皂荚ISSR-PCR反应体系的建立与优化  

Establishment and optimization of ISSR-PCR reaction system of Gleditsia sinensis

作　　者：张蕊 齐钦辉 朱文瑛 李保会[2] 张芹[1] ZHANG Rui;QI Qinhui;ZHU Wenying;LI Baohui;ZHANG Qin(College of Landscape Architecture and Tourism,Hebei Agricultural University,Baoding 071000,China;College of Forestry,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学园林与旅游学院,河北保定071000 [2]河北农业大学林学院,河北保定071000

出　　处：《林业与生态科学》2024年第3期264-272,共9页Forestry and Ecological Sciences

基　　金：河北省重点研发计划项目(21326312D-9);河北省林业和草原科学技术研究项目(2112054)。

摘　　要：建立皂荚ISSR-PCR反应体系,为皂荚遗传多样性分析、种质资源鉴定提供技术支持,以皂荚DNA为模板,对影响PCR扩增反应的4个因素(模板DNA浓度、引物浓度、Master Mix用量、PCR反应循环数)设置6个梯度并从3个水平上进行单因素优化,后采用L 9(34)正交设计,对各处理组合进行电泳检测后进行评分,对评分结果进行极差分析和方差分析,从而筛选出皂荚最佳的ISSR-PCR反应体系和扩增程序以及得出各因素对反应体系的影响程度。通过设置不同的退火温度,利用优化后的皂荚ISSR-PCR反应体系及扩增程序对已公布的100条ISSR通用引物进行筛选。研究结果表明,皂荚ISSR-PCR最佳反应体系为:在25μL的反应体系中,DNA模板浓度35 ng,引物浓度5μmol/L,Master Mix用量12μL,30个循环;各因素影响大小依次是:DNA模板浓度>Master Mix用量>引物浓度>PCR反应循环数。最后在该体系下共筛选出了22条条带清晰、多态性好的引物。To establish an ISSR-PCR reaction system of Gleditsia sinensis and provide technical support for genetic diversity analysis and germplasm resource identification,DNA of G.sinensis was used as template in this study.Six gradients were set for the four factors affecting PCR amplification reaction(template DNA concentration,primer concentration,Master Mix dosage,and number of PCR reaction cycles),and single factor optimization was carried out at three levels.Then L 9(34)orthogonal design was used to score each treatment combination after electrophoresis detection.Range analysis and variance analysis were carried out on the score results,to screen out the best ISSR-PCR reaction system and amplification program of G.sinensis and get the influence degree of each factor on the reaction system,then by setting different annealing temperatures,100 published universal primers of G.sinensis were screened using the optimized ISSR-PCR system and amplification program.The results showed that optimal reaction system was as follows:in 25μL reaction system,DNA template concentration was 35 ng,primer concentration was 5μmol/L,Master Mix dosage was 12μL,30 cycles were performed.The influence of each factor was successively:DNA template concentration>Master Mix dosage>primer concentration>number of PCR reaction cycles.Finally,22 primers with clear bands and good polymorphism were selected under this system.

关 键 词：皂荚 ISSR-PCR 单因素优化 正交优化 引物筛选 

分 类 号：S722.8[农业科学—林木遗传育种]