不同大小规格墨西哥湾扇贝(Argopecten irradians concentricus)转录组分析及生长相关基因筛选
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摘要
墨西哥湾扇贝(Argopecten irradians concentricus)是中国双壳贝类养殖的重要经济品种,具有较高的研究价值,然而,转录组学及基因组学信息的匮乏,严重制约了该贝的遗传改良进程。本研究基于Illumina Hi SeqTM2000平台,对5月龄大规格(平均体质量16.05 g)和小规格(平均体质量5.57 g)墨西哥湾扇贝进行转录组测序,经de nove组装,大、小规格墨西哥湾扇贝分别获得120 563和118 794条高质量unigenes,unigene的平均长度分别为944 bp和969 bp;按照FDR≤0.001且|log2Ratio|≥1的原则,共筛选出9 901个差异表达基因,其中,大规格组相对于小规格组,上调基因4 976个(50.26%),下调基因4 925个(49.74%);六大公共数据库Blast比对结果显示,差异表达基因在Nr数据库的注释率最高,其次是GO数据库;从已获功能注释的差异表达基因中筛选得到47个生长相关候选基因,涉及多个生长因子及其受体;GO功能富集结果显示,膜、L-氨基酸运输和糖基键水解酶活性分别是细胞组分、生物过程和分子功能富集unigene最多的条目;Pathway富集分析发现,3 699条差异表达基因被成功注释到252条代谢通路中,其中33条为显著性富集通路(p<0.05),富集度最高的是焦点粘附;大规格组中共鉴定出10 870个SSRs和242 551个SNPs,小规格组中共鉴定出10 857个SSRs和228 121个SNPs。研究结果为墨西哥湾扇贝分子标记批量开发、功能相关基因挖掘以及分子遗传育种提供了理论数据支撑。
Argopecten irradians concentricus is a commercially important bivalve species for aquaculture in China,with high research value. However, the scarcity of transcriptome and genomic resources have severely constrained the progress of its genetic improvement. This study was based on the Illumina HiSeqTM2000 platform to perform transcriptome sequencing for the large size(average body weight was 16.05 g) and the small size(average body weight was 5.57 g) of A. i. concentricus. By de nove assembly, large and small sizes of A. i. concentricus obtained120 563 and 118 794 high-quality unigenes, and the average length of these unigenes were 944 bp and 969 bp,respectively; According to the principle of FDR≤0.001 and |log2Ratio|≥1, a total of 9 901 differentially expressed genes(DEGs) were screened. Among them, the large-size group compared with the small-size group, there were 4 976(50.26%) up-regulated genes, 4 925(49.74%) down-regulated genes. The results of blast comparison between six major public databases showed that the DEGs have the highest annotation rate in Nr database, followed by GO database. 47 growth-related candidate genes were screened from the DEGs that have been functionally annotated,involving multiple growth factors and their receptors. GO functional enrichment results showed that membrane,L-amino acid transport and sugar-based key hydrolase activity were enriched with the largest number of unigene entries of cell composition, biological processes and molecular function. Pathway enrichment analysis showed that 3 699 differentially expressed genes were successfully annotated into 252 metabolic pathways, 33 of which were significantly enriched(p<0.05), and the highest enrichment was focal adhesion. In the large size group, a total of10 870 SSRs and 242 551 SNPs were identified, and 10 857 SSRs and 228 121 SNPs were identified in the small size group. The results could provide theoretical data support for A. i. concentricus on batch development of molecular markers, deep excavation of functional related genes and molecular genetic breeding.
