Abstract:

Nowadays, there are two methods to optimize ISSR-PCR amplification system, one is a single factor test, and the other is orthogonal design. In this study, two methods were used to optimize ISSR-PCR amplification system on Tilia amurensis in three levels of four factors(Taq DNA polymerase, Mg²⁺ , dNTP, primer)respectively. From the results we found that there were some differences between the two methods in suitable levels of factors. Through the deep analyze, a suitable ISSR-PCR reaction system was established, namely 20 μL reaction system containing 1.0 U Taq DNA polymerase, 2.0 mmol·L^-1 Mg²⁺ , 0.20 mmol·L^-1 dNTP, 0.4 μmol·L^-1 primer, 1×PCR buffer, 30 ng DNA template. 14 primers with stable amplification and rich polymorphism for ISSR were screened. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. The result provided a standardizing ISSR-PCR program for the analysis of genetic diversity of T. amurensis.

Key words: Tilia amurensis, ISSR-PCR, orthogonal design, single factor tests, gradient PCR