BACKGROUND
all cereal crop species are members of the grass  family that is the fourth largest family of flowering plants.
with about  <dig>  <dig> species growing under nearly all climates and latitudes, grasses exceed all other plant families in ecological dominance and economic importance.
in terms of genome organisation they represent a very diverse family with basic chromosome numbers ranging from  <dig> to  <dig> and genome sizes ranging from  <dig> mb to  <dig> gb  <cit> .
fossil data and phylogenetic studies have estimated that the grasses have diverged from a common ancestor  <dig> to  <dig> million years ago   <cit> .
archaeological records suggest that farming started concomitantly in at least three widely separated regions between  <dig> 000- <dig>  <dig> years ago during the late neolithic period.
the three most important cereals were independently domesticated in three centres: wheat in south western asia in the 'fertile crescent' region, maize in mexico and rice in both south east asia and west africa  <cit> .
within the poaceae, the genera aegilops and triticum include several diploid species  that, via allopolyploidization, produced several tetraploid and hexaploid wheat species, most of which have been domesticated  <cit> .
t. turgidum  was derived from a hybridization event that happened  between t. urartu, , the diploid donor of the a genome , and another unknown species of the sitopsis section, donor of the b genome , for which the closest known relative is ae.
speltoides  <cit> .
the hexaploid wheat  originated from an additional polyploidization event between the early-domesticated tetraploid t. turgidum ssp dicoccum and the diploid donor of the d genome , ae.
tauschii ,  <dig>  <dig> to  <dig>  <dig> years ago .
several wheat phylogeny studies have tried to identify the progenitor of the b genome of polyploid wheat based on cytology  <cit> , nuclear and mitochondrial dna sequences  <cit>  as well as chromosome rearrangement studies   <cit> .
it remains controversial from those studies whether the progenitor of the b genome is a unique aegilops species  or whether this genome resulted from an introgression of several parental aegilops species .
more recent and representative molecular comparisons using germplasm collections have shown that the b genome could be related to several ae.
speltoides lines but not to other species of the sitopsis section  <cit> .
transposable elements  have been shown since the seventies to be well represented in the wheat genome, ~80%  <cit> .
comparative studies have shown that beside the general conservation in coding sequences, no te insertions are conserved between the a, b and d genomes of wheat whereas important proportion of te insertions are shared between the a or d genomes of polyploid wheat and their respective progenitors t. urartu and ae.
tauschii  <cit> .
no such studies have been yet reported comparing the b genome of these polyploid wheat species to that of its closest known diploid relative, i.e.
ae.
speltoides.
in the present study, we compared for the first time coding and non-coding sequences as well as dynamics of te insertions between the s genome of ae.
speltoïdes and that of the a, b and d co-resident in the hexaploid wheat .
the spa  locus region, belonging to bzip , located on chromosome 1bl  <cit> , has been chosen because of its importance as trans-acting elements of seed storage protein and its conservation in several other cereals such as maize , rice , and barley .
updating phylogeny relationships and insights onto the origin of the b genome are discussed.
RESULTS
organization of spa locus region in the a, b, d and s genomes
three bac clones spanning the spa gene of the a, b and d genomes of t. aestivum were screened from cv renan bac library with pcr markers specific for each of the three spa genes  <cit> .
sequencing resulted in  <dig>  <dig>   <dig>  <dig> and  <dig>  <dig> bp for, respectively, the a, b and d genomes.
screening of an ae.
speltoides pooled bac library with the same spa-specific pcr markers allowed us to identify and sequence a bac clone of  <dig>  <dig> bp sequence spanning the spa locus gene.
annotation has been performed to identify and compare gene and repeat contents of the four available genome sequences, graphically presented in figure 1a.
more details are also presented in additional file  <dig>  as expected for wheat, the four genomic sequences are very rich in tes.
overall, the  <dig>  <dig> bp a genome sequence is structured as  <dig>  <dig> bp  of class i te,  <dig>  <dig> bp  of class ii elements and  <dig> % of unclassified te.
fourteen class i tes are identified as one incompletely sequenced , five truncated ,  <dig> relics , one fragmented  and three complete elements.
the class ii tes is represented as a complete cacta element  and three mites .
besides the identification of tes a pseudo tubulin gene separated by  <dig>  <dig> bp from the spa gene was also identified, both genes covering  <dig> % of the sequence.
the  <dig>  <dig> bp b genome sequence is structured as  <dig>  <dig> bp  of class i tes,  <dig>  <dig> bp  of class ii elements and  <dig> % of unclassified elements.
twelve class i elements are identified as two incompletes, six truncated, two relics, one fragmented and one complete element.
