基于RNA-seq的两种柳树转录组微卫星特征比较分析
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摘要
随着高通量测序技术的飞速发展,转录组测序为EST-SSR标记的开发提供了重要的信息资源。为阐明柳树中EST-SSRs的变化情况,本研究以垂柳和簸箕柳转录组序列为来源,分析并比较了两种柳树中EST-SSR的分布规律及特征。结果表明:垂柳与簸箕柳中EST-SSR发生频率分别为3.6%和4.8%,SSR的重复数量和碱基类型呈负相关,而重复数量与EST-SSR总长度或EST-SSRs总数量呈正相关。二、三碱基重复在两种柳树中均为主要类型,三碱基重复占比最大(垂柳为47.5%,箕柳簸为47.8%),五碱基重复占比最小(垂柳为2.4%,簸箕柳为1.9%)。两种柳树中,(AG/CT)、(AAG/CTT)、(AAAT/ATTT)和(AAAAG/CTTTT)分别为不同碱基重复中最丰富的基元类型,而(AAACCG/CGGTTT)和(AGAGGG/CCCTCT)分别为垂柳和簸箕柳六碱基重复中最主要类型。通过侧翼序列分析,EST-SSR趋向产生于A/T富集区域。此外,共有1 305个垂柳EST-SSR和2 463个簸箕柳EST-SSR被成功定位到毛果杨染色体上。本研究将为后期柳树EST-SSR标记的开发及分子遗传学的研究提供科学依据。
With the rapid development of high-throughput sequencing techniques, RNA-seq provides valuable information resources of expressed sequence tags(ESTs) for developing SSRs. To investigate the variation of EST-SSRs in Salix, the transcriptome sequences of S. babylonica and S. suchowensis were selected as research sources and the distribution rules and characteristics of EST-SSR in two Salix were analyzed and compared. The results indicated that the occurrence frequency of EST-SSR in S. babylonica and S. suchowensis were 3.6% and 4.8%, respectively. The correlation between the repeat number and the motif type was negative, while the correlation between the repeat number and the full length of EST-SSR or the total number of SSRs was positive.Di-nucleotide and tri-nucleotide repeats were the most abundant motif types in two Salix. The proportion of tri-nucleotide repeats was the largest(47.5% for S. babylonica and 47.8% for S. suchowensis), while the proportion of penta-nucleotide repeats was the least(2.4% for S. babylonica and 1.9% for S. suchowensis). AG/CT, AAG/CTT,AAAT/ATTT and AAAAG/CTTTT were the most abundant di-, tri-, tetraand penta-nucleotide repeat motifs in S.babylonica and S. suchowensis, respectively. AAACCG/CGGTTT and AGA GGG/CCCTCT were the most frequent hexa-nucleotide repeat motifs in S. babylonica and S. suchowensis, respectively. Through the analysis of flanking sequences, EST-SSRs tended to occur in A/T-rich regions. Furthermore, 1 305 EST-SSRs from S.babylonica and 2 642 EST-SSRs from S. suchowensis were successfully mapped onto chromosomes of Populus trichocarpa. This study might provide scientific reference for the development of EST-SSR markers and the research on molecular genetics in Salix.
With the rapid development of high-throughput sequencing techniques, RNA-seq provides valuable information resources of expressed sequence tags(ESTs) for developing SSRs. To investigate the variation of EST-SSRs in Salix, the transcriptome sequences of S. babylonica and S. suchowensis were selected as research sources and the distribution rules and characteristics of EST-SSR in two Salix were analyzed and compared. The results indicated that the occurrence frequency of EST-SSR in S. babylonica and S. suchowensis were 3.6% and 4.8%, respectively. The correlation between the repeat number and the motif type was negative, while the correlation between the repeat number and the full length of EST-SSR or the total number of SSRs was positive.Di-nucleotide and tri-nucleotide repeats were the most abundant motif types in two Salix. The proportion of tri-nucleotide repeats was the largest(47.5% for S. babylonica and 47.8% for S. suchowensis), while the proportion of penta-nucleotide repeats was the least(2.4% for S. babylonica and 1.9% for S. suchowensis). AG/CT, AAG/CTT,AAAT/ATTT and AAAAG/CTTTT were the most abundant di-, tri-, tetraand penta-nucleotide repeat motifs in S.babylonica and S. suchowensis, respectively. AAACCG/CGGTTT and AGA GGG/CCCTCT were the most frequent hexa-nucleotide repeat motifs in S. babylonica and S. suchowensis, respectively. Through the analysis of flanking sequences, EST-SSRs tended to occur in A/T-rich regions. Furthermore, 1 305 EST-SSRs from S.babylonica and 2 642 EST-SSRs from S. suchowensis were successfully mapped onto chromosomes of Populus trichocarpa. This study might provide scientific reference for the development of EST-SSR markers and the research on molecular genetics in Salix.
