Genome-wide association study and fine-mapping identify a major quantitative trait locus controlling hundred-seed weight in soybean

Background Hundred-seed weight (HSW) is a critical yield component in soybean that directly influences productivity and seed quality. Despite its agronomic importance, the genetic architecture underlying natural variation in seed weight remains incompletely understood. Methods We conducted a comprehensive genome-wide association study (GWAS) using 554 globally diverse soybean accessions, comprising 453 Chinese varieties (81.8%) and 101 international accessions (18.2%) from 15 countries. Accessions were evaluated across three consecutive years (2022-2024) and genotyped with 78,050 high-quality single-nucleotide polymorphisms (SNPs). Results Mixed linear model (MLM) analysis revealed a major QTL on Chr.20 that consistently explained the largest proportion of phenotypic variation across all environments. This QTL demonstrated exceptional temporal stability, maintaining genome-wide significance with peak -log 10 (P) values of 13.4, 12.1, and 10.2 across the three evaluation years. Fine mapping narrowed the critical interval to 493.69 kb containing 25 annotated genes. The lead SNP within Glyma.20G223200 explained 8-12% of phenotypic variance, while multi-SNP models incorporating five high-priority candidates cumulatively explained 14-18% of variance. Expression analysis of candidate genes revealed differential patterns between large-seeded and small-seeded varieties during seed development, with up to 32-fold expression differences. Conclusions The environmentally stable Chr. 20 QTL provides immediate opportunities for marker-assisted selection (MAS) in soybean breeding programs. Genomic prediction modeling suggests 35% greater genetic gain compared to phenotypic selection alone, supporting broad applicability for global soybean improvement programs.