BACKGROUND
the major histocompatibility complex  is a gene-dense genomic region within which many genes play a role in vertebrate immune response.
mhc class i genes encode surface receptors in most nucleated cell types and facilitate immune responses to intracellular pathogens.
mhc class ii genes also encode receptors but are restricted to antigen presenting cells of the immune system where they play a role in combating extracellular pathogens.
after the binding of antigens, class i and class ii proteins present them to cd <dig> and cd <dig> t cells, respectively.
this presentation in turn triggers the adaptive immune response against the antigen.
polymorphism at mhc loci facilitates binding of a diversity of pathogens and this evolutionary selection pressure is thought to contribute to the high genetic variation in mhc loci  <cit> .
mhc genes are perhaps the most thoroughly studied example of adaptive molecular evolution, representing a classic example of balancing selection  <cit> .
mhc genes have also played an important role in studies of molecular ecology with mhc genotype influencing patterns of mate choice , local adaptation  <cit> , disease resistance  <cit> , and the expression of sexually selected ornaments  <cit> .
an mhc has been identified in all jawed vertebrates studied to date.
a core set of genes, including tap, tapbp, tnxb and cenp-a, are syntenic to the mhc across vertebrates that have been characterized  <cit> .
therefore, the presence of these genes, along with class i and class ii genes can be used to define the mhc region.
in addition to class i and class ii genes, many of the other genes in the mhc region also play a role in immune response.
like class i and class ii genes, cd <dig> genes  play a role in the adaptive immune response.
cd <dig> molecules present lipid, glycolipid and lipopeptides to t and nkt cells  <cit> .
cd <dig> genes are in fact evolutionarily related to class i and class ii genes  <cit> .
some mhc-linked genes encode proteins that interact with mhc molecules.
tap <dig> and tap <dig> genes, for example, are involved in the loading of peptides onto class i molecules for transport to the cell surface  <cit> .
the interaction between class i and tap is itself mediated by tapbp .
while mhcs share many structural features there is also tremendous variation in their organization among species.
among birds, the chicken gallus gallus has been most intensively studied, and its mhc  has a rather remarkable structure: a minimal essential mhc  <cit> .
in contrast to humans, in which the mhc spans four megabases  and consists of over  <dig> genes, the chicken mhc consists of only about  <dig> genes spanning only a few hundred kb on chromosome  <dig>  <cit> .
in addition to the mhc-b, chicken mhc class i and class ii genes are also present in a separate and unlinked cluster called the mhc-y  region  <cit> .
even when considering both the mhc-b and mhc-y together, the chicken mhc has fewer total genes, gene duplicates, pseudogenes, repetitive sequences, and shorter genes and introns than its mammalian counterparts.
tight linkage of genes in the mhc may facilitate their coordinated coexpression  <cit>  and coevolution in the chicken b-complex  <cit> .
furthermore, the suppression of recombination among mhc genes is thought to contribute to the evolution of gene complexes coadapted to particular pathogens and environments  <cit> .
interacting tap and class i genes are more closely linked in the chicken than in mammals and these genes in particular are thought to coevolve in birds  <cit> .
some of the strongest genotype/disease resistance correlations have been identified in the chicken  and the simple architecture of the chicken mhc, with few highly expressed mhc genes, likely contributes to this pattern  <cit> .
due to the limited taxonomic and genomic sampling of mhc regions in birds, however, it remains unclear whether the streamlining of the mhc reflects the broader trend of reduced genome size in birds  <cit>  and whether a small mhc represents the ancestral condition for birds.
alternatively, a small mhc may be a highly derived condition unique to the biology of galliforms.
the zebra finch genome, representing the taxonomically diverse passerine clade , offers the opportunity to characterize mhc structure in an avian lineage highly divergent from the chicken.
molecular estimates of divergence between passerines and galliforms indicate that they diverged between about  <dig> and  <dig> million years ago .
among birds, only two galliform mhcs, the domestic chicken and japanese quail coturnix japonica, have been well characterized  <cit> .
the mhc-b complex of another galliform species, the turkey meleagris gallopavo has also recently been sequenced and appears similar to the chicken in structure spanning about  <dig> kb  <cit> .
mhc polymorphism surveys in passerines suggest that their mhc may differ from the structure seen in galliforms  <cit> .
class iib genes in particular appear to have been extensively duplicated in passerine birds, although little is known regarding the expression of these genes.
initial attempts to characterize the passerine mhc regions using genomic sequence data have uncovered pseudogenes and have revealed a much lower gene density than the chicken  <cit> .
the number of expressed class i genes in some songbirds also appears greater than in the chicken  <cit> .
none of the core mhc-associated genes described above have been characterized in passerines making it unclear whether classical mhc regions have been sequenced.
in this study we used the draft assembly of the zebra finch genome  <cit>  in combination with targeted bac sequencing, fluorescence in situ hybridization  mapping, and restriction fragment length polymorphism  analysis to describe the fundamental features of the zebra finch mhc.
RESULTS
genome assembly analysis
in our scan of the zebra finch genome assembly we found one or more homologous loci for  <dig> of  <dig> investigated chicken mhc related genes .
these represent whole coding sequence  or fragments of genes .
since several of the genes we queried had multiple loci in the zebra finch assembly, our set of sequences comprises a total of  <dig> manually curated mhc genes and eight putative pseudogenes .
genes are listed in order according to their organisation on chicken chromosome  <dig>  duplicated genes are given a numbered locus id if they appear functional.
putative pseudogenes are marked with a ψ and given a lettered locus id.
loci are classified based on sequence similarity such that identical sequences found in multiple places in the genome assembly are given the same locus id.
where ensembl ids have been assigned these are also given.
three genes outside of the mhc region but with a related function  are also included.
we found only one functional mhc class i gene, which was situated on chromosome 22_random .
in addition, there are also  <dig> contigs unincorporated into the genome assembly, and one contig on chromosome 16_random, that contain fragments of class i genes .
sequence differences suggest that these genes correspond to at least three different pseudogenes.
in the case of mhc class iib, we found  <dig> contigs on chromosome un , and three hits on chromosomes 22_random and 7_random, containing parts of the cds.
four distinct sequences with an open reading frame spanning exons two and three appear to be functional.
