空心李RNA-seq SSR信息分析及分子标记开发
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摘要
为开发适用于空心李的目的基因SSR分子标记。基于空心李果实中果皮转录组测序数据,利用Trinity软件组装45 389条Unigene,其中32 822条获得功能注释。通过KEGG代谢通路分析发现817条Unigene与碳水化合物代谢相关。利用MISA软件在所有Unigene中共检测到9 797个SSR位点,分布于7 963条Unigene中,其占总Unigene数的17.54%。优势重复基序为二核苷酸(占总SSR位点数的40.57%)、单核苷酸(39.34%)和三核苷酸(18.58%)。AG/CT是二核苷酸中最多的重复类型(32.40%),三核苷酸重复类型以AAG/CTT为主(5.87%)。根据已注释的与果实品质(大小,糖和酸合成等)相关Uningene序列,挖掘到47个目的基因SSR分子标记,以‘李子王30号’空心李种质叶片基因组DNA为模板对这些SSR标记进行有效性验证,39对引物能扩增出清晰条带,其中11对引物在5份不同空心李种质中表现出多态性。开发的空心李目的基因SSR标记为空心李种质遗传多样性分析、指纹图谱构建和果实品质性状关联分析提供有效标记。
In order to develop the SSR molecular markers for the target gene of Kongxinli, 45 389 Unigenes were assembled by Trinity software based on the transcriptome sequencing data of pericarp in Kongxinli fruit, of which32 822 were functional annotations. Analysis of KEGG metabolic pathway revealed that 817 Unigenes were related to carbohydrate metabolism. A total of 9 797 SSR loci were detected in 7 963 U nigenes using MISA software,accounting for 17.54% of the total number of Unigenes. The dominant repeat motifs were dinucleotides(40.57% of total SSR loci), single nucleotides(39.34%) and trinucleotides(18.58%). AG/CT was the most common duplicate type of dinucleotides(32.40%) and AAG/CTT was the main duplicate type of trinucleotides(5.87%). According to the annotated Unigene sequence related to fruit quality(size, sugar and acid synthesis), 47 target gene SSR markers were identified. The validity of these SSR markers was validated by using genomic DNA of leaves of 'Liziwang No. 30' Kongxinli germplasm as template. 39 pairs of primers could amplify clear bands, and 11 pairs of primers showed polymorphism in 5 different hollow plum germplasms. The SSR markers for target gene developed in this study could provide effective markers for genetic diversity analysis, fingerprint map construction and traits association of Kongxinli.
In order to develop the SSR molecular markers for the target gene of Kongxinli, 45 389 Unigenes were assembled by Trinity software based on the transcriptome sequencing data of pericarp in Kongxinli fruit, of which32 822 were functional annotations. Analysis of KEGG metabolic pathway revealed that 817 Unigenes were related to carbohydrate metabolism. A total of 9 797 SSR loci were detected in 7 963 U nigenes using MISA software,accounting for 17.54% of the total number of Unigenes. The dominant repeat motifs were dinucleotides(40.57% of total SSR loci), single nucleotides(39.34%) and trinucleotides(18.58%). AG/CT was the most common duplicate type of dinucleotides(32.40%) and AAG/CTT was the main duplicate type of trinucleotides(5.87%). According to the annotated Unigene sequence related to fruit quality(size, sugar and acid synthesis), 47 target gene SSR markers were identified. The validity of these SSR markers was validated by using genomic DNA of leaves of 'Liziwang No. 30' Kongxinli germplasm as template. 39 pairs of primers could amplify clear bands, and 11 pairs of primers showed polymorphism in 5 different hollow plum germplasms. The SSR markers for target gene developed in this study could provide effective markers for genetic diversity analysis, fingerprint map construction and traits association of Kongxinli.
