摘要
对影响PCR反应的主要因子——模板DNA、引物、dNTPs和Mg^2＋浓度进行优化，建立起适合于萝卜基因组DNA的RAID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，Mg^2＋ 2．0mmol／L，模板DNA10～40ng，TaqDNA聚合酶0．8U；ISSR反应体系（16-）为引物0．5μmol／L，dNTPs 0．16mmol／L，Mg^2＋ 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RAID．PCR和ISSR-PCR反应：应用优化的RAPD和ISSR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。
The effect of different concentrations of template DNA, primer, dNTPs and Mg^2＋ 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 umol/L of RAPD primer,0. 15 - 0.2 mmol/L of dNTPs,2.0 mmol/L of Mg^2＋ ,genomic DNA 10 -40 ng and 0.8 U TaqDNA polymerase. The ISSR-PCR reaction was perfolmed in a 16 ul solution containing 0.5 umol/L of primer,0.16 mmol/L of dNTPs,2.5 mmol/L of Mg^2＋ ,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 BAPD and ISSRPCR in radish,respectively 17 radish caltivars were fingerprinted with the optimized systems.The systems would play an important role in molecular breeding,cultivar fingerprinting and seed purity testing in radish.
出处
《种子》
CSCD
北大核心
2007年第2期1-5,共5页
Seed
基金
国家自然科学基金(30300238
30571193)
江苏省自然科学基金(创新人才BK2004418)
江苏省高技术研究计划(BG2005314)项目资助部分内容
上海市科技兴农重点攻关(沪农科攻字2004第9-1号)项目。