the genome assembly suggests that some mhc-associated genes may not be as clustered in the zebra finch mhc as they are in the chicken.
blec <dig>  nkr and trim <dig>  for example, map to the z chromosome in the zebra finch genome assembly, while two cd <dig> loci map to chromosome  <dig>  many of the other genes for which we searched, however, mapped to chromosome un or were not found in the assembly .
bac screening, sequencing and gene prediction
we further characterized the zebra finch mhc by isolating and sequencing mhc-containing bac clones.
we first identified  <dig> clones that hybridized strongly with a probe targeted to exon  <dig> of an mhc class iib gene.
four of these bacs were selected for sequencing .
because of the large number of class iib positive clones, we conducted further screening using overgo probes targeted to five conserved genes linked to the mhc across a diversity of taxa .
for this second screening, we screened a different bac library derived from the same zebra finch individual as the whole genome sequence .
positive clones were found for each of the five genes: mhc class i , kifc , cenp-a , tap <dig> , and tnxb .
probes for three pairs of genes were found to cohybridize to individual bac clones: mhc class i and kifc, mhc class i and tnxb, and tnxb and tap .
one bac clone containing each of these three gene pairs was chosen for 6× sequencing .
no clones were positive for both tap <dig> and mhc class i, suggesting that these two genes are not closely linked in the zebra finch as they are in the chicken.
cenp-a probes also did not cohybridize with any of the other mhc genes, again indicating a lack of close linkage observed in other species .
we did not sequence any of the cenp-a positive clones.
cohybridization patterns link all genes but cenp-a.
the clone that was positive for tnxb and class i appears to contain a class i pseudogene.
each clone was assembled into multiple ordered contigs .
the fragmented nature of the bac assemblies is expected given the coverage, but was exacerbated by high repeat content .
sequence analysis of two class i clones, tgac-86i <dig> and tgac-167e <dig>  revealed extensive sequence overlap and thus were assembled together .
to improve the assembly for the class ii clones we generated additional sequencing reads.
because the class i clones were derived from the same zebra finch as the whole genome sequence, we were also able to incorporate sequence reads from the whole genome sequencing effort to improve the assembly of these bacs.
most of the bac assemblies contained at least a fragment of the genes expected based on the probes used to identify them .
class i clones contained a number of genes of interest.
the assembly of clones tgac-86i <dig> and tgac-167e <dig> contained sequences with similarity to tap <dig>  tap <dig>  and tnxb genes, and clone tgac-102m <dig> contained kifc, daxx, tubb, class i, and flot .
although numerous genes separate kifc and class i genes in chicken  we did not find evidence for these genes within this bac.
daxx, tubb and flot have not yet been identified in the chicken mhc but are associated with the mhc in other vertebrates .
because of gaps in the bac assemblies complete coding sequences could not always be reconstructed .
although tgac-86i <dig> hybridized with both class i and tnxb probes, sequencing only revealed a small region with similarity to the class i 3' utr in the great reed warbler .
polymerase chain reaction  screening of this clone also identified a stretch of a short exon  <dig> sequence  that is identical to the expressed locus.
the utr region, however, is distinctive in sequence from the expressed zebra finch class i 3' utr and blast searches of brain expressed sequence tags  and  <dig> sequencing data from multiple tissues suggest that this locus is not expressed  <cit> .
together this suggests that clone tgac-86i <dig> contains an mhc class i pseudogene.
class ii clones contained numerous predicted genes with sequence similarity to zinc finger genes, as well as gag and pol proteins .
aside from these and the expected class iib sequences, however, only one other gene of interest was found.
a gene whose best blast hit matched the first four exons of the turkey tapbp gene  was found in clone tgaa-157b <dig>  sequence conservation mapping using zpicture  <cit>  of this clone and a previously sequence red winged blackbird agelaius phoenicius class ii region  <cit> , highlight sequence similarities in the coding and utrs of predicted genes, as well as in some putative intergenic regions .
fish mapping of bac clones to zebra finch chromosomes
single-color fish mapping experiments revealed that sequenced class ii bacs  hybridized to several pairs of microchromosomes each , likely due to the high repeat content in these clones .
in contrast, sequenced class i bacs  each hybridized to one pair of small microchromosomes.
some bacs also cross-hybridized to repeats in the centromeric and telomeric regions of macrochromosomes .
linkage analysis by dual color fish demonstrated that bacs tgac-102m <dig> , and three class ii bacs  shared hybridization to one pair of small microchromosomes.
sequenced bacs tgac-86i <dig> and tgac-167e <dig>  and 157b <dig> , however, hybridized to a different pair of small microchromosomes .
mhc genes are thus found in two linkage groups on separate chromosomes in zebra finch.
to further test whether tap and mhc class i genes are syntenic, we conducted five additional two-color fish experiments with bac clones that were positive for tap <dig> and mhc class i. while some mhc class i probes hybridized to multiple microchromosomes, in only one case did we find colocalisation of class i and tap <dig> probes .
in this case, mhc class i probes hybridized to multiple microchromosomes, and the colocalisation occurred on the w sex chromosome.
it is therefore likely that this colocalisation is due to nonspecific binding, and the repetitive nature of the avian w chromosome.
in total we have four cases in which tap <dig> probes hybridize unambiguously to a single microchromosome and in all of these, class i maps to a different chromosome.
* clones sequenced in this study
most clones mapped to a single microchromosome  where as some showed non specific binding to multiple microchromosomes .
a few clones mapped to sex chromosomes , and one mapped to the centromeric region of a macrochromosome .
in only one cases did tap <dig> and class i clones colocalise, and that was on the w chromosome.
in order to identify the zebra finch chromosomes corresponding to the two zebra finch linkage groups, we performed dual-color fish experiments in which one bac from one of the two linkage groups  was co-hybridized with a non-mhc bac with known chromosomal location .
these experiments covered all microchromosomes for which bacs are currently available .
neither of the two mhc linkage groups mapped to these chromosomes, suggesting that both microchromosomes to which the mhc bacs mapped may indeed correspond to parts of zebra finch chromosome  <dig>  the only chromosome for which we do not have known bacs.
polymorphism survey via rflp/southern blot
to test our findings on the numbers of class i and class iib genes, and as a preliminary survey of gene number and intraspecific polymorphism, we conducted a rflp analysis using probes targeted to these loci.