the class ii tes consists of two complete, one fragmented and one truncated cacta  as well as three mites.
the spa gene is the only gene identified on the b genome sequence, representing  <dig> % of the sequence.
the  <dig>  <dig> bp d genome sequence is structured as  <dig>  <dig> bp  of class i tes,  <dig>  <dig> bp  of class ii elements.
twenty-two class i tes are identified as two incomplete, eight truncated, eight relics, two fragmented and two complete elements.
class ii tes are represented as three truncated cacta elements , one mutator relic and one mite.
three genes have been annotated on the d genome sequence, the spa gene, a putative kinesin and a putative cortical cell-delineating gene, covering  <dig> % of a  <dig>  <dig> bp interval.
the  <dig>  <dig> bp s genome sequence is structured as  <dig>  <dig> bp  of class i tes, and a single mite class ii te.
thirteen class i tes are identified as one incomplete, six truncated, four fragmented and two complete tes .
as in the b genome sequence, only the spa gene, covering  <dig> % of the annotated sequence, has been identified on the s genome sequence.
identification and characterization of conserved sequences
alignment of the four genomic regions allows the identification of the 'spa orthologous region', which we have defined as the shared common regions delimitated by conserved non-coding sequence  stretches  that do not correspond to tes.
the 'spa orthologous region' spans respectively  <dig>  <dig> bp,  <dig>  <dig> bp,  <dig>  <dig> bp and  <dig>  <dig> bp for the a, b, d and the s genomes .
dot plot analysis performed between ae.
speltoides gs  and the t. aestivum ga, -gb, -gd genome  sequences, allows the identification of four conserved sequence stretches, highlighted by blue dotted circles in the figure  <dig>  the majority of the remaining dna within the 'spa orthologous region'  is composed of class i and class ii tes that were differentially inserted and/or deleted in each of the four genomes .
the cumulative length of the conserved sequence stretches, within the 'spa orthologous region' of the four genomes are approximately similar between the genomes pairs ga/gb , ga/gd , ga/gs , gb/gd , gb/gs, gd/gs .
these could be considered as the aegilops-triticum 'ancestral spa locus' covering  <dig>  <dig> bp of cumulative length considering sequences stretches conserved between at least two of the compared sequence.
other stretches of sequence conservation were observed outside the 'spa orthologous region' when comparing pairs of genomes but these sequences were not determined in the available bac clone sequences of the other genomes .
as we cannot rule out whether these sequences were not covered in the sequenced bac clones or were not really conserved across the four genomes, they were not considered in the evolutionary relationship analysis.
no genes, other than spa can be predicted from these four conserved sequence stretches.
as coding and non-coding sequences can evolve at different rates, we perform evolutionary analysis separately for the spa cds  and the remaining conserved non-coding sequences .
conserved non-coding sequences  analysis
the conserved non-coding sequences consist of the four shared sequence stretches, excluding the spa gene itself .
the gb/gs genome comparison shows the highest sequence identity and cumulative length  compared to the other sequence comparisons, i.e.
ga/gb , ga/gd , ga/gs , gb/gd , and gd/gs  .
nevertheless, only a  <dig> bp sequence was shown to be conserved between gs/gb  and absent from other genomes .
on the contrary, three sequence stretches  are conserved between the s, the a and/or d genomes and absent from the b genome .
moreover, although it represents the majority of the cns comparisons, sequence conservation was not always the highest between the s and b genomes across the cns as  <dig> small stretches  of sequences were more conserved between the s and the a and/or d genomes than with the b genome .
non-coding 'spa orthologous loci' sequences.
detailed features obtained for the  <dig> pairwised alignments of the  <dig> spa orthologous regions excluding the spa gene itself are mentioned with the cns length, percentage of sequence identity, ks and ka values and rate.
as an example, alignment of the ga and gb spa orthologous regions of respectively  <dig>  <dig> bp and  <dig>  <dig> bp correspond to a cumulative cns length of  <dig>  <dig> bp with  <dig> % of sequence identity and  <dig> ,  <dig> ,  <dig>  values for respectively ks, ka and ks/ka.
coding 'spa orthologous loci' sequences.
