Diversity of major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes and their interactions in domestic horses.

The immunogenome is the part of the genome that underlies immune mechanisms and evolves under various selective pressures. Two complex regions of the immunogenome, major histocompatibility complex (MHC) and natural killer cell receptor (NKR) genes, play an important role in the response to selective pressures of pathogens. Their importance is expressed by their genetic polymorphism at the molecular level, and their diversity associated with different types of diseases at the population level. Findings of associations between specific combinations of MHC/NKR haplotypes with different diseases in model species suggest that these gene complexes did not evolve independently. No such associations have been described in horses so far. The aim of the study was to detect associations between MHC and NKR gene/microsatellite haplotypes in three horse breed groups (Camargue, African, and Romanian) by statistical methods; chi-square test, Fisher's exact test, Pearson's goodness-of-fit test and logistic regression. Associations were detected for both MHC/NKR genes and microsatellites; the most significant associations were found between the most variable KLRA3 gene and the EQCA-1 or EQCA-2 genes. This finding supports the assumption that the KLRA3 is an important receptor for MHC I and that interactions of these molecules play important roles in the horse immunity and reproduction. Despite some limitations of the study such as low numbers of horses or lack of knowledge of the selected genes functions, the results were consistent across different statistical methods and remained significant even after overconservative Bonferroni corrections. We therefore consider them biologically plausible.

Diversity of selected toll-like receptor genes in cheetahs (Acinonyx jubatus) and African leopards (Panthera pardus pardus).

The anthropogenic impact on wildlife is ever increasing. With shrinking habitats, wild populations are being pushed to co-exist in proximity to humans leading to an increased threat of infectious diseases. Therefore, understanding the immune system of a species is key to assess its resilience in a changing environment. The innate immune system (IIS) is the body's first line of defense against pathogens. High variability in IIS genes, like toll-like receptor (TLR) genes, appears to be associated with resistance to infectious diseases. However, few studies have investigated diversity in TLR genes in vulnerable species for conservation. Large predators are threatened globally including leopards and cheetahs, both listed as 'vulnerable' by IUCN. To examine IIS diversity in these sympatric species, we used next-generation-sequencing to compare selected TLR genes in African leopards and cheetahs. Despite differences, both species show some TLR haplotype similarity. Historic cheetahs from all subspecies exhibit greater genetic diversity than modern Southern African cheetahs. The diversity in investigated TLR genes is lower in modern Southern African cheetahs than in African leopards. Compared to historic cheetah data and other subspecies, a more recent population decline might explain the observed genetic impoverishment of TLR genes in modern Southern African cheetahs. However, this may not yet impact the health of this cheetah subspecies.

Comparative genomics of the Leukocyte Receptor Complex in carnivores.

Background: The mammalian Leukocyte Receptor Complex (LRC) chromosomal region may contain gene families for the killer cell immunoglobulin-like receptor (KIR) and/or leukocyte immunoglobulin-like receptor (LILR) collections as well as various framing genes. This complex region is well described in humans, mice, and some domestic animals. Although single KIR genes are known in some Carnivora, their complements of LILR genes remain largely unknown due to obstacles in the assembly of regions of high homology in short-read based genomes. Methods: As part of the analysis of felid immunogenomes, this study focuses on the search for LRC genes in reference genomes and the annotation of LILR genes in Felidae. Chromosome-level genomes based on single-molecule long-read sequencing were preferentially sought and compared to representatives of the Carnivora. Results: Seven putatively functional LILR genes were found across the Felidae and in the Californian sea lion, four to five genes in Canidae, and four to nine genes in Mustelidae. They form two lineages, as seen in the Bovidae. The ratio of functional genes for activating LILRs to inhibitory LILRs is slightly in favor of inhibitory genes in the Felidae and the Canidae; the reverse is seen in the Californian sea lion. This ratio is even in all of the Mustelidae except the Eurasian otter, which has a predominance of activating LILRs. Various numbers of LILR pseudogenes were identified. Conclusions: The structure of the LRC is rather conservative in felids and the other Carnivora studied. The LILR sub-region is conserved within the Felidae and has slight differences in the Canidae, but it has taken various evolutionary paths in the Mustelidae. Overall, the process of pseudogenization of LILR genes seems to be more frequent for activating receptors. Phylogenetic analysis found no direct orthologues across the Carnivora which corroborate the rapid evolution of LILRs seen in mammals.

