BACKGROUND
wheat  is one of the major food crops in the world, providing 20% of the total food calories and protein in human nutrition  <cit>  but its large genome size  and complexity  has hindered genome sequencing studies  <cit> .
to date, the largest sequenced, assembled and annotated genomic sequence from bac clones from triticeae genomes is  <dig> kb  <cit> .
the recent publication of the physical map of the largest wheat chromosome 3b  <cit> , along with other mapping projects initiated within the international wheat genome sequencing consortium , has established a platform for more extensive sequencing studies.
the high complexity of the wheat genome produced by such factors as retrotransposable element insertion  <cit>  and polyploidy, has complicated comparisons between the genomes of closely related species.
gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat  <cit> , have resulted in loss of colinearity at a micro  level  <cit> .
in view of genomic changes that are possible within a given time span, conservation of gene structure, order and orientation between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure  <cit> .
the ctg <dig> sequence characterized in the present study is a good example of such high conservation of structure.
the availability of finished, whole genome sequences for rice   <cit>  and sorghum bicolor  <cit> , as well as extensive sequencing of the maize  and brachypodium  genomes, has provided a basis for a detailed analysis of colinearity.
due to a lack of whole genome sequence data for wheat, micro-colinearity has been confirmed on individual sequenced genetic loci of agronomically important genes  <cit>  but little work has been carried out to identify any detailed relationships in regions of low gene density.
gene islands exist in grass genomes  <cit>  and gene-densities of over one gene every  <dig> kb have been reported  <cit> .
on wheat chromosome 3b, charles et al.
<cit>  carried out random bac sequencing to identify transposable element distribution and annotated the very few active genes within distal regions of the long-arm of the chromosome.
only one gene island  was identified out of the  <dig> annotated bac clones.
in this study we report on the sequencing of a group of  <dig> bacs located within fpc ctg <dig> from the hexaploid wheat chromosome 3b physical map  <cit> , and their assembly into a  <dig>  bp genomic sequence.
this large genomic sequence is among the first reported for the long arm of chromosome 3b and is also one of the largest and most complete genomic sequences described for wheat.
the  <dig>  bp genomic sequence was initially studied because it was assigned to the sr <dig> resistance locus region, however detailed mapping studies subsequently assigned it to the long arm of 3b and revealed its unusual features.
annotation of ctg <dig> through bioinformatics analysis identified a conserved gene island containing three genes of different gene function.
comparative analysis with five other cereal genomes revealed the highly conserved nature of the gene island despite no apparent co-expression or other shared functional attributes being identified.
RESULTS
ctg <dig> sequence assembly and chromosome 3b mapping
the sequencing of chromosome 3b-ctg <dig> from triticum aestivum cv.
chinese spring was initially carried out using a bac-by-bac shotgun method at a 6× sequencing coverage.
twelve bacs  were originally selected from the chromosome 3b physical map generated by fpc mapping  <cit>  .
gap closure was carried out by identifying sequence read pairs and designing primers to extend the genomic sequence.
a second set of four bac clones  was sequenced at 10× coverage in an attempt to close all remaining genomic sequence gaps .
the sixteen wheat bacs were assembled using the phred/phrap sequence assembly package  <cit> .
bac contigs were ordered and assembled into a scaffold sequence by using target site duplications  located at the ends of transposable elements .
tsds associated with a particular transposable element allowed this element to be annotated even if nested insertions had occurred within it to complicate its internal structure.
the bac contigs sequenced in this study were initially linked to bacs located in the region of the sr <dig> locus  of chromosome 3bs and the contig was of interest because of the identification of a defensin gene within the gene island.
however, when no overlap could be found within assembled contigs, markers were tested to re-examine the chromosome location of the bacs.
two insertion site based polymorphism  markers  <cit>  were identified in the sequence and used to screen the cranbrook × halberd mapping population  <cit>  to find the chromosomal location of ctg <dig> on chromosome 3b.
figure  <dig> shows the genetic mapping analysis results on the cranbrook × halberd mapping population which confirmed earlier indications from studies using a french  mapping population that contig  <dig> was co-located with molecular markers previously assigned to bin 3bl- <dig>  on the long arm of 3b.
confirmation of the location in bin 3bl <dig> is provided in figure 2b using the same isbp markers used for the genetic mapping.
the isbp sc3- <dig> marker primers  showed multiple pcr bands with the band of the expected size missing from lanes  <dig> and  <dig>  for isbp sc3- <dig>  the pcr showed a single band of the expected size  and this band was missing in lanes  <dig> and  <dig>  since lanes  <dig> and  <dig> are deletion lines 3bl <dig> and 3bl <dig> the analysis indicated that the locus containing the isbp is located on 3bl <dig>  <cit> .
transposable element annotation of ctg1034
transposable elements  were annotated in this study using blastn and blastx  <cit>  sequence homology searches against the non-redundant nucleotide  and protein  sets of the triticeae repetitive element  database.
alignments to tes located within ctg <dig> were confirmed using the dotter dot-matrix program  <cit>  and were used to identify target site duplications  at either end of the element.
table  <dig> lists the te families that are distributed within ctg <dig> and their sequence proportions compared to the rest of the assembled contig.
class i
overall, tes made up  <dig> % of the entire ctg <dig> chromosome 3b sequence.
the repetitive element content was dominated by retrotransposable elements , considered to transpose through the action of a rna intermediate.
this was compared to the dna transposons , which comprised only  <dig> % of all tes .
the gypsy family of long terminal repeat  retrotransposable elements were by far the most abundant type of class i-te, with  <dig> out of the  <dig> transposons found  and occupying more than half the total sequence of ctg <dig> .
two gypsy elements, romani located at  <dig>  -  <dig> bp and latidu located at  <dig>  -  <dig> bp in ctg <dig>  were also found to contain significant matches to chromodomains, which are located in the c-terminal region of the integrase  protein domain of the gypsy internal polyprotein  domain  <cit> .
a profile hmm  <cit>  constructed using consensus sequences of reverse transcriptase  and in domains in  <cit>  was used to identify potential chromodomain matches .
