BACKGROUND
barley is an important crop but also a model plant for temperate cereals because of its diploid nature and rich genetics.
extensive genomics resources have been developed, and a number of important genes were already isolated by map-based cloning  <cit> .
since the grain is the agriculturally most important part of the plant many studies have been carried out on barley seed development and germination .
however, we still know little about the genes determining important traits and the regulatory networks underlying developmental processes.
prime candidates for members of signal transduction chains and regulatory networks are transcription factors , kinases and other experimentally verified regulators.
different approaches have been used to gain insight into the relationship between genotype and phenotype in mutant studies and the analysis of transgenic plants.
one additional promising approach is the more recently developed concept of genetical genomics, which combines gene expression studies with genetic linkage analysis  <cit> .
differences in transcript levels are used to map the responsible chromosal regions thus eventually allowing the identification of the causative gene.
in species whose genomes have not yet been sequenced such as barley, the feasibility of this strategy depends on the number of genetically mapped genes.
based on our long-term dedication to understand seed development and seed storage product accumulation as well as to study agronomical traits by extensively using marker-based techniques we set out to first define and characterize a set of genes with known or putative regulatory functions during grain development.
in a second step snp marker were developed for as many as possible of those genes and used to anchor them on the barley linkage map.
thus we provide new data on regulatory genes and their putative joint action based on cluster analysis of expression data  and place part of them on the barley linkage map to promote marker-assisted studies in barley especially on the biology of barley grains.
RESULTS
expression map of selected regulatory genes
to define a set of barley genes putatively involved in regulating preferentially grain development sequences from cdna libraries of developing seeds  <cit>  were used in similarity searches against rice and arabidopsis databases in search for homologs of regulatory genes.
from the total set, those transcription factors  and signalling related genes were selected, which were shown to be exclusively expressed during seed development or alternatively characterized by a spatio-temporal pattern of gene expression, which is characteristic for both seed development and across plant ontogeny  <cit> .
in a first step  <dig> regulatory sequences  were selected based on their homology to known regulatory genes in other model species  and on identified homologous sequences from the affymetrix barley <dig> genechip.
these genes were further characterized.
expression patterns were extracted and normalized from  <dig> published experiments concerning samples from both endosperm and embryo fractions of developing and imbibed seeds of cultivar barke  <cit>  as well as selected stages of seed development, coleoptile, radicle, root, crown and leaf from germinating seeds and immature inflorescence, floral bracts, pistil and anthers from reproductive tissues of cultivars morex and golden promise  <cit> .
a hierarchical clustering method identified major clusters with defined co-expressed regulatory genes that are associated either with specific tissues during seed development or found to be expressed in both developing seeds as well as during other stages of plant ontogeny .
individual clades of the hierarchical tree were subjected to k-mean clustering to get more refined sub clusters.
as a result,  <dig> regulatory genes were found to be preferentially expressed in one of the three studied seed tissues  during seed development.
they are represented in cluster groups  <dig>   <dig> and  <dig>  respectively .
a total of  <dig> regulators are characteristically expressed in multiple tissues undergoing cell differentiation events during seed development, also during the shift from germination to seedling establishment as well during the transition from vegetative to generative development .
in addition  <dig> regulators were found to be expressed during both seed development and germination .
the first cluster contains two subclusters  <dig> and  <dig>  subcluster  <dig> contains  <dig> regulatory genes that are preferentially activated in pericarp during seed development as well as partly in reproductive tissues .
examples are members of the following gene families: an auxin-responsive aux/iaa gene , ap <dig> , bhlh , c2h <dig> zincfinger , homeobox , a well characterized crabs claw gene  from the yabby family and tf mads  <dig> , which is homologous to the arabidopsis sepallata gene agl <dig>  whereas in arabidopsis crabs claw and agl <dig> members were shown to be required for carpel development  <cit> , the homologous genes in barley are expressed in the pericarp during early seed developmen, as well as in the pistil.
