基于转录组的黑果枸杞SSR分布特征分析及引物设计
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摘要
为了全面了解独特、珍稀植物黑果枸杞果实中简单重复序列的分布特征及分布规律,为西北特有的珍稀种质资源的保存及合理的开发和利用提供遗传学和分子生物学理论依据,本研究利用Illumina Hi Seq TM4000高通量测序平台对黑果枸杞进行RNA-seq测序,再通过专业数据分析MISA对测序所获得的Unigene进行SSR深入挖掘和特征分析。结果显示,黑果枸杞转录组测序共获得101 466条Unigenes,筛选搜索到含SSR位点的序列15 494条,共得到20 269个SSR,发生频率为19.98%。平均约每4 kb出现1个SSR位点。黑果枸杞SSR中主要重复单元类型为单碱基和二碱基重复,分别占SSR总数的74.83%和12.68%,在统计的46种重复类型中(C/G)n所占比例最高(14 652, 72.15%),其次是(AG/CT)n(1 195, 5.90%)、(AT/AT)n(3.72%)和(AC/GT)n(3.04%)。在SSR序列长度方面,长度变化范围最大的为三碱基重复(15～39 bp),其次是单碱基重复(10～32 bp),SSR位点的频率与分布长度呈负相关。黑果枸杞转录组中SSR位点分布频率较高,重复单元类型丰富,分布密度大,具有较高的特异性和多态性,为研究黑果枸杞的遗传多样性及分子标记辅助育种等方面提供可靠的理论依据。
In order to comprehensively understand the distribution characteristics and regular pattern of simple repeat sequences in the fruit of Lycium ruthenicum Murr., a unique and rare plant, to provide a theoretical basis of genetics and molecular biology for cherishing the preservation of the unique germplasm resources with rational development and utilization in Northwest China. RNA-seq sequencing of Lycium ruthenicum Murr. by using Illumina Hi SeqTM4000 high-throughput sequencing platform. Then the SSR were deeply sought and characterized by using the professional data analysis software MISA from the obtained Unigenes. The results showed that 101 466 Unigenes were obtained from the transcriptome sequencing of Lycium ruthenicum Murr. and 15 494 SSR containing sequences were searched for a total of 20 269 SSRs with a frequency of distribution of 19.98%. On average,approximately 1 SSR site per 4 kb. Main repeating unit types in the SSR of Lycium ruthenicum Murr. were single base and two base repeats, accounting for 74.83% and 12.68% of total SSR, respectively.(C/G)naccounted for the highest proportion(14 652, 72.15%), in the statistics of 46 repetition types, then followed by(AG/CT)n(1 195, 5.90%),(AT/AT)n(3.72%), and(AC/GT)n(3.04%). In terms of SSR sequence length, the largest range of length variation was three base repeats(15～39 bp), then followed by single base repeats(10～32 bp) and the frequency of SSR loci was inversely correlated with the distribution length. The SSR loci in the transcriptome of Lycium ruthenicum Murr. has a high frequency of distribution, rich in repeating unit types, with large distribution density, which was provided with high specificity and polymorphism and it can provide a reliable theoretical basis in studying the genetic diversity and molecular marker-assisted breeding.
In order to comprehensively understand the distribution characteristics and regular pattern of simple repeat sequences in the fruit of Lycium ruthenicum Murr., a unique and rare plant, to provide a theoretical basis of genetics and molecular biology for cherishing the preservation of the unique germplasm resources with rational development and utilization in Northwest China. RNA-seq sequencing of Lycium ruthenicum Murr. by using Illumina Hi SeqTM4000 high-throughput sequencing platform. Then the SSR were deeply sought and characterized by using the professional data analysis software MISA from the obtained Unigenes. The results showed that 101 466 Unigenes were obtained from the transcriptome sequencing of Lycium ruthenicum Murr. and 15 494 SSR containing sequences were searched for a total of 20 269 SSRs with a frequency of distribution of 19.98%. On average,approximately 1 SSR site per 4 kb. Main repeating unit types in the SSR of Lycium ruthenicum Murr. were single base and two base repeats, accounting for 74.83% and 12.68% of total SSR, respectively.(C/G)naccounted for the highest proportion(14 652, 72.15%), in the statistics of 46 repetition types, then followed by(AG/CT)n(1 195, 5.90%),(AT/AT)n(3.72%), and(AC/GT)n(3.04%). In terms of SSR sequence length, the largest range of length variation was three base repeats(15～39 bp), then followed by single base repeats(10～32 bp) and the frequency of SSR loci was inversely correlated with the distribution length. The SSR loci in the transcriptome of Lycium ruthenicum Murr. has a high frequency of distribution, rich in repeating unit types, with large distribution density, which was provided with high specificity and polymorphism and it can provide a reliable theoretical basis in studying the genetic diversity and molecular marker-assisted breeding.
