Optimization for ISSR-PCR reaction system of tea plant

The orthogonal design was used to optimize ISSR amplification system of tea plant in five factors (Taq polymerase, Mg2+, DNA templet, dNTP, primer) at four levels respectively. The best level of each factor were selected, a suitable ISSR reaction system was established, namely 20 μL reaction system containing 1.0 U/(20 μL) Taq DNA polymerase, 2.0 mmol/L Mg2+, 40 ng/(20 μL) DNA templet, 0.20 mmol/L dNTP, 0.25 μmol/L primer. The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR, showing that the most suitable temperature of primer ISSR1, UBC881 was 52.4 ℃, 59.0 ℃.