Soybean is a primary source of plant-based oil and protein for human diets. Seed size and weight are important agronomic traits that significantly influence soybean yield. Despite their importance, the genetic mechanisms underlying soybean seed size and weight remain to be fully elucidated. In order to identify additional, major quantitative trait loci (QTL) associated with seed size and weight, we developed segregating populations by crossing a large-seeded soybean variety “Kebaliang” with a small-seeded soybean variety “SUZUMARU”. We evaluated seed length, width, thickness, and hundred-seed weight across two generations, F4 and F5, in 2022 and 2023. Employing bulked segregate analysis with whole-genome resequencing (BSA-seq), we detected 18 QTLs in the F4 population and 12 QTLs in the F5 population. Notably, six QTLs showed high stability between the two generations, with five derived from two pleiotropic loci (qSS4-1 and qSS20-1) and one specific to seed width (qSW14-1). Further validation and refinement of these loci were carried out through linkage mapping using molecular markers in the F5 population. Additionally, we identified 18 candidate genes within these stable loci and analyzed their sequence variations and expression profiles. Together, our findings offered a foundational reference for further soybean seed size research and unveiled novel genetic loci and candidate genes that could be harnessed for the genetic enhancement of soybean production.
