BACKGROUND
major histocompatibility complex  class i and class ii molecules are vital parts of the cellular immune system presenting self and/or foreign peptides to cd <dig> positive and cd <dig> positive t cells.
both classes of genes reside in a  <dig> mb gene dense region on human chromosome  <dig> shared with many other immune genes  <cit> .
atlantic salmon and rainbow trout genomes encode one major mhc class i locus designated uba in addition to the major mhc class ii alpha and beta genes designated daa and dab respectively  <cit> .
for uba, the main polymorphism resides in the alpha  <dig> and alpha  <dig> domains with up to 60% sequence divergence between these antigen binding domains.
added variability for uba is produced by shuffling of exon  <dig> onto different exon  <dig> and downstream regions through recombination occurring in intron  <dig>  <cit> .
additional class i loci and lineages have been described in both atlantic salmon as well as in rainbow trout.
the majority of reported salmonid mhc class i molecules are classified into a u-lineage consisting of both uba as well as non-classical mhc molecules  <cit> .
two other described mhc class i-like lineages are ze described by miller et al.
<cit>  and l described by dijkstra et al.
<cit> .
in all teleosts studied so far including salmonids the mhc class i and class ii regions are unlinked  <cit> .
sequence data on the mhc class i region is available from zebrafish  <cit> , fugu  <cit> , medaka  <cit>  and rainbow trout  <cit> .
a general feature of these four mhc class i regions is a core region containing genes for the proteasome components  and the transporter for antigen processing  being flanked by various numbers of mhc class i loci in addition to many other genes also residing in the human mhc region located on chromosome  <dig>  data from medaka and zebrafish indicate that other fish orthologs of the mammalian mhc-encoded genes are dispersed on several different chromosomes  <cit> , similar to the paralogue mhc regions described on human chromosomes  <dig>   <dig> and  <dig>  <cit> .
salmonids are seen as partially tetraploid with a unique whole genome duplication occurring between  <dig> and  <dig> million years ago  with remnants of tetraploidy visible also today  <cit> .
shiina et al.
<cit>  sequenced two duplicated core mhc regions of rainbow trout.
based on sequence divergence they estimated the duplication event to have taken place approx.
<dig> mya, in agreement with the salmonid whole genome duplication theory.
the classical or ia region contained the major expressed classical mhc class i uba locus while the duplicated region denoted ib contained the four onmy-uca, -uda, -uea and -ufa class i loci.
based on expression and polymorphism data, onmy-uca, -uda and -uea were defined as non-classical loci and -ufa as a pseudogene due to an incapacitating mutation in exon  <dig>  <cit> .
data is rapidly emerging on associations between mhc and resistance to salmonid pathogens.
in atlantic salmon, uba genotypes have been found to provide resistance towards aeromonas salmonicida and infectious salmon anaemia virus  <cit> .
class ib, but not class ia was found associated with susceptibility towards infectious hematopoietic necrosis virus  in atlantic salmon and towards infectious pancreatic necrosis virus  in rainbow trout  <cit> .
both trout and salmon are main aquaculture species and understanding their immune systems will improve our understanding of how these regions influence disease resistance and thus improve our breeding schemes for the trait.
atlantic salmon and rainbow trout are estimated to have split approx.
<dig> mya  <cit> .
as atlantic salmon is a major aquaculture species and displays some differences in response to pathogens when compared to rainbow trout  <cit> , we ventured into sequencing of the two duplicated mhc class i regions of atlantic salmon.
here we describe the gene organization of these two mhc class i regions comprising approx.
<dig> kb each and compare our results to data from other teleosts.
RESULTS
the aim of this study was to characterize the gene organization and identify new genes potentially contributing to disease resistance in the two mhc class i regions of atlantic salmon.
characterization and sequencing of bac clones
sasa-uba and tap <dig> probes hybridized to  <dig> bac clones, where  <dig> clones were positive for both probes.
the  <dig> bac clones were ordered into three contigs using restriction fragment analysis together with grasp hindiii fingerprint information  <cit> .
the two contigs that were positive for uba, tap <dig>  psmb <dig> and psmb <dig> by southern hybridization, were tested for presence of a polymorphic dinucleotide repeat located in the 3'utr of the uba locus  <cit> .
only bac clones from one of the two contigs gave pcr-products, thus this contig was defined as the ia region, and the other contig remained a candidate for the duplicated ib region.
the bac clones in the third contig hybridized to the uba probe as well as a mixed uba exon  <dig> probe.
these clones also tested positive for a u-lineage ula locus that has previously been found closely linked to uba  <cit> .
three bacs were sequenced from the ia region.
the bac clones 92i <dig> and 714p <dig> indicated allelic variants based on variation in the uba 3'utr marker  with 523m <dig> as a continuation of 714p <dig>  from the duplicated ib region we chose 8i <dig>  424m <dig>  15l <dig> and 189m <dig> for sequencing.
30c <dig> was chosen as a candidate from the third contig and was extended  <dig> kb with the sequence of 868o <dig>  the selected bac clones were subcloned, sequenced, and assembled into continuous sequences.
the atlantic salmon ia region consisted of the bac clones 30c <dig>  868o <dig>  92i <dig>  714p <dig> and 523m <dig> covering  <dig> bp, while the ib region consisted of 8i <dig>  424m <dig>  15l <dig> and 189m <dig> totaling  <dig> bp.
gene organization of the atlantic salmon mhc class i regions
we have adopted the nomenclature described by shiina et al.
<cit>  with ia covering the uba locus region and ib for the duplicated region.
thus the genes identified in the regions will be named accordingly; the ia proteasome subunits are given an extension of a  and the ib genes have an extension of b .
the previous symbol abcb <dig> has been withdrawn for the transporter for antigen processing  <dig>  so we have used the current symbol tap <dig>  <cit> .
the gene organization of the ia and ib mhc regions is shown in fig.