there are clearly a larger number of rflp bands for mhc class iib  than for class i  and this also suggests that there are more class iib genes than class i genes in zebra finches .
this difference in gene number is not likely due to differences in the sequence similarity of probe and target for class i and iib probes as we would expect that the longer class i probe  should hybridize to a larger number of fragments than the class iib probe .
we repeated this hybridization twice using different class i and ii probes .
for mhc class i there are two to four rflp fragments in the captive zebra finches from the us  and two to three fragments in the zebra finches from sweden .
for mhc class iib there are  <dig> to  <dig> rflp fragments in the zebra finches from the us and as many as  <dig> fragments in the three zebra finches from sweden.
comparative analysis of mhc genes
in order to explore the evolution of the compact avian mhc structure observed in chicken, we estimated the average gene density in quail, chicken, zebra finch and human.
interestingly, the estimated gene density in the zebra finch is similar to that in humans, and distinctive from both quail and chicken .
repeat content also differs markedly between the chicken mhc region and the zebra finch bacs.
long interspersed nuclear elements  occur at frequency of  <dig>  per kb in chicken versus  <dig>  per kb across the  <dig> kb of zebra finch bac sequence.
long terminal repeat  content, specifically in the form of erv <dig> elements, was exceptionally high in zebra finch class ii sequences, occurring at frequency of  <dig>  per kb, whereas the chicken mhc is depauperate in ltr at  <dig>  per kb .
zebra finch class i clones sequenced here more closely resemble the chicken content, but still had higher ltr content .
to examine the evolutionary relationships among mhc genes, we placed exons  <dig> and  <dig> of four putatively functional zebra finch mhc class iib sequences in a phylogenetic context by comparing them with other passerine sequences from genbank.
exon  <dig> of the class iib gene encodes the protein that forms the peptide binding region of the class ii protein, and exons  <dig> and  <dig> have been amplified and sequenced in a diversity of bird taxa.
bayesian phylogenetic analyses using both exons concatenated together grouped three zebra finch sequences in a highly supported clade .
using outgroups to the passerine sequences , a divergent zebra finch locus was placed basal to all passerine sequences, between passerine and non-passerine sequences.
thus for analyses of passerine sequences we rooted the tree at this zebra finch sequence .
we also analyzed exons  <dig> and  <dig> separately.
these results reflect previously described differences among the exons  so are not described further here.
phylogenetic analyses of exon  <dig> sequences from mhc class i also placed zebra finch class i sequences in a strongly supported clade .
passerine sequences were analyzed using phylogenetic analysis using maximum likelihood   <cit> , to test for the influence of positive selection on mhc class i exon  <dig>  and class iib exon  <dig>  two pairs of models were compared in codeml, m <dig>  versus m <dig>  and m <dig>  versus m <dig>   <cit> .
for class i exons both tests indicate that models incorporating positive selection provide a better fit to the data than do neutral models .
bayes empirical bayes analyses  <cit>  identified  <dig> sites with ω >  <dig> .
analyses of mhc class iib exon  <dig> also suggest a strong influence of positive selection  with  <dig> sites with ω >  <dig> .
in both class i and class iib, these sites correspond well with the peptide binding region  from the human mhc  <cit>  and with selected sites identified in birds of prey  <cit> .
discussion
we have provided here a detailed characterization of the zebra finch mhc.
there is clear cytogenetic evidence that mhc genes map to at least two different chromosome pairs in the zebra finch.
if the chicken mhc represents the ancestral state, the situation in the zebra finch may have arisen through fission of chromosome  <dig> or a translocation of part of it to another pair of microchromosomes.
the hypothesis of chromosomal fission is consistent with the finding that the mhc bacs did not map to zebra finch chromosomes  <dig> to  <dig> or  <dig> to  <dig>  and that the microchromosomes recognized by these probes were small.
the finding of mhc genes on two chromosomes in the zebra finch is particularly intriguing because tap genes map to one of them, whereas an expressed class i gene  maps to a distinct chromosome .
this finding is unexpected because tap and class i genes functionally interact and are syntenic in most mhcs studied to date including both chicken and humans .
in chicken this tight linkage is thought to result in coevolution between tap and class i genes and strong correlations between mhc haplotype and disease resistance .
tap genes in mammals, while generally syntenic, are not as closely linked to class i as they are in galliform birds.
the separation of tap and class i in mammals has been hypothesized to have resulted in their evolutionary independence and in turn led to high levels of duplication and divergence in class i genes  <cit> .
this dissociation is perhaps most clearly illustrated by the tammar wallaby macropus eugenii in which class i sequences have been found dispersed across seven chromosomes  <cit> .
the separation of tap and class i genes in the zebra finch may therefore represent convergent dissociation of these genes.
an alternative explanation for the separation of class i and tap genes in the zebra finch is that the regions sequenced here could represent duplication blocks.
the sequenced class i locus could even be related to the mhc-y region of chicken.
phylogenetic analyses of zebra finch class i and chicken class i , however, suggest that our sequenced class i gene is not the ortholog of a chicken mhc-y gene as chicken  and zebra finch sequences are reciprocally monophyletic .
it is also possible that a second class i gene resides on the same chromosome as tap <dig> and tap <dig> and therefore, that class i and tap are actually syntenic.
in fact, a sequenced bac was positive for both mhc class i and tnxb; another four clones were positive for tnxb and tap <dig> suggesting a possible linkage between these mhc class i and tap <dig>  based on a divergent sequence and a lack of expression, we suggest that this class i sequence is a pseudogene.
even if it were not a pseudogene, tap and this class i gene would be much more distantly located in zebra finch than they are in chicken and would be free of the linkage seen in the chicken.
the whole genome assembly, digital expression profiling  <cit>  and est data suggest only one full-length, expressed, class i gene.
it is also possible that there is a second set of tap genes that we have not sequenced.
given the extremely low coverage of tap genes in the genome trace archives , it is unlikely that tap genes have been duplicated.
fish mapping of five pairs of putative tap <dig> and mhc class i clones further supports the lack of synteny among tap and class i genes .
together these findings suggest that the class i and tap are not linked in the zebra finch.
in addition to class i loci identified in the bacs, we identified three distinct class i sequences that appear to be pseudogenes.
