ISSR Research Of Germplasm Resources Of Zanthoxylum Bungeanum

Zanthoxylum bungeanum Maxim is deciduous small arbor or shrub, which belong to Rutaceae Zanthoxylum. At present it is an important ecotype economic tree in project of conversion of cropland to forest. In Gansu province, because long-term evolution and domestication, Zanthoxylum bungeanum formed many strains, for instance DaHongPao, XiaoHongPao, MeiHuaJiao, DouJiao,GouJiao,LiuYueJiao and BaYueJiao etc, the many names because of different classification basis. There are exist problem of varietal complexity and name confusion reflect in scientific research and production. Present, Zanthoxylum bungeanum′s research main concentrarion in cultivation and physiological, molecular biology field there were isozyme research and RAPD technology have roported,but ISSR technology have none roport,it is greatly restriction the Zanthoxylum bungeanum germplasm resources development and utilization.The research take for 11 varieties of Zanthoxylum bungeanum as material,which main producing in Gansu province. At molecular level analyze Zanthoxylum bungeanum′s genetic relationship and genetic diversity, useing ISSR technology and orthogonal test, combined with SPSS and POPGENE software. In order to provide genetic background for new Zanthoxylum bungeanum variety cultivation , provide scientific basis for Zanthoxylum bungeanum resources protection and rational utilization. The main research results as follows:(1)Orthogonal design was used to optimize ISSR-PCR amplification system in three levels of four factors(TaqDNA polymerase, Mg2+, dNTP, primer)on Zanthoxylum bungeanum in this study. L9(34) scheme obtained the best level of influence factors.The results showed that a suitable ISSR-PCR reaction system was for Zanthoxylum bungeanum established:20μL reaction system containing 1.0UTaqDNA polymerase e,2.0mmol·L^-1 Mg 2+,0.1mmol·L^-1dNTP,1.0μmol·L^-1primer,10×PCR buffer2.5μL, 50 ng DNA template. The optimal annealing temperature and cycle number for ISSR-PCR reaction was proposed by gradient PCR.(2)Ten primers produced 131 bands,of them 112 band(s85.5%) were polymorphic. The average effective number of alleles,Nei's gene diversity and Shannon's information index were 1.4664,0.2198 and 0.3028,respectively,suggesting that the different kinds of Zanthoxylum bungeanum was comparatively great genetic diversity.(3)UPGMA cluster analysis based on POPGENE divided the populations into three main groups with the similarity coefficients of 0.675,QinanDaHongPao,QinanⅠand DouJiao formed one group,GouJiao,YouJiao,MeiHuaJiao,WuDouDaHongPao,ErHongPao,BaYueJiao andYeLiCang formed one group, ChuanShanHuaJiao formed the thired group.(4)Based on the Proportion of Polymorphic loci, Nei's index , Shannon index and genetic differentiation, fom the 11 varieties of Zanthoxylum bungeanum WuDouDaHongPao and QinanDaHongPao got high level genetic diversity. QinanⅠgot low level genetic diversity. Different varieties of Zanthoxylum bungeanum have great difference in genetic diversity.