Genetic relationships among species of the genus Diplotaxis (Brassicaceae) using inter-simple sequence repeat markers

Inter-simple sequence repeat (ISSR) amplification was evaluated for its applicability as a genetic marker system to establish relationships among ten Diplotaxis species. ISSR amplification generated multiple banding profiles with the 12 primers from all DNA samples, with an average of 41.2 fragments per primer. This average was clearly higher for the 5� triple-anchored primers than for other primers. The banding profiles were highly repeatable across separate PCR runs. DNA mixing procedures were found to be appropriate strategies to generate banding patterns representative of each species studied. Similarity values were calculated considering 494 ISSR bands, and a dendrogram was constructed based on the similarity matrix. The ten Diplotaxis species were clustered into two major groups. The first group consists of five species, Diplotaxis tenuifolia and Diplotaxis cretacea , and Diplotaxis muralis with their putative parents ( D. tenuifolia and Diplotaxis viminea ). In the second group three species are clustered that are closely related ( Diplotaxis virgata , Diplotaxis catholica and Diplotaxis siettiana ), in addition to Diplotaxis harra , and Diplotaxis erucoides , which has lowest similarity values with the rest of the species studied. The two groups defined in the present work may be concordant with the idea suggested by several authors of a biphyletic origin for Diplotaxis . The genetic relationships among the ten Diplotaxis species estimated by the polymorphism of ISSR markers are in agreement with those previously inferred by other morphological, biochemical and molecular data, indicating the reliability of the ISSR approach for this purpose.