one of the putative pseudogenes only contains exon  <dig>  one only contain exons  <dig> to  <dig>  while the third contains exons  <dig> to  <dig>  because the probes used in rflp analyses target exon  <dig> only one of these pseudogenes would be reflected in the rflp banding patterns.
while the zebra finch appears to possess only one expressed class i locus, the great reed warbler acrocephalus arundinaceous, another passerine species, expresses multiple class i loci  <cit> .
an intriguing possibility is that the dissociation of tap and class i in ancestral passerines preceded the radiation of class i genes in some passerine groups  <cit>  as has been suggested for the wallaby  <cit> .
class iib genes in zebra finch are highly duplicated as evidenced by the genome assembly, bac sequencing and the rflp analysis.
we identified  <dig> distinct class iib sequences in the genome assembly  some of which appear to be pseudogenes.
these findings corroborate previous surveys of class iib variation in other passerine birds  <cit> .
another feature of zebra finch class iib regions is their high ltr content, mostly in the form of erv elements .
the finding of multiple zinc-finger genes and retroelements in proximity to class ii genes was also presaged by multikilobase mhc sequences from red-winged blackbirds, which showed a similar pattern  <cit> .
given the large number of class iib duplicates and pseudogenes we speculate that duplication may have been related to the presence of retroviral sequences.
thus, the passerine mhc class iib may have been invaded by endogenous retroviruses much like the primate class i  <cit> .
endogenous retroviruses have also been implicated in the duplication of wallaby class i genes and their spread across multiple chromosomes  <cit> .
given the fish mapping results and the whole genome assembly, mhc genes appear to be located on even more than two chromosomes.
the genome assembly suggests that homologs of chicken mhc genes have been dispersed in the genome.
there are at least three possible explanations for this: 1) there have been chromosome rearrangements for these genes between the chicken and zebra finch; 2) the contigs containing these genes have been misplaced in either the chicken or the zebra finch genome assembly; 3) the zebra finch gene identified is not the true ortholog of the chicken gene.
chicken mhc genes placed on different chromosomes in the zebra finch assembly compared to the chicken include mhc class i , cd <dig> and cd <dig> , and nkr, blec <dig> and trim <dig>  .
the mhc class i gene placed on chromosome  <dig> and its surrounding region in the assembly is essentially identical to that in our sequenced bac.
this sequenced bac did not cohybridize with two known chromosome  <dig> bacs , so the placement of this class i region on chromosome  <dig> appears to be an assembly artifact.
rather, the fish mapping results suggest that these genes are in fact on chromosome  <dig> as they are in chicken.
the genome assembly data underlying the placement of cd <dig> genes on chromosome  <dig> is also somewhat uncertain, with no bac-end sequences linking contigs containing these genes to chromosome  <dig>  further work will be needed to test whether the genome assembly has properly placed these genes.
contigs containing, blec <dig>  nkr and trim <dig>  however, are linked by bac-end sequence pairs to the z chromosome, making it likely that these are appropriately placed in the assembly.
a number of core mhc-associated genes including dma, bg, c <dig>  tnxb, tap <dig> and tapbp are conspicuous by their absence in the zebra finch genome assembly .
there is no reason, however, to believe that these are truly absent in the zebra finch as they are present in a wide range of other vertebrates and are crucial for mhc function.
more likely, these genes cannot be identified due to the incomplete assembly of zebra finch chromosome  <dig>  tap <dig>  tapbp and tnxb-like sequences, for example, were found in the bac sequences but are not represented in the genome assembly.
many of the zebra finch mhc-related genes identified in the genome scan map to linkage groups in chromosome unknown.
this again appears to be a result of the incomplete assembly of chromosome  <dig>  the problem of assembling chromosome  <dig> is likely due in part to the highly duplicated mhc region in combination with the high repeat content in these regions.
bac sequencing revealed two genes, flot and daxx, that are mhc-linked in non-avian vertebrates  <cit> , but have not been described in chicken.
the relatively close linkage to mhc class i and ii genes of flot, tubb and daxx in the zebra finch is actually more similar to the organisation in some teleost mhcs  than it is to either xenopus or the human mhc, where daxx is physically distant from the flot and tubb genes.
chicken chromosome  <dig>  like the zebra finch, is not well assembled at this point so it is possible that these genes will be found as the chicken assembly continues to improve.
phylogenetic analyses highlight the clustering of class iib loci by species rather than by orthology relationships, suggesting a history of concerted evolution, at least on portions of the genes  <cit> .
we did, however, identify a unique class iib lineage that falls at the base of all other passerine class ii sequences.
this appears to be a novel locus that has not previously been sequenced in birds and it is unknown whether it is expressed and/or polymorphic.
further analysis will be needed to clarify the role of this locus but its discovery underscores the utility of genomic approaches  for characterizing mhc genes in birds.
tests of selection using zebra finch and other passerine mhc sequences support a strong role of selection in shaping patterns of polymorphism in the peptide binding region of class i and class ii genes in passerines.
the specific sites under positive selection are similar to those previously identified for other bird groups  <cit>  and they closely match the peptide binding regions in humans  <cit> .
high variability among individuals in rflp banding patterns support the prediction that mhc class iib genes are influenced by balancing selection.
among birds, there is tremendous variation among lineages in the number of mhc genes.
in quail  <cit> , red-winged blackbird  <cit>  and the zebra finch, there are multiple class ii genes.
most non-passerine species, in contrast, appear to have only between one and three loci  <cit> .
given the derived phylogenetic position of passerines  <cit> , these patterns imply that in terms of class ii genes, a minimal mhc may be ancestral for birds  <cit> .
because of the extensive variation among avian lineages in the number of class i genes , it remains unclear what the ancestral condition for class i genes might be.
CONCLUSIONS
we have made significant progress towards the understanding of the complex structure of the zebra finch mhc, the first such analysis from a representative of the diverse passerine radiation.
although the genome assembly and bac sequencing are fragmentary, the zebra finch appears to possess an mhc differing markedly from previously described avian mhcs.
the genomic architecture of the zebra finch mhc highlights the dynamic nature of mhc evolution.
the evidence for gene duplication, pseudogenization and the distribution of mhc genes on multiple chromosomes in the zebra finch are particularly striking when measured against the compact mhc of the chicken present on a single chicken microchromosome.
further genomic characterization of mhcs from a broader diversity of birds, as well as further refinement of the zebra finch mhc assembly, will continue to refine our picture of mhc evolution in birds.