Newly identified variability of the antigen binding site coding sequences of the equine major histocompatibility complex class I and class II genes.

The major histocompatibility complex (MHC) with its class I and II genes plays a crucial role in the immune response to pathogens by presenting oligopeptide antigens to various immune response effector cells. In order to counteract the vast variability of infectious agents, MHC class I and II genes usually retain high levels of SNPs mainly concentrated in the exons encoding the antigen binding sites. The aim of the study was to reveal new variability of selected MHC genes with a special focus on MHC class I physical haplotypes. Long-range NGS to was used to identify exon 2-exon 3 alleles in three genetically distinct horse breeds. A total of 116 allelic variants were found in the MHC class I genes Eqca-1, Eqca-2, Eqca-7 and Eqca-Ψ, 112 of which were novel. The MHC class II DRA locus was confirmed to comprise five exon 2 alleles, and no new sequences were observed. Additional variability in terms of 15 novel exon 2 alleles was identified in the DQA1 locus. Extensive overall variability across the entire MHC region was confirmed by an analysis of MHC-linked microsatellite loci. Both diversifying and purifying selection were detected within the MHC class I and II loci analyzed.

A chromosome-scale high-contiguity genome assembly of the cheetah (Acinonyx jubatus).

The cheetah (Acinonyx jubatus, SCHREBER 1775) is a large felid and is considered the fastest land animal. Historically, it inhabited open grassland across Africa, the Arabian Peninsula, and southwestern Asia; however, only small and fragmented populations remain today. Here, we present a de novo genome assembly of the cheetah based on PacBio continuous long reads and Hi-C proximity ligation data. The final assembly (VMU_Ajub_asm_v1.0) has a total length of 2.38 Gb, of which 99.7% are anchored into the expected 19 chromosome-scale scaffolds. The contig and scaffold N50 values of 96.8 Mb and 144.4 Mb, respectively, a BUSCO completeness of 95.4% and a k-mer completeness of 98.4%, emphasize the high quality of the assembly. Furthermore, annotation of the assembly identified 23,622 genes and a repeat content of 40.4%. This new highly contiguous and chromosome-scale assembly will greatly benefit conservation and evolutionary genomic analyses and will be a valuable resource, e.g., to gain a detailed understanding of the function and diversity of immune response genes in felids.

The potential and shortcomings of mitochondrial DNA analysis for cheetah conservation management.

There are only about 7,100 adolescent and adult cheetahs (Acinonyx jubatus) remaining in the wild. With the majority occurring outside protected areas, their numbers are rapidly declining. Evidence-based conservation measures are essential for the survival of this species. Genetic data is routinely used to inform conservation strategies, e.g., by establishing conservation units (CU). A commonly used marker in conservation genetics is mitochondrial DNA (mtDNA). Here, we investigated the cheetah's phylogeography using a large-scale mtDNA data set to refine subspecies distributions and better assign individuals to CUs. Our dataset mostly consisted of historic samples to cover the cheetah's whole range as the species has been extinct in most of its former distribution. While our genetic data largely agree with geography-based subspecies assignments, several geographic regions show conflicting mtDNA signals. Our analyses support previous findings that evolutionary forces such as incomplete lineage sorting or mitochondrial capture likely confound the mitochondrial phylogeography of this species, especially in East and, to some extent, in Northeast Africa. We caution that subspecies assignments solely based on mtDNA should be treated carefully and argue for an additional standardized nuclear single nucleotide polymorphism (SNP) marker set for subspecies identification and monitoring. However, the detection of the A. j. soemmeringii specific haplogroup by a newly designed Amplification-Refractory Mutation System (ARMS) can already provide support for conservation measures. Supplementary Information: The online version contains supplementary material available at 10.1007/s10592-022-01483-1.

Microsatellite markers of the major histocompatibility complex genomic region of domestic camels.

We identified and characterized 11 polymorphic microsatellite markers suitable for routine testing (three in the MHC class I sub-region, four in MHC class II and four in the MHC class III sub-region) of dromedaries and Bactrian camels. In total, 38 dromedaries and 33 Bactrian camels were genotyped, and interspecific differences were observed in the numbers of alleles and in allelic frequencies, as well as in the observed heterozygosity. These loci may be used as markers to study the adaptive genetic diversity of the MHC region in Old World camels.

The natural cytotoxicity receptor genes in the family Felidae.

