Domestication does not narrow MHC diversity in Sus scrofa

The Major Histocompatibility Complex (MHC) is a multigene family of outstanding polymorphism. MHC molecules bind antigenic peptides in the peptide-binding region (PBR) that consists of five binding pockets (P). In this study, we compared the genetic diversity of domestic pigs to that of the modern representatives of their wild ancestors, the wild boar, in two MHC loci, the oligomorphic DQA and the polymorphic DRB1. MHC nucleotide polymorphism was compared with the actual functional polymorphism in the PBR and the binding pockets P1, P4, P6, P7, and P9. The analysis of approximately 200 wild boars collected throughout Europe and 120 domestic pigs from four breeds (three pureblood, Pietrain, Leicoma, and Landrace, and one mixed Danbred) revealed that wild boars and domestic pigs share the same levels of nucleotide and amino acid polymorphism, allelic richness, and heterozygosity. Domestication did not appear to act as a bottleneck that would narrow MHC diversity. Although the pattern of polymorphism was uniform between the two loci, the magnitude of polymorphism was different. For both loci, most of the polymorphism was located in the PBR region and the presence of positive selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous substitutions in the PBR. P4 and P6 were the most polymorphic binding pockets. Functional polymorphism, i.e., the number and the distribution of pocket variants within and among populations, was significantly narrower than genetic polymorphism, indicative of a hierarchical action of selection pressures on MHC loci.     

Allelic diversity at the Mhc-DQA locus of woodmouse populations (Apodemus sylvaticus) present in the islands and mainland of the northern Mediterranean

Aim Polymorphism at neutral markers and at MHC loci in rodent populations living on islands is generally low. The main genetic factors that may contribute to a reduced level of genetic variability are genetic drift, reduced gene flow and founder events. We investigated the pattern of polymorphism at the second exon of the Mhc‐DQA gene in island populations of Apodemus sylvaticus and in their mainland counterparts to investigate the pattern of MHC polymorphism in a phylogeographical context and to assess the impact of insularity on diversity at this locus. Location Eight north Mediterranean populations of Apodemus sylvaticus were studied, including five island populations (Majorca, Minorca, Porquerolles, Port‐Cros and Sicily) and three mainland populations. Methods cDNA sequencing and nucleotide sequences analyses. Synonymous and non‐synonymous substitutions were examined at the PBR and non‐PBR sites. The DQA allelic distribution in populations was compared with the woodmouse phylogeography. Results This study presents novel DQA alleles. High polymorphism of the DQA locus is recorded in natural populations of A. sylvaticus with 13 alleles being widely distributed irrespective of the geographical origin and palaeoclimatic history of populations. The DQA locus does not show the expected pattern for non‐synonymous substitutions at the PBR sites. However, island populations show a weak loss of polymorphism in comparison with their mainland counterparts. Main conclusions The DQA locus in the woodmouse seems to be subject to weak selection and does not allow resolution of phylogeographical relationships among European woodmouse populations. The presence of at least three alleles in island populations and the maintenance of five alleles between the two European lineages over 1.5 Myr suggest that balancing selection may act within populations, and more precisely within island populations, to maintain genetic variability. This study shows that phylogeographical studies are a prerequisite for any genetic investigation of selected genes in natural populations.     

Genetic variation in the major histocompatibility complex of the European brown hare (Lepus europaeus) across distinct phylogeographic areas

The major histocompatibility complex is one of the best studied systems in vertebrates providing evidence for the long-term action of selection. Here, we examined the intra- and inter-population genetic diversity of the MHC class II DRB locus in European brown hare (Lepus europaeus) and correlated the results with genetic variability already estimated from the MHC DQA locus and from maternally (mitochondrial DNA (mtDNA)) and biparentally (allozymes, microsatellites) inherited loci. L. europaeus showed remarkable genetic polymorphism in both DQA and DRB1 loci. The Anatolian populations exhibited the highest genetic polymorphism for both loci. Balancing selection has established increased variability in the European populations despite the founder effects after the last glaciation. Different evolutionary rates were traced for DRB1 and DQA loci, as evidenced by the higher number of common DRB1 than DQA alleles and the greater differences between DRB1 alleles with common origin in comparison with DQA alleles. The high number of rare alleles with low frequencies detected implies that frequency-dependent selection drives MHC evolution in the brown hare through the advantage of rare alleles. Both loci were under the influence of positive selection within the peptide-binding region. The functional polymorphism, recorded as amino acid substitutions within the binding pockets, fell also within distinct geographic patterns, yet it was much narrower than the genetic polymorphism. We hypothesize that certain structural and functional characteristics of the binding pockets set limitations to the actual shape of genetic polymorphism in MHC.     

