BACKGROUND
cereal plant crops are vital to the overall health of the world's population and genome sequencing is an important step in the genetic improvement of crops.
while hexaploid wheat  accounts for nearly one-fifth of the entire world's daily calories  <cit> , the sequencing of its genome has been restricted by high sequencing costs associated with its large genome size  and high  repetitive content  <cit> .
the published physical map of the largest wheat chromosome 3b  <cit> , which itself is twice the size of the entire rice genome, has allowed researchers to target specific regions that have been identified to contain agronomically important traits such as fungal resistance or grain quality.
projects co-coordinated within the international wheat genome sequencing consortium  on chromosome 3b aim to tackle the challenges associated with genome sequencing through collaboration, and facilitate the study of significant multi-gene families.
one such multi-gene family found extensively in the wheat and other cereal genomes are the expansins.
it has been estimated that the hexaploid wheat genome contains more than  <dig> expressed members  <cit> , much higher than the rice genome.
expansins belong to a large group of proteins found within the structure of plant cell walls and are considered to be involved in the expansion of cell walls as a tissue grows  <cit> .
the proposed model of expansin action is that these proteins modify the cell-wall matrix to enable growth and development of plant cells  <cit>  and, as a result, expansins have been implicated in providing resistance to certain diseases  <cit> .
the latter was of particular interest because it was located in a region of the wheat genome being sequenced in order to define disease resistance genes in the region.
expansins were originally isolated from cucumber seedlings and have 'acid growth' characteristics, where they can stimulate cell enlargement in the response to acid ph  <cit> .
expansins have now been reported in many plants such as cotton , tomato , arabidopsis  and pea .
cdna clones have also been isolated from wheat  <cit>  and barley .
the multi-gene expansin family can be divided up into two main groups, namely, α-expansin  and beta-expansin proteins , which share very limited  amino acid similarity even though both are associated with cell-loosening activity  <cit> .
the beta-expansin proteins were originally viewed exclusively as group  <dig> pollen allergens but are now considered to be important in cell wall changes during growth in vegetative tissues of grasses and dicotyledon plants  <cit> , most notably in development and growth zones of tissues such as roots  <cit> .
the group  <dig> pollen allergen domain-containing beta-expansin proteins are highly expressed in mature pollen of grass species and are thought to have a role in pollen tube penetration  <cit> .
pollen-triggered allergic reactions  affect up to 25% of adults in industrialized nations  <cit> .
group  <dig> allergens bind to group  <dig> specific ige antibodies  <cit>  and a well studied example is the pollen allergen, phl p  <dig> in timothy grass  where a specific protein domain has been identified as a binding site  <cit> .
a previous study analysed the sera of patients that had undergone positive double blind, placebo-controlled food challenge to hexaploid wheat and identified the gene taexpb <dig> as one of  <dig> genes that encoded proteins binding to the ige from wheat sensitive patients  <cit> .
in the present study, we present the identification of three beta-expansin genes through a genome sequencing study of the short-arm of chromosome 3b in hexaploid wheat .
structural analyses of the three taexpb11-chinese spring variants indicate a localised gene duplication producing non-coding gene copies.
re-analysis of recombinant lines using specific markers from the expansin genes indicated that they were not linked to the sr <dig> resistance phenotype of wheat cv.
hope, arguing against an involvement in the resistance.
comparative sequence analysis was also undertaken on selected cultivars and a t. spelta wheat accession demonstrating changes within the gene-coding sequence of taexpb <dig> produced protein structural changes.
expression of taexpb <dig>  experimentally and within est databases, was also assessed.
our study provides novel insights into the structure of beta-expansins and their variation across different wheat genomes.
RESULTS
ctg <dig> wheat genome sequencing
the physical map of chromosome 3bs of hexaploid wheat  has been compiled through dna fingerprinting of the flow-sorted chromosome 3b bac library and anchoring bac contigs to genetic maps  <cit> .
the shotgun sequencing of bacs located within a minimum tilling path of ca  <dig>  mb across the sr <dig> locus on chromosome 3bs  <cit>  was carried out to identify possible candidate genes for this disease resistance locus .
the bac clones characterized in this study, and related work , indicated the presence of an active expansin gene which was a candidate for the sr <dig> locus because, as a cell wall component, the product of the gene has been associated with disease resistance  <cit> .
a  <dig> kb region the ctg <dig> genome sequence containing three beta-expansin genes was annotated in detail for the present study
the  <dig> kb ctg <dig> sequence contained nine complete or partial gene-coding regions  giving it a gene density of one gene per  <dig>  kb.
the sequence contained  <dig>  bp  repetitive dna in the form of transposable elements  and a  <dig>  bp mitochondrial genome insert.
the presence of the mitochondrial dna insert in normal genomic dna has been confirmed by assaying the genomic sequence for mtdna-chromosomal dna junctions .
further genome sequencing was carried out using 454-technology sequencing of bac clone 3b_036_i <dig> , which contained two full-length beta-expansin genes and the mitochondrial insert, in order to confirm the presence of the mitochondrial insert and improve the sequence assembly in some regions.
