雷公笋SRAP-PCR体系建立及有效引物筛选

Establishment of SRAP-PCR System and Selection of Effective Primer for Costus speciosus

为构建雷公笋最佳SRAP-PCR反应体系以及评价并筛选雷公笋SRAP引物的有效性,本研究通过单因素试验和正交试验相结合的方法,对模板DNA、dNTPs、引物及Taq DNA聚合酶4种影响雷公笋SRAP-PCR扩增结果进行分析。单因素试验结果表明:雷公笋基因组DNA浓度越高扩增效率越好,低浓度d NTPs利于扩增产物,而Taq DNA聚合酶和引物扩增高效率偏于中高浓度。正交试验结果表明:各因素对海南雷公笋SRAP-PCR扩增影响大小先后顺序分别为:引物=dNTPs>模板DNA>TaqDNA聚合酶;最优总体系为25μL时,4种影响扩增效率的因素最佳用量各为:模板DNA 100 ng、TaqDNA聚合酶6.40 U、dNTPs浓度0.12 mmol/L、引物浓度2.0μmol/L。利用SRAP-PCR最佳反应体系,从100对SRAP引物对中筛选出30对条带清晰的多态性引物,多态性比率平均值达到48.77%。研究结果可为海南雷公笋的遗传多样性分析、种质资源鉴定等研究提供参考依据。

In order to construct the optimal SRAP-PCR reaction system and to evaluate and screen the effectiveness of SRAP primers for Costus speciosus,this study was conducted to analyze the results of the amplification of Costus speciosus SRAP-PCR by a combination of single-factor test and orthogonal test,in which the template DNA,dNTPs,primers and Taq DNA polymerase were used to influence the amplification results of Cos tus speciosus SRAP-PCRSR.The results of the single-factor test showed that the higher the concentration of Costus speciosus genomic DNA,the better the amplification efficiency,the lower the concentration of dNTPs,and the Taq DNA polymerase and primer amplification efficiency favoured the medium to high concentration.The results of the orthogonal test showed that:the order of influence of each factor on the SRAP-PCR amplification of Hainan Costus speciosus was:primers=dNTPstemplate DNA Taq DNA polymerase;the optimal total system was 25μL,and the optimal amount of each of the four factors affecting the amplification efficiency was:100 ng of template DNA,6.40 U of Taq DNA polymerase,dNTPs concentration 0.12 mmol/L,and primer concentration of 2.0μmol/L.Using the optimal SRAP-PCR reaction system,30 pairs of primers with clear bands were selected from 100 pairs of SRAP primer pairs,and the average polymorphism ratio reached 48.77%.The results of this experiment can be used as a reference for genetic diversity analysis and germplasm identification of Hainan Costus speciosus.