茶树SRAP反应体系优化及引物筛选

Optimization of SRAP System and Selection of Primers for Tea Plant [Camellia sinensis (L. ) O. Kuntz]

以茶树叶片DNA为模板,采用正交试验设计,以Mg2+、引物、dNTP、TaqDNA聚合酶、模板DNA 5种因素5个水平,对茶树SRAP反应体系进行了研究,建立了茶树SRAP最佳反应体系。结果表明,茶树SRAP最佳反应体系为:Mg+22.0mmol/L、引物各0.5μmol/L、dNTP 0.1mmol/L、TaqDNA聚合酶0.5U/μL、模板DNA 4ng/μL,总体积为10μL。运用该体系,从128个SRAP引物组合中筛选出扩增条带清晰、多态性丰富的56个引物组合。这一体系的建立及引物组合的筛选为今后利用SRAP标记技术进行茶树分子遗传学研究提供了理论依据。

The orthogonal design was used to optimize SRAP-PCR system with five factors （Mg2＋, primer, dNTP, Taq polymerase and template DNA ） at five levels respectively. The results showed the optimized SRAP-PCR system for tea plant was ： Mg＋2 2.0 mmol/L, primer 0.5 μ mol/L, dNTP 0.1 mmol/L, Taq DNA polymerase 0.SU/μ L, 4ng/μ L template DNA with total 10 μ L. Through this optimized SRAP-PCR system, fifty-six primer combinations were se- lected with abundant polymorphism from 128 primer combinations. The optimized SRAP-PCR system and polymor- phism primer combinations could be applied to molecular genetics research of tea plant.