<dig> pairwised comparisons of the spa gene between a/b b/d d/a a/s b/s d/s sequences are associated with the number of substitutions, the number of transition, the number of transversion, the transition/transversion ratio, the ks value, the ka value and speciation date .
we also estimated divergence times on the basis of the number of base substitutions  accumulated after the split-time from the ancestor genome.
ks values were obtained for the  <dig> pairwise alignment combinations .
the lowest and highest ks values correspond respectively to the gb/gs , and gb/gd .
conserved coding sequences analysis: spa gene structure and evolution
spa genes are structured as six exons .
in wheat, spa gene  are respectively  <dig>  <dig> bp,  <dig>  <dig> bp,  <dig>  <dig> bp,  <dig>  <dig> bp, long for a, b, d and s genes .
these spa genes are composed of six exons ranging in size from  <dig>  to  <dig> bp  and five introns ranging in size from  <dig>  to  <dig> bp .
all of exon-intron junction sites obey the gt/ag rule as identified in other eukaryotic genes.
the relative organization of the exons and introns is the same for the others spa-like bzip protein genes characterized to date in cereal, i.e.
the number of exons and introns is conserved and individual introns occur at relatively the same sites for the maize o <dig>  <cit> , sorghum o <dig>  <cit> , and barley blz <dig> genes  <cit> .
it is interesting to note that the first and fifth introns of the homoeologous spa genes are respectively much shorter and larger, compare to the other cereal spa-like bzip protein genes .
we conducted a phylogenic analysis based on spa cds of the four wheat genomes as well as that available from other cereals.
a graphical representation of these data is shown in the figure  <dig> with a classical phylogenic tree including spa homologs available from other cereals  and illustrates that wheat spa and barley blz <dig> consists in the same triticeae subfamily in which ae.
speltoides and t. aestivum-gb spa sequences are linked on the same branch.
such phylogenic analysis shows that the lowest synonymous  and non-synonymous  substitution rates were obtained between ae.
speltoides and t. aestivum -gb, with ks  and ka  values corresponding to a  <dig>  to  <dig>  mya divergence time, while rates obtained when ae.
speltoides is compared to -ga and -gd are respectively ks  and ka  values corresponding to  <dig>  to  <dig>  mya divergence time; and ks  and ka  values corresponding to  <dig>  to  <dig>  mya divergence time .
this result strongly suggests that, despite the strong nucleotide conservation between the  <dig> homoeologous copies of the spa cds in t. aestivum, ae.
speltoides cds is closest to the t. aestivum spa-gb than the two other homoeologous -ga and -gd sequences.
as reported by guillaumie et al.
<cit> , a stop codon tga  site had been identified in the spa-gb sequence suggesting that it might be no more functional.
no proof of expression could be also provided for the spa gb haplotype presenting this stop codon as we were unable to find any corresponding ests.
in order to clarify the apparition of the tga stop codon in the b genome, the stop codon allele distribution was analyzed using  <dig> wheat genotypes which cover,  <dig> diploid genome s ,  <dig> tetraploid  and  <dig> hexaploid  genotypes.
genotyping data demonstrate that the tga allele is present at 50% in hexaploid wheat  and for the first time in one tetraploid  genotype over  <dig> tested and absent in ae.
longissima .
differential transposable elements insertions and evolution
size discrepancies of the 'spa orthologous regions' can be attributed to differential te insertions or eliminations , which occurred after the four genomes divergence.
hence, the size increase observed for the 'spa orthologous region' in ae.
speltoides  when compared to t. aestivum-gb  is due to  <dig> class i elements, i.e.
<dig> truncated angela solo-ltrs , one complete angela , one truncated rada ,  <dig> fragmented lines  and one mite .
these tes may correspond to insertions, which occurred in the ae.
speltoides genome after its divergence from the ancestor of the b genome as they are dispersed between cns stretches and not present in the b genome of t. aestivum.
occurrence of eight class i tes displaying complete ltr and tsd , identified in the four annotated genomes  allows to estimate the insertion dates, based on nucleotide substitution pattern analysis .
thus, the complete angela_ <dig> identified in ae.
speltoides  located in the 'spa orthologous region' exhibits a transition and tranversion value of  <dig>  +/-  <dig>  respectively associated with an estimated insertion time of  <dig>  to  <dig>  mya.
the youngest insertion time was observed for the angela_ <dig> element annotated outside the 'spa orthologous region' in the ae.
speltoides sequence, i.e.
<dig>  to  <dig>  mya.
discussion
we sequenced for the first time an ae.
speltoides genomic region  and compared it to orthologous regions of the a, b and d genomes coresident in the hexaploid wheat t. aestivum at the spa cds, the cns and the te insertion dynamics levels.
spa gene structure comparison and haplotype variability
the spa gene is the only gene conserved across the four genomes.
a phylogenic analysis involving spa protein sequences from t. aestivum, ae.
speltoides, rice, barley, maize, sorghum, arabidopsis thaliana, nicotiana tabacum, petroselinum crispum, clearly identified a triticeae outgroup in which ae.
speltoides spa sequence is more closely related to t. aestivum-gb spa than any other sequence involved in the tree.
