怀地黄SRAP扩增体系的建立与引物的筛选

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。

Establishment of SRAP amplification system for Rehmannia glutinosa and primer screening

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,Mg2+and dNTP).SRAP amplification system was established as follows:2.5 mmol/L Mg2+,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.