论文部分内容阅读
为获得南瓜ISSR最优扩增体系,为后续南瓜的遗传多样性分析和亲缘关系鉴定奠定基础,本研究以‘密本’南瓜为筛选体系材料,采用单因素试验,对ISSR反应体系的Mg2+、dNTP、Taq酶、引物和DNA浓度等5个因素进行优化。研究结果表明,南瓜ISSR 25μL最佳反应体系为:2.5μL 10×Buffer,2.0 mmol/L Mg2+,0.3mmol/LdNTP,1UTaq酶,0.48mmol/L引物,75ng的DNA。在此基础上,对筛选出的12条引物进行退火温度的优化,最终确定每条引物的最佳退火温度。利用该反应体系,选用引物891对85个南瓜品种进行所确立扩增体系的验证,结果显示扩增产物条带清晰明亮、多态性丰富,且特异性强、重复性好,表明本研究所确定的反应体系适用于南瓜的ISSR分子标记。
In order to obtain the optimal ISSR amplification system for pumpkin, which laid the foundation for genetic diversity analysis and genetic relationship identification of pumpkin in the follow-up study. In this study, we used ’pumpkin’ pumpkin as screening material and single factor test, dNTP, Taq enzyme, primer and DNA concentration were optimized. The results showed that the best reaction system of pumpkin ISSR 25μL was 2.5μL 10 × Buffer, 2.0mmol / L Mg2 +, 0.3mmol / LdNTP, 1UTaq enzyme, 0.48mmol / L primer and 75ng DNA. On this basis, the annealing temperature of the 12 selected primers was optimized, and the optimal annealing temperature of each primer was finally determined. Using this reaction system, 851 pumpkin cultivars were selected by primer 891 to validate the established amplification system. The results showed that the amplification product bands were clear and bright, rich in polymorphism, strong specificity and good repeatability, indicating that the Institute The identified reaction system is suitable for the ISSR molecular marker of pumpkin.