interestingly, in this study we showed that the stop codon tga allele,  <dig> bases downstream the atg transcription initiation site, previously identified in the b genome of hexaploid wheat  <cit> , is also present in the tetraploid t. turgidum.
this indicates that the stop-codon tga spa allele has been generated before the allohexaploidization event.
the presence of both stop tga and tca spa alleles in tetraploid and hexaploid wheat accessions provides further evidences for the hypothesis of  recurrent hexapolyploidization events or  gene flow through introgression between the different wheat species with different ploidy levels  <cit> .
differential pattern of cns conservation
our results reveal that, a large proportion of the remaining non-genes and non-transposable elements sequences are highly conserved between the four genomes .
at the 'spa orthologous region', excluding the spa gene itself, the gb/gs genome comparison shows the highest sequence identity and cumulative length as well as the lowest ks value  compared to the other sequences .
thus, the s genome was confirmed to be the closest to the b genome in term of cumulative conserved sequence length as well as identity as compared to any other pairwise genome combinations.
small stretches of sequences, which were more conserved between the s and/or the a and d genomes , do not contradict with the general pattern of an overall higher cns conservation between the s and b genomes.
this is the first time that we precisely report close relationships between the s and b genomes based on both coding and non-coding sequence comparisons.
cns , have been recently surveyed in cereals  and mammals   <cit> .
it has been shown that cnss are more abundant in loci embedding regulatory genes such as transcription factors  and that despite divergence from a common ancestors, grass genes have dramatically fewer  and smaller cnss than mammalian genes.
one possible explanation is that, in contrast to vertebrate genomes, plant genomes have been subjected to more rounds of whole genome duplications  events that have profoundly affected their organisation, the subfunctionalisation of duplicated genes leading to a greater per gene loss of cns  <cit> .
differential te insertion dynamics
no class i or class ii te annotated within or outside the 'spa orthologous region' is common when comparing any two-genome combinations.
the two wis retrotransposons, displaying similar apparent insertion positions in the 5' spa locus boundaries of the a and d genomes correspond to independent insertions as target site duplication  signature-motifs are distinct .
this is also confirmed by estimation of their insertion dates with a transition and transversion ratio of  <dig> +/- <dig>   and  <dig> +/- <dig>   for respectively the a and d genome sequences .
the differential insertion of tes is surprisingly the case of the b and s genomes.
overall, we count six  and eight  tes differentially inserted in the b and s genomes respectively .
the 'spa orthologous region' of the s genome has been invaded by retrotransposons, whereas outside the 'spa orthologous region' the b genome seems to have a specific site for the insertion of class ii tes .
overall, we were able to estimate insertion dates for  <dig> retrotransposons.
out of them, only one  has been inserted into the 'spa orthologous region' of ae.
speltoides, .
thus, the differential insertions of tes in the s genome might be posterior to the s and b genome progenitors divergence from a common ancestor  <dig>  mya,  <dig>  in the present study.
figure  <dig> retraces the process of te differential insertion-deletions from a suggested triticum-aegilops 'ancestral spa locus' sequence of  <dig>  <dig> bp that has been subjected to intensive te insertions in the a, d and s genomes as compared to the b genome analysed in the present study.
the progenitor enigma of the b genome of polyploid wheat species
according to the two allopolyploidization events that gave rise to t. aestivum, the d genomes of the hexaploid wheat have diverged relatively recently from that of its donnor ae.
tauschii  whereas divergence of the a and b genomes from their respective progenitors occurred much more earlier   <cit> .
for almost  <dig> years, it remained controversial whether the source of the b genome is unique  related to ae.
speltoides or whether this genome resulted from an introgression of several parental aegilops species   <cit> .
recent data on molecular comparisons using germplasm collections clearly show that the b genome could be related to several ae.
speltoides lines but not to other species of the sitopsis section  <cit> .
comparison between the a genome of polyploid wheat species to that of its progenitor t.urartu at the psr <dig> region  <cit>  has shown a very high cds conservation .
moreover, dvorak et al.
<cit>  found in the  <dig> kb intergenic sequences four conserved tes  whereas four and one other tes were respectively inserted in the a genome of t. urartu and that of t. durum, after their divergence from a common ancestor.
our present comparison based on cds and cns confirms that the b genome is closer to the s genome of ae.
speltoides than the a and d genomes.
however, spa sequence divergence and the differential insertions/deletions of tes, none of which is conserved between the two genomes, indicate that ae.
speltoids have diverged very early  from the b genome progenitor.
