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

摘要： 利用ISSR技术对叉孢苏铁(Cycas segmentifida)遗传多样性进行研究,对影响PCR反应的DNA模板浓度、dNTP浓度、Taq酶含量、引物浓度、Mg²⁺浓度和退火温度等进行了条件优化。叉孢苏铁的ISSR最佳反应扩增体系为20μl反应体积2μl 10×PCR buffer,模板DNA20ng,1UTaq酶,1.5~2.75mmol/L MgCl₂,0.1mmol/L 4×dNTP,0.22μmol/L引物,2%甲酰胺。适宜的退火温度为50~52℃。从103条引物中筛选出12条用于所有样品的扩增,共扩增出122条带,其中多态性条带为86条,占总条带数的70.49%。POPGENE分析结果表明,种级水平上,叉孢苏铁具有中等稍高的遗传多样性水平,且各居群间有着很高的遗传分化度。

关键词: 叉孢苏铁, ISSR, 条件优化, 初步应用