chromodomains were originally identified in the centromeric heterochromatin proteins of drosophila  <cit>  and have been argued to target heterochromatin by recognizing histone modifications  <cit> .
the internal regions of both the romani and latidu ltr gypsy elements were also searched using blastx against the characterised internal domains of the gypsy mobile genetic element database   <cit> .
both elements contained significant matches  to the integrase domain of the o.sativa ltr gypsy element retrosat- <dig>
the cacta dna transposons and copia ltr families were also well represented with  <dig> % and  <dig> % of the total ctg <dig> sequence respectively.
the cacta dna transposons had a high proportion of elements  that contained nested insertions.
gypsy ltr retrotransposons had a slightly lower proportion of nested elements .
the copia ltr elements were found to be the most intact with only one element containing a nested insertion and all identified to be complete with tsds.
tandem repeats considered to be located within heterochromatic regions in triticeae genomes were also assayed to identify matches to ctg <dig>  two matches were genome specific' aegilops squarrosa repetitive dna sequence   <cit>  and the other being an aegilops tauschii so-called centromeric-specific tandem repeat from clone 6c6- <dig>   <cit> .
the well-studied d <dig> repeat  <cit>  is from the afa <dig> family of tandem repeats  and is made up of  <dig> bp units, only one of which was located in the ctg <dig> sequence between  <dig>  and  <dig>  bp .
the ay <dig> repeat was almost a perfect match  between  <dig>  and  <dig>  bp.
both these representatives of tandem repeat families were located within tes, with the d <dig> repetitive dna sequence found within the cacta dna transposon conan and the ay <dig> tandem repeat found within the gypsy ltr retrotransposon cereba.
gene content annotation of ctg1034
the  <dig>  bp ctg <dig> sequence was masked for repetitive elements with the repeat masking program repeatmasker http://www.repeatmasker.org run using the triticeae repetitive element  database http://wheat.pw.usda.gov/itmi/repeats.
the masked sequence was then passed through gene prediction programs fgenesh http://linux <dig> softberry.com/berry.phtml and glimmerhmm  <cit> .
predicted gene models from both programs were screened against the michigan state university  o. sativa annotation project's http://rice.plantbiology.msu.edu/ protein database and ncbi non-redundant amino-acid database using blastp to identify non-redundant gene matches.
a database of  <dig>  full-length wheat cdna sequences from the komugi wheat genetic resource http://www.shigen.nig.ac.jp/wheat/komugi/ was also screened to identify any direct matches to wheat cdnas.
all annotated genes followed the standard gt-ag eukaryotic intron splice site model.
four gene models of significant homology to characterised protein matches in ncbi non-redundant amino acid and msu rice protein database were all found within a  <dig> kb window between  <dig> - <dig>  bp of the ctg <dig> contig.
the first gene model found at the very beginning of the  <dig> kb region was identified to be a fragment  of the msu rice gene hit, loc_os <dig> g <dig>  with no significant wheat est or cdna match and was not considered further.
the other three gene models were annotated as gene-coding sequences and all had hits to the ncbi non-redundant protein database.
all three genes were annotated using blastx alignments to the msu rice protein annotations  <cit>  and their intron-exon structure was confirmed using both a wheat subset of expressed sequence tags  from ncbi and rice full-length cdna information  <cit> .
the overall gene-density of the ctg <dig> sequence was  <dig> gene per  <dig>  bp.
the first of the three genes, named taep <dig>  was identified to be  <dig>  bp in length and encoded a predicted protein of  <dig> amino acids over  <dig> exons .
the gene has a high similarity at the amino acid level  to an expressed protein in rice  as well as a catalytic domain-containing protein in arabidopsis  .
the gene was supported by multiple wheat full-length ests  all with a greater than 95% nucleotide identity.
the second gene  was quite small at only  <dig> bp in length and encoded a predicted protein of  <dig> amino acids over  <dig> exons .
the gene was well supported by multiple full-length ests , which all had a greater than 95% nucleotide identity.
the predicted protein of the gene was found to have homology to an expressed protein in rice , which appears in the msu rice community annotation as a small, cysteine-rich protein named crp <dig> <cit> .
in previous work involving legumes  <cit> , small cysteine rich peptides  were classified as different classes of defensins, proteins that are active in conferring resistance to bacteria, fungi and viruses.
the cx-cxccx-cx-ccx-cx-cx-cxcx  arrangement of the cysteine domain in tacrp1corresponds to the ma ternally expressed gene  family.
the last gene matched the ncbi non-redundant amino acid database was annotated as a zinc finger protein .
this gene was  <dig>  bp, has a 7-exon gene structure and encodes a predicted protein of  <dig> amino acids .
the gene was shown to have high homology  to the rice zinc finger ccch domain-containing protein zfn-like  <dig>  and well supported by wheat ests  and a full-length wheat cdna taken from the komugi wheat genetic resources resource http://www.shigen.nig.ac.jp/wheat/komugi/.
tazfn <dig> was the only one of the four identified gene models that had a match to a full-length wheat cdna; the cdna match to the coding sequence of tazfn <dig> was almost 100%, with only  <dig> base differences.
the ncbi unigene est profiles http://www.ncbi.nlm.nih.gov/unigene of each of the genes were compared in table  <dig>  showing the expression of each of the genes in wheat, barley  and rice tissue-specific est pools.
there was a low-level of transcription of the ep <dig> gene and no tissue specificity could be inferred from the data across all species.
while there was a low-level of transcription found from the est data for the crp <dig> gene, there was clear seed-tissue specificity across all species relative to the expression in leaf and root tissues.
the zfn <dig> gene displayed much higher transcription levels than the previous two genes but like ep <dig> showed no tissue-specificity could be inferred.
