Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

Fisher, Colleen A.; Bhattarai, Eric K.; Osterstock, Jason B.; Dowd, Scot E.; Seabury, Paul M.; Vikram, Meenu; Whitlock, Robert H.; Schukken, Ynte H.; Schnabel, Robert D.; Taylor, Jeremy F.; Womack, James E.; Seabury, Christopher M.

Of the 276 validated SNPs, 71 were predicted to encode nonsynonymous substitutions (nsSNPs), and one was predicted to encode a nonsense mutation in bovine TLR5 (AA substitution R125*; SNP C2332T). For the validated SNPs detected via pyrosequencing (n = 244), we investigated the relationship between minor allele frequencies (MAFs) estimated from the analysis of pyrosequencing data, as compared to corresponding allele frequencies derived from individual fluorescent allele-specific genotyping assays, and found significant correlations across all 10 TLR genes (discovery panel; Table 1 ). Moreover, an analysis performed across all genes (n = 244 SNPs) revealed that there was little or no bias in the estimates of allele frequencies produced via targeted pyrosequencing (P = 0.999846; Ho: slope = 1; Figure 1 ). Collectively, 266 SNPs and 4 indels were successfully incorporated into 243 unique haplotypes via Bayesian reconstructions [30, 31] (Table 2) , which included one discrete haplotype carrying the putative TLR5 nonsense SNP. Ten SNPs (TLR2: 9431, 10047, 12121; TLR3: 3624, 3804, 5201, 6382; TLR4: 8166; TLR5: 1562, 1685; see Table S2 ) could not be incorporated into discrete haplotypes with best-pair phase probabilities$0.90. Summary data representing the total number of predicted haplotypes, number of cattle with phase probabilities$0.90, total number of variable sites with MAF#0.10, genic distributions of validated variable sites, size of the investigated regions, and average estimates of linkage disequilibrium (LD; r 2 ) between adjacent variable sites are depicted in Table 2 . Across all investigated loci (n = 549 cattle; 31 breeds), the MAF spectrum derived from allelespecific genotyping assays ranged from 0.001 to 0.498, with 64% of the validated SNPs possessing MAFs#0.10 ( Table 2) .