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园艺学报 ›› 2021, Vol. 48 ›› Issue (6): 1094-1106.doi: 10.16420/j.issn.0513-353x.2020-0542

• 研究论文 • 上一篇    下一篇

大白菜抗根肿病基因位点CRs的KASP标记开发及应用

杨双娟, 原玉香, 赵艳艳, 魏小春, 王志勇, 赵肖斌, 张晓伟()   

  1. 河南省农业科学院园艺研究所,郑州 450002
  • 收稿日期:2020-12-18 修回日期:2021-02-17 出版日期:2021-06-25 发布日期:2021-07-07
  • 通讯作者: 张晓伟 E-mail:xiaowei5737@163.com
  • 基金资助:
    国家自然科学(31872945);国家自然科学(31801874);河南省中原千人计划中原学者项目(202101510003);国家现代农业产业技术体系建设专项资金项目(CARS-23-G-16);河南省农业科学院自主创新项目(2020ZC23)

Development and Application of KASP Markers for Clubroot Resistance Gene CRs in Chinese Cabbage

YANG Shuangjuan, YUAN Yuxiang, ZHAO Yanyan, WEI Xiaochun, WANG Zhiyong, ZHAO Xiaobin, ZHANG Xiaowei()   

  1. Institute of Horticulture,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China
  • Received:2020-12-18 Revised:2021-02-17 Online:2021-06-25 Published:2021-07-07
  • Contact: ZHANG Xiaowei E-mail:xiaowei5737@163.com

摘要:

CRs为最近发现的1个抗根肿病基因。以连锁的4个标记作为引物,以8个抗病和8个感病大白菜材料为模板,进行PCR扩增和测序。结果发现,抗病和感病材料中存在6个序列变异位点,其中5个(A08染色体9 956 808、9 956 888、9 956 904、10 707 299和10 707 385 bp)为新的变异位点,1个(10 539 495 bp处的G/A SNP变异)与前人研究结果一致。针对4个新的变异位点(因9 956 888 bp与9 956 904 bp处变异距离很近,故后者未分析)和10 539 495 bp的变异位点设计开发出5个竞争性等位基因特异性PCR(Kompetitive allele specific PCR,KASP)标记Probe55K1、Probe55K2、Probe64K1、Probe64K2和Probe60K1。结果表明,设计的5个KASP标记均可有效将抗病材料和感病材料分为2组。利用标记Probe55K1和Probe64K1在包含45个抗病单株和48个感病单株的BC1P1群体中进行验证,结果发现,这2个标记在各个单株的基因型和抗病性表型一致率高达94.62%。因此,本研究中开发的KASP标记可以高效用于根肿病的分子标记辅助育种。

关键词: 大白菜, 根肿病, CRs基因, KASP标记

Abstract:

CRs is an recently discovered clubroot resistance gene in Chinese cabbage. In this study,through PCR amplication and sequencing of four CRs linked markers in eight resistant materials and eight susceptible materials,we found six sequence variations between resistant and susceptible materials. Among which,five variations(9 956 808,9 956 888,9 956 904,10 707 299 and 10 707 385 bp in chromosome A08)were novel,G/A SNP(Single Nucleotide Polymorphisms)variation in 10 539 495 bp of A08 is consistent with previous study. Five KASP(Kompetitive allele specific PCR)markers, Probe55K1,Probe55K2,Probe64K1,Probe64K2 and Probe60K1,were developed in terms of variations in 9 956 808,9 956 888,10 707 299,10 707 385 and 10 539 495 bp. It turned out that all the five KASP markers can successfully differentiate the resistance from the susceptible materials. Probe55K1 and Probe64K1 were further genotyped in 45 resistant and 48 susceptible individuals, which were part of a BC1P1 population. The results showed that the consistency rate between genotypes and phenotypes of the above 93 individuals was high to 94.62%. Thus,the KASP markers developed in this study can be efficently used for marker-assisted selection(MAS)of clubroot resistant gene in breeding.

Key words: Chinese cabbage, clubroot, CRs gene, KASP marker

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