additional bac sequencing was also carried out on the syntenic ctg <dig> region of wheat cv.
hope .
structural characterisation and validation of three beta-expansin genes in ctg11
the  <dig> kb subsequence of ctg <dig> was masked for repetitive elements by running repeatmasker  using the triticeae repeat sequence  database http://wheat.pw.usda.gov/itmi/repeats/.
gene predictions were then searched against a protein subsection of the trep database using blastp to ensure no repetitive predicted proteins were included in the analysis.
two highly similar gene models were predicted and found within  <dig>  bp of each other with a third similar truncated copy located over  <dig> kb away .
annotation of the genomic sequence identified that the three copies of sequences were related to the beta-expansin family  <cit> , with the second copy of the three genes being very similar to a published taexpb <dig> cdna   <cit> .
the published taexpb <dig> sequence was recovered from an endosperm cdna library from wheat cv.
wyuna  <cit> .
the coding regions of the beta-expansin that has the highest sequence similarity to the published taexpb <dig> cdna has  <dig> amino acid differences  resulting from single nucleotide polymorphism  differences; these snps are not unexpected considering the published taexpb <dig> derived from wheat cv.
wyuna and the genomic sequence from wheat cv.
chinese spring.
we propose to name this gene taexpb11cs <dig> with the names of copies located upstream being taexpb11cs <dig> and taexpb11cs <dig> respectively.
the taexpb <dig> from wheat cv.
wyuna  <cit>  will be referred to as taexpb11wy in this study.
taexpb11cs <dig> had a lower sequence similarity  to the taexpb11wy cdna and a slightly lower similarity to the identified rice homolog osexpb <dig> .
the taexpb11cs <dig> gene was very closely related to taexpb11wy except for the fact that the gene is truncated, containing only one exon .
the coding regions of all the genes were found to have high sequence similarity  to a full-length wheat cdna from a recently published database of  <dig>  full-length wheat cdna sequences .
comparing the nucleotide sequence to taexpb11wy identified the intron-exon structure of taexpb11cs <dig> .
taexpb11cs <dig> and taexpb11cs <dig>  both have three exons with intron  <dig>  being more than twice the size of intron  <dig> , while the truncated gene taexpb11cs <dig> does not contain any introns.
taexpb11cs <dig> and has a  <dig> bp insertion at position 90- <dig>  within the first exon of the gene when compared to taexpb11cs <dig>  this insertion in taexpb11cs <dig> contains a stop codon which suggests that it is probably a pseudo-gene.
the intron  <dig> sequences from taexpb11cs <dig> and taexpb11cs <dig> were compared to identify whether or not they represented recent duplications.
both genes sequences have a 92% sequence similarity over  <dig>  bp.
outside the coding sequence of the genes,  <dig> bp of sequence 5' from the start codon and  <dig> bp 3' from the end codon, are conserved between taexpb11cs <dig> and taexpb11cs <dig>  the comparison in figure  <dig> between taexpb11cs <dig> and taexpb11cs <dig> demonstrates that the first intron is found to be well conserved between both sequences, as well as  <dig> bp of genomic sequence 5' to the start of the first exon.
the beta-expansin genes amplified from the genomic dna of a number of varieties of wheat , showed that the respective taexpb11cs2-type genes were readily distinguished from taexpb11cs <dig> based on sequence divergence in intron  <dig>  intron  <dig> was not as diagnostic for distinguishing the taexpb11cs <dig> and taexpb11cs <dig> gene categories.
exon  <dig> in taexpb11cs <dig> showed a characteristic  <dig> bp insertion that was not present in any of the other taexp11cs type genes.
the ages of both taexpb11cs <dig> and taexpb11cs <dig>  were estimated using methods used to date ltr retrotransposons  <cit> .
the nucleotide sequence used for comparison between taexpb11cs <dig> and the two proposed gene duplications was from the start codon of each gene or gene fragment , covering all of exon  <dig> and part of the intron  <dig> sequence .
the taexpb11cs <dig> duplication was identified to have duplicated  <dig>  million years ago  with a standard deviation of  <dig>  mya, while the taexpb11cs <dig> duplication was much younger at only  <dig>  mya  of ca  <dig> bp, in reasonable agreement with the expected sizes.
the unusual structure of taexpb11cs <dig>  was shown to exist in genomic dna by amplifying a dna fragment using primers from within the respective insertions and which were predicted to generate a  <dig> bp fragment.
the results  indicated that the fragment obtained was slightly larger than  <dig> bp using agarose gel electrophoresis, in agreement with the expected size.
the analysis of nulli-tetra stocks of wheat showed that taexpb11cs <dig> was present only on chromosome 3b since the pcr product was missing when chromosome 3b was missing from the wheat line analysed .
the transcribed gene, taexpb11cs <dig>  was shown to be present on chromosomes 3a, 3b and 3d using the same nulli-tetra stocks illustrated in figure  <dig>  generated pcr products of the same size from the three chromosomes .