Argopecten irradians concentricus is a commercially important bivalve species for aquaculture in China,with high research value. However, the scarcity of transcriptome and genomic resources have severely constrained the progress of its genetic improvement. This study was based on the Illumina HiSeqTM2000 platform to perform transcriptome sequencing for the large size(average body weight was 16.05 g) and the small size(average body weight was 5.57 g) of A. i. concentricus. By de nove assembly, large and small sizes of A. i. concentricus obtained120 563 and 118 794 high-quality unigenes, and the average length of these unigenes were 944 bp and 969 bp,respectively; According to the principle of FDR≤0.001 and |log2Ratio|≥1, a total of 9 901 differentially expressed genes(DEGs) were screened. Among them, the large-size group compared with the small-size group, there were 4 976(50.26%) up-regulated genes, 4 925(49.74%) down-regulated genes. The results of blast comparison between six major public databases showed that the DEGs have the highest annotation rate in Nr database, followed by GO database. 47 growth-related candidate genes were screened from the DEGs that have been functionally annotated,involving multiple growth factors and their receptors. GO functional enrichment results showed that membrane,L-amino acid transport and sugar-based key hydrolase activity were enriched with the largest number of unigene entries of cell composition, biological processes and molecular function. Pathway enrichment analysis showed that 3 699 differentially expressed genes were successfully annotated into 252 metabolic pathways, 33 of which were significantly enriched(p<0.05), and the highest enrichment was focal adhesion. In the large size group, a total of10 870 SSRs and 242 551 SNPs were identified, and 10 857 SSRs and 228 121 SNPs were identified in the small size group. The results could provide theoretical data support for A. i. concentricus on batch development of molecular markers, deep excavation of functional related genes and molecular genetic breeding.
引文
Artigaud S.,Thorne M.A.S.,Richard J.,Lavaud R.,Jean F.,FlyeSainte-Marie J.,Peck L.S.,Pichereau V.,and Clark M.S.,2014,Deep sequencing of the mantle transcriptome of the great scallop pecten maximus,Mar.Genomics.,15:3-4
Dong Y.H.,2012,Transcriptome analysis using 454 pyrosequencing and cloning and expression of growth-related genes for the blood clam Tegillarca granosa,Dissertation for Ph.D.,Ocean University of China,Supervisor:Bao Z.M.,pp.48(董迎辉,2012,泥蚶高通量转录组分析及生长相关基因的克隆与表达研究,博士学位论文,中国海洋大学,导师:包振民,pp.48)
Fan S.G.,Zhang D.L.,Guo Y.H.,Liu B.S.,and Yu D.H.,2015,Characterization of the bay scallop(Argopecten irradians concentricus Say)transcriptome and identification of growthrelated genes,Marine Genomics,24(1):225-227
Feng Z.P.,Zhang Z.,Kesteren R.E.,Straub V.A.,van Nierop P.,Jin K.,Nejatbakhsh N.,Goldberg J.I.,Spencer G.E.,Yeoman M.S.,Wildering W.,Coorssen J.R.,Croll R.P.,Buck L.T.,Syed N.I.,and Smit A.B.,2009,Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis,BMC Genomics,10:451
