以东方百合商品种‘索蚌’(‘Sorbonne’)和‘康斯坦撒’(‘Constantsa’)及其F1代杂交群体为实验材料,从100对SRAP引物中筛选出59对扩增产物丰富、具有较高多态性的引物,同时对PCR反应体系进行了优化。采用正交设计,对Mg2+、dNTPs、Taq聚合酶及引物浓度进行4因素4水平优化,并在此基础上对Mg2+、引物浓度和退火温度进行了单因素优化。结果表明,东方百合SRAP的最佳体系为:1×Buffer,Mg2+浓度2.7 mmol/L,dNTPs浓度 0.25 mmol/L,引物浓度 0.8 μmol/L,Taq聚合酶1.5 μmol/min, 模板DNA 60～100 ng。SRAP两步反应最佳退火温度为第1步35 ℃,第2步退火59.3 ℃。最后用‘索蚌’、‘康斯坦撒’和部分F1植株对优化体系进行稳定性检测,发现所筛选出的59对引物在优化的体系下均有稳定的扩增产物。
Abstract
One hundred pairs of SRAP primers were screened by using Oriental lily varieties including‘Sorbonne’,‘Constantsa’ and their hybrid offspring as materials. As results,59 pairs which had rich products and high polymorphism were selected,and the PCR reaction system were also optimized. First of all, an orthogonal design with four factors and four levels was adopted to optimize the concentration of Mg2+, dNTPs, Taq DNA polymerase and primers. Then the concentration of Mg2+, primers and annealing temperature was analyzed with a single-factor design. It was revealed that the optimal reaction system was: 1×Buffer,Mg2+ 2.7 mmol/L,dNTPs 0.25 mmol/L, primers 0.8 μmol/L, Taq DNA polymerase 1.5 μmol/min, and DNA 60-100 ng. The optimal annealing temperature of SRAP two-step reaction was 35 ℃(first step)and 59.3 ℃(second step).
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基金
收稿日期:2011-11-20 修回日期:2012-10-26
基金项目:国家自然科学基金项目(30972407); 江苏省农业科技自主创新资金项目(CX(12)2018)
第一作者:邱帅,博士生。*通信作者:席梦利,副教授。E-mail:ximemglinjfu@126.com; 施季森,教授。E-mail: jshi_1221@163.com。
引文格式:邱帅,丁信誉,席梦利,等. 东方百合SRAP体系优化及引物筛选[J]. 南京林业大学学报:自然科学版,2013,37(3):6-10.
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