Major Histocompatibility Complex Class IIB Genes Research Articles

Ancient duplication, coevolution, and selection at the MHC class IIA and IIB genes of birds.

The Major Histocompatibility Complex (MHC) of vertebrates is a dynamically evolving multigene family primarily responsible for recognizing non-self peptide antigens and triggering a pathogen-specific adaptive immune response. In birds, the MHC was previously thought to evolve via concerted evolution with high degree of gene homogenization and the rapid loss of orthology. However, the discovery of two ancient avian MHC-IIB gene lineages (DAB1 and DAB2) originating before the radiation of extant birds indicated that despite the action of concerted evolution, orthology may be detectable for long evolutionary periods. Here, we take advantage of rapidly accumulating digital genomic resources to search for the signal of an ancient duplication at the avian MHC-IIA genes, as well as to compare phylogenetic distribution and selection between MHC-IIA and IIB gene lineages. The analysis of MHC sequences from over 230 species representing ca. 70 bird families revealed the presence of two ancient MHC-IIA gene lineages (DAA1 and DAA2) and showed that their phylogenetic distribution matches exactly the distribution of DAB1 and DAB2 lineages, suggesting tight coevolution. The early post-duplication divergence of DAA1 and DAA2 was driven by positive selection fixing radical amino acid differences within the membrane-proximal domain and, most probably, being functionally related to the interactions between α2 and β2 chains of the MHC-II heterodimer. We detected no evidence for an overall (gene-wide) relaxation or intensification of selection at either DAA1/DAB1 or DAA2/DAB2, but codon-specific differences in selection signature were found at the peptide-binding sites between the two gene lineages, perhaps implying specialization to different pathogen regimes. Our results suggest that specific pairing of MHC-II α and β chains may have an adaptive significance, a conclusion that advances knowledge on the macroevolution of the avian MHC-II and opens exciting novel directions for future research.

Trans-specific polymorphism and the convergent evolution of supertypes in major histocompatibility complex class II genes in darters (Etheostoma).

Major Histocompatibility Complex (MHC) genes are one of the most polymorphic gene groups known in vertebrates. MHC genes also exhibit allelic variants that are shared among taxa, referred to as trans‐specific polymorphism (TSP). The role that selection plays in maintaining such high diversity within species, as well as TSP, is an ongoing discussion in biology. In this study, we used deep‐sequencing techniques to characterize MHC class IIb gene diversity in three sympatric species of darters. We found at least 5 copies of the MHC gene in darters, with 126 genetic variants encoding 122 unique amino acid sequences. We identified four supertypes based on the binding properties of proteins encoded by the sequences. Although each species had a unique pool of variants, many variants were shared between species pairs and across all three species. Phylogenetic analysis showed that the variants did not group together monophyletically based on species identity or on supertype. An expanded phylogenetic analysis showed that some darter alleles grouped together with alleles from other percid fishes. Our findings show that TSP occurs in darters, which suggests that balancing selection is acting at the genotype level. Supertypes, however, are most likely evolving convergently, as evidenced by the fact that alleles do not form monophyletic groups based on supertype. Our research demonstrates that selection may be acting differently on MHC genes at the genotype and supertype levels, selecting for the maintenance of high genotypic diversity while driving the convergent evolution of similar MHC phenotypes across different species.

MHC Class IIB gene polymorphisms associated with resistance/susceptibility to Streptococcus agalactiae in Nile tilapia Oreochromis niloticus.