Major histocompatibility complex variation and evolution at a single, expressed DQA locus in two genera of elephants

Genes of the vertebrate major histocompatibility complex (MHC) are crucial to defense against infectious disease, provide an important measure of functional genetic diversity, and have been implicated in mate choice and kin recognition. As a result, MHC loci have been characterized for a number of vertebrate species, especially mammals; however, elephants are a notable exception. Our study is the first to characterize patterns of genetic diversity and natural selection in the elephant MHC. We did so using DNA sequences from a single, expressed DQA locus in elephants. We characterized six alleles in 30 African elephants (Loxodonta africana) and four alleles in three Asian elephants (Elephas maximus). In addition, for two of the African alleles and three of the Asian alleles, we characterized complete coding sequences (exons 1–5) and nearly complete non-coding sequences (introns 2–4) for the class II DQA loci. Compared to DQA in other wild mammals, we found moderate polymorphism and allelic diversity and similar patterns of selection; patterns of non-synonymous and synonymous substitutions were consistent with balancing selection acting on the peptides involved in antigen binding in the second exon. In addition, balancing selection has led to strong trans-species allelism that has maintained multiple allelic lineages across both genera of extant elephants for at least 6 million years. We discuss our results in the context of MHC diversity in other mammals and patterns of evolution in elephants.     

Sequence diversity of the MHC DRB gene in the Eurasian beaver (Castor fiber)

Major histocompatibility complex (MHC) genes, coding molecules which play an important role in immune response, are the most polymorphic genes known in vertebrates. However, MHC polymorphism in some species is limited. MHC monomorphism at several MHC class I and II loci was previously reported for two neighbouring northern European populations of the Eurasian beaver (Castor fiber) and reduced selection for polymorphism has been hypothesized. Here, we analysed a partial sequence of the second exon of the MHC II DRB locus from seven relict European and Asian beaver populations. We detected 10 unique alleles among 76 beavers analysed. Only a western Siberian population was polymorphic, with four alleles detected in 10 individuals. Each of the remaining populations was fixed for a different allele. Sequences showed considerable divergence, suggesting the long persistence of allelic lineages. A significant excess of nonsynonymous substitutions was detected at the antigen binding sites, indicating that sequence evolution of beaver DRB was driven by positive selection. Current MHC monomorphism in the majority of populations may be the result of the superimposition of the recent bottleneck on pre-existing genetic structure resulting from population subdivision and differential pathogen pressure.     

Trans-species polymorphism and evidence of selection on class II MHC loci in tuco-tucos (Rodentia: Ctenomyidae)

Balancing selection acting over the evolutionary history of a lineage can result in the retention of alleles among species for longer than expected under neutral evolution. The associated pattern of trans-species polymorphism, in which similar or even identical alleles are shared among species, is often used to infer that balancing selection has occurred. The genes of the major histocompatibility complex (MHC) are thought to be subject to balancing selection that maintains alleles associated with response to specific pathogens. To explore the role of balancing selection in shaping MHC diversity in ctenomyid rodents, we examined allelic variability at the class II DRB and DQA loci in 18 species in the genus Ctenomys. Previous studies of four of these species had revealed significant within-population evidence of positive selection on MHC loci. The current study expands upon these analyses to (1) evaluate among-species evidence of positive selection and (2) explore the potential for balancing selection on MHC genes. Interspecific nucleotide sequence variation revealed significant evidence of positive selection on the DRB and DQA loci. At the same time, comparisons of phylogenetic trees for these MHC loci with a putative species tree based on mitochondrial sequence data revealed multiple examples of trans-specific polymorphism, including sharing of identical DRB and DQA alleles among distantly related species of Ctenomys. These findings suggest that MHC genes in these animals have historically been subject to balancing selection and yield new insights into the complex suite of forces shaping MHC diversity in free-living vertebrates.     