Natural killer (NK) cells belong to the innate immune system. The germline-encoded natural killer cell receptors represent activating and inhibitory receptors regulating multiple NK cell activities. The natural cytotoxicity receptors (NCRs) are activating natural cytotoxicity triggering receptors 1, 2, and 3 (NKp46, NKp44, and NKp30), encoded by the genes NCR1, NCR2, and NCR3, respectively. NCRs may be expressed in different cell types engaged in mechanisms of innate and adaptive immunity. The family Felidae, comprising the domestic cat and a wide variety of free-ranging species represents a well-suited model for biomedical and evolutionary studies. We characterized the NCR1, NCR2, and NCR3 genes in a panel of felid species. We confirmed the presence of potentially functional genes NCR1, NCR2, and NCR3 in all species. All three genes are conserved within the family and are similar to other phylogenetically related mammalian families. The NCR1 and NCR2 phylogenetic trees based on both nucleotide and protein sequences corresponded to the current zoological taxonomy, with some exceptions suggesting effects of different selection pressures in some species. Highly conserved NCR3 sequences did not allow a robust phylogenetic analysis. Most interspecific differences both at the nucleotide and protein level were found in NCR2. Within species, the most polymorphic CDS was detected in NCR1. Selection analyses indicated the effects of purifying selection on individual amino acid sites in all three genes. In stray cats, a rather high intraspecific diversity was observed.

Shedding persistency and intensity patterns of feline coronavirus (FCoV) in feces of cats living in breeding catteries in the Czech Republic.

Infection with feline coronavirus (FCoV) is a major problem in multiple-cat households, where many cats are kept together in a small space such as catteries and shelters. Sixty cats from 19 breeding catteries included in the study were evaluated for their shedding persistency and intensity patterns using qPCR identification of FCoV in feces. Cats were identified based on shedding persistency as non-shedders (NS) if all four samples negative, intermittent shedders (IS) when at least one positive and one negative sampling followed by another positive sampling, persistent shedders (PS) if all four samples positive and shedders with unclear status (US) if the shedding patterns could not be determined based on only 4 samples. There were 11 NS (18%), 15 IS (25%) and 15 PS (25%) and in 19/60 cats (32%), the shedding patterns could not be determined based only on four samplings. The intensity of shedding was evaluated based on the total number of FCoV particles shed during the 12 months of the study. There were 11 non-shedders (18%), 2 very low intensity shedders (3%), 9 low intensity shedders (15%), 25 medium intensity shedders (42%) and 13 high intensity shedders (22%). Intermittent shedders were shedding significantly lower FCoV particles compared to the persistent shedders (p = 0.0082). Permanent shedders represent the most important source of FCoV infection in multi-cat households and identifying permanent shedders in is the key to minimize the viral load in the environment to control FCoV in a shelters and breeding catteries.

Genomic analyses show extremely perilous conservation status of African and Asiatic cheetahs (Acinonyx jubatus).

We live in a world characterized by biodiversity loss and global environmental change. The extinction of large carnivores can have ramifying effects on ecosystems like an uncontrolled increase in wild herbivores, which in turn can have knock-on impacts on vegetation regeneration and communities. Cheetahs (Acinonyx jubatus) serve important ecosystem functions as apex predators; yet, they are quickly heading towards an uncertain future. Threatened by habitat loss, human-wildlife conflict and illegal trafficking, there are only approximately 7100 individuals remaining in nature. We present the most comprehensive genome-wide analysis of cheetah phylogeography and conservation genomics to date, assembling samples from nearly the entire current and past species' range. We show that their phylogeography is more complex than previously thought, and that East African cheetahs (A. j. raineyi) are genetically distinct from Southern African individuals (A. j. jubatus), warranting their recognition as a distinct subspecies. We found strong genetic differentiation between all classically recognized subspecies, thus refuting earlier findings that cheetahs show only little differentiation. The strongest differentiation was observed between the Asiatic and all the African subspecies. We detected high inbreeding in the Critically Endangered Iranian (A. j. venaticus) and North-western (A. j. hecki) subspecies, and show that overall cheetahs, along with snow leopards, have the lowest genome-wide heterozygosity of all the big cats. This further emphasizes the cheetah's perilous conservation status. Our results provide novel and important information on cheetah phylogeography that can support evidence-based conservation policy decisions to help protect this species. This is especially relevant in light of ongoing and proposed translocations across subspecies boundaries, and the increasing threats of illegal trafficking.

Comparative Genomics of the Major Histocompatibility Complex (MHC) of Felids.