Genetic variation in the major histocompatibility complex (MHC) Class IIB was tested in Nile tilapia Oreochromis niloticus, and the association between the MHC IIB alleles and disease resistance was also studied. F3 fry offspring (n = 1200) from 12 full-sib families were challenged with Streptococcus agalactiae, which caused significantly different mortalities in different Nile tilapia families (11.00-81.10%). Twenty fry (F1) from each of the 12 families were selected to study the polymorphisms of the MHC Class IIB gene using PCR followed by cloning and sequencing methods. The results showed that the size of the amplified fragment was 770-797 bp. Thirty-seven sequences from 240 individuals revealed 22 different alleles, which belonged to 9 major allele types. Up to 63.58% of nucleotide positions were variable, while the proportion of the amino acid variable positions was up to 68.73%. According to the survival rate of offspring (F3) from 12 full-sib families, we deduced that the alleles Orni-DAB*0107, Orni-DAB*0201 and Orni-DAB*0302 were highly associated with resistance to S. agalactiae, while the allele Orni-DAB*0701 was associated with susceptibility to S. agalactiae. In addition, our previous study found that the allele Orni-DAB*0201 was more frequently distributed in the disease-resistant groups. Therefore, the allele Orni-DAB*0201 could be used as an S. agalactiae resistance-related MHC marker in molecular marker-assisted selective breeding programs for S. agalactiae-resistant Nile tilapia.

Karakterisasi Molekuler Ikan Gurami Soang (Osphronemus gouramy Lac.) yang Mati pada Rentang Waktu Berbeda Menggunakan PCR-RFLP Gen Major Histocompatibility Complex Kelas II B

Gurami (Osphronemus gouramy Lac.) is a popular fish species among Indonesian people. Several Gurami strains have been cultivated by fish farmer, one of which is Gurami Soang. This strain is belived to have a faster growth rate compared to other strains. However, like other strains, the fingerling of Soang strain have also a low survival and suceptible to disease, especially that caused by Aeromonas hydrophila infection. It has been proved that seeds from a single spawning event show varibale disease resistance. The difference in resistance among individuals is suggested related to the difference in their genetic component. One of the genes responsible for resistance is Major Histocompatibility Complex (MHC) class II B gene. Variability in resistance can be analyzed by using PCR - RFLP technique. PCR-RFLP is a technique that can produce a specific DNA fragments by PCR, followed by cutting the PCR product using restriction enzymes to describe the presence or absence of restriction sites in DNA fragments. This research aims to determine genetic marker to differiantiate between resitant and irresistant individual of Gurami Soang infected by A. hydrophila which die at a different time priod based on PCR-RFLP MHC class IIB gene. The study used survey method with purposive random sampling. The Data of PCR-RFLP band patterns were analyzed descriptively. The result indicated that cutting of the MHC class II B gene using HinfI produce two RFLP bands with 300 bp and 100 bp length in all samples. Meanwhile, the MHC IIB gene was not cuted by PstI, HindIII, BamHI and EcoRI enzymes forall samples. These mean that MHC II gene in all individuals were monomorphic. Therefore,it can be concluded that there is no specific genetic marker to differentiate gurami soang individulas which was dying in different time periods.

Genetic variation of major histocompatibility complex genes in the endangered red-crowned crane.

Populations that have drastically decreased in the past often have low genetic variation, which may increase the risk of extinction. The genes of major histocompatibility complex (MHC) play an important role in the adaptive immune response of jawed vertebrates. Maintenance of adaptive genetic diversity such as that of MHC genes is important for wildlife conservation. Here, we determined genotypes of exon 3 of MHC class IA genes (MHCIA) and exon 2 of MHC class IIB genes (MHCIIB) to evaluate genetic variation of the endangered red-crowned crane population on Hokkaido Island, Japan, which experienced severe population decline in the past. We identified 16 and 6 alleles of MHCIA and MHCIIB, respectively, from 152 individuals. We found evidence of a positive selection at the antigen-binding sites in MHCIA exon 3 and MHCIIB exon 2. The phylogenetic analyses indicated evidence of trans-species polymorphism among the crane MHC genes. The genetic variability in both classes of MHC genes at the population level was low. No geographic structure was found based on the genetic diversity of microsatellite and MHC genes. Our study provides useful data for the optimal management of the red-crowned crane population in Hokkaido and can contribute to future studies on MHC genes of the continental populations of the red-crowned crane and other crane species.

