Seed protein content is a critical trait in soybean breeding, as it provides a primary source of high-quality protein for both human consumption and animal feed. This study aimed to enhance molecular marker-assisted selection for high-protein soybean varieties by developing Kompetitive Allele-Specific Polymerase Chain Reaction (KASP) markers targeted at loci associated with seed protein content. Nineteen markers with high genotyping efficacy were identified through screening. Utilizing SN76 (a high-protein line) as the male parent and SN49 and DS1 (both low-protein lines) as female parents, 484 F₆ generation individuals from these hybrid combinations were selected to validate the predictive accuracy of the 19 KASP markers. Notably, KASP-Pro-1, KASP-Pro-2, and KASP-Pro-3 effectively distinguished genotypes associated with high and low protein content, with prediction accuracies of 68.4%, 75.0%, and 83.3%, respectively. These results underscore the reliability and practical utility of the selected molecular markers, which are located within the genes Glyma.03G219900, Glyma.14G119000, and Glyma.17G074400, respectively. Haplotype analysis and gene pyramiding indicate that these three genes may influence seed protein content. Consequently, these KASP markers can be effectively integrated into genetic and genomic research on soybean seed protein content as well as into marker-assisted breeding.

Seed oil content is a key trait in soybean, yet its genetic basis remains largely unresolved. Here, we identify GmABHD6 , an α/β‐hydrolase domain‐containing gene, as the causal gene underlying a major quantitative trait locus (QTL) for seed oil content on chromosome 16. Through fine‐mapping in a chromosome segment substitution population derived from Glycine max (SN14) and wild Glycine soja (ZYD00006), we fine‐mapped the QTL to a 150‐kb interval. Functional analyses reveal that overexpression of GmABHD6 significantly reduces seed oil content, whereas gene editing to truncate GmABHD6 led to increased oil accumulation. These modifications also alter seed protein levels, seed size and 100‐seed weight, indicating pleiotropic effects on seed composition and yield traits. Integrated transcriptomic and metabolomic profiling of transgenic lines highlighted widespread changes in lipid metabolism and energy pathways associated with GmABHD6 activity. Further functional characterisation indicates that the ERF transcription factor GmERF physically binds to the GmABHD6 promoter region to regulate its expression. Population genetic analyses show strong signatures of selection at the GmABHD6 locus, suggesting that favourable alleles are enriched during soybean domestication and improvement. Our findings establish GmABHD6 as a domestication‐related gene that negatively regulates seed oil content and provide new insights into the genetic mechanisms driving soybean seed composition and evolution.

Soybean is the major global source of edible oils and vegetable proteins. Seed size and weight are crucial traits determining the soybean yield. Understanding the molecular regulatory mechanism underlying the seed weight and size is helpful for improving soybean genetic breeding. The molecular regulatory pathways controlling the seed weight and size were investigated in this study. The 100-seed weight, seed length, seed width, and seed weight per plant of a chromosome segment substitution line (CSSL) R217 increased compared with those of its recurrent parent ‘Suinong14’ (SN14). Transcriptomic and proteomic analyses of R217 and SN14 were performed at the seed developmental stages S15 and S20. In total, 2643 differentially expressed genes (DEGs) and 208 differentially accumulated proteins (DAPs) were detected at S15, and 1943 DEGs and 1248 DAPs were detected at S20. Furthermore, integrated transcriptomic and proteomic analyses revealed that mitogen-activated protein kinase signaling and cell wall biosynthesis and modification were potential pathways associated with seed weight and size control. Finally, 59 candidate genes that might control seed weight and size were identified. Among them, 25 genes were located on the substituted segments of R217. Two critical pathways controlling seed weight were uncovered in our work. These findings provided new insights into the seed weight-related regulatory network in soybean.

PIN-LIKES (PILS) auxin transport genes play key roles in plant development, but their functions and molecular mechanism in soybean yield remain unclear. Here, we characterized the 44-member soybean GmPILS genes via comprehensive analyses. Phylogenetic analysis classified GmPILS into three subfamilies, with most proteins being hydrophobic, stable, and membrane-localized. Chromosomal distribution showed random scattering across 17 chromosomes, with gene duplication driving family expansion. Expression profiling identified GmPILS36 and GmPILS40 as seed-specific and differentially expressed between cultivated Suinong14 (SN14) and wild ZYD00006 (ZYD06) soybeans. Population genetic analyses revealed GmPILS40 experienced a domestication bottleneck without yield-related superior haplotypes, while GmPILS36 underwent selection during landrace-to-improved variety domestication. A coding region CC/TT natural variation in GmPILS36 (S/A substitution) was significantly associated with seed weight per plant and 100-seed weight, with the TT genotype conferring superior traits. This study provides insights into GmPILS genes’ evolution and identifies GmPILS36 as an important candidate gene for further functional study and investigation of the molecular mechanisms regulating soybean yield.