Small RNA深度测序鉴定甘薯种质的甘薯曲叶病毒
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摘要
本研究利用Small RNA深度测序对长沙、永州、邵阳等地采集的甘薯种质资源病毒症状样品进行鉴定,发现样品中含有与甘薯曲叶病毒同源的系列。设计引物进行PCR扩增,鉴定出24个SPLCV-AC1基因片段序列,经NCBI-Blast比对,与10个不同株系SPLCV同源性达96%以上。为进一步获得SPLCV全长序列,设计背靠背引物,经PCR扩增、鉴定,获得3个SPLCV全长序列。系列进化分析显示,这3个SPLCV分离物聚为同一分支,其中SPLCV (76)序列大小2 774 bp,与甘薯乔治亚曲叶病毒中国分离物、甘薯金脉病毒具有较高同源性;SPLCV (47)序列大小2 777 bp,与甘薯河南曲叶病毒分离物具有较高同源性;SPLCV (68)序列大小2 832 bp,与甘薯广西曲叶病毒分离物具有96%的同源性。以上同源系列与Small RNA测序显示结果相一致。本研究结果为甘薯病毒病防控提供理论依据。
In this study, the virus symptom samples of sweet potato germplasm resources collected from Changsha, Yongzhou and Shaoyang were identified by small RNA deep sequencing, and it was found that the samples contained a series of homology sequences with sweet potato leaf curl virus. 24 SPLCV-AC1 gene fragments sequences were identified after amplified by PCR with designed primers, which had over 96% homology with 10 different lines SPLCV by NCBI-Blast comparison. In order to further obtain the full-length sequence of SPLCV,back-to-back primers were designed, and then three full-length sequences of SPLCV were obtained by PCR amplification and identification. A series of evolutionary analysis showed that the three SPLCV isolates were clustered into the same branch. Among them, the sequence size of SPLCV(76) was 2 774 bp, which had high homology with Chinese isolates of sweet potato leaf curl Georgia virus and golden vein virus in sweet potato. The sequence of SPLCV(47) was 2 777 bp, which had high homology with the isolates of sweet potato leaf curl Henan virus. The SPLCV(68) sequence was 2 832 bp, which had 96% homology with the isolates of sweet potato leaf curl Guangxi virus. These homologous sequences were consistent with the results of Small RNA sequencing. The results of this study provide theoretical basis for prevention and control of sweet potato virus disease.
In this study, the virus symptom samples of sweet potato germplasm resources collected from Changsha, Yongzhou and Shaoyang were identified by small RNA deep sequencing, and it was found that the samples contained a series of homology sequences with sweet potato leaf curl virus. 24 SPLCV-AC1 gene fragments sequences were identified after amplified by PCR with designed primers, which had over 96% homology with 10 different lines SPLCV by NCBI-Blast comparison. In order to further obtain the full-length sequence of SPLCV,back-to-back primers were designed, and then three full-length sequences of SPLCV were obtained by PCR amplification and identification. A series of evolutionary analysis showed that the three SPLCV isolates were clustered into the same branch. Among them, the sequence size of SPLCV(76) was 2 774 bp, which had high homology with Chinese isolates of sweet potato leaf curl Georgia virus and golden vein virus in sweet potato. The sequence of SPLCV(47) was 2 777 bp, which had high homology with the isolates of sweet potato leaf curl Henan virus. The SPLCV(68) sequence was 2 832 bp, which had 96% homology with the isolates of sweet potato leaf curl Guangxi virus. These homologous sequences were consistent with the results of Small RNA sequencing. The results of this study provide theoretical basis for prevention and control of sweet potato virus disease.
