| 木薯SCoT-PCR反应体系优化及引物筛选 |
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| 引用本文： | 严华兵,周慧文,单建伟,谢向誉. 木薯SCoT-PCR反应体系优化及引物筛选[J]. 南方农业学报, 2016, 47(10): 1648-1652. DOI: 10.3969/jissn.2095-1191.2016.10.1648 |
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| 作者姓名： | 严华兵 周慧文 单建伟 谢向誉 |
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| 作者单位： | 广西作物遗传改良生物技术重点开放实验室,南宁,530007 |
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| 基金项目： | 国家自然科学基金项目(31301378)；广西八桂学者专项项目(桂人才通字[2011]4号) |
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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。
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| 关 键 词： | 木薯 SCoT-PCR反应体系 正交试验设计 引物筛选 |
Optimization of SCoT-PCR reaction system and screening primers for cassava |
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| YAN Hua-bing,ZHOU Hui-wen,SHAN Jian-wei,XIE Xiang-yu. Optimization of SCoT-PCR reaction system and screening primers for cassava[J]. Journal of Southern Agriculture, 2016, 47(10): 1648-1652. DOI: 10.3969/jissn.2095-1191.2016.10.1648 |
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| Authors: | YAN Hua-bing ZHOU Hui-wen SHAN Jian-wei XIE Xiang-yu |
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| Abstract: | 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. |
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| Keywords: | cassava SCoT-PCR reaction system orthogonal experimental design primer screening |
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