several snp differences between taexpb11cs <dig> on chromosomes 3a, 3b and 3d were detected by sequencing a genome pcr product directly .
sequencing of the pcr products from the respective nulli-tetra lines allowed the assignment of the base pair differences to a chromosome.
in figure  <dig>  an example is presented for exon  <dig> where several base positions show a mixed base-call in the lower panel  where chromosome 3b is present .
in the genetic stocks where 3b is missing  the mixed base-call is resolved indicating that the base which is removed in the top panel is located on the chromosome 3b site for expansin exbp <dig>  although only two examples are shown in figure  <dig>  all possible nulli-tetra combinations for 3a, 3b and 3d were analysed.
based on snp analyses, the 3b gene was more similar to the published taexb <dig> than the genes on 3a and 3d but the relationship was not unambiguous because of the background of snps expected between wheat cv.
chinese spring  and cv.
wyuna .
comparative sequence analysis in the beta-expansin gene sequence from selected wheat species
the beta-expansin gene pcr products from wheat cv.
westonia, cv.
hope and cv.
wyuna , and a t. spelta line were compared to taexpb11cs <dig> in figure  <dig>  the cv westonia and t. spelta survey sequences were recovered from genomic dna using primers positioned just inside the first exon and  <dig> bp before the end of the gene in order to amplify exon and intron sequences.
the resulting sequences amplified from the dna samples was ~ <dig>  bp in length and were specific as judged from direct sequencing of the pcr product.
in the genomic bac sequencing of wheat cv.
hope , an orf from the sequence was identified and named taexpb11hp as it contained the three predicted exons and was shown to have high sequence similarity to taexpb11wy as well as taexpb11cs <dig>  exon  <dig> of this taexpb11hp gene was truncated due to a point mutation causing a premature end to the coding sequence.
overall the nucleotide sequence similarity of taexpb11hp and taexpb11cs <dig> was 90%.
the wheat cv.
westonia beta-expansin  and t. spelta  accessions showed  <dig> % sequence similarity.
transcription of the taexpb11cs genes
assessing the data sets available from the ncbi unigene est profile  indicated that two taexpb <dig> ests were present in libraries comprising  <dig>  est clusters.
the ests were identified as being expressed from seed  and flower .
no evidence could be found for the expression of taexpb11cs <dig>  using the insertion sequence in exon  <dig> that is unique to this gene, as a probe.
the endosperm expression of taexpb11cs <dig> was confirmed by rt-pcr .
the tissue analysed was from developing grain with endosperm and embryo tissues separated by hand.
the pericarp tissue was retained as an example of maternal tissue.
in a study of the time course of expression, using tissues collected  <dig>   <dig>   <dig>   <dig> and  <dig> days post anthesis, strong expression was found for the three tissues at all stages of development.
quantitative analyses of the rt-pcr data indicated that the relatively lower expression for the maternal tissue suggestive in figure  <dig> was not significant .
no evidence for the transcription of taexpb11cs <dig> could be found in these rt-pcr experiments consistent with the analysis of available est databases.
protein domain characterisation
the well-characterised maize beta-expansin gene  purified from maize pollen, and its crystal structure  <cit>  was used to validate the two protein domains commonly identified in expansins, within the full-length taexpb11cs <dig> and taexpb11hp genes.
both genes contained conserved cysteine residues that create disulphide bonds between the domain folds as well as the characteristic 'hfd' motif that is the catalytic site of the distantly related family- <dig> endoglucanases   <cit>  .
domain  <dig> is a lipoprotein a -like double-psi beta-barrel family domain  commonly found at the n-terminus of pollen allergens.
domain  <dig>  is a pollen_allerg_ <dig> family , grass type- <dig> pollen allergen domain originally characterised in timothy grass   <cit> .
the conserved residues in domain  <dig> outlined in  <cit>  could also be identified although, when compared to the maize expb <dig> sequence, its amino acid sequence is shown to be more diverged  compared to domain  <dig> .
as mentioned previously , domain  <dig> is truncated in taexpb11hp due to a stop codon terminating the coding sequence.
discussion
expansins, a cell-wall loosening class of proteins, are a multigene family found in grass genomes that are considered to play important roles in growth and development in wheat  <cit> ; est mining has estimated that there exist at least  <dig> and  <dig> alpha- and beta-expansins, respectively  <cit>  in wheat.
the higher beta-expansin gene estimate compared to alpha-expansins within wheat is consistent with the estimates in other grasses such as maize  <cit> .
different expression characteristics suggest that the two expansin classes play different functional roles within the cell wall structure  <cit> .
in the present study we identified and characterised three beta-expansin genes from wheat cv.
chinese spring located in a ~ <dig> kb region of chromosome 3bs.
only one of these genes was found to be the taexpb <dig> gene, coding for an ige binding protein identified from the sera of patients that had undergone positive double-blind, placebo-controlled food challenge to wheat  <cit> .
