BACKGROUND
the major histocompatibility complex  is a highly polymorphic gene-dense region, spanning an area of approximately  <dig> mbp in the human genome  <cit> .
since its first discovery in mice  <cit> , the mhc has been intensely studied in many species due to its association with immune related functions .
in domestic animals, this has included the evolutionary relationship of the mhc in different species, the genetic diversity of animals subjected to domestication, its role in the immune response to parasites, its association with infectious and parasitic diseases and the development of vaccines .
in sheep, characterisation of the mhc has been based predominantly on analysis of orthologous loci from the respective human and cattle mhcs.
early studies have assumed that the basic structure of the sheep mhc was similar to that of other mammals, consisting of the telomeric class i, central class iii and centromeric class ii.
a later study of mhc structure in chinese merino sheep reported that, like the cattle mhc  <cit> , the sheep class ii region is sub-divided into two distinct iia and iib regions and is most likely derived from a common ancestral partial chromosomal inversion  <cit> .
in recent years, low resolution physical maps of sheep mhc class ii and iii regions have been constructed using a combination of sub-cloning and partial sequencing of bacterial artificial chromosome  clones known to contain mhc sequences  <cit> .
in addition, a panel of single nucleotide polymorphisms  spanning the sheep mhc class ii and iii regions have also been developed  <cit> .
these have provided a framework for the identification and analysis of haplotypes.
however, a relative paucity in the knowledge regarding the sheep mhc still exists, in particular the class i region in terms of its gene content, structural organisation and genetic variation.
research into the mhc of sheep is currently limited in comparison with other domestic animals, especially cattle and swine .
for instance, there is a lack of understanding of its haplotype structure.
although dinucleotide microsatellite loci such as ohcci  <cit>  have been widely used in association studies in sheep  there is still a lack of understanding and characterisation of haplotypes in this important genetic region.
the better understood human mhc map indicates that the class i region is rich in pseudogenes, duplicated genes and genes showing copy number variation  <cit> .
recently, a physical map of sheep mhc derived from a chinese merino sheep has been published by gao and colleagues  <cit> .
annotation of this chinese merino physical map led to the identification of a total of  <dig> genes, among which  <dig> of the genes were apparently not previously identified in sheep and  <dig> described as unique to sheep  <cit> .
from their study,  <dig> genes were reported in the mhc class i region.
twenty two predicted genes had either high sequence similarity to other known gene sequences, or contained a predicted open reading frame  having low sequence similarity with sequences from other species  <cit> .
three novel sheep-specific genes were also reported with no apparent sequence homology to any known mammalian sequences  <cit> .
the objective of this study was to reanalyse existing and new information regarding the sheep class i region and produce a comprehensive and updated version of the sheep mhc class i map.
this was achieved through the mapping of genes sequenced from chori bacs and comparing the result with the cattle reference genome  <cit> , the previously published chinese merino sheep map  <cit>  and the very recently available sheep reference genome  <cit> .
in this study, we sub-cloned chori bacs known to contain class i sequences and re-assembled the sequences in order to annotate genes present within the sheep mhc class i region.
in addition, we reanalysed, re-assembled and re-annotated the chinese merino bacs published by gao and colleagues  <cit> .
annotation of the chori bac and chinese merino bac sequences was then used to generate a revised contig map.
comparison with the recent annotation of chromosome  <dig> from the sheep genome reference sequence  <cit>  was then used to further inform this map.
RESULTS
re-analysis of chinese merino mhc contig map
initial analysis with geneious  <dig>  software produced  <dig> distinct contigs instead of the single contig reported by gao et al.
.
the geneious assembly also revealed that some of the bac sequences were not in the correct  orientation .
four reads were not incorporated into the assembly; genbank: fj <dig>  genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig>  comparison with the published chinese merino map  in the 5′ to 3′ direction: contig  <dig> assembles four reads - genbank: fj <dig>  genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig>  however, an overlap between genbank: fj <dig> and either genbank: fj <dig> or genbank: fj <dig> was not detected.
contig  <dig> assembles  <dig> reads - genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig>  genbank: fj <dig> was not included in the assembly with genbank: fj <dig> and genbank: fj <dig>  no overlap between genbank: fj <dig> and genbank: fj <dig> was identified.
contig  <dig> assembles genbank: fj <dig> and genbank: fj <dig>  analysis using geneious did not identify any overlap between genbank: fj <dig> and genbank: fj <dig>  contig  <dig> assembles genbank: fj <dig>  genbank: fj <dig>  genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig>  no overlap between genbank: fj <dig> and genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig> was identified.
contig  <dig> assembles genbank: fj <dig> and genbank: fj <dig>  no overlap between genbank: fj <dig> and genbank: fj <dig> was detected by the geneious algorithm.
contigs  <dig>   <dig> and  <dig> are in the opposite orientation when compared to the published chinese merino map  <cit>  .
further analyses using the ncbi blast option to align two sequences and the chaos/dialign software detected an overlapping region between the chinese merino bacs  that was not reported by geneious.
in addition, analyses of bacs genbank: fj <dig>  genbank: fj <dig>  and genbank: fj <dig> that had been omitted in the initial assembly using the geneious analysis showed overlapping sequence alignment with other chinese merino bacs; genbank: fj <dig> overlaps with genbank: fj <dig> and genbank: fj <dig>  genbank: fj <dig> overlaps with genbank: fj <dig>  and genbank: fj <dig> overlaps with genbank: fj <dig>  figures s <dig> and s <dig> in additional file  <dig> provides the results of these analyses as a series of pairwise dotplots for the bacs along with an interpretive comment.
the regions of overlaps between the bac sequences are detailed in table  <dig> table  <dig> geneious assembly of  <dig> bac clones published by gao et al.
indicates assembly of sequence in the forward  direction.
indicates assembly of the reverse complement sequence
fig.
<dig> comparison of the old and new tiling path of chinese merino bacs.
a original tiling path of the chinese merino mhc map .
b new tiling path of chinese merino bac sequences proposed in this study.
a vertical red line between bac contigs indicates a gap region
precede bac id numerals with ‘fj9858’ to determine ncbi genbank accession.
appended ‘rc’ indicates the bac was reverse complemented before alignment.
‘-‘ indicates there was no overlap identified.
afirst 765 bp of genbank: fj985864rc do not overlap with genbank: fj985854rc, this is most likely due to contig misassembly in this region
a pictorial view of our revised tiling map of the chinese merino bac sequences is shown in fig.
1b.
based on this analysis, there are gaps between bacs genbank: fj <dig> and genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig>  and genbank: fj <dig> and genbank: fj <dig>  blast analysis of the first  <dig> nucleotides of the reverse complemented genbank: fj <dig> indicated that nucleotides 1– <dig> aligned perfectly, beginning  <dig>  nucleotides downstream in the same bac, and nucleotides 403– <dig> aligned perfectly, beginning  <dig>  nucleotides downstream also in the same bac, with a gap of approximately 8900 bp between the two alignments .
the sequence between bp  <dig> and  <dig> is a string of undefined nucleotides, hence no alignment was obtained in this region.
we conclude that these  <dig> nucleotides have not been assembled correctly.
subsequent dot plot alignment of the chinese merino bacs with the mhc class i region of the reference sheep chromosome  <dig> is presented in the "comparative analysis of chori bac contigs map with mhc class i maps" results section below.
analysis of sheep mhc class i gene content
initial analysis of chori bac sub-clone sequences identified  <dig> additional loci in the sheep mhc class i region that were not recognised in the chinese merino mhc map  <cit>  but were found in both the cattle and sheep reference maps.
comparison of mhc class i gene content between the cattle reference map, sheep reference map and the published chinese merino map  <cit>  is given in fig.
<dig>  the additional loci identified through chori bac sub-clones include ribonuclease p 21-like isoform  <dig> , guanine nucleotide-binding protein-like  <dig> , atp-binding cassette sub-family f member  <dig> , chromosome  <dig> open reading frame  <dig> ortholog , deah  box polypeptide  <dig> , nuclear envelope membrane protein or nurim , general transcription factor ii h subunit  <dig> , surfactant associated protein g , transcription factor  <dig>  and pou class  <dig> homeobox  <dig> .
the relative locations of these additional loci were deduced using blast analysis.
figure  <dig> shows the positions of the additional loci identified through sequencing of these chori bac subclones that had not been identified in the previously published chinese merino mhc map  <cit> .fig.
<dig> comparison of cattle , sheep  and chinese merino mhc class i maps.
loci highlighted in red indicate additional loci identified through sequencing of chori bac clones.
additional file 3: table s <dig> and s <dig> provides the detailing of gene symbol, gene name and gene description in the class i region of the sheep  and cattle  reference genomes at ncbi, respectively
fig.
<dig> loci identified through chori bac sub-cloning and their relative position within the chinese merino map.
identity of these loci was confirmed by sequence homology using ncbi blast.
loci highlighted in red indicate loci previously not present on the map proposed by gao et al.
our re-analysis of the mhc ci chinese merino map indicates that the majority of genes identified appear to have the same gene arrangement with that previously observed in cattle, along with a high level of nucleotide similarity to their cattle orthologues.
the name and location of genes identified in the chinese merino bac sequences are all tabulated in table  <dig>  identical gene predictions between adjacent bac sequences corroborates with overlapping regions identified through dotplot and pairwise sequence alignment analysis.
likewise, non-overlapping regions show no similarity in gene content.table  <dig> summary of gene content on each of the chinese merino bac sequences
†first exon not in bac.