引文
Alisoltani A.,Ebrahimi S.,Azarian S.,Hematyar M.,Shiran B.,Jahanbazi H.,Fallahi H.,Mousavi-Fard S.,and Rafiei F.,2016,Parallel consideration of SSRs and differentially expressed genes under abiotic stress for targeted development of functional markers in almond and related Prunus species,Sci.Hort.,198(26):462-472
Ammara I.B.,Sonnanteb G.,and Dridi B.A.,2015,Genetic variability in wild cardoon(Cynara cardunculus L.var.sylvestris)revealed by SSR markers and morphological traits,Sci.Hort.,185:76-81
Bhandawat A.,Sharma V.,Sharma H.,Sood A.,and Sharma R.K.,2015,Development and crosstransferability of functionally relevant microsatellite markers in Dendrocalamus latiflorus and related bamboo species,J.Genet.,94(S):48-55
Chen L.N.,Ma Q.G.,Zhang J.P.,Zhou B.B.,and Pei D.,2014,Development of BAC-SSR markers in walnut and its application in genetic diversity analysis,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),36(6):24-29(陈凌娜,马庆国,张俊佩,周贝贝,裴东,2014,核桃BES-SSR的开发及在遗传多样性分析中的应用,北京林业大学学报,36(6):24-29)
Chen Y.N.,Dai X.G.,Hou J.,Guan H.W.,Wang Y.X.,Li Y.,and Yin T.M.,2016,DNA fingerprinting of oil camellia cultivars with SSR markers,Tree Genetics&Genomes,12(1):7
Chhotaray C.,Mishra J.,Sahoo P.K.,Das S.,Mahapatra K.D.,Saha J.N.,Baranski M.,Robinson N.,and Das P.,2015,Development of twenty-seven genic SSR markers and screening for their association with resistance to Aeromonas hydrophila infection in rohu(Labeo rohita,Hamilton),Biochem.Syst.Ecol.,58(6):85-90
Conesa A.,Gotz S.,Garcia-Gomez J.M.,Terol J.,Talon M.,and Robles M.,2005,Blast2GO:a universal tool for annotation,visualization and analysis in functional genomics research,Bioinformatics,21(18):3674-3676
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
Fang Z.Z.,Ye X.F.,Zhou D.R.,Jiang C.C.,and Pan S.L.,2016,Analysis on SSR information in'Furongli'plum transcriptome and development of molecular markers in Prunus salicina Lindl.,Guoshu Xuebao(Journal of Fruit Science),33(4):416-424(方智振,叶新福,周丹蓉,姜翠翠,潘少霖,2016,‘芙蓉李’转录组SSR信息分析与分子标记开发,果树学报,33(4):416-424)
Fang Z.Z.,Zhou D.R.,Jiang C.C.,Liao R.Y.,and Ye X.F.,2014,Cloning and bioinformatic analysis of Ran genes from Prunus salicina Lindl.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(4):780-787(方智振,周丹蓉,姜翠翠,廖汝玉,叶新福,2014,李Ran基因的克隆与生物信息学分析,分子植物育种,12(4):780-787)
Feng C.J.,2005,Phylogenetic relationship and genetic diversity revealed by RAPD,SSR,ISSR markers in Prunus,Thesis for M.S.,Hebei Agricultural University,Supervisor:Yang J.M.,pp.24-25(冯晨静,2005,李种质资源RAPD、SSR、IS-SR亲缘关系鉴定及遗传多样性研究,硕士学位论文,河北农业大学,导师:杨建民,pp.24-25)
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thompson D.A.,Amit I.,Adiconis X.,Fan L.,Raychowdhury R.,Zeng Q.D.,Chen Z.H.,Mauceli E.,Hacohen N.,Gnirke A.,Rhind N.,Palma F.D.,Birren B.W.,Nusbaum C.,Lindblad-Toh K.,Friedman N.,and Regev A.,2011,Full-length transcriptome assembly from RNA-Seq data without a reference genome,Nat.Biotechnol.,29(7):644-652
Kanehisa M.,Goto S.,Kawashima S.,Okuno Y.,and Hattori M.,2004,The KEGG resource for deciphering the genome,Nucleic Acids Res.,32(D):277-280