Freer A.,Bridgett S.,Jiang J.,and Cusack M.,2014,Biomineral proteins from mytilus edulis mantle tissue transcriptome,Mar.Biotechnol.,16(1):34-45
Guo H.,Ye C.X.,Wang A.L.,Xian J.A.,Liao S.A.,Miao Y.T.,and Zhang S.P.,2013,Trascriptome analysis of the Pacific white shrimp Litopenaeus vannamei exposed tonitrite by RNA-seq,Fish Shellfish Immunol.,35(6):2008-2016
Hou R.,Bao Z.,Wang S.,Su H.,Li Y.,Du H.,Hu J.,Wang S.,and Hu X.,2011,Transcriptome sequencing and de novo analysis for Yesso scallop(Patinopecten yessoensis)using 454GS FLX,PLoS One,6(6):e21560
Huan P.,Wang H.X.,and Liu B.Z.,2012,Transcriptomic analysis of the clam Meretrix meretrix on different larval stages,Marine Biotechnology,14(1):69-78
Kanehisa M.,Araki M.,Goto S.,Hattori M.,Hirakawa M.,Itoh M.,Katayama T.,Kawashima S.,Okuda S.,Tokimatsu T.,and Yamanishi Y.,2008,KEGG for linking genomes to life and the environment,Nucleic Acids Research,36(suppl 1):D480-D484
Lei C.,Bei W.L.,Wang X.Y.,Du X.D.,Jiao Y.,Huang R.L.,Wang Q.H.,and Deng Y.W.,2017,Development of SSRmarker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii,Electronic Journal of Biotechnology,25:70-74
Li J.Y.,Pan L.Q.,Miao J.J.,Xu R.Y.,and Xu W.J.,2016,De novo assembly and characterization of the ovarian transcriptome reveal mechanisms of the final maturation stage in Chinese scallop Chlamys farreri,Comparative Biochemistry and Physiology Part D:Genomics and Proteomics,20:118-124
Li R.Q.,Li Y.R.,Fang X.D.,Yang H.M.,Wang J.,Kristiansen K.,and Wang J.,2009,SNP detection for massively parallel whole-genome resequencing,Genome Research,19(6):1124-1132
Liang C.,Liu X.,Siuming Y.,and Bao L.L.,2013,De novo assembly and characterization of Camelina sativa transcriptome by paired-end sequencing,BMC Genomics,14(1):146
Liu Z.G.,Wang.H.,Li Z.M.,and Zheng Y.L.,2007,Upper incipient lethal temperature of Argopecten irradians concentricus Say,Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),14(5):778-785(刘志刚,王辉,栗志民,郑云龙,2007,墨西哥湾扇贝高起始致死温度的研究,中国水产科学,14(5):778-785)
Liu Z.G.,Zhang Q.Z.,Zhu X.W.,Wang H.,Chen J.,and Sun X.Z.,2013,Effect of selection for two traits on the adductor weight of the bayscallop,Argopeten irradians concentricus(Say),Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),20(4):771-777(刘志刚,章启忠,朱晓闻,王辉,陈静,孙小真,2013,海湾扇贝南部亚种闭壳肌质量的双性状选择效应,中国水产科学,20(4):771-777)
Luo H.,Ye H.,Xiao S.J.,Zheng S.M.,Wang X.Q.,and Wang Z.Y.,2015,Application of transcriptomics technology to aquatic animal research,Shuichan Xuebao(Journal of Fisheries of China),39(4):598-607(罗辉,叶华,肖世俊,郑曙明,王晓清,王志勇,2015,转录组学技术在水产动物研究中的运用,水产学报,39(4):598-607)
Lu X.,Wang H.X.,Liu B.Z.,and Xiang J.H.,2012,Three EST-SSR markers associated with QTL for the growth of the clam Meretrix meretrix revealed by selective genotyping,Marine Biotechnology,15(1):16-25
Mortazavi A.,Williams B.A.,McCue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mammalian transcriptomes by RNA-Seq,Nature Methods,5(7):621-628
