节瓜ISSR-PCR反应体系的建立与正交优化

Establishment and Optimization of an ISSR-PCR Reaction System for Chieh-qua Using Othogonal Design

旨在开展节瓜种质资源分类鉴定与遗传多样性研究。通过正交试验设计与单因素分析相结合的方法,对节瓜ISSR-PCR反应体系5个因素(模板DNA浓度、dNTP浓度、Mg2+浓度、引物浓度与Taq聚合酶浓度)在4个水平上进行优化分析,建立了节瓜稳定可靠且具丰富多态性的最佳反应体系,进而对引物退火温度进行梯度试验分析。结果表明,20μL节瓜ISSRPCR最佳反应体系为70 ng模板DNA、0.2 mmol/L dNTP、1.2 mmol/L Mg2+浓度、0.96μmol/L引物、0.8 U Taq DNA聚合酶和2.0μL10×buffer;引物IS807最佳退火温度为53℃。以此为基础,利用4条引物对4份节瓜种质进行最佳反应体系稳定性验证,证明该体系稳定可靠、扩增谱带清晰、多态性丰富且重复性较好。

It was to identify and classify chieh-qua germplasm resources. The main factors affected ISSR reaction was tested to optimize ISSR amplification system. In this research, the orthogonal design and single-factor gradient method were used to establish and screen chieh-qua ISSR-PCR system for 4 levels of 5 factors（DNA template, dNTPs, Mg^2＋ concentration, primer, and Taq DNA polymerase）respectively, and then annealing temperatures were proposed by gradient PCR based on the optimal reaction system established. The results showed that the optimal ISSR-PCR system mixture（20 μL）including 70 ng template DNA, 0.2 mmol/L dNTPs, 1.2 mmol/L Mg^2＋ concentration, 0.96 μmol/L primer, 0.8 U Taq DNA polymerase and 2.0 μL 10×buffer. The suitable annealing temperature of primer IS807 was 53℃. Four primers were applied to four chieh-qua materials for testing and verifying stability of the established reaction system, which showed that the ISSR reaction system was proved to be stable, credible and of clear bands and rich polymorphism.