湖北道地药材贝母ISSR反应体系优化及多态性引物筛选  

ISSR Reaction Optimization and Primer Selection for Species Authentication of Fritillaria hupehensis Hsiao et K.C.Hsia

作　　者：蒋小刚 王华 郭坤元 张美德 JIANG Xiaogang;WANG Hua;GUO Kunyuan;ZHANG Meide(Institute of Chinese Medicinal Materials,Hubei Academy of Agricultural Sciences/Enshi Comprehensive Test Station,National Traditional Chinese Medicine Industry Technology System/Chinese Medicinal Materials Sub-center,Hubei Agricultural Science and Technology Innovation Center,Enshi,Hubei 445000,China)

机构地区：[1]湖北省农业科学院中药材研究所/国家中药材产业技术体系恩施综合试验站/湖北省农业科技创新中心中药材分中心,湖北恩施445000

出　　处：《福建农业学报》2021年第6期642-650,共9页Fujian Journal of Agricultural Sciences

基　　金：湖北省重点研发计划项目(2020BCA059);湖北省农业科技创新中心2020年重大科技研发项目(2020-620-000-002-04);国家现代农业产业技术体系建设专项(CARS-21);第三批“湖北省青年英才开发计划”项目[鄂青联发(2020)1号];湖北省农业科学院中药材研究所技术创新重大项目(2019ZYCZD01);湖北省中央引导地方科技发展专项资金(2019ZYYD064);湖北省技术创新专项(2019AKB092);湖北省农业科学院青年科学基金(2021NKYJJ19)。

摘　　要：【目的】优化湖北贝母ISSR-PCR体系,并筛选适用于湖北贝母的多态性引物,为后续湖北贝母的分子辅助育种及遗传多样性和亲缘关系分析等提供科学依据。【方法】采用U12(43)均匀设计和单因素试验结合的方法,获得PCR体系各组分(2×Taq Master Mix,模板DNA,引物)的最佳用量,在此基础上通过梯度退火温度实验探究引物的最佳退火温度。【结果】湖北贝母ISSR-PCR最佳反应体系为:20μL反应体系中,2×Taq Master Mix 10.5μL,模板DNA 0.8μL(40.0 ng),引物2.2μL(5.5μmol),ddH2O 6.5μL,ISSR-PCR扩增程序为:94℃预变性5 min;94℃变性0.75 min,54.3℃~60.0℃退火0.75 min,72℃延伸1.5 min,40个循环;72℃延伸10 min,4℃保存。利用优化的反应体系筛选了6条多态性较好、扩增稳定的引物(UBC848、UBC850、UBC853、UBC857、UBC859、UBC866),其最佳退火温度分别为59.3、58.2、56.9、54.3、59.3和60.0℃。用优化的ISSR-PCR体系对12份不同产地的湖北贝母资源进行PCR扩增,结果表明,该反应体系稳定可靠,不同产地湖北贝母具有较丰富的遗传多样性。【结论】优化的湖北贝母ISSR-PCR反应体系可用于湖北贝母种质资源鉴定、亲缘关系和遗传多样性分析等研究。【Objective】The optimized ISSR-PCR reaction system and appropriate polymorphic primers were established to facilitate the authentication,breeding,and genetic studies on Fritillaria hupehensis Hsiao et K.C.Hsia.【Method】A U12(43)uniform design method combined with a single factor screening test was employed to optimize the dosages of 2×Taq Master Mix,template DNA,and primer applied for ISSR-PCR and followed by a gradient temperature experiment to determine the primer annealing temperature.【Result】The optimal ISSR reaction system used 10.5μL of 2×Taq Master Mix,0.8μL of template DNA(40.0 ng),2.2μL(2.5μmol·L-1)of primers,and 6.5 uL of ddH2O for a 5 min sequencing at 94℃and 40 cycles at 94℃for 0.75 min,54.3–60.0℃for 0.75 min,and 72℃for 1.5 min,followed by 10 min at 72℃prior to storage at 4℃.Six stable polymorphic primers,UBC848,UBC850,UBC853,UBC857,UBC859,and UBC866 with the optimal annealing temperatures of 59.3,58.2,56.9,54.3,59.3,and 60.0℃,respectively,were selected.On 12 F.hupehensis germplasms from various locations,the optimized ISSR-PCR yielded stable and reliable results that showed abundant genetic diversity.【Conclusion】The optimized ISSR-PCR reaction system could be satisfactorily applied for the resource authentication and genetic analysis on F.hupehensis.

关 键 词：湖北贝母 ISSR 分子标记 体系优化 

分 类 号：S682[农业科学—观赏园艺]