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.

Bovine TLR pyrosequencing, SNP detection, variant validation, and haplotype inference For 96 elite bovine sires representing 31 domestic cattle breeds (B. t. taurus; B. t. indicus; and composites), we generated and purified 81 amplicons targeting all 10 bovine TLR genes (n = 7,776 total amplicon targets; see methods). The majority of the amplicons were pooled (n = 6,816) to form a normalized fragment library (Table S1 ) which was subjected to a workflow involving Roche 454 Titanium pyrosequencing with downstream variant detection using the Neighborhood Quality Standard algorithm as recently described [29] , and the remaining purified amplicons (n = 960) were analyzed by standard dye-terminator cycle sequencing (Sanger) with alignment-based variant detection [23] [24] [25] . Sanger sequencing was necessary for amplicons that were intolerant to the addition of 59 oligonucleotide barcodes for PCR amplification. In total, 474 variable sites were predicted from intragenic analyses of all sequence data, which included 212 previously validated SNPs [30] , 4 known insertion-deletion mutations (indels) [30] , and 258 new putative SNPs. Evaluation of the genic distributions of all newly predicted TLR variable sites detected within the pyrosequencing data revealed that$62% of the 258 new putative SNPs were located either within or immediately flanking homopolymer repeats. Nevertheless, to allow for inclusion of all possible SNPs in downstream analyses, we investigated all 474 variable sites via fluorescent allele-specific genotyping assays [30] . Collectively, we validated 280 biallelic TLR variants (276 SNPs + 4 indels; Table S2 ) using custom genotyping assays applied to the sequencing discovery panel (n = 96 elite sires; 31 breeds), a panel of Holstein dairy cattle (n = 405; 3 herds), and a panel of purebred Angus beef cattle from a single herd (n = 48).