<dig>  a core region was identified in both regions which included mhc class i genes, together with the proteasome subunits genes psmb <dig> , psmb <dig> , psmb9-like , psmb <dig>  and tap <dig>  the gene order and orientation of the atlantic salmon psmbs and tap <dig> was very similar to that found in rainbow trout and other teleost .
for the ia region, the main difference between atlantic salmon and rainbow trout is that the rainbow trout psmb8a gene is a pseudogene.
the ia region contained the major mhc class i sasa-uba locus and the recently reported sasa-ula locus residing approximately  <dig> kb upstream.
the duplicated class ib region contained an mhc class i locus resembling the rainbow trout uca locus, but although transcribed it was a pseudogene.
no other mhc class i-like genes were detected in the two duplicated regions.
outside the core region we found  <dig> atlantic salmon orthologs of genes residing in the extended human mhc class ii region.
alternative nomenclature for these genes is described in table  <dig>  the following genes were found in both the ia and ib regions; kifc <dig>  zbtb <dig>  daxx, tapbp, brd <dig>  col11a <dig>  rxrb and slc39a <dig>  three orthologs found in the ia region only were ring <dig>  rps <dig> and vps <dig>  for other teleosts, the gene organization of the extended mhc class i region is partly known for zebrafish  <cit> , fugu  <cit>  and medaka  <cit> .
the tapbp, daxx, zbtb <dig> and kifc <dig> genes are conserved in the same order in both fish and human .
as described in medaka we also found a gene for znf <dig> in the ia region, which is located on chromosome  <dig> in human.
mostly one match is listed per gene.
current abbreviation vs. older nomenclature is listed for each gene.
all est/cdna sequences are from genbank .
hsd17b <dig>  which resides in between slc39a <dig> and ring <dig> in the extended human class ii region, was found in the ib region only and showed more than 81% identity towards counterparts in tilapia , zebrafish  and medaka .
hsd17b <dig> has thus been deleted from the atlantic salmon ia region as it is also present in other fish mhc class i regions .
three orthologs of genes located in the human class i region were identified in the ia region; tcf <dig>  tubb and flot <dig>  atlantic salmon tubulin is highly conserved and showed more than 94% identity towards mammalian counterparts.
another highly conserved gene is rps <dig>  which showed 98% identity towards mammalian sequences.
a gene that was predicted by digit in the ia region had one est match , but no homology to annotated proteins and is thus denoted unknown in fig.
<dig>  however, some sequence identity was found towards a protein in zebrafish located on chromosome  <dig>  as well as to a tetraodon nigroviridis protein , which could indicate a molecule unique to teleosts.
in addition to the genes described above we identified genes for pvrl <dig>  rt, vshv-induced gene and a novel gene similar to a non-vertebrate chitin synthase protein that are not mhc linked in humans.
the human pvrl <dig> is located on chromosome  <dig> .
a homologue of this gene is also found on zebrafish chromosome  <dig> .
a  <dig> kb insertion was found in the ib region in between the rxrb and slc39a <dig> genes containing three copies of a chitin synthase gene approx.
<dig> kb apart .
chitin synthase is involved in the synthesis of chitin, which is a main structural component of the fungal cell wall.
a similar protein has also been identified in zebrafish .
no chitin synthase genes were present in the ia region nor are chitin synthase genes found in any other teleost mhc regions suggestive of a single insertion of this gene in the ib region with two subsequent duplications .
most genes in both regions are supported by matching cdnas apart from tcf <dig> and col11a <dig> where no match has been found so far .
other open reading frames were also identified, but were associated with transposon related repetitive elements.
comparison of the ia and ib regions
the two allelic bacs 92i <dig> and 714p <dig> had an overall sequences identity of  <dig> % over  <dig> bp, with similar exon intron organization for all genes.
the major differences between the two allelic regions resided in the uba α <dig> and α <dig> exons and in differences in number of repeats .
dotplot analysis of 714p <dig> or 92i <dig> against themselves showed no extended regions of local similarity, with the exception of the tap <dig> region which showed similarity due to a duplicated tap <dig> exon  <dig> .
a dot plot analysis of more than  <dig> kb of the ia and ib regions showed four regions with high sequence similarity consisting of subregion one containing genes for kifc <dig> to tapbp, subregion two ranging from psmb <dig> to tap <dig>  subregion three covering brd <dig> to rxrb and subregion four containing slc39a <dig> .
the conserved regions in ia and ib have  <dig> % identity over  <dig> bp.
in total, repeats constituted approximately 24% of the sequence in both regions, and 17% of the repeats were fish-specific dna elements.
mhc class i genes
sasa-uba
the promoter, leader and α <dig> exons of sasa-uba were identified in 30c23/868o <dig>  while the remaining exons of sasa-uba were found in 92i <dig> and 714p <dig>  the leader and α <dig> exons found in 30c23/868o <dig> were identical to the pcr amplified uba* <dig> allele  as well as to the leader and α <dig> exons of another salmon full-length cdna .
together with the α <dig> and α <dig> exons of 92i <dig> they collectively provide a bona fida uba* <dig> allele.
the uba α <dig> exon and downstream sequences of the two allelic bacs 92i <dig> and 714p <dig> have complete sequence identity to the sasa-uba*0201/* <dig> and sasa-uba* <dig> alleles respectively.
uba* <dig> and uba* <dig> are prime examples of the recombination shown to occur within intron  <dig> of salmonid uba alleles  <cit>  showing complete sequence identity in the α <dig> and downstream regions, but highly divergent α <dig> exons.
the predicted amino acid sequences of uba, ula and the two open reading frames of ucaψ were aligned for comparison of the mhc class i genes encoded in the two regions .
analysis of the promoter sequence of uba* <dig> in 30c23/868o <dig> showed high similarity to a rainbow trout uba* <dig> promoter  <cit> , both containing similar regulatory elements typical for mhc class i promoters such as an interferon stimulated response element , w/s-box and enhancer b  .
the uba* <dig> promoter contains a potential site α element according to the core sequence   <cit>  while a sequence more resembling an x2-box has been found in rainbow trout .
both the site α and the homologous x2-box found in mammalian mhc class i and mhc class ii promoters respectively, are involved in regulation of transcription and bind atf/creb family transcription factors  <cit> .
a potential tata-box was also identified in the promoter sequence of uba* <dig>  <cit> .
a salmon uba* <dig> promoter published by jorgensen et al.
