3016 10412 19809
393: 19384 19382 682 6463 1021 6039 6041 6057 7898 7896 13532 13533
8037 9356 16887 16866 7150 7148 11880 11903 7084 7083 3381 13816
13817 12463 4530 2311 2312 10624 10623 15054 2256 2257 14016 14018
8801 6140 6142 8800 6138 8357 8375 8360 3666 3668 11044 11022
9473 9494 5001 5000 4831 4836 18327 19232 5812 5815 9999 7941
7942 749 3079 7426 7451 8001 7999 8005 19635 4051 15885 12205
2860 786 784 15576 15575 12310 12307 2306 3461 7640 10242
394: 12840 13577 8132 1535 5905 3211 3797 12558 6843 17949 15017 9231
1310 17041 6367 18764 8461 15888 6866 6864 7983 8980 10079 15489
11102 9933 7325 1749 8441 7720 17727
Example 3 Consensus Sequence Build ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 198 and its homologs, through SEQ ID NO: 394 and its 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. The consensus sequence of SEQ ID NO: 227 and its 25 homologs were derived according to the procedure described above and is displayed in FIG. 1.
Example 4 Identification of Amino Acid Domain by Pfam Analysis This example illustrates the identification of amino acid domain by Pfam analysis.
The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein families for the proteins of SEQ ID NO: 198 through 394 are shown in Table 16. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 207 is characterized by two Pfam domains, i.e. “TFIIS_M” and “TFIIS_C”. See also the protein with amino acids of SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”. In Table 16 “score” is the gathering score for the Hidden Markov Model of the domain which exceeds the gathering cutoff reported in Table 17.
TABLE 16
PEP
SEQ Pfam domain
ID NO GENE ID name begin stop score E-value
198 CGPG106 CMAS 69 306 −149.8 5.00E−05
198 CGPG106 Methyltransf_11 131 229 109.1 1.20E−29
198 CGPG106 Methyltransf_12 131 227 54.6 3.10E−13
200 CGPG117 SNF5 14 252 66.9 5.90E−17
202 CGPG1288 Cupin_3 57 131 130.8 3.40E−36
204 CGPG1458 Phosphorylase 180 958 1454.7 0
205 CGPG1542 LMBR1 11 488 715.1 4.40E−212
206 CGPG170 Metallophos 54 249 140.4 4.50E−39
207 CGPG1828 TFIIS_M 206 327 203.5 4.50E−58
207 CGPG1828 TFIIS_C 338 376 83.3 7.00E−22
209 CGPG2217 ORMDL 14 156 356.8 3.20E−104
210 CGPG2292 Tim17 52 186 16.1 4.20E−05
210 CGPG2292 SAM_1 192 253 13.8 0.019
212 CGPG2499 Glycolytic 4 339 588.8 4.50E−174
213 CGPG2653 WD40 135 172 40.3 6.20E−09
213 CGPG2653 WD40 235 271 40.7 4.60E−09
213 CGPG2653 WD40 276 313 38 3.00E−08
214 CGPG2813 HLH 204 251 34.1 4.60E−07
215 CGPG3002 eIF-4B 5 451 −50.3 1.90E−10
216 CGPG3154 C2 14 102 101.8 1.80E−27
216 CGPG3154 C2 198 272 57.8 3.20E−14
217 CGPG3235 B_lectin 66 176 156.8 5.30E−44
217 CGPG3235 S_locus_glycop 189 315 227 3.90E−65
217 CGPG3235 PAN_2 332 402 103.9 4.20E−28
217 CGPG3235 Pkinase_Tyr 488 757 75.6 2.10E−20
217 CGPG3235 Pkinase 488 757 109 1.30E−29
218 CGPG3274 Allene_ox_cyc 74 253 401.6 1.10E−117
219 CGPG3275 Auxin_inducible 50 155 135.6 1.30E−37
220 CGPG3363 zf-AN1 21 58 10.5 0.0033
220 CGPG3363 zf-AN1 103 148 12.4 0.0019
221 CGPG3367 TFIIA 9 375 556 3.40E−164
222 CGPG3375 Tryp_alpha_amyl 33 111 55.2 1.90E−13
224 CGPG3534 Ank 42 74 13.6 0.31
224 CGPG3534 Ank 75 107 36.8 6.90E−08
224 CGPG3534 Ank 108 140 1.6 17
224 CGPG3534 Pkinase_Tyr 160 413 202.4 9.90E−58
224 CGPG3534 Pkinase 160 413 198.5 1.40E−56
225 CGPG3638 Ribonuclease_T2 30 219 367.3 2.20E−107
226 CGPG3918 Metallophos 60 255 159.1 1.10E−44
229 CGPG3972 14-3-3 8 243 501.1 1.20E−147
230 CGPG3990 zf-C3HC4 259 299 47.3 4.60E−11
231 CGPG3994 Pkinase 554 802 207 4.10E−59
231 CGPG3994 Pkinase_Tyr 554 802 233 5.90E−67
232 CGPG4026 CMAS 59 320 −174.5 0.003
232 CGPG4026 Methyltransf_11 122 220 96.3 8.10E−26
232 CGPG4026 Methyltransf_12 122 218 37 5.90E−08
232 CGPG4026 Sterol_MT_C 229 358 301.1 1.80E−87
233 CGPG4048 Pkinase 21 275 342.1 8.80E−100
233 CGPG4048 Pkinase_Tyr 21 275 65.1 1.20E−19
233 CGPG4048 NAF 304 364 104.5 2.80E−28
234 CGPG4052 DUF298 127 242 222.4 9.20E−64
236 CGPG4058 Asp 99 437 −128.4 4.60E−06
237 CGPG4069 adh_short 31 218 −16.7 4.80E−05
239 CGPG4088 SWIB 324 399 96.4 7.80E−26
241 CGPG4121 Cyclin_N 58 190 133.7 4.80E−37
241 CGPG4121 Cyclin_C 192 314 44.3 3.70E−10
242 CGPG4122 zf-C3HC4 100 141 27.6 4.10E−05
243 CGPG4140 p450 48 484 140.8 3.30E−39
244 CGPG4154 zf-CCCH 90 115 22.6 0.0012
245 CGPG4311 FBPase 70 395 347.9 1.50E−101
247 CGPG4369 BolA 10 79 92.1 1.60E−24
248 CGPG442 2-Hacid_dh 85 394 140.9 3.20E−39
248 CGPG442 2-Hacid_dh_C 187 362 293 5.20E−85
248 CGPG442 ACT 551 621 39.9 8.10E−09
