Phenomics, RNA sequencing and weighted gene co-expression network analysis reveals key regulatory networks and genes involved in the determination of seed hardness in Vicia sativa

Background Wild-type Vicia sativa L. demonstrates superior agronomic traits, including high yield, elevated crude protein content, enhanced reproductive efficiency, and tolerance to nutrient-poor soils, rendering it a valuable genetic resource for crop improvement. However, its utilization in germplasm enhancement is severely constrained by seed hardness, whose underlying physiological and molecular regulatory mechanisms remain poorly characterized. Results Phenomics studies reveal that wild-type seeds exhibited significantly higher hardness indices and lignin content compared to cultivated varieties, whereas cultivated seeds contain significantly greater levels of soluble sugars, soluble proteins, and starch. During the same developmental period, cultivated seeds demonstrate significantly larger diameters than wild-type seeds. However, wild-type seeds show significantly thicker palisade layers and higher palisade-layer-to-seed-coat thickness ratios. Notably, the cuticle thickness of wild-type seeds is significantly greater. Scanning electron microscopy further indicated that cultivated varieties have significantly larger hilum width and length, whereas wild-type seeds display significantly deeper micropyle depth. Through WGCNA analysis, three key candidate genes (TRINITY_DN3402_c0_g1, TRINITY_DN13607_c0_g1, and TRINITY_DN6606_c0_g1) involved in regulating seed hardness were identified. The expression pattern analysis results indicated that, the expression levels of TRINITY_DN3402_c0_g1, TRINITY_DN13607_c0_g1, and TRINITY_DN6606_c0_g1 in the wild hard-seeded type vicia sativa (W12) were significantly higher than those in the cultivated type vicia sativa (W30). Conclusions ​Our study elucidates key physical determinants and molecular mechanisms underlying seed hardness in Vicia sativa L., providing critical insights for crop improvement.