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 highquality 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 mu L, 2.5 mu L 10xbuffer, 1.0 mmol.L--(1) Mg2+, 0.15 mmol.L--(1) dNTPs, 0.1 umol.L-1 primer, 1.2 U Taq DNA polymerase, and 80 ng template DNA. The reaction procedure was as follows: pre-degeneration at 94 degrees C for 5 min, degeneration at 94 degrees C for 1 min, annealing at 51.0 degrees C similar to 59.2 degrees C for 1 min (annealing temperature depend on different primer), extension at 72 degrees C for 1 min, 40 cycles, final extension at 72 degrees C for 5 min and preservation at 4 degrees C.