similarly, mads box gene transcripts, known to be involved in floral transition  <cit> , are here found to be abundantly expressed not only in reproductive organs such as pistil and anther but also in pericarp and endosperm fractions during the onset of seed development and not in the embryo  .
this indicates an unexplored distinct role of mads box tfs in specific tissues during grain development.
similar transcript profiles were observed among signaling related genes: a two-component response regulator , the receptor protein kinase perk <dig> , the abc <dig> family protein kinase  and the gibberellin response modulator rga <dig> .
regulatory genes expressed preferentially during seed maturation in the endosperm are represented in subcluster  <dig>  we especially note preferential expression of the well-known regulators abi3/vp <dig>  and snf <dig>  in endosperm during the maturation phase but not in any other tissue during plant ontogeny .
also preferentially expressed during seed maturation in endosperm tissue and to some extent in embryo ) were the aba response element binding factors abf <dig>  and abi <dig> .
since in a recent study we found abre elements in the snf <dig> kinase promoter region  <cit>  it seems likely that aba plays a role in triggering endosperm maturation events and that the hormone acts through snf <dig> mediated by aba via abf3/abi <dig> and abi <dig>  probably by the same way aba is involved in controlling starch biosynthesis during endosperm maturation  <cit> .
also present in subcluster  <dig> is the barley prolamin binding factor transcript .
the respective protein is a member of the dof family of tfs known to activate storage protein hordein b transcription during the main endosperm storage period  <cit> .
coexpressed with the prolamin binding factor in cluster  <dig> are two members of the ccaat tf family .
this family contains the well characterized lec <dig> tf of arabidopsis, a major seed maturation control factor of the arabidopsis hap <dig> family  <cit> .
taken together, most of the maturation control genes discovered in arabidopsis were also found to be preferentially expressed during maturation events in barley.
along with these gene sets we also noticed calcium signaling related genes possibly participating in a genetic framework for seed maturation , which needs further investigation.
subcluster  <dig> of cluster  <dig> encodes  <dig> regulators such as transcription factor x <dig> , a putative leucine zipper protein , calmodulin-binding proteins , a receptor protein kinase perk1-like protein , a wpk <dig> protein kinase  and an unclassified protein kinase  all expressed preferentially in the embryo during seed maturation .
the fourth cluster contains  <dig> regulators found to be expressed in pericarp, endosperm and embryo during the cell differentiation events in the developing seed.
its expression is reduced during the peak of storage phase as well in the mature seeds of the endosperm .
interestingly, gene expression patterns of these regulators are found to be expressed throughout embryo development.
they are also expressed in the immature inflorescence, the pistil as well as in early germinating seedling tissues such as crown and coleoptile  where cell differentiation occurs.
this cluster is enriched with a nucleosome/chromatin assembly factor , a set-domain transcriptional regulator-like protein  and wd- <dig> proteins .
wd- <dig> members belong to chromatin complex subunits of the snf <dig>  family.
the suppression of its homologues in arabidopsis results in small cotyledonary embryos with limited cell expansion  <cit> .
we furthermore observe the expression of two members of mcm genes , which are homologous to prolifera, a protein required for dna replication during seed development  <cit>  as well as hmg family members  known to promote meristematic activities during plant development  <cit> .
g-protein signalling components are also expressed.
interestingly,  <dig> regulators which were found to be expressed in the developing seed were also ubiquitously expressed in all tissues of germinating seedlings as well as in reproductive tissues.
examples are ap2-erebp, bzip, hmg and myb tf family members and calcium, g-protein, receptor kinases and serine/threonine protein kinase family members representing signaling components .
snp-marker development and anchoring to the linkage map of barley
based on the analyzed expression profiles an initial list of  <dig> transcription factors and  <dig> kinases was derived and the sequences used for snp detection, marker development and anchoring on the barley linkage map.
of the  <dig> primer pairs designed,  <dig>  yielded a single copy-pcr product and  <dig> of them were sequenced from both sides for each parental line amounting to a maximal read depth of fourteen reads resulting in an average consensus length  of  <dig> bp amounting to  <dig>  kb sequenced in total.
