BACKGROUND
strawberry  is one of the most important berry crops in the world; in  <dig> its production was approximately  <dig> million metric tons  <cit> .
the benefits that strawberry fruit consumption has on cardiovascular, neurodegenerative, and other diseases like aging, obesity, and cancer has been a subject of increased study over recent years  <cit> .
the strawberry belongs to the family rosaceae in the genus fragaria.
there are four basic fertility groups in fragaria that are associated primarily with their ploidy level or chromosome number.
the most common native species, f. vesca l., has  <dig> chromosomes and it is considered to be a diploid and proposed as model for the genus  <cit> .
the most important cultivated strawberry is a perennial and herbaceous octoploid plant, with fifty six chromosomes , that stems from the cross of the octoploids f. virginiana duchesne from eastern north america, which was noted for its fine flavour, and f. chiloensis  mill.
from chile, noted for its large size  <cit> .
numerous varieties of strawberries have been developed in the temperate zones of the world by different breeding programs.
strawberry has been considered a non-climacteric fruit, since there is no concomitant burst of respiration and production of the hormone ethylene that triggers the ripening process  <cit> .
the berry results from the development of the flower receptacle that consists of a pith at the centre, a fleshy cortex, an epidermis, and a ring of vascular bundles with branches leading to the achenes, the true fruits.
each achene contains a single seed and a hard pericarp.
the achenes are attached to the receptacle by vascular strands.
when classifying the strawberry as non-climacteric, no distinction was made between the receptacle and the achenes.
growth and ripening of strawberry fruits is an important field of research, which includes the role played by hormones, the synthesis of anthocyanins and flavour compounds, and the cell wall changes occurring during the late stages of ripening.
it is reasonable to think that those changes that are important for fruit quality, like anthocyanins and flavour content, as well as fruit softening, mostly rest on the receptacle, whereas hormone control of the process might be supported by the achenes.
therefore, the generation of tools to distinguish the functional roles of these two parts in the growth and ripening of the whole berry is important.
the hormone auxin, which is supplied by the achenes, is considered as a key regulator of growth and ripening.
removal the achenes from the receptacle has different effects depending on the developmental stage.
in the early green stage it stops receptacle growth, whereas in the late green and white stages it accelerates ripening  <cit> .
interestingly, both effects are suppressed by the exogenous application of auxin restoring normal development  <cit> ,  <cit> .
therefore the role of ethylene in fruit ripening has been considered as negligible.
recently, however, it has been reported that the achenes of red fruits produce ethylene at low concentrations, although its role in fruit ripening is unclear  <cit> .
genes related to biochemical processes and metabolites, such as the health promoting metabolites anthocyanin  <cit>  and vitamin c  <cit> , with important roles in modulating fruit quality have been studied.
the aroma, an important criterion defining strawberry quality is dependent on more than  <dig> volatile compounds, many of them esters, whose synthesis is dependent on the strawberry alcohol acyltransferase  activity encoded by the fasaat gene  <cit> .
of all the volatiles, furaneol  is the main one responsible of the aroma of the strawberry fruit  <cit> .
the genes of two enzymes related to the biosynthesis of hdmf have been cloned  <cit> ,  <cit> .
due to the importance of the cell wall in the integrity of the strawberry fruit, genes encoding for cell wall modifying enzymes have been analysed, including expansins  <cit> , cellulases  <cit> , beta-galactosidase  <cit> , pectate lyases  <cit> ,  <cit> , and pectinmethylesterases  <cit> ,  <cit> .
despite all the previous molecular studies, including a recent report on metabolic changes during fruit growth and ripening  <cit> , information on regulatory genes involved in the strawberry fruit development is still scarce.
the development of genomic tools will, no doubt, constitute important input that will facilitate strawberry research.
in recent years molecular markers for this species have been developed  <cit> ,  <cit> , and microarray gene expression experiments during fruit ripening  <cit> ,  <cit> , and in relation to fruit firmness have been reported  <cit> .
one of the most useful tools in the gene discovery, and further assignment of function, is the availability of expressed sequence tags .
these sequences stem from cdna libraries constructed from different tissues and organs, under different environmental conditions and stages of development, so they represent a broad set of expressed genes.
ests collections have been used in gene expression studies  <cit>  and to saturate genetic maps with simple sequence repeats   <cit>  or single nucleotide polymorphisms   <cit> .
they also allow the identification of mirna precursors and targets  <cit> , and massive transcriptome analysis using microarrays  <cit> ,  <cit> .
