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.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第6期59-65,共7页
Biotechnology Bulletin
基金
国家牧草产业技术体系
关键词
朝鲜碱茅
ISSR
单因子试验
体系优化
PuccineUia chinampoensis ISSR Single factor tests Optimization