the remaining sixteen loci showed an indel-polymorphism or a microsatellite polymorphism between the parental lines at least in one of the four mapping populations on agarose gels.
for  <dig> sequenced loci we detected polymorphisms between the parental lines in at least one out of four mapping populations .
these mapping populations comprised the two doubled haploid standard populations steptoe/morex  and oregon wolf barley   <cit> , as well as two bc <dig> advanced backcross populations brenda/hs <dig>  and brenda/hs <dig>  which were used for qtl detection elsewhere  <cit> .
due to their population structure the advanced backcross populations are not useful for mapping of previously unassigned markers, but were only used for genotyping of markers mapped in other mapping populations.
we detected  <dig> snps in  <dig> sequenced polymorphic pcr products resulting in a snp-density of  <dig>  snp per kb .
seventy-three loci did not show a sequence polymorphism between the seven barley varieties.
all detected snps were dinucleotide polymorphisms except the locus gbs <dig> that showed a trinucleotide snp.
with respect to snps between the parental lines of the mapping populations the oregon wolfe barleys showed  <dig> snp in  <dig> loci or genes, respectively.
the number of polymorphic loci and the total number of snps in the oregon wolfe barley was more than twice as found for the brenda/hs <dig> population .
remarkably, the lowest numbers of polymorphic loci were found between the parental lines of the advanced backcross populations comprising the elite variety 'brenda' and two hordeum spontaneum accessions.
this low number of polymorphic loci entails snp-densities of  <dig>  and  <dig>  snps per kb in hs <dig> and hs <dig> versus 'brenda', compared to a snp density of  <dig>  and  <dig>  for the oregon wolfe barley and the steptoe/morex populations .
in contrast, we found a higher number of snps within polymorphic genes in the brenda/hs <dig> and brenda/hs <dig> population of  <dig>  and  <dig>  per gene, whereas the snp density in oregon wolfe barley and the steptoe/morex population amounted to  <dig>  snp and  <dig>  snp per locus, respectively.
the detected percentage of 22% polymorphic loci for the steptoe/morex population was below the value of 36% reported for the same population in  <cit> .
a low polymorphism rate of 15% was also reported for microsatellite markers in the brenda/hs <dig> population  <cit> , while 46% of  <dig> tested microsatellite markers were polymorphic in the brenda/hs <dig> population  <cit> .
twenty-eight out of  <dig> snps, for which markers have been developed, are located in exons, whereof  <dig> snps could be assigned to utrs .
the representative examples includes utr regions with assigned snp-sites belonged, for instance, to a well known dof transcription factor , an activator of prolamin gene transcription expressed preferentially in endosperm during storage phase .
the other snp-sites assigned to specific utr-motifs includes marker gbs <dig>  and gbs <dig> , which were located in an ires  motif that is supposed to be located in 5' utrs  <cit> .
the internal mrna ribosome binding is a mechanism of translation initiation alternative to the conventional 5'-cap mechanism.
the ires motifs are  <dig> and  <dig> bp in size, respectively.
both clones clustered in the expression analysis in the first cluster group with specific expression in pericarp during seed development and partly in reproductive tissues .
the number of exonic snps involving transitions exceeds the number of transversions , whereas the ratio of transitions vs. transversions appeared to be balanced at intronic snp-sites .
altogether, we developed  <dig> marker assays for  <dig> transcription factors and kinases showing a polymorphism at least in one out of four mapping populations .
information about primer sequences, expected fragment lengths as well as the crest ids for the cluster-sequences that have been used as template sequence for primer design  can be found in additional files  <dig> and  <dig>
in total, we mapped  <dig> markers onto the sm- and owb-maps that have been used for the construction of the barley consensus map in  <cit>  .
thirty-eight markers have been placed onto the owb-framework map and  <dig> markers onto the sm-framework map.
eight markers could not be assigned to a barley linkage group.
