Cross-population QTL mapping and association analysis identify breeding-ready markers for high seed protein content...

Soybean [Glycine max (L.) Merr.] is a major crop for global protein and oil production, yet most cultivated varieties contain only 35-42% protein. HJ117 is a soybean accession with high protein content (52.99%), but the genetic basis of this trait is not well characterized. Identifying the major loci controlling protein accumulation in HJ117 is necessary to develop improved high-protein varieties through marker-assisted breeding. We conducted QTL mapping and genome-wide association studies (GWAS) using two recombinant inbred line (RIL) populations: XH1617 (183 lines from Xudou16 × HJ117) and QH3417 (171 lines from Qihuang34 × HJ117). Seed protein and oil content were evaluated across three growing seasons (2019, 2021, 2022). Both traits showed continuous distributions with high broad-sense heritability (h²b > 0.91), confirming their quantitative genetic basis. Quantitative trait locus (QTL) mapping identified nine stable loci across both populations, including three major loci, qtl-15, qtl-20, and qtl-20-1, on the chromosome. 15 and 20, consistently detected across populations and years. These loci explained 7.20-41.50% of phenotypic variance (LOD = 2.97-19.82). GWAS analysis of the same 357 accessions, comprising both RIL populations and three parental lines, improved mapping resolution within linkage-derived intervals, identifying 168 significant SNPs, including chromosome_15_943943_T_C, that colocalized with Locus15 and provide markers suitable for marker-assisted selection. Additive effect analysis revealed that 96.70% of protein-enhancing alleles originated from HJ117. The stable QTL regions harbor candidate genes (GmSWEET10 (Glycine max SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 10), GmSop20 (Glycine max Seed oil-to-protein 20), and POWR1 (Protein and Oil With Regulatory role 1)) involved in sugar transport and nutrient partitioning during seed development. These results provide validated genetic markers and positional data for the major loci controlling protein content in HJ117, with potential utility for marker-assisted selection in high-protein soybean breeding programs.