The genome sequence of Atlantic cod reveals a unique immune system

Journal name:
Nature
Volume:
477,
Pages:
207–210
Date published:
DOI:
doi:10.1038/nature10342
Received
Accepted
Published online

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture1, 2. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC)II is a conserved feature of the adaptive immune system of jawed vertebrates3, 4, but we show that Atlantic cod has lost the genes for MHCII, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions5. We find a highly expanded number of MHCI genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHCII. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

At a glance

Figures

  1. Synteny between Atlantic cod and selected teleosts.
    Figure 1: Synteny between Atlantic cod and selected teleosts.

    The co-occurrence of orthologous genes (with a minimum of 50% sequence identity over 50% of the alignment, sphere size indicates the numbers of syntenic genes) in 23 Atlantic cod linkage groups8 (x-axis) reveals synteny with the chromosomes of four teleosts (y-axis). Several genes located on the stickleback chromosome XIV, tetraodon chromosome 4 and medaka chromosome 12 indicate a lineage-specific chromosomal rearrangement in Atlantic cod.

  2. Functional haemoglobin polymorphisms in Atlantic cod.
    Figure 2: Functional haemoglobin polymorphisms in Atlantic cod.

    a, Schematic of the head-to-head organized α1 and β1 globin genes, the intergenic promoter region and transcription start sites (red arrows). A promoter polymorphism consisting of a 73-bp indel (red box) segregates in linkage disequilibrium with two amino-acid-substitution polymorphisms (vertical lines) at positions 55 and 62 in β1 globin that affect its oxygen-binding affinity. This linkage disequilibrium results in two predominant haplotypes, long–Val–Ala and short–Met–Lys. b, Normalized luciferase luminescence ratios in salmon kidney cells. Cells were transfected using the long promoter (black circles) or the short promoter (white circles) and incubated at 4°C, 15°C or 20°C (n = 3 for each treatment level). Error bars show 95% confidence intervals.

  3. MHC[thinsp]I diversity in Atlantic cod.
    Figure 3: MHCI diversity in Atlantic cod.

    a, Copy-number estimates of the MHCI α3 domain. Estimates are based on qPCR ratios (see Supplementary Note 28) of the MHCI α3 domain and a single-copy reference gene. For Atlantic cod, β2-microglobulin and topoisomeraseIII-α (*) were used as reference genes; for human and stickleback, β2-microglobulin was used. The estimates for human and stickleback agree with the expected number of α3 domains found in both reference genomes (Supplementary Table 15). Black dots indicate 95% confidence intervals calculated by bootstrapping (n = 50,000). b, Phylogeny of amino-acid sequences of MHCI α1–α3 domains in teleosts. The Atlantic cod sequences are derived from cDNA and comprise classical U-lineage MHCI only. The other teleost sequences were obtained from Ensembl and NCBI, and contain classical and non-classical U-lineage MHCI. Alignments were visually inspected and corrected where necessary. Maximum likelihood (ML) values and Bayesian posterior probabilities (dots) support the main branches on the ML topology. Distance represents the number of substitutions per site (scale bar). The ratio of non-synonymous to synonymous variable sites (Ka/Ks), the average nucleotide diversity per site (π) and Tajima’s D (D) were calculated for the two main clades in Atlantic cod.

  4. Phylogeny of TLR families in Atlantic cod.
    Figure 4: Phylogeny of TLR families in Atlantic cod.

    TLR protein sequences were selected on the basis of the conserved Toll-IL-1 receptor (TIR) domain for Atlantic cod, including known sequences from stickleback, zebrafish, tetraodon, fugu, medaka and human as references. TLR clades with (*) or without () Atlantic cod sequences are denoted according to human or teleost orthologues (summary tree topology, top left panel). Distance represents the average number of substitutions per site (scale bar). ML values and Bayesian posterior probabilities greater than 75/0.75 support the ML topology. Detailed topologies of TLR7 (blue), TLR8 (purple), TLR9 (green) and TLR22 (grey) show gene expansions for Atlantic cod (red). Multiple TLR copies within species are subdivided by letters, and follow Ensembl nomenclature for D.rerio.

