辣椒SRAP分子标记的优化及在航椒4号种子纯度检测中的应用

为了对SRAP分子标记在辣椒中的应用进行优化,试验采用正交设计L16（45）,在4个水平上对影响辣椒SRAP反应体系的Taq酶浓度、Mg2＋含量、模板DNA用量、dNTP浓度及引物用量等5个因素进行了优化,并用单因素完全随机试验筛选各反应因素的最佳水平,建立了辣椒SRAP分子标记的最佳体系。结果表明,在10μL反应体系中,Taq酶最适浓度为1.5U、Mg2＋最适浓度为1.5 mmol/L、模板DNA最适用量为100ng、dNTP最适用量为0.3 mmol/L、引物最适浓度为0.1μmol/L。利用20个辣椒材料来验证此反应体系,6%变性聚丙烯酰胺凝胶电泳检测结果显示,扩增产物在350～750 bp之间多态性高,且反应体系的稳定性和重复性好。通过优化的SRAP分子标记对F1代辣椒种子航椒4号进行纯度检测,检测结果为98%,与其田间检测结果100%十分接近。表明了SRAP分子标记技术是鉴定辣椒一代杂种纯度的有效方法,具有准确、可靠、快速的特点,在辣椒杂交种子纯度室内快速检测中有很大的应用前景。

Optimization of SRAP Marker in Pepper and Its Application in Purity Testing of Hangjiao No.4 Seeds

The experiment was conducted to investigate the impact of Taq enzyme concentration,Mg2＋ content,amount of template DNA,concentration of dNTP and amount of primer on SRAP reaction system for pepper by using of L16（45） orthogonal design.The fully random single factor experiment design was used to select the optimum level for each factor to optimize the SRAP amplification system.The results showed that in the 10 μL reaction system,the optimum concentration of Taq enzyme,Mg2＋,template DNA,dNTP and primers are 1.5 U,1.5 mmol/L,100 ng,0.3 mmol/L and 0.1 μmol/L,respectively.The established amplification system was tested on 20 pepper strains and 6% denaturing polyacrylamide gel electrophoresis results showed that the amplified products were in the range of 350-750 bp polymorphism,and the reaction system was proved in good stability and reproducibility.Using the optimized SRAP markers to test seeds of Hangjiao No.4,the results was 98%,and the purities were very close to the purities field tested 100%,which indicated that SRAP markers would have a wide range of application in rapid lab test of seed purities of water melon hybrid cultivars.