大白菜抗TuMV显性位点F_2的QTL-seq分析
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摘要
为了挖掘大白菜抗TuMV基因的多样性,制定抗TuMV的遗传改良策略和综合防治措施,对抗TuMV的显性位点进行QTL分析。以大白菜对TuMV高抗的‘89B’和高感的‘强势’为亲本,构建F_2群体。采用人工磨擦接种TuMV法对F_2群体进行单株接种鉴定,经卡平方测验F_2群体抗、感植株分离比例不符合3︰1(χ~2=4.8>χ_(0.05)~2=3.84),非单基因遗传,为数量位点控制。选取表型高抗和高感的F_2单株各40株进行混池,对2个极端池以及2个亲本进行重测序分析。通过计算抗、感池与亲本间的△(SNP-index),获得两个抗TuMV位点区域,物理位置为A07染色体的13.9～14.4 Mb和A08染色体的16.4～17.4 Mb。上述两个区域共筛选出具有多态性位点的基因68个,其中6个基因与植物抗性有关,其编号分别为Bra028499、Bra028500、Bra016311、Bra016312、Bra016313和Bra016314,其中Bra028499和Bra028500编码抗病蛋白,Bra016311、Bra016312、Bra016313和Bra016314编码抗TMV蛋白。Bra028499、Bra028500、Bra016311和Bra016314含有TIR-NBS-LRR特殊结构域。在已定位克隆的植物抗病基因中,大约80%属于NBS基因家族,因此推测这些基因可能与大白菜抗TuMV有关。
In order to excavate the diversity of TuMV resistant genes and make the genetic improvement strategy and measures for the prevention and control in Chinese cabbage,QTL analysis of the dominant locus was conducted in the study,which would be helpful to cultivate the broad-spectrum resistance of Chinese cabbage varieties. The F_2 population was constructed from the parents ‘89 B'(resistant)and‘Qiangshi'(susceptible),which were the highly inbred Chinese cabbage lines. The F_2 population individuals were inoculated by TuMV C4 isolate,and the segregation from F_2 did not fit the expected segregation for a Mendelian model 3︰1(χ~2 = 4.8 > χ_(0.05)~2 = 3.84),so quantitative trait locus controlled the character,not a single gene. Choosing 40 highly resistant/susceptible individuals from F_2 population,respectively,to mix the pools,and the two parents and two pools were re-sequenced. By calculation analysis of △(SNP-index) between parent and pools,two main regions were obtained(A07 chromosome:13.9–14.4 Mb and A08 chromosome:16.4–17.4 Mb). There were 68 genes in the above two regions,and 6 out of 68 were associated with resistance in plants(Bra028499,Bra028500,Bra016311,Bra016312,Bra016313 and Bra016314). In addition,Bra028499 and Bra028500 coded the disease-resistant proteins,and Bra016311,Bra016312,Bra016313 and Bra016314 coded TMV-resistant proteins. Bra028499,Bra028500,Bra016311 and Bra016314 contained the TIR-NBS-LRR domains.About 80% of the resistance genes cloned in plants belonged to the NBS gene families,indicating that these genes may be associated with Chinese cabbage resistance to TuMV.
