MUTATIONS IN MADS-BOX GENES AND USES THEREOF

Info

Publication number: 20220002740
Type: Application
Filed: May 17, 2018
Publication Date: Jan 6, 2022
Applicant: Cold Spring Harbor Laboratory (Cold Spring Harbor, NY)
Inventors: Zachary Lippman (North Bellmore, NY), Sebastian Soyk (Cold Spring Harbor, NY), Zachary Hartford Lemmon (Cold Spring Harbor, NY)
Application Number: 16/613,660

Abstract

Aspects of the disclosure relate to plants, such as Solanaceae plants containing one or more of a mutant Solyc04g005320 gene (or a homolog thereof), a mutant Solycl2g038510 gene (or a homolog thereof), and a mutant Solyc03gl14840 gene (or a homolog thereof), as well as methods of producing such plants. In some aspects, such plants have one or more improved traits, such as modified inflorescence architecture, modified flower number, modified fruit number, higher yield, higher quality products, and higher fruit productivity.

Description

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 62/507,369, filed on May 17, 2017. The entire contents of this referenced application are incorporated by reference herein.

GOVERNMENT SUPPORT

This invention was made with government support under IOS-1523423 and IOS-1237880 awarded by the National Science Foundation. The government has certain rights in the invention.

BACKGROUND

The architectures of plant reproductive shoot systems—inflorescences—are major determinants of crop yield, and modified inflorescence complexity was a recurring target during crop domestication and improvement (Doebley et al., 2006; Meyer and Purugganan, 2013). Prominent examples include the cereal crops barley, maize, rice and wheat, for which humans selected variants with greater branching to increase flower and grain production (Ashikari et al., 2005; Boden et al., 2015; Doebley et al., 1997; Huang et al., 2009; Jiao et al., 2010; Komatsuda et al., 2007; Ramsay et al., 2011). Yet, for many crops, particularly fruit-bearing species such as grape and tomato, inflorescence architecture has changed little from wild ancestors, and therefore has been underexploited in breeding (Lippman et al., 2008; Mullins et al., 1992; Peralta and Spooner, 2005).

SUMMARY

Aspects of the present disclosure relate to compositions, such as novel genetic variants of plants, and methods for generating the compositions, which have favorable traits, such as yield-related traits. In some aspects, the combination of mutations in the novel genetic variants increase inflorescence and fruit production. In other aspects, mutations in one or more of the genes of the genetic variants can be used to create a quantitative range of inflorescence types, such as the development of weakly branched genetic variants that results in higher flower and fruit production.

In some aspects, the disclosure provides a genetically-altered Solanaceae plant (e.g., a tomato plant) comprising a mutant Solyc04g005320 gene or a homolog thereof. In some embodiments, the mutant Solyc04g005320 gene or homolog thereof is a null allele or a hypomorphic allele. In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) is heterozygous or homozygous for the mutant Solyc04g005320 gene or homolog thereof.

In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) further comprises a mutant Solyc12g038510 gene or a homolog thereof, a mutant Solyc03g114840 gene or a homolog thereof, or both a mutant Solyc12g038510 gene or a homolog thereof and a mutant Solyc03g114840 gene or a homolog thereof.

In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) further comprises a mutant Solyc12g038510 gene or homolog thereof and the mutant Solyc12g038510 gene or homolog thereof is a null allele or a hypomorphic allele. In some embodiments, the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc12g038510 gene or homolog thereof.

In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) further comprises a mutant Solyc03g114840 gene or a homolog thereof and the mutant Solyc03g114840 gene or homolog thereof is a null allele or a hypomorphic allele. In some embodiments, the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc03g114840 gene or homolog thereof.

In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) further comprises both a mutant Solyc12g038510 gene or a homolog thereof and a mutant Solyc03g114840 gene or a homolog thereof, each of which are independently a null allele or a hypomorphic allele. In some embodiments, the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc12g038510 gene or homolog thereof and is heterozygous or homozygous for the mutant Solyc03g114840 gene or homolog thereof.

In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) comprises the mutant Solyc04g005320 gene or homolog thereof, the mutant Solyc12g038510 gene or homolog thereof, and the mutant Solyc03g114840 gene or homolog thereof, and wherein each is a hypomorphic allele. In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) is heterozygous or homozygous for the mutant Solyc04g005320 gene or homolog thereof, is heterozygous or homozygous for the mutant Solyc03g114840 gene or homolog thereof and is heterozygous or homozygous for the mutant Solyc03g114840 gene or homolog thereof.

In some embodiments, the mutant Solyc04g005320 gene or homolog thereof is a hypermorphic allele. In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) is heterozygous or homozygous for the mutant Solyc04g005320 gene or homolog thereof.

In some embodiments, the genetically-altered Solanaceae plant (e.g., tomato plant) further comprises a mutant Solyc12g038510 gene or a homolog thereof, a mutant Solyc03g114840 gene or a homolog thereof, or both the mutant Solyc12g038510 gene or homolog thereof and the mutant Solyc03g114840 gene or homolog thereof.

In other aspects, the disclosure provides a genetically-altered Solanaceae plant (e.g., a tomato plant), comprising a mutant Solyc12g038510 gene or a homolog thereof and a mutant Solyc03g114840 gene or a homolog thereof, wherein the genetically-altered Solanaceae plant is homozygous for the mutant Solyc12g038510 gene or homolog thereof and heterozygous for the mutant Solyc03g114840 gene or homolog thereof. In some embodiments, the mutant Solyc12g038510 gene or homolog thereof is a null allele or a hypomorphic allele and the mutant Solyc03g114840 gene or homolog thereof is a null allele or a hypomorphic allele.

In some embodiments of any one of the genetically-altered Solanaceae plants (e.g., a tomato plant) provided herein, the mutant Solyc04g005320 gene or homolog thereof, the mutant Solyc12g038510 gene or homolog thereof, and/or the mutant Solyc03g114840 gene or homolog thereof is introduced by technical means. In some embodiments of any one of the genetically-altered Solanaceae plants (e.g., a tomato plant) provided herein, the mutant Solyc04g005320 gene or homolog thereof, the mutant Solyc12g038510 gene or homolog thereof, and/or the mutant Solyc03g114840 gene or homolog thereof is introduced by chemical or physical means. In some embodiments of any one of the genetically-altered Solanaceae plants (e.g., a tomato plant) provided herein, the mutant Solyc04g005320 gene or homolog thereof, the mutant Solyc12g038510 gene or homolog thereof, and/or the mutant Solyc03g114840 gene or homolog thereof is introduced using CRISPR/Cas9, chemical mutagenesis, radiation, Agrobacterium-mediated recombination, viral-vector mediated recombination, or transposon mutagenesis. In some embodiments of any one of the genetically-altered Solanaceae plants (e.g., a tomato plant) provided herein, the plants are provided with the provision that plants exclusively obtained by means of an essentially biological process are excluded.

In other aspects, the disclosure provides a crop harvested from a genetically-altered Solanaceae plant (e.g., a tomato plant) of any one of the above embodiments or of any other embodiment described herein.

In yet other aspects, the disclosure provides a seed for producing a genetically-altered Solanaceae plant (e.g., a tomato plant) of any one of the above embodiments or of any other embodiment described herein.

In other aspects, the disclosure provides a method for producing a genetically-altered Solanaceae plant (e.g., a tomato plant), the method comprising introducing a mutation into a Solyc04g005320 gene or a homolog thereof in a Solanaceae plant, thereby producing a genetically-altered Solanaceae plant containing a mutant Solyc04g005320 gene or homolog thereof. In some embodiments, the mutation is introduced using CRISPR/Cas9. In some embodiments, the mutation produces a null allele or a hypomorphic allele of the Solyc04g005320 gene or homolog thereof.

In some embodiments of any one of the methods provided herein, the method further comprises introducing into the Solanaceae plant a mutation into a Solyc12g038510 gene or a homolog thereof, introducing a mutation into a Solyc03g114840 gene or a homolog thereof, or introducing the mutation into the Solyc12g038510 gene or homolog thereof and introducing the mutation into the Solyc03g114840 gene or homolog thereof. In some embodiments, the mutation(s) is/are introduced using CRISPR/Cas9.

In some embodiments of any one of the methods provided herein, the genetically-altered Solanaceae plant (e.g., a tomato plant) containing the mutant Solyc04g005320 gene or homolog thereof is crossed to another genetically-altered Solanaceae plant (e.g., a tomato plant) comprising a mutant Solyc12g038510 gene or homolog thereof, a mutant Solyc03g114840 gene or homolog thereof, or both the mutant Solyc12g038510 gene or homolog thereof and the mutant Solyc03g114840 gene or homolog thereof, thereby producing a genetically-altered Solanaceae plant (e.g., a tomato plant) containing the mutant Solyc04g005320 gene or homolog thereof and the mutant Solyc12g038510 gene or homolog thereof, the mutant Solyc03g114840 gene or homolog thereof, or both the mutant Solyc12g038510 gene or homolog thereof and the mutant Solyc03g114840 gene or homolog thereof.

In other aspects, the disclosure provides a genetically-altered Solanaceae plant (e.g., a tomato plant) produced or obtainable by a method of any one of the above embodiments or of any other embodiment described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

FIGS. 1A-1K show the s2 inflorescence architecture variant branches due to delayed meristem maturation. FIG. 1A shows a typical wild type (WT) tomato plant with unbranched, multi-flowered inflorescences and jointed pedicels (dotted asterisk in inset). Numbers in FIGS. 1A-1C indicate flowers per inflorescence (mean±SEM, N=number of inflorescences). Striped arrowheads indicate successive inflorescences. P: two-tailed, two-sample t-test compared to WT. FIG. 1B shows the highly branched inflorescences and jointed pedicels of s mutants. White arrowheads indicate branch points. FIG. 1C shows the s2 mutant with moderately branched inflorescences and jointless pedicels (asterisk). FIG. 1D shows quantification of inflorescence branching events in WT, s, and s2. FIG. 1E shows phenotypic classes in a WT×s2 F2 population. The segregation ratio for the jointless pedicel phenotype and the branched inflorescence phenotype (s2) is given. Asterisks mark jointless pedicels. Scale bars in FIGS. 1A-1C and lE=1 cm. FIGS. 1F-1H show the transition meristem (TM), sympodial inflorescence meristem (SIM), and floral meristem (FM) from WT (FIG. 1F), s (FIG. 1G), and s2 (FIG. 1H). Scale bars in FIGS. 1F-1H represent 100 μm. L, leaf. F, flower. Schematics depict developing inflorescences. Lines, internodes; circles, FMs/flowers; arrowheads, SIMs. Overproliferating branches are indicated in bolded line. FIG. 1I shows PCA of 2,582 dynamically expressed genes in the vegetative meristem (VM), TM, SIM, and FM of WT, s, and s2, determined by RNA-seq. FIGS. 1J-1K show expression (z-score normalized) of TM (FIG. 1J) and FM (FIG. 1K) marker genes in the vegetative (VM) meristem, TM and FM stage of meristem maturation of WT and mutant (s and s2). Cluster of genes with moderately (left) and strongly (right) delayed expression pattern are shown. Dashed lines indicate median expression with dot-filled-in area representing the 5^thand 95^thquantile.

FIGS. 2A-2N show that mutations in two SEPALLATA MADS-box genes cause s2 branching. FIG. 2A shows mapping-by-sequencing of s2. Ratio of SNP-ratios (s2/M82) between different pools of segregating phenotypic classes (top: s2/WT; middle: s2/j2; bottom: j2/WT) is shown for chromosome 3 and 12. FIG. 2B shows the j2 mapping interval includes the SEP4 homolog Solyc12g038510. FIG. 2C shows Genomic Illumina-sequence reads showing a breakpoint in Solyc12g038510 (left), and PCR showing a Copia/Rider transposon insertion in the first intron of Solyc12g038510 in s2 mutants (right). The sequence corresponds to SEQ ID NO: 89. FIG. 2D shows Sashimi plots of normalized RNA-seq reads (reads per million, RPM) for Solyc12g038510 in WT (top) and s2 (bottom) floral meristems. An intronic transcriptional start site leads to out-of-frame Solyc12g038510 transcripts in s2 mutants. Numbers indicate reads supporting splice-junctions and alternative splicing in s2 is highlighted in the bottom panel by diagonal line filling. FIG. 2E shows the generation of j2^CRnull mutations by CRISPR/Cas9 using two single-guide RNAs (sgRNA, target1 and target2; arrows). Black arrows indicate forward (F) and reverse (R) primers used for genotyping and sequencing. Sequences of j2^CRallele 1 (a1) and a2 are shown. sgRNA targets and protospacer-adjacent motif (PAM) are indicated in bold font and deletions by dashes. Insertions are indicated in italic font and sequence gap length is shown in parentheses. From top to bottom, sequences correspond to SEQ ID NOs: 90-92. FIG. 2F shows inflorescences and fruits from WT and j2^CRmutants showing unbranched inflorescence with jointless pedicels for j2^CR. White and dotted asterisks indicate jointed and jointless pedicels, respectively. FIG. 2G shows a complementation test between j2^CRand j2^TE(jointless pedicels; asterisks). FIG. 2H shows that the ej2 mapping interval includes the SEP4 homolog Solyc03g114840. FIG. 2I shows the Genomic Illumina-sequence reads showing a breakpoint in Solyc03g114840 and PCR revealing a 564 bp insertion in the 5th intron of Solyc03g114840 in s2 mutants. The sequence corresponds to SEQ ID NO: 93. FIG. 2J shows Sashimi plots for Solyc03g114840 RNA-seq reads in WT and s2 floral meristems indicating partial exon skipping and intron retention in s2 mutants. FIG. 2K shows the generation of ej2^CRnull mutations by CRISPR/Cas9. From top to bottom, sequences correspond to SEQ ID NOs: 94-97. FIG. 2L shows unbranched ej2^CRmutant inflorescences with extremely long sepals (arrowheads) and pear-shaped fruits. Scale bars=1 cm. FIG. 2M shows unopened flowers showing the weak natural ej2^wallele causes longer sepals and fails to complement ej2^CR. FIG. 2N shows quantification of relative sepal length (sepal length/petal length f SEM, N=number of flowers) for genotypes in FIG. 2M. P: two-tailed, two-sample t-test compared to WT.

FIGS. 3A-3F show the ej2^wvariant arose during domestication and was selected during breeding of large-fruited cultivars. FIG. 3A shows distribution of the ej2^wallele in wild tomato species, early domesticates (landraces, S. lyc. var. cerasiforme), and cultivars (S. lycopersicum)(N=number of accessions). FIG. 3B shows relative sepal length (sepal length/petal length) from a subset of accessions in FIG. 3A homozygous EJ2 and ej2^w. FIG. 3C shows relative sepal length in a subset of confirmed landraces (Blanca et al., 2015). FIG. 3D shows PCR genotyping for the ej2^wallele in 10 landraces with the longest and shortest sepals. S. pimpinellifolium (S. pim) was used as a WT control. FIG. 3E shows inflorescences and flowers (inset) of the accessions with the shortest and longest sepals. See asterisks in FIG. 3D. Numbers indicate relative sepal length. FIG. 3F shows PCR genotyping in 258 cultivars shows enrichment of the ej2^wallele in large-fruited types. Data in FIGS. 3B, 3C, and 3E are means (±SEM, n=10 flowers per accession). N=number of accessions. P: two-tailed, two-sample t-test. Scale bars=1 cm.

FIGS. 4A-4D show that breeders overcame negative epistasis between j2 and ej2 by selecting suppressors of s2 branching in elite germplasm. FIG. 4A shows PCR genotyping of 153 elite breeding lines for j2^TEand ej2^wreveals the jointless germplasm is dominated by the j2 transposon allele and contains many j2^TEej2^wdouble mutants. Number of accessions is indicated in parenthesis. FIG. 4B shows PCR genotyping of 31 jointless inbreds and hybrids from 4 major seed companies for ej2^w. Asterisks indicate j2^TEej2^wdouble mutants. FIG. 4C shows representative images of phenotypic classes found inj2^TEej2^wdouble mutants isolated from an S. pimpinellifolium×s2 F2 population. N indicates number of plants and percentage of plants in each phenotypic class is indicated in parentheses. FIG. 4D shows mapping-by-sequencing a suppressor of s2 to a 3 Mbp interval on chromosome 2 containing 167 genes. DNA from pools of s2 and suppressed s2 plants was sequenced and the ratio (suppressed s2/s2) of the SNP-ratios (S.pin/s2) is presented.

FIGS. 5A-5I show that redundancy among three SEP4 genes regulates inflorescence branching and flower development. FIG. 5A shows the phylogenetic tree of SEP proteins in Arabidopsis and tomato. Bootstrap values (%) for 1000 replicates are shown. FIG. 5B shows normalized gene expression (RPKM) for TM5 and TM29 (left) and the SEP4 sub-clade (right) during meristem maturation (VM, vegetative meristem; TM, transition meristem; FM, floral meristem; SIM, sympodial inflorescence meristem; SYM, sympodial shoot meristem). FIG. 5C shows yeast two-hybrid assays showing heteromeric interactions for Solyc04g005320, J2, and EJ2, and homomeric interactions for Solyc04g005320 and J2 (3-AT, 3-amino-1,2,4-triazole; L, leucine; T, tryptophan; H, histidine; e.v., empty vector). FIG. 5D shows the summary of results in FIG. 5C; (−) no interaction; (+) interaction; (++) strong interaction. FIG. 5E shows the longer inflorescence of a Solyc04g005320^CRmutant (hereafter referred to as long inflorescence^CR; lin^CR) compared to WT. Numbers indicate flowers per inflorescence (mean±SEM, N=10 inflorescences). P: two-tailed, two-sample t-test. Scale bar=1 cm. FIG. 5F shows the longer inflorescence of a Solyc04g005320^CRmutant in S. pimpineiolium (S. pim lin^CR) compared to S. pimpinellifolium WT. FIG. 5G shows j2^CRej2^CRdouble mutant plant (left) and inflorescence (right) showing SIM overproliferation and few flowers late in development, respectively. FIG. 5H shows j2^CRej2^CRZi^CRtriple mutant. Stereoscope images (insets) of j2^CRej2^CRZin^CRtriple mutants showing massive SIM overproliferation and no floral termination. FIG. 5I shows j2^CRe j2^CRZin^CRtriple mutant in S. pimpinellifolium as in FIG. 5H showing massive SIM overproliferation and no floral termination. Striped arrowheads indicate successive inflorescences. Scale bars represent 1 cm and 1 mm for photographs and stereoscopic images, respectively.

FIGS. 6A-6D show the exploiting dosage effects of key meristem maturation genes to improve flower production and fruit yield. FIG. 6A shows representative inflorescences from different genotypic combinations of natural and engineered j2 and ej2 mutations in M82. Red arrowheads indicate branching events. FIG. 6B shows the percentage of inflorescences with 1 to 5 or greater branching events for the indicated genotypes. Circled, lower-case letters match genotypes shown in FIG. 6A. Weakly branched genotypes are highlighted with bolded black circles. FIG. 6C shows representative weakly branched inflorescence of a s^classic/+ heterozygote. FIG. 6D shows the percentage of inflorescences with branching events for s^classic/+, s^multiflora/+, and sⁿ⁵⁵⁶⁸/+ heterozygous genotypes. White arrowheads in FIGS. 6A and 6C mark inflorescence branch points. Nindicates number of inflorescences (FIGS. 6B and 6D). Scale bars in FIGS. 6A and 6C indicate 1 cm.

FIGS. 7A-7K show that s2 inflorescence branching variants are allelic, fail to complement the classical j2 mutant, and are genetically additive with s. FIGS. 7A-7C show the accessions LA0315 (FIG. 7A), LA3226 (FIG. 7B), and the X-ray-induced mutant frondea (FIG. 7C) (Stubbe, 1972) develop highly proliferated inflorescences that bear flowers and fruits with jointless pedicels (white asterisks). FIGS. 7D-7F show stereoscope images of primary meristems in LA0315 (FIG. 7D), LA3226 (FIG. 7E), and frondea (FIG. 7F), showing the first inflorescence branching event (white arrowhead) at the base of the first flower (F1). SYM: sympodial shoot meristem; L8: leaf 8. FIGS. 7G-7I show representative inflorescences of F₁progeny from the crosses LA0315×s2 (FIG. 7G), LA3226×s2 (FIG. 7H), and fro×LA0315 (FIG. 7I) showing all four accessions (mutants) are allelic. Scale bars in FIGS. 7A-7C, 7G-7I, and 7D-7F indicate 5 cm and 500 μm, respectively. FIG. 7J shows inflorescences of s (left), s2 (middle), and the s s2 higher-order mutant (right). Greater inflorescence complexity in the s s2 higher-order mutant suggests additivity. FIG. 7K shows a complementation test using an s2-derived jointless mutant plants and the classical j2 mutant. Jointed fruits (dotted asterisk) of WT plants and jointless fruits (white asterisk) of F₁progeny from a cross of s2-derived j2 and j2 are shown. Scale bar=1 cm.

FIGS. 8A-8C show the rate of meristem maturation in s2 mutants is less delayed than in s. FIG. 8A shows the clustering of 2,582 genes that were dynamically expressed during the early (EVM), middle (MVM), and late (LVM) vegetative meristem, the transition meristem (TM) and floral meristem (FM) stage of meristem maturation in the WT (see STAR Methods). Genes in Cluster 06 and Cluster 08 (solid line boxes) were selected as TM and FM marker genes, respectively. Thick black lines indicate median expression with dotted area representing the 5^thand 95^thquantile. N=number of genes. FIGS. 8B and 8C show WT, s (top), and s2 (bottom) z-score normalized expression of TM marker genes in vegetative (VM), transition (TM), and floral (FM) meristem stages. Cluster in dotted line boxes and solid line boxes were selected as moderately and strongly delayed genes, respectively.

FIGS. 9A-9J show that mapping-by-sequencing reveals s2 branching is caused by mutations in two tomato homologs of the SEPALLATA MADS-box genes (J2 and EJ2). FIGS. 9A and 9B show representative images of the phenotypic classes found in the M82×s2 F₂(FIG. 9A) and S. pimpinellifolium×s2 F₂populations (FIG. 9B). Asterisks mark jointless pedicels and arrowheads mark inflorescence branching events. Scale bars=1 cm. FIG. 9C shows segregation ratios of the s2 branching phenotype in the two F₂populations. Note that in the M82×s2 F₂, the j2 and s2 phenotypes segregated ¼ and 1/16, respectively. FIG. 9D shows mapping-by-sequencing of the loci underlying s2 in an M82×s2 F₂population. Pooled DNA from WT, j2 and s2 plants was sequenced and the ratios of the SNP-ratios (s2/M82) between different phenotypic classes (top: s2/WT; middle: s2/j2; bottom: jointless/WT) are shown. FIG. 9E shows mapping-by-sequencing of the loci underlying s2 in a S. pimpinellifolium×s2 F₂population. Pooled DNA from WT,j2, and s2 plants was sequenced and ratios of the SNP-ratios (S.lyc/S.pim) are shown as in FIG. 9D. FIG. 9F shows partial sequence alignment of J2 (Solyc12g038510) from M82, the jointless S. cheesmaniae (S. che) accession LA0166, the classical j2 accession (LA0315) and the s2 accession (LA4371). A S. cheesmaniae SNP in the second exon leads to a premature stop-codon (asterisk). Allele designated as j2^stop. From top to bottom, sequences correspond to SEQ ID NOs: 98, 98, 99, 100, and 101. FIG. 9G shows the CAPS marker PCR genotyping assay for j2^stopin accessions from FIG. 9F. Positions of WT and mutant (mut) bands are indicated. FIG. 9H shows gene models showing the position of the Copia/Rider transposable element (TE) insertion in j2^TEand the S. cheesmaniae SNP in j2^stop. Predicted RNA transcripts are shown below. The j2^stopallele results in a premature stop codon in the second exon. The j2^TEallele results in an intronic transcriptional start site and an early stop codon. FIG. 9I shows representative inflorescences of WT, ej2^w, ej2^CR, and e j2^CR×ej2^wF₁progeny are shown. Scale bar=1 cm. FIG. 9J shows genotyping of s2, LA0315, LA3226,frondea (fro), and WT plants using diagnostic PCR markers for j2^TE,j2^stop, and ej2^w. Note that both s2 and LA3226 carry the j2^TEand ej2^walleles, whereas LA0315 carries j2^stopand ej2^w. The frondea mutant carries ej2^TE, however, failed J2 amplification in frondea using both j2 markers suggest a large structural variant has disrupted the gene (SV). Band sizes are in kilobase pairs (kbp).

FIGS. 10A-10S show that the three SEP4 genes J2, EJ2 and Solyc04g005320/LIN interact to regulate branching and flower development. FIG. 10A shows normalized gene expression (RPKM) for TM5 and TM29 (left) and the SEP4 sub-clade (right) in major tissues.

FIG. 10B shows yeast two-hybrid assays showing heteromeric interaction of Solyc04g005320, RIN, J2, and EJ2, and homomeric interaction of Solyc04g005320, RIN and J2 (3-AT, 3-amino-1,2,4-triazole; L, leucine; T, tryptophan; H, histidine; A, adenine; e.v., empty vector). FIG. 10C shows the summary of results in FIG. 10B; (−) no interaction; (+) interaction; (++) strong interaction. FIG. 10D shows CRISPR/Cas9 targeting of Solyc04g005320. Sequences of Solyc04g005320^CRallele 1 (a1) and a2 in S. lycopersicum cv. M82 are shown (top). Three independent first-generation (T₀) chimeric S. pimpinellifolium transgenics were sequenced and 5 reads were obtained per plant (bottom). All sequenced alleles carried mutations, revealing putative biallelic (T₀#4), homozygous (T₀#8), and chimeric (T₀#9) plants. From top to bottom, sequences correspond to SEQ ID NOs: 102-111. FIG. 10E shows the quantification of flowers per inflorescence for WT and 3 independent lin^CRT₀transgenics. N=number of inflorescences. FIG. 10F shows the quantification of internode length between flowers of the same plants as in FIG. 10E. N=number of internodes. FIG. 10G shows representative lin mutant plant with elongated and weakly branched inflorescences. White arrowheads indicate branch points. Inset shows lin fruit with jointed pedicel. FIG. 10H shows quantification of flowers per inflorescence for WT and lin. N=number of inflorescences. FIG. 10I shows quantification of inflorescence branching events in WT and lin. FIGS. 10J and 10K show mapping-by-sequencing of the lin mutation in a lin×S. pim F₂population to a 0.5 Mbp mapping interval on chromosome 4 containing 80 genes including Solyc04g005320. Reads mapping to chromosome 4 indicate a translocation in Solyc04g005320, which was assayed by PCR (FIG. 10K). The sequence in FIG. 10J corresponds to SEQ ID NO: 112. The WT allele (wt) was amplified with primer-F1 and primer-R2, which bind 5′ and 3′ to the translocation site, respectively. The lin mutant allele (m) was amplified with primer-F3, which binds the 3′ border of the translocated sequence, and primer-R2. FIG. 10L shows semi-quantitative RT-PCR of Solyc04g005320 in WT and lin showing loss of Solyc04g005320 transcript in the lin mutant. UBIQUITIN(UBI) was used as control. FIG. 10M shows j2^CRlin double mutant with elongated, weakly branched inflorescences and jointless pedicel (white asterisk). White arrowheads mark branch points. FIG. 10N shows ej2^CRlin double mutant with long inflorescences, extremely enlarged sepals, and inner floral organ defects (inset). FIG. 10O shows simultaneous targeting of LIN, J2 and E/2 by CRISPR/Cas9 with two single-guide RNAs. sgRNA, Target 1 and Target 2 on each respective gene model is shown. Note that sgRNA-1 targets all three genes. Black arrows indicate forward (F) and reverse (R) primers used for PCR genotyping and sequencing (see STAR Methods). Sequencing results of second-generation (Ti) transgenicj2^CRej2^CRlin^CRtriple mutant plants generated in M82 (top) and S. pimpinellifolium (bottom). All three genes carry homozygous mutations. From top to bottom, sequences correspond to SEQ ID NOs: 113-124. FIG. 10P shows CRISPR/Cas9 targeting of LIN in the elite cherry cultivar Sweet 100. Sequences of lin^CR-allele 1 (a1) and a2 in the first-generation (T₀) lin^CRplant #1. Five reads were obtained per plant. All sequenced alleles carried mutations, including a complex rearrangement (italicized font). From top to bottom, sequences correspond to SEQ ID NOs: 125-127. FIG. 10Q shows representative images of Sweet 100 and Sweet 100 lin^CRT₀#1 mutant inflorescences showing different degrees of branching. FIGS. 10R and 10S show quantification of flowers per inflorescence (FIG. 10R) and inflorescence branching events (FIG. 10S) for Sweet 100 and Sweet 100 lin^CRT₀#1. N=number of inflorescences. Bar graphs in FIGS. 10E, 10F., 10H, 10I, 10R, and 10S show means (±SEM). P-values determined by two-tailed, two-sample t-tests. Scale bars represent 1 cm.

SEQUENCES

Below is a brief description of certain sequences described herein.

SEQ ID NO: 1 is a nucleic acid sequence of a wild-type Solyc04g005320 gene.

SEQ ID NO: 2 is a nucleic acid sequence of a wild-type Solyc04g005320 coding sequence.

SEQ ID NO: 3 is a nucleic acid sequence for a mutant Solyc04g005320 gene allele lin^trans. The border sequences of a translocation site are shown in bold italic letters, with the translocation sequence being represented by the NNNNNN(N*X)NNNNNN sequence.

SEQ ID NO: 4 is a nucleic acid sequence for a mutant Solyc04g005320 gene allele lin^CR-allele 1.

SEQ ID NO: 5 is a nucleic acid sequence for a mutant Solyc04g005320 gene allele lin^CR-allele 2.

SEQ ID NO: 6 is a nucleic acid sequence of a wild-type Solyc12g038510 gene.

SEQ ID NO: 7 is a nucleic acid sequence of a wild-type Solyc12g038510 coding sequence.

SEQ ID NO: 8 is a nucleic acid sequence for a mutant Solyc12g038510 gene allele j2^TE. The border sequences of a transposable element insertion site are shown in bold italic letters, with the transposable element sequence being represented by the NNNNNN(N*X)NNNNNN sequence.

SEQ ID NO: 9 is a nucleic acid sequence of a mutant Solyc12g038510 gene allele j2 stop SEQ ID NO: 10 is a nucleic acid sequence for a mutant Solyc04g005320 gene allele j2^CR-allele 1.

SEQ ID NO: 11 is a nucleic acid sequence for a mutant Solyc04g005320 gene allele j2^CR-allele 2.

SEQ ID NO: 12 is a nucleic acid sequence of a wild-type Solyc03g114840 gene.

SEQ ID NO: 13 is a nucleic acid sequence of a wild-type Solyc03g114840 coding sequence.

SEQ ID NO: 14 is a nucleic acid sequence for a mutant Solyc03g114840 gene allele ej2^W.

SEQ ID NO: 15 is a nucleic acid sequence for a mutant Solyc04g005320 gene allele ej2^CR-allele 1.

SEQ ID NO: 16 is a nucleic acid sequence for a mutant Solyc04g005320 gene allele ej2^CR-allele 3.

DETAILED DESCRIPTION

Variation in inflorescence architecture is based on changes in the activity of meristems, small groups of stem cells located at the tips of shoots (Kyozuka et al., 2014; Park et al., 2014a). During the transition to flowering, vegetative meristems gradually mature to a reproductive state and, depending on the species, terminate immediately in a flower or give rise to a variable number of new inflorescence meristems that become additional flowers or flower-bearing branches (Prusinkiewicz et al., 2007). In domesticated tomato (Solanum lycopersicum) and its wild progenitor S. pimpinellifolium, a new inflorescence meristem emerges at the flank of each previous meristem. Several reiterations of this process give rise to inflorescences with multiple flowers arranged in a zigzag pattern, resulting in the familiar “tomatoes on the vine” architecture (FIG. 1A)(Park et al., 2012).

Improving tomato inflorescence architecture to boost flower production and yield has remained surprisingly challenging, despite a rich resource of wild relatives that develop weakly branched inflorescences with high fertility (Lemmon et al., 2016; Lippman et al., 2008; Park et al., 2012; Zamir, 2001). However, genetic incompatibilities and the challenge of transferring complex polygenic traits without undesired effects from linked genes has precluded exploiting wild species to improve inflorescence architecture (MacArthur and Chiasson, 1947). Another source of potentially valuable inflorescence variation is rare natural and induced highly branched mutants in domesticated germplasm. It was previously shown that branching in one of these variants and branching in a wild species is due to an extended meristem maturation schedule, which allows additional inflorescence meristems to form (Lemmon et al., 2016; Park et al., 2012). This suggested subtle modification of meristem maturation could provide beneficial changes in inflorescence architecture (Park et al., 2014a). Yet, breeders typically select against even moderate branching, primarily due to an imbalance in source-sink relationships that results in high flower abortion and low fruit production, especially in large-fruited varieties (Stephenson, 1981).

In some aspects, the present disclosure relates to the discovery of the identity of mutations in two closely related MADS-box transcription factor genes, one of which arose during domestication and the other within the last century of crop improvement. Each mutant was selected separately based on the phenotype of improved flower morphology and fruit retention traits without knowledge of the locations of the mutations and, therefore, the underlying genes affected by the mutations. However, combining these two mutants revealed some redundancy in controlling meristem maturation, which caused undesirable branching. Breeders overcame this negative epistasis by selecting suppressors of branching, but in so doing limited the potential to improve flower production through weak branching.

As described herein, the identification of the mutations in MADS-box transcription factor genes and the dissection of the interaction between the MADS-box genes by Applicants revealed a dosage relationship among natural and gene-edited mutations in multiple regulators of meristem maturation. Combining two or more of the mutations in the MADS-box genes in homozygous and heterozygous combinations allowed for the creation of a quantitative range of inflorescence types, and the development of weakly branched hybrids with desirable traits, such as higher flower and fruit production. In particular, data described herein in tomato plants demonstrates the utility of mutant MADS-box genes, such as mutant SEP4 homologs, and the interaction between such mutant genes to alter inflorescence phenotypes. In particular, mutants of the MADS-box gene Solyc12g038510, mutants of the MADS-box gene Solyc03g114840, and mutants of the MADS-box gene Solyc04g005320, each of which are homologs of Arabidopsis SEPALLATA4 (SEP4), were shown to be capable of altering inflorescence phenotypes in tomato plants. Specifically, it was found that mixing and matching these mutations in various homozygous and heterozygous combinations resulted in a quantitative range of inflorescence phenotypes and the development of weakly branched hybrids with higher flower and fruit production.

Accordingly, in some aspects, the present disclosure relates to plants (e.g., Solanaceae plants) comprising one or more mutant MADS-box genes such as mutant SEPALLATA4 (SEP4) homologs, which may provide a range of inflorescence phenotypes and may result in improved inflorescence architecture and yield.

In some aspects, provided herein are genetically-altered Solanaceae plants, such as genetically-altered Solanaceae (e.g., Solanum lycopersicum) plants comprising one or more of a mutant Solyc04g005320 gene (or a homolog thereof), a mutant Solyc12g038510 gene (or a homolog thereof), and a mutant Solyc03g114840 gene (or a homolog thereof), which exhibit characteristics different from a reference plant such as a corresponding plant that has not been genetically altered (also referred to herein as “wild-type”) or a corresponding plant comprising a null mutation of one or more of the Solyc04g005320 gene, the Solyc12g038510 gene, and the Solyc03g114840 gene. The characteristics include, but are not limited to, one or more of the following: modified inflorescence architecture, modified flower number, higher yield, higher quality products (e.g., fruits), and modified fruit productivity (e.g., modified such as higher fruit number).

In some embodiments, genetically-altered Solanaceae plants, e.g., tomato plants (such as Solanum lycopersicum), comprise one or more of a mutant Solyc04g005320 gene (heterozygous or homozygous), a mutant Solyc12g038510 gene (heterozygous or homozygous), and a mutant Solyc03g114840 gene (heterozygous or homozygous). In some embodiments, the plants comprise a variety of combinations of the different mutant alleles, such as, for example, mutant Solyc04g005320 with mutant Solyc12g038510; mutant Solyc04g005320 with mutant Solyc03g114840; or mutant Solyc04g005320 with mutant Solyc12g038510 and mutant Solyc03g114840. The genetically-altered plants may be heterozygotes or homozygotes and, in some embodiments, may be double heterozygotes, double homozygotes, triple heterozygotes, or triple homozygotes. In some embodiments, such a plant comprises a mutant Solyc04g005320 gene as described herein. In some embodiments, such a plant comprises a mutant Solyc04g005320 gene as described herein and a mutant Solyc12g038510 gene as described herein. In some embodiments, such a plant comprises a mutant Solyc04g005320 gene as described herein and a mutant Solyc03g114840 gene as described herein. In some embodiments, such a plant comprises a mutant Solyc04g005320 as described herein with a mutant Solyc12g038510 as described herein and a mutant Solyc03g114840 as described herein.

Mutant Solyc04g005320 Gene

Aspects of the disclosure relate to mutants of the Solyc04g005320 gene (or a homolog thereof) as well as plants, plant cells, seeds, and nucleic acids comprising such mutant genes. The Solyc04g005320 gene is also referred to herein as Long Inflorescence or LIN. The Solyc04g005320 gene is a homolog of SEP4 in Arabidopsis.

In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc04g005320 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have long inflorescences, e.g., producing an average of at least 15 flowers (e.g., 9 to 30 flowers) on each inflorescence per plant. In some embodiments, the number of flowers per inflorescence may vary by variety (e.g. for plum varieties 9-15 flowers and for cherry varieties 20-40 flowers). In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc04g005320 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have longer inflorescences than a plant comprising a wild-type Solyc04g005320 gene (or a wild-type homolog thereof). In some embodiments, the mutant Solyc04g005320 gene (or homolog thereof) is a hypomorphic allele that, when crossed to a null allele of the Solyc04g005320 gene (or homolog thereof), does not restore a wild-type Solyc04g005320 gene (or a wild-type homolog thereof) phenotype (such as producing an average of 8 flowers (e.g., 6 to 10 flowers) on each inflorescence per plant). In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc04g005320 gene (or a homolog thereof), such as a hypermorphic allele, have short inflorescences, e.g., producing an average of less than 5 flowers (e.g., 2 to 6 flowers) on each inflorescence per plant. In some embodiments, plants comprising a mutant Solyc04g005320 gene, such as a hypermorphic allele, have shorter inflorescence than a plant comprising a wild-type Solyc04g005320 gene.

In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc04g005320 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have more branches per inflorescence, e.g., producing 2 or more branches per inflorescence. In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc04g005320 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have more branches than a plant comprising a wild-type Solyc04g005320 gene (or a wild-type homolog thereof). In some embodiments, the mutant Solyc04g005320 gene (or homolog thereof) is a hypomorphic allele that, when crossed to a null allele of the Solyc04g005320 gene, does not restore a wild-type Solyc04g005320 gene (or a wild-type homolog thereof) phenotype (such as producing an average of 1 branch per inflorescence).

In some embodiments, the mutant Solyc04g005320 gene (or homolog thereof) contains a mutation in a regulatory region, a coding region or both (e.g., a missense, nonsense, insertion, deletion, duplication, inversion, indel, or translocation mutation in such a region). In some embodiments, the regulatory region is a promoter. In some embodiments, the mutation in the coding region is in an exon. In some embodiments, the mutation is a translocation in the first intron (e.g., lin^trans, which contains a translocation in the first intron that eliminates transcription). In some embodiments, the mutation is a null mutation in which the coding sequence has been deleted (e.g., lin^CRwhich is a null allele produced by CRISPR/Cas9).

In some embodiments, the mutant Solyc04g005320 gene (or homolog thereof) is a hypomorphic allele or a null allele. In some embodiments, a hypomorphic allele is an allele that results in an mRNA or protein expression level of the gene of interest that is at least 30% lower (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%) than results from an allele of the gene of interest that does not contain the mutation (e.g., a wild-type allele). As used herein, a “null allele” refers to an allele of a gene of interest in which transcription into RNA does not occur, translation into a functional protein does not occur or neither occurs due to a mutation which may be located within the coding sequence, in a regulatory region of the gene, or in both (e.g., a missense, nonsense, insertion, deletion, duplication, inversion, indel, or translocation). In some embodiments, the null allele is a knock-out allele. As used herein, a “knock out allele” refers to an allele of a gene in which transcription into RNA does not occur, translation into a functional protein does not occur or neither occurs as a result of a deletion of some portion or all of the coding sequence of the gene, e.g., using homologous recombination. One non-limiting approach to creating null mutations is to use CRISPR-Cas9 mutagenesis to target exons that encode functional protein domains or to target a large portion (e.g., at least 80%) of the coding sequence (see, e.g., Shi et al. Nature Biotechnology. (2015) 33(6): 661-667 and Online Methods).

In some embodiments, the mutant Solyc04g005320 gene (or homolog thereof) is a hypermorphic allele. In some embodiments, a hypermorphic allele is an allele that results in an mRNA or protein expression level of the gene of interest that is at least 30% greater (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200% or more) than results from an allele of the gene of interest that does not contain the mutation (e.g., a wild-type allele). mRNA and protein levels can be measured using any method known in the art or described herein, e.g., using qRT-PCR for mRNA levels or an immunoassay for protein levels.

In some embodiments, a Solanaceae plant (e.g., Solanum lycopersicum) comprising the mutant Solyc04g005320 gene, or homolog thereof, (e.g., a hypomorphic, knock-out or null allele described herein) is heterozygous for the mutant gene. In some embodiments, a Solanaceae plant (e.g., Solanum lycopersicum) comprising the mutant Solyc04g005320 gene, or homolog thereof, (e.g., a hypomorphic, knock-out or null allele described herein) is homozygous for the mutant gene.

In some embodiments, the Solyc04g005320 gene homolog (a) has a sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 1 or 2 and (b) is not a Solanum lycopersicum gene.

In some embodiments, the mutant lin^transgene comprises, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 3; a portion of SEQ ID NO: 3 that exhibits substantially the same activity (e.g., encoding the same polypeptide or substantially the same polypeptide that has the same activity) as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 3; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 3; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 3.

In some embodiments, the mutant lin^CRgene comprises, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 4 or 5; a portion of SEQ ID NO: 4 or 5 that exhibits substantially the same activity (e.g., encoding the same polypeptide or substantially the same polypeptide that has the same activity) as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 4 or 5; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 4 or 5; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 4 or 5.

Mutant Solyc12g038510 Gene

Other aspects of the disclosure relate to mutants of the Solyc12g038510 gene (or a homolog thereof) as well as plants, plant cells, seeds, and nucleic acids comprising such mutant genes. The Solyc12g038510 gene is also referred to herein as Jointless-2 or J2. The Solyc12g038510 gene is a homolog of SEP4 in Arabidopsis.

In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc12g038510 gene (or homolog thereof), such as a hypomorphic allele or null allele, have more branches, e.g., producing 2 or more branches per inflorescence. In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc12g038510 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have more branches than a plant comprising a wild-type Solyc12g038510 gene. In some embodiments, the mutant Solyc12g038510 gene (or homolog thereof) is a hypomorphic allele that, when crossed to a null allele of the Solyc12g038510 gene (or homolog thereof), does not restore a wild-type Solyc12g038510 gene (or a wild-type homolog thereof) phenotype (such as producing an average of 1 branch per inflorescence). In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc12g038510 gene (or a homolog thereof), such as a hypomorphic allele or null allele, lack the abscission zone on the stems (pedicels) of flowers known as the joint (this absence of the abscission zone is also referred to herein as “jointless pedicels”) or produce a visible abscission zone (i.e. joint) but abscission does not occur or requires more force (e.g., hand harvesting) to separate the fruit from the pedicel, providing better fruit retention properties. In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc12g038510 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have more jointless pedicels than a plant comprising a wild-type Solyc12g038510 gene (or a wild-type homolog thereof). In some embodiments, the mutant Solyc12g038510 gene (or homolog thereof) is a hypomorphic allele that, when crossed to a null allele of the Solyc12g038510 gene (or homolog thereof), does not restore a wild-type Solyc12g038510 gene (or a wild-type homolog thereof) phenotype (such as having a normal abscission zone on the pedicels).

In some embodiments, the mutant Solyc12g038510 gene (or homolog thereof) contains a mutation in a regulatory region, a coding region or both (e.g., a missense, nonsense, insertion, deletion, duplication, inversion, indel, or translocation mutation in such a region). In some embodiments, the regulatory region is a promoter. In some embodiments, the mutation in the coding region is in an exon. In some embodiments, the mutation is in the first intron (e.g., j2^TEwhich contains a Copia/Rider-type transposable element (TE) in the first intron). In some embodiments, the mutation is a nonsense mutation that results in an early stop codon (e.g., j2^stophas an early nonsense mutation). In some embodiments, the mutation is a null mutation in which the coding sequence has been deleted (e.g., j2^CRwhich is a null allele produced by CRISPR/Cas9).

In some embodiments, the mutant Solyc12g038510 gene (or homolog thereof) is a hypomorphic allele or a null allele. In some embodiments, a hypomorphic allele is an allele that results in an mRNA or protein expression level of the gene of interest that is at least 30% lower (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%) than results from an allele of the gene of interest that does not contain the mutation (e.g., a wild-type allele).

