CRISPR/Cas9-Mediated Targeted Mutagenesis of <i>GmEOD1</i> Enhances Seed Size of Soybean

Seed size is a critical agronomic trait that influences the yield and appearance quality of soybeans, making it a primary breeding objective with significant economic value. While the molecular mechanisms that regulate soybean seed size remain largely unknown, several functional molecular targets have been applied in breeding to create larger grain size materials. In this study, we utilized the CRISPR/Cas9 system to induce the targeted mutagenesis of <i>GmEOD1</i>, which encodes the E3 ubiquitin ligase. The resulting homozygous soybean mutant of <i>GmEOD1</i> exhibited larger seed size and 100-seed weight, with no significant change in the average seed weight per plant. The sum of crude protein and oil content increased significantly in mutants while fatty acid composition remained unchanged. We identified six haplotypes among 156 soybean cultivars, with Hap1 and Hap2 representing the majority of cultivars with relatively higher 100-seed weight, suggesting that sequence variations of <i>GmEOD1</i> may correlate with seed weight. Transcriptomic analysis across five stages of seed development revealed that stages one–three mainly focused on cell cycle, growth, wall synthesis and modification, photosynthesis, and sugar metabolism; promoting cell growth, reproduction, and carbon accumulation; and providing key intermediates for substance synthesis. Stages four–five focused on polysaccharide catabolism, xylan metabolism, and nutrient pool activity, promoting the accumulation of dry matter, such as sugars, proteins, and lipids in seeds. Weighted gene co-expression network analysis (WGCNA) of modules related to seed size revealed 13 hub genes involved in seed development regulation. This study provides a valuable theoretical basis and excellent opportunities for genetic editing of germplasm cells with subsequent molecular soybean seed size breeding, facilitating easier seed selection to improve soybean quality.