‡last exon not in bac.
*alignment to known homolog shows high percent identity with no evidence of wrong or missing exons.
# not included in fig.
<dig>  shaded regions indicate genes identified in overlapping regions between clones.
## identified from additional file 3: table s <dig> .
gene ids not matching our gene symbols were surmised based on gene description and/or alternate gene symbols provided in the ncbi gene database along with relative gene position compared to our analysis
the majority of gene predictions in the overlapping regions of the chinese merino bacs were identical, however there were a few exceptions.
in the overlap region between genbank: fj985852rc and genbank: fj985875rc, there were differences observed in the vars <dig> and dpcr1-like predictions.
exon  <dig> of the vars <dig> gene prediction is longer in genbank: fj985852rc than in genbank: fj985875rc due to a single bp deletion in the exon region in genbank: fj985875rc that results in a change in the sequence reading frame and hence a premature stop to the exon.
alignment with reference sequences indicates that the vars <dig> gene prediction from genbank: fj985852rc is correct .
exon  <dig> of the dpcr1-like gene prediction is shorter in genbank: fj985852rc than in genbank: fj985875rc due to a significant insertion of ~370 bp in genbank: fj985875rc compared to genbank: fj985852rc.
alignment with reference sequences indicates that the genbank: fj985852rc prediction is more similar and hence more likely to be correct.
in the overlapping region between genbank: fj985859rc and genbank: fj <dig>  there is a single amino acid length difference noted in the genbank: fj <dig> mhc class i-like predicted peptide compared to the genbank: fj985859rc peptide due to a 3 bp deletion in the region of the first exon.
an alignment with reference sequences failed to indicate which sequence is correct since the difference occurs in an area of low complexity repeats within the signal peptide.
a comparison between genes reported in the chinese merino map by gao et al.
<cit>  and our analysis showed that twenty-nine of the sixty-eight genes identified in this study were not previously annotated.
the ten genes identified through sub-cloning and sequencing of the chori bac sequences were annotated in the re-analysis of chinese merino bac sequences.
table  <dig> includes a list of gene identifications reported by gao et al.
<cit>  for the thirty-nine genes identified in both studies.
sequence comparison of chinese merino bacs with mhc class i region of reference sheep chromosome 20
chinese merino bacs covering the mhc class i region were aligned via dotplot analysis with a portion of the sheep chromosome  <dig> from the mhc class i region downloaded from ncbi .
the results of this dotplot analysis are presented in additional file 2: figure s <dig>
bacs genbank: fj <dig>  genbank: fj <dig> and genbank: fj <dig> align well with the sheep reference genome sequence in this region, there are no obvious large indels and no significant interspersed repetitive regions.
a large indel is observed in the alignment between genbank: fj <dig>  and genbank: nc_ <dig> due to the presence of ~ <dig>  bp in the sheep reference sequence not found in the genbank: fj <dig> bac.
the insertion in the sheep genome is approximately from position  <dig> to  <dig> in genbank: nc_ <dig>  within this region there are two annotated genes - described as an ubiquitin d-like gene and c19orf <dig> homolog – that were not located in the genbank: fj <dig> bac.
smaller indels and gaps in the alignment diagonal appear to correspond to runs of undefined nucleotides within the sheep reference sequence.
shorter runs of undefined nucleotides are also present in the bac sequence.
bacs genbank: fj <dig> and genbank: fj <dig> overlap over the majority of their sequence, and this is evident when comparing their alignments to genbank: nc_ <dig>  there are several indels, and the dotplot shows parallel lines indicating large interspersed repeating regions.
these bacs contain several mhc class i histocompatibility antigen-like loci, which share enough sequence similarity to create an interspersed pattern of diagonals.
within the corresponding region on genbank: nc_ <dig>  larger gaps in the alignment are evidence of possible mis-assembly in either the bac or sheep reference chromosome  <dig>  or could also represent breed specific differences in the region.
the alignment of fj <dig> with genbank: nc_ <dig> also reveals that the first  <dig> nucleotides align in two different segments 5′ to the main alignment diagonal, with a gap of approximately 9000 bp between, confirming the observation from blast analysis of this region that these first  <dig> nucleotides are mis-assembled .
the alignment of genbank: fj <dig> with genbank: nc_ <dig> reveals a segmental inversion covering an area of approximately  <dig>  bp.
within this region are four genes that have been predicted in reverse orientation within the two sequences – abcf <dig>  prr <dig>  gnl <dig> and rpp <dig> .
the inverted sequence may be a result of mis-assembly in this region in either the sheep reference chromosome  <dig> or the chinese merino bac; alternatively, it may represent a genuine breed specific difference.
in cattle and in the chinese merino bac sequence, the genes are annotated in the order rpp <dig>  gnl <dig>  prr <dig>  abcf <dig>  this order was chosen for our proposed map  as it is consistent with the order annotated  in other species including bos taurus, sus scrofa and mus musculus.fig.
<dig> the new sheep mhc ci map proposed based on findings in this study.
position of each locus is based on the comparative analysis between chori bacs, chinese merino bacs, cattle reference sequence for chromosome  <dig>  and sheep reference sequence for chromosome  <dig>
the dotplot analysis confirmed a gap between chinese merino bac sequences genbank: fj <dig> and genbank: fj <dig> of  <dig>  bp, lying in the region from 27555826– <dig> in genbank: nc_ <dig>  annotated within this region in the sheep reference genome are two mhc class i histocompatibility antigen–like genes  and an envelope glycoprotein-like gene.
the reverse complement of genbank: fj <dig> aligns well with sheep reference chromosome  <dig> for ~ <dig>  bp.
there is no significant alignment from ~ <dig>  bp to the end of the sequence - ~ <dig>  bp.
genbank: nc_ <dig> has a 5000 bp run of undefined nucleotides spanning the region from ~ <dig>  to  <dig>  bp in genbank: fj <dig> and  <dig> to  <dig> in genbank: nc_ <dig>  the remaining sequence in genbank: fj <dig> represents inserted sequence not currently present in the sheep reference genome.
this is confirmed in the dotplot alignment with the reverse complement of genbank: fj <dig>  which spans a region in genbank: nc_ <dig> from bp  <dig> to  <dig>  five genes are annotated in the region from bp  <dig>  to the end of genbank: fj <dig>  including two mhc class i-like genes, two mucin-like genes and eukaryotic translation elongation factor  <dig> alpha  <dig> .
mhc class i and mucin-like genes would be expected in the mhc class i region, however eef1a <dig> has not been annotated in the mhc class i region in genomes from other closely related species, including cow, pig, mouse and human.
the first  <dig>  nucleotides of the reverse complement of genbank: fj <dig> do not align with genbank: nc_ <dig>  5000 bp may be accounted for by undefined nucleotides in genbank: nc_ <dig>  however the remainder represents an inserted region of ~ <dig>  bp not currently present in the sheep reference genome.
within this region in genbank: fj <dig> one mhc class i histocompatibility antigen gene has been predicted.
several interspersed repeat regions are indicated in the alignment bp of the reverse complement of genbank: fj <dig> in a ~9000 bp span between bp  <dig>  and  <dig> .
within this region an mhc class i-like gene has been predicted.
comparison of the dotplot alignments between the reverse complement of genbank: fj <dig> and genbank: nc_ <dig> and between genbank: fj <dig> and genbank: nc_ <dig> indicates an overlap between genbank: fj <dig> and genbank: fj <dig> of  <dig>  bp.
the dotplot alignment of genbank: fj <dig> with genbank: nc_ <dig> shows a mostly unbroken alignment diagonal.
there are two noticeable indels of approximately 1500 bp that are the result of undefined nucleotides in the sheep reference genome sequence.
a number of interspersed repeats are indicated in the first ~ <dig>  bp of genbank: fj <dig>  within this region there are four predicted mhc class i – like genes.
analysis of the dotplot alignments confirms the overlaps of the chinese merino bac sequences as indicated in fig.
1b.
these are summarised in table  <dig>  dotplot analysis provides no evidence of overlap between genbank: fj <dig> and genbank: fj <dig> or between genbank: fj <dig> and genbank: fj <dig>  we were unable to estimate the size of the gap between genbank: fj <dig> and genbank: fj <dig> table  <dig> overlaps of bacs published by gao et al.
with sheep reference genome sequence genbank: nc_019477
comparative analysis of chori bac contigs map with mhc class i maps
comparative analysis of the assembled, sub-cloned chori bac sequences revealed the coverage of each bac and its position with respect to the cattle reference sequence for chromosome  <dig> , chinese merino bac sequences  <cit>  and the sheep reference sequence for chromosome  <dig>  .table  <dig> blast analysis of chori bac end sequences available in genbank.
the sequences were aligned with cattle and sheep reference sequences from chromosome  <dig> and  <dig>  respectively, and with the chinese merino bac sequences
the telomeric end sequence of chori 243-390h <dig>  aligns between two mhc class i-like loci in both the cattle and sheep reference sequences, but shows no significant similarity to any of the chinese merino bac sequences.
it appears that this chori bac end sequence lies within a gap region between chinese merino bac genbank: fj <dig> and genbank: fj <dig>  the centromeric end sequence of chori 243–390h <dig>  aligns within the predicted tubb locus in both cattle and sheep reference sequences and in chinese merino bac genbank: fj <dig>
the chori 243–454e <dig> bac appears to span a region beginning between loci ddr <dig> and ier <dig>  and ending within a trim 39-like locus in the cattle and sheep reference sequences and in the chinese merino bacs.
chori 243–390h <dig> overlaps with chori 243–454e <dig> and was located in the middle of mhc class i region whereas, chori 243–269 m <dig> was located further away towards the centromeric end.
both the telomeric and centromeric ends of chori 243–269 m <dig> are located between  <dig> mhc class i-like genes with respect to the cattle and sheep reference sequences and in the chinese merino bacs.
using this information fig.