<cit>  had lower sequence identity to the uba* <dig> promoter, but both promoters are supported by complete identity to 5'utr cdna sequences of bona fida uba alleles suggesting that atlantic salmon uba alleles have different promoters.
the functional consequences of these differences are being investigated.
the 30c23/868o <dig> and 92i <dig> bacs jointly have an intron sequence of  <dig> kb while in rainbow trout, the intron between the uba α <dig> and α <dig> exons is  <dig> kb  <cit>  suggesting we lack approximately  <dig> kb to have a continuous genomic sequence of the entire uba region.
pcr and cloning of the gap was performed multiple times, but despite successful pcr amplification no fragments covering the gap have been cloned suggestive of an unclonable region.
the amplified products support an intron sequence of approx.
<dig> kb.
unfortunately no mrna or cdna is available from the bac library fish, preventing verification of expressed uba alleles in this animal.
to verify the linkage between 92i <dig> and 30c <dig> fluorescent in situ hybridization was undertaken and showed that both bacs hybridized to the same region of one of the smallest chromosomes, potentially chromosome  <dig> .
the close linkage described by miller et al.
<cit>  between ula and uba also supported 30c23/868o <dig> being an extension of the ia region.
sasa-ula
the ula locus residing approximately  <dig> kb upstream of the uba locus matched a partial ula* <dig> sequence  described by miller et al.
<cit> .
another est in the cgrasp database  <cit>  provided us with a full-length match .
the exon encoding the transmembrane domain is missing, suggestive of a secreted mhc class i molecule .
similar secretory class i molecules are also found for human class i molecules and the potential role of secretory hla-g is currently being deciphered and holds promise for an interesting function.
the 30c <dig> ula gene has an α <dig> exon with highest sequence identity to uba* <dig> while α <dig> and downstream exons have highest identity to uba* <dig>  no ests for ula have been identified in rainbow trout, and a negative pcr-based survey for this gene in rainbow trout by miller et al.
<cit>  suggest this gene may be unique to atlantic salmon.
sasa-ucaψ
only one mhc class i locus was identified in the four bacs representing the ib region.
this locus found in 8i <dig> showed highest sequence identity to the onmy uca* <dig> allele and was thus denoted sasa-uca.
multiple salmon ests with a polymorphic pattern resembling that of onmy-uca sequences were found in databases.
however, both the 8i <dig> uca orf sequence as well as matching ests , contained an internal stop codon in exon  <dig> making sasa-uca an expressed pseudogene.
the exon intron organization of the uba, ula and ucaψ loci are quite similar apart from the enlarged first intron in ula, the even larger second intron of uba and the missing transmembrane exon of ula .
antigen presenting genes
previously reported cdnas for tap <dig>  which were assumed to reside in the ia and ib region and denoted tap2b  and tap2a   <cit>  respectively, indeed did match the tap <dig> in the ia and ib bac sequences.
to avoid nomenclature confusion we hereby rename our ia uba-linked tap <dig> locus to tap2a and the ib ucaψ linked tap <dig> locus to tap2b.
we adapted a similar nomenclature for the reported rainbow trout tap <dig> sequences, and suggest that tap <dig> sequences without locus identification should be denoted tap <dig> only .
atlantic salmon ia and ib tap <dig> sequences have more than 90% aa sequence identity and a similar identity to the rainbow trout ia and ib tap <dig> sequences described by shiina et al.
<cit> .
other tap <dig> ests were also found in databases, which were difficult to define as tap2a or tap2b variants such as the grasptap2- <dig> in fig.
<dig>  attempts to decipher locus origin including rainbow trout information shows that the tap2a and tap2b sequences described by shiina et al.
<cit>  resembles the tap2b sequence identified in atlantic salmon containing for instance the characteristic fca motif at position  <dig> and the two aa deletion at position  <dig> .
a rainbow trout tap2a   sequence described by hansen et al.
<cit> , shown by in situ hybridization to reside in the ia region  <cit> , resembles the atlantic salmon ia tap2a sequence and does not contain these motifs mentioned above.
thus, rainbow trout has a polymorphic tap2a locus and the confusing sequence identities between the two tap <dig> loci may suggest that these genes are exposed to recombination or gene conversion mechanisms.
locus designation of either salmon or trout tap <dig> sequences therefore can not be performed on sequence alone, but must be verified by linkage mapping.
other more divergent atlantic salmon tap <dig> ests  have approx.
50% sequence identity to all above described ia and ib tap <dig> sequences , but has 94% sequence identity to a rainbow trout tap <dig> variant described by hansen et al.
<cit>   .
if these sequences represent an additional tap <dig> locus, i.e.
a tap2c locus, or are allelic variants of the tap2a/b loci is currently unknown.
ancient lineages of divergent mhc class i, tap <dig>  tap <dig> and lmp <dig> haplotypes have been described in xenopus where the sequence identity between allelic tap2s was less than 76%  <cit> .
similar ancient lineages of uba and tap2a may also exist in salmonids, where we were unfortunate enough to sequence allelic variants belonging to similar lineages.
all psmbs genes in the salmon ia and ib regions were fully intact, as opposed to the ia region in rainbow trout where psmb <dig> was a pseudogene, lacking the first three exons.
the core region psmb <dig>  psmb <dig>  psmb9-like, psmb <dig> and tap <dig> loci were found to be organized in a similar fashion in both regions and also displayed a high amount of sequence identity .
some nucleotide differences were found between the allelic bacs 92i <dig> and 714p <dig> genes that resulted in amino acid changes; psmb10a* <dig> and psmb10a* <dig> had  <dig> aa differences located in the propeptide at position  <dig>  and  <dig> .
the proteasome subunits therefore seem to be non-restrictive in providing peptides for uba molecules as opposed to xenopus where psmb <dig>  segregates in lineages with mhc class i, tap <dig> and tap <dig>  <cit> .
the table shows comparison of % nucleotide  and % amino acid  identity between the genes found respectively in the atlantic salmon ia vs ib region and in the ia and ib region of atlantic salmon versus rainbow trout  <cit> .
tapasin  is a key member of mhc class i antigen-loading complexes, linking the class i molecule to the tap.
a full-length cdna  recently described by jorgensen et al.
