响应面法优化余甘子种质资源ISSR反应体系  

Response Surface Optimization of ISSR-PCR Reaction for Genetic Study on Phyllanthus Emblica

作　　者：王建超[1,2] 何银莺 黄旭萍 沈朝贵 陈发兴[2] 郭林榕[1] WANG Jianchao;HE Yinying;HUANG Xuping;SHEN Chaogui;CHEN Faxing;Guo Linrong(Fruit Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350013,China;College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)

机构地区：[1]福建省农业科学院果树研究所,福建福州350013 [2]福建农林大学园艺学院,福建福州350002

出　　处：《福建农业学报》2021年第7期759-765,共7页Fujian Journal of Agricultural Sciences

基　　金：福建省科技计划公益类专项(2019R1028-11);国家热带植物种质资源库(余甘子种质资源分库)(NTPGRC2021-011);农业农村部物种品种(热带作物)资源保护项目(151821301354052701)。

摘　　要：【目的】优化余甘子种质资源ISSR-PCR反应体系,为余甘子种质资源遗传多样性及亲缘关系研究提供基础。【方法】以缅甸、印度、广东、云南、福建等5份来源不同的余甘子种质资源的基因组DNA组成混合的DNA模板,综合单因素试验和响应面分析法,分析引物浓度、2×Taq Master Mix添加量、DNA模板量、退火温度等反应条件对ISSR-PCR反应体系的影响,优化建立余甘子ISSR-PCR反应体系。【结果】引物浓度和2×Taq Master Mix添加量对扩增效果有较大影响,DNA模板量影响较小;引物浓度和DNA模板量交互作用明显;余甘子ISSR反应体系为引物浓度0.4μmol·L^(-1),2×Taq Master Mix添加量13μL,DNA模板量30 ng,扩增结果与响应面分析模型理论值相对误差仅为9.39%;退火温度为50.5~52.7℃时,随着温度的升高,条带质量变好,数量变多,退火温度为52.7℃时可获得多样性好的清晰条带。【结论】获得ISSR-PCR反应体系为引物浓度0.4μmol·L^(-1),2×Taq Master Mix添加量13μL,DNA模板量30 ng,退火温度52.7℃,扩增循环数35循环,扩增获得的条带清晰、稳定,多样性好,该体系适于余甘子种质资源的遗传多样性和亲缘关系等分析研究。【Objective】ISSR-PCR reaction system for genetic study on Phyllanthus emblica germplasms was optimized.【Methods】A mixed DNA template composed of genomic DNA of P.emblica germplasms came from Myanmar,India,Guangdong,Yunnan,and Fujian was obtained.The single factor test and response surface analysis were used to optimize the ISSR-PCR reaction conditions including primer concentration,amount of 2×Taq Master Mix,DNA template amount,and annealing temperature.【Result】The primer concentration and addition amount of 2×Taq Master Mix had a greater impact on the amplification than did the DNA template amount.A significant interaction between the primer concentration and DNA template amount was found.The optimized system with a primer concentration of 0.4μmol·L^(-1),a 2×Taq Master Mix of 13μL,and a DNA template concentration of 30 ng was established that achieved a low relative error of 9.39% in predicting the theoretical response.Within the annealing temperature between 50.5℃ and 52.7℃,increasing temperature improved the number and quality of bands.When the annealing temperature is 50.5-52.7℃,the number of strips increases,and the quality of the strips becomes better with the increase of temperature.The annealing temperature is 52.7℃ to obtain good diversity and clear bands.【Conclusion】The optimized ISSR-PCR reaction system was established applying a primer concentration of 0.4μmol·L^(-1),a 2×Taq Master Mix of 13 L,a DNA template concentration of 30 ng at the annealing temperature of 52.7℃ for 35 amplification cycles.The methodology could be used to study the genetic diversity and relationship of P.emblica germplasms.

关 键 词：余甘子 ISSR-PCR 响应面 体系优化 

分 类 号：S682.310.36[农业科学—观赏园艺]