<dig> shows a revised sheep mhc class i map based upon our reanalysis.
comparative analysis of mhc class i histocompatibility antigen genes
of particular interest was the determination of the number and distribution of the class i histocompatibility antigen loci.
in cattle, there is evidence for at least six classical and four nonclassical discrete mhc class i loci .
in sheep, evidence exists for at least six discrete class i loci, with at least two being classical  <cit> .
the number and expression levels of class i loci have been demonstrated to be haplotype specific in the scottish blackface sheep breed  <cit> , so loci identified in the chinese merino bacs are expected to differ from the sheep reference genome .
correct assembly of these highly repetitive gene sequences is bioinformatically a difficult task, compounded by variant numbers of loci in different haplotypes.
mhc class i genes were predicted in the chinese merino bacs as detailed in the "re-analysis of chinese merino sheep mhc map" methods section and are included table  <dig>  predicted mhc class i protein sequences from both the chinese merino bacs and the sheep reference sequence for chromosome  <dig> were aligned with selected reference sequences taken from the ipd-mhc database .
in addition, functional domain analysis was carried out for each sequence.
tables  <dig> and  <dig> list the class i genes predicted in the chinese merino bac sequences and corresponding sheep reference chromosome  <dig> sequences, respectively, and include details on the location of signal peptides and functional domains identified.
the mhc class i genes with identifiable transmembrane and cytoplasmic domains are denoted as classical  or nonclassical  according to sequence criteria previously used in cattle  <cit>  and sheep  <cit> : namely the presence of a vpi, ipi or vlik motif in the transmembrane domain and/or a truncated cytoplasmic domain.
a sequence alignment of the predicted class i proteins from the chinese merino bac sequences in comparison with a selection of reference sequences representing different loci can be seen in , a similar alignment with the predicted class i proteins from the sheep reference chromosome  <dig> sequences can be seen in .
sequences considered to most likely represent functional class i genes are aligned in fig.
<dig>  the location of functional domains are also indicated for each of these alignments.table  <dig> mhc class 1-like genes predicted from genomic bac sequences published by gao et al.
fj985874c1bc
fj985874c1b2c
s: strand.
#: locus number in telomeric to centromeric direction.
#ex: number of exons identified.
aa len: length of predicted amino acid sequence.
sp: signal peptide.
α1: alpha  <dig> domain.
α2: alpha  <dig> domain.
α3: alpha  <dig> domain.
tm: transmembrane domain.
c-term: c-terminus end.
homologue indicates sequence used as homologous protein sequence with fgenesh+
aterminal exon not predicted.
bweak match.
calternative transcript predictions
s: strand.
#: locus number in telomeric to centromeric direction.
#ex: number of cds exons identified.
aa len: length of predicted amino acid sequence.
sp: signal peptide.
α1: alpha  <dig> domain.
α2: alpha  <dig> domain.
α3: alpha  <dig> domain.
tm: transmembrane domain.
c-term: c-terminus end.
type: ia – classical, ib – nonclassical.
protein acc: accession of protein sequence in ncbi database
atranscripts differ only at the c-terminal end
fig.
<dig> shows an alignment of mhc class i amino acid sequences predicted from selected bac sequences published by gao et al.,  <dig> in comparison with selected class i loci from the sheep reference chromosome  <dig>  and six class i reference sequences from the ipd database  plus an alternative transcript sequence aaa <dig>  nomenclature for the predicted genes from bac sequences derived as follows: ncbi accession number, plus ‘rc’ if the sequence was reverse complemented before analysis, plus the class i gene identifier within the bac.
sheep reference sequences identified by gene symbol in the ncbi gene database.
ipd sequences identified according to ipd allele name in the ipd-mhc database .
aaa <dig> identified as an allele of locus  <dig>  by ballinghall, et al.
<dig>  vertical lines delineate functional domains.
a vertical box surrounds a motif in the transmembrane region important in classification of class i sequences as classical or nonclassical in cattle and sheep
in summary,  <dig> putative mhc class i-like genes were located in the chinese merino bacs .
three were found in bacs fj <dig> and fj985870; in each case the corresponding gene from these two bac sequences was identical.
this was not unexpected as these two bac sequences overlap.
bac sequences fj <dig>  fj <dig>  fj <dig> contained two, three and four putative mhc class i genes respectively.
the last gene in bac fj <dig> sequence is identical to the first gene identified in bac fj <dig> except for a  <dig> nucleotide indel, which most likely represents an allelic variation.
pairwise alignment of the two bacs indicates an overlap in the region containing the putative mhc class i gene.
predicted genes identified as fj985874_c1b and fj985874_c1b <dig> represent alternative transcripts of the same gene.
similar analysis of the sheep reference chromosome  <dig> annotated class i sequences is shown in table  <dig>  ten putative mhc class i loci have been identified.
based on the analysis of the protein sequences, one locus  appeared to be a classical class i gene and there were three nonclassical genes based upon the criteria described above.
six putative genes were shown to be missing an identifiable mhc class i c-terminal domain .
discussion
the work described in this study clarifies the physical map of the sheep mhc class i region and provides an updated gene annotation.
it is important for future studies that a reliable map and gene content of the mhc is available.
this study details the gene content and arrangement within the class i region obtained by sub-cloning and sequencing of chori bacs that contain class i sequences.
in addition, we used extensive manual, rather than automated, gene prediction analyses to determine the identity and location of mhc genes in the class i region within chinese merino bacs previously published by gao, et al.
<cit> .
these analyses provide a more detailed description of gene content within the sheep mhc class i region and allows a comparison between the currently available genome sequence data in the ncbi database to be performed.
this work also clarifies ambiguous information related to the mhc class i region that is available to date for public access; this will be of use to other researchers with an interest in the sheep mhc class i region and is essential for future targeted next generational re-sequencing of the mhc and fine-mapping the causal mutations for disease susceptibility.
re-assembly of the bac sequences used in the previously published chinese merino map together with annotation of the genes within was a necessary step to enable comparison with the chori bac based clones mapped in this study.
gao and colleagues reported the location of each gene within the chinese merino map relative to their reportedly contiguous map; however the complete sequence map was not published in a public database  <cit> .
instead, the individual bac sequences used for construction of the chinese merino map were published without identifying the overlapping regions between bac sequences  <cit> .
such overlaps would have enabled confirmation of the final contiguous architecture reported by this group  <cit> .
assembly of the chinese merino bac sequences with geneious resulted in five contigs rather than the single long contig reported by gao et al.
<cit> .
these multiple contigs indicate the probable presence of gaps in the map inferred by gao and colleagues.
analysis of overlapping regions between the chinese merino bac sequences using manual methods  suggested that there are actually six contigs in the sheep mhc map published by gao et al.
<cit> .
three gaps are present in the class i region while the remaining two gaps were identified in the class iia region.
the comparison of alignments generated by geneious and a manual method suggests that the latter produced a better contig tiling path.
this may be explained by the low sensitivity of geneious for a sequence containing a string of undefined nucleotides , possibly resulting in omission of the complete bac sequence from the assembly.
it seems that geneious is not always capable of discriminating between real contigs and false positives.
for instance, geneious failed to include four bac sequences  in the contig tiling path and indicated that there is an overlap between genbank: fj <dig> and genbank: fj <dig>  closer manual examination of the overlapping region between genbank: fj <dig> and genbank: fj <dig> revealed that there is a potential gap in this region, which encompasses a region between trim <dig> and basp <dig> .
the initial 764 bp in the 5′ end of bac sequence genbank: fj <dig> does not align with the 3′ overlapping region of genbank: fj <dig>  but aligns from bp  <dig> onwards.
blast analysis indicates that the 764 bp sequence at the 5′ end of bac genbank: fj <dig> does not show a contiguous alignment with the downstream bac sequence when examining the matching alignments with two bac sequences from cattle.
instead this region shows a match with a region elsewhere in the same cattle bac sequence.
self dotplot and blast analysis of genbank: fj <dig> indicates an alignment of bp 1– <dig> approximately  <dig>  downstream and bp 403– <dig> approximately  <dig>  bp downstream, with an intervening string of undefined nucleotides between bp 360– <dig>  dotplot alignment with the sheep reference chromosome  <dig> also indicates two alignments 5′ of the main diagonal separated by approximately 9000 bp.
the 764 bp sequence in the 5′ end of bac genbank: fj <dig> could possibly be due to a mistake introduced in the initial re-assembly of the bac sequence.
each of the  <dig> bac sequences present in the chinese merino map was sequenced through a dna shotgun sequencing method, which involves sub-cloning, sequencing and assembling randomised  <dig> – <dig>  kbp small fragments of dna to form a full-length bac sequence  <cit> .
if the 764 bp ambiguity is not present on bac genbank: fj <dig>  the rest of the bac would align with no gap with bac genbank: fj <dig>  despite the differences in the result produced by geneious and the manual method, both showed that the mhc map published by gao et al.