引文
Albuquerque L.C.,Inoue-Nagata A.K.,Pinheiro B.,Resende R.O.,Moriones E.,and Navas-Castillo J.,2012,Genetic diversity and recombination analysis of sweepoviruses from Brazil,Virol.J.,9:241
Arkorful E.,and Addae-Frimpomaah F.,2015,Detection of sweet potato(Ipomoea batatas L.)leaf curl virus(SPLCV)in Ghana using visual symptomatology and PCR technique,International Journal of Plant Breeding and Genetics,9(3):106-115
Bi H.,and Zhang P.,2012,Molecular characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in Nicotiana benthamiana,Arch.Virol.,157(3):441-454
Clark C.A.,and Hoy M.W.,2006,Effects of common viruses on yield and quality of beauregard sweetpotato in Louisiana,Plant Dis.,90(1):83-88
Esterhuizen L.L.,Van Heerden S.W.,Rey M.E.,and Van Heerden H.,2012,Genetic identification of two sweet-potato-infecting begomoviruses in South Africa,Arch.Virol.,157(11):2241-2245
Gu Y.H.,Tao X.,Lai X.J.,Wang H.Y.,and Zhang Y.Z.,2014,Exploring the polyadenylated RNA virome of sweet potato through high-throughput sequencing,PLoS One,9(6):e98884
Huang L.L.,Pham B.,He F.L.,Liu Y.J.,Liu Y.M.,Chen B.S.,and Liao Y.M.,2016,The pathogenic virus species detection of sweet potato viral diseases in Guangxi,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(5):1213-1218(黄利利,Pham B.,何芳练,刘奕君,刘义明,陈保善,廖咏梅,2016,广西甘薯病毒病的病原病毒种类检测,基因组学与应用生物学,35(5):1213-1218)
Kim J.,Kil E.J.,Kim S.,Seo H.,Byun H.S.,Park J.,Chung M.N.,Kawk H.R.,Kim K.M.,Kim C.S.,Yang J.W.,Lee K.Y.,Choi H.S.,and Lee S.,2015,Seed transmission of Sweet potato leaf curl virus in sweet potato(Ipomoea batatas),Plant Pathol.,64(6):1284-1291
Kim J.,Yang J.W.,Kwak H.R.,Kim M.K.,Seo J.K.,Chung M.N.,Lee H.U.,Lee K.B.,Nam S.S.,Kim C.S.,Lee G.S.,Kim J.S.,Lee S.,and Choi H.S.,2017,Virus incidence of sweet potato in Korea from 2011 to 2014,Plant Pathol.J.,33(5):467-477
Kreuze J.F.,Perez A.,Untiveros M.,Quspe D.,Fuentes S.,Barker I.,and Simon R.,2009,Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNA:a generic method for diagnosis,discovery and sequencing of viruses,Virology,388(1):l-7
Kwak H.R.,Kim M.K.,Chung M.N.,Lee S.H.,Park J.W.,Kim K.H.,and Choi H.S.,2006,Virus disease incidences of sweet potatoes in Korea,Plant Pathol.J.,22(3):239-247
Li Y.,Wang H.,Zhang C.,and Ma X.J.,2015,Advance in deep sequencin g of sm all RNAs of virus identification and discovery,Bingdu Xuebao(Chinese Journal of Virology),31(4):457-462(李洋,王昊,张晨,马学军,2015,基于小RNA(sRNA)深度测序技术进行病毒鉴定和发现的研究进展,病毒学报,31(4):457-462)
Liu Q.,Zhang Z.,Qiao Q.,Qin Y.,Zhang D.,Tian Y.,Wang S,and Wang Y.,2013,Complete genome sequence of a novel monopartite begomovirus infecting sweet potato in China,Virus Genes,47(3):591-594
Liu Q.L.,Zhang J.X.,Li X.X.,Shi M.W.,and Ou X.Q.,2017,Advances in research of DNA virus infecing sweet potato,Zhiwu Baohu(Plant Protection),43(3):36-42(刘起丽,张建新,李学成,石明旺,欧行奇,2017,侵染甘薯的DNA病毒研究进展,植物保护,43(3):36-42)