*the number of tissue-specific ests is shown and the extrapolated transcripts per million  are shown in brackets
**barley est profiles are also shown on the basis of blastn homology searches against the ncbi est dataset.
comparative analysis using the gene-island-coding sequences from cereal genomes
the three genes found as a small cluster or island within the wheat ctg <dig> region of chromosome 3b were used to search against the rice  and sorghum bicolor genome sequences and each genome was found to be syntenic over the region, showing both conserved gene order and orientation.
the rice msu version  <dig> annotation of chromosome  <dig> between  <dig> , <dig> to  <dig> , <dig> bp  in the rice genome was used along with the sorghum sbi <dig>  annotation models  from chromosome  <dig> of sorghum bicolor from between  <dig> , <dig> and  <dig> , <dig> bp .
the three genes from ctg <dig> were also searched against the brachypodium  line bd <dig> and maize  genome sequences using blastn and the top hits of all three were once again found to have conserved gene order and orientation.
the equivalent of the wheat  <dig> kb region in the draft brachypodium super <dig> scaffold sequence  contained the conserved colinear genes and was annotated using the same protocol used to annotate the ctg <dig> wheat genome sequence.
the three genes were named bdep <dig>  bdcrp <dig> and bdzfn <dig> based on their rice protein matches in the msu version  <dig> annotations .
a small genomic sequence gap within the draft sequence  located inside exon  <dig> and intron  <dig> of bdzfn <dig> was filled on request to the brachypodium genome-sequencing project  and reduced the size of the syntenic gene island sequence to  <dig> kb.
a syntenic region containing the taep <dig>  tacrp <dig> and tazfn <dig> genes in maize was located within the ctg <dig> fpc contig on maize chromosome  <dig>  the maize genomic bac clone containing all three genes  was accessed from the maize genome sequence project website http://maizesequence.org/ and were annotated for all genes  based on alignments to sorghum bicolor gene annotations http://www.phytozome.net/sorghum with intron-exon structures confirmed using ncbi maize ests.
the three genes were named zmep <dig>  zmcrp <dig> and zmzfn <dig>  transposable elements were annotated using blastn against the tigr zea repeat database  <cit> .
*denotes the brachypodium and maize genes that were annotated along with wheat in this study.
taep <dig> showed the highest level of amino-acid conservation  among the ep <dig> genes in the cereal genomes.
the highest similarity was with brachypodium bdep <dig> , followed by rice loc_os <dig> g <dig> , s. bicolor sb <dig> g <dig> and maize zmep <dig> .
taep <dig> also had 49% amino acid identity to a peptidase protein at3g <dig> in arabidopsis.
the brachypodium bdep <dig> also had a good conservation of non-coding nucleotide sequence within the intron of taep <dig>  the intron sequence of bdep <dig> was 70% identical over  <dig> nucleotides with 85% sequence coverage compared to the rice comparison, which had only 16% coverage over the  <dig> nucleotides.
there was a small region of  <dig> bp near the start of the intron that did not show any homology.
the intron sequence of maize zmep <dig> compared to s. bicolor sb <dig> g <dig> was also highly conserved .
the large 7-exon tazfn <dig> gene, much like taep <dig>  showed significant homology to its brachypodium orthologous sequence.
the amino acid sequences of both genes showed 89% identity over  <dig> amino acids, with only a small section located within the first exon lacking homology.
both s. bicolor  and rice  showed 82% identity compared to tazfn <dig>  with maize showing the least similarity at amino acid level .
the amino acids from the first exon  seemed to contain the most sequence divergence when compared to the rest of the genome sequences.
as was found in taep <dig>  there was a significant conservation of the non-coding sequence between tazfn <dig> and bdzfn <dig>  especially in introns  <dig>   <dig> and  <dig> which all have a nucleotide sequence coverage of >62%.
the zfn <dig> gene sequence between maize and s. bicolor was also well conserved  except for two small inserts contained within the fifth intron of zmzfn <dig>  one insert of  <dig>  bp and another of  <dig> bp.
the larger  <dig>  bp insert contained two small transposable elements casine  and f <dig>  classified as sine elements from the non-ltr retrotransposable element group.
by comparison, the tacrp <dig> showed the least amount of homology across the genomes when analysed at the amino acid level  relative to taep <dig> and tazfn <dig>  once again, brachypodium had the highest amino acid homology , followed by rice , s. bicolor  and maize .
the low amino acid identity was due to insertions within the rice, maize and s. bicolor amino acid sequences.
s. bicolorsb <dig> g <dig> had a  <dig> amino acid insertion located just after the signal peptide and before the cysteine domain.
it is also the only crp <dig> sequence out of the five syntenic gene copies that had only one exon .
the cysteine domain located in the last  <dig> amino acids of the gene was conserved in all genomes .
the only significant difference in the cysteine-rich domain across all genomes was a six amino acid insertion  within the rice loc_os <dig> g <dig> sequence.
exon  <dig> in all sequences that contained multiple exons showed little nucleotide and amino acid sequence homology and exon  <dig> was the only region found to have significant sequence conservation.
exon  <dig> contained the coding sequence of the cysteine-rich domain.
no significant intron sequence similarity could be identified across the genomes.
the syntenic gene island region across five genomes
the ctg <dig>  <dig> kb gene island was analysed across the available genomic sequences including arabidopsis genome sequence  <cit> , the grapevine  genome sequence  <cit>  and the wheat, brachypodium, s. bicolor, rice and maize genome sequences.
the  <dig> kb, three-gene region containing taep <dig>  tacrp <dig> and tazfn <dig> did not show any conservation of colinearity in the arabidopsis genome sequence.
chromosome  <dig> of the grapevine genome did have homologs of tacrp <dig> and tazfn <dig> located within a  <dig> kb window  but also contained a small  gene found in between.
this small gene coding for a  <dig> aa protein has no rice or arabidopsis blastx hit and could possibly be within a transposable element.
there are large size differences of the gene-island regions between wheat  and both maize  and rice  resulted from an insertion-deletion event  in both cases.
comparing the genome sequences of rice against the wheat gene island region shows a 6- <dig> kb sequence variation  between the oscrp <dig> and oszfn <dig> genes .
the proposed indel within the rice genome contains a hypothetical protein annotation loc_os <dig> g <dig> and contained many mite and dna transposons found in the tigr oryza repeat database v <dig>   including a mule or mutator-like element .
the loc_os <dig> g <dig> open-reading frame was identified using fgenesh and contained a high amount of repetitive elements.
furthermore, the match to sequences in the est databases of rice indicated the largest region of significant homology  was only  <dig> bp and thus it seems unlikely to be an active entity.
