马蔺ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Iris lacteal

根据正交试验设计原理,设计探索正交试验和细调正交试验以确定马蔺(Iris lactealPall.var.chinensis(Fisch.)Koidz)ISSR-PCR反应体系中各成分的浓度,从dNTP、Taq酶、Pri mers、Mg2+4种因素对马蔺ISSR反应体系进行了优化,得到适合马蔺的ISSR-PCR最佳反应体系,即25μl的反应体系中含有dNTP 0.3 mmol/L、Taq酶1.5 U、Pri mers 0.4μmol/L、Mg2+2.5 mmol/L以及1×buffer和50 ng模板DNA。通过对马蔺ISSR-PCR最佳反应体系进行梯度退火试验,得到这条引物的最佳退火温度为52℃。这一体系的建立为今后利用ISSR标记技术研究马蔺遗传多样性奠定了基础。

In this paper,two orthogonal experiments were conducted to optimize ISSR amplification system of Iris lacteal Pall.var.chinensis（Fisch.） Koidz.Results show that the optimum concentrations of reactants in 25 μl reaction mixture were as follows： dNTP 0.3 mmol/L,Taq DNA polymerase 1.5 U,Primers 0.4 μmol/L,Mg^2＋ 2.5mmol/L,1×buffer and 50 ng template DNA.The optimal annealing temperature of this primer was proposed by gradient PCR at 52℃ for ISSR-PCR reaction.The result provided a standardized ISSR-PCR program for the analysis of genetic diversity of I.lacteal var.chinensis.