云南高海拔地区桃种质资源ISSR体系的建立和优化

Establishment and Optimization of ISSR System for Peach Germplasm Resources in High Altitude Areas of Yunnan Province

本试验以云南高海拔地区桃种质资源的样本为试验材料,从提取的100余份DNA样品中随机抽取1份,再用初筛的引物UBC-891,用于ISSR体系的优化。根据单因素试验和正交试验设计,本研究发现了最佳ISSR-PCR反应体系。研究结果表明:25μL的ISSR反应体系中,2.5μL 10×PCR buffer(含 Mg^(2+)),1.5 mmol/L Mg^(2+),2.0 U Taq-DNA聚合酶,0.10 mmol/L dNTPs,0.40μmol/L引物,45 ng DNA组合最佳。Taq-DNA聚合酶浓度对反应影响最大。本研究为引物筛选和云南高海拔地区桃种质资源ISSR遗传多样性研究提供了理论基础。

In this r esearch,we used peach germplasm resources from high altitude areas of Yunnan as the test material,randomly selected one of the 100 extracted DNA samples,and used the preliminary screening primer UBC-891 for the optimization of ISSR system.Based on the single factor test and the orthogonal experiment design,we found the best ISSR-PCR reaction system.The results showed that 2.5 μL 10×PCR buffer（containing Mg2＋）,1.5 mmol/L Mg2＋,2.0 U Taq-DNA polymerase,0.10 mmol/L dNTPs,0.40 μmol/L primers and 45 ng DNA were the best combination in 25 μL ISSR-PCR reaction system.The concentration of Taq-DNA polymerase had the most significant effect on the reaction.This research might provide theoretical foundation for primer selection and ISSR genetic diversity research of peach germplasm resources in Yunnan high altitude areas.