CONCLUSIONS
the present study based on detailed cds, cns and te dynamics comparisons, clearly shows that evolutionary relationship between the b genome and the s genome of ae.
speltoides is not as close as it has been reported in the literature for the a genome of polyploid wheat species compared to its identified progenitor, t. urartu.
thus, a b genome progenitor remains to be identified.

 all cereal crop species are members of the grass family that is the fourth largest family of flowering plants. with about species growing under nearly all climates and latitudes, grasses exceed all other plant families in ecological dominance and economic importance. in terms of genome organisation they represent a very diverse family with basic chromosome numbers ranging from to and genome sizes ranging from mb to gb. fossil data and phylogenetic studies have estimated that the grasses have diverged from a common ancestor to million years ago. archaeological records suggest that farming started concomitantly in at least three widely separated regions between 000- years ago during the late neolithic period. the three most important cereals were independently domesticated in three centres: wheat in south western asia in the'fertile crescent' region, maize in mexico and rice in both south east asia and west africa. within the poaceae, the genera aegilops and triticum include several diploid species that, via allopolyploidization, produced several tetraploid and hexaploid wheat species, most of which have been domesticated. t. turgidum was derived from a hybridization event that happened between t. urartu,, the diploid donor of the a genome, and another unknown species of the sitopsis section, donor of the b genome, for which the closest known relative is ae. speltoides. the hexaploid wheat originated from an additional polyploidization event between the early-domesticated tetraploid t. turgidum ssp dicoccum and the diploid donor of the d genome, ae. tauschii, to years ago. several wheat phylogeny studies have tried to identify the progenitor of the b genome of polyploid wheat based on cytology, nuclear and mitochondrial dna sequences as well as chromosome rearrangement studies. it remains controversial from those studies whether the progenitor of the b genome is a unique aegilops species or whether this genome resulted from an introgression of several parental aegilops species. more recent and representative molecular comparisons using germplasm collections have shown that the b genome could be related to several ae. speltoides lines but not to other species of the sitopsis section. transposable elements have been shown since the seventies to be well represented in the wheat genome, ~80%. comparative studies have shown that beside the general conservation in coding sequences, no te insertions are conserved between the a, b and d genomes of wheat whereas important proportion of te insertions are shared between the a or d genomes of polyploid wheat and their respective progenitors t. urartu and ae. tauschii. no such studies have been yet reported comparing the b genome of these polyploid wheat species to that of its closest known diploid relative, i.e. ae. speltoides. in the present study, we compared for the first time coding and non-coding sequences as well as dynamics of te insertions between the s genome of ae. speltoïdes and that of the a, b and d co-resident in the hexaploid wheat. the spa locus region, belonging to bzip, located on chromosome 1bl, has been chosen because of its importance as trans-acting elements of seed storage protein and its conservation in several other cereals such as maize, rice, and barley. updating phylogeny relationships and insights onto the origin of the b genome are discussed.  organization of spa locus region in the a, b, d and s genomes three bac clones spanning the spa gene of the a, b and d genomes of t. aestivum were screened from cv renan bac library with pcr markers specific for each of the three spa genes. sequencing resulted in and bp for, respectively, the a, b and d genomes. screening of an ae. speltoides pooled bac library with the same spa-specific pcr markers allowed us to identify and sequence a bac clone of bp sequence spanning the spa locus gene. annotation has been performed to identify and compare gene and repeat contents of the four available genome sequences, graphically presented in figure 1a. more details are also presented in additional file as expected for wheat, the four genomic sequences are very rich in tes. overall, the bp a genome sequence is structured as bp of class i te, bp of class ii elements and % of unclassified te. fourteen class i tes are identified as one incompletely sequenced, five truncated, relics, one fragmented and three complete elements. the class ii tes is represented as a complete cacta element and three mites. besides the identification of tes a pseudo tubulin gene separated by bp from the spa gene was also identified, both genes covering % of the sequence. the bp b genome sequence is structured as bp of class i tes, bp of class ii elements and % of unclassified elements. twelve class i elements are identified as two incompletes, six truncated, two relics, one fragmented and one complete element. the class ii tes consists of two complete, one fragmented and one truncated cacta as well as three mites. the spa gene is the only gene identified on the b genome sequence, representing % of the sequence. the bp d genome sequence is structured as bp of class i tes, bp of class ii elements. twenty-two class i tes are identified as two incomplete, eight truncated, eight relics, two fragmented and two complete elements. class ii tes are represented as three truncated cacta elements, one mutator relic and one mite. three genes have been annotated on the d genome sequence, the spa gene, a putative