This review summarizes the current knowledge on the major histocompatibility complex (MHC) of the family Felidae. This family comprises an important domestic species, the cat, as well as a variety of free-living felids, including several endangered species. As such, the Felidae have the potential to be an informative model for studying different aspects of the biological functions of MHC genes, such as their role in disease mechanisms and adaptation to different environments, as well as the importance of genetic diversity for conservation issues in free-ranging or captive populations. Despite this potential, the current knowledge on the MHC in the family as a whole is fragmentary and based mostly on studies of the domestic cat and selected species of big cats. The overall structure of the domestic cat MHC is similar to other mammalian MHCs following the general scheme "centromere-MHC class I-MHC class III-MHC class II" with some differences in the gene contents. An unambiguously defined orthologue of the non-classical class I HLA-E gene has not been identified so far and the class II DQ and DP genes are missing or pseudogenized, respectively. A comparison with available genomes of other felids showed a generally high level of structural and sequence conservation of the MHC region. Very little and fragmentary information on in vitro and/or in vivo biological functions of felid MHC genes is available. So far, no association studies have indicated effects of MHC genetic diversity on a particular disease. No information is available on the role of MHC class I molecules in interactions with Natural Killer (NK) cell receptors or on the putative evolutionary interactions (co-evolution) of the underlying genes. A comparison of complex genomic regions encoding NK cell receptors (the Leukocyte Receptor Complex, LRC and the Natural Killer Cell Complex, NKC) in the available felid genomes showed a higher variability in the NKC compared to the LRC and the MHC regions. Studies of the genetic diversity of domestic cat populations and/or specific breeds have focused mainly on DRB genes. Not surprisingly, higher levels of MHC diversity were observed in stray cats compared to pure breeds, as evaluated by DRB sequencing as well as by MHC-linked microsatellite typing. Immunogenetic analysis in wild felids has only been performed on MHC class I and II loci in tigers, Namibian leopards and cheetahs. This information is important as part of current conservation tasks to assess the adaptive potential of endangered wild species at the human-wildlife interface, which will be essential for preserving biodiversity in a functional ecosystem.

The deadly face of felid killer cells: The cytotoxic proteins and their genes.

Natural killer cells and cytotoxic T lymphocytes are the main cell populations of the immune system able to directly kill target cells via cytotoxic granules. Different mammalian species may differ in specific features of their pore-forming protein (perforin) and granule-bound serine proteases (granzymes). One perforin gene (PRF1) and four genes encoding granzymes A, B, H, and K (GZMA, GZMB, GZMH, GZMK) were identified in the reference genomes of felids. The objective of this work was to characterize the genes PRF1, GZMA and GZMB in a panel of 17 felid species by next-generation re-sequencing. A search of available felid genomes (17 species) retrieved the coding sequences of these genes for comparison to our data. Both sets of sequences or their combinations (23 species) were used for phylogenetic and selection analyses. Nucleotide PRF1, GZMA and GZMB sequences showed high similarities between felid species (over 95% identity). All trees derived from coding sequences expressed phylogenetic relationships corresponding to the zoological taxonomy of the Felidae, except GZMA. No effects of positive selection were detected in the genes studied, however, effects of purifying selection were observed for PRF1 and GZMA. The conservation of PRF1 is in agreement with its critical biological function. The differentiation observed between granzyme sub-families may reflect an adaptation to pathogen variation. The need to maintain important gene functions and at the same time cope with various pathogens may lead to an equilibrium between positive and negative selective pressures acting on GZMB. The within-species variability in wild felid populations merits further investigation.

The Population Diversity of Candidate Genes for Resistance/Susceptibility to Coronavirus Infection in Domestic Cats: An Inter-Breed Comparison.

Feline coronavirus (FCoV) is a complex pathogen causing feline infectious peritonitis (FIP). Host genetics represents a factor contributing to the pathogenesis of the disease. Differential susceptibility of various breeds to FIP was reported with controversial results. The objective of this study was to compare the genetic diversity of different breeds on a panel of candidate genes potentially affecting FCoV infection. One hundred thirteen cats of six breeds were genotyped on a panel of sixteen candidate genes. SNP allelic/haplotype frequencies were calculated; pairwise FST and molecular variance analyses were performed. Principal coordinate (PCoA) and STRUCTURE analyses were used to infer population structure. Interbreed differences in allele frequencies were observed. PCoA analysis performed for all genes of the panel indicated no population substructure. In contrast to the full marker set, PCoA of SNP markers associated with FCoV shedding (NCR1 and SLX4IP) showed three clusters containing only alleles associated with susceptibility to FCoV shedding, homozygotes and heterozygotes for the susceptibility alleles, and all three genotypes, respectively. Each cluster contained cats of multiple breeds. Three clusters of haplotypes were identified by PCoA, two clusters by STRUCTURE. Haplotypes of a single gene (SNX5) differed significantly between the PCoA clusters.