Lotrakul P.,Valverde R.A.,Clark C.A.,Sim J.,and De La Torre R.,1998,Detection of a geminivirus infecting sweet potato in the United States,Plant Dis.,82(11):1253-1257
Lozano G.,Trenado H.P.,Valverde R.A.,and Navas-Castillo J.,2009,Novel begomovirus species of recombinant nature in sweet potato(Ipomoea batatas)and Ipomoea indica:taxonomic and phylogenetic implications,J.Gen.Virol.,90(Pt10):2550-2562
Luan Y.S.,Zhang J.,and An L.J.,2007,First report of Sweet potato leaf curl virus in China,Plant Dis.,90(8):1111
Paprotka T.,Boiteux L.S.,Fonseca M.E.,Resende R.O.,Jeske H.,Faria J.C.,and Ribeiro S.G.,2010,Genomic diversity of sweet potato geminiviruses in a Brazilian germplasm bank,Virus Res.,149(2):224-233
Pardina P.R.,Luque A.,Nome C.,Colomba E.L.,Delgado S.F.,and Feo L.D.,2012,First report of Sweet potato leaf curl virus infecting sweet potato in Argentina,Australasian Plant Disease Notes,7(1):157-160
Qin Y.H.,Zhang Z.C.,Qiao Q.,Zhang D.S.,Tian Y.T.,and Wang Y.J.,2013,Molecular variability of sweet potato chlorotic stunt virus(SPCSV)and five potyviruses infecting sweet potato in China,Arch.Virol.,158(2):491-495
Valverde R.A.,Sim J.,and Lotrakul P.,2004,Whitefly transmission of sweet potato viruses,Virus Res.,99(1):123-128
Zhang S.C.,and Ling K.S.,2011,Genetic diversity of sweet potato begomoviruses in the United States and identification of a natural recombinant between sweet potato leaf curl virus and sweet potato leaf curl Georgia virus,Arch.Virol.,156(6):955-968
Arkorful E.,and Addae-Frimpomaah F.,2015,Detection of sweet potato(Ipomoea batatas L.)leaf curl virus(SPLCV)in Ghana using visual symptomatology and PCR technique,International Journal of Plant Breeding and Genetics,9(3):106-115
Bi H.,and Zhang P.,2012,Molecular characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in Nicotiana benthamiana,Arch.Virol.,157(3):441-454
Clark C.A.,and Hoy M.W.,2006,Effects of common viruses on yield and quality of beauregard sweetpotato in Louisiana,Plant Dis.,90(1):83-88
Esterhuizen L.L.,Van Heerden S.W.,Rey M.E.,and Van Heerden H.,2012,Genetic identification of two sweet-potato-infecting begomoviruses in South Africa,Arch.Virol.,157(11):2241-2245
Gu Y.H.,Tao X.,Lai X.J.,Wang H.Y.,and Zhang Y.Z.,2014,Exploring the polyadenylated RNA virome of sweet potato through high-throughput sequencing,PLoS One,9(6):e98884
Huang L.L.,Pham B.,He F.L.,Liu Y.J.,Liu Y.M.,Chen B.S.,and Liao Y.M.,2016,The pathogenic virus species detection of sweet potato viral diseases in Guangxi,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(5):1213-1218(黄利利,Pham B.,何芳练,刘奕君,刘义明,陈保善,廖咏梅,2016,广西甘薯病毒病的病原病毒种类检测,基因组学与应用生物学,35(5):1213-1218)
Kim J.,Kil E.J.,Kim S.,Seo H.,Byun H.S.,Park J.,Chung M.N.,Kawk H.R.,Kim K.M.,Kim C.S.,Yang J.W.,Lee K.Y.,Choi H.S.,and Lee S.,2015,Seed transmission of Sweet potato leaf curl virus in sweet potato(Ipomoea batatas),Plant Pathol.,64(6):1284-1291