Molecular characterization of MHC class IIB genes of sympatric Neotropical cichlids

BackgroundThe Major Histocompatibility Complex (MHC) is a key component of the adaptive immune system of all vertebrates and consists of the most polymorphic genes known to date. Due to this complexity, however, MHC remains to be characterized in many species including any Neotropical cichlid fish. Neotropical crater lake cichlids are ideal models to study evolutionary processes as they display one of the most convincing examples of sympatric and repeated parallel radiation events within and among isolated crater lakes.ResultsHere, we characterized the genes of MHC class IIB chain of the Midas cichlid species complex (Amphilophus cf. citrinellus) including fish from five lakes in Nicaragua. We designed 19 new specific primers anchored in a stepwise fashion in order to detect all alleles present. We obtained 866 genomic DNA (gDNA) sequences from thirteen individuals and 756 additional sequences from complementary DNA (cDNA) of seven of those individuals. We identified 69 distinct alleles with up to 25 alleles per individual. We also found considerable intron length variation and mismatches of alleles detected in cDNA and gDNA suggesting that some loci have undergone pseudogenization. Lastly, we created a model of protein structure homology for each allele and identified their key structural components.ConclusionsOverall, the Midas cichlid has one of the most diverse repertoires of MHC class IIB genes known, which could serve as a powerful tool to elucidate the process of divergent radiations, colonization and speciation in sympatry.

Does the parasite-mediated selection drive the MHC class IIB diversity in wild populations of European chub (Squalius cephalus)?

The genes of major histocompatibility complex (MHC) provide an excellent opportunity to study host-parasite relationships because they are expected to evolve in response to parasites and variation in parasite communities. In this study, we investigated the potential role of parasite-mediated selection acting on MHC class IIB (DAB) genes in European chub (Squalius cephalus) natural populations. We found significant differences between populations in metazoan parasites, neutral and adaptive genetic diversities. The analyses based on pairwise data revealed that populations with dissimilar MHC allelic profiles were geographically distant populations with significantly different diversity in microsatellites and a dissimilar composition of parasite communities. The results from the generalized estimating equations method (GEE) on the level of individuals revealed that metazoan parasite load in European chub was influenced by the diversity of DAB alleles as well as by the diversity of neutral genetic markers and host traits reflecting condition and immunocompetence. The multivariate co-inertia analysis showed specific associations between DAB alleles and parasite species. DAB1-like alleles were more involved in associations with ectoparasites, while DAB3-like alleles were positively associated with endoparasites which could suggest potential differences between DAB genes caused by different selection pressure. Our study revealed that parasite-mediated selection is not the only variable affecting MHC diversity in European chub; however, we strongly support the role of neutral processes as the main driver of DAB diversity across populations. In addition, our study contributes to the understanding of the evolution of MHC genes in wild living fish.

Genetic polymorphism of major histocompatibility complex class IIB alleles and pathogen resistance in the giant spiny frog Quasipaa spinosa

Major histocompatibility complex (MHC) genes are candidates for determining disease susceptibility due to their pivotal role in both innate and adaptive immune responses. Accordingly, the association between the genetic variation of MHC genes and the pathogen resistance has been investigated in numerous vertebrates. To date, however, little is reported in amphibians. In this study, we investigate the genetic variation at the MHC class IIB gene in the giant spiny frog Quasipaa spinosa, which has high commercial value in China. The full length of MHC class IIB cDNA was cloned from Q. spinosa by homology cloning and rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR). Two MHC class IIB loci were identified in Q. spinosa. We also developed PCR primers for a portion of the second exon of the MHC class IIB gene. A total of 26 MHC class IIB alleles were identified. The dN rate was significantly higher than the dS rate in the putative peptide-binding region, thereby proving the positive selection hypothesis. In addition, individuals intraperitoneally injected with Aeromonas hydrophila were used to study the association between MHC class IIB alleles and pathogen resistance/susceptibility, to explore the specific alleles in balancing selection. Eighty frogs were used after exposure to A. hydrophila infection. Nine alleles were used to study the association between the alleles and disease resistance. Two alleles, namely, Pasa-DAB∗1301 and Pasa-DAB∗0901, were significantly associated with resistance against A. hydrophila. This study provides valuable information on the structure of the MHC class IIB gene and confirms the association between MHC class IIB gene alleles and disease resistance to bacterial infection in Q. spinosa. Moreover, pathogen resistance-related MHC markers can be used for the selective breeding of the giant spiny frog.