In order to excavate the diversity of TuMV resistant genes and make the genetic improvement strategy and measures for the prevention and control in Chinese cabbage,QTL analysis of the dominant locus was conducted in the study,which would be helpful to cultivate the broad-spectrum resistance of Chinese cabbage varieties. The F_2 population was constructed from the parents ‘89 B'(resistant)and‘Qiangshi'(susceptible),which were the highly inbred Chinese cabbage lines. The F_2 population individuals were inoculated by TuMV C4 isolate,and the segregation from F_2 did not fit the expected segregation for a Mendelian model 3︰1(χ~2 = 4.8 > χ_(0.05)~2 = 3.84),so quantitative trait locus controlled the character,not a single gene. Choosing 40 highly resistant/susceptible individuals from F_2 population,respectively,to mix the pools,and the two parents and two pools were re-sequenced. By calculation analysis of △(SNP-index) between parent and pools,two main regions were obtained(A07 chromosome:13.9–14.4 Mb and A08 chromosome:16.4–17.4 Mb). There were 68 genes in the above two regions,and 6 out of 68 were associated with resistance in plants(Bra028499,Bra028500,Bra016311,Bra016312,Bra016313 and Bra016314). In addition,Bra028499 and Bra028500 coded the disease-resistant proteins,and Bra016311,Bra016312,Bra016313 and Bra016314 coded TMV-resistant proteins. Bra028499,Bra028500,Bra016311 and Bra016314 contained the TIR-NBS-LRR domains.About 80% of the resistance genes cloned in plants belonged to the NBS gene families,indicating that these genes may be associated with Chinese cabbage resistance to TuMV.
引文
Azhar M T,Amin I,Bashir A.2011.Characterization of resistance gene analogs from Gossypium arboreum and their evolutionary relationships with homologs from tetraploid cottons.Euphytica,178(3):351-362.
Chung H,Jeong Y M,Mun J H,Lee S S,Chung W H,Yu H J.2014.Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa.Molecular Genetics&Genomics,289(2):149-160.
Dangl J L,Jones J D.2001.Plant pathogens and integrated defence responses to infection.Nature,411(6839):826-833.
Ding Guohua.2004.Cucumber disease resistance gene cloning homologous sequence and the study of downy mildew resistance gene markers[Ph.D.Dissertation].Harbin:Northeast Agricultural University.(in Chinese)丁国华.2004.黄瓜抗病基因同源序列的克隆及其对霜霉病抗病基因标记的研究[博士论文].哈尔滨:东北农业大学.
Guo G,Wang S,Liu J,Pan B,Diao W,Ge W,Snyder J C.2017.Rapid identification of QTLs underlying resistance to Cucumber mosaic virus in pepper(Capsicum frutescens).Theoretical and Applied Genetics,130(1):41-52.
Harris C J,Slootweg E J,Goverse A.2013.Stepwise artificial evolution of a plant disease resistance gene.Proc Natl Acad Sci,110(52):21189-21194.
Huang S W,Li R Q,Zhang Z H.2009.The genome of the cucumber,Cucumis sativus L.Nature Genetics,41(12):1275-2752.
Hughes S L,Hunter P J,Sharpe A G,Kearsey M J,Lydiate D J,Walsh J A.2003.Genetic mapping of the novel Turnip mosaic virus resistance gene Turb03 in Brassica napus.Theoretical and Applied Genetics,107(7):1169-1173.
Jenner C E,Sb S F N,Foster G D,Ponz F,Walsh J A.2000.The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene Turb01.Molecular Plant-Microbe Interactions,13(10):1102-1108.
Jenner C E,Wang X,Ponz F,Walsh J A.2002.A fitness cost for Turnip mosaic virus to overcome host resistance.Virus Research,86(1):1-6.
Jin M,Lee S S,Ke L,Kim J S,Seo M S,Sohn S H.2014.Identification and mapping of a novel dominant resistance gene,Turb07,to Turnip mosaic virus in Brassica rapa.Theoretical and Applied Genetics,127(2):509-519.
Li G L,Qian W,Zhang S,Li F,Zhang S,Zhang H,Wu J,Wang X,Sun R F.2016.Development of gene‐based markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa.Plant Breeding,135(4):466-470.
Li Guoliang.2016.Investigation of interaction mechanism of the eIF(iso)4E gene to TuMV in Brassica rapa L.[M.D.Dissertation].Beijing:Chinese Academy of Agricultural Sciences.(in Chinese)李国亮.2016.白菜e IF(iso)4E基因对TuMV的抗性机制研究[硕士论文].北京:中国农业科学院.
