柽柳cpSSR-PCR反应体系的建立和优化

Establishment and Optimization of cpSSR-PCR Reaction System of Tamarix chinensis

[目的]建立并优化柽柳cpSSR-PCR反应体系和反应条件。[方法]对影响PCR反应的5个变量(Mg^(2+)浓度、dNTPs浓度、Taq DNA聚合酶浓度、引物浓度、模板DNA浓度)进行L_(16)(4~5)正交试验设计,并对引物退火温度进行梯度筛选。[结果]最优反应体系:Mg^(2+) 2.0 mmol/L、dNTPs 0.125 mmol/L、Taq DNA聚合酶0.25 U、引物0.25μmol/L、模板DNA 20 ng,共10μL。反应程序:94℃预变性4 min;94℃30 s,引物退火温度30 s,72℃30 s,30个循环;72℃延伸10 min。[结论]该反应体系成功扩增1个柽柳天然群体的23个个体,为柽柳群体扩散路线的确定奠定基础。

[Objective] To establish and optimize the cpSSR-PCR reaction system and amplification condition of Tamarix chinensis .[Method] L 16 (4 5) orthogonal design was used to identify the optimum Mg^ 2+ concentration, dNTPs concentration, Taq concentration, primers concentration, and DNA concentration for cpSSR-PCR amplification of T. chinensis , and gradient annealing temperature test was also conducted.[Result] The optimized system was as follows: a 10 uL reaction volume including 2.0 mmol/L Mg ^2+, 0.125 mmol/L dNTPs, 0.25 U Taq , 0.25 μmol/L primers, 20 ng DNA. The suitable procedure was 4 min denaturation at 94 ℃, followed by 30 cycles of 30 s denaturation at 94 ℃, 30 s annealing step at 56.5 ℃, 30s elongation at 72 ℃, and a final extension step at 72 ℃ for 10 min.[Conclusion] The optimized cpSSR-PCR system successfully amplified the 23 individuas from a natural T. chinensis population and could be applied to study on population dispersal of T. chinensis .