In some embodiments, a Solanaceae plant (e.g., Solanum lycopersicum) comprising the mutant Solyc12g038510 gene, or homolog thereof, (e.g., a hypomorphic, knock-out or null allele described herein) is heterozygous for the mutant gene. In some embodiments, a Solanaceae plant (e.g., Solanum lycopersicum) comprising the mutant Solyc12g038510 gene, or homolog thereof, (e.g., a hypomorphic, knock-out or null allele described herein) is homozygous for the mutant gene.

In some embodiments, the Solyc12g038510 gene homolog (a) has a sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 6 or 7 and (b) is not a Solanum lycopersicum gene.

In some embodiments, the mutant j2^TEgene comprises, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 8; a portion of SEQ ID NO: 8 that exhibits substantially the same activity (e.g., encoding the same polypeptide or substantially the same polypeptide that has the same activity) as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 8; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 8; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 8.

In some embodiments, the mutant j2^stopgene comprises, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 9; a portion of SEQ ID NO: 9 that exhibits substantially the same activity (e.g., encoding the same polypeptide or substantially the same polypeptide that has the same activity) as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 9; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 9; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 9.

In some embodiments, the mutant j2^CRgene comprises, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 10 or 11; a portion of SEQ ID NO: 10 or 11 that exhibits substantially the same activity (e.g., encoding the same polypeptide or substantially the same polypeptide that has the same activity) as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 10 or 11; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 10 or 11; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 10 or 11.

Mutant Solyc03g114840 Gene

Other aspects of the disclosure relate to mutants of the Solyc03g114840 gene (or a homolog thereof) as well as plants, plant cells, seeds, and nucleic acids comprising such mutant genes. The Solyc03g114840 gene is also referred to herein as Enhancer-of-Jointless-2 or EJ2. The Solyc03g114840 gene is a homolog of SEP4 in Arabidopsis.

In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc03g114840 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have more branches, e.g., producing 2 or more branches per inflorescence. In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc03g114840 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have more branches than a plant comprising a wild-type Solyc03g114840 gene (or a wild-type homolog thereof). In some embodiments, the mutant Solyc03g114840 gene (or homolog thereof) is a hypomorphic allele that, when crossed to a null allele of the Solyc03g114840 gene (or homolog thereof), does not restore a wild-type Solyc03g114840 gene (or a wild-type homolog thereof) phenotype (such as producing an average of 1 branch per inflorescence). In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc03g114840 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have long sepals resulting in larger calyxes, e.g., that are an average sepal to petal ratio (sepal length/petal length) of at least 1.2. In some embodiments, Solanaceae plants (e.g., Solanum lycopersicum) comprising a mutant Solyc03g114840 gene (or a homolog thereof), such as a hypomorphic allele or null allele, have longer sepals than a plant comprising a wild-type Solyc03g114840 gene (or a wild-type homolog thereof). In some embodiments, the mutant Solyc03g114840 gene (or homolog thereof) is a hypomorphic allele that, when crossed to a null allele of the Solyc03g114840 gene (or homolog thereof), does not restore a wild-type Solyc03g114840 gene (or wild-type homolog thereof) phenotype (such as having an average sepal to petal ratio (sepal length/petal length) of not more than 0.8).

In some embodiments, the mutant Solyc03g114840 gene (or homolog thereof) contains a mutation in a regulatory region, a coding region or both (e.g., a missense, nonsense, insertion, deletion, duplication, inversion, indel, or translocation mutation in such a region). In some embodiments, the regulatory region is a promoter. In some embodiments, the mutation is a null mutation in which the coding sequence has been deleted (e.g., ej2^CRwhich is a null allele produced by CRISPR/Cas9). In some embodiments, the mutation is an insertion mutation in the 5^thintron (e.g., ej2^Wwhich is a hypomorphic allele with a 564 bp insertion in the 5th intron).

In some embodiments, the mutant Solyc03g114840 gene (or homolog thereof) is a hypomorphic allele or a null allele. In some embodiments, a hypomorphic allele is an allele that results in an mRNA or protein expression level of the gene of interest that is at least 30% lower (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%) than results from an allele of the gene of interest that does not contain the mutation (e.g., a wild-type allele).

In some embodiments, a Solanaceae plant (e.g., Solanum lycopersicum) comprising the mutant Solyc03g114840 gene, or homolog thereof, (e.g., a hypomorphic, knock-out or null allele described herein) is heterozygous for the mutant gene. In some embodiments, a Solanaceae plant (e.g., Solanum lycopersicum) comprising the mutant Solyc03g114840 gene, or homolog thereof, (e.g., a hypomorphic, knock-out or null allele described herein) is homozygous for the mutant gene.

In some embodiments, the Solyc03g114840 gene homolog (a) has a sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 12 or 13 and (b) is not a Solanum lycopersicum gene.

In some embodiments, the mutant ej2^wgene comprises, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 14; a portion of SEQ ID NO: 14 that exhibits substantially the same activity (e.g., encoding the same polypeptide or substantially the same polypeptide that has the same activity) as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 14; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 14; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 14.

In some embodiments, the mutant ej2^CRgene comprises, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 15 or 16; a portion of SEQ ID NO: 15 or 16 that exhibits substantially the same activity (e.g., encoding the same polypeptide or substantially the same polypeptide that has the same activity) as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 15 or 16; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 15 or 16; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 15 or 16.

Solanaceae Plants Comprising Mutant Genes

Higher yield, higher quality products (e.g., fruits) and products (e.g., fruits) with different compositions (e.g., brix, also known as enhanced soluble solids or sugar concentration in the fruits), can be manipulated in a wide variety of types of Solanaceae plants that comprise a mutant gene, such as a mutant Solyc04g005320 gene (or homolog thereof), a mutant Solyc12g038510 gene (or homolog thereof), or a mutant Solyc03g114840 gene (or homolog thereof); or two mutant genes, such as both a mutant Solyc04g005320 gene (or homolog thereof) and a mutant Solyc12g038510 gene (or homolog thereof), both a mutant Solyc04g005320 gene (or homolog thereof) and a mutant Solyc03g114840 gene (or homolog thereof), or both a mutant Solyc12g038510 gene (or homolog thereof) and a mutant Solyc03g114840 gene (or homolog thereof); or three mutant genes, such as a mutant Solyc04g005320 gene (or homolog thereof), a mutant Solyc12g038510 gene (or homolog thereof), and a mutant Solyc03g114840 gene (or homolog thereof). In some embodiments, the Solanaceae plant is a genetically-altered Solanaceae plant. In some embodiments, a “genetically-altered” plant includes a plant that has had introduced into it (or introduced into a plant that is used to produce the plant, such as introduced into a parental line) at least one mutation by chemical or physical means (e.g., using CRISPR/Cas9, chemical mutagenesis, radiation, Agrobacterium-mediated recombination, viral-vector mediated recombination, or transposon mutagenesis).

The mutant Solyc04g005320 gene (or homolog thereof) can be any of the mutant Solyc04g005320 genes (or homologs thereof) described herein. The mutant Solyc12g038510 gene (or homolog thereof) can be any of the mutant Solyc12g038510 genes (or homologs thereof) described herein. The mutant Solyc03g114840 gene (or homolog thereof) can be any of the mutant Solyc03g114840 genes (or homologs thereof) described herein.

The genetically-altered Solanaceae plant can be, for example, inbred, isogenic or hybrid, as long as the plant comprises a mutant gene, such as a mutant Solyc04g005320 gene (or homolog thereof), a mutant Solyc12g038510 gene (or homolog thereof), or a mutant Solyc03g114840 gene (or homolog thereof); or two mutant genes, such as both a mutant Solyc04g005320 gene (or homolog thereof) and a mutant Solyc12g038510 gene (or homolog thereof), both a mutant Solyc04g005320 gene (or homolog thereof) and a mutant Solyc03g114840 gene (or homolog thereof), or both a mutant Solyc12g038510 gene (or homolog thereof) and a mutant Solyc03g114840 gene (or homolog thereof); or three mutant genes, such as a mutant Solyc04g005320 gene (or homolog thereof), a mutant Solyc12g038510 gene (or homolog thereof), and a mutant Solyc03g114840 gene (or homolog thereof).

Plants in the Solanaceae family include, e.g., tomato, potato, eggplant, petunia, tobacco, and pepper. In some embodiments, the Solanaceae plant is a tomato plant. In some embodiments, the Solanaceae plant, e.g. tomato plant, is not a variety.

In some embodiments, the genetically-altered Solanaceae plant comprises one wild-type (WT) copy of the SOLYC04G005320 gene (or homolog thereof) and one mutant copy of the Solyc04g005320 gene (or homolog thereof) as described herein (is heterozygous for the mutant Solyc04g005320 gene or homolog thereof). In some embodiments, the Solanaceae plant comprises two copies of a mutant Solyc04g005320 gene (or homolog thereof) as described herein (is homozygous for the mutant Solyc04g005320 gene or homolog thereof). In some embodiments, the Solanaceae plant comprises a first mutant Solyc04g005320 gene (or homolog thereof) as described herein and a second mutant Solyc04g005320 gene (or homolog thereof) as described herein, wherein the first mutant Solyc04g005320 gene (or homolog thereof) and the second mutant Solyc04g005320 gene (or homolog thereof) are different. In some embodiments, the Solanaceae plant comprises one copy of a mutant Solyc04g005320 gene (or homolog thereof) as described herein and one copy of a mutant Solyc12g038510 gene (or homolog thereof) as described herein (is heterozygous for the mutant Solyc04g005320 gene, or homolog thereof, and heterozygous for the mutant Solyc12g038510 gene, or homolog thereof). In some embodiments, the Solanaceae plant comprises one copy of a mutant Solyc04g005320 gene (or homolog thereof) as described herein and two copies of a mutant Solyc12g038510 gene (or homolog thereof) as described herein (is heterozygous for the mutant Solyc04g005320 gene, or homolog thereof and homozygous for the mutant Solyc12g038510 gene, or homolog thereof). In some embodiments, the Solanaceae plant comprises two copies of a mutant Solyc04g005320 gene (or homolog thereof) as described herein and two copies of a mutant Solyc12g038510 gene (or homolog thereof) as described herein (is homozygous for the mutant Solyc04g005320 gene, or homolog thereof, and homozygous for the mutant Solyc12g038510 gene, or homolog thereof).

In some embodiments, the genetically-altered Solanaceae plant comprises one WT copy of a SOLYC03G114840 gene (or homolog thereof) and one mutant copy of a Solyc03g114840 gene (or homolog thereof) as described herein (is heterozygous for the mutant Solyc03g114840 gene, or homolog thereof). In some embodiments, the Solanaceae plant comprises two copies of a mutant Solyc03g114840 gene (or homolog thereof) as described herein (is homozygous for the mutant Solyc03g114840 gene or homolog thereof). In some embodiments, the Solanaceae plant comprises one copy of a mutant Solyc03g114840 gene (or homolog thereof) as described herein and one copy of a mutant Solyc04g005320 gene (or homolog thereof) as described herein (is heterozygous for the mutant Solyc03g114840 gene, or homolog thereof, and heterozygous for the mutant Solyc04g005320 gene, or homolog thereof). In some embodiments, the Solanaceae plant comprises one copy of a mutant Solyc03g114840 gene (or homolog thereof) as described herein and two copies of a mutant Solyc04g005320 gene (or homolog thereof) as described herein (is heterozygous for the mutant Solyc03g114840 gene, or homolog thereof, and homozygous for the mutant Solyc04g005320 gene, or homolog thereof). In some embodiments, the Solanaceae plant comprises two copies of a mutant Solyc03g114840 gene (or homolog thereof) as described herein and two copies of a mutant Solyc04g005320 gene (or homolog thereof) as described herein (is homozygous for the mutant Solyc03g114840 gene, or homolog thereof, and homozygous for the mutant Solyc04g005320 gene, or homolog thereof).

In some embodiments, the genetically-altered Solanaceae plant comprises one WT copy of a SOLYC03G114840 gene and one mutant copy of a Solyc03g114840 gene as described herein (is heterozygous for the mutant Solyc03g114840 gene) and comprises one WT copy of the SOLYC12G038510 gene and one mutant copy of the Solyc12g038510 gene as described herein (is heterozygous for the mutant Solyc12g038510 gene). In some embodiments, the Solanaceae plant comprises two copies of a mutant Solyc03g114840 gene as described herein (is homozygous for the mutant Solyc03g114840 gene) and comprises two copies of a mutant Solyc12g038510 gene as described herein (is homozygous for the mutant Solyc12g038510 gene). In some embodiments, the Solanaceae plant comprising a mutant Solyc03g114840 gene (one or two copies) as described herein and a mutant Solyc12g038510 gene (one or two copies) further comprises one copy of a mutant Solyc04g005320 gene as described herein (is heterozygous or homozygous for the mutant Solyc03g114840 gene and the mutant Solyc12g038510 gene and heterozygous for the mutant Solyc04g005320 gene). In some embodiments, the Solanaceae plant further comprises two copies of a mutant Solyc04g005320 gene as described herein (is homozygous for the mutant Solyc04g005320 gene).

Other, non-limiting example genotype combinations which a Solanaceae (e.g., Solanum lycopersicum) plant may comprise are displayed in Table 1. The combinations in Table 1 may also be with homologs of the genes.

TABLE 1 Example genotype combinations. Combination Solyc12g038510 Solyc03g114840 Solyc04g005320 No. (J2) Genotype (EJ2) Genotype (LIN) Genotype 1 j2^TE/j2^TE ej2^W/ej2^W lin^trans/lin^trans 2 j2^TE/j2^TE ej2^W/+ lin^trans/lin^trans 3 j2^TE/j2^TE +/+ lin^trans/lin^trans 4 j2^TE/j2^TE ej2^W/ej2^W lin^trans/+ 5 j2^TE/j2^TE ej2^W/+ lin^trans/+ 6 j2^TE/j2^TE +/+ lin^trans/+ 7 j2^TE/j2^TE ej2^W/ej2^W +/+ 8 j2^TE/j2^TE ej2^W/+ +/+ 9 j2^TE/j2^TE +/+ +/+ 10 j2^stop/j2^stop ej2^W/ej2^W lin^trans/lin^trans 11 j2^stop/j2^stop ej2^W/+ lin^trans/lin^trans 12 j2^stop/j2^stop +/+ lin^trans/lin^trans 13 j2^stop/j2^stop ej2^W/ej2^W lin^trans/+ 14 j2^stop/j2^stop ej2^W/+ lin^trans/+ 15 j2^stop/j2^stop +/+ lin^trans/+ 16 j2^stop/j2^stop ej2^W/ej2^W +/+ 17 j2^stop/j2^stop ej2^W/+ +/+ 18 j2^stop/j2^stop +/+ +/+ 19 j2^CR/j2^CR ej2^W/ej2^W lin^trans/lin^trans 20 j2^CR/j2^CR ej2^W/+ lin^trans/lin^trans 21 j2^CR/j2^CR +/+ lin^trans/lin^trans 22 j2^CR/j2^CR ej2^W/ej2^W lin^trans/+ 23 j2^CR/j2^CR ej2^W/+ lin^trans/+ 24 j2^CR/j2^CR +/+ lin^trans/+ 25 j2^CR/j2^CR ej2^W/ej2^W +/+ 26 j2^CR/j2^CR ej2^W/+ +/+ 27 j2^CR/j2^CR +/+ +/+ 28 j2^TE/+ ej2^W/ej2^W lin^trans/lin^trans 29 j2^TE/+ ej2^W/+ lin^trans/lin^trans 30 j2^TE/+ +/+ lin^trans/lin^trans 31 j2^TE/+ ej2^W/ej2^W lin^trans/+ 32 j2^TE/+ ej2^W/+ lin^trans/+ 33 j2^TE/+ +/+ lin^trans/+ 34 j2^TE/+ ej2^W/ej2^W +/+ 35 j2^TE/+ ej2^W/+ +/+ 36 j2^TE/+ +/+ +/+ 37 j2^stop/+ ej2^W/ej2^W lin^trans/lin^trans 38 j2^stop/+ ej2^W/+ lin^trans/lin^trans 39 j2^stop/+ +/+ lin^trans/lin^trans 40 j2^stop/+ ej2^W/ej2^W lin^trans/+ 41 j2^stop/+ ej2^W/+ lin^trans/+ 42 j2^stop/+ +/+ lin^trans/+ 43 j2^stop/+ ej2^W/ej2^W +/+ 44 j2^stop/+ ej2^W/+ +/+ 45 j2^stop/+ +/+ +/+ 46 j2^CR/+ ej2^W/ej2^W lin^trans/lin^trans 47 j2^CR/+ ej2^W/+ lin^trans/lin^trans 48 j2^CR/+ +/+ lin^trans/lin^trans 49 j2^CR/+ ej2^W/ej2^W lin^trans/+ 50 j2^CR/+ ej2^W/+ lin^trans/+ 51 j2^CR/+ +/+ lin^trans/+ 52 j2^CR/+ ej2^W/ej2^W +/+ 53 j2^CR/+ ej2^W/+ +/+ 54 j2^CR/+ +/+ +/+ 55 +/+ ej2^W/ej2^W lin^trans/lin^trans 56 +/+ ej2^W/+ lin^trans/lin^trans 57 +/+ +/+ lin^trans/lin^trans 58 +/+ ej2^W/ej2^W lin^trans/+ 59 +/+ ej2^W/+ lin^trans/+ 60 +/+ +/+ lin^trans/+ 61 +/+ ej2^W/ej2^W +/+ 62 +/+ ej2^W/+ +/+ 63 +/+ +/+ +/+ 64 j2^TE/j2^TE ej2^CR/ej2^CR lin^trans/lin^trans 65 j2^TE/j2^TE ej2^CR/+ lin^trans/lin^trans 66 j2^TE/j2^TE +/+ lin^trans/lin^trans 67 j2^TE/j2^TE ej2^CR/ej2^CR lin^trans/+ 68 j2^TE/j2^TE ej2^CR/+ lin^trans/+ 69 j2^TE/j2^TE +/+ lin^trans/+ 70 j2^TE/j2^TE ej2^CR/ej2^CR +/+ 71 j2^TE/j2^TE ej2^CR/+ +/+ 72 j2^TE/j2^TE +/+ +/+ 73 j2^stop/j2^stop ej2^CR/ej2^CR lin^trans/lin^trans 74 j2^stop/j2^stop ej2^CR/+ lin^trans/lin^trans 75 j2^stop/j2^stop +/+ lin^trans/lin^trans 76 j2^stop/j2^stop ej2^CR/ej2^CR lin^trans/+ 77 j2^stop/j2^stop ej2^CR/+ lin^trans/+ 78 j2^stop/j2^stop +/+ lin^trans/+ 79 j2^stop/j2^stop ej2^CR/ej2^CR +/+ 80 j2^stop/j2^stop ej2^CR/+ +/+ 81 j2^stop/j2^stop +/+ +/+ 82 j2^CR/j2^CR ej2^CR/ej2^CR lin^trans/lin^trans 83 j2^CR/j2^CR ej2^CR/+ lin^trans/lin^trans 84 j2^CR/j2^CR +/+ lin^trans/lin^trans 85 j2^CR/j2^CR ej2^CR/ej2^CR lin^trans/+ 86 j2^CR/j2^CR ej2^CR/+ lin^trans/+ 87 j2^CR/j2^CR +/+ lin^trans/+ 88 j2^CR/j2^CR ej2^CR/ej2^CR +/+ 89 j2^CR/j2^CR ej2^CR/+ +/+ 90 j2^CR/j2^CR +/+ +/+ 91 j2^TE/+ ej2^CR/ej2^CR lin^trans/lin^trans 92 j2^TE/+ ej2^CR/+ lin^trans/lin^trans 93 j2^TE/+ +/+ lin^trans/lin^trans 94 j2^TE/+ ej2^CR/ej2^CR lin^trans/+ 95 j2^TE/+ ej2^CR/+ lin^trans/+ 96 j2^TE/+ +/+ lin^trans/+ 97 j2^TE/+ ej2^CR/ej2^CR +/+ 98 j2^TE/+ ej2^CR/+ +/+ 99 j2^TE/+ +/+ +/+ 100 j2^stop/+ ej2^CR/ej2^CR lin^trans/lin^trans 101 j2^stop/+ ej2^CR/+ lin^trans/lin^trans 102 j2^stop/+ +/+ lin^trans/lin^trans 103 j2^stop/+ ej2^CR/ej2^CR lin^trans/+ 104 j2^stop/+ ej2^CR/+ lin^trans/+ 105 j2^stop/+ +/+ lin^trans/+ 106 j2^stop/+ ej2^CR/ej2^CR +/+ 107 j2^stop/+ ej2^CR/+ +/+ 108 j2^stop/+ +/+ +/+ 109 j2^CR/+ ej2^CR/ej2^CR lin^trans/lin^trans 110 j2^CR/+ ej2^CR/+ lin^trans/lin^trans 111 j2^CR/+ +/+ lin^trans/lin^trans 112 j2^CR/+ ej2^CR/ej2^CR lin^trans/+ 113 j2^CR/+ ej2^CR/+ lin^trans/+ 114 j2^CR/+ +/+ lin^trans/+ 115 j2^CR/+ ej2^CR/ej2^CR +/+ 116 j2^CR/+ ej2^CR/+ +/+ 117 j2^CR/+ +/+ +/+ 118 +/+ ej2^CR/ej2^CR lin^trans/lin^trans 119 +/+ ej2^CR/+ lin^trans/lin^trans 120 +/+ +/+ lin^trans/lin^trans 121 +/+ ej2^CR/ej2^CR lin^trans/+ 122 +/+ ej2^CR/+ lin^trans/+ 123 +/+ +/+ lin^trans/+ 124 +/+ ej2^CR/ej2^CR +/+ 125 +/+ ej2^CR/+ +/+ 126 j2^TE/j2^TE ej2^W/ej2^W lin^CR/lin^CR 127 j2^TE/j2^TE ej2^W/+ lin^CR/lin^CR 128 j2^TE/j2^TE +/+ lin^CR/lin^CR 129 j2^TE/j2^TE ej2^W/ej2^W lin^CR/+ 130 j2^TE/j2^TE ej2^W/+ lin^CR/+ 131 j2^TE/j2^TE +/+ lin^CR/+ 132 j2^TE/j2^TE ej2^W/ej2^W +/+ 133 j2^TE/j2^TE ej2^W/+ +/+ 134 j2^TE/j2^TE +/+ +/+ 135 j2^stop/j2^stop ej2^W/ej2^W lin^CR/lin^CR 136 j2^stop/j2^stop ej2^W/+ lin^CR/lin^CR 137 j2^stop/j2^stop +/+ lin^CR/lin^CR 138 j2^stop/j2^stop ej2^W/ej2^W lin^CR/+ 139 j2^stop/j2^stop ej2^W/+ lin^CR/+ 140 j2^stop/j2^stop +/+ lin^CR/+ 141 j2^stop/j2^stop ej2^W/ej2^W +/+ 142 j2^stop/j2^stop ej2^W/+ +/+ 143 j2^stop/j2^stop +/+ +/+ 144 j2^CR/j2^CR ej2^W/ej2^W lin^CR/lin^CR 145 j2^CR/j2^CR ej2^W/+ lin^CR/lin^CR 146 j2^CR/j2^CR +/+ lin^CR/lin^CR 147 j2^CR/j2^CR ej2^W/ej2^W lin^CR/+ 148 j2^CR/j2^CR ej2^W/+ lin^CR/+ 149 j2^CR/j2^CR +/+ lin^CR/+ 150 j2^CR/j2^CR ej2^W/ej2^W +/+ 151 j2^CR/j2^CR ej2^W/+ +/+ 152 j2^CR/j2^CR +/+ +/+ 153 j2^TE/+ ej2^W/ej2^W lin^CR/lin^CR 154 j2^TE/+ ej2^W/+ lin^CR/lin^CR 155 j2^TE/+ +/+ lin^CR/lin^CR 156 j2^TE/+ ej2^W/ej2^W lin^CR/+ 157 j2^TE/+ ej2^W/+ lin^CR/+ 158 j2^TE/+ +/+ lin^CR/+ 159 j2^TE/+ ej2^W/ej2^W +/+ 160 j2^TE/+ ej2^W/+ +/+ 161 j2^TE/+ +/+ +/+ 162 j2^stop/+ ej2^W/ej2^W lin^CR/lin^CR 163 j2^stop/+ ej2^W/+ lin^CR/lin^CR 164 j2^stop/+ +/+ lin^CR/lin^CR 165 j2^stop/+ ej2^W/ej2^W lin^CR/+ 166 j2^stop/+ ej2^W/+ lin^CR/+ 167 j2^stop/+ +/+ lin^CR/+ 168 j2^stop/+ ej2^W/ej2^W +/+ 169 j2^stop/+ ej2^W/+ +/+ 170 j2^stop/+ +/+ +/+ 171 j2^CR/+ ej2^W/ej2^W lin^CR/lin^CR 172 j2^CR/+ ej2^W/+ lin^CR/lin^CR 173 j2^CR/+ +/+ lin^CR/lin^CR 174 j2^CR/+ ej2^W/ej2^W lin^CR/+ 175 j2^CR/+ ej2^W/+ lin^CR/+ 176 j2^CR/+ +/+ lin^CR/+ 177 j2^CR/+ ej2^W/ej2^W +/+ 178 j2^CR/+ ej2^W/+ +/+ 179 j2^CR/+ +/+ +/+ 180 +/+ ej2^W/ej2^W lin^CR/lin^CR 181 +/+ ej2^W/+ lin^CR/lin^CR 182 +/+ +/+ lin^CR/lin^CR 183 +/+ ej2^W/ej2^W lin^CR/+ 184 +/+ ej2^W/+ lin^CR/+ 185 +/+ +/+ lin^CR/+ 186 +/+ ej2^W/ej2^W +/+ 187 +/+ ej2^W/+ +/+ 188 +/+ +/+ +/+ 189 j2^TE/j2^TE ej2^CR/ej2^CR lin^CR/lin^CR 190 j2^TE/j2^TE ej2^CR/+ lin^CR/lin^CR 191 j2^TE/j2^TE +/+ lin^CR/lin^CR 192 j2^TE/j2^TE ej2^CR/ej2^CR lin^CR/+ 193 j2^TE/j2^TE ej2^CR/+ lin^CR/+ 194 j2^TE/j2^TE +/+ lin^CR/+ 195 j2^TE/j2^TE ej2^CR/ej2^CR +/+ 196 j2^TE/j2^TE ej2^CR/+ +/+ 197 j2^TE/j2^TE +/+ +/+ 198 j2^stop/j2^stop ej2^CR/ej2^CR lin^CR/lin^CR 199 j2^stop/j2^stop ej2^CR/+ lin^CR/lin^CR 200 j2^stop/j2^stop +/+ lin^CR/lin^CR 201 j2^stop/j2^stop ej2^CR/ej2^CR lin^CR/+ 202 j2^stop/j2^stop ej2^CR/+ lin^CR/+ 203 j2^stop/j2^stop +/+ lin^CR/+ 204 j2^stop/j2^stop ej2^CR/ej2^CR +/+ 205 j2^stop/j2^stop ej2^CR/+ +/+ 206 j2^stop/j2^stop +/+ +/+ 207 j2^CR/j2^CR ej2^CR/ej2^CR lin^CR/lin^CR 208 j2^CR/j2^CR ej2^CR/+ lin^CR/lin^CR 209 j2^CR/j2^CR +/+ lin^CR/lin^CR 210 j2^CR/j2^CR ej2^CR/ej2^CR lin^CR/+ 211 j2^CR/j2^CR ej2^CR/+ lin^CR/+ 212 j2^CR/j2^CR +/+ lin^CR/+ 213 j2^CR/j2^CR ej2^CR/ej2^CR +/+ 214 j2^CR/j2^CR ej2^CR/+ +/+ 215 j2^CR/j2^CR +/+ +/+ 216 j2^TE/+ ej2^CR/ej2^CR lin^CR/lin^CR 217 j2^TE/+ ej2^CR/+ lin^CR/lin^CR 218 j2^TE/+ +/+ lin^CR/lin^CR 219 j2^TE/+ ej2^CR/ej2^CR lin^CR/+ 220 j2^TE/+ ej2^CR/+ lin^CR/+ 221 j2^TE/+ +/+ lin^CR/+ 222 j2^TE/+ ej2^CR/ej2^CR +/+ 223 j2^TE/+ ej2^CR/+ +/+ 224 j2^TE/+ +/+ +/+ 225 j2^stop/+ ej2^CR/ej2^CR lin^CR/lin^CR 226 j2^stop/+ ej2^CR/+ lin^CR/lin^CR 227 j2^stop/+ +/+ lin^CR/lin^CR 228 j2^stop/+ ej2^CR/ej2^CR lin^CR/+ 229 j2^stop/+ ej2^CR/+ lin^CR/+ 230 j2^stop/+ +/+ lin^CR/+ 231 j2^stop/+ ej2^CR/ej2^CR +/+ 232 j2^stop/+ ej2^CR/+ +/+ 233 j2^stop/+ +/+ +/+ 234 j2^CR/+ ej2^CR/ej2^CR lin^CR/lin^CR 235 j2^CR/+ ej2^CR/+ lin^CR/lin^CR 236 j2^CR/+ +/+ lin^CR/lin^CR 237 j2^CR/+ ej2^CR/ej2^CR lin^CR/+ 238 j2^CR/+ ej2^CR/+ lin^CR/+ 239 j2^CR/+ +/+ lin^CR/+ 240 j2^CR/+ ej2^CR/ej2^CR +/+ 241 j2^CR/+ ej2^CR/+ +/+ 242 j2^CR/+ +/+ +/+ 243 +/+ ej2^CR/ej2^CR lin^CR/lin^CR 244 +/+ ej2^CR/+ lin^CR/lin^CR 245 +/+ +/+ lin^CR/lin^CR 246 +/+ ej2^CR/ej2^CR lin^CR/+ 247 +/+ ej2^CR/+ lin^CR/+ 248 +/+ +/+ lin^CR/+ 249 +/+ ej2^CR/ej2^CR +/+ 250 +/+ ej2^CR/+ +/+

Solanaceae plant cells are also contemplated herein. A Solanaceae plant cell may comprise any genotype described herein, e.g., as shown without limitation in Table 1, in the context of the Solanaceae plant (e.g., a Solanaceae plant cell heterozygous for a mutant Solyc03g114840 gene, or a homolog thereof, and a mutant Solyc12g038510 gene, or a homolog thereof, or a Solanaceae plant cell homozygous for a mutant Solyc12g038510 gene, or a homolog thereof, and a mutant Solyc04g005320 gene, or a homolog thereof). In some embodiments, the Solanaceae plant cell is isolated. In some embodiments, the Solanaceae plant cell is a non-replicating plant cell.

In some embodiments, any of the Solanaceae plants described herein may an altered phenotype compared to a WT Solanaceae plant (e.g., a Solanaceae plant comprising two copies or one copy of the corresponding WT gene). In some embodiments, any of the Solanaceae plants described herein have a higher yield than a corresponding WT Solanaceae plant. In some embodiments, any of the Solanaceae plants described herein have one or more of the following characteristics: longer sepals, larger calyxes, a different fruit shape, fewer branches, jointless pedicels, long inflorescences, or larger fruit compared to a corresponding WT Solanaceae plant. In some embodiments, such characteristics are appealing to consumers (e.g., products of the Solanaceae plant look fresher) and are advantageous for growers (e.g., products of the Solanaceae plant stay attached to the plant for a longer period of time).

Food products are also contemplated herein. Such food products comprise a Solanaceae plant part, such as a fruit (e.g., a tomato fruit). Non-limiting examples of food products include sauces (e.g., tomato sauce or ketchup), purees, pastes, juices, canned fruits, and soups. Food products may be produced or producible by using methods known in the art.

Isolated polynucleotides are also described herein, including WT and mutant alleles of the Solyc04g005320 gene, or a homolog thereof, e.g., lin^transand lin^CR. Isolated polynucleotides including WT and mutant alleles of the Solyc12g038510 gene, or a homolog thereof, are also contemplated, e.g., j2^CRj2^TEand j2^stop. Isolated polynucleotides including WT and mutant alleles of the Solyc03g114840 gene, or a homolog thereof, are also contemplated, e.g., ej2^CRand ej2^W.

Isolated polynucleotides can comprise, for example, a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 3, 4, 5, 8, 9, 10, 11, 14, 15 or 16; a portion of SEQ ID NO: 3, 4, 5, 8, 9, 10, 11, 14, 15 or 16 that exhibits substantially the same activity as a nucleic acid (e.g., DNA) having the sequence of SEQ ID NO: 3, 4, 5, 8, 9, 10, 11, 14, 15 or 16; a nucleic acid (e.g., DNA) having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 3, 4, 5, 8, 9, 10, 11, 14, 15 or 16; an orthologue or homologue of the nucleic acid having the sequence of SEQ ID NO: 3, 4, 5, 8, 9, 10, 11, 14, 15 or 16. In some embodiments, the isolated polynucleotide is a cDNA. Such isolated polynucleotides can be used, for example, in methods of producing genetically-altered plants.

Other aspects of the disclosure relate to seeds for producing a Solanaceae plant as described herein, e.g., a mutant Solyc04g005320 gene (or a homolog thereof), a mutant Solyc12g038510 gene (or a homolog thereof), or a mutant Solyc03g114840 gene (or a homolog thereof).

Methods of Producing Plants

In other aspects, the disclosure provides methods for producing a genetically-altered Solanaceae plant. In some embodiments, the method comprises introducing a mutation into a Solyc04g005320 gene (or a homolog thereof), into a Solyc12g038510 gene (or a homolog thereof), or into a Solyc03g114840 gene (or a homolog thereof) in the Solanaceae plant, thereby producing a genetically-altered Solanaceae plant containing a mutant version of the gene. In some embodiments, the method comprises introducing a mutation into a Solyc04g005320 gene (or a homolog thereof), into a Solyc12g038510 gene (or a homolog thereof), or into a Solyc03g114840 gene (or a homolog thereof) in the Solanaceae plant part, maintaining the plant part under conditions and for sufficient time for production of a genetically-altered Solanaceae plant, thereby producing a genetically-altered Solanaceae plant (or a homolog thereof) containing a mutant version of the gene. In some embodiments, mutations are introduced into two or all three of a Solyc04g005320 gene (or a homolog thereof), a Solyc12g038510 gene (or a homolog thereof), and a Solyc03g114840 gene (or a homolog thereof).

In any of the methods described herein, the mutant gene can be introduced into a Solanaceae plant or a plant part or produced in a Solanaceae plant or plant part by any method described herein or known to those of skill in the art, such as Agrobacterium-mediated recombination, viral-vector mediated recombination, microinjection, gene gun bombardment/biolistic particle delivery, electroporation, mutagenesis (e.g., by ethyl methanesulfonate or fast neutron irradiation), TILLING (Targeting Induced Local Lesions in Genomes), conventional marker-assisted introgression, and nuclease mediated recombination (e.g., use of custom-made restriction enzymes for targeting mutagenesis by gene replacement, see, e.g., CRISPR-Cas9: Genome engineering using the CRISPR-Cas9 system. Ran F A, Hsu P D, Wright J, Agarwala V, Scott D A, Zhang F. Nat Protoc. 2013 November; 8(11):2281-308; TALEN endonucleases: Nucleic Acids Res. 2011 July; 39(12):e82. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Cermak T, Doyle E L, Christian M, Wang L, Zhang Y, Schmidt C, Baller J A, Somia N V, Bogdanove A J, Voytas D F and Plant Biotechnol J. 2012 May; 10(4):373-89. Genome modifications in plant cells by custom-made restriction enzymes. Tzfira T, Weinthal D, Marton I, Zeevi V, Zuker A, Vainstein A.). Genetically-altered Solanaceae plants produced by or producible by a method described herein are also claimed.

In some embodiments, the mutation produces a null allele, a hypomorphic allele, or a hypermorphic allele of a Solyc04g005320 gene (or a homolog thereof), a Solyc12g038510 gene (or a homolog thereof), or a Solyc03g114840 gene (or a homolog thereof) as described herein. In some embodiments, the mutation is a null mutation of a Solyc04g005320 gene (or a homolog thereof), a Solyc12g038510 gene (or a homolog thereof), or a Solyc03g114840 gene (or a homolog thereof) that is introduced using CRISPR/Cas9.

Alternatively, a method of producing a genetically-altered Solanaceae plant comprises a reducing (partially or completely) function of a wild-type Solyc04g005320 gene (or a homolog thereof), a wild-type Solyc12g038510 gene (or a homolog thereof), or a wild-type Solyc03g114840 gene (or a homolog thereof) in the plant or plant part. In some embodiments, reducing the function comprises performing any of the following methods of RNA-interference (e.g., administering to the Solanaceae plant a micro-RNA or a small interfering (si)-RNA or hairpin RNA) or translational blocking (e.g., administering to the Solanaceae plant a morpholino). Methods of RNA-interference and translational blocking are well-known in the art. Methods of producing micro-RNAs, si-RNAs, and morpholinos are well-known in the art and can involve use of the nucleotides sequences provided herein.

In some embodiments, the method comprises crossing a produced genetically-altered Solanaceae plant containing a mutant Solyc04g005320 gene (or a homolog thereof) to another genetically-altered Solanaceae plant comprising a mutant Solyc12g038510 gene (or a homolog thereof), a mutant Solyc03g114840 gene (or a homolog thereof), or both a mutant Solyc12g038510 gene (or a homolog thereof) and a mutant Solyc03g114840 gene (or a homolog thereof). In some embodiments, the method comprises crossing a produced genetically-altered Solanaceae plant containing a mutant Solyc12g038510 gene (or a homolog thereof) to another genetically-altered Solanaceae plant comprising a mutant Solyc04g005320 gene (or a homolog thereof), a mutant Solyc03g114840 gene (or a homolog thereof), or both a mutant Solyc04g005320 gene (or a homolog thereof) and a mutant Solyc03g114840 gene (or a homolog thereof). In some embodiments, the method comprises crossing a produced genetically-altered Solanaceae plant containing a mutant Solyc03g114840 gene (or a homolog thereof) to another genetically-altered Solanaceae plant comprising a mutant Solyc12g038510 gene (or a homolog thereof), a mutant Solyc04g005320 gene (or a homolog thereof), or both a mutant Solyc12g038510 gene (or a homolog thereof) and a mutant Solyc04g005320 gene (or a homolog thereof).

