Stage-specific GWAS identifies a pleiotropic ankyrin repeat locus near GmSALT3 for salinity tolerance in soybean

GWAS identified 20 salinity-tolerance SNPs in soybean, including a pleiotropic locus near GmSALT3. A validated KASP marker for rs.Gm03.39710939 enables early screening and marker-assisted breeding. Soybean is a vital global source of protein and oil, yet its productivity is severely affected by soil salinity, which hampers germination and seedling growth, leading to stunted development and yield reductions. In this study, 198 diverse soybean genotypes were assessed for salinity tolerance at the germination and seedling stages under 200 mM NaCl stress. Seven phenotypic traits were evaluated, and delta percentages (Δ%) across two experiments (E1 and E2) were calculated. Genome-wide association studies (GWASs) were conducted via two complementary models (BLINK and FarmCPU), identifying 66 significant SNPs, with 20 consistently detected across both experiments and models, confirming their robustness. SNP × SNP interaction analysis revealed 953 significant epistatic interactions, highlighting the complex genetic basis of salinity tolerance. Among these SNPs, rs.Gm03.39710939, linked with the gene Glyma.03G170501 on chromosome 3, had strong pleiotropic effects on three major traits: delta percentage of chlorophyll content (Δ%_SPAD_200mM), mean of leaf scorch score (LSS_MEAN), and mean of seedling-to-flowering survival (SFS_MEAN). This gene encodes an ankyrin repeat (ANK) protein, which plays a crucial role in salt tolerance mechanisms. Structurally, Glyma.03G170501 is located approximately 73 kb upstream of GmCHX1/GmSALT3 (Glyma.03G171600), a well-recognized salt tolerance locus. On the basis of this pleiotropic SNP, a Kompetitive Allele-Specific PCR (KASP) marker was successfully developed and validated. These genomic resources offer valuable tools for improving soybean breeding strategies and developing salt-tolerant varieties.