关键词: 节瓜, ISSR-PCR, 正交优化, 梯度PCR, 退火温度

Abstract: 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, Mg2+ 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 Mg2+ 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.

Key words: Chieh-qua, ISSR-PCR , Orthogonal optimization , Gradient PCR , Annealing temperature