sequence analysis of specific pcr products using taexpb <dig> primers from the wheat cultivars and a t. spelta wheat accession validated the identification of the wheat cv.
chinese spring variant of taexpb <dig> cdna  <cit> , originally obtained from wheat cv.
wyuna  <cit> .
it is proposed that sequence variants of taexpb <dig> be designated taexpb11wy, taexpb11cs, taexpb11ws, taexpb11hp.
the high sequence similarity between the taexpb <dig> cdna and taexpb11cs <dig> provided good evidence for assigning the two sequences to be alleles of the same gene.
even though a large family of expansin genes has been reported in wheat  <cit> , the primers used to assay the transcripts  and gene locations did not provide any evidence of the presence of other copies of this particular gene elsewhere in the genome.
the primers are evidently specific enough to assay only the taexpb <dig> gene category on homoeologous locations on chromosome 3a, 3b and 3d.
utilizing snps identified in the present study, expansin was ruled out as a candidate gene for sr <dig> resistance because re-examination of recombinant chinese spring-hope lines studied by kota et al  <cit>  identified a recombination event between the sr <dig> phenotype and taexpb11hp .
we note that in the region examined, wheat cv hope  is missing the mitochondrial dna insert found in wheat cv chinese spring.
this mitochondrial dna segment encodes a gene nad <dig>  which is a subunit of nadh dehydrogenase that is active in producing reactive oxygen species.
although this is not genetically linked to the sr <dig> resistance gene, its close proximity may indicate it is part of a gene network controlling levels of active oxygen  <cit>  .
the taexpb11cs genes identified from the genome sequencing of the ctg <dig> contig on the small arm of wheat chromosome 3b could be characterized in detail.
the taexpb11cs <dig> gene fragment contained only one coding exon and showed an extremely high sequence similarity to taexpb11cs <dig>  this sequence similarity extended not only over its coding sequence but also the  <dig> bp 5' upstream from the start of the coding sequence of its only exon, as well as part of the sequence corresponding to the first intron sequence.
the high sequence similarity over the coding and non-coding regions of the two sequences indicated that taexpb11cs <dig> is a very recent duplication of taexpb11cs <dig> with an estimated duplication age of  <dig>  mya.
direct genome analysis confirmed the existence of taexpb11cs <dig>  the neighbouring cacta dna transposon 'caspar' found within a base pair of the duplicated taexpb11cs <dig> fragment suggests the possibility that this genomic duplication was mediated by this te or was the result of a te-mediated recombination event  <cit> .
while cacta dna transposons have not yet been shown to be involved in gene fragment duplication and the creation of chimeric genes, repetitive elements such as pack-mules found in multiple copies within the rice genome, have been shown to capture gene fragments and other genomic dna to create chimeric orfs  <cit> .
the taexpb11cs <dig> gene appeared to be an older duplication of taexpb11cs <dig> with an estimated age of the duplication being  <dig>  mya.
there was significant conserved nucleotide sequence either side of the gene coding sequences and a high level of sequence similarity between taexpb11cs <dig> and taexpb11cs <dig> intron <dig> sequences.
the characteristic insertions within taexpb11cs <dig>  were validated by direct genome pcr and the pseudo-gene was shown to exist only on chromosome 3b using nulli-tetra mapping lines of wheat.
at the level of the snp analysis it is clear that very similar genes exist as homoeologues on chromosomes 3a, 3b and 3d.
each of these homoeologous genes could be contributing to the mrna assayed in figure  <dig>  the snp analysis in figure  <dig> is consistent with taexpb11cs <dig> representing the cdna identified as taexpb <dig>
a well-characterised maize beta-expansin gene  was used as a comparison with taexpb11cs <dig>  to identify the particular motifs that are conserved between the protein domains.
the three wheat beta-expansin genes, shown in figure  <dig>  contained the conserved cysteine residues that form disulphide bonds between the domain folds and the 'hfd' motif that is common in the catalytic site of the distantly related family- <dig> endoglucanases   <cit> .
they also contained many conserved residues in domain  <dig> outlined in  <cit> , but when compared to the maize expb <dig> sequence, this allergen domain amino acids sequence is shown to be more diverged  compared to domain  <dig> , .
similar folding patterns were identified in taexpb11hp except that the amino acid sequence in the pollen allergen domain was largely missing.
CONCLUSIONS
sequence analysis and annotation of  <dig> kb of chromosome 3b genomic sequence identified three beta-expansin genes, one of which was identified to be taexpb <dig>  originally from a cdna identified from wheat cv.
wyuna.
through the analysis of intron sequences of the three wheat cv chinese spring genes, we propose that two of these beta-expansin genes are duplications of the taexpb <dig> gene.
comparative sequence analysis with two other wheat cultivars  and a t. spelta accession validated the identification of the wheat cv.
chinese spring variant of taexpb <dig>  est and rt-pcr experiments confirmed the expression in maternal and grain tissues.
the variation established in this gene suggests it is a clearly identifiable member of a gene family and reflects the dynamic features of the wheat genome as it adapted to a range of different environments and uses.

