正交设计优化青蒿SRAP-PCR反应体系及引物筛选

Optimization of SRAP-PCR System on Artemisia annua L.Using Orthogonal Design and Selection of Primers

为了建立青蒿的SRAP最佳扩增体系,并筛选出SRAP多态性引物,本研究以青蒿叶片DNA为模板,采用正交试验设计,以Mg^2+、dNTP Mix、Taq DNA聚合酶、引物和DNA模板5种因素5个水平,对青蒿SRAP反应体系进行研究。结果表明,青蒿SRAP-PCR最佳反应体系为:引物0.6μmol/L、Mg^2+2.0 mmol/L、模板DNA 5.1 ng、Taq DNA聚合酶2.0 U、dNTPs 0.25 mmol/L,总体积为25μL。各因素对扩增反应均有不同影响,其中引物浓度的影响最大,dNTPs的影响最小。运用该体系对不同种质资源的青蒿进行验证,证明该体系稳定可靠,并在30个引物组合中筛选出了25对扩增条带清晰,多态性丰富的引物组合。这一结论为今后利用SRAP标记技术进行青蒿分子遗传学研究提供了科学依据。

In order to establish the best SRAP amplification system of Artemisia annua.and to screen the SRAP polymorphism primer.In this study,DNA of Artemisia annua leaves was used as template,and the SRAP reaction system of Artemisia annua was studied by orthogonal design with five levels of Mg^2+,dNTP Mix,Taq DNA polymerase,primers and DNA template.The results showed that the optimum reaction system of Artemisia annua SRAP-PCR was as follow:primer 0.6μmol/L,Mg^2+2.0 mmol/L,template DNA 5.1 ng,Taq DNA polymerase 2.0 U,dNTPs 0.25 mmol/L in a total of 25μL reaction solution.All the factors had different effects on the amplification reaction,among which the concentration of primers had the greatest effect and the effect of dNTPs was the least.The optimized SRAP-PCR system was used to verify the stability and reliability of Artemisia annua from different germline resources,and 25 pairs of primers with clear amplification bands and rich polymorphism were selected from 30 primer combinations.This conclusion provides a scientific basis for the study of molecular genetics of Artemisia annua.by SRAP markers in the future.