PCR-RFLP and genetic diversity analysis of cpDNA in some species of the genus Salvia L.

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Long Accurate polymerase chain reaction restriction fragment length polymorphism (LA-PCR-RFLP) of the chloroplast DNA (cpDNA) was used to investigate phylogenetic relationships between 54 species of Salvia L. Two Ocimum L. species were used as an out group. A pair of universal primers in LA-PCR protocol resulted in long cpDNA of an expected size (∼15 kilo base pair (kb)) from each Salvia and Ocimum species, except S. x superba (hybrid; S. sylvestris x S. amplexicaulis or S. nemorosa). LA-PCR products were separately digested with each of EcoRI, HindIII, PvuII, NdeI, SmaI or MfeI restriction enzymes. S. roemeriana Scheele showed amplifi ed fragment length difference (AFLD). S. roemeriana and/or S. lyrata L. could be identifi ed with any of the enzymes used except PvuII. Identifi cation of Ocimum species from Salvia could be carried out with NdeI, SmaI or MfeI. EcoRI and MfeI exhibited more polymorphic and informative patterns than HindIII, PvuII, NdeI or SmaI. LA-PCR-RFLP analysis generated 28 polymorphic restriction fragments. Fragments were visually detected, photographed and used to build a genetic similarity data matrix based on Nei and Li (1979) method. PHYLIP software package (Felsenstein 2005) and the genetic similarity matrix were exploited to construct a phylogenetic tree. The phenogram generated by UPGMA clustering analysis separated Salvia species based on genetic distance into distinct groups obviously dependent on origin.