Abstract:

The establishment of the ISSR-PCR amplification system could lay a good foundation for identification and classification and analysis on the genetic diversity of germplasm resources of Huperzia javanica using ISSR molecular marker techniques. In this paper，the orthogonal design was used to optimize the ISSR-PCR amplification system of Huperzia javanica in four factors (the concentration of dNTP, Taq DNA polymerase, Primers and Mg2+) respectively. Then, based on the optimal ISSR-PCR amplification system, the concentration of template DNA was proposed by gradient PCR. The results showed that a suitable ISSR-PCR amplification system of Huperzia javanica was established. In a total volume of 20 μL ISSR-PCR amplification system, it contained 1×PCR buffer, 300 μmol/ L dNTP, 0.2 μmol/ L primer, 2.0 mmol/ L Mg2+, 1.75 U Taq DNA polymerase and 40 ng template DNA. The optimized system laid the groundwork for ISSR molecular analysis of Huperzia javanica.