关键词: 叉叶苏铁, ISSR-PCR, 正交设计, 单因子试验

Abstract: CTAB method and SDS method were used to extract genomic DNA from Cycas micholitzii and CTAB method was testified to be more effective to C.micholitzii.The Orthogonal design was used to optimize ISSR-PCR amplification system in three levels of four factors dNTP, Taq DNA plymerase,Primer and Mg²⁺. Base on the pre-liminary experiment,other three factors（amount of DNA，anneal-ling temperature，circular times）were further optimized. A sutable ISSR-PCR reaction system was established, namely 20 μL reaction system contaning:1.0 U Taq DNA polymerase, 2.5 mmol·L^-1 Mg²⁺, 250 μmol·L^-1 dNTPs, 0.2 μmol·L^-1 Primer, 1×Buffer, 75 ng DNA. Programmed for an initial step of 5 min at 94℃, followed by 40 cycles of 1 min at 94℃, 1 min at 52℃ and 2 min at 72℃,and a final 10 min extension at 72℃. The result afford a reference to the research on molecular biology and molecular systematics of Cycadopcida.

Key words: Cycas micholitzii, ISSR-PCR, orthogonal design, single factor test