Characterization of the peripheral blood transcriptome and adaptive evolution of the MHC I and TLR gene families in the wolf (Canis lupus)

Guangshuai Liu, Honghai Zhang, Guolei Sun, Chao Zhao, Shuai Shang, Xiaodong Gao, Tian Xia, Xiufeng Yang

The wolf (Canis lupus) is one of the most widely distributed terrestrial mammals, because it is well adapted to various ecological niches and their corresponding pathogen environments. Immunological competence is a crucial factor involved in adapting to a changing environment and fighting pathogen infection in animals. In this study, the peripheral blood transcriptome of wolves was generated via RNA-seq to advance understanding of the wolf immunome, with a special focus on the major histocompatibility complex class I (MHC I) and toll-like receptor (TLR) gene families, which are involved in pathogen recognition and defense. The blood transcriptomic libraries of eight wolves originating from Tibet and Inner Mongolia were sequenced, and approximately 383 million reads were generated. Using a genome-guided assembly strategy, we obtained 123,851 unigenes, with a mean length of 845 bp and an N50 length of 1121 bp. On the basis of BLAST searches against the NCBI non-redundant protein database (Nr), a total of 36,192 (29.22%) unigenes were annotated. For functional classification, 24,663 unigenes were assigned to 13,016 Gene Ontology (GO) terms belonging to 51 sub-categories of the three main GO categories. Additionally, 7682 unigenes were classified into 6 Kyoto Encyclopedia of Genes and Genomes (KEGG) categories, in which the most represented functional sub-categories were signal transduction and the immune system, and 16,238 unigenes were functionally classified into 25 Eukaryotic Orthologous Groups (KOG) categories. We observed an overall higher ω (d N/d S) value at antigen-binding sites (ABSs) than at non-ABS regions as well as clear evidence of intergenic/intragenic recombination events at wolf MHC I loci. Additionally, our analysis revealed that carnivorous TLRs were dominated by purifying selection, with mean ω values at each TLR locus ranging from 0.173 to 0.527. However, we also found significant instances of positive selection that acted on several codons in pathogen recognition domains and were linked to species-specific differences in pathogen recognition. This study represents the first attempt to characterize the blood transcriptome of the wolf and to highlight the value of investigating the immune system. Balancing selection and recombination have contributed to the historical evolution of wolf MHC I genes. Moreover, TLRs in carnivores have undergone adaptive evolution against the background of purifying selection, and a high level of adaptive evolution was detected in the wolf TLR system.