松江鲈鱼ISSR-PCR反应体系的建立及优化研究

目的]建立及优化松江鲈鱼ISSR-PCR的反应体系。方法]采用ISSR-63引物,通过正交试验设计,分别对TaqDNA聚合酶、Mg2+d、NTPs和引物浓度等4种PCR原料进行优化,以确立松江鲈鱼的ISSR-PCR反应最佳体系,并通过温度梯度PCR,筛选适宜的退火温度。结果]确定了松江鲈鱼的最适ISSR-PCR反应体系,20μl反应体系中含2.5 mmol/L Mg2+,250μmol/L dNTPs,0.25μmol/L引物,1.0UTaqDNA聚合酶,30 ng DNA模板及1×PCR buffer,确立退火温度为50.8℃。利用所建立的ISSR-PCR反应体系,通过对24份野生松江鲈鱼样本的检验,获得了清晰、重复性好、多态性高的DNA谱带。结论]该研究为松江鲈鱼的遗传多样性分析及种质资源研究奠定了一定的基础。

Establishment and Optimization of ISSR-PCR System in Trachidermus fasciatus Heckel

Objective] The aim of this study is to establish and optimize the ISSR-PCR system in roughskin sculpin(Trachidermus fasciatus Heckel).Method] By using the primer ISSR-63,the concentrations of Taq DNA polymerase,Mg2+,dNTPs and primer were optimized by orthogonal design for establishing the suitable ISSR-PCR system in roughskin sculpin;moreover,the suitable anneal temperature was yielded from gradient PCR on temperature.Result] The optimized ISSR-PCR system(20 μl reaction volume) in roughskin sculpin was proved to be: 2.5 mmol/L Mg2+,250 μmol/L dNTPs,0.25 μmol/L primer,1 U Taq DNA polymerase,30 ng DNA template and 1×PCR buffer;and suitable anneal temperature was determined to be 50.8℃.The established system was further confirmed in 24 wild roughskin sculpin samples.Conclusion] The results lay a foundation for the analysis of genetic diversity and germplasm resources of roughskin sculpin.