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.

backgroundWild-type Vicia sativa L. exhibits superior agronomic traits, including high yield, elevated crude protein content, improved reproductive efficiency, and tolerance to nutrient-poor soils, positioning it as a valuable genetic resource for crop improvement. However, its utilization in germplasm enhancement is severely hindered by seed hardness, the underlying physiological and molecular regulatory mechanisms of which remain poorly understood.

resultsPhenomic analyses have revealed that wild-type V. sativa seeds exhibit significantly higher hardness indices and lignin content compared to cultivated varieties, whereas cultivated seeds display elevated levels of soluble sugars, soluble proteins, and starch. During the same developmental stage, cultivated seeds demonstrate notably larger diameters than their wild-type counterparts. Conversely, wild-type seeds possess significantly thicker palisade layers and higher palisade-layer-to-seed-coat thickness ratios. Notably, the cuticle of wild-type seeds is markedly thicker. The SEM further revealed that cultivated varieties exhibit significantly wider and longer hilums, while wild-type seeds display deeper micropyles. WGCNA identified three key candidate genes, TRINITY_DN3402_c0_g1, TRINITY_DN13607_c0_g1, and TRINITY_DN6606_c0_g1, involved in seed hardness regulation. Expression pattern analysis further revealed that these three genes showed significantly higher transcript levels in the wild hard-seeded genotype V. sativa W12 compared to the cultivated genotype V. sativa W30.

conclusionsOur study elucidates the physical determinants and key candidate genes underpinning seed hardness in V. sativa, thereby providing critical insights to advance crop improvement.