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Identification of Genes Involved in Immune Response, Microsatellite, and SNP Markers from Expressed Sequence Tags Generated from Hemocytes of Freshwater Pearl Mussel (Hyriopsis cumingii)

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Abstract

Triangle sail mussel (Hyriopsis cumingii) is the most important mussel species commercially exploited for freshwater pearl production in China. However, its genome research is still at the infantry. Genomic resources for this species are largely not available. The objectives of this study was to generate expressed sequence tags from a hemocyte cDNA library, to identify genes involved in defense mechanisms, and to identify polymorphic markers from the expressed sequence tag (EST) resources for genetic analysis. A total of 5,290 ESTs were sequenced, obtaining 481 contigs and 1,165 singletons. BLAST similarity analysis indicated almost half (46.5%) of these ESTs were homologs of known genes while 53.5% were transcripts of unknown identities. Based on sequence similarities, 50 genes were identified as putative genes involved in immune and defense functions such as hemocyte immune process, stress proteins, adhesive proteins, proteases and protease regulators, antimicrobial peptides, lysosomal enzymes, cell apoptosis, and cell cycle proteins. A total of 201 microsatellites were identified from these ESTs, with 31 having sufficient flanking sequences for primer design. Polymerase chain reaction amplification was successful for 18 primer pairs and 14 of them were polymorphic. A total of 987 putative single nucleotide polymorphisms were identified including 204 transitions, 611 transversions, and 172 indels; 12 of them were involved in nine genes of defense mechanisms. These resources provide the material basis for future marker validation and genetic linkage and quantitative trait loci analysis in the freshwater pearl mussel.

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References

  • Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF et al (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252:1651–1656

    Article  PubMed  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  • Altschul SF, Madden TL, Schaffer AA, Zhang J (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Ashburner M, Ball C, Eppig J, Harris M (2000) Gene ontology: tool for the unification of biology. Nat Genet 25:25–29

    Article  PubMed  CAS  Google Scholar 

  • Bachere E, Chagot D, Grizel H (1988) Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation. Dev Comp Immunol 12:549–559

    Article  PubMed  CAS  Google Scholar 

  • Bachere E, Hervio D, Mialhe E (1991) Luminol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas. Dis Aquat Org 6:131–136

    Google Scholar 

  • Beck G, Ellis TW, Habicht GS, Schluter SF, Marchalonis JJ (2002) Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule. Dev Comp Immunol 26:11–26

    Article  PubMed  CAS  Google Scholar 

  • Boutet I, Tanguy A, Moraga D (2004) Response of the Pacific oyster Crassostrea gigas to hydrocarbon contamination under experimental conditions. Gene 329:147–157

    Article  PubMed  CAS  Google Scholar 

  • Cheng W, Hsiao IS, Hsu CH, Chen JC (2004) Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus. Fish Shellfish Immunol 17:235–243

    Article  PubMed  CAS  Google Scholar 

  • Chistiakov DA, Tsigenopoulos CS, Lagnel J, Guo Y-M, Hellemans B, Haley CS, Volckaert FA, Kotoulas G. (2008) A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L. Anim Genet 39:623–634

    Google Scholar 

  • David E, Tanguy A, Pichavant K, Moraga D (2005) Response of the Pacific oyster Crassostrea gigas to hypoxia exposure under experimental conditions. The FEBS Journal 272:5635–5652

    Article  PubMed  CAS  Google Scholar 

  • Ewing B, Hillier LD, Wendl MC, Green P (1998a) Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res 8:175–185

    PubMed  CAS  Google Scholar 

  • Ewing B, Hillier LD, Wendl MC, Green P (1998b) Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res 8:186–194

    PubMed  CAS  Google Scholar 

  • Fujiwara S, Hirokawa Y, Takatsuka Y, Suda K, Asamizu E, Takayanagi T, Shibata D, Tabata S, Tsuzuki M (2007) Gene expression profiling of coccolith-bearing cells and naked cells in haptophyte Pleurochrysis haptonemofera with a cDNA macroarray system. Mar Biotechnol 9:550–560

    Article  PubMed  CAS  Google Scholar 

  • Gueguen Y, Cadoret JP, Flament D, Barreau-Roumiguiere C, Girardot AL, Garnier J, Hoareau A, Bachere E, Escoubas JM (2003) Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas. Gene 303:139–145