the marker locations appeared to be randomly distributed over the barley genome, although a slight accumulation is seen on chromosome 5h .
in addition to the mapping of polymorphic loci, we assigned the chromosomal location for  <dig> loci with the aid of wheat-barley chromosomal addition lines  <cit> .
altogether,  <dig> markers were allocated to the barley chromosomes of which  <dig> markers are based on genes which were found specifically expressed in developing seeds or expressed in developing seeds as well as in other tissues .
another  <dig> markers could be genotyped on the advanced backcross populations brenda/hs <dig> and brenda/hs <dig>  these two populations have been used in the past for qtl-analysis of agronomic important traits including grain-related traits, such as thousand-grain weight  <cit> .
in the future we want to extend our studies and include transcriptome-based e-qtls of grain development.
synteny to rice
there are extensive syntenic relationships between barley and rice chromosomes that are well documented  <cit> .
for instance,  <dig> out of  <dig> est-based markers in the consensus map of  <cit>  mapped to known co-linear linkage groups in rice.
to assess the synteny of the developed markers to the rice genome we performed a blastn-analyis of the marker sequences to the rice genome in order to detect putative orthologs in rice.
then, we analysed the publicly available information on map position and blastn-analysis of the consensus map published by  <cit>  and counted the markers of the barley consensus map, whose best blastn-hit mapped to the same rice chromosome as for the developed markers.
for  <dig> mapped markers and  <dig> markers assigned to chromosomes in wheat barley addition lines we found a significant blastn-hit  in rice, whereas for  <dig> markers we did not find a putative ortholog in rice .
we tested the accumulation of blastn-hits of developed and published barley markers  <cit>  on specific rice chromosomes with a χ2-test assuming n markers on the linkage group divided by the number of rice-chromosomes under the null-hypothesis.
based on this test, fourty-one developed markers obey macro co-linear relationships between barley and rice .
some highlighting examples include ethylene signal transduction etr <dig> gene gbs <dig> ; members of the ap2/erebp family gbs <dig> , gbs <dig> , gbs <dig> , gbm <dig> ; and other signaling genes.
figure  <dig> shows an example, where the developed markers gbs <dig>  gbs <dig> gbs <dig>  gbs <dig>  gbs <dig> and gbm <dig> mapped on the barley chromosome 3h show their putative ortholog in rice on chromosome os <dig> as the majority of published markers, whereas no syntenic relationship can be observed for the markers gbs <dig>  gbs <dig> and gbs <dig> with orthologous sites on os <dig>  os <dig> and os <dig>  only three markers  did not yield a significant χ <dig> statistics but showed a syntenic relationship in a smaller region between barley and rice.
additionally, we detected for the marker gbs <dig> a syntenic relationship to rice for the second best blastn-hit.
this observation may result from different evolutionary fates of gene duplications occurring in rice after the divergence of the triticeae and oryzeae.
for the remaining twelve markers we did not find an indication for a syntenic relationship based on marker statistics.
they were predominantly located on chromosome 5h but were also found on chromosome 1h, 2h, 3h and 7h.
CONCLUSIONS
we identified expression patterns of major transcription factor genes and signaling related genes expressed during barley ontogeny and selected putative regulators expressed preferentially in pericarp, endosperm and embryo during seed development and germination based on clustering and possible functions.
for a total of  <dig> candidate genes snp and indel-marker development was conducted and allowed the anchoring of  <dig> markers on the genetic linkage map of barley.
exploration of the macrosynteny to rice revealed that the chromosomal order of the putative regulatory factors is in most cases conserved during evolution.
the combined linkage map and reference expression map of regulators defined in the present study offers the possibility for further directed research on the nature and role of regulators during seed development and germination in barley.