249 CGPG4454 p450 45 447 −35.5 2.10E−09
250 CGPG4456 p450 75 502 103.6 5.30E−28
252 CGPG4588 Auxin_inducible 1 102 151.5 2.00E−42
253 CGPG4765 DUF868 28 304 175.5 1.20E−49
255 CGPG4912 WD40 243 281 31 3.80E−06
256 CGPG4926 WD40 42 79 22.8 0.0012
256 CGPG4926 WD40 126 163 25.8 0.00014
257 CGPG4967 Asp 71 424 −94.7 4.70E−08
258 CGPG4977 Usp 3 157 85.3 1.80E−22
259 CGPG5001 adh_short 5 181 6.4 1.40E−06
260 CGPG5025 adh_short 30 212 5.7 1.50E−06
261 CGPG5041 DUF26 77 132 84.3 3.40E−22
261 CGPG5041 DUF26 188 242 100.7 4.00E−27
261 CGPG5041 Pkinase 333 558 60.3 5.70E−15
262 CGPG5116 ArfGap 15 137 174.1 3.20E−49
262 CGPG5116 C2 182 261 101.4 2.40E−27
263 CGPG5144 p450 61 535 157.9 2.40E−44
264 CGPG5171 p450 35 496 137 4.60E−38
265 CGPG5194 DUF1191 25 308 616.2 2.60E−182
268 CGPG5221 p450 30 499 135.4 1.40E−37
269 CGPG5269 PCI 297 401 108.9 1.30E−29
270 CGPG5404 Peptidase_S10 68 480 683.3 1.70E−202
271 CGPG5432 MtN3_slv 12 99 145.4 1.40E−40
271 CGPG5432 MtN3_slv 133 219 140.4 4.40E−39
272 CGPG5518 Ribosomal_S8e 1 238 325.9 6.30E−95
273 CGPG5535 WD40 335 371 28.8 1.80E−05
273 CGPG5535 WD40 434 471 37.1 5.50E−08
273 CGPG5535 WD40 476 513 47.2 5.20E−11
273 CGPG5535 WD40 518 555 35.6 1.60E−07
273 CGPG5535 WD40 567 604 36.1 1.10E−07
273 CGPG5535 WD40 621 658 34.9 2.50E−07
273 CGPG5535 WD40 663 706 35.5 1.60E−07
274 CGPG5540 ESCRT-III 21 207 265.7 8.40E−77
275 CGPG5568 AA_permease 69 521 −67.1 0.00013
276 CGPG5577 SMC_N 21 1049 −2.8 1.50E−11
277 CGPG5587 Thioredoxin 78 199 0.1 0.00015
278 CGPG5594 Histone 27 100 99.6 8.80E−27
279 CGPG5633 PGAM 91 277 153.2 6.30E−43
280 CGPG5640 Aminotran_1_2 30 385 362.1 8.00E−106
281 CGPG5646 iPGM_N 2 363 876.8 9.10E−261
281 CGPG5646 Metalloenzyme 373 488 173.3 5.40E−49
282 CGPG5656 Gln-synt_N 17 99 85.7 1.30E−22
282 CGPG5656 Gln-synt_C 105 358 528.1 8.50E−156
283 CGPG5659 Aminotran_3 35 361 321.7 1.20E−93
284 CGPG5661 PK 1 345 702 3.80E−208
284 CGPG5661 PK_C 355 469 115.4 1.50E−31
285 CGPG5684 Glycolytic 4 339 578.6 5.60E−171
286 CGPG5694 TIM 4 246 465.9 4.50E−137
287 CGPG5704 NDK 2 136 329 7.30E−96
288 CGPG5714 NDK 4 138 266.4 5.20E−77
289 CGPG5721 Rib_5-P_isom_A 47 215 354.4 1.70E−103
290 CGPG5728 zf-CCCH 80 106 39.3 1.20E−08
291 CGPG5757 Sad1_UNC 203 330 195.6 1.00E−55
292 CGPG5764 Actin 11 140 36.4 6.60E−08
293 CGPG5783 TFT 272 442 71.5 2.40E−18
294 CGPG5791 AA_permease 114 588 704.9 5.40E−209
295 CGPG5799 Aa_trans 206 601 453 3.60E−133
296 CGPG5856 Pkinase 79 353 159.5 8.00E−45
296 CGPG5856 Pkinase_Tyr 79 353 149.4 9.00E−42
297 CGPG5927 AAA 58 248 252.4 8.70E−73
297 CGPG5927 AAA 322 510 290.8 2.40E−84
298 CGPG5941 PfkB 42 336 86.2 9.00E−23
299 CGPG5957 CBM_20 86 178 23 5.70E−07
300 CGPG5967 DUF822 2 147 307.1 2.90E−89
301 CGPG6040 LEA_3 1 88 178.7 1.30E−50
302 CGPG607 PurA 28 275 44.4 5.80E−12
303 CGPG6178 DUF1336 53 267 427.9 1.30E−125
304 CGPG6185 UQ_con 7 148 197.9 2.10E−56
305 CGPG6306 APC8 1 161 401.5 1.10E−117
305 CGPG6306 TPR_1 339 372 34.7 2.90E−07
305 CGPG6306 TPR_2 339 372 23.8 0.00058
305 CGPG6306 TPR_2 373 406 23.7 0.00062
305 CGPG6306 TPR_1 373 406 34.1 4.40E−07
305 CGPG6306 TPR_2 407 440 22 0.0019
305 CGPG6306 TPR_1 407 440 24.3 0.00039
306 CGPG6318 MFS_1 31 496 35.3 1.90E−07
306 CGPG6318 PTR2 92 484 208.6 1.30E−59
307 CGPG6326 Kelch_1 34 79 45 2.40E−10
307 CGPG6326 Kelch_2 34 79 43.5 6.80E−10
307 CGPG6326 Kelch_1 152 198 26.7 7.70E−05
307 CGPG6326 Kelch_2 152 198 32.1 1.80E−06
307 CGPG6326 Kelch_2 203 249 20.2 0.0067
307 CGPG6326 Kelch_1 204 248 7.9 0.45
308 CGPG6370 Gp_dh_N 3 151 326 5.80E−95
308 CGPG6370 Gp_dh_C 156 313 362 8.80E−106
309 CGPG6429 ADH_N 25 135 159.4 8.40E−45
309 CGPG6429 ADH_zinc_N 166 305 105.1 1.90E−28
310 CGPG6440 PK 1 345 789.4 1.90E−234
310 CGPG6440 PK_C 357 471 167.5 3.10E−47
310 CGPG6440 PEP-utilizers 486 575 134 3.60E−37
311 CGPG6516 Aldedh 19 478 778.3 4.10E−231
312 CGPG6653 LRRNT_2 23 66 49.3 1.20E−11
312 CGPG6653 LRR_1 71 93 12.1 1.4
312 CGPG6653 LRR_1 95 117 10.5 2.8
312 CGPG6653 LRR_1 119 142 13 0.93
312 CGPG6653 LRR_1 144 166 19.5 0.011
312 CGPG6653 LRR_1 168 190 10.6 2.7
312 CGPG6653 LRR_1 192 214 8.8 5.7
312 CGPG6653 LRR_1 289 311 17.4 0.046
312 CGPG6653 LRR_1 313 335 10.8 2.4
312 CGPG6653 LRR_1 337 359 10.7 2.5
312 CGPG6653 LRR_1 361 384 12 1.5
312 CGPG6653 LRR_1 409 431 10.4 2.9
312 CGPG6653 LRR_1 457 479 11.9 1.5
312 CGPG6653 LRR_1 481 503 10.4 3
312 CGPG6653 LRR_1 505 527 10.5 2.8
312 CGPG6653 LRR_1 529 551 11.1 2.2
312 CGPG6653 LRR_1 553 575 9.3 4.7
312 CGPG6653 LRR_1 577 598 11.1 2.1
312 CGPG6653 Pkinase 695 966 134.8 2.20E−37
