Phylogenetic Relationships And Genetic Diversity Revealed By RAPD,SSR,ISSR Markers In Prunus

In order to identify the germplasm resources to discriminate duplicated or mislabelled collections, evaluate the diversity to facilitate using in breeding and further collection and analysing the genetic relationship of plum species. , three molecular markers such as RAPD, SSR and ISSR were employed to detect the genetic diversity in above 60 accessions of plum and its related species sampling. Some main conclusions in this study are as follows:(1) RAPD-PCR reaction mixtures had a total volume of 20 ul containing 80 ng DNA template, 0.1 μ M primer, 0.15-0.2 mMdNTPs, 0.5-1.0 units of Taq DNA polymerase, 2.5-3.0 mM MgCI, 2 μl 10×buffer (20 mM MgSO₄, 100 mM KCl, 80 mM (NH4) SO₄, 100 mM Tris-HCl, 0.5%NP-40, pH9.0). Reaction mixtures were overlayed with 50μl mineral oil.Amplifications were performed on a DNA Thermal Cycler 9700 (Perkin Elmer) with the following temperature profile: a pre-denaturation step of 5 min at 94℃ followed by 45 cycles of denaturation 1 min at 94℃; annealing 1 min at 39.2-39.8 ℃; extension 2 min at 72 ℃. A final extension was allowed for 5 min at 72 ℃.(2) ISSR-PCR reaction mixtures had a total volume of 20 μl containing 20-80 ng DNA template, 0.15-0.2 μ M primer, 0.1-0.15 mM dNTPs, 0.5-1.0 units of Taq DNA polymerase, 2.5-3.0 mM MgCI, 2 μl 10×buffer (20 mM MgSO₄,100 mM KCl, 80 mM(NH₄) SO₄, 100 mM Tris-HCl, 0.5%NP-40, pH9.0). Reaction mixtures were overlayed with 50μl mineral oil.Amplifications were performed on a DNA Thermal Cycler 9700 (Perkin Elmer) with the following temperature profile: a pre-denaturation step of 5 min at 94℃ followed by 40 cycles of denaturation 45s at 94℃; annealing 1 min at 50-50.8℃; extension 1.5 min at 72 ℃. A final extension was allowed for 5 min at 72 ℃.(3) The clustering results showed that 60 accessions tested could be classed into four groups :"apricot and hybrids between apricot and plum", "Chinese plums and the hybrids with other diploid plum species", "European plums", and "other plums( Wanhei, an European cultivar with unknown name)".(4) Some accession-specific band patterns could be found apparently when scrutinizing the electrophoretic gels.(5) There are three accessions (8, 11 and 49) with same name, Xiangjiaoli, and they belong to 3 different species, P. simonii, P. salicina and P. ussuriensis respectively.