朝鲜碱茅ISSR-PCR反应体系的建立与优化  

Establishment and Optimization of ISSR-PCR Reaction System for Puccinellia chinampoensis

作　　者：任伟[1] 徐安凯[1] 徐博[2] 于洪柱[1] 王志锋[1] 

机构地区：[1]吉林省农业科学院,公主岭136100 [2]中国农业科学院草原研究所,呼和浩特010010

出　　处：《生物技术通报》2012年第6期59-65,共7页Biotechnology Bulletin

基　　金：国家牧草产业技术体系

摘　　要：为进一步开展朝鲜碱茅种质资源遗传多样性的研究,以野生朝鲜碱茅(Puccinellia chinampoensis)为材料,通过单因子试验对ISSR-PCR反应进行优化。确立最佳的PCR反应体系:在20μL反应体系中,含有模板DNA 40 ng,dNTPs 0.2 mmol/L,引物0.8μmol/L,TaqDNA聚合酶1 U,MgCl22.5 mmol/L和10×PCR Buffer(Mg2+free)2μL。此外,还筛选到10条扩增稳定、条带丰富的候选引物,并确定了各自的最佳退火温度。It is necessary to establish and optimize the ISSR-PCR reaction system for studying the genetic diversity of Puccinellia chinampoensis. To obtain the best amplification result of Puccinellia chinampoensis with clear, repeatable and rich polymorphism bands, the ISSR reaction system including dNTPs, Mg , primer, template DNA and Taq DNA polymerase was optimized. Results showed that the optimum concentrations of five reactants in 20 μL reaction mixture were as follows： genomic DNA 40 ng, dNTPs 0.2 mmol/L, primer 0.8 μtmol/L, Taq DNA polymerase l U, MgC12 2.5 mmol/L and 10 x PCR Buffer （ Mg^2＋ free ） 2 μL. 10 primers with stable amplification bands and rich polymorphism for ISSR-PCR were selected from 100 candidate primers, which the optimal annealing temperature was also found. This optimized ISSR reaction system would provide reference for genetic diversity analysis, germplasm resources classification and map construction.

关 键 词：朝鲜碱茅 ISSR 单因子试验 体系优化 

分 类 号：S543.9[农业科学—作物学]