Li Guoliang,Qian Wei,Zhang Shujiang,Li Fei,Zhang Shifan,Zhang Hui,Xie Lulu,Wu Jian,Wang Xiaowu,Sun Rifei.2017.Analysis of the protein interaction of e IF(iso)4E.a/c with TuMV-C4/UK1 in Brassica rapa ssp.chinensis.Acta Horticulturae Sinica,44(7):1299-1308.(in Chinese)李国亮,钱伟,张淑江,李菲,章时蕃,张慧,谢露露,武剑,王晓武,孙日飞.2017.白菜翻译起始因子e IF(iso)4E.a/c与芜菁花叶病毒TuMV-C4/UK1蛋白互作分析.园艺学报,44(7):1299-1308.
Li Q,Zhang X,Zeng Q,Zhang Z,Liu S,Pei Y.2014.Identification and mapping of a novel Turnip mosaic virus resistance gene TURBCS01 in Chinese cabbage(Brassica rapa).Plant Breeding,134(2):221-225.
Li Qiaoyun,Zhang Zhigang,Liu Shuantao.2012.Chinese cabbage Turnip mosaic virus disease resistance genetic analysis.North China Agriculture Report,27(4):135-139.(in Chinese)李巧云,张志刚,刘栓桃.2012.大白菜芜菁花叶病毒病抗性遗传分析.华北农学报,27(4):135-139.
Liu Dengquan,Wang Yuanxiu,Cui Chaoyu,Qin Shuanglin,Ouyang Hui,Jiang Junxi.2016.Variety of Wenzhou mandarin NBS LRR-class disease-resistant gene cloning and analysis of homologous sequences.Journal of Jiangxi Agricultural University,38(1):83-89.(in Chinese)刘登全,王园秀,崔朝宇,秦双林,欧阳慧,蒋军喜.2016.晚熟温州蜜柑NBS-LRR类抗病基因同源序列的克隆及分析.江西农业大学学报,38(1):83-89.
Liu Shuantao,Zhang Zhigang,Li Qiaoyun,Wang Shufen,Lu Jindong,Zhang Xiaoyan.2014.Chinese cabbage e IF(iso)4E.a pseudogene mutant identification and development of related inspection tag.China Agriculture Journal,30(4):101-107.(in Chinese)刘栓桃,张志刚,李巧云,王淑芬,卢金东,张晓燕.2014.大白菜e IF(iso)4E.a假基因突变体的鉴别及相关检测标记的开发.中国农学通报,30(4):101-107.
Lu H,Lin T,Klein J,Wang S,Qi J,Zhou Q.2014.QTL-seq identifies an early flowering QTL located near flowering locus T in cucumber.Theoretical&Applied Genetics,127(7):1491-1499.
Meyers B C,Kozik A,Griego A.2003.Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis.Plant Cell,15(4):809-834.
Nellist C F,Wei Q,Jenner C E,Moore J D,Zhang S,Wang X,Briggs W H,Barker G C,Sun R,Walsh J A.2014.Multiple copies of eukaryotic translation initiation factors in Brassica rapa,facilitate redundancy,enabling diversification through variation in splicing and broad-spectrum virus resistance.The Plant Journal,77:261-268.
Parida S K,Srivastava R,Bajaj D.2017.A genome-wide mQTL-seq scan identifies potential molecular signatures regulating plant height in chickpea.Plant Molecular Biology Reporter,35(2):273-286.
Qian W,Zhang S J,Zhang S F,Li F,Zhang H,Wu J,Wang X W,Walsh J A,Sun R F.2013.Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage(Brassica rapa L.).Theoretical and Applied Genetics,126:179-188.
Qian Wei.2013.TuMV retr02 resistance gene cloning and resistance mechanism research in Brassica rapa[Ph.D.Dissertation].Beijing:Chinese Academy of Agricultural Sciences.(in Chinese)钱伟.2013.大白菜TuMV抗性基因retr02的克隆与抗性机制研究[博士论文].北京:中国农业科学院.