<cit>  is not contiguous and appears incomplete.
the bac sequence genbank: fj <dig> does not overlap with either genbank: fj <dig> or genbank: fj <dig> and the gap size in this region relative to the cattle reference sequence map is expected to be approximately  <dig> kbp, however dotplot alignment of the bacs with the sheep reference chromosome  <dig> indicates the gap is more likely to be approximately  <dig> kbp.
it is likely that there are several genes missing in this region due to the gap.
within the sheep reference chromosome  <dig> there are two mhc class i like genes and an envelope glycoprotein-like gene predicted in the region of the gap.
comparison of the class i region in other species suggests that the gap region may account for at least one peptide-presenting mhc class i gene  <cit> .
the gap between the ovar-mhci and trim <dig> loci is notable.
the other gap in the class i region is located between genbank: fj <dig> and genbank: fj <dig>  which is between eef1a <dig> and an adjacent ovar-mhci locus.
the size of this gap may be only a few thousand bp relative to the cattle map but the actual size is not known.
a direct comparison to the sheep reference genome in this region is not possible because there are indels observed in the alignments between both genbank: fj <dig> and genbank: fj <dig> with the sheep reference sequence, indicating an inserted region of nucleotides in the sheep genome analysed by gao et al.
<cit>  or a deleted  region in the sheep reference genome.
misassembly in this region of one or both genomes is another possibility.
the size of the gap between genbank: fj <dig> and genbank: fj <dig> and the genes at either end of the gap is not known because the class iia region is yet to be annotated.
the size of the gap in the class iia region between genbank: fj <dig> and genbank: fj <dig> is also unknown.
sequencing and re-assembly of chori bac sub-clones provided a low resolution physical map of two separate areas spanning approximately  <dig> kbp within the class i region.
identification of ten new genes in this study adds significantly to the incomplete annotation in the original chinese merino map.
these ten genes account for approximately 14 % of gene content within the class i region relative to the cattle reference map.
the genes identified in chori bac sequences are also present in the class i region of other mammals such as cattle, horse, human and pig .
the sheep mhc map derived from chinese merino published by gao et al.
<cit> , predicted  <dig> orthologous genes that have yet to be mapped to the cattle mhc.
in contrast, annotation of genes within the bac sequences reported above for the class i region showed that there was a high level of sequence identity between genes within the chinese merino bac sequences and known genes previously reported in sheep  and cattle .
amongst the  <dig> genes predicted in this study using the chinese merino bac sequences,  <dig>  were reported by gao et al.
<cit> .
conversely, and not taking into consideration novel and predicted genes, of the  <dig> genes reported by gao et al.
<cit>  in the class i region,  <dig> were not identified in this study.
the ten genes identified in the chori bac sequences were confirmed through re-analysis of the chinese merino bac sequences, indicating that the gene prediction methods  used by gao et al.
<cit>  were not entirely accurate.
our revised map of the mhc class i region annotates  <dig> genes, from gabbr <dig> to mccd <dig> in a telomeric to centromeric direction, and represents a consensus map taking into consideration our re-annotation of the chinese merino bacs, the sheep reference chromosome  <dig> and the cattle reference chromosome  <dig>  fifty two of the annotated genes have been identified in both the chinese merino bacs and the sheep reference chromosome  <dig>  nine genes have been identified in the chinese merino bacs, but not the sheep reference chromosome  <dig> and four genes are present in the sheep reference chromosome  <dig> but not identified in the chinese merino bacs.
the four genes not identified in the chinese merino bacs are all located in indel regions identified by dot plot sequence alignment .
a ribosome production factor  <dig> homolog -like gene  is annotated between trim <dig> and loc <dig>  on the sheep reference chromosome  <dig>  loc <dig> is annotated within an apparent insertion of 3934 bp in the reference chromosome  <dig> sequence that is not present in the corresponding chinese merino bac sequence, as indicated in an alignment with bac genbank: fj <dig>  the gap is located ~8000 bp from the 5′ end of the reverse complemented genbank: fj <dig> sequence .
rpf <dig> has not previously been identified in the mhc class i region in other closely related species.
the gene is annotated on chromosome  <dig>   <dig> and  <dig> in cattle, house mouse and pig, respectively.
rpf <dig> has also been annotated within the sheep reference genome on chromosome  <dig>  the structure of the two genes differs markedly; however, the translated protein sequences are identical.
the mrnas differ by only one bp in their corresponding sequences; however, the gene located on chromosome  <dig> has longer 5′ and 3′ untranslated regions annotated.
the rpf <dig> on chromosome  <dig> in sheep is annotated on the reverse complement strand and has ten exons; the exon size and distribution are the same as that annotated for the gene in the cattle, mouse, human and pig genomes.
genes 5′ on the same strand are general transcription factor iiic, polypeptide  <dig>  and adenosylmethionine decarboxylase  <dig> .
genes 3′ on the same strand are solute carrier family  <dig>  member  <dig>   and kiaa <dig>  this is the same gene order seen in the cattle, mouse and human genomes.
in the pig genome, gtf3c <dig> and amdi are 5′ to rpf <dig>  but slc16a <dig> and kiaa <dig> have not been annotated 3′ to the gene.
loc <dig> is annotated on the forward strand with two exons.
based on an alignment of mrna and genomic sequence from the two genes, the first exon of loc <dig> appears to be a concatenation of the first nine exons of rpf <dig>  whereas the intron and second exon correspond to the ninth intron and tenth exon of rpf <dig> .
due to this altered gene structure, we suggest that loc <dig> may be the result of a gene duplication event involving retrotransposition.
this may represent a breed specific gene duplication, which will require further investigation to clarify.
three genes have been identified in the sheep reference chromosome  <dig> within the apparent gap region between chinese merino bacs genbank: fj <dig> and genbank: fj <dig>  these include an envelope glycoprotein-like gene  and two mhc class i-like genes.
one of the nine genes identified in the chinese merino bacs but not annotated on sheep reference chromosome  <dig> - c20h6orf <dig> - was found in a region of high sequence similarity between bac genbank: fj <dig> and the sheep reference sequence.
analysis of this region on sheep chromosome  <dig> using fgenesh+ with the predicted c20h6orf <dig> as homolog indicates that the gene is present, and lies between genes zfp <dig> and znrd <dig> on the forward strand .
the remaining eight genes in our map that are not annotated in the sheep reference sequence occur in sequence that is present in genbank: fj <dig> but not in the sheep reference chromosome  <dig>  genes found in this region of genbank: fj <dig> include one dpcr1-like gene, three mucin-like genes, three mhc class i-like genes and eukaryotic translation elongation factor  <dig> alpha  <dig> .
of these, only eef1a <dig> has not been annotated in the mhc class i region in other closely related species.
a search of the ncbi databases indicates that eef1a <dig> partial mrnas have been isolated in sheep, but the gene has not been mapped to a chromosome to date.
the gene is annotated on chromosome  <dig> in both cattle and house mouse, chromosome  <dig> in rats and chromosome  <dig> in humans but outside of the mhc region.
blast analysis of the eef1a <dig> gene sequence from genbank: fj <dig> against the sheep genome on the ucsc genome browser revealed near continous alignments with  <dig>  % identity on chromosomes  <dig>   <dig> and  <dig>  eef1a <dig> refseq mrnas from a number of species map to the same regions on these chromosomes .
more broken alignments matching exon regions from eef1a <dig> annotation tracks from other species were observed on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig> .
it was noted that eef1a <dig> refseq mrnas from cattle, mouse, rat and humans all mapped to the alignment on sheep chromosome 8; eight exons appear to be present in this region in these species.
this would appear to be the most likely location for the functional gene in sheep.
an alignment on chromosome  <dig> in a region matching annotation tracks from eef1a <dig> genes in other species was not observed.
sequencing of additional sheep genomes from various breeds is required to determine the accuracy of the current assemblies.
the revised sequence annotation shows that the general structure and gene content in the sheep mhc class i region is more similar to that of other mammals than previously suggested.
there is however, a slight difference in the actual gene arrangement .
the class i genes involved in peptide presentation in some mammalian species such as chimpanzee  <cit> , human  <cit> , rhesus macaque  <cit>  and horse  <cit> , are often clustered within three distinct locations designated as the alpha , beta  and kappa  blocks.
however, the clustering of peptide-presenting mhc class i genes in sheep does not fit entirely into the alpha, beta and kappa block framework.
in a previous study of the sheep mhc, the presence of an additional novel block located between gtf2h <dig> and cdsn was suggested  <cit> .
analysis of gene organisation within the class i region in this study confirms the presence of such a novel block.
in this study, there are at least two definite mhc class i and two mhc class i-like genes between gtf2h <dig> and cdsn.
the exact number of peptide-presenting mhc class i genes is not known due to the presence of a gap in this block.
in addition, this study reveals that there is no evidence for the presence of peptide-presenting mhc class i genes between mog and ppp1r <dig>  as reported in other organisms.
this finding is also in agreement with the previously reported sheep mhc study by liu et al.