Li H.,Ruan C.J.,Wang L.,Ding J.,and Tian X.J.,2017,Development of RNA-Seq SSR markers and application to genetic relationship analysis among sea buckthorn germplasm,J.Am.Soc.Hort.Sci.,142(3):200-208
Pan L.,Zhang J.,Chen N.,Chen M.,Wang M.,Wang T.,Chi X.,Yuan M.,Wan Y.,Yu S.,and Liu F.,2017,Molecular characterization and expression profiling of the phosphoenolpyruvate carboxylase genes in peanut(Arachis hypogaea L.),Russ.J.Plant Physiol.,64(4):576-587
Rai A.,Yamazaki M.,Takahashi H.,Nakamura M.,Kojoma M.,Suzuki H.,and Saito K.,2016,RNA-seq transcriptome analysis of Panax japonicus,and its comparison with other panax species to identify potential genes involved in the saponins biosynthesis,Front.Plant Sci.,7:481
Shangguan L.F.,Li X.Y.,Ning N.,Wang Y.Z.,Zhang Z.,and Fang J.G.,2011,Development of EST-SSR markers in apricot,Yuanyi Xuebao(Acta Horticulturae Sinica),38(1):43-54(上官凌飞,李晓颖,宁宁,王玉柱,章镇,房经贵,2011,杏EST-SSR标记的开发,园艺学报,38(1):43-54)
Sun L.L.,Mu Y.H.,Zhen C.F.,and Ye W.J.,2017,Genetic diversity of SSR analysis of plum and apricot in cold region,Beifang Yuanyi(Northern Horticulture),(13):50-55(孙莉莉,牟蕴慧,甄灿福,叶万军,2017,寒地李和杏遗传多样性SSR分析,北方园艺,(13):50-55)
Tatusov R.L.,Natale D.A.,Garkavtsev I.V.,Tatusova T.A.,Shankavaram U.T.,Rao B.S.,Kiryutin B.,Galperin M.Y.,Fedorova N.D.,and Koonin E.V.,2001,The COG database:new developments in phylogenetic classification of proteins from complete genomes,Nucleic Acids Res.,29(1):22-28
Yan R.,Ruan C.J.,Du W.,Ding J.,Wu B.,and Liu L.Y.,2018,Development of SSR markers for target-genes derived from Camellia oleifera by RNA-seq technology,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(8):2540-2548(闫蕊,阮成江,杜维,丁健,吴波,刘祾悦,2018,RNA-seq技术开发油茶目的基因SSR标记,分子植物育种,16(8):2540-2548)
Ye J.,Fang L.,Zheng H.,Zhang Y.,Chen J.,Zhang Z.,Wang J.,Li S.,Li R.,Bolund L.,and Wand J.,2006,WEGO:a web tool for plotting GO annotations,Nucleic Acids Res.,34(W):293-297
Zhang H.,Wei L.,Miao H.,Zhang T.,and Wang C.,2012,Development and validation of genic-SSR markers in sesame by RNA-seq,BMC Genomics,13(1):316
Zhang M.,Zhou C.H.,Chen T.,Dai W.S.,and Zhang C.,2017,Analysis of SSR information in transcriptome and development of molecular markers in Torreya grandis,Guoshu Xuebao(Journal of Fruit Science),34(10):1258-1265(张敏,周彩红,陈焘,戴文圣,张迟,2017,榧树转录组SSR信息分析及其分子标记开发,果树学报,34(10):1258-1265)
Zhang M.F.,Jiang L.M.,Zhang D.M.,and Jia G.X.,2015,De novo transcriptome characterization of Lilium'Sorbonne'and key enzymes related to the flavonoid biosynthesis,Mol.Genet.Genomics,290(1):399-412
Zhao H.,Fang Y.L.,Zhang Y.,Fan X.C.,Sun H.S.,Jiang J.F.,and Liu C.H.,2018,Application of trait-marker association analysis in fruit breeding research,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(7):2291-2299(赵惠,房玉林,张颖,樊秀彩,孙海生,姜建福,刘崇怀,2018,关联分析及其在果树育种研究中的应用,分子植物育种,16(7):2291-2299)