 the major histocompatibility complex is a gene-dense genomic region within which many genes play a role in vertebrate immune response. mhc class i genes encode surface receptors in most nucleated cell types and facilitate immune responses to intracellular pathogens. mhc class ii genes also encode receptors but are restricted to antigen presenting cells of the immune system where they play a role in combating extracellular pathogens. after the binding of antigens, class i and class ii proteins present them to cd and cd t cells, respectively. this presentation in turn triggers the adaptive immune response against the antigen. polymorphism at mhc loci facilitates binding of a diversity of pathogens and this evolutionary selection pressure is thought to contribute to the high genetic variation in mhc loci. mhc genes are perhaps the most thoroughly studied example of adaptive molecular evolution, representing a classic example of balancing selection. mhc genes have also played an important role in studies of molecular ecology with mhc genotype influencing patterns of mate choice, local adaptation, disease resistance, and the expression of sexually selected ornaments. an mhc has been identified in all jawed vertebrates studied to date. a core set of genes, including tap, tapbp, tnxb and cenp-a, are syntenic to the mhc across vertebrates that have been characterized. therefore, the presence of these genes, along with class i and class ii genes can be used to define the mhc region. in addition to class i and class ii genes, many of the other genes in the mhc region also play a role in immune response. like class i and class ii genes, cd genes play a role in the adaptive immune response. cd molecules present lipid, glycolipid and lipopeptides to t and nkt cells. cd genes are in fact evolutionarily related to class i and class ii genes. some mhc-linked genes encode proteins that interact with mhc molecules. tap and tap genes, for example, are involved in the loading of peptides onto class i molecules for transport to the cell surface. the interaction between class i and tap is itself mediated by tapbp. while mhcs share many structural features there is also tremendous variation in their organization among species. among birds, the chicken gallus gallus has been most intensively studied, and its mhc has a rather remarkable structure: a minimal essential mhc. in contrast to humans, in which the mhc spans four megabases and consists of over genes, the chicken mhc consists of only about genes spanning only a few hundred kb on chromosome. in addition to the mhc-b, chicken mhc class i and class ii genes are also present in a separate and unlinked cluster called the mhc-y region. even when considering both the mhc-b and mhc-y together, the chicken mhc has fewer total genes, gene duplicates, pseudogenes, repetitive sequences, and shorter genes and introns than its mammalian counterparts. tight linkage of genes in the mhc may facilitate their coordinated coexpression and coevolution in the chicken b-complex. furthermore, the suppression of recombination among mhc genes is thought to contribute to the evolution of gene complexes coadapted to particular pathogens and environments. interacting tap and class i genes are more closely linked in the chicken than in mammals and these genes in particular are thought to coevolve in birds. some of the strongest genotype/disease resistance correlations have been identified in the chicken and the simple architecture of the chicken mhc, with few highly expressed mhc genes, likely contributes to this pattern. due to the limited taxonomic and genomic sampling of mhc regions in birds, however, it remains unclear whether the streamlining of the mhc reflects the broader trend of reduced genome size in birds and whether a small mhc represents the ancestral condition for birds. alternatively, a small mhc may be a highly derived condition unique to the biology of galliforms. the zebra finch genome, representing the taxonomically diverse passerine clade, offers the opportunity to characterize mhc structure in an avian lineage highly divergent from the chicken. molecular estimates of divergence between passerines and galliforms indicate that they diverged between about and million years ago. among birds, only two galliform mhcs, the domestic chicken and japanese quail coturnix japonica, have been well characterized. the mhc-b complex of another galliform species, the turkey meleagris gallopavo has also recently been sequenced and appears similar to the chicken in structure spanning about kb. mhc polymorphism surveys in passerines suggest that their mhc may differ from the structure seen in galliforms. class iib genes in particular appear to have been extensively duplicated in passerine birds, although little is known regarding the expression of these genes. initial attempts to characterize the passerine mhc regions using genomic sequence data have uncovered pseudogenes and have revealed a much lower gene density than the chicken. the number of expressed class i genes in some songbirds also appears greater than in the chicken. none of the core mhc-associated genes described above have been characterized in passerines making it unclear whether classical mhc regions have been sequenced. in this study we used the draft assembly of the zebra finch genome in combination with targeted bac sequencing, fluorescence in situ hybridization mapping, and restriction fragment length polymorphism analysis to describe the fundamental features of the zebra finch mhc.  genome assembly analysis in our scan of the zebra finch genome assembly we found one or more homologous loci for of investigated chicken mhc related genes. these represent whole coding sequence or fragments of genes. since several of the genes we queried had multiple loci in the zebra finch assembly, our set of sequences comprises a total of manually curated mhc genes and eight putative pseudogenes. genes are listed in order according to their organisation on chicken chromosome duplicated genes are given a numbered locus id if they appear functional. putative pseudogenes are marked with a ψ and given a lettered locus id. loci are classified based on sequence similarity such that identical sequences found in multiple places in the genome assembly are given the same locus id. where ensembl ids have been assigned these are also given. three genes outside of the mhc region but with a related function are also included. we found only one functional mhc class i gene, which was situated on chromosome 22_random. in addition, there are also contigs unincorporated into the genome assembly, and one contig on chromosome 16_random, that contain fragments of class i genes. sequence differences suggest that these genes correspond to at least three different pseudogenes. in the case of mhc class iib, we found contigs on chromosome un, and three hits on chromosomes 22_random and 7_random, containing parts of the cds. four distinct sequences with an open reading frame spanning exons two and three appear to be functional. the genome assembly suggests that some mhc-associated genes may not be as clustered in the zebra finch mhc as they are in the chicken. blec nkr and trim for example, map to the z chromosome in the zebra finch genome assembly, while two cd loci map to chromosome many of the other genes for which we searched, however, mapped to chromosome un or were not found in the assembly. bac screening, sequencing and gene prediction we further characterized the zebra finch mhc by isolating and sequencing mhc-containing bac clones. we first identified clones that hybridized strongly with a probe targeted to exon of an mhc class iib gene. four of these bacs were selected for sequencing. because of the large number of class iib positive clones, we conducted further screening using overgo probes targeted to five conserved genes linked to the mhc across a diversity of taxa. for this