Identification of polymorphisms in bovine TLR2 and CARD15,associations between CARD15 polymorphisms and milk somatic cell score in Canadian Holsteins, and functional relevance of SNP c.3020A>T.

Abstract

Toll-like receptor-2 (TLR2) and caspase recruitmentdomain 15 (CARD15) are important pattern recognition receptors that play a role in the initiation of the inflammatory and subsequent immune response. They have been previously identified as susceptibility loci for inflammatory bowel diseases in humans and are, therefore, suitable candidate genes for inflammatory disease resistance in cattle. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the bovine TLR2 and CARD15 and evaluate the association of these SNPs with health and production traits in a population of Canadian Holstein bulls. A selective DNA pool was constructed based on the estimated breeding values (EBVs) for somatic cell score (SCS). Gene segments were amplified from this pool in PCR reactions and the amplicons sequenced to reveal polymorphisms. A total of four SNPs, including one in intron 10 (c.2886-14A>G) and three in exon 12 (c.3020A>T, c.4500A>C and c.4950C>T)were identified in CARD15; nonewere identified in TLR2. Canadian Holstein bulls (n=338) were genotyped and haplotypes were reconstructed. Two SNPs, c.3020A>T and c.4500A>C, were associated with EBVs for health and production traits. The SNP, c.3020A>T for example, was associated with SCS EBVs (p = 0.0097) with an allele substitution effect of 0.07 score. When compared to the most frequent haplotype Hap12(AC), Hap22(TC) was associated with increased milk (p < 0.0001) and protein (p = 0.0007) yield EBVs, and hap21(TA) was significantly associated with increased SCS EBV (p = 0.0120). All significant comparison-wise associations retained significance at 8% experimental-wise level by permutation test. The role of SNP c.3020A>T in MDP induced IL-1beta expression was investigated by real-time quantitative reverse transcription-PCR (real-time quantitative RT-PCR). The induction of IL-1beta by MDP was highly variable between individuals, and no association was observed between IL-1beta expression and SNP c.3020A>T genotypes. In summary, the association study indicates that SNP c.3020A>T might play a role in the host response against mastitis; however, it is not the sole determinant of MDP induced IL-1beta expression in blood leukocytes. Further detailed studies are needed to understand the functional implications of SNP c.3020A>T.