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
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摘要
以黄瓜/酸黄瓜渐渗系‘IL77’为抗病亲本,栽培种‘8419’为感病亲本,通过亲本及F2:6群体筛选,构建了包含137对SSR标记和6对InDel标记的遗传图谱,结合2017年春、秋两季F2:6群体表型统计结果对抗蔓枯病主效QTL进行定位;同时构建极端混池并进行QTL-seq,最终将抗病基因定位到1号染色体24.6～27.1 Mb范围内;通过与参考基因组9930比对发现该区段存在13个基因在外显子区域发生非同义突变,其中Csa1G654870参与RNAi抗性反应,可能与抗蔓枯病过程相关。通过qRT-PCR验证发现,接种前后Csa1G654870在抗病亲本‘IL77’中表达量明显高于感病亲本‘8419’,由此进一步推断Csa1G654870是抗蔓枯病基因。
In this study,a linkage map containing 137 pairs of SSR markers and 6 pairs of InDel markers was constructed using recombinant inbred lines F2:6 derived from a cross between parental lines 'IL77'(resistant line from the cross of C. hystrix × C. sativus)and '8419'(susceptible line). Genetic analysis indicated that the resistance to gummy stem blight(GSB)in'IL77'was quantitative. Both mapping and BSA-seq analysis were used to detect quantitative trait loci(QTLs)conferring GSB resistance in cucumber leaves. The results showed that the GSB resistance genes were located in the 24.6–27.1 Mb region of chromosome 1. There were 13 candidate genes with exonic nonsynonymous SNP mutation predicted as GSB-resistance related genes in this region. Moreover,bioinformatics analysis and qRT-PCR results provided strong evidence that Csa1 G654870 was closely related to GSB resistance process.
In this study,a linkage map containing 137 pairs of SSR markers and 6 pairs of InDel markers was constructed using recombinant inbred lines F2:6 derived from a cross between parental lines 'IL77'(resistant line from the cross of C. hystrix × C. sativus)and '8419'(susceptible line). Genetic analysis indicated that the resistance to gummy stem blight(GSB)in'IL77'was quantitative. Both mapping and BSA-seq analysis were used to detect quantitative trait loci(QTLs)conferring GSB resistance in cucumber leaves. The results showed that the GSB resistance genes were located in the 24.6–27.1 Mb region of chromosome 1. There were 13 candidate genes with exonic nonsynonymous SNP mutation predicted as GSB-resistance related genes in this region. Moreover,bioinformatics analysis and qRT-PCR results provided strong evidence that Csa1 G654870 was closely related to GSB resistance process.
引文
Basim E,Basim H,Abdulai M,Baki D,?ztürk N.2016.Identification and characterization of Didymella bryoniae causing gummy stem blight disease of watermelon(Citrullus lanatus)in Turkey.Crop Protection,90:150-156.
Chen J F,Staub J E,Tashiro Y,Isshiki S,Miyazaki S.1997.Successful interspecific hybridization between Cucumis sativus L.and C.C-hystrix Chakr.Euphytica,96(3):413-419.
Chen L,Jin L,Li M,Tian S,Che T,Tang Q,Lan J,Jiang Z,Li R,Gu Y,Li X,Wang J.2014.Snapshot of structural variations in the tibetan wild boar genome at single-nucleotide resolution.Journal of Genetics and Genomics,41(12):653-657.
Cordero T,Cerdan L,Carbonell A,Katsarou K,Kalantidis K,Daròs J A.2017.Dicer-Like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.Molecular Plant-Microbe Interactions,30(1):63-71.
dos Santos G R,Le?o E U,Garcia M,Maluf,W R,Cardon C H,Gon?alves,C G,do Nascimento.2013.Reaction of experimental watermelon genotypes to gummy stem blight.Horticultura Brasileira,31(4):540-548.
Duan Meng-meng,Wang Ning,Mao Sheng-li,Zhang Zheng-hai,Wang Li-hao,Zhang Bao-xi.2014.Construction of the intraspecific genetic linkage map and QTL analysis for main agronomic traits of Capsicum annuum.Acta Horticulturae Sinica,41(12):2497-2506.(in Chinese)段蒙蒙,王宁,毛胜利,张正海,王立浩,张宝玺.2014.辣椒种内遗传图谱的构建及主要农艺性状的QTL分析.园艺学报,41(12):2497-2506.
Fazio G,Staub J E,Stevens M R.2003.Genetic mapping and QTL analysis of horticultural traits in cucumber(Cucumis sativus L.)using recombinant inbred lines.Theoretical and Applied Genetics,107(5):864-874.
