Molecular Identification And Genetic Diversity Analysis Of Radix Bupleuri

This thesis firstly briefly introduced the general situation of Bupleurum germplasm, and the research results of its genetic diversity by morphology, cytology, biochemical research and DNA molecular marker. What's more, the significance, novations and contents of this paper were presented.Bupleurum germplasm is the Chinese traditional medicine, which has important medical and economic value. It is the dried root of "Bupleurum chinese DC." and "Bupleurum scorzonerifolium Willd." that are recorded in "Pharmacopoeia of the People's Republic of China" (2005 edition). In fact, the source of Radix Bupleuri is very complicated, and there are a good many factors which lead that the quality of Radix Bupleuri and its product is unstable and the identification of Radix Bupleuri is difficult. As a result, it is difficult for Bupleurum resources research, preservation, evaluation, use and exploitation of the new germplasm.RAPD was used to analysis the 14 dried roots Radix Bupleuri samples. Firstly we optimized the parameter containing the concentration of template DNA, primer concentration, dNTP concentration and Taq enzyme amount. Then RAPD fingerprinting and the dendrogram was obtained by differential primers OPD-8 selected under the optimization. The results showed that:all the samples were clustered into one category respectively when the similarity coefficient was 0.930, so they could be distinguished by one primer, and we got the specific RAPD markers of superior variety ZQ-T which was tagged OPD-82500.Furthermore, we used a new data processing method, namely the each lane of RAPD fingerprint electrophoresis was converted to optical density curves by the Gene-Snap software. The different samples had the different curves, with each peak represented a band. When the Rf was 0.365, the sample no other than ZQ-T had the obvious peak, which could represent the specific RAPD markers of superior variety ZQ-T(OPD-82500).This could be as important molecular characteristic for the variety identification rapidly, which can provide a reference for the identification of Radix Bupleuri dried roots at the molecular level and the establishment of a new identification method for Chinese traditional medicine.Besides, we also have studied the genetic diversity and relative relationship of different species and different cultivated samples by AFLP molecular marker, with the medicinal dried roots as material. AFLP fingerprinting of the 14 dried roots samples were obtained by seleced primers E-AAC/M-CTA, E-AGG/M-CTG and E-AGG/M-CAA. The results showed that:14 samples have amplified 106 clearly differentiable bands, of which 90 bands were polymorphic, the rate of polymorphic was 84.91%. Moreover, the primer combination E-AGG/M-CTG had the maximal rate of polymorphic, which was 86.96%. The coefficient of genetic similarity of 14 samples ranged from 0.326 to 0.848, of which the coefficient between No.7(LC-L) and No.8 (LC-T) was 0.848. They were from the same source lingchuan and respectively cultivated in Shanxi Lingchuan and Taigu, so it showed that they had the relative genetic stability. Furthermore, the samples were clustering analysis by UPGMA, the results showed that when the similarity coefficient was 0.480,14 samples were clustered into two categories, No.10 and 11 (Bupleurum scorzonerifolium Willd.) clustered together, the remaining 12 samples were clustered into another category, and No.7 and 8 were clustered firstly which had the closest relative relationship. It is indicated that the Bupleurum chinese DC. and Bupleurum scorzonerifolium Willd. could be distinguished by AFLP, which was same with the taxology and the differences between species were more visible than intra-specific.