SRAP标记体系优化及在茶树种质资源研究中的应用

Optimization of SRAP Analysis and Its Application in Germplasm Research of Camellia sinensis(L.) O. Kuntze

以Me3和Em5正反向引物组合对凤凰水仙茶树品种进行了SRAP分析体系的优化。结果表明:茶树SRAP-PCR最佳反应体系为:Mg2+浓度3.0 mmol.L-1;Taq酶2.0 U;dNTPs浓度0.2 mmol.L-1;引物浓度2.0μmol.L-1;10×Buffer 2.0μL;模板DNA 20 ng;反应总体积为25μL。经过重复试验,确定茶树SRAP-PCR扩增程序为:94℃预变性5 min;94℃变性1 min,35℃复性1 min,72℃延伸100 s,5个循环;94℃变性1 min,50℃复性1 min,72℃延伸100 s,35个循环;最后72℃延伸5 min。采用已优化的体系对30份茶树品种进行SRAP分析,经2%的琼脂糖凝胶电泳得到了清晰且重复性好的扩增谱带,说明该反应体系稳定可靠,适用于茶树种质资源的分子标记分析。

An optimal system of SRAP analysis for Camellia sinensis（L.） O.Kuntze cultivar Fenghuang Shuixian was established with the primer combination of Me3 and Em5,the optimum reaction of which was composed of Mg2＋ 3.0 mmol·L-1,Taq 2.0 U,dNTPs 0.2 mmol·L-1,2.0 μmol·L-1 of each primer,10×Buffer 2.0 μL,20 ng DNA in a total volume of 25 μL.The optimized program was pre-denaturing at 94℃ for 5 min,then denaturing at 94℃ for 1 min,annealing at 35℃ for 1 min and extending at 72℃ for 100 s for the first five cycles; followed hy another 35 cycles with annealing temperature at 50℃, and finally terminated by post-extending at 72℃ for 5 min. Primer combinations used herein gave rise to distinct bands with abundant polymorphism, indicating that SRAP could be used for identification of genetic diversity, elucidation of origin and classification of Camellia sinensis （L.） O. Kuntze.