<cit> .
the blocks of peptide-presenting genes are separated by numerous other class i genes with immune and non-immune related functions.
the organisation of other sheep genes in the class i region is similar to the closely related cattle mhc.
the organisation of mhc class i peptide-presenting genes in distinct blocks, which are interspersed between other genes located within the class i region, is most likely due to segment or tandem block duplication .
the framework hypothesis suggests that the mhc class i region is a “conserved ordered segment” that represents a dense region of genes with essential functions, whose alterations are deleterious  <cit> .
this study has assembled a single haplotype and shown that it is more similar to the reference cattle sequence.
however, there is considerable diversity among mhc haplotypes in other species  <cit> .
therefore additional haplotypes will need to be sequenced and assembled to provide a true picture of mhc diversity and structural evolution.
CONCLUSIONS
the analysis performed in this study updates the existing sheep mhc map and enhances annotation of the genes present in the mhc class i region.
this study also provides useful knowledge to complement the publicly available sequence information on ncbi regarding the chinese merino bacs, so that the information can be easily interpreted for future studies.
in particular, the telomeric to centromeric orientation of bacs used by gao and colleagues  <cit>  has been resolved, overlapping sequence regions identified, gaps in the sheep mhc class i map mapped and the putative position of loci within each bac encompassing mhc class i region detailed.
the updated sequence map provides a reference for future studies and will simplify the use of next generation sequences and snp chips for multiple mhc studies including determination of the gene/genes responsible for resistance to infectious and parasitic diseases.

 the major histocompatibility complex is a highly polymorphic gene-dense region, spanning an area of approximately mbp in the human genome. since its first discovery in mice, the mhc has been intensely studied in many species due to its association with immune related functions. in domestic animals, this has included the evolutionary relationship of the mhc in different species, the genetic diversity of animals subjected to domestication, its role in the immune response to parasites, its association with infectious and parasitic diseases and the development of vaccines. in sheep, characterisation of the mhc has been based predominantly on analysis of orthologous loci from the respective human and cattle mhcs. early studies have assumed that the basic structure of the sheep mhc was similar to that of other mammals, consisting of the telomeric class i, central class iii and centromeric class ii. a later study of mhc structure in chinese merino sheep reported that, like the cattle mhc, the sheep class ii region is sub-divided into two distinct iia and iib regions and is most likely derived from a common ancestral partial chromosomal inversion. in recent years, low resolution physical maps of sheep mhc class ii and iii regions have been constructed using a combination of sub-cloning and partial sequencing of bacterial artificial chromosome clones known to contain mhc sequences. in addition, a panel of single nucleotide polymorphisms spanning the sheep mhc class ii and iii regions have also been developed. these have provided a framework for the identification and analysis of haplotypes. however, a relative paucity in the knowledge regarding the sheep mhc still exists, in particular the class i region in terms of its gene content, structural organisation and genetic variation. research into the mhc of sheep is currently limited in comparison with other domestic animals, especially cattle and swine. for instance, there is a lack of understanding of its haplotype structure. although dinucleotide microsatellite loci such as ohcci have been widely used in association studies in sheep there is still a lack of understanding and characterisation of haplotypes in this important genetic region. the better understood human mhc map indicates that the class i region is rich in pseudogenes, duplicated genes and genes showing copy number variation. recently, a physical map of sheep mhc derived from a chinese merino sheep has been published by gao and colleagues. annotation of this chinese merino physical map led to the identification of a total of genes, among which of the genes were apparently not previously identified in sheep and described as unique to sheep. from their study, genes were reported in the mhc class i region. twenty two predicted genes had either high sequence similarity to other known gene sequences, or contained a predicted open reading frame having low sequence similarity with sequences from other species. three novel sheep-specific genes were also reported with no apparent sequence homology to any known mammalian sequences. the objective of this study was to reanalyse existing and new information regarding the sheep class i region and produce a comprehensive and updated version of the sheep mhc class i map. this was achieved through the mapping of genes sequenced from chori bacs and comparing the result with the cattle reference genome, the previously published chinese merino sheep map and the very recently available sheep reference genome. in this study, we sub-cloned chori bacs known to contain class i sequences and re-assembled the sequences in order to annotate genes present within the sheep mhc class i region. in addition, we reanalysed, re-assembled and re-annotated the chinese merino bacs published by gao and colleagues. annotation of the chori bac and chinese merino bac sequences was then used to generate a revised contig map. comparison with the recent annotation of chromosome from the sheep genome reference sequence was then used to further inform this map.  re-analysis of chinese merino mhc contig map initial analysis with geneious software produced distinct contigs instead of the single contig reported by gao et al.. the geneious assembly also revealed that some of the bac sequences were not in the correct orientation. four reads were not incorporated into the assembly; genbank: fj genbank: fj genbank: fj and genbank: fj comparison with the published chinese merino map in the 5′ to 3′ direction: contig assembles four reads - genbank: fj genbank: fj genbank: fj and genbank: fj however, an overlap between genbank: fj and either genbank: fj or genbank: fj was not detected. contig assembles reads - genbank: fj genbank: fj and genbank: fj genbank: fj was not included in the assembly with genbank: fj and genbank: fj no overlap between genbank: fj and genbank: fj was identified. contig assembles genbank: fj and genbank: fj analysis using geneious did not identify any overlap between genbank: fj and genbank: fj contig assembles genbank: fj genbank: fj genbank: fj genbank: fj and genbank: fj no overlap between genbank: fj and genbank: fj genbank: fj and genbank: fj genbank: fj and genbank: fj was identified. contig assembles genbank: fj and genbank: fj no overlap between genbank: fj and genbank: fj was detected by the geneious algorithm. contigs and are in the opposite orientation when compared to the published chinese merino map. further analyses using the ncbi blast option to align two sequences and the chaos/dialign software detected an overlapping region between the chinese merino bacs that was not reported by geneious. in addition, analyses of bacs genbank: fj genbank: fj and genbank: fj that had been omitted in the initial assembly using the geneious analysis showed overlapping sequence alignment with other chinese merino bacs; genbank: fj overlaps with genbank: fj and genbank: fj genbank: fj overlaps with genbank: fj and genbank: fj overlaps with genbank: fj figures s and s in additional file provides the results of these analyses as a series of pairwise dotplots for the bacs along with an interpretive comment. the regions of overlaps between the bac sequences are detailed in table  table geneious assembly of bac clones published by gao et al. indicates assembly of sequence in the forward direction. indicates assembly of the reverse complement sequence fig. comparison of the old and new tiling path of chinese merino bacs. a original tiling path of the chinese merino mhc map. b new tiling path of chinese merino bac sequences proposed in this study. a vertical red line between bac contigs indicates a gap region precede bac id numerals with ‘fj9858’ to determine ncbi genbank accession. appended ‘rc’ indicates the bac was reverse complemented before alignment. ‘-‘ indicates there was no overlap identified. afirst 765 bp of genbank: fj985864rc do not overlap with genbank: fj985854rc, this is most likely due to contig misassembly in this region a pictorial view of our revised tiling map of the chinese merino bac sequences is shown in fig. 1b. based on this analysis, there are gaps between bacs genbank: fj and genbank: fj genbank: fj and genbank: fj genbank: fj and genbank: fj and genbank: fj and genbank: fj blast analysis of the first nucleotides of the reverse complemented genbank: fj indicated that nucleotides 1– aligned perfectly, beginning nucleotides downstream in the same bac, and nucleotides 403– aligned perfectly, beginning nucleotides downstream also in the same bac, with a gap of approximately 8900 bp between the two alignments. the sequence between bp and is a string of undefined nucleotides, hence no alignment was obtained in this region. we conclude that these nucleotides have not been assembled correctly. subsequent dot plot alignment of the chinese merino bacs with the mhc class i region of the reference sheep chromosome is presented in the "comparative analysis of chori bac contigs map with mhc class i maps" results section below. analysis of sheep mhc class i gene content initial analysis of chori bac sub-clone sequences identified additional loci in the sheep mhc class i region that were not recognised in the chinese merino mhc map but were found in both the cattle and sheep reference maps. comparison of mhc class i gene content between the cattle reference map, sheep reference map and the published chinese merino map is given in fig.  the additional loci identified through chori bac sub-clones include ribonuclease p 21-like isoform, guanine nucleotide-binding protein-like, atp-binding cassette sub-family f member, chromosome open reading frame ortholog, deah box polypeptide, nuclear envelope membrane protein or nurim, general transcription factor ii h subunit, surfactant associated protein g, transcription factor and pou class homeobox. the relative locations of these additional loci were deduced using blast analysis. figure  shows the positions of the additional loci identified through sequencing of these chori bac subclones that had not been identified in the previously published chinese merino mhc map.fig. comparison of cattle, sheep and chinese merino mhc class i maps. loci highlighted in red indicate additional loci identified through sequencing of chori bac clones. additional file 3: table s and s provides the detailing of gene symbol, gene name and gene description in the class i region of the sheep and cattle reference genomes at ncbi, respectively fig. loci identified through chori bac sub-cloning and their relative position within the chinese merino map. identity of these loci was confirmed by sequence homology using ncbi blast. loci highlighted in red indicate loci previously not present on the map proposed by gao et al. our re-analysis of the mhc ci chinese merino map indicates that the majority of genes identified appear to have the same gene arrangement with that previously observed in cattle, along with a high level of nucleotide similarity to their cattle orthologues. the name and location of genes identified in the chinese merino bac