<cit>  matched the tapbpa locus in ia and another est matched the tapbpb locus in ib.
as opposed to atlantic salmon, the rainbow trout tapbp in the ib region was described as a pseudogene both by shiina et al.
<cit>  as well as by landis et al.
<cit>  due to a deletion of the last  <dig> or  <dig> exons respectively.
landis et al.
did however find transcripts of the first  <dig> exons.
different rainbow trout strains were used in the two studies, potentially accounting for the observed differences in deleted exons.
the core ia region in atlantic salmon, ranging from the uba α <dig> exon and downstream including tap <dig>  shows  <dig> % sequence identity over  <dig> bp to the same region in rainbow trout.
a comparison of the salmon and rainbow trout ib region sequences from psmb <dig> to tap <dig> show  <dig>  % identity over  <dig> bp.
this would be in accordance with the general perception that uba lineages are ancient while the polymorphism of the duplicated ib region has evolved after the duplication event.
salmonid mhc evolution and function
in the atlantic salmon ib region we found only one mhc class i pseudo locus denoted ucaΨ, which is still being transcribed and shows a polymorphic pattern similar to that of rainbow trout uca and uda  <cit> .
the rainbow trout ib region contained four mhc class i loci denoted uca, uda, uea and ufaΨ  <cit> .
as suggested by shiina et al.
<cit>  there has been a primordial salmonid mhc region containing three mhc class i loci  where uea and uba have been deleted from the atlantic salmon ib region and uca and uea have been deleted from the atlantic salmon ia region.
the trout ib uda locus is a duplication of uca that occured in trout only.
once the extended trout ia region is sequenced we will see if the uba to ula duplication occurred in both species and if the uca and uea homologues have been retained in this region of trout.
the salmonid whole-genome duplication was estimated to have occurred between  <dig> and  <dig> mya  <cit>  while the study of shiina et al.
<cit>  estimate the duplication to have occurred  <dig> mya based on sequence identity of the mhc class i regions.
evolving from a tetraploid to a diploid state includes not only accumulation of mutations, but also random rearrangements and recombinations as exemplified by the multiple deletions that have occurred in the atlantic salmon ia and ib regions.
with a sequence identity between the atlantic salmon ia and ib regions of approximately  <dig> percent, recombination may even be occurring between the two duplicates today.
salmonids are also known for using recombination within the second intron of the uba locus to generate "new" alleles using exons already tested for functionality  <cit> .
as recombination was not observed in  <dig> siblings  <cit>  the recombination frequency is probably low.
one way of reducing the risk of recombination between duplicates may be insertions such as the  <dig> kb insertion with three copies of chitin synthase genes in the ib region.
another example of differences between atlantic salmon and rainbow trout is the chromosomal location of the ia region.
in both species, the ib region is located on chromosome  <dig>  <cit>  , while the ia region is located on chromosome  <dig> in rainbow trout and on one of the smaller chromosomes, potentially chromosome  <dig>  in atlantic salmon   <cit> .
in atlantic salmon, the ia and ib regions map to linkage groups  <dig> and  <dig> respectively  <cit> , while in rainbow trout they map to linkage groups  <dig> and  <dig>  <cit>  supporting the differences.
atlantic salmon and rainbow trout have diploid chromosome numbers ranging from  <dig> to  <dig>  <cit> .
most likely, different centric fusions have occurred in the diploidization processes of atlantic salmon and rainbow trout leading to ia residing on one arm of a metacentric chromosome in rainbow trout while on an acrocentric chromosome in atlantic salmon.
why the atlantic salmon ib region has undergone more deletions than trout is unknown, but it has functional consequences.
the ib region has been identified as a major qtl for resistance towards ihnv in atlantic salmon and ipnv in rainbow trout where the polymorphic uca, uda or uea loci were suggested as prime candidates for the observed effects  <cit> .
as our study indicates that the atlantic salmon ib region only contains a uca pseudolocus, there must either be other genes flanking our bacs which contribute to resistance or there could be haplotype variation in number of class i loci between norwegian and canadian atlantic salmon.
the ia region was not found associated with resistance towards ihnv in atlantic salmon nor ipnv in rainbow trout.
atlantic salmon uba genotypes have however been shown to provide resistance towards the viral pathogen causing infectious salmon anaemia   <cit> .
an ongoing study will identify the role of atlantic salmon ia and ib in providing resistance towards ipnv, enabling us to decipher between differences in pathogens versus genetic organization.
apart from the potential tap2a and uba lineages, limited polymorphism in psmbs and other linked loci suggest that the observed linkage between sasa-uba and disease resistance in norwegian atlantic salmon  <cit>  is caused by sasa-uba alleles or genotypes and not closely linked genes.
however, the psmbs and tap <dig> molecules residing in the ib region might still influence the overall peptide repertoire available for presentation by uba alleles.
due to the pseudo status of sasa-uca, the psmbs and tap2b in the ib region will most likely devolve over time.
