Optimization for ISSR-PCR reaction system in Impatiens macrovexilla Y. L. Chen using orthogonal design

【Objective】The present experiment was conducted to explore the best ISSR-PCR reaction for genetic diversity analysis of Impatiens macrovexilla Y. L. Chen. 【Method】Orthogonal design method was used to optimize the ISSR-PCR amplification system of Impatiens macrovexilla Y. L. Chen in five factors(concentration of Mg2+, dNTP, primers, Taq DNA polymerase, and template DNA) at four levels. Then, based on the optimal ISSR-PCR amplification system, annealing temperature was screened by gradient PCR. 【Result】An optimal reaction system(25.0 μL) was established with containing 1× PCR Buffer,1.5 mmol/L Mg2+, 0.25 mmol/L dNTP, 0.8 μmol/L primers, 0.75-1.00 U Taq DNA polymerase, and 100 ng temple DNA. Six ISSR primers with better effects were screened by this reaction system. 【Conclusion】This optimal ISSR-PCR reaction system showed a very stable result, which could be used for ISSR analysis of genetiec diversity in Impatiens macrovexilla Y. L. Chen.