 cereal plant crops are vital to the overall health of the world's population and genome sequencing is an important step in the genetic improvement of crops. while hexaploid wheat accounts for nearly one-fifth of the entire world's daily calories, the sequencing of its genome has been restricted by high sequencing costs associated with its large genome size and high repetitive content. the published physical map of the largest wheat chromosome 3b, which itself is twice the size of the entire rice genome, has allowed researchers to target specific regions that have been identified to contain agronomically important traits such as fungal resistance or grain quality. projects co-coordinated within the international wheat genome sequencing consortium on chromosome 3b aim to tackle the challenges associated with genome sequencing through collaboration, and facilitate the study of significant multi-gene families. one such multi-gene family found extensively in the wheat and other cereal genomes are the expansins. it has been estimated that the hexaploid wheat genome contains more than expressed members, much higher than the rice genome. expansins belong to a large group of proteins found within the structure of plant cell walls and are considered to be involved in the expansion of cell walls as a tissue grows. the proposed model of expansin action is that these proteins modify the cell-wall matrix to enable growth and development of plant cells and, as a result, expansins have been implicated in providing resistance to certain diseases. the latter was of particular interest because it was located in a region of the wheat genome being sequenced in order to define disease resistance genes in the region. expansins were originally isolated from cucumber seedlings and have'acid growth' characteristics, where they can stimulate cell enlargement in the response to acid ph. expansins have now been reported in many plants such as cotton, tomato, arabidopsis and pea. cdna clones have also been isolated from wheat and barley. the multi-gene expansin family can be divided up into two main groups, namely, α-expansin and beta-expansin proteins, which share very limited amino acid similarity even though both are associated with cell-loosening activity. the beta-expansin proteins were originally viewed exclusively as group pollen allergens but are now considered to be important in cell wall changes during growth in vegetative tissues of grasses and dicotyledon plants, most notably in development and growth zones of tissues such as roots. the group pollen allergen domain-containing beta-expansin proteins are highly expressed in mature pollen of grass species and are thought to have a role in pollen tube penetration. pollen-triggered allergic reactions affect up to 25% of adults in industrialized nations. group allergens bind to group specific ige antibodies and a well studied example is the pollen allergen, phl p in timothy grass where a specific protein domain has been identified as a binding site. a previous study analysed the sera of patients that had undergone positive double blind, placebo-controlled food challenge to hexaploid wheat and identified the gene taexpb as one of genes that encoded proteins binding to the ige from wheat sensitive patients. in the present study, we present the identification of three beta-expansin genes through a genome sequencing study of the short-arm of chromosome 3b in hexaploid wheat. structural analyses of the three taexpb11-chinese spring variants indicate a localised gene duplication producing non-coding gene copies. re-analysis of recombinant lines using specific markers from the expansin genes indicated that they were not linked to the sr resistance phenotype of wheat cv. hope, arguing against an involvement in the resistance. comparative sequence analysis was also undertaken on selected cultivars and a t. spelta wheat accession demonstrating changes within the gene-coding sequence of taexpb produced protein structural changes. expression of taexpb experimentally and within est databases, was also assessed. our study provides novel insights into the structure of beta-expansins and their variation across different wheat genomes.  ctg wheat genome sequencing the physical map of chromosome 3bs of hexaploid wheat has been compiled through dna fingerprinting of the flow-sorted chromosome 3b bac library and anchoring bac contigs to genetic maps. the shotgun sequencing of bacs located within a minimum tilling path of ca mb across the sr locus on chromosome 3bs was carried out to identify possible candidate genes for this disease resistance locus. the bac clones characterized in this study, and related work, indicated the presence of an active expansin gene which was a candidate for the sr locus because, as a cell wall component, the product of the gene has been associated with disease resistance. a kb region the ctg genome sequence containing three beta-expansin genes was annotated in detail for the present study the kb ctg sequence contained nine complete or partial gene-coding regions giving it a gene density of one gene per kb. the sequence contained bp repetitive dna in the form of transposable elements and a bp mitochondrial genome insert. the presence of the mitochondrial dna insert in normal genomic dna has been confirmed by assaying the genomic sequence for mtdna-chromosomal dna junctions. further genome sequencing was carried out using 454-technology sequencing of bac clone 3b_036_i, which contained two full-length beta-expansin genes and the mitochondrial insert, in order to confirm the presence of the mitochondrial insert and improve the sequence assembly in some regions. additional bac sequencing was also carried out on the syntenic ctg region of wheat cv. hope. structural characterisation and validation of three beta-expansin genes in ctg11 the kb subsequence of ctg was masked for repetitive elements by running repeatmasker using the triticeae repeat sequence database http://wheat.pw.usda.gov/itmi/repeats/. gene predictions were then searched against a protein subsection of the trep database using blastp to ensure