引文
An M.,Deng M.,Zheng S.S.,and Song Y.G.,2016,De novo transcriptome assembly and development of SSR markers of oaks Quercus austrocochinchinensis and Q.kerrii(Fagaceae),Tree Genet.Genomes,12(6):103
Bozzi J.A.,Liepelt S.,Ohneiser S.,Gallo L.A.,Marchelli P.,Leyear I.,Ziegenhagen B.,and Mengel C.,2015,Characterization of 23 polymorphic SSR markers in Salix humboldtiana(Salicaceae)using next-generation sequencing and cross-amplification from related species,Appl.Plant Sci.,3(4):1-4
Dai X.G.,Hu Q.J.,Cai Q.L.,Feng K.,Ye N.,Tuskan G.A.,Milne R.,Chen Y.N.,Wang Z.B.,Wang Z.F.,Luo W.C.,Wang K.,Wan D.S.,Wang M.X.,Wang J.,Liu J.Q.,and Yin T.M.,2014,The willow genome and divergent evolution from poplar after the common genome duplication,Cell Res.,24(10):1274-1277
Dai X.G.,Zhu T.W.,Li X.P.,and Yin T.M.,2017,Gene discovery and marker resource development by transcriptome sequencing from a short-rotation coppice willow,Salix suchowensis,Plant Breeding,136(2):1-8
Dong S.B.,Liu Y.L.,Niu J.,Ning Y.,Lin S.Z.,and Zhang Z.X.,2014,De novo transcriptome analysis of the Siberian apricot(Prunus sibirica L.)and search for potential SSR markers by454 pyrosequencing,Gene,544(2):220-227
Du F.K.,Xu F.,Qu H.,Feng S.S.,Tang J.J.,and Wu R.L.,2013,Exploiting the transcriptome of Euphrates poplar,Populus euphratica(Salicaceae)to develop and characterize new EST-SSR markers and construct an EST-SSR database,PLoS One,8(4):e61337
Guo Q.,Wang J.X.,Su L.Z.,Lv W.,Sun Y.H.,and Li Y.,2017,Development and evaluation of a novel set of EST-SSRmarkers based on transcriptome sequences of black locust(Robinia pseudoacacia L.),Genes,8(7):177
Han S.M.,Wu Z.J.,Jin Y.,Yang W.N.,and Shi H.Z.,2015,RNA-Seq analysis for transcriptome assembly,gene identification,and SSR mining in ginkgo(Ginkgo biloba L.),Tree Genet.Genomes,11(3):37
Hanley S.J.,Mallott M.D.,and Karp A.,2006,Alignment of a Salix linkage map to the Populus genomic sequence reveals macrosynteny between willow and poplar genomes,Tree Genet.Genomes,3(1):35-48
He X.D.,Zheng J.W.,Serapiglia M.,Smart L.,Shi S.Z.,and Wang B.S.,2014,Development,characterization and cross-amplification of eight EST-derived microsatellites in Salix,Silvae Genetica,63(3):113-115
He X.D.,Zheng J.W.,Zhou J.,He K.Y.,Shi S.Z.,and Wang B.S.,2015,Characterization and comparison of EST-SSRs in Salix,Populus,and Eucalyptus,Tree Genet.Genomes,11(1):820
Jia H.X.,Yang H.F.,Sun P.,Li J.B.,Zhang J.,Guo Y.H.,Han X.J.,Zhan g G.S.,Lu M.Z.,and Hu J.J.,2016,De novo transcriptome assembly,development of EST-SSR markers and population genetic analyses for the desert biomass willow,Salix psammophila,Sci.Rep.,6:39591
Kalia R.K.,Rai M.K.,Kalia S.,Singh R.,and Dhawan A.K.,2011,Microsatellite markers:an overview of the recent progress in plants,Euphytica,177(3):309-334
Kumpatla S.P.,and Mukhopadhyay S.,2005,Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species,Genome,48(6):985-998
Li D.J.,Deng Z.,Qin B.,Liu X.H.,and Men Z.H.,2012,De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree(Hevea brasiliensis Muell.Arg.),BMC Genomics,13(1):192
