观赏海棠SRAP-PCR反应体系优化及引物筛选

Optimization of SRAP-PCR System and Selection of Primers for Ornamental Crabapple

以观赏海棠叶片DNA为模板,采用正交试验设计,以Mg2+、dNTP、引物、模板DNA和Taq DNA聚合酶5种因素4个水平,对观赏海棠的SRAP反应体系进行了研究,建立了观赏海棠的SRAP最佳反应体系。结果表明,观赏海棠SRAP-PCR最佳反应体系为:Mg2+2.50 mmol·L-1、dNTP 0.2 mmol·L-1、引物0.4μmol·L-1、Taq DNA聚合酶2U、10 ng模板DNA,总体积为25μL。各因素对扩增反应结果均有不同影响,其中以dNTP浓度的影响最大,模板浓度的影响最小。运用该体系对观赏海棠4个种进行验证,证明该体系稳定可靠,并从70个SRAP引物组合中筛选出扩增条带清晰、多态性丰富的26个引物组合。这一体系的建立及多态性引物组合的筛选为今后利用SRAP标记技术进行观赏海棠的分子遗传学研究提供了科学的依据。

The orthogonal design was used to optimize SRAP-PCR system with five factors （Mg^2＋, dNTP, primer,template DNA and Taq polymerase） at four levels respectively. The results showed the optimized SRAP-PCR system for ornamental crabapple was： Mg^2＋ 2.50mmol· L^-1,dNTP 0.2mmol·L^-1, primer 0.4 μmol·L^-1, Taq DNA polymerase 2U,10 ng template DNA with total 25 μL reaction solution. Each factor had different effect on the result of PCR. The concentration of dNTP had the greatest effect and template DNA had the least effect on the result. The optimized SRAP-PCR system was also carried out on four crabapple genus which showed that this system was stable and reliable. Twenty-six primer combinations were selected with abundant polymorphism from 70 primer combinations. The optimized SRAP-PCR system and polymorphism primer combinations could be applied to molecular genetics research of ornamental crabapple.