Example Nucleic Acid Sequences of the Disclosure

Wild-type Solyc04g005320 gene (SEQ ID NO: 1) ATGGGAAGAGGTAAGGTAGAATTGAAGAGAATAGAAAATAAGATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGATTACTCAAAAAAGCTTATGAGCTTTCTATTTTGTGTGAAGCTGAAGTTGCTCTTATCATTTTCTCTAAT AGAGGCAAACTCTATGAATTTTGCAGTACCTCTAGGTAATATTTTTATGTTTATGTCGTTCCGTTTAAGCTTTAC ATTTACGTTTTTATACGCAAAACTTTAAATTAGTTCTAAATGTATTAAAAAATTGAAATTTTGAGATTTAATTTC AAAATCTATGGTTAAACGAATGTTTATATGCATTATGATTTTGTTATCTTCTTTTTTTTTAAAAAAAGAAATAAA ATATATTGATGTTATAGATCTGAGTGAGAATAGAGTTTTTGGTACATTTATTAAGGGTGAATAATCAAATGTTTC ATTTGATTAGATCTAGGTTTTCTTGAACATTAAAATTGTTAAAAAAATTAGTTCATTTTATGAGGTAAATTTTGT TATGATTTGATGTTCCACCTCCATTTTTTCTTATTTTTATTATAAATAAATAAGTTTTAAAATATCCTTACTTTT ATATGTTCTTTTAAGTACAGACACATGAATCAAAAAGAAGTTTTATAATATGAATTGAATTAAAGCTGGTTGAAT TTCTATCTTCAGTTTTTGAAAACAACTAAAAACTTTGAAAAGGAATTTGATTTTATTATTTATGGCAACAAATAA CACCTAACTACTTATCGAGTCGGAATTGACGATATGAATCCTTTAACTTTTCATTTAAGCTCAATTTATATAGAA AATTCTGTATTGTGGATTGAAGTAATTTCTGGAGTTGATCAATTCTATTTAAAAAATTATTTAATTAATAATCAT TATCCCAAAAAATTATATTGAAATTAAAAAATAATATTAATTTTTTTAAATAACAAACTTATTAATTGAGTGACC ATCTAAATCGTCTTTTTCTTAAAGTTAGGGTCTTGCCTTTCATCTAATTTTGATAGTAATGTTCTTGAACCGACA AATTTTGTCATTTACTCTTATCTGTTATAATTTATGTGATTCGAGTTTTACGAATCAATTTTTGTTTATAATTTC AATCATGTATAAGAAGTATTTTAAGTTATAATAATTAACAATTTTAAGAAAGCATAATCAAGATCAAATAACTTA GTAGAAATAATATTGGTTTATGTAACCTCTATGCATTGACAATATAGTGTTTTTTTTATACTATCAAGTCATTTA TTGGATAATTATAATTAAAGAATATTAACTAATGAGTAAATCAATAGTTTAATATTAATGAGTTATCATAGTAGC GTATACTTATTACTCGATATTTGTAATCTAAACATTTTCAATATGCTTAAACTTGATTTTTTTATTTGGATCAAG TATACAATTTTTTTGTTAATAATAAATGACATTGAAACTTATAACTAATTTTATTTAAACAATTTTCTTTCTTTC TTTCCTCAAGGAGAGCATAGTTCTAATTATTATCAATATCATTATTATTATTATCTCTATGTTTATTTTATTATT ACTGTTGTTTCTTTTACTTGGATTGTCTGTACTATTTTTACTTCATGGACTTTAATTTTTTGTCTATCGTATTTT TATCATAGTTTTTACTCTTGTATTGGCTAAACCTAGTTTTGAAATTGTTTTTCATAAGCTGAAAGAGTCTATCAA AAACAACTTCTCACGAGATAGAAATAAAGTTTACGTATATTCCATTCTTCTCAAACCCCACTTATGAGATTATAC TGAAATGTTACTATTATTATTATACTTTGTAACATGCTAAAAAAACTAGTAATAATTACACTTCTTGCCAAAGAG TAAATAAAGTATGATCCTTTAATAAGTTGAAAATCCCTCTAAATCAAATTATCACTTTTGTGCAACTTGTCTTCT TTTTTTTCTTCTAGTATGTCTGATACACTGGAGAGATACCATAGATGCAGCTATGGTGACCTTGAAACTGGCCAG TCTTCAAAGGATTCACAGGTTACTTCATCTTCCTCAGAATTACAATTTACTAATAAATTTAACTTATATACTCTG ACACAGTATCGATGCAATTTAAACCTTTTATAACAGATTATCTGTTTTTATTTTAATTTCTTCGTAAATAATTAA TAAGTCGATATTGATAACTAACGCCAAGCACCCTATCTTCATCTAACTAATTAGTGTTATTATGCAATAGAATAA CTACCAAGAGTATATGAAGCTGAAAGCAAGAGTTGAAGTGCTACAACAGTCACAAAGGTGATACATTATTTGTTT TAAAAACACTTTTACTTGTCTCATTTTGATTGGCTCATCTGAACACCTGAACCGGTCTAGAAGTATTTTGAACAT GCATAATTGGACATGTTCAATCATGCGTTTGTTTGATCAGGTTCAGGATGTTTAGATGAGACCTCGTAAAATAAA TTAAGGGGAGGCTTTTTAATATGATATTTGTGTCTCAAATATATCACTTTTCTACCCTAATTCTTAATAACATTG TATTTACTTAATTATTCTTAATCTTTAAGGCATATACTTGGAGAGGACTTAGGACAATTAAACACAAAAGATTTG GAACAGCTTGAGCGTCAACTGGATTCATCTTTGAGGCTAATAAGATCAAGAAGGGTATGTTCTATGCACCTTCAA TTTATTTGTCAAATTTTAGGCTTTCAGATCATGTCTTAATCTTAATGTCCGATGACAGTTTCAGTGGCGGAATTA GAAATTTATGCAAGACAATTCAAGCAATATTATATATTATAGAATGTCAGACTTGAAATTTGAACTTGAGACATT GAACCTCTTTACAAATACACTAATATCTAACCTCGTATCAACGGGGTTCAACAATTTATATATATATAAAAAACA CTTAATTTTGCCCTATTTGGTGTAATATATAATTTTATCAAAGGTATGTTGGGAAAATGATAAAAATTACTTATG AATAATATCCAAATGGAATAATATAATAACAATTACTTACTATTACTTGATAGTGCCACAAAACTACTAAACCTT AAAATAAGTTCTTTTATTTTACATAATTCATTATAATCTTTGGCATGAATTTACTCAAGCATTGCTTCAGAATGA TCAAAGCCTCCTTAATATTTTTGGGTACAGACATAAAGTCTAGACATGCAATCAAAGATATAGATGCACGAGATG ACTAATCAAAGGAAACAATAGGAACGATCAAAAAAATTGAAATTGAAAATATATTTTTTTTAAACTAAAGGTAAG TCAAGATTACCAAGTAAGTGTATTATTGTAACTTTTGTATTATTTATCCTAAGTAAACATGTATCAAAAACATAC ACAAATTTACTTTCTCTTTTATTACTAACATCAACTTACATGCTAATTATAAATAATTAAAGGGTAAATAGTTGG TTGCATGATTTGGTAAAAGAAGTTGTTAACCTACTCTTTGATAACATATATGTTTTCAGACACAAAACATGCTTG ATCAACTTTCTGATCTTCAACAAAAGGTATGTATTGTATAATATAATCCCTTAAGTTGACAATTAAATAGATTGT TCAATTGTTAATTTGACATTGTATGTGTTCTTTTTTCTTTTTTTCTACAGGAACAATCTCTTCTTGAAATCAACA GATCCTTGAAAACAAAGGTACAAAGCACACATTTTGGACCTTTTATGAGTTTTTTAGGGCGTGTTTGATTTATTT ATTTTTTCTGAATTTTTTCATGTTTGGTTGATCTAAATTCTGGGAAAATACTTTTTTCTATGAAAGTAAGTTTTT TAAAAATGACTTAGCCAGTGGAAGTAGGGAAAACAAGTTGTGACGACATTCCACGTTGATTGTTTTCTCTCGATC TTCCTACACACCTTAAGTTCGCCACCACCTCTCGCAGTATTTGTTTAGATTATATAAAAATGTATCAAGAATGAC ACTTTTTATTTGTGTACATAATAAAAGAAAATAAGTAAGAAACCGAACATTTTCCCATGGAAAATATTATTTTTC ATACCAAACACACCCTTAGTCTTTGTTTTAGGGTATATGACTAATTTGTTCTCCATTTCGGATATTTAGATTCGT ATGGGTTTTTCTTTGATGTCTAACTTATCGTACTTTTTACGCGATTTTATGAAATTCTTATAATAGTTGGAAGAA AACTCTGTAGCACATTGGCATATCACTGGAGAGCAAAATGTACAATTCAGACAACAACCTGCTCAGTCAGAGGGG TTCTTTCAGCCTTTACAATGCAATACTAATATAGTGCCAAACAGGTAACATATAATTTTATGTTTTCTTTTTTTC CTTTAAATAGCATATTTTTTGCAACATTTTAAATTGAACCGTTGAATTGAGTCGTTGAGAGGTGAATTCAGAATC TGAAGTAACAGATACATTCAAATTAATTTCTTTGTGTATTTATCGAAGTGAGTCAAGTCGTAAGTCTAGAGGTGA ATTTAGAATCTAAAGTAATAGATACAGTCGAATCAATCTCTTTGTGTATTTATTGAAGTGAGTCGTTTTGTAAAG TTTGAGACGAATTCAAAACCTAAAGTAATACTAAATACATACATTCAAAATAATTTCTAAAGCGAGTTATGTTGG AAGTCGAGAGACGAAAGTATATTATATATGGATCAATTCAAATTAATTTCTTAATGTATTTGATGAGCGTTGTTG TAGGGGCGAATTCAGAATCTGAAGTTCATGTAAGTACAGGTACAATGTGGCTCCATTGGATAGTATAGAACCATC AACACAGAATGCTACTGGAATTTTACCAGGATGGATGCTTTGA Wild-type Solyc04g005320 coding sequence (SEQ ID NO: 2) ATGGGAAGAGGTAAGGTAGAATTGAAGAGAATAGAAAATAAGATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGATTACTCAAAAAAGCTTATGAGCTTTCTATTTTGTGTGAAGCTGAAGTTGCTCTTATCATTTTCTCTAAT AGAGGCAAACTCTATGAATTTTGCAGTACCTCTAGTATGTCTGATACACTGGAGAGATACCATAGATGCAGCTAT GGTGACCTTGAAACTGGCCAGTCTTCAAAGGATTCACAGAATAACTACCAAGAGTATATGAAGCTGAAAGCAAGA GTTGAAGTGCTACAACAGTCACAAAGGCATATACTTGGAGAGGACTTAGGACAATTAAACACAAAAGATTTGGAA CAGCTTGAGCGTCAACTGGATTCATCTTTGAGGCTAATAAGATCAAGAAGGACACAAAACATGCTTGATCAACTT TCTGATCTTCAACAAAAGGAACAATCTCTTCTTGAAATCAACAGATCCTTGAAAACAAAGTTGGAAGAAAACTCT GTAGCACATTGGCATATCACTGGAGAGCAAAATGTACAATTCAGACAACAACCTGCTCAGTCAGAGGGGTTCTTT CAGCCTTTACAATGCAATACTAATATAGTGCCAAACAGGTACAATGTGGCTCCATTGGATAGTATAGAACCATCA ACACAGAATGCTACTGGAATTTTACCAGGATGGATGCTTTGA Mutant Solyc04g005320 gene allele lin^trans (SEQ ID NO: 3) ATGGGAAGAGGTAAGGTAGAATTGAAGAGAATAGAAAATAAGATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGATTACTCAAAAAAGCTTATGAGCTTTCTATTTTGTGTGAAGCTGAAGTTGCTCTTATCATTTTCTCTAAT AGAGGCAAACTCTATGAATTTTGCAGTACCTCTAGGTAATATTTTTATGTTTATGTCGTTCCGTTTAAGCTTTAC ATTTACGTTTTTATACGCAAAACTTTAAATTAGTTCTAAATGTATTAAAAAATTGAAATTTTGAGATTTAATTTC AAAATCTATGGTTAAACGAATGTTTATATGCATTATGATTTTGTTATCTTCTTTTTTTTTAAAAAAAGAAATAAA ATATATTGATGTTATAGATCTGAGTGAGAATAGAGTTTTTGGTACATTTATTAAGGGTGAATAATCAAATGTTTC ATTTGATTAGATCTAGGTTTTCTTGAACATTAAAATTGTTAAAAAAATT TTCATTTTATGAGGTAAATTTTGTTATGAT TTGATGTTCCACCTCCATTTTTTCTTATTTTTATTATAAATAAATAAGTTTTAAAATATCCTTACTTTTATATGT TCTTTTAAGTACAGACACATGAATCAAAAAGAAGTTTTATAATATGAATTGAATTAAAGCTGGTTGAATTTCTAT CTTCAGTTTTTGAAAACAACTAAAAACTTTGAAAAGGAATTTGATTTTATTATTTATGGCAACAAATAACACCTA ACTACTTATCGAGTCGGAATTGACGATATGAATCCTTTAACTTTTCATTTAAGCTCAATTTATATAGAAAATTCT GTATTGTGGATTGAAGTAATTTCTGGAGTTGATCAATTCTATTTAAAAAATTATTTAATTAATAATCATTATCCC AAAAAATTATATTGAAATTAAAAAATAATATTAATTTTTTTAAATAACAAACTTATTAATTGAGTGACCATCTAA ATCGTCTTTTTCTTAAAGTTAGGGTCTTGCCTTTCATCTAATTTTGATAGTAATGTTCTTGAACCGACAAATTTT GTCATTTACTCTTATCTGTTATAATTTATGTGATTCGAGTTTTACGAATCAATTTTTGTTTATAATTTCAATCAT GTATAAGAAGTATTTTAAGTTATAATAATTAACAATTTTAAGAAAGCATAATCAAGATCAAATAACTTAGTAGAA ATAATATTGGTTTATGTAACCTCTATGCATTGACAATATAGTGTTTTTTTTATACTATCAAGTCATTTATTGGAT AATTATAATTAAAGAATATTAACTAATGAGTAAATCAATAGTTTAATATTAATGAGTTATCATAGTAGCGTATAC TTATTACTCGATATTTGTAATCTAAACATTTTCAATATGCTTAAACTTGATTTTTTTATTTGGATCAAGTATACA ATTTTTTTGTTAATAATAAATGACATTGAAACTTATAACTAATTTTATTTAAACAATTTTCTTTCTTTCTTTCCT CAAGGAGAGCATAGTTCTAATTATTATCAATATCATTATTATTATTATCTCTATGTTTATTTTATTATTACTGTT GTTTCTTTTACTTGGATTGTCTGTACTATTTTTACTTCATGGACTTTAATTTTTTGTCTATCGTATTTTTATCAT AGTTTTTACTCTTGTATTGGCTAAACCTAGTTTTGAAATTGTTTTTCATAAGCTGAAAGAGTCTATCAAAAACAA CTTCTCACGAGATAGAAATAAAGTTTACGTATATTCCATTCTTCTCAAACCCCACTTATGAGATTATACTGAAAT GTTACTATTATTATTATACTTTGTAACATGCTAAAAAAACTAGTAATAATTACACTTCTTGCCAAAGAGTAAATA AAGTATGATCCTTTAATAAGTTGAAAATCCCTCTAAATCAAATTATCACTTTTGTGCAACTTGTCTTCTTTTTTT TCTTCTAGTATGTCTGATACACTGGAGAGATACCATAGATGCAGCTATGGTGACCTTGAAACTGGCCAGTCTTCA AAGGATTCACAGGTTACTTCATCTTCCTCAGAATTACAATTTACTAATAAATTTAACTTATATACTCTGACACAG TATCGATGCAATTTAAACCTTTTATAACAGATTATCTGTTTTTATTTTAATTTCTTCGTAAATAATTAATAAGTC GATATTGATAACTAACGCCAAGCACCCTATCTTCATCTAACTAATTAGTGTTATTATGCAATAGAATAACTACCA AGAGTATATGAAGCTGAAAGCAAGAGTTGAAGTGCTACAACAGTCACAAAGGTGATACATTATTTGTTTTAAAAA CACTTTTACTTGTCTCATTTTGATTGGCTCATCTGAACACCTGAACCGGTCTAGAAGTATTTTGAACATGCATAA TTGGACATGTTCAATCATGCGTTTGTTTGATCAGGTTCAGGATGTTTAGATGAGACCTCGTAAAATAAATTAAGG GGAGGCTTTTTAATATGATATTTGTGTCTCAAATATATCACTTTTCTACCCTAATTCTTAATAACATTGTATTTA CTTAATTATTCTTAATCTTTAAGGCATATACTTGGAGAGGACTTAGGACAATTAAACACAAAAGATTTGGAACAG CTTGAGCGTCAACTGGATTCATCTTTGAGGCTAATAAGATCAAGAAGGGTATGTTCTATGCACCTTCAATTTATT TGTCAAATTTTAGGCTTTCAGATCATGTCTTAATCTTAATGTCCGATGACAGTTTCAGTGGCGGAATTAGAAATT TATGCAAGACAATTCAAGCAATATTATATATTATAGAATGTCAGACTTGAAATTTGAACTTGAGACATTGAACCT CTTTACAAATACACTAATATCTAACCTCGTATCAACGGGGTTCAACAATTTATATATATATAAAAAACACTTAAT TTTGCCCTATTTGGTGTAATATATAATTTTATCAAAGGTATGTTGGGAAAATGATAAAAATTACTTATGAATAAT ATCCAAATGGAATAATATAATAACAATTACTTACTATTACTTGATAGTGCCACAAAACTACTAAACCTTAAAATA AGTTCTTTTATTTTACATAATTCATTATAATCTTTGGCATGAATTTACTCAAGCATTGCTTCAGAATGATCAAAG CCTCCTTAATATTTTTGGGTACAGACATAAAGTCTAGACATGCAATCAAAGATATAGATGCACGAGATGACTAAT CAAAGGAAACAATAGGAACGATCAAAAAAATTGAAATTGAAAATATATTTTTTTTAAACTAAAGGTAAGTCAAGA TTACCAAGTAAGTGTATTATTGTAACTTTTGTATTATTTATCCTAAGTAAACATGTATCAAAAACATACACAAAT TTACTTTCTCTTTTATTACTAACATCAACTTACATGCTAATTATAAATAATTAAAGGGTAAATAGTTGGTTGCAT GATTTGGTAAAAGAAGTTGTTAACCTACTCTTTGATAACATATATGTTTTCAGACACAAAACATGCTTGATCAAC TTTCTGATCTTCAACAAAAGGTATGTATTGTATAATATAATCCCTTAAGTTGACAATTAAATAGATTGTTCAATT GTTAATTTGACATTGTATGTGTTCTTTTTTCTTTTTTTCTACAGGAACAATCTCTTCTTGAAATCAACAGATCCT TGAAAACAAAGGTACAAAGCACACATTTTGGACCTTTTATGAGTTTTTTAGGGCGTGTTTGATTTATTTATTTTT TCTGAATTTTTTCATGTTTGGTTGATCTAAATTCTGGGAAAATACTTTTTTCTATGAAAGTAAGTTTTTTAAAAA TGACTTAGCCAGTGGAAGTAGGGAAAACAAGTTGTGACGACATTCCACGTTGATTGTTTTCTCTCGATCTTCCTA CACACCTTAAGTTCGCCACCACCTCTCGCAGTATTTGTTTAGATTATATAAAAATGTATCAAGAATGACACTTTT TATTTGTGTACATAATAAAAGAAAATAAGTAAGAAACCGAACATTTTCCCATGGAAAATATTATTTTTCATACCA AACACACCCTTAGTCTTTGTTTTAGGGTATATGACTAATTTGTTCTCCATTTCGGATATTTAGATTCGTATGGGT TTTTCTTTGATGTCTAACTTATCGTACTTTTTACGCGATTTTATGAAATTCTTATAATAGTTGGAAGAAAACTCT GTAGCACATTGGCATATCACTGGAGAGCAAAATGTACAATTCAGACAACAACCTGCTCAGTCAGAGGGGTTCTTT CAGCCTTTACAATGCAATACTAATATAGTGCCAAACAGGTAACATATAATTTTATGTTTTCTTTTTTTCCTTTAA ATAGCATATTTTTTGCAACATTTTAAATTGAACCGTTGAATTGAGTCGTTGAGAGGTGAATTCAGAATCTGAAGT AACAGATACATTCAAATTAATTTCTTTGTGTATTTATCGAAGTGAGTCAAGTCGTAAGTCTAGAGGTGAATTTAG AATCTAAAGTAATAGATACAGTCGAATCAATCTCTTTGTGTATTTATTGAAGTGAGTCGTTTTGTAAAGTTTGAG ACGAATTCAAAACCTAAAGTAATACTAAATACATACATTCAAAATAATTTCTAAAGCGAGTTATGTTGGAAGTCG AGAGACGAAAGTATATTATATATGGATCAATTCAAATTAATTTCTTAATGTATTTGATGAGCGTTGTTGTAGGGG CGAATTCAGAATCTGAAGTTCATGTAAGTACAGGTACAATGTGGCTCCATTGGATAGTATAGAACCATCAACACA GAATGCTACTGGAATTTTACCAGGATGGATGCTTTGA Mutant Solyc04g005320 gene allele lin^CR >allele-1 (SEQ ID NO: 4) ATGGGAAGAGGTAAGGTAGAATTGAAGAGAATAGAAAATAAGATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGATTACTCAAAAAAGCTTATGAGCTTTCTATTTTGTGTGAAGCTGAAGTTGCTCTTATCATTTTCTCTAAT AGAGGCAAACTCTATGAATTTTGCAGTACCTCTAGGTAATATTTTTATGTTTATGTCGTTCCGTTTAAGCTTTAC ATTTACGTTTTTATACGCAAAACTTTAAATTAGTTCTAAATGTATTAAAAAATTGAAATTTTGAGATTTAATTTC AAAATCTATGGTTAAACGAATGTTTATATGCATTATGATTTTGTTATCTTCTTTTTTTTTAAAAAAAGAAATAAA ATATATTGATGTTATAGATCTGAGTGAGAATAGAGTTTTTGGTACATTTATTAAGGGTGAATAATCAAATGTTTC ATTTGATTAGATCTAGGTTTTCTTGAACATTAAAATTGTTAAAAAAATTAGTTCATTTTATGAGGTAAATTTTGT TATGATTTGATGTTCCACCTCCATTTTTTCTTATTTTTATTATAAATAAATAAGTTTTAAAATATCCTTACTTTT ATATGTTCTTTTAAGTACAGACACATGAATCAAAAAGAAGTTTTATAATATGAATTGAATTAAAGCTGGTTGAAT TTCTATCTTCAGTTTTTGAAAACAACTAAAAACTTTGAAAAGGAATTTGATTTTATTATTTATGGCAACAAATAA CACCTAACTACTTATCGAGTCGGAATTGACGATATGAATCCTTTAACTTTTCATTTAAGCTCAATTTATATAGAA AATTCTGTATTGTGGATTGAAGTAATTTCTGGAGTTGATCAATTCTATTTAAAAAATTATTTAATTAATAATCAT TATCCCAAAAAATTATATTGAAATTAAAAAATAATATTAATTTTTTTAAATAACAAACTTATTAATTGAGTGACC ATCTAAATCGTCTTTTTCTTAAAGTTAGGGTCTTGCCTTTCATCTAATTTTGATAGTAATGTTCTTGAACCGACA AATTTTGTCATTTACTCTTATCTGTTATAATTTATGTGATTCGAGTTTTACGAATCAATTTTTGTTTATAATTTC AATCATGTATAAGAAGTATTTTAAGTTATAATAATTAACAATTTTAAGAAAGCATAATCAAGATCAAATAACTTA GTAGAAATAATATTGGTTTATGTAACCTCTATGCATTGACAATATAGTGTTTTTTTTATACTATCAAGTCATTTA TTGGATAATTATAATTAAAGAATATTAACTAATGAGTAAATCAATAGTTTAATATTAATGAGTTATCATAGTAGC GTATACTTATTACTCGATATTTGTAATCTAAACATTTTCAATATGCTTAAACTTGATTTTTTTATTTGGATCAAG TATACAATTTTTTTGTTAATAATAAATGACATTGAAACTTATAACTAATTTTATTTAAACAATTTTCTTTCTTTC TTTCCTCAAGGAGAGCATAGTTCTAATTATTATCAATATCATTATTATTATTATCTCTATGTTTATTTTATTATT ACTGTTGTTTCTTTTACTTGGATTGTCTGTACTATTTTTACTTCATGGACTTTAATTTTTTGTCTATCGTATTTT TATCATAGTTTTTACTCTTGTATTGGCTAAACCTAGTTTTGAAATTGTTTTTCATAAGCTGAAAGAGTCTATCAA AAACAACTTCTCACGAGATAGAAATAAAGTTTACGTATATTCCATTCTTCTCAAACCCCACTTATGAGATTATAC TGAAATGTTACTATTATTATTATACTTTGTAACATGCTAAAAAAACTAGTAATAATTACACTTCTTGCCAAAGAG TAAATAAAGTATGATCCTTTAATAAGTTGAAAATCCCTCTAAATCAAATTATCACTTTTGTGCAACTTGTCTTCT TTTTTTTCTTCTAGTATGTCCCATAGATGCAGCTATGGTGACCTTGAAACTGGCCAGTCTTCAAAGGATTCACAG GTTACTTCATCTTCCTCAGAATTACAATTTACTAATAAATTTAACTTATATACTCTGACACAGTATCGATGCAAT TTAAACCTTTTATAACAGATTATCTGTTTTTATTTTAATTTCTTCGTAAATAATTAATAAGTCGATATTGATAAC TAACGCCAAGCACCCTATCTTCATCTAACTAATTAGTGTTATTATGCAATAGAATAACTACCAAGAGTATATGAA GCTGAAAGCAAGAGTTGAAGTGCTACAACAGTCACAAAGGTGATACATTATTTGTTTTAAAAACACTTTTACTTG TCTCATTTTGATTGGCTCATCTGAACACCTGAACCGGTCTAGAAGTATTTTGAACATGCATAATTGGACATGTTC AATCATGCGTTTGTTTGATCAGGTTCAGGATGTTTAGATGAGACCTCGTAAAATAAATTAAGGGGAGGCTTTTTA ATATGATATTTGTGTCTCAAATATATCACTTTTCTACCCTAATTCTTAATAACATTGTATTTACTTAATTATTCT TAATCTTTAAGGCATATACTTGGAGAGGACTTAGGACAATTAAACACAAAAGATTTGGAACAGCTTGAGCAACTG GATTCATCTTTGAGGCTAATAAGATCAAGAAGGGTATGTTCTATGCACCTTCAATTTATTTGTCAAATTTTAGGC TTTCAGATCATGTCTTAATCTTAATGTCCGATGACAGTTTCAGTGGCGGAATTAGAAATTTATGCAAGACAATTC AAGCAATATTATATATTATAGAATGTCAGACTTGAAATTTGAACTTGAGACATTGAACCTCTTTACAAATACACT AATATCTAACCTCGTATCAACGGGGTTCAACAATTTATATATATATAAAAAACACTTAATTTTGCCCTATTTGGT GTAATATATAATTTTATCAAAGGTATGTTGGGAAAATGATAAAAATTACTTATGAATAATATCCAAATGGAATAA TATAATAACAATTACTTACTATTACTTGATAGTGCCACAAAACTACTAAACCTTAAAATAAGTTCTTTTATTTTA CATAATTCATTATAATCTTTGGCATGAATTTACTCAAGCATTGCTTCAGAATGATCAAAGCCTCCTTAATATTTT TGGGTACAGACATAAAGTCTAGACATGCAATCAAAGATATAGATGCACGAGATGACTAATCAAAGGAAACAATAG GAACGATCAAAAAAATTGAAATTGAAAATATATTTTTTTTAAACTAAAGGTAAGTCAAGATTACCAAGTAAGTGT ATTATTGTAACTTTTGTATTATTTATCCTAAGTAAACATGTATCAAAAACATACACAAATTTACTTTCTCTTTTA TTACTAACATCAACTTACATGCTAATTATAAATAATTAAAGGGTAAATAGTTGGTTGCATGATTTGGTAAAAGAA GTTGTTAACCTACTCTTTGATAACATATATGTTTTCAGACACAAAACATGCTTGATCAACTTTCTGATCTTCAAC AAAAGGTATGTATTGTATAATATAATCCCTTAAGTTGACAATTAAATAGATTGTTCAATTGTTAATTTGACATTG TATGTGTTCTTTTTTCTTTTTTTCTACAGGAACAATCTCTTCTTGAAATCAACAGATCCTTGAAAACAAAGGTAC AAAGCACACATTTTGGACCTTTTATGAGTTTTTTAGGGCGTGTTTGATTTATTTATTTTTTCTGAATTTTTTCAT GTTTGGTTGATCTAAATTCTGGGAAAATACTTTTTTCTATGAAAGTAAGTTTTTTAAAAATGACTTAGCCAGTGG AAGTAGGGAAAACAAGTTGTGACGACATTCCACGTTGATTGTTTTCTCTCGATCTTCCTACACACCTTAAGTTCG CCACCACCTCTCGCAGTATTTGTTTAGATTATATAAAAATGTATCAAGAATGACACTTTTTATTTGTGTACATAA TAAAAGAAAATAAGTAAGAAACCGAACATTTTCCCATGGAAAATATTATTTTTCATACCAAACACACCCTTAGTC TTTGTTTTAGGGTATATGACTAATTTGTTCTCCATTTCGGATATTTAGATTCGTATGGGTTTTTCTTTGATGTCT AACTTATCGTACTTTTTACGCGATTTTATGAAATTCTTATAATAGTTGGAAGAAAACTCTGTAGCACATTGGCAT ATCACTGGAGAGCAAAATGTACAATTCAGACAACAACCTGCTCAGTCAGAGGGGTTCTTTCAGCCTTTACAATGC AATACTAATATAGTGCCAAACAGGTAACATATAATTTTATGTTTTCTTTTTTTCCTTTAAATAGCATATTTTTTG CAACATTTTAAATTGAACCGTTGAATTGAGTCGTTGAGAGGTGAATTCAGAATCTGAAGTAACAGATACATTCAA ATTAATTTCTTTGTGTATTTATCGAAGTGAGTCAAGTCGTAAGTCTAGAGGTGAATTTAGAATCTAAAGTAATAG ATACAGTCGAATCAATCTCTTTGTGTATTTATTGAAGTGAGTCGTTTTGTAAAGTTTGAGACGAATTCAAAACCT AAAGTAATACTAAATACATACATTCAAAATAATTTCTAAAGCGAGTTATGTTGGAAGTCGAGAGACGAAAGTATA TTATATATGGATCAATTCAAATTAATTTCTTAATGTATTTGATGAGCGTTGTTGTAGGGGCGAATTCAGAATCTG AAGTTCATGTAAGTACAGGTACAATGTGGCTCCATTGGATAGTATAGAACCATCAACACAGAATGCTACTGGAAT TTTACCAGGATGGATGCTTTGA >allele-2 (SEQ ID NO: 5) ATGGGAAGAGGTAAGGTAGAATTGAAGAGAATAGAAAATAAGATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGATTACTCAAAAAAGCTTATGAGCTTTCTATTTTGTGTGAAGCTGAAGTTGCTCTTATCATTTTCTCTAAT AGAGGCAAACTCTATGAATTTTGCAGTACCTCTAGGTAATATTTTTATGTTTATGTCGTTCCGTTTAAGCTTTAC ATTTACGTTTTTATACGCAAAACTTTAAATTAGTTCTAAATGTATTAAAAAATTGAAATTTTGAGATTTAATTTC AAAATCTATGGTTAAACGAATGTTTATATGCATTATGATTTTGTTATCTTCTTTTTTTTTAAAAAAAGAAATAAA ATATATTGATGTTATAGATCTGAGTGAGAATAGAGTTTTTGGTACATTTATTAAGGGTGAATAATCAAATGTTTC ATTTGATTAGATCTAGGTTTTCTTGAACATTAAAATTGTTAAAAAAATTAGTTCATTTTATGAGGTAAATTTTGT TATGATTTGATGTTCCACCTCCATTTTTTCTTATTTTTATTATAAATAAATAAGTTTTAAAATATCCTTACTTTT ATATGTTCTTTTAAGTACAGACACATGAATCAAAAAGAAGTTTTATAATATGAATTGAATTAAAGCTGGTTGAAT TTCTATCTTCAGTTTTTGAAAACAACTAAAAACTTTGAAAAGGAATTTGATTTTATTATTTATGGCAACAAATAA CACCTAACTACTTATCGAGTCGGAATTGACGATATGAATCCTTTAACTTTTCATTTAAGCTCAATTTATATAGAA AATTCTGTATTGTGGATTGAAGTAATTTCTGGAGTTGATCAATTCTATTTAAAAAATTATTTAATTAATAATCAT TATCCCAAAAAATTATATTGAAATTAAAAAATAATATTAATTTTTTTAAATAACAAACTTATTAATTGAGTGACC ATCTAAATCGTCTTTTTCTTAAAGTTAGGGTCTTGCCTTTCATCTAATTTTGATAGTAATGTTCTTGAACCGACA AATTTTGTCATTTACTCTTATCTGTTATAATTTATGTGATTCGAGTTTTACGAATCAATTTTTGTTTATAATTTC AATCATGTATAAGAAGTATTTTAAGTTATAATAATTAACAATTTTAAGAAAGCATAATCAAGATCAAATAACTTA GTAGAAATAATATTGGTTTATGTAACCTCTATGCATTGACAATATAGTGTTTTTTTTATACTATCAAGTCATTTA TTGGATAATTATAATTAAAGAATATTAACTAATGAGTAAATCAATAGTTTAATATTAATGAGTTATCATAGTAGC GTATACTTATTACTCGATATTTGTAATCTAAACATTTTCAATATGCTTAAACTTGATTTTTTTATTTGGATCAAG TATACAATTTTTTTGTTAATAATAAATGACATTGAAACTTATAACTAATTTTATTTAAACAATTTTCTTTCTTTC TTTCCTCAAGGAGAGCATAGTTCTAATTATTATCAATATCATTATTATTATTATCTCTATGTTTATTTTATTATT ACTGTTGTTTCTTTTACTTGGATTGTCTGTACTATTTTTACTTCATGGACTTTAATTTTTTGTCTATCGTATTTT TATCATAGTTTTTACTCTTGTATTGGCTAAACCTAGTTTTGAAATTGTTTTTCATAAGCTGAAAGAGTCTATCAA AAACAACTTCTCACGAGATAGAAATAAAGTTTACGTATATTCCATTCTTCTCAAACCCCACTTATGAGATTATAC TGAAATGTTACTATTATTATTATACTTTGTAACATGCTAAAAAAACTAGTAATAATTACACTTCTTGCCAAAGAG TAAATAAAGTATGATCCTTTAATAAGTTGAAAATCCCTCTAAATCAAATTATCACTTTTGTGCAACTTGTCTTCT TTTTTTTCTTCTAGTATGTCTGATACACTGGAGAGATACCATAGATGCAGCTATGGTGACCTTGAAACTGGCCAG TCTTCAAAGGATTCACAGGTTACTTCATCTTCCTCAGAATTACAATTTACTAATAAATTTAACTTATATACTCTG ACACAGTATCGATGCAATTTAAACCTTTTATAACAGATTATCTGTTTTTATTTTAATTTCTTCGTAAATAATTAA TAAGTCGATATTGATAACTAACGCCAAGCACCCTATCTTCATCTAACTAATTAGTGTTATTATGCAATAGAATAA CTACCAAGAGTATATGAAGCTGAAAGCAAGAGTTGAAGTGCTACAACAGTCACAAAGGTGATACATTATTTGTTT TAAAAACACTTTTACTTGTCTCATTTTGATTGGCTCATCTGAACACCTGAACCGGTCTAGAAGTATTTTGAACAT GCATAATTGGACATGTTCAATCATGCGTTTGTTTGATCAGGTTCAGGATGTTTAGATGAGACCTCGTAAAATAAA TTAAGGGGAGGCTTTTTAATATGATATTTGTGTCTCAAATATATCACTTTTCTACCCTAATTCTTAATAACATTG TATTTACTTAATTATTCTTAATCTTTAAGGCATATACTTGGAGAGGACTTAGGACAATTAAACACAAAAGATTTG GAAAACTGGATTCATCTTTGAGGCTAATAAGATCAAGAAGGGTATGTTCTATGCACCTTCAATTTATTTGTCAAA TTTTAGGCTTTCAGATCATGTCTTAATCTTAATGTCCGATGACAGTTTCAGTGGCGGAATTAGAAATTTATGCAA GACAATTCAAGCAATATTATATATTATAGAATGTCAGACTTGAAATTTGAACTTGAGACATTGAACCTCTTTACA AATACACTAATATCTAACCTCGTATCAACGGGGTTCAACAATTTATATATATATAAAAAACACTTAATTTTGCCC TATTTGGTGTAATATATAATTTTATCAAAGGTATGTTGGGAAAATGATAAAAATTACTTATGAATAATATCCAAA TGGAATAATATAATAACAATTACTTACTATTACTTGATAGTGCCACAAAACTACTAAACCTTAAAATAAGTTCTT TTATTTTACATAATTCATTATAATCTTTGGCATGAATTTACTCAAGCATTGCTTCAGAATGATCAAAGCCTCCTT AATATTTTTGGGTACAGACATAAAGTCTAGACATGCAATCAAAGATATAGATGCACGAGATGACTAATCAAAGGA AACAATAGGAACGATCAAAAAAATTGAAATTGAAAATATATTTTTTTTAAACTAAAGGTAAGTCAAGATTACCAA GTAAGTGTATTATTGTAACTTTTGTATTATTTATCCTAAGTAAACATGTATCAAAAACATACACAAATTTACTTT CTCTTTTATTACTAACATCAACTTACATGCTAATTATAAATAATTAAAGGGTAAATAGTTGGTTGCATGATTTGG TAAAAGAAGTTGTTAACCTACTCTTTGATAACATATATGTTTTCAGACACAAAACATGCTTGATCAACTTTCTGA TCTTCAACAAAAGGTATGTATTGTATAATATAATCCCTTAAGTTGACAATTAAATAGATTGTTCAATTGTTAATT TGACATTGTATGTGTTCTTTTTTCTTTTTTTCTACAGGAACAATCTCTTCTTGAAATCAACAGATCCTTGAAAAC AAAGGTACAAAGCACACATTTTGGACCTTTTATGAGTTTTTTAGGGCGTGTTTGATTTATTTATTTTTTCTGAAT TTTTTCATGTTTGGTTGATCTAAATTCTGGGAAAATACTTTTTTCTATGAAAGTAAGTTTTTTAAAAATGACTTA GCCAGTGGAAGTAGGGAAAACAAGTTGTGACGACATTCCACGTTGATTGTTTTCTCTCGATCTTCCTACACACCT TAAGTTCGCCACCACCTCTCGCAGTATTTGTTTAGATTATATAAAAATGTATCAAGAATGACACTTTTTATTTGT GTACATAATAAAAGAAAATAAGTAAGAAACCGAACATTTTCCCATGGAAAATATTATTTTTCATACCAAACACAC CCTTAGTCTTTGTTTTAGGGTATATGACTAATTTGTTCTCCATTTCGGATATTTAGATTCGTATGGGTTTTTCTT TGATGTCTAACTTATCGTACTTTTTACGCGATTTTATGAAATTCTTATAATAGTTGGAAGAAAACTCTGTAGCAC ATTGGCATATCACTGGAGAGCAAAATGTACAATTCAGACAACAACCTGCTCAGTCAGAGGGGTTCTTTCAGCCTT TACAATGCAATACTAATATAGTGCCAAACAGGTAACATATAATTTTATGTTTTCTTTTTTTCCTTTAAATAGCAT ATTTTTTGCAACATTTTAAATTGAACCGTTGAATTGAGTCGTTGAGAGGTGAATTCAGAATCTGAAGTAACAGAT ACATTCAAATTAATTTCTTTGTGTATTTATCGAAGTGAGTCAAGTCGTAAGTCTAGAGGTGAATTTAGAATCTAA AGTAATAGATACAGTCGAATCAATCTCTTTGTGTATTTATTGAAGTGAGTCGTTTTGTAAAGTTTGAGACGAATT CAAAACCTAAAGTAATACTAAATACATACATTCAAAATAATTTCTAAAGCGAGTTATGTTGGAAGTCGAGAGACG AAAGTATATTATATATGGATCAATTCAAATTAATTTCTTAATGTATTTGATGAGCGTTGTTGTAGGGGCGAATTC AGAATCTGAAGTTCATGTAAGTACAGGTACAATGTGGCTCCATTGGATAGTATAGAACCATCAACACAGAATGCT ACTGGAATTTTACCAGGATGGATGCTTTGA Wild-type Solyc12g038510 gene (SEQ ID NO: 6) ATGGGAAGAGGAAGAGTAGAACTAAAGAGAATAGAGAACAAAATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGACTTCTTAAGAAAGCTTATGAGTTATCTATACTTTGTGATGCTGAAGTTGCTCTCATCATCTTCTCTAGC CGCGGAAAACTCTATGAGTTTTCAAGTGCTTCCAGGTATATATATATATACATATGTTTTTCTTCTTTTTGTGTG TGCGTATGTGTTTACTTACTTTCATTAATTAACTCAACCATATATATACATCTCTCACCTCAATTATATATATGT TTGAGATCTGAATGTCTACGGACTCCATTTAGGTACATATCTTTGTTTAGATCATAAATCATCTATCTTCATTCC TAAGATCTACTAATATATATGTATAAGAAGATCCATCCATCTATTAGGTTTTTCAACAACATATACAGTGAAATC TTATATGTGGGCCCACGTATAGCCATATGAGAAAATAGTGTGCACGTAAACATTATCATTACTTAATTATAGGAA TATACATCCATTAGGTTTATCAACAACAATAAAATCCTCTAAATGGAGTCTAGTCATAGGTCTAGCCGTTTGAAA ATGTAAAATATATGCCGATCTTATCACTATGTCATAATAATAGATATGTTGTTATTGAAAGATTCTCAATCTTTT TTTTTCTTCAAGGTAGAGATTCTTAAGTGGATTCATGTTTTTTTTATCAAAAAAGAAAAAAACAAAAGTGTCCAT TTGTTCATCTAATGGGTTTTCCATGTTACCAATTCACTACACTGTTGAGATTTGATTATCAGATGTGTCAAGTTT CGTTTGGTTCCCTAGAAGGGAGAAAAGGCTGCTTATGCAGGCAGGGTATTAAAGATGATATTAATATCTGCAGTA ATCAGTAACAGAATATATAAACTTAATAATAAACTTGAAGGTACTTAATTATCCAGCAGATAATCTTCTGTCTCA CCGTACACTTTTGTTATATCATAAGCATAAGAATTGTTTTATCAAATATTACCAAACAAAACTTAGTTTTGTTTG GTAATATTTTATAAAATATGTTACCGAAAGTTACTTCCTATAACATATTTTATAAAGAAAAAAATTAAAAACTCC ATATACCTAAGAAATGTAACCCCCCCTCCATAACAACAATTTAACAAAAATAAAAACCTACTTTTTTTGAATTTG GTAAATTAGTTTTCTATCCTTTTTAGTAACTTCCTTTCTTATTTTCTTTTTATATTGGTAAAGTTTAATATTACA CATTATTTTAACATGTTATAATTTTTTGTGATGCTTAATTATTTGATACATGTAATAAACCATATATTAGAGCTA TAAATCAATGACAATGCATGTAGATACAACTCATTTATGATATATTTTGTTTATATATATAACCAATTAGATAAT TTGTCTGCGCTTTGTGCAGTCATAAATAATAATTGCATTGAACTTGCAAATATTTTTTTTTAATATCCATACATT AAAAAAAAAGAAAGAGGAAAATTGGTTCCTAAAATATTAGCAATATTCAAACATTTATTTGATTATTAATCATTA TCACATAACTTAAGAACGTCTAATGAATGAATTATTCACGAAATAATAAATCATTGGTTCTAAAAAGGAATTTCG TAATAAAATAAAAATTTAAGTTACCATATTCAAAAAAAGAAATTGTGCTTGAACATGAAAATAATTATAATTTTT GAACTTGTATAATGAATTTCTTCAATTCATAAGTGGGAAATTTCATATTTATGTAATAATAGATAATATGTAAGC TCTAATATAGTACTTTAGGTTATAGAATTTAATATAAAATATCAAAACATGAATTCTTGAAATTGAGTAGAGTAA TTATTTTCTGCACAATGAATCGGAGACAATAACTTTGAAGAAATATAAACAATAGAGTTCAAAAGATGTAGTCAA AAACAACAATTAATATCATAAGAATAAATTAATGAGTGTAAAAATGCATACCACGATATGTAAAAACAGAATGGA ATATAATAAAAAAAATCGAGTTCACTGAATACACAATGTTCCTTTAAGAAAATTATTCTCCTCCAATACCAACGA GATTACATCCTCTAAGGATGGAAATGATTTCATTCCCCAACTTATCCATATAAAAATAGTGGTGTTAGTATGTAA CTCAATAGGAGTAAAATACACAAATATTTAATTTTGCGAAAGTAGAAGAAGAAGATCATATTTTTTTTTTAAAAT GAGAGGATATATCACTATTTTTAAACAACAAAGGGTAGTGTTAACAAATTTTTATTGTGTCTTGTCTAAAAGGTT ACAGCTATTTGAAAAAGTTACAACACTTCGAAAAGTGAACAACATTTCATAAAAGTCGTAACTTTTCATAAAGTC GTAACTCTTCATAAATGTCGCAACTCTTCATAAAAATTACAACTATTGATAAAAGTCACCACTCTTGATAAAGAT CACCACTCTTCATTGAAGTTGCAACTTTTCATAAAAATCACATCTTTTAATAAAAAAGAAAGACTAGTTTTTGGA ATAAATTAATTTAAAAGAAAATTTTTGTTTGTGGTGGGGCGCCAAGTAGGCAGGCGTAGGGTTCTTTTTATATAA ATATATATGATATATGATTCAATATTTGATATATATATATATAGAGAGAGAGATGACAATATAAGACAATTGCAA AAAATAAAATAAAAAACTAATCGAGTAAGTAGGCAAAAAATTATTTATAAAATATATGTAGAATTTCTTTATCAG ATATGACTGCCCAAATCTTATATTCAAACTAAAATGCAAGATCAATGGTGCTATATATAGGGTTTTACACAAAAA TCAAGATCTAGTCTTGCAAATTTAAATAAAAAACAGTGGTTTACGATGAGATAATGTAGCTTTTGTAAACAATAA AACTAGAAAAATAAATGCAAAGGCATTTTAAAGGATATAATAATGAAGATCAAAGGCAGAGAAGGGAAGAGGCAG CAATATAATGAAGGTAACATCATGGTTCCATTCTAATATATATGCTATTTTTCTTTAGTAAATTTCAAAAATAAT GATACATTTTCATATTTGATAAATATTTAATGATACTATCAACATTTTATCTATATTGAGTTCCATTTATTTGAC CAAAACCTCACAAAGATGTGCTCTTCGATCTATTCAAAATTTATTCAATTTAAGGATAGCTTTAAAACATGACAA AGTTTTCTCATATATTTCTTAAATTTTATATCCAGTCTAAATACGTATATAAACTAAAATGAAGAGAATAATATG AAGCTTTATTTGATGACATTGTTGAAATAACCAAAAGCTATAAGTGATACAATAGTAAATTTACCATTGGTCAAT TCAGAATTATTTAAAAGCTAAAAAAGTCATATAAGTTGGGGTTGCTCAATGTATAGTTTTTGGCTTGTTTTAAGC ATTTTAAAACTTTTTTTAAGCGCTTTTTAACATTGCTAAACACTCAAAAAATGATAAATAGTATTTAAATTTGAT ATGATTAGCTTAAAAGTGAACTCATATACCTTCAAAGTAAAAATCCCCAATTCGAGCTTTCAAACCACTTGATTT TGTGGATGAAATTATACTGAAGTTGAATATATCACTATTTATAGGGGTTAGTGAACTAATACCTTTGATTATTTG GTAGAAATATGTATCTTAGATCACCCTAATGAGCTCCCACTTTTAAAATAGGAAAAACCTCATATGAAGTTCATC ACTGTTCATTATATATCACTTTTATTCAAAAACGTTTACAAATGTTCATTGTGACTAAATACCCTTGAGTGTCGA GTTTTCACACCAATAAGGCCTAATTAATAGGTAAACAAAACTATGTCAATCTTCAAAACGCAAATCTAATTATAT TTTTAACAAGATTAGAGGTATATATACATATTCTCTTATGTTAACTCTTATTCATTATTGAACAAACTAAGTAAG TGTACCCAAGGTCTCAAACAACAGTTGGTACATTCTTTGTATGTCTTCCTTTGTCTCTTAATAGTCGTCTCCTCC TGTCGATGATTCCTCCAAATACATTAATCAAAGGAAAATCTTTCGCCCTCAACTTGCAAACTTGTCTATCTAAAA TTGTTAACAAAGTTTCTTCATTAGAGAAACTATGATTTCTTGAATGTAGCAATTTGATGTGCCATGACTATCATC TTGATCAACATGCTTCTTAACCATCAAAAGATCCTAAACTAGATGCATGTCATGTTAGGAGACATATTAAGCTTG TATATAACTACACCAACATGCTTTAGGATCTCATAAGATCCAAAATTTCTTATTTGGGAGATTTTCAATCCAACA ACCATCATAATGAGCAACGTGATGTTATAACATCTCTCTCACACTGCCAGAACAGTCTTATACCTTGTCGGAGTG AAGGACATCCTTAACTAAGTAGATTCACTAAGCTATACTTAAAAAGCAATAAGGAATCATCTAAAATGTGTGACT CTTAACCCATATTGGCATACATGGTTTATGGGGGTTATTAATTGTCTGAACACTCCCCCATATAAATCAGTGATC AATATTAATCCCAATAATATACACTATTATGATTTGAGACTACACCCTGGAAGTGGCCGGCTCTCAAGAACCATT GCTGATCTCCAAGCCAAACCCTCATTCTGGTTGACTACAAGCTGAAGGCAAACTCAAGTATACAAAGCTTAAAAC ATAATAAAAATAATATACTCAACTCGCCACAAAATAGGCATTTAAGTCTTTAAAACATTTTTAAAAATAAATGAA ACAAACTTCTCAAACTGTAATGTATATCTATGAAGCCTCTAAATGAAAAAAATGAAGGCAGATGAGACATACGGC ATCCTAACAACTGATATAACTAAGAGTACAAGTGGAGCCCTTCGGATGTAAGGAGGCTCATCAAAGCTAATGTGA ACTCCATGTGGTATCAATGAAGCACCTATTGATGACCGTGAATACATGTATCTGCATCATGAAACGATGCAGGCC AAAGGGCTTAGTACGTGAAATGTACGAGCATGTAAAGGGAATTCAAATACATAAACATAGGCTTGAACTTTGATA TAAAGGAAACATACTTACCTATTTTTAACTCAAGAATAAAAAACATAGTTCAACTCAATGAAAAGACACTCAAGT CAGTGAAATAGGCCGCAACTCAATAATAAGATATTCGACTATGGGTAATCAACTCTGGGTACTCTATTCAATATA AAGTAAGAATACAAATGCATTATATGGAAAGACTTTAAAACGGTAGAAAACAACTCAATGTATTGAAAATTCAAT AGTAAATTAGTTTGTATGTAAGGAACAATATAAACTTTGTTTGTATATGAAAATACAAAATAAACTTTGTGTATA TAAAAGTACAAAATATCTCTGTGAAAGTTTCTCTAACCAACAACCATCACTATGAGCTTTCTGATAATACCACGT TTCGCCCATGATGTCAGAACTGTCCTATGATTTTCCAGTTCATAAGACCTACTCACTAAGTGGATCCACAAGTCT ATGCTAAAAAATATTTAAGGAATCGTCTAAAAAGTATGACTCATTCTACCCACGTTGGCTACATGATTTATGGGG GTCGTAAGTTATCTAAACTCTCCTCCATATCGATGCGTAATGCTACTCACAAATATACTAGCTCACATGTTTAAA AATATAACTCGTTTTGTTTGAGATCATTACTCAAAATCCTTCTCTTAAAAGAGATGATACTCAAACTGCTCAAAA