【摘要】 对影响PCR反应的主要因子———模板DNA、引物、dNTPs和Mg2+浓度进行优化,建立起适合于萝卜基因组DNA的RAPD和ISSR反应体系。RAPD反应体系(18μl)为随机引物0.4~0.6μmol/L,dNTPs0.15~0.2mmol/L,Mg2+2.0mmol/L,模板DNA10~40ng,TaqDNA聚合酶0.8U;ISSR反应体系(16μl)为引物0.5μmol/L,dNTPs0.16mmol/L,Mg2+2.5mmol/L,模板DNA10~40ng,TaqDNA聚合酶0.64U。对扩增程序中复性温度进行了梯度PCR试验,表明35~42℃(37℃)与52.4~58.3℃(55℃)分别适于萝卜基因组DNA的RAPD-PCR和ISSR-PCR反应;应用优化的RAPD和IS-SR反应体系对17个萝卜品种进行了鉴定分析。研究结果将为萝卜分子育种、品种鉴定与种子纯度检测提供重要的技术基础。更多还原

【Abstract】 The effect of different concentrations of template DNA,primer,dNTPs and Mg2+on the PCR reaction was analyzed and the optimized RAPD and ISSR-PCR systems in radish were established.The RAPD-PCR reaction was performed in a 18 L solution containing 0.4-0.6 μmol/L of RAPD primer,0.15-0.2 mmol/L of dNTPs,2.0 mmol/L of Mg2+,genomic DNA 10-40 ng and 0.8 U TaqDNA polymerase.The ISSR-PCR reaction was performed in a 16 μl solution containing 0.5 μmol/L of primer,0.16 mmol/L of dNTPs,2.5 mmol/L of Mg2+,10-40 ng template DNA and 0.64 U TaqDNA polymerase.Gradient PCR experiment showed,The annealing temperature of 35-42 ℃ (37 ℃) and 52.4-58.3 ℃(55 ℃) are suitable for RAPD and ISSR-PCR in radish,respectively.17 radish cultivars were fingerprinted with the optimized systems.The systems would play an important role in molecular breeding,cultivar fingerprinting and seed purity testing in radish.更多还原