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以10个烟草栽培品种为试验材料,研究了烟草DNA的提取方法以及对建立烟草SRAP-PCR反应体系的影响因子设置梯度实验,筛选和建立可扩增多态性高、重复性好、带型清晰的最佳SRAP-PCR反应条件:在25μL的反应体系中,MgCl22.0mmol、dNTP200μmol,上下引物各30ng、DNA模板40ng、DNA聚合酶1.5U,扩增程序为:在94℃预变性5min,反应前5个循环在94℃1min,33℃1min,72℃1min条件下运行;随后的30个循环复性温度提高到53℃,最后72℃延伸5min。本文还讨论了不同分子标记在烟草中应用的优缺点以及SRAP分子标记在烟草上的应用前景,提出了应用SRAP分子标记构建烟草遗传图谱的可行性。
Ten tobacco cultivars were used as experimental materials to study the extraction methods of tobacco DNA and to set up gradient experiments on the factors influencing the establishment of tobacco SRAP-PCR reaction system. Screening and establishment of high reproducible polymorphism, good repeatability, Clear and optimal SRAP-PCR reaction conditions: In 25μL of the reaction system, MgCl22.0mmol, dNTP200μmol, the upper and lower primers 30ng, DNA template 40ng, DNA polymerase 1.5U, amplification procedure: pre-denaturation at 94 ℃ 5min, The first five cycles of the reaction were run at 94 ° C for 1 min, 33 ° C for 1 min, and 72 ° C for 1 min. The subsequent 30 cycles renaturation temperature increased to 53 ° C and finally 72 ° C for 5 min. In this paper, the advantages and disadvantages of different molecular markers in tobacco and the application prospects of SRAP markers in tobacco are also discussed. The feasibility of using SRAP molecular markers to construct tobacco genetic map is also proposed.