BACKGROUND
on a worldwide basis, grasslands occupy twice the land area of grain crops  <cit> .
tall fescue  is a major cool season forage and turf grass with a genome size of approximately  <dig> ×  <dig> mbp and an out-crossing mode of reproduction  <cit> .
tall fescue is a hexaploid species that contains three genomes .
the p  genome originates from f. pratensis while the g <dig> and g <dig>  genomes are derived from f. arundinacea var 'glaucescens'  <cit> .
tall fescue is closely related to a number of lolium species including perennial ryegrass  and annual ryegrass .
the festuca-lolium complex contains well-adapted, highly productive grass species that are widely distributed in many parts of the world  <cit> .
these cultivated forage grasses provide numerous benefits to humans, including providing feed and fodder for millions of dairy and beef cattle, horses, sheep, and countless wild animals  <cit> .
turf grass production for use in golf courses and lawns is a multi-billion dollar u.s. industry.
besides the direct economic benefits gained from forage and turf grasses, their contributions in soil conservation, environmental protection, recreation, and aesthetics are substantial.
to date, complete genome sequences are available for only two plant species, arabidopsis thaliana and oryza sativa, both with relatively small genome sizes compared to most crop plants.
the genome of tall fescue is approximately  <dig> times larger than that of rice.
it is unlikely that a complete genome sequence will be available for tall fescue or any other forage or turf grass species in the near future.
for grass species with large genomes, focused large-scale development and analysis of ests can provide a basis for gene discovery and the determination of gene function  <cit> .
the out-crossing nature of reproduction and genome complexity of tall fescue make conventional molecular studies difficult and inefficient.
thus molecular studies in tall fescue have lagged far behind those of major cereal species.
tall fescue est and database resources will be useful for comparative genomic analyses of this important plant species with other major grass species, including rice  <cit> , and help cross-species transfer of genetic knowledge from the well characterized species  to those less studied.
here we report the generation of  <dig>  tall fescue ests characterized from nine cdna libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors.
we also report the results of inter-library and library-specific in silico est expression analyses.
RESULTS
festuca cdna libraries and generation of unigene sets
more than  <dig>  est sequences were generated from nine tall fescue cdna libraries constructed from tissues representing various tissue types , growth stages , and abiotic stress factors  .
dna sequencing success rates varied between  <dig> – 97% for all libraries with an overall average length of  <dig> bp.
the young seedling  and heat stressed shoot  libraries had the highest  and lowest  average trimmed est lengths, respectively .
sequences less than  <dig> nucleotides in length , low quality sequences , chimeric sequences , and contaminated sequences  were removed from the data set.
a total of  <dig>  est sequences were deposited in the genbank dbest.
adetails on plant growing conditions, tissue sampling, and library construction for each cdna library are available at ncbi.
bnumber within parenthesis indicates percent sequencing success rate.
cnumber of tentative consensus  sequences generated from est library members.
dpercentage of the total number of ests in each library.
enumber of unique tcs assembled from ests only present in each library.
fnumber of unique sequences only present in each library, including singleton and unique tc.
g fraction of unique sequences in this library to all  <dig>  unique sequences.
in collaboration with the institute for genomic research  the festuca gene index  containing  <dig>  high-quality ests was established  <cit> .
a library-based breakdown of the ests is shown in table  <dig>  cluster analysis revealed  <dig>  unigene sequences that included  <dig>  singletons and  <dig>  tentative consensus  sequences assembled from  <dig>  ests .
more than 67% of the unique sequences or 29% of all ests were singletons.
the number of ests in the tcs ranged from two to  <dig> ests with an average of five ests per tc .
more than 99% of tcs are less than  <dig> kb in length, including tcs < <dig> kb  and those between  <dig> to  <dig> kb  .
the longest tentative consensus  was  <dig>  bp and encodes a rice beta-galactosidase homologue .
approximately 30% of unique sequences are expressed at a low to medium level, i.e.
they are represented by tcs assembled from two to nine ests and accounted for 42% of all sequences.
only  <dig> % of unique sequences are highly expressed as they are represented by tcs comprising more than ten ests.
the highly expressed genes covered 30% of the ests .
among them were  <dig> tcs that consist of more than  <dig> ests each.
eight of these highly expressed transcripts, derived from stem or leaf tissue libraries, demonstrate sequence similarities to genes with known function, most of which are involved in carbon fixation  and carbon metabolism.
tc <dig>  comprising the largest number of ests , is most similar to a rice hypothetical protein of unknown function.
a unique sequences = tcs + singletons
to compare ests characterized from each library, the numbers of singletons, ests in unique tcs and unique ests were calculated.
singleton percentages were calculated by dividing singletons by the total number of ests in each library.
singleton percentages ranged from  <dig> % to 61% in the dr <dig> and fss libraries, respectively.
only  <dig> % of ests assembled into unique tcs in the fss library.
the percentage of unique ests, which include both singleton and ests in unique tcs, is based on ests present in each library, and therefore, suggests library specificity of ests.
almost two-thirds of the ests from the fss library are unique to this library .
annotation of the unigene sets
blastx  <cit>  was conducted against the genbank non-redundant protein  database to assign putative identity to the festuca unigene set.