Accession codes

Primary accessions

EMBL/GenBank/DDBJ

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Author information

Affiliations

  1. Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, University of Oslo, PO Box 1066, Blindern, N-0316 Oslo, Norway

    • Bastiaan Star,
    • Alexander J. Nederbragt,
    • Sissel Jentoft,
    • Unni Grimholt,
    • Martin Malmstrøm,
    • Trine B. Rounge,
    • Jonas Paulsen,
    • Monica H. Solbakken,
    • Karin Lagesen,
    • Ave Tooming-Klunderud,
    • Kirubakaran G. Tina,
    • Mari Espelund,
    • Morten Skage,
    • Paul R. Berg,
    • Stig W. Omholt,
    • Nils Chr. Stenseth &
    • Kjetill S. Jakobsen
  2. Department of Molecular Biosciences, Centre for Immune Regulation, University of Oslo, Blindern, N-0316 Oslo, Norway

    • Tone F. Gregers
  3. Bioinformatics Core Facility, Institute for Medical Informatics, Oslo University Hospital, Montebello, N-0310 Oslo, Norway

    • Jonas Paulsen
  4. Department of Informatics, University of Bergen, N-5020 Bergen, Norway

    • Animesh Sharma,
    • Chirag Nepal &
    • Inge Jonassen
  5. Department of Natural Sciences and Technology, Hedmark University College, P.O. Box 4010, Bedriftsenteret, N-2306 Hamar, Norway

    • Ola F. Wetten
  6. Department of Animal and Aquacultural Sciences, University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway

    • Ola F. Wetten
  7. Department of Biology, Centre for Geobiology, University of Bergen, N-5020 Bergen, Norway

    • Anders Lanzén
  8. Computational Biology Unit, Uni Computing, Uni Research AS, N-5020 Bergen, Norway

    • Anders Lanzén,
    • Chirag Nepal,
    • Christopher Previti &
    • Inge Jonassen
  9. 454 Life Sciences, 15 Commercial Street, Branford, Connecticut 06405, USA

    • Roger Winer,
    • James Knight &
    • Lei Du
  10. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK

    • Jan-Hinnerk Vogel,
    • Bronwen Aken &
    • Steve Searle
  11. Nofima Marine, P.O. Box 5010, N-1430 Ås, Norway

    • Øivind Andersen
  12. Institute of Marine Research, P.O., Box 1870, Nordnes, N-5817 Bergen, Norway

    • Rolf B. Edvardsen &
    • Ketil Malde
  13. Department of Animal and Aquacultural Sciences, CIGENE, Centre for Integrative Genetics, Norwegian University of Life Sciences, PO Box 5003, 1432 Ås, Norway

    • Kirubakaran G. Tina,
    • Sigbjørn Lien &
    • Stig W. Omholt
  14. Faculty of Biosciences and Aquaculture, University of Nordland, N-8049 Bodø, Norway

    • Bård Ove Karlsen,
    • Truls Moum &
    • Steinar D. Johansen
  15. Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, N-0316 Oslo, Norway

    • Tor Gjøen
  16. Max Planck Institute for Molecular Genetics, Ihnestrasse 63-73, D-14195 Berlin-Dahlem, Germany

    • Heiner Kuhl &
    • Richard Reinhardt
  17. Institute for Basic Sciences and Aquatic Medicine, School of Veterinary Sciences, N-0033 Oslo, Norway

    • Jim Thorsen
  18. Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, N-9037 Tromsø, Norway

    • Steinar D. Johansen
  19. Department of Biology, PO Box 7803, University of Bergen, N-5020 Bergen, Norway

    • Frank Nilsen

Contributions

DNA and RNA isolation, library construction and sequencing: A.T.-K., M.S., M.H.S., T.B.R., M.M., M.E., B.S., A.J.N. and J.T. Sanger BAC (end-) sequencing: H.K. and R.R. Assembly: A.J.N., B.S., A.S. and A.L. Linkage map analyses: K.G.T. and B.S. SNP analyses: K.G.T., P.R.B., S.L. and A.J.N. Annotation: J.-H.V., B.A. and S.S. Repeat analyses: B.S. Synteny analyses: J.P. and B.S. Haemoglobin analyses: Ø.A., O.F.W., B.S. and T.G. Bioinformatics: A.J.N., B.S., A.S., T.B.R., J.P., C.P., C.N., R.B.E., R.W., J.K., K.L., A.L., I.J., M.M., K.M., P.R.B., K.G.T. and M.H.S. Immune analyses: U.G., M.M., M.H.S., M.E., B.S., B.O.K., T.M., K.L., S.D.J. and T.B.R. Interpretation of immune results: U.G., T.F.G., S.J., B.S. and K.S.J. 454 contributions: L.D. Revisions: Ø.A., T.M., S.D.J., F.N., I.J., S.J., N.C.S. and S.W.O. Project initiation: S.W.O., I.J., F.N., S.L., N.C.S. and K.S.J. Project coordination: S.J. Consortium leader: K.S.J. This manuscript is dedicated to the memory of L. Pilström and R. J. M. Stet. Their research inspired our work to understand further the Atlantic cod immune system.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The unassembled sequencing reads and Newbler assembly have been deposited at ENA-EMBL under the accession numbers CAEA01000001–CAEA01554869. The annotation is available through Ensembl at http://www.ensembl.org/index.html. These and more resources are also available through http://codgenome.no.