Li S.X.,Zhang X.Y.,and Yin T.M.,2010,Characteristics of microsatellites in the transcript sequences of the Laccaria bicolor genome,J.Microbiol.Biotechnol.,20(3):474-479
Li Y.C.,Korol A.B.,Fahima T.,and Nevo E.,2004,Microsatellites within genes:structure,function,and evolution,Mol.Biol.Evol.,21(6):991-1007
Li Y.C.,Korol A.B.,Fahima T.,Beiles A.,and Nevo E.,2002,Microsatellites:genomic distribution,putative functions and mutational mechanisms:a review,Mol.Ecol.,11(12):2453-2465
Marioni J.C.,Mason C.E.,Mane S.M.,Stephens M.,and Gilad Y.,2008,RNA-seq:an assessment of technical reproducibility and comparison with gene expression arrays,Genome Res.,18(9):1509-1517
Metzgar D.,Bytof J.,and Wills C.,2000,Selection against frame shift mutations limits microsatellite expansion in coding DNA,Genome Res.,10(1):72-80
Ranade S.S.,Lin Y.C.,Zuccolo A.,de Peer Y.V.,and Garcia-gil M.R.,2014,Comparative in silico analysis of EST-SSRs in angiosperm and gymnosperm tree genera,BMC Plant Biol.,14(1):220
Smart L.B.,and Cameron K.D.,eds,2008,Genetic improvement of willow(Salix spp.)as a dedicated bioenergy crop,Springer,New York,pp.347-376
Tuler A.C.,Carrijo T.T.,Nóia L.R.,Ferreira A.,Peixoto A.L.,and da Silva Ferreira M.F.,2015,SSR markers:a tool for species identification in Psidium(Myrtaceae),Mol.Biol.Rep.,42(11):1501-1513
Varshney R.K.,Graner A.,and Sorrells M.E.,2005,Genic microsatellite markers in plants:features and applications,Trends Biotechnol.,23(1):48-55
Wang J.Y.,Liang Y.L.,Hai M.R.,Chen J.W.,Gao Z.J.,Hu Q.Q.,Zhang G.H.,and Yang S.C.,2016,Genome-Wide transcriptional excavation of Dipsacus asperoides unmasked both cryptic asperosaponin biosynthetic genes and SSR markers,Front.Plant Sci.,7(348):339
Wang Y.M.,Wei Z.G.,and Yang C.P.,2008,Data mining for SSRs in ESTs and EST-SSR marker development in Betula platyphylla,Linye Kexue(Scientia Silvae Sinicae),44(2):78-84(王艳敏,魏志刚,杨传平,2008,白桦EST-SSR信息分析与标记的开发,林业科学,44(2):78-84)
Wu H.L.,Chen D.,Li J.X.,Yu B.,Qiao X.Y.,Huang H.L.,and He Y.M.,2013,De Novo characterization of leaf transcriptome using 454 sequencing and development of EST-SSRmarkers in tea(Camellia sinensis),Plant Mol.Biol.Rep.,31(3):524-538
Xiao Y.,Zhou L.X.,Xia W.,Mason A.S.,Yang Y.D.,Ma Z.L.,and Peng M.,2014,Exploiting transcriptome data for the development and characterization of gene-based SSR markers related to cold tolerance in oil palm(Elaeis guineensis),BMC Plant Biol.,14(1):384
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zalapa J.E.,Cuevas H.,Zhu H.,Steffan S.,Senalik D.,Zeldin E.,Mccown B.,Harbut R.,and Simon P.,2012,Using next-generation sequencing approaches to isolate simple sequence repeat(SSR)loci in the plant sciences,Am.J.Bot.,99(2):193-208
Zeng S.H.,Xiao G.,Guo J.,Fei Z.F.,Xu Y.Q.,Roe B.A.,and Wang Y.,2010,Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant,Epimedium sagittatum(Sieb.Et Zucc.)Maxim,BMCGenomics,11(1):94
Zhang H.Y.,Wei L.B.,Miao H.M.,Zhang T.D.,and Wang C.Y.,2012,Development and validation of genic-SSR markers in sesame by RNA-seq,BMC Genomics,13(1):316
