摘要
为了建立适宜甜菜的SRAP反应体系,以40个不同类型的甜菜品系为试验材料,研究了PCR反应体系的主要成分对SRAP扩增结果的影响。对SRAP反应体系中的DNA模板浓度、Taq DNA聚合酶浓度、引物浓度以及dNTP浓度进行了探索,确立了适合甜菜的SRAP反应体系为:在20μL反应体系中,模板DNA为40ng,Taq DNA聚合酶1.0U,引物60ng,dNTP(2.5mmol/L)为1.6μL,扩增程序为94℃预变性3min,反应前5个循环在94℃1min,35℃1min,72℃1min的条件下运行;随后的35个循环复性温度提高到50℃,最后72℃延伸10min。并利用该反应体系对甜菜40个不同品系进行SRAP反应,用6%的变性聚丙烯酰胺凝胶电泳检测,不同品系间DNA谱带多态性丰富,证实该体系稳定可靠,可以用于甜菜的分子标记研究。
In this study, SRAP-PCR amplified condition was optimized on 40 different sugarbeet lines and analyzed effects of the concentration of DNA template, Taq DNA polymerase, primer, dNTP on PCR results. The results showed that 40 ng template DNA, 1.0 U Taq polymerase, 60 ng primers and 1.6 μL dNTP （2.5 mmol/L） in 20 μL SRAP reaction system were the best suitable PCR system, 3 min of denaturing at 94℃, five cycles of three steps： 1 min of denaturing at 94℃, 1 min of annealing at 35℃ and 1 min of elongation at 72%. In the following 35 cycles the annealing temperature was increased up to 50℃, with a final elongation step of 10 min at 72℃. SRAP of 40 different sugarbeet lines were obtained and detected. Polymorphism between different lines abundantly detected by 6% denaturing polyacrylamide gel which showed this system was suitable and stable.
出处
《中国糖料》
2007年第2期1-4,共4页
Sugar Crops of China
基金
国家高技术研究发展计划(863计划)项目(2001AA241192)资助
