Establishment and Condition Optimization of ISSR-PCR Amplification System of Hypericum perforatum Linn.

[Objective] To establish the ISSR-PCR amplification system for Hypericum perforatum Linn., and to optimize the condition. [Method] With genomic DNA of H. perforatum as the template, L16(45) orthogonal test system was adopted; the effects of primer concentration, Taq DNA polymerase concentration, Mg2+ concentration, dNTP concentration, and template DNA concentration on the amplification results of ISSR-PCR were researched. [Result] Orthogonal test could be used to establish the ISSR-PCR reaction system of H. perforatum. And the optimal condition of ISSR-PCR reaction system was as follows: 10×PCR buffer, 1.2 mmol/L Mg2+, 50 U/mL Taq DNA polymerase, 20 ng/μL DNA, 250 μmol/L dNTP, and 0.75 μmol/L primer in 20 μL ISSR-PCR reaction system. [Conclusion] The established ISSR-PCR reaction system for H. perforatum had good repeatability, high resolution ratio, and stable and reliable results.