CONCLUSIONS
we have characterized the gene organization of more than  <dig> kb of the two duplicated mhc regions in atlantic salmon.
although atlantic salmon and rainbow trout are closely related, the gene organization of their ib region has undergone extensive gene rearrangements.
the atlantic salmon had only one identified mhc class i uca pseudo gene in the ib region while this region in trout contained the four mhc class i loci uca, uda, uea and ufaψ .
the atlantic salmon ib region also contained a  <dig> kb insertion as compared to the ia region potentially limiting recombination between the two regions.
the large difference in gene content and most likely function of salmon and trout class ib regions clearly argues that sequencing of salmon will not necessarily provide information relevant for trout and vice versa.

 major histocompatibility complex class i and class ii molecules are vital parts of the cellular immune system presenting self and/or foreign peptides to cd positive and cd positive t cells. both classes of genes reside in a mb gene dense region on human chromosome shared with many other immune genes. atlantic salmon and rainbow trout genomes encode one major mhc class i locus designated uba in addition to the major mhc class ii alpha and beta genes designated daa and dab respectively. for uba, the main polymorphism resides in the alpha and alpha domains with up to 60% sequence divergence between these antigen binding domains. added variability for uba is produced by shuffling of exon onto different exon and downstream regions through recombination occurring in intron. additional class i loci and lineages have been described in both atlantic salmon as well as in rainbow trout. the majority of reported salmonid mhc class i molecules are classified into a u-lineage consisting of both uba as well as non-classical mhc molecules. two other described mhc class i-like lineages are ze described by miller et al. and l described by dijkstra et al.. in all teleosts studied so far including salmonids the mhc class i and class ii regions are unlinked. sequence data on the mhc class i region is available from zebrafish, fugu, medaka and rainbow trout. a general feature of these four mhc class i regions is a core region containing genes for the proteasome components and the transporter for antigen processing being flanked by various numbers of mhc class i loci in addition to many other genes also residing in the human mhc region located on chromosome data from medaka and zebrafish indicate that other fish orthologs of the mammalian mhc-encoded genes are dispersed on several different chromosomes, similar to the paralogue mhc regions described on human chromosomes and. salmonids are seen as partially tetraploid with a unique whole genome duplication occurring between and million years ago with remnants of tetraploidy visible also today. shiina et al. sequenced two duplicated core mhc regions of rainbow trout. based on sequence divergence they estimated the duplication event to have taken place approx. mya, in agreement with the salmonid whole genome duplication theory. the classical or ia region contained the major expressed classical mhc class i uba locus while the duplicated region denoted ib contained the four onmy-uca, -uda, -uea and -ufa class i loci. based on expression and polymorphism data, onmy-uca, -uda and -uea were defined as non-classical loci and -ufa as a pseudogene due to an incapacitating mutation in exon. data is rapidly emerging on associations between mhc and resistance to salmonid pathogens. in atlantic salmon, uba genotypes have been found to provide resistance towards aeromonas salmonicida and infectious salmon anaemia virus. class ib, but not class ia was found associated with susceptibility towards infectious hematopoietic necrosis virus in atlantic salmon and towards infectious pancreatic necrosis virus in rainbow trout. both trout and salmon are main aquaculture species and understanding their immune systems will improve our understanding of how these regions influence disease resistance and thus improve our breeding schemes for the trait. atlantic salmon and rainbow trout are estimated to have split approx. mya. as atlantic salmon is a major aquaculture species and displays some differences in response to pathogens when compared to rainbow trout, we ventured into sequencing of the two duplicated mhc class i regions of atlantic salmon. here we describe the gene organization of these two mhc class i regions comprising approx. kb each and compare our results to data from other teleosts.  the aim of this study was to characterize the gene organization and identify new genes potentially contributing to disease resistance in the two mhc class i regions of atlantic salmon. characterization and sequencing of bac clones sasa-uba and tap probes hybridized to bac clones, where clones were positive for both probes. the bac clones were ordered into three contigs using restriction fragment analysis together with grasp hindiii fingerprint information. the two contigs that were positive for uba, tap psmb and psmb by southern hybridization, were tested for presence of a polymorphic dinucleotide repeat located in the 3'utr of the uba locus. only bac clones from one of the two contigs gave pcr-products, thus this contig was defined as the ia region, and the other contig remained a candidate for the duplicated ib region. the bac clones in the third contig hybridized to the uba probe as well as a mixed uba exon probe. these clones also tested positive for a u-lineage ula locus that has previously been found closely linked to uba. three bacs were sequenced from the ia region. the bac clones 92i and 714p indicated allelic variants based on variation in the uba 3'utr marker with 523m as a continuation of 714p from the duplicated ib region we chose 8i 424m 15l and 189m for sequencing. 30c was chosen as a candidate from the third contig and was extended kb with the sequence of 868o the selected bac clones were subcloned, sequenced, and assembled into continuous sequences. the atlantic salmon ia region consisted of the bac clones 30c 868o 92i 714p and 523m covering bp, while the ib region consisted of 8i 424m 15l and 189m totaling bp. gene organization of the atlantic salmon mhc class i regions we have adopted the nomenclature described by shiina et al. with ia covering the uba locus region and ib for the duplicated region. thus the genes identified in the regions will be named accordingly; the ia proteasome subunits are given an extension of a and the ib genes have an extension of b. the previous symbol abcb has been withdrawn for the transporter for antigen processing so we have used the current symbol tap. the gene organization of the ia and ib mhc regions is shown in fig. a core region was identified in both regions which included mhc class i genes, together with the proteasome subunits genes psmb, psmb, psmb9-like, psmb and tap the gene order and orientation of the atlantic salmon psmbs and tap was very similar to that found in rainbow trout and other teleost. for the ia region, the main difference between atlantic salmon and rainbow trout is that the rainbow trout psmb8a gene is a pseudogene. the ia region contained the major mhc class i sasa-uba locus and the recently reported sasa-ula locus residing