no repetitive predicted proteins were included in the analysis. two highly similar gene models were predicted and found within bp of each other with a third similar truncated copy located over kb away. annotation of the genomic sequence identified that the three copies of sequences were related to the beta-expansin family, with the second copy of the three genes being very similar to a published taexpb cdna. the published taexpb sequence was recovered from an endosperm cdna library from wheat cv. wyuna. the coding regions of the beta-expansin that has the highest sequence similarity to the published taexpb cdna has amino acid differences resulting from single nucleotide polymorphism differences; these snps are not unexpected considering the published taexpb derived from wheat cv. wyuna and the genomic sequence from wheat cv. chinese spring. we propose to name this gene taexpb11cs with the names of copies located upstream being taexpb11cs and taexpb11cs respectively. the taexpb from wheat cv. wyuna will be referred to as taexpb11wy in this study. taexpb11cs had a lower sequence similarity to the taexpb11wy cdna and a slightly lower similarity to the identified rice homolog osexpb. the taexpb11cs gene was very closely related to taexpb11wy except for the fact that the gene is truncated, containing only one exon. the coding regions of all the genes were found to have high sequence similarity to a full-length wheat cdna from a recently published database of full-length wheat cdna sequences. comparing the nucleotide sequence to taexpb11wy identified the intron-exon structure of taexpb11cs. taexpb11cs and taexpb11cs both have three exons with intron being more than twice the size of intron, while the truncated gene taexpb11cs does not contain any introns. taexpb11cs and has a bp insertion at position 90- within the first exon of the gene when compared to taexpb11cs this insertion in taexpb11cs contains a stop codon which suggests that it is probably a pseudo-gene. the intron sequences from taexpb11cs and taexpb11cs were compared to identify whether or not they represented recent duplications. both genes sequences have a 92% sequence similarity over bp. outside the coding sequence of the genes, bp of sequence 5' from the start codon and bp 3' from the end codon, are conserved between taexpb11cs and taexpb11cs the comparison in figure between taexpb11cs and taexpb11cs demonstrates that the first intron is found to be well conserved between both sequences, as well as bp of genomic sequence 5' to the start of the first exon. the beta-expansin genes amplified from the genomic dna of a number of varieties of wheat, showed that the respective taexpb11cs2-type genes were readily distinguished from taexpb11cs based on sequence divergence in intron intron was not as diagnostic for distinguishing the taexpb11cs and taexpb11cs gene categories. exon in taexpb11cs showed a characteristic bp insertion that was not present in any of the other taexp11cs type genes. the ages of both taexpb11cs and taexpb11cs were estimated using methods used to date ltr retrotransposons. the nucleotide sequence used for comparison between taexpb11cs and the two proposed gene duplications was from the start codon of each gene or gene fragment, covering all of exon and part of the intron sequence. the taexpb11cs duplication was identified to have duplicated million years ago with a standard deviation of mya, while the taexpb11cs duplication was much younger at only mya of ca bp, in reasonable agreement with the expected sizes. the unusual structure of taexpb11cs was shown to exist in genomic dna by amplifying a dna fragment using primers from within the respective insertions and which were predicted to generate a bp fragment. the results indicated that the fragment obtained was slightly larger than bp using agarose gel electrophoresis, in agreement with the expected size. the analysis of nulli-tetra stocks of wheat showed that taexpb11cs was present only on chromosome 3b since the pcr product was missing when chromosome 3b was missing from the wheat line analysed. the transcribed gene, taexpb11cs was shown to be present on chromosomes 3a, 3b and 3d using the same nulli-tetra stocks illustrated in figure generated pcr products of the same size from the three chromosomes. several snp differences between taexpb11cs on chromosomes 3a, 3b and 3d were detected by sequencing a genome pcr product directly. sequencing of the pcr products from the respective nulli-tetra lines allowed the assignment of the base pair differences to a chromosome. in figure an example is presented for exon where several base positions show a mixed base-call in the lower panel where chromosome 3b is present. in the genetic stocks where 3b is missing the mixed base-call is resolved indicating that the base which is removed in the top panel is located on the chromosome 3b site for expansin exbp although only two examples are shown in figure all possible nulli-tetra combinations for 3a, 3b and 3d were analysed. based on snp analyses, the 3b gene was more similar to the published taexb than the genes on 3a and 3d but the relationship was not unambiguous because of the background of snps expected between wheat cv. chinese spring and cv. wyuna. comparative sequence analysis in the beta-expansin gene sequence from selected wheat species the beta-expansin gene pcr products from wheat cv. westonia, cv. hope and cv. wyuna, and a t. spelta line were compared to taexpb11cs in figure the cv westonia and t. spelta survey sequences were recovered from genomic dna using primers positioned just inside the first exon and bp before the end of the gene in order to amplify exon and intron sequences. the resulting sequences amplified from the dna samples was ~ bp in length and were specific as judged from direct sequencing of the pcr product. in the genomic bac sequencing of wheat cv. hope, an orf from the sequence was identified and named taexpb11hp as it contained the three predicted exons and was shown to have high sequence similarity to taexpb11wy as well as taexpb11cs exon of this taexpb11hp gene was truncated due to a point mutation