second screening, we screened a different bac library derived from the same zebra finch individual as the whole genome sequence. positive clones were found for each of the five genes: mhc class i, kifc, cenp-a, tap, and tnxb. probes for three pairs of genes were found to cohybridize to individual bac clones: mhc class i and kifc, mhc class i and tnxb, and tnxb and tap. one bac clone containing each of these three gene pairs was chosen for 6× sequencing. no clones were positive for both tap and mhc class i, suggesting that these two genes are not closely linked in the zebra finch as they are in the chicken. cenp-a probes also did not cohybridize with any of the other mhc genes, again indicating a lack of close linkage observed in other species. we did not sequence any of the cenp-a positive clones. cohybridization patterns link all genes but cenp-a. the clone that was positive for tnxb and class i appears to contain a class i pseudogene. each clone was assembled into multiple ordered contigs. the fragmented nature of the bac assemblies is expected given the coverage, but was exacerbated by high repeat content. sequence analysis of two class i clones, tgac-86i and tgac-167e revealed extensive sequence overlap and thus were assembled together. to improve the assembly for the class ii clones we generated additional sequencing reads. because the class i clones were derived from the same zebra finch as the whole genome sequence, we were also able to incorporate sequence reads from the whole genome sequencing effort to improve the assembly of these bacs. most of the bac assemblies contained at least a fragment of the genes expected based on the probes used to identify them. class i clones contained a number of genes of interest. the assembly of clones tgac-86i and tgac-167e contained sequences with similarity to tap tap and tnxb genes, and clone tgac-102m contained kifc, daxx, tubb, class i, and flot. although numerous genes separate kifc and class i genes in chicken we did not find evidence for these genes within this bac. daxx, tubb and flot have not yet been identified in the chicken mhc but are associated with the mhc in other vertebrates. because of gaps in the bac assemblies complete coding sequences could not always be reconstructed. although tgac-86i hybridized with both class i and tnxb probes, sequencing only revealed a small region with similarity to the class i 3' utr in the great reed warbler. polymerase chain reaction screening of this clone also identified a stretch of a short exon sequence that is identical to the expressed locus. the utr region, however, is distinctive in sequence from the expressed zebra finch class i 3' utr and blast searches of brain expressed sequence tags and sequencing data from multiple tissues suggest that this locus is not expressed. together this suggests that clone tgac-86i contains an mhc class i pseudogene. class ii clones contained numerous predicted genes with sequence similarity to zinc finger genes, as well as gag and pol proteins. aside from these and the expected class iib sequences, however, only one other gene of interest was found. a gene whose best blast hit matched the first four exons of the turkey tapbp gene was found in clone tgaa-157b sequence conservation mapping using zpicture of this clone and a previously sequence red winged blackbird agelaius phoenicius class ii region, highlight sequence similarities in the coding and utrs of predicted genes, as well as in some putative intergenic regions. fish mapping of bac clones to zebra finch chromosomes single-color fish mapping experiments revealed that sequenced class ii bacs hybridized to several pairs of microchromosomes each, likely due to the high repeat content in these clones. in contrast, sequenced class i bacs each hybridized to one pair of small microchromosomes. some bacs also cross-hybridized to repeats in the centromeric and telomeric regions of macrochromosomes. linkage analysis by dual color fish demonstrated that bacs tgac-102m, and three class ii bacs shared hybridization to one pair of small microchromosomes. sequenced bacs tgac-86i and tgac-167e and 157b, however, hybridized to a different pair of small microchromosomes. mhc genes are thus found in two linkage groups on separate chromosomes in zebra finch. to further test whether tap and mhc class i genes are syntenic, we conducted five additional two-color fish experiments with bac clones that were positive for tap and mhc class i. while some mhc class i probes hybridized to multiple microchromosomes, in only one case did we find colocalisation of class i and tap probes. in this case, mhc class i probes hybridized to multiple microchromosomes, and the colocalisation occurred on the w sex chromosome. it is therefore likely that this colocalisation is due to nonspecific binding, and the repetitive nature of the avian w chromosome. in total we have four cases in which tap probes hybridize unambiguously to a single microchromosome and in all of these, class i maps to a different chromosome. * clones sequenced in this study most clones mapped to a single microchromosome where as some showed non specific binding to multiple microchromosomes. a few clones mapped to sex chromosomes, and one mapped to the centromeric region of a macrochromosome. in only one cases did tap and class i clones colocalise, and that was on the w chromosome. in order to identify the zebra finch chromosomes corresponding to the two zebra finch linkage groups, we performed dual-color fish experiments in which one bac from one of the two linkage groups was co-hybridized with a non-mhc bac with known chromosomal location. these experiments covered all microchromosomes for which bacs are currently available. neither of the two mhc linkage groups mapped to these chromosomes, suggesting that both microchromosomes to which the mhc bacs mapped may indeed correspond to parts of zebra finch chromosome the only chromosome for which we do not have known bacs. polymorphism survey via rflp/southern blot to test our findings on the numbers of class i and class iib genes, and as a preliminary survey of gene number and intraspecific polymorphism, we conducted a rflp analysis using probes targeted to these loci. there are clearly a larger number of rflp bands for mhc class iib than for class i and this also suggests that there are more class iib genes than class i genes in zebra finches. this difference in gene number is not likely due to differences in the sequence similarity of probe and target for class i and iib probes as we would expect that the longer class i probe should hybridize to a larger number of fragments than the class iib probe. we repeated this hybridization twice using different class i and ii probes. for mhc class i there are two to four rflp fragments in the captive zebra finches from the us and two to three fragments in the zebra finches from sweden. for mhc class iib there are to rflp fragments in the zebra finches from the us and as many as fragments in the three zebra finches from sweden. comparative analysis of mhc genes in order to explore the evolution of the compact avian mhc structure observed in chicken, we estimated the average gene density in quail, chicken, zebra finch and human. interestingly, the estimated gene density in the zebra finch is similar to that in humans, and distinctive from both quail and chicken. repeat content also differs markedly between the chicken mhc region and the zebra finch bacs. long interspersed nuclear elements occur at frequency of per kb in chicken versus per kb across the kb of zebra finch bac sequence. long terminal repeat content, specifically in the form of erv elements, was exceptionally high in zebra finch class ii sequences, occurring at frequency of per kb, whereas the chicken mhc is depauperate in ltr at per kb. zebra finch class i clones sequenced here more closely resemble the chicken content, but still had higher ltr content. to