US-UK BBSRC-NIFA Collab-Reassembly of cattle immune gene clusters for quantitative analysis

Since livestock were first domesticated approximately 10,000 years ago they have been selectively bred for desirable traits. Traditional genetic improvement using measurable traits and animal pedigrees has been very successful, particularly to increase production in important livestock species. The result today is a plethora of different breeds that are particularly suited for different environments or types of production, e.g. dairy and beef cattle.

However within most livestock populations there is considerable amount of variation that has never been exploited during selective breeding. As the global demand for food increases rapidly, the demand for livestock improvement is escalating. As a consequence of this demand and recent advances in technology, it is now possible to inform breeding strategies based on the animal's genome sequence. In cattle this has been made possible through the characterisation of nearly 800,000 single nucleotide polymorphisms (SNPs) identified in cattle genomes. Rather than having to sequence the whole genome of each animal, rapid identification of SNPs that are associated with parentage, productive traits or breed composition allows for breeding decisions to be made earlier in an animal's life. Unknown animals with no phenotypic data can then be assessed solely on their SNP genotype and their breeding values calculated. This method of genomic selection is now widely used by cattle breeding companies.

However, as with any young technology, problems remain. If regions of the genome are very variable between individuals and/or very repetitive it is difficult to identify SNPs that can be screened by the genotyping technology. There are several highly variable and repetitive immune gene complexes in mammalian genomes which have a fundamental role in disease resistance and responses to vaccines. Moreover these regions have evolved this complexity, at least in part, to combat rapidly evolving pathogens. In cattle, we have identified that the current SNPs do not cover three large and vital immune gene complexes, and to a large extent these complexes have not been assembled in the current genome builds. The validation of SNPs for use in genotyping relies upon an accurate genome assembly over the region the SNP is located; therefore this further compounds the problem. Ultimately the current technology is not yet able to type for genetic markers associated with important immune genes that are likely to influence health and disease resistance traits.

Cattle possess a pool of natural genetic diversity that has evolved to counter rapidly evolving pathogens that cannot yet be selected for using genomics. We propose to develop the tools to utilise this diversity to improve health and disease resistance traits in cattle. Building on our initial assemblies of these gene complexes, we will assemble these genomic regions in many individuals to characterise the extent a large structural variation. Existing short whole genome sequence reads from > 30 individuals will then be aligned to these larger regions, alongside other publically available sequence datasets. By targeting these regions, it will be possible to identify and validate suitable SNPs, even those at low frequency, which will then be incorporated into a genotyping platform. The utility of this tool will then be tested by genotyping a herd of cattle that display differential disease resistance to bovine tuberculosis, a complex disease that is known to involve a genetic component and is influenced by the gene complexes we are targeting in this study. Ultimately we envisage that these markers can then be incorporated into current and future genotyping technologies to improve disease resistance in cattle through selective breeding.

This project will target and sequence three highly variable immune gene clusters in cattle, the major histocompatibility complex (MHC), the leukocyte receptor complex (LRC) and the natural killer complex (NKC). The highly polymorphic molecules encoded in these regions control and influence a diverse array of fundamental immune functions. However, these regions remain largely absent or misassembled in the current builds of the cattle genome, and very little information regarding polymorphism has been gathered. This in turn has impacted the SNP density on the current single nucleotide polymorphism (SNP) typing platforms that are being used to genotype cattle to improve production and health traits, as well as performing genome wide association studies. Consequently, it is not possible to determine if these fundamental immune genes are associated with any health or disease traits in cattle, including responses to vaccines.

Using PacBio (SMRT) sequencing of BAC and fosmid clones we aim to de novo assemble these regions in five dairy cattle. Existing whole genome data from 40 Holstein and 20 Angus bulls can then mapped with confidence to these regions, revealing the position and pattern of polymorphisms. This will enable the development of a bespoke SNP typing platform which for the first time will allow the genotypes over these regions to be determined and examined alongside the SNP genotypes from the rest of the genome. Finally, as a proof of principle experiment, we will test this SNP platform on approximately 1200 cattle that are differentially susceptible to bovine tuberculosis. This experimental cohort was selected due to a priori knowledge of the involvement of genes within the immune complexes being studied with this complex disease. Additionally, this cohort has already been genotyped using the most advanced current SNP typing platform allowing us to combine these datasets.

Single nucleotide polymorphism genotyping is both the most contemporary and the most cost effective method used to improve desirable traits through genomic selection. However, important regions of the genome that encode fundamental genes of the immune system are not currently interrogated by these platforms. By targeting these gene complexes this research will enhance the ability of SNP genotyping to examine immune and health traits. We will also test the utility of these new genetic markers by studying their involvement in resistance to bovine tuberculosis. This disease is a massive economic burden to the UK, and the immune response to Mycobacterium bovis is known to involve genes encoded for in complexes targeted in this proposal. Therefore, as a proof of principle experiment, resistance to bovine tuberculosis offers an excellent opportunity to highlight the role of these immune complexes, add to our epidemiological understanding of this disease and maximise the impact of this research.
The impact of the immediate outputs of this research is broad. The initial genomic assemblies will include novel regions that remain unassembled in the current cattle genome builds. As we are targeting one of the two related animals that are the subjects of the cattle genome assembly, the impact of providing and annotating regions that are not yet assembled, and correcting regions that contain misassembly will be of benefit to the entire global community using this pivotal reference genome. Moreover, characterising the variation within these regions and including this alongside the reference genome will ensure that this important diversity can be exploited by all potential parties. This will be of significant value for comparative genomics researchers as well as the cattle breeding and animal research community. The improved genome structure will allow for better cross-species comparisons and tracking of evolutionary changes in ruminants. It will allow for the tracking of SNP associated with health and fitness traits in the animal breeding communities. Agri-genomic SNP genotyping companies will benefit from the identification of new useful markers that will be deployed to improve health and fitness traits in cattle.
Therefore the potential impact is very high for the cattle industry. In addition, any identification of bTB associated markers will allow for the creation of low-cost genotyping chips that could identify highly susceptible animals in a herd. The extension to other diseases would be straightforward. The financial savings of such a strategy are enormous.

This research will produce a highly skilled cross disciplinary researcher. Producing such skilled researchers with expertise in livestock immunology and bioinformatics is of significant benefit to the UK academic and non-academic communities. The significant level of collaboration with researchers in the US will make this an outstanding opportunity for a PDRA.
Ultimately, this research will provide information for farmers and breeders to help make decisions on herd management and breeding. This could have enormous benefit by reducing the cost of disease management and increasing sustainability. Any advancement in understanding disease resistance will be of benefit by improving productivity and hence wealth creation. As part of improving food security this research will have a beneficial impact on UK society in general and ultimately the rest of the world. Any effect on reducing the burden of disease will have a major beneficial effect on social welfare, wealth creation through development of livestock industries and the removal of barriers to trade. As such, this project directly addresses BBSRC strategic priority areas in Food Security and therefore contributes to meeting its targets. This project also facilitates data sharing within the animal genetics community, and several other bioscience areas, to facilitate global research within the food security agenda.