kinesin and a putative cortical cell-delineating gene, covering % of a bp interval. the bp s genome sequence is structured as bp of class i tes, and a single mite class ii te. thirteen class i tes are identified as one incomplete, six truncated, four fragmented and two complete tes. as in the b genome sequence, only the spa gene, covering % of the annotated sequence, has been identified on the s genome sequence. identification and characterization of conserved sequences alignment of the four genomic regions allows the identification of the'spa orthologous region', which we have defined as the shared common regions delimitated by conserved non-coding sequence stretches that do not correspond to tes. the'spa orthologous region' spans respectively bp, bp, bp and bp for the a, b, d and the s genomes. dot plot analysis performed between ae. speltoides gs and the t. aestivum ga, -gb, -gd genome sequences, allows the identification of four conserved sequence stretches, highlighted by blue dotted circles in the figure the majority of the remaining dna within the'spa orthologous region' is composed of class i and class ii tes that were differentially inserted and/or deleted in each of the four genomes. the cumulative length of the conserved sequence stretches, within the'spa orthologous region' of the four genomes are approximately similar between the genomes pairs ga/gb, ga/gd, ga/gs, gb/gd, gb/gs, gd/gs. these could be considered as the aegilops-triticum'ancestral spa locus' covering bp of cumulative length considering sequences stretches conserved between at least two of the compared sequence. other stretches of sequence conservation were observed outside the'spa orthologous region' when comparing pairs of genomes but these sequences were not determined in the available bac clone sequences of the other genomes. as we cannot rule out whether these sequences were not covered in the sequenced bac clones or were not really conserved across the four genomes, they were not considered in the evolutionary relationship analysis. no genes, other than spa can be predicted from these four conserved sequence stretches. as coding and non-coding sequences can evolve at different rates, we perform evolutionary analysis separately for the spa cds and the remaining conserved non-coding sequences. conserved non-coding sequences analysis the conserved non-coding sequences consist of the four shared sequence stretches, excluding the spa gene itself. the gb/gs genome comparison shows the highest sequence identity and cumulative length compared to the other sequence comparisons, i.e. ga/gb, ga/gd, ga/gs, gb/gd, and gd/gs. nevertheless, only a bp sequence was shown to be conserved between gs/gb and absent from other genomes. on the contrary, three sequence stretches are conserved between the s, the a and/or d genomes and absent from the b genome. moreover, although it represents the majority of the cns comparisons, sequence conservation was not always the highest between the s and b genomes across the cns as small stretches of sequences were more conserved between the s and the a and/or d genomes than with the b genome. non-coding'spa orthologous loci' sequences. detailed features obtained for the pairwised alignments of the spa orthologous regions excluding the spa gene itself are mentioned with the cns length, percentage of sequence identity, ks and ka values and rate. as an example, alignment of the ga and gb spa orthologous regions of respectively bp and bp correspond to a cumulative cns length of bp with % of sequence identity and,, values for respectively ks, ka and ks/ka. coding'spa orthologous loci' sequences. pairwised comparisons of the spa gene between a/b b/d d/a a/s b/s d/s sequences are associated with the number of substitutions, the number of transition, the number of transversion, the transition/transversion ratio, the ks value, the ka value and speciation date. we also estimated divergence times on the basis of the number of base substitutions accumulated after the split-time from the ancestor genome. ks values were obtained for the pairwise alignment combinations. the lowest and highest ks values correspond respectively to the gb/gs, and gb/gd. conserved coding sequences analysis: spa gene structure and evolution spa genes are structured as six exons. in wheat, spa gene are respectively bp, bp, bp, bp, long for a, b, d and s genes. these spa genes are composed of six exons ranging in size from to bp and five introns ranging in size from to bp. all of exon-intron junction sites obey the gt/ag rule as identified in other eukaryotic genes. the relative organization of the exons and introns is the same for the others spa-like bzip protein genes characterized to date in cereal, i.e. the number of exons and introns is conserved and individual introns occur at relatively the same sites for the maize o, sorghum o, and barley blz genes. it is interesting to note that the first and fifth introns of the homoeologous spa genes are respectively much shorter and larger, compare to the other cereal spa-like bzip protein genes. we conducted a phylogenic analysis based on spa cds of the four wheat genomes as well as that available from other cereals. a graphical representation of these data is shown in the figure with a classical phylogenic tree including spa homologs available from other cereals and illustrates that wheat spa and barley blz consists in the same triticeae subfamily in which ae. speltoides and t. aestivum-gb spa sequences are linked on the same branch. such phylogenic analysis shows that the lowest synonymous and non-synonymous substitution rates were obtained between ae. speltoides and t. aestivum -gb, with ks and ka values corresponding to a to mya divergence time, while rates obtained when ae. speltoides is compared to -ga and -gd are respectively ks and ka values