DNA Extraction and Optimization of ISSR-PCR Reaction System for Pyracantha

Three different DNA extraction methods (CTAB, improved CTAB, and nuclear DNA method) were compared in order to isolate high-quality genomic DNA from fresh leaves of Pryacantha. The concentration of Mg2+, Taq polymerase, dNTPs, primer, and template DNA, greatly influencing ISSR-PCR of Pyracantha, were optimized by orthogonal design in this study, and the annealing temperature was also improved. The results showed that the high-quality genomic DNA was obtained by the improved CTAB method and was suitable for ISSR study. The optimal PCR system for ISSR analysis was as follows: total volume 25 µL, 2.5 µL 10×buffer, 1.0 mmolL−1 Mg2+, 0.15 mmolL−1 dNTPs, 0.1 umolL−1 primer, 1.2 U Taq DNA polymerase, and 80 ng template DNA. The reaction procedure was as follows: pre-degeneration at 94 °C for 5 min, degeneration at 94 °C for 1 min, annealing at 51.0 °C~59.2 °C for 1 min (annealing temperature depend on different primer), extension at 72 °C for 1 min, 40 cycles, final extension at 72 °C for 5 min and preservation at 4 °C.