西藏光核桃SRAP-PCR反应体系的优化和引物筛选

Optimization for SRAP-PCR System and Selection of Primers on Prunusmira Koehne from Tibet

以西藏12份光核桃种质为试材,采用正交设计,从dNTPs、Mg^(2+)、引物、模板DNA和Taq DNA聚合酶5种因素5个水平来优化SRAP-PCR反应体系,并对引物进行了筛选。结果表明:光核桃25μL的SRAP反应体系的最佳组分包括2.5μL 10×buffer,0.35 mmol/L dNTPs,1.5 mmol/L Mg^(2+),0.4μmol/L引物,20 ng模板DNA和2.5 U Taq DNA聚合酶。各因素对扩增反应结果均有不同影响,其中以dNTPs浓度影响最大,模板DNA的影响最小。应用该体系从40个引物组合中共筛选出扩增条带清晰、多态性丰富的SRAP引物组合23个。这一体系的建立及多态性引物组合的筛选为利用SRAP标记技术进行光核桃遗传多样性研究提供了依据。

The orthogonal design was used to optimize the sequence-related amplified polymorphism（SRAP）reaction system for Prunus mira Koehne, which involved 5 factors, i. e. d NTPs, Mg2＋ , primer, template DNA and Taq DNA polymerase,each at 5 levels, and primers were screened. The results showed that an optimal 25 μL reaction system of SRAP for Prunus mira Koehne included 2. 5μL 10 × buffer,0. 35 mmol/L dNTPs, 1.5 mmol/L Mg2＋ ,0. 4 t,mol/L primer,20 ng template DNA and 2. 5 U Taq DNA polymerase. Each factor had a different effect on the results of PCR. Concentration of dNTPs had the greatest effect and template DNA had the least effect. At the same time, 23 primer combinations were selected with the optimized system among 40 primer combinations,which had abundant polymorphism bands. The optimized SRAP-PCR system and polymorphism primer combinations could be applied, to molecular genetics research of Prunus mira Koehne.