based on an e-value cutoff of ≤  <dig> e- <dig>  68%  of the festuca unigenes showed significant levels of similarity to nr.
approximately 28%  of these protein homologues were annotated as unknown, hypothetical, or expressed proteins, while the remaining  correspond to proteins with putative known functions.
functional annotation was assigned by mapping unigenes onto the gene ontology consortium  <cit>  structure using the fagi .
unigenes with assigned putative functions were classified into three ontologies: molecular function, biological process, and cellular component by controlled go vocabulary  <cit> .
in total,  <dig>  unigenes  were mapped to one or more ontologies, with multiple assignments possible for a given protein within a single ontology.
thus,  <dig>  assignments were made to the molecular function ontology, with more than 75% of these in the catalytic activity and binding category annotations such ligase, transferase, helicase, and nucleotide binding proteins .
branch child terms for transporter and transcription regulator activities revealed several genes implicated in water channel , carbohydrate transporter , as well as predicted transcription factors with putative roles in stress responses .
under the biological process ontology, the majority of the  <dig>  assignments were to the physiological process  and cellular process  categories, with frequent sub-classification into the response to stress, response to external stimulus, and cell growth and maintenance categories .
the abundance of stress-related annotations is not surprising, considering that a significant portion of the ests were generated from tissues subjected to abiotic stresses.
of the  <dig>  unigenes mapped into cellular component ontologies, the largest groups were assigned into the cell  and organelle  categories .
in silico analysis of gene expression
to identify putative differentially expressed genes, in silico expression analysis were conducted by hierarchical clustering  <cit>  of expression levels of all  <dig>  tcs in the nine cdna libraries, represented as est counts normalized according to library size, using genespring  <dig> .
the libraries were separated into four arbitrary groups based on clustering analysis .
five libraries, including ds <dig>  lf <dig>  sd <dig>  fss, and hss formed the largest group by step further relationships.
three libraries  were from stressed above ground tissues, and therefore, may share similar gene expression memberships.
another group contains rt <dig> and st <dig> libraries, which contain apical meristem tissues active in cell division, elongation, and differentiation.
expression of ests generated from stressed root tissue  significantly differed from other libraries, and was therefore grouped into a third block.
as expected, tfm  library, representing ests from reproductive tissue showed unique expression that differentiated from all other tissue type libraries.
genes sharing quantitatively and functionally related expression patterns were also identified.
based on their library specificity, tcs were classified into nine major clusters  as shown in figure  <dig>  each library was assigned a major cluster containing genes specifically expressed in the particular tissue type or stress treatment of the library.
number of tcs included in each cluster was from  <dig>  to  <dig>   .
the majority of the genes in cluster a were generated from leaf tissue-derived ests.
not surprisingly, numerous photosynthesis and carbon fixation genes were highly expressed, e.g., protein homologue of chloroplast carbonic anhydrase and rubisco activase gene transcripts were represented by more than  <dig> and  <dig> ests, respectively from this library.
in addition, more than  <dig> tcs in cluster a were the homologues of chlorophyll a/b binding  and photosystem ii  proteins.
abundant stem tissue ests were grouped in cluster c. included in this cluster are homologues of enzymes involved in cytosolic glycolysis.
for example, ests of genes that encode for sucrose synthase, glucose dehydrogenase, malate dehydrogenase, and fructose  <dig> -bisphosphate aldolase were present at  <dig> or greater copies in the stem library.
cluster d includes genes specifically expressed in roots  and contains high-copy number ests encoding different classes of methionine synthases and methyl transferases.
ests of a barley metallothionein-like protein  homologue were highly expressed in the root  library and were clustered into six tcs comprised of  <dig> copies.
mt <dig>  one of the four classes of metallothioneins was previously reported to express significantly higher in root than in other tissues such as leaf and flower  <cit> .
the genes associated with clusters e and g are mainly expressed in actively dividing  tissues.
products of genes in these clusters are necessary for cell cycle regulation, transcription and translation.
germination-specific cluster e contains a number of genes coding for transcriptional factors .
in addition, genes involved in photosynthesis were also highly expressed in young seedlings.
g, the largest cluster comprised of  <dig> tcs, contains significant numbers of floral meristem ribosomal and histone protein isoforms that comprise approximately 8% of the members of this cluster.
histone h <dig>  a meristem-specific gene homologue, was present in  <dig> tcs from this library.
histone h <dig> mrna is transiently accumulated during a period of the cell cycle that mostly overlaps the s phase  <cit> .
our results show increased expression of these transcripts which may be indicative of active cell division in floral meristem tissues  <cit> .
cluster b, f, h, and i represent drought-stressed , heat-stressed shoot , drought stressed root , and field stressed shoot  libraries, respectively.
a total of  <dig>  tcs were contained in these four clusters, and accounted for 40% of all contigs.
large numbers of stress response genes were found in these four clusters, including homologues of heat-shock and oxidative-stress proteins, and various classes of transcription factors.
differences in stress-related gene expression were also observed among libraries.
perhaps this is due to differences in stress mechanisms and in other environmental and biological factors among libraries.
for example,  <dig> tcs coding for different classes of heat-shock proteins  were found in the heat-stressed shoot library.
