BSA-Seq-Based Discovery of Functional InDel Markers for Seed Size Selection in Litchi (Litchi chinensis Sonn.)

As a globally significant fruit crop, litchi (Litchi chinensis Sonn.) exhibits substantial variation in seed size, which is a key determinant of fruit quality. However, the lack of molecular markers closely associated with seed-related traits has hindered targeted breeding efforts. In this study, we systematically evaluated six critical traits—single fruit weight, seed weight, seed length, seed width, edible rate, and seed-to-fruit weight ratio—across 131 early-maturing litchi accessions. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) revealed a clear bifurcation of these accessions into two distinct groups based on seed size-related traits. Using bulked segregant analysis sequencing (BSA-seq), we identified a candidate genomic region (24.93–25.69 Mb) on chromosome 5, potentially regulating litchi seed size. Within this region, 1600 single-nucleotide polymorphisms (SNPs) and 314 insertion/deletion mutations (InDels) exhibited significant divergences between the extreme pools. To validate these findings, we performed PCR-based screening on 87 litchi accessions. Two InDel markers demonstrated strong phenotypic associations: Chr5_25610680_InDel showed highly significant correlations with seed weight, edible rate, seed length, seed width, and seed-to-fruit weight ratio, explaining 22.60–35.54% of phenotypic variation. Meanwhile, Chr5_25585686_InDel was significantly associated with seed weight and edible rate, accounting for 18.66% and 18.94% of the phenotypic variation, respectively. These findings provide valuable molecular markers for marker-assisted breeding of litchi seed size, offering a promising avenue to advance precision breeding in this economically important crop.