Zhao Y.L.,Williams R.,Prakash C.S.,and He G.H.,2013,Identification and characterization of gene-based SSR markers in date palm(Phoenix dactylifera L.),BMC Plant Biol.,12(1):1-8
Bozzi J.A.,Liepelt S.,Ohneiser S.,Gallo L.A.,Marchelli P.,Leyear I.,Ziegenhagen B.,and Mengel C.,2015,Characterization of 23 polymorphic SSR markers in Salix humboldtiana(Salicaceae)using next-generation sequencing and cross-amplification from related species,Appl.Plant Sci.,3(4):1-4
Dai X.G.,Hu Q.J.,Cai Q.L.,Feng K.,Ye N.,Tuskan G.A.,Milne R.,Chen Y.N.,Wang Z.B.,Wang Z.F.,Luo W.C.,Wang K.,Wan D.S.,Wang M.X.,Wang J.,Liu J.Q.,and Yin T.M.,2014,The willow genome and divergent evolution from poplar after the common genome duplication,Cell Res.,24(10):1274-1277
Dai X.G.,Zhu T.W.,Li X.P.,and Yin T.M.,2017,Gene discovery and marker resource development by transcriptome sequencing from a short-rotation coppice willow,Salix suchowensis,Plant Breeding,136(2):1-8
Dong S.B.,Liu Y.L.,Niu J.,Ning Y.,Lin S.Z.,and Zhang Z.X.,2014,De novo transcriptome analysis of the Siberian apricot(Prunus sibirica L.)and search for potential SSR markers by454 pyrosequencing,Gene,544(2):220-227
Du F.K.,Xu F.,Qu H.,Feng S.S.,Tang J.J.,and Wu R.L.,2013,Exploiting the transcriptome of Euphrates poplar,Populus euphratica(Salicaceae)to develop and characterize new EST-SSR markers and construct an EST-SSR database,PLoS One,8(4):e61337
Guo Q.,Wang J.X.,Su L.Z.,Lv W.,Sun Y.H.,and Li Y.,2017,Development and evaluation of a novel set of EST-SSRmarkers based on transcriptome sequences of black locust(Robinia pseudoacacia L.),Genes,8(7):177
Han S.M.,Wu Z.J.,Jin Y.,Yang W.N.,and Shi H.Z.,2015,RNA-Seq analysis for transcriptome assembly,gene identification,and SSR mining in ginkgo(Ginkgo biloba L.),Tree Genet.Genomes,11(3):37
Hanley S.J.,Mallott M.D.,and Karp A.,2006,Alignment of a Salix linkage map to the Populus genomic sequence reveals macrosynteny between willow and poplar genomes,Tree Genet.Genomes,3(1):35-48
He X.D.,Zheng J.W.,Serapiglia M.,Smart L.,Shi S.Z.,and Wang B.S.,2014,Development,characterization and cross-amplification of eight EST-derived microsatellites in Salix,Silvae Genetica,63(3):113-115
He X.D.,Zheng J.W.,Zhou J.,He K.Y.,Shi S.Z.,and Wang B.S.,2015,Characterization and comparison of EST-SSRs in Salix,Populus,and Eucalyptus,Tree Genet.Genomes,11(1):820
Jia H.X.,Yang H.F.,Sun P.,Li J.B.,Zhang J.,Guo Y.H.,Han X.J.,Zhan g G.S.,Lu M.Z.,and Hu J.J.,2016,De novo transcriptome assembly,development of EST-SSR markers and population genetic analyses for the desert biomass willow,Salix psammophila,Sci.Rep.,6:39591
Kalia R.K.,Rai M.K.,Kalia S.,Singh R.,and Dhawan A.K.,2011,Microsatellite markers:an overview of the recent progress in plants,Euphytica,177(3):309-334
Kumpatla S.P.,and Mukhopadhyay S.,2005,Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species,Genome,48(6):985-998
Li D.J.,Deng Z.,Qin B.,Liu X.H.,and Men Z.H.,2012,De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree(Hevea brasiliensis Muell.Arg.),BMC Genomics,13(1):192
Li S.X.,Zhang X.Y.,and Yin T.M.,2010,Characteristics of microsatellites in the transcript sequences of the Laccaria bicolor genome,J.Microbiol.Biotechnol.,20(3):474-479