this is significantly higher than the number of heat-shock tcs observed in other stress libraries.
heat stress induced by high temperatures can result in damage to the photosynthetic apparatus  <cit>  thus many chloroplast and photosynthesis related genes found in this cluster were expected.
tc <dig> had high est numbers in both drought-stressed shoot  and drought-stressed root  libraries, but was not detected in other libraries .
this novel drought-specific tc has no similarity to any gene in the nr database.
another highly expressed tc, a homologue of a dna binding protein, contained  <dig>  ests in drought-stressed shoot and  <dig> est in drought-stressed root libraries.
environmental conditions and gene expression
hot dry summers limit the production of cool season forage and turf grasses including tall fescue, therefore, abiotic stresses constitute challenges to forage production.
heat and drought stress typically overlap during summer tall fescue production, and both stresses may induce plant responses leading to similar physiological changes.
to identify genes induced under stress conditions, ests were generated from four cdna libraries constructed from drought stressed shoot , drought stressed root , heat stressed shoot , and field stressed shoot  tissues.
ests were assigned go molecular function annotations, and est percentages were calculated based on comparison to libraries representing non-stressed, greenhouse-grown tissues to identify stressed-associated genes .
the field-stressed shoot library was constructed using the entire shoot and leaf tissues harvested from field-grown plants during mid-july with high daily average air temperatures ranging from  <dig> °c to  <dig> °c.
plants were under environmental stresses including heat, drought as well as possible field pathogens, representing severe summer pasture growth conditions in the u.s. southern great plains.
fss ests were compared to the greenhouse-grown leaf library to examine stress-response of above ground tissues.
many ests identified in the field-stressed library include a number of stress-related gene homologoues .
in addition, the percentage of ests involved in enzyme regulator activity, based on ontology, was much higher than those from non-stressed leaf  .
however, a higher percentage of ests having transporter, binding, and catalytic activity go function may suggest active growth of non-stressed leaf tissue.
large numbers of genes involved in photosynthesis and carbon fixation were found only in the greenhouse-grown leaf library.
comparison of drought stressed  vs. non-stressed, greenhouse-grown root libraries revealed an elevation in transcription regulator ests in drought stressed roots .
ests of genes coding for components of signal transduction pathways , transcription factors , and hormone-mediated signalling proteins  are included in this group.
homologues of the rice s-adenosylmethionine synthetase gene were highly induced in drought-stressed roots , while only four ests of this gene were found in non-stressed root tissues.
ontology analyses indicate that antioxidant, translation regulator, transporter, binding, and catalytic activities of drought stressed root were suppressed in comparison to greenhouse grown roots .
the expression of root-specific metallothionein-like protein genes were significantly decreased in drought-stressed root tissues  when compared to roots from greenhouse-grown plants .
this may suggest physiological processes, including chelation of toxic metal ions and protection against oxidative damage during the process of cellular death and degradation  <cit> , may have been reduced in stressed roots.
ests unique to drought-stress were also identified.
a drought-specific gene , which was highly expressed only in drought-stressed root and shoot libraries  showed no similarity to sequences in nr.
between library est comparisons were made to examine changes in shoot tissue ests from tall fescue plants subjected to drought-, heat-, or field-stress .
under multiple stress conditions, field stressed shoots expressed fewer classes of genes compared to heat-  or drought-  stress alone .
however, many ests expressed in at least two of the three libraries were found.
proline-rich protein ests were expressed in both ds <dig> and fss libraries, and may suggest a common mechanism of producing osmoprotectants in response to drought under either condition.
among the transcripts expressed in field- and heat-stressed shoots, transcripts encoding mitochondrial proteins such as various subunits of nadh dehydrogenase and cytochrome c oxidase were identified.
expression of these transcripts correlated with the enhanced respiratory activity  <cit>  detected in plants subjected to heat stress or field stress.
as expected, large numbers of hsps genes were expressed under both environments.
transcripts expressed under all three treatments include genes for transcriptional factors, oxidative burst, and stress-related homologues .
our results indicate universal expression of these genes in response to stress conditions, similar to those reported in other studies  <cit> .
heat shock proteins and beyond
due to the important roles of heat shock proteins  in multi-stress conditions, we compared the expression of hsps in all nine cdna libraries .
a total  <dig> hsp gene homologues were identified, representing four of the five classes of heat shock proteins  <cit> .
based on their predicted molecular mass, tall fescue hsps were grouped into low molecular weight , hsp <dig> , high molecular weight , and unclassified hsps , each contains  <dig>   <dig>   <dig>  and  <dig> hsps, respectively.
although special focus was placed on stress treated tissues, hsps ests were also present in rapidly developing tissues of floral meristems, roots, and young seedlings.
aunigene id.
dt = singleton, tc = tentative consensus, drought stressed root = dr <dig>  drought stressed shoot = ds <dig>  field stressed shoot = fss, floral meristem = tfm, heat stressed shoot = hss, greenhouse grown leaf = lf <dig>  greenhouse grown root = rt <dig>  field grown stem = st <dig>  young seedling = sd <dig>
b numbers of normalized ests found matching known hsps or hsfs were listed in individual library.
generation of low molecular weight  or small  heat shock proteins is one of the unique aspects of the heat shock response in plants  <cit> .
six classes of smhsps have been identified in stressed plants  <cit> .
although the function of smhsps has not been defined, evidence suggests that they serve as molecular chaperones to protect cells from stress damage, but that they are not required for normal cellular function  <cit> .
our est analysis showed that  <dig> lmw-hsp genes were expressed in the heat stressed shoot library, which is significantly different from their expression in other libraries .
the expression of five lmw-hsp genes in field-stressed shoot tissues may reflect the high temperatures encountered during summer.
previously we found that lmw-hsp genes were highly induced in heat treated tall fescue but not detectable in non-stressed plants  <cit> .
sun et al.
