72个大白菜杂交种DNA指纹构建  

DNA Fingerprint Construction for 72 Chinese Cabbage(Brassica rapa ssp.pekinensis)Hybrids

作　　者：刘栓桃[1] 张志刚[1] 李巧云[1] 王荣花 王立华 王树彬 赵智中[1] LIU Shuan-Tao;ZHANG Zhi-Gang;LI Qiao-Yun;WANG Rong-Hua;WANG Li-Hua;WANG Shu-Bin;ZHAO Zhi-Zhong(Shandong Branch of National Center for Vegetables Improvement/Vegetable and Flower Institute,Shandong Academy of Agricultural Sciences/Shandong Key Laboratory for Biology of Greenhouse Vegetables/Huang-Huai-Hai Region Scientific Observation and Experimental Station of Vegetables(Shandong),Ministry of Agriculture and Rural Affairs,Ji'nan 250100,China)

机构地区：[1]国家蔬菜改良中心山东分中心/山东省农业科学院蔬菜花卉研究所/山东省设施蔬菜生物学重点实验室/农业农村部黄淮地区蔬菜农业科学观测实验站(山东),济南250100

出　　处：《农业生物技术学报》2021年第2期240-250,共11页Journal of Agricultural Biotechnology

基　　金：十三五国家重点研发计划(2016YFD0101701);山东省农业重大应用技术创新项目(2018-2021项目);山东省农业良种工程项目(2019LZGC006,2019LZGC01703)。

摘　　要：生产实践中大白菜(Brassica rapa ssp.pekinensis)杂交种的鉴定多依赖田间种植和表型鉴定,效率低、准确率不高,在分子标记研究技术越来越成熟的条件下,有必要尽快建立以分子标记技术为基础的大白菜杂交种快速、高效、精准鉴定技术。鉴于此,本研究采用86对插入/缺失(insertion/deletion,InDel)分子标记引物对40个大白菜杂交种进行普通PCR扩增和多态性检测,用Powermarker V3.25软件对检测结果进行比较分析,用MEGA7.0软件对40个杂交种进行聚类分析,通过比较每对引物在每个类群中的杂合率百分比,筛选适合不同类型大白菜杂交种DNA指纹构建的核心引物。结果显示,共得到189个多态性位点,每对引物平均可以检测出2.2个多态性位点;引物的杂合度指数介于0~0.6842,平均为0.3042;主要等位基因频率介于0.3919~1.0000,平均为0.7011;引物所能检测到的等位基因数目介于1~4个,有74对引物都只能检测到2个等位基因。聚类分析结果表明,40个杂交种基本按照产地分为进口和国产品种群,国产品种群又进一步依据抱合方式的不同分为叠抱、直筒和合抱类群;各类群之间杂合态百分比≥50%的引物数目及其在染色体上的分布差异较大。分别从各类群中筛选5对杂合态百分比≥50%且仅能检测出2个等位基因的引物组成20对核心引物,对另外32个近年来市场销售的杂交种进行PCR扩增和电泳检测,纯合位点检测结果根据迁移率大小被赋予0或1、杂合位点则被赋予H,所有核心引物按所在染色体从A01~10排列,相同染色体上的引物按其所在物理位置由小到大排列,构建了72个大白菜杂交种的DNA指纹。本研究所筛选的20对核心引物对未知新品种的DNA指纹构建具有通用性和兼容性,研究结果可为大白菜种质筛选和杂交种鉴定提供参考依据。In production practice,the identification of Chinese cabbage(Brassica rapa ssp.pekinensis)hybrids mostly rely on field planting and phenotypic identification,which is inefficient and inaccurate.Under the condition of more and more mature molecular marker research technology,it is necessary to establish a rapid,efficient and accurate identification technology of Chinese cabbage hybrids based on molecular marker technology.Considering this,a total of 86 pairs of insertion/deletion(InDel)primers were used to identify 40 Chinese cabbage hybrids by PCR amplification and polymorphism detection.Powermarker V3.0 software were used to analyze the polymorphic results.MEGA 7.0 software were performed for cluster analysis of hybrids.In order to screen core primers suitable for DNA fingerprint construction of different kind of Chinese cabbage hybrids,the percentage of heterozygous state for each pair of primers were compared in every group.The results showed that,189 polymorphic loci were obtained,with an average of 2.2 alleles for each primer combination.The heterozygosity index ranged from 0 to 0.6482 and averaged at 0.3042.The major allele frequency ranged from 0.3919 to 1.0000 and averaged at 0.7011.The number of alleles detected by each pair of primers ranged from 1 to 4,of which 74 pairs of primers could only detect 2 alleles.Then,cluster analysis revealed that,based on producing area,the 40 hybrids were clearly divided into imported and domestic varieties,among which the domestic varieties could be further divided into folded-,cylindric-,and fluffy-type varieties.The number of loci with heterozygous state≥50%and their distribution in genome was significantly different in the 4 groups.Finally,5 pairs of primers with heterozygous state≥50%and allele number equal to 2 were selected from each group for construction of core primers.Other 32 Chinese cabbage hybrid varieties which were sold in market in recent years were detected with core primers.The detection results of pure loci were assigned a binary numeral 0 or 1 ac

关 键 词：大白菜 杂交种 DNA指纹 插入/缺失(InDel)标记 

分 类 号：S634.1[农业科学—蔬菜学]