Zong Y.,Wang Y.,Zhu Y.Y.,Shao X.,Li Y.Q.,and Guo W.D.,2016,Development and validation of SSR markers based on transcriptomic data of Chinese cherry(Prunus pseudocerasus),Yuanyi Xuebao(Acta Horticulturae Sinica),43(8):1566-1576(宗宇,王月,朱友银,邵姁,李永强,郭卫东,2016,基于中国樱桃转录组的SSR分子标记开发与鉴定,园艺学报,43(8):1566-1576)
Ammara I.B.,Sonnanteb G.,and Dridi B.A.,2015,Genetic variability in wild cardoon(Cynara cardunculus L.var.sylvestris)revealed by SSR markers and morphological traits,Sci.Hort.,185:76-81
Bhandawat A.,Sharma V.,Sharma H.,Sood A.,and Sharma R.K.,2015,Development and crosstransferability of functionally relevant microsatellite markers in Dendrocalamus latiflorus and related bamboo species,J.Genet.,94(S):48-55
Chen L.N.,Ma Q.G.,Zhang J.P.,Zhou B.B.,and Pei D.,2014,Development of BAC-SSR markers in walnut and its application in genetic diversity analysis,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),36(6):24-29(陈凌娜,马庆国,张俊佩,周贝贝,裴东,2014,核桃BES-SSR的开发及在遗传多样性分析中的应用,北京林业大学学报,36(6):24-29)
Chen Y.N.,Dai X.G.,Hou J.,Guan H.W.,Wang Y.X.,Li Y.,and Yin T.M.,2016,DNA fingerprinting of oil camellia cultivars with SSR markers,Tree Genetics&Genomes,12(1):7
Chhotaray C.,Mishra J.,Sahoo P.K.,Das S.,Mahapatra K.D.,Saha J.N.,Baranski M.,Robinson N.,and Das P.,2015,Development of twenty-seven genic SSR markers and screening for their association with resistance to Aeromonas hydrophila infection in rohu(Labeo rohita,Hamilton),Biochem.Syst.Ecol.,58(6):85-90
Conesa A.,Gotz S.,Garcia-Gomez J.M.,Terol J.,Talon M.,and Robles M.,2005,Blast2GO:a universal tool for annotation,visualization and analysis in functional genomics research,Bioinformatics,21(18):3674-3676
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
Fang Z.Z.,Ye X.F.,Zhou D.R.,Jiang C.C.,and Pan S.L.,2016,Analysis on SSR information in'Furongli'plum transcriptome and development of molecular markers in Prunus salicina Lindl.,Guoshu Xuebao(Journal of Fruit Science),33(4):416-424(方智振,叶新福,周丹蓉,姜翠翠,潘少霖,2016,‘芙蓉李’转录组SSR信息分析与分子标记开发,果树学报,33(4):416-424)
Fang Z.Z.,Zhou D.R.,Jiang C.C.,Liao R.Y.,and Ye X.F.,2014,Cloning and bioinformatic analysis of Ran genes from Prunus salicina Lindl.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(4):780-787(方智振,周丹蓉,姜翠翠,廖汝玉,叶新福,2014,李Ran基因的克隆与生物信息学分析,分子植物育种,12(4):780-787)
Feng C.J.,2005,Phylogenetic relationship and genetic diversity revealed by RAPD,SSR,ISSR markers in Prunus,Thesis for M.S.,Hebei Agricultural University,Supervisor:Yang J.M.,pp.24-25(冯晨静,2005,李种质资源RAPD、SSR、IS-SR亲缘关系鉴定及遗传多样性研究,硕士学位论文,河北农业大学,导师:杨建民,pp.24-25)
Grabherr M.G.,Haas B.J.,Yassour M.,Levin J.Z.,Thompson D.A.,Amit I.,Adiconis X.,Fan L.,Raychowdhury R.,Zeng Q.D.,Chen Z.H.,Mauceli E.,Hacohen N.,Gnirke A.,Rhind N.,Palma F.D.,Birren B.W.,Nusbaum C.,Lindblad-Toh K.,Friedman N.,and Regev A.,2011,Full-length transcriptome assembly from RNA-Seq data without a reference genome,Nat.Biotechnol.,29(7):644-652
Kanehisa M.,Goto S.,Kawashima S.,Okuno Y.,and Hattori M.,2004,The KEGG resource for deciphering the genome,Nucleic Acids Res.,32(D):277-280
Li H.,Ruan C.J.,Wang L.,Ding J.,and Tian X.J.,2017,Development of RNA-Seq SSR markers and application to genetic relationship analysis among sea buckthorn germplasm,J.Am.Soc.Hort.Sci.,142(3):200-208