CTCTTTTGGAAATCTCAAATTCGTCTCATCTTAAATGTAAAAATATTTACTCTTGGGAATACATAGTTATCATAT ATCATTTTAAAGAAAATGAACTCAACTCTGTTCTTTCTCAACTCAAGTGCTCAGTCTTAAACCAAATTAAAAAAA AGACTTCTCAAAATAAAGTTTATGTCGAATTATGGACGTGAACAATTCAATTCAAAGTTTTCGATAACCATAACT AAAACTAAATACTCGAGACTCAACATCTTAGAACTCAAGAACTTAAATGGTAATACTTCTTTCAAGAATGCTCGA CTCAGAAGGTTAATGCAGAATAATGTGCATGAATTACTCAACTAAAGGACTCACTGATACTACTCAATCTCAAGA TTGCTCGACTCGTAGGGTTAATGCAGAATTATGTGCATGAACTACTCAACTCAAAGACCTTCATAGGTAACATGT AGTAGCCCCATGATTTGGAATATAATCCCAAAATGATTAGGAACTCAATACTCAGGACTTAGAACTTGAAGATAA TACTACTTCTCTCAAAGATACCCAACTGACGGAGTTCATGCAGAATTTATGGGCATGAACTACTCGACTCAAGAG TCTAAAACACAATATGACACTCATGTATATAACTCTTCTCATTCTAATACTTGTTTTCTCAAAACTCGGTTTAAC TAAATAGTTGATCTCAAAGGATTCACAATTGAACTCAAAGACTTTCTTTGACTCCACTCTTAATTCTCTCTTAAA TTTGTATTTGAATTATGAATTTAAGAGTTATGATTCATGATATGGGGAATCTCAATAACAATATAGAAATTTGAT AATTAGGAATAGTACTTTTAAAAGAAAACATGAATTCAACTTAAAATCAACTTATCTAAAAAATATTCAAATATA GGGAAAGTATCCTAGACTACTGTGCTACTGATCTGAAAGTAGATGTAGGATGTGAGGATGAACTAGTCCAACACT ATGATAGCCTTACATACCTGGAATAACGAGGTTCTTGGAAAATCTTCACTTGAAGAAGAACTTGATTAGAAGCCT TGAAACCTAGCTTGAAGGTAAACAATCAAGAAAACCTTTCTTAAGATTCTTGAATTAGTTTATGAAAATCTCTAT GACCAAGCATTTTGATTTTCACTAGTGATTCATAATTGTATGGAGGAATTTGAATTGAAAAAGATGAAATGCTTG GAGAAAAGCTATCTTTGAAGAAGCTTGAAAAAGATTGGAAAGTCCTGTACTTTGATTTTCCCTTAGGATTTTGTC TTAGGGTTTGAGATAGAAAAGAATGATGGACTAAAAGATGAAAATCTAATTGTTTGGATCCTTTTTCAGCCAAGA AATCCGTTTAGGGTTTTCTTGGAGACAAACAAAATAAAAAAGACCATTTTTAATATTTTTCCGTCGGCTAATTCG TAATAACATTGTATCATGTTATTGAAAGAGTCATAACTTTTTACTCAAAAATTGGATTGATGCGAAATTAGTGGT GTTGGAAAGTAGATTCAAGTACCTCTAATTGGATAGGTTATTCCCTACATAAGTCTTTATATTCTAAAAGATATG GTTGTTTGCACTTGACCTAAGTAGAATTTTACATGAAAACTTAATAGAGAAGGAAACTTCAAGAACTCATCAAGA AATTTCAATTGCTCAATATTTATGGATAAATTTGTAGAAGAAACTCATGATTGACATGCGGGTGAATAAACCCAA CACTATGGAAGCTTACATACCTCAAAGAACTAGGTTCTTGGCGAAATCTTGAATTTCTTCAACGAACGCTTGAAA CTTTGAACTTTTTCTCTTCTTGAACTCTCAACTAAAACCCTAGGCGTATATTAGGATTATAAAAGTTAACATGAT AGGATTAGACCTTTAAAAACTTTCTAAAATGAATTAAATCTGATTTAGCATGAAAAAGACCAAAATACCCCTTAC TATTTTCGGATAACTTTTCTTAATTGGACTGCCTGACTTCAAAAAGGTATATCTCACTCATCCGACCTCAAAATT TAGCAAATTCAGTGGCGTTAGAAAGCTAATTTAAACACCTTTCATTTTCCATCTCATGGCACACATAACTCATTC TTTAAAGAGAGCTATGATCGTTCAAATTAACTCAAATCTTAGAAGAATTTAGGAATGTCTTGAACGAGCTACATC TAGTGACCTTAACACTTTGGAAAATTTTAAATTTCTTAGTAAAAACTTACTCACTATGAAGGATGGTTCAAGTCT TAGCTCAAAATTTTCCTAAGTTGCTATATATACTCATGCTCATATGTTTAAAACCAAAACCCTTCCTCGATTTGA ATTAATTACCAAAAAGATTCTCTTAAAAAGATAATGCTCAAAACTCCCCCTAAACTCATTTGGAAATCTAGGTTT CCCTTGTTTTAAATATAAAAACATTTACTCTTGGAAATATTTAGTTCTCAGATATTCACTTGAAAAAAATTAAAC TCGACTCTCATCATCTTCATACTCAAGTGCTCAAGTCCTAAAACAATTTATAACTAATTGTATAAGACTTCTCAA AATAGGGTTCATTCCGAATTATGGACGTGAACGACTCAATTCAAGGATTTCAATAACCATATATATAACTCAATA ATAGGAACTCAACAACTCCAGAACTCAATGATACTACTCATCTCAAGAATGCTCGACTCACAGGGTCTTTGCGAA ATTATTGGGCATGAACAACTCAACTCAAAGACCTTCATTTATACCATATGGTAGTCCCATAATAGGAATATAATC CCAAAAAAATTAGGAACTCAATACTCAAAAACTTAGAACTCGAAGATATTACTCATCTCAAAGATATTCAATTTA TGGAATTCATGCTGAATTATGAGCATGAACGACTTGACTCAAGGATCTCAATAATAATGTAGACTCATGAATACA CTCTTCTCATTCTCATACTCACATACTCGAGTATTAAAATAAATTATAAGTAATTGCAGAAGACTCCTTGAACAG ACTCAAAAGGACTCCTTCGAATTTTACTCTTAATGCTACCTGAATTTTGTATTATAAATTTAAGGATCATGATTA TGATATAAAGAATTTCTCAGCATATATGAAATGAACGAATTTGAGCATTGAACGTCTAACCTCATTTTTTAATTA TTGTGATATGTAGAGTGGTGCAAAATCACAGATACCTCTCTTGATGCATTTCTATAGTTACGTTGATGTGAGATT ATATATAGTTCAGCAGCAGCATGTTGGGAAAATTACTAATAACTCTTCTTTTATATCAAATTGTTGAAGCATGAT GACAACACTTGAAAAGTATCAACAATGCAGTTACGCATCTTTGGACCCGATGTTACCGGTTAGTGATACTCAGGT ATTGTTTATCTACTTTATCATGTCGTAAGTATATTATTTGTAAAGATATATATCAAGATAGTTCGATTGCGTACA CTTACATTTTGATTATGTTTGGTGAATACTATTCTAATACCTTTTTTTTTCCTAAAGCCTAACAAATAAAGATAA TTAAGATGGGAACGTAATTCAAGTACAACATGGTTCCATACGTGACATATTTACACATATAGTGGAACCAAAAGA GCAATTTTTCCTAATATCATTTTCTAAATATCACGTGTGCCCGTGATTCTTTTTTATGGACATGAATTTTTTTTT TAATATGAGTGGAAGTAAGGTTCGATCTTTCTATCTGCTTTGATATCATATTGAATCGTGTGATTGTCTCTTTAA AAAATTAAGCAAGAGCATATTTTATTAATTAATTGTCTTTCTCGACGTTTTTCTCTTTCAACAGATGAACTACAA TGAGTATGTGAGGCTAAAAGCTAGAGTTGAGCTCCTTCAACGTTCTCAAAGGTAAGATATTAGTGATGTAATTAA ATGATTTTAGTTAGATTTACATAAGTTTTTAATAAGTGAAAATTAATAGACATATTCTTGGAGAGGATTTGGGCA CACTAAACTCGAAAGAACTTGAGCAGCTTGAGCACCAATTGGATGCATCTTTGAAGAAAGTTAGATCAAAAAAGG TATATCCAAATACTATAACTTAAATATATTGTAACGATTTAATTAATAGCATGTGTCACGTTCATCTATTCTTTA GTCACAATATATAGGGGCATGTCCTTAACAACGTGCCATGCCTCGATAGTCATTTTTGTCTTTTTGTGCGTATGA ATTTAACTTTGACACAAATTTTTGTAGTAATAATAACTCATGCTTTAGCATCTTAGGAAGCAGTCATATGAAAAA CAGAAGCATATATATATATTACATGAGTTAATTTAATTTAATATAAAATTTAATAAAATTGTGTCTCGCTATAAA TAATTTTATTAAAAAATTATATAAATATATTATTTTTTTAACTGGCCGCAAAGTTATATAAATTGATAGAGAAAG AGGTTTTGGTGTAAGGTTCATTTTCCAACAATTAGTTTTATAATTTGTAAGTGCACACTTTATCAGACTCAATCT ATGCTGGATCAGCTGGCAGACCTTCAAGAAAAGGTACACTGCCTTAACATTACAAAATTAATTTATTTCATCAAA AGCATATCATAAAATTCTGACAAATAAATATATTAGGAGCAAATGCTGGAAGAAGCAAATAAACAACTAAAAAAC AAGGTACATATCTATATATGTGTGTTAATTAATTAAGTTGATTTTGTATTTTTGTTTAATGAATAATTGTTTGTG ATCATCAGCTGGAAGAAAGTGCAGCTAGAATTCCACTTGGATTGTCATGGGGAAATAATGGAGGACAAACAATGG AATACAATCGACTCCCTCCACAAACTACTGCACAACCTTTCTTTCAACCTCTCCGTTTGAATTCTTCATCGCCTC AATTCGGGTAAGTATCTTATTTTATATGACTTAGTTTGACTTGACATAAAGTTTAATAAAGAAAGAAAGACTTTT AAAACTTATAGTGTAAAATAAGTGAATAGATATATATGTGGTTGTACTAACACTACAACAAAAATAATTTTCAGC GGCATTAAATATTGACATTAATAATGAGTGCTAAAGACTTTATCGGTATTAGTTAAGTGTCATTAGGATCAATGT CGTTAAAGGCTTCACGGACATATACAAAGAGTGACAATTGCCGCTAATGATTATTTTTGTTGTAGTGAAAATGAG TATTTTAAAGTTAAATTGTTACATAATATAGAAATATGTCAGAAACAGGACAAATATACCACCGAACTATCATAT ATGTTATGGAGATATTCTCAGTCATACTTCTGCGACATTGGTACTCATGTCGTCCAAAAACTAGAACATATATAT ACCCTTTATATATTAACGAAGATACAAGTGTCATAATCTTATGCACCGATTCGATATTTATTAAATATCGAATCG ACGGATAAAATTATGTCACGTGTCCCTATTAAGTCTTCTATTAGAGTAAAAAGCATATATTCTCTAGTTTTTGAA CGAAAAAAGGTATTAATGTCTCAAAAGTATAACGAAAAGCATTTGCATACAATTTATGATAATTTGGGGCATATT AATTTATCATTCCCCCTTTTTTTGGCACTGATTAAAAAGAAAAAGAAAGTTATAAAAATTGGGATAGAGGGAATA ATTGTTTCATAGGGAAAACTTAGAAGCTTCTCAGTATGTCAGTGAGAATGTGTTTCCTAATTAGTGAACTATGGT TTGGTGAAAAATAAAGAGAAAAAAATCAGTACAAATTTTCCACTGATTAGCAATGAGAAAAATATTTGTTTCTAG TAGTATGAGGAGAGGATAGTCCGCATAAATAATCCTTAAATTTGTGGATAAATAAACTATTTTCAATAGATTATC GTCTCAAAATAAAATAAAATGATTGCAAGAAAAGAATAATAGGTATGCTGGTAATATGTATAATACACTCAAATT TATTTGCTGTCCATGCAGATACAATCCAAATATGGGTGCAAATGATCATGAGGTTAATGCAGCAACAACTGCTCA TAATATTAATGGATTTATTCCAGGGTGGATGCTCTAA Wild-type Solyc12g038510 coding sequence (SEQ ID NO: 7) ATGGGAAGAGGAAGAGTAGAACTAAAGAGAATAGAGAACAAAATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGACTTCTTAAGAAAGCTTATGAGTTATCTATACTTTGTGATGCTGAAGTTGCTCTCATCATCTTCTCTAGC CGCGGAAAACTCTATGAGTTTTCAAGTGCTTCCAGCATGATGACAACACTTGAAAAGTATCAACAATGCAGTTAC GCATCTTTGGACCCGATGTTACCGGTTAGTGATACTCAGATGAACTACAATGAGTATGTGAGGCTAAAAGCTAGA GTTGAGCTCCTTCAACGTTCTCAAAGACATATTCTTGGAGAGGATTTGGGCACACTAAACTCGAAAGAACTTGAG CAGCTTGAGCACCAATTGGATGCATCTTTGAAGAAAGTTAGATCAAAAAAGACTCAATCTATGCTGGATCAGCTG GCAGACCTTCAAGAAAAGGAGCAAATGCTGGAAGAAGCAAATAAACAACTAAAAAACAAGCTGGAAGAAAGTGCA GCTAGAATTCCACTTGGATTGTCATGGGGAAATAATGGAGGACAAACAATGGAATACAATCGACTCCCTCCACAA ACTACTGCACAACCTTTCTTTCAACCTCTCCGTTTGAATTCTTCATCGCCTCAATTCGGATACAATCCAAATATG GGTGCAAATGATCATGAGGTTAATGCAGCAACAACTGCTCATAATATTAATGGATTTATTCCAGGGTGGATGCTC TAA Mutant Solyc12g038510 gene allele j2^TE (SEQ ID NO: 8) ATGGGAAGAGGAAGAGTAGAACTAAAGAGAATAGAGAACAAAATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGACTTCTTAAGAAAGCTTATGAGTTATCTATACTTTGTGATGCTGAAGTTGCTCTCATCATCTTCTCTAGC CGCGGAAAACTCTATGAGTTTTCAAGTGCTTCCAGGTATATATATATATACATATG ATA TGTTTTTCTTCTTTTTGTGTGTGCGTATGTGTTTACTTACTTTCATTAATTAACTCAACCATATATATACATCTC TCACCTCAATTATATATATGTTTGAGATCTGAATGTCTACGGACTCCATTTAGGTACATATCTTTGTTTAGATCA TAAATCATCTATCTTCATTCCTAAGATCTACTAATATATATGTATAAGAAGATCCATCCATCTATTAGGTTTTTC AACAACATATACAGTGAAATCTTATATGTGGGCCCACGTATAGCCATATGAGAAAATAGTGTGCACGTAAACATT ATCATTACTTAATTATAGGAATATACATCCATTAGGTTTATCAACAACAATAAAATCCTCTAAATGGAGTCTAGT CATAGGTCTAGCCGTTTGAAAATGTAAAATATATGCCGATCTTATCACTATGTCATAATAATAGATATGTTGTTA TTGAAAGATTCTCAATCTTTTTTTTTCTTCAAGGTAGAGATTCTTAAGTGGATTCATGTTTTTTTTATCAAAAAA GAAAAAAACAAAAGTGTCCATTTGTTCATCTAATGGGTTTTCCATGTTACCAATTCACTACACTGTTGAGATTTG ATTATCAGATGTGTCAAGTTTCGTTTGGTTCCCTAGAAGGGAGAAAAGGCTGCTTATGCAGGCAGGGTATTAAAG ATGATATTAATATCTGCAGTAATCAGTAACAGAATATATAAACTTAATAATAAACTTGAAGGTACTTARTTATCC AGCAGATAATCTTCTGTCTCACCGTACACTTTTGTTATATCATAAGCATAAGAATTGTTTTATCAAATATTACCA AACAAAACTTAGTTTTGTTTGGTAATATTTTATAAAATATGTTACCGAAAGTTACTTCCTATAACATATTTTATA AAGAAAAAAATTAAAAACTCCATATACCTAAGAAATGTAACCCCCCCTCCATAACAACAATTTAACAAAAATAAA AACCTACTTTTTTTGAATTTGGTAAATTAGTTTTCTATCCTTTTTAGTAACTTCCTTTCTTATTTTCTTTTTATA TTGGTAAAGTTTAATATTACACATTATTTTAACATGTTATAATTTTTTGTGATGCTTAATTATTTGATACATGTA ATAAACCATATATTAGAGCTATAAATCAATGACAATGCATGTAGATACAACTCATTTATGATATATTTTGTTTAT ATATATAACCAATTAGATAATTTGTCTGCGCTTTGTGCAGTCATAAATAATAATTGCATTGAACTTGCAAATATT TTTTTTTAATATCCATACATTAAAAAAAAAGAAAGAGGAAAATTGGTTCCTAAAATATTAGCAATATTCAAACAT TTATTTGATTATTAATCATTATCACATAACTTAAGAACGTCTAATGAATGAATTATTCACGAAATAATAAATCAT TGGTTCTAAAAAGGAATTTCGTAATAAAATAAAAATTTAAGTTACCATATTCAAAAAAAGAAATTGTGCTTGAAC ATGAAAATAATTATAATTTTTGAACTTGTATAATGAATTTCTTCAATTCATAAGTGGGAAATTTCATATTTATGT AATAATAGATAATATGTAAGCTCTAATATAGTACTTTAGGTTATAGAATTTAATATAAAATATCAAAACATGAAT TCTTGAAATTGAGTAGAGTAATTATTTTCTGCACAATGAATCGGAGACAATAACTTTGAAGAAATATAAACAATA GAGTTCAAAAGATGTAGTCAAAAACAACAATTAATATCATAAGAATAAATTAATGAGTGTAAAAATGCATACCAC GATATGTAAAAACAGAATGGAATATAATAAAAAAAATCGAGTTCACTGAATACACAATGTTCCTTTAAGAAAATT ATTCTCCTCCAATACCAACGAGATTACATCCTCTAAGGATGGAAATGATTTCATTCCCCAACTTATCCATATAAA AATAGTGGTGTTAGTATGTAACTCAATAGGAGTAAAATACACAAATATTTAATTTTGCGAAAGTAGAAGAAGAAG ATCATATTTTTTTTTTAAAATGAGAGGATATATCACTATTTTTAAACAACAAAGGGTAGTGTTAACAAATTTTTA TTGTGTCTTGTCTAAAAGGTTACAGCTATTTGAAAAAGTTACAACACTTCGAAAAGTGAACAACATTTCATAAAA GTCGTAACTTTTCATAAAGTCGTAACTCTTCATAAATGTCGCAACTCTTCATAAAAATTACAACTATTGATAAAA GTCACCACTCTTGATAAAGATCACCACTCTTCATTGAAGTTGCAACTTTTCATAAAAATCACATCTTTTAATAAA AAAGAAAGACTAGTTTTTGGAATAAATTAATTTAAAAGAAAATTTTTGTTTGTGGTGGGGCGCCAAGTAGGCAGG CGTAGGGTTCTTTTTATATAAATATATATGATATATGATTCAATATTTGATATATATATATATAGAGAGAGAGAT GACAATATAAGACAATTGCAAAAAATAAAATAAAAAACTAATCGAGTAAGTAGGCAAAAAATTATTTATAAAATA TATGTAGAATTTCTTTATCAGATATGACTGCCCAAATCTTATATTCAAACTAAAATGCAAGATCAATGGTGCTAT ATATAGGGTTTTACACAAAAATCAAGATCTAGTCTTGCAAATTTAAATAAAAAACAGTGGTTTACGATGAGATAA TGTAGCTTTTGTAAACAATAAAACTAGAAAAATAAATGCAAAGGCATTTTAAAGGATATAATAATGAAGATCAAA GGCAGAGAAGGGAAGAGGCAGCAATATAATGAAGGTAACATCATGGTTCCATTCTAATATATATGCTATTTTTCT TTAGTAAATTTCAAAAATAATGATACATTTTCATATTTGATAAATATTTAATGATACTATCAACATTTTATCTAT ATTGAGTTCCATTTATTTGACCAAAACCTCACAAAGATGTGCTCTTCGATCTATTCAAAATTTATTCAATTTAAG GATAGCTTTAAAACATGACAAAGTTTTCTCATATATTTCTTAAATTTTATATCCAGTCTAAATACGTATATAAAC TAAAATGAAGAGAATAATATGAAGCTTTATTTGATGACATTGTTGAAATAACCAAAAGCTATAAGTGATACAATA GTAAATTTACCATTGGTCAATTCAGAATTATTTAAAAGCTAAAAAAGTCATATAAGTTGGGGTTGCTCAATGTAT AGTTTTTGGCTTGTTTTAAGCATTTTAAAACTTTTTTTAAGCGCTTTTTAACATTGCTAAACACTCAAAAAATGA TAAATAGTATTTAAATTTGATATGATTAGCTTAAAAGTGAACTCATATACCTTCAAAGTAAAAATCCCCAATTCG AGCTTTCAAACCACTTGATTTTGTGGATGAAATTATACTGAAGTTGAATATATCACTATTTATAGGGGTTAGTGA ACTAATACCTTTGATTATTTGGTAGAAATATGTATCTTAGATCACCCTAATGAGCTCCCACTTTTAAAATAGGAA AAACCTCATATGAAGTTCATCACTGTTCATTATATATCACTTTTATTCAAAAACGTTTACAAATGTTCATTGTGA CTAAATACCCTTGAGTGTCGAGTTTTCACACCAATAAGGCCTAATTAATAGGTAAACAAAACTATGTCAATCTTC AAAACGCAAATCTAATTATATTTTTAACAAGATTAGAGGTATATATACATATTCTCTTATGTTAACTCTTATTCA TTATTGAACAAACTAAGTAAGTGTACCCAAGGTCTCAAACAACAGTTGGTACATTCTTTGTATGTCTTCCTTTGT CTCTTAATAGTCGTCTCCTCCTGTCGATGATTCCTCCAAATACATTAATCAAAGGAAAATCTTTCGCCCTCAACT TGCAAACTTGTCTATCTAAAATTGTTAACAAAGTTTCTTCATTAGAGAAACTATGATTTCTTGAATGTAGCAATT TGATGTGCCATGACTATCATCTTGATCAACATGCTTCTTAACCATCAAAAGATCCTAAACTAGATGCATGTCATG TTAGGAGACATATTAAGCTTGTATATAACTACACCAACATGCTTTAGGATCTCATAAGATCCAAAATTTCTTATT TGGGAGATTTTCAATCCAACAACCATCATAATGAGCAACGTGATGTTATAACATCTCTCTCACACTGCCAGAACA GTCTTATACCTTGTCGGAGTGAAGGACATCCTTAACTAAGTAGATTCACTAAGCTATACTTAAAAAGCAATAAGG AATCATCTAAAATGTGTGACTCTTAACCCATATTGGCATACATGGTTTATGGGGGTTATTAATTGTCTGAACACT CCCCCATATAAATCAGTGATCAATATTAATCCCAATAATATACACTATTATGATTTGAGACTACACCCTGGAAGT GGCCGGCTCTCAAGAACCATTGCTGATCTCCAAGCCAAACCCTCATTCTGGTTGACTACAAGCTGAAGGCAAACT CAAGTATACAAAGCTTAAAACATAATAAAAATAATATACTCAACTCGCCACAAAATAGGCATTTAAGTCTTTAAA ACATTTTTAAAAATAAATGAAACAAACTTCTCAAACTGTAATGTATATCTATGAAGCCTCTAAATGAAAAAAATG AAGGCAGATGAGACATACGGCATCCTAACAACTGATATAACTAAGAGTACAAGTGGAGCCCTTCGGATGTAAGGA GGCTCATCAAAGCTAATGTGAACTCCATGTGGTATCAATGAAGCACCTATTGATGACCGTGAATACATGTATCTG CATCATGAAACGATGCAGGCCAAAGGGCTTAGTACGTGAAATGTACGAGCATGTAAAGGGAATTCAAATACATAA ACATAGGCTTGAACTTTGATATAAAGGAAACATACTTACCTATTTTTAACTCAAGAATAAAAAACATAGTTCAAC TCAATGAAAAGACACTCAAGTCAGTGAAATAGGCCGCAACTCAATAATAAGATATTCGACTATGGGTAATCAACT CTGGGTACTCTATTCAATATAAAGTAAGAATACAAATGCATTATATGGAAAGACTTTAAAACGGTAGAAAACAAC TCAATGTATTGAAAATTCAATAGTAAATTAGTTTGTATGTAAGGAACAATATAAACTTTGTTTGTATATGAAAAT ACAAAATAAACTTTGTGTATATAAAAGTACAAAATATCTCTGTGAAAGTTTCTCTAACCAACAACCATCACTATG AGCTTTCTGATAATACCACGTTTCGCCCATGATGTCAGAACTGTCCTATGATTTTCCAGTTCATAAGACCTACTC ACTAAGTGGATCCACAAGTCTATGCTAAAAAATATTTAAGGAATCGTCTAAAAAGTATGACTCATTCTACCCACG TTGGCTACATGATTTATGGGGGTCGTAAGTTATCTAAACTCTCCTCCATATCGATGCGTAATGCTACTCACAAAT ATACTAGCTCACATGTTTAAAAATATAACTCGTTTTGTTTGAGATCATTACTCAAAATCCTTCTCTTAAAAGAGA TGATACTCAAACTGCTCAAAACTCTTTTGGAAATCTCAAATTCGTCTCATCTTAAATGTAAAAATATTTACTCTT GGGAATACATAGTTATCATATATCATTTTAAAGAAAATGAACTCAACTCTGTTCTTTCTCAACTCAAGTGCTCAG TCTTAAACCAAATTAAAAAAAAGACTTCTCAAAATAAAGTTTATGTCGAATTATGGACGTGAACAATTCAATTCA AAGTTTTCGATAACCATAACTAAAACTAAATACTCGAGACTCAACATCTTAGAACTCAAGAACTTAAATGGTAAT ACTTCTTTCAAGAATGCTCGACTCAGAAGGTTAATGCAGAATAATGTGCATGAATTACTCAACTAAAGGACTCAC TGATACTACTCAATCTCAAGATTGCTCGACTCGTAGGGTTAATGCAGAATTATGTGCATGAACTACTCAACTCAA AGACCTTCATAGGTAACATGTAGTAGCCCCATGATTTGGAATATAATCCCAAAATGATTAGGAACTCAATACTCA GGACTTAGAACTTGAAGATAATACTACTTCTCTCAAAGATACCCAACTGACGGAGTTCATGCAGAATTTATGGGC ATGAACTACTCGACTCAAGAGTCTAAAACACAATATGACACTCATGTATATAACTCTTCTCATTCTAATACTTGT TTTCTCAAAACTCGGTTTAACTAAATAGTTGATCTCAAAGGATTCACAATTGAACTCAAAGACTTTCTTTGACTC CACTCTTAATTCTCTCTTAAATTTGTATTTGAATTATGAATTTAAGAGTTATGATTCATGATATGGGGAATCTCA ATAACAATATAGAAATTTGATAATTAGGAATAGTACTTTTAAAAGAAAACATGAATTCAACTTAAAATCAACTTA TCTAAAAAATATTCAAATATAGGGAAAGTATCCTAGACTACTGTGCTACTGATCTGAAAGTAGATGTAGGATGTG AGGATGAACTAGTCCAACACTATGATAGCCTTACATACCTGGAATAACGAGGTTCTTGGAAAATCTTCACTTGAA GAAGAACTTGATTAGAAGCCTTGAAACCTAGCTTGAAGGTAAACAATCAAGAAAACCTTTCTTAAGATTCTTGAA TTAGTTTATGAAAATCTCTATGACCAAGCATTTTGATTTTCACTAGTGATTCATAATTGTATGGAGGAATTTGAA TTGAAAAAGATGAAATGCTTGGAGAAAAGCTATCTTTGAAGAAGCTTGAAAAAGATTGGAAAGTCCTGTACTTTG ATTTTCCCTTAGGATTTTGTCTTAGGGTTTGAGATAGAAAAGAATGATGGACTAAAAGATGAAAATCTAATTGTT TGGATCCTTTTTCAGCCAAGAAATCCGTTTAGGGTTTTCTTGGAGACAAACAAAATAAAAAAGACCATTTTTAAT ATTTTTCCGTCGGCTAATTCGTAATAACATTGTATCATGTTATTGAAAGAGTCATAACTTTTTACTCAAAAATTG GATTGATGCGAAATTAGTGGTGTTGGAAAGTAGATTCAAGTACCTCTAATTGGATAGGTTATTCCCTACATAAGT CTTTATATTCTAAAAGATATGGTTGTTTGCACTTGACCTAAGTAGAATTTTACATGAAAACTTAATAGAGAAGGA AACTTCAAGAACTCATCAAGAAATTTCAATTGCTCAATATTTATGGATAAATTTGTAGAAGAAACTCATGATTGA CATGCGGGTGAATAAACCCAACACTATGGAAGCTTACATACCTCAAAGAACTAGGTTCTTGGCGAAATCTTGAAT TTCTTCAACGAACGCTTGAAACTTTGAACTTTTTCTCTTCTTGAACTCTCAACTAAAACCCTAGGCGTATATTAG GATTATAAAAGTTAACATGATAGGATTAGACCTTTAAAAACTTTCTAAAATGAATTAAATCTGATTTAGCATGAA AAAGACCAAAATACCCCTTACTATTTTCGGATAACTTTTCTTAATTGGACTGCCTGACTTCAAAAAGGTATATCT CACTCATCCGACCTCAAAATTTAGCAAATTCAGTGGCGTTAGAAAGCTAATTTAAACACCTTTCATTTTCCATCT CATGGCACACATAACTCATTCTTTAAAGAGAGCTATGATCGTTCAAATTAACTCAAATCTTAGAAGAATTTAGGA ATGTCTTGAACGAGCTACATCTAGTGACCTTAACACTTTGGAAAATTTTAAATTTCTTAGTAAAAACTTACTCAC TATGAAGGATGGTTCAAGTCTTAGCTCAAAATTTTCCTAAGTTGCTATATATACTCATGCTCATATGTTTAAAAC CAAAACCCTTCCTCGATTTGAATTAATTACCAAAAAGATTCTCTTAAAAAGATAATGCTCAAAACTCCCCCTAAA CTCATTTGGAAATCTAGGTTTCCCTTGTTTTAAATATAAAAACATTTACTCTTGGAAATATTTAGTTCTCAGATA TTCACTTGAAAAAAATTAAACTCGACTCTCATCATCTTCATACTCAAGTGCTCAAGTCCTAAAACAATTTATAAC TAATTGTATAAGACTTCTCAAAATAGGGTTCATTCCGAATTATGGACGTGAACGACTCAATTCAAGGATTTCAAT AACCATATATATAACTCAATAATAGGAACTCAACAACTCCAGAACTCAATGATACTACTCATCTCAAGAATGCTC GACTCACAGGGTCTTTGCGAAATTATTGGGCATGAACAACTCAACTCAAAGACCTTCATTTATACCATATGGTAG TCCCATAATAGGAATATAATCCCAAAAAAATTAGGAACTCAATACTCAAAAACTTAGAACTCGAAGATATTACTC ATCTCAAAGATATTCAATTTATGGAATTCATGCTGAATTATGAGCATGAACGACTTGACTCAAGGATCTCAATAA TAATGTAGACTCATGAATACACTCTTCTCATTCTCATACTCACATACTCGAGTATTAAAATAAATTATAAGTAAT TGCAGAAGACTCCTTGAACAGACTCAAAAGGACTCCTTCGAATTTTACTCTTAATGCTACCTGAATTTTGTATTA TAAATTTAAGGATCATGATTATGATATAAAGAATTTCTCAGCATATATGAAATGAACGAATTTGAGCATTGAACG TCTAACCTCATTTTTTAATTATTGTGATATGTAGAGTGGTGCAAAATCACAGATACCTCTCTTGATGCATTTCTA TAGTTACGTTGATGTGAGATTATATATAGTTCAGCAGCAGCATGTTGGGAAAATTACTAATAACTCTTCTTTTAT ATCAAATTGTTGAAGCATGATGACAACACTTGAAAAGTATCAACAATGCAGTTACGCATCTTTGGACCCGATGTT ACCGGTTAGTGATACTCAGGTATTGTTTATCTACTTTATCATGTCGTAAGTATATTATTTGTAAAGATATATATC AAGATAGTTCGATTGCGTACACTTACATTTTGATTATGTTTGGTGAATACTATTCTAATACCTTTTTTTTTCCTA AAGCCTAACAAATAAAGATAATTAAGATGGGAACGTAATTCAAGTACAACATGGTTCCATACGTGACATATTTAC ACATATAGTGGAACCAAAAGAGCAATTTTTCCTAATATCATTTTCTAAATATCACGTGTGCCCGTGATTCTTTTT TATGGACATGAATTTTTTTTTTAATATGAGTGGAAGTAAGGTTCGATCTTTCTATCTGCTTTGATATCATATTGA ATCGTGTGATTGTCTCTTTAAAAAATTAAGCAAGAGCATATTTTATTAATTAATTGTCTTTCTCGACGTTTTTCT CTTTCAACAGATGAACTACAATGAGTATGTGAGGCTAAAAGCTAGAGTTGAGCTCCTTCAACGTTCTCAAAGGTA AGATATTAGTGATGTAATTAAATGATTTTAGTTAGATTTACATAAGTTTTTAATAAGTGAAAATTAATAGACATA TTCTTGGAGAGGATTTGGGCACACTAAACTCGAAAGAACTTGAGCAGCTTGAGCACCAATTGGATGCATCTTTGA AGAAAGTTAGATCAAAAAAGGTATATCCAAATACTATAACTTAAATATATTGTAACGATTTAATTAATAGCATGT GTCACGTTCATCTATTCTTTAGTCACAATATATAGGGGCATGTCCTTAACAACGTGCCATGCCTCGATAGTCATT TTTGTCTTTTTGTGCGTATGAATTTAACTTTGACACAAATTTTTGTAGTAATAATAACTCATGCTTTAGCATCTT AGGAAGCAGTCATATGAAAAACAGAAGCATATATATATATTACATGAGTTAATTTAATTTAATATAAAATTTAAT AAAATTGTGTCTCGCTATAAATAATTTTATTAAAAAATTATATAAATATATTATTTTTTTAACTGGCCGCAAAGT TATATAAATTGATAGAGAAAGAGGTTTTGGTGTAAGGTTCATTTTCCAACAATTAGTTTTATAATTTGTAAGTGC ACACTTTATCAGACTCAATCTATGCTGGATCAGCTGGCAGACCTTCAAGAAAAGGTACACTGCCTTAACATTACA AAATTAATTTATTTCATCAAAAGCATATCATAAAATTCTGACAAATAAATATATTAGGAGCAAATGCTGGAAGAA GCAAATAAACAACTAAAAAACAAGGTACATATCTATATATGTGTGTTAATTAATTAAGTTGATTTTGTATTTTTG TTTAATGAATAATTGTTTGTGATCATCAGCTGGAAGAAAGTGCAGCTAGAATTCCACTTGGATTGTCATGGGGAA ATAATGGAGGACAAACAATGGAATACAATCGACTCCCTCCACAAACTACTGCACAACCTTTCTTTCAACCTCTCC GTTTGAATTCTTCATCGCCTCAATTCGGGTAAGTATCTTATTTTATATGACTTAGTTTGACTTGACATAAAGTTT AATAAAGAAAGAAAGACTTTTAAAACTTATAGTGTAAAATAAGTGAATAGATATATATGTGGTTGTACTAACACT ACAACAAAAATAATTTTCAGCGGCATTAAATATTGACATTAATAATGAGTGCTAAAGACTTTATCGGTATTAGTT AAGTGTCATTAGGATCAATGTCGTTAAAGGCTTCACGGACATATACAAAGAGTGACAATTGCCGCTAATGATTAT TTTTGTTGTAGTGAAAATGAGTATTTTAAAGTTAAATTGTTACATAATATAGAAATATGTCAGAAACAGGACAAA TATACCACCGAACTATCATATATGTTATGGAGATATTCTCAGTCATACTTCTGCGACATTGGTACTCATGTCGTC CAAAAACTAGAACATATATATACCCTTTATATATTAACGAAGATACAAGTGTCATAATCTTATGCACCGATTCGA TATTTATTAAATATCGAATCGACGGATAAAATTATGTCACGTGTCCCTATTAAGTCTTCTATTAGAGTAAAAAGC ATATATTCTCTAGTTTTTGAACGAAAAAAGGTATTAATGTCTCAAAAGTATAACGAAAAGCATTTGCATACAATT TATGATAATTTGGGGCATATTAATTTATCATTCCCCCTTTTTTTGGCACTGATTAAAAAGAAAAAGAAAGTTATA AAAATTGGGATAGAGGGAATAATTGTTTCATAGGGAAAACTTAGAAGCTTCTCAGTATGTCAGTGAGAATGTGTT TCCTAATTAGTGAACTATGGTTTGGTGAAAAATAAAGAGAAAAAAATCAGTACAAATTTTCCACTGATTAGCAAT GAGAAAAATATTTGTTTCTAGTAGTATGAGGAGAGGATAGTCCGCATAAATAATCCTTAAATTTGTGGATAAATA AACTATTTTCAATAGATTATCGTCTCAAAATAAAATAAAATGATTGCAAGAAAAGAATAATAGGTATGCTGGTAA TATGTATAATACACTCAAATTTATTTGCTGTCCATGCAGATACAATCCAAATATGGGTGCAAATGATCATGAGGT TAATGCAGCAACAACTGCTCATAATATTAATGGATTTATTCCAGGGTGGATGCTCTAA Mutant Solyc12g038510 gene allele j2^stop (SEQ ID NO: 9) ATGGGAAGAGGAAGAGTAGAACTAAAGAGAATAGAGAACAAAATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGACTTCTTAAGAAAGCTTATGAGTTATCTATACTTTGTGATGCTGAAGTTGCTCTCATCATCTTCTCTAGC CGCGGAAAACTCTATGAGTTTTCAAGTGCTTCCAGGTATATATATATATACATATGTTTTTCTTCTTTTTGTGTG TGCGTATGTGTTTACTTACTTTCATTAATTAACTCAACCATATATATACATCTCTCACCTCAATTATATATATGT TTGAGATCTGAATGTCTACGGACTCCATTTAGGTACATATCTTTGTTTAGATCATAAATCATCTATCTTCATTCC TAAGATCTACTAATATATATGTATAAGAAGATCCATCCATCTATTAGGTTTTTCAACAACATATACAGTGAAATC TTATATGTGGGCCCACGTATAGCCATATGAGAAAATAGTGTGCACGTAAACATTATCATTACTTAATTATAGGAA TATACATCCATTAGGTTTATCAACAACAATAAAATCCTCTAAATGGAGTCTAGTCATAGGTCTAGCCGTTTGAAA ATGTAAAATATATGCCGATCTTATCACTATGTCATAATAATAGATATGTTGTTATTGAAAGATTCTCAATCTTTT TTTTTCTTCAAGGTAGAGATTCTTAAGTGGATTCATGTTTTTTTTATCAAAAAAGAAAAAAACAAAAGTGTCCAT TTGTTCATCTAATGGGTTTTCCATGTTACCAATTCACTACACTGTTGAGATTTGATTATCAGATGTGTCAAGTTT CGTTTGGTTCCCTAGAAGGGAGAAAAGGCTGCTTATGCAGGCAGGGTATTAAAGATGATATTAATATCTGCAGTA ATCAGTAACAGAATATATAAACTTAATAATAAACTTGAAGGTACTTAATTATCCAGCAGATAATCTTCTGTCTCA CCGTACACTTTTGTTATATCATAAGCATAAGAATTGTTTTATCAAATATTACCAAACAAAACTTAGTTTTGTTTG GTAATATTTTATAAAATATGTTACCGAAAGTTACTTCCTATAACATATTTTATAAAGAAAAAAATTAAAAACTCC ATATACCTAAGAAATGTAACCCCCCCTCCATAACAACAATTTAACAAAAATAAAAACCTACTTTTTTTGAATTTG GTAAATTAGTTTTCTATCCTTTTTAGTAACTTCCTTTCTTATTTTCTTTTTATATTGGTAAAGTTTAATATTACA CATTATTTTAACATGTTATAATTTTTTGTGATGCTTAATTATTTGATACATGTAATAAACCATATATTAGAGCTA TAAATCAATGACAATGCATGTAGATACAACTCATTTATGATATATTTTGTTTATATATATAACCAATTAGATAAT TTGTCTGCGCTTTGTGCAGTCATAAATAATAATTGCATTGAACTTGCAAATATTTTTTTTTAATATCCATACATT AAAAAAAAAGAAAGAGGAAAATTGGTTCCTAAAATATTAGCAATATTCAAACATTTATTTGATTATTAATCATTA TCACATAACTTAAGAACGTCTAATGAATGAATTATTCACGAAATAATAAATCATTGGTTCTAAAAAGGAATTTCG TAATAAAATAAAAATTTAAGTTACCATATTCAAAAAAAGAAATTGTGCTTGAACATGAAAATAATTATAATTTTT GAACTTGTATAATGAATTTCTTCAATTCATAAGTGGGAAATTTCATATTTATGTAATAATAGATAATATGTAAGC TCTAATATAGTACTTTAGGTTATAGAATTTAATATAAAATATCAAAACATGAATTCTTGAAATTGAGTAGAGTAA TTATTTTCTGCACAATGAATCGGAGACAATAACTTTGAAGAAATATAAACAATAGAGTTCAAAAGATGTAGTCAA AAACAACAATTAATATCATAAGAATAAATTAATGAGTGTAAAAATGCATACCACGATATGTAAAAACAGAATGGA ATATAATAAAAAAAATCGAGTTCACTGAATACACAATGTTCCTTTAAGAAAATTATTCTCCTCCAATACCAACGA GATTACATCCTCTAAGGATGGAAATGATTTCATTCCCCAACTTATCCATATAAAAATAGTGGTGTTAGTATGTAA CTCAATAGGAGTAAAATACACAAATATTTAATTTTGCGAAAGTAGAAGAAGAAGATCATATTTTTTTTTTAAAAT GAGAGGATATATCACTATTTTTAAACAACAAAGGGTAGTGTTAACAAATTTTTATTGTGTCTTGTCTAAAAGGTT ACAGCTATTTGAAAAAGTTACAACACTTCGAAAAGTGAACAACATTTCATAAAAGTCGTAACTTTTCATAAAGTC GTAACTCTTCATAAATGTCGCAACTCTTCATAAAAATTACAACTATTGATAAAAGTCACCACTCTTGATAAAGAT CACCACTCTTCATTGAAGTTGCAACTTTTCATAAAAATCACATCTTTTAATAAAAAAGAAAGACTAGTTTTTGGA ATAAATTAATTTAAAAGAAAATTTTTGTTTGTGGTGGGGCGCCAAGTAGGCAGGCGTAGGGTTCTTTTTATATAA ATATATATGATATATGATTCAATATTTGATATATATATATATAGAGAGAGAGATGACAATATAAGACAATTGCAA AAAATAAAATAAAAAACTAATCGAGTAAGTAGGCAAAAAATTATTTATAAAATATATGTAGAATTTCTTTATCAG ATATGACTGCCCAAATCTTATATTCAAACTAAAATGCAAGATCAATGGTGCTATATATAGGGTTTTACACAAAAA TCAAGATCTAGTCTTGCAAATTTAAATAAAAAACAGTGGTTTACGATGAGATAATGTAGCTTTTGTAAACAATAA AACTAGAAAAATAAATGCAAAGGCATTTTAAAGGATATAATAATGAAGATCAAAGGCAGAGAAGGGAAGAGGCAG CAATATAATGAAGGTAACATCATGGTTCCATTCTAATATATATGCTATTTTTCTTTAGTAAATTTCAAAAATAAT GATACATTTTCATATTTGATAAATATTTAATGATACTATCAACATTTTATCTATATTGAGTTCCATTTATTTGAC CAAAACCTCACAAAGATGTGCTCTTCGATCTATTCAAAATTTATTCAATTTAAGGATAGCTTTAAAACATGACAA AGTTTTCTCATATATTTCTTAAATTTTATATCCAGTCTAAATACGTATATAAACTAAAATGAAGAGAATAATATG AAGCTTTATTTGATGACATTGTTGAAATAACCAAAAGCTATAAGTGATACAATAGTAAATTTACCATTGGTCAAT TCAGAATTATTTAAAAGCTAAAAAAGTCATATAAGTTGGGGTTGCTCAATGTATAGTTTTTGGCTTGTTTTAAGC ATTTTAAAACTTTTTTTAAGCGCTTTTTAACATTGCTAAACACTCAAAAAATGATAAATAGTATTTAAATTTGAT ATGATTAGCTTAAAAGTGAACTCATATACCTTCAAAGTAAAAATCCCCAATTCGAGCTTTCAAACCACTTGATTT TGTGGATGAAATTATACTGAAGTTGAATATATCACTATTTATAGGGGTTAGTGAACTAATACCTTTGATTATTTG GTAGAAATATGTATCTTAGATCACCCTAATGAGCTCCCACTTTTAAAATAGGAAAAACCTCATATGAAGTTCATC ACTGTTCATTATATATCACTTTTATTCAAAAACGTTTACAAATGTTCATTGTGACTAAATACCCTTGAGTGTCGA GTTTTCACACCAATAAGGCCTAATTAATAGGTAAACAAAACTATGTCAATCTTCAAAACGCAAATCTAATTATAT TTTTAACAAGATTAGAGGTATATATACATATTCTCTTATGTTAACTCTTATTCATTATTGAACAAACTAAGTAAG TGTACCCAAGGTCTCAAACAACAGTTGGTACATTCTTTGTATGTCTTCCTTTGTCTCTTAATAGTCGTCTCCTCC TGTCGATGATTCCTCCAAATACATTAATCAAAGGAAAATCTTTCGCCCTCAACTTGCAAACTTGTCTATCTAAAA TTGTTAACAAAGTTTCTTCATTAGAGAAACTATGATTTCTTGAATGTAGCAATTTGATGTGCCATGACTATCATC TTGATCAACATGCTTCTTAACCATCAAAAGATCCTAAACTAGATGCATGTCATGTTAGGAGACATATTAAGCTTG TATATAACTACACCAACATGCTTTAGGATCTCATAAGATCCAAAATTTCTTATTTGGGAGATTTTCAATCCAACA ACCATCATAATGAGCAACGTGATGTTATAACATCTCTCTCACACTGCCAGAACAGTCTTATACCTTGTCGGAGTG AAGGACATCCTTAACTAAGTAGATTCACTAAGCTATACTTAAAAAGCAATAAGGAATCATCTAAAATGTGTGACT CTTAACCCATATTGGCATACATGGTTTATGGGGGTTATTAATTGTCTGAACACTCCCCCATATAAATCAGTGATC AATATTAATCCCAATAATATACACTATTATGATTTGAGACTACACCCTGGAAGTGGCCGGCTCTCAAGAACCATT GCTGATCTCCAAGCCAAACCCTCATTCTGGTTGACTACAAGCTGAAGGCAAACTCAAGTATACAAAGCTTAAAAC ATAATAAAAATAATATACTCAACTCGCCACAAAATAGGCATTTAAGTCTTTAAAACATTTTTAAAAATAAATGAA ACAAACTTCTCAAACTGTAATGTATATCTATGAAGCCTCTAAATGAAAAAAATGAAGGCAGATGAGACATACGGC ATCCTAACAACTGATATAACTAAGAGTACAAGTGGAGCCCTTCGGATGTAAGGAGGCTCATCAAAGCTAATGTGA ACTCCATGTGGTATCAATGAAGCACCTATTGATGACCGTGAATACATGTATCTGCATCATGAAACGATGCAGGCC AAAGGGCTTAGTACGTGAAATGTACGAGCATGTAAAGGGAATTCAAATACATAAACATAGGCTTGAACTTTGATA TAAAGGAAACATACTTACCTATTTTTAACTCAAGAATAAAAAACATAGTTCAACTCAATGAAAAGACACTCAAGT CAGTGAAATAGGCCGCAACTCAATAATAAGATATTCGACTATGGGTAATCAACTCTGGGTACTCTATTCAATATA AAGTAAGAATACAAATGCATTATATGGAAAGACTTTAAAACGGTAGAAAACAACTCAATGTATTGAAAATTCAAT AGTAAATTAGTTTGTATGTAAGGAACAATATAAACTTTGTTTGTATATGAAAATACAAAATAAACTTTGTGTATA TAAAAGTACAAAATATCTCTGTGAAAGTTTCTCTAACCAACAACCATCACTATGAGCTTTCTGATAATACCACGT TTCGCCCATGATGTCAGAACTGTCCTATGATTTTCCAGTTCATAAGACCTACTCACTAAGTGGATCCACAAGTCT ATGCTAAAAAATATTTAAGGAATCGTCTAAAAAGTATGACTCATTCTACCCACGTTGGCTACATGATTTATGGGG GTCGTAAGTTATCTAAACTCTCCTCCATATCGATGCGTAATGCTACTCACAAATATACTAGCTCACATGTTTAAA AATATAACTCGTTTTGTTTGAGATCATTACTCAAAATCCTTCTCTTAAAAGAGATGATACTCAAACTGCTCAAAA CTCTTTTGGAAATCTCAAATTCGTCTCATCTTAAATGTAAAAATATTTACTCTTGGGAATACATAGTTATCATAT ATCATTTTAAAGAAAATGAACTCAACTCTGTTCTTTCTCAACTCAAGTGCTCAGTCTTAAACCAAATTAAAAAAA AGACTTCTCAAAATAAAGTTTATGTCGAATTATGGACGTGAACAATTCAATTCAAAGTTTTCGATAACCATAACT AAAACTAAATACTCGAGACTCAACATCTTAGAACTCAAGAACTTAAATGGTAATACTTCTTTCAAGAATGCTCGA CTCAGAAGGTTAATGCAGAATAATGTGCATGAATTACTCAACTAAAGGACTCACTGATACTACTCAATCTCAAGA TTGCTCGACTCGTAGGGTTAATGCAGAATTATGTGCATGAACTACTCAACTCAAAGACCTTCATAGGTAACATGT AGTAGCCCCATGATTTGGAATATAATCCCAAAATGATTAGGAACTCAATACTCAGGACTTAGAACTTGAAGATAA TACTACTTCTCTCAAAGATACCCAACTGACGGAGTTCATGCAGAATTTATGGGCATGAACTACTCGACTCAAGAG TCTAAAACACAATATGACACTCATGTATATAACTCTTCTCATTCTAATACTTGTTTTCTCAAAACTCGGTTTAAC TAAATAGTTGATCTCAAAGGATTCACAATTGAACTCAAAGACTTTCTTTGACTCCACTCTTAATTCTCTCTTAAA TTTGTATTTGAATTATGAATTTAAGAGTTATGATTCATGATATGGGGAATCTCAATAACAATATAGAAATTTGAT AATTAGGAATAGTACTTTTAAAAGAAAACATGAATTCAACTTAAAATCAACTTATCTAAAAAATATTCAAATATA GGGAAAGTATCCTAGACTACTGTGCTACTGATCTGAAAGTAGATGTAGGATGTGAGGATGAACTAGTCCAACACT ATGATAGCCTTACATACCTGGAATAACGAGGTTCTTGGAAAATCTTCACTTGAAGAAGAACTTGATTAGAAGCCT TGAAACCTAGCTTGAAGGTAAACAATCAAGAAAACCTTTCTTAAGATTCTTGAATTAGTTTATGAAAATCTCTAT GACCAAGCATTTTGATTTTCACTAGTGATTCATAATTGTATGGAGGAATTTGAATTGAAAAAGATGAAATGCTTG GAGAAAAGCTATCTTTGAAGAAGCTTGAAAAAGATTGGAAAGTCCTGTACTTTGATTTTCCCTTAGGATTTTGTC TTAGGGTTTGAGATAGAAAAGAATGATGGACTAAAAGATGAAAATCTAATTGTTTGGATCCTTTTTCAGCCAAGA AATCCGTTTAGGGTTTTCTTGGAGACAAACAAAATAAAAAAGACCATTTTTAATATTTTTCCGTCGGCTAATTCG TAATAACATTGTATCATGTTATTGAAAGAGTCATAACTTTTTACTCAAAAATTGGATTGATGCGAAATTAGTGGT GTTGGAAAGTAGATTCAAGTACCTCTAATTGGATAGGTTATTCCCTACATAAGTCTTTATATTCTAAAAGATATG GTTGTTTGCACTTGACCTAAGTAGAATTTTACATGAAAACTTAATAGAGAAGGAAACTTCAAGAACTCATCAAGA AATTTCAATTGCTCAATATTTATGGATAAATTTGTAGAAGAAACTCATGATTGACATGCGGGTGAATAAACCCAA