Increase in tomato locule number is controlled by two single-nucleotide polymorphisms located near WUSCHEL

Abstract

In tomato (Solanum lycopersicum) fruit, the number of locules (cavities containing seeds that are derived from carpels) varies from two to up to 10 or more. Locule number affects fruit shape and size and is controlled by several quantitative trait loci (QTLs). The large majority of the phenotypic variation is explained by two of these QTLs, fasciated (fas) and locule number (lc), that interact epistatically with one another. FAS has been cloned, and mutations in the gene are described as key factors leading to the increase in fruit size in modern varieties. Here, we report the map-based cloning of lc. The lc QTL includes a 1,600-bp region that is located 1,080 bp from the 3' end of WUSCHEL, which encodes a homeodomain protein that regulates stem cell fate in plants. The molecular evolution of lc showed a reduction of diversity in cultivated accessions with the exception of two single-nucleotide polymorphisms. These two single-nucleotide polymorphisms were shown to be responsible for the increase in locule number. An evolutionary model of locule number is proposed herein, suggesting that the fas mutation appeared after the mutation in the lc locus to confer the extreme high-locule-number phenotype.

Figure 1.

Phenotypic analysis and mendelization of the locule number QTL (lc). A, Fruit morphology of lines used in the study. The parental lines Cervil and Levovil were crossed to obtain the F1 hybrid and the near-isogenic recombinant F8 lines CF12-C and CF13-L. The L2 line was derived from Levovil, and its lc region of chromosome 2 containing the Cervil allele has been introgressed by marker-assisted selection. B, Phenotypic effect of the QTL. The average locule number of F2 heterozygous plants (H) or F3 plants homozygous for either the Cervil (C) or the Levovil (L) allele was determined. The plants were selected based on genotyping data, and heterozygous plants were selected by analyzing genotypic and phenotypic segregation in their corresponding F3 progeny. Each histogram, with its sd, is partitioned according to the proportion of fruits with two locules (white), three locules (black), and four or more locules (gray). A Student’s t test indicated that the means are significantly different between C and H (P < 0.001), between H and L (P < 0.001), and between H and C (P < 0.01). [See online article for color version of this figure.]

Figure 2.

Fine mapping (A), ultra-high-resolution mapping (B), and polymorphism (C) of lc QTL. A, All sequences highlighted in green represent tomato genes syntenic with the At2g18000 region in Arabidopsis. Markers highlighted in yellow were used to screen for recombinant plants. The PA205-2 and JB149-9 recombinant lines allowed the identification of the BAC clone containing the QTL. This BAC, LeHBa139K19, contains 11 putative ORFs, with some showing homology to known genes or expressed sequences. lc is located in the region between z1416 and z1420. B, The z1416 and z1420 markers were used to screen for recombinant plants. Newly designed markers finally localized lc between z1497 and z1499. Seven plants with recombination events between these two markers were identified. The molecular characterization of these seven lines restricted lc to a 1,608-bp noncoding DNA region. The orientation (from ATG to stop codon) and the exons (in blue) of the cDNAs coding for WUSCHEL and the WD40 protein are shown. C, Sequence alignment of the two alleles of lc from Cervil and Levovil. The 14 polymorphic sites are highlighted in gray. The two underlined SNPs are responsible for lc (Fig. 5). [See online article for color version of this figure.]

Figure 3.

Expression analysis of the two candidate genes. RT-PCR was performed on total RNA extracted from floral buds, leaves, or fruits. Primers were designed on WUSCHEL (accession no. AJ538329) and the WD40 repeat protein (tomato Unigene no. SGN-U585584). eIF4A2 (tomato Unigene no. SGN-U593757) was used as a reference gene. DAA, Days after anthesis.

Figure 4.

Effect of lc on locule number (A), flower size (B), and petal number (C). Cervil and Levovil are the parental lines. L2 and L4 have been obtained by marker-assisted selection from Levovil; they are both identical to Levovil except for a region of chromosome 4 from Cervil (L4) and a region of chromosome 2 from Cervil that contains lc (L2). L2 and L4 must be compared with Levovil. CF12-C and CF13-L are a pair of identical isogenic lines except for a 30-cM region that contains lc. JB1538 and JB1546 are identical to CF12-C and CF13-L, respectively, but differ for less than 30 cM. PA205-2 and JB149-9, on the same principle, differ from one another by a 28-kb region surrounding lc. The lines are all homozygous at the lc locus and contain either the alleles from Cervil, which produce fruits with low locule number, or from Levovil, which produce fruits with high locule number. The genetic background is either Cervil (C) or Levovil (L); it is recombinant (R) between Cervil and Levovil for the isogenic lines. The mean of each line has been compared with Cervil. Statistical significance of the t test is indicated as ns (not significant) or *** (P < 0.001).

Figure 5.

Molecular diversity of the lc locus. A, Tomato haplotypic structure of lc and other loci on chromosome 2. Each block corresponds to one of the 24 amplicons (average length of 500 bp). Columns and rows represent individuals and SNPs, respectively. With Heinz1706 used as a reference, polymorphisms are indicated either in gray (identical allele) or in black (different allele). B, Tajima test over the whole chromosome and within lc. Stars indicate significant departures from the neutrality hypothesis. A significant positive value of Tajima’s D for lc indicated either a balancing selection of the locus or a population decrease. An analysis of the entire chromosome showed that lc evolved differently from the entire chromosome: lc underwent a balancing selection, whereas chromosome 2 evolved following a population expansion. Dashed lines represent mean and se for the whole chromosome. C, Molecular diversity of lc in wild and cultivated lines. The diversity (π) of the locus was drastically reduced in cultivated lines (black lines) except for two SNPs (arrow) in comparison with wild accessions (gray lines). A total of 17 cultivated and 11 wild lines were used in this analysis.

Figure 6.

Model of locule number evolution in tomato fruits during domestication. S. pimpinellifolium is considered the wild ancestor of the cultivated tomato S. lycopersicum. Based on the analysis of 267 tomato accessions, we propose a model that could explain the history of locule number evolution during tomato domestication. In our study, only 4.6% of the high-locule-number accessions (i.e. those with more than three locules) had the low-locule allele of lc, and 96.9% of the fasciated accessions (i.e. those with more than six locules) had the high-locule allele of lc, but only 49.4% of them had the fas allele. These results indicate that lc was required for the increase in locule number in tomato fruits during domestication. The lc locus could have appeared before the fas locus. These two QTLs are the major loci controlling locule number. Modern breeding has used other loci to expand phenotypic diversity. [See online article for color version of this figure.]