Rusholme R L,Higgins E E,Walsh J A,Lydiate D J.2007.Genetic control of broad-spectrum resistance to Turnip mosaic virus in Brassica rapa(Chinese cabbage).Journal of General Virology,88(11):3177-3186.
Song Hongyuan,Cao Bihao.2002.Head cabbage resistance to TuMV related gene cloning.Journal of Genetics,29(7):646-652.(in Chinese)宋洪元,曹必好.2002.结球甘蓝抗TuMV相关基因的克隆.遗传学报,29(7):646-652.
Takagi H,Abe A,Yoshida K,Kosugi S,Natsume S,Mitsuoka C,Innan H.2013.QTL-seq:rapid mapping of quantitative trait loci in rice by whole genome re-sequencing of DNA from two bulked populations.The Plant Journal,74(1):174-183.
Walsh J A,Jenner C E.2002.Turnip mosaic virus and the quest for durable resistance.Molecular Plant Pathology,3(5):289-300.
Walsh J A,Sharpe A G,Jenner C E,Lydiate D J.1999.Characterization of resistance to Turnip mosaic virus in oilseed rape(Brassica napus)and genetic mapping of TuRB01.Theoretical and Applied Genetics,99(7):1149-1154.
Wang X,Wang H,Wang J,Sun R,Wu J,Liu S,Huang S.2011.The genome of the mesopolyploid crop species Brassica rapa.Nature Genetics,43:1035-1039.
Wang Xue,Liu Yumei,Li Hanxia,Zhang Yangyong,Fang Zhiyuan.2005.Advancein research on TuMV-resistance breeding of Brassica crops.Acta Horticulturae Sinica,32(5):939-946.(in Chinese)王雪,刘玉梅,李汉霞,张扬勇,方智远.2005.芸薹属作物抗芜菁花叶病毒育种研究进展.园艺学报,32(5):939-946.
Zhang Xu,Xu Jian,Li Ji,Lou Qunfeng,Chen Jinfeng.2018.QTL mapping and identification of candidate gene for resistance to gummy stem blight in Cucumis sativus/hystrix introgression line‘IL77’.Acta Horticulturae Sinica,45(11):2141-2152.(in Chinese)张旭,徐建,李季,娄群峰,陈劲枫.2018.黄瓜/酸黄瓜渐渗系‘IL77’抗蔓枯病主效QTL定位及候选基因鉴定.园艺学报,45(11):2141-2152.
Chung H,Jeong Y M,Mun J H,Lee S S,Chung W H,Yu H J.2014.Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa.Molecular Genetics&Genomics,289(2):149-160.
Dangl J L,Jones J D.2001.Plant pathogens and integrated defence responses to infection.Nature,411(6839):826-833.
Ding Guohua.2004.Cucumber disease resistance gene cloning homologous sequence and the study of downy mildew resistance gene markers[Ph.D.Dissertation].Harbin:Northeast Agricultural University.(in Chinese)丁国华.2004.黄瓜抗病基因同源序列的克隆及其对霜霉病抗病基因标记的研究[博士论文].哈尔滨:东北农业大学.
Guo G,Wang S,Liu J,Pan B,Diao W,Ge W,Snyder J C.2017.Rapid identification of QTLs underlying resistance to Cucumber mosaic virus in pepper(Capsicum frutescens).Theoretical and Applied Genetics,130(1):41-52.
Harris C J,Slootweg E J,Goverse A.2013.Stepwise artificial evolution of a plant disease resistance gene.Proc Natl Acad Sci,110(52):21189-21194.
Huang S W,Li R Q,Zhang Z H.2009.The genome of the cucumber,Cucumis sativus L.Nature Genetics,41(12):1275-2752.