CACTATGGAAGCTTACATACCTCAAAGAACTAGGTTCTTGGCGAAATCTTGAATTTCTTCAACGAACGCTTGAAA CTTTGAACTTTTTCTCTTCTTGAACTCTCAACTAAAACCCTAGGCGTATATTAGGATTATAAAAGTTAACATGAT AGGATTAGACCTTTAAAAACTTTCTAAAATGAATTAAATCTGATTTAGCATGAAAAAGACCAAAATACCCCTTAC TATTTTCGGATAACTTTTCTTAATTGGACTGCCTGACTTCAAAAAGGTATATCTCACTCATCCGACCTCAAAATT TAGCAAATTCAGTGGCGTTAGAAAGCTAATTTAAACACCTTTCATTTTCCATCTCATGGCACACATAACTCATTC TTTAAAGAGAGCTATGATCGTTCAAATTAACTCAAATCTTAGAAGAATTTAGGAATGTCTTGAACGAGCTACATC TAGTGACCTTAACACTTTGGAAAATTTTAAATTTCTTAGTAAAAACTTACTCACTATGAAGGATGGTTCAAGTCT TAGCTCAAAATTTTCCTAAGTTGCTATATATACTCATGCTCATATGTTTAAAACCAAAACCCTTCCTCGATTTGA ATTAATTACCAAAAAGATTCTCTTAAAAAGATAATGCTCAAAACTCCCCCTAAACTCATTTGGAAATCTAGGTTT CCCTTGTTTTAAATATAAAAACATTTACTCTTGGAAATATTTAGTTCTCAGATATTCACTTGAAAAAAATTAAAC TCGACTCTCATCATCTTCATACTCAAGTGCTCAAGTCCTAAAACAATTTATAACTAATTGTATAAGACTTCTCAA AATAGGGTTCATTCCGAATTATGGACGTGAACGACTCAATTCAAGGATTTCAATAACCATATATATAACTCAATA ATAGGAACTCAACAACTCCAGAACTCAATGATACTACTCATCTCAAGAATGCTCGACTCACAGGGTCTTTGCGAA ATTATTGGGCATGAACAACTCAACTCAAAGACCTTCATTTATACCATATGGTAGTCCCATAATAGGAATATAATC CCAAAAAAATTAGGAACTCAATACTCAAAAACTTAGAACTCGAAGATATTACTCATCTCAAAGATATTCAATTTA TGGAATTCATGCTGAATTATGAGCATGAACGACTTGACTCAAGGATCTCAATAATAATGTAGACTCATGAATACA CTCTTCTCATTCTCATACTCACATACTCGAGTATTAAAATAAATTATAAGTAATTGCAGAAGACTCCTTGAACAG ACTCAAAAGGACTCCTTCGAATTTTACTCTTAATGCTACCTGAATTTTGTATTATAAATTTAAGGATCATGATTA TGATATAAAGAATTTCTCAGCATATATGAAATGAACGAATTTGAGCATTGAACGTCTAACCTCATTTTTTAATTA TTGTGATATGTAGAGTGGTGCAAAATCACAGATACCTCTCTTGATGCATTTCTATAGTTACGTTGATGTGAGATT ATATATAGTTCAGCAGCAGCATGTTGGGAAAATTACTAATAACTCTTCTTTTATATCAAATTGTTGAAGCATGAT GACAACACTTGAAAAGTATCAACAATGCAGTTACGCATCTTTGGACCCGATGT ACCGGTTAGTGATACTCAGGT ATTGTTTATCTACTTTATCATGTCGTAAGTATATTATTTGTAAAGATATATATCAAGATAGTTCGATTGCGTACA CTTACATTTTGATTATGTTTGGTGAATACTATTCTAATACCTTTTTTTTTCCTAAAGCCTAACAAATAAAGATAA TTAAGATGGGAACGTAATTCAAGTACAACATGGTTCCATACGTGACATATTTACACATATAGTGGAACCAAAAGA GCAATTTTTCCTAATATCATTTTCTAAATATCACGTGTGCCCGTGATTCTTTTTTATGGACATGAATTTTTTTTT TAATATGAGTGGAAGTAAGGTTCGATCTTTCTATCTGCTTTGATATCATATTGAATCGTGTGATTGTCTCTTTAA AAAATTAAGCAAGAGCATATTTTATTAATTAATTGTCTTTCTCGACGTTTTTCTCTTTCAACAGATGAACTACAA TGAGTATGTGAGGCTAAAAGCTAGAGTTGAGCTCCTTCAACGTTCTCAAAGGTAAGATATTAGTGATGTAATTAA ATGATTTTAGTTAGATTTACATAAGTTTTTAATAAGTGAAAATTAATAGACATATTCTTGGAGAGGATTTGGGCA CACTAAACTCGAAAGAACTTGAGCAGCTTGAGCACCAATTGGATGCATCTTTGAAGAAAGTTAGATCAAAAAAGG TATATCCAAATACTATAACTTAAATATATTGTAACGATTTAATTAATAGCATGTGTCACGTTCATCTATTCTTTA GTCACAATATATAGGGGCATGTCCTTAACAACGTGCCATGCCTCGATAGTCATTTTTGTCTTTTTGTGCGTATGA ATTTAACTTTGACACAAATTTTTGTAGTAATAATAACTCATGCTTTAGCATCTTAGGAAGCAGTCATATGAAAAA CAGAAGCATATATATATATTACATGAGTTAATTTAATTTAATATAAAATTTAATAAAATTGTGTCTCGCTATAAA TAATTTTATTAAAAAATTATATAAATATATTATTTTTTTAACTGGCCGCAAAGTTATATAAATTGATAGAGAAAG AGGTTTTGGTGTAAGGTTCATTTTCCAACAATTAGTTTTATAATTTGTAAGTGCACACTTTATCAGACTCAATCT ATGCTGGATCAGCTGGCAGACCTTCAAGAAAAGGTACACTGCCTTAACATTACAAAATTAATTTATTTCATCAAA AGCATATCATAAAATTCTGACAAATAAATATATTAGGAGCAAATGCTGGAAGAAGCAAATAAACAACTAAAAAAC AAGGTACATATCTATATATGTGTGTTAATTAATTAAGTTGATTTTGTATTTTTGTTTAATGAATAATTGTTTGTG ATCATCAGCTGGAAGAAAGTGCAGCTAGAATTCCACTTGGATTGTCATGGGGAAATAATGGAGGACAAACAATGG AATACAATCGACTCCCTCCACAAACTACTGCACAACCTTTCTTTCAACCTCTCCGTTTGAATTCTTCATCGCCTC AATTCGGGTAAGTATCTTATTTTATATGACTTAGTTTGACTTGACATAAAGTTTAATAAAGAAAGAAAGACTTTT AAAACTTATAGTGTAAAATAAGTGAATAGATATATATGTGGTTGTACTAACACTACAACAAAAATAATTTTCAGC GGCATTAAATATTGACATTAATAATGAGTGCTAAAGACTTTATCGGTATTAGTTAAGTGTCATTAGGATCAATGT CGTTAAAGGCTTCACGGACATATACAAAGAGTGACAATTGCCGCTAATGATTATTTTTGTTGTAGTGAAAATGAG TATTTTAAACTTAAATTGTTACATAATATAGAAATATGTCAGAAACAGGACAAATATACCACCGAACTATCATAT ATGTTATGGAGATATTCTCAGTCATACTTCTGCGACATTGGTACTCATGTCGTCCAAAAACTAGAACATATATAT ACCCTTTATATATTAACGAAGATACAAGTGTCATAATCTTATGCACCGATTCGATATTTATTAAATATCGAATCG ACGGATAAAATTATGTCACGTGTCCCTATTAAGTCTTCTATTAGAGTAAAAAGCATATATTCTCTAGTTTTTGAA CGAAAAAAGGTATTAATGTCTCAAAAGTATAACGAAAAGCATTTGCATACAATTTATGATAATTTGGGGCATATT AATTTATCATTCCCCCTTTTTTTGGCACTGATTAAAAAGAAAAAGAAAGTTATAAAAATTGGGATAGAGGGAATA ATTGTTTCATAGGGAAAACTTAGAAGCTTCTCAGTATGTCAGTGAGAATGTGTTTCCTAATTAGTGAACTATGGT TTGGTGAAAAATAAAGAGAAAAAAATCAGTACAAATTTTCCACTGATTAGCAATGAGAAAAATATTTGTTTCTAG TAGTATGAGGAGAGGATAGTCCGCATAAATAATCCTTAAATTTGTGGATAAATAAACTATTTTCAATAGATTATC GTCTCAAAATAAAATAAAATGATTGCAAGAAAAGAATAATAGGTATGCTGGTAATATGTATAATACACTCAAATT TATTTGCTGTCCATGCAGATACAATCCAAATATGGGTGCAAATGATCATGAGGTTAATGCAGCAACAACTGCTCA TAATATTAATGGATTTATTCCAGGGTGGATGCTCTAA Mutant Solyc12g038510 gene allele j2^CR >allele-1 (SEQ ID NO: 10) ATGGGAAGAGGAAGAGTAGAACTAAAGAGAATAGAGAACAAAATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGACTTCTTAAGAAAGCTTATGAGTTATCTATACTTTGTGATGCTGAAGTTGCTCTCATCATCTTCTCTAGC CGCGGAAAACTCTATGAGTTTTCAAGTGCTTCCAGGTATATATATATATACATATGTTTTTCTTCTTTTTGTGTG TGCGTATGTGTTTACTTACTTTCATTAATTAACTCAACCATATATATACATCTCTCACCTCAATTATATATATGT TTGAGATCTGAATGTCTACGGACTCCATTTAGGTACATATCTTTGTTTAGATCATAAATCATCTATCTTCATTCC TAAGATCTACTAATATATATGTATAAGAAGATCCATCCATCTATTAGGTTTTTCAACAACATATACAGTGAAATC TTATATGTGGGCCCACGTATAGCCATATGAGAAAATAGTGTGCACGTAAACATTATCATTACTTAATTATAGGAA TATACATCCATTAGGTTTATCAACAACAATAAAATCCTCTAAATGGAGTCTAGTCATAGGTCTAGCCGTTTGAAA ATGTAAAATATATGCCGATCTTATCACTATGTCATAATAATAGATATGTTGTTATTGAAAGATTCTCAATCTTTT TTTTTCTTCAAGGTAGAGATTCTTAAGTGGATTCATGTTTTTTTTATCAAAAAAGAAAAAAACAAAAGTGTCCAT TTGTTCATCTAATGGGTTTTCCATGTTACCAATTCACTACACTGTTGAGATTTGATTATCAGATGTGTCAAGTTT CGTTTGGTTCCCTAGAAGGGAGAAAAGGCTGCTTATGCAGGCAGGGTATTAAAGATGATATTAATATCTGCAGTA ATCAGTAACAGAATATATAAACTTAATAATAAACTTGAAGGTACTTAATTATCCAGCAGATAATCTTCTGTCTCA CCGTACACTTTTGTTATATCATAAGCATAAGAATTGTTTTATCAAATATTACCAAACAAAACTTAGTTTTGTTTG GTAATATTTTATAAAATATGTTACCGAAAGTTACTTCCTATAACATATTTTATAAAGAAAAAAATTAAAAACTCC ATATACCTAAGAAATGTAACCCCCCCTCCATAACAACAATTTAACAAAAATAAAAACCTACTTTTTTTGAATTTG GTAAATTAGTTTTCTATCCTTTTTAGTAACTTCCTTTCTTATTTTCTTTTTATATTGGTAAAGTTTAATATTACA CATTATTTTAACATGTTATAATTTTTTGTGATGCTTAATTATTTGATACATGTAATAAACCATATATTAGAGCTA TAAATCAATGACAATGCATGTAGATACAACTCATTTATGATATATTTTGTTTATATATATAACCAATTAGATAAT TTGTCTGCGCTTTGTGCAGTCATAAATAATAATTGCATTGAACTTGCAAATATTTTTTTTTAATATCCATACATT AAAAAAAAAGAAAGAGGAAAATTGGTTCCTAAAATATTAGCAATATTCAAACATTTATTTGATTATTAATCATTA TCACATAACTTAAGAACGTCTAATGAATGAATTATTCACGAAATAATAAATCATTGGTTCTAAAAAGGAATTTCG TAATAAAATAAAAATTTAAGTTACCATATTCAAAAAAAGAAATTGTGCTTGAACATGAAAATAATTATAATTTTT GAACTTGTATAATGAATTTCTTCAATTCATAAGTGGGAAATTTCATATTTATGTAATAATAGATAATATGTAAGC TCTAATATAGTACTTTAGGTTATAGAATTTAATATAAAATATCAAAACATGAATTCTTGAAATTGAGTAGAGTAA TTATTTTCTGCACAATGAATCGGAGACAATAACTTTGAAGAAATATAAACAATAGAGTTCAAAAGATGTAGTCAA AAACAACAATTAATATCATAAGAATAAATTAATGAGTGTAAAAATGCATACCACGATATGTAAAAACAGAATGGA ATATAATAAAAAAAATCGAGTTCACTGAATACACAATGTTCCTTTAAGAAAATTATTCTCCTCCAATACCAACGA GATTACATCCTCTAAGGATGGAAATGATTTCATTCCCCAACTTATCCATATAAAAATAGTGGTGTTAGTATGTAA CTCAATAGGAGTAAAATACACAAATATTTAATTTTGCGAAAGTAGAAGAAGAAGATCATATTTTTTTTTTAAAAT GAGAGGATATATCACTATTTTTAAACAACAAAGGGTAGTGTTAACAAATTTTTATTGTGTCTTGTCTAAAAGGTT ACAGCTATTTGAAAAAGTTACAACACTTCGAAAAGTGAACAACATTTCATAAAAGTCGTAACTTTTCATAAAGTC GTAACTCTTCATAAATGTCGCAACTCTTCATAAAAATTACAACTATTGATAAAAGTCACCACTCTTGATAAAGAT CACCACTCTTCATTGAAGTTGCAACTTTTCATAAAAATCACATCTTTTAATAAAAAAGAAAGACTAGTTTTTGGA ATAAATTAATTTAAAAGAAAATTTTTGTTTGTGGTGGGGCGCCAAGTAGGCAGGCGTAGGGTTCTTTTTATATAA ATATATATGATATATGATTCAATATTTGATATATATATATATAGAGAGAGAGATGACAATATAAGACAATTGCAA AAAATAAAATAAAAAACTAATCGAGTAAGTAGGCAAAAAATTATTTATAAAATATATGTAGAATTTCTTTATCAG ATATGACTGCCCAAATCTTATATTCAAACTAAAATGCAAGATCAATGGTGCTATATATAGGGTTTTACACAAAAA TCAAGATCTAGTCTTGCAAATTTAAATAAAAAACAGTGGTTTACGATGAGATAATGTAGCTTTTGTAAACAATAA AACTAGAAAAATAAATGCAAAGGCATTTTAAAGGATATAATAATGAAGATCAAAGGCAGAGAAGGGAAGAGGCAG CAATATAATGAAGGTAACATCATGGTTCCATTCTAATATATATGCTATTTTTCTTTAGTAAATTTCAAAAATAAT GATACATTTTCATATTTGATAAATATTTAATGATACTATCAACATTTTATCTATATTGAGTTCCATTTATTTGAC CAAAACCTCACAAAGATGTGCTCTTCGATCTATTCAAPATTTATTCAATTTAAGGATAGCTTTAAAACATGACAA AGTTTTCTCATATATTTCTTAAATTTTATATCCAGTCTAAATACGTATATAAACTAAAATGAAGAGAATAATATG AAGCTTTATTTGATGACATTGTTGAAATAACCAAAAGCTATAAGTGATACAATAGTAAATTTACCATTGGTCAAT TCAGAATTATTTAAAAGCTAAAAAAGTCATATAAGTTGGGGTTGCTCAATGTATAGTTTTTGGCTTGTTTTAAGC ATTTTAAAACTTTTTTTAAGCGCTTTTTAACATTGCTAAACACTCAAAAAATGATAAATAGTATTTAAATTTGAT ATGATTAGCTTAAAAGTGAACTCATATACCTTCAAAGTAAAAATCCCCAATTCGAGCTTTCAAACCACTTGATTT TGTGGATGAAATTATACTGAAGTTGAATATATCACTATTTATAGGGGTTAGTGAACTAATACCTTTGATTATTTG GTAGAAATATGTATCTTAGATCACCCTAATGAGCTCCCACTTTTAAAATAGGAAAAACCTCATATGAAGTTCATC ACTGTTCATTATATATCACTTTTATTCAAAAACGTTTACAAATGTTCATTGTGACTAAATACCCTTGAGTGTCGA GTTTTCACACCAATAAGGCCTAATTAATAGGTAAACAAAACTATGTCAATCTTCAAAACGCAAATCTAATTATAT TTTTAACAAGATTAGAGGTATATATACATATTCTCTTATGTTAACTCTTATTCATTATTGAACAAACTAAGTAAG TGTACCCAAGGTCTCAAACAACAGTTGGTACATTCTTTGTATGTCTTCCTTTGTCTCTTAATAGTCGTCTCCTCC TGTCGATGATTCCTCCAAATACATTAATCAAAGGAAAATCTTTCGCCCTCAACTTGCAAACTTGTCTATCTAAAA TTGTTAACAAAGTTTCTTCATTAGAGAAACTATGATTTCTTGAATGTAGCAATTTGATGTGCCATGACTATCATC TTGATCAACATGCTTCTTAACCATCAAAAGATCCTAAACTAGATGCATGTCATGTTAGGAGACATATTAAGCTTG TATATAACTACACCAACATGCTTTAGGATCTCATAAGATCCAAAATTTCTTATTTGGGAGATTTTCAATCCAACA ACCATCATAATGAGCAACGTGATGTTATAACATCTCTCTCACACTGCCAGAACAGTCTTATACCTTGTCGGAGTG AAGGACATCCTTAACTAAGTAGATTCACTAAGCTATACTTAAAAAGCAATAAGGAATCATCTAAAATGTGTGACT CTTAACCCATATTGGCATACATGGTTTATGGGGGTTATTAATTGTCTGAACACTCCCCCATATAAATCAGTGATC AATATTAATCCCAATAATATACACTATTATGATTTGAGACTACACCCTGGAAGTGGCCGGCTCTCAAGAACCATT GCTGATCTCCAAGCCAAACCCTCATTCTGGTTGACTACAAGCTGAAGGCAAACTCAAGTATACAAAGCTTAAAAC ATAATAAAAATAATATACTCAACTCGCCACAAAATAGGCATTTAAGTCTTTAAAACATTTTTAAAAATAAATGAA ACAAACTTCTCAAACTGTAATGTATATCTATGAAGCCTCTAAATGAAAAAAATGAAGGCAGATGAGACATACGGC ATCCTAACAACTGATATAACTAAGAGTACAAGTGGAGCCCTTCGGATGTAAGGAGGCTCATCAAAGCTAATGTGA ACTCCATGTGGTATCAATGAAGCACCTATTGATGACCGTGAATACATGTATCTGCATCATGAAACGATGCAGGCC AAAGGGCTTAGTACGTGAAATGTACGAGCATGTAAAGGGAATTCAAATACATAAACATAGGCTTGAACTTTGATA TAAAGGAAACATACTTACCTATTTTTAACTCAAGAATAAAAAACATAGTTCAACTCAATGAAAAGACACTCAAGT CAGTGAAATAGGCCGCAACTCAATAATAAGATATTCGACTATGGGTAATCAACTCTGGGTACTCTATTCAATATA AAGTAAGAATACAAATGCATTATATGGAAAGACTTTAAAACGGTAGAAAACAACTCAATGTATTGAAAATTCAAT AGTAAATTAGTTTGTATGTAAGGAACAATATAAACTTTGTTTGTATATGAAAATACAAAATAAACTTTGTGTATA TAAAAGTACAAAATATCTCTGTGAAAGTTTCTCTAACCAACAACCATCACTATGAGCTTTCTGATAATACCACGT TTCGCCCATGATGTCAGAACTGTCCTATGATTTTCCAGTTCATAAGACCTACTCACTAAGTGGATCCACAAGTCT ATGCTAAAAAATATTTAAGGAATCGTCTAAAAAGTATGACTCATTCTACCCACGTTGGCTACATGATTTATGGGG GTCGTAAGTTATCTAAACTCTCCTCCATATCGATGCGTAATGCTACTCACAAATATACTAGCTCACATGTTTAAA AATATAACTCGTTTTGTTTGAGATCATTACTCAAAATCCTTCTCTTAAAAGAGATGATACTCAAACTGCTCAAAA CTCTTTTGGAAATCTCAAATTCGTCTCATCTTAAATGTAAAAATATTTACTCTTGGGAATACATAGTTATCATAT ATCATTTTAAAGAAAATGAACTCAACTCTGTTCTTTCTCAACTCAAGTGCTCAGTCTTAAACCAAATTAAAAAAA AGACTTCTCAAAATAAAGTTTATGTCGAATTATGGACGTGAACAATTCAATTCAAAGTTTTCGATAACCATAACT AAAACTAAATACTCGAGACTCAACATCTTAGAACTCAAGAACTTAAATGGTAATACTTCTTTCAAGAATGCTCGA CTCAGAAGGTTAATGCAGAATAATGTGCATGAATTACTCAACTAAAGGACTCACTGATACTACTCAATCTCAAGA TTGCTCGACTCGTAGGGTTAATGCAGAATTATGTGCATGAACTACTCAACTCAAAGACCTTCATAGGTAACATGT AGTAGCCCCATGATTTGGAATATAATCCCAAAATGATTAGGAACTCAATACTCAGGACTTAGAACTTGAAGATAA TACTACTTCTCTCAAAGATACCCAACTGACGGAGTTCATGCAGAATTTATGGGCATGAACTACTCGACTCAAGAG TCTAAAACACAATATGACACTCATGTATATAACTCTTCTCATTCTAATACTTGTTTTCTCAAAACTCGGTTTAAC TAAATAGTTGATCTCAAAGGATTCACAATTGAACTCAAAGACTTTCTTTGACTCCACTCTTAATTCTCTCTTAAA TTTGTATTTGAATTATGAATTTAAGAGTTATGATTCATGATATGGGGAATCTCAATAACAATATAGAAATTTGAT AATTAGGAATAGTACTTTTAAAAGAAAACATGAATTCAACTTAAAATCAACTTATCTAAAAAATATTCAAATATA GGGAAAGTATCCTAGACTACTGTGCTACTGATCTGAAAGTAGATGTAGGATGTGAGGATGAACTAGTCCAACACT ATGATAGCCTTACATACCTGGAATAACGAGGTTCTTGGAAAATCTTCACTTGAAGAAGAACTTGATTAGAAGCCT TGAAACCTAGCTTGAAGGTAAACAATCAAGAAAACCTTTCTTAAGATTCTTGAATTAGTTTATGAAAATCTCTAT GACCAAGCATTTTGATTTTCACTAGTGATTCATAATTGTATGGAGGAATTTGAATTGAAAAAGATGAAATGCTTG GAGAAAAGCTATCTTTGAAGAAGCTTGAAAAAGATTGGAAAGTCCTGTACTTTGATTTTCCCTTAGGATTTTGTC TTAGGGTTTGAGATAGAAAAGAATGATGGACTAAAAGATGAAAATCTAATTGTTTGGATCCTTTTTCAGCCAAGA AATCCGTTTAGGGTTTTCTTGGAGACAAACAAAATAAAAAAGACCATTTTTAATATTTTTCCGTCGGCTAATTCG TAATAACATTGTATCATGTTATTGAAAGAGTCATAACTTTTTACTCAAAAATTGGATTGATGCGAAATTAGTGGT GTTGGAAAGTAGATTCAAGTACCTCTAATTGGATAGGTTATTCCCTACATAAGTCTTTATATTCTAAAAGATATG GTTGTTTGCACTTGACCTAAGTAGAATTTTACATGAAAACTTAATAGAGAAGGAAACTTCAAGAACTCATCAAGA AATTTCAATTGCTCAATATTTATGGATAAATTTGTAGAAGAAACTCATGATTGACATGCGGGTGAATAAACCCAA CACTATGGAAGCTTACATACCTCAAAGAACTAGGTTCTTGGCGAAATCTTGAATTTCTTCAACGAACGCTTGAAA CTTTGAACTTTTTCTCTTCTTGAACTCTCAACTAAAACCCTAGGCGTATATTAGGATTATAAAAGTTAACATGAT AGGATTAGACCTTTAAAAACTTTCTAAAATGAATTAAATCTGATTTAGCATGAAAAAGACCAAAATACCCCTTAC TATTTTCGGATAACTTTTCTTAATTGGACTGCCTGACTTCAAAAAGGTATATCTCACTCATCCGACCTCAAAATT TAGCAAATTCAGTGGCGTTAGAAAGCTAATTTAAACACCTTTCATTTTCCATCTCATGGCACACATAACTCATTC TTTAAAGAGAGCTATGATCGTTCAAATTAACTCAAATCTTAGAAGAATTTAGGAATGTCTTGAACGAGCTACATC TAGTGACCTTAACACTTTGGAAAATTTTAAATTTCTTAGTAAAAACTTACTCACTATGAAGGATGGTTCAAGTCT TAGCTCAAAATTTTCCTAAGTTGCTATATATACTCATGCTCATATGTTTAAAACCAAAACCCTTCCTCGATTTGA ATTAATTACCAAAAAGATTCTCTTAAAAAGATAATGCTCAAAACTCCCCCTAAACTCATTTGGAAATCTAGGTTT CCCTTGTTTTAAATATAAAAACATTTACTCTTGGAAATATTTAGTTCTCAGATATTCACTTGAAAAAAATTAAAC TCGACTCTCATCATCTTCATACTCAAGTGCTCAAGTCCTAAAACAATTTATAACTAATTGTATAAGACTTCTCAA AATAGGGTTCATTCCGAATTATGGACGTGAACGACTCAATTCAAGGATTTCAATAACCATATATATAACTCAATA ATAGGAACTCAACAACTCCAGAACTCAATGATACTACTCATCTCAAGAATGCTCGACTCACAGGGTCTTTGCGAA ATTATTGGGCATGAACAACTCAACTCAAAGACCTTCATTTATACCATATGGTAGTCCCATAATAGGAATATAATC CCAAAAAAATTAGGAACTCAATACTCAAAAACTTAGAACTCGAAGATATTACTCATCTCAAAGATATTCAATTTA TGGAATTCATGCTGAATTATGAGCATGAACGACTTGACTCAAGGATCTCAATAATAATGTAGACTCATGAATACA CTCTTCTCATTCTCATACTCACATACTCGAGTATTAAAATAAATTATAAGTAATTGCAGAAGACTCCTTGAACAG ACTCAAAAGGACTCCTTCGAATTTTACTCTTAATGCTACCTGAATTTTGTATTATAAATTTAAGGATCATGATTA TGATATAAAGAATTTCTCAGCATATATGAAATGAACGAATTTGAGCATTGAACGTCTAACCTCATTTTTTAATTA TTGTGATATGTAGAGTGGTGCAAAATCACAGATACCTCTCTTGATGCATTTCTATAGTTACGTTGATGTGAGATT ATATATAGTTCAGCAGCAGCATGTTGGGAAAATTACTAATAACTCTTCTTTTATATCAAATTGTTGAAGCATGAT GACAACACTTGAAAAGTATCAACAATGCAGTTACGCATCTTTGGACCCGATGTTACCGGTTAGTGATACTCAGGT ATTGTTTATCTACTTTATCATGTCGTAAGTATATTATTTGTAAAGATATATATCAAGATAGTTCGATTGCGTACA CTTACATTTTGATTATGTTTGGTGAATACTATTCTAATACCTTTTTTTTTCCTAAAGCCTAACAAATAAAGATAA TTAAGATGGGAACGTAATTCAAGTACAACATGGTTCCATACGTGACATATTTACACATATAGTGGAACCAAAAGA GCAATTTTTCCTAATATCATTTTCTAAATATCACGTGTGCCCGTGATTCTTTTTTATGGACATGAATTTTTTTTT TAATATGAGTGGAAGTAAGGTTCGATCTTTCTATCTGCTTTGATATCATATTGAATCGTGTGATTGTCTCTTTAA AAAATTAAGCAAGAGCATATTTTATTAATTAATTGTCTTTCTCGACGTTTTTCTCTTTCAACAGATGAACTACAA TGAGTATGTGAGGCTAAAAGCTAGAGTTGAGCTCCTTCAACGTTCTTCAAAGGTAAGATATTAGTGATGTAATTA AATGATTTTAGTTAGATTTACATAAGTTTTTAATAAGTGAAAATTAATAGACATATTCTTGGAGATTTGGGCACA CTAAACTCGAAAGAACTTGAGCAGCTTGAGCACCAATTGGATGCATCTTTGAAGAAAGTTAGATCAAAAAAGGTA TATCCAAATACTATAACTTAAATATATTGTAACGATTTAATTAATAGCATGTGTCACGTTCATCTATTCTTTAGT CACAATATATAGGGGCATGTCCTTAACAACGTGCCATGCCTCGATAGTCATTTTTGTCTTTTTGTGCGTATGAAT TTAACTTTGACACAAATTTTTGTAGTAATAATAACTCATGCTTTAGCATCTTAGGAAGCAGTCATATGAAAAACA GAAGCATATATATATATTACATGAGTTAATTTAATTTAATATAAAATTTAATAAAATTGTGTCTCGCTATAAATA ATTTTATTAAAAAATTATATAAATATATTATTTTTTTAACTGGCCGCAAAGTTATATAAATTGATAGAGAAAGAG GTTTTGGTGTAAGGTTCATTTTCCAACAATTAGTTTTATAATTTGTAAGTGCACACTTTATCAGACTCAATCTAT GCTGGATCAGCTGGCAGACCTTCAAGAAAAGGTACACTGCCTTAACATTACAAAATTAATTTATTTCATCAAAAG CATATCATAAAATTCTGACAAATAAATATATTAGGAGCAAATGCTGGAAGAAGCAAATAAACAACTAAAAAACAA GGTACATATCTATATATGTGTGTTAATTAATTAAGTTGATTTTGTATTTTTGTTTAATGAATAATTGTTTGTGAT CATCAGCTGGAAGAAAGTGCAGCTAGAATTCCACTTGGATTGTCATGGGGAAATAATGGAGGACAAACAATGGAA TACAATCGACTCCCTCCACAAACTACTGCACAACCTTTCTTTCAACCTCTCCGTTTGAATTCTTCATCGCCTCAA TTCGGGTAAGTATCTTATTTTATATGACTTAGTTTGACTTGACATAAAGTTTAATAAAGAAAGAAAGACTTTTAA AACTTATAGTGTAAAATAAGTGAATAGATATATATGTGGTTGTACTAACACTACAACAAAAATAATTTTCAGCGG CATTAAATATTGACATTAATAATGAGTGCTAAAGACTTTATCGGTATTAGTTAAGTGTCATTAGGATCAATGTCG TTAAAGGCTTCACGGACATATACAAAGAGTGACAATTGCCGCTAATGATTATTTTTGTTGTAGTGAAAATGAGTA TTTTAAAGTTAAATTGTTACATAATATAGAAATATGTCAGAAACAGGACAAATATACCACCGAACTATCATATAT GTTATGGAGATATTCTCAGTCATACTTCTGCGACATTGGTACTCATGTCGTCCAAAAACTAGAACATATATATAC CCTTTATATATTAACGAAGATACAAGTGTCATAATCTTATGCACCGATTCGATATTTATTAAATATCGAATCGAC GGATAAAATTATGTCACGTGTCCCTATTAAGTCTTCTATTAGAGTAAAAAGCATATATTCTCTAGTTTTTGAACG AAAAAAGGTATTAATGTCTCAAAAGTATAACGAAAAGCATTTGCATACAATTTATGATAATTTGGGGCATATTAA TTTATCATTCCCCCTTTTTTTGGCACTGATTAAAAAGAAAAAGAAAGTTATAAAAATTGGGATAGAGGGAATAAT TGTTTCATAGGGAAAACTTAGAAGCTTCTCAGTATGTCAGTGAGAATGTGTTTCCTAATTAGTGAACTATGGTTT GGTGAAAAATAAAGAGAAAAAAATCAGTACAAATTTTCCACTGATTAGCAATGAGAAAAATATTTGTTTCTAGTA GTATGAGGAGAGGATAGTCCGCATAAATAATCCTTAAATTTGTGGATAAATAAACTATTTTCAATAGATTATCGT CTCAAAATAAAATAAAATGATTGCAAGAAAAGAATAATAGGTATGCTGGTAATATGTATAATACACTCAAATTTA TTTGCTGTCCATGCAGATACAATCCAAATATGGGTGCAAATGATCATGAGGTTAATGCAGCAACAACTGCTCATA ATATTAATGGATTTATTCCAGGGTGGATGCTCTAA >allele-2 (SEQ ID NO: 11) ATGGGAAGAGGAAGAGTAGAACTAAAGAGAATAGAGAACAAAATAAACAGGCAAGTTACTTTTGCTAAGAGAAGA AATGGACTTCTTAAGAAAGCTTATGAGTTATCTATACTTTGTGATGCTGAAGTTGCTCTCATCATCTTCTCTAGC CGCGGAAAACTCTATGAGTTTTCAAGTGCTTCCAGGTATATATATATATACATATGTTTTTCTTCTTTTTGTGTG TGCGTATGTGTTTACTTACTTTCATTAATTAACTCAACCATATATATACATCTCTCACCTCAATTATATATATGT TTGAGATCTGAATGTCTACGGACTCCATTTAGGTACATATCTTTGTTTAGATCATAAATCATCTATCTTCATTCC TAAGATCTACTAATATATATGTATAAGAAGATCCATCCATCTATTAGGTTTTTCAACAACATATACAGTGAAATC TTATATGTGGGCCCACGTATAGCCATATGAGAAAATAGTGTGCACGTAAACATTATCATTACTTAATTATAGGAA TATACATCCATTAGGTTTATCAACAACAATAAAATCCTCTAAATGGAGTCTAGTCATAGGTCTAGCCGTTTGAAA ATGTAAAATATATGCCGATCTTATCACTATGTCATAATAATAGATATGTTGTTATTGAAAGATTCTCAATCTTTT TTTTTCTTCAAGGTAGAGATTCTTAAGTGGATTCATGTTTTTTTTATCAAAAAAGAAAAAAACAAAAGTGTCCAT TTGTTCATCTAATGGGTTTTCCATGTTACCAATTCACTACACTGTTGAGATTTGATTATCAGATGTGTCAAGTTT CGTTTGGTTCCCTAGAAGGGAGAAAAGGCTGCTTATGCAGGCAGGGTATTAAAGATGATATTAATATCTGCAGTA ATCAGTAACAGAATATATAAACTTAATAATAAACTTGAAGGTACTTAATTATCCAGCAGATAATCTTCTGTCTCA CCGTACACTTTTGTTATATCATAAGCATAAGAATTGTTTTATCAAATATTACCAAACAAAACTTAGTTTTGTTTG GTAATATTTTATAAAATATGTTACCGAAAGTTACTTCCTATAACATATTTTATAAAGAAAAAAATTAAAAACTCC ATATACCTAAGAAATGTAACCCCCCCTCCATAACAACAATTTAACAAAAATAAAAACCTACTTTTTTTGAATTTG GTAAATTAGTTTTCTATCCTTTTTAGTAACTTCCTTTCTTATTTTCTTTTTATATTGGTAAAGTTTAATATTACA CATTATTTTAACATGTTATAATTTTTTGTGATGCTTAATTATTTGATACATGTAATAAACCATATATTAGAGCTA TAAATCAATGACAATGCATGTAGATACAACTCATTTATGATATATTTTGTTTATATATATAACCAATTAGATAAT TTGTCTGCGCTTTGTGCAGTCATAAATAATAATTGCATTGAACTTGCAAATATTTTTTTTTAATATCCATACATT AAAAAAAAAGAAAGAGGAAAATTGGTTCCTAAAATATTAGCAATATTCAAACATTTATTTGATTATTAATCATTA TCACATAACTTAAGAACGTCTAATGAATGAATTATTCACGAAATAATAAATCATTGGTTCTAAAAAGGAATTTCG TAATAAAATAAAAATTTAAGTTACCATATTCAAAAAAAGAAATTGTGCTTGAACATGAAAATAATTATAATTTTT GAACTTGTATAATGAATTTCTTCAATTCATAAGTGGGAAATTTCATATTTATGTAATAATAGATAATATGTAAGC TCTAATATAGTACTTTAGGTTATAGAATTTAATATAAAATATCAAAACATGAATTCTTGAAATTGAGTAGAGTAA TTATTTTCTGCACAATGAATCGGAGACAATAACTTTGAAGAAATATAAACAATAGAGTTCAAAAGATGTAGTCAA AAACAACAATTAATATCATAAGAATAAATTAATGAGTGTAAAAATGCATACCACGATATGTAAAAACAGAATGGA ATATAATAAAAAAAATCGAGTTCACTGAATACACAATGTTCCTTTAAGAAAATTATTCTCCTCCAATACCAACGA GATTACATCCTCTAAGGATGGAAATGATTTCATTCCCCAACTTATCCATATAAAAATAGTGGTGTTAGTATGTAA CTCAATAGGAGTAAAATACACAAATATTTAATTTTGCGAAAGTAGAAGAAGAAGATCATATTTTTTTTTTAAAAT GAGAGGATATATCACTATTTTTAAACAACAAAGGGTAGTGTTAACAAATTTTTATTGTGTCTTGTCTAAAAGGTT ACAGCTATTTGAAAAAGTTACAACACTTCGAAAAGTGAACAACATTTCATAAAAGTCGTAACTTTTCATAAAGTC GTAACTCTTCATAAATGTCGCAACTCTTCATAAAAATTACAACTATTGATAAAAGTCACCACTCTTGATAAAGAT CACCACTCTTCATTGAAGTTGCAACTTTTCATAAAAATCACATCTTTTAATAAAAAAGAAAGACTAGTTTTTGGA ATAAATTAATTTAAAAGAAAATTTTTGTTTGTGGTGGGGCGCCAAGTAGGCAGGCGTAGGGTTCTTTTTATATAA ATATATATGATATATGATTCAATATTTGATATATATATATATAGAGAGAGAGATGACAATATAAGACAATTGCAA AAAATAAAATAAAAAACTAATCGAGTAAGTAGGCAAAAAATTATTTATAAAATATATGTAGAATTTCTTTATCAG ATATGACTGCCCAAATCTTATATTCAAACTAAAATGCAAGATCAATGGTGCTATATATAGGGTTTTACACAAAAA TCAAGATCTAGTCTTGCAAATTTAAATAAAAAACAGTGGTTTACGATGAGATAATGTAGCTTTTGTAAACAATAA AACTAGAAAAATAAATGCAAAGGCATTTTAAAGGATATAATAATGAAGATCAAAGGCAGAGAAGGGAAGAGGCAG CAATATAATGAAGGTAACATCATGGTTCCATTCTAATATATATGCTATTTTTCTTTAGTAAATTTCAAAAATAAT GATACATTTTCATATTTGATAAATATTTAATGATACTATCAACATTTTATCTATATTGAGTTCCATTTATTTGAC CAAAACCTCACAAAGATGTGCTCTTCGATCTATTCAAAATTTATTCAATTTAAGGATAGCTTTAAAACATGACAA AGTTTTCTCATATATTTCTTAAATTTTATATCCAGTCTAAATACGTATATAAACTAAAATGAAGAGAATAATATG AAGCTTTATTTGATGACATTGTTGAAATAACCAAAAGCTATAAGTGATACAATAGTAAATTTACCATTGGTCAAT TCAGAATTATTTAAAAGCTAAAAAAGTCATATAAGTTGGGGTTGCTCAATGTATAGTTTTTGGCTTGTTTTAAGC ATTTTAAAACTTTTTTTAAGCGCTTTTTAACATTGCTAAACACTCAAAAAATGATAAATAGTATTTAAATTTGAT ATGATTAGCTTAAAAGTGAACTCATATACCTTCAAAGTAAAAATCCCCAATTCGAGCTTTCAAACCACTTGATTT TGTGGATGAAATTATACTGAAGTTGAATATATCACTATTTATAGGGGTTAGTGAACTAATACCTTTGATTATTTG GTAGAAATATGTATCTTAGATCACCCTAATGAGCTCCCACTTTTAAAATAGGAAAAACCTCATATGAAGTTCATC ACTGTTCATTATATATCACTTTTATTCAAAAACGTTTACAAATGTTCATTGTGACTAAATACCCTTGAGTGTCGA GTTTTCACACCAATAAGGCCTAATTAATAGGTAAACAAAACTATGTCAATCTTCAAAACGCAAATCTAATTATAT TTTTAACAAGATTAGAGGTATATATACATATTCTCTTATGTTAACTCTTATTCATTATTGAACAAACTAAGTAAG TGTACCCAAGGTCTCAAACAACAGTTGGTACATTCTTTGTATGTCTTCCTTTGTCTCTTAATAGTCGTCTCCTCC TGTCGATGATTCCTCCAAATACATTAATCAAAGGAAAATCTTTCGCCCTCAACTTGCAAACTTGTCTATCTAAAA TTGTTAACAAAGTTTCTTCATTAGAGAAACTATGATTTCTTGAATGTAGCAATTTGATGTGCCATGACTATCATC TTGATCAACATGCTTCTTAACCATCAAAAGATCCTAAACTAGATGCATGTCATGTTAGGAGACATATTAAGCTTG TATATAACTACACCAACATGCTTTAGGATCTCATAAGATCCAAAATTTCTTATTTGGGAGATTTTCAATCCAACA ACCATCATAATGAGCAACGTGATGTTATAACATCTCTCTCACACTGCCAGAACAGTCTTATACCTTGTCGGAGTG AAGGACATCCTTAACTAAGTAGATTCACTAAGCTATACTTAAAAAGCAATAAGGAATCATCTAAAATGTGTGACT CTTAACCCATATTGGCATACATGGTTTATGGGGGTTATTAATTGTCTGAACACTCCCCCATATAAATCAGTGATC AATATTAATCCCAATAATATACACTATTATGATTTGAGACTACACCCTGGAAGTGGCCGGCTCTCAAGAACCATT GCTGATCTCCAAGCCAAACCCTCATTCTGGTTGACTACAAGCTGAAGGCAAACTCAAGTATACAAAGCTTAAAAC ATAATAAAAATAATATACTCAACTCGCCACAAAATAGGCATTTAAGTCTTTAAAACATTTTTAAAAATAAATGAA ACAAACTTCTCAAACTGTAATGTATATCTATGAAGCCTCTAAATGAAAAAAATGAAGGCAGATGAGACATACGGC ATCCTAACAACTGATATAACTAAGAGTACAAGTGGAGCCCTTCGGATGTAAGGAGGCTCATCAAAGCTAATGTGA ACTCCATGTGGTATCAATGAAGCACCTATTGATGACCGTGAATACATGTATCTGCATCATGAAACGATGCAGGCC AAAGGGCTTAGTACGTGAAATGTACGAGCATGTAAAGGGAATTCAAATACATAAACATAGGCTTGAACTTTGATA TAAAGGAAACATACTTACCTATTTTTAACTCAAGAATAAAAAACATAGTTCAACTCAATGAAAAGACACTCAAGT CAGTGAAATAGGCCGCAACTCAATAATAAGATATTCGACTATGGGTAATCAACTCTGGGTACTCTATTCAATATA AAGTAAGAATACAAATGCATTATATGGAAAGACTTTAAAACGGTAGAAAACAACTCAATGTATTGAAAATTCAAT AGTAAATTAGTTTGTATGTAAGGAACAATATAAACTTTGTTTGTATATGAAAATACAAAATAAACTTTGTGTATA TAAAAGTACAAAATATCTCTGTGAAAGTTTCTCTAACCAACAACCATCACTATGAGCTTTCTGATAATACCACGT TTCGCCCATGATGTCAGAACTGTCCTATGATTTTCCAGTTCATAAGACCTACTCACTAAGTGGATCCACAAGTCT ATGCTAAAAAATATTTAAGGAATCGTCTAAAAAGTATGACTCATTCTACCCACGTTGGCTACATGATTTATGGGG GTCGTAAGTTATCTAAACTCTCCTCCATATCGATGCGTAATGCTACTCACAAATATACTAGCTCACATGTTTAAA AATATAACTCGTTTTGTTTGAGATCATTACTCAAAATCCTTCTCTTAAAAGAGATGATACTCAAACTGCTCAAAA CTCTTTTGGAAATCTCAAATTCGTCTCATCTTAAATGTAAAAATATTTACTCTTGGGAATACATAGTTATCATAT ATCATTTTAAAGAAAATGAACTCAACTCTGTTCTTTCTCAACTCAAGTGCTCAGTCTTAAACCAAATTAAAAAAA AGACTTCTCAAAATAAAGTTTATGTCGAATTATGGACGTGAACAATTCAATTCAAAGTTTTCGATAACCATAACT AAAACTAAATACTCGAGACTCAACATCTTAGAACTCAAGAACTTAAATGGTAATACTTCTTTCAAGAATGCTCGA CTCAGAAGGTTAATGCAGAATAATGTGCATGAATTACTCAACTAAAGGACTCACTGATACTACTCAATCTCAAGA TTGCTCGACTCGTAGGGTTAATGCAGAATTATGTGCATGAACTACTCAACTCAAAGACCTTCATAGGTAACATGT AGTAGCCCCATGATTTGGAATATAATCCCAAAATGATTAGGAACTCAATACTCAGGACTTAGAACTTGAAGATAA TACTACTTCTCTCAAAGATACCCAACTGACGGAGTTCATGCAGAATTTATGGGCATGAACTACTCGACTCAAGAG TCTAAAACACAATATGACACTCATGTATATAACTCTTCTCATTCTAATACTTGTTTTCTCAAAACTCGGTTTAAC TAAATAGTTGATCTCAAAGGATTCACAATTGAACTCAAAGACTTTCTTTGACTCCACTCTTAATTCTCTCTTAAA TTTGTATTTGAATTATGAATTTAAGAGTTATGATTCATGATATGGGGAATCTCAATAACAATATAGAAATTTGAT AATTAGGAATAGTACTTTTAAAAGAAAACATGAATTCAACTTAAAATCAACTTATCTAAAAAATATTCAAATATA GGGAAAGTATCCTAGACTACTGTGCTACTGATCTGAAAGTAGATGTAGGATGTGAGGATGAACTAGTCCAACACT ATGATAGCCTTACATACCTGGAATAACGAGGTTCTTGGAAAATCTTCACTTGAAGAAGAACTTGATTAGAAGCCT TGAAACCTAGCTTGAAGGTAAACAATCAAGAAAACCTTTCTTAAGATTCTTGAATTAGTTTATGAAAATCTCTAT GACCAAGCATTTTGATTTTCACTAGTGATTCATAATTGTATGGAGGAATTTGAATTGAAAAAGATGAAATGCTTG GAGAAAAGCTATCTTTGAAGAAGCTTGAAAAAGATTGGAAAGTCCTGTACTTTGATTTTCCCTTAGGATTTTGTC TTAGGGTTTGAGATAGAAAAGAATGATGGACTAAAAGATGAAAATCTAATTGTTTGGATCCTTTTTCAGCCAAGA AATCCGTTTAGGGTTTTCTTGGAGACAAACAAAATAAAAAAGACCATTTTTAATATTTTTCCGTCGGCTAATTCG TAATAACATTGTATCATGTTATTGAAAGAGTCATAACTTTTTACTCAAAAATTGGATTGATGCGAAATTAGTGGT GTTGGAAAGTAGATTCAAGTACCTCTAATTGGATAGGTTATTCCCTACATAAGTCTTTATATTCTAAAAGATATG GTTGTTTGCACTTGACCTAAGTAGAATTTTACATGAAAACTTAATAGAGAAGGAAACTTCAAGAACTCATCAAGA AATTTCAATTGCTCAATATTTATGGATAAATTTGTAGAAGAAACTCATGATTGACATGCGGGTGAATAAACCCAA CACTATGGAAGCTTACATACCTCAAAGAACTAGGTTCTTGGCGAAATCTTGAATTTCTTCAACGAACGCTTGAAA CTTTGAACTTTTTCTCTTCTTGAACTCTCAACTAAAACCCTAGGCGTATATTAGGATTATAAAAGTTAACATGAT AGGATTAGACCTTTAAAAACTTTCTAAAATGAATTAAATCTGATTTAGCATGAAAAAGACCAAAATACCCCTTAC TATTTTCGGATAACTTTTCTTAATTGGACTGCCTGACTTCAAAAAGGTATATCTCACTCATCCGACCTCAAAATT TAGCAAATTCAGTGGCGTTAGAAAGCTAATTTAAACACCTTTCATTTTCCATCTCATGGCACACATAACTCATTC TTTAAAGAGAGCTATGATCGTTCAAATTAACTCAAATCTTAGAAGAATTTAGGAATGTCTTGAACGAGCTACATC TAGTGACCTTAACACTTTGGAAAATTTTAAATTTCTTAGTAAAAACTTACTCACTATGAAGGATGGTTCAAGTCT TAGCTCAAAATTTTCCTAAGTTGCTATATATACTCATGCTCATATGTTTAAAACCAAAACCCTTCCTCGATTTGA ATTAATTACCAAAAAGATTCTCTTAAAAAGATAATGCTCAAAACTCCCCCTAAACTCATTTGGAAATCTAGGTTT CCCTTGTTTTAAATATAAAAACATTTACTCTTGGAAATATTTAGTTCTCAGATATTCACTTGAAAAAAATTAAAC TCGACTCTCATCATCTTCATACTCAAGTGCTCAAGTCCTAAAACAATTTATAACTAATTGTATAAGACTTCTCAA AATAGGGTTCATTCCGAATTATGGACGTGAACGACTCAATTCAAGGATTTCAATAACCATATATATAACTCAATA ATAGGAACTCAACAACTCCAGAACTCAATGATACTACTCATCTCAAGAATGCTCGACTCACAGGGTCTTTGCGAA ATTATTGGGCATGAACAACTCAACTCAAAGACCTTCATTTATACCATATGGTAGTCCCATAATAGGAATATAATC CCAAAAAAATTAGGAACTCAATACTCAAAAACTTAGAACTCGAAGATATTACTCATCTCAAAGATATTCAATTTA TGGAATTCATGCTGAATTATGAGCATGAACGACTTGACTCAAGGATCTCAATAATAATGTAGACTCATGAATACA CTCTTCTCATTCTCATACTCACATACTCGAGTATTAAAATAAATTATAAGTAATTGCAGAAGACTCCTTGAACAG ACTCAAAAGGACTCCTTCGAATTTTACTCTTAATGCTACCTGAATTTTGTATTATAAATTTAAGGATCATGATTA TGATATAAAGAATTTCTCAGCATATATGAAATGAACGAATTTGAGCATTGAACGTCTAACCTCATTTTTTAATTA TTGTGATATGTAGAGTGGTGCAAAATCACAGATACCTCTCTTGATGCATTTCTATAGTTACGTTGATGTGAGATT ATATATAGTTCAGCAGCAGCATGTTGGGAAAATTACTAATAACTCTTCTTTTATATCAAATTGTTGAAGCATGAT GACAACACTTGAAAAGTATCAACAATGCAGTTACGCATCTTTGGACCCGATGTTACCGGTTAGTGATACTCAGGT ATTGTTTATCTACTTTATCATGTCGTAAGTATATTATTTGTAAAGATATATATCAAGATAGTTCGATTGCGTACA CTTACATTTTGATTATGTTTGGTGAATACTATTCTAATACCTTTTTTTTTCCTAAAGCCTAACAAATAAAGATAA TTAAGATGGGAACGTAATTCAAGTACAACATGGTTCCATACGTGACATATTTACACATATAGTGGAACCAAAAGA GCAATTTTTCCTAATATCATTTTCTAAATATCACGTGTGCCCGTGATTCTTTTTTATGGACATGAATTTTTTTTT TAATATGAGTGGAAGTAAGGTTCGATCTTTCTATCTGCTTTGATATCATATTGAATCGTGTGATTGTCTCTTTAA AAAATTAAGCAAGAGCATATTTTATTAATTAATTGTCTTTCTCGACGTTTTTCTCTTTCAACAGATGAACTACAA TGAGTATGTGAGGCTAAAAGCTAGAGTTGAGCTCCTTCAACGTTC TCAAAGGTAA GATATTAGTGATGTAATTAAATGATTTTAGTTAGATTTACATAAGTTTTTAATAAGTGAAAATTAATAGACATAT TCTTGGAGAGATTTGGGCACACTAAACTCGAAAGAACTTGAGCAGCTTGAGCACCAATTGGATGCATCTTTGAAG AAAGTTAGATCAAAAAAGGTATATCCAAATACTATAACTTAAATATATTGTAACGATTTAATTAATAGCATGTGT CACGTTCATCTATTCTTTAGTCACAATATATAGGGGCATGTCCTTAACAACGTGCCATGCCTCGATAGTCATTTT TGTCTTTTTGTGCGTATGAATTTAACTTTGACACAAATTTTTGTAGTAATAATAACTCATGCTTTAGCATCTTAG