大白菜软腐病抗性相关基因分子标记的开发  

Molecular Marker Development of Soft Rot Disease Defense-related Genes in Chinese Cabbage (Brassica rapa ssp. pekinensis)

作　　者：陈昌龙 赵雪[1] 孙旺旺 李晓颖 田宇 李未然 谢华[1] CHEN Chang-Long;ZHAO Xue SUN;Wang-Wang;LI Xiao-Ying;TIAN Yu;LI Wei-Ran;XIE Hua(Beijing Agro-Biotechnology Research Center/Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing 100097, China)

机构地区：[1]北京农业生物技术研究中心/北京市农林科学院/农业基因资源与生物技术北京市重点实验室

出　　处：《农业生物技术学报》2019年第6期982-992,共11页Journal of Agricultural Biotechnology

基　　金：现代农业产业技术体系北京市叶类蔬菜创新团队(No.BAIC07-2019)

摘　　要：细菌性软腐病对大白菜(Brassica rapa ssp. pekinensis)的危害严重,胡萝卜果胶杆菌(Pectobacterium carotovorum, Pc)是主要致病菌。本研究基于大白菜软腐病防御反应相关基因,拟开发软腐病抗性基因的分子标记。首先利用生物信息学方法对具有自主产权的大白菜抗软腐病相关基因的表达序列标签(expressed sequence tags, ESTs)数据进行分析,设计合成56对特异性引物,在大白菜软腐病抗病材料(A19-2)和感病材料(A32-2)亲本间筛选有多态性的EST-PCR和EST-CAPS (cleaved amplified polymorphic sequence)标记。在两亲本F1代自交产生的F2代群体中(142个单株),统计标记位点的基因型,进行标记的分离分析。56对设计合成的特异性引物均能在亲本中扩增出清晰条带,其中具有多态性的标记共鉴定出30个:11个EST-PCR标记,19个EST-CAPS标记(其中有9对引物和限制性内切酶的组合不唯一);14个显性标记,16个共显性标记。将上述30个标记位点在F2代群体142个单株中的基因型进行统计,结果显示符合3∶1或1∶2∶1分离比例的位点共有24个,频率为80%;表现偏分离(不符合孟德尔分离比, P<0.05)的位点有6个,频率为20%。本研究利用大白菜抗软腐病相关的ESTs资源,开发了EST-PCR和EST-CAPS分子标记,对于加速大白菜ESTs资源的开发利用、大白菜抗性基因定位和分子标记辅助育种技术等研究具有一定的意义。Bacterial soft rot disease causes severe damage to Chinese cabbage (Brassica rapa ssp. pekinensis), and Pectobacterium carotovorum (Pc) is a major pathogen. Based on defense-related genes in Chinese cabbage to soft rot disease, molecular markers were developed in this study, which would be very important for molecular marker-assisted breeding and studying defense mechanism of Chinese cabbage to soft rot disease. Firstly, the ESTs (expressed sequence tags) database related to soft rot resistance of Chinese cabbage with independent property rights were analyzed by bioinformatics method, and 56 pairs of specific PCR primers were designed and used to test the polymorphism between resistant line (A19-2) and sensitive line (A32-2) of Chinese cabbage to soft rot disease. The genotypes of the F2 population (containing 142 Chinese cabbage individuals) generated by F1 self-breeding were recorded using the markers and the segregation analysis was conducted. All the 56 pairs of specific PCR primers obtained successful PCR-amplification. There were 30 markers which had polymorphism in the resistant and sensitive parents, including 11 EST-PCR markers and 19 EST-CAPS (cleaved amplified polymorphic sequence) markers, and 9 pairs could match with more than one restriction enzymes. There were 14 dominant markers and 16 co-dominant markers. In addition, the genotypes of 142 individuals of the F2 population were statistically analyzed using the above 30 marker loci. The result showed that 24 loci (80%) had the segregation ratio of 3∶1 or 1∶2∶1, and 6 loci (20%) had the genetic distortion (P<0.05) which was inconsistent with Mendelian ratio. This research developed defense-related EST-PCR and EST-CAPS molecular markers of Chinese cabbage to soft rot disease based on the ESTs resources of Chinese cabbage, which have certain significance in the study on utilization of ESTs resources, localization of defense-related genes and molecular marker-assisted breeding.

关 键 词：大白菜 细菌性软腐病 EST EST-PCR EST-CAPS 

分 类 号：S432.21[农业科学—植物病理学] S431.192[农业科学—农业昆虫与害虫防治]