Li Y.C.,Korol A.B.,Fahima T.,and Nevo E.,2004,Microsatellites within genes:structure,function,and evolution,Mol.Biol.Evol.,21(6):991-1007
Li Y.C.,Korol A.B.,Fahima T.,Beiles A.,and Nevo E.,2002,Microsatellites:genomic distribution,putative functions and mutational mechanisms:a review,Mol.Ecol.,11(12):2453-2465
Marioni J.C.,Mason C.E.,Mane S.M.,Stephens M.,and Gilad Y.,2008,RNA-seq:an assessment of technical reproducibility and comparison with gene expression arrays,Genome Res.,18(9):1509-1517
Metzgar D.,Bytof J.,and Wills C.,2000,Selection against frame shift mutations limits microsatellite expansion in coding DNA,Genome Res.,10(1):72-80
Ranade S.S.,Lin Y.C.,Zuccolo A.,de Peer Y.V.,and Garcia-gil M.R.,2014,Comparative in silico analysis of EST-SSRs in angiosperm and gymnosperm tree genera,BMC Plant Biol.,14(1):220
Smart L.B.,and Cameron K.D.,eds,2008,Genetic improvement of willow(Salix spp.)as a dedicated bioenergy crop,Springer,New York,pp.347-376
Tuler A.C.,Carrijo T.T.,Nóia L.R.,Ferreira A.,Peixoto A.L.,and da Silva Ferreira M.F.,2015,SSR markers:a tool for species identification in Psidium(Myrtaceae),Mol.Biol.Rep.,42(11):1501-1513
Varshney R.K.,Graner A.,and Sorrells M.E.,2005,Genic microsatellite markers in plants:features and applications,Trends Biotechnol.,23(1):48-55
Wang J.Y.,Liang Y.L.,Hai M.R.,Chen J.W.,Gao Z.J.,Hu Q.Q.,Zhang G.H.,and Yang S.C.,2016,Genome-Wide transcriptional excavation of Dipsacus asperoides unmasked both cryptic asperosaponin biosynthetic genes and SSR markers,Front.Plant Sci.,7(348):339
Wang Y.M.,Wei Z.G.,and Yang C.P.,2008,Data mining for SSRs in ESTs and EST-SSR marker development in Betula platyphylla,Linye Kexue(Scientia Silvae Sinicae),44(2):78-84(王艳敏,魏志刚,杨传平,2008,白桦EST-SSR信息分析与标记的开发,林业科学,44(2):78-84)
Wu H.L.,Chen D.,Li J.X.,Yu B.,Qiao X.Y.,Huang H.L.,and He Y.M.,2013,De Novo characterization of leaf transcriptome using 454 sequencing and development of EST-SSRmarkers in tea(Camellia sinensis),Plant Mol.Biol.Rep.,31(3):524-538
Xiao Y.,Zhou L.X.,Xia W.,Mason A.S.,Yang Y.D.,Ma Z.L.,and Peng M.,2014,Exploiting transcriptome data for the development and characterization of gene-based SSR markers related to cold tolerance in oil palm(Elaeis guineensis),BMC Plant Biol.,14(1):384
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Zalapa J.E.,Cuevas H.,Zhu H.,Steffan S.,Senalik D.,Zeldin E.,Mccown B.,Harbut R.,and Simon P.,2012,Using next-generation sequencing approaches to isolate simple sequence repeat(SSR)loci in the plant sciences,Am.J.Bot.,99(2):193-208
Zeng S.H.,Xiao G.,Guo J.,Fei Z.F.,Xu Y.Q.,Roe B.A.,and Wang Y.,2010,Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant,Epimedium sagittatum(Sieb.Et Zucc.)Maxim,BMCGenomics,11(1):94
Zhang H.Y.,Wei L.B.,Miao H.M.,Zhang T.D.,and Wang C.Y.,2012,Development and validation of genic-SSR markers in sesame by RNA-seq,BMC Genomics,13(1):316
Zhao Y.L.,Williams R.,Prakash C.S.,and He G.H.,2013,Identification and characterization of gene-based SSR markers in date palm(Phoenix dactylifera L.),BMC Plant Biol.,12(1):1-8