sequences are all tabulated in table  identical gene predictions between adjacent bac sequences corroborates with overlapping regions identified through dotplot and pairwise sequence alignment analysis. likewise, non-overlapping regions show no similarity in gene content.table summary of gene content on each of the chinese merino bac sequences †first exon not in bac. ‡last exon not in bac. *alignment to known homolog shows high percent identity with no evidence of wrong or missing exons. # not included in fig.  shaded regions indicate genes identified in overlapping regions between clones. ## identified from additional file 3: table s. gene ids not matching our gene symbols were surmised based on gene description and/or alternate gene symbols provided in the ncbi gene database along with relative gene position compared to our analysis the majority of gene predictions in the overlapping regions of the chinese merino bacs were identical, however there were a few exceptions. in the overlap region between genbank: fj985852rc and genbank: fj985875rc, there were differences observed in the vars and dpcr1-like predictions. exon of the vars gene prediction is longer in genbank: fj985852rc than in genbank: fj985875rc due to a single bp deletion in the exon region in genbank: fj985875rc that results in a change in the sequence reading frame and hence a premature stop to the exon. alignment with reference sequences indicates that the vars gene prediction from genbank: fj985852rc is correct. exon of the dpcr1-like gene prediction is shorter in genbank: fj985852rc than in genbank: fj985875rc due to a significant insertion of ~370 bp in genbank: fj985875rc compared to genbank: fj985852rc. alignment with reference sequences indicates that the genbank: fj985852rc prediction is more similar and hence more likely to be correct. in the overlapping region between genbank: fj985859rc and genbank: fj there is a single amino acid length difference noted in the genbank: fj mhc class i-like predicted peptide compared to the genbank: fj985859rc peptide due to a 3 bp deletion in the region of the first exon. an alignment with reference sequences failed to indicate which sequence is correct since the difference occurs in an area of low complexity repeats within the signal peptide. a comparison between genes reported in the chinese merino map by gao et al. and our analysis showed that twenty-nine of the sixty-eight genes identified in this study were not previously annotated. the ten genes identified through sub-cloning and sequencing of the chori bac sequences were annotated in the re-analysis of chinese merino bac sequences. table  includes a list of gene identifications reported by gao et al. for the thirty-nine genes identified in both studies. sequence comparison of chinese merino bacs with mhc class i region of reference sheep chromosome 20 chinese merino bacs covering the mhc class i region were aligned via dotplot analysis with a portion of the sheep chromosome from the mhc class i region downloaded from ncbi. the results of this dotplot analysis are presented in additional file 2: figure s bacs genbank: fj genbank: fj and genbank: fj align well with the sheep reference genome sequence in this region, there are no obvious large indels and no significant interspersed repetitive regions. a large indel is observed in the alignment between genbank: fj and genbank: nc_ due to the presence of ~  bp in the sheep reference sequence not found in the genbank: fj bac. the insertion in the sheep genome is approximately from position to in genbank: nc_ within this region there are two annotated genes - described as an ubiquitin d-like gene and c19orf homolog – that were not located in the genbank: fj bac. smaller indels and gaps in the alignment diagonal appear to correspond to runs of undefined nucleotides within the sheep reference sequence. shorter runs of undefined nucleotides are also present in the bac sequence. bacs genbank: fj and genbank: fj overlap over the majority of their sequence, and this is evident when comparing their alignments to genbank: nc_ there are several indels, and the dotplot shows parallel lines indicating large interspersed repeating regions. these bacs contain several mhc class i histocompatibility antigen-like loci, which share enough sequence similarity to create an interspersed pattern of diagonals. within the corresponding region on genbank: nc_ larger gaps in the alignment are evidence of possible mis-assembly in either the bac or sheep reference chromosome or could also represent breed specific differences in the region. the alignment of fj with genbank: nc_ also reveals that the first nucleotides align in two different segments 5′ to the main alignment diagonal, with a gap of approximately 9000 bp between, confirming the observation from blast analysis of this region that these first nucleotides are mis-assembled. the alignment of genbank: fj with genbank: nc_ reveals a segmental inversion covering an area of approximately  bp. within this region are four genes that have been predicted in reverse orientation within the two sequences – abcf prr gnl and rpp. the inverted sequence may be a result of mis-assembly in this region in either the sheep reference chromosome or the chinese merino bac; alternatively, it may represent a genuine breed specific difference. in cattle and in the chinese merino bac sequence, the genes are annotated in the order rpp gnl prr abcf this order was chosen for our proposed map as it is consistent with the order annotated in other species including bos taurus, sus scrofa and mus musculus.fig. the new sheep mhc ci map proposed based on findings in this study. position of each locus is based on the comparative analysis between chori bacs, chinese merino bacs, cattle reference sequence for chromosome and sheep reference sequence for chromosome the dotplot analysis confirmed a gap between chinese merino bac sequences genbank: fj and genbank: fj of  bp, lying in the region from 27555826– in genbank: nc_ annotated within this region in the sheep reference genome are two mhc class i histocompatibility antigen–like genes and an envelope glycoprotein-like gene. the reverse complement of genbank: fj aligns well with sheep reference chromosome for ~  bp. there is no significant alignment from ~  bp to the end of the sequence - ~  bp. genbank: nc_ has a 5000 bp run of undefined nucleotides spanning the region from ~ to  bp in genbank: fj and to in genbank: nc_ the remaining sequence in genbank: fj represents inserted sequence not currently present in the sheep reference genome. this is confirmed in the dotplot alignment with the reverse complement of genbank: fj which spans a region in genbank: nc_ from bp to five genes are annotated in the region from bp to the end of genbank: fj including two mhc class i-like genes, two mucin-like genes and eukaryotic translation elongation factor alpha. mhc class i and mucin-like genes would be expected in the mhc class i region, however eef1a has not been annotated in the mhc class i region in genomes from other closely related species, including cow, pig, mouse and human. the first nucleotides of the reverse complement of genbank: fj do not align with genbank: nc_ 5000 bp may be accounted for by undefined nucleotides in genbank: nc_ however the remainder represents an inserted region of ~  bp not currently present in the sheep reference genome. within this region in genbank: fj one mhc class i histocompatibility antigen gene has been predicted. several interspersed repeat regions are indicated in the alignment bp of the reverse complement of genbank: fj in a ~9000 bp span between bp and. within this region an mhc class i-like gene has been predicted. comparison of the dotplot alignments between the reverse complement of genbank: fj and genbank: nc_ and between genbank: fj and genbank: nc_ indicates an overlap between genbank: fj and genbank: fj of  bp. the dotplot alignment of genbank: fj with genbank: nc_ shows a mostly unbroken alignment diagonal. there are two noticeable indels of approximately 1500 bp that are the result of undefined nucleotides in the sheep reference genome sequence. a number of interspersed repeats are indicated in the first ~  bp of genbank: fj within this region there are four predicted mhc class i – like genes. analysis of the dotplot alignments confirms the overlaps of the chinese merino bac sequences as indicated in fig. 1b. these are summarised in table  dotplot analysis provides no evidence of overlap between genbank: fj and genbank: fj or between genbank: fj and genbank: fj we were unable to estimate the size of the gap between genbank: fj and genbank: fj table overlaps of bacs published by gao et al. with sheep reference genome sequence genbank: nc_019477 comparative analysis of chori bac contigs map with mhc class i maps comparative analysis of the assembled, sub-cloned chori bac sequences revealed the coverage of each bac and its position with respect to the cattle reference sequence for chromosome, chinese merino bac sequences and the sheep reference sequence for chromosome.table blast analysis of chori bac end sequences available in genbank. the sequences were aligned with cattle and sheep reference sequences from chromosome and respectively, and with the chinese merino bac sequences the telomeric end sequence of chori 243-390h aligns between two mhc class i-like loci in both the cattle and sheep reference sequences, but shows no significant similarity to any of the chinese merino bac sequences. it appears that this chori bac end sequence lies within a gap region between chinese merino bac genbank: fj and genbank: fj the centromeric end sequence of chori 243–390h aligns within the predicted tubb locus in both cattle and sheep reference sequences and in chinese merino bac genbank: fj the chori 243–454e bac appears to span a region beginning between loci ddr and ier and ending within a trim 39-like locus in the cattle and sheep reference sequences and in the chinese merino bacs. chori 243–390h overlaps with chori 243–454e and was located in the middle of mhc class i region whereas, chori 243–269 m was located further away towards the centromeric end. both the telomeric and centromeric ends of chori 243–269 m are located between mhc class i-like genes with respect to the cattle and sheep reference sequences and in the chinese merino bacs. using this information fig.  shows a revised sheep mhc class i map based upon our reanalysis. comparative analysis of mhc class i histocompatibility antigen genes of particular interest was the determination of the number and distribution of the class i histocompatibility antigen loci. in cattle, there is evidence for at least six classical and four nonclassical discrete mhc class i loci. in sheep, evidence exists for at least six discrete class i loci, with at least two being classical. the number and expression levels of class i loci have been demonstrated to be haplotype specific in the scottish blackface sheep breed, so loci identified in the chinese merino bacs are expected to differ from the sheep reference genome. correct assembly of these highly repetitive gene sequences is bioinformatically a difficult task, compounded by variant numbers of loci in different haplotypes. mhc class i genes were predicted in the chinese merino bacs as detailed in the "re-analysis of chinese merino sheep mhc map" methods section and are included table  predicted mhc class i protein sequences from both the chinese merino bacs and the sheep reference sequence for chromosome were aligned with selected reference sequences taken from the ipd-mhc database. in addition, functional domain analysis was carried out for each sequence. tables  and list the class i genes predicted in the chinese merino bac sequences and corresponding sheep reference chromosome sequences, respectively, and include details on the location of signal peptides and functional domains identified. the