Determination of Genetic Variations of Toll-like Receptor (TLR) 2, 4 and 6 with Next Generation Sequencing in Native Cattle Breeds of Anatolia and Holstein Friesian

Abstract

Abstract:In recent years, the focus of disease resistance and susceptibility studies in cattle have been on determining patterns in the innate immune response of key proteins, such as Toll-like receptors (TLR). In the bovine genome, there are 10 TLR family members and, of these, TLR2, TLR4 and TLR6 are specialized in recognition of bacterial ligands. Indigenous cattle breeds of Anatolia have been reported to show fewer signs of clinical bacterial infections, such as bovine tuberculosis and mastitis, and it is hypothesized that this might be due to a less stringent genetic selection during breeding.In contrast, Holstein-Friesian cattle have been under strong selection for milk production, which may have resulted in greater susceptibility to diseases. To test this hypothesis, we have compared the TLR2, TLR4 and TLR6 genes of Anatolian Black (AB), East Anatolian Red (EAR), South Anatolian Red (SAR), Turkish Grey (TG), and Holstein (HOL) cattle using Next Generation Sequencing. The SAR breed had the most variations overall, followed by EAR, AB, TG and HOL. TG had the most variations for TLR2 whereas SAR had the most variations in TLR4 and TLR6. We compared these variants with those associated with disease and susceptibility traits. We used exon variants to construct haplotypes, investigated shared haplotypes within breeds and determined candidate haplotypes for disease resistance phenotype in Anatolian cattle breeds.

Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

“…One synonymous SNP at mRNA position 202 could not be classified with any available database, was described by Jann et al [47]and is potentially novel. Of all SNPs identified across the breeds, two of the non-synonymous SNPs have been already reported in the literature [44,47,55] and were only observed in the BS animals. One identified SNP variant, rs55617172, leading to an amino acid change of aspartic acid (D) to glutamic acid (E), both negatively charged, at amino acid position 63, has been significantly associated with TB resistance in a TB case-control cattle study [44].…”

“…Among the Bos indicus breeds, all Sahiwal samples in our study were homozygous for the H326Q SNP and except for two samples, half of the Boran samples were either homozygous or heterozygous for this variant, confirming a strong presence of this SNP in the Bos indicus samples tested. This is interesting given that these breeds are known to be less extensively bred for production traits [55] and more resistant to several diseases, including bovine tuberculosis [56] compared with Bos taurus. Within the BS breed, more than half were heterozygous for the SNP.…”

“…However, SNP rs68343167, was only identified in BS isolates in this study, and results in an amino acid change of the positively charged histidine (H) to the uncharged glutamine (Q) at amino acid position 326 (H326Q). This amino acid position has been suggested to be of relevance for ligand binding and functionality of human TLR2 [47,57], bovine TLR2 and was additionally identified in the Anatolian Black Bos taurus breed [55]. In addition, position 326 resides in leucine rich repeat 11 (LRR11) which together with LLR12 are the key domains involved in ligand biding and heterodimerisation of TLR2 with TLR1 and TLR6 [57].…”

“…TLR2 is an important PRR involved in the recognition and uptake of MTBC family members. Key SNPs in TLR2 have been identified that affect susceptibility to mycobacterial infection for both humans and cattle [55,56]. The CDS of the bovine tlr2 gene of six BS and four HF cattle were cloned and compared to the bovine NCBI reference sequence genome (RefSeq) comprised of that of a Bos taurus Hereford breed bull (NM_174197.2, NCBI, USA, accessed April 2017) in order to identify potential functional SNP sites.…”

“…We utilised a previously published HEK cell assay to express the different TLR2 constructs in the absence of additional PRR [54]. As well as the BS H326Q TLR2 variant, we included the Hereford reference variant and a L306P SNP variant that has been identified by others [47,55] but not identified in the present study. We were also interested to assess the functionality of TLR2 across species, and therefore included the huTLR2 reference sequence (NCBI RefSeq Accession Number NG_016229.1).…”