鼠曲草简单重复序列间扩增反应体系的建立及优化

Establish and optimization of inter-simple sequence repeat PCR reaction system of Gnaphalium affine

目的通过建立并优化鼠曲草简单重复序列间扩增(ISSR-PCR)反应体系,为鼠曲草后续遗传多样性研究提供实验依据。方法本研究首先采用单因素实验法和全面实验法优化鼠曲草ISSR-PCR反应体系,然后在最适体系下,分步进行引物及其退火温度的筛选,最后验证该体系的可行性。结果ISSR-PCR最适反应体系包括10μl Premix Taq DNA聚合酶、0.3μmol/L引物、10 ng DNA模板、灭菌水加至20μl;从100条通用引物中最终筛选出10条引物;验证结果表明该体系稳定性高、重复性好,且选定引物多态性好。结论本研究首次建立了鼠曲草ISSR-PCR扩增体系,并筛选出合适的引物,为鼠曲草后续遗传多样性研究夯实了基础。

Objective To provide the experimental basis for the subsequent genetic diversity research through establishing and optimizing the inter-simple sequence repeat PCR(ISSR-PCR)reaction system of Gnaphalium affine.Methods The single-factor experimental method and full experimental method were used to optimize the ISSR-PCR reaction system of Gnaphalium affine.Under the optimal system,after screening primers and corresponding annealing temperatures,the systematic feasibility was verified.Results The optimal ISSR-PCR reaction system was consisted of 10μl Premix Taq DNA polymerase,0.3μmol/L primer,10 ng DNA template,and sterilized water added to 20μl.Finally,10 primers were screened from 100 universal primers,and verification results indicated the system had high stability,good reproducibility,and the selected primers had good polymorphism.Conclusion The ISSR-PCR amplification system of Gnaphalium affine was established for the first time and the primers with appropriate annealing temperatures were filtered out,which provided a reference for the subsequent genetic diversity research of Gnaphalium affine.