causing a premature end to the coding sequence. overall the nucleotide sequence similarity of taexpb11hp and taexpb11cs was 90%. the wheat cv. westonia beta-expansin and t. spelta accessions showed % sequence similarity. transcription of the taexpb11cs genes assessing the data sets available from the ncbi unigene est profile indicated that two taexpb ests were present in libraries comprising est clusters. the ests were identified as being expressed from seed and flower. no evidence could be found for the expression of taexpb11cs using the insertion sequence in exon that is unique to this gene, as a probe. the endosperm expression of taexpb11cs was confirmed by rt-pcr. the tissue analysed was from developing grain with endosperm and embryo tissues separated by hand. the pericarp tissue was retained as an example of maternal tissue. in a study of the time course of expression, using tissues collected and days post anthesis, strong expression was found for the three tissues at all stages of development. quantitative analyses of the rt-pcr data indicated that the relatively lower expression for the maternal tissue suggestive in figure was not significant. no evidence for the transcription of taexpb11cs could be found in these rt-pcr experiments consistent with the analysis of available est databases. protein domain characterisation the well-characterised maize beta-expansin gene purified from maize pollen, and its crystal structure was used to validate the two protein domains commonly identified in expansins, within the full-length taexpb11cs and taexpb11hp genes. both genes contained conserved cysteine residues that create disulphide bonds between the domain folds as well as the characteristic'hfd' motif that is the catalytic site of the distantly related family- endoglucanases. domain is a lipoprotein a -like double-psi beta-barrel family domain commonly found at the n-terminus of pollen allergens. domain is a pollen_allerg_ family, grass type- pollen allergen domain originally characterised in timothy grass. the conserved residues in domain outlined in could also be identified although, when compared to the maize expb sequence, its amino acid sequence is shown to be more diverged compared to domain. as mentioned previously, domain is truncated in taexpb11hp due to a stop codon terminating the coding sequence. discussion expansins, a cell-wall loosening class of proteins, are a multigene family found in grass genomes that are considered to play important roles in growth and development in wheat; est mining has estimated that there exist at least and alpha- and beta-expansins, respectively in wheat. the higher beta-expansin gene estimate compared to alpha-expansins within wheat is consistent with the estimates in other grasses such as maize. different expression characteristics suggest that the two expansin classes play different functional roles within the cell wall structure. in the present study we identified and characterised three beta-expansin genes from wheat cv. chinese spring located in a ~ kb region of chromosome 3bs. only one of these genes was found to be the taexpb gene, coding for an ige binding protein identified from the sera of patients that had undergone positive double-blind, placebo-controlled food challenge to wheat. sequence analysis of specific pcr products using taexpb primers from the wheat cultivars and a t. spelta wheat accession validated the identification of the wheat cv. chinese spring variant of taexpb cdna, originally obtained from wheat cv. wyuna. it is proposed that sequence variants of taexpb be designated taexpb11wy, taexpb11cs, taexpb11ws, taexpb11hp. the high sequence similarity between the taexpb cdna and taexpb11cs provided good evidence for assigning the two sequences to be alleles of the same gene. even though a large family of expansin genes has been reported in wheat, the primers used to assay the transcripts and gene locations did not provide any evidence of the presence of other copies of this particular gene elsewhere in the genome. the primers are evidently specific enough to assay only the taexpb gene category on homoeologous locations on chromosome 3a, 3b and 3d. utilizing snps identified in the present study, expansin was ruled out as a candidate gene for sr resistance because re-examination of recombinant chinese spring-hope lines studied by kota et al identified a recombination event between the sr phenotype and taexpb11hp. we note that in the region examined, wheat cv hope is missing the mitochondrial dna insert found in wheat cv chinese spring. this mitochondrial dna segment encodes a gene nad which is a subunit of nadh dehydrogenase that is active in producing reactive oxygen species. although this is not genetically linked to the sr resistance gene, its close proximity may indicate it is part of a gene network controlling levels of active oxygen. the taexpb11cs genes identified from the genome sequencing of the ctg contig on the small arm of wheat chromosome 3b could be characterized in detail. the taexpb11cs gene fragment contained only one coding exon and showed an extremely high sequence similarity to taexpb11cs this sequence similarity extended not only over its coding sequence but also the bp 5' upstream from the start of the coding sequence of its only exon, as well as part of the sequence corresponding to the first intron sequence. the high sequence similarity over the coding and non-coding regions of the two sequences indicated that taexpb11cs is a very recent duplication of taexpb11cs with an estimated duplication age of mya. direct genome analysis confirmed the existence of taexpb11cs the neighbouring cacta dna transposon'caspar' found within a base pair of the duplicated taexpb11cs fragment suggests the possibility that this genomic duplication was mediated by this te or was the result of a te-mediated recombination event. while cacta dna transposons have not yet been shown to be involved in gene fragment duplication and the creation of chimeric genes, repetitive elements such as pack-mules found in multiple copies within the rice genome, have been shown to capture gene