红椿SRAP反应体系优化及引物筛选

Optimization and Primers Selection of SRAP-PCR System in Toona ciliata Roem

[目的]优化相关序列扩增多态性(SRAP)体系内的不同组分,建立适用于红椿SRAP分子标记的反应体系,并进一步从SRAP引物组合中筛选出稳定、多态性好的引物组合,为红椿遗传多样性研究奠定试验基础。[方法]针对SRAP-PCR反应体系中5个因素各设置8个水平,先利用单因素试验确定浓度梯度,后在确定的梯度范围内选定4个水平,按照正交试验L16(45)进行优化,结合正交直观分析法和新复极差法对各因素进行优化筛选。[结果]确定最优体系为总体系25μL,模板DNA 25 ng,上下游引物各0.3μmol·L^(-1),Taq DNA聚合酶1U,Mg2+2.5 mmol·L^(-1),d NTP 0.3 mmol·L^(-1)。利用稳定的SRAP-PCR体系,从1 505对SRAP引物组合中筛选出30对优质引物组合。[结论]通过不同种源红椿基因组DNA的重复验证,获得了稳定清晰、多态性较强的扩增条带,表明所确定的最优体系稳定可靠,适用性较强,可用于不同种源红椿遗传多样性研究的后续实验。

[ Objective] To optimize the different components of sequence-related amplified polymorphism （SRAP） and to establish suitable SRAP-PCR system for Toona ciliata Roem. , and to select high-stability polymorphic band SRAP primer combinations. [ Method ] The experiment basis for genetic diversity of T. ciliate was established. Five factors each with eight concentration levels were screened to the suitable concentration range in the PCR reaction system using single factor experiment. After that, four levels were selected in each range of the five factors. The or- thogonal experiment of L^16 （4^5 ） was carried out for optimization. Combining with orthogonal design-direct analysis and SSR to optimize these factors and the optimized system was determined. [ Result I The optimal SRAP-PCR system was established, containing 25 ng template DNA, 0.3 mmol ~ L- 1 each dNTP, 0.3 p^mol ~ L- 1 each prim- er, 2.5 mmol ~ L-1 each Mg2~ and 1 U Taq DNA polymerase in a total volume of 25 p^L. 30 primer combinations were selected from 1 505 s using the stability of the PCR system. [ Conclusion] Through repeated validations using DNA of different T. ciliata provenances, the clear polymorphic bands were obtained. The result showed that the optimal SRAP-PCR system can be used for the follow-up experiments on T. ciliata genetic diversity research in virtue of its stable, reliable, and good applicability.