corresponding to to mya divergence time; and ks and ka values corresponding to to mya divergence time. this result strongly suggests that, despite the strong nucleotide conservation between the homoeologous copies of the spa cds in t. aestivum, ae. speltoides cds is closest to the t. aestivum spa-gb than the two other homoeologous -ga and -gd sequences. as reported by guillaumie et al., a stop codon tga site had been identified in the spa-gb sequence suggesting that it might be no more functional. no proof of expression could be also provided for the spa gb haplotype presenting this stop codon as we were unable to find any corresponding ests. in order to clarify the apparition of the tga stop codon in the b genome, the stop codon allele distribution was analyzed using wheat genotypes which cover, diploid genome s, tetraploid and hexaploid genotypes. genotyping data demonstrate that the tga allele is present at 50% in hexaploid wheat and for the first time in one tetraploid genotype over tested and absent in ae. longissima. differential transposable elements insertions and evolution size discrepancies of the'spa orthologous regions' can be attributed to differential te insertions or eliminations, which occurred after the four genomes divergence. hence, the size increase observed for the'spa orthologous region' in ae. speltoides when compared to t. aestivum-gb is due to class i elements, i.e. truncated angela solo-ltrs, one complete angela, one truncated rada, fragmented lines and one mite. these tes may correspond to insertions, which occurred in the ae. speltoides genome after its divergence from the ancestor of the b genome as they are dispersed between cns stretches and not present in the b genome of t. aestivum. occurrence of eight class i tes displaying complete ltr and tsd, identified in the four annotated genomes allows to estimate the insertion dates, based on nucleotide substitution pattern analysis. thus, the complete angela_ identified in ae. speltoides located in the'spa orthologous region' exhibits a transition and tranversion value of +/- respectively associated with an estimated insertion time of to mya. the youngest insertion time was observed for the angela_ element annotated outside the'spa orthologous region' in the ae. speltoides sequence, i.e. to mya. discussion we sequenced for the first time an ae. speltoides genomic region and compared it to orthologous regions of the a, b and d genomes coresident in the hexaploid wheat t. aestivum at the spa cds, the cns and the te insertion dynamics levels. spa gene structure comparison and haplotype variability the spa gene is the only gene conserved across the four genomes. a phylogenic analysis involving spa protein sequences from t. aestivum, ae. speltoides, rice, barley, maize, sorghum, arabidopsis thaliana, nicotiana tabacum, petroselinum crispum, clearly identified a triticeae outgroup in which ae. speltoides spa sequence is more closely related to t. aestivum-gb spa than any other sequence involved in the tree. interestingly, in this study we showed that the stop codon tga allele, bases downstream the atg transcription initiation site, previously identified in the b genome of hexaploid wheat, is also present in the tetraploid t. turgidum. this indicates that the stop-codon tga spa allele has been generated before the allohexaploidization event. the presence of both stop tga and tca spa alleles in tetraploid and hexaploid wheat accessions provides further evidences for the hypothesis of recurrent hexapolyploidization events or gene flow through introgression between the different wheat species with different ploidy levels. differential pattern of cns conservation our results reveal that, a large proportion of the remaining non-genes and non-transposable elements sequences are highly conserved between the four genomes. at the'spa orthologous region', excluding the spa gene itself, the gb/gs genome comparison shows the highest sequence identity and cumulative length as well as the lowest ks value compared to the other sequences. thus, the s genome was confirmed to be the closest to the b genome in term of cumulative conserved sequence length as well as identity as compared to any other pairwise genome combinations. small stretches of sequences, which were more conserved between the s and/or the a and d genomes, do not contradict with the general pattern of an overall higher cns conservation between the s and b genomes. this is the first time that we precisely report close relationships between the s and b genomes based on both coding and non-coding sequence comparisons. cns, have been recently surveyed in cereals and mammals. it has been shown that cnss are more abundant in loci embedding regulatory genes such as transcription factors and that despite divergence from a common ancestors, grass genes have dramatically fewer and smaller cnss than mammalian genes. one possible explanation is that, in contrast to vertebrate genomes, plant genomes have been subjected to more rounds of whole genome duplications events that have profoundly affected their organisation, the subfunctionalisation of duplicated genes leading to a greater per gene loss of cns. differential te insertion dynamics no class i or class ii te annotated within or outside the'spa orthologous region' is common when comparing any two-genome combinations. the two wis retrotransposons, displaying similar apparent insertion positions in the 5' spa locus boundaries of the a and d genomes correspond to independent insertions as target site duplication signature-motifs are distinct. this is also confirmed by estimation of their insertion dates with a transition and transversion ratio of +/- and +/- for respectively the a and d genome sequences. the differential insertion of tes is surprisingly the case of the b and s genomes. overall, we