引文
Altintas A.,Kosar M.,Kirimer N.,Baser K.H.C.,and Demirci B.,2006,Composition of the essential oils of Lycium barbarum,and L.ruthenicum fruits,Chemistry of Natural Compounds,42(1):24-25
Chen L.,Li L.N.,Yang G.D.,and Dai Y.P.,2016,Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):959-965(陈林,李龙娜,杨国栋,戴亚平,2016,特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析,分子植物育种,14(4):959-965)
Chen S.Y.,Ning D.L.,Wu T.,Xiao L.J.,Lin H.D.,Zhu Y.F.,He N.,and Pan L.,2017,Development of SSR markers in Juglans sigillata and its application in genetic diversity analysis,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),32(3):91-96(陈少瑜,宁德鲁,吴涛,肖良俊,林红丁,朱云凤,贺娜,潘莉,2017,泡核桃SSR标记开发及在遗传多样性研究中的应用,西北林学院学报,32(3):91-96)
Graham I.A.,Besser K.,Blumer S.,Branigan C.A.,Czechowski T.,Elias L.,Guterman I.,Harvey D.,Isaac P.G.,Khan A.M.,Larson T.R.,Li Y.,Pawson T.,Penfield T.,Rae A.M.,Rathbone D.A.,Reid S.,Ross J.,Smallwood M.F,Segura V.,Townsend T.,Vyas D.,Winzer T.,and Bowles D.,2010,The genetic map of Artemisia annua L.identifies loci affecting yield of the antimalarial drug artemisini,Science,327(5963):328-331
Huang H.Y.,Du H.Y.,Wuyun T.N.,and Liu P.F.,2013,Development of SSR molecular markers based on transcriptome sequencing of Eucommia ulmoides,Linye Kexue(Scientia Silvae Sinicae),49(5):176-181(黄海燕,杜红岩,乌云塔娜,刘攀峰,2013,基于杜仲转录组序列的SSR分子标记的开发,林业科学,49(5):176-181)
Harr B.,and Schlotterer C.,2000,Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times,which cause their genome-wide underrepresentation,Genetics,155(3):1213
Jiang D.,Zhong G.Y.,and Hong Q.B.,2006,Analysis of microsatellites in citrus unigenes,Acta Genetica Sinica,33(4):345-353
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
Luo R.,Wu W.L.,Zhang Y.,and Li Y.H.,2010,SSR marker and its application to crop genetics and breeding,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(1):137-143(罗冉,吴委林,张旸,李玉花,2010,SSR分子标记在作物遗传育种中的应用,基因组学与应用生物学,29(1):137-143)
Lin S.,Zhang C.M.,Pan T.L.,Zhang H.Y.,and Li K.P.,2017,Information analysis of SSR locus in transcriptome of herbal tea plant Microcos paniculata,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1654-1659(林爽,张初梅,潘天玲,张宏意,李坤平,2017,凉茶植物布渣叶转录组SSR位点信息分析,基因组学与应用生物学,36(4):1654-1659)
Li T.Q.,Liu X.F.,Wan Y.M.,Li Z.H.,Li Y.Y.,Liu X.X.,and Ma H.,2017,Characteristic analysis of microsatellites in the transcriptome of Rhododendron longipedicellatum,an endangered species endemic to southeastern Yunnan,China,Linye Kexue Yanjiu(Forest Research),30(4):533-541(李太强,刘雄芳,万友名,李正红,李钰莹,刘秀贤,马宏,2017,滇东南濒危植物长梗杜鹃转录组微卫星特征分析,林业科学研究,30(4):533-541)
Potterat O.,2010,Goji(Lycium barbarum and L.chinense):phytochemistry,pharmacology and safety in the perspective of traditional uses and recent popularity,Planta Medica,76(1):7-19
Ruan Z.Y.,Wang B.Y.,Ouyang Z.Q.,Liao L.B.,Su T.W.,and Qiao L.,2016,Characterization of microsatellites in genome of Pinus squamata,a critically endangered species in the world,Zhiwu Yanjiu(Bulletin of Botanical Research),36(5):775-781(阮桢媛,王兵益,欧阳志勤,廖礼彬,苏腾伟,乔璐,2016,极度濒危植物巧家五针松基因组微卫星特征分析,植物研究,36(5):775-781)
