结缕草属植物ISSR-PCR反应体系研究

Optimization of ISSR-PCR System on Zoysia spp.

以SDS法提取的结缕草(Zoysia spp.)叶片DNA为模板,应用正交试验设计,从Mg2+、dNTP、引物和DNA聚合酶并通过比较不同浓度的模板DNA对PCR扩增的影响,确立结缕草属植物ISSR-PCR反应的最佳体系。结果表明:各因素不同浓度对PCR反应结果有显著影响,正交设计可以用于ISSR反应体系建立,该方法建立的结缕草属植物ISSR-PCR优化反应体系为10X Buffer7、0 ng模板DNA、Mg2+2.0 mmol.L-1、dNTP 150μmol.L-1、Primer 0.3μmol.L-1、Taq DNA聚合酶1.0 U,总体积20μL;经对11份结缕草属植物进行检验,证明该体系稳定可靠。该体系的建立可为今后利用ISSR标记技术进行结缕草属遗传多样性研究、种质资源鉴定和亲缘关系分析等提供依据。

The ISSR-PCR system was optimized and the effects of template DNA with different concentrations on the PCR amplification were studied by an orthogonal experimental design with 3 different levels of 4 factors,e.g.Mg2＋,dNTP,primers,and Taq DNA polymerase.The results show that each factor in different levels had significant effect on the amplification result of PCR and the orthogonal design was useful to optimize the ISSR-PCR system.The optimized PCR reaction system for Zoysia spp.was 10X Buffer,70 ng genomic DNA template,2.0 mmol·L-1 Mg2＋,150 μmol·L-1 dNTP,0.3 μmol·L-1 Primer,1.0 U Taq DNA polymerase with total reaction solution of 20 μL.The system was proved stable and credible according to the result of 11 Zoysia spp.and the result of this study could be useful to provide the basis in the analysis of genetic diversity,germplasm identification,and kinship of Zoysia spp.