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 vitality is a key factor for successful germination of seeds and successful root establishment of crops. However, a cold environment can severely hinder the germination of soybean seeds, resulting in a significant decrease in yield. In this study, the cold tolerance of 205 chromosome segment substitution lines (CSSL) during the germination process was evaluated. CSSL_R22 exhibited higher seed vitality under low-temperature conditions. Five quantitative trait loci (QTL) related to cold tolerance during the germination stage were detected. By combining the QTL analysis results with transcriptome data, we determined that GmKAN1 (Glyma.20G108600) is an important regulatory factor for cold tolerance during seed germination. Preliminary studies have shown that GmKAN1, as a transcriptional repressor of GmARF2 and GmARF8, can regulate auxin synthesis to enhance the tolerance of seeds to cold stress. These results provide valuable insights into the regulatory network related to cold tolerance during soybean seed germination.