at present there are more than  <dig> million ests in the genbank database, a quarter of which are from plants.
although fruit crops have been less studied than other plants like arabidopsis, rice, soybean, maize or pine, there is a significant number of ests obtained from fruits like tomato  <cit> , grape  <cit> , apple  <cit> , citrus  <cit>  and melon  <cit> .
in this report we have analysed around  <dig>  ests from f. xananassa,  <dig>  of which originated from our own sequencing project, and  <dig>  are from the genbank database.
these ests have been processed, clustered, annotated and classified into different functional categories.
we have searched for ssrs and snps in the ests set in order to evaluate their potential in marker-assisted breeding programs.
creation of a gene index  <cit>  and comparisons with other species enabled the conclusion that the highest average sequence identity was with the wild diploid relative f. vesca, up to a value of  <dig> % between sequences of orthologous genes.
expression studies of selected ests using qrt-pcr allowed investigating on the possible involvement of hormones like auxin, ethylene, and brassinosteroid in strawberry fruit ripening.
in addition, the set of non-redundant sequences from f. xananassa jointly with an equivalent number of sequences from f. vesca has been used to design and perform a microarrays-based expression studies in ripe receptacle of these two species.
RESULTS
est sequencing and clustering
more than  <dig>  clones were sequenced from six cdna libraries prepared from fruits of several varieties of the cultivated strawberry  .
because we are interested in fruit ripening, transcripts were extracted from two ripening stages, green and red, and two different fruit parts: achenes and receptacle.
in addition, transcripts from red fruits were favoured vs. transcripts from green fruits using two different subtraction procedures .
also, sequences were obtained from transcripts corresponding to genes differentially expressed in ethylene-treated ripe fruits.
a web-accessible database containing all the est sequences, contigs, and bioinformatic tools for their analysis and data mining has been created and named frest http://fresa.uco.uma.es/srs <dig>  the set was completed with the dbest genbank sequences of f. xananassa.
in total,  <dig>  sequences were analyzed in the present study .
* these libraries  were pooled for their analysis and named here after as sgbl
clustering and determination of the consensus sequences were performed through the tgicl pipeline as described in the methods section.
the raw sequences were processed to remove vector and adaptor sequences and to discard sequences with either more than 3% of n or being less than  <dig> bp in length.
the mean length ranged from  <dig> to  <dig> bp, and the accuracy was evaluated by the frequency of appearance of an undetermined nucleotide, and changed from an average of once every  <dig> to  <dig> bp .
all ests  that passed the quality control were used for clustering.
a total of  <dig>  singletons and  <dig>  contigs/tentative consensuses were obtained, resulting in  <dig>  unigenes/non redundant sequences .
some genes were represented by multiple ests as shown in the table  <dig> that includes the contigs with more than  <dig> ests.
in the case of contigs corresponding to metallothionein-like and prunin, with more than  <dig> ests for each one, it is notable that they are overrepresented in the m <dig>  and m <dig>  libraries, respectively .
this is related to the high expression level of these genes in these fruit parts, i.e.
the receptacle and achenes, at this developmental stage.
abundancy
abundancy
functional annotation
a summary of the different parameters studied in the annotation of the complete set of unigenes from f. xananassa is shown in table  <dig>  the number of chimeras was very low based on the blastx sequence searches against the arabidopsis tair database.
annotation included not only sequence homology comparisons in the genbank at two e-value cutoff , but also search for domains using interproscan , signal peptides using signalp tool , association to gene ontology  terms , and numbers of the enzyme commission  .
in total, a  <dig> % unigenes were annotated in at least one of the categories of table  <dig>  this means that there is still a  <dig> % of the unigenes that remained unknown for any putative function.
further, a global analysis by gene ontology  groups was performed with the blast2go software  <cit> .
blast2go uses different tools as blastx and interproscan to annotate sequences.
figure  <dig> shows the result of this analysis.
metabolic processes account for almost 60% of the annotated sequences, including primary, macromolecule, and cellular metabolic processes.
remarkable is the dominance of biosynthetic processes  over catabolic processes .
proteins involved in transport are represented by  <dig> %, and other groups of proteins encoded by est correspond to a wide variety of biological processes.
sequence analyses allowed the identification of genes involved in metabolic and regulatory processes of fruit ripening
we were interested in the fruit ripening process; therefore the rna used in the preparation of the cdna libraries of the present publication was extracted from two parts of the berry at two developmental stages, as it was ripening-enriched by subtraction .