312 CGPG6653 Pkinase_Tyr 695 966 134.5 2.60E−37
313 CGPG6712 PGAM 91 277 154.6 2.30E−43
314 CGPG6737 PGAM 92 253 173 6.80E−49
315 CGPG6747 FBPase 106 429 448.6 7.20E−132
316 CGPG6796 Alpha-amylase 14 452 199.5 7.00E−57
318 CGPG6810 GH3 12 561 1261.5 0
320 CGPG6953 Ank 121 153 50.4 5.60E−12
321 CGPG7121 L51_S25_CI-B8 20 93 108.9 1.40E−29
322 CGPG7163 Prenylcys_lyase 149 500 788 5.20E−234
324 CGPG7206 Aldo_ket_red 14 298 389.4 4.80E−114
325 CGPG7225 Subtilisin_N 48 125 84.3 3.30E−22
326 CGPG7267 DUF588 34 164 154.6 2.40E−43
327 CGPG7272 DUF1005 50 254 524.8 8.50E−155
328 CGPG7281 FA_hydroxylase 86 229 361.9 9.30E−106
329 CGPG7308 CoA_binding 1 100 −10.8 0.04
329 CGPG7308 NAD_Gly3P_dh_N 4 147 −0.6 2.00E−06
329 CGPG7308 F420_oxidored 5 251 282.5 7.50E−82
330 CGPG7316 Anti-silence 1 155 419.9 3.20E−123
331 CGPG7371 Response_reg 29 157 94.8 2.30E−25
332 CGPG7457 PfkB 114 408 146.6 6.00E−41
335 CGPG7636 LSM 13 81 78.2 2.40E−20
338 CGPG7804 FAR1 62 279 364.9 1.20E−106
338 CGPG7804 SWIM 556 589 37.3 4.90E−08
339 CGPG7823 Rotamase 104 188 91.8 1.90E−24
339 CGPG7823 Rhodanese 203 298 44.1 4.40E−10
340 CGPG7828 DnaJ 12 81 66.1 1.00E−16
340 CGPG7828 zf-CSL 96 174 25.2 0.00021
343 CGPG7986 F-box 48 96 31.2 3.40E−06
343 CGPG7986 LRR_1 189 216 9 5.4
343 CGPG7986 LRR_1 428 451 8.2 7.7
343 CGPG7986 LRR_1 561 584 8.4 6.9
345 CGPG8015 zf-CCHC 18 35 24.2 7.90E−05
349 CGPG8083 Tryp_alpha_amyl 28 105 37.6 3.90E−08
350 CGPG8106 BURP 56 280 380 3.20E−111
353 CGPG8152 PAP_fibrillin 9 124 45.9 1.20E−10
354 CGPG8166 PBD 118 166 49.9 7.80E−12
355 CGPG8377 Oleosin 30 109 55 2.20E−13
356 CGPG8976 Ceramidase_alk 50 795 1545.5 0
357 CGPG8987 FH2 439 839 552.9 3.10E−163
358 CGPG9013 NAD_binding_1 234 350 138.6 1.50E−38
359 CGPG9080 EGF_CA 315 357 42.2 1.60E−09
359 CGPG9080 Pkinase 433 716 120.3 4.90E−33
359 CGPG9080 Pkinase_Tyr 433 704 113.1 7.40E−31
360 CGPG9081 DUF676 30 247 319 7.50E−93
361 CGPG9130 MMR_HSR1 266 369 71.9 1.80E−18
362 CGPG9133 PPR 125 159 13.2 0.19
362 CGPG9133 PPR 161 195 2.8 3.2
362 CGPG9133 PPR 196 230 22.7 0.0012
362 CGPG9133 PPR 232 266 42.1 1.70E−09
362 CGPG9133 PPR 267 301 29.4 1.20E−05
362 CGPG9133 PPR 302 336 49.4 1.10E−11
362 CGPG9133 PPR 337 371 32.9 1.00E−06
362 CGPG9133 PPR 372 407 7.9 0.8
362 CGPG9133 PPR 408 442 49.7 8.80E−12
362 CGPG9133 PPR 443 477 22.6 0.0013
362 CGPG9133 PPR 478 512 37.8 3.40E−08
362 CGPG9133 PPR 513 547 42.9 1.00E−09
362 CGPG9133 PPR 548 582 30.2 6.70E−06
362 CGPG9133 PPR 583 617 38.4 2.20E−08
363 CGPG9134 HD 91 232 46.2 1.00E−10
364 CGPG9137 RmaAD 65 337 122.2 1.30E−33
365 CGPG9141 Pantoate_transf 40 306 402.9 4.40E−118
366 CGPG9145 Lung_7-TM_R 168 423 385.2 8.80E−113
367 CGPG9147 DNA_pol_E_B 178 389 249.9 5.00E−72
368 CGPG9148 p450 36 502 128.8 1.40E−35
369 CGPG9155 Pkinase 86 347 56.9 6.10E−14
369 CGPG9155 Pkinase_Tyr 86 351 73.7 2.90E−20
370 CGPG9163 Na_H_Exchanger 12 378 280.1 3.80E−81
370 CGPG9163 TrkA_N 416 531 118.8 1.50E−32
371 CGPG9170 Complex1_30 kDa 90 158 103.8 4.70E−28
371 CGPG9170 Complex1_49 kDa 298 537 2.8 6.10E−13
373 CGPG9183 HTH_11 1 56 72.4 1.30E−18
373 CGPG9183 BPL_LipA_LipB 84 182 94.5 2.90E−25
373 CGPG9183 BPL_C 275 322 43.3 7.80E−10
374 CGPG9186 DHBP_synthase 8 203 370.6 2.20E−108
374 CGPG9186 GTP_cyclohydro2 208 366 −2.3 3.80E−10
375 CGPG9205 NTP_transferase 4 288 421 1.50E−123
375 CGPG9205 MannoseP_isomer 299 465 350.8 2.00E−102
375 CGPG9205 Cupin_2 380 450 55.3 1.80E−13
376 CGPG9207 HTH_11 6 59 50.9 3.90E−12
376 CGPG9207 BPL_LipA_LipB 83 180 104.5 2.80E−28
376 CGPG9207 BPL_C 271 317 46.5 8.50E−11
377 CGPG9219 Complex1_30 kDa 107 175 99.8 7.60E−27
377 CGPG9219 Complex1_49 kDa 311 537 −11.6 5.90E−12
378 CGPG9220 GDC-P 3 443 700.9 8.30E−208
379 CGPG9230 Peptidase_S10 88 488 657.2 1.20E−194
381 CGPG9238 Tryp_alpha_amyl 36 114 56 1.20E−13
382 CGPG9259 Mit_rib_S27 14 93 135.3 1.50E−37
383 CGPG9271 NPH3 215 418 189.9 5.60E−54
384 CGPG9275 ETC_C1_NDUFA5 35 91 112.8 9.00E−31
386 CGPG9283 DUF1195 6 161 180.3 4.30E−51
387 CGPG9309 MAP65_ASE1 38 575 52.8 1.10E−12
389 CGPG9322 Pkinase 103 383 170 5.40E−48
389 CGPG9322 Pkinase_Tyr 103 383 158.7 1.30E−44
390 CGPG9335 Sugar_tr 7 726 210.6 3.30E−60
390 CGPG9335 MFS_1 11 685 107.7 3.00E−29
391 CGPG9341 RMMBL 531 573 35.9 1.20E−07
392 CGPG9344 TPR_2 531 564 23.9 0.00051
393 CGPG9345 UPF0261 5 432 483.7 2.00E−142
394 CGPG976 Glyco_transf_8 216 533 401.3 1.30E−117
TABLE 17
Pfam domain accession gathering
name number cutoff domain description
14-3-3 PF00244.9 25 14-3-3 protein