Feng H,Xu M,Liu Y Y,Dong R,Gao X,Huang L.2017.Dicer-like genes are required for H2O2 and KCl stress responses,pathogenicity and small RNA generation in Valsa mali.Frontiers in Microbiology,8:1166.
Frantz J D,Jahn M M.2004.Five independent loci each control monogenic resistance to gummy stem blight in melon(Cucumis melo L.).Theoretical and Applied Genetics,108(6):1033-1038.
Gan D F,Zhan M D,Yang F,Zhang Q Q,Hu K L,Xu W J,Lu,Q H,Zhang L,Liang D D.2017.Expression analysis of argonaute,Dicer-like,and RNA-dependent RNA polymerase genes in cucumber(Cucumis sativus L.)in response to abiotic stress.Journal of Genetics,96(2):235-249.
Ge Hai-yan,Liu Yang,Chen Huo-ying.2015.QTL analysis of fruit-associated traits in eggplant.Acta Horticulturae Sinica,42(11):2197-2205.(in Chinese)葛海燕,刘杨,陈火英.2015.茄子果实性状相关基因的QTL定位.园艺学报,42(11):2197-2205.
Horejsi T,Staub J E.1999.Genetic variation in cucumber(Cucumis sativus L.)as assessed by random amplified polymorphic DNA.Genetic Resources and Crop Evolution,46(4):337-350.
Ito L A,Charlo H C D O,Castoldi R,Braz L T,Camargo M.2009.Rootstocks slection to gummy stem blight resistance and their effect on the yeildof melon‘Bonus no 2’.Revista Brasileira De Fruticultura,31(1):262-267.
Jagadeesan R,Fotheringham A,Ebert P R,Schlipalius D I.2013.Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle,Tribolium castaneum.BMC Genomics,14:650.
Ji Yi,Xu Qiang,Miao Minmin.2009.Construction of genetic linkage map and QTLs analysis for the related traits of plant height in cucumber.Acta Horticulturae Sinica,36(10):1450-1456.(in Chinese)嵇怡,徐强,缪旻珉.2009.黄瓜遗传图谱构建及株高相关性状的QTL定位.园艺学报,36(10):1450-1456.
Keinath A P.2011.From native plants in central Europe to cultivated crops worldwide:The emergence of Didymella bryoniae as a cucurbit pathogen.HortScience,46(4):532-535.
Keinath A P.2014.Differential susceptibility of nine cucurbit species to the foliar blight and crown canker phases of gummy stem blight.Plant Disease,98(2):247-254.
Li H,Zhang L,Wang J.2010.Analysis and answers to frequently asked questions in quantitative trait locus mapping.Acta Agronomica Sinica,36(6):918-931.
Li Wei,Tian Yi-ke,Wang Cai-hong,Bai Mu-dan,Hou Dong-liang.2015.Screening of SNP markers tightly linked to PcDw locus determining pear dwarf trait using HRM technology.Acta Horticulturae Sinica,42(2):214-220.(in Chinese)李炜,田义轲,王彩虹,白牡丹,侯董亮.2015.通过HRM技术筛查与梨矮生性状决定位点PcDw紧密连锁的SNP标记.园艺学报,42(2):214-220.
Liu S,Shi Y,Miao H,Wang M,Li B,Gu X,Zhang S.2017.Genetic analysis and QTL mapping of resistance to gummy stem blight in Cucumis sativus seedling stage.Plant Disease,101(7).
Lou L,Wang H,Qian C,Liu J,Bai Y,Chen J.2013.Genetic mapping of gummy stem blight(Didymella bryoniae)resistance genes in Cucumis sativus-hystrix introgression lines.Euphytica,192(3):359-369.
Lu H,Lin T,Klein J,Wang S,Qi J,Zhou Q,Sun J,Zhang Z,Weng Y,Huang S.2014.QTL-seq identifies an early flowering QTL located near flowering locus T in cucumber.Theoretical and Applied Genetics,127(7):1491-1499.
Murray M G,Thompson W F.1980.Rapid isolation of high molecular weight plant DNA.Nucleic acids research,8(19):4321-4325.
Ou Chenggang,Sun Tingting,Liu Xinyan,Li Chengjiang,Xu Donghui,Zhao Zhiwei,Zhuang Feiyun.2017.Fine mapping of QTLs related to main carotenoids in carrot root.Acta Horticulturae Sinica,44(2):288-296.(in Chinese)欧承刚,孙婷婷,刘新艳,李成江,徐东辉,赵志伟,庄飞云.2017.胡萝卜根中主要类胡萝卜素含量相关QTL的精细定位.园艺学报,44(2):288-296.