<cit>  found the expression of hsp <dig> a, a cytoplasmic class ii smhsp gene in arabidopsis was triggered by changed water potential and was critical in osmotic stress tolerance.
tc <dig> codes for a wheat smhsp <dig>  homologue that was highly expressed in drought-stressed roots , however, no expression was detected in drought-stressed shoot.
hsp <dig> proteins have been proven to be essential for normal cell function  <cit> .
some members of the hsp <dig> family are expressed constitutively while others are induced by heat or cold  <cit> .
tc <dig> was expressed in seven of the nine libraries .
interestingly, more than twenty ests of this tc were found in floral meristem and greenhouse grown root libraries- tissues active in cell division and elongation.
divergent from lmw-hsp and hsp <dig> gene transcripts, expression of high molecular weight hsp genes occurred mostly in developing tissues  rather than in stressed tissues.
for example, tc <dig>  which encodes a barley cytosolic hsp <dig> homologue, was represented by multiple ests in all developing tissue libraries.
as a chaperone complex in plants, hsp <dig> functions in response to external stimuli  and it is involved in phenotypic plasticity, developmental stability, and buffering of genetic variation  <cit> .
a gene homologue of clpb, a subfamily of hsp <dig>  <cit> , had mid-level expression in young seedling tissues in addition to expression in heat-stressed shoots of tall fescue.
a previous study has shown this nuclear-localized protein has a negative influence to the growth rate of the primary root in addition to its role in thermotolerance in maize  <cit> .
because the expression of hsps is regulated by the activity of heat shock transcription factors   <cit> , the expression pattern of hsf genes was examined for tall fescue.
we identified ten hsf genes representing eight classes of hsfs, all of which had homologues in o. sativa.
it is well accepted that at least two families of heat shock transcription factors  exist in plants: class a hsfs are primarily responsible for stress-inducible activation of heat shock genes whereas some of the class b hsfs may be specialized for repression, or down-regulation, of the heat shock response  <cit> .
this may explain the difference of the expression patterns for ten tall fescue hsf genes.
for example, dt <dig> which codes for hsf <dig> homologue was expressed in heat-stressed shoot tissue only.
this is similar to findings regarding athsf <dig>  which functions as a key regulator of the immediate stress-induced activation of heat shock gene transcription in arabidopsis  <cit> .
a hsf <dig> gene homologue  had high levels of expression in drought-stressed roots, heat-stressed shoots, and young seedlings, and therefore, may function in both stressed and developing tissues.
hsfa <dig> is a transcription factor critically involved in developmental activation of hsp <dig> g <dig> and specifically expressed during embryogenesis in sunflower in the absence of environmental stress  <cit> .
a tall fescue homologue of rhsf <dig> was expressed in greenhouse grown root tissues.
expression of ascorbate peroxidase , a defence enzyme controlled by hsfs and elevated during heat stress was also examined  <cit> .
apx isoenzymes are critical components that prevent oxidative stress in photosynthetic organisms by removing reactive oxygen intermediates  <cit> .
six genes coding for apx homologues were identified in our tall fescue est gene index, all of which were expressed in heat stressed shoot.
expression of these genes was also present in eight of the nine cdna libraries, but not in field stressed shoot library .
a previous study has shown that the expression of apx was induced shortly after tall fescue plants were exposed to heat   <cit> .
however, apx expression was reduced with increased temperatures and exposure time.
therefore, it is not surprising to find low  expression of apx in plants grown under prolonged high-stress  conditions.
CONCLUSIONS
prior to this study, little or no genomic information was available for tall fescue.
in this study, we analyzed  <dig>  tall fescue ests from nine cdna libraries representing different abiotic stresses, tissue types and developmental stages.
a large number of known stress response gene ests were identified from stressed tissue libraries.
these ests represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families.
the est database reported herein has been used for the development of  <dig> est-ssr markers  <cit>  that have been used to generate a genetic linkage map of festuca  <cit> .
the development of additional est-ssr markers is underway  <cit> .
a study using tall fescue est-ssr markers in a phylogenetic analysis of  <dig> cool-season forage grass species representing eight genera of four tribes from two subfamilies of poaceae demonstrated the utility of these markers for comparative genomics studies among grass species  <cit> .
the est characterized in this study can be used for the development of microarrays and mined for single nucleotide polymorphism markers.
this database provides a basis for further gene discovery and the determination of gene function.
additional cdna libraries representing tissues subjected to other abiotic stress factors, e.g., phosphate starvation, high salt stress, summer field stress, not included in this study will enrich this database.
moreover, an important growth component of tall fescue, endophyte-host interactions, has not been studied in relation to gene expression.
development of ests from fungal endophyte-infected tall fescue tissue may assist in better understanding of plant-fungal symbiotic relationships.