approximately kb upstream. the duplicated class ib region contained an mhc class i locus resembling the rainbow trout uca locus, but although transcribed it was a pseudogene. no other mhc class i-like genes were detected in the two duplicated regions. outside the core region we found atlantic salmon orthologs of genes residing in the extended human mhc class ii region. alternative nomenclature for these genes is described in table the following genes were found in both the ia and ib regions; kifc zbtb daxx, tapbp, brd col11a rxrb and slc39a three orthologs found in the ia region only were ring rps and vps for other teleosts, the gene organization of the extended mhc class i region is partly known for zebrafish, fugu and medaka. the tapbp, daxx, zbtb and kifc genes are conserved in the same order in both fish and human. as described in medaka we also found a gene for znf in the ia region, which is located on chromosome in human. mostly one match is listed per gene. current abbreviation vs. older nomenclature is listed for each gene. all est/cdna sequences are from genbank. hsd17b which resides in between slc39a and ring in the extended human class ii region, was found in the ib region only and showed more than 81% identity towards counterparts in tilapia, zebrafish and medaka. hsd17b has thus been deleted from the atlantic salmon ia region as it is also present in other fish mhc class i regions. three orthologs of genes located in the human class i region were identified in the ia region; tcf tubb and flot atlantic salmon tubulin is highly conserved and showed more than 94% identity towards mammalian counterparts. another highly conserved gene is rps which showed 98% identity towards mammalian sequences. a gene that was predicted by digit in the ia region had one est match, but no homology to annotated proteins and is thus denoted unknown in fig. however, some sequence identity was found towards a protein in zebrafish located on chromosome as well as to a tetraodon nigroviridis protein, which could indicate a molecule unique to teleosts. in addition to the genes described above we identified genes for pvrl rt, vshv-induced gene and a novel gene similar to a non-vertebrate chitin synthase protein that are not mhc linked in humans. the human pvrl is located on chromosome. a homologue of this gene is also found on zebrafish chromosome. a kb insertion was found in the ib region in between the rxrb and slc39a genes containing three copies of a chitin synthase gene approx. kb apart. chitin synthase is involved in the synthesis of chitin, which is a main structural component of the fungal cell wall. a similar protein has also been identified in zebrafish. no chitin synthase genes were present in the ia region nor are chitin synthase genes found in any other teleost mhc regions suggestive of a single insertion of this gene in the ib region with two subsequent duplications. most genes in both regions are supported by matching cdnas apart from tcf and col11a where no match has been found so far. other open reading frames were also identified, but were associated with transposon related repetitive elements. comparison of the ia and ib regions the two allelic bacs 92i and 714p had an overall sequences identity of % over bp, with similar exon intron organization for all genes. the major differences between the two allelic regions resided in the uba α and α exons and in differences in number of repeats. dotplot analysis of 714p or 92i against themselves showed no extended regions of local similarity, with the exception of the tap region which showed similarity due to a duplicated tap exon. a dot plot analysis of more than kb of the ia and ib regions showed four regions with high sequence similarity consisting of subregion one containing genes for kifc to tapbp, subregion two ranging from psmb to tap subregion three covering brd to rxrb and subregion four containing slc39a. the conserved regions in ia and ib have % identity over bp. in total, repeats constituted approximately 24% of the sequence in both regions, and 17% of the repeats were fish-specific dna elements. mhc class i genes sasa-uba the promoter, leader and α exons of sasa-uba were identified in 30c23/868o while the remaining exons of sasa-uba were found in 92i and 714p the leader and α exons found in 30c23/868o were identical to the pcr amplified uba* allele as well as to the leader and α exons of another salmon full-length cdna. together with the α and α exons of 92i they collectively provide a bona fida uba* allele. the uba α exon and downstream sequences of the two allelic bacs 92i and 714p have complete sequence identity to the sasa-uba*0201/* and sasa-uba* alleles respectively. uba* and uba* are prime examples of the recombination shown to occur within intron of salmonid uba alleles showing complete sequence identity in the α and downstream regions, but highly divergent α exons. the predicted amino acid sequences of uba, ula and the two open reading frames of ucaψ were aligned for comparison of the mhc class i genes encoded in the two regions. analysis of the promoter sequence of uba* in 30c23/868o showed high similarity to a rainbow trout uba* promoter, both containing similar regulatory elements typical for mhc class i promoters such as an interferon stimulated response element, w/s-box and enhancer b. the uba* promoter contains a potential site α element according to the core sequence while a sequence more resembling an x2-box has been found in rainbow trout. both the site α and the homologous x2-box found in mammalian mhc class i and mhc class ii promoters respectively, are involved in regulation of transcription and bind atf/creb family transcription factors. a potential tata-box was also identified in the promoter sequence of uba*. a salmon uba* promoter published by jorgensen et al. had lower sequence identity to the uba* promoter, but both promoters are supported by complete identity to 5'utr cdna sequences of bona fida uba alleles suggesting that atlantic salmon uba alleles have different promoters. the functional consequences of these differences are being investigated. the 30c23/868o and 92i bacs jointly have an intron sequence of kb while in rainbow trout, the intron between the uba α and α exons is kb suggesting we lack approximately kb to have a continuous genomic sequence of the entire uba region. pcr and cloning of the gap was performed multiple times, but despite successful pcr amplification no fragments covering the gap have been cloned suggestive of an unclonable region. the amplified products support an intron sequence of approx. kb. unfortunately no mrna or cdna is available from the bac library fish, preventing verification of expressed uba alleles in this animal. to verify the linkage between 92i and 30c fluorescent in situ hybridization was undertaken and showed that both bacs hybridized to the same region of one of the smallest chromosomes, potentially chromosome. the close linkage described by miller et al. between ula and uba also supported 30c23/868o being an extension of the ia region. sasa-ula the ula locus residing approximately kb upstream of the uba locus matched a partial ula* sequence described by miller et al.. another est in the cgrasp database provided us with a full-length match. the exon encoding the transmembrane domain is