花生MITE反应体系优化及其遗传多样性分析  

Optimization of Peanut MITE Reaction System and Analysis of Genetic Diversity

作　　者：徐彪 王佰众[2] 李玉发[2] 杨翔宇 王伟[2] 牛海龙[2] 李伟堂[2] 刘红欣[2] 吴松权[1] 何中国[2] Xu Biao;Wang Baizhong;Li Yufa;Yang Xiangyu;Wang Wei;Niu Hailong;Li Weitang;Liu Hongxin;Wu Songquan;He Zhongguo(Agronomy College of Yanbian University,Yanji 133000,China;Peanut Institute,Jilin Academy of Agricultural Sciences,Gongzhuling 136110,China)

机构地区：[1]延边大学农学院,吉林延吉133000 [2]吉林省农业科学院花生研究所,吉林公主岭136110

出　　处：《山东农业科学》2022年第1期1-6,共6页Shandong Agricultural Sciences

基　　金：吉林省现代农业产业技术示范推广项目“油料作物新品种及配套高产高效栽培技术示范推广”(202106);吉林省农业科技创新工程人才基金“研究生基金”(C02103506)。

摘　　要：为了建立适宜不同来源花生种质遗传差异分析的MITE反应体系,本研究从反应体系总量、Taq PCRMix用量、引物浓度、模板DNA浓度及退火温度、循环次数方面对反应体系进行优化,得到的最佳反应体系为:反应总体积10μL,模板DNA20ng,引物(15μmol/L)2μL,TaqPCRMix5μL。反应程序为95℃预变性3min;95℃变性30s,58℃退火40s,72℃延伸30s,循环32次;72℃延伸10min;4℃保存。利用优化的反应体系从50对AhMITE引物中筛选出11对多态性高的引物,并用于对22份不同来源的花生种质进行遗传多样性分析。结果表明,共扩增出23条带,其中,多态性带21条,多态性比例为91.3%,每个引物平均扩增的多态性带为1.91条。种质间遗传相似系数在0.222~0.957之间,平均值为0.644;通过NTSYS-pcVersion 2.1软件基于UPGMA法进行聚类图绘制,在遗传相似系数为0.62时可将22份花生种质分为3大类。表明经过优化的AhMITE反应体系可以有效地用于不同来源花生种质的遗传多样性分析,表现出良好的多态性,可为进一步使用AhMITE标记对花生品种鉴定和遗传图谱构建奠定基础。In order to establish a MITE reaction system suitable for genetic diversity analysis of peanut germplasms from different sources,we optimized the reaction system from total volume of reaction system,Taq PCR Mix dosage,primer concentration,template DNA concentration,annealing temperature and cycle times.The best reaction system was obtained:total reaction volume 10μL,template DNA 20 ng,primers(15μmol/L)2μL and Taq PCR Mix 5μL.The reaction program was pre-denaturation at 95℃for 3 min;denaturation at 95℃for 30 s,annealing at 58℃for 40 s,extension at 72℃for 30 s,32 cycles;extension at 72℃for 10 min;storage at 4℃.Using the optimized reaction system,11 pairs of highly polymorphic primers were screened out from 50 pairs of AhMITE primers and were used to analyze the genetic diversity of 22 peanut germplasms from different sources.The results showed that a total of 23 bands were amplified,of which,21 ones were polymorphic bands with the polymorphism ratio as 91.3%,and the average number of polymorphic bands amplified by each primer was 1.91.The genetic similarity coefficient between germplasms was between 0.222 and 0.957 with an average value of 0.644.The cluster map was drawn by the NTSYS-pc Version 2.1 software based on the UPGMA method.When the genetic similarity coefficient was 0.62,the 22 peanut resources could be divided into 3 categories.It showed that the optimized AhMITE reaction system could be effectively used for the genetic diversity analysis of peanut germplasms from different sources showing good polymorphism,which could lay foundations for further use of AhMITE markers to identify peanut varieties and construct genetic maps.

关 键 词：花生 AhMITE标记 反应体系优化 遗传多样性分析 

分 类 号：S565.203[农业科学—作物学]