Multi-Locus Genome-Wide Association Study Identifies Stable Quantitative Trait Nucleotides and Candidate Genes for Seed Protein Content in an Early-Maturing Soybean Recombinant Inbred Line Population

Soybean seed protein content is a polygenic quantitative trait, and dissecting its genetic architecture is critical for advancing high-protein soybean molecular breeding. In this study, an F5 recombinant inbred line (RIL) population with 174 lines derived from a cross between high-protein cultivar Kedou 48 and low-protein cultivar Kedou 61 was phenotyped for seed protein content across four distinct growing environments. Genotyping was performed using 25,663 high-quality single nucleotide polymorphisms (SNPs), followed by multi-locus genome-wide association studies (GWAS). Phenotypic data revealed seed protein content ranged from 31.24% to 46.65% across the population, with a broad-sense heritability of 0.89. In total, 54 quantitative trait nucleotides (QTNs) linked to seed protein content were detected via 11 multi-locus GWAS algorithms; among these, 16 QTNs were classified as stable QTNs because they were detected by at least two GWAS models within the same environment and/or in at least two environments, and 33 QTNs overlapped with previously published seed protein quantitative trait loci (QTLs). Integrating linkage disequilibrium (LD) decay estimation, seed tissue expression profiles, parental resequencing-derived non-synonymous variants, and germplasm haplotype characterization, three candidate genes were prioritized: Glyma.08G246400 (AFP3/NINJA-like transcriptional repressor), Glyma.09G154600 (LRR receptor-like protein kinase), and Glyma.19G224000 (translation initiation factor IF-2). Haplotype analyses using eligible accessions from the collaborative 2898-accession soybean resequencing panel identified significant haplotype–protein associations for all three genes after adjustment for germplasm type, with divergent haplotype distributions among Soja, landraces, and improved cultivars. This work provides stable QTNs supported by repeated detection, three biologically plausible candidate genes, and protein-associated haplotypes for further mechanistic and breeding evaluation in high-protein early-maturing soybean.