mhc class i genes with identifiable transmembrane and cytoplasmic domains are denoted as classical or nonclassical according to sequence criteria previously used in cattle and sheep: namely the presence of a vpi, ipi or vlik motif in the transmembrane domain and/or a truncated cytoplasmic domain. a sequence alignment of the predicted class i proteins from the chinese merino bac sequences in comparison with a selection of reference sequences representing different loci can be seen in, a similar alignment with the predicted class i proteins from the sheep reference chromosome sequences can be seen in. sequences considered to most likely represent functional class i genes are aligned in fig.  the location of functional domains are also indicated for each of these alignments.table mhc class 1-like genes predicted from genomic bac sequences published by gao et al. fj985874c1bc fj985874c1b2c s: strand. #: locus number in telomeric to centromeric direction. #ex: number of exons identified. aa len: length of predicted amino acid sequence. sp: signal peptide. α1: alpha domain. α2: alpha domain. α3: alpha domain. tm: transmembrane domain. c-term: c-terminus end. homologue indicates sequence used as homologous protein sequence with fgenesh+ aterminal exon not predicted. bweak match. calternative transcript predictions s: strand. #: locus number in telomeric to centromeric direction. #ex: number of cds exons identified. aa len: length of predicted amino acid sequence. sp: signal peptide. α1: alpha domain. α2: alpha domain. α3: alpha domain. tm: transmembrane domain. c-term: c-terminus end. type: ia – classical, ib – nonclassical. protein acc: accession of protein sequence in ncbi database atranscripts differ only at the c-terminal end fig. shows an alignment of mhc class i amino acid sequences predicted from selected bac sequences published by gao et al., in comparison with selected class i loci from the sheep reference chromosome and six class i reference sequences from the ipd database plus an alternative transcript sequence aaa nomenclature for the predicted genes from bac sequences derived as follows: ncbi accession number, plus ‘rc’ if the sequence was reverse complemented before analysis, plus the class i gene identifier within the bac. sheep reference sequences identified by gene symbol in the ncbi gene database. ipd sequences identified according to ipd allele name in the ipd-mhc database. aaa identified as an allele of locus by ballinghall, et al. vertical lines delineate functional domains. a vertical box surrounds a motif in the transmembrane region important in classification of class i sequences as classical or nonclassical in cattle and sheep in summary, putative mhc class i-like genes were located in the chinese merino bacs. three were found in bacs fj and fj985870; in each case the corresponding gene from these two bac sequences was identical. this was not unexpected as these two bac sequences overlap. bac sequences fj fj fj contained two, three and four putative mhc class i genes respectively. the last gene in bac fj sequence is identical to the first gene identified in bac fj except for a nucleotide indel, which most likely represents an allelic variation. pairwise alignment of the two bacs indicates an overlap in the region containing the putative mhc class i gene. predicted genes identified as fj985874_c1b and fj985874_c1b represent alternative transcripts of the same gene. similar analysis of the sheep reference chromosome annotated class i sequences is shown in table  ten putative mhc class i loci have been identified. based on the analysis of the protein sequences, one locus appeared to be a classical class i gene and there were three nonclassical genes based upon the criteria described above. six putative genes were shown to be missing an identifiable mhc class i c-terminal domain. discussion the work described in this study clarifies the physical map of the sheep mhc class i region and provides an updated gene annotation. it is important for future studies that a reliable map and gene content of the mhc is available. this study details the gene content and arrangement within the class i region obtained by sub-cloning and sequencing of chori bacs that contain class i sequences. in addition, we used extensive manual, rather than automated, gene prediction analyses to determine the identity and location of mhc genes in the class i region within chinese merino bacs previously published by gao, et al.. these analyses provide a more detailed description of gene content within the sheep mhc class i region and allows a comparison between the currently available genome sequence data in the ncbi database to be performed. this work also clarifies ambiguous information related to the mhc class i region that is available to date for public access; this will be of use to other researchers with an interest in the sheep mhc class i region and is essential for future targeted next generational re-sequencing of the mhc and fine-mapping the causal mutations for disease susceptibility. re-assembly of the bac sequences used in the previously published chinese merino map together with annotation of the genes within was a necessary step to enable comparison with the chori bac based clones mapped in this study. gao and colleagues reported the location of each gene within the chinese merino map relative to their reportedly contiguous map; however the complete sequence map was not published in a public database. instead, the individual bac sequences used for construction of the chinese merino map were published without identifying the overlapping regions between bac sequences. such overlaps would have enabled confirmation of the final contiguous architecture reported by this group. assembly of the chinese merino bac sequences with geneious resulted in five contigs rather than the single long contig reported by gao et al.. these multiple contigs indicate the probable presence of gaps in the map inferred by gao and colleagues. analysis of overlapping regions between the chinese merino bac sequences using manual methods suggested that there are actually six contigs in the sheep mhc map published by gao et al.. three gaps are present in the class i region while the remaining two gaps were identified in the class iia region. the comparison of alignments generated by geneious and a manual method suggests that the latter produced a better contig tiling path. this may be explained by the low sensitivity of geneious for a sequence containing a string of undefined nucleotides, possibly resulting in omission of the complete bac sequence from the assembly. it seems that geneious is not always capable of discriminating between real contigs and false positives. for instance, geneious failed to include four bac sequences in the contig tiling path and indicated that there is an overlap between genbank: fj and genbank: fj closer manual examination of the overlapping region between genbank: fj and genbank: fj revealed that there is a potential gap in this region, which encompasses a region between trim and basp. the initial 764 bp in the 5′ end of bac sequence genbank: fj does not align with the 3′ overlapping region of genbank: fj but aligns from bp onwards. blast analysis indicates that the 764 bp sequence at the 5′ end of bac genbank: fj does not show a contiguous alignment with the downstream bac sequence when examining the matching alignments with two bac sequences from cattle. instead this region shows a match with a region elsewhere in the same cattle bac sequence. self dotplot and blast analysis of genbank: fj indicates an alignment of bp 1– approximately downstream and bp 403– approximately  bp downstream, with an intervening string of undefined nucleotides between bp 360– dotplot alignment with the sheep reference chromosome also indicates two alignments 5′ of the main diagonal separated by approximately 9000 bp. the 764 bp sequence in the 5′ end of bac genbank: fj could possibly be due to a mistake introduced in the initial re-assembly of the bac sequence. each of the bac sequences present in the chinese merino map was sequenced through a dna shotgun sequencing method, which involves sub-cloning, sequencing and assembling randomised – kbp small fragments of dna to form a full-length bac sequence. if the 764 bp ambiguity is not present on bac genbank: fj the rest of the bac would align with no gap with bac genbank: fj despite the differences in the result produced by geneious and the manual method, both showed that the mhc map published by gao et al. is not contiguous and appears incomplete. the bac sequence genbank: fj does not overlap with either genbank: fj or genbank: fj and the gap size in this region relative to the cattle reference sequence map is expected to be approximately kbp, however dotplot alignment of the bacs with the sheep reference chromosome indicates the gap is more likely to be approximately kbp. it is likely that there are several genes missing in this region due to the gap. within the sheep reference chromosome there are two mhc class i like genes and an envelope glycoprotein-like gene predicted in the region of the gap. comparison of the class i region in other species suggests that the gap region may account for at least one peptide-presenting mhc class i gene. the gap between the ovar-mhci and trim loci is notable. the other gap in the class i region is located between genbank: fj and genbank: fj which is between eef1a and an adjacent ovar-mhci locus. the size of this gap may be only a few thousand bp relative to the cattle map but the actual size is not known. a direct comparison to the sheep reference genome in this region is not possible because there are indels observed in the alignments between both genbank: fj and genbank: fj with the sheep reference sequence, indicating an inserted region of nucleotides in the sheep genome analysed by gao et al. or a deleted region in the sheep reference genome. misassembly in this region of one or both genomes is another possibility. the size of the gap between genbank: fj and genbank: fj and the genes at either end of the gap is not known because the class iia region is yet to be annotated. the size of the gap in the class iia region between genbank: fj and genbank: fj is also unknown. sequencing and re-assembly of chori bac sub-clones provided a low resolution physical map of two separate areas spanning approximately kbp within the class i region. identification of ten new genes in this study adds significantly to the incomplete annotation in the original chinese merino map. these ten genes account for approximately 14 % of gene content within the class i region relative to the cattle reference map. the genes identified in chori bac sequences are also present in the class i region of other mammals such as cattle, horse, human and pig. the sheep mhc map derived from chinese merino published by gao et al., predicted orthologous genes that have yet to be mapped to the cattle mhc. in contrast, annotation of genes within the bac sequences reported above for the class i region showed that there was a high level of sequence identity between genes within the chinese merino bac sequences and known genes previously reported in sheep and cattle. amongst the genes predicted in this study using the chinese merino bac sequences, were reported by gao et al.. conversely, and not taking into consideration novel and predicted genes, of the genes reported by gao et al. in the class i region, were not identified in this study. the ten genes identified in the chori bac sequences were confirmed through re-analysis of the chinese merino bac sequences, indicating that the gene prediction methods used by gao et al. were not entirely accurate. our revised map of the mhc class i region annotates genes, from gabbr to mccd in a telomeric to centromeric direction, and represents a consensus map taking into consideration our re-annotation of the chinese