PCR‐based isolation of multigene families: lessons from the avian MHC class IIB

The amount of sequence data available today highly facilitates the access to genes from many gene families. Primers amplifying the desired genes over a range of species are readily obtained by aligning conserved gene regions, and laborious gene isolation procedures can often be replaced by quicker PCR-based approaches. However, in the case of multigene families, PCR-based approaches bear the often ignored risk of incomplete isolation of family members. This problem is most prominent in gene families with highly variable and thus unpredictable number of gene copies among species, such as in the major histocompatibility complex (MHC). In this study, we (i) report new primers for the isolation of the MHC class IIB (MHCIIB) gene family in birds and (ii) share our experience with isolating MHCIIB genes from an unprecedented number of avian species from all over the avian phylogeny. We report important and usually underappreciated problems encountered during PCR-based multigene family isolation and provide a collection of measures to help significantly improving the chance of successfully isolating complete multigene families using PCR-based approaches.

Differential modes of MHC class IIB gene evolution in cichlid fishes

Cichlid fishes are emblematic models for the study of adaptive radiation, driven by natural and sexual selection. Parasite mediated selection is an important component in these processes, and the evolution of their immune system therefore merits special attention. In this study, light is shed on the phylogeny of the b family of cichlid major histocompatibility complex (MHC) class IIB genes. Full-length coding sequences were used to reconstruct phylogenies using criteria of maximum parsimony, maximum likelihood and Bayesian inference. All analyses suggest monophyly of the b family of cichlid MHC class IIB genes, although sequences of the cichlid sister taxa are currently not available. Two evolutionary lineages of these genes, respectively encompassing the recently defined genomic regions DBB-DEB-DFB and DCB-DDB, show highly contrasting levels of differentiation. To explore putative causes for these differences, exon 2 sequences were screened for variation in recombination rate and strength of selection. The more diversified lineage of cichlid MHC class IIB b genes was found to have higher levels of both recombination and selection. This is consistent with the observation in other taxa that recombination facilitates the horizontal spread of positively selected sites across MHC loci and hence contributes to fast sequence evolution. In contrast, the lineage that showed low diversification might either be under stabilizing selection or is evolutionary constrained by its low recombination rate. We speculate whether this lineage might include MHC genes with non-classical functions.

Molecular cloning and expression analysis of major histocompatibility complex class I, IIA and IIB genes of blunt snout bream (Megalobrama amblycephala)

Major histocompatibility complex (MHC) plays an important role in the immune response of vertebrates. In this study, we isolated MHC class IIA and IIB genes from blunt snout bream (Megalobrama amblycephala) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). In order to study the function of the MHC genes in M. amblycephala, tissue distribution and immune response of the MHC genes to bacterial challenge were analyzed. All the characteristic features of MHC class II chain structure could be identified in the deduced amino sequences of MHC IIA and IIB, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. The deduced amino acid sequence of the MHC IIA and IIB molecules shared from 48% to 88% and from 65% to 77% similarity with those of other teleosts, respectively. Quantitative real-time PCR (qRT-PCR) demonstrated that MHC I and II genes were ubiquitously expressed in ten tissues, with high level in immune related tissues, including kidney, intestine, gill and spleen. Challenge of M. amblycephala with the extracellular pathogen, Aeromonas hydrophila, resulted in a significant increase in the expression of MHC I, MHC IIA and IIB mRNA within 72h after infection in gill, kidney, intestine and liver, followed by a recovery to normal level after 120h. The changes of expression levels for MHC IIA and IIB in most tissues were significantly higher than that of MHC I in the corresponding tissues at most time points (P<0.05). These results demonstrated the MHC genes played an important role in response to bacterial infection in M. amblycephala; however, MHC class I and II genes showed different functional activity, which need be further investigated in teleost.

