Abstract
The TLR genes coding for Toll-like receptors of antibacterial series, namely TLR1, -2, -4, -5 and -6, were re-sequenced in Czech Simmental (Czech Red) cattle using HiSeq and PacBio technologies. Hybrid sequencing allowed to determine SNPs for individual genotyping with primer extension method.
Haplotypes were established within the range of the designed PacBio amplicons, especially for TLR2. A more general statistical reconstruction of haplotypes from individual genotyping was carried out in parallel.
The directly determined haplotypes from PacBio reads demonstrated randomly distributed frequencies of haplotypes in the amplicon 2 - 5 of TLR2, however, 15 haplotypes in amplicon 1 in the proximal part of the transcript formed 2 distinct groups. The results were consistent with the distribution of haplotypes obtained by statistical reconstruction in TLR2.
Similarly, the bimodal distribution was detectable in TLR5 and other TLRs. In all cases, the trend for bimodal distribution was expressed stronger in proximal regions of the transcripts.
The clustering of TLR haplotypes has been reported earlier in a panel of world breeds (Fisher C.A. et al., Plos One 6:e27744, 2011; Bilgen N. et al., Diversity 8:23, 2016), however, the origin of this disequilibrium is still not documented. Alternating infectious agents are feasible (Bilgen et al., 2016) along with 2 different essential functions performed by a TLR gene or its product.
An example of a dual function might be formation of 2 kinds of products differing in specificity. The association of the groups of haplotypes with the transcript proximal region suggests the selection target in the 5'- regulatory regions of the TLR genes, although functional interactions in the proximal part of the transcript cannot be excluded.
The confirmation of the haplotype reconstruction data by targeted genotyping and new resequencing technologies is in progress in the set of bulls studied.