GAAGCAGTCATATGAAAAACAGAAGCATATATATATATTACATGAGTTAATTTAATTTAATATAAAATTTAATAA AATTGTGTCTCGCTATAAATAATTTTATTAAAAAATTATATAAATATATTATTTTTTTAACTGGCCGCAAAGTTA TATAAATTGATAGAGAAAGAGGTTTTGGTGTAAGGTTCATTTTCCAACAATTAGTTTTATAATTTGTAAGTGCAC ACTTTATCAGACTCAATCTATGCTGGATCAGCTGGCAGACCTTCAAGAAAAGGTACACTGCCTTAACATTACAAA ATTAATTTATTTCATCAAAAGCATATCATAAAATTCTGACAAATAAATATATTAGGAGCAAATGCTGGAAGAAGC AAATAAACAACTAAAAAACAAGGTACATATCTATATATGTGTGTTAATTAATTAAGTTGATTTTGTATTTTTGTT TAATGAATAATTGTTTGTGATCATCAGCTGGAAGAAAGTGCAGCTAGAATTCCACTTGGATTGTCATGGGGAAAT AATGGAGGACAAACAATGGAATACAATCGACTCCCTCCACAAACTACTGCACAACCTTTCTTTCAACCTCTCCGT TTGAATTCTTCATCGCCTCAATTCGGGTAAGTATCTTATTTTATATGACTTAGTTTGACTTGACATAAAGTTTAA TAAAGAAAGAAAGACTTTTAAAACTTATAGTGTAAAATAAGTGAATAGATATATATGTGGTTGTACTAACACTAC AACAAAAATAATTTTCAGCGGCATTAAATATTGACATTAATAATGAGTGCTAAAGACTTTATCGGTATTAGTTAA GTGTCATTAGGATCAATGTCGTTAAAGGCTTCACGGACATATACAAAGAGTGACAATTGCCGCTAATGATTATTT TTGTTGTAGTGAAAATGAGTATTTTAAAGTTAAATTGTTACATAATATAGAAATATGTCAGAAACAGGACAAATA TACCACCGAACTATCATATATGTTATGGAGATATTCTCAGTCATACTTCTGCGACATTGGTACTCATGTCGTCCA AAAACTAGAACATATATATACCCTTTATATATTAACGAAGATACAAGTGTCATAATCTTATGCACCGATTCGATA TTTATTAAATATCGAATCGACGGATAAAATTATGTCACGTGTCCCTATTAAGTCTTCTATTAGAGTAAAAAGCAT ATATTCTCTAGTTTTTGAACGAAAAAAGGTATTAATGTCTCAAAAGTATAACGAAAAGCATTTGCATACAATTTA TGATAATTTGGGGCATATTAATTTATCATTCCCCCTTTTTTTGGCACTGATTAAAAAGAAAAAGAAAGTTATAAA AATTGGGATAGAGGGAATAATTGTTTCATAGGGAAAACTTAGAAGCTTCTCAGTATGTCAGTGAGAATGTGTTTC CTAATTAGTGAACTATGGTTTGGTGAAAAATAAAGAGAAAAAAATCAGTACAAATTTTCCACTGATTAGCAATGA GAAAAATATTTGTTTCTAGTAGTATGAGGAGAGGATAGTCCGCATAAATAATCCTTAAATTTGTGGATAAATAAA CTATTTTCAATAGATTATCGTCTCAAAATAAAATAAAATGATTGCAAGAAAAGAATAATAGGTATGCTGGTAATA TGTATAATACACTCAAATTTATTTGCTGTCCATGCAGATACAATCCAAATATGGGTGCAAATGATCATGAGGTTA ATGCAGCAACAACTGCTCATAATATTAATGGATTTATTCCAGGGTGGATGCTCTAA Wild-type Solyc03g114840 gene (SEQ ID NO: 12) ATGGGAAGAGGAAGAGTTGAGCTTAAGAGAATAGAAAATAAAATAAATAGGCAAGTCACTTTTGCTAAGAGAAGA AATGGACTTCTTAAAAAAGCTTATGAACTTTCTGTTCTTTGTGATGCTGAAGTTGCCCTTATAATCTTCTCTAAT AGGGGTAAACTCTATGAATTTTGCAGCACTTCAAGGTATTTTTTATTTTATTATATTAACATCAAAGATTTTATT TTTTTAAAAAAAACCTTAAGTCCTTCATTACCAAAACCCTTAATTGATTTACAAAGTACTTTCATTAAATTTAGT AATTCTTTTTTTTTTTATCTCTGACTTCAATTATAATGCAAGATCTATGTTGTCTTTATATATATTGAATTATAT ATGTACTGTATTTTTACTATATACATATAAGATCCTTTTTTCTTTTTTTTCTGTCTCTTTATATAAATATATTTT AAATAGTTGATTTTGAAAGATCTACTAATGTATATTTATTTTTGGAACTTTTGTGTATATGGAATTTTTTTCTTT TTTATGTTTTTTTTTTGTTCTAATTGTTTTAAAAGCGTTTAAGATCAGAATGTTCTTGATTATTCTTTTAGGAAA AAGATTTCCCATACATTGAGTTATTTTTTGATCTGTAGATTGAATTTTTTTAATGAGTTCCGATAGATTTTCGTT CAATTTTTCAATGAAACTATTGAGGGTTGATGATTAGATAATTACTCGATTGAAAGTTTTTATTTCAAAAAAATT ATAATTCTTCTTAATTTTATATTTATGAGATAGAGTTAGTTTAGTGATTATATGAAAAATCGTATCAGATTATTG GGAATCGAAACTTAAAAATTCTGAAAATATTATTATAAATTTTACATGTTACAATATTTTTACTGTTAAGATTTG ATTTGCAGACTAGGTGTCATGTTTGACAGTTGATAAAAAATCTGTTATTTTTGTTCTTTAATTCCCAAGACGGAT AAACAAAGGCTGCTTATGTTGGTTTCCAATAAGCAGCCATAATTTTAAATATTTTTGTTAAGATTAATTAATAAC AATTATTTCCACCAGATAATTTTCAAAATTTGTGACCCCGAGTTCATATAAATTGTTAATTTTACTGCTAGAAAT TACATCGATAATAATTTATTTAGTGTAATCTTATAAATACGAGGGCAGTAGTGTATAGACTGTTTTTTATTAATC CTGACTCAAAGTGAGGTAAGTTAAGTATATTTTGATTAAAAGGACTACATTTCATTTATGTATGTTTAATTAATA TTATTTTGTAAGTCAATAAATCTAAACAACATGAGTTTATCTAGACCCTTAATTATGCACCTTCATTATCAATTT TTTCAATACTCTCCTCAGAACATATGCTTCTCTATAATTTTGTGCACGAGTTAATCAATTCTTCCTTTTCAATAA TTAAATATGTGATTTATGTTTAGCACTTATTTTTCGGTTAGTTAATTGATAATAGGAAAAAGCCTCTTTTTTTTT GTGTGTGTGGTAATTAGGATCTTTATTGAATTTAAAATGACCTACTATAGAACTTGGGAGTTTTTCTTCATAATA ATGCACTGCAACGTGTTAAAAAAAAAGAATCAAATGAAATTAATAGATGTTTACTGGATTGCCATGGTAAAGTGA TAAGTATTAATTTCGCTTTAACTAAGAGATCATTATATTCAAGTCCCCTTGATACAAACTTGCCTTTGTAAATAA GTGTTTTATTTTTCAATGTGAAACTTTCGCTGTTAATTTAAATTTAATTATACTTCTATATAAATACCAAACAAT AATGTAATAAAACAAAAAATAAAAGAGTAGATGTTTCATATTGTTAATGCAGCATGGTGAAAACAATTGAAAAGT ACCAACGTTGCAGCTATGCTACTTTGGAAGCCAACCAATCAGTTACTGATACTCAGGTACTGCTTTATATTTTAA TTTATTTGGCTTTTTTTTAAAAAAATAATTAGTTTTGATTAATATGCATCATTTTATTTATTTTTGGCAACTCTT TATTTATCAGTAATAAGTAATAACTTTTTAACTAGTATATTTAAAAATCACAAAATTTAAGAATATTTTAATAGA TTCGACATATTTTAGTTTAAAAATAACAAATTAAATTATGTTTTTAATTTTTTAAATATTCTTACTATAATTATC ATGTACTCTTTGATCTGTTCATCTTTTCCATGATAATATTATTTGGTCAGTTAGTGACATAAGAGTTTGAAATTT AGAAAAAAGGAATATTTGGAGAAAACTGAAATGGATATTTAGAAATGAAAGTTATTTAATATAAATATAAGTATG GGCTGCTGAGTTGGGAATCCACGCTGGAGATCTCAAGTTTGAAGCGTCTCACAAACAATAGTAATGTCTTTTTGG TCGAGTTTGTCGGATTGGACTTGTCCGTGGCCTGTGGGTTACTTTTCCTATATGGTTTGCAAGCTATCGGGAATT TTATCCTGGCGCACCCAAATTTGAGTTATTTTTGAGTTTTTATATGAAATAGCTTTGTGAATTCATCGAACTCCC GAAAACATTGAACTTTACTCCAAGTTGAATTGCAGTAAAATAATAGTAGCGATTCTTTAATTTATCCTAACAGTT TTTCGAAATAATAATCCCAAAAAAGTTTAAAATAACCATACCATAAACTTACTGGGTAAGATATTATCTGTCTAA TAATATATAGTAGTTTCTTTTGTTTTATTAGTTTATCTAATCCATATTTCATTTCTTGATAAGTTATTCTTAATA GGAAAATAAACTTATTTCGAAAAACTGTTTTTAAAATTTTCTTGAGTTGAGTCTTGGATGAAAAATAGTTAATTT TGCATTAATTAATTTTGTTCTAACAAAAACTAATTAAATTTTTTTGAAGCGCATATTCACTCAAAAAATAAATAA AAACCATCATGCATACAGGAAATGTTCTTTTTTTAATTTATTTTTTCATTGGAGCCCTGACTAATTTTATATCGG TTCATACTTTCATAAATTACAAAAAGTTCAAAATTTAAACTAACCATATAAGTGAATAAAATAAATCAACAAAAT ATTCACCACATAATACTTTTTAAATAGAATTTTTCATACCAAAGACCTTACTTTAATTAATTAGGGTGAGAGAAT CCTATAAGTCAATGCAAAACAATTCTATCTATCGGATTATAATCGTTGATTCATAAAATTTTAAAATCGACGATT TTCATTTAAATGACCCTTTTTTTTCTTTCATTTTTTATTGTTATTCATCTATTTAACTTGTGAGCATCTTTCATA TTGATATTTCAGACCCTTAAATTAATTGTTTTCTTACAGAATAACTACCACGAATATCTGAGGCTAAAAGCTAGA GTTGAGCTCCTCCAACGATCTCAGAGGTAATTTCTGTTCACTATCTTTATCTCAAATGAATTCTCATGTTTTTAT TTTTCGAGATTCAGATTAAATATAATTTGATGTATTATTAATTTAAATACGTTATTTAATATGGTCCTTATGTCC AACCATTGATTTAATTTGATATTTTTTTAATGAAAATTACACAGAAACTTTCTTGGTGAAGATTTGGGCACGTTA AGCTCGAAGGACCTTGAGCAGCTTGAGAATCAATTAGAGTCTTCCTTAAAGCAAATCAGGTCAAGGAAGGTAAAT TATTTAATCTAATTATACAGAAAAATCATCTAAAAGTTACCTTAATTGCTAGCCCAATAAGTTTGCTATCTGTTG ATCCTCACATTATTTTACTCACAGAAATTCACAATACCTTTATTTTTGTTTGAGTTTGAAGTATACAATTTCTTT AAAATGTAAAATTTGAAATCTCAACAATAAGATATGTTATTGATCCTTGCAATTATGGGTAGATTGCGAATTAAA CTATCTTGTCTTTGCTTACAACAGTCATTTTGTTTATAAACTAATTATACATAAATCCTAACTGATAGATAGTTT ATAAAGATGAATAATGAACATAGGTCATATATTAAAAAAACAAAAAACAAAAAAAAACTAAACAAGATGAGCGAG TCAAAAATAGTCTTAACAAAAGAATATATATATATGTATATATCATATTTGATTTGTCTATTTTTAATTTTGAAA AAACTAAGTTAATCGATATATAATATGAAGGCATAATGCATAAATATGTCCTTTAACTTGGTTTTAAATCACATT TATACCTCTTCGACTTTGGGTGTATACAAACAAACACTTAAACTTATATAATGTTGAACAAATAGATATATATGT CCTACATGTCATTTTTCGTCCTAAATGGTGTCCTAAGTGTATTGTGTCACGCAGGACTCATGTGTCTATTTGTTC AAATTTATACAAGTTTAAGTGCTTACTTATGTATAAACAAAGTTGAATGACATAAATGTGAAATAAAATCAAATT AAAGGGCATATTTATGCATTATACCTAATACGAAAATCCATATTATTCACTAAAAAATGAGTCGGATTATATGAT TACTTTTTTATTCATTTTGCCAATCGTATCCTACGACATTGTTTTTAATTTGCAGACACAATTCATGCTGGATCA GCTTGCAGATCTTCAACAAAAGGTAATTATAAAATTCTACAAATTTCCAATAATTAATAAATGGAATAATTATGC GCGAGAAATTTATCTATTTAAAATTTACGATGAATTTTAATTTTACAGGAGCAAATGCTTGCAGAATCTAATAGA TTACTCCGTAGAAAGGTAAACTAACTTGATAGCCGTGCGTAATGAATAACTTATTTTATTTTCAAAATTATAAAT CTAAATACTTAGGTAACTCGATAACATAAGAAGTATTTATACTGATGATATTGGTGTTGTGTTTTTTTTTATTAG TTAGAAGAAAGTGTAGCTGGATTTCCACTTCGATTGTGTTGGGAAGATGGAGGTGATCATCAACTTATGCATCAA CAAAATCGTCTCCCTAACACAGAGGGTTTCTTTCAGCCTCTTGGATTGCATTCTTCTTCTCCACATTTTGGGTAA TTACTTTTATTATTATTAAAAATAATTTCAATTTTTTTTACTTTTATTTCGATTAATAAATCAATGTGCACCAAG GTACGGTCTAACATAAACAAAAATGTGGGGAATGCTCTTAAAGCCCTAACAAAAGTTATTTGGTACGTGTACTAA TGTAATCGTACTATATATCTTACTTGATTAGTGGATGGACAGTACTGGGCACACACAATTGACATAAGTTATTAT AAGGAAAAAAAAAGGCCAATAATCAATATAGTCCAACATTACATTATTTATTATAACAGGTCACTCTAGATTAAA TGTTAATGAATAACAAAAAGTCTCATATTGATGATTAATGTGATGGGTGGGCTTCTTATAAGGCTTTGACAATCC TACTCTCTTTGAGCTAGTTTTGGGGGTGTGACCTAATTCAACAGAACGTAGTTAAGATTGTGAAGTAAAGTTGAT CATTGTTATAACAGGTTTAAATACTTCTAGTAAAAATAGTTCCTAGATAATCCATCGCAAAATAGCTCCTATATA GTTAGTTGGATTTTCATATAATCTATAGCTTATACATAGCTAAATGGGAATAGATGAGAGTTTCTGTTGTTTAGA TATGATATTTGATCGGTTTCTAAATCGTTACTATCATGTAGTGAATAATTTTCATGTTATTACTATTACATTTGA TTGTTTCTGTGGTTATTTTTTTTTCTAGGTACAATCCTGTTAATACAGATGAGGTGAATGCAGCGGCAACTGCAC ACAATATGAATGGATTTATTCATGGATGGATGCTTTAA Wild-type Solyc03g114840 coding sequence (SEQ ID NO: 13) ATGGGAAGAGGAAGAGTTGAGCTTAAGAGAATAGAAAATAAAATAAATAGGCAAGTCACTTTTGCTAAGAGAAGA AATGGACTTCTTAAAAAAGCTTATGAACTTTCTGTTCTTTGTGATGCTGAAGTTGCCCTTATAATCTTCTCTAAT AGGGGTAAACTCTATGAATTTTGCAGCACTTCAAGCATGGTGAAAACAATTGAAAAGTACCAACGTTGCAGCTAT GCTACTTTGGAAGCCAACCAATCAGTTACTGATACTCAGAATAACTACCACGAATATCTGAGGCTAAAAGCTAGA GTTGAGCTCCTCCAACGATCTCAGAGAAACTTTCTTGGTGAAGATTTGGGCACGTTAAGCTCGAAGGACCTTGAG CAGCTTGAGAATCAATTAGAGTCTTCCTTAAAGCAAATCAGGTCAAGGAAGACACAATTCATGCTGGATCAGCTT GCAGATCTTCAACAAAAGGAGCAAATGCTTGCAGAATCTAATAGATTACTCCGTAGAAAGTTAGAAGAAAGTGTA GCTGGATTTCCACTTCGATTGTGTTGGGAAGATGGAGGTGATCATCAACTTATGCATCAACAAAATCGTCTCCCT AACACAGAGGGTTTCTTTCAGCCTCTTGGATTGCATTCTTCTTCTCCACATTTTGGGTACAATCCTGTTAATACA GATGAGGTGAATGCAGCGGCAACTGCACACAATATGAATGGATTTATTCATGGATGGATGCTTTAA Mutant Solyc03g114840 gene allele ej2^W (SEQ ID NO: 14) ATGGGAAGAGGAAGAGTTGAGCTTAAGAGAATAGAAAATAAAATAAATAGGCAAGTCACTTTTGCTAAGAGAAGA AATGGACTTCTTAAAAAAGCTTATGAACTTTCTGTTCTTTGTGATGCTGAAGTTGCCCTTATAATCTTCTCTAAT AGGGGTAAACTCTATGAATTTTGCAGCACTTCAAGGTATTTTTTATTTTATTATATTAACATCAAAGATTTTATT TTTTTAAAAAAAACCTTAAGTCCTTCATTACCAAAACCCTTAATTGATTTACAAAGTACTTTCATTAAATTTAGT AATTCTTTTTTTTTTTATCTCTGACTTCAATTATAATGCAAGATCTATGTTGTCTTTATATATATTGAATTATAT ATGTACTGTATTTTTACTATATACATATAAGATCCTTTTTTCTTTTTTTTCTGTCTCTTTATATAAATATATTTT AAATAGTTGATTTTGAAAGATCTACTAATGTATATTTATTTTTGGAACTTTTGTGTATATGGAATTTTTTTCTTT TTTATGTTTTTTTTTTGTTCTAATTGTTTTAAAAGCGTTTAAGATCAGAATGTTCTTGATTATTCTTTTAGGAAA AAGATTTCCCATACATTGAGTTATTTTTTGATCTGTAGATTGAATTTTTTTAATGAGTTCCGATAGATTTTCGTT CAATTTTTCAATGAAACTATTGAGGGTTGATGATTAGATAATTACTCGATTGAAAGTTTTTATTTCAAAAAAATT ATAATTCTTCTTAATTTTATATTTATGAGATAGAGTTAGTTTAGTGATTATATGAAAAATCGTATCAGATTATTG GGAATCGAAACTTAAAAATTCTGAAAATATTATTATAAATTTTACATGTTACAATATTTTTACTGTTAAGATTTG ATTTGCAGACTAGGTGTCATGTTTGACAGTTGATAAAAAATCTGTTATTTTTGTTCTTTAATTCCCAAGACGGAT AAACAAAGGCTGCTTATGTTGGTTTCCAATAAGCAGCCATAATTTTAAATATTTTTGTTAAGATTAATTAATAAC AATTATTTCCACCAGATAATTTTCAAAATTTGTGACCCCGAGTTCATATAAATTGTTAATTTTACTGCTAGAAAT TACATCGATAATAATTTATTTAGTGTAATCTTATAAATACGAGGGCAGTAGTGTATAGACTGTTTTTTATTAATC CTGACTCAAAGTGAGGTAAGTTAAGTATATTTTGATTAAAAGGACTACATTTCATTTATGTATGTTTAATTAATA TTATTTTGTAAGTCAATAAATCTAAACAACATGAGTTTATCTAGACCCTTAATTATGCACCTTCATTATCAATTT TTTCAATACTCTCCTCAGAACATATGCTTCTCTATAATTTTGTGCACGAGTTAATCAATTCTTCCTTTTCAATAA TTAAATATGTGATTTATGTTTAGCACTTATTTTTCGGTTAGTTAATTGATAATAGGAAAAAGCCTCTTTTTTTTT GTGTGTGTGGTAATTAGGATCTTTATTGAATTTAAAATGACCTACTATAGAACTTGGGAGTTTTTCTTCATAATA ATGCACTGCAACGTGTTAAAAAAAAAGAATCAAATGAAATTAATAGATGTTTACTGGATTGCCATGGTAAAGTGA TAAGTATTAATTTCGCTTTAACTAAGAGATCATTATATTCAAGTCCCCTTGATACAAACTTGCCTTTGTAAATAA GTGTTTTATTTTTCAATGTGAAACTTTCGCTGTTAATTTAAATTTAATTATACTTCTATATAAATACCAAACAAT AATGTAATAAAACAAAAAATAAAAGAGTAGATGTTTCATATTGTTAATGCAGCATGGTGAAAACAATTGAAAAGT ACCAACGTTGCAGCTATGCTACTTTGGAAGCCAACCAATCAGTTACTGATACTCAGGTACTGCTTTATATTTTAA TTTATTTGGCTTTTTTTTAAAAAAATAATTAGTTTTGATTAATATGCATCATTTTATTTATTTTTGGCAACTCTT TATTTATCAGTAATAAGTAATAACTTTTTAACTAGTATATTTAAAAATCACAAAATTTAAGAATATTTTAATAGA TTCGACATATTTTAGTTTAAAAATAACAAATTAAATTATGTTTTTAATTTTTTAAATATTCTTACTATAATTATC ATGTACTCTTTGATCTGTTCATCTTTTCCATGATAATATTATTTGGTCAGTTAGTGACATAAGAGTTTGAAATTT AGAAAAAAGGAATATTTGGAGAAAACTGAAATGGATATTTAGAAATGAAAGTTATTTAATATAAATATAAGTATG GGCTGCTGAGTTGGGAATCCACGCTGGAGATCTCAAGTTTGAAGCGTCTCACAAACAATAGTAATGTCTTTTTGG TCGAGTTTGTCGGATTGGACTTGTCCGTGGCCTGTGGGTTACTTTTCCTATATGGTTTGCAAGCTATCGGGAATT TTATCCTGGCGCACCCAAATTTGAGTTATTTTTGAGTTTTTATATGAAATAGCTTTGTGAATTCATCGAACTCCC GAAAACATTGAACTTTACTCCAAGTTGAATTGCAGTAAAATAATAGTAGCGATTCTTTAATTTATCCTAACAGTT TTTCGAAATAATAATCCCAAAAAAGTTTAAAATAACCATACCATAAACTTACTGGGTAAGATATTATCTGTCTAA TAATATATAGTAGTTTCTTTTGTTTTATTAGTTTATCTAATCCATATTTCATTTCTTGATAAGTTATTCTTAATA GGAAAATAAACTTATTTCGAAAAACTGTTTTTAAAATTTTCTTGAGTTGAGTCTTGGATGAAAAATAGTTAATTT TGCATTAATTAATTTTGTTCTAACAAAAACTAATTAAATTTTTTTGAAGCGCATATTCACTCAAAAAATAAATAA AAACCATCATGCATACAGGAAATGTTCTTTTTTTAATTTATTTTTTCATTGGAGCCCTGACTAATTTTATATCGG TTCATACTTTCATAAATTACAAAAAGTTCAAAATTTAAACTAACCATATAAGTGAATAAAATAAATCAACAAAAT ATTCACCACATAATACTTTTTAAATAGAATTTTTCATACCAAAGACCTTACTTTAATTAATTAGGGTGAGAGAAT CCTATAAGTCAATGCAAAACAATTCTATCTATCGGATTATAATCGTTGATTCATAAAATTTTAAAATCGACGATT TTCATTTAAATGACCCTTTTTTTTCTTTCATTTTTTATTGTTATTCATCTATTTAACTTGTGAGCATCTTTCATA TTGATATTTCAGACCCTTAAATTAATTGTTTTCTTACAGAATAACTACCACGAATATCTGAGGCTAAAAGCTAGA GTTGAGCTCCTCCAACGATCTCAGAGGTAATTTCTGTTCACTATCTTTATCTCAAATGAATTCTCATGTTTTTAT TTTTCGAGATTCAGATTAAATATAATTTGATGTATTATTAATTTAAATACGTTATTTAATATGGTCCTTATGTCC AACCATTGATTTAATTTGATATTTTTTTAATGAAAATTACACAGAAACTTTCTTGGTGAAGATTTGGGCACGTTA AGCTCGAAGGACCTTGAGCAGCTTGAGAATCAATTAGAGTCTTCCTTAAAGCAAATCAGGTCAAGGAAGGTAAAT TATTTAATCTAATTATACAGAAAAATCATCTAAAAGTTACCTTAATTGCTAGCCCAATAAGTTTGCTATCTGTTG ATCCTCACATTATTTTACTCACAGAAATTCACAATACCTTTATTTTTGTTTGAGTTTGAAGTATACAATTTCTTT AAAATGTAAAATTTGAAATCTCAACAATAAGATATGTTATTGATCCTTGCAATTATGGGTAGATTGCGAATTAAA CTATCTTGTCTTTGCTTACAACAGTCATTTTGTTTATAAACTAATTATACATAAATCCTAACTGATAGATAGTTT ATAAAGATGAATAATGAACATAGGTCATATATTAAAAAAACAAAAAACAAAAAAAAACTAAACAAGATGAGCGAG TCAAAAATAGTCTTAACAAAAGAATATATATATATGTATATATCATATTTGATTTGTCTATTTTTAATTTTGAAA AAACTAAGTTAATCGATATATAATATGAAGGCATAATGCATAAATATGTCCTTTAACTTGGTTTTAAATCACATT TATACCTCTTCGACTTTGGGTGTATACAAACAAACACTTAAACTTATATAATGTTGAACAAATAGATATATATGT CCTACATGTCATTTTTCGTCCTAAATGGTGTCCTAAGTGTATTGTGTCACGCAGGACTCATGTGTCTATTTGTTC AAATTTATACAAGTTTAAGTGCTTACTTATGTATAAACAAAGTTGAATGACATAAATGTGAAATAAAATCAAATT AAAGGGCATATTTATGCATTATACCTAATACGAAAATCCATATTATTCACTAAAAAATGAGTCGGATTATATGAT TACTTTTTTATTCATTTTGCCAATCGTATCCTACGACATTGTTTTTAATTTGCAGACACAATTCATGCTGGATCA GCTTGCAGATCTTCAACAAAAGGTAATTATAAAATTCTACAAATTTCCAATAATTAATAAATGGAATAATTATGC GCGAGAAAT CTACCCTATGTAGGCGGAATCCTCTTTTCGACTCTG ACTCTCCCACTCCAGTCGTGAAAAAACAACAAACTAGTCAAAGGACAGCCTGCCTTATTCTTCTCCCGTTCGGGA CCCCTATTTTCTCGGAGATAGCCTGGTCTGAGCTAGAACAGCAGATTCGTGAGCAAGAGCGTATTTCACAGCTGA TTCAACAACAGCCATTTTTTCTGGGACCCGCAATTCCGTAGAAAGACATCACGATTCCTTGTGGACGGGGAATCG GCAGAAAGAGATGGGTCGGATACTGGAATCTGCCCAAAAGTCCTGACTTCTATTTAAAATTTACGATGAATTTTA ATTTTACAGGAGCAAATGCTTGCAGAATCTAATAGATTACTCCGTAGAAAGGTAAACTAACTTGATAGCCGTGCG TAATGAATAACTTATTTTATTTTCAAAATTATAAATCTAAATACTTAGGTAACTCGATAACATAAGAAGTATTTA TACTGATGATATTGGTGTTGTGTTTTTTTTTATTAGTTAGAAGAAAGTGTAGCTGGATTTCCACTTCGATTGTGT TGGGAAGATGGAGGTGATCATCAACTTATGCATCAACAAAATCGTCTCCCTAACACAGAGGGTTTCTTTCAGCCT CTTGGATTGCATTCTTCTTCTCCACATTTTGGGTAATTACTTTTATTATTATTAAAAATAATTTCAATTTTTTTT ACTTTTATTTCGATTAATAAATCAATGTGCACCAAGGTACGGTCTAACATAAACAAAAATGTGGGGAATGCTCTT AAAGCCCTAACAAAAGTTATTTGGTACGTGTACTAATGTAATCGTACTATATATCTTACTTGATTAGTGGATGGA CAGTACTGGGCACACACAATTGACATAAGTTATTATAAGGAAAAAAAAAGGCCAATAATCAATATAGTCCAACAT TACATTATTTATTATAACAGGTCACTCTAGATTAAATGTTAATGAATAACAAAAAGTCTCATATTGATGATTAAT GTGATGGGTGGGCTTCTTATAAGGCTTTGACAATCCTACTCTCTTTGAGCTAGTTTTGGGGGTGTGACCTAATTC AACAGAACGTAGTTAAGATTGTGAAGTAAAGTTGATCATTGTTATAACAGGTTTAAATACTTCTAGTAAAAATAG TTCCTAGATAATCCATCGCAAAATAGCTCCTATATAGTTAGTTGGATTTTCATATAATCTATAGCTTATACATAG CTAAATGGGAATAGATGAGAGTTTCTGTTGTTTAGATATGATATTTGATCGGTTTCTAAATCGTTACTATCATGT AGTGAATAATTTTCATGTTATTACTATTACATTTGATTGTTTCTGTGGTTATTTTTTTTTCTAGGTACAATCCTG TTAATACAGATGAGGTGAATGCAGCGGCAACTGCACACAATATGAATGGATTTATTCATGGATGGATGCTTTAA Mutant Solyc03g114840 gene allele ej2^CR >allele-1 (SEQ ID NO: 15) ATGGGAAGAGGAAGAGTTGAGCTTAAGAGAATAGAAAATAAAATAAATAGGCAAGTCACTTTTGCTAAGAGAAGA AATGGACTTCTTAAAAAAGCTTATGAACTTTCTGTTCTTTGTGATGCTGAAGTTGCCCTTATAATCTTCTCTAAT AGGGGTAAACTCTATGAATTTTGCAGCACTTCAAGGTATTTTTTATTTTATTATATTAACATCAAAGATTTTATT TTTTTAAAAAAAACCTTAAGTCCTTCATTACCAAAACCCTTAATTGATTTACAAAGTACTTTCATTAAATTTAGT AATTCTTTTTTTTTTTATCTCTGACTTCAATTATAATGCAAGATCTATGTTGTCTTTATATATATTGAATTATAT ATGTACTGTATTTTTACTATATACATATAAGATCCTTTTTTCTTTTTTTTCTGTCTCTTTATATAAATATATTTT AAATAGTTGATTTTGAAAGATCTACTAATGTATATTTATTTTTGGAACTTTTGTGTATATGGAATTTTTTTCTTT TTTATGTTTTTTTTTTGTTCTAATTGTTTTAAAAGCGTTTAAGATCAGAATGTTCTTGATTATTCTTTTAGGAAA AAGATTTCCCATACATTGAGTTATTTTTTGATCTGTAGATTGAATTTTTTTAATGAGTTCCGATAGATTTTCGTT CAATTTTTCAATGAAACTATTGAGGGTTGATGATTAGATAATTACTCGATTGAAAGTTTTTATTTCAAAAAAATT ATAATTCTTCTTAATTTTATATTTATGAGATAGAGTTAGTTTAGTGATTATATGAAAAATCGTATCAGATTATTG GGAATCGAAACTTAAAAATTCTGAAAATATTATTATAAATTTTACATGTTACAATATTTTTACTGTTAAGATTTG ATTTGCAGACTAGGTGTCATGTTTGACAGTTGATAAAAAATCTGTTATTTTTGTTCTTTAATTCCCAAGACGGAT AAACAAAGGCTGCTTATGTTGGTTTCCAATAAGCAGCCATAATTTTAAATATTTTTGTTAAGATTAATTAATAAC AATTATTTCCACCAGATAATTTTCAAAATTTGTGACCCCGAGTTCATATAAATTGTTAATTTTACTGCTAGAAAT TACATCGATAATAATTTATTTAGTGTAATCTTATAAATACGAGGGCAGTAGTGTATAGACTGTTTTTTATTAATC CTGACTCAAAGTGAGGTAAGTTAAGTATATTTTGATTAAAAGGACTACATTTCATTTATGTATGTTTAATTAATA TTATTTTGTAAGTCAATAAATCTAAACAACATGAGTTTATCTAGACCCTTAATTATGCACCTTCATTATCAATTT TTTCAATACTCTCCTCAGAACATATGCTTCTCTATAATTTTGTGCACGAGTTAATCAATTCTTCCTTTTCAATAA TTAAATATGTGATTTATGTTTAGCACTTATTTTTCGGTTAGTTAATTGATAATAGGAAAAAGCCTCTTTTTTTTT GTGTGTGTGGTAATTAGGATCTTTATTGAATTTAAAATGACCTACTATAGAACTTGGGAGTTTTTCTTCATAATA ATGCACTGCAACGTGTTAAAAAAAAAGAATCAAATGAAATTAATAGATGTTTACTGGATTGCCATGGTAAAGTGA TAAGTATTAATTTCGCTTTAACTAAGAGATCATTATATTCAAGTCCCCTTGATACAAACTTGCCTTTGTAAATAA GTGTTTTATTTTTCAATGTGAAACTTTCGCTGTTAATTTAAATTTAATTATACTTCTATATAAATACCAAACAAT AATGTAATAAAACAAAAAATAAAAGAGTAGATGTTTCATATTGTTAATGCAGCATGGTGAAAACAATTGAAAAGT ACCAACGTTGCAGCTATGCTACTTTGGAAGCCAACCAATCAGTTACTGATACTCAGGTACTGCTTTATATTTTAA TTTATTTGGCTTTTTTTTAAAAAAATAATTAGTTTTGATTAATATGCATCATTTTATTTATTTTTGGCAACTCTT TATTTATCAGTAATAAGTAATAACTTTTTAACTAGTATATTTAAAAATCACAAAATTTAAGAATATTTTAATAGA TTCGACATATTTTAGTTTAAAAATAACAAATTAAATTATGTTTTTAATTTTTTAAATATTCTTACTATAATTATC ATGTACTCTTTGATCTGTTCATCTTTTCCATGATAATATTATTTGGTCAGTTAGTGACATAAGAGTTTGAAATTT AGAAAAAAGGAATATTTGGAGAAAACTGAAATGGATATTTAGAAATGAAAGTTATTTAATATAAATATAAGTATG GGCTGCTGAGTTGGGAATCCACGCTGGAGATCTCAAGTTTGAAGCGTCTCACAAACAATAGTAATGTCTTTTTGG TCGAGTTTGTCGGATTGGACTTGTCCGTGGCCTGTGGGTTACTTTTCCTATATGGTTTGCAAGCTATCGGGAATT TTATCCTGGCGCACCCAAATTTGAGTTATTTTTGAGTTTTTATATGAAATAGCTTTGTGAATTCATCGAACTCCC GAAAACATTGAACTTTACTCCAAGTTGAATTGCAGTAAAATAATAGTAGCGATTCTTTAATTTATCCTAACAGTT TTTCGAAATAATAATCCCAAAAAAGTTTAAAATAACCATACCATAAACTTACTGGGTAAGATATTATCTGTCTAA TAATATATAGTAGTTTCTTTTGTTTTATTAGTTTATCTAATCCATATTTCATTTCTTGATAAGTTATTCTTAATA GGAAAATAAACTTATTTCGAAAAACTGTTTTTAAAATTTTCTTGAGTTGAGTCTTGGATGAAAAATAGTTAATTT TGCATTAATTAATTTTGTTCTAACAAAAACTAATTAAATTTTTTTGAAGCGCATATTCACTCAAAAAATAAATAA AAACCATCATGCATACAGGAAATGTTCTTTTTTTAATTTATTTTTTCATTGGAGCCCTGACTAATTTTATATCGG TTCATACTTTCATAAATTACAAAAAGTTCAAAATTTAAACTAACCATATAAGTGAATAAAATAAATCAACAAAAT ATTCACCACATAATACTTTTTAAATAGAATTTTTCATACCAAAGACCTTACTTTAATTAATTAGGGTGAGAGAAT CCTATAAGTCAATGCAAAACAATTCTATCTATCGGATTATAATCGTTGATTCATAAAATTTTAAAATCGACGATT TTCATTTAAATGACCCTTTTTTTTCTTTCATTTTTTATTGTTATTCATCTATTTAACTTGTGAGCATCTTTCATA TTGATATTTCAGACCCTTAAATTAATTGTTTTCTTACAGAATAACTACCACGAATATCTGAGGCTAAAAGCTAGA GTTGAGCTCCTCCAACGATCTCAGAGGTAATTTCTGTTCACTATCTTTATCTCAAATGAATTCTCATGTTTTTAT TTTTCGAGATTCAGATTAAATATAATTTGATGTATTATTAATTTAAATACGTTATTTAATATGGTCCTTATGTCC AACCATTGATTTAATTTGATATTTTTTTAATGAAAATTACACAGAAACTTTCTTGGTGAAGATTTGGGCACCTTG AGCAGCTTGAGAATCAATTAGAGTCTTCCTTAAAGTCAAGGAAGGTAAATTATTTAATCTAATTATACAGAAAAA TCATCTAAAAGTTACCTTAATTGCTAGCCCAATAAGTTTGCTATCTGTTGATCCTCACATTATTTTACTCACAGA AATTCACAATACCTTTATTTTTGTTTGAGTTTGAAGTATACAATTTCTTTAAAATGTAAAATTTGAAATCTCAAC AATAAGATATGTTATTGATCCTTGCAATTATGGGTAGATTGCGAATTAAACTATCTTGTCTTTGCTTACAACAGT CATTTTGTTTATAAACTAATTATACATAAATCCTAACTGATAGATAGTTTATAAAGATGAATAATGAACATAGGT CATATATTAAAAAAACAAAAAACAAAAAAAAACTAAACAAGATGAGCGAGTCAAAAATAGTCTTAACAAAAGAAT ATATATATATGTATATATCATATTTGATTTGTCTATTTTTAATTTTGAAAAAACTAAGTTAATCGATATATAATA TGAAGGCATAATGCATAAATATGTCCTTTAACTTGGTTTTAAATCACATTTATACCTCTTCGACTTTGGGTGTAT ACAAACAAACACTTAAACTTATATAATGTTGAACAAATAGATATATATGTCCTACATGTCATTTTTCGTCCTAAA TGGTGTCCTAAGTGTATTGTGTCACGCAGGACTCATGTGTCTATTTGTTCAAATTTATACAAGTTTAAGTGCTTA CTTATGTATAAACAAAGTTGAATGACATAAATGTGAAATAAAATCAAATTAAAGGGCATATTTATGCATTATACC TAATACGAAAATCCATATTATTCACTAAAAAATGAGTCGGATTATATGATTACTTTTTTATTCATTTTGCCAATC GTATCCTACGACATTGTTTTTAATTTGCAGACACAATTCATGCTGGATCAGCTTGCAGATCTTCAACAAAAGGTA ATTATAAAATTCTACAAATTTCCAATAATTAATAAATGGAATAATTATGCGCGAGAAATTTATCTATTTAAAATT TACGATGAATTTTAATTTTACAGGAGCAAATGCTTGCAGAATCTAATAGATTACTCCGTAGAAAGGTAAACTAAC TTGATAGCCGTGCGTAATGAATAACTTATTTTATTTTCAAAATTATAAATCTAAATACTTAGGTAACTCGATAAC ATAAGAAGTATTTATACTGATGATATTGGTGTTGTGTTTTTTTTTATTAGTTAGAAGAAAGTGTAGCTGGATTTC CACTTCGATTGTGTTGGGAAGATGGAGGTGATCATCAACTTATGCATCAACAAAATCGTCTCCCTAACACAGAGG GTTTCTTTCAGCCTCTTGGATTGCATTCTTCTTCTCCACATTTTGGGTAATTACTTTTATTATTATTAAAAATAA TTTCAATTTTTTTTACTTTTATTTCGATTAATAAATCAATGTGCACCAAGGTACGGTCTAACATAAACAAAAATG TGGGGAATGCTCTTAAAGCCCTAACAAAAGTTATTTGGTACGTGTACTAATGTAATCGTACTATATATCTTACTT GATTAGTGGATGGACAGTACTGGGCACACACAATTGACATAAGTTATTATAAGGAAAAAAAAAGGCCAATAATCA ATATAGTCCAACATTACATTATTTATTATAACAGGTCACTCTAGATTAAATGTTAATGAATAACAAAAAGTCTCA TATTGATGATTAATGTGATGGGTGGGCTTCTTATAAGGCTTTGACAATCCTACTCTCTTTGAGCTAGTTTTGGGG GTGTGACCTAATTCAACAGAACGTAGTTAAGATTGTGAAGTAAAGTTGATCATTGTTATAACAGGTTTAAATACT TCTAGTAAAAATAGTTCCTAGATAATCCATCGCAAAATAGCTCCTATATAGTTAGTTGGATTTTCATATAATCTA TAGCTTATACATAGCTAAATGGGAATAGATGAGAGTTTCTGTTGTTTAGATATGATATTTGATCGGTTTCTAAAT CGTTACTATCATGTAGTGAATAATTTTCATGTTATTACTATTACATTTGATTGTTTCTGTGGTTATTTTTTTTTC TAGGTACAATCCTGTTAATACAGATGAGGTGAATGCAGCGGCAACTGCACACAATATGAATGGATTTATTCATGG ATGGATGCTTTAA >allele-3 (SEQ ID NO: 16) ATGGGAAGAGGAAGAGTTGAGCTTAAGAGAATAGAAAATAAAATAAATAGGCAAGTCACTTTTGCTAAGAGAAGA AATGGACTTCTTAAAAAAGCTTATGAACTTTCTGTTCTTTGTGATGCTGAAGTTGCCCTTATAATCTTCTCTAAT AGGGGTAAACTCTATGAATTTTGCAGCACTTCAAGGTATTTTTTATTTTATTATATTAACATCAAAGATTTTATT TTTTTAAAAAAAACCTTAAGTCCTTCATTACCAAAACCCTTAATTGATTTACAAAGTACTTTCATTAAATTTAGT AATTCTTTTTTTTTTTATCTCTGACTTCAATTATAATGCAAGATCTATGTTGTCTTTATATATATTGAATTATAT ATGTACTGTATTTTTACTATATACATATAAGATCCTTTTTTCTTTTTTTTCTGTCTCTTTATATAAATATATTTT AAATAGTTGATTTTGAAAGATCTACTAATGTATATTTATTTTTGGAACTTTTGTGTATATGGAATTTTTTTCTTT TTTATGTTTTTTTTTTGTTCTAATTGTTTTAAAAGCGTTTAAGATCAGAATGTTCTTGATTATTCTTTTAGGAAA AAGATTTCCCATACATTGAGTTATTTTTTGATCTGTAGATTGAATTTTTTTAATGAGTTCCGATAGATTTTCGTT CAATTTTTCAATGAAACTATTGAGGGTTGATGATTAGATAATTACTCGATTGAAAGTTTTTATTTCAAAAAAATT ATAATTCTTCTTAATTTTATATTTATGAGATAGAGTTAGTTTAGTGATTATATGAAAAATCGTATCAGATTATTG GGAATCGAAACTTAAAAATTCTGAAAATATTATTATAAATTTTACATGTTACAATATTTTTACTGTTAAGATTTG ATTTGCAGACTAGGTGTCATGTTTGACAGTTGATAAAAAATCTGTTATTTTTGTTCTTTAATTCCCAAGACGGAT AAACAAAGGCTGCTTATGTTGGTTTCCAATAAGCAGCCATAATTTTAAATATTTTTGTTAAGATTAATTAATAAC AATTATTTCCACCAGATAATTTTCAAAATTTGTGACCCCGAGTTCATATAAATTGTTAATTTTACTGCTAGAAAT TACATCGATAATAATTTATTTAGTGTAATCTTATAAATACGAGGGCAGTAGTGTATAGACTGTTTTTTATTAATC CTGACTCAAAGTGAGGTAAGTTAAGTATATTTTGATTAAAAGGACTACATTTCATTTATGTATGTTTAATTAATA TTATTTTGTAAGTCAATAAATCTAAACAACATGAGTTTATCTAGACCCTTAATTATGCACCTTCATTATCAATTT TTTCAATACTCTCCTCAGAACATATGCTTCTCTATAATTTTGTGCACGAGTTAATCAATTCTTCCTTTTCAATAA TTAAATATGTGATTTATGTTTAGCACTTATTTTTCGGTTAGTTAATTGATAATAGGAAAAAGCCTCTTTTTTTTT GTGTGTGTGGTAATTAGGATCTTTATTGAATTTAAAATGACCTACTATAGAACTTGGGAGTTTTTCTTCATAATA ATGCACTGCAACGTGTTAAAAAAAAAGAATCAAATGAAATTAATAGATGTTTACTGGATTGCCATGGTAAAGTGA TAAGTATTAATTTCGCTTTAACTAAGAGATCATTATATTCAAGTCCCCTTGATACAAACTTGCCTTTGTAAATAA GTGTTTTATTTTTCAATGTGAAACTTTCGCTGTTAATTTAAATTTAATTATACTTCTATATAAATACCAAACAAT AATGTAATAAAACAAAAAATAAAAGAGTAGATGTTTCATATTGTTAATGCAGCATGGTGAAAACAATTGAAAAGT ACCAACGTTGCAGCTATGCTACTTTGGAAGCCAACCAATCAGTTACTGATACTCAGGTACTGCTTTATATTTTAA TTTATTTGGCTTTTTTTTAAAAAAATAATTAGTTTTGATTAATATGCATCATTTTATTTATTTTTGGCAACTCTT TATTTATCAGTAATAAGTAATAACTTTTTAACTAGTATATTTAAAAATCACAAAATTTAAGAATATTTTAATAGA TTCGACATATTTTAGTTTAAAAATAACAAATTAAATTATGTTTTTAATTTTTTAAATATTCTTACTATAATTATC ATGTACTCTTTGATCTGTTCATCTTTTCCATGATAATATTATTTGGTCAGTTAGTGACATAAGAGTTTGAAATTT AGAAAAAAGGAATATTTGGAGAAAACTGAAATGGATATTTAGAAATGAAAGTTATTTAATATAAATATAAGTATG GGCTGCTGAGTTGGGAATCCACGCTGGAGATCTCAAGTTTGAAGCGTCTCACAAACAATAGTAATGTCTTTTTGG TCGAGTTTGTCGGATTGGACTTGTCCGTGGCCTGTGGGTTACTTTTCCTATATGGTTTGCAAGCTATCGGGAATT TTATCCTGGCGCACCCAAATTTGAGTTATTTTTGAGTTTTTATATGAAATAGCTTTGTGAATTCATCGAACTCCC GAAAACATTGAACTTTACTCCAAGTTGAATTGCAGTAAAATAATAGTAGCGATTCTTTAATTTATCCTAACAGTT TTTCGAAATAATAATCCCAAAAAAGTTTAAAATAACCATACCATAAACTTACTGGGTAAGATATTATCTGTCTAA TAATATATAGTAGTTTCTTTTGTTTTATTAGTTTATCTAATCCATATTTCATTTCTTGATAAGTTATTCTTAATA GGAAAATAAACTTATTTCGAAAAACTGTTTTTAAAATTTTCTTGAGTTGAGTCTTGGATGAAAAATAGTTAATTT TGCATTAATTAATTTTGTTCTAACAAAAACTAATTAAATTTTTTTGAAGCGCATATTCACTCAAAAAATAAATAA AAACCATCATGCATACAGGAAATGTTCTTTTTTTAATTTATTTTTTCATTGGAGCCCTGACTAATTTTATATCGG TTCATACTTTCATAAATTACAAAAAGTTCAAAATTTAAACTAACCATATAAGTGAATAAAATAAATCAACAAAAT ATTCACCACATAATACTTTTTAAATAGAATTTTTCATACCAAAGACCTTACTTTAATTAATTAGGGTGAGAGAAT CCTATAAGTCAATGCAAAACAATTCTATCTATCGGATTATAATCGTTGATTCATAAAATTTTAAAATCGACGATT TTCATTTAAATGACCCTTTTTTTTCTTTCATTTTTTATTGTTATTCATCTATTTAACTTGTGAGCATCTTTCATA TTGATATTTCAGACCCTTAAATTAATTGTTTTCTTACAGAATAACTACCACGAATATCTGAGGCTAAAAGCTAGA GTTGAGCTCCTCCAACGATCTCAGAGGTAATTTCTGTTCACTATCTTTATCTCAAATGAATTCTCATGTTTTTAT TTTTCGAGATTCAGATTAAATATAATTTGATGTATTATTAATTTAAATACGTTATTTAATATGGTCCTTATGTCC AACCATTGATTTAATTTGATATTTTTTTAATGAAAATTACACAGAAACTTTCTTGGTGAAGATTTGGGCACGTTA AGCTTCGAAGGACCTTGAGCAGCTTGAGAATCAATTAGAGTCTTCCTTAAAGCAAATCAGGTCAAGGAAGGTAAA TTATTTAATCTAATTATACAGAAAAATCATCTAAAAGTTACCTTAATTGCTAGCCCAATAAGTTTGCTATCTGTT GATCCTCACATTATTTTACTCACAGAAATTCACAATACCTTTATTTTTGTTTGAGTTTGAAGTATACAATTTCTT TAAAATGTAAAATTTGAAATCTCAACAATAAGATATGTTATTGATCCTTGCAATTATGGGTAGATTGCGAATTAA ACTATCTTGTCTTTGCTTACAACAGTCATTTTGTTTATAAACTAATTATACATAAATCCTAACTGATAGATAGTT TATAAAGATGAATAATGAACATAGGTCATATATTAAAAAAACAAAAAACAAAAAAAAACTAAACAAGATGAGCGA GTCAAAAATAGTCTTAACAAAAGAATATATATATATGTATATATCATATTTGATTTGTCTATTTTTAATTTTGAA AAAACTAAGTTAATCGATATATAATATGAAGGCATAATGCATAAATATGTCCTTTAACTTGGTTTTAAATCACAT TTATACCTCTTCGACTTTGGGTGTATACAAACAAACACTTAAACTTATATAATGTTGAACAAATAGATATATATG TCCTACATGTCATTTTTCGTCCTAAATGGTGTCCTAAGTGTATTGTGTCACGCAGGACTCATGTGTCTATTTGTT CAAATTTATACAAGTTTAAGTGCTTACTTATGTATAAACAAAGTTGAATGACATAAATGTGAAATAAAATCAAAT TAAAGGGCATATTTATGCATTATACCTAATACGAAAATCCATATTATTCACTAAAAAATGAGTCGGATTATATGA TTACTTTTTTATTCATTTTGCCAATCGTATCCTACGACATTGTTTTTAATTTGCAGACACAATTCATGCTGGATC AGCTTGCAGATCTTCAACAAAAGGTAATTATAAAATTCTACAAATTTCCAATAATTAATAAATGGAATAATTATG CGCGAGAAATTTATCTATTTAAAATTTACGATGAATTTTAATTTTACAGGAGCAAATGCTTGCAGAATCTAATAG ATTACTCCGTAGAAAGGTAAACTAACTTGATAGCCGTGCGTAATGAATAACTTATTTTATTTTCAAAATTATAAA TCTAAATACTTAGGTAACTCGATAACATAAGAAGTATTTATACTGATGATATTGGTGTTGTGTTTTTTTTTATTA GTTAGAAGAAAGTGTAGCTGGATTTCCACTTCGATTGTGTTGGGAAGATGGAGGTGATCATCAACTTATGCATCA ACAAAATCGTCTCCCTAACACAGAGGGTTTCTTTCAGCCTCTTGGATTGCATTCTTCTTCTCCACATTTTGGGTA ATTACTTTTATTATTATTAAAAATAATTTCAATTTTTTTTACTTTTATTTCGATTAATAAATCAATGTGCACCAA GGTACGGTCTAACATAAACAAAAATGTGGGGAATGCTCTTAAAGCCCTAACAAAAGTTATTTGGTACGTGTACTA ATGTAATCGTACTATATATCTTACTTGATTAGTGGATGGACAGTACTGGGCACACACAATTGACATAAGTTATTA TAAGGAAAAAAAAAGGCCAATAATCAATATAGTCCAACATTACATTATTTATTATAACAGGTCACTCTAGATTAA ATGTTAATGAATAACAAAAAGTCTCATATTGATGATTAATGTGATGGGTGGGCTTCTTATAAGGCTTTGACAATC CTACTCTCTTTGAGCTAGTTTTGGGGGTGTGACCTAATTCAACAGAACGTAGTTAAGATTGTGAAGTAAAGTTGA TCATTGTTATAACAGGTTTAAATACTTCTAGTAAAAATAGTTCCTAGATAATCCATCGCAAAATAGCTCCTATAT AGTTAGTTGGATTTTCATATAATCTATAGCTTATACATAGCTAAATGGGAATAGATGAGAGTTTCTGTTGTTTAG ATATGATATTTGATCGGTTTCTAAATCGTTACTATCATGTAGTGAATAATTTTCATGTTATTACTATTACATTTG ATTGTTTCTGTGGTTATTTTTTTTTCTAGGTACAATCCTGTTAATACAGATGAGGTGAATGCAGCGGCAACTGCA CACAATATGAATGGATTTATTCATGGATGGATGCTTTAA