2-Hacid_dh PF00389.19 11.2 D-isomer specific 2-hydroxyacid
dehydrogenase, catalytic domain
2-Hacid_dh_C PF02826.7 −82.2 D-isomer specific 2-hydroxyacid
dehydrogenase, NAD binding domain
AAA PF00004.18 12.3 ATPase family associated with various
cellular activities (AAA)
AA_permease PF00324.10 −120.8 Amino acid permease
ACT PF01842.13 0 ACT domain
ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain
ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase
APC8 PF04049.3 −19.8 Anaphase promoting complex subunit 8/
Cdc23
Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporter
protein
Actin PF00022.8 −30 Actin
Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family
Aldo_ket_red PF00248.10 −97 Aldo/keto reductase family
Allene_ox_cyc PF06351.2 25 Allene oxide cyclase
Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domain
Aminotran_1_2 PF00155.10 −57.5 Aminotransferase class I and II
Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III
Ank PF00023.18 0 Ankyrin repeat
Anti-silence PF04729.4 25 Anti-silencing protein, ASF1-like
ArfGap PF01412.8 −17 Putative GTPase activating protein for Arf
Asp PF00026.13 −186.1 Eukaryotic aspartyl protease
Auxin_inducible PF02519.4 −15 Auxin responsive protein
BPL_C PF02237.6 16 Biotin protein ligase C terminal domain
BPL_LipA_LipB PF03099.8 −0.2 Biotin/lipoate A/B protein ligase family
BURP PF03181.5 −52 BURP domain
B_lectin PF01453.14 28.2 D-mannose binding lectin
BolA PF01722.7 23 BolA-like protein
C2 PF00168.18 3.7 C2 domain
CBM_20 PF00686.9 −3 Starch binding domain
CMAS PF02353.10 −177.9 Cyclopropane-fatty-acyl-phospholipid
synthase
Ceramidase_alk PF04734.3 25 Neutral/alkaline non-lysosomal ceramidase
CoA_binding PF02629.8 −12.8 CoA binding domain
Complex1_30kDa PF00329.8 −3 Respiratory-chain NADH dehydrogenase, 30 Kd
subunit
Complex1_49kDa PF00346.8 −108 Respiratory-chain NADH dehydrogenase, 49 Kd
subunit
Cupin_2 PF07883.1 16.6 Cupin domain
Cupin_3 PF05899.2 4.4 Protein of unknown function (DUF861)
Cyclin_C PF02984.8 −13 Cyclin, C-terminal domain
Cyclin_N PF00134.13 −14.7 Cyclin, N-terminal domain
DHBP_synthase PF00926.10 −116 3,4-dihydroxy-2-butanone 4-phosphate
synthase
DNA_pol_E_B PF04042.5 −47.5 DNA polymerase alpha/epsilon subunit B
DUF1005 PF06219.2 25 Protein of unknown function (DUF1005)
DUF1191 PF06697.2 25 Protein of unknown function (DUF1191)
DUF1195 PF06708.1 25 Protein of unknown function (DUF1195)
DUF1336 PF07059.2 −78.2 Protein of unknown function (DUF1336)
DUF26 PF01657.7 0 Domain of unknown function DUF26
DUF298 PF03556.6 25 Domain of unknown function (DUF298)
DUF588 PF04535.2 25 Domain of unknown function (DUF588)
DUF676 PF05057.4 −60.7 Putative serine esterase (DUF676)
DUF822 PF05687.3 25 Plant protein of unknown function (DUF822)
DUF868 PF05910.2 25 Plant protein of unknown function (DUF868)
DnaJ PF00226.19 −8 DnaJ domain
EGF_CA PF07645.4 24.5 Calcium binding EGF domain
ESCRT-III PF03357.10 −35.4 ESCRT-III complex subunit
ETC_C1_NDUFA5 PF04716.3 25 ETC complex I subunit conserved region
F-box PF00646.21 13.6 F-box domain
F420_oxidored PF03807.6 −34.5 NADP oxidoreductase coenzyme F420-
dependent
FAR1 PF03101.4 0 FAR1 family
FA_hydroxylase PF04116.2 −64.1 Fatty acid hydroxylase
FBPase PF00316.10 −170.3 Fructose-1-6-bisphosphatase
FH2 PF02181.13 −98.3 Formin Homology 2 Domain
GDC-P PF02347.5 −306.2 Glycine cleavage system P-protein
GH3 PF03321.3 −336 GH3 auxin-responsive promoter
GTP_cyclohydro2 PF00925.11 −49 GTP cyclohydrolase II
Gln-synt_C PF00120.14 −124 Glutamine synthetase, catalytic domain
Gln-synt_N PF03951.9 9 Glutamine synthetase, beta-Grasp domain
Glyco_transf_8 PF01501.9 −43.2 Glycosyl transferase family 8
Glycolytic PF00274.9 −174.5 Fructose-bisphosphate aldolase class-I
Gp_dh_C PF02800.9 −64.1 Glyceraldehyde 3-phosphate dehydrogenase,
C-terminal domain
Gp_dh_N PF00044.12 −74.2 Glyceraldehyde 3-phosphate dehydrogenase,
NAD binding domain
HD PF01966.11 18 HD domain
HLH PF00010.15 8.2 Helix-loop-helix DNA-binding domain
HTH_11 PF08279.1 11.3 HTH domain
Histone PF00125.13 17.4 Core histone H2A/H2B/H3/H4
Kelch_1 PF01344.14 7.8 Kelch motif
Kelch_2 PF07646.4 14 Kelch motif
L51_S25_CI-B8 PF05047.5 25 Mitochondrial ribosomal protein L51/S25/
CI-B8 domain
LEA_3 PF03242.3 25 Late embryogenesis abundant protein
LMBR1 PF04791.5 −116.3 LMBR1-like membrane protein
LRRNT_2 PF08263.2 18.6 Leucine rich repeat N-terminal domain
LRR_1 PF00560.21 7.7 Leucine Rich Repeat
LSM PF01423.12 13.7 LSM domain
