木薯SCoT-PCR反应体系优化及引物筛选

【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。

Optimization of SCoT-PCR reaction system and screening primers for cassava

Objective]SCoT-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. Method]Taking 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. Result]Results 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. Conclusion]The 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.