Abstract: Orthogonal design was used to optimize ISSR-PCR amplification system of Calanthe tsoongiana in five factors (Mg²⁺, dNTP, primer, DNA template and Taq DNA polymerase) at four levels, respectively. The results showed that the suitable ISSR-PCR system was performed in a 25 μL volume containing 3.0 mmol·L^-1 Mg²⁺, 0.3 mmol·L^-1 dNTP, 0.4 μmol·L^-1 primer, 2.5 ng·μL^-1 DNA template, 0.08 U·μL^-1 Taq DNA polymerase and 1×PCR buffer. The optimized augmentation procedure was predenaturation at 94℃ for 5 min, followed by 40 cycles of 94℃ for 1 min, annealing at suitable temperature for different primers for 1 min, 72℃ for 1 min, extension at 72℃ for 10 min, and preservation at 12℃. This optimized ISSR-PCR system would play an important role in further research of molecular systematics and genetic diversity in C.tsoongiana.

Key words: Calanthe tsoongiana, ISSR, orthogonal design