Hughes S L,Hunter P J,Sharpe A G,Kearsey M J,Lydiate D J,Walsh J A.2003.Genetic mapping of the novel Turnip mosaic virus resistance gene Turb03 in Brassica napus.Theoretical and Applied Genetics,107(7):1169-1173.
Jenner C E,Sb S F N,Foster G D,Ponz F,Walsh J A.2000.The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene Turb01.Molecular Plant-Microbe Interactions,13(10):1102-1108.
Jenner C E,Wang X,Ponz F,Walsh J A.2002.A fitness cost for Turnip mosaic virus to overcome host resistance.Virus Research,86(1):1-6.
Jin M,Lee S S,Ke L,Kim J S,Seo M S,Sohn S H.2014.Identification and mapping of a novel dominant resistance gene,Turb07,to Turnip mosaic virus in Brassica rapa.Theoretical and Applied Genetics,127(2):509-519.
Li G L,Qian W,Zhang S,Li F,Zhang S,Zhang H,Wu J,Wang X,Sun R F.2016.Development of gene‐based markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa.Plant Breeding,135(4):466-470.
Li Guoliang.2016.Investigation of interaction mechanism of the eIF(iso)4E gene to TuMV in Brassica rapa L.[M.D.Dissertation].Beijing:Chinese Academy of Agricultural Sciences.(in Chinese)李国亮.2016.白菜e IF(iso)4E基因对TuMV的抗性机制研究[硕士论文].北京:中国农业科学院.
Li Guoliang,Qian Wei,Zhang Shujiang,Li Fei,Zhang Shifan,Zhang Hui,Xie Lulu,Wu Jian,Wang Xiaowu,Sun Rifei.2017.Analysis of the protein interaction of e IF(iso)4E.a/c with TuMV-C4/UK1 in Brassica rapa ssp.chinensis.Acta Horticulturae Sinica,44(7):1299-1308.(in Chinese)李国亮,钱伟,张淑江,李菲,章时蕃,张慧,谢露露,武剑,王晓武,孙日飞.2017.白菜翻译起始因子e IF(iso)4E.a/c与芜菁花叶病毒TuMV-C4/UK1蛋白互作分析.园艺学报,44(7):1299-1308.
Li Q,Zhang X,Zeng Q,Zhang Z,Liu S,Pei Y.2014.Identification and mapping of a novel Turnip mosaic virus resistance gene TURBCS01 in Chinese cabbage(Brassica rapa).Plant Breeding,134(2):221-225.
Li Qiaoyun,Zhang Zhigang,Liu Shuantao.2012.Chinese cabbage Turnip mosaic virus disease resistance genetic analysis.North China Agriculture Report,27(4):135-139.(in Chinese)李巧云,张志刚,刘栓桃.2012.大白菜芜菁花叶病毒病抗性遗传分析.华北农学报,27(4):135-139.
Liu Dengquan,Wang Yuanxiu,Cui Chaoyu,Qin Shuanglin,Ouyang Hui,Jiang Junxi.2016.Variety of Wenzhou mandarin NBS LRR-class disease-resistant gene cloning and analysis of homologous sequences.Journal of Jiangxi Agricultural University,38(1):83-89.(in Chinese)刘登全,王园秀,崔朝宇,秦双林,欧阳慧,蒋军喜.2016.晚熟温州蜜柑NBS-LRR类抗病基因同源序列的克隆及分析.江西农业大学学报,38(1):83-89.
Liu Shuantao,Zhang Zhigang,Li Qiaoyun,Wang Shufen,Lu Jindong,Zhang Xiaoyan.2014.Chinese cabbage e IF(iso)4E.a pseudogene mutant identification and development of related inspection tag.China Agriculture Journal,30(4):101-107.(in Chinese)刘栓桃,张志刚,李巧云,王淑芬,卢金东,张晓燕.2014.大白菜e IF(iso)4E.a假基因突变体的鉴别及相关检测标记的开发.中国农学通报,30(4):101-107.