 on a worldwide basis, grasslands occupy twice the land area of grain crops. tall fescue is a major cool season forage and turf grass with a genome size of approximately × mbp and an out-crossing mode of reproduction. tall fescue is a hexaploid species that contains three genomes. the p genome originates from f. pratensis while the g and g genomes are derived from f. arundinacea var'glaucescens'. tall fescue is closely related to a number of lolium species including perennial ryegrass and annual ryegrass. the festuca-lolium complex contains well-adapted, highly productive grass species that are widely distributed in many parts of the world. these cultivated forage grasses provide numerous benefits to humans, including providing feed and fodder for millions of dairy and beef cattle, horses, sheep, and countless wild animals. turf grass production for use in golf courses and lawns is a multi-billion dollar u.s. industry. besides the direct economic benefits gained from forage and turf grasses, their contributions in soil conservation, environmental protection, recreation, and aesthetics are substantial. to date, complete genome sequences are available for only two plant species, arabidopsis thaliana and oryza sativa, both with relatively small genome sizes compared to most crop plants. the genome of tall fescue is approximately times larger than that of rice. it is unlikely that a complete genome sequence will be available for tall fescue or any other forage or turf grass species in the near future. for grass species with large genomes, focused large-scale development and analysis of ests can provide a basis for gene discovery and the determination of gene function. the out-crossing nature of reproduction and genome complexity of tall fescue make conventional molecular studies difficult and inefficient. thus molecular studies in tall fescue have lagged far behind those of major cereal species. tall fescue est and database resources will be useful for comparative genomic analyses of this important plant species with other major grass species, including rice, and help cross-species transfer of genetic knowledge from the well characterized species to those less studied. here we report the generation of tall fescue ests characterized from nine cdna libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. we also report the results of inter-library and library-specific in silico est expression analyses.  festuca cdna libraries and generation of unigene sets more than est sequences were generated from nine tall fescue cdna libraries constructed from tissues representing various tissue types, growth stages, and abiotic stress factors. dna sequencing success rates varied between – 97% for all libraries with an overall average length of bp. the young seedling and heat stressed shoot libraries had the highest and lowest average trimmed est lengths, respectively. sequences less than nucleotides in length, low quality sequences, chimeric sequences, and contaminated sequences were removed from the data set. a total of est sequences were deposited in the genbank dbest. adetails on plant growing conditions, tissue sampling, and library construction for each cdna library are available at ncbi. bnumber within parenthesis indicates percent sequencing success rate. cnumber of tentative consensus sequences generated from est library members. dpercentage of the total number of ests in each library. enumber of unique tcs assembled from ests only present in each library. fnumber of unique sequences only present in each library, including singleton and unique tc. g fraction of unique sequences in this library to all unique sequences. in collaboration with the institute for genomic research the festuca gene index containing high-quality ests was established. a library-based breakdown of the ests is shown in table cluster analysis revealed unigene sequences that included singletons and tentative consensus sequences assembled from ests. more than 67% of the unique sequences or 29% of all ests were singletons. the number of ests in the tcs ranged from two to ests with an average of five ests per tc. more than 99% of tcs are less than kb in length, including tcs < kb and those between to kb. the longest tentative consensus was bp and encodes a rice beta-galactosidase homologue. approximately 30% of unique sequences are expressed at a low to medium level, i.e. they are represented by tcs assembled from two to nine ests and accounted for 42% of all sequences. only % of unique sequences are highly expressed as they are represented by tcs comprising more than ten ests. the highly expressed genes covered 30% of the ests. among them were tcs that consist of more than ests each. eight of these highly expressed transcripts, derived from stem or leaf tissue libraries, demonstrate sequence similarities to genes with known function, most of which are involved in carbon fixation and carbon metabolism. tc comprising the largest number of ests, is most similar to a rice hypothetical protein of unknown function. a unique sequences = tcs + singletons to compare ests characterized from each library, the numbers of singletons, ests in unique tcs and unique ests were calculated. singleton percentages were calculated by dividing singletons by the total number of ests in each library. singleton percentages ranged from % to 61% in the dr and fss libraries, respectively. only % of ests assembled into unique tcs in the fss library. the percentage of unique ests, which include both singleton and ests in unique tcs, is based on ests present in each library, and therefore, suggests library specificity of ests. almost two-thirds of the ests from the fss library are unique to this library. annotation of the unigene sets blastx was conducted against the genbank non-redundant protein database to assign putative identity to the festuca unigene set. based on an e-value cutoff of ≤ e- 68% of the festuca unigenes showed significant levels of similarity to nr. approximately 28% of these protein homologues were annotated as unknown, hypothetical, or expressed proteins, while the remaining correspond to proteins with putative known functions. functional annotation was assigned by mapping unigenes onto the gene ontology consortium structure using the fagi. unigenes with assigned putative functions were classified into three ontologies: molecular function, biological process, and cellular component by controlled go vocabulary. in total, unigenes were mapped to one or more ontologies, with multiple assignments possible for a given protein within a single ontology. thus, assignments were made to the molecular function ontology, with more than 75% of these in the catalytic activity and binding category annotations such ligase, transferase, helicase, and