Lung_7-TM_R PF06814.3 25 Lung seven transmembrane receptor
MAP65_ASE1 PF03999.2 −134.8 Microtubule associated protein (MAP65/ASE1
family)
MFS_1 PF07690.5 23.5 Major Facilitator Superfamily
MMR_HSR1 PF01926.11 31.2 GTPase of unknown function
MannoseP_isomer PF01050.8 −70 Mannose-6-phosphate isomerase
Metalloenzyme PF01676.7 −14.4 Metalloenzyme superfamily
Metallophos PF00149.17 22 Calcineurin-like phosphoesterase
Methyltransf_11 PF08241.1 17.1 Methyltransferase domain
Methyltransf_12 PF08242.1 21.4 Methyltransferase domain
Mit_rib_S27 PF08293.1 25 Mitochondrial ribosomal subunit S27
MtN3_slv PF03083.5 −0.8 MtN3/saliva family
NAD_Gly3P_dh_N PF01210.12 −44 NAD-dependent glycerol-3-phosphate
dehydrogenase N-terminus
NAD_binding_1 PF00175.10 −3.9 Oxidoreductase NAD-binding domain
NAF PF03822.4 4.5 NAF domain
NDK PF00334.9 −59.9 Nucleoside diphosphate kinase
NPH3 PF03000.4 25 NPH3 family
NTP_transferase PF00483.12 −90.5 Nucleotidyl transferase
Na_H_Exchanger PF00999.10 −67.9 Sodium/hydrogen exchanger family
ORMDL PF04061.4 25 ORMDL family
Oleosin PF01277.7 −27 Oleosin
PAN_2 PF08276.2 −4.9 PAN-like domain
PAP_fibrillin PF04755.2 25 PAP_fibrillin
PBD PF00786.17 12.2 P21-Rho-binding domain
PCI PF01399.15 25 PCI domain
PEP-utilizers PF00391.12 10 PEP-utilising enzyme, mobile domain
PGAM PF00300.12 −3 Phosphoglycerate mutase family
PK PF00224.10 −244 Pyruvate kinase, barrel domain
PK_C PF02887.5 −44 Pyruvate kinase, alpha/beta domain
PPR PF01535.11 0 PPR repeat
PTR2 PF00854.12 −50 POT family
Pantoate_transf PF02548.5 −93 Ketopantoate hydroxymethyltransferase
Peptidase_S10 PF00450.11 −198 Serine carboxypeptidase
PfkB PF00294.13 −67.8 pfkB family carbohydrate kinase
Phosphorylase PF00343.9 −601.1 Carbohydrate phosphorylase
Pkinase PF00069.14 −70.8 Protein kinase domain
Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase
Prenylcys_lyase PF07156.3 −164.1 Prenylcysteine lyase
PurA PF04845.3 25 PurA ssDNA and RNA-binding protein
RMMBL PF07521.1 18.5 RNA-metabolising metallo-beta-lactamase
Response_reg PF00072.12 4 Response regulator receiver domain
Rhodanese PF00581.9 25 Rhodanese-like domain
Rib_5-P_isom_A PF06026.4 25 Ribose 5-phosphate isomerase A
(phosphoriboisomerase A)
Ribonuclease_T2 PF00445.8 −53 Ribonuclease T2 family
Ribosomal_S8e PF01201.11 25 Ribosomal protein S8e
Rotamase PF00639.10 4 PPIC-type PPIASE domain
RrnaAD PF00398.9 −73.3 Ribosomal RNA adenine dimethylase
SAM_1 PF00536.18 11.3 SAM domain (Sterile alpha motif)
SMC_N PF02463.8 −95.8 RecF/RecN/SMC N terminal domain
SNF5 PF04855.3 25 SNF5/SMARCB1/INI1
SWIB PF02201.9 −7 SWIB/MDM2 domain
SWIM PF04434.7 10 SWIM zinc finger
S_locus_glycop PF00954.11 −12.7 S-locus glycoprotein family
Sad1_UNC PF07738.2 −20.4 Sad1/UNC-like C-terminal
Sterol_MT_C PF08498.1 30.5 Sterol methyltransferase C-terminal
Subtilisin_N PF05922.6 26.1 Subtilisin N-terminal Region
Sugar_tr PF00083.13 −85 Sugar (and other) transporter
TFIIA PF03153.4 25 Transcription factor IIA, alpha/beta subunit
TFIIS_C PF01096.9 15 Transcription factor S-II (TFIIS)
TFIIS_M PF07500.3 7.4 Transcription factor S-II (TFIIS), central
domain
TIM PF00121.8 −97 Triosephosphate isomerase
TPR_1 PF00515.16 7.7 Tetratricopeptide repeat
TPR_2 PF07719.5 20.1 Tetratricopeptide repeat
TPT PF03151.7 −15.3 Triose-phosphate Transporter family
Thioredoxin PF00085.9 −25.7 Thioredoxin
Tim17 PF02466.8 2.7 Tim17/Tim22/Tim23 family
TrkA_N PF02254.7 4.7 TrkA-N domain
Tryp_alpha_amyl PF00234.10 −4 Protease inhibitor/seed storage/LTP family
UPF0261 PF06792.1 25 Uncharacterised protein family (UPF0261)
UQ_con PF00179.16 −30 Ubiquitin-conjugating enzyme
Usp PF00582.16 36.1 Universal stress protein family
WD40 PF00400.20 21.5 WD domain, G-beta repeat
adh_short PF00106.14 −17 short chain dehydrogenase
eIF-4B PF06273.1 −205.7 Plant specific eukaryotic initiation factor 4B
iPGM_N PF06415.3 −263.4 BPG-independent PGAM N-terminus
(iPGM_N)
p450 PF00067.11 −105 Cytochrome P450
zf-AN1 PF01428.6 0 AN1-like Zinc finger
zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger)
zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and
similar)
zf-CCHC PF00098.12 17.9 Zinc knuckle
zf-CSL PF05207.3 2.9 CSL zinc finger
Example 5. Plasmid Construction for Transferring Recombinant DNA This example illustrates the construction of plasmids for transferring recombinant DNA into the nucleus of a plant cell which can be regenerated into a transgenic crop plant of this invention. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector.
