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Analysis of a deep transcriptome from the mantle tissue of Patella vulgata Linnaeus (Mollusca: Gastropoda: Patellidae) reveals candidate biomineralising genes

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Abstract

The gastropod Patella vulgata is abundant on rocky shores in Northern Europe and a significant grazer of intertidal algae. Here we report the application of Illumina sequencing to develop a transcriptome from the adult mantle tissue of P. vulgata. We obtained 47,237,104 paired-end reads of 51 bp, trialled de novo assembly methods and settled on the additive multiple K method followed by redundancy removal as resulting in the most comprehensive assembly. This yielded 29,489 contigs of at least 500 bp in length. We then used three methods to search for candidate genes relevant to biomineralisation: searches via BLAST and Hidden Markov Models for homologues of biomineralising genes from other molluscs, searches for predicted proteins containing tandem repeats and searches for secreted proteins that lacked a transmembrane domain. From the results of these searches we selected 15 contigs for verification by RT-PCR, of which 14 were successfully amplified and cloned. These included homologues of Pif-177/BSMP, Perlustrin, SPARC, AP24, Follistatin-like and Carbonic anhydrase, as well as three containing extensive G-X-Y repeats as found in nacrein. We selected two for further verification by in situ hybridisation, demonstrating expression in the larval shell field. We conclude that de novo assembly of Illumina data offers a cheap and rapid route to a predicted transcriptome that can be used as a resource for further biological study.

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References

  • Bendtsen JD, Nielsen H, Von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–95

    Article  PubMed  Google Scholar 

  • Bradshaw AD (2009) The role of SPARC in extracellular matrix assembly. J Cell Commun Signal 3:239–46

    Article  PubMed  Google Scholar 

  • Bragdon B, Moseychuk O, Saldanha S, King D, Julian J, Nohe A (2011) Bone morphogenetic proteins: a critical review. Cell Signal 23:609–20

    Article  PubMed  CAS  Google Scholar 

  • Clark MS, Thorne MA, Vieira FA, Cardoso JC, Power DM, Peck LS (2010) Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing. BMC Genomics 11:362

    Article  PubMed  Google Scholar 

  • Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–6

    Article  PubMed  CAS  Google Scholar 

  • Delany AM, Hankenson KD (2009) Thrombospondin-2 and SPARC/osteonectin are critical regulators of bone remodeling. J Cell Commun Signal 3:227–38

    Article  PubMed  Google Scholar 

  • Donachy JE, Drake B, Sikes CS (1992) Sequence and atomic-force microscopy analysis of a matrix protein from the shell of the oyster Crassostrea virginica. Mar Biol 114:423–428

    Article  CAS  Google Scholar 

  • Emanuelsson O, Brunak S, Von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–71

    Article  PubMed  CAS  Google Scholar 

  • Emanuelsson O, Nielsen H, Brunak S, Von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–16

    Article  PubMed  CAS  Google Scholar 

  • Fang D, Xu G, Hu Y, Pan C, Xie L, Zhang R (2011) Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata. PLoS One 6:e21860

    Article  PubMed  CAS  Google Scholar 

  • Feldmeyer B, Wheat CW, Krezdorn N, Rotter B, Pfenninger M (2011) Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance. BMC Genomics 12:317

    Article  PubMed  Google Scholar 

  • Finn RD, Clements J, Eddy SR (2011) HMMER web server: interactive sequence similarity searching. Nucleic Acids Res 39:W29–37

    Article  PubMed  CAS  Google Scholar 

  • Freeman JA, Wilbur KM (1948) Carbonic anhydrase in molluscs. Biol Bull 94:55–9

    Article  PubMed  CAS  Google Scholar 

  • Fuchigami T, Sasaki T (2005) The shell structure of the recent Patellogastropoda (Mollusca: Gastropda). Palaeontological Research 9:143–168

    Article  Google Scholar 

  • Hansen KD, Brenner SE, Dudoit S (2010) Biases in Illumina transcriptome sequencing caused by random hexamer priming. Nucleic Acids Res 38:e131

