Identification of Functional Polymorphisms in the Coding Sequence of Swine Toll-like Receptors

Toll-like receptors (TLRs) play a key role in innate immunity and are thus of relevance for animal breeding and welfare. With the aim to identify genetic variation that might be relevant in the susceptibility to pathogens, we designed an Agilent genome capture assay to selectively sequence the ten TLR genes annotated in the pig genome. The assay captures 40,953 bp of coding sequence. We have sequenced the TLRs of 286 pigs from ten breeds and four commercial lines using an Illumina HiSeq2000 system. Based on the current genome annotation, we identified 307 coding Single Nucleotide Polymorphisms (SNP), of which 101 (33%) were not annotated in dbSNP (July 2014) and can thus be considered as novel. No indels were identified with enough technical confidence. We further predicted the impact of these variants on the protein sequence. 7 SNPs were predicted to have high impact on protein sequence (non-sense, splice site acceptor/donor), 141 SNPs showed a moderate impact potential (non-synonymous changes) and 159 were classified as low impact (silent or start gained). These polymorphisms affect all the pig annotated TLRs. We have further designed a genotyping assay to validate the most relevant polymorphisms in the Open Array™ platform. This assay contains six high, 107 moderate and 10 low impact variants covering the ten TLRs. Moreover, the chip includes three 3’-UTR SNPs and two SRY SNPs for sex identification. We will use this assay to validate these variants and to undertake association studies in relevant populations with trait records on infection and immunity.