nucleotide binding proteins. branch child terms for transporter and transcription regulator activities revealed several genes implicated in water channel, carbohydrate transporter, as well as predicted transcription factors with putative roles in stress responses. under the biological process ontology, the majority of the assignments were to the physiological process and cellular process categories, with frequent sub-classification into the response to stress, response to external stimulus, and cell growth and maintenance categories. the abundance of stress-related annotations is not surprising, considering that a significant portion of the ests were generated from tissues subjected to abiotic stresses. of the unigenes mapped into cellular component ontologies, the largest groups were assigned into the cell and organelle categories. in silico analysis of gene expression to identify putative differentially expressed genes, in silico expression analysis were conducted by hierarchical clustering of expression levels of all tcs in the nine cdna libraries, represented as est counts normalized according to library size, using genespring. the libraries were separated into four arbitrary groups based on clustering analysis. five libraries, including ds lf sd fss, and hss formed the largest group by step further relationships. three libraries were from stressed above ground tissues, and therefore, may share similar gene expression memberships. another group contains rt and st libraries, which contain apical meristem tissues active in cell division, elongation, and differentiation. expression of ests generated from stressed root tissue significantly differed from other libraries, and was therefore grouped into a third block. as expected, tfm library, representing ests from reproductive tissue showed unique expression that differentiated from all other tissue type libraries. genes sharing quantitatively and functionally related expression patterns were also identified. based on their library specificity, tcs were classified into nine major clusters as shown in figure each library was assigned a major cluster containing genes specifically expressed in the particular tissue type or stress treatment of the library. number of tcs included in each cluster was from to. the majority of the genes in cluster a were generated from leaf tissue-derived ests. not surprisingly, numerous photosynthesis and carbon fixation genes were highly expressed, e.g., protein homologue of chloroplast carbonic anhydrase and rubisco activase gene transcripts were represented by more than and ests, respectively from this library. in addition, more than tcs in cluster a were the homologues of chlorophyll a/b binding and photosystem ii proteins. abundant stem tissue ests were grouped in cluster c. included in this cluster are homologues of enzymes involved in cytosolic glycolysis. for example, ests of genes that encode for sucrose synthase, glucose dehydrogenase, malate dehydrogenase, and fructose -bisphosphate aldolase were present at or greater copies in the stem library. cluster d includes genes specifically expressed in roots and contains high-copy number ests encoding different classes of methionine synthases and methyl transferases. ests of a barley metallothionein-like protein homologue were highly expressed in the root library and were clustered into six tcs comprised of copies. mt one of the four classes of metallothioneins was previously reported to express significantly higher in root than in other tissues such as leaf and flower. the genes associated with clusters e and g are mainly expressed in actively dividing tissues. products of genes in these clusters are necessary for cell cycle regulation, transcription and translation. germination-specific cluster e contains a number of genes coding for transcriptional factors. in addition, genes involved in photosynthesis were also highly expressed in young seedlings. g, the largest cluster comprised of tcs, contains significant numbers of floral meristem ribosomal and histone protein isoforms that comprise approximately 8% of the members of this cluster. histone h a meristem-specific gene homologue, was present in tcs from this library. histone h mrna is transiently accumulated during a period of the cell cycle that mostly overlaps the s phase. our results show increased expression of these transcripts which may be indicative of active cell division in floral meristem tissues. cluster b, f, h, and i represent drought-stressed, heat-stressed shoot, drought stressed root, and field stressed shoot libraries, respectively. a total of tcs were contained in these four clusters, and accounted for 40% of all contigs. large numbers of stress response genes were found in these four clusters, including homologues of heat-shock and oxidative-stress proteins, and various classes of transcription factors. differences in stress-related gene expression were also observed among libraries. perhaps this is due to differences in stress mechanisms and in other environmental and biological factors among libraries. for example, tcs coding for different classes of heat-shock proteins were found in the heat-stressed shoot library. this is significantly higher than the number of heat-shock tcs observed in other stress libraries. heat stress induced by high temperatures can result in damage to the photosynthetic apparatus thus many chloroplast and photosynthesis related genes found in this cluster were expected. tc had high est numbers in both drought-stressed shoot and drought-stressed root libraries, but was not detected in other libraries. this novel drought-specific tc has no similarity to any gene in the nr database. another highly expressed tc, a homologue of a dna binding protein, contained ests in drought-stressed shoot and est in drought-stressed root libraries. environmental conditions and gene expression hot dry summers limit the production of cool season forage and turf grasses including tall fescue, therefore, abiotic stresses constitute challenges to forage production. heat and drought stress typically overlap during summer tall fescue production, and both stresses may induce plant responses leading to similar physiological changes. to identify genes induced under stress conditions, ests were generated from four cdna libraries constructed from drought stressed shoot, drought stressed root, heat stressed shoot, and field stressed shoot tissues. ests were assigned go molecular function annotations, and est percentages were calculated based on comparison to libraries representing non-stressed, greenhouse-grown tissues to identify stressed-associated genes. the field-stressed shoot library was constructed using the entire shoot and leaf tissues harvested from field-grown plants during mid-july