    Article  PubMed  Google Scholar 

  • Heyland A, Vue Z, Voolstra CR, Medina M, Moroz LL (2011) Developmental transcriptome of Aplysia californica. J Exp Zool B Mol Dev Evol 316B:113–34

    Article  PubMed  Google Scholar 

  • Hodgson AN, Le Quesne WJF, Hawkins SJ, Bishop JDD (2007) Factors affecting fertilization success in two species of patellid limpet (Mollusca: Gastropoda) and development of fertilization kinetics models. Mar Biol 150:415–426

    Article  Google Scholar 

  • Iijima M, Takeuchi T, Sarashina I, Endo K (2008) Expression patterns of engrailed and dpp in the gastropod Lymnaea stagnalis. Dev Genes Evol 218:237–51

    Article  PubMed  CAS  Google Scholar 

  • Jackson DJ, Mcdougall C, Green K, Simpson F, Worheide G, Degnan BM (2006) A rapidly evolving secretome builds and patterns a sea shell. BMC Biol 4:40

    Article  PubMed  Google Scholar 

  • Jackson DJ, Mcdougall C, Woodcroft B, Moase P, Rose RA, Kube M, Reinhardt R, Rokhsar DS, Montagnani C, Joubert C, Piquemal D, Degnan BM (2010) Parallel evolution of nacre building gene sets in molluscs. Mol Biol Evol 27:591–608

    Article  PubMed  CAS  Google Scholar 

  • Jackson DJ, Worheide G, Degnan BM (2007) Dynamic expression of ancient and novel molluscan shell genes during ecological transitions. BMC Evol Biol 7:160

    Article  PubMed  Google Scholar 

  • Joubert C, Piquemal D, Marie B, Manchon L, Pierrat F, Zanella-Cleon I, Cochennec-Laureau N, Gueguen Y, Montagnani C (2010) Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization. BMC Genomics 11:613

    Article  PubMed  Google Scholar 

  • Kalamajski S, Oldberg A (2010) The role of small leucine-rich proteoglycans in collagen fibrillogenesis. Matrix Biol 29:248–53

    Article  PubMed  CAS  Google Scholar 

  • Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–98

    Article  PubMed  CAS  Google Scholar 

  • Kin K, Kakoi S, Wada H (2009) A novel role for dpp in the shaping of bivalve shells revealed in a conserved molluscan developmental program. Dev Biol 329:152–66

    Article  PubMed  CAS  Google Scholar 

  • Kleypas J, Feely R, Fabry V, Langdon C, Sabine C, Robbins L (2006) Imapcts of ocean acidification on coral reefs and other marine calcifiers. Report of the workshop sponsored by NSF, NOAA and USGS

    Google Scholar 

  • Kniprath E (1981) Ontogeny of the molluscan shell field—a review. Zoologica Scripta 10:61–79

    Article  Google Scholar 

  • Koop D, Richards GS, Wanninger A, Gunter HM, Degnan BM (2007) The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina. Dev Biol 311:200–12

    Article  PubMed  CAS  Google Scholar 

  • Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T, Schultz J, Ponting CP, Bork P (2004) SMART 4.0: towards genomic data integration. Nucleic Acids Res 32:D142–4

    Article  PubMed  CAS  Google Scholar 

  • Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–9

    Article  PubMed  CAS  Google Scholar 

  • Marie B, Marie A, Jackson DJ, Dubost L, Degnan BM, Milet C, Marin F (2010) Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell. Proteome Sci 8:54

    Article  PubMed  CAS  Google Scholar 

  • Marin F, Amons R, Guichard N, Stigter M, Hecker A, Luquet G, Layrolle P, Alcaraz G, Riondet C, Westbroek P (2005) Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis. J Biol Chem 280:33895–908