 barley is an important crop but also a model plant for temperate cereals because of its diploid nature and rich genetics. extensive genomics resources have been developed, and a number of important genes were already isolated by map-based cloning. since the grain is the agriculturally most important part of the plant many studies have been carried out on barley seed development and germination. however, we still know little about the genes determining important traits and the regulatory networks underlying developmental processes. prime candidates for members of signal transduction chains and regulatory networks are transcription factors, kinases and other experimentally verified regulators. different approaches have been used to gain insight into the relationship between genotype and phenotype in mutant studies and the analysis of transgenic plants. one additional promising approach is the more recently developed concept of genetical genomics, which combines gene expression studies with genetic linkage analysis. differences in transcript levels are used to map the responsible chromosal regions thus eventually allowing the identification of the causative gene. in species whose genomes have not yet been sequenced such as barley, the feasibility of this strategy depends on the number of genetically mapped genes. based on our long-term dedication to understand seed development and seed storage product accumulation as well as to study agronomical traits by extensively using marker-based techniques we set out to first define and characterize a set of genes with known or putative regulatory functions during grain development. in a second step snp marker were developed for as many as possible of those genes and used to anchor them on the barley linkage map. thus we provide new data on regulatory genes and their putative joint action based on cluster analysis of expression data and place part of them on the barley linkage map to promote marker-assisted studies in barley especially on the biology of barley grains.  expression map of selected regulatory genes to define a set of barley genes putatively involved in regulating preferentially grain development sequences from cdna libraries of developing seeds were used in similarity searches against rice and arabidopsis databases in search for homologs of regulatory genes. from the total set, those transcription factors and signalling related genes were selected, which were shown to be exclusively expressed during seed development or alternatively characterized by a spatio-temporal pattern of gene expression, which is characteristic for both seed development and across plant ontogeny. in a first step regulatory sequences were selected based on their homology to known regulatory genes in other model species and on identified homologous sequences from the affymetrix barley genechip. these genes were further characterized. expression patterns were extracted and normalized from published experiments concerning samples from both endosperm and embryo fractions of developing and imbibed seeds of cultivar barke as well as selected stages of seed development, coleoptile, radicle, root, crown and leaf from germinating seeds and immature inflorescence, floral bracts, pistil and anthers from reproductive tissues of cultivars morex and golden promise. a hierarchical clustering method identified major clusters with defined co-expressed regulatory genes that are associated either with specific tissues during seed development or found to be expressed in both developing seeds as well as during other stages of plant ontogeny. individual clades of the hierarchical tree were subjected to k-mean clustering to get more refined sub clusters. as a result, regulatory genes were found to be preferentially expressed in one of the three studied seed tissues during seed development. they are represented in cluster groups and respectively. a total of regulators are characteristically expressed in multiple tissues undergoing cell differentiation events during seed development, also during the shift from germination to seedling establishment as well during the transition from vegetative to generative development. in addition regulators were found to be expressed during both seed development and germination. the first cluster contains two subclusters and subcluster contains regulatory genes that are preferentially activated in pericarp during seed development as well as partly in reproductive tissues. examples are members of the following gene families: an auxin-responsive aux/iaa gene, ap, bhlh, c2h zincfinger, homeobox, a well characterized crabs claw gene from the yabby family and tf mads, which is homologous to the arabidopsis sepallata gene agl whereas in arabidopsis crabs claw and agl members were shown to be required for carpel development, the homologous genes in barley are expressed in the pericarp during early seed developmen, as well as in the pistil. similarly, mads box gene transcripts, known to be involved in floral transition, are here found to be abundantly expressed not only in reproductive organs such as pistil and anther but also in pericarp and endosperm fractions during the onset of seed development and not in the embryo. this indicates an unexplored distinct role of mads box tfs in specific tissues during grain development. similar transcript profiles were observed among signaling related genes: a two-component response regulator, the receptor protein kinase perk, the abc family protein kinase and the gibberellin response modulator rga. regulatory genes expressed preferentially during seed maturation in the endosperm are represented in subcluster we especially note preferential expression of the well-known regulators abi3/vp and snf in endosperm during the maturation phase but not in any other tissue during plant ontogeny. also preferentially expressed during