Combining molecular evolution and environmental genomics to unravel adaptive processes of MHC class IIB diversity in European minnows (Phoxinus phoxinus)

Host–pathogen interactions are a major evolutionary force promoting local adaptation. Genes of the major histocompatibility complex (MHC) represent unique candidates to investigate evolutionary processes driving local adaptation to parasite communities. The present study aimed at identifying the relative roles of neutral and adaptive processes driving the evolution of MHC class IIB (MHCIIB) genes in natural populations of European minnows (Phoxinus phoxinus). To this end, we isolated and genotyped exon 2 of two MHCIIB gene duplicates (DAB1 and DAB3) and 1′665 amplified fragment length polymorphism (AFLP) markers in nine populations, and characterized local bacterial communities by 16S rDNA barcoding using 454 amplicon sequencing. Both MHCIIB loci exhibited signs of historical balancing selection. Whereas genetic differentiation exceeded that of neutral markers at both loci, the populations' genetic diversities were positively correlated with local pathogen diversities only at DAB3. Overall, our results suggest pathogen-mediated local adaptation in European minnows at both MHCIIB loci. While at DAB1 selection appears to favor different alleles among populations, this is only partially the case in DAB3, which appears to be locally adapted to pathogen communities in terms of genetic diversity. These results provide new insights into the importance of host–pathogen interactions in driving local adaptation in the European minnow, and highlight that the importance of adaptive processes driving MHCIIB gene evolution may differ among duplicates within species, presumably as a consequence of alternative selective regimes or different genomic context.Using next-generation sequencing, the present manuscript identifies the relative roles of neutral and adaptive processes driving the evolution of MHC class IIB (MHCIIB) genes in natural populations of a cyprinid fish: the European minnow (Phoxinus phoxinus). We highlight that the relative importance of neutral versus adaptive processes in shaping immune competence may differ between duplicates as a consequence of alternative selective regimes or different genomic contexts.

Evolutionary history of black grouse major histocompatibility complex class IIB genes revealed through single locus sequence-based genotyping

BackgroundGene duplications are frequently observed in the Major Histocompatibility Complex (MHC) of many species, and as a consequence loci belonging to the same MHC class are often too similar to tell apart. In birds, single locus genotyping of MHC genes has proven difficult due to concerted evolution homogenizing sequences at different loci. But studies on evolutionary history, mode of selection and heterozygosity correlations on the MHC cannot be performed before it is possible to analyse duplicated genes separately. In this study we investigate the architecture and evolution of the MHC class IIB genes in black grouse. We developed a sequence-based genotyping method for separate amplification of the two black grouse MHC class IIB genes BLB1 and BLB2. Based on this approach we are able to study differences in structure and selection between the two genes in black grouse and relate these results to the chicken MHC structure and organization.ResultsSequences were obtained from 12 individuals and separated into alleles using the software PHASE. We compared nucleotide diversity measures and employed selection tests for BLB1 and BLB2 to explore their modes of selection. Both BLB1 and BLB2 are transcribed and display classic characteristics of balancing selection as predicted for expressed MHC class IIB genes. We found evidence for both intra- and interlocus recombination or gene conversion, as well as indication for positive but differential selection at both loci. Moreover, the two loci appear to be linked. Phylogenetic analyses revealed orthology of the black grouse MHC class IIB genes to the respective BLB loci in chicken.ConclusionsThe results indicate that the duplication of the BLB gene occurred before the species divergence into black grouse, chicken and pheasant. Further, we conclude that BLB1 and BLB2 in black grouse are subjected to homogenizing concerted evolution due to interlocus genetic exchange after species divergence. The loci are in linkage disequilibrium, which is in line with the theory of tightly coevolving genes within the MHC under the minimal essential MHC hypothesis. Our results support the conclusion that MHC form and function in birds derived from studies on the domesticated chicken are not artefacts of the domestication process.

Maintenance of MHC Class IIB diversity in a recently established songbird population.

We examined variation at MHC Class IIB genes in a recently established population of dark-eyed juncos (Junco hyemalis) in a coastal urban environment in southern California, USA relative to an ancestral-range population from a nearby species-typical montane environment. The founding population is estimated to have been quite small, but we predicted that variation at the major histocompatibility complex (MHC) among the founders would nevertheless be preserved owing to the high functional significance of MHC. Previous studies of MHC in songbirds have had varying degrees of success in isolating loci, as passerines show extensive MHC gene duplication. In order to compare diversity in the two populations, we employed two published approaches to sequencing MHC Class II exon 2: direct sequencing with exon-based primers, and traditional cloning and sequencing with intron-based primers. Results from both methods show that the colonist population has maintained high levels of variation. Our results also indicate varying numbers of alleles across individuals, corroborating evidence for gene duplication in songbird MHC. While future studies in songbirds may need to take a genomic approach to fully understand the structure of MHC in this lineage, our results show that it is possible to use traditional methods to reveal functional variation across populations.

