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 AA substitution phenotypes were predicted using PolyPhen [38] and SIFT [39] (http://genetics.bwh.harvard.edu/ pph/; http://genetics.bwh.harvard.edu/pph/pph_help.html; http:// sift.jcvi.org/; http://sift.jcvi.org/www/SIFT_help.html) with the default settings. Results other than ''benign'' or ''tolerated'' were categorized as substitutions predicted to impact protein function [30, 38, 39] . To assess the potential for functional and/or selective constraint across the entire bovine TLR gene family, a goodness of fit test (x 2 ) was performed assuming equal probabilities for benign or tolerated AA phenotypes versus those predicted to impact protein function. Frequency distribution tests, including Tajima's D [43] and Fu's F S [44] , were performed in DnaSP v4.90.1 [67] using all validated SNPs. Significance levels for frequency distribution tests were defined by confidence intervals estimated for each test statistic via coalescent simulation (10,000 replicates) [67] . Simulations were performed given the observed number of segregating sites, both with and without recombination [67, 68] .