Semagn K.,Bjornstad A.,and Ndjiondjop M.N.,2006,An overview of molecular marker methods for plants,African Journal of Biotechnology,5(25):2540-2568
Simon S.A.,Zhai J.X.,Nandety R.S.,McCormick K.P.,Zeng J.,Mejia D.,and Meyers B.C.,2009,Short-read sequencing technologies for transcriptional analyses,Anunu.Rev.Plamt Biol.,60(1):305-333
Thao D.V.,Yamashita M.,Watanabe A.,and Shiraishi S.,2013,Development of tetranucleotide microsatellite markers in Pinus kesiya Royle ex Gordon,Conservation Genetics Resources,5(2):405-407
Temnykh S.,DeClerck G.,Lukashova A.,Lipovich L.,Cartinhour S.,and McCouch S.,2001,Computational and experi mental analysis of microsatellites in rice(Oryza sativa L.):frequency,length variation,transposon associations,and genetic marker potential,Genome Research,59(19):4001-4009
Zheng J.,Ding C.X.,Wang L.S.,Li G.L.,Shi J.Y.,Li H.,Wang H.L.,and Suo Y.R.,2011,Anthocyanins composition and antioxidant activity of wild Lycium ruthenicum Murr.from Qinghai-Tibet Plateau,Food Chemistry,126(3):859-865
Zeng S.H.,Wu M.,Zou C.Y.,Liu X.M.,Shen X.F.,Hayward A.,Liu C.Z.,and Wang Y.,2014,Comparative analysis of anthocyanin biosynthesis during fruit development in two Lycium species,Physiologia Plantarum,150(4):505-516
Zhang Z.,Zhang H.G.,Mo C.,and Zhang L.,2015,Transcriptome sequencing analysis and development of EST-SSRmarkers for Pinus koraiensis,Linye Kexue(Scientia Silvae Sinicae),51(8):114-120(张振,张含国,莫迟,张磊,2015,红松转录组SSR分析及EST-SSR标记开发,林业科学,51(8):114-120)
Chen L.,Li L.N.,Yang G.D.,and Dai Y.P.,2016,Microsatellites characteristics of transcriptomic sequences from Osmanthus serrulatus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):959-965(陈林,李龙娜,杨国栋,戴亚平,2016,特有植物短丝木犀(Osmanthus serrulatus)转录组微卫星特征分析,分子植物育种,14(4):959-965)
Chen S.Y.,Ning D.L.,Wu T.,Xiao L.J.,Lin H.D.,Zhu Y.F.,He N.,and Pan L.,2017,Development of SSR markers in Juglans sigillata and its application in genetic diversity analysis,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),32(3):91-96(陈少瑜,宁德鲁,吴涛,肖良俊,林红丁,朱云凤,贺娜,潘莉,2017,泡核桃SSR标记开发及在遗传多样性研究中的应用,西北林学院学报,32(3):91-96)
Graham I.A.,Besser K.,Blumer S.,Branigan C.A.,Czechowski T.,Elias L.,Guterman I.,Harvey D.,Isaac P.G.,Khan A.M.,Larson T.R.,Li Y.,Pawson T.,Penfield T.,Rae A.M.,Rathbone D.A.,Reid S.,Ross J.,Smallwood M.F,Segura V.,Townsend T.,Vyas D.,Winzer T.,and Bowles D.,2010,The genetic map of Artemisia annua L.identifies loci affecting yield of the antimalarial drug artemisini,Science,327(5963):328-331
Huang H.Y.,Du H.Y.,Wuyun T.N.,and Liu P.F.,2013,Development of SSR molecular markers based on transcriptome sequencing of Eucommia ulmoides,Linye Kexue(Scientia Silvae Sinicae),49(5):176-181(黄海燕,杜红岩,乌云塔娜,刘攀峰,2013,基于杜仲转录组序列的SSR分子标记的开发,林业科学,49(5):176-181)
Harr B.,and Schlotterer C.,2000,Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times,which cause their genome-wide underrepresentation,Genetics,155(3):1213