    Article  PubMed  CAS  Google Scholar 

  • Hedgecock D, Gaffney PM, Glletquer P, Guo X, Reece K, Warr GW (2005) The case for sequencing the pacific oyster genome. J Shellfish Res 24:429–441

    Google Scholar 

  • Huvet A, Herpin A, Degremont L, Labreuche Y, Samain JF, Cunningham C (2004) The identification of genes from the oyster Crassostrea gigas that are differentially expressed in progeny exhibiting opposed susceptibility to summer mortality. Gene 343:211–220

    Article  PubMed  CAS  Google Scholar 

  • Hwang DM, Dempsey A, Tan KT, Liew CC (1996) A modular domain of NifU, a nitrogen fixation cluster protein, is highly conserved in evolution. J Mol Evol 43:536–540

    Article  PubMed  CAS  Google Scholar 

  • Hwang HY, Duvall MR, Tomlinson S, Boackle RJ (2008) Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment. Mol Immunol 45:2570–2580

    Article  PubMed  CAS  Google Scholar 

  • Kanekiyo M, Itoh N, Kawasaki A, Matsuyama A, Matsuda K, Nakanishi T, Tanaka K (2002) Metallothionein modulates lipopolysaccharide-stimulated tumour necrosis factor expression in mouse peritoneal macrophages. Biochem J 361:363–369

    Article  PubMed  CAS  Google Scholar 

  • Kang YS, Kim YM, Park KI, Kim Cho S, Choi KS, Cho M (2006) Analysis of EST and lectin expressions in hemocytes of Manila clams (Ruditapes philippinarum) (Bivalvia: Mollusca) infected with Perkinsus olseni. Dev Comp Immunol 30:1119–1131

    Article  PubMed  CAS  Google Scholar 

  • Lacoste A, De Cian MC, Cueff A, Poulet SA (2001) Noradrenaline and alpha-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells. J Cell Sci 114:3557–3564

    PubMed  CAS  Google Scholar 

  • Lacoste A, Cueff A, Poulet SA (2002) P35-sensitive caspases, MAP kinases and Rho modulate beta-adrenergic induction of apoptosis in mollusc immune cells. J Cell Sci 115:761–768

    PubMed  CAS  Google Scholar 

  • Lee J, Guo X (2006) Mining EST database for single-nucleotide polymorphisms in the eastern oyster (Crassostrea virginica). J Shellfish Res 25:748–749

    Google Scholar 

  • Li JL (2007) Exploitation and protection of genetic resources of freshwater pearl mussel. Sci Fish Farming 6:1–2

    Google Scholar 

  • Li JL, Qian RH, Bao BL, Wang GL, Qi WD (2005) RAPD analysis on genetic diversity among the stocks of Hyriopsis cumingii from the five large lakes of China. J Shanghai Fish Univ 14:1–5

    Google Scholar 

  • Li JL, Bai ZY, Qian RH (2006) Comparative study on growth performance of cultured populations of Zhuji city and populations of Hyriopsis cumingii from the five lakes of China. Fish Sci Technol Inf 33:243–246

    Google Scholar 

  • Liu YY (1979) Economic fauna of China (freshwater mollusk). Science, Beijing

    Google Scholar 

  • Liu ZJ, Cordes JF (2004) DNA marker technologies and their applications in aquaculture genetics. Aquaculture 238:1–37

    Article  CAS  Google Scholar 

  • Medzhitov R, Janeway C Jr (2000) Innate immune recognition: mechanisms and pathways. Immunol Rev 173:89–97

    Article  PubMed  CAS  Google Scholar 

  • Montagnani C, Le Roux F, Berthe F, Escoubas JM (2001) Cg-TIMP, an inducible tissue inhibitor of metalloproteinase from the Pacific oyster Crassostrea gigas with a potential role in wound healing and defense mechanisms(1). FEBS Lett 500:64–70

    Article  PubMed  CAS  Google Scholar 

  • Nair SV, Del Valle H, Gross PS, Terwilliger DP, Smith LC (2005) Macroarray analysis of coelomocyte gene expression in response to LPS in the sea urchin. Identification of unexpected immune diversity in an invertebrate. Physiol Genomics 22:33–47

    Article  PubMed  CAS  Google Scholar 

  • Parrino J, Hotchkiss RS, Bray M (2007) Prevention of immune cell apoptosis as potential therapeutic strategy for severe infections. Emerg Infect Dis 13:191–198

