Barley is an economically important cereal crop especially for feed and malt production, but its value as food is increasing due to various health benefits. Wild barley is the progenitor of modern day barley cultivars possessing a rich source of genetic variation for various biotic and abiotic stresses. Species‐specific molecular markers have great potential for efficient introgression of these important traits from wild to cultivated barley. In the present study, 140 microsatellite markers were screened to assess the genetic variation and species‐specific markers between wild and cultivated germplasm. Of these 140, a polymorphic set of 48 genomic (gSSR) and 16 EST‐SSRs amplified a total of 685 alleles. Cluster analysis discriminated all 47 accessions and classified wild and cultivated genotypes into two distinct groups, according to their geographic origin. Our analysis indicated that gSSRs were more informative than EST‐based SSRs. Results from PCoA analysis for species‐specific alleles clearly suggest that wild barley genotypes contain a higher number of unique alleles.

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