怀地黄SRAP扩增体系的建立与引物的筛选

Establishment of SRAP amplification system for Rehmannia glutinosa and primer screening

为建立适合怀地黄SRAP-PCR分子标记技术体系,通过单因子实验分别研究了DNA模板浓度、TaqDNA聚合酶浓度、Mg2+浓度、引物浓度以及dNTP浓度对怀地黄SRAP扩增反应的影响,确立了适合怀地黄SRAP最佳反应体系为:在25μL的反应体系中,模板DNA量20ng/25μL、2.5mmol/LMg2+、0.32μmol/L的上下游引物、0.30μmol/L的dNTP以及2.5UTaq酶,并利用确定的体系从88个引物组合中筛选出12对适合怀地黄SRAP-PCR反应的引物。

The single factor design was used to optimize SRAP amplification system of 22 Rehmannia glutinosa lines in five factors（the concentration of template DNA,Taq DNA polymerase,primer,Mg^2＋and dNTP）.SRAP amplification system was established as follows：2.5 mmol/L Mg^2＋,0.30 mmol/L dNTP mixture,2.5 U Taq DNA polymerase,0.32 μmol/L each primer,20 ng template DNA and 2.5 μL 10 X PCR buffer in 25 μL SRAP reaction system were the best suitable PCR system.12 primers were collected from 88 primers using the optimized amplification system above.