Lu H,Lin T,Klein J,Wang S,Qi J,Zhou Q.2014.QTL-seq identifies an early flowering QTL located near flowering locus T in cucumber.Theoretical&Applied Genetics,127(7):1491-1499.
Meyers B C,Kozik A,Griego A.2003.Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis.Plant Cell,15(4):809-834.
Nellist C F,Wei Q,Jenner C E,Moore J D,Zhang S,Wang X,Briggs W H,Barker G C,Sun R,Walsh J A.2014.Multiple copies of eukaryotic translation initiation factors in Brassica rapa,facilitate redundancy,enabling diversification through variation in splicing and broad-spectrum virus resistance.The Plant Journal,77:261-268.
Parida S K,Srivastava R,Bajaj D.2017.A genome-wide mQTL-seq scan identifies potential molecular signatures regulating plant height in chickpea.Plant Molecular Biology Reporter,35(2):273-286.
Qian W,Zhang S J,Zhang S F,Li F,Zhang H,Wu J,Wang X W,Walsh J A,Sun R F.2013.Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage(Brassica rapa L.).Theoretical and Applied Genetics,126:179-188.
Qian Wei.2013.TuMV retr02 resistance gene cloning and resistance mechanism research in Brassica rapa[Ph.D.Dissertation].Beijing:Chinese Academy of Agricultural Sciences.(in Chinese)钱伟.2013.大白菜TuMV抗性基因retr02的克隆与抗性机制研究[博士论文].北京:中国农业科学院.
Rusholme R L,Higgins E E,Walsh J A,Lydiate D J.2007.Genetic control of broad-spectrum resistance to Turnip mosaic virus in Brassica rapa(Chinese cabbage).Journal of General Virology,88(11):3177-3186.
Song Hongyuan,Cao Bihao.2002.Head cabbage resistance to TuMV related gene cloning.Journal of Genetics,29(7):646-652.(in Chinese)宋洪元,曹必好.2002.结球甘蓝抗TuMV相关基因的克隆.遗传学报,29(7):646-652.
Takagi H,Abe A,Yoshida K,Kosugi S,Natsume S,Mitsuoka C,Innan H.2013.QTL-seq:rapid mapping of quantitative trait loci in rice by whole genome re-sequencing of DNA from two bulked populations.The Plant Journal,74(1):174-183.
Walsh J A,Jenner C E.2002.Turnip mosaic virus and the quest for durable resistance.Molecular Plant Pathology,3(5):289-300.
Walsh J A,Sharpe A G,Jenner C E,Lydiate D J.1999.Characterization of resistance to Turnip mosaic virus in oilseed rape(Brassica napus)and genetic mapping of TuRB01.Theoretical and Applied Genetics,99(7):1149-1154.
Wang X,Wang H,Wang J,Sun R,Wu J,Liu S,Huang S.2011.The genome of the mesopolyploid crop species Brassica rapa.Nature Genetics,43:1035-1039.
Wang Xue,Liu Yumei,Li Hanxia,Zhang Yangyong,Fang Zhiyuan.2005.Advancein research on TuMV-resistance breeding of Brassica crops.Acta Horticulturae Sinica,32(5):939-946.(in Chinese)王雪,刘玉梅,李汉霞,张扬勇,方智远.2005.芸薹属作物抗芜菁花叶病毒育种研究进展.园艺学报,32(5):939-946.
Zhang Xu,Xu Jian,Li Ji,Lou Qunfeng,Chen Jinfeng.2018.QTL mapping and identification of candidate gene for resistance to gummy stem blight in Cucumis sativus/hystrix introgression line‘IL77’.Acta Horticulturae Sinica,45(11):2141-2152.(in Chinese)张旭,徐建,李季,娄群峰,陈劲枫.2018.黄瓜/酸黄瓜渐渗系‘IL77’抗蔓枯病主效QTL定位及候选基因鉴定.园艺学报,45(11):2141-2152.
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