南药益智SRAP-PCR体系优化及引物筛选

Optimization of SRAP-PCR System and Primers Screening for Southern Chinese Herbs Alpinia oxyphylla Miquel

为了建立适用于南药益智的SRAP-PCR体系,并筛选特异性引物用于研究不同地理居群的南药益智的遗传多样性,采集了海南不同地理居群的益智资源,利用植物基因组DNA提取试剂盒提取益智基因组DNA,并检测其纯度和浓度。采用正交试验对SRAP-PCR体系进行优化,最终建立了最优反应体系(总体积为25μL):Taq酶0.5 U,d NTPs 4mmol/L,Mg^(2+)2 mmol/L,引物各2μmol/L,DNA模板10 ng,10×PCR buffer 2.5μL。再利用最优反应体系对144对引物进行筛选,共获得7对特异性引物,其扩增片段的大小均在100~2 000 bp以内且分布较均匀,多态性条带比率皆达85%以上。该研究结果为南药益智遗传多样性的研究奠定了基础。

In order to establish the optimal SRAP-PCR system for southern Chinese herbs Alpinia oxyphylla Miquel and screen the specific primers for studying the genetic diversity of Alpinia oxyphylla Miquel in different geographical groups,the different geographical groups of Alpinia oxyphylla resources in Hainan were collected,and the genomic DNA of Alpinia oxyphylla Miquel was extracted by plant genome DNA extraction kit and then the purity and concentration of the DNA samples were detected. The optimal reaction system（ total volume of 25 μL） was obtained by orthogonal test： Taq DNA polymerase 0. 5 U,d NTPs 4 mmol/L,Mg^（2＋）2 mmol/L,primers each 2 μmol/L,DNA templates 10 ng,10×PCR buffer 2.5 μL. 7 pairs of specific primers were screened by the optimal reaction system from 144 primers,and the sizes of their amplified fragments were within 100 ~ 2 000 bp and evenly distributed. And the ratios of polymorphic bands were above 85%. The results of this study laid a foundation for the study on genetic diversity of southern Chinese herbs Alpinia oxyphylla Miquel.