    Article  PubMed  CAS  Google Scholar 

  • Pipe RK (1990) Hydrolytic enzymes associated with the granular haemocytes of the marine mussel Mytilus edulis. Histochem J 22:595–603

    Article  PubMed  CAS  Google Scholar 

  • Quilang J, Wang S, Li P, Abernathy J, Peatman E, Wang Y, Wang L, Shi Y, Wallace R, Guo X, Liu Z (2007) Generation and analysis of ESTs fom the eastern oyster, Crassostrea virginica Gmelin and identification of microsatellite and SNP markers. BMC Genomics 8:157

    Google Scholar 

  • Reusch TB, Veron AS, Preuss C, Weiner J, Wissler L, Beck A, Klages S, Kube M, Reinhardt R, Bornberg-Bauer E (2008) Comparative analysis of expressed sequence tag (EST) libraries in the seagrass Zostera marina subjected to temperature stress. Mar Biotechnol 10:297–309

    Article  PubMed  CAS  Google Scholar 

  • Saavedra C, Bachere E (2006) Bivalve genomics. Aquaculture 256:1–14

    Article  CAS  Google Scholar 

  • Santos CVD, Cuine S, Rouhier N, Rey P (2005) The arabidopsis plastidic methionine sulfoxide reductase B proteins. sequence and activity characteristics, comparison of the expression with plastidic methionine sulfoxide reductase A, and induction by photooxidative stress. Plant Physiol 138:909–922

    Article  CAS  Google Scholar 

  • Sarropoulou E, Franch R, Louro B, Power DM, Bargelloni L, Magoulas A, Senger F, Tsalavouta M, Patarnello T, Galibert F, Kotoulas G, Geisler R (2007) A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis. BMC Genomics 8:44

    Article  PubMed  CAS  Google Scholar 

  • Sarropoulou E, Nousdili D, Magoulas A, Kotoulas G (2008) Linking the genomes of nonmodel teleosts through comparative genomics. Mar Biotechnol 10:227–233

    Article  PubMed  CAS  Google Scholar 

  • Senger F, Priat C, Hitte C, Sarropoulou E, Franch R, Geisler R, Bargelloni L, Power D, Galibert F (2006) The first radiation hybrid map of a perch-like fish: the gilthead seabream (Sparus aurata L). Genomics 87:793–800

    Article  PubMed  CAS  Google Scholar 

  • Shao J, Xiang L, Li Y, Zhang M, Mao S (1995) Histopathological studies on the plague disease of Hyriopsis Cumingii. J Fisheries China 19:1–7

    Google Scholar 

  • Smith LC, Chang L, Britten RJ, Davidson EH (1996) Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. J Immunol 156:593–602

    PubMed  CAS  Google Scholar 

  • Suzuki T, Shin IT, Kohara Y, Kasahara M (2004) Transcriptome analysis of hagfish leukocytes: a framework for understanding the immune system of jawless fishes. Dev Comp Immunol 28:993–1003

    Article  PubMed  Google Scholar 

  • Tanguy A, Bierne N, Saavedra C, Pina B, Bachere E, Kube M, Bazin E, Bonhomme F, Boudry P, Boulo V, Boutet I, Cancela L, Dossat C, Favrel P, Huvet A, Jarque S, Jollivet D, Klages S, Lapegue S, Leite R, Moal J, Moraga D, Reinhardt R, Samain JF, Zouros E, Canario A (2008) Increasing genomic information in bivalves through new EST collections in four species: Development of new genetic markers for environmental studies and genome evolution. Gene 408:27–36

    Article  PubMed  CAS  Google Scholar 

  • Tincu JA, Taylor SW (2004) Antimicrobial peptides from marine invertebrates. Antimicrob Agents Chemother 48:3645–3654

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Guo X (2007) Development and characterization of EST–SSR markers in the eastern oyster Crassostrea virginica. Mar Biotechnol 9:500–511

    Article  PubMed  CAS  Google Scholar 

  • Wang GL, Wang JJ, Li JL (2006) Preliminary study on applicability of microsatellite primers developed from Crassostrea gigas to genomic analysis of Hyriopsis cumingii. J Fisheries China 1:30–38

    Google Scholar 

  • Wang GL, Yuan YM, Li JL (2007) SSR analysis of genetic diversity and phylogenetic relationships among different populations of Hyriopsis cumingii from the five lakes of China. J Fisheries China 31:152–158

