Identification of candidate loci regulating seed-associated traits in soybean using genome-wide association study and image-based high-throughput phenotyping

Seed-associated traits such as flowering, maturity, and seed size are critical determinants of yield and seed morphology in soybean. However, their reliable evaluation is often hindered by polygenic control and strong environmental influences. In this study, we performed a genome-wide association study (GWAS) across 374 soybean accessions evaluated under field conditions during 2020 and 2021 growing seasons in Rural Development Administration, Jeonju, South Korea. The genetic architecture of eight traits was investigated including two reproductive traits: days to flowering (DtF) and days to maturity (DtM), and six seed size-related traits: seed length (SL), seed width (SW), seed thickness (ST), seed area (SA), seed volume (SV), and 100-seed weight (HSW). Among these, SL, SW, ST, SA, and SV were measured using an image-based high-throughput phenotyping platform. Correlation analysis revealed strong associations within reproductive traits and within seed size traits, with relatively weak correlations between reproductive and seed size traits (r ≤ 0.45) suggesting largely independent genetic regulation. GWAS identified six significant pleiotropic SNPs: one on chromosome 19 for DtF and DtM, and five on chromosomes 4, 13, and 20 for seed size traits (SL, SW, ST, SV, and HSW). The linkage disequilibrium interval (± 153 kbp) was defined as the stable QTL region for candidate gene mining. One novel QTL, <i>qSS13</i> was also identified for seed size. Haplotype analysis revealed three alleles at <i>qSS4.2</i> (Hap4B_1, Hap4B_2, Hap4B_3) and two at <i>qSS13</i> (Hap13_1, Hap13_2), each regulating SV and HSW. Within the six QTLs, 23 putative genes were identified, including 16 specifically associated with seed size traits. Among these, six genes showed distinct expression patterns between large and small-seeded accessions, suggesting their roles as strong candidates for seed size regulation. Collectively, these findings provide new insights into the genetic control of seed size and offer valuable targets for marker-assisted selection and molecular breeding to improve soybean yield.