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Sequence-based genotyping of the sheep MHC class II DRB1 locus

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Abstract

The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.

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References

  • Arrieta-Aguirre I, Garcia-Etxebarria K, Jugo BM (2006) Optimization of the MhcOvar-DRB1 gene typing. Tissue Antigens 67:222–228

    Article  CAS  PubMed  Google Scholar 

  • Ballingall KT, Wright H, Redmond J, Dutia BM, Hopkins J, Lang J, Deverson EV, Howard JC, Puri N, Haig D (1992) Expression and characterization of ovine major histocompatibility complex class II (OLA-DR) genes. Anim Genet 23:347–359

    Article  CAS  PubMed  Google Scholar 

  • Ballingall KT, Fardoe K, McKeever DJ (2008) Genomic organisation and allelic diversity within coding and non-coding regions of the Ovar-DRB1 locus. Immunogenetics 60:95–103

    Article  CAS  PubMed  Google Scholar 

  • Baxter R, Hastings N, Law A, Glass EJ (2008) A rapid and robust sequence-based genotyping method for BoLA-DRB3 alleles in large numbers of heterozygous cattle. Anim Genet 39:561–563

    Article  CAS  PubMed  Google Scholar 

  • Deverson EV, Wright H, Watson S, Ballingall K, Huskisson N, Diamond AG, Howard JC (1991) Class II major histocompatibility complex genes of the sheep. Anim Genet 22:211–225

    CAS  PubMed  Google Scholar 

  • Ellis SA, Bontrop RE, Antczak DF, Ballingall K, Davies CJ, Kaufman J, Kennedy LJ, Robinson J, Smith DM, Stear MJ, Stet RJM, Waller MJ, Walter L, Marsh SGE (2006) ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005. Immunogenetics 57:953–958

    Article  PubMed  Google Scholar 

  • Fremont DH, Hendrickson WA, Marrack P, Kappler J (1996) Structures of an MHC class II molecule with covalently bound single peptides. Science 272:1001–1004

    Article  CAS  PubMed  Google Scholar 

  • Gruszczynska J, Brokowska K, Charon KM, Swiderek WP (2005) Restriction fragment length polymorphism of exon 2 Ovar-DRB1 gene in Polish Heath Sheep and Polish Lowland Sheep. J Appl Genet 46:311–314

    PubMed  Google Scholar 

  • Herrmann-Hoesing LM, White SN, Kappmeyer LS, Herndon DR, Knowles DP (2008a) Genomic analysis of Ovis aries (Ovar) MHC class IIa loci. Immunogenetics 60:167–176

    Article  CAS  Google Scholar 

  • Herrmann-Hoesing LM, White SN, Mousel MR, Lewis GS, Knowles DP (2008b) Ovine progressive pneumonia provirus levels associate with breed and Ovar-DRB1. Immunogenetics 60:749–758

    Article  Google Scholar 

  • Konnai S, Nagaoka Y, Takesima S, Onuma M, Aida Y (2003a) Technical note: DNA typing for ovine MHC DRB1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). J Dairy Sci 86:3362–3365

    CAS  Google Scholar 

  • Konnai S, Nagaoka Y, Takeshima S, Onuma M, Aida Y (2003b) Sequences and diversity of 17 new Ovar-DRB1 alleles from three breeds of sheep. Eur J Immunogenet 30:275–282

    Article  CAS  Google Scholar 

  • Kostia S, Kantanen J, Kolkkala M, Varvio SL (1998) Applicability of SSCP analysis for MHC genotyping: fingerprinting of Ovar-DRB1 exon 2 alleles from Finnish and Russian breeds. Anim Genet 29:453–455

    Article  CAS  PubMed  Google Scholar 

  • Miltiadou D, Ballingall KT, Ellis SA, Russell GC, McKeever DJ (2005) Haplotype characterization of transcribed ovine major histocompatibility complex (MHC) class I genes. Immunogenetics 57:499–509

    Article  CAS  PubMed  Google Scholar 

  • Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1984) Nomenclature for incompletely specified bases in nucleic acid sequences. Recommendations. Biochem J 229:281–286

    Google Scholar 

  • Sayers G, Good B, Hanrahan JP, Ryan M, Angles JM, Sweeney T (2005) Major histocompatibility complex DRB1 gene: its role in nematode resistance in Suffolk and Texel sheep breeds. Parasitology 131:403–409

    Article  CAS  PubMed  Google Scholar 

  • Schwaiger FW, Epplen JT (1995) Exonic MHC-DRB polymorphisms and intronic simple repeat sequences: Janus’ faces of DNA sequence evolution. Immunol Rev 143:199–224

    Article  CAS  PubMed  Google Scholar 

  • Schwaiger FW, Weyers E, Buitkamp J, Ede AJ, Crawford A, Epplen JT (1994) Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls. Mol Biol Evol 11:239–249

    CAS  PubMed  Google Scholar 

  • Scott PC, Maddox JF, Gogolin-Ewens KJ, Brandon MR (1991) The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB. Immunogenetics 34:80–87

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to acknowledge the help of all those who provided genetic material from the range of sheep breeds analyzed here, specifically Chris Cousens and Craig Watkins of the Moredun Research Institute who provided the bulk of the DNA samples. This work was funded in part by the Scottish Government and RT was funded through the EU Framework 7, Leonardo da Vinci, Vocational Education and Training Project, Vets in Europe V.

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  1. Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, EH26 OPZ, Scotland, UK

    Keith T. Ballingall & Riccardo Tassi

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  1. Keith T. Ballingall
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  2. Riccardo Tassi
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Correspondence to Keith T. Ballingall.

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Ballingall, K.T., Tassi, R. Sequence-based genotyping of the sheep MHC class II DRB1 locus. Immunogenetics 62, 31–39 (2010). https://doi.org/10.1007/s00251-009-0410-y

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