Mu Y.,Li M.,Ding F.,Ding Y.,Ao J.,Hu S.,and Chen X.,2014,De novo characterization of the spleen transcriptome of the large yellow croaker(Pseudosciaena crocea)and analysis of the immune relevant genes and pathways involved in the antiviral response,PLoS One,9(5):e97471
Nguyen C.,Nguyen T.G.,Nguyen L.V.,Pham Q.H.,Nguyen T.H.,Pham H.T.,Nguyen H.T.,Ha T.T.,Dau T.H.,Vu H.T.,Nguyen D.D.,Nguyen T.T.,Nguyen H.H.,Quyen D.V.,Chu H.H.,and Dinh K.D.,2016,De novo,assembly and transcriptome characterization of major growth-related genes in various tissues of penaeus monodon,Aquaculture,464:545-553
Sookruksawong S.,Sun F.,Liu Z.,and Tassanakajon A.,2013,RNA-seq analysis reveals genes associated with resistance to taura syndrome virus(TSV)in the Pacific white shrimp Litopenaeus vannamei,Dev.Comp.Immuno.,41(4):523-533
Sun S.,Xuan F.,Fu H.,Zhu J.,Ge X.,and Gu Z.,2015,Transciptomic and histological analysis of hepatopancreas,muscle and gill tissues of oriental river prawn(Macrobrachium nipponense)in response to chronic hypoxia,BMC Genomics,16(1):491
Truebano M.,Burns G.,Thorne M.A.S.,Hillyard G.,Peck L.S.,Skibinski D.O.F.,and Clack M.S.,2010,Transcriptional response to heat stress in the Antarctic`bivalve Laternula elliptica,Journal of Experimental Marine Biology and Ecology,391(1-2):65-72
Valenzuela-Miranda D.,Del Río-Portilla M.A.,and Gallardo-Escárate C.,2015,Characterization of the growth-related transcriptome in California red abalone(Haliotis rufescens)through RNA-Seq analysis,Marine Genomics,24:199-202
Wang L.,Feng Z.,Wang X.,Wang X.,and Zhang X.,2010,DEGseq:an R package for identifying differentially expressed genes from RNA-seq data,Bioinformatics,26(1):136-138
Wilbur A.E.,and Gaffney P.M.,1997,Agenetic basis for geographic variation in shell morphology in the bay scallop,Argopecten irradians,Marine Biology,128(1):97-105
Yang Y.,Yu H.,Li H.,and Wang A.L.,2016,Transcriptome profiling of grass carp(Ctenopharyngodon idellus)infected with Aeromonas hydrophila,Fish and shellfish immunology,51:329-336
Ye J.,Fang L.,Zheng H.,Zhang Y.,Chen J.,Zhang Z.J.,Wang J.,Li S.T.,Li R.Q.,Bolund L.,and Wang J.,2006,WEGO:a web tool for plotting GO annotations,Nucleic Acids Research,34(Web Server issue):293-297
Zhang F.S.,He Y.C.,Qi L.X.,Sun L.N.,and Liu B.Z.,1994,Introduction and spat-rearing of the F1generation of Argopecten irradians concentricus,Haiyang Yu Huzhao(O-ceanologiaceanologia Et Limnologia Sinica),25(4):372-377(张福绥,何义朝,亓铃欣,孙鲁宁,刘保忠,1994,墨西哥湾扇贝的引种和子一代苗种培育,海洋与湖沼,25(4):372-377)
Zhu J.J.,Li C.,Ao Q.W.,Tan Y.,Luo Y.J.,Guo Y.F.,Lan G.Q.,Jing H.S.,and Gan X.,2015,Trancriptomic profiling revealed the signatures of acute immune response in tilapia(Oreochromis niloticus)following streptococcus iniae challenge,Fish and shellfish immunology,46(2):346-353
Dong Y.H.,2012,Transcriptome analysis using 454 pyrosequencing and cloning and expression of growth-related genes for the blood clam Tegillarca granosa,Dissertation for Ph.D.,Ocean University of China,Supervisor:Bao Z.M.,pp.48(董迎辉,2012,泥蚶高通量转录组分析及生长相关基因的克隆与表达研究,博士学位论文,中国海洋大学,导师:包振民,pp.48)