merino bacs, the sheep reference chromosome and the cattle reference chromosome fifty two of the annotated genes have been identified in both the chinese merino bacs and the sheep reference chromosome nine genes have been identified in the chinese merino bacs, but not the sheep reference chromosome and four genes are present in the sheep reference chromosome but not identified in the chinese merino bacs. the four genes not identified in the chinese merino bacs are all located in indel regions identified by dot plot sequence alignment. a ribosome production factor homolog -like gene is annotated between trim and loc on the sheep reference chromosome loc is annotated within an apparent insertion of 3934 bp in the reference chromosome sequence that is not present in the corresponding chinese merino bac sequence, as indicated in an alignment with bac genbank: fj the gap is located ~8000 bp from the 5′ end of the reverse complemented genbank: fj sequence. rpf has not previously been identified in the mhc class i region in other closely related species. the gene is annotated on chromosome and in cattle, house mouse and pig, respectively. rpf has also been annotated within the sheep reference genome on chromosome the structure of the two genes differs markedly; however, the translated protein sequences are identical. the mrnas differ by only one bp in their corresponding sequences; however, the gene located on chromosome has longer 5′ and 3′ untranslated regions annotated. the rpf on chromosome in sheep is annotated on the reverse complement strand and has ten exons; the exon size and distribution are the same as that annotated for the gene in the cattle, mouse, human and pig genomes. genes 5′ on the same strand are general transcription factor iiic, polypeptide and adenosylmethionine decarboxylase. genes 3′ on the same strand are solute carrier family member and kiaa this is the same gene order seen in the cattle, mouse and human genomes. in the pig genome, gtf3c and amdi are 5′ to rpf but slc16a and kiaa have not been annotated 3′ to the gene. loc is annotated on the forward strand with two exons. based on an alignment of mrna and genomic sequence from the two genes, the first exon of loc appears to be a concatenation of the first nine exons of rpf whereas the intron and second exon correspond to the ninth intron and tenth exon of rpf. due to this altered gene structure, we suggest that loc may be the result of a gene duplication event involving retrotransposition. this may represent a breed specific gene duplication, which will require further investigation to clarify. three genes have been identified in the sheep reference chromosome within the apparent gap region between chinese merino bacs genbank: fj and genbank: fj these include an envelope glycoprotein-like gene and two mhc class i-like genes. one of the nine genes identified in the chinese merino bacs but not annotated on sheep reference chromosome - c20h6orf - was found in a region of high sequence similarity between bac genbank: fj and the sheep reference sequence. analysis of this region on sheep chromosome using fgenesh+ with the predicted c20h6orf as homolog indicates that the gene is present, and lies between genes zfp and znrd on the forward strand. the remaining eight genes in our map that are not annotated in the sheep reference sequence occur in sequence that is present in genbank: fj but not in the sheep reference chromosome genes found in this region of genbank: fj include one dpcr1-like gene, three mucin-like genes, three mhc class i-like genes and eukaryotic translation elongation factor alpha. of these, only eef1a has not been annotated in the mhc class i region in other closely related species. a search of the ncbi databases indicates that eef1a partial mrnas have been isolated in sheep, but the gene has not been mapped to a chromosome to date. the gene is annotated on chromosome in both cattle and house mouse, chromosome in rats and chromosome in humans but outside of the mhc region. blast analysis of the eef1a gene sequence from genbank: fj against the sheep genome on the ucsc genome browser revealed near continous alignments with  % identity on chromosomes and eef1a refseq mrnas from a number of species map to the same regions on these chromosomes. more broken alignments matching exon regions from eef1a annotation tracks from other species were observed on chromosomes and. it was noted that eef1a refseq mrnas from cattle, mouse, rat and humans all mapped to the alignment on sheep chromosome 8; eight exons appear to be present in this region in these species. this would appear to be the most likely location for the functional gene in sheep. an alignment on chromosome in a region matching annotation tracks from eef1a genes in other species was not observed. sequencing of additional sheep genomes from various breeds is required to determine the accuracy of the current assemblies. the revised sequence annotation shows that the general structure and gene content in the sheep mhc class i region is more similar to that of other mammals than previously suggested. there is however, a slight difference in the actual gene arrangement. the class i genes involved in peptide presentation in some mammalian species such as chimpanzee, human, rhesus macaque and horse, are often clustered within three distinct locations designated as the alpha, beta and kappa blocks. however, the clustering of peptide-presenting mhc class i genes in sheep does not fit entirely into the alpha, beta and kappa block framework. in a previous study of the sheep mhc, the presence of an additional novel block located between gtf2h and cdsn was suggested. analysis of gene organisation within the class i region in this study confirms the presence of such a novel block. in this study, there are at least two definite mhc class i and two mhc class i-like genes between gtf2h and cdsn. the exact number of peptide-presenting mhc class i genes is not known due to the presence of a gap in this block. in addition, this study reveals that there is no evidence for the presence of peptide-presenting mhc class i genes between mog and ppp1r as reported in other organisms. this finding is also in agreement with the previously reported sheep mhc study by liu et al.. the blocks of peptide-presenting genes are separated by numerous other class i genes with immune and non-immune related functions. the organisation of other sheep genes in the class i region is similar to the closely related cattle mhc. the organisation of mhc class i peptide-presenting genes in distinct blocks, which are interspersed between other genes located within the class i region, is most likely due to segment or tandem block duplication. the framework hypothesis suggests that the mhc class i region is a “conserved ordered segment” that represents a dense region of genes with essential functions, whose alterations are deleterious. this study has assembled a single haplotype and shown that it is more similar to the reference cattle sequence. however, there is considerable diversity among mhc haplotypes in other species. therefore additional haplotypes will need to be sequenced and assembled to provide a true picture of mhc diversity and structural evolution.  the analysis performed in this study updates the existing sheep mhc map and enhances annotation of the genes present in the mhc class i region. this study also provides useful knowledge to complement the publicly available sequence information on ncbi regarding the chinese merino bacs, so that the information can be easily interpreted for future studies. in particular, the telomeric to centromeric orientation of bacs used by gao and colleagues has been resolved, overlapping sequence regions identified, gaps in the sheep mhc class i map mapped and the putative position of loci within each bac encompassing mhc class i region detailed. the updated sequence map provides a reference for future studies and will simplify the use of next generation sequences and snp chips for multiple mhc studies including determination of the gene/genes responsible for resistance to infectious and parasitic diseases. 
the comprehensive physical map of the sheep mhc class i region enhances our understanding of the genetic architecture of the class i mhc region in sheep and will facilitate future studies of mhc function. chori bacterial artificial chromosomes known to contain sequences from the sheep mhc class i region were sub-cloned, and the clones partially sequenced. the low resolution mhc class i physical map was then compared to the cattle reference genome, the chinese merino sheep mhc map published by gao, et al. the revised map is more similar to bovidae maps than the previous sheep maps and also includes several genes previously not annotated in the chinese merino bac assembly and others not currently annotated in the sheep reference chromosome in particular, the organisation of nonclassical mhc class i genes is similar to that present in the cattle mhc. sequence analysis and prediction of amino acid sequences of mhc class i classical and nonclassical genes was performed and it was observed that the map contained one classical and eight nonclassical genes together with three possible pseudogenes. major histocompatibility complex mhc class isheepmappingissue-copyright-statement© the author 2015 immune related class i genes are clustered into blocks; beta, kappa and a novel block not previously identified in other organisms. the major histocompatibility complex is a chromosomal region that regulates immune responsiveness in vertebrates.
 the major histocompatibility complex is a chromosomal region that regulates immune responsiveness in vertebrates. this region is one of the most important for disease resistance because it has been associated with resistance or susceptibility to a wide variety of diseases and because the mhc often accounts for more of the variance than other loci. selective breeding for disease resistance is becoming increasingly common in livestock industries, and it is important to determine how this will influence mhc polymorphism and resistance to diseases that are not targeted for selection. however, in sheep the order and sequence of the protein coding genes is controversial. yet this information is needed to determine precisely how the mhc influences resistance and susceptibility to disease. methods chori bacterial artificial chromosomes known to contain sequences from the sheep mhc class i region were sub-cloned, and the clones partially sequenced. the resulting sequences were analysed and re-assembled to identify gene content and organisation within each bac. the low resolution mhc class i physical map was then compared to the cattle reference genome, the chinese merino sheep mhc map published by gao, et al. and the recently available sheep reference genome.  immune related class i genes are clustered into blocks; beta, kappa and a novel block not previously identified in other organisms. the revised map is more similar to bovidae maps than the previous sheep maps and also includes several genes previously not annotated in the chinese merino bac assembly and others not currently annotated in the sheep reference chromosome in particular, the organisation of nonclassical mhc class i genes is similar to that present in the cattle mhc. sequence analysis and prediction of amino acid sequences of mhc class i classical and nonclassical genes was performed and it was observed that the map contained one classical and eight nonclassical genes together with three possible pseudogenes.  the comprehensive physical map of the sheep mhc class i region enhances our understanding of the genetic architecture of the class i mhc region in sheep and will facilitate future studies of mhc function. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords major histocompatibility complex mhc class isheepmappingissue-copyright-statement© the author 2015 