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Supplementary information

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  1. Supplementary Information (8.1M)

    The file contains Supplementary Text, Supplementary Figures 1-22 with legends, Supplementary Tables 1-19 and additional references.

Comments

  1. Report this comment #26050

    Johannes M. Dijkstra said:

    Dear Star and Co-workers,

    Thank you for your important contribution to fish research. Based on your data the typical MHC class II pathways seem absent or at least diminished in Atlantic cod indeed. However, I do have a few comments and a question.

    In the Atlantic cod CD4 region sequence in scaffold ATLCOD1Bs1550564 of the Celera assembly, between the genes USP5 and COPS7A, a CD4 Ig-like domain 2 coding sequence can be found in proper location and orientation. This CD4 fragment was not mentioned by the authors. It encodes NLSRGLSQSCIVQGPRHGLQPQIHWRSPKIEILARGPGTHVEWTCVSTSVGMATYAETSVPVV; please note the family-characteristic EWTC motif in the tentative F-strand. The sequence N-terminal of the first cysteine is short and atypical (it lacks the otherwise conserved GxxVxLxC or similar motif), but probably there are not enough arguments to conclude with certainty that this is a pseudogene locus.

    As for CD4 genes in the stickleback genome. In contrast to what the article states (only one CD4 gene in stickleback), stickleback has both CD4-1 (probably the figure relates to CD4-1 because this gene is easier to detect, though the figure position suggests CD4-2 identity) and CD4-2 (encoding e.g. LASLSAASKVVLISPGQKVTLECGVKTFKTLQWHQGNDLIHSVSMSGFPRKGLTEIIQRTKVRNTD

    LEIVNVKEEDTGTFICTADRKREEQTLLVVS) genes in the CD4-region. Stickleback CD4-2 may be incapacitated, but such conclusion would need additional analysis.

    I also have a question. What is the definition used for ''classical'' in the sentence Notably, Atlantic cod has about 100 classical MHCI loci, which is a highly expanded number compared to other teleosts?

    Sincerely,

    J.M. Dijkstra
    Institute for Comprehensive Medical Science
    Fujita Health University
    Toyoake, Aichi-ken, Japan
    E-mail: dijkstra@fujita-hu.ac.jp

  2. Report this comment #26260

    Bastiaan Star said:

    Dear Dr. Johannes Dijkstra,

    Thank you for your valuable comments regarding the CD4 region in Atlantic cod. The Ig-like region you have pointed out is certainly interesting, though it has unusual characteristics as you mention. Modeling the tertiary structure using Phyre2 shows limited overall confidence (47%) for the Immunoglobulin-like beta-sandwich C2 set domain. None of the residues are modeled at >90% confidence, cf. in stickleback the comparative region is modeled at 99.8% confidence for the C2 set, with 95% of the residues modeled >90% (similar scores are obtained by using other teleost CD4 regions). Full-length (CD4-1) proteins contain four of these Ig domains, rather than only one. Shorter CD4-like proteins (2 domains) have been found by you and colleagues in salmon (Moore et a.l. 2009) and several other teleosts, in addition to the four domain CD4. Moreover, the typically conserved intron-exon structure of CD4 is lacking in Atlantic cod. Therefore, we are confident that our current conclusion of lack of functional CD4 proteins in Atlantic cod genome remains correct.

    To infer synteny with other teleosts, we selected teleost genes based on protein annotation available in the Ensembl database. It appears that the stickleback sequence you mention has not been given a protein translation yet, thus it was not included. It is indeed possible that stickleback possesses another functional CD4 (like) gene.

    Finally, in response to your question, the sequences we consider as "classical" are MHCI U-lineage class Ia genes. The Atlantic cod genome also contains several ZE-lineage genes (considered ?unclassical?), these have not been included in any analyses.

    Thank you for your interest,
    On behalf of my coauthors,
    Bastiaan Star

    Protein structure prediction on the web:
    A case study using the Phyre server. Kelley LA and Sternberg MJE
    Nature Protocols 4, 363-371 (2009).

    Moore et al. (2009) CD4 homologues in Atlantic salmon. Fish and Shellfish Immunology 26; 10-18

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