Molecular Mapping and CRISPR/Cas9 Validation Reveal That SlVQ10 Regulates Seed Size in Tomato

Seed size affects seed vigor, seedling establishment, and seed utilization in tomato. Yet the genetic basis of this trait remains poorly defined in tomato itself. Here, we describe a stable small-seed mutant, T31, isolated from an ethyl methanesulfonate (EMS)-mutagenized population of the elite inbred line DL5. Relative to the wild type, T31 showed a 24% reduction in seed width, whereas vegetative growth and major fruit traits were largely unchanged. Throughout this study, 'seed size' refers to seed width, which was used as the principal index of seed size because tomato seeds are oblate. Genetic analysis of six populations (P₁, P₂, F₁, F₂, BC₁, and BC₂) indicated that the phenotype is controlled by a single recessive locus, designated ssm. Bulked segregant analysis sequencing (BSA-seq) placed ssm within a 1.77 -Mb interval on chromosome 4. KASP-based fine mapping reduced this interval to 390 kb and identified six EMS-type SNPs. Only one of these SNPs was located in an exon of Solyc04g073950.2, where it caused a Pro337Ser substitution. This gene encodes a VQ motif-containing protein and was designated SlVQ10. To test gene function, we generated CRISPR/Cas9 knockout lines in the DL5 background. Two independent homozygous knockout lines reproduced the small-seed phenotype. Seed size was reduced by 31-33%, and thousand-seed weight decreased by 33-35%. Histological analysis further showed reduced seed-coat cell expansion in the mutants. Together, the genetic and genome-editing data support SlVQ10 as the gene underlying ssm and indicate that it promotes seed size in tomato.