missing, suggestive of a secreted mhc class i molecule. similar secretory class i molecules are also found for human class i molecules and the potential role of secretory hla-g is currently being deciphered and holds promise for an interesting function. the 30c ula gene has an α exon with highest sequence identity to uba* while α and downstream exons have highest identity to uba* no ests for ula have been identified in rainbow trout, and a negative pcr-based survey for this gene in rainbow trout by miller et al. suggest this gene may be unique to atlantic salmon. sasa-ucaψ only one mhc class i locus was identified in the four bacs representing the ib region. this locus found in 8i showed highest sequence identity to the onmy uca* allele and was thus denoted sasa-uca. multiple salmon ests with a polymorphic pattern resembling that of onmy-uca sequences were found in databases. however, both the 8i uca orf sequence as well as matching ests, contained an internal stop codon in exon making sasa-uca an expressed pseudogene. the exon intron organization of the uba, ula and ucaψ loci are quite similar apart from the enlarged first intron in ula, the even larger second intron of uba and the missing transmembrane exon of ula. antigen presenting genes previously reported cdnas for tap which were assumed to reside in the ia and ib region and denoted tap2b and tap2a respectively, indeed did match the tap in the ia and ib bac sequences. to avoid nomenclature confusion we hereby rename our ia uba-linked tap locus to tap2a and the ib ucaψ linked tap locus to tap2b. we adapted a similar nomenclature for the reported rainbow trout tap sequences, and suggest that tap sequences without locus identification should be denoted tap only. atlantic salmon ia and ib tap sequences have more than 90% aa sequence identity and a similar identity to the rainbow trout ia and ib tap sequences described by shiina et al.. other tap ests were also found in databases, which were difficult to define as tap2a or tap2b variants such as the grasptap2- in fig. attempts to decipher locus origin including rainbow trout information shows that the tap2a and tap2b sequences described by shiina et al. resembles the tap2b sequence identified in atlantic salmon containing for instance the characteristic fca motif at position and the two aa deletion at position. a rainbow trout tap2a sequence described by hansen et al., shown by in situ hybridization to reside in the ia region, resembles the atlantic salmon ia tap2a sequence and does not contain these motifs mentioned above. thus, rainbow trout has a polymorphic tap2a locus and the confusing sequence identities between the two tap loci may suggest that these genes are exposed to recombination or gene conversion mechanisms. locus designation of either salmon or trout tap sequences therefore can not be performed on sequence alone, but must be verified by linkage mapping. other more divergent atlantic salmon tap ests have approx. 50% sequence identity to all above described ia and ib tap sequences, but has 94% sequence identity to a rainbow trout tap variant described by hansen et al.. if these sequences represent an additional tap locus, i.e. a tap2c locus, or are allelic variants of the tap2a/b loci is currently unknown. ancient lineages of divergent mhc class i, tap tap and lmp haplotypes have been described in xenopus where the sequence identity between allelic tap2s was less than 76%. similar ancient lineages of uba and tap2a may also exist in salmonids, where we were unfortunate enough to sequence allelic variants belonging to similar lineages. all psmbs genes in the salmon ia and ib regions were fully intact, as opposed to the ia region in rainbow trout where psmb was a pseudogene, lacking the first three exons. the core region psmb psmb psmb9-like, psmb and tap loci were found to be organized in a similar fashion in both regions and also displayed a high amount of sequence identity. some nucleotide differences were found between the allelic bacs 92i and 714p genes that resulted in amino acid changes; psmb10a* and psmb10a* had aa differences located in the propeptide at position and. the proteasome subunits therefore seem to be non-restrictive in providing peptides for uba molecules as opposed to xenopus where psmb segregates in lineages with mhc class i, tap and tap. the table shows comparison of % nucleotide and % amino acid identity between the genes found respectively in the atlantic salmon ia vs ib region and in the ia and ib region of atlantic salmon versus rainbow trout. tapasin is a key member of mhc class i antigen-loading complexes, linking the class i molecule to the tap. a full-length cdna recently described by jorgensen et al. matched the tapbpa locus in ia and another est matched the tapbpb locus in ib. as opposed to atlantic salmon, the rainbow trout tapbp in the ib region was described as a pseudogene both by shiina et al. as well as by landis et al. due to a deletion of the last or exons respectively. landis et al. did however find transcripts of the first exons. different rainbow trout strains were used in the two studies, potentially accounting for the observed differences in deleted exons. the core ia region in atlantic salmon, ranging from the uba α exon and downstream including tap shows % sequence identity over bp to the same region in rainbow trout. a comparison of the salmon and rainbow trout ib region sequences from psmb to tap show % identity over bp. this would be in accordance with the general perception that uba lineages are ancient while the polymorphism of the duplicated ib region has evolved after the duplication event. salmonid mhc evolution and function in the atlantic salmon ib region we found only one mhc class i pseudo locus denoted ucaΨ, which is still being transcribed and shows a polymorphic pattern similar to that of rainbow trout uca and uda. the rainbow trout ib region contained four mhc class i loci denoted uca, uda, uea and ufaΨ. as suggested by shiina et al. there has been a primordial salmonid mhc region containing three mhc class i loci where uea and uba have been deleted from the atlantic salmon ib region and uca and uea have been deleted from the atlantic salmon ia region. the trout ib uda locus is a duplication of uca that occured in trout only. once the extended trout ia region is sequenced we will see if the uba to ula duplication occurred in both species and if the uca and uea homologues have been retained in this region of trout. the salmonid whole-genome duplication was estimated to have occurred between and mya while the study of shiina et al. estimate the duplication to have occurred mya based on sequence identity of the mhc class i regions. evolving from a tetraploid to a diploid state includes not only accumulation of mutations, but also random rearrangements and recombinations as exemplified by the multiple deletions that have occurred in the atlantic salmon ia and ib regions. with a sequence identity between the atlantic salmon ia and ib regions of approximately percent, recombination may even be occurring between the two duplicates today. salmonids are also known for using recombination within the second intron of the uba locus to generate "new" alleles using exons already tested for functionality. as recombination was not observed in siblings the recombination