Establishment and optimization of SSRPCR system of Salix oritrepha

In this study, the SSRPCR system of Salix oritrepha from four altitudes was optimized. The main factors of the system were studied using orthogonal design and single factor experiment. Meanwhile， the suitable annealing temperature of primer SHUK123 was optimized. The results of this study showed the optimized system was: 1 L Mg2+ (2.50 mmolL-1), 1.5 L dNTPs concentration (0.10 mmolL-1), 1 L Taq polymerase (0.5 U), 1 L DNA template (20 ngL-1), 2 L each primer concentration (0.5 molL-1), 2 L 10 Taq Buffer in a mixture of 20 L. The PCR procedure included an initial step of 94 ℃ for 3 min, followed by 30 cycles of 94 ℃ for 45 s, annealing 45 s (annealing temperature varies with the primers), 72 ℃ extension 30 s, and a final extension at 72 ℃ for 5 min. The products of PCR were saved at 4 ℃. The optimized annealing temperature of primer SHUK123 was 56 ℃. The optimized SSRPCR system was stable and repeatable, which will be a good choice in the study of S. oritrepha.