Kim J.,Yang J.W.,Kwak H.R.,Kim M.K.,Seo J.K.,Chung M.N.,Lee H.U.,Lee K.B.,Nam S.S.,Kim C.S.,Lee G.S.,Kim J.S.,Lee S.,and Choi H.S.,2017,Virus incidence of sweet potato in Korea from 2011 to 2014,Plant Pathol.J.,33(5):467-477
Kreuze J.F.,Perez A.,Untiveros M.,Quspe D.,Fuentes S.,Barker I.,and Simon R.,2009,Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNA:a generic method for diagnosis,discovery and sequencing of viruses,Virology,388(1):l-7
Kwak H.R.,Kim M.K.,Chung M.N.,Lee S.H.,Park J.W.,Kim K.H.,and Choi H.S.,2006,Virus disease incidences of sweet potatoes in Korea,Plant Pathol.J.,22(3):239-247
Li Y.,Wang H.,Zhang C.,and Ma X.J.,2015,Advance in deep sequencin g of sm all RNAs of virus identification and discovery,Bingdu Xuebao(Chinese Journal of Virology),31(4):457-462(李洋,王昊,张晨,马学军,2015,基于小RNA(sRNA)深度测序技术进行病毒鉴定和发现的研究进展,病毒学报,31(4):457-462)
Liu Q.,Zhang Z.,Qiao Q.,Qin Y.,Zhang D.,Tian Y.,Wang S,and Wang Y.,2013,Complete genome sequence of a novel monopartite begomovirus infecting sweet potato in China,Virus Genes,47(3):591-594
Liu Q.L.,Zhang J.X.,Li X.X.,Shi M.W.,and Ou X.Q.,2017,Advances in research of DNA virus infecing sweet potato,Zhiwu Baohu(Plant Protection),43(3):36-42(刘起丽,张建新,李学成,石明旺,欧行奇,2017,侵染甘薯的DNA病毒研究进展,植物保护,43(3):36-42)
Lotrakul P.,Valverde R.A.,Clark C.A.,Sim J.,and De La Torre R.,1998,Detection of a geminivirus infecting sweet potato in the United States,Plant Dis.,82(11):1253-1257
Lozano G.,Trenado H.P.,Valverde R.A.,and Navas-Castillo J.,2009,Novel begomovirus species of recombinant nature in sweet potato(Ipomoea batatas)and Ipomoea indica:taxonomic and phylogenetic implications,J.Gen.Virol.,90(Pt10):2550-2562
Luan Y.S.,Zhang J.,and An L.J.,2007,First report of Sweet potato leaf curl virus in China,Plant Dis.,90(8):1111
Paprotka T.,Boiteux L.S.,Fonseca M.E.,Resende R.O.,Jeske H.,Faria J.C.,and Ribeiro S.G.,2010,Genomic diversity of sweet potato geminiviruses in a Brazilian germplasm bank,Virus Res.,149(2):224-233
Pardina P.R.,Luque A.,Nome C.,Colomba E.L.,Delgado S.F.,and Feo L.D.,2012,First report of Sweet potato leaf curl virus infecting sweet potato in Argentina,Australasian Plant Disease Notes,7(1):157-160
Qin Y.H.,Zhang Z.C.,Qiao Q.,Zhang D.S.,Tian Y.T.,and Wang Y.J.,2013,Molecular variability of sweet potato chlorotic stunt virus(SPCSV)and five potyviruses infecting sweet potato in China,Arch.Virol.,158(2):491-495
Valverde R.A.,Sim J.,and Lotrakul P.,2004,Whitefly transmission of sweet potato viruses,Virus Res.,99(1):123-128
Zhang S.C.,and Ling K.S.,2011,Genetic diversity of sweet potato begomoviruses in the United States and identification of a natural recombinant between sweet potato leaf curl virus and sweet potato leaf curl Georgia virus,Arch.Virol.,156(6):955-968