examine the evolutionary relationships among mhc genes, we placed exons and of four putatively functional zebra finch mhc class iib sequences in a phylogenetic context by comparing them with other passerine sequences from genbank. exon of the class iib gene encodes the protein that forms the peptide binding region of the class ii protein, and exons and have been amplified and sequenced in a diversity of bird taxa. bayesian phylogenetic analyses using both exons concatenated together grouped three zebra finch sequences in a highly supported clade. using outgroups to the passerine sequences, a divergent zebra finch locus was placed basal to all passerine sequences, between passerine and non-passerine sequences. thus for analyses of passerine sequences we rooted the tree at this zebra finch sequence. we also analyzed exons and separately. these results reflect previously described differences among the exons so are not described further here. phylogenetic analyses of exon sequences from mhc class i also placed zebra finch class i sequences in a strongly supported clade. passerine sequences were analyzed using phylogenetic analysis using maximum likelihood, to test for the influence of positive selection on mhc class i exon and class iib exon two pairs of models were compared in codeml, m versus m and m versus m. for class i exons both tests indicate that models incorporating positive selection provide a better fit to the data than do neutral models. bayes empirical bayes analyses identified sites with ω >. analyses of mhc class iib exon also suggest a strong influence of positive selection with sites with ω >. in both class i and class iib, these sites correspond well with the peptide binding region from the human mhc and with selected sites identified in birds of prey. discussion we have provided here a detailed characterization of the zebra finch mhc. there is clear cytogenetic evidence that mhc genes map to at least two different chromosome pairs in the zebra finch. if the chicken mhc represents the ancestral state, the situation in the zebra finch may have arisen through fission of chromosome or a translocation of part of it to another pair of microchromosomes. the hypothesis of chromosomal fission is consistent with the finding that the mhc bacs did not map to zebra finch chromosomes to or to and that the microchromosomes recognized by these probes were small. the finding of mhc genes on two chromosomes in the zebra finch is particularly intriguing because tap genes map to one of them, whereas an expressed class i gene maps to a distinct chromosome. this finding is unexpected because tap and class i genes functionally interact and are syntenic in most mhcs studied to date including both chicken and humans. in chicken this tight linkage is thought to result in coevolution between tap and class i genes and strong correlations between mhc haplotype and disease resistance. tap genes in mammals, while generally syntenic, are not as closely linked to class i as they are in galliform birds. the separation of tap and class i in mammals has been hypothesized to have resulted in their evolutionary independence and in turn led to high levels of duplication and divergence in class i genes. this dissociation is perhaps most clearly illustrated by the tammar wallaby macropus eugenii in which class i sequences have been found dispersed across seven chromosomes. the separation of tap and class i genes in the zebra finch may therefore represent convergent dissociation of these genes. an alternative explanation for the separation of class i and tap genes in the zebra finch is that the regions sequenced here could represent duplication blocks. the sequenced class i locus could even be related to the mhc-y region of chicken. phylogenetic analyses of zebra finch class i and chicken class i, however, suggest that our sequenced class i gene is not the ortholog of a chicken mhc-y gene as chicken and zebra finch sequences are reciprocally monophyletic. it is also possible that a second class i gene resides on the same chromosome as tap and tap and therefore, that class i and tap are actually syntenic. in fact, a sequenced bac was positive for both mhc class i and tnxb; another four clones were positive for tnxb and tap suggesting a possible linkage between these mhc class i and tap based on a divergent sequence and a lack of expression, we suggest that this class i sequence is a pseudogene. even if it were not a pseudogene, tap and this class i gene would be much more distantly located in zebra finch than they are in chicken and would be free of the linkage seen in the chicken. the whole genome assembly, digital expression profiling and est data suggest only one full-length, expressed, class i gene. it is also possible that there is a second set of tap genes that we have not sequenced. given the extremely low coverage of tap genes in the genome trace archives, it is unlikely that tap genes have been duplicated. fish mapping of five pairs of putative tap and mhc class i clones further supports the lack of synteny among tap and class i genes. together these findings suggest that the class i and tap are not linked in the zebra finch. in addition to class i loci identified in the bacs, we identified three distinct class i sequences that appear to be pseudogenes. one of the putative pseudogenes only contains exon one only contain exons to while the third contains exons to because the probes used in rflp analyses target exon only one of these pseudogenes would be reflected in the rflp banding patterns. while the zebra finch appears to possess only one expressed class i locus, the great reed warbler acrocephalus arundinaceous, another passerine species, expresses multiple class i loci. an intriguing possibility is that the dissociation of tap and class i in ancestral passerines preceded the radiation of class i genes in some passerine groups as has been suggested for the wallaby. class iib genes in zebra finch are highly duplicated as evidenced by the genome assembly, bac sequencing and the rflp analysis. we identified distinct class iib sequences in the genome assembly some of which appear to be pseudogenes. these findings corroborate previous surveys of class iib variation in other passerine birds. another feature of zebra finch class iib regions is their high ltr content, mostly in the form of erv elements. the finding of multiple zinc-finger genes and retroelements in proximity to class ii genes was also presaged by multikilobase mhc sequences from red-winged blackbirds, which showed a similar pattern. given the large number of class iib duplicates and pseudogenes we speculate that duplication may have been related to the presence of retroviral sequences. thus, the passerine mhc class iib may have been invaded by endogenous retroviruses much like the primate class i. endogenous retroviruses have also been implicated in the duplication of wallaby class i genes and their spread across multiple chromosomes. given the fish mapping results and the whole genome assembly, mhc genes appear to be located on even more than two chromosomes. the genome assembly suggests that homologs of chicken mhc genes have been dispersed in the genome. there are at least three possible explanations for this: 1) there have been chromosome rearrangements for these genes between the chicken and zebra finch; 2) the contigs containing these genes have been misplaced in either the chicken or the zebra finch genome assembly; 3) the zebra finch gene identified is not the true ortholog of the chicken gene. chicken mhc genes placed on different chromosomes in the zebra finch assembly compared to the chicken include mhc class i, cd and cd, and nkr, blec and trim. the mhc class i gene placed on chromosome and its surrounding region in the assembly is essentially identical to that in our sequenced bac. this sequenced bac did not cohybridize with two known chromosome bacs, so the placement