Poland J,Endelman J,Dawson J,Rutkoski J,Wu S,Manes Y,Dreisigacker S,Crossa J,Sánchez-Villeda H,Sorrells M,Jannink L L.2012.Genomic selection in wheat breeding using genotyping-by-sequencing.Plant Genome,5(3):103-113.
Soto J C,Ortiz J F,Perlaza-Jiménez L,Vásquez A X,Lopez-Lavalle L A B,Mathew B,Léon J,Bernal A J,Ballvora A,López C E.2015.Agenetic map of cassava(Manihot esculenta Crantz)with integrated physical mapping of immunity-related genes.Bmc Genomics,16.
SpInDel J,Wright M,Chen C,Cobb J,Gage J,Harrington S,Lorieux M,Ahmadi N,McCouch S.2013.Bridging the genotyping gap:using genotyping by sequencing(GBS)to add high-density SNP markers and new value to traditional bi-parental mapping and breeding populations.Theoretical and Applied Genetics,126(11):2699-2716.
Stamand P C,Wehner T C.1995.Greenhous,detached-leaf,and field testing methods to determine cucumber resistance to gummy stem blight.Journal of the American Society for Horticultural Science,120(4):673-680.
Takagi H,Tamiru M,Abe A,Yoshida K,Uemura A,Yaegashi H,Obara T,Oikawa K,Utsushi H,Kanzaki E,Mitsuoka C,Natsume S,Kosugi S,Kanzaki H,Matsumura H,Urasaki N,Kamoun S,Terauchi R.2015.MutMap accelerates breeding of a salt-tolerant rice cultivar.Nature Biotechnology,33(5):445-449.
Wang Q N,An B,Hou X R,Guo Y,Luo H,He C.2018.Dicer-like proteins regulate the growth,conidiation,and pathogenicity of colletotrichum gloeosporioides from hevea brasiliensis.Frontiers in Microbiology,8.
Wei Q,Wang Y,Qin X,Zhang Y,Zhang Z,Wang J,Li J,Lou Q,Chen J.2014.An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment(SLAF)sequencing.Bmc Genomics,15.
Wolukau J N,Zhou X,Chen J.2009.Identification of amplified fragment length polymorphism markers linked to gummy stem blight(Didymella bryoniae)resistance in melon(Cucumis melo L.)PI 420145.Hortscience,44(1):32-34.
Xu Yan,Wang Yuejin,Zhou Peng,Zhang Jianxia.2003.Identification of molecular genetic markers tightly linked to white rot resistant genes in hinese wild grape.Acta Horticulturae Sinica,30(1):6-10.(in Chinese)徐炎,王跃进,周鹏,张剑侠.2003.中国野生葡萄果实抗白腐病基因的分子标记.园艺学报,30(1):6-10.
Zhang S,Liu S,Miao H,Shi Y,Wang M,Wang Y,Li B,Gu X.2017.Inheritance and QTL mapping of resistance to gummy stem blight in cucumber stem.Molecular Breeding,37:49.
Zhang S P,Liu M M,Miao H,Zhang S Q,Yang Y H,Xie B Y,Gu X F.2013.Chromosomal mapping and QTL analysis of resistance to downy mildew in Cucumis sativus.Plant Disease,97(2):245-251.
Zhang W W,Pan J S,He H L,Zhang C,Li Z,Zhao J L,Yuan X J,Zhu L H,Huang S W,Cai R.2012.Construction of a high density integrated genetic map for cucumber(Cucumis sativus L.).Theoretical and Applied Genetics,124(2):249-259.
Chen J F,Staub J E,Tashiro Y,Isshiki S,Miyazaki S.1997.Successful interspecific hybridization between Cucumis sativus L.and C.C-hystrix Chakr.Euphytica,96(3):413-419.
Chen L,Jin L,Li M,Tian S,Che T,Tang Q,Lan J,Jiang Z,Li R,Gu Y,Li X,Wang J.2014.Snapshot of structural variations in the tibetan wild boar genome at single-nucleotide resolution.Journal of Genetics and Genomics,41(12):653-657.
Cordero T,Cerdan L,Carbonell A,Katsarou K,Kalantidis K,Daròs J A.2017.Dicer-Like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.Molecular Plant-Microbe Interactions,30(1):63-71.
dos Santos G R,Le?o E U,Garcia M,Maluf,W R,Cardon C H,Gon?alves,C G,do Nascimento.2013.Reaction of experimental watermelon genotypes to gummy stem blight.Horticultura Brasileira,31(4):540-548.
