| Tsoongiodendron odorum Chun belongs to magnoliaceae. It has many important scientific value for the flora、the ancient geography and climate. Nowadays, the number of natural population of T. odorum has declined, and most populations only have one or several trees. It is in peril of extinction and has been classified as the state second class protected plant in China. Therefore, studying of the genetic diversity and protecting the genetic resource of T. odorum is particularly important and urgent. The main studies are as follows:(1)The important factors of the inter-simple sequence repeat PCR(ISSR-PCR) amplification system, such as DNA template, primers, dNTPs, Mg2+concentration, dose of TaqDNA polymerase and annealing temperature for T.odorum were optimized using the orthogonal design experiments. The results show that the optimal reacting system with the volume of20μL was established as follows:50ng DNA template,1.25U Taq DNA polymerase,2.5mmol/LMgcl2,0.25mmol/L dNTPs,0.4μmol/L primers. The PCR procedure was:predenaturing at94℃for5minutes,35cycles of denaturation at94℃for1minutes, annealing at55℃for30seconds and extension at72℃for90seconds, with7minutes final extension at72℃and then stored at4℃.(2)The genetic diversity of255individuals from14natural populations of this plant was assessed using ISSR-PCR amplification system. The results showed that182total ISSR discernible bands with8ISSR primers,145were polymorphic. The percentage of polymorphic bands was79.67%. At the population level with the percent of polymorphic bands ranged from9.34%to62.64%, the average value was43.45%. At the population level with the Shannon’s information index (I) from0.0565to0.2873, the average value was0.2054. The result showed there existed high level genetic diversity both at population and species level. The highest genetic diversity of populations was NKS of guangdong province(T=62.64,I=0.2873), and the lowest genetic diversity of populations was LTJ of guangdong province(P=9.34%,I=0.0565). Analysis of molecular variance (AMOVA) revealed that among and within-population genetic variation accounted for37.58%and62.42%of the total genetic variation, respectively. Cluster analysis showed that collected from guizhou、hunan and guangxi populations clustered together, the most population of jiangxi clustered together. CBL and FX populations together with DN population. It is a further indicator that genetic variation of populations and its geographic pattern is basic consistency. (3)The results of nature and ex-situ conservation population of T. odorum showed that the percentage of polymorphic bands(P) and Shannon information index(I) of natural populations(59.60%,0.2847) were much higher than those of ex-situ conservation populations (44.78%,0.2333). It is justified to assume the narrow genetic base for plantations of T. odorum. Analysis of molecular variance (AMOVA) revealed that the variation of nature and ex-situ conservation population of this species have occupied3.52%(df=1, P>0.05) in total variation. The result showed that genetic structure between those both populations have not obviously diversity.(4)Our results indicated that human activities may be one of the main endangered reasons of this species, environmental selection pressure resulted in genetic differentiation among populations. Established ex-situ is not an efficient gene pool in protect genetic diversity of natural populations. We suggest that we should take some measures to protect natural populations of T. odorum. At the same time, planted populations are an important conservation resource for the future, and planting material collection strategies for ex-situ should include many different populations of this species. We present suggestions strengthening research on the reproductive biology of T. odorum. In some degree, the species endangered situation should be eased, in order to maximize the protection of the genetic diversity and maintain its survival and reproduction. |
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