Integrating morphological and molecular characterization to reveal genetic diversity and breeding potential in Turkish local alfalfa (Medicago sativa L.) populations

Alfalfa (Medicago sativa L.) is one of the most important forage crops due to its high crude protein content, nitrogen fixation ability, and adaptability to diverse ecological conditions. This study aimed to evaluate the morphological, phenological, yield, quality, and molecular diversity of 60 local alfalfa populations, together with 4 control varieties (64 genotypes in total), collected as clones from the Lakes Region of Türkiye (Isparta, Burdur, Afyonkarahisar, and Konya) between 2011 and 2013. Twenty-seven agronomic, morphological, and quality traits were analyzed, revealing substantial phenotypic variation among genotypes in terms of growth habit, regrowth rate, dormancy level, forage yield, and quality. Heatmap, PCA-biplot, and correlation analyses identified five major morphological groups with distinct yield–quality relationships. Molecular clustering and PCoA analyses supported these groupings and revealed strong genetic differentiation shaped by eco-geographic isolation and local adaptation. The combined evaluation of morphological and molecular data demonstrated that several genotypes possess both high agronomic performance and distinct genetic structure. In particular, genotypes LC3, LC17, LC24, LC29, LC39, LC49, LC53, LC54, LC57, and LC60 were identified as the most promising candidates for synthetic variety development due to their superior yield, quality traits, and representation of different genetic clusters. Overall, the findings highlight the significant breeding potential of local alfalfa germplasm and provide a reliable framework for developing high-yielding and high-quality varieties adapted to regional conditions.