overall, in five of the genome sequences used in this study, there was a highly conserved gene sequence, order and orientation, when compared to the wheat ctg <dig> gene island , even though the size of the region differed considerably; greatest in the maize genome , followed by rice , s. bicolor  and brachypodium  .
the 4× draft brachypodium super <dig> scaffold sequence showed the highest nucleotide coverage , followed by rice , s. bicolor  and maize  when each sequence was compared to the  <dig> kb gene island wheat genomic sequence using ncbi blast  <dig> sequences   <cit> .
a detailed analysis of the differences in length in this region between the genomes analysed  is provided in the additional file  <dig>
discussion
in this study a group of twelve hexaploid wheat  bac clones from chromosome 3b were assembled into a  <dig>  bp genomic sequence  that was then analysed for biological features.
diverse gene functions in a conserved gene-island
the three gene-coding sequences on ctg <dig> , located within a  <dig> kb sequence, all show different sequence characteristics and a range of expression patterns and functions based on rice annotations.
taep <dig> is highly conserved in plant genomes  but does not show any distinct characterised function in sequenced genomes such as s. bicolor and rice.
tacrp <dig>  on the other hand, is a small cysteine-rich protein, a protein from a diverse, well characterised protein family that is involved in pathogen defence  <cit> .
tazfn <dig> was like tacrp <dig>  a well-characterised protein from a large family of transcription factors that have been extensively studied.
furthermore multiple full-length cdna sequences of tazfn <dig> in rice indicate alternative splicing forms of the gene product  exist.
est expression profiles from  <dig> did not provide any clear indication of co-expression of the three genes; tazfn <dig> was striking in its relatively high levels of expression in a range of tissue compared to taep <dig> and tacrp <dig>  this is consistent with the genes being involved in independent pathways within cells.
gene island structure is maintained over the evolution of plant genomes despite the occurrence of indels
conservation of colinearity between grass species has been reported extensively at a micro-level  <cit>  but even in colinear regions, gene order and orientation may not be conserved  <cit> .
the conserved gene-islands described between wheat and other major crop species such as rice  <cit> , have been limited because of the use of est and single bac sequences , with the associated lack resolution making it difficult to reach definitive conclusions about the level of conservation.
while conserved gene islands at gene-rich orthologous loci have been shown, small genetic rearrangements were found to dramatically change gene order in maize and s. bicolor  <cit>  and a mosaic of rearrangements between rice and wheat were also reported, with the conservation of gene orientation only limited to local duplications in one species  <cit> .
the gene island provides a contrast to the concepts developing from mammalian genome studies that repeat sequence regions may be hot-spots for structural change in gene regions  <cit> .
while the relative order and orientation of the genes within the ctg <dig> gene island has been maintained, the intergenic region was not immune to changes during the course of its evolution.
figure  <dig> shows a summary evolutionary tree deduced from colinearity and sequence similarity seen in this study over the ctg <dig> gene island.
this evolutionary tree is consistent with taxonomic relationships  <cit> .
despite the large amount of repetitive elements that surround the island, only the rice and maize genome  sequences showed post-divergence te insertions.
an insert within maize is not unusual, as  <cit>  has shown that even maize inbred lines can show intra-specific variation in genetic colinearity.
high sequence diversity in maize can be caused by tes such as helitrons  <cit> .
the 6- <dig> kb rice insertion mostly contained miniature inverted tes , known to insert into and be associated with gene-containing regions  <cit>  and  <cit>  suggested that these small tes might also have a role in modification of expression in neighbouring genes.
another te found within the insert was a mule transposon.
so called pack-mules are extremely common in the rice genome  <cit>  and have been implicated in the evolution of genes within higher plant genomes through their ability to capture and carry fragments of genomic dna to create new open reading frames.
gene island location on chromosome 3bl
the gene density in this study was identified to be quite low at  <dig> gene per  <dig> kb.
in contrast, gene densities in triticeae genomes  have been reported to be much higher , possibly reflecting the initial targeting of important agronomic genes in distal regions of chromosomes  <cit>  rather than a random representation of the genome sequence.
random wheat bac sequencing  <cit>  indicated that even in gene-rich regions of wheat, the gene density is considerably lower than previously thought .
genetic mapping studies showed sc3- <dig> and sc3- <dig> isbp markers from ctg <dig> were located within the terminal deletion bin 3bl- <dig> on chromosome 3b , a deletion bin known to contain two major c-bands  <cit> .
previous cytogenetic studies of the wheat chromosomes suggested a large variation of gene-density over the entire length of chromosomes  <cit>  and it is feasible that particularly low gene densities occur within c band regions.
the low gene density found in this study is more consistent with studies in triticeae genomes of more proximal regions of chromosome arms  and regions of low recombination .
the two representatives of tandem repeat families located within the first  <dig> kb of the ctg <dig> sequence also indicate a relationship to heterochromatin, with one  being previously identified to be a d-genome centromeric heterochromatin tandem repeat sequence  <cit> .
the presence of a representative of this d-genome specific repeat in the chromosome 3b sequence suggest specific amplification of the sequence family must have occurred in the d genome even though single copy representatives were located elsewhere.
the presence of representatives of sequences normally located as tandem arrays in heterochromatin and the presence of high gypsy ltr content support our proposal that this ctg <dig> genomic sequence is located within a c-banding region in deletion bin  <dig> of chromosome 3bl.
the highly repetitive element content in ctg <dig>  was not unusual in wheat sequences, as studies have shown repetitive element contents of greater than 70%  <cit> , however the high content of the gypsy-like ltr retrotransposon is significant in this study .
a previously sequenced wheat bac clone from the same chromosome 3bl- <dig> deletion bin  <cit>  also found a similar high proportion of gypsy-like ltr-retrotransposons.
in the present study, chromodomains were shown to be located within the internal polyprotein domain of two gypsy-ltr retrotransposons, and are thus potentially key components defining the condensed nature of heterochromatin  <cit> .
one of the potential chromodomains  was located immediately adjacent to the gene island in ctg <dig>
the unusual properties of heterochromatin that would contribute to the unique conservation of both the gene order and relative gene orientation found in this study include:  the low levels of recombination and gene activity associated with heterochromatin  <cit> ;  the high level of transcript processing by the rnai machinery that mediates chromatin structure  <cit> .
these properties could contribute to the relative isolation of the gene island from the normal processes that lead to genome rearrangements.