A. Corn Transformation Vector
Elements of an exemplary common expression vector, pMON93093 are illustrated in Table 18. The exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art. The DNA of interest are inserted in a expression vector at the insertion site between the intron 1 of rice act 1 gene and the termination sequence of PinII gene.
TABLE 18
pMON93093
Coordinates of
SEQ ID NO:
function name annotation 19940
Agro B-AGRtu.right border Agro right border 11364-11720
transforamtion sequence, essential for
transfer of T-DNA.
Gene of E-Os.Act1 upstream promoter 19-775
interest region of the rice actin
expression 1 gene
cassette E-CaMV.35S.2xA1-B3 duplicated35S A1-B3 788-1120
domain without TATA
box
P-Os.Act1 promoter region of the 1125-1204
rice actin 1 gene
L-Ta.Lhcb1 5′ untranslated leader 1210-1270
of wheat major
chlorophyll a/b binding
protein
I-Os.Act1 first intron and flanking 1287-1766
UTR exon sequences
from the rice actin 1
gene
T-St.Pis4 3′ non-translated region 1838-2780
of the potato proteinase
inhibitor II gene which
functions to direct
polyadenylation of the
mRNA
Plant P-Os.Act1 Promoter from the rice 2830-3670
selectable actin 1 gene
marker L-Os.Act1 first exon of the rice 3671-3750
expression actin 1 gene
cassette I-Os.Act1 first intron and flanking 3751-4228
UTR exon sequences
from the rice actin 1
gene
TS-At.ShkG-CTP2 Transit peptide region 4238-4465
of Arabidopsis EPSPS
CR-AGRtu.aroA- Synthetic CP4 coding 4466-5833
CP4.nat region with dicot
preferred codon usage.
T-AGRtu.nos A 3′ non-translated 5849-6101
region of the nopaline
synthase gene of
Agrobacterium
tumefaciens Ti plasmid
which functions to
direct polyadenylation
of the mRNA.
Agro B-AGRtu.left border Agro left border 6168-6609
transformation sequence, essential for
transfer of T-DNA.
Maintenance OR-Ec.oriV-RK2 The vegetative origin of 6696-7092
in E. coli replication from
plasmid RK2.
CR-Ec.rop Coding region for 8601-8792
repressor of primer
from the ColE1
plasmid. Expression of
this gene product
interferes with primer
binding at the origin of
replication, keeping
plasmid copy number
low.
OR-Ec.ori-ColE1 The minimal origin of 9220-9808
replication from the E. coli
plasmid ColE1.
P-Ec.aadA-SPC/STR romoter for Tn7 10339-10380
adenylyltransferase
(AAD(3″))
CR-Ec.aadA-SPC/STR Coding region for Tn7 10381-11169
adenylyltransferase
(AAD(3″)) conferring
spectinomycin and
streptomycin resistance.
T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 11170-11227
adenylyltransferase
(AAD(3″)) gene of E. coli.
B. Soybean Transformation Vector Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 19 below. This exemplary soybean or canola transformation base vector illustrated in FIG. 3 is assembled using the technology known in the art. Recobminant DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, is cloned and amplified by PCR prior to insertion into the insertion site the base vector at the insertion site between the enhanced 35S CaMV promoter and the termination sequence of cotton E6 gene.
TABLE 19
pMON82053
Coordinates of
SEQ ID NO:
function name annotation 19941
Agro B-AGRtu.left Agro left border sequence, essential 6144-6585
transforamtion border for transfer of T-DNA.
Plant P-At.Act7 Promoter from the arabidopsis actin 6624-7861
selectable 7 gene
marker L-At.Act7 5′UTR of Arabidopsis Act7 gene
expression I-At.Act7 Intron from the Arabidopsis actin7
cassette gene
TS-At.ShkG- Transit peptide region of Arabidopsis 7864-8091
CTP2 EPSPS
CR-AGRtu.aroA- Synthetic CP4 coding region with 8092-9459
CP4.nno_At dicot preferred codon usage.
T-AGRtu.nos A 3′ non-translated region of the 9466-9718
nopaline synthase gene of
Agrobacterium tumefaciens Ti
plasmid which functions to direct
polyadenylation of the mRNA.
Gene of P-CaMV.35S-enh Promoter for 35S RNA from CaMV 1-613
interest containing a duplication of the −90 to
expression −350 region.
cassette T-Gb.E6-3b 3′ untranslated region from the fiber 688-1002
protein E6 gene of sea-island cotton;
Agro B-AGRtu.right Agro right border sequence, essential 1033-1389
transformation border for transfer of T-DNA.
Maintenance OR-Ec.oriV-RK2 The vegetative origin of replication 5661-6057
in E. coli from plasmid RK2.
CR-Ec.rop Coding region for repressor of primer 3961-4152
from the ColE1 plasmid. Expression
of this gene product interferes with
primer binding at the origin of
replication, keeping plasmid copy
number low.
