Genetic diversity and genetic structure of alfalfa (Medicago sativa L.) genotypes as revealed by Start Codon Targeted (SCoT) markers

Understanding the genetic diversity of alfalfa (Medicago sativa L.) is fundamental for the development of improved cultivars and the implementation of effective conservation strategies. In the present study, the genetic diversity and population structure of 85 alfalfa genotypes, comprising both cultivated and wild accessions collected from various regions, were evaluated using 10 Start Codon Targeted (SCoT) molecular markers. A total of 92 scorable bands were generated, with an average polymorphism information content (PIC) value of 0.34. Analysis of molecular variance (AMOVA) revealed that the majority of genetic variation (74%) was partitioned within genotypes, whereas 26% of the variation was distributed among three genetic clusters, as determined by STRUCTURE analysis. Principal Coordinate Analysis (PCoA) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering further confirmed the grouping pattern, indicating low to moderate genetic differentiation among clusters (pairwise Fst values ranging from 0.048 to 0.103). Notably, the SCoT-2 and SCoT-3 primers exhibited the highest levels of polymorphism and discriminatory power. These results underscore the effectiveness of SCoT markers for assessing genetic diversity and delineating population structure in alfalfa, providing an essential foundation for future breeding programs aimed at broadening the genetic base and improving agronomic traits.