Innate and Adaptive Immune Genes Associated with MERS-CoV Infection in Dromedaries.

The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses, only eight years after the emergence of another zoonotic betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I (HLA-A-24-like) and II (HLA-DPB1-like), and innate immune response (PTPN4, MAGOHB), and in cilia coating the respiratory tract (DNAH7). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses, although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.

A Deadly Cargo: Gene Repertoire of Cytotoxic Effector Proteins in the Camelidae.

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).

Crimean-Congo Hemorrhagic Fever Virus Past Infections Are Associated with Two Innate Immune Response Candidate Genes in Dromedaries.

Dromedaries are an important livestock, used as beasts of burden and for meat and milk production. However, they can act as an intermediate source or vector for transmitting zoonotic viruses to humans, such as the Middle East respiratory syndrome coronavirus (MERS-CoV) or Crimean-Congo hemorrhagic fever virus (CCHFV). After several outbreaks of CCHFV in the Arabian Peninsula, recent studies have demonstrated that CCHFV is endemic in dromedaries and camel ticks in the United Arab Emirates (UAE). There is no apparent disease in dromedaries after the bite of infected ticks; in contrast, fever, myalgia, lymphadenopathy, and petechial hemorrhaging are common symptoms in humans, with a case fatality ratio of up to 40%. We used the in-solution hybridization capture of 100 annotated immune genes to genotype 121 dromedaries from the UAE tested for seropositivity to CCHFV. Through univariate linear regression analysis, we identified two candidate genes belonging to the innate immune system: FCAR and CLEC2B. These genes have important functions in the host defense against viral infections and in stimulating natural killer cells, respectively. This study opens doors for future research into immune defense mechanisms in an enzootic host against an important zoonotic disease.

Candidate Gene Markers Associated with Fecal Shedding of the Feline Enteric Coronavirus (FECV).

The Feline coronavirus (FCoV) can cause a fatal disease, the Feline Infectious Peritonitis. Persistent shedders represent the most important source of infection. The role of the host in FCoV fecal shedding is unknown. The objective of this study was to develop gene markers and to test their associations with FCoV shedding patterns. Fecal samples were taken from 57 cats of 12 breeds on the day 0 and after 2, 4 and 12 months. Variation from persistent and/or high-intensity shedding to no shedding was observed. Thirteen immunity-related genes were selected as functional and positional/functional candidates. Positional candidates were selected in a candidate region detected by a GWAS analysis. Tens to hundreds of single nucleotide polymorphisms (SNPs) per gene were identified using next generation sequencing. Associations with different phenotypes were assessed by chi-square and Fisher's exact tests. SNPs of one functional and one positional candidate (NCR1 and SLX4IP, respectively) and haplotypes of four genes (SNX5, NCR2, SLX4IP, NCR1) were associated with FCoV shedding at pcorected < 0.01. Highly significant associations were observed for extreme phenotypes (persistent/high-intensity shedders and non-shedders) suggesting that there are two major phenotypes associated with different genotypes, highly susceptible cats permanently shedding high amounts of viral particles and resistant non-shedders.

Genetic diversity, evolution and selection in the major histocompatibility complex DRB and DQB loci in the family Equidae.