OR-Ec.ori-ColE1 The minimal origin of replication 2945-3533
from the E. coli plasmid ColE1.
P-Ec.aadA- romoter for Tn7 adenylyltransferase 2373-2414
SPC/STR (AAD(3″))
CR-Ec.aadA- Coding region for Tn7 1584-2372
SPC/STR adenylyltransferase (AAD(3″))
conferring spectinomycin and
streptomycin resistance.
T-Ec.aadA- 3′ UTR from the Tn7 1526-1583
SPC/STR adenylyltransferase (AAD(3″)) gene
of E. coli.
C. Cotton Transformation Vector Plasmids for use in transformation of cotton are also prepared. Elements of an exemplary common expression vector plasmid pMON99053 are shown in Table 20 below and FIG. 4. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 1 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of one of the base.
TABLE 20
Coordinates
of SEQ ID
function name annotation NO: 19942
Agro B-AGRtu.right border Agro right border sequence, 11364-11720
transforamtion essential for transfer of T-
DNA.
Gene of interest Exp-CaMV.35S- Enhanced version of the 35S 7794-8497
expression enh + ph.DnaK RNA promoter from CaMV
cassette plus the petunia hsp70 5′
untranslated region
T-Ps.RbcS2-E9 The 3′ non-translated region 67-699
of the pea RbcS2 gene which
functions to direct
polyadenylation of the
mRNA.
Plant selectable Exp-CaMV.35S Promoter and 5′ untranslated 730-1053
marker region of the 35S RNA from
expression CaMV
cassette CR-Ec.nptII-Tn5 Neomycin 1087-1881
Phosphotransferase II gene
that confers resistance to
neomycin and kanamycin
T-AGRtu.nos A 3′ non-translated region of 1913-2165
the nopaline synthase gene of
Agrobacterium tumefaciens
Ti plasmid which functions to
direct polyadenylation of the
mRNA.
Agro B-AGRtu.left border Agro left border sequence, 2211-2652
transformation essential for transfer of T-
DNA.
Maintenance in OR-Ec.oriV-RK2 The vegetative origin of 2739-3135
E. coli replication from plasmid
RK2.
CR-Ec.rop Coding region for repressor 4644-4835
of primer from the ColE1
plasmid. Expression of this
gene product interferes with
primer binding at the origin
of replication, keeping
plasmid copy number low.
OR-Ec.ori-ColE1 The minimal origin of 5263-5851
replication from the E. coli
plasmid ColE1.
P-Ec.aadA-SPC/STR romoter for Tn7 6382-6423
adenylyltransferase
(AAD(3″))
CR-Ec.aadA-SPC/STR Coding region for Tn7 6424-7212
adenylyltransferase
(AAD(3″)) conferring
spectinomycin and
streptomycin resistance.
T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 7213-7270
adenylyltransferase
(AAD(3″)) gene of E. coli.
Example 6. Corn Plant Transformation This example illustrates the production and identification of transgenic corn cells in seed of transgenic corn plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic corn cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the corn transformation vectors pMON93093 as disclosed in Example 6. Corn transformation is effected using methods disclosed in U.S. Patent Application Publication 2004/0344075 A1 where corn embryos are inoculated and co-cultured with the Agrobacterium tumefaciens strain ABI and the corn transformation vector. To regenerate transgenic corn plants the transgenic callus resulting from transformation is placed on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber followed by a mist bench before transplanting to pots where plants are grown to maturity. The plants are self fertilized and seed is harvested for screening as seed, seedlings or progeny R2 plants or hybrids, e.g., for yield trials in the screens indicated above.
Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.
Example 7. Soybean Plant Transformation This example illustrates the production and identification of transgenic soybean cells in seed of transgenic soybean plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic soybean cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the soybean transformation vectors pMON82053 disclosed in Example 7. Soybean transformation is effected using methods disclosed in U.S. Pat. No. 6,384,301 where soybean meristem explants are wounded then inoculated and co-cultured with the soybean transformation vector, then transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots.
The transformation is repeated for each of the protein encoding DNAs identified in Table 1.
Transgenic shoots producing roots are transferred to the greenhouse and potted in soil. Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.
Example 8. Canola Transformation This example illustrates plant transformation useful in producing the transgenic canola plants of this invention and the production and identification of transgenic seed for transgenic canola having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
Tissues from in vitro grown canola seedlings are prepared and inoculated with overnight-grown Agrobacterium cells containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette. Following co-cultivation with Agrobacterium, the infected tissues are allowed to grow on selection to promote growth of transgenic shoots, followed by growth of roots from the transgenic shoots. The selected plantlets are then transferred to the greenhouse and potted in soil. Molecular characterization are performed to confirm the presence of the gene of interest, and its expression in transgenic plants and progenies. Progeny transgenic plants are selected from a population of transgenic canola events under specified growing conditions and are compared with control canola plants. Control canola plants are substantially the same canola genotype but without the recombinant DNA, for example, either a parental canola plant of the same genotype that is not transformed with the identical recombinant DNA or a negative isoline of the transformed plant
Transgenic canola plant cells are transformed with recombinant DNA from each of the genes identified in Table 1. Transgenic progeny plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as reported in Example 9.
Example 9. Selection of Transgenic Plants with Enhanced Agronomic Trait(s) This example illustrates identification of nuclei of the invention by screening derived plants and seeds for an enhanced trait identified below.
Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Populations of transgenic seed and plants prepared in Examples 6 and 7 are screened to identify those transgenic events providing transgenic plant cells with a nucleus having recombinant DNA imparting an enhanced trait. Each population is screened for enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and heat, increased level of oil and protein in seed using assays described below. Plant cell nuclei having recombinant DNA with each of the genes identified in Table 1 and the identified homologs are identified in plants and seeds with at least one of the enhanced traits.
A. Selection for Enhanced Nitrogen Use Efficiency Transgenic corn plants with nuclei of the invention are planted in fields with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 pounds per acre N), medium level (80 pounds per acre N) and high level (180 pounds per acre N). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied in the low level treatment, the soil should still be disturbed in the same fashion as the treated area. Transgenic plants and control plants can be grouped by genotype and construct with controls arranged randomly within genotype blocks. For improved statistical analysis each type of transgenic plant can be tested by 3 replications and across 4 locations. Nitrogen levels in the fields are analyzed before planting by collecting sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus (P), potassium (K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.
