Phenotypic and genomic insights into alfalfa diversity: Identifying critical loci for enhanced resilience

Alfalfa (Medicago sativa L.) is a globally important forage crop, yet the relationship between its phenotypic variation and underlying genetic structure remains poorly resolved. In this study, we combined spatially adjusted, multiyear phenotyping with high-density single-nucleotide polymorphism genotyping in a panel of 395 cultivated accessions to (i) characterize the structure of phenotypic diversity across a broad range of agronomic traits, (ii) quantify the correspondence between phenotypic and genetic structure, and (iii) identify specific traits and genomic regions associated with genetic differentiation. Phenotypic data were collected across multiple locations and years, while genotypic data were obtained using genotyping by sequencing. Multivariate and feature selection analyses revealed nine phenotypic groups, with 78 traits confirmed as important, especially anthracnose resistance and lodging susceptibility. However, phenotypic clustering showed only moderate correspondence with genetic structure (Mantel r = 0.17, p = 0.001). Genome-wide scans for genetic groups differentiation (F_ST) identified discrete genomic regions enriched for candidate genes linked to disease resistance, stress tolerance, and reproductive processes, including loci potentially involved in self-incompatibility. These results highlight the complexity of genotype-phenotype relationships in alfalfa and pinpoint specific genomic targets for future breeding efforts aiming to enhance resilience, productivity, and adaptability.