朝鲜淫羊藿ISSR-PCR反应体系优化及引物筛选  

Optimization of ISSR-PCR Reaction System and Primer Selection of Epimedium koreanum Nakai

作　　者：吴望蕊 王英哲[1] 国坤[1] 容路生 孟芳芳 孙云龙[1] 肖井雷[1] 姜大成[1] Wu Wangrui;Wang Yingzhe;Guo Kun;Rong Lusheng;Meng Fangfang;Sun Yunlong;Xiao Jinglei;Jiang Dacheng(College of Pharmacy,Changchun University of Traditional Chinese Medicine,Changchun,130117)

机构地区：[1]长春中医药大学药学院,长春130117

出　　处：《分子植物育种》2023年第7期2318-2324,共7页Molecular Plant Breeding

基　　金：吉林省科学技术厅项目(20200404001YY);长春中医药大学2021年度研究生“橘井杯”学术科研创新项目(YK202109)共同资助。

摘　　要：本研究采用L16(43)正交试验设计,优化了朝鲜淫羊藿ISSR-PCR反应体系中的模板DNA加入量、引物加入量、2×Taq Master Mix加入量3个参数,建立了最佳反应体系。采用优化后的反应体系,从100条ISSR引物中筛选出7条多态性引物,并对每条引物的退火温度逐个优化。试验结果表明朝鲜淫羊藿ISSR-PCR最佳反应体系为:3μL模板DNA(8.7 ng/μL),3.5μL引物(2.5μmol/L),12μL 2×Taq Master Mix,双蒸水补足至体积为20μL。PCR扩增步骤:在94℃条件下先预变性4 min,后变性1 min,于57℃退火,72℃延伸均进行40 s,共循环35次,然后于72℃下补齐5 min,产物保存在4℃冰箱。经验证表明该反应体系稳定,引物条带清晰且多态性良好,该研究为朝鲜淫羊藿的群体遗传学研究奠定了前期基础。In this study,the L_(16)_(4^(3))orthogonal design was used to optimize the template DNA addition amount,pr-imer addition amount,and 2×Taq Master Mix addition amount in the ISSR-PCR reaction system of Epimedium koreanum Nakai,and the optimal reaction was established.Using the optimized reaction system,7 polymorphic pr-imers were screened from 100 ISSR primers,and the annealing temperature of each primer was optimized one by one.The test results showed that the best reaction system for ISSR-PCR of E.koreanum.was:3μL template DNA(8.7 ng/μL),3.5μL primer(2.5μmol/L),12μL 2×Taq Master Mix,double distilled water to make up to the volume 20μL.PCR amplification procedure:pre-denatured at 94℃for 4 min,then denatured for 1 minute.Annealing at 57℃and elongation at 72℃were carried out for 40 s,a total of 35 cycles,and then supplemented at 72℃for 5 min.The product was stored in a refrigerator at 4℃.The verification showed that the reaction system was stable,the primer bands were clear and the polymorphism was good.This study laid the foundation for the population genetics of E.koreanum.

关 键 词：朝鲜淫羊藿 ISSR-PCR 分子标记 体系优化 引物筛选 

分 类 号：S567.239[农业科学—中草药栽培]