EXAMPLES Example 1. Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene Abstract

Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. The present disclosure relates to the finding of branched variants that carry mutations in two related transcription factors that had been selected independently. As described herein, one founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing “jointless” fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms.

Methods Experimental Model and Subject Details Plant Material and Growth Conditions

Seeds of the standard S. lycopersicum cultivar M82 (LA3475) were from the present stocks. Core collection germplasm (www.eu-sol.wur.nl) was from the seed stocks of Z. Lippman, D. Zamir, and S. Huang (Lin et al., 2014). Seeds of the jointless S. cheesmaniae accession LA0166 were obtained from the Charles M. Rick Tomato Genetics Resource Center (TGRC) at the University of California, Davis. The frondea mutant was obtained from the gene bank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) in Gatersleben, Germany. Seed of the long inflorescence (lin) mutant in the Micro-tom background (TOM-JPG5091) was provided by the University of Tsukuba, Gene Research Center, through the National Bio-Resource Project (NBRP) of the AMED, Japan (tomatoma.nbrp.jp/). The lin mutant was backcrossed four times to the standard M82 cultivar. The landrace collection (S. lycopersicunm var. cerasifornme) was from the seed stocks of E. van der Knaap. Tissue samples, DNA, or seed of elite breeding lines were obtained from Syngenta, Nunhems, Monsanto, Lipman Seeds, Johnny's Seeds, and TomatoGrowers.

Seeds were either pre-germinated on moistened Whatman paper at 28° C. in complete darkness or directly sown and germinated in soil in 96-cell plastic flats. Plants were grown under long-day conditions (16-h light/8-h dark) in a greenhouse under natural light supplemented with artificial light from high-pressure sodium bulbs (˜250 μmol m⁻²s⁻¹). Daytime and nighttime temperatures were 26-28° C. and 18-20° C., respectively, with a relative humidity of 40-60%.

Analyses of inflorescence architecture, sepal length, fruit type, and productivity traits were conducted on plants grown in the fields at Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., the Cornell Long Island Horticultural Experiment Station in Riverhead, N.Y., and net houses in Hatzav, Israel. Analyses of sepal length in the landraces were conducted on plants grown in the fields of the Durham horticulture farm at the University of Georgia, Athens, Ga. Seeds were germinated in 96-cell flats and grown for 32 d in the greenhouse before being transplanted to the field. Plants were grown under drip irrigation and standard fertilizer regimes. Damaged or diseased plants were marked throughout the season and were excluded from the analyses.

Method Details Plant Phenotyping

For analyses of sepal length, the length of sepals and petals of 10 closed flower buds per accession were manually measured and the sepal/petal ratio was calculated. Mature floral buds of similar developmental stage were collected (1-2 days before anthesis, i.e. before flower opening). For analyses of inflorescence complexity, the number of branching events was counted on at least 5 inflorescences on each replicate plant.

Yeast Two-Hybrid Analysis

Protein interaction assays in yeast were performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech) as described before (Park et al., 2014b). The coding sequences for bait proteins were cloned into the pGBKT7 vector, and the resulting vectors were transformed into the Y2HGold yeast strain. The coding sequences for prey proteins were cloned into the pGADT7 AD vector, and the resulting vectors were transformed into the Y187 yeast strain. After mating the two yeast strains expressing bait and prey proteins, diploid yeast cells were selected and grown on dropout medium without leucine and tryptophan. To assay protein-protein interactions, clones were grown on triple-dropout medium without leucine, tryptophan, and histidine for 3 d at 30° C. To block auto-activation, 3 mM 3-amino-1,2,4-triazole (3-AT) were added or adenine was removed from the triple-dropout medium. All primer sequences used for cloning can be found in Table 2.

Meristem Imaging

Live meristems were imaged using a Nikon SMZ1500 stereomicroscope (Nikon). Shoot apices were dissected from seedlings and older leaf primordia were removed to expose meristems. Immediately after dissection, sequences of optical layers were imaged using a Nikon DS-Ri1 digital camera (Nikon) mounted on the stereomicroscope. Z-stacks of optical sections were aligned and merged to produce final focused images using the NIS Elements BR3.2 software (Nikon).

Meristem Transcriptome Profiling

Meristem collection, RNA extraction, and library preparation for s2 mutant plants was performed as previously described (Park et al., 2012). Briefly, seedling shoots were collected at the vegetative meristem (VM), transition meristem (TM), sympodial inflorescence meristem (SIM), and floral meristem (FM) stage of meristem maturation, and immediately fixed them in ice-cold acetone. Meristems were manually dissected under a stereoscope and two biological replicates consisting of 30-50 meristems from independent plants were generated. Total RNA was extracted with the PicoPure RNA Extraction kit (Arcturus) and mRNA was purified with Dynabeads mRNA Purification kits (Thermo Fisher). Barcoded libraries were prepared using the NEBNext Ultra RNA library prep kit for Illumina according to the manufacturer's instructions, and assessed for size distribution and concentration with a Bioanalyzer 2100 (Agilent) and the Kapa Library quantification kit (Kapa Biosystems), respectively. Libraries were sequenced on a single Illumina Hiseq 2500 lane (222,279,510 million paired-end reads) at the Genome Center of Cold Spring Harbor Laboratories, Cold Spring Harbor.

Previously collected reads for wild-type tomato cultivar M82, compound inflorescence (s) mutant (Lemmon et al., 2016; Park et al., 2012), and reads for the s2 mutant were trimmed by quality using Trimmomatic (Bolger et al., 2014b) and aligned to the reference genome sequence of tomato (SL2.50) (Consortium, 2012) using Tophat2 (Kim et al., 2013). Alignments were sorted with samtools (Li et al., 2009) and gene expression quantified as unique read pairs aligned to reference annotated gene features (ITAG2.4) using HTSeq-count (Anders et al., 2015).

All statistical analyses of gene expression were conducted in R (RTeam, 2015). Expression of individual genes is shown as transcripts per million (TPM). Significant differential expression between meristem stages in wild-type tomato cultivar M82 was identified for 2,582 genes with edgeR (Robinson et al., 2009) using 2-foldchange, average 1 CPM, and FDR≤0.10 cutoffs (Lemmon et al., 2016). To compare expression dynamics by principal component analysis (PCA), z-score normalization of raw counts was used within genotype to minimize the impact of the different sequencing lengths (50 bp vs. 100 bp) and platforms (GAIIx and HiSeq2500). PCA was conducted on normalized expression values for the 2,582 dynamic genes in wild-type tomato cultivar M82, s, and s2 using the prcomp function in R (RTeam, 2015). The first two principal components were then plotted to assess acceleration or delay of the meristem maturation process in mutant samples. The proportion of TM and FM marker genes with moderate and severely delayed expression was assessed by a two-step k-means clustering. First, normalized WT expression was grouped into twelve clusters and the two clusters with the most specific TM and FM expression were designated as markers. Mutant expression from TM and FM marker genes was normalized with WT, producing WT:s and WT:s2 normalized expression datasets. Finally, k-means clustering (12 clusters) was performed on s and s2 normalized expression alone and clusters with delays in activation compared to WT were identified by hand.

Mapping-by-Sequencing

To map the causal mutations in the s2 mutant, two second-generation (F₂) populations were generated by crossing s2 with the S. lycopersicum cultivar M82, and s2 with S. pimpinellifolium. From a total of 464 s2×M82 F₂plants, 25 s2 mutants, 20 j2 mutants, and 13 WT siblings were selected for tissue collection, nuclei isolation, and DNA extraction. An equal amount of tissue from each plant (˜0.2 g) was pooled for DNA extraction using standard protocols. Libraries were prepared with the Illumina TruSeq DNA PCR-free prep kit from 2 μg genomic DNA sheared to 550 bp insert size. From a total of 576 s2×S. pimpinellifolium F₂plants, 16 s2 mutants, 9 j2 mutants, and 13 wild-type siblings were selected for DNA extraction. DNA was also extracted from the s2 parent (LA4371). Libraries were prepared with the Illumina TruSeq Nano DNA prep kit from 200 ng genomic DNA sheared to 550 bp insert size and 8 cycles of final amplification. All DNA libraries were sequenced on an Illumina NextSeq platform at the Cold Spring Harbor Laboratory Genome Center (Woodbury, N.Y.). For the s2×M82 F₂population, 62,317,992, 73,496,741, and 79,699,274 paired-end 151-bp reads were obtained for the s2 mutant, j2 mutant, and the WT sibling samples, respectively. For the s2×S. pimpinellifolium F₂population, 32,979,728, 82,439,796, and 50,763,441 paired-end 151-bp reads were obtained for pools of s2,j2, and the WT siblings, respectively. For the s2 parent 48,281,689 paired-end 151-bp reads were obtained.

To map the causal mutation in the in mutant, a F₂population was generated by crossing the in mutant with S. pimpinellifolium. From a total of 216 F₂plants, 8 lin mutant plants were selected with the most strongly branched inflorescences and 17 WT siblings for tissue collection. An equal amount of tissue from each plant (˜0.2 g) was pooled for nuclei isolation and DNA extraction using standard protocols. Barcoded libraries were prepared with the Illumina TruSeq DNA PCR-free prep kit from 2 μg genomic DNA sheared to 550 bp insert size and sequenced as above. 4,624,816 and 5,063,861 paired-end 101-bp reads were obtained for the in mutant and the WT sibling pools, respectively. To find the in mutation, a pool of 7 lin×S. pimpinellifolium F₂mutant plants was resequenced on the Illumina HiSeq2500 platform, and an additional 161,827,433 paired-end 101-bp reads were obtained.

To map s2 suppressor loci in S. pimpinellifolium, 1,536 S. pimpinellifolium×s2 F₂plants were regrown and 92 homozygous j2^TEej2 double mutants were selected by PCR genotyping. Primers are listed in Table 2. 18 s2 mutants, 6 moderately suppressed s2 mutants, and 2 strongly suppressed s2 mutants were selected for tissue collection, nuclei isolation, and DNA extraction. Libraries were prepared with the Illumina TruSeq DNA PCR-free prep kit from 2 μg genomic DNA sheared to 550 bp insert size, and sequenced as above. 38,060,212, 38,044,727 and 52,426,078 paired-end 151-bp reads were obtained for the pools of s2, moderately suppressed s2, and the strongly suppressed s2 plants, respectively.

Genomic DNA reads were trimmed by quality using Trimmomatic and paired reads mapped to the reference tomato genome (SL2.50) using BWA-MEM (Li, 2013; Li and Durbin, 2009). Alignments were then sorted with samtools and duplicates marked with PicardTools (Li et al., 2009, broadinstitute.github.io/picard). SNPs were called with samtools/bcftools (Li, 2011; Li et al., 2009) using read alignments for the various genomic DNA sequencing pools from this project in addition to reference M82 (Bolger et al., 2014a) and S. pimpinellifolium (Consortium, 2012) reads. Called SNPs were then filtered for bi-allelic high quality SNPs at least 100 bp from a called indel using bcftools (Li, 2011). Following read alignment and SNP calling, all statistics and calculations were done in R (RTeam, 2015). Read depth for each allele at segregating bi-allelic SNPs in 1 Mb sliding windows (by 100 kb) was summed for the various mutant (s2,j2^TEor suppression of s2) and wild-type sequencing pools and mutant:non-mutant SNP ratios were calculated. Finally, mutant SNP ratio was divided by wild-type SNP ratio (+0.5) and plotted across the 12 tomato chromosomes.

Tissue Collection and RNA Extraction

For semi-quantitative RT-PCR, seeds were germinated on moistened Whatman paper at 28° C. in complete darkness. Seedlings at similar germination stages were transferred to soil in 72-cell plastic flats and grown in the greenhouse. Shoot apices were collected at the floral meristem (FM) stage of meristem maturation (Park et al., 2012), and immediately flash-frozen in liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) and treated with the RNase Free DNase Set (Qiagen), or the Arcturus PicoPure RNA Extraction kit (Thermo Fisher) according to the manufacturer's instructions. 100 ng to 1 μg of total RNA was used for cDNA synthesis using the SuperScript III First-Strand Synthesis System (Invitrogen). All primer sequences can be found in Table 2.

Phylogenetic Analyses and Sequence Analyses

Sequences of tomato and Arabidopsis SEP family members were obtained from the Phytozome v11 database (phytozome.net) and aligned using the ClustalW function in MEGA. Phylogenetic trees for proteins with 1,000 bootstrap replicates were constructed using the maximum likelihood method in MEGA6 (Tamura et al., 2013). Homologous proteins in the clades containing Arabidopsis SEP1/2, SEP3, and SEP4 were assigned as SEP1/2-, SEP3-, and SEP4-homologs, respectively.

For analysing linkage between EJ2 and FW3.2, the M9 SNP was genotyped at position SL2.50ch03:64799226 (Chakrabarti et al., 2013) (G in S. pimpinellifolium (FW3.2) and A in S. lycopersicum cv. M82 (fw3.2)) in accessions of the tomato core collection using published genome sequencing data (Lin et al., 2014; Tieman et al., 2017).

CRISPR/Cas9 Mutagenesis, Plant Transformation, and Selection of Mutant Alleles

CRISPR/Cas9 mutagenesis and generation of transgenic plants was performed following the standard protocol (Belhaj et al., 2013; Brooks et al., 2014). Briefly, two single-guide (sg)RNAs binding in the coding sequence of the target gene were designed using the CRISPR-P tool (cbi.hzau.edu.cn/cgi-bin/CRISPR) (Lei et al., 2014). Vectors were assembled using the Golden Gate cloning system (Werner et al., 2012). The sgRNA-1 and sgRNA-2 were cloned downstream of the Arabidopsis U6 promoter in the Level 1 acceptors pICH47751 and pICH47761, respectively. The Level1 constructs pICH47731-NOSpro::NPTII, pICH47742-35S:Cas9, pICH47751-AtU6pro:sgRNA-1, and pICH47761-AtU6::sgRNA-2 were assembled in the binary Level 2 vector pAGM4723. Fifteen-μl restriction-ligation reactions were performed in a thermocycler (3 min at 37° C. and 4 min at 16° for 20 cycles, 5 min at 50° C., 5 min at 80° C., and final storage at 4° C.). All sgRNA sequences are listed in Table 2.

Final binary vectors were transformed into the tomato cultivar M82 and the tomato wild species S. pimpinelifolium by Agrobacterium tumefaciens-mediated transformation (Gupta, S. and Van Eck, 2016). After in-vitro regeneration, culture medium was washed from the root system and plants transplanted into soil. For acclimation, plants were covered with transparent plastic domes and maintained in a shaded area for 5 days. A total of 8 first-generation (To) transgenics were genotyped for induced lesions using forward and reverse primer flanking the sgRNA target sites. PCR products were separated on agarose gels and selected products were cloned into pSC-A-amp/kan vector (StrataClone Blunt PCR Cloning Kit, Stratagene). At least 6 clones per PCR product were sequenced using M13-F and M13-R primer. T₀plants with lesions were backcrossed to wild type and the F₁generation was genotyped for desirable large deletion alleles and presence/absence of the CRISPR/Cas9 transgene using primer binding the 3′ of the 35S promoter and the 5′ of the Cas9 transgene, respectively. All primers are listed in Table 2. Plants heterozygous for the engineered deletion alleles and lacking the transgene were self-pollinated to isolate homozygous, non-transgenic null mutants from the F₂generation.

Generation of Parental and Hybrid Lines for Cherry Tomato Breeding and Yield Trials Under Agricultural Greenhouse Conditions

To test the potential of j2 ej2 and s genotypes for fresh-market tomato breeding, hybrids were generated by crossing near-isogenic lines isolated from a breeding population that was developed for breeding high-yielding, indeterminate cherry tomato cultivars with a range of fruit shapes (Dani Zamir). Depending on genotype, near-isogenic lines were generated by backcrossing once to the respective cherry parents (BC₁) followed by inbreeding for 3 generations (F₃) or by inbreeding for 3-6 generations (F₃-F₆). Fruit shapes, inflorescence types, and yield characteristics were evaluated and selected each generation. Ten replicate plants per parental and hybrid line were grown in a randomized plot design in net houses in Hatzav, Israel in the year 2017. Damaged or diseased plants were marked throughout the season and were excluded from the analyses.

j2 Ej2 Hybrid Experiment

A jointless (j2^TE) processing inbred (F₆) wild type for EJ2 (j2 EJ2) served as parent (P-6022) for generating test and control hybrids. Test parents were isolated from a jointless (j2^TE) cherry inbred population (BC₁F3), which segregated for ej2^w. Twoj2^TEparents (P-6086-2 and P-6086-9) and two j2^TEej2 parents (P-6086-4 and P-6086-8) were selected by ej2^wgenotyping, and were crossed to P-6022. Control hybrids were generated by crossing the j2^TEtest parents (P-6086-2 for trail-I and P-6086-9 for trial-2) to the j2^TEparent (P-6022). Test hybrids were generated by bulk crossing the j2^TEej2^wtest parents (P-6086-4 for trail-1 and P-6086-8 for trial-2) to the j2^TEparent (P-6022).

s Hybrid Experiment

An indeterminate cocktail inbred (F₃) and a determinate cherry inbred (F₃) served as parents (P-6097 and P-6105, respectively) for generating test and control hybrids. Test parents were isolated from an indeterminate cherry-type F₅inbred line that segregated the s mutation. One parent wild type for S (P-6089) and one s mutant parent (P-6090) were selected by phenotyping and self-fertilized. The F₆generation was stable for unbranched (P-6089) and compound inflorescences (P-6090). Control and test hybrids were generated by bulk crossing the S parents (P-6097 for trail-1 and P-6105 for trial-2) to the S (P-6089) and the s (P-6090) test parents, respectively.

For analyses of yield component traits, mature green (MG) and red fruits (MR) were collected from 6 subsequent individual inflorescences and MG fruit number (MGFN), MR fruit number (MRFN), MG fruit weight (MGFW), and MR fruit weight (MRFW) was determined per inflorescence. Total fruit number (TFN) was the sum of MGFN and MRFN from each plant. Total yield (TY) was the sum of MGFW and MRFW from each plant. The average fruit weight (FW) was calculated by dividing MRFW by MRFN. From each plant, 7-10 fruits from at least one inflorescence were randomly selected to determine total soluble sugar content (Brix) in fruit juice. Brix value (percent) was quantified with a digital Brix refractometer (ATAGO Palette). For each measured yield parameter, mean values and percentage difference to the control hybrid were statistically compared using two tailed, two-sample t-tests.

Quantification and Statistical Analyses Sampling

For quantitative analyses of flower number per inflorescence and inflorescence internode length, at least 10 inflorescences were analyzed per genotype. For quantitative analyses of inflorescence complexity at least 5 inflorescences each from 6 individual replicate plants were analyzed per genotype. For quantitative analyses of relative sepal length, at least flowers were analyzed per genotype or ecotype. Hybrid inflorescence traits (number of branching events per inflorescence, total number of branches and flowers per plant) were determined for 6 subsequent inflorescences per individual plant and 9-10 individual plants per hybrid line. Total number of mature green and red fruits per individual plant was determined from 6 subsequent inflorescences per plant and 9-10 individual plants per hybrid line. Exact numbers of individuals (N) are presented in all FIGs. Statistical calculations were performed using R and Microsoft Excel. Mean values for each measured parameter were compared using two-tailed, two-samples Student's t-test.

Transcriptome Quantification

Reads for the wild-type M82, compound inflorescence (s) mutant (Lemmon et al., 2016; Park et al., 2012), and the s2 mutant were trimmed by quality using Trimmomatic v0.32 (HiSeq2500 parameters: ILLUMINACLIP:TruSeq3-PE-2.fa:2:40:15:1:FALSE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36; GAIIx parameters: ILLUMINACLIP:TruSeq2-PE.fa:2:30:10:1:FALSE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 TOPHRED33) (Bolger et al., 2014b) and aligned to the reference genome sequence of tomato (SL2.50) (Consortium, 2012) using Tophat2 v2.0.127 (parameters: —b2-very-sensitive—read-mismatches 2—read-edit-dist 2—min-anchor 8—splice-mismatches 0—min-intron-length 50—max-intron-length 50000—max-multihits 20) (Kim et al., 2013). Alignments were sorted with samtools (Li et al., 2009) and gene expression quantified as unique read pairs aligned to reference annotated gene features (ITAG2.4) using HTSeq-count v0.6.08 (parameters: —format=bam—order=name—stranded=no—type=exon—idattr=Parent) (Anders et al., 2015).