    Article  PubMed  CAS  Google Scholar 

  • Marin F, Luquet G (2004) Molluscan shell proteins. CR Palevol 3:469–492

    Article  Google Scholar 

  • Marxen JC, Nimtz M, Becker W, Mann K (2003) The major soluble 19.6 kDa protein of the organic shell matrix of the freshwater snail Biomphalaria glabrata is an N-glycosylated dermatopontin. Biochim Biophys Acta 1650:92–8

    Article  PubMed  CAS  Google Scholar 

  • Michenfelder M, Fu G, Lawrence C, Weaver JC, Wustman BA, Taranto L, Evans JS, Morse DE (2003) Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains. Biopolymers 70:522–33

    Article  PubMed  CAS  Google Scholar 

  • Milan M, Coppe A, Reinhardt R, Cancela LM, Leite RB, Saavedra C, Ciofi C, Chelazzi G, Patarnello T, Bortoluzzi S, Bargelloni L (2011) Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring. BMC Genomics 12:234

    Article  PubMed  CAS  Google Scholar 

  • Miyamoto H, Miyashita T, Okushima M, Nakano S, Morita T, Matsushiro A (1996) A carbonic anhydrase from the nacreous layer in oyster pearls. Proc Natl Acad Sci USA 93:9657–60

    Article  PubMed  CAS  Google Scholar 

  • Miyamoto H, Miyoshi F, Kohno J (2005) The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusc Pinctada fucata. Zoolog Sci 22:311–5

    Article  PubMed  CAS  Google Scholar 

  • Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35:W182–5

    Article  PubMed  Google Scholar 

  • Moroz LL, Edwards JR, Puthanveettil SV, Kohn AB, Ha T, Heyland A, Knudsen B, Sahni A, Yu F, Liu L, Jezzini S, Lovell P, Iannucculli W, Chen M, Nguyen T, Sheng H, Shaw R, Kalachikov S, Panchin YV, Farmerie W, Russo JJ, Ju J, Kandel ER (2006) Neuronal transcriptome of Aplysia: neuronal compartments and circuitry. Cell 127:1453–67

    Article  PubMed  CAS  Google Scholar 

  • Nederbragt AJ, Van Loon AE, Dictus WJ (2002) Expression of Patella vulgata orthologs of engrailed and dpp-BMP2/4 in adjacent domains during molluscan shell development suggests a conserved compartment boundary mechanism. Dev Biol 246:341–55

    Article  PubMed  CAS  Google Scholar 

  • Newman AM, Cooper JB (2007) XSTREAM: a practical algorithm for identification and architecture modeling of tandem repeats in protein sequences. BMC Bioinforma 8:382

    Article  Google Scholar 

  • Orr JC, Fabry VJ, Aumont O, Bopp L, Doney SC, Feely RA, Gnanadesikan A, Gruber N, Ishida A, Joos F, Key RM, Lindsay K, Maier-Reimer E, Matear R, Monfray P, Mouchet A, Najjar RG, Plattner GK, Rodgers KB, Sabine CL, Sarmiento JL, Schlitzer R, Slater RD, Totterdell IJ, Weirig MF, Yamanaka Y, Yool A (2005) Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437:681–6

    Article  PubMed  CAS  Google Scholar 

  • Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, Lee Y, White J, Cheung F, Parvizi B, Tsai J, Quackenbush J (2003) TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics 19:651–2

    Article  PubMed  CAS  Google Scholar 

  • Ren J, Wen LP, Gao XJ, Jin CJ, Xue Y, Yao XB (2009) DOG 1.0: illustrator of protein domain structures. Cell Research 19:271–273

    Article  PubMed  CAS  Google Scholar 

  • Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–4

    Article  PubMed  CAS  Google Scholar 

  • Ruppert EE, Fox RS, Barnes RD (2004) Invertebrate zoology. Thomson learning Inc., Belmont

    Google Scholar 

  • Sarashina I, Yamaguchi H, Haga T, Iijima M, Chiba S, Endo K (2006) Molecular evolution and functionally important structures of molluscan Dermatopontin: implications for the origins of molluscan shell matrix proteins. J Mol Evol 62:307–18

