Karyotype characterization and cytogenetic diversity of Medicago sativa L. (Leguminosae) populations from the Lake Region of Türkiye

Medicago sativa Linnaeus, 1753 (Leguminosae) is a globally important autotetraploid forage crop with a complex genome. Local populations adapted to specific ecological niches, such as those in Türkiye’s Lake Region, represent valuable genetic resources for breeding. However, their cytogenetic diversity remains poorly characterized. This study aimed to perform detailed karyotype analyses of eight alfalfa populations from this region to assess chromosome number, morphology, asymmetry, and structural variants. Root tips from eight populations collected from Afyonkarahisar, Burdur, Isparta, and Konya provinces were analyzed using aceto-iron-hematoxylin staining and the squash technique. Chromosome measurements, arm ratios, centromeric indices, karyotype formulas, and asymmetry indices (intrachromosomal A1 and interchromosomal A2) were determined. Descriptive parameters (TF%, DRL, DI) and clustering analyses were used to compare cytogenetic diversity among populations. In accordance with current karyomorphological recommendations, mean centromeric asymmetry (MCA), coefficient of variation of chromosome length (CVCL), and coefficient of variation of centromeric index (CVCI) were additionally reconstructed from the chromosome-pair mean measurements reported in the paper. All populations were confirmed as autotetraploid (2n = 4x = 32). Karyotypes consisted predominantly of metacentric chromosomes, with fewer submetacentrics, and all possessed four satellite chromosomes. Reconstructed MCA ranged from 11.86% (Bolvadin-3) to 15.36% (Yalvaç-3), CVCL ranged from 8.81% (Akşehir-2) to 12.69% (Bolvadin-3), and CVCI ranged from 7.77% (Bucak-2) to 11.63% (Yalvaç-3). A1 ranged from 0.208 (Bolvadin-3) to 0.258 (Hüyük-2), and A2 ranged from 0.096 (Yeşilova-3) to 0.149 (Hüyük-2). B-chromosomes (2n = 32 + 2B) were detected exclusively in Bucak-2, and heteromorphic chromosome pairs were observed in Hüyük-2 and Uluborlu-1. Hüyük-2 remained the most structurally distinctive population because of its high A1 and A2 values, heteromorphic chromosome pair, enlarged satellite, and Stebbins 2C classification. Cluster analysis based on asymmetry indices separated Hüyük-2 as a distinct, highly asymmetric group. Substantial cytogenetic diversity exists among Turkish Lake Region alfalfa populations, including rare features such as B-chromosomes and heteromorphic pairs. These findings highlight the value of local germplasm as genetic resources for conservation and breeding. The Hüyük-2 population, with its highly asymmetric karyotype, is particularly noteworthy for further evolutionary and applied studies.