with high daily average air temperatures ranging from °c to °c. plants were under environmental stresses including heat, drought as well as possible field pathogens, representing severe summer pasture growth conditions in the u.s. southern great plains. fss ests were compared to the greenhouse-grown leaf library to examine stress-response of above ground tissues. many ests identified in the field-stressed library include a number of stress-related gene homologoues. in addition, the percentage of ests involved in enzyme regulator activity, based on ontology, was much higher than those from non-stressed leaf. however, a higher percentage of ests having transporter, binding, and catalytic activity go function may suggest active growth of non-stressed leaf tissue. large numbers of genes involved in photosynthesis and carbon fixation were found only in the greenhouse-grown leaf library. comparison of drought stressed vs. non-stressed, greenhouse-grown root libraries revealed an elevation in transcription regulator ests in drought stressed roots. ests of genes coding for components of signal transduction pathways, transcription factors, and hormone-mediated signalling proteins are included in this group. homologues of the rice s-adenosylmethionine synthetase gene were highly induced in drought-stressed roots, while only four ests of this gene were found in non-stressed root tissues. ontology analyses indicate that antioxidant, translation regulator, transporter, binding, and catalytic activities of drought stressed root were suppressed in comparison to greenhouse grown roots. the expression of root-specific metallothionein-like protein genes were significantly decreased in drought-stressed root tissues when compared to roots from greenhouse-grown plants. this may suggest physiological processes, including chelation of toxic metal ions and protection against oxidative damage during the process of cellular death and degradation, may have been reduced in stressed roots. ests unique to drought-stress were also identified. a drought-specific gene, which was highly expressed only in drought-stressed root and shoot libraries showed no similarity to sequences in nr. between library est comparisons were made to examine changes in shoot tissue ests from tall fescue plants subjected to drought-, heat-, or field-stress. under multiple stress conditions, field stressed shoots expressed fewer classes of genes compared to heat- or drought- stress alone. however, many ests expressed in at least two of the three libraries were found. proline-rich protein ests were expressed in both ds and fss libraries, and may suggest a common mechanism of producing osmoprotectants in response to drought under either condition. among the transcripts expressed in field- and heat-stressed shoots, transcripts encoding mitochondrial proteins such as various subunits of nadh dehydrogenase and cytochrome c oxidase were identified. expression of these transcripts correlated with the enhanced respiratory activity detected in plants subjected to heat stress or field stress. as expected, large numbers of hsps genes were expressed under both environments. transcripts expressed under all three treatments include genes for transcriptional factors, oxidative burst, and stress-related homologues. our results indicate universal expression of these genes in response to stress conditions, similar to those reported in other studies. heat shock proteins and beyond due to the important roles of heat shock proteins in multi-stress conditions, we compared the expression of hsps in all nine cdna libraries. a total hsp gene homologues were identified, representing four of the five classes of heat shock proteins. based on their predicted molecular mass, tall fescue hsps were grouped into low molecular weight, hsp, high molecular weight, and unclassified hsps, each contains and hsps, respectively. although special focus was placed on stress treated tissues, hsps ests were also present in rapidly developing tissues of floral meristems, roots, and young seedlings. aunigene id. dt = singleton, tc = tentative consensus, drought stressed root = dr drought stressed shoot = ds field stressed shoot = fss, floral meristem = tfm, heat stressed shoot = hss, greenhouse grown leaf = lf greenhouse grown root = rt field grown stem = st young seedling = sd b numbers of normalized ests found matching known hsps or hsfs were listed in individual library. generation of low molecular weight or small heat shock proteins is one of the unique aspects of the heat shock response in plants. six classes of smhsps have been identified in stressed plants. although the function of smhsps has not been defined, evidence suggests that they serve as molecular chaperones to protect cells from stress damage, but that they are not required for normal cellular function. our est analysis showed that lmw-hsp genes were expressed in the heat stressed shoot library, which is significantly different from their expression in other libraries. the expression of five lmw-hsp genes in field-stressed shoot tissues may reflect the high temperatures encountered during summer. previously we found that lmw-hsp genes were highly induced in heat treated tall fescue but not detectable in non-stressed plants. sun et al. found the expression of hsp a, a cytoplasmic class ii smhsp gene in arabidopsis was triggered by changed water potential and was critical in osmotic stress tolerance. tc codes for a wheat smhsp homologue that was highly expressed in drought-stressed roots, however, no expression was detected in drought-stressed shoot. hsp proteins have been proven to be essential for normal cell function. some members of the hsp family are expressed constitutively while others are induced by heat or cold. tc was expressed in seven of the nine libraries. interestingly, more than twenty ests of this tc were found in floral meristem and greenhouse grown root libraries- tissues active in cell division and elongation. divergent from lmw-hsp and hsp gene transcripts, expression of high molecular weight hsp genes occurred mostly in developing tissues rather than in stressed tissues. for example, tc which encodes a barley cytosolic hsp homologue, was represented by multiple ests in all developing tissue libraries. as a chaperone complex in plants, hsp functions in response to external stimuli and it is involved in phenotypic plasticity, developmental stability, and buffering of genetic variation. a gene homologue of clpb, a subfamily of hsp, had mid-level expression in young seedling tissues in addition to expression in heat-stressed shoots of tall fescue. a previous study has shown this nuclear-localized protein has a negative influence to the growth rate of the primary root in addition to its role in thermotolerance