CONCLUSIONS
bac shotgun sequencing of the fpc physical map contig ctg <dig> from the genome of chromosome 3b has provided the largest assembly of a wheat genome region to date .
annotation and comparative analysis with four other plant genomes of  identified an island of three genes showing complete conservation of gene order and orientation.
this conservation could not be readily accounted for by shared functions between the genes and it is proposed that ctg <dig> is located within a heterochromatic c-band region of deletion bin 3bl <dig> based on the identification of representatives of heterochromatin tandem repeats and presence of significant matches to chromodomain-containing gypsy ltr retrotransposable elements.
we speculate that this location, among other highly repetitive sequences, may account for the relative stability of the gene island through insulating the gene island from normal recombination processes.

 wheat is one of the major food crops in the world, providing 20% of the total food calories and protein in human nutrition but its large genome size and complexity has hindered genome sequencing studies. to date, the largest sequenced, assembled and annotated genomic sequence from bac clones from triticeae genomes is kb. the recent publication of the physical map of the largest wheat chromosome 3b, along with other mapping projects initiated within the international wheat genome sequencing consortium, has established a platform for more extensive sequencing studies. the high complexity of the wheat genome produced by such factors as retrotransposable element insertion and polyploidy, has complicated comparisons between the genomes of closely related species. gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat, have resulted in loss of colinearity at a micro level. in view of genomic changes that are possible within a given time span, conservation of gene structure, order and orientation between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. the ctg sequence characterized in the present study is a good example of such high conservation of structure. the availability of finished, whole genome sequences for rice and sorghum bicolor, as well as extensive sequencing of the maize and brachypodium genomes, has provided a basis for a detailed analysis of colinearity. due to a lack of whole genome sequence data for wheat, micro-colinearity has been confirmed on individual sequenced genetic loci of agronomically important genes but little work has been carried out to identify any detailed relationships in regions of low gene density. gene islands exist in grass genomes and gene-densities of over one gene every kb have been reported. on wheat chromosome 3b, charles et al. carried out random bac sequencing to identify transposable element distribution and annotated the very few active genes within distal regions of the long-arm of the chromosome. only one gene island was identified out of the annotated bac clones. in this study we report on the sequencing of a group of bacs located within fpc ctg from the hexaploid wheat chromosome 3b physical map, and their assembly into a bp genomic sequence. this large genomic sequence is among the first reported for the long arm of chromosome 3b and is also one of the largest and most complete genomic sequences described for wheat. the bp genomic sequence was initially studied because it was assigned to the sr resistance locus region, however detailed mapping studies subsequently assigned it to the long arm of 3b and revealed its unusual features. annotation of ctg through bioinformatics analysis identified a conserved gene island containing three genes of different gene function. comparative analysis with five other cereal genomes revealed the highly conserved nature of the gene island despite no apparent co-expression or other shared functional attributes being identified.  ctg sequence assembly and chromosome 3b mapping the sequencing of chromosome 3b-ctg from triticum aestivum cv. chinese spring was initially carried out using a bac-by-bac shotgun method at a 6× sequencing coverage. twelve bacs were originally selected from the chromosome 3b physical map generated by fpc mapping. gap closure was carried out by identifying sequence read pairs and designing primers to extend the genomic sequence. a second set of four bac clones was sequenced at 10× coverage in an attempt to close all remaining genomic sequence gaps. the sixteen wheat bacs were assembled using the phred/phrap sequence assembly package. bac contigs were ordered and assembled into a scaffold sequence by using target site duplications located at the ends of transposable elements. tsds associated with a particular transposable element allowed this element to be annotated even if nested insertions had occurred within it to complicate its internal structure. the bac contigs sequenced in this study were initially linked to bacs located in the region of the sr locus of chromosome 3bs and the contig was of interest because of the identification of a defensin gene within the gene island. however, when no overlap could be found within assembled contigs, markers were tested to re-examine the chromosome location of the bacs. two insertion site based polymorphism markers were identified in the sequence and used to screen the cranbrook × halberd mapping population to find the chromosomal location of ctg on chromosome 3b. figure shows the genetic mapping analysis results on the cranbrook × halberd mapping population which confirmed earlier indications from studies using a french mapping population that contig was co-located with molecular markers previously assigned to bin 3bl- on the long arm of 3b. confirmation of the location in bin 3bl is provided in figure 2b using the same isbp markers used for the genetic mapping. the isbp sc3- marker primers showed multiple pcr bands with the band of the expected size missing from lanes and for isbp sc3- the pcr showed a single band of the expected size and this band was missing in lanes and since lanes and are deletion lines 3bl and 3bl the analysis indicated that the locus containing the isbp is located on 3bl. transposable element annotation of ctg1034 transposable elements were annotated in this study using blastn and blastx sequence homology searches against the non-redundant nucleotide and protein sets of the triticeae repetitive element database. alignments to tes located within ctg were confirmed using the dotter dot-matrix program and were used to identify target site duplications at either end of the element. table lists the te families that are distributed within ctg and their sequence proportions compared to the rest of the assembled contig. class i overall, tes made up % of the entire ctg chromosome 3b sequence. the repetitive element content was dominated by retrotransposable elements, considered to transpose through the action of a rna intermediate. this was compared to the dna transposons, which comprised only % of all tes. the gypsy family of long terminal repeat retrotransposable elements were by far the most abundant type of class i-te, with out of the transposons found and occupying more than half the total sequence of ctg. two gypsy elements, romani located at - bp and latidu located at - bp in ctg were also found to contain significant matches to chromodomains, which are located in the c-terminal region of the integrase protein domain of the gypsy internal polyprotein domain. a profile hmm constructed using consensus sequences of reverse transcriptase and in domains in was used to identify potential chromodomain matches. chromodomains were originally identified in the centromeric heterochromatin proteins of drosophila and have been argued to target heterochromatin by recognizing histone modifications. the internal regions of both the romani and latidu ltr gypsy elements were also searched using blastx against the characterised internal