葛根SCoT-PCR反应体系优化及引物筛选  

Optimization of SCoT-PCR reaction system and primer selection for Pueraria DC.

作　　者：尚小红[1] 严华兵[2] 曹升 张尚文[3] 谢向誉[1] 王艳[3] 欧昆鹏[3] SHANG Xiao-hong;YAN Hua-bing;CAO Sheng;ZHANG Shang-wen;XIE Xiang-yu;WANG Yan;OU Kun-peng(Guangxi Crop Genetic Improvement and Biotechnology Key Lab,Nanning 530007,China;Cash Crops Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,China;Biotechnology Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,China)

机构地区：[1]广西作物遗传改良生物技术重点开放实验室,南宁530007 [2]广西农业科学院经济作物研究所,南宁530007 [3]广西农业科学院生物技术研究所,南宁530007

出　　处：《南方农业学报》2018年第1期1-7,共7页Journal of Southern Agriculture

基　　金：广西重点研发计划项目(桂科AB16380087);广西藤县粉葛试验站项目(桂TS201431);广西科技基地和人才专项项目(桂科AD17195072)

摘　　要：【目的】优化葛根SCoT-PCR反应体系,筛选适用于葛根的SCoT引物,为利用SCoT分子标记进行葛根种质资源鉴定、遗传多样性分析及分子标记辅助育种提供技术支持。【方法】采用正交设计对SCoT-PCR反应体系中的DNA模板量、dNTPs浓度、Mg^(2+)浓度、Taq DNA聚合酶量和引物浓度5个因素进行优化,利用最佳SCoT-PCR反应体系筛选出适用于葛根的SCoT引物,并对该体系进行验证。【结果】最佳葛根SCoT-PCR反应体系20.00μL:DNA模板50.00 ng,dNTPs 0.25 mmol/L,Mg^(2+)1.50 mmol/L,引物0.80μmol/L,Taq DNA聚合酶0.50 U。利用该体系从36条SCoT引物中筛选出35条可从葛根中扩增出清晰条带的引物。使用3条引物对18个葛根材料进行PCR扩增,PCR产物表现出良好的重复性、稳定性和丰富的多态性。【结论】建立的最佳葛根SCoT-PCR反应体系和筛选获得的35条SCoT引物可适用于葛根种质资种鉴定、遗传多样性分析、相关分子标记开发等研究。【Objective】SCoT-PCR reaction system of Pueraria DC.was optimized and SCoT primers that suitable for Pueraria were screened to provide reference for using SCoT molecular marker in Pueraria germplasm resources identification,genetic diversity and molecular mark assisted breeding.【Method】This study carried out orthogonal design to optimize five factors including DNA template amount,dNTPs concentration,Mg2+concentration,Taq DNA polymerase amount and primer concentration.SCoT primers that suitable for Pueraria were screened out by the optimal SCoT-PCR reaction system,and this system was testified later.【Result】The results showed that,the optimized SCoT-PCR reaction system(20.00μL)for Pueraria was composed of 50.00 ng DNA template,0.25 mmol/L dNTPs,1.50 mmol/L Mg2+,0.80μmol/L primer and 0.50 U Taq DNA polymerase.Out of 36 primers,35 SCoT primers were selected which could amplified clear bands from Pueraria based on the optimized reaction system.Using three primers to amplify 18 Puerariae materials,the PCR products showed fine repeatability,stability and polymorphism.【Conclusion】The optimal SCoT-PCR reaction system for Pueraria obtained and 35 selected SCoT primers can be used for germplasm resources identification,genetic diversity analysis and molecular marker development.

关 键 词：葛根 SCoT-PCR 反应体系优化 引物筛选 

分 类 号：S567.03[农业科学—中草药栽培]