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[目的]优化适合柑橘的SCoT反应体系,并利用优化的SCoT体系分析无籽沙糖橘和有籽沙糖橘的遗传多样性。[方法]采用正交设计方法分别对反应体系中DNA模板、Mg~(2+)、引物、dNTPs、Taq DNA聚合酶的浓度等进行优化。利用优化的体系,选用60条SCoT引物对有籽沙糖橘和无籽沙糖橘进行扩增,扩增出有差异条带进行SCAR转化。[结果]优化的柑橘ScoT反应体系为:Mg~(2+)1.5 mmol/L,dNTPs0.35 mmol/L,引物0.25μmol/L,Taq酶0.5 U,模板DNA 30 ng,总反应体系为20μl。最适退火温度为50.6℃。利用优化的体系,选用60条SCoT引物对有籽沙糖橘和无籽沙糖橘进行分析,经过引物筛选,获得了较好扩增的SCoT引物共42条,通过42条SCoT引物对有籽沙糖橘和无籽沙糖橘的筛选,获得了1条有差异条带引物。[结论]有籽沙糖橘和无籽沙糖橘基因组水平上存在差异。
[Objective] The research aimed to optimize the SCoT reaction system suitable for citrus and to analyze the genetic diversity of seedless sugarcane and fructose-fructose orange with the optimized SCoT system. [Method] The orthogonal design was used to optimize the concentration of DNA template, Mg 2+, primers, dNTPs and Taq DNA polymerase in the reaction system. Using the optimized system, 60 SCoT primers were used to amplify the seeds of Sesame and Sesame, and different bands were amplified for SCAR transformation. [Result] Optimized citrus ScoT reaction system was 1.5 mmol / L Mg 2+, 0.35 mmol / L dNTPs, 0.25 μmol / L primer, 0.5 U Taq DNA polymerase and 30 ng template DNA. The total reaction system was 20 μl . The optimum annealing temperature is 50.6 ℃. Using the optimized system, 60 SCoT primers were used to analyze the seeds of Sesame and Sesame, and 42 primer pairs were screened by primer selection. A total of 42 SCoT primers were amplified, Shatang tangerine and seedless Shatang tangerine screening, access to a differential strip primers. [Conclusion] There are differences in the genomic level of Fructus Juniperus and Fructus.