Duan Meng-meng,Wang Ning,Mao Sheng-li,Zhang Zheng-hai,Wang Li-hao,Zhang Bao-xi.2014.Construction of the intraspecific genetic linkage map and QTL analysis for main agronomic traits of Capsicum annuum.Acta Horticulturae Sinica,41(12):2497-2506.(in Chinese)段蒙蒙,王宁,毛胜利,张正海,王立浩,张宝玺.2014.辣椒种内遗传图谱的构建及主要农艺性状的QTL分析.园艺学报,41(12):2497-2506.
Fazio G,Staub J E,Stevens M R.2003.Genetic mapping and QTL analysis of horticultural traits in cucumber(Cucumis sativus L.)using recombinant inbred lines.Theoretical and Applied Genetics,107(5):864-874.
Feng H,Xu M,Liu Y Y,Dong R,Gao X,Huang L.2017.Dicer-like genes are required for H2O2 and KCl stress responses,pathogenicity and small RNA generation in Valsa mali.Frontiers in Microbiology,8:1166.
Frantz J D,Jahn M M.2004.Five independent loci each control monogenic resistance to gummy stem blight in melon(Cucumis melo L.).Theoretical and Applied Genetics,108(6):1033-1038.
Gan D F,Zhan M D,Yang F,Zhang Q Q,Hu K L,Xu W J,Lu,Q H,Zhang L,Liang D D.2017.Expression analysis of argonaute,Dicer-like,and RNA-dependent RNA polymerase genes in cucumber(Cucumis sativus L.)in response to abiotic stress.Journal of Genetics,96(2):235-249.
Ge Hai-yan,Liu Yang,Chen Huo-ying.2015.QTL analysis of fruit-associated traits in eggplant.Acta Horticulturae Sinica,42(11):2197-2205.(in Chinese)葛海燕,刘杨,陈火英.2015.茄子果实性状相关基因的QTL定位.园艺学报,42(11):2197-2205.
Horejsi T,Staub J E.1999.Genetic variation in cucumber(Cucumis sativus L.)as assessed by random amplified polymorphic DNA.Genetic Resources and Crop Evolution,46(4):337-350.
Ito L A,Charlo H C D O,Castoldi R,Braz L T,Camargo M.2009.Rootstocks slection to gummy stem blight resistance and their effect on the yeildof melon‘Bonus no 2’.Revista Brasileira De Fruticultura,31(1):262-267.
Jagadeesan R,Fotheringham A,Ebert P R,Schlipalius D I.2013.Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle,Tribolium castaneum.BMC Genomics,14:650.
Ji Yi,Xu Qiang,Miao Minmin.2009.Construction of genetic linkage map and QTLs analysis for the related traits of plant height in cucumber.Acta Horticulturae Sinica,36(10):1450-1456.(in Chinese)嵇怡,徐强,缪旻珉.2009.黄瓜遗传图谱构建及株高相关性状的QTL定位.园艺学报,36(10):1450-1456.
Keinath A P.2011.From native plants in central Europe to cultivated crops worldwide:The emergence of Didymella bryoniae as a cucurbit pathogen.HortScience,46(4):532-535.
Keinath A P.2014.Differential susceptibility of nine cucurbit species to the foliar blight and crown canker phases of gummy stem blight.Plant Disease,98(2):247-254.
Li H,Zhang L,Wang J.2010.Analysis and answers to frequently asked questions in quantitative trait locus mapping.Acta Agronomica Sinica,36(6):918-931.
Li Wei,Tian Yi-ke,Wang Cai-hong,Bai Mu-dan,Hou Dong-liang.2015.Screening of SNP markers tightly linked to PcDw locus determining pear dwarf trait using HRM technology.Acta Horticulturae Sinica,42(2):214-220.(in Chinese)李炜,田义轲,王彩虹,白牡丹,侯董亮.2015.通过HRM技术筛查与梨矮生性状决定位点PcDw紧密连锁的SNP标记.园艺学报,42(2):214-220.
Liu S,Shi Y,Miao H,Wang M,Li B,Gu X,Zhang S.2017.Genetic analysis and QTL mapping of resistance to gummy stem blight in Cucumis sativus seedling stage.Plant Disease,101(7).
Lou L,Wang H,Qian C,Liu J,Bai Y,Chen J.2013.Genetic mapping of gummy stem blight(Didymella bryoniae)resistance genes in Cucumis sativus-hystrix introgression lines.Euphytica,192(3):359-369.
