木薯SCoT-PCR反应体系优化及引物筛选
Optimization of SCoT-PCR reaction system and screening primers for cassava
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摘要: 【目的】优化木薯SCoT-PCR反应体系并进行SCoT引物筛选,为木薯辅助育种提供技术支持。【方法】以木薯品种新选048为材料,利用正交试验设计对DNA模板量、Mg2+浓度、dNTPs浓度、引物浓度、Taq DNA聚合酶量等因素进行优化,确定木薯最佳SCoT-PCR反应体系,并用其进行SCoT引物筛选。【结果】木薯最佳SCoT-PCR反应体系(20.0μL):模板DNA 60 ng,引物浓度0.30μmol/L,TaqDNA聚合酶1.5 U,Mg2+1.50 mmol/L,dNTPs 0.25 mmol/L。利用该体系筛选出19条SCoT引物,能稳定扩增出数量多、清晰可辨且多态性丰富的条带。【结论】优化的木薯SCoT-PCR反应体系检测结果稳定,重复性好,且筛选出的19条引物均适用于木薯遗传多样性分析。Abstract: ObjectiveSCoT-PCR reaction system of cassava was optimized and SCoT primers were screened in order to provide technical support for applying SCoT molecular marker technique in cassava molecule breeding. MethodTaking cassava variety(Xinxuan 048) as materials,the orthogonal design method was used to optimize five factors including DNA template amount,Mg2+concentrations,dNTPs concentrations,primer concentration and Taq DNA polymerase amount. Then the optimized SCoT-PCR reaction system was used to screen SCoT primers for cassava. ResultResults showed that,the optimal SCoT-PCR reaction system(20 μL) was composed of 60 ng DNA template,0.30 μmol/L primer,1.5 U Taq DNA polymerase,1.50 mmol/L Mg2+ and 0.25 mmol/L dNTPs. Nineteen primers were selected from 36 SCoT primers based on optimal SCoT-PCR reaction system,which could amplify steadily a large number of clear,distinguishable and polymorphic bands. ConclusionThe optimal SCoT-PCR reaction system shows stable detection result and good reproducibility ,and 19 selected primers can be used for genetic diversity analysis of cassava.
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