| Salix oritrepha is unique to china and only distribute in Gansu, Sichuan and Qinghai province, china, which is mainly distributed on mountain ridges in the altitude of3000-3700m, is considered as protective forest, fuel forest, soil and water conservation forest and timber forest of main afforestation tree species, it is also the important energy forest species, which has a high ecological and economic value. It is a perennial shrub with high product and feed value etc, Which is the pioneer plant of wind-breaking and sand-fixing. In this study, SSR markers were used to detected genetic diversity and relationship among7populations of Salix oritrepha which were collected from different producing area. The objectives of this study are to provide basis for Salix oritrepha genetics and breeding research. The results showed as follows:1. In this paper, the SSR-PCR reaction system of Salix oritrepha was optimized. In all the factors of each component changes caused by the effect of PCR-SSR, were studied by single factor and orthogonal design. The results showed that polymerase chain reaction (PCR) system optimum condition for:20μL system,1μL Mg2+(2.50m mol/L),1.5μL dNTPs concentration (0.10mmol/L),1μL Taq polymerase (0.5U),1μL DNA template (20ng/U L),2μL each primer concentration (0.5μmol/L),2μL10×Taq Buffer, then add ddH2O to20μL The PCR procedure was an initial step of94℃for3min, followed by94℃for45s, annealing45s (annealing temperature varies with the primers),72℃extension30s,30cycles, final72℃extension5min, saving for4℃.The optimized SSR-PCR system was stable and universality, The results indicated that the SSR reaction system would be more productive and repeatability with Salix oritrepha PCR reaction system.2. Also in this study, the diversity and genetic relationship among7Salix oritrepha geographical populations consisting of140individuals sampled across their main distribution were investigated by using the simple sequence repeats (SSRs) markers in these eco-geographical populations, in order to estimate genetic variability and compare the level of diversity. A total of173alleles were detected on15SSR loci, and the number of alleles on each locus ranged from7to16, with an average of11.53. The average polymorphic rate (P) for the species was89.02%, The total gene diversity (Ht=0.4177) and the coefficient of population differentiation (GST=0.0761), all the results showed that Salix oritrepha had a relatively high level of genetic variation, and the genetic variation was mainly distributed within the populations. The gene flow among the populations of Salix oritrepha (Nm=6.0745) was more than that of the common anemophytes (Nm=5.24). Cluster analysis showed no direct correlation between genetic distance and geographic distance, suggesting that genetic diversity of Salix oritrepha was possibly related to species traits, communities and the climate. Implications of these results for future Salix oritrepha collection, evaluation and conservation were discussed.3. Genetic diversity and genetic structure of nine altitudes of Salix oritrepha from Qilian mountain,(totally about180individuals), were analyzed by SSR (Simple Sequence Repeat) markers. Fifteen highly stable and repeatable SSRs (selected from47SSRs of populus) detected a total of211alleles of which176were polymorphic(85%of the total). At the species level Shannon index (Ⅰ) was0.4192, Nei’s gene diversity index (H) was0.2796, the coefficient of gene differentiation (Gst) was0.0935, for the gene flow(Nm) was4.8487. The results also showed that there was more genetic differentiation within altitudes than between altitudes. All of the results include P, H, I showed that S. oritrepha has a high genetic diversity in Gansu Qilian mountain. Cluster analysis showed direct correlation between genetic distance and geographic distance. Genetic diversity of S. oritrepha was notably affected by the latitude and altitude, the higher the altitude gradient is, the more abundant the genetic diversity of S. oritrepha. |
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