seed maturation in endosperm tissue and to some extent in embryo ) were the aba response element binding factors abf and abi. since in a recent study we found abre elements in the snf kinase promoter region it seems likely that aba plays a role in triggering endosperm maturation events and that the hormone acts through snf mediated by aba via abf3/abi and abi probably by the same way aba is involved in controlling starch biosynthesis during endosperm maturation. also present in subcluster is the barley prolamin binding factor transcript. the respective protein is a member of the dof family of tfs known to activate storage protein hordein b transcription during the main endosperm storage period. coexpressed with the prolamin binding factor in cluster are two members of the ccaat tf family. this family contains the well characterized lec tf of arabidopsis, a major seed maturation control factor of the arabidopsis hap family. taken together, most of the maturation control genes discovered in arabidopsis were also found to be preferentially expressed during maturation events in barley. along with these gene sets we also noticed calcium signaling related genes possibly participating in a genetic framework for seed maturation, which needs further investigation. subcluster of cluster encodes regulators such as transcription factor x, a putative leucine zipper protein, calmodulin-binding proteins, a receptor protein kinase perk1-like protein, a wpk protein kinase and an unclassified protein kinase all expressed preferentially in the embryo during seed maturation. the fourth cluster contains regulators found to be expressed in pericarp, endosperm and embryo during the cell differentiation events in the developing seed. its expression is reduced during the peak of storage phase as well in the mature seeds of the endosperm. interestingly, gene expression patterns of these regulators are found to be expressed throughout embryo development. they are also expressed in the immature inflorescence, the pistil as well as in early germinating seedling tissues such as crown and coleoptile where cell differentiation occurs. this cluster is enriched with a nucleosome/chromatin assembly factor, a set-domain transcriptional regulator-like protein and wd- proteins. wd- members belong to chromatin complex subunits of the snf family. the suppression of its homologues in arabidopsis results in small cotyledonary embryos with limited cell expansion. we furthermore observe the expression of two members of mcm genes, which are homologous to prolifera, a protein required for dna replication during seed development as well as hmg family members known to promote meristematic activities during plant development. g-protein signalling components are also expressed. interestingly, regulators which were found to be expressed in the developing seed were also ubiquitously expressed in all tissues of germinating seedlings as well as in reproductive tissues. examples are ap2-erebp, bzip, hmg and myb tf family members and calcium, g-protein, receptor kinases and serine/threonine protein kinase family members representing signaling components. snp-marker development and anchoring to the linkage map of barley based on the analyzed expression profiles an initial list of transcription factors and kinases was derived and the sequences used for snp detection, marker development and anchoring on the barley linkage map. of the primer pairs designed, yielded a single copy-pcr product and of them were sequenced from both sides for each parental line amounting to a maximal read depth of fourteen reads resulting in an average consensus length of bp amounting to kb sequenced in total. the remaining sixteen loci showed an indel-polymorphism or a microsatellite polymorphism between the parental lines at least in one of the four mapping populations on agarose gels. for sequenced loci we detected polymorphisms between the parental lines in at least one out of four mapping populations. these mapping populations comprised the two doubled haploid standard populations steptoe/morex and oregon wolf barley, as well as two bc advanced backcross populations brenda/hs and brenda/hs which were used for qtl detection elsewhere. due to their population structure the advanced backcross populations are not useful for mapping of previously unassigned markers, but were only used for genotyping of markers mapped in other mapping populations. we detected snps in sequenced polymorphic pcr products resulting in a snp-density of snp per kb. seventy-three loci did not show a sequence polymorphism between the seven barley varieties. all detected snps were dinucleotide polymorphisms except the locus gbs that showed a trinucleotide snp. with respect to snps between the parental lines of the mapping populations the oregon wolfe barleys showed snp in loci or genes, respectively. the number of polymorphic loci and the total number of snps in the oregon wolfe barley was more than twice as found for the brenda/hs population. remarkably, the lowest numbers of polymorphic loci were found between the parental lines of the advanced backcross populations comprising the elite variety'brenda' and two hordeum spontaneum accessions. this low number of polymorphic loci entails snp-densities of and snps per kb in hs and hs versus'brenda', compared to a snp density of and for the oregon wolfe barley and the steptoe/morex populations. in contrast, we found a higher number of snps within polymorphic genes in the brenda/hs and brenda/hs population of and per gene, whereas the snp density in oregon wolfe barley and the steptoe/morex population amounted to snp and snp per locus, respectively. the detected percentage of 22% polymorphic loci for the steptoe/morex population was below the value of 36% reported for the same population in. a low polymorphism rate of 15% was also reported for microsatellite