of this class i region on chromosome appears to be an assembly artifact. rather, the fish mapping results suggest that these genes are in fact on chromosome as they are in chicken. the genome assembly data underlying the placement of cd genes on chromosome is also somewhat uncertain, with no bac-end sequences linking contigs containing these genes to chromosome further work will be needed to test whether the genome assembly has properly placed these genes. contigs containing, blec nkr and trim however, are linked by bac-end sequence pairs to the z chromosome, making it likely that these are appropriately placed in the assembly. a number of core mhc-associated genes including dma, bg, c tnxb, tap and tapbp are conspicuous by their absence in the zebra finch genome assembly. there is no reason, however, to believe that these are truly absent in the zebra finch as they are present in a wide range of other vertebrates and are crucial for mhc function. more likely, these genes cannot be identified due to the incomplete assembly of zebra finch chromosome tap tapbp and tnxb-like sequences, for example, were found in the bac sequences but are not represented in the genome assembly. many of the zebra finch mhc-related genes identified in the genome scan map to linkage groups in chromosome unknown. this again appears to be a result of the incomplete assembly of chromosome the problem of assembling chromosome is likely due in part to the highly duplicated mhc region in combination with the high repeat content in these regions. bac sequencing revealed two genes, flot and daxx, that are mhc-linked in non-avian vertebrates, but have not been described in chicken. the relatively close linkage to mhc class i and ii genes of flot, tubb and daxx in the zebra finch is actually more similar to the organisation in some teleost mhcs than it is to either xenopus or the human mhc, where daxx is physically distant from the flot and tubb genes. chicken chromosome like the zebra finch, is not well assembled at this point so it is possible that these genes will be found as the chicken assembly continues to improve. phylogenetic analyses highlight the clustering of class iib loci by species rather than by orthology relationships, suggesting a history of concerted evolution, at least on portions of the genes. we did, however, identify a unique class iib lineage that falls at the base of all other passerine class ii sequences. this appears to be a novel locus that has not previously been sequenced in birds and it is unknown whether it is expressed and/or polymorphic. further analysis will be needed to clarify the role of this locus but its discovery underscores the utility of genomic approaches for characterizing mhc genes in birds. tests of selection using zebra finch and other passerine mhc sequences support a strong role of selection in shaping patterns of polymorphism in the peptide binding region of class i and class ii genes in passerines. the specific sites under positive selection are similar to those previously identified for other bird groups and they closely match the peptide binding regions in humans. high variability among individuals in rflp banding patterns support the prediction that mhc class iib genes are influenced by balancing selection. among birds, there is tremendous variation among lineages in the number of mhc genes. in quail, red-winged blackbird and the zebra finch, there are multiple class ii genes. most non-passerine species, in contrast, appear to have only between one and three loci. given the derived phylogenetic position of passerines, these patterns imply that in terms of class ii genes, a minimal mhc may be ancestral for birds. because of the extensive variation among avian lineages in the number of class i genes, it remains unclear what the ancestral condition for class i genes might be.  we have made significant progress towards the understanding of the complex structure of the zebra finch mhc, the first such analysis from a representative of the diverse passerine radiation. although the genome assembly and bac sequencing are fragmentary, the zebra finch appears to possess an mhc differing markedly from previously described avian mhcs. the genomic architecture of the zebra finch mhc highlights the dynamic nature of mhc evolution. the evidence for gene duplication, pseudogenization and the distribution of mhc genes on multiple chromosomes in the zebra finch are particularly striking when measured against the compact mhc of the chicken present on a single chicken microchromosome. further genomic characterization of mhcs from a broader diversity of birds, as well as further refinement of the zebra finch mhc assembly, will continue to refine our picture of mhc evolution in birds. 
the zebra finch mhc includes multiple class i and class ii genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken mhc, as evidenced by the presence of mhc genes on each of seven bacs spanning kb. cytogenetic evidence and the genome assembly itself place core mhc genes on as many as four chromosomes with tap and class i genes mapping to different chromosomes. it remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. the zebra finch mhc exhibits a complex structure and history involving gene duplication and fragmentation. avian mhc organization is of particular interest because the chicken gallus gallus, the avian species with the best characterized mhc, possesses a highly streamlined minimal essential mhc, which is linked to resistance against specific pathogens. our analyses of the zebra finch mhc suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the mhc in birds, and highlight striking differences in mhc structure and organization among avian lineages. lastly, we find strong evidence of selection acting on sites within passerine mhc class i and class ii genes. the zebra finch mhc differs markedly from that of the chicken, the only other bird species with a complete genome sequence. due to its high polymorphism and importance for disease resistance, the major histocompatibility complex has been an important focus of many vertebrate genome projects.
 due to its high polymorphism and importance for disease resistance, the major histocompatibility complex has been an important focus of many vertebrate genome projects. avian mhc organization is of particular interest because the chicken gallus gallus, the avian species with the best characterized mhc, possesses a highly streamlined minimal essential mhc, which is linked to resistance against specific pathogens. it remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. the sequencing of the zebra finch taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome sequencing, has allowed us to characterize an mhc from a highly divergent and diverse avian lineage, the passerines.  the zebra finch mhc exhibits a complex structure and history involving gene duplication and fragmentation. the zebra finch mhc includes multiple class i and class ii genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken mhc, as evidenced by the presence of mhc genes on each of seven bacs spanning kb. cytogenetic evidence and the genome assembly itself place core mhc genes on as many as four chromosomes with tap and class i genes mapping to different chromosomes. mhc class ii regions are further characterized by high endogenous retroviral content. lastly, we find strong evidence of selection acting on sites within passerine mhc class i and class ii genes.  the zebra finch mhc differs markedly from that of the chicken, the only other bird species with a complete genome sequence. the apparent lack of synteny between tap and the expressed mhc class i locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. our analyses of the zebra finch mhc suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the mhc in birds, and highlight striking differences in mhc structure and organization among avian lineages. 