Establishment and Optimization of ISSR-PCR Reaction System of Anoectochilus emeiensis

The effects of different levels of factors on ISSR-PCR reaction results of Anoectochilus omeiensis were discussed by orthogonal design with 5 factors and 4 levels, and a stable and reproducible ISSR-PCR reaction system of Anoectochilus omeiensis was established. That is, the 25 μL system contained 40ng DNA template, 0.4μmol/L primer, 0.4 mmol/L dNTPs, 0.8 U Taq DNA polymerse, 2 mmol/L Mg²⁺, and 2.0 μL 10×Buffer μL in 25 μL reaction volume. At the same time, in order to determine the optimum Tm （annealing temperature）, gradient PCR test was carried out, and finally ISSR-PCR experimental reaction system suitable for Anoectochilus omeiensis was optimized and established.