正交设计优化槟榔SSR-PCR反应体系及引物筛选

Optimization of SSR-PCR System by Orthongonal Design and Primers Screening for Areca catechu L.

优化槟榔SSR-PCR反应体系,筛选适用于槟榔的SSR引物。以槟榔(Areca catechu L.)叶片DNA为模板,利用正交设计方法对槟榔SSR反应体系中的Mg^(2+)、TaqDNA聚合酶、dNTPs和引物等4种因子三个水平进行了优化,并通过比较模板DNA浓度对PCR扩增结果进行研究,确定了最佳反应体系。利用该体系筛选出适用于槟榔的SSR引物,对该体系稳定性进行验证。槟榔L9(34)正交试验设计的SSR反应体系的最优条件为,即20μL反应体系中含Mg^(2+)Buffer 2.0 mmol/L、dNTPs 0.15 mmol/L、Taq DNA聚合酶1.0 U、引物0.4μmol/L、模板DNA量为60 ng为最优的反应体系。利用6份不同地理来源的槟榔资源DNA样品对50对SSR引物进行筛选,得到条带清晰、多态性好的引物6对。优化后的槟榔SSR-PCR反应体系和筛选的多态性引物可应用于槟榔种质资源遗传多样性分析、分子标记辅助育种等研究。

SSR-PCR reaction system of areca-nut was optimized,and SSR primers that suitable for areca-nut were screened.Using the DNA extract from leaf of Areca catechu L.as a template,an orthogonal design was used to optimize SSR amplification reaction,which employed three levels and four factors content(Mg2+,dNTPs,Taq DNA polymerase and primer,respectively)on the result of SSR,and the SSR reaction also optimized by comparing with concentrations of template DNA on application,to detect the best SSR-PCR system,SSR primers that suitable for areca-nut were screened by the optimal reaction system to verify the stability of system.The result were shown as follows:A suitable 20μL SSR reaction system contained 2.0 mmol/L Mg2+,0.15 mmol/L dNTPs,1.0 U Taq DNA polymerase,0.4μmol/L primer,DNA template 60 ng.Six polymorphism primers were screened from 50 primers existing in areca-nut by using six different geographically resource DNA samples,which band clearly and stability.The optimal SSR-PCR reaction system for areca-nut obtained and polymorphism SSR primers can be used in research for genetic diversity analysis and molecular marker assisted breeding.