朝鲜碱茅ISSR-PCR反应体系的建立与优化

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。

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

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,Mg2+,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 μmol/L,Taq DNA polymerase 1 U,MgCl2 2.5 mmol/L and 10×PCR Buffer(Mg2+ 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.