灰色家鸽ISSR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Columba domestica

本研究旨在通过正交优化得出灰色家鸽的ISSR-PCR反应的最佳体系。以灰色家鸽为试验材料,使用引物ISSR807,采用正交优化方法对影响PCR反应的Mg2+、dNTPs、引物、Taq DNA聚合酶、模板进行了体系优化,同时对引物的最佳退火温度进行了选择,建立了适合灰色家鸽的ISSR-PCR分析的最佳体系。结果表明,灰色家鸽ISSR-PCR反应的最优体系为:25μL总体积中包括10×PCR Buffer 2.5μL,Mg2+2.6mmol/L,dNTPs 0.2mmol/L,引物0.4μmol/L,TaqDNA聚合酶0.5U,DNA模板1.2ng/μL,最佳退火温度为56.2℃。通过对ISSR-PCR反应体系的优化,能为利用该技术进行灰色家鸽遗传多样性评价、不同种源鉴定及亲缘关系分析奠定基础。

This experiment was conducted to obtain the suitable ISSR-PCR reaction system of Columba domestica by orthogonal design. Columba domestica was chosen to study the affecting factors of ISSR-PCR amplification. Orthogonal design of five factors （Mg2＋ , dNTPs, primer, Taq DNA polymerase, template） at four levels was used to establish the suitable ISSR-PCR system by primer ISSRS07 in Columba domestica, and the suitable anneal temperature was yielded from gradient PCR on temperature. The results showed that the optimized ISSR-PCR system（25 μL reaction volume） in Columba domestica was proved to be ： 10 × PCR Buffer 2.5 μL, 2.6 mmol/L Mg2 ＋ , 0.2 mool/L dNTPs, 0.4 μmol/L primer, 0.5 U Taq DNA polymerase, DNA template 1.2 ng/μL; and suitable anneal temperature was 56.2 ℃. According to the optimum ISSR-PCR reaction system, the results lay a foundation for the analyses of genetic diversity, germplasm resources and genetic relationship of Colurnba domestica.