Lu H,Lin T,Klein J,Wang S,Qi J,Zhou Q,Sun J,Zhang Z,Weng Y,Huang S.2014.QTL-seq identifies an early flowering QTL located near flowering locus T in cucumber.Theoretical and Applied Genetics,127(7):1491-1499.
Murray M G,Thompson W F.1980.Rapid isolation of high molecular weight plant DNA.Nucleic acids research,8(19):4321-4325.
Ou Chenggang,Sun Tingting,Liu Xinyan,Li Chengjiang,Xu Donghui,Zhao Zhiwei,Zhuang Feiyun.2017.Fine mapping of QTLs related to main carotenoids in carrot root.Acta Horticulturae Sinica,44(2):288-296.(in Chinese)欧承刚,孙婷婷,刘新艳,李成江,徐东辉,赵志伟,庄飞云.2017.胡萝卜根中主要类胡萝卜素含量相关QTL的精细定位.园艺学报,44(2):288-296.
Poland J,Endelman J,Dawson J,Rutkoski J,Wu S,Manes Y,Dreisigacker S,Crossa J,Sánchez-Villeda H,Sorrells M,Jannink L L.2012.Genomic selection in wheat breeding using genotyping-by-sequencing.Plant Genome,5(3):103-113.
Soto J C,Ortiz J F,Perlaza-Jiménez L,Vásquez A X,Lopez-Lavalle L A B,Mathew B,Léon J,Bernal A J,Ballvora A,López C E.2015.Agenetic map of cassava(Manihot esculenta Crantz)with integrated physical mapping of immunity-related genes.Bmc Genomics,16.
SpInDel J,Wright M,Chen C,Cobb J,Gage J,Harrington S,Lorieux M,Ahmadi N,McCouch S.2013.Bridging the genotyping gap:using genotyping by sequencing(GBS)to add high-density SNP markers and new value to traditional bi-parental mapping and breeding populations.Theoretical and Applied Genetics,126(11):2699-2716.
Stamand P C,Wehner T C.1995.Greenhous,detached-leaf,and field testing methods to determine cucumber resistance to gummy stem blight.Journal of the American Society for Horticultural Science,120(4):673-680.
Takagi H,Tamiru M,Abe A,Yoshida K,Uemura A,Yaegashi H,Obara T,Oikawa K,Utsushi H,Kanzaki E,Mitsuoka C,Natsume S,Kosugi S,Kanzaki H,Matsumura H,Urasaki N,Kamoun S,Terauchi R.2015.MutMap accelerates breeding of a salt-tolerant rice cultivar.Nature Biotechnology,33(5):445-449.
Wang Q N,An B,Hou X R,Guo Y,Luo H,He C.2018.Dicer-like proteins regulate the growth,conidiation,and pathogenicity of colletotrichum gloeosporioides from hevea brasiliensis.Frontiers in Microbiology,8.
Wei Q,Wang Y,Qin X,Zhang Y,Zhang Z,Wang J,Li J,Lou Q,Chen J.2014.An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment(SLAF)sequencing.Bmc Genomics,15.
Wolukau J N,Zhou X,Chen J.2009.Identification of amplified fragment length polymorphism markers linked to gummy stem blight(Didymella bryoniae)resistance in melon(Cucumis melo L.)PI 420145.Hortscience,44(1):32-34.
Xu Yan,Wang Yuejin,Zhou Peng,Zhang Jianxia.2003.Identification of molecular genetic markers tightly linked to white rot resistant genes in hinese wild grape.Acta Horticulturae Sinica,30(1):6-10.(in Chinese)徐炎,王跃进,周鹏,张剑侠.2003.中国野生葡萄果实抗白腐病基因的分子标记.园艺学报,30(1):6-10.
Zhang S,Liu S,Miao H,Shi Y,Wang M,Wang Y,Li B,Gu X.2017.Inheritance and QTL mapping of resistance to gummy stem blight in cucumber stem.Molecular Breeding,37:49.
Zhang S P,Liu M M,Miao H,Zhang S Q,Yang Y H,Xie B Y,Gu X F.2013.Chromosomal mapping and QTL analysis of resistance to downy mildew in Cucumis sativus.Plant Disease,97(2):245-251.
Zhang W W,Pan J S,He H L,Zhang C,Li Z,Zhao J L,Yuan X J,Zhu L H,Huang S W,Cai R.2012.Construction of a high density integrated genetic map for cucumber(Cucumis sativus L.).Theoretical and Applied Genetics,124(2):249-259.