Resequencing the Bovine and Equine Toll-like Receptor Genes for Applications in Translational and Functional Genomics

NON-TECHNICAL SUMMARY: Advances in agricultural genomics have often been fueled by the supposition that improving host genetics through selective breeding may ultimately help suppress important diseases affecting agricultural species. Because the innate immune system in mammals provides host defense against a diverse spectrum of pathogens without requiring prior exposure, host genes modulating innate immunity, such as the Toll-like receptors (hereafter TLR), have been considered putative candidates for potentially improving host genetics among agricultural species. Moreover, as a first line of defense against invading pathogens, failure to properly recognize those pathogens may potentially provide a plausible explanation for differential susceptibility to certain infectious diseases commonly affecting agricultural species. Therefore, this project proposes to develop and validate high-density variable genetic markers within all 10 bovine (cow) and equine (horse) TLR genes, with considerable preliminary data generated to date.<P>

Importantly, several bovine TLR genes either overlap with, or are proximal to, regions of the bovine genome which have been implicated in the modulation of differential susceptibility to diseases such clinical mastitis and bovine spongiform encephalopathy. Notably, the most robust genetic profiling tool currently available for cattle (Illumina Bovine SNP50 Assay) will not facilitate fine-scale investigation of the bovine genomic regions implicated. Therefore, additional variable genetic markers are necessary to establish and fully translate the relationship between bovine genotype and differential susceptibility to disease. Moreover, little or no information currently exists regarding potentially important genetic variation within the equine TLR genes. For these reasons, a need currently exists to comprehensively characterization and validate TLR genetic variation for both cattle and horses. In doing so, we will also endeavor to computationally predict regions of the bovine and equine TLR genes that may potentially influence expression (called promoter regions), as well as genetic variants that may potentially alter structural features of the host TLR proteins.<P>

In summary, discovery and validation of variable genetic markers within the bovine and equine TLR genes provides deliverables that directly enhance and/or facilitate fine mapping and genome-wide association studies for health-related traits of importance in both cattle and horses (Animal Genome 43.0 Priorities).

<P>APPROACH: To characterize the frequency and distribution of SNPs and indel polymorphisms within all 10 bovine and equine TLR loci we will utilize large, diverse DNA panels and cost-effective next generation sequencing. Two DNA panels (96 cattle sires, 96 horses of both sexes, 30 and 45 breeds, respectively) will be utilized for molecular interrogation of TLRs 1-10. DNA working stocks are standardized to 50 ng/ul for consistency and to prevent imbalanced representation during downstream pyrosequencing. Amplification of cattle and equine TLR genes via Fusion (FLX) Primers with barcodes will follow concentrations and thermal cycling parameters optimized in our laboratory. All TLR amplicons will be visualized via agarose gel electrophoresis. Bovine and equine TLR amplicons will be purified using Agencourt Ampure Beads, and all (approx. 100) purified TLR amplicons resulting from a single animal will be pooled by equal volume, with this process repeated for every individual investigated. <P>

Because our working stock DNAs are standardized in concentration, the resulting purified amplicon concentrations are very similar (CM Seabury; unpublished). In total, approximately 19,200 purified amplicons will be created (9600 for cattle/9600 for horse), pooled by animal, clonally (emulsion) PCR amplified, and sequenced using optimized pyrosequencing methods (454 GSFLX). One bi-directional pyrosequencing run delivering approximately 750,000 reads 400 bp in length will be executed for the cattle TLR amplicons, with a second run executed for the equine amplicons. These two runs will deliver approximately 39X coverage for each amplicon. Sequence variants will be detected using 454 GSFLX software. Individual sequences for all animals will be compared to a TLR reference sequence, and a variant report will be generated for each animal. The overall frequency and distribution of polymorphism will be determined for all 10 cattle and equine TLR genes. SNPs and indels will be considered validated by coverage, whereby the variant is present in 5 or more discrete sequencing reads for a given animal. Estimates for bovine and equine TLR haplotype frequencies as well as the most likely pairs of haplotypes for each individual will be estimated using a Bayesian statistical approach. Estimates of recombination within TLR genes will be computed using two or more established statistical procedures. A minimal set of polymorphisms that collectively tag all observed haplotypes predicted on four or more chromosomes will be identified using publicly available software. Bovine and equine promoter prediction will entail the use of two publicly available promoter prediction algorithms.<P>

An online utility (http://smart.embl-heidelberg.ed/) will be used for comparative prediction of protein domains, and to evaluate how amino acid replacements encoded by nonsynonymous SNPs influence protein domain predictions. SNPs, indels, and haplotype data will be collaboratively made available to the International Bovine and Equine SNP Consortium for validation in QTL mapping populations and to augment existing SNP Assays. All sequences, SNPs, and indels will be deposited in appropriate online databases.