正交设计优化草地早熟禾SRAP-PCR反应体系及引物筛选

采用L9(34)正交试验设计方法，对草地早熟禾(Poa pratensis)基因组DNA SRAP PCR反应体系中的Taq DNA聚合酶、Mg2+、引物及dNTP四因素的用量进行优化，并比较不同模板DNA用量对扩增的影响，建立草地早熟禾SRAP PCR最佳反应体系，同时，利用该体系对SRAP引物进行筛选。结果表明，草地早熟禾SRAP PCR最佳反应体系为Taq DNA聚合酶1.0 U、Mg2+ 1.75 mmol·L-1、引物0.25 μmol·L-1、dNTP 220 μmol·L-1、40 ng模板DNA、2 μL 10×PCR buffer，总体积20 μL。运用该体系从100对SRAP引物中筛选出43对引物能够产生清晰稳定的扩增条带且多态性丰富。优化体系的建立及引物的筛选可为今后利用SRAP标记技术对草地早熟禾进行遗传多样性分析、图谱构建、种质资源鉴定奠定技术基础。

Optimization of SRAP-PCR system on Poa pratensis using orthogonal design and selection of primers

An orthogonal design was used to optimize a SRAP-PCR system for Poa pratensis with 4 factors(Mg2+,dNTP,primer and Taq polymerase) at 3 levels plus the concentration of template DNA.The optimized SRAP-PCR system was: 2 μL 10 × PCR buffer,40 ng template DNA,Mg2+1.75 mmol·L-1,dNTP 220 μmol·L-1,primer 0.25 μmol·L-1,Taq DNA polymerase 1.0 U in a total of 20 μL reaction mixtures.With the optimized system,43 primer combinations were selected among 100 primer combinations,which produced abundant polymorphism bands.This study optimized SRAP-PCR system and selected the proper primers in P.pratensis,which would play an important role in genetic diversity analyses,map construction,germplasm identification in P.pratensis with SRAP markers.