markers in the brenda/hs population, while 46% of tested microsatellite markers were polymorphic in the brenda/hs population. twenty-eight out of snps, for which markers have been developed, are located in exons, whereof snps could be assigned to utrs. the representative examples includes utr regions with assigned snp-sites belonged, for instance, to a well known dof transcription factor, an activator of prolamin gene transcription expressed preferentially in endosperm during storage phase. the other snp-sites assigned to specific utr-motifs includes marker gbs and gbs, which were located in an ires motif that is supposed to be located in 5' utrs. the internal mrna ribosome binding is a mechanism of translation initiation alternative to the conventional 5'-cap mechanism. the ires motifs are and bp in size, respectively. both clones clustered in the expression analysis in the first cluster group with specific expression in pericarp during seed development and partly in reproductive tissues. the number of exonic snps involving transitions exceeds the number of transversions, whereas the ratio of transitions vs. transversions appeared to be balanced at intronic snp-sites. altogether, we developed marker assays for transcription factors and kinases showing a polymorphism at least in one out of four mapping populations. information about primer sequences, expected fragment lengths as well as the crest ids for the cluster-sequences that have been used as template sequence for primer design can be found in additional files and in total, we mapped markers onto the sm- and owb-maps that have been used for the construction of the barley consensus map in. thirty-eight markers have been placed onto the owb-framework map and markers onto the sm-framework map. eight markers could not be assigned to a barley linkage group. the marker locations appeared to be randomly distributed over the barley genome, although a slight accumulation is seen on chromosome 5h. in addition to the mapping of polymorphic loci, we assigned the chromosomal location for loci with the aid of wheat-barley chromosomal addition lines. altogether, markers were allocated to the barley chromosomes of which markers are based on genes which were found specifically expressed in developing seeds or expressed in developing seeds as well as in other tissues. another markers could be genotyped on the advanced backcross populations brenda/hs and brenda/hs these two populations have been used in the past for qtl-analysis of agronomic important traits including grain-related traits, such as thousand-grain weight. in the future we want to extend our studies and include transcriptome-based e-qtls of grain development. synteny to rice there are extensive syntenic relationships between barley and rice chromosomes that are well documented. for instance, out of est-based markers in the consensus map of mapped to known co-linear linkage groups in rice. to assess the synteny of the developed markers to the rice genome we performed a blastn-analyis of the marker sequences to the rice genome in order to detect putative orthologs in rice. then, we analysed the publicly available information on map position and blastn-analysis of the consensus map published by and counted the markers of the barley consensus map, whose best blastn-hit mapped to the same rice chromosome as for the developed markers. for mapped markers and markers assigned to chromosomes in wheat barley addition lines we found a significant blastn-hit in rice, whereas for markers we did not find a putative ortholog in rice. we tested the accumulation of blastn-hits of developed and published barley markers on specific rice chromosomes with a χ2-test assuming n markers on the linkage group divided by the number of rice-chromosomes under the null-hypothesis. based on this test, fourty-one developed markers obey macro co-linear relationships between barley and rice. some highlighting examples include ethylene signal transduction etr gene gbs; members of the ap2/erebp family gbs, gbs, gbs, gbm; and other signaling genes. figure shows an example, where the developed markers gbs gbs gbs gbs gbs and gbm mapped on the barley chromosome 3h show their putative ortholog in rice on chromosome os as the majority of published markers, whereas no syntenic relationship can be observed for the markers gbs gbs and gbs with orthologous sites on os os and os only three markers did not yield a significant χ statistics but showed a syntenic relationship in a smaller region between barley and rice. additionally, we detected for the marker gbs a syntenic relationship to rice for the second best blastn-hit. this observation may result from different evolutionary fates of gene duplications occurring in rice after the divergence of the triticeae and oryzeae. for the remaining twelve markers we did not find an indication for a syntenic relationship based on marker statistics. they were predominantly located on chromosome 5h but were also found on chromosome 1h, 2h, 3h and 7h.  we identified expression patterns of major transcription factor genes and signaling related genes expressed during barley ontogeny and selected putative regulators expressed preferentially in pericarp, endosperm and embryo during seed development and germination based on clustering and possible functions. for a total of candidate genes snp and indel-marker development was conducted and allowed the anchoring of markers on the genetic linkage map of barley. exploration of the macrosynteny to rice revealed that the chromosomal order of the putative regulatory factors is in most cases conserved during evolution. the combined linkage map and reference expression map of regulators defined in the present study offers the possibility for further directed research on the nature and role of regulators during seed development and germination in barley. 