Diversity of immune genes and associated gill microbes of European plaice Pleuronectes platessa

Genetic variability of marine fish species is much higher than in most other vertebrates. Nevertheless, some species with large population sizes including flatfish such as European plaice Pleuronectes platessa show signs of population collapse and inbreeding. Taking plaice as a flagship example for fisheries-induced genetic changes also affecting the Wadden Sea, we determined the amount of genetic variability at antigen-presenting genes of the Major Histocompatibility Complex (MHC) and its potential interaction with the microbiota associated to gill tissue using a next-generation parallel tag sequencing approach. Genetic variation at MHC class IIB genes was extremely large, with 97 alleles found in 40 fish from different age cohorts. Although a strong signal of positive selection was present (dN/dS = 4.01) and we found significantly higher allelic diversity in 0+ fish than in older age classes, the amount of genetic variation maintained within the population may not have exceeded neutral expectations derived from mitochondrial markers. Associated microbes revealed significant spatiotemporal structure with 0+ fish displaying the highest microbial diversity as well as the highest diversity of potentially pathogenic genera. Overall the correlation between MHC genotypes and bacterial abundance was weak, and only few alleles significantly correlated with certain bacterial genera. These associations all conferred susceptibility (i.e. presence of an allele correlated to higher number of bacteria), either suggesting age-dependent selection on common alleles or weak selection on resistance against these bacterial genera. Taken together, our data suggest that selection coefficients of balancing selection maintaining immunogenetic diversity may be relatively small in large marine populations. However, if population sizes are further reduced by overharvesting, the response to increasing balancing selection coefficients will be largely unpredictable and may also negatively influence the adaptive potential of populations.

Molecular cloning, polymorphism and tissue distribution of the MHC class IIB gene in the Chinese goose (Anser cygnoides)

1. The goose major histocompatibility complex (MHC) class IIB cDNA (Ancy-MHCII) was cloned by homology cloning and rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR), and the genomic structure and tissue expression were investigated. 2. Three different 5′-RACE sequences (Ancy-MHC II5′-1, Ancy-MHC II5′-2, Ancy-MHC II5′-3), one 3′-RACE sequence (Ancy-MHC II-3′) and two different full length Ancy-MHC IIB cDNA sequences (Ancy-CD01, Ancy-CD02), which came from different alleles at one locus or different loci, were determined. 3. The genomic organisation is composed of 6 exons and 5 introns, with a longer intron region than that of the chicken. The alleles encode 259 and 260 amino acids in the mature protein. 4. The number of non-synonymous substitutions (dN) in the peptide-binding region of exon 2 from 8 alleles was higher than that of the synonymous substitutions (dS). 5. Tissue-specific expression of Ancy-MHC II mRNA was detected in an adult goose using RT-PCR. These results showed that Ancy-MHC II mRNA was expressed in the lung, spleen, liver, intestine, heart, kidney, pancreas, brain, skin and muscle. This is consistent with the expression of MHC class IIB in various tissues from the chicken. 6. Sequences from goose, snipe and duck clustered together when compared with known MHC class IIB sequences from the other species, significantly differing from mammals and aquatic species, indicating a pattern consistent with accepted evolutionary pathways.