frequency is probably low. one way of reducing the risk of recombination between duplicates may be insertions such as the kb insertion with three copies of chitin synthase genes in the ib region. another example of differences between atlantic salmon and rainbow trout is the chromosomal location of the ia region. in both species, the ib region is located on chromosome, while the ia region is located on chromosome in rainbow trout and on one of the smaller chromosomes, potentially chromosome in atlantic salmon. in atlantic salmon, the ia and ib regions map to linkage groups and respectively, while in rainbow trout they map to linkage groups and supporting the differences. atlantic salmon and rainbow trout have diploid chromosome numbers ranging from to. most likely, different centric fusions have occurred in the diploidization processes of atlantic salmon and rainbow trout leading to ia residing on one arm of a metacentric chromosome in rainbow trout while on an acrocentric chromosome in atlantic salmon. why the atlantic salmon ib region has undergone more deletions than trout is unknown, but it has functional consequences. the ib region has been identified as a major qtl for resistance towards ihnv in atlantic salmon and ipnv in rainbow trout where the polymorphic uca, uda or uea loci were suggested as prime candidates for the observed effects. as our study indicates that the atlantic salmon ib region only contains a uca pseudolocus, there must either be other genes flanking our bacs which contribute to resistance or there could be haplotype variation in number of class i loci between norwegian and canadian atlantic salmon. the ia region was not found associated with resistance towards ihnv in atlantic salmon nor ipnv in rainbow trout. atlantic salmon uba genotypes have however been shown to provide resistance towards the viral pathogen causing infectious salmon anaemia. an ongoing study will identify the role of atlantic salmon ia and ib in providing resistance towards ipnv, enabling us to decipher between differences in pathogens versus genetic organization. apart from the potential tap2a and uba lineages, limited polymorphism in psmbs and other linked loci suggest that the observed linkage between sasa-uba and disease resistance in norwegian atlantic salmon is caused by sasa-uba alleles or genotypes and not closely linked genes. however, the psmbs and tap molecules residing in the ib region might still influence the overall peptide repertoire available for presentation by uba alleles. due to the pseudo status of sasa-uca, the psmbs and tap2b in the ib region will most likely devolve over time.  we have characterized the gene organization of more than kb of the two duplicated mhc regions in atlantic salmon. although atlantic salmon and rainbow trout are closely related, the gene organization of their ib region has undergone extensive gene rearrangements. the atlantic salmon had only one identified mhc class i uca pseudo gene in the ib region while this region in trout contained the four mhc class i loci uca, uda, uea and ufaψ. the atlantic salmon ib region also contained a kb insertion as compared to the ia region potentially limiting recombination between the two regions. the large difference in gene content and most likely function of salmon and trout class ib regions clearly argues that sequencing of salmon will not necessarily provide information relevant for trout and vice versa. 
the large differences in gene content and most likely function of the salmon and trout class ib region clearly argues that sequencing of salmon will not necessarily provide information relevant for trout and vice versa. although atlantic salmon and rainbow trout are closely related, the gene organization of their ib region has undergone extensive gene rearrangements. the atlantic salmon ib region had an insert of kb in comparison to the ia region containing three chitin synthase genes. the duplicated class ib region contained an mhc class i locus resembling the rainbow trout uca locus, but although transcribed it was a pseudogene. both regions contained the proteasome components psmb psmb psmb9-like and psmb in addition to the transporter for antigen processing tap as well as genes for kifc zbtb daxx, tapbp, brd col11a rxrb and slc39a the ia region contained the recently reported mhc class i sasa-ula locus residing approximately kb upstream of the major sasa-uba locus. two allelic bacs spanning the uba locus had % identity over kb, while the ia region showed % identity over kb to the ib region. no other mhc class i-like genes were detected in the two duplicated regions. nine bacs covering more than kb of the two duplicated mhc class i regions of atlantic salmon were sequenced and the gene organizations characterized. we have characterized the gene organization of more than kb of the two duplicated mhc class i regions in atlantic salmon. we have previously identified associations between major histocompatibility complex class i and resistance towards bacterial and viral pathogens in atlantic salmon. to evaluate if only mhc or also closely linked genes contributed to the observed resistance we ventured into sequencing of the duplicated mhc class i regions of atlantic salmon. the atlantic salmon has only one class i uca pseudogene in the ib region while trout contains the four mhc uca, uda, uea and ufa class i loci.
 we have previously identified associations between major histocompatibility complex class i and resistance towards bacterial and viral pathogens in atlantic salmon. to evaluate if only mhc or also closely linked genes contributed to the observed resistance we ventured into sequencing of the duplicated mhc class i regions of atlantic salmon.  nine bacs covering more than kb of the two duplicated mhc class i regions of atlantic salmon were sequenced and the gene organizations characterized. both regions contained the proteasome components psmb psmb psmb9-like and psmb in addition to the transporter for antigen processing tap as well as genes for kifc zbtb daxx, tapbp, brd col11a rxrb and slc39a the ia region contained the recently reported mhc class i sasa-ula locus residing approximately kb upstream of the major sasa-uba locus. the duplicated class ib region contained an mhc class i locus resembling the rainbow trout uca locus, but although transcribed it was a pseudogene. no other mhc class i-like genes were detected in the two duplicated regions. two allelic bacs spanning the uba locus had % identity over kb, while the ia region showed % identity over kb to the ib region. the atlantic salmon ib region had an insert of kb in comparison to the ia region containing three chitin synthase genes.  we have characterized the gene organization of more than kb of the two duplicated mhc class i regions in atlantic salmon. although atlantic salmon and rainbow trout are closely related, the gene organization of their ib region has undergone extensive gene rearrangements. the atlantic salmon has only one class i uca pseudogene in the ib region while trout contains the four mhc uca, uda, uea and ufa class i loci. the large differences in gene content and most likely function of the salmon and trout class ib region clearly argues that sequencing of salmon will not necessarily provide information relevant for trout and vice versa. 