    Article  PubMed  CAS  Google Scholar 

  • Shen X, Belcher AM, Hansma PK, Stucky GD, Morse DE (1997) Molecular cloning and characterization of lustrin A, a matrix protein from shell and pearl nacre of Haliotis rufescens. J Biol Chem 272:32472–81

    Article  PubMed  CAS  Google Scholar 

  • Shimeld SM, Boyle MJ, Brunet T, Luke GN, Seaver E (2010) Clustered Fox genes in molluscs and annelids and the evolution of mesoderm. Dev Biol 340:234–248

    Google Scholar 

  • Shimizu K, Sarashina I, Kagi H, Endo K (2011) Possible functions of Dpp in gastropod shell formation and shell coiling. Dev Genes Evol 221:59–68

    Article  PubMed  CAS  Google Scholar 

  • Smith FGW (1935) The development of Patella vulgata. Phil Trans Royal Soc B 225:95–125

    Article  Google Scholar 

  • Surget-Groba Y, Montoya-Burgos JI (2010) Optimization of de novo transcriptome assembly from next-generation sequencing data. Genome Res 20:1432–40

    Article  PubMed  CAS  Google Scholar 

  • Suzuki M, Iwashima A, Tsutsui N, Ohira T, Kogure T, Nagasawa H (2011) Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer. ChemBioChem 16:2478–2487

    Article  Google Scholar 

  • Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura T, Kato T, Nagasawa H (2009) An acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 325:1388–90

    Article  PubMed  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–9

    Article  PubMed  CAS  Google Scholar 

  • Tellam RL, Wijffels G, Willadsen P (1999) Peritrophic matrix proteins. Insect Biochem Mol Biol 29:87–101

    Article  PubMed  CAS  Google Scholar 

  • Veis A (2003) Mineralization in organic matrix frameworks. Biomineralization 54:249–289

    CAS  Google Scholar 

  • Weiss IM, Kaufmann S, Mann K, Fritz M (2000) Purification and characterization of perlucin and perlustrin, two new proteins from the shell of the mollusc Haliotis laevigata. Biochem Biophys Res Commun 267:17–21

    Article  PubMed  CAS  Google Scholar 

  • Wilt FH (2005) Developmental biology meets materials science: morphogenesis of biomineralized structures. Dev Biol 280:15–25

    Article  PubMed  CAS  Google Scholar 

  • Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–9

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank The Elizabeth Hannah Jenkinson fund for financial support for the sequencing, and the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics in Oxford for conducting the Illumina GAII sequencing. We also thank Dr. Paul Naylor for Fig. 1a, and Dr. Helen Thompson for Fig. 9a. SG was supported by the EU Lifelong Learning Programme. GDAW was supported by the Huygens Scholarship Programme. PG was supported by an EPSRC studentship.

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Author notes

  1. Gijsbert D. A. Werner

    Present address: Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands

  2. Stefanie Grosser

    Present address: Landcare Research—Manaaki Whenua, 231 Morrin Road, St Johns, Auckland, 1072, New Zealand

Authors and Affiliations

  1. Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK

    Gijsbert D. A. Werner, Patrick Gemmell, Stefanie Grosser & Sebastian M. Shimeld

  2. Life Sciences Interface Doctoral Training Centre, University of Oxford, Oxford, UK

    Patrick Gemmell

  3. Department of Statistics, University of Oxford, 1 South Parks Road, Oxford, OX1 3TG, UK

    Rebecca Hamer

  4. Doctoral Training Centre, Rex Richards Building, South Parks Road, Oxford, OX1 3QU, UK

    Rebecca Hamer

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  1. Gijsbert D. A. Werner
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Correspondence to Sebastian M. Shimeld.

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Werner, G.D.A., Gemmell, P., Grosser, S. et al. Analysis of a deep transcriptome from the mantle tissue of Patella vulgata Linnaeus (Mollusca: Gastropoda: Patellidae) reveals candidate biomineralising genes. Mar Biotechnol 15, 230–243 (2013). https://doi.org/10.1007/s10126-012-9481-0

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