Molecular cloning, expression pattern, and 3D structural analysis of the MHC class IIB gene in the Chinese longsnout catfish ( Leiocassis longirostris)

Major histocompatibility complex (MHC) class I and class II molecules encode glycoproteins which mediate the specificity of the vertebrate adaptive immune response. In this study, MHC class IIB gene from the Chinese longsnout catfish ( Leiocassis longirostris) was cloned and sequenced, which encoded a predicted protein of 248 amino acids (28.06 kDa) containing a signal peptide, a beta 1 domain, a beta 2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. Using PCR with primers designed from known fish MHC class IIB sequences followed by elongation of the 5′ and 3′ ends using rapid amplification of cDNA ends (RACE), the full-length cDNA of longsnout catfish MHC class IIB was identified to be 1293 bp, consisting of a 26 bp 5′-terminal untranslated region (UTR), a 520 bp 3′-UTR, and a 747 bp open reading frame (ORF) bearing characteristics of the immunoglobulin C-type 1 (IGc1) family. The deduced amino acid sequences of the Chinese longsnout catfish MHC class IIB gene had 58–75% identity with those of other fishes. Six class IIB alleles were identified from five individuals. At most two different alleles observed in each individual may infer the existence of a single locus of class IIB gene in the Chinese longsnout catfish genome. An extensive study of polymorphism was examined in 60 individuals. A total of 11 haplotypes of exon 2 were detected in the sampled Chinese longsnout catfish. The rates of nonsynonymous substitutions ( d N) occurred at a higher frequency than that of synonymous substitutions ( d S), suggesting the polymorphism of exon 2 seemed to be maintained by the balancing selection. By using long PCR technique, the genomic sequence was further identified to be 2345 bp in length, which contained six exons and five introns. Interestingly, a 98 bp intron 5 cut the 3′-UTR into two parts. Real-time quantitative RT-PCR demonstrated high expression of MHC IIB in gills, spleen, head kidney, and intestine, moderate expression in liver and stomach, and low or negligible expression in heart. Homology modelling has been applied to create a 3D model of longsnout catfish MHC class IIB, with features evaluated and discussed to investigate its interaction with CD4 participating in antigen recognition. The present findings will provide valuable information for understanding structure, function, expression, and molecular polymorphism of MHC class IIB in adaptive immunity of the Chinese longsnout catfish and teleost.

454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

BackgroundBecause of their functional significance, the Major Histocompatibility Complex (MHC) class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping.ResultsHere we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family.Conclusions454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective, genotyping of MHC systems of virtually any complexity.

Polymorphism of sheep MHC Class IIb gene TAPASIN

The Major Histocompatibility Complex (MHC) is one of the most gene dense regions in the genome and studies in several species have shown significant associations between the MHC and disease. The endoplasmic reticular glycoprotein, tapasin, is involved in the MHC class I antigen presentation pathway. Sheep TAPASIN is located in the class IIb region of the MHC. Sheep TAPASIN was subcloned from BAC and cosmid genomic clones and DNA sequenced. TAPASIN is 9549 bp in length and encodes a protein of 447 amino acids. The structure of sheep TAPASIN was similar to other mammals and consisted of eight exons with a distinctively larger intron between exon three and four. Sheep TAPASIN gene had high sequence identity with other mammalian TAPASINs. The TAPASIN gene sequence is conserved across many mammalian species and is possibly maintained through purifying selection with the average ratio of ds/dn of 3.9. Twenty-six SNPs in sheep TAPASIN were identified.

Parasite mediated homogenizing selection at the MHC in guppies

Understanding the selective forces influencing genetic diversity is a fundamental goal of evolutionary ecology. The genes of the major histocompatibility complex (MHC) play a key role in the adaptive immune response of vertebrates and thus provide an excellent opportunity to examine the agents of selection on a functionally important gene. Here we examine the genetic architecture of the MHC class IIB genes in 10 wild populations of guppies (Poecilia reticulata) in Northern Trinidad. We have previously shown that these populations are significantly less diverged at the class IIB locus than expected based on neutral (microsatellite) loci. We now survey infection by Gyrodactylus turnbulli and G. bullatarudis, common parasitic worms that infect guppies, as a potential agent of homogenizing selection. We used a genetic algorithm to partition both additive and non-additive genetic effects of the five most common MHC allele types as well as a rare allele category. Although we found no evidence for non-additive effects, across the populations we found that one allele type (the a-type) had a significant negative additive effect on parasite load. Thus, individuals who had more copies of the a-type allele were infected with fewer gyrodactylus than individuals with fewer copies of the allele. These results not only link parasite infection with MHC genotype, they provide a mechanism of homogenizing selection across these otherwise disparate populations.