Pan L.,Zhang J.,Chen N.,Chen M.,Wang M.,Wang T.,Chi X.,Yuan M.,Wan Y.,Yu S.,and Liu F.,2017,Molecular characterization and expression profiling of the phosphoenolpyruvate carboxylase genes in peanut(Arachis hypogaea L.),Russ.J.Plant Physiol.,64(4):576-587
Rai A.,Yamazaki M.,Takahashi H.,Nakamura M.,Kojoma M.,Suzuki H.,and Saito K.,2016,RNA-seq transcriptome analysis of Panax japonicus,and its comparison with other panax species to identify potential genes involved in the saponins biosynthesis,Front.Plant Sci.,7:481
Shangguan L.F.,Li X.Y.,Ning N.,Wang Y.Z.,Zhang Z.,and Fang J.G.,2011,Development of EST-SSR markers in apricot,Yuanyi Xuebao(Acta Horticulturae Sinica),38(1):43-54(上官凌飞,李晓颖,宁宁,王玉柱,章镇,房经贵,2011,杏EST-SSR标记的开发,园艺学报,38(1):43-54)
Sun L.L.,Mu Y.H.,Zhen C.F.,and Ye W.J.,2017,Genetic diversity of SSR analysis of plum and apricot in cold region,Beifang Yuanyi(Northern Horticulture),(13):50-55(孙莉莉,牟蕴慧,甄灿福,叶万军,2017,寒地李和杏遗传多样性SSR分析,北方园艺,(13):50-55)
Tatusov R.L.,Natale D.A.,Garkavtsev I.V.,Tatusova T.A.,Shankavaram U.T.,Rao B.S.,Kiryutin B.,Galperin M.Y.,Fedorova N.D.,and Koonin E.V.,2001,The COG database:new developments in phylogenetic classification of proteins from complete genomes,Nucleic Acids Res.,29(1):22-28
Yan R.,Ruan C.J.,Du W.,Ding J.,Wu B.,and Liu L.Y.,2018,Development of SSR markers for target-genes derived from Camellia oleifera by RNA-seq technology,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(8):2540-2548(闫蕊,阮成江,杜维,丁健,吴波,刘祾悦,2018,RNA-seq技术开发油茶目的基因SSR标记,分子植物育种,16(8):2540-2548)
Ye J.,Fang L.,Zheng H.,Zhang Y.,Chen J.,Zhang Z.,Wang J.,Li S.,Li R.,Bolund L.,and Wand J.,2006,WEGO:a web tool for plotting GO annotations,Nucleic Acids Res.,34(W):293-297
Zhang H.,Wei L.,Miao H.,Zhang T.,and Wang C.,2012,Development and validation of genic-SSR markers in sesame by RNA-seq,BMC Genomics,13(1):316
Zhang M.,Zhou C.H.,Chen T.,Dai W.S.,and Zhang C.,2017,Analysis of SSR information in transcriptome and development of molecular markers in Torreya grandis,Guoshu Xuebao(Journal of Fruit Science),34(10):1258-1265(张敏,周彩红,陈焘,戴文圣,张迟,2017,榧树转录组SSR信息分析及其分子标记开发,果树学报,34(10):1258-1265)
Zhang M.F.,Jiang L.M.,Zhang D.M.,and Jia G.X.,2015,De novo transcriptome characterization of Lilium'Sorbonne'and key enzymes related to the flavonoid biosynthesis,Mol.Genet.Genomics,290(1):399-412
Zhao H.,Fang Y.L.,Zhang Y.,Fan X.C.,Sun H.S.,Jiang J.F.,and Liu C.H.,2018,Application of trait-marker association analysis in fruit breeding research,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(7):2291-2299(赵惠,房玉林,张颖,樊秀彩,孙海生,姜建福,刘崇怀,2018,关联分析及其在果树育种研究中的应用,分子植物育种,16(7):2291-2299)
Zong Y.,Wang Y.,Zhu Y.Y.,Shao X.,Li Y.Q.,and Guo W.D.,2016,Development and validation of SSR markers based on transcriptomic data of Chinese cherry(Prunus pseudocerasus),Yuanyi Xuebao(Acta Horticulturae Sinica),43(8):1566-1576(宗宇,王月,朱友银,邵姁,李永强,郭卫东,2016,基于中国樱桃转录组的SSR分子标记开发与鉴定,园艺学报,43(8):1566-1576)