Fan S.G.,Zhang D.L.,Guo Y.H.,Liu B.S.,and Yu D.H.,2015,Characterization of the bay scallop(Argopecten irradians concentricus Say)transcriptome and identification of growthrelated genes,Marine Genomics,24(1):225-227
Feng Z.P.,Zhang Z.,Kesteren R.E.,Straub V.A.,van Nierop P.,Jin K.,Nejatbakhsh N.,Goldberg J.I.,Spencer G.E.,Yeoman M.S.,Wildering W.,Coorssen J.R.,Croll R.P.,Buck L.T.,Syed N.I.,and Smit A.B.,2009,Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis,BMC Genomics,10:451
Freer A.,Bridgett S.,Jiang J.,and Cusack M.,2014,Biomineral proteins from mytilus edulis mantle tissue transcriptome,Mar.Biotechnol.,16(1):34-45
Guo H.,Ye C.X.,Wang A.L.,Xian J.A.,Liao S.A.,Miao Y.T.,and Zhang S.P.,2013,Trascriptome analysis of the Pacific white shrimp Litopenaeus vannamei exposed tonitrite by RNA-seq,Fish Shellfish Immunol.,35(6):2008-2016
Hou R.,Bao Z.,Wang S.,Su H.,Li Y.,Du H.,Hu J.,Wang S.,and Hu X.,2011,Transcriptome sequencing and de novo analysis for Yesso scallop(Patinopecten yessoensis)using 454GS FLX,PLoS One,6(6):e21560
Huan P.,Wang H.X.,and Liu B.Z.,2012,Transcriptomic analysis of the clam Meretrix meretrix on different larval stages,Marine Biotechnology,14(1):69-78
Kanehisa M.,Araki M.,Goto S.,Hattori M.,Hirakawa M.,Itoh M.,Katayama T.,Kawashima S.,Okuda S.,Tokimatsu T.,and Yamanishi Y.,2008,KEGG for linking genomes to life and the environment,Nucleic Acids Research,36(suppl 1):D480-D484
Lei C.,Bei W.L.,Wang X.Y.,Du X.D.,Jiao Y.,Huang R.L.,Wang Q.H.,and Deng Y.W.,2017,Development of SSRmarker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii,Electronic Journal of Biotechnology,25:70-74
Li J.Y.,Pan L.Q.,Miao J.J.,Xu R.Y.,and Xu W.J.,2016,De novo assembly and characterization of the ovarian transcriptome reveal mechanisms of the final maturation stage in Chinese scallop Chlamys farreri,Comparative Biochemistry and Physiology Part D:Genomics and Proteomics,20:118-124
Li R.Q.,Li Y.R.,Fang X.D.,Yang H.M.,Wang J.,Kristiansen K.,and Wang J.,2009,SNP detection for massively parallel whole-genome resequencing,Genome Research,19(6):1124-1132
Liang C.,Liu X.,Siuming Y.,and Bao L.L.,2013,De novo assembly and characterization of Camelina sativa transcriptome by paired-end sequencing,BMC Genomics,14(1):146
Liu Z.G.,Wang.H.,Li Z.M.,and Zheng Y.L.,2007,Upper incipient lethal temperature of Argopecten irradians concentricus Say,Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),14(5):778-785(刘志刚,王辉,栗志民,郑云龙,2007,墨西哥湾扇贝高起始致死温度的研究,中国水产科学,14(5):778-785)
Liu Z.G.,Zhang Q.Z.,Zhu X.W.,Wang H.,Chen J.,and Sun X.Z.,2013,Effect of selection for two traits on the adductor weight of the bayscallop,Argopeten irradians concentricus(Say),Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),20(4):771-777(刘志刚,章启忠,朱晓闻,王辉,陈静,孙小真,2013,海湾扇贝南部亚种闭壳肌质量的双性状选择效应,中国水产科学,20(4):771-777)
Luo H.,Ye H.,Xiao S.J.,Zheng S.M.,Wang X.Q.,and Wang Z.Y.,2015,Application of transcriptomics technology to aquatic animal research,Shuichan Xuebao(Journal of Fisheries of China),39(4):598-607(罗辉,叶华,肖世俊,郑曙明,王晓清,王志勇,2015,转录组学技术在水产动物研究中的运用,水产学报,39(4):598-607)
