褐飞虱SRAP-PCR体系的优化与确立

Optimization and establishment of SRAP-PCR reaction system for the brown planthopper

采用L16(45)正交试验设计,对褐飞虱SRAP-PCR反应体系中的Mg2+、dNTPs、TaqDNA聚合酶、引物浓度和DNA模板浓度5个因素进行优化,确立了褐飞虱SRAP-PCR最佳的反应体系。结果表明:褐飞虱SRAP-PCR最佳反应体系为:总体积10μL,Mg2+浓度3mmol/L、dNTPs为150μmol/L、TaqDNA聚合酶2U、正反向引物各0.3μmol/L、DNA用量25ng以及1×PCR Buffer。使用生物型1雌虫与生物型2雄虫单对杂交F2群体对优化后的褐飞虱SRAP-PCR反应体系进行验证,获得了条带清晰、多态性丰富的图谱,并发现共显性条带,表明确定的反应体系稳定可靠。

By the orthogonal experimental design L16 （45）,five factors,including Mg2＋,dNTPs,Taq DNA polymerase,primer concentration and DNA template concentrations in Nilaparvata lugens SRAP-PCR reaction system,were optimized,and the optimal reaction system of the brown planthoppcr SRAP-PCR was established.The results showed that a suitable SRAP-PCR reaction system was constructed with 10 μL volume containing 25 ng of template DNA,0.3 μmol/L of each of the two primers,150 μmol/L deoxynucleotide triphosphates （dNTPs）,3 mmol/L MgCl2,1 × PCR buffer,and 2 unit of Taq DNA polymerase.We used the females of biotypes 1 and the males of biotype 2 monomer hybridization population for the optimal reaction system.The results showed that the bands were clear,with rich polymorphism and co-dominant bands.It showed that the reaction system is stable and reliable.