Diversity and evolutionary history of the MHC DQA gene in leporids

The European rabbit (Oryctolagus cuniculus) is used as a model for many human diseases, yet comparatively little is known of its genetics, particularly at important loci such as the major histocompatibility complex (MHC). This study investigated genetic diversity and evolutionary history of the DQA gene in a range of leporid species by analysing coding sequence diversity of exon 2 and intron 2 in 53 individuals of 16 different species. Fifty leporid DQA alleles were detected, including 13 novel European rabbit alleles. In the rabbit, the highest levels of diversity were observed in wild rabbits from Portugal, with wild rabbits from England and domestic rabbits showing less diversity. Within the sample, several recombination events were detected and trans-specific evolution of alleles was evidenced, both being general characteristics of mammalian MHC genes. Positive selection is implicated as operating on six codons within exon 2, which are also subject to positive selection in other mammals. Some of these positions are putative antigen recognition sites and underline the importance of pathogen-driven selection on these MHC genes.     

Ancestral polymorphism at the major histocompatibility complex (MHCIIbeta) in the Nesospiza bunting species complex and its sister species (Rowettia goughensis)

ABSTRACT: BACKGROUND: The major histocompatibility complex (MHC) is an important component of the vertebrate immune system and is frequently used to characterise adaptive variation in wild populations due to its co-evolution with pathogens. Passerine birds have an exceptionally diverse MHC with multiple gene copies and large numbers of alleles compared to other avian taxa. The Nesospiza bunting species complex (two species on Nightingale Island; one species with three sub-species on Inaccessible Island) represents a rapid adaptive radiation at a small, isolated archipelago, and is thus an excellent model for the study of adaptation and speciation. In this first study of MHC in Nesospiza buntings, we aim to characterize MHCIIbeta variation, determine the strength of selection acting at this gene region and assess the level of shared polymorphism between the Nesospiza species complex and its putative sister taxon, Rowettia goughensis, from Gough Island. RESULTS: In total, 23 unique alleles were found in 14 Nesospiza and 2 R. goughensis individuals encoding at least four presumably functional loci and two pseudogenes. There was no evidence of ongoing selection on the peptide binding region (PBR). Of the 23 alleles, 15 were found on both the islands inhabited by Nesospiza species, and seven in both Nesospiza and Rowettia; indications of shared, ancestral polymorphism. A gene tree of Nesospiza MHCIIbeta alleles with several other passerine birds shows three highly supported Nesospiza-specific groups. All R. goughensis alleles were shared with Nesospiza, and these alleles were found in all three Nesospiza sequence groups in the gene tree, suggesting that most of the observed variation predates their phylogenetic split. CONCLUSIONS: Lack of evidence of selection on the PBR, together with shared polymorphism across the gene tree, suggests that population variation of MHCIIbeta among Nesospiza and Rowettia is due to ancestral polymorphism rather than local selective forces. Weak or no selection pressure could be attributed to low parasite load at these isolated Atlantic islands. The deep divergence between the highly supported Nesospiza-specific sequence Groups 2 and 3, and the clustering of Group 3 close to the distantly related passerines, provide strong support for preserved ancestral polymorphism, and present evidence of one of the rare cases of extensive ancestral polymorphism in birds.     

Major histocompatibility complex class II genetic variation in forest musk deer (Moschus berezovskii) in China

The major histocompatibility complex (MHC) plays an important role in the immune system of vertebrates. We used the second exon of four MHC class II genes (DRA, DQA1, DQA2 and DRB3) to assess the overall MHC variation in forest musk deer (Moschus berezovskii). We also compared the MHC variation in captive and wild populations. We observed 22 alleles at four loci (four at DRA, four at DQA1, four at DQA2 and 10 at DRB3), 15 of which were newly identified alleles. Results suggest that forest musk deer maintain relatively high MHC variation, which may result from balancing selection. Moreover, considerable diversity was observed at the DRA locus. We found a high frequency of Mobe‐DRA*02, Mobe‐DQA1*01 and Mobe‐DQA2*05 alleles, which may be important for pathogen resistance. A Ewens–Watterson test showed that the DRB3 locus in the wild population had experienced recent balancing selection. We detected a small divergence at the DRA locus, suggesting the effect of weak positive selection on the DRA gene. Alternatively, this locus may be young and not yet adapted a wide spectrum of alleles for pathogen resistance. The significant heterozygosity deficit observed at the DQA1 and DRB3 loci in the captive population and at all four loci in the wild population may be the result of a population bottleneck. Additionally, MHC genetic diversity was higher in the wild population than in the captive, suggesting that the wild population may have the ability to respond to a wider range of pathogens.     