Jiang D.,Zhong G.Y.,and Hong Q.B.,2006,Analysis of microsatellites in citrus unigenes,Acta Genetica Sinica,33(4):345-353
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
Luo R.,Wu W.L.,Zhang Y.,and Li Y.H.,2010,SSR marker and its application to crop genetics and breeding,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),29(1):137-143(罗冉,吴委林,张旸,李玉花,2010,SSR分子标记在作物遗传育种中的应用,基因组学与应用生物学,29(1):137-143)
Lin S.,Zhang C.M.,Pan T.L.,Zhang H.Y.,and Li K.P.,2017,Information analysis of SSR locus in transcriptome of herbal tea plant Microcos paniculata,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1654-1659(林爽,张初梅,潘天玲,张宏意,李坤平,2017,凉茶植物布渣叶转录组SSR位点信息分析,基因组学与应用生物学,36(4):1654-1659)
Li T.Q.,Liu X.F.,Wan Y.M.,Li Z.H.,Li Y.Y.,Liu X.X.,and Ma H.,2017,Characteristic analysis of microsatellites in the transcriptome of Rhododendron longipedicellatum,an endangered species endemic to southeastern Yunnan,China,Linye Kexue Yanjiu(Forest Research),30(4):533-541(李太强,刘雄芳,万友名,李正红,李钰莹,刘秀贤,马宏,2017,滇东南濒危植物长梗杜鹃转录组微卫星特征分析,林业科学研究,30(4):533-541)
Potterat O.,2010,Goji(Lycium barbarum and L.chinense):phytochemistry,pharmacology and safety in the perspective of traditional uses and recent popularity,Planta Medica,76(1):7-19
Ruan Z.Y.,Wang B.Y.,Ouyang Z.Q.,Liao L.B.,Su T.W.,and Qiao L.,2016,Characterization of microsatellites in genome of Pinus squamata,a critically endangered species in the world,Zhiwu Yanjiu(Bulletin of Botanical Research),36(5):775-781(阮桢媛,王兵益,欧阳志勤,廖礼彬,苏腾伟,乔璐,2016,极度濒危植物巧家五针松基因组微卫星特征分析,植物研究,36(5):775-781)
Semagn K.,Bjornstad A.,and Ndjiondjop M.N.,2006,An overview of molecular marker methods for plants,African Journal of Biotechnology,5(25):2540-2568
Simon S.A.,Zhai J.X.,Nandety R.S.,McCormick K.P.,Zeng J.,Mejia D.,and Meyers B.C.,2009,Short-read sequencing technologies for transcriptional analyses,Anunu.Rev.Plamt Biol.,60(1):305-333
Thao D.V.,Yamashita M.,Watanabe A.,and Shiraishi S.,2013,Development of tetranucleotide microsatellite markers in Pinus kesiya Royle ex Gordon,Conservation Genetics Resources,5(2):405-407
Temnykh S.,DeClerck G.,Lukashova A.,Lipovich L.,Cartinhour S.,and McCouch S.,2001,Computational and experi mental analysis of microsatellites in rice(Oryza sativa L.):frequency,length variation,transposon associations,and genetic marker potential,Genome Research,59(19):4001-4009
Zheng J.,Ding C.X.,Wang L.S.,Li G.L.,Shi J.Y.,Li H.,Wang H.L.,and Suo Y.R.,2011,Anthocyanins composition and antioxidant activity of wild Lycium ruthenicum Murr.from Qinghai-Tibet Plateau,Food Chemistry,126(3):859-865
Zeng S.H.,Wu M.,Zou C.Y.,Liu X.M.,Shen X.F.,Hayward A.,Liu C.Z.,and Wang Y.,2014,Comparative analysis of anthocyanin biosynthesis during fruit development in two Lycium species,Physiologia Plantarum,150(4):505-516
Zhang Z.,Zhang H.G.,Mo C.,and Zhang L.,2015,Transcriptome sequencing analysis and development of EST-SSRmarkers for Pinus koraiensis,Linye Kexue(Scientia Silvae Sinicae),51(8):114-120(张振,张含国,莫迟,张磊,2015,红松转录组SSR分析及EST-SSR标记开发,林业科学,51(8):114-120)