In cattle, there are six classical MHC class I genes that are variably present between different haplotypes. Almost all known haplotypes contain between one and three genes, with an allele of Gene 2 present on the vast majority. However, very little is known about the sequence and therefore structure and evolutionary history of this genomic region. To address this, we have refined the MHC class I region in the Hereford cattle genome assembly and sequenced a complete A14 haplotype from a homozygous Holstein. Comparison of the two haplotypes revealed extensive variation within the MHC class Ia region, but not within the flanking regions, with each gene contained within a conserved 63- to 68-kb sequence block. This variable region appears to have undergone block gene duplication and likely deletion at regular breakpoints, suggestive of a site-specific mechanism. Phylogenetic analysis using complete gene sequences provided evidence of allelic diversification via gene conversion, with breakpoints between each of the extracellular domains that were associated with high guanine-cytosine (GC) content. Advancing our knowledge of cattle MHC class I evolution will help inform investigations of cattle genetic diversity and disease resistance.

The leukocyte receptor complex (LRC) encodes numerous immunoglobulin (Ig)-like receptors involved in innate immunity. These include the killer-cell Ig-like receptors (KIR) and the leukocyte Ig-like receptors (LILR) which can be polymorphic and vary greatly in number between species. Using the recent long-read genome assembly, Sscrofa11.1, we have characterized the porcine LRC on chromosome 6. We identified a ~ 197-kb region containing numerous LILR genes that were missing in previous assemblies. Out of 17 such LILR genes and fragments, six encode functional proteins, of which three are inhibitory and three are activating, while the majority of pseudogenes had the potential to encode activating receptors. Elsewhere in the LRC, between FCAR and GP6, we identified a novel gene that encodes two Ig-like domains and a long inhibitory intracellular tail. Comparison with two other porcine assemblies revealed a second, nearly identical, non-functional gene encoding a short intracellular tail with ambiguous function. These novel genes were found in a diverse range of mammalian species, including a pseudogene in humans, and typically consist of a single long-tailed receptor and a variable number of short-tailed receptors. Using porcine transcriptome data, both the novel inhibitory gene and the LILR were highly expressed in peripheral blood, while the single KIR gene, KIR2DL1, was either very poorly expressed or not at all. These observations are a prerequisite for improved understanding of immune cell functions in the pig and other species.

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20-30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination.

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.