Characterisation of the central region of the sheep major histocompatibility complex

Abstract

The major histocompatibility complex (MHC) is a chromosomal region encoding molecules controlling adaptive immune response in vertebrates. In farm animals, many associations between MHC loci and productivity traits including disease susceptibility have been described. However, current knowledge about the structure and function of the MHC in domestic animals, especially sheep, is very limited. Characterization of the sheep MHC may potentially facilitate breeding for enhanced disease-resistant animals through use of marker assisted selection. The main aim of this project has been to provide insights into the organization of the genomic content of the central region of the sheep MHC. The work described herein has utilized subcloning of a sheep BAC genomic library in conjunction with DNA sequencing to generate a map of the central region of the sheep MHC covering ≈700 kbp. Within this map the relative order and identity of twenty five recognized loci were established. For some loci the intergenic distances were also determined. The final map is the most accurate map of this region reported to date and shows a high degree of similarity to the analogous region of the human MHC. This work has been published and a copy of the paper is included in Appendix 1. During the course of this work detailed genomic sequences were obtained for several sheep central region loci. Complete nucleotide sequences were generated for the complement factor B locus (CFB) and the TNFα locus and a comparative analysis of these sequences confirmed their homology with other vertebrate orthologues. Extensive partial sequences for complement components C2 and C4 were also obtained and reported to GenBank.In addition, a previously identified short tandem repeat locus designated BfMs believed to be in the CFB locus was mapped to an intron within the adjacent SKI2VL locus. Single nucleotide polymorphisms (SNPs) were identified by analysing homologous sequences from a minimum of five individual sheep. In total 33 SNPs were discovered distributed over eleven distinct loci. Allele frequencies for SNPs from ten of these loci were determined and reported for a panel of 71 sheep comprising 58 unrelated sheep from the Rylington Merino flock plus a further 13 unrelated parental animals from a three generation half sibling sheep pedigree. The availability of an independently confirmed pedigree constructed from a three generation half sibling sheep family permitted the identification by deduction of central region MHC haplotypes based on a panel of SNPs derived from 10 loci. This is the first reporting of haplotypes covering this region of the sheep MHC. Analysis of SNP panel genotypes in the cohort of 71 unrelated sheep using the expectation maximization algorithm permitted the prediction of a group of approximately 20 haplotypes, which accounted for more than 90% of the expected haplotype distribution. Four of these predicted haplotypes were also present in the known haplotype cohort deduced from the sheep pedigree. Analysis of pairwise linkage disequilibrium between SNP loci in the cohort of 71 unrelated sheep showed a centre-most region displaying relatively high levels of linkage disequilibrium which was bounded by two regions displaying more variable linkage disequilibrium.It is hypothesised that this mid region of the central region of the sheep MHC may be a block like structure characterized by low recombination similar to those that have been widely described in the human and mouse genomes. The discoveries reported in this thesis provide a more accurate and detailed description of the central region of the sheep MHC together with a panel of SNPs, which reflect the diversity of this important genomic region which is known to be associated with immune responsiveness. The description, for the first time, of central region haplotypes provides a practical means of seeking candidate loci associated with disease resistance and productivity traits. The application of molecular techniques will enhance the rate at which the genomic composition of this region is elucidated and the work described in this thesis will contribute to final characterization of this important complex in health and disease.