Genetic drift outweighs balancing selection in shaping post-bottleneck major histocompatibility complex variation in New Zealand robins (Petroicidae)

The Chatham Island black robin, Petroica traversi, is a highly inbred, endangered passerine with extremely low levels of variation at hypervariable neutral DNA markers. In this study we investigated variation in major histocompatibility complex (MHC) class II genes in both the black robin and its nonendangered relative, the South Island robin Petroica australis australis. Previous studies have shown that Petroica have at least four expressed class II B MHC genes. In this study, the sequences of introns flanking exon 2 of these loci were characterized to design primers for peptide-binding region (PBR) sequence analysis. Intron sequences were comprised of varying numbers of repeated units, with highly conserved regions immediately flanking exon 2. Polymerase chain reaction primers designed to this region amplified three or four sequences per black robin individual, and eight to 14 sequences per South Island robin individual. MHC genes are fitness-related genes thought to be under balancing selection, so they may be more likely to retain variation in bottlenecked populations. To test this, we compared MHC variation in the black robin with artificially bottlenecked populations of South Island robin, and with their respective source populations, using restriction fragment length polymorphism analyses and DNA sequencing of the PBR. Our results indicate that the black robin is monomorphic at class II B MHC loci, while both source and bottlenecked populations of South Island robin have retained moderate levels of variation. Comparison of MHC variation with minisatellite DNA variation indicates that genetic drift outweighs balancing selection in determining MHC diversity in the bottlenecked populations. However, balancing selection appears to influence MHC diversity over evolutionary timescales, and the effects of gene conversion are evident.     

Ancestral polymorphism in exon 2 of bluethroat (Luscinia svecica) MHC class II B genes

The genes of the major histocompatibility complex (MHC) are important model genes for understanding selective forces in evolution. Here, we document, using a cloning and sequencing approach, high polymorphism at the exon 2 of the MHC class II B (MHCIIB) genes in the bluethroat (Luscinia svecica); a minimum of 61 unique alleles were detected in 20 individuals, and at least 11 functional loci. In addition, several pseudogenes were revealed. The specimens originated from three different bluethroat subspecies (azuricollis, cyanecula and svecica), and we also analysed four specimens of the closely related thrush nightingale (L. luscinia) for comparison. Phylogenetic analyses of the functional alleles revealed 258 equally parsimonious trees with poor statistical support for the majority of nodes. The distribution of the sequences in the trees point to an ancestral origin of the polymorphism in MHC class II B genes, a portion of which predated the phylogenetic split between the bluethroat and the thrush nightingale. Strong signatures of balancing selection were uncovered for the codons coding for the peptide‐binding residues of the functional MHCIIB exon 2 alleles. Our results highlight the importance of duplication and recombination events for shaping passerine MHC and give insights in the evolutionary dynamics of MHC variation among closely related taxa.     

Cryptic MHC polymorphism revealed but not explained by selection on the class IIb peptide-binding region