BACKGROUND: The mammalian Major Histocompatibility Complex (MHC) is a genetic region containing highly polymorphic genes with immunological functions. MHC class I and class II genes encode antigen-presenting molecules expressed on the cell surface. The MHC class II sub-region contains genes expressed in antigen presenting cells. The antigen binding site is encoded by the second exon of genes encoding antigen presenting molecules. The exon 2 sequences of these MHC genes have evolved under the selective pressure of pathogens. Interspecific differences can be observed in the class II sub-region. The family Equidae includes a variety of domesticated, and free-ranging species inhabiting a range of habitats exposed to different pathogens and represents a model for studying this important part of the immunogenome. While equine MHC class II DRA and DQA loci have received attention, the genetic diversity and effects of selection on DRB and DQB loci have been largely overlooked. This study aimed to provide the first in-depth analysis of the MHC class II DRB and DQB loci in the Equidae family. RESULTS: Three DRB and two DQB genes were identified in the genomes of all equids. The genes DRB2, DRB3 and DQB3 showed high sequence conservation, while polymorphisms were more frequent at DRB1 and DQB1 across all species analyzed. DQB2 was not found in the genome of the Asiatic asses Equus hemionus kulan and E. h. onager. The bioinformatic analysis of non-zero-coverage-bases of DRB and DQB genes in 14 equine individual genomes revealed differences among individual genes. Evidence for recombination was found for DRB1, DRB2, DQB1 and DQB2 genes. Trans-species allele sharing was identified in all genes except DRB1. Site-specific selection analysis predicted genes evolving under positive selection both at DRB and DQB loci. No selected amino acid sites were identified in DQB3. CONCLUSIONS: The organization of the MHC class II sub-region of equids is similar across all species of the family. Genomic sequences, along with phylogenetic trees suggesting effects of selection as well as trans-species polymorphism support the contention that pathogen-driven positive selection has shaped the MHC class II DRB/DQB sub-regions in the Equidae.

Nucleotide diversity of functionally different groups of immune response genes in Old World camels based on newly annotated and reference-guided assemblies.

BACKGROUND: Immune-response (IR) genes have an important role in the defense against highly variable pathogens, and therefore, diversity in these genomic regions is essential for species' survival and adaptation. Although current genome assemblies from Old World camelids are very useful for investigating genome-wide diversity, demography and population structure, they have inconsistencies and gaps that limit analyses at local genomic scales. Improved and more accurate genome assemblies and annotations are needed to study complex genomic regions like adaptive and innate IR genes. RESULTS: In this work, we improved the genome assemblies of the three Old World camel species - domestic dromedary and Bactrian camel, and the two-humped wild camel - via different computational methods. The newly annotated dromedary genome assembly CamDro3 served as reference to scaffold the NCBI RefSeq genomes of domestic Bactrian and wild camels. These upgraded assemblies were then used to assess nucleotide diversity of IR genes within and between species, and to compare the diversity found in immune genes and the rest of the genes in the genome. We detected differences in the nucleotide diversity among the three Old World camelid species and between IR gene groups, i.e., innate versus adaptive. Among the three species, domestic Bactrian camels showed the highest mean nucleotide diversity. Among the functionally different IR gene groups, the highest mean nucleotide diversity was observed in the major histocompatibility complex. CONCLUSIONS: The new camel genome assemblies were greatly improved in terms of contiguity and increased size with fewer scaffolds, which is of general value for the scientific community. This allowed us to perform in-depth studies on genetic diversity in immunity-related regions of the genome. Our results suggest that differences of diversity across classes of genes appear compatible with a combined role of population history and differential exposures to pathogens, and consequent different selective pressures.

Complex variation in the KLRA (LY49) immunity-related genomic region in horses.

Natural killer (NK) cells play important roles in innate and adaptive immunity, as well as in the reproduction of placental mammals. Ly49 (KLRA) molecules represent a lectin-like type of NK cell receptor encoded within a complex genomic region, the NK cell complex. In rodents and horses, an expansion of the genes encoding Ly49 receptors leading to the formation of a gene family was observed. High sequence similarities and frequent high polymorphism of multiple family members represent an obstacle both for their individual identification and for annotation in the reference genomes of their respective species. Here, we focused on resolving complex variation of the KLRA gene family observed in domestic and Przewalski's horses. The KLRA (LY49) genomic region contains six genes (KLRA2-KLRA7) and one putative pseudogene, KLRA1. Two types of polymorphism were observed in the horses analyzed. Copy number variation between haplotypes was documented for the gene KLRA7 by polymerase chain reaction. As expected, the major source of variation of all KLRA genes, including KLRA7, is because of single nucleotide polymorphisms, many of them being nonsynonymous substitutions. Extensive allelic variability of the expanded KLRA (LY49) genes was observed. For four out of the six functional KLRA, high numbers of novel allelic amino acid sequence variants were identified in the genes studied, suggesting that this variation might be of functional importance, especially in the context of high polymorphism of their presumed ligands encoded by major histocompatibility complex class I genes. In fact, polymorphic amino acid sites were mostly found in the ligand-binding C-type lectin-like domain of the putative receptor molecule.