Lu X.,Wang H.X.,Liu B.Z.,and Xiang J.H.,2012,Three EST-SSR markers associated with QTL for the growth of the clam Meretrix meretrix revealed by selective genotyping,Marine Biotechnology,15(1):16-25
Mortazavi A.,Williams B.A.,McCue K.,Schaeffer L.,and Wold B.,2008,Mapping and quantifying mammalian transcriptomes by RNA-Seq,Nature Methods,5(7):621-628
Mu Y.,Li M.,Ding F.,Ding Y.,Ao J.,Hu S.,and Chen X.,2014,De novo characterization of the spleen transcriptome of the large yellow croaker(Pseudosciaena crocea)and analysis of the immune relevant genes and pathways involved in the antiviral response,PLoS One,9(5):e97471
Nguyen C.,Nguyen T.G.,Nguyen L.V.,Pham Q.H.,Nguyen T.H.,Pham H.T.,Nguyen H.T.,Ha T.T.,Dau T.H.,Vu H.T.,Nguyen D.D.,Nguyen T.T.,Nguyen H.H.,Quyen D.V.,Chu H.H.,and Dinh K.D.,2016,De novo,assembly and transcriptome characterization of major growth-related genes in various tissues of penaeus monodon,Aquaculture,464:545-553
Sookruksawong S.,Sun F.,Liu Z.,and Tassanakajon A.,2013,RNA-seq analysis reveals genes associated with resistance to taura syndrome virus(TSV)in the Pacific white shrimp Litopenaeus vannamei,Dev.Comp.Immuno.,41(4):523-533
Sun S.,Xuan F.,Fu H.,Zhu J.,Ge X.,and Gu Z.,2015,Transciptomic and histological analysis of hepatopancreas,muscle and gill tissues of oriental river prawn(Macrobrachium nipponense)in response to chronic hypoxia,BMC Genomics,16(1):491
Truebano M.,Burns G.,Thorne M.A.S.,Hillyard G.,Peck L.S.,Skibinski D.O.F.,and Clack M.S.,2010,Transcriptional response to heat stress in the Antarctic`bivalve Laternula elliptica,Journal of Experimental Marine Biology and Ecology,391(1-2):65-72
Valenzuela-Miranda D.,Del Río-Portilla M.A.,and Gallardo-Escárate C.,2015,Characterization of the growth-related transcriptome in California red abalone(Haliotis rufescens)through RNA-Seq analysis,Marine Genomics,24:199-202
Wang L.,Feng Z.,Wang X.,Wang X.,and Zhang X.,2010,DEGseq:an R package for identifying differentially expressed genes from RNA-seq data,Bioinformatics,26(1):136-138
Wilbur A.E.,and Gaffney P.M.,1997,Agenetic basis for geographic variation in shell morphology in the bay scallop,Argopecten irradians,Marine Biology,128(1):97-105
Yang Y.,Yu H.,Li H.,and Wang A.L.,2016,Transcriptome profiling of grass carp(Ctenopharyngodon idellus)infected with Aeromonas hydrophila,Fish and shellfish immunology,51:329-336
Ye J.,Fang L.,Zheng H.,Zhang Y.,Chen J.,Zhang Z.J.,Wang J.,Li S.T.,Li R.Q.,Bolund L.,and Wang J.,2006,WEGO:a web tool for plotting GO annotations,Nucleic Acids Research,34(Web Server issue):293-297
Zhang F.S.,He Y.C.,Qi L.X.,Sun L.N.,and Liu B.Z.,1994,Introduction and spat-rearing of the F1generation of Argopecten irradians concentricus,Haiyang Yu Huzhao(O-ceanologiaceanologia Et Limnologia Sinica),25(4):372-377(张福绥,何义朝,亓铃欣,孙鲁宁,刘保忠,1994,墨西哥湾扇贝的引种和子一代苗种培育,海洋与湖沼,25(4):372-377)
Zhu J.J.,Li C.,Ao Q.W.,Tan Y.,Luo Y.J.,Guo Y.F.,Lan G.Q.,Jing H.S.,and Gan X.,2015,Trancriptomic profiling revealed the signatures of acute immune response in tilapia(Oreochromis niloticus)following streptococcus iniae challenge,Fish and shellfish immunology,46(2):346-353