The immune genes of the major histocompatibility complex (MHC) are characterized by extraordinarily high levels of nucleotide and haplotype diversity. This variation is maintained by pathogen-mediated balancing selection that is operating on the peptide-binding region (PBR). Several recent studies have found, however, that some populations possess large clusters of alleles that are translated into virtually identical proteins. Here, we address the question of how this nucleotide polymorphism is maintained with little or no functional variation for selection to operate on. We investigate circa 750-850 bp of MHC class II DAB genes in four wild populations of the guppy Poecilia reticulata. By sequencing an extended region, we uncovered 40.9% more sequences (alleles), which would have been missed if we had amplified the exon 2 alone. We found evidence of several gene conversion events that may have homogenized sequence variation. This reduces the visible copy number variation (CNV) and can result in a systematic underestimation of the CNV in studies of the MHC and perhaps other multigene families. We then focus on a single cluster, which comprises 27 (of a total of 66) sequences. These sequences are virtually identical and show no signal of selection. We use microsatellites to reconstruct the populations' demography and employ simulations to examine whether so many similar nucleotide sequences can be maintained in the populations. Simulations show that this variation does not behave neutrally. We propose that selection operates outside the PBR, for example, on linked immune genes or on the "sheltered load" that is thought to be associated to the MHC. Future studies on the MHC would benefit from extending the amplicon size to include polymorphisms outside the exon with the PBR. This may capture otherwise cryptic haplotype variation and CNV, and it may help detect other regions in the MHC that are under selection.     

Polymorphisms in major histocompatibility complex class IIα genes are associated with resistance to infectious hematopoietic necrosis in rainbow trout,Oncorhynchus mykiss (Walbaum, 1792)

Infectious hematopoietic necrosis is a serious viral disease of salmonids, including rainbow trout Oncorhynchus mykiss, and causes tremendous economic losses to the rainbow trout farming industry. Major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity in vertebrate organisms and have been linked with the resistance to numerous pathogenic diseases. In this study, polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by cloning and sequencing were used to examine polymorphisms in the DAA genes (specifically DAA exon 2 of MHC class IIα) of rainbow trout and investigate their association with the infectious hematopoietic necrosis virus (IHNV) resistance in rainbow trout. Seventeen alleles were resolved, including 13 novel alleles. Individuals possessed between two and five alleles, indicating that the genome harbours at least three closely‐related DAA exon 2 loci. The ratio of non‐synonymous to synonymous nucleotide substitutions suggested that DAA exon 2 is under positive selection. A greater variability of amino acids and non‐synonymous nucleotide substitution rate was evident in the peptide‐binding region (PBR) than in the non‐PBR (27.75%). Importantly, the analyses revealed that certain MHC class IIα alleles appear to confer resistance to IHNV in rainbow trout, while others confer susceptibility. The most common alleles in the resistant populations of rainbow trout, Onmy‐DAA*1301 and Onmy‐DAA*0304, confer resistance to IHNV and were not present in the susceptible population. Hence, these alleles may be ideal molecular markers that can assist the breeding of IHNV resistance in rainbow trout.     

Genetic variation of the MHC DQB locus in the finless porpoise (Neophocaena phocaenoides)

The Major Histocompatibility Complex (MHC) is a large multigene coding for glycoproteins that play a key role in the initiation of immune responses in vertebrates. The exon 2 region of the MHC DQB locus was analyzed using 160 finless porpoises from 5 populations in Japanese waters. The 5 populations were based on a previous mitochondrial DNA control region analysis, which showed distinct geographical separation. Eight DQB alleles were detected, and the geographical distribution of the alleles indicated that most of them are shared among the populations. Heterozygosity of the DQB alleles in each population ranged from 0.55 to 0.78, and for all 5 populations was 0.78. Low MHC variability is not a common feature in marine mammals, but the finless porpoise populations inhabiting coastal waters had a relatively high MHC heterozygosity. Balancing selection in the MHC DQB alleles of the finless porpoise was indicated by the higher rate of nonsynonymous than synonymous substitutions for PBR; however, an excess of hetrozygotes compared to expectation was not observed. This suggests that the MHC DQB locus in the finless porpoise may have been under balancing selection for a long evolutionary time period, and is influenced by genetic drift beyond the effect of balancing selection for short time periods in small local populations.     

