Different Origin The Bupleurum Of Issr Molecular Markers And Quality Of Research

Objective:To characterize nine individuals of Bupleurum by inter simple sequence repeat(ISSR)technique and to assess the germplasm genetic diversity and studying the genetic relationship among varieties of molecular evidences for its breeding.Methods:A total of nine samples including seven Bupleurum chinense DC isolated from four areas,one Bupleurum falcatum L.and one B.yinchowense shan et Y.Li.The radix of each sample of Bupleurum were detected by HPLC.DNA was extracted from leaves of all the samples and analyed by by inter simple sequence repeat(ISSR) technique.To calculate Nei measure and draw dendrograms by using genetic distance UPGMA method.A HPLC method was established for the determination of Saikosaponin a and Saikosaponin d in "Thorowax".Many parameters of HPLC technique was studied by a series of experiments.Finally a BonChrom C18 column was used with acetonitrile-water(40:60)as the mobile phase and in a flow rate of 1.0 ml·min-1. The detecting wavelength was 204nm.The column temperature was 25℃.Commercial Kit methods was used to extract DNA from leaves.Five parameters of PCR were studied by orthogonal experimental designa and series of annealing temperature were comparered.Seven appropriate ISSR primer were selected from a total of 60 ISSR ones for ISSR PCR amplification.Result:The HPLC experimental results indicated that Thorowax Shangluo contented of highest Saikosaponin a and Saikosaponin d.Than it is Thorowax Gansu,Bupleurum falcatum L,Thorowax Zhongchai,Thorowax Shanxi and Thorowax Baokang.The average recovery rates were 99.35%,99.52%,respectively while RSD were 1.14%,1.18%.Assay methods were reliable and accurate in the research of content.The ISSR analysis showed that there were significant genetic diversity among the 9 samples.A total of 81 bands was amplified by the 7 primers,97.5%of the total bands was polymorphic.Two common bands were amplified by primer 808 and primer 828.Three bands were unique of Bupleurum falcatum L.and which were amplified by primer 808,primer 828 and primer 856.The results of cluster analysis by using UPGMA method showed that all the tested accessions can be differentiated by ISSR markers,and classified as 3 groups.Of which,Bupleurum falcatum L.was classified as one group.B.yinchowense shan et Y.Li.was classified together with three samples of Bupleurum chinense DC as one group,indicate that there may be genetic aberrance in B.yinchowense shan et Y.Li. from Gansu.Conclusion:The Thorowax Shangluo had highest quality among all the individuals.The germplasm of Thorowax collected from Baokang Anjiawan was good for the sample to classified together with two high quality Thorowax.However, Thorowax collected from Baokang Tangerhe has bad quality and germplasm.