domains of the gypsy mobile genetic element database. both elements contained significant matches to the integrase domain of the o.sativa ltr gypsy element retrosat- the cacta dna transposons and copia ltr families were also well represented with % and % of the total ctg sequence respectively. the cacta dna transposons had a high proportion of elements that contained nested insertions. gypsy ltr retrotransposons had a slightly lower proportion of nested elements. the copia ltr elements were found to be the most intact with only one element containing a nested insertion and all identified to be complete with tsds. tandem repeats considered to be located within heterochromatic regions in triticeae genomes were also assayed to identify matches to ctg two matches were genome specific' aegilops squarrosa repetitive dna sequence and the other being an aegilops tauschii so-called centromeric-specific tandem repeat from clone 6c6-. the well-studied d repeat is from the afa family of tandem repeats and is made up of bp units, only one of which was located in the ctg sequence between and bp. the ay repeat was almost a perfect match between and bp. both these representatives of tandem repeat families were located within tes, with the d repetitive dna sequence found within the cacta dna transposon conan and the ay tandem repeat found within the gypsy ltr retrotransposon cereba. gene content annotation of ctg1034 the bp ctg sequence was masked for repetitive elements with the repeat masking program repeatmasker http://www.repeatmasker.org run using the triticeae repetitive element database http://wheat.pw.usda.gov/itmi/repeats. the masked sequence was then passed through gene prediction programs fgenesh http://linux softberry.com/berry.phtml and glimmerhmm. predicted gene models from both programs were screened against the michigan state university o. sativa annotation project's http://rice.plantbiology.msu.edu/ protein database and ncbi non-redundant amino-acid database using blastp to identify non-redundant gene matches. a database of full-length wheat cdna sequences from the komugi wheat genetic resource http://www.shigen.nig.ac.jp/wheat/komugi/ was also screened to identify any direct matches to wheat cdnas. all annotated genes followed the standard gt-ag eukaryotic intron splice site model. four gene models of significant homology to characterised protein matches in ncbi non-redundant amino acid and msu rice protein database were all found within a kb window between - bp of the ctg contig. the first gene model found at the very beginning of the kb region was identified to be a fragment of the msu rice gene hit, loc_os g with no significant wheat est or cdna match and was not considered further. the other three gene models were annotated as gene-coding sequences and all had hits to the ncbi non-redundant protein database. all three genes were annotated using blastx alignments to the msu rice protein annotations and their intron-exon structure was confirmed using both a wheat subset of expressed sequence tags from ncbi and rice full-length cdna information. the overall gene-density of the ctg sequence was gene per bp. the first of the three genes, named taep was identified to be bp in length and encoded a predicted protein of amino acids over exons. the gene has a high similarity at the amino acid level to an expressed protein in rice as well as a catalytic domain-containing protein in arabidopsis. the gene was supported by multiple wheat full-length ests all with a greater than 95% nucleotide identity. the second gene was quite small at only bp in length and encoded a predicted protein of amino acids over exons. the gene was well supported by multiple full-length ests, which all had a greater than 95% nucleotide identity. the predicted protein of the gene was found to have homology to an expressed protein in rice, which appears in the msu rice community annotation as a small, cysteine-rich protein named crp. in previous work involving legumes, small cysteine rich peptides were classified as different classes of defensins, proteins that are active in conferring resistance to bacteria, fungi and viruses. the cx-cxccx-cx-ccx-cx-cx-cxcx arrangement of the cysteine domain in tacrp1corresponds to the ma ternally expressed gene family. the last gene matched the ncbi non-redundant amino acid database was annotated as a zinc finger protein. this gene was bp, has a 7-exon gene structure and encodes a predicted protein of amino acids. the gene was shown to have high homology to the rice zinc finger ccch domain-containing protein zfn-like and well supported by wheat ests and a full-length wheat cdna taken from the komugi wheat genetic resources resource http://www.shigen.nig.ac.jp/wheat/komugi/. tazfn was the only one of the four identified gene models that had a match to a full-length wheat cdna; the cdna match to the coding sequence of tazfn was almost 100%, with only base differences. the ncbi unigene est profiles http://www.ncbi.nlm.nih.gov/unigene of each of the genes were compared in table showing the expression of each of the genes in wheat, barley and rice tissue-specific est pools. there was a low-level of transcription of the ep gene and no tissue specificity could be inferred from the data across all species. while there was a low-level of transcription found from the est data for the crp gene, there was clear seed-tissue specificity across all species relative to the expression in leaf and root tissues. the zfn gene displayed much higher transcription levels than the previous two genes but like ep showed no tissue-specificity could be inferred. *the number of tissue-specific ests is shown and the extrapolated transcripts per million are shown in brackets **barley est profiles are also shown on the basis of blastn homology searches against the ncbi est dataset. comparative analysis using the gene-island-coding sequences from cereal genomes the three genes found as a small cluster or island within the wheat ctg region of chromosome 3b were used to search against the rice and sorghum bicolor genome sequences and each genome was found to be syntenic over the region, showing both conserved gene order and orientation. the rice msu version annotation of chromosome between, to, bp in the rice genome was used along with the sorghum sbi annotation models from chromosome of sorghum bicolor from between, and, bp. the three genes from ctg were also searched against the brachypodium line bd and maize genome sequences using blastn and the top hits of all three were once again found to have conserved gene order and orientation. the equivalent of the wheat kb region in the draft brachypodium super scaffold sequence contained the conserved colinear genes and was annotated using the same protocol used to annotate the ctg wheat genome sequence. the three genes were named bdep bdcrp and bdzfn based on their rice protein matches in the msu version annotations. a small genomic sequence gap within the draft sequence located inside exon and intron of bdzfn was filled on request to the brachypodium genome-sequencing project and reduced the size of the syntenic gene island sequence to kb. a syntenic region containing the taep tacrp and tazfn genes in maize was located within the ctg fpc contig on maize chromosome the maize genomic bac clone containing all three genes was accessed from the maize genome sequence project website http://maizesequence.org/ and were annotated for all genes based on alignments to sorghum bicolor gene annotations http://www.phytozome.net/sorghum with intron-exon structures confirmed using ncbi maize ests. the three genes were named zmep zmcrp and zmzfn transposable elements were annotated using blastn against the tigr zea repeat database. *denotes the brachypodium and maize genes that were annotated along with wheat in this study. taep showed the highest level of amino-acid conservation among the ep genes in the