All statistical analyses of gene expression were conducted in R (RTeam, 2015). Significant differential expression between meristem stages in wild-type M82 was identified for 2,582 genes with edgeR (Robinson et al., 2009) using 2-foldchange, average 1 CPM, and FDR≤0.10 cutoffs (Lemmon et al., 2016). To compare expression dynamics between genotypes, z-score normalization was used within genotype to minimize the impact of the different sequencing lengths (50 bp vs. 100 bp) and platforms (GAIIx and HiSeq2500). A principal component analysis (PCA) was conducted on these normalized expression values for the 2,582 dynamic genes including wild-type M82, s, and s2 using the prcomp function in R (RTeam, 2015). The first two principal components were then plotted to assess modified maturation schedules in the mutant samples. The proportion of TM and FM marker genes with moderate and strongly delayed expression was assessed by a two-step k-means clustering. First, WT expression (TPM) was z-score normalized and clustered into twelve groups using the kmeans2 function from the Mfuzz package (Futschik, 2015) in R. The two clusters with the most specific TM and FM expression (clusters 06 and 08, respectively; FIG. 8A) were designated as marker clusters. Mutant s and s2 expression (TPM) from the 277 TM and 241 FM marker genes was z-score normalized with WT expression, producing a WT:s normalized expression and WT:s2 normalized expression dataset. Finally, k-means clustering (12 clusters) was performed on s (FIG. 8B) and s2 (FIG. 8C) expression alone (normalized by WT expression levels) and clusters with moderate and severe delays in activation compared to WT were manually identified.

Mapping

For mapping-by-sequencing of the various mutants, reads were trimmed by quality using Trimmomatic v0.32 (HiSeq 2500 read parameters: ILLUMINACLIP:TruSeq3-PE-2.fa:2:40:15:1:FALSE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36; GAIIx read parameters: ILLUMINACLIP:TruSeq2-PE.fa:2:30:10:1:FALSE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 TOPHRED33) and paired reads mapped to the reference tomato genome (SL2.50) using BWA-MEM v0.7.10-r789 (parameters: -M) (Li, 2013). Alignments were then sorted with samtools and duplicates marked with PicardTools vi.126 (parameters: VALIDATION_STRINGENCY=LENIENT) (Li et al., 2009, broadinstitute.github.io/picard). SNPs were called with samtools/bcftools v1.3.1 (samtools mpileup parameters: —ignore-RG—max-depth 1000000—output-tags DP,AN—min-BQ 0—no-BAQ—uncompressed—BCF; bcftools call parameters: —multiallelic-caller—variants-only—output-type z) (Li, 2011; Li et al., 2009) using read alignments for the various sequencing pools from this project in addition to reference M82 (Bolger et al., 2014a) and S. pimpinellifolium (Consortium, 2012) reads. Called SNPs were then filtered for bi-allelic high quality (MQ>=50) SNPs at least 100 bp from a called indel using bcftools (Li, 2011). Following read alignment and SNP calling and filtering, all mapping statistics and calculations were done using R (RTeam, 2015). Read depth for each allele at segregating bi-allelic SNPs in 1 Mb sliding windows (by 100 kb) was summed for the various mutant (lin, s2,j2, suppression of s2) and wild-type sequencing pools and mutant:non-mutant SNP ratios were calculated. Finally, mutant SNP ratio was divided by wild-type SNP ratio (+0.5) and plotted across the tomato genome.

Data and Software Availability

Raw sequencing reads generated in this study have been deposited at the Sequence Read Archive (ncbi.nlm.nih.gov/sra) under BioProject SRP100435.

Additional resources for the tomato core collection (please see e.g., unity.phenome-networks.com), for CRISPR design (please see e.g., cbi.hzau.edu.cn/cgi-bin/CRISPR), for sequence retrieval (please see e.g., phytozome.jgi.doe.gov/) and for data deposition (please see e.g., ncbi.nlm.nih.gov/sra) are also available to one of ordinary skill in the art.

Results

The s2 Variants Produce Branched Inflorescences and Flowers with Jointless Pedicels

To explore the challenges with improving tomato inflorescences, a core collection of 4,193 wild and domesticated accessions was screened for deviation from the typical inflorescence architecture of multiple flowers arranged along a single branch (FIG. 1A) (unity.phenome-networks.com, see STAR Methods). Twenty-three extremely branched accessions were previously reported that were all defective in the gene COMPOUND INFLORESCENCE (S, homolog of Arabidopsis WUSCHEL-RELATED HOMEOBOX 9, WOX9) (FIG. 1B) (Lippman et al., 2008). However, three rare variants not allelic to s that branched less frequently and also lacked the abscission zone on the stems (pedicels) of flowers known as the “joint” were also found (FIGS. 1C, 1D, and 7A-7F). Searching other germplasm sources provided one additional branched jointless mutant derived from an X-ray mutagenesis (FIGS. 7C and 7F) (Stubbe, 1972). Crosses among all four accessions failed to complement (FIGS. 7G-71). Consequently, these accessions were collectively named compound inflorescence 2 (s2).

One s2 accession was designated as a reference (LA4371), and an analysis of higher-order mutants with s showed an additive genetic relationship, indicating the gene(s) underlying s2 function separately from the S gene (FIGS. 1C and 7J). It was noted during the generation of s s2 plants that s2 segregated at a ratio of ˜ 1/16 (FIG. 1E), suggesting two unlinked recessive mutations underlie s2 phenotypes. Consistent with this, jointless plants (unbranched and branched) segregated as a single recessive mutation. This jointless trait resembled two classicaljointless-2 (j2) mutants reported 50 years ago. The original j2 was discovered in the unbranched wild tomato species S. cheesmaniae from the Galapagos Islands (Rick, 1956a). A second allele arose spontaneously in an agricultural field, but this mutation was also associated with inflorescence branching that caused excessive flower production and poor fruit set due to epistatic interactions with the domesticated germplasm (Reynard, 1961; Rick, 1956b). Breeders selected and utilized unbranched j2, because it reduced fruit dropping and enabled large-scale machine harvesting of processing tomatoes, while maintaining good fruit set (Robinson, 1980; Zahara and Scheuerman, 1988). Notably, the jointless phenotype of s2 was allelic to j2 (FIG. 7K), and s2 plants with normal pedicels were not found, suggesting branching required the j2 mutation. Therefore, the second locus was designated enhancer-of-jointless2 (ej2).

To better understand the developmental basis of s2 branching, the stages of meristem maturation during early inflorescence development were examined. Tomato inflorescences develop according to the sympodial growth program (Park et al., 2014a), in which each vegetative meristem matures into a transition meristem (TM) and terminates in a floral meristem (FM) that produces the first flower of the inflorescence. Additional flowers arise from iterative formation of specialized axillary (sympodial) inflorescence meristems (SIM), resulting in a multi-flowered inflorescence (FIG. 1F). In s mutants, both TM and SIM maturation are severely delayed, allowing multiple SIMs to form at each cycle (FIG. 1G) (Lippman et al., 2008; Park et al., 2012). Additional SIMs also formed in s2 plants, but less than in s (FIG. 1H). To determine if s2 was delayed in maturation, RNA-seq was performed on sequential s2 meristem maturation stages and compared transcriptome dynamics with existing maturation profiles for s and WT (see STAR Methods) (Park et al., 2012). A principal component analysis (PCA) using 2,582 maturation marker genes (Lemmon et al., 2016) showed fewer TM and FM marker genes were delayed during s2 meristem maturation compared to s, consistent with less branching in s2 inflorescences (FIGS. 1I-1K and 8).

Mutations in Two related MADS-Box Genes Cause s2 Branching

The j2 mutant was previously mapped to the centromere of chromosome 12, but poor recombination prevented identification of the responsible gene (Budiman et al., 2004; Yang et al., 2005). To clone the genes underlying j2 and ej2, two F₂populations were generated from crossing s2 with the jointed (J2/J2) cultivar M82 and the wild ancestor of tomato, S. pimpinellifolium. In the intra-species F₂population, s2 plants segregated at the expected ratio of ˜ 1/16, but this segregation was substantially lower in the S. pimpinellifolium population, suggesting unknown modifier loci can suppress s2 branching (FIGS. 9A-9C). To map j2 and ej2 simultaneously, genome sequencing was performed on pools of DNA from s2,j2, and WT F₂segregating plants (see STAR Methods). Comparing SNP ratios between s2 and WT pools in both populations revealed a region near the bottom of chromosome 3 and the centromere of chromosome 12 with a strong bias for SNPs from the s2 parent (FIGS. 2A, 9D, and 9E). SNP ratios between s2 and j2 revealed a bias only at the bottom of chromosome 3. These results confirmed j2 is located near the chromosome 12 centromere and revealed ej2 resides on chromosome 3.

MADS-box transcription factors are known to contribute to pedicel abscission zone development in tomato (Liu et al., 2014; Mao et al., 2000; Nakano et al., 2012; Shalit et al., 2009). The jointless1 mutant (j1) was mapped to chromosome 11 and found to be mutated in a homolog of the Arabidopsis MADS-box flowering regulator SHORT VEGETATIVE PHASE (SVP) (Hartmann et al., 2000; Mao et al., 2000). Therefore, the ˜6 Mbp j2 mapping interval for MADS-box genes was searched, and among the 164 genes in this region only one candidate was found, Solyc12g038510, a homolog of the Arabidopsis floral organ identity MADS-box gene SEPALLATA4 (SEP4) (FIG. 2B) (Ditta et al., 2004). Previous transcriptional silencing of Solyc12g038510 resulted in jointless pedicels, but it was suggested Solyc12g038510 and J2 were different genes, because the published j2 mapping interval did not coincide with Solyc12g038510, likely from unreliable centromeric marker resolution (Budiman et al., 2004; Liu et al., 2014). However, the genomic sequencing of s2 and j2 mutants exposed a Copia/Rider-type transposable element (TE) in the first intron of Solyc12g038510 that was absent in WT (FIG. 2C). Furthermore, the s2 RNA-seq revealed that most Solyc12g038510 transcripts initiated in the first intron, resulting in an early nonsense mutation (FIGS. 2D and 9H). To validate that Solyc12g038510 is J2, CRISPR/Cas9 was used to engineer loss-of-function mutations, and the resulting j2^CRplants developed jointless unbranched inflorescences (FIGS. 2E and 2F). Moreover, progeny from crossing j2^CRwith s2-derived j2 had jointless and unbranched inflorescences (FIG. 2G), and sequencing Solyc12g038510 in the original j2 S. cheesmaniae accession revealed an early stop codon (FIGS. 9F-9H). Thus, the SEP4 gene Solyc12g038510 is J2, and two natural mutations arose independently (hereafter designated j2^TEand j2^stop) (Reynard, 1961; Rick, 1956a).

Both j2 and ej2 are required for s2 branching, suggesting the underlying genes function redundantly, similar to SEP genes in Arabidopsis that control floral organ identity (Ditta et al., 2004; Pelaz et al., 2000). The 66 genes were searched in the 500 kbp ej2 mapping interval for MADS-box genes and the tandemly arranged Solyc03g114830 and Solyc03g114840 were found (FIG. 2H). Solyc03g114830 is a homolog of Arabidopsis FRUITFULL and transcriptional knockdown of this gene causes defects in fruit ripening (Bemer et al., 2012; Wang et al., 2014). The genomic sequencing of s2 mutants did not reveal any Solyc03g114830 coding or noncoding SNPs, or large indels, and s2 fruits ripened normally. In contrast, Solyc03g114840 is another homolog of SEP4, and a 564 bp insertion was found in the 5^thintron of s2 mutants, which was absent in WT (FIG. 2I). Notably, RNA-seq reads from s2 revealed a third of Solyc03g114840 transcripts were misspliced, suggesting the insertion caused a partial loss of function (FIG. 2J). To test this and uncover the phenotypic consequences of strong loss of EJ2 function, new alleles were engineered with CRISPR/Cas9 and e j2^CRinflorescences were found to be unbranched, but the sepals (outermost leaf-like organs of the flowers) were exceptionally large and fruits were pear-shaped (FIGS. 2K and 2L). To determine if the original ej2 mutation impacted flower and/or fruit morphology, ej2 was backcrossed into M82 and relative sepal length (defined by sepal/petal length ratio) was measured. Notably, whereas there was no obvious change in fruit shape or size, ej2 sepals were 50% longer than WT but shorter than ej2^CR, consistent with a weak allele (FIGS. 2M, 2N, and 9I). Importantly, flowers of F₁progeny from crossing ej2 and ej2^CRalso developed long sepals. Thus, Solyc03g114840 is E12, and the natural ej2 mutation is a weak loss-of-function allele (hereafter designated ej2^w).

Finally, it was verified that the other s2 accessions carried mutations in both j2 and ej2. PCR genotyping showed all but one accession was double mutant for ej2^wand either j2^TEor j2^stop(FIG. 9J). The last accession was homozygous for ej2^w, but J2 could not be amplified, consistent with having originated from an X-ray mutagenesis (Stubbe, 1972). Thus, the prolonged meristem maturation underlying s2 inflorescence branching is caused by mutations in two redundantly acting SEP MADS-box genes.

Ej2^wArose During Domestication and Hindered j2 Utilization for Breeding

In modern breeding programs, the value of jointless varieties was recognized for their potential to reduce fruit drop and post-harvest damage during mechanical harvesting for the processing tomato industry. Yet, plants carrying j1 yield poorly due to reversion of inflorescences to vegetative growth after developing a few flowers (Butler, 1936; Mao et al., 2000). Thus, j2 was widely favored over the last 50 years of breeding. However, breeders frequently experienced problems with excessive inflorescence branching and low yield upon introducing j2 into different cultivated backgrounds (Robinson, 1980), probably because of negative epistasis with ej2^w. To determine to what extent ej2^whindered j2 utilization in breeding, 568 wild and domesticated accessions were genotyped from the tomato core collection and more than half were found to be homozygous for the ej2^wallele (FIG. 3A). Notably, ej2^wwas absent from S. pimpinellifolium, but 40% of early domesticates (landraces) were homozygous for the mutation, and the percentage doubled in cultivars. Most importantly, ej2^wwas strongly associated with long sepals, including within a subset of confirmed landraces (Blanca et al., 2015), suggesting selection during domestication (FIGS. 3B-3E). In support of this, ej2^wis in close proximity (<46 Kbp) to a previously reported domestication and improvement selective sweep (Lin et al., 2014). Notably, a minor fruit weight QTL (fv3.2) that also arose in the landraces is in close proximity (˜85 Kbp) to EJ2 (Chakrabarti et al., 2013; Zhang et al., 2012). Among 62 landraces, accessions were found that carried ej2^wbut not fw3.2 (ej2^w/FW3.2: 7%) and vice versa (EJ2/fv3.2: 9%), suggesting that each mutant allele arose independently and were likely combined early in domestication. It was also found that not all cultivated lines carried both alleles (ej2^w/FW3.2: 2%; EJ2/fv3.2: 11%), indicating that both mutations were either passed on independently during domestication and improvement, or were co-selected and then separated later by breeding.

One explanation for the early selection of ej2^wand its subsequent spread in the cultivated germplasm is that larger sepals provided an enlarged calyx that was concomitantly selected as fruit size increased, perhaps with fw3.2. Such a trait would not necessarily have been selected for improved productivity by increasing fruit size or number per se, but instead could have provided improved fruit support, strong local source tissue, or simply aesthetic value for larger fruits. To determine if ej2^wwas selected during domestication and breeding of larger fruits, the frequency of the ej2^wallele was evaluated in 258 cultivars representing five fruit sizes ranging from small “cherry” tomatoes (<5 g) to extremely large “beefsteak” varieties (>500 g). Remarkably, the frequency of the allele increased with fruit size, and nearly all (>90%) large-fruited accessions were homozygous for ej2^w, including 88% of vintage heirloom cultivars (Male, 1999). These results show that the ej2^wallele was already widespread in larger fruit types before j2 was discovered and adopted in modern breeding (FIG. 3F). Since EJ2 is also expressed in developing fruits (FIG. 10A) and e j2^CRfruits are elongated (FIG. 2L), it is also possible the ej2^wallele impacts other fruit traits such as size/shape and/or ripening, especially in the presence of other QTL that impact these traits.

Elite breeding germplasm carries both j2^TEand ej2^w, but branching is suppressed

Because ej2^wbecame widespread in tomato germplasm and j2 arose much later, introducing either of the j2 alleles into most cultivars would have resulted in undesirable branching and low yield. However, it was reported these adverse effects could be overcome by breeding (Robinson, 1980). One possibility is that ej2^wwas segregated away through crosses. Alternatively, breeders could have identified and selected natural suppressors of branching. To test this, 153 unbranched jointed and jointless elite inbreds and hybrids were obtained from major seed companies and public breeders (see STAR Methods), and genotyped for both mutations. All jointless lines were homozygous for j2^TE, indicating the allele that arose in the domesticated germplasm was favored in breeding. Since new tomato varieties for processing and fresh-market production are developed in separate breeding programs, it was asked if j2^TEwas utilized in both. The value of the jointless trait is most recognized for mechanical harvesting of processing types, and in support of this the j2^TEallele was present in 74% of sampled processing lines. Although less widespread, j2^TEwas also found in 34% of fresh-market lines, indicating that j2^TEcontinues to be utilized in both breeding programs.

Remarkably, it was found that more than 60% of j2^TEhomozygotes in both processing and fresh-market lines were also homozygous ej2^w(FIGS. 4A and 4B), supporting the hypothesis that suppressors were selected during improvement. This was reminiscent of the reduced segregation of s2 in the S. pimpinellifolium F₂mapping population (FIGS. 9B and 9C). To map potential suppressor loci, 1,536 F₂plants were regrown, and of 92 plants homozygous for both mutations, 24% showed various degrees of suppression (FIG. 4C).

Using genome sequencing, one large-effect suppressor was mapped near the end of chromosome 2 in the same region as a previously reported suppressor in the domesticated germplasm (FIG. 4D) (Robinson, 1980). However, given that only a small percentage of j2^TEej2^wF₂plants displayed unbranched inflorescences, additional suppressors from breeding germplasm are likely involved, which together were needed to achieve complete suppression.

Three meristem expressed SEP4 genes modulate inflorescence complexity

The dissection of the negative epistasis underlying s2 branching exposed two tomato SEP4 genes that act redundantly to control meristem maturation and inflorescence development. This led to the question of to what extent these genes work with other tomato SEP family members to regulate inflorescence architecture and flower production, and could have potential for agricultural application. In Arabidopsis, a family of four redundant SEP genes is required to establish floral organ identity (Ditta et al., 2004; Pelaz et al., 2000). Tomato has an expanded SEP family of six members (Consortium, 2012), and a phylogenetic analysis of protein sequences showed Arabidopsis SEP1, 2, and 3 have two tomato homologs (Solyc05g015750/TM5 and Solyc02g089200/TM29) (FIG. 5A). In contrast, there are four homologs of SEP4, and among them is the RIPENING INHIBITOR (RIN) gene. A classical mutation in RIN blocks ripening and is widely used in hybrid breeding due to a heterozygous dosage effect that causes fruits to remain firm and ripen over a protracted period, improving shelf life (Klee and Giovannoni, 2011; Vrebalov et al., 2002).

To investigate individual and combined roles of tomato SEP genes in inflorescence development, expression patterns were first analyzed using the meristem maturation atlas and transcriptome data from other major tissues (Consortium, 2012; Park et al., 2012). Both TM5 and TM29 (SEP1/2/3 homologs) were expressed only later in reproductive development, beginning in floral meristems and extending into flowers and fruits (FIGS. 5B and 10A), supporting previously characterized roles in floral organ identity (Ampomah-Dwamena et al., 2002; Pnueli et al., 1994). RIN was only expressed in fruits, consistent with its role in ripening (FIG. 10A) (Vrebalov et al., 2002). In contrast, expression of J2, EJ2, and the fourth SEP4 homolog (Solyc04g005320) began earlier, in the TM stage of meristem maturation and in SIMs (FIG. 5B). This suggested Solyc04g005320 could function with J2 and EJ2 in meristem maturation. Moreover, given that Arabidopsis SEP redundancy is based on formation of multimeric protein complexes (Theissen et al., 2016), interactions were tested among all four tomato SEP4 proteins in yeast two-hybrid assays and J2, EJ2, and Solyc04g005320 were found to interact with each other and themselves, except for homomeric EJ2. These results validated previous findings (Leseberg et al., 2008), and further revealed that J2 and EJ2 interact with each other, supporting redundancy in the control of meristem maturation and inflorescence architecture (FIGS. 5C, 5D, 10B and 10C).

To test if Solyc04g005320 contributes to inflorescence architecture and flower production, CRISPR/Cas9 was used to engineer plants with null mutations, which resulted in exceptionally long inflorescences with nearly twice as many flowers as WT and longer internodes (FIGS. 5E and 10D). Weak branching late in inflorescence development was also frequently observed. Whether similar effects occur in genotypes that already have long inflorescences was tested by mutating Solyc04g005320 in S. pimpinellifolium, which produces 15-20 flowers on each inflorescence. Remarkably, internode length and flower number doubled (FIGS. 5F, 10D-10F). These phenotypes were reminiscent of a gamma-irradiation mutant designated long inflorescence (lin) that was previously mapped to an interval on chromosome 4 containing Solyc04g005320 (FIGS. 10G-10J)(see STAR Methods). Sequencing Solyc04g005320 from the lin mutant revealed a translocation in the first intron that eliminated transcription (FIGS. 10J-10L, also referred to herein as lin^trans) and crosses with a CRISPR allele failed to complement the long inflorescence phenotype.

The increase in inflorescence complexity in lin mutants is modest compared to j2 ej2^wdouble mutants. To study the extent of redundancy and potential dosage relationships among the three genes, strong alleles were used in the same background to create all combinations of higher-order mutants (see STAR Methods). Whereas j2^CRwas largely additive with lin (FIG. 10M), ej2^CRand lin were synergistic for floral organ development; double-mutants had long inflorescences with more flowers that developed extremely enlarged sepals, but inner floral organs did not fully develop and fruits failed to form (FIG. 10N). As expected, j2^CRand ej2^CRwere also synergistic, but unlike the moderately branched, highly floral inflorescences of the original j2^TE/stopej2^wnatural double mutants (s2), inflorescences from j2^CRe j2^CRplants were extraordinarily branched and rarely produced normal fertile flowers (FIG. 5G). Finally, combining all three mutants resulted in massively over proliferated SIMs without forming flowers (FIGS. 5H and 10O). The same effect was observed in S. pimpinellifolium j2^CRej2^CRlin^CRplants (FIGS. 5I and 10O). The sequences for S. pimpinellifolium j2^CRej2^CRlin^CRare shown below. Thus, J2 and E/2 have distinct roles in floral development, but all three SEP4 genes have overlapping roles in meristem maturation and inflorescence development.

Dosage of Meristem Maturation Transcription factors can be Exploited to Improve Inflorescence Architecture and Yield

The individual and combined mutations in J2, EJ2, and LIN provided a series of three forms of increased inflorescence complexity ranging from weak (lin single mutants) to extremely severe (j2 ej2 lin triple mutants), indicating quantitative relationships among these SEP4 genes. It was previously demonstrated that dosage relationships among genes in the florigen pathway could be exploited to create a quantitative range of plant architectures that translated to improved productivity in determinate field-grown tomatoes (Park et al., 2014b; Soyk et al., 2016). It was reasoned that dosage sensitivity could be similarly used to fine-tune inflorescence architecture and flower production. To test this, a series of homozygous and heterozygous combinations of j2 strong alleles with ej2^wor ej2^CRin the isogenic M82 background was first created (FIGS. 6A and 6B). All double heterozygotes (e.g. j2/+ej2^w/+; j2/+ej2^CR/+) and plants heterozygous for j2 and homozygous for ej2^w(j2/+ej2^w) produced unbranched inflorescences like the single mutants. In contrast, heterozygosity for ej2^win a j2 background (j2 ej2^w/+) conferred weak branching, as did j2/+ej2^CR. Notably, heterozygosity for the null e j2^CRallele in the null j2 background (j2 ej2^CR/+) resulted in branching that matched s2 inflorescences (j2 ej2^w), further validating that ej2^wis a weak allele and confirming a sensitive dosage relationship between these genes. Given these results, it was reasoned that other meristem maturation regulators might have similar dosage-sensitivity on inflorescence architecture and this was tested with S, a member of the WOX protein family (Graaff et al., 2009; Lippman et al., 2008). Indeed, plants heterozygous for three s mutant alleles were also mildly branched (FIGS. 6C and 6D), demonstrating dosage-sensitivity of independent meristem maturation genes allows for quantitative tuning of inflorescence architecture.

DISCUSSION Dose-Dependent Quantitative Variation, Weak Alleles, and Crop Improvement

This study involved exploration of the potential of genes and alleles underlying natural variation in inflorescence complexity to improve productivity. By analyzing the s2 branching variant, it was found that multiple members of the SEP4 subfamily of tomato MADS-box genes play critical redundant roles in modulating meristem maturation and inflorescence architecture. The first MADS-box family member involved in tomato domestication was further described, highlighting the growing significance of this transcription factor family in contributing to domestication and improvement of diverse crops (Singh et al., 2013; Vrebalov et al., 2002; Zhao et al., 2011). By dissecting interactions between meristem expressed SEP4 genes dosage relationships were uncovered among an allelic series of MADS-box mutations with potential for breeding. This collection of alleles, including mutations in S, comprises a toolkit to manipulate inflorescence architecture, which can now be expanded to additional regulators of meristem maturation, such as LIN. To demonstrate this, CRISPR/Cas9 was used to target LIN in the elite cherry tomato cultivar Sweet 100 and mutant lines were generated with moderately branched inflorescences and increased flower production (FIGS. 10P-10S).

The present approach for creating desirable phenotypic variation in major yield traits relies on combining specific heterozygous and homozygous mutations to obtain a quantitative range of dosage effects (Park et al., 2014b). However, exploiting gene dosage may be limited by the availability of weak alleles that confer quantitative trait modifications. For example, longer sepals and weak branching were achieved through different levels of reduced EJ2 dosage from homozygosity and heterozygosity for ej2′, respectively. In nature, similar dosage effects often arise from mutations in transcriptional control regions (e.g., in cis-regulatory DNA). Such alleles were widely favored in crop domestication and improvement for their subtle phenotypic changes compared to null alleles that frequently display deleterious pleiotropic effects (Meyer and Purugganan, 2013; Purugganan and Fuller, 2009). For example, increased fruit size during tomato domestication depended in part on transcriptional alleles of multiple components in the classical CLAVATA-WUSCHEL stem cell circuit (Xu et al., 2015). A potentially powerful approach to engineer novel weak alleles that are being explored (Swinnen et al., 2016) is exploiting gene-editing technology to mutate cis-regulatory control regions of productivity genes. A promising target identified in this study is LIN. CRISPR/Cas9-induced weak transcriptional alleles that confer reduced LIN expression may provide subtle increases in flower production, which may be especially valuable in large-fruited cultivars where branching often negatively impacts fruit weight and yield. Notably, a rice homolog ofLINand other meristem maturation genes control panicle architecture and grain production (Kobayashi et al., 2010, 2012; Liu et al., 2013), suggesting the present findings have broad agricultural potential. New gene-editing tools should enable the engineering of diverse types and strengths of alleles that can provide customized gene dosage effects to improve a wide range of agronomic traits in many crops.

Epistasis in Evolution, Domestication, and Breeding

Progress in breeding is largely driven by loci with predictable additive effects. For example, the majority of flowering time variation in maize is determined by thousands of small additive quantitative trait loci (QTL) (Buckler et al., 2009), and the same is true for traits in other crops (Doust et al., 2014; Gao et al., 2015). Yet, epistatic interactions, both positive and negative, are also important in breeding, particularly when working with disparate germplasm. For example, interactions between interspecific quantitative trait loci (QTL) in rice can improve aluminum tolerance (Famoso et al., 2011), whereas stacking multiple wild species-derived QTL affecting the same yield traits in tomato results in less-than-additive or “diminishing returns” epistasis (Eshed and Zamir, 1996).

In recent years, several cases of negative epistasis have emerged in diverse organisms involving clashes between newly evolved and established alleles, or upon bringing together distinct genomes, either through natural or artificial means. Examples include compromised fitness gains upon combining interacting alleles in bacteria and yeast (Chou et al., 2011; Heck et al., 2006; Khan et al., 2011; Kvitek and Sherlock, 2011), hybrid necrosis between distinct accessions of Arabidopsis (Chae et al., 2014), and loss of protection from malaria in humans when two common resistance variants are co-inherited (Williams et al., 2005). Compared to negative epistasis in evolution and natural selection, the intense artificial selection imposed by humans during domestication and breeding could drive more frequent occurrences of epistasis. While dramatic cases like the one described in this study could be overcome through selection against interactions or suppression with modifiers, there may be many undiscovered negative interactions in agriculture with more subtle phenotypic consequences that may remain challenging to detect and dissect until high throughput quantitative phenotyping platforms (phenomics) and power in genome-wide association studies (GWAS) improves.

The present dissection of the extreme negative epistasis underlying the s2 branching syndrome has highlighted an underappreciated challenge for the next generation of crop breeding. Specifically, using rapidly advancing gene-editing technologies to introduce precise novel allelic variation for specific genes into existing germplasm may not provide desirable phenotypic outcomes, and could potentially result in negative consequences due to interactions with alleles selected and stabilized during domestication and early breeding (Mackay, 2013). That the present example of negative epistasis involved two closely related MADS-box genes suggests that engineering new alleles in gene families or related developmental pathways that already played a role in domestication and improvement may be particularly sensitive to unexpected epistatic consequences, perhaps explaining other as yet uncharacterized examples of negative epistasis in agriculture (Bomblies and Weigel, 2007; Matsubara et al., 2015; Shang et al., 2016; Zhang et al., 2011). Elucidating, neutralizing, and potentially exploiting negative epistasis, as done in the present study, could have a significant impact in helping break productivity barriers in breeding of both plants and animals.

TABLE 2 Oligos used in this study Yeast two-hybrid assays Gene Forward Reverse Restr. name Gene ID primer primer enzyme LIN Solyc04 CACCGAATTCA TTCGGATCCTCA EcoRI + g005320 TGGGAAGAGGT AAGCATCCATCC BamHI AAGGTAGAA TGGTAA (SEQ (SEQ ID NO: ID NO: 18) 17) J2 Solyc12 CACCGAATTCA TTCGGATCCTTA EcoRI + g038510 TGGGAAGAGGA GAGCATCCACCC BamH I AGAGTAGAAC TGGAAT (SEQ (SEQ ID NO: ID NO: 20) 19) EJ2 Solyc03 CACCGAATTCA TTCGGATCCTTA EcoRI + g114840 TGGGAAGAGGA AAGCATCCATCC BamHI AGAGTTGAG ATGAATAAATC (SEQ ID NO: (SEQ ID NO: 21) 22) RIN Solyc05 CACCGAATTCA TTCGGATCCTCA EcoRI + g012020 TGGGTAGAGGG AAGCATCCATCC BamH I AAAGTAGAA AGGTACA (SEQ (SEQ ID NO: ID NO: 24) 23) Natural and induced mutant alleles analyzed in this study, and respective genotyping markers Gene Forward Reverse Allele Restr. name Gene ID primer primer name Type WT Mutant enzyme LIN Solyc04 GCAAAACTTTA CTTTTTGATTCA lin indel 396 — — g005320 AATTAGTTCTA TGTGTCTGTAC ATG (SEQ (SEQ ID NO: ID NO: 25) 26) LIN Solyc04 AATATCGTGTT CTTTTTGATTCA lin indel — 358 — g005320 AGAATGTGACA TGTGTCTGTAC C (SEQ ID (SEQ ID NO: NO: 27) 28) J2 Solyc12 TTACTTTTGCT CCGTCCTTTCTG j2-TE Indel — 193 — g038510 AAGAGAAGAAA TTTGTAGC bp TGG (SEQ ID (SEQ ID NO: NO: 29) 30) J2 Solyc12 TTACTTTTGCT GAATCCACTTAA j2-TE Indel 709 — g038510 AAGAGAAGAAA GAATCTCTACC bp TGG (SEQ ID (SEQ ID NO: NO: 31) 32) J2 Solyc12 TATTGTGATAT AATACCTGAGTA j2- dCAPS 206 230 HpaI g038510 GTAGAGTGGTG TCACTAACCGTT classic bp + bp C (SEQ ID (SEQ ID NO: 24 NO: 33) 34) bp EJ2 Solyc03 CACAATTCATG CGGAGTAATCTA ej2-w Indel 177 738 — g114840 CTGGATCAGC TTAGATTCTGC bp bp (SEQ ID NO: (SEQ ID NO: 35) 36) LIN Solyc04 CCTTTAATAAG TTGAAGGTGCAT CR-lin- Indel 855 1390 — g005320 TTGAAAATCCC AGAACATACC a1 bp bp TC (SEQ ID (SEQ ID NO: NO: 37) 38) LIN Solyc04 CCTTTAATAAG TTGAAGGTGCAT CR-lin- CAPS 796 855 Hinc g005320 TTGAAAATCCC AGAACATACC a2 bp bp II TC (SEQ ID (SEQ ID NO: 59 NO: 39) 40) bp J2 Solyc12 ATATTGAATCG TAACTTTCTTCA CR-j2- Indel 316 411 — g038510 TGTGATTGTCT AAGATGCATCC a1 bp bp C (SEQ ID (SEQ ID NO: NO: 41) 42) J2 Solyc12 ATATTGAATCG TAACTTTCTTCA CR-j2- CAPS 316 178 MboI g038510 TGTGATTGTCT AAGATGCATCC a2 bp bp I C (SEQ ID (SEQ ID NO: 139 NO: 43) 44) bp EJ2 Solyc03 AATATGGTCCT TAGCAAACTTAT CR-ej2- Indel 236 211 — g114840 TATGTCCAACC TGGGCTAGC a1 bp bp (SEQ ID NO: (SEQ ID NO: 45) 46) EJ2 Solyc03 AATATGGTCCT TAGCAAACTTAT CR-ej2- CAPS 236 144 Hind g114840 TATGTCCAACC TGGGCTAGC a2 bp bp III (SEQ ID NO: (SEQ ID NO: 94 47) 48) bp Cas9 — CTGACGTAAGG CATCTCATTACT — T- — 446 — GATGACGCAC AAAGATCTCC DNA bp (SEQ ID NO: (SEQ ID NO: 49) 50) RT-PCR Gene Forward Reverse name Gene ID primer primer LIN Solyc04 ATGGGAAGAGG TCAAAGCATCCA g005320 TAAGGTAGAA TCCTGGTAAA (SEQ ID NO: (SEQ ID NO: 51) 52) J2 Solyc12 ATGGGAAGAGG TTAGAGCATCCA g038510 AAGAGTAGAAC CCCTGGAAT (SEQ ID NO: (SEQ ID NO: 53) 54) EJ2 Solyc03 ATGGGAAGAGG TTAAAGCATCCA g114840 AAGAGTTGAG TCCATGAATAAA (SEQ ID NO: TC (SEQ ID 55) NO: 56) UBI Solyc01 CGTGGTGGTGC ACGAAGCCTCTG g056940 TAAGAAGAG AACCTTTC (SEQ ID NO: (SEQ ID NO: 57) 58) CRISPR/Cas9 genome-editing sgRNA Forward Reverse sgRNA name Gene ID primer primer sequence LIN- Solyc04 TGTGGTCTCAA TGTGGTCTCAAG TTCTAGT sgRNA- g005320 TTTTCTAGTAT CGTAATGCCAAC ATGTCTG 1 GTCTGATACAC TTTGTAC (SEQ ATACAC GTTTTAGAGCT ID NO: 60) (SEQ ID AGAAATAGCAA NO: 81) G (SEQ ID NO: 59) LIN- Solyc04 TGTGGTCTCAA TGTGGTCTCAAG GGAACAG sgRNA- g005320 TTGGAACAGCT CGTAATGCCAAC CTTGAGC 2 TGAGCGTCAAC TTTGTAC (SEQ GTCAAC GTTTTAGAGCT ID NO: 62) (SEQ ID AGAAATAGCAA NO: 82) G (SEQ ID NO: 61) J2- Solyc12 TGTGGTCTCAA TGTGGTCTCAAG AGCTCCT sgRNA- g038510 TTAGCTCCTTC CGTAATGCCAAC TCAACGT 1 AACGTTCTCAA TTTGTAC (SEQ TCTCAA GTTTTAGAGCT ID NO: 64) (SEQ ID AGAAATAGCAA NO: 83) G (SEQ ID NO: 63) J2- Solyc12 TGTGGTCTCAA TGTGGTCTCAAG ACATATT sgRNA- g038510 TTACATATTCT CGTAATGCCAAC CTTGGAG 2 TGGAGAGGATT TTTGTAC (SEQ AGGATT GTTTTAGAGCT ID NO: 66) (SEQ ID AGAAATAGCAA NO: 84) G (SEQ ID NO: 65) EJ2- Solyc03 TGTGGTCTCAA TGTGGTCTCAAG TTTGGGC sgRNA- g114840 TTTTTGGGCAC CGTAATGCCAAC ACGTTAA 1 GTTAAGCTCGA TTTGTAC (SEQ GCTCGA GTTTTAGAGCT ID NO: 68) (SEQ ID AGAAATAGCAA NO: 85) G (SEQ ID NO: 67) E32- Solyc03 TGTGGTCTCAA TGTGGTCTCAAG CCTTAAA sgRNA- g114840 TTCCTTAAAGC CGTAATGCCAAC GCAAATC 2 AAATCAGGTCA TTTGTAC (SEQ AGGTCA GTTTTAGAGCT ID NO: 70) (SEQ ID AGAAATAGCAA NO: 86) G (SEQ ID NO: 69) LIN/ Solyc12 TGTGGTCTCAA TGTGGTCTCAAG GCTTTTG J2/ g038510; TTGCTTTTGCT CGTAATGCCAAC CTAAGAG EJ2- Solyc04 AAGAGAAGAAA TTTGTAC (SEQ AGAA sgRNA- g005320; GTTTTAGAGCT ID NO: 72) (SEQ ID 1 Solyc03 AGAAATAGCAA NO: 87) g114840 G (SEQ ID NO: 71) LIN/ Solyc04 TGTGGTCTCAA TGTGGTCTCAAG GCAGTCT J2/ g005320 TTGCAGTCTTC CGTAATGCCAAC TCAAAGG EJ2- AAAGGATTCAC TTTGTAC (SEQ ATTCAC sgRNA- GTTTTAGAGCT ID NO: 74) (SEQ ID 2 AGAAATAGCAA NO: 88) G (SEQ ID NO: 73) Sequencing Forward Reverse Target primer primer pSC- GTAAAACGACG CAGGAAACAGCT B- GCCAG (SEQ ATGAC (SEQ amp/ ID NO: 75) ID NO: 76) kan pICH TCCTGTCAAAC TAATGTACTGGG 47761 ACTGATAG GTGGATGCAG (SEQ ID NO: (SEQ ID NO: 77) 78) pAGM ATAAGCCCATC CGGATAAACCTT 4723 AGGGAGCAG TTCACGCC (SEQ ID NO: (SEQ ID NO: 79) 80)

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From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

1. A genetically-altered Solanaceae plant comprising a mutant Solyc04g005320 gene or a homolog thereof.

2. The genetically-altered Solanaceae plant of claim 1, wherein the mutant Solyc04g005320 gene or homolog thereof is a null allele or a hypomorphic allele.

3. The genetically-altered Solanaceae plant of claim 1, wherein the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc04g005320 gene or homolog thereof.

4. The genetically-altered Solanaceae plant of claim 1, wherein the genetically-altered Solanaceae plant further comprises a mutant Solyc12g038510 gene or a homolog thereof, a mutant Solyc03g114840 gene or a homolog thereof, or both a mutant Solyc12g038510 gene or a homolog thereof and a mutant Solyc03g114840 gene or a homolog thereof.

5. The genetically-altered Solanaceae plant of claim 1, wherein the plant further comprises a mutant Solyc12g038510 gene or homolog thereof and the mutant Solyc12g038510 gene or homolog thereof is a null allele or a hypomorphic allele.

6. The genetically-altered Solanaceae plant of claim 5, wherein the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc12g038510 gene or homolog thereof.

7. The genetically-altered Solanaceae plant of claim 1, wherein the plant further comprises a mutant Solyc03g114840 gene or a homolog thereof and the mutant Solyc03g114840 gene or homolog thereof is a null allele or a hypomorphic allele.

8. The genetically-altered Solanaceae plant of claim 7, wherein the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc03g114840 gene or homolog thereof.

9. The genetically-altered Solanaceae plant of claim 1, wherein the plant further comprises both a mutant Solyc12g038510 gene or a homolog thereof and a mutant Solyc03g114840 gene or a homolog thereof, each of which are independently a null allele or a hypomorphic allele.

10. The genetically-altered Solanaceae plant of claim 9, wherein the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc12g038510 gene or homolog thereof and is heterozygous or homozygous for the mutant Solyc03g114840 gene or homolog thereof.

11. The genetically-altered Solanaceae plant of claim 1, wherein the genetically-altered Solanaceae plant further comprises a mutant Solyc12g038510 gene or homolog thereof, and a mutant Solyc03g114840 gene or homolog thereof, and wherein each is a hypomorphic allele.

12. A genetically-altered Solanaceae plant, comprising a mutant Solyc12g038510 gene or a homolog thereof and a mutant Solyc03g114840 gene or a homolog thereof, wherein the genetically-altered Solanaceae plant is homozygous for the mutant Solyc12g038510 gene or homolog thereof and heterozygous for the mutant Solyc03g114840 gene or homolog thereof.

13. The genetically-altered Solanaceae plant of claim 12, wherein the mutant Solyc12g038510 gene or homolog thereof is a null allele or a hypomorphic allele and the mutant Solyc03g114840 gene or homolog thereof is a null allele or a hypomorphic allele.

14. The genetically-altered Solanaceae plant of claim 1, wherein the genetically-altered Solanaceae plant is a tomato (Solanum lycopersicum) plant.

15. The genetically-altered Solanaceae plant of claim 4, wherein the mutant Solyc04g005320 gene or homolog thereof, the mutant Solyc12g038510 gene or homolog thereof, and/or the mutant Solyc03g114840 gene or homolog thereof is introduced by technical means.

16. The genetically-altered Solanaceae plant of claim 4, wherein the mutant Solyc04g005320 gene or homolog thereof, the mutant Solyc12g038510 gene or homolog thereof, and/or the mutant Solyc03g114840 gene or homolog thereof is introduced by chemical or physical means.

17. The genetically-altered Solanaceae plant of claim 16, wherein the mutant Solyc04g005320 gene or homolog thereof, the mutant Solyc12g038510 gene or homolog thereof, and/or the mutant Solyc03g114840 gene or homolog thereof is introduced using CRISPR/Cas9, chemical mutagenesis, radiation, Agrobacterium-mediated recombination, viral-vector mediated recombination, or transposon mutagenesis.

18. (canceled)

19. A crop harvested from genetically-altered Solanaceae plants as defined in claim 1.

20. A seed for producing a genetically-altered Solanaceae plant of claim 1.

21. A method for producing a genetically-altered Solanaceae plant, the method comprising introducing a mutation into a Solyc04g005320 gene or a homolog thereof in a Solanaceae plant, thereby producing a genetically-altered Solanaceae plant containing a mutant Solyc04g005320 gene or homolog thereof.

22. The method of claim 21, wherein the mutation is introduced using CRISPR/Cas9.

23. The method of claim 21, wherein the mutation produces a null allele or a hypomorphic allele of the Solyc04g005320 gene or homolog thereof.

24. The method of claim 21, wherein the method further comprises introducing into the Solanaceae plant a mutation into a Solyc12g038510 gene or a homolog thereof, introducing a mutation into a Solyc03g114840 gene or a homolog thereof, or introducing the mutation into the Solyc12g038510 gene or homolog thereof and introducing the mutation into the Solyc03g114840 gene or homolog thereof.

25. The method of claim 24, wherein the mutation(s) is/are introduced using CRISPR/Cas9.

26. The method of claim 21, wherein the genetically-altered Solanaceae plant containing the mutant Solyc04g005320 gene or homolog thereof is crossed to another genetically-altered Solanaceae plant comprising a mutant Solyc12g038510 gene or homolog thereof, a mutant Solyc03g114840 gene or homolog thereof, or both the mutant Solyc12g038510 gene or homolog thereof and the mutant Solyc03g114840 gene or homolog thereof, thereby producing a genetically-altered Solanaceae plant containing the mutant Solyc04g005320 gene or homolog thereof and the mutant Solyc12g038510 gene or homolog thereof, the mutant Solyc03g114840 gene or homolog thereof, or both the mutant Solyc12g038510 gene or homolog thereof and the mutant Solyc03g114840 gene or homolog thereof.

27. The method of claim 21, wherein the genetically-altered Solanaceae plant is a tomato (Solanum lycopersicum) plant.

28. A genetically-altered Solanaceae plant produced or obtainable by the method of claim 21.

29. The genetically-altered Solanaceae plant of claim 1, wherein the mutant Solyc04g005320 gene or homolog thereof is a hypermorphic allele.

30. The genetically-altered Solanaceae plant of claim 29, wherein the genetically-altered Solanaceae plant is heterozygous or homozygous for the mutant Solyc04g005320 gene or homolog thereof.

31. (canceled)