Evolutionary history of an MHC gene in two leporid species: characterisation of <Emphasis Type="Italic">Mhc-DQA</Emphasis> in the European brown hare and comparison with the European rabbit

We surveyed the genetic diversity of the expressed major histocompatibility complex class II DQA locus in natural populations of European brown hares, Lepus europaeus, from Austria and Belgium (267 individuals in total). Based on cDNA sequences, we designed hare-specific primers to amplify the highly variable second exon of the DQA gene. Using cloning–sequencing methodology and capillary electrophoresis single-strand conformation polymorphism, we found ten alleles of the DQA exon 2 locus across these two European regions, of which eight are described for the first time. To search for signals of selection and recombination in the evolution of the DQA gene within the leporids, we augmented our sample with orthologous DQA alleles from the European rabbit, Oryctolagus cuniculus, in order to carry out a species level, species pairwise comparison. We found evidence of recombination in the history of the DQA sequences in leporids with some recombinant alleles bridging the species divide. In both species, selection on peptide binding site codons can be detected, though stronger for the rabbit. This result suggests that there may be a differential selection pressure in the deeper evolutionary history of these two species due to differences in several demographic and ecological traits likely subjecting them to differential selection by parasites. Finally, evolutionary relationships show a widespread and statistically significant intermingling of alleles from the two species. The many macroparasites shared between hares and rabbits may explain this pattern of trans-species polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper have been submitted to Genbank and have been assigned the accession numbers FJ225335–FJ225346.     

Structure and Evolution of a New Avian MHC Class II B Gene in a Sub-Antarctic Seabird,the Thin-Billed Prion (Procellariiformes: <Emphasis Type="Italic">Pachyptila belcheri</Emphasis>)

The major histocompatibility complex encodes molecules that present foreign peptides to T cells of the immune system. The peptide binding region (PBR) of these molecules is among the most polymorphic regions found in vertebrate taxa. Genomic cloning approaches are improving our understanding of the evolution of this multigene family in nonmodel avian groups. By building a cosmid library, a new MHC class II B gene, Pabe-DAB1, was isolated and characterized at the genomic level in a sub-Antarctic seabird, the thin-billed prion (Pachyptila belcheri). Pabe-DAB1 exhibits the hallmark structural features of functional MHC class II loci. Direct sequencing of the PBR encoding exon in a panel of prions revealed significantly higher levels of genetic diversity compared to two noncoding neutral loci, with most alleles differing by at least one replacement substitution in the peptide binding codons. We estimated evolutionary dynamics for Pabe-DAB1 using a variety of Bayesian and other approaches. Evidence for balancing selection comes from a spatially variable ratio of nonsynonymous-to-synonymous substitutions (mean d _N/d _S = 2.87) in the PBR, with sites predicted to be functionally relevant exhibiting the highest ω values. We estimate the population recombination rate to be approximately 0.3 per site per generation, indicating an important role for recombination in generating polymorphism at this locus. Pabe-DAB1 is among the few avian class II loci characterized at the genomic level and with a known intron-exon structure, a feature that greatly facilitated the amplification and sequencing of a single MHC locus in this species.     

Evolution of MHC class IIB in the genome of wild and ornamental guppies, Poecilia reticulata

This is the first study to quantify genomic sequence variation of the major histocompatibility complex (MHC) in wild and ornamental guppies, Poecilia reticulata. We sequenced 196-219 bp of exon 2 MHC class IIB (DAB) in 56 wild Trinidadian guppies and 14 ornamental strain guppies. Each of two natural populations possessed high allelic richness (15-16 alleles), whereas only three or fewer DAB alleles were amplified from ornamental guppies. The disparity in allelic richness between wild and ornamental fish cannot be fully explained by fixation of alleles by inbreeding, nor by the presence of non-amplified sequences (ie null alleles). Rather, we suggest that the same allele is fixed at duplicated MHC DAB loci owing to gene conversion. Alternatively, the number of loci in the ornamental strains has contracted during >100 generations in captivity, a hypothesis consistent with the accordion model of MHC evolution. We furthermore analysed the substitution patterns by making pairwise comparisons of sequence variation at the putative peptide binding region (PBR). The rate of non-synonymous substitutions (dN) only marginally exceeded synonymous substitutions (dS) in PBR codons. Highly diverged sequences showed no evidence for diversifying selection, possibly because synonymous substitutions have accumulated since their divergence. Also, the substitution pattern of similar alleles did not show evidence for diversifying selection, plausibly because advantageous non-synonymous substitutions have not yet accumulated. Intermediately diverged sequences showed the highest relative rate of non-synonymous substitutions, with dN/dS>14 in some pairwise comparisons. Consequently, a curvilinear relationship was observed between the dN/dS ratio and the level of sequence divergence.