cereal genomes. the highest similarity was with brachypodium bdep, followed by rice loc_os g, s. bicolor sb g and maize zmep. taep also had 49% amino acid identity to a peptidase protein at3g in arabidopsis. the brachypodium bdep also had a good conservation of non-coding nucleotide sequence within the intron of taep the intron sequence of bdep was 70% identical over nucleotides with 85% sequence coverage compared to the rice comparison, which had only 16% coverage over the nucleotides. there was a small region of bp near the start of the intron that did not show any homology. the intron sequence of maize zmep compared to s. bicolor sb g was also highly conserved. the large 7-exon tazfn gene, much like taep showed significant homology to its brachypodium orthologous sequence. the amino acid sequences of both genes showed 89% identity over amino acids, with only a small section located within the first exon lacking homology. both s. bicolor and rice showed 82% identity compared to tazfn with maize showing the least similarity at amino acid level. the amino acids from the first exon seemed to contain the most sequence divergence when compared to the rest of the genome sequences. as was found in taep there was a significant conservation of the non-coding sequence between tazfn and bdzfn especially in introns and which all have a nucleotide sequence coverage of >62%. the zfn gene sequence between maize and s. bicolor was also well conserved except for two small inserts contained within the fifth intron of zmzfn one insert of bp and another of bp. the larger bp insert contained two small transposable elements casine and f classified as sine elements from the non-ltr retrotransposable element group. by comparison, the tacrp showed the least amount of homology across the genomes when analysed at the amino acid level relative to taep and tazfn once again, brachypodium had the highest amino acid homology, followed by rice, s. bicolor and maize. the low amino acid identity was due to insertions within the rice, maize and s. bicolor amino acid sequences. s. bicolorsb g had a amino acid insertion located just after the signal peptide and before the cysteine domain. it is also the only crp sequence out of the five syntenic gene copies that had only one exon. the cysteine domain located in the last amino acids of the gene was conserved in all genomes. the only significant difference in the cysteine-rich domain across all genomes was a six amino acid insertion within the rice loc_os g sequence. exon in all sequences that contained multiple exons showed little nucleotide and amino acid sequence homology and exon was the only region found to have significant sequence conservation. exon contained the coding sequence of the cysteine-rich domain. no significant intron sequence similarity could be identified across the genomes. the syntenic gene island region across five genomes the ctg kb gene island was analysed across the available genomic sequences including arabidopsis genome sequence, the grapevine genome sequence and the wheat, brachypodium, s. bicolor, rice and maize genome sequences. the kb, three-gene region containing taep tacrp and tazfn did not show any conservation of colinearity in the arabidopsis genome sequence. chromosome of the grapevine genome did have homologs of tacrp and tazfn located within a kb window but also contained a small gene found in between. this small gene coding for a aa protein has no rice or arabidopsis blastx hit and could possibly be within a transposable element. there are large size differences of the gene-island regions between wheat and both maize and rice resulted from an insertion-deletion event in both cases. comparing the genome sequences of rice against the wheat gene island region shows a 6- kb sequence variation between the oscrp and oszfn genes. the proposed indel within the rice genome contains a hypothetical protein annotation loc_os g and contained many mite and dna transposons found in the tigr oryza repeat database v including a mule or mutator-like element. the loc_os g open-reading frame was identified using fgenesh and contained a high amount of repetitive elements. furthermore, the match to sequences in the est databases of rice indicated the largest region of significant homology was only bp and thus it seems unlikely to be an active entity. overall, in five of the genome sequences used in this study, there was a highly conserved gene sequence, order and orientation, when compared to the wheat ctg gene island, even though the size of the region differed considerably; greatest in the maize genome, followed by rice, s. bicolor and brachypodium. the 4× draft brachypodium super scaffold sequence showed the highest nucleotide coverage, followed by rice, s. bicolor and maize when each sequence was compared to the kb gene island wheat genomic sequence using ncbi blast sequences. a detailed analysis of the differences in length in this region between the genomes analysed is provided in the additional file discussion in this study a group of twelve hexaploid wheat bac clones from chromosome 3b were assembled into a bp genomic sequence that was then analysed for biological features. diverse gene functions in a conserved gene-island the three gene-coding sequences on ctg, located within a kb sequence, all show different sequence characteristics and a range of expression patterns and functions based on rice annotations. taep is highly conserved in plant genomes but does not show any distinct characterised function in sequenced genomes such as s. bicolor and rice. tacrp on the other hand, is a small cysteine-rich protein, a protein from a diverse, well characterised protein family that is involved in pathogen defence. tazfn was like tacrp a well-characterised protein from a large family of transcription factors that have been extensively studied. furthermore multiple full-length cdna sequences of tazfn in rice indicate alternative splicing forms of the gene product exist. est expression profiles from did not provide any clear indication of co-expression of the three genes; tazfn was striking in its relatively high levels of expression in a range of tissue compared to taep and tacrp this is consistent with the genes being involved in independent pathways within cells. gene island structure is maintained over the evolution of plant genomes despite the occurrence of indels conservation of colinearity between grass species has been reported extensively at a micro-level but even in colinear regions, gene order and orientation may not be conserved. the conserved gene-islands described between wheat and other major crop species such as rice, have been limited because of the use of est and single bac sequences, with the associated lack resolution making it difficult to reach definitive conclusions about the level of conservation. while conserved gene islands at gene-rich orthologous loci have been shown, small genetic rearrangements were found to dramatically change gene order in maize and s. bicolor and a mosaic of rearrangements between rice and wheat were also reported, with the conservation of gene orientation only limited to local duplications in one species. the gene island provides a contrast to the concepts developing from mammalian genome studies that repeat sequence regions may be hot-spots for structural change in gene regions. while the relative order and orientation of the genes within the ctg gene island has been maintained, the intergenic region was not immune to changes during the course of its evolution. figure shows a summary evolutionary tree deduced from colinearity and sequence similarity seen in this study over the ctg gene island. this evolutionary tree is consistent with taxonomic relationships. despite the large amount of repetitive elements that surround the island, only the rice and maize genome sequences showed post-divergence te insertions. an insert within