利用正交试验设计的方法,从Mg2+浓度、dNTPs浓度、引物浓度和Taq酶浓度4因素对菊芋ISSR-PCR反应体系进行优化分析,并在此基础上对模板DNA浓度及退火温度进行梯度检测.结果表明:菊芋20μl最佳反应体系包括10×PCR buffer,200μmol/L dNTP,0.5 μmol/L引物,1.5 mmol/L Mg2+,1.0 U Taq DNA聚合酶和50 ng模板DNA.这一优化系统的建立,将为菊芋种质资源鉴定及遗传多样性的研究奠定基础.
Establishment and Optimization of ISSR-PCR Reaction System for Helianthus tuberosus L.
In order to optimizing ISSR-PCR reaction system in Heliaruhus tuberosus L. , the concentrations of Mg2+ , dNTPs, primer DNA, and Taq DNA polymerase were optimized at three levels by orthogonal design. Then, based on the optimal ISSR-PCR amplification the concentration of DNA template and annealing temperature were selected. As a result, the optimal PCR(20 pi) mixture contained 10 x PCR Buffer, 200 |xmol/L dNTP.0.5 jimol/L primer, 1.5 mmol/L MgCl2 , 1.0 U Taq polymerase and 50 ng DNA template. This optimized system would play an important role in further research on germplasm identification and the genetic diversity of H. Tuberosus by using ISSR molecular marker techniques.
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