fragments and other genomic dna to create chimeric orfs. the taexpb11cs gene appeared to be an older duplication of taexpb11cs with an estimated age of the duplication being mya. there was significant conserved nucleotide sequence either side of the gene coding sequences and a high level of sequence similarity between taexpb11cs and taexpb11cs intron sequences. the characteristic insertions within taexpb11cs were validated by direct genome pcr and the pseudo-gene was shown to exist only on chromosome 3b using nulli-tetra mapping lines of wheat. at the level of the snp analysis it is clear that very similar genes exist as homoeologues on chromosomes 3a, 3b and 3d. each of these homoeologous genes could be contributing to the mrna assayed in figure the snp analysis in figure is consistent with taexpb11cs representing the cdna identified as taexpb a well-characterised maize beta-expansin gene was used as a comparison with taexpb11cs to identify the particular motifs that are conserved between the protein domains. the three wheat beta-expansin genes, shown in figure contained the conserved cysteine residues that form disulphide bonds between the domain folds and the'hfd' motif that is common in the catalytic site of the distantly related family- endoglucanases. they also contained many conserved residues in domain outlined in, but when compared to the maize expb sequence, this allergen domain amino acids sequence is shown to be more diverged compared to domain,. similar folding patterns were identified in taexpb11hp except that the amino acid sequence in the pollen allergen domain was largely missing.  sequence analysis and annotation of kb of chromosome 3b genomic sequence identified three beta-expansin genes, one of which was identified to be taexpb originally from a cdna identified from wheat cv. wyuna. through the analysis of intron sequences of the three wheat cv chinese spring genes, we propose that two of these beta-expansin genes are duplications of the taexpb gene. comparative sequence analysis with two other wheat cultivars and a t. spelta accession validated the identification of the wheat cv. chinese spring variant of taexpb est and rt-pcr experiments confirmed the expression in maternal and grain tissues. the variation established in this gene suggests it is a clearly identifiable member of a gene family and reflects the dynamic features of the wheat genome as it adapted to a range of different environments and uses. 
through the analysis of intron sequences of the three wheat cv. spelta line validated the identification of the chinese spring variant of taexpb the expression in maternal and grain tissues was confirmed by examining est databases and carrying out rt-pcr experiments. the expansin family can be divided up into two main groups, namely, alpha-expansin and beta-expansin proteins, with the expb group being of particular interest as group 1-pollen allergens. expansins form a large multi-gene family found in wheat and other cereal genomes that are involved in the expansion of cell walls as a tissue grows. the variation established in this gene suggests it is a clearly identifiable member of a gene family and reflects the dynamic features of the wheat genome as it adapted to a range of different environments and uses. the analysis of a kb sub-sequence of fpc contig ctg identified one beta-expansin genes to be taexpb originally identified as a cdna from the wheat cv wyuna. in this study, three beta-expansin genes were identified and characterized from a newly sequenced region of the triticum aestivum cv. chinese spring genes, we propose that two of these beta-expansin genes are duplications of the taexpb gene. chinese spring chromosome 3b physical map at the sr locus. comparative sequence analysis with two other wheat cultivars and a triticum aestivum var. through 3-d structural protein comparisons with zea mays expb we proposed that variations within the coding sequence of taexpb in wheats may produce a functional change within features such as domain related to possible involvement in cell wall structure and domain defining the pollen allergen domain and binding to ige protein.
 expansins form a large multi-gene family found in wheat and other cereal genomes that are involved in the expansion of cell walls as a tissue grows. the expansin family can be divided up into two main groups, namely, alpha-expansin and beta-expansin proteins, with the expb group being of particular interest as group 1-pollen allergens.  in this study, three beta-expansin genes were identified and characterized from a newly sequenced region of the triticum aestivum cv. chinese spring chromosome 3b physical map at the sr locus. the analysis of a kb sub-sequence of fpc contig ctg identified one beta-expansin genes to be taexpb originally identified as a cdna from the wheat cv wyuna. through the analysis of intron sequences of the three wheat cv. chinese spring genes, we propose that two of these beta-expansin genes are duplications of the taexpb gene. comparative sequence analysis with two other wheat cultivars and a triticum aestivum var. spelta line validated the identification of the chinese spring variant of taexpb the expression in maternal and grain tissues was confirmed by examining est databases and carrying out rt-pcr experiments. detailed examination of the position of taexpb relative to the locus encoding sr disease resistance ruled out the possibility of this gene directly contributing to the resistance phenotype.  through 3-d structural protein comparisons with zea mays expb we proposed that variations within the coding sequence of taexpb in wheats may produce a functional change within features such as domain related to possible involvement in cell wall structure and domain defining the pollen allergen domain and binding to ige protein. the variation established in this gene suggests it is a clearly identifiable member of a gene family and reflects the dynamic features of the wheat genome as it adapted to a range of different environments and uses. accession numbers: ctg =fn564426 survey sequences of taexpb11ws and tsexpb are provided request. 