maize is not unusual, as has shown that even maize inbred lines can show intra-specific variation in genetic colinearity. high sequence diversity in maize can be caused by tes such as helitrons. the 6- kb rice insertion mostly contained miniature inverted tes, known to insert into and be associated with gene-containing regions and suggested that these small tes might also have a role in modification of expression in neighbouring genes. another te found within the insert was a mule transposon. so called pack-mules are extremely common in the rice genome and have been implicated in the evolution of genes within higher plant genomes through their ability to capture and carry fragments of genomic dna to create new open reading frames. gene island location on chromosome 3bl the gene density in this study was identified to be quite low at gene per kb. in contrast, gene densities in triticeae genomes have been reported to be much higher, possibly reflecting the initial targeting of important agronomic genes in distal regions of chromosomes rather than a random representation of the genome sequence. random wheat bac sequencing indicated that even in gene-rich regions of wheat, the gene density is considerably lower than previously thought. genetic mapping studies showed sc3- and sc3- isbp markers from ctg were located within the terminal deletion bin 3bl- on chromosome 3b, a deletion bin known to contain two major c-bands. previous cytogenetic studies of the wheat chromosomes suggested a large variation of gene-density over the entire length of chromosomes and it is feasible that particularly low gene densities occur within c band regions. the low gene density found in this study is more consistent with studies in triticeae genomes of more proximal regions of chromosome arms and regions of low recombination. the two representatives of tandem repeat families located within the first kb of the ctg sequence also indicate a relationship to heterochromatin, with one being previously identified to be a d-genome centromeric heterochromatin tandem repeat sequence. the presence of a representative of this d-genome specific repeat in the chromosome 3b sequence suggest specific amplification of the sequence family must have occurred in the d genome even though single copy representatives were located elsewhere. the presence of representatives of sequences normally located as tandem arrays in heterochromatin and the presence of high gypsy ltr content support our proposal that this ctg genomic sequence is located within a c-banding region in deletion bin of chromosome 3bl. the highly repetitive element content in ctg was not unusual in wheat sequences, as studies have shown repetitive element contents of greater than 70%, however the high content of the gypsy-like ltr retrotransposon is significant in this study. a previously sequenced wheat bac clone from the same chromosome 3bl- deletion bin also found a similar high proportion of gypsy-like ltr-retrotransposons. in the present study, chromodomains were shown to be located within the internal polyprotein domain of two gypsy-ltr retrotransposons, and are thus potentially key components defining the condensed nature of heterochromatin. one of the potential chromodomains was located immediately adjacent to the gene island in ctg the unusual properties of heterochromatin that would contribute to the unique conservation of both the gene order and relative gene orientation found in this study include: the low levels of recombination and gene activity associated with heterochromatin; the high level of transcript processing by the rnai machinery that mediates chromatin structure. these properties could contribute to the relative isolation of the gene island from the normal processes that lead to genome rearrangements.  bac shotgun sequencing of the fpc physical map contig ctg from the genome of chromosome 3b has provided the largest assembly of a wheat genome region to date. annotation and comparative analysis with four other plant genomes of identified an island of three genes showing complete conservation of gene order and orientation. this conservation could not be readily accounted for by shared functions between the genes and it is proposed that ctg is located within a heterochromatic c-band region of deletion bin 3bl based on the identification of representatives of heterochromatin tandem repeats and presence of significant matches to chromodomain-containing gypsy ltr retrotransposable elements. we speculate that this location, among other highly repetitive sequences, may account for the relative stability of the gene island through insulating the gene island from normal recombination processes. 
in view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro level among the cereals. the ctg gene island also displayed complete conservation of gene order and orientation with syntenic gene islands found in publicly available genome sequences of brachypodium distachyon, oryza sativa, sorghum bicolor and zea mays, even though the intergenic space and introns were divergent. bac shotgun sequencing of the hexaploid wheat genome has been used to assemble a group of wheat bacs from the chromosome 3b physical map fpc contig ctg into a bp genomic sequence. this ctg sequence was annotated for biological features such as genes and transposable elements. we propose that ctg is located within the heterochromatic c-band region of deletion bin 3bl based on the identification of heterochromatic tandem repeats and presence of significant matches to chromodomain-containing gypsy ltr retrotransposable elements. we also speculate that this location, among other highly repetitive sequences, may account for the relative stability in gene order and orientation within the gene island. the ctg contig completed in this paper was initially studied because it was assigned to the sr resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3b and revealed its unusual features. a three-gene island was identified among >80% repetitive dna sequence. using bioinformatics analysis there were no observable similarity in their gene functions. the complexity of the wheat genome has resulted from waves of retrotransposable element insertions.
 the complexity of the wheat genome has resulted from waves of retrotransposable element insertions. gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro level among the cereals. in view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. the ctg contig completed in this paper was initially studied because it was assigned to the sr resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3b and revealed its unusual features.  bac shotgun sequencing of the hexaploid wheat genome has been used to assemble a group of wheat bacs from the chromosome 3b physical map fpc contig ctg into a bp genomic sequence. this ctg sequence was annotated for biological features such as genes and transposable elements. a three-gene island was identified among >80% repetitive dna sequence. using bioinformatics analysis there were no observable similarity in their gene functions. the ctg gene island also displayed complete conservation of gene order and orientation with syntenic gene islands found in publicly available genome sequences of brachypodium distachyon, oryza sativa, sorghum bicolor and zea mays, even though the intergenic space and introns were divergent.  we propose that ctg is located within the heterochromatic c-band region of deletion bin 3bl based on the identification of heterochromatic tandem repeats and presence of significant matches to chromodomain-containing gypsy ltr retrotransposable elements. we also speculate that this location, among other highly repetitive sequences, may account for the relative stability in gene order and orientation within the gene island. sequence data from this article have been deposited with the genbank data libraries under accession no. gq422824 