in maize. because the expression of hsps is regulated by the activity of heat shock transcription factors, the expression pattern of hsf genes was examined for tall fescue. we identified ten hsf genes representing eight classes of hsfs, all of which had homologues in o. sativa. it is well accepted that at least two families of heat shock transcription factors exist in plants: class a hsfs are primarily responsible for stress-inducible activation of heat shock genes whereas some of the class b hsfs may be specialized for repression, or down-regulation, of the heat shock response. this may explain the difference of the expression patterns for ten tall fescue hsf genes. for example, dt which codes for hsf homologue was expressed in heat-stressed shoot tissue only. this is similar to findings regarding athsf which functions as a key regulator of the immediate stress-induced activation of heat shock gene transcription in arabidopsis. a hsf gene homologue had high levels of expression in drought-stressed roots, heat-stressed shoots, and young seedlings, and therefore, may function in both stressed and developing tissues. hsfa is a transcription factor critically involved in developmental activation of hsp g and specifically expressed during embryogenesis in sunflower in the absence of environmental stress. a tall fescue homologue of rhsf was expressed in greenhouse grown root tissues. expression of ascorbate peroxidase, a defence enzyme controlled by hsfs and elevated during heat stress was also examined. apx isoenzymes are critical components that prevent oxidative stress in photosynthetic organisms by removing reactive oxygen intermediates. six genes coding for apx homologues were identified in our tall fescue est gene index, all of which were expressed in heat stressed shoot. expression of these genes was also present in eight of the nine cdna libraries, but not in field stressed shoot library. a previous study has shown that the expression of apx was induced shortly after tall fescue plants were exposed to heat. however, apx expression was reduced with increased temperatures and exposure time. therefore, it is not surprising to find low expression of apx in plants grown under prolonged high-stress conditions.  prior to this study, little or no genomic information was available for tall fescue. in this study, we analyzed tall fescue ests from nine cdna libraries representing different abiotic stresses, tissue types and developmental stages. a large number of known stress response gene ests were identified from stressed tissue libraries. these ests represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families. the est database reported herein has been used for the development of est-ssr markers that have been used to generate a genetic linkage map of festuca. the development of additional est-ssr markers is underway. a study using tall fescue est-ssr markers in a phylogenetic analysis of cool-season forage grass species representing eight genera of four tribes from two subfamilies of poaceae demonstrated the utility of these markers for comparative genomics studies among grass species. the est characterized in this study can be used for the development of microarrays and mined for single nucleotide polymorphism markers. this database provides a basis for further gene discovery and the determination of gene function. additional cdna libraries representing tissues subjected to other abiotic stress factors, e.g., phosphate starvation, high salt stress, summer field stress, not included in this study will enrich this database. moreover, an important growth component of tall fescue, endophyte-host interactions, has not been studied in relation to gene expression. development of ests from fungal endophyte-infected tall fescue tissue may assist in better understanding of plant-fungal symbiotic relationships. 
the festuca gene index has been established. in silico gene expression studies using these ests were performed to understand stress responses in tall fescue. in this paper we report the generation of a tall fescue expressed sequence tag database developed from nine cdna libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. a total of ests were generated from nine cdna libraries of tall fescue representing tissues from different plant organs, developmental stages, and abiotic stress conditions. tall fescue is a major cool season forage and turf grass species grown in the temperate regions of the world. fagi provides a useful resource for genomics studies of tall fescue and other closely related forage and turf grass species. the results of inter-library and library-specific in silico expression analyses of these ests are also reported. comparative genomic analyses between tall fescue and other grass species, including ryegrasses, meadow fescue and tetraploid fescue will benefit from this database. these ests represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families. a large number of ests of known stress response gene were identified from stressed tissue libraries. to date, this represents the first publicly available tall fescue est database. highly expressed ests representing genes of unknown functions were also identified in the stressed tissue libraries.
 tall fescue is a major cool season forage and turf grass species grown in the temperate regions of the world. in this paper we report the generation of a tall fescue expressed sequence tag database developed from nine cdna libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. the results of inter-library and library-specific in silico expression analyses of these ests are also reported.  a total of ests were generated from nine cdna libraries of tall fescue representing tissues from different plant organs, developmental stages, and abiotic stress conditions. the festuca gene index has been established. to date, this represents the first publicly available tall fescue est database. in silico gene expression studies using these ests were performed to understand stress responses in tall fescue. a large number of ests of known stress response gene were identified from stressed tissue libraries. these ests represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families. highly expressed ests representing genes of unknown functions were also identified in the stressed tissue libraries.  fagi provides a useful resource for genomics studies of tall fescue and other closely related forage and turf grass species. comparative genomic analyses between tall fescue and other grass species, including ryegrasses, meadow fescue and tetraploid fescue will benefit from this database. these ests are an excellent resource for the development of simple sequence repeat and single nucleotide polymorphism pcr-based molecular markers. 