半枫荷ISSR-PCR体系优化及引物筛选

Optimization and Primers Screening of ISSR-PCR Reaction System for Semiliquidambar cathayensis Chang

半枫荷(Semiliquidambar cathayensis Chang)是中国特有濒危保护植物。为了利用ISSR分子标记对其开展遗传多样性研究,本研究针对Mg^(2+)浓度、引物浓度、d NTPs浓度、DNA模板量、Taq DNA聚合酶总量这5个因素,通过单因素试验结合L_(16)(4^(5))正交试验对半枫荷ISSR-PCR反应体系进行优化。得到20μL最优体系为:Mg^(2+)2.5 mmol/L、引物0.3μmol/L、dNTPs 0.2 mmol/L、模板DNA 40 ng、Taq DNA聚合酶2 U、2μL 10×PCR Buffer。利用该体系筛选出7条扩增产物稳定、多态性好的引物(UBC811,UBC815,UBC817,UBC826,UBC827,UBC848,UBC849)作为ISSR-PCR扩增引物。在此基础上,对4份半枫荷材料进行扩增验证,共扩增出63个条带,其中多态性条带58个,多态性百分率为92.06%,平均Nei’s基因多样性指数为0.384 9,Shannon’s信息多样性指数为0.559 2,结果表明优化体系稳定性强,扩增效果较好,可用于半枫荷遗传多样性研究。

Semiliquidambar cathayensis Chang is an endangered and endemic species of China.In order to explore genetic diversity of S.cathayensis by ISSR marker,the single factor test and L_(16)(4^(5)) orthogonal design experiment were used to optimize the amount of Mg^(2+),primers,d NTPs,DNA template,Taq DNA polymerase in its ISSR-PCR reaction system.The optimal ISSR-PCR reaction system was established as the following:20 μL PCR reaction system containing 2.5 mmol/L Mg^(2+),0.3 μmol/L primers,0.2 mmol/L dNTPs,40 ng DNA template,2 U Taq DNA polymerase and 2 μL 10×PCR Buffer.Using this optimal system,7 primers(UBC811,UBC815,UBC817,UBC826,UBC827,UBC848 and UBC849) with stable and abundant polymorphism were selected for ISSR-PCR amplification.On this basis,four S.cathayensis samples were selected to verify the reliability of this system.63 bands were amplified,of which 58 were polymorphic bands.The percentage of polymorphic bands was 92.06%.The mean Nei’s gene diversity and Shannon’s information index were 0.384 9 and 0.559 2 respectively.The results indicated that the optimum reaction system was effective and stable,suggesting it could be applied to further study on genetic diversity of S.cathayensis.