    CAS  Google Scholar 

  • Wang X, Zhang Q, Sun X, Chen Y, Zhai T, Zhuang W, Qi J, Wang Z (2008) Fosmid library construction and initial analysis of end sequences in female half-smooth tongue sole (Cynoglossus semilaevis). Mar Biotechnol (in press). doi:10.1007/s10126-008-9137-2

  • Whitaker HA, Mcandrew BJ, Taggart JB (2006) Construction and characterization of a BAC library for the European sea bass Dicentrarchus labrax. Anim Genet 37:526

    Article  PubMed  CAS  Google Scholar 

  • Wynne JW, O'sullivan MG, Cook MT, Stone G, Nowak BF, Lovell DR, Elliott NG (2008) Transcriptome analyses of amoebic gill disease-affected Atlantic salmon (Salmo salar) tissues reveal localized host gene suppression. Mar Biotechnol 10:388–403

    Article  PubMed  CAS  Google Scholar 

  • Yazawa R, Yasuike M, Leong J, Von Schalburg KR, Cooper GA, Beetz-Sargent M, Robb A, Davidson WS, Jones SR, Koop BF (2008) EST and mitochondrial DNA sequences support a distinct pacific form of salmon louse, Lepeophtheirus salmonis. Mar Biotechnol 10:741–749

    Article  PubMed  CAS  Google Scholar 

  • Yu J, Farjo R, Macnee SP, Baehr W, Stambolian DE, Swaroop A (2003) Annotation and analysis of 10,000 expressed sequence tags from developing mouse eye and adult retina. Genome Biology 4:R65

    Article  PubMed  Google Scholar 

  • Zasloff M (1992) Antibiotic peptides as mediators of innate immunity. Curr Opin Immunol 4:3–7

    Article  PubMed  CAS  Google Scholar 

  • Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–848

    Article  PubMed  CAS  Google Scholar 

  • Zhang G, Fang A, Zeng X, Lu X, Du X, Li H (2003) Study on the disease epidemiology of Hyriopsis cumingii. J Shanghai Fish Univ 12:243–248

    CAS  Google Scholar 

  • Zhang B, Jin W, Zeng Y, Su Z, Hu S, Yu J (2004) EST-based analysis of gene expression in the porcine brain. Genomics Proteomics Bioinformatics/Beijing Genomics Institute 2:237–244

    CAS  Google Scholar 

  • Zhang G, Wu X, Li J (2005) Advances of the studies on diseases of Hyriopsis cumingii and its control. J Shanghai Fish Univ 14:313–318

    Google Scholar 

  • Zou H, Song L, Xu W, Yang G (2005) Molecular cloning and characterization analysis of cDNA encoding g-type lysozyme from scallop (Argopecten irradians). High Technol Lett 15:101–106

    CAS  Google Scholar 

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Acknowledgments

This study was supported by the Key Basic Research Project (06DJ14003) from Science and Technology Commission of Shanghai Municipal Government, the National Science and Technology Pillar Program (2006BAD01A13) from the Ministry of Science and Technology, the Specialized Project for the Key Technology Research of Agricultural Restructuring (06-05-05B) from the Ministry of Agriculture, and the Youth Research Program (2008CG56) from Shanghai Educational Development Foundation.

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Authors and Affiliations

  1. Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecology Certificated by Ministry of Agriculture, E-Institute of Shanghai Universities, Shanghai Ocean University, Shanghai, 201306, China

    Zhiyi Bai, Guiling Wang & Jiale Li

  2. Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100029, China

    Yuxin Yin, Songnian Hu & Xiaowei Zhang

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  1. Zhiyi Bai
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  2. Yuxin Yin
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  3. Songnian Hu
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  4. Guiling Wang
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  5. Xiaowei Zhang
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  6. Jiale Li
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Correspondence to Xiaowei Zhang or Jiale Li.

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Zhiyi Bai and Yuxin Yin contributed equally to this study.

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Bai, Z., Yin, Y., Hu, S. et al. Identification of Genes Involved in Immune Response, Microsatellite, and SNP Markers from Expressed Sequence Tags Generated from Hemocytes of Freshwater Pearl Mussel (Hyriopsis cumingii). Mar Biotechnol 11, 520–530 (2009). https://doi.org/10.1007/s10126-008-9163-0

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