华山松SSR-PCR反应体系优化  

Optimization of the SSR-PCR reaction system for Pinus armandii Franch.

作　　者：高丽云 徐剑 李伟 陈乔稳 王凡 董章宏 瞿绍宏 常晓勇 辛培尧[1] GAO Li-yun;XU Jian;LIWei;CHEN Qiao-wen;WANG Fan;DONG Zhang-hong;QU Shao-hong;CHANG Xiao-yong;XIN Pei-yao(Southwest Forestry University Key Laboratory of Education Ministry for Forest Resources Conservation and Use in the Southwest Mountains of China,Kunming 650224,China;Yunnan Jicheng Landscape Technology Co.,Ltd.,Mile,Yunnan 652399,China;Haikou Forest Farm,Kunming 650114,China)

机构地区：[1]西南林业大学/西南山地森林资源保育与利用教育部重点实验室,昆明650224 [2]云南吉成园林科技股份有限公司,云南弥勒652399 [3]昆明市海口林场,昆明650114

出　　处：《南方农业学报》2020年第4期879-886,共8页Journal of Southern Agriculture

基　　金：云南省科技计划重点研发项目(2018BB005);云南省省级林业科技推广项目(〔2018〕ts04)。

摘　　要：【目的】建立华山松SSR-PCR最佳反应体系,为华山松种质资源的分子标记辅助育种、遗传多样性分析及遗传图谱构建研究提供理论参考。【方法】以华山松幼嫩针叶为材料,分别采用试剂盒法、SDS法和改良CTAB法提取华山松基因组DNA,确定华山松DNA最佳的提取方法。通过L16(44)正交试验设计对华山松SSR-PCR反应体系中引物用量(A)、dNTP用量(B)、Taq DNA聚合酶用量(C)和DNA模板用量(D)进行优化,获得华山松SSR-PCR最佳反应体系。【结果】改良CTAB法提取的基因组DNA浓度为92.1~1786.3 ng/μL,OD260/OD280为1.80~2.07,OD260/OD230比值约2.0,条带明亮且清晰,无弥散现象,表明该方法提取效果最佳。正交试验极差分析结果显示,4个因素影响华山松SSR-PCR反应体系扩增效果的主次顺序为A>D>B>C,最优水平组合为A4B2C2D2。正交试验方差分析结果显示,4个因素影响华山松SSR-PCR反应体系扩增效果的主次顺序为A>D>C>B,最优水平组合为A4D2C2B2。结合正交试验16个组合的评分结果,最终确定华山松SSR-PCR最佳反应体系(20.00μL):10μmol/L引物0.35μL,50 ng/μL DNA模板1.00μL,10 mmol/L dNTP 2.00μL,5 U/μL Taq DNA聚合酶1.20μL,10×PCR Buffer(含Mg^2+15 mmol/L)2.00μL,用ddH2O补足至20.00μL。【结论】优化获得的华山松SSR-PCR最佳反应体系可应用于华山松种质资源评价及分子标记辅助育种等研究。【Objective】To establish the optimal SSR-PCR reaction system for Pinus armandii Franch.,and provide the theoretical and practical guidance for the germplasm resources evaluation and molecular marker-assisted breeding of P.armandii.【Method】The young planting needles of P.armandii were as materials.P.armandii genome DNA were extracted by DNA extraction kit,SDS method and modified CTAB method to find the best DNA extraction method of P.armandii by comparing the three results.Through the L16(44)orthogonal design,four factors of Taq DNA polymerase amount,dNTPs amount,primer amount and template DNA amount in the SSR-PCR reaction system of P.armandii were optimized to obtain the best P.armandii SSR-PCR reaction system.【Result】The concentration of genomic DNA,which extracted by the improved CTAB method,was 92.1-1786.3 ng/μL,and OD260/OD230 was 1.80-2.07,the ratio of OD260/OD230 was about 2.0,and the bands were bright and clear,without dispersion phenomenon,indicating that this method had the best extraction effect.The range analysis results of orthogonal test showed that the four factors which affected the amplification effect of P.armandii SSR-PCR reaction system were:A>D>B>C,and the optimal level combination was:A4B2C2D2.Orthogonal test analysis of variance showed that the four factors affecting the amplification effect of P.armandii SSR-PCR reaction system were:A>D>C>B,and the optimal level combination was:A4D2C2B2.Combining the scoring results of 16 combinations of orthogonal experiments,the best reaction system for SSR-PCR of P.armandii(20.00μL)was as follows:0.35μL primer(10μmol/L);1.00μL template DNA(50 ng/μL);2.00μL dNTP(10 mmol/L),1.20μL(5 U/μL)Taq DNA polymerase and 2.00μL of 10×PCR Buffer(containing Mg^2+15 mmol/L),then added to a volume of 20.00μL with ddH2O.【Conclusion】The optimal reaction system of P.armandii SSR-PCR can be applied to the evaluation of germplasm resources and molecular marker-assisted breeding of P.armandii.

关 键 词：华山松 SSR-PCR反应体系 优化 DNA提取 

分 类 号：S791.241[农业科学—林木遗传育种]