Transgenic corn plants prepared in Example 6 and which exhibit a 2 to 5% yield increase as compared to control plants when grown in the high nitrogen field are selected as having nuclei of the invention. Transgenic corn plants which have at least the same or higher yield as compared to control plants when grown in the medium nitrogen field are selected as having nuclei of the invention. Transgenic corn plants having a nucleus with DNA identified in Table 3 as imparting nitrogen use efficiency (LN) and homologous DNA are selected from a nitrogen use efficiency screen as having a nucleus of this invention.
B. Selection for Increased Yield Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and 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 selection of enhanced yielding transgenic corn events 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. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. 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. High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre. Each of the transgenic corn plants and soybean plants with a nucleus of the invention prepared in Examples 6 and 7 are screened for yield enhancement. At least one event from each of the corn and soybean plants is selected as having at least between 3 and 5% increase in yield as compared to a control plant as having a nucleus of this invention.
C. Selection for Enhanced Water Use Efficiency (WUE) The following is a high-throughput method for screening for water use efficiency in a greenhouse to identify the transgenic corn plants with a nucleus of this invention. This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant height are measured at three time points. The first is taken just prior to the onset drought when the plant is 11 days old, which is the shoot initial height (SIH). The plant height is also measured halfway throughout the drought/re-water regimen, on day 18 after planting, to give rise to the shoot mid-drought height (SMH). Upon the completion of the final drought cycle on day 26 after planting, the shoot portion of the plant is harvested and measured for a final height, which is the shoot wilt height (SWH) and also measured for shoot wilted biomass (SWM). The shoot is placed in water at 40 degree Celsius in the dark. Three days later, the shoot is weighted to give rise to the shoot turgid weight (STM). After drying in an oven for four days, the shoots are weighted for shoot dry biomass (SDM). The shoot average height (SAH) is the mean plant height across the 3 height measurements. The procedure described above may be adjusted for +/−˜one day for each step given the situation.
To correct for slight differences between plants, a size corrected growth value is derived from SIH and SWH. This is the Relative Growth Rate (RGR). Relative Growth Rate (RGR) is calculated for each shoot using the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)×100]. Relative water content (RWC) is a measurement of how much (%) of the plant was water at harvest. Water Content (RWC) is calculated for each shoot using the formula [RWC %=(SWM−SDM)/(STM−SDM)×100]. Fully watered corn plants of this age run around 98% RWC.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 1% increase in RGR and RWC as compared to control plants are identified as having enhanced water used efficiency and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting drought tolerance improvement (DS) and homologous DNA are identified as showing increased water use efficiency as compared to control plants and are selected as having a nucleus of this invention.
D. Selection for Growth Under Cold Stress Cold germination assay—Three sets of seeds are used for the assay. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.
Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C for 24 days (no light) in a Convrion® growth chamber (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and fifty millilters of deionized water are added to each germination tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.
The germination index is calculated as per:
Germination index=(Σ([T+1−ni]*[Pi−Pi-1]))/T
where T is the total number of days for which the germination assay is performed. The number of days after planting is defined by n. “i” indicated the number of times the germination had been counted, including the current day. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between transgenic events and wild type control. After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold screen is conducted in the same manner of the primary selection only increasing the number of repetitions to ten. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 5% increase in germination index as compared to control plants are identified as having enhanced cold stress tolerance and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting cold tolerance improvement (CK or CS) and homologous DNA are identified as showing increased cold stress tolerance as compared to control plants and are selected as having a nucleus of this invention.
E. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels
The following is a high-throughput selection method for identifying plant seeds with improvement in seed composition using the Infratec® 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample. Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan. An NIR calibration for the analytes of interest is used to predict the values of an unknown sample. The NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.
Infratec® Model 1221, 1225, or 1227 analyzer with transport module by Foss North America is used with cuvette, item #1000-4033, Foss North America or for small samples with small cell cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and soy check samples of varying composition maintained in check cell cuvettes are supplied by Leon Girard Co. NIT collection software is provided by Maximum Consulting Inc. Calculations are performed automatically by the software. Seed samples are received in packets or containers with barcode labels from the customer. The seed is poured into the cuvettes and analyzed as received.
TABLE 21
Typical sample(s): Whole grain corn and soybean seeds
Analytical time to run method: Less than 0.75 min per sample
Total elapsed time per run: 1.5 minute per sample
Typical and minimum sample size: Corn typical: 50 cc; minimum 30 cc
Soybean typical: 50 cc; minimum
5 cc
Typical analytical range: Determined in part by the specific
calibration.
Corn - moisture 5-15%, oil 5-20%,
protein 5-30%, starch 50-75%, and
density 1.0-1.3%.
Soybean - moisture 5-15%, oil
15-25%, and protein 35-50%.
Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for increased protein and oil in seed. Transgenic inbred corn and soybean plants having an increase of at least 1 percentage point in the total percent seed protein or at least 0.3 percentage point in total seed oil and transgenic hybrid corn plants having an increase of at least 0.4 percentage point in the total percent seed protein as compared to control plants are identified as having enhanced seed protein or enhanced seed oil and are selected as having a nucleus of this invention.
Example 10. Cotton Transgenic Plants with Enhanced Agronomic Traits Cotton transformation is performed as generally described in WO036911 and in U.S. Pat. No. 5,846,797. Transgenic cotton plants containing each of the recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 197 are obtained by transforming with recombinant DNA from each of the genes identified in Table 1. Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA. The specified culture conditions are growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.
The transgenic cotton plants of this invention are identified from among the transgenic cotton plants by agronomic trait screening as having increased yield and enhanced water use efficiency.
Example 11. Monocot and Dicot Plant Transformation for the Suppression of Endogeneous Protein This example illustrates monocot and dicot plant transformation to produce nuclei of this invention in cells of a transgenic plant by transformation where the recombinant DNA suppresses the expression of an endogenous protein identified by Pfam, SNF5, LMBR1, TFIIS_M, TFIIS_C, or Glyco_transf_8. Corn callus and soybean tissue are transformed as describe in Examples 6 and 7 using recombinant DNA in the nucleus with DNA that transcribes to RNA that forms double-stranded RNA targeted to an endogenous gene with DNA encoding the protein. The genes for which the double-stranded RNAs are targeted are the native gene in corn and soybean that are homolog of the genes encoding the protein with an amino acid sequence of SEQ ID NO:200, 201, 205, 207, 211, and 394.
Populations of transgenic corn plants and soybean plants prepared in Examples 6 and 7 with DNA for suppressing a gene identified in Table 3 as providing an enhanced trait by gene suppression are screened to identify an event from those plants with a nucleus of the invention by selecting the trait identified in this specification.
