Genetic Diversity among Selected <i>Medicago sativa</i> Cultivars Using Inter-Retrotransposon-Amplified Polymorphism, Chloroplast DNA Barcodes and Morpho-Agronomic Trait Analyses

Alfalfa (<i>Medicago sativa</i> L.) is a major forage crop of family Fabaceae and is frequently cultivated in Egypt. The present study is concerned with the genetic discrimination of fifteen alfalfa cultivars from three different countries (Egypt, Australia, and USA) using two molecular approaches: inter-retrotransposon-amplified polymorphism (IRAP) markers and two chloroplast DNA barcodes <i>mat</i>K and the <i>trn</i>H in addition to the analysis of fifteen morpho-agronomic traits. The genetic relatedness, based on analysis of IRAP marker polymorphism and produced using eleven primers by clustering via principal component analysis (PCA) and multivariate heatmap biostatistical methods differentiated the two Egyptian cultivars EGY1-Ismailia1 and EGY2-Nubaria1 from the three Australian and seven American cultivars, with some distinction of the cv. USA6-SW9720 and cv. AUS4-SuperFast. The results were also supported by the sequence analysis of the <i>mat</i>K and the <i>trn</i>H genes on the genetic relatedness between eight cultivars. Moreover, it might be suggested that breeding lines from <i>M. sativa</i> cultivars may provide novel insights and a better understanding of the domestication of <i>M. sativa</i> genetic diversity. The classification of the eight cultivars, as revealed by morpho-agronomic traits, confirmed the close genetic relationship between the two Egyptian cultivars and indicated some resemblance between them and the AUS2-Siri Nafa, whereas the two American cultivars, USA1-Super supreme and USA4-Cuf101, were clearly isolated from a cluster of other three cultivars USA7-SW9628, USA8-Magna901, and USA9-Perfect. The results are useful sources of genetic information for future breeding programs in crop development and open new possibilities of producing <i>M. sativa</i> lines harboring high forage quality, productivity, and resistance to biotic and abiotic stresses.