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.

Soybean, a major source of oil and protein, has seen an annual increase in consumption when used in soybean-derived products and the broadening of its cultivation range. The demand for soybean necessitates a better understanding of the regulatory networks driving storage protein accumulation and oil biosynthesis to broaden its positive impact on human health. In this study, we selected a chromosome segment substitution line (CSSL) with high protein and low oil contents to investigate the underlying effect of donor introgression on seed storage through multi-omics analysis. In total, 1479 differentially expressed genes (DEGs), 82 differentially expressed proteins (DEPs), and 34 differentially expressed metabolites (DEMs) were identified in the CSSL compared to the recurrent parent. Based on Gene Ontology (GO) term analysis and the Kyoto Encyclopedia of Genes and Genomes enrichment (KEGG), integrated analysis indicated that 31 DEGs, 24 DEPs, and 13 DEMs were related to seed storage functionality. Integrated analysis further showed a significant decrease in the contents of the seed storage lipids LysoPG 16:0 and LysoPC 18:4 as well as an increase in the contents of organic acids such as L-malic acid. Taken together, these results offer new insights into the molecular mechanisms of seed storage and provide guidance for the molecular breeding of new favorable soybean varieties.

The Kunitz trypsin inhibitor (KTI) gene family encompasses a category of trypsin inhibitors, and the KTI proteins are important components of the 2S storage protein fraction in soybeans. In this study, fifty members of the GmKTI family were identified in the soybean genome, and their physicochemical properties, domain compositions, phylogenetic relationships, gene structures, and expression patterns were comprehensively analyzed to explore their impact on soybean seed protein content. The results revealed significant gene expansion within the GmKTI family in soybean. The gene structures and conserved motifs of GmKTI members exhibited both regularity and diversity, with distinct expression patterns across different soybean tissues. Haplotype analysis identified 7 GmKTI genes significantly associated with seed storage protein content, and the combination of superior haplotypes was found to enhance seed storage protein content. This is crucial for the improvement of soybean varieties and the enhancement of storage protein content. Additionally, the GmKTI family demonstrated evolutionary conservation, with its functions likely linked to light induction, biotic stress, and growth development. This study characterizes the structure, expression, genomic haplotypes, and molecular features of the soybean KTI domain for the first time, providing a foundation for functional analyses of the GmKTI domain in soybean and other plants.

Background/Objectives: The significant negative correlation between protein and oil content in soybean seeds is a long-standing bottleneck for conventional breeding. Its root cause lies in insufficient understanding of related molecular regulatory processes. Methods: We selected the CSSL_R19, a chromosome segment substitution line, to thoroughly investigate the intrinsic effects of the substituted segment on the high seed storage protein (SSP) and low fatty acid (FA) phenotype. Transcriptomic, proteomic, and metabolomic analyses were performed on the recurrent parent and R19. Results: A total of 1821 differentially expressed genes (DEGs), 12 differentially expressed proteins (DEPs), and 10 differentially accumulated metabolites (DEMs) were detected. Subsequently, an integrative examination of the data demonstrated that 28 DEGs, 5 DEPs, and 4 DEMs participated in biological processes such as carbohydrate metabolism, lipid degradation, as well as protein synthesis and transport. Mechanistically, down-regulation of PGM reduces the carbon source supply for FA synthesis; up-regulation of LOX, LACS, ACX, and KAT promotes FA degradation. SRP, SAR1, and HSP70 are involved in the synthesis and transport of SSP. Crucially, qRT-PCR validation performed on all 28 core DEGs showed that their expression trends were highly consistent with the transcriptome data, confirming the reliability of the findings. Conclusions: In conclusion, we propose a potential regulatory network that enhances SSP accumulation and reduces FA content. Altogether, these findings advance our understanding of storage compound accumulation in soybeans and guide future breeding strategies.

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.