Identification of Genes Associated with Seed Weight and Development of Functional Markers for GmUBP15 in Glycine max

Seed weight is a key agronomic trait determining soybean yield and quality, yet only a few of genes regulating this trait have been functionally characterized to date. In this study, we identified 155 homologous genes in the soybean genome through BLAST searches using 78 functionally validated rice grain weight-related genes as queries. Haplotype analysis prioritized 40 candidate genes exhibiting significant differences in seed weight between haplotypes. To further refine the candidate list, we integrated haplotype frequency analysis, expression-trait association mapping, and tissue-specific expression profiling, ultimately delineating eight key genes. Given the established role of ubiquitination in seed development, we focused on homologs of OsUBP15 and identified three candidate genes, GmUBP5, GmUBP11, and GmUBP33, that exhibited significant haplotype-dependent variation in seed weight. Subcellular localization assays confirmed their nuclear localization. Haplotype frequency analysis revealed that the superior haplotypes of these genes have been preferentially retained during modern breeding and are widely distributed across major soybean-producing regions. Leveraging non-synonymous SNP variants, we developed and validated robust KASP markers that efficiently discriminate germplasm with contrasting seed weight phenotypes. Collectively, our study provides not only high-confidence genetic targets and actionable molecular markers but also insights into pyramiding breeding strategies for improving seed weight in soybean.