count six and eight tes differentially inserted in the b and s genomes respectively. the'spa orthologous region' of the s genome has been invaded by retrotransposons, whereas outside the'spa orthologous region' the b genome seems to have a specific site for the insertion of class ii tes. overall, we were able to estimate insertion dates for retrotransposons. out of them, only one has been inserted into the'spa orthologous region' of ae. speltoides,. thus, the differential insertions of tes in the s genome might be posterior to the s and b genome progenitors divergence from a common ancestor mya, in the present study. figure retraces the process of te differential insertion-deletions from a suggested triticum-aegilops'ancestral spa locus' sequence of bp that has been subjected to intensive te insertions in the a, d and s genomes as compared to the b genome analysed in the present study. the progenitor enigma of the b genome of polyploid wheat species according to the two allopolyploidization events that gave rise to t. aestivum, the d genomes of the hexaploid wheat have diverged relatively recently from that of its donnor ae. tauschii whereas divergence of the a and b genomes from their respective progenitors occurred much more earlier. for almost years, it remained controversial whether the source of the b genome is unique related to ae. speltoides or whether this genome resulted from an introgression of several parental aegilops species. recent data on molecular comparisons using germplasm collections clearly show that the b genome could be related to several ae. speltoides lines but not to other species of the sitopsis section. comparison between the a genome of polyploid wheat species to that of its progenitor t.urartu at the psr region has shown a very high cds conservation. moreover, dvorak et al. found in the kb intergenic sequences four conserved tes whereas four and one other tes were respectively inserted in the a genome of t. urartu and that of t. durum, after their divergence from a common ancestor. our present comparison based on cds and cns confirms that the b genome is closer to the s genome of ae. speltoides than the a and d genomes. however, spa sequence divergence and the differential insertions/deletions of tes, none of which is conserved between the two genomes, indicate that ae. speltoids have diverged very early from the b genome progenitor.  the present study based on detailed cds, cns and te dynamics comparisons, clearly shows that evolutionary relationship between the b genome and the s genome of ae. speltoides is not as close as it has been reported in the literature for the a genome of polyploid wheat species compared to its identified progenitor, t. urartu. thus, a b genome progenitor remains to be identified. 
sequence length discrepancies within and outside the spa orthologous regions are the result of non-shared transposable elements insertions, all of which inserted after the progenitors of the four genomes divergence. on the basis of conserved sequence length as well as identity of the shared non-te regions and the spa coding sequence, ae speltoides appears to be more evolutionary related to the b genome of t. aestivum than the a and d genomes. several studies suggested that the diploid ancestor of the b genome of tetraploid and hexaploid wheat species belongs to the sitopsis section, having aegilops speltoides as the closest identified relative. speltoides to that of the a, b and d genomes co-resident in the hexaploid wheat species. four bac clones, spanning the spa locus of respectively the a, b, d and s genomes, were isolated and sequenced. speltoides has diverged very early from the progenitor of the b genome which remains to be identified. in an attempt to clarify these relationships, we compared, in this study, sequences of the storage protein activator locus region of the s genome of ae. orthologous genomic regions were identified as delimited by shared non-transposable elements and non-coding sequences surrounding the spa gene and correspond to and bp for the a, b, d and s genomes, respectively. however, the differential insertions of tes, none of which are conserved between the two genomes led to the conclusion that the s genome of ae.
 several studies suggested that the diploid ancestor of the b genome of tetraploid and hexaploid wheat species belongs to the sitopsis section, having aegilops speltoides as the closest identified relative. however molecular relationships based on genomic sequence comparison, including both coding and non-coding dna, have never been investigated. in an attempt to clarify these relationships, we compared, in this study, sequences of the storage protein activator locus region of the s genome of ae. speltoides to that of the a, b and d genomes co-resident in the hexaploid wheat species.  four bac clones, spanning the spa locus of respectively the a, b, d and s genomes, were isolated and sequenced. orthologous genomic regions were identified as delimited by shared non-transposable elements and non-coding sequences surrounding the spa gene and correspond to and bp for the a, b, d and s genomes, respectively. sequence length discrepancies within and outside the spa orthologous regions are the result of non-shared transposable elements insertions, all of which inserted after the progenitors of the four genomes divergence.  on the basis of conserved sequence length as well as identity of the shared non-te regions and the spa coding sequence, ae speltoides appears to be more evolutionary related to the b genome of t. aestivum than the a and d genomes. however, the differential insertions of tes, none of which are conserved between the two genomes led to the conclusion that the s genome of ae. speltoides has diverged very early from the progenitor of the b genome which remains to be identified. 