关键词: 叉孢苏铁, ISSR, 条件优化, 初步应用

Abstract: Cycas segmentifida genomic DNA, extracted from its leaves by CTAB method, was used as ISSR-PCR amplification template. By adjusting the influencing factors of ISSR, such as template DNA concentration, dNTP concentration, Taq polymerase content, primer concentration, Mg²⁺ concentration and annealing temperature, the PCR amplication conditions were optimized. The optimal experiment conditions were as follows:PCR reaction volume of 20μl contained 2μl 10×PCR buffer, 20ng template DNA, 1U Taq polymerase, 1.5~2.75mmol/L MgCl₂, 0.1mmol/L 4×dNTP, 0.22μmol/L primer and 2% formamide; proper annealing temperature was 50~52℃. 12 primers, which could produce more reproducible and polymorphic bands, were used to amplify all of the samples,and 122 DNA bands were generated, of which 86(70.49%) were polymorphic. POPGENE analysis indicated that moderate or slightly high levels of genetic diversity existed at species level, and the value of gene diversity coefficient(Gst) among populations was very high.

Key words: Cycas segmentifida, ISSR, optimization, preliminary application