snp-marker development for those regulators resulted in anchoring markers on the genetic linkage map of barley and the chromosomal assignment of another loci by using wheat-barley addition lines. barley seed development is a highly regulated process with fine-tuned interaction of various tissues controlling distinct physiological events during prestorage, storage and dessication phase. by a hierachical clustering of the expression profiles of potential regulatory genes expressed in different tissues, we found regulators preferentially expressed in one of the three grain tissue fractions pericarp, endosperm and embryo during seed development. in addition, regulators found to be expressed during both seed development and germination and additional regulators are characteristically expressed in multiple tissues undergoing cell differentiation events during barley plant ontogeny. the combined linkage map and reference expression map of regulators defined in the present study offers the possibility of further directed research of the functional role of regulators during seed development in barley. another regulators were, beside in the developing seed, ubiquitously expressed among all tissues of germinating seedlings as well as in reproductive tissues. we identified expression patterns of major transcription factors and signaling related genes expressed during barley ontogeny and further assigned possible functions based on likely orthologs functionally well characterized in model plant species. as potential regulators involved within this process we studied transcription factors and kinases for their expression behaviour and anchored a subset of them to the barley linkage map to promote marker-assisted studies on barley grains. exploration of macrosynteny to rice revealed that the chromosomal orders of the mapped putative regulatory factors were predominantly conserved during evolution.
 barley seed development is a highly regulated process with fine-tuned interaction of various tissues controlling distinct physiological events during prestorage, storage and dessication phase. as potential regulators involved within this process we studied transcription factors and kinases for their expression behaviour and anchored a subset of them to the barley linkage map to promote marker-assisted studies on barley grains.  by a hierachical clustering of the expression profiles of potential regulatory genes expressed in different tissues, we found regulators preferentially expressed in one of the three grain tissue fractions pericarp, endosperm and embryo during seed development. in addition, regulators found to be expressed during both seed development and germination and additional regulators are characteristically expressed in multiple tissues undergoing cell differentiation events during barley plant ontogeny. another regulators were, beside in the developing seed, ubiquitously expressed among all tissues of germinating seedlings as well as in reproductive tissues. snp-marker development for those regulators resulted in anchoring markers on the genetic linkage map of barley and the chromosomal assignment of another loci by using wheat-barley addition lines. the snp frequency ranged from to snp/kb in the parents of the various mapping populations and was snp/kb over all eight lines tested. exploration of macrosynteny to rice revealed that the chromosomal orders of the mapped putative regulatory factors were predominantly conserved during evolution.  we identified expression patterns of major transcription factors and signaling related genes expressed during barley ontogeny and further assigned possible functions based on likely orthologs functionally well characterized in model plant species. the combined linkage map and reference expression map of regulators defined in the present study offers the possibility of further directed research of the functional role of regulators during seed development in barley. 