there has been previous sequencing project in strawberry focused on the fruit ripening  <cit>  and we now complete this previous information.
we have performed manual assignment of f. xananassa unigenes to specific metabolic and signalling pathways  providing an exhaustive catalogue of f. xananassa sequences to be further used in specific research projects.
we summarize in table  <dig> the contribution of the new sequences to the information previously available on strawberry genes relevant for the fruit ripening process.
in  <dig> of the  <dig> metabolic pathways the new genes account for more than  <dig> percent of the total number of genes known.
more interestingly, in hormone signalling the information on new genes is very significant, being over  <dig> percent in  <dig> of the  <dig> pathways.
in some cases, like brassinosteroid, gibberellins and abscisic acid, there is not previous information on gene sequences of the corresponding signalling pathways.
overall comparison of f. xananassa sequences with other species.
gene index
we determined the f. xananassa gene index and related it to different plant species using the dfci gene index database , and "ad hoc" gene indices created from the genbank dbest .
the homology search was performed using the blastn tool against non-redundant sequences and true orthologues were considered as having e values of ≤ 1e- <dig>  results of this analysis are shown in table  <dig>  the analysis of these orthologous groups was made from three different perspectives.
percentage of orthologous unigenes of each species relative to f. xananassa unigenes, percentage of f. xananassa orthologous unigenes relative to each species unigenes, and the average identity, after the alignments, of unigenes from each species with the corresponding f. xananassa orthologous unigene.
values for the first two comparisons  are highly dependent on both the number and the length of the available sequences for the species compared.
we focus on the values obtained for f. vesca, the wild diploid species of the same genus of f. xananassa.
it is noteworthy that there is a  <dig>  percent of unigenes from cultivated strawberry that show no putative orthologues to f. vesca unigenes .
thus, although the number of ests available for f. vesca is more than four-fold the number of ests for f. xananassa there are a high number of sequences of cultivated strawberry that have not been revealed in the f. vesca sequencing projects.
high homology to strawberry
high homology to x species
the alignments
the average identity was calculated after the alignments of these putative orthologous sequences from different species with the f. xananassa sequences .
as expected, the highest value was for f. vesca reaching the  <dig>  percent, as the genome of this species probably shares a common ancestor with f. xananassa  <cit> .
the order of the species in this column reflects the taxonomic proximity with close relatives, having rosa hybrid, prunus and malus the highest values.
however, this is not an analysis of phylogeny, but the result of the multiple alignments of sequences available in the databases for the different species.
therefore, it is not possible to gain taxonomic information from the results here presented on species out of the rosaceae family
actual polymorphisms evaluation inside the est collection
microsatellites, or simple sequence repeats , are stretches of dna consisting of tandem repeated short units of 1- <dig> base pairs in length.
the uniqueness and the value of microsatellites as molecular markers arise from their multiallelic nature, co dominant inheritance, relative abundance, extensive genome coverage and simple detection by pcr.
three hundred eighty three  ssrs were identified in  <dig> of the  <dig>  unigenes.
fifty sequences contained more than  <dig> ssr and  <dig> of them with less than  <dig> bp between  <dig> consecutive ssrs.
the frequency of ssr was one every  <dig>  kb of the sequence.
as shown in table  <dig>  dinucleotides are the most frequent motifs , followed by trinucleotides .
other nucleotide combinations are poorly represented .
most of the ssrs found were on the 5' non-coding regions upstream of putative orfs, close to the initial atg.
a total of  <dig> ssrs have been amplified and  <dig> have already been used for studies of f. xananassa varieties and fragaria species  <cit> .
the number of di-, tri-, tetra and pentanucleotides repeats identified in the strawberry database is shown for the complete set of putative ssrs
of the  <dig>  contigs generated in the present study,  <dig> contained a minimum of two alleles,  <dig> of them with potential snps.
in these contigs the changes corresponded to  <dig> potential snps and  <dig> indels.
the final number of good quality true-snps was  <dig>   <dig> of them were transitions,  <dig> were transversions,  <dig> were tri-allelic polymorphisms, and  <dig> were indels.
the frequency of snp was one every  <dig> bp, and a mean value of  <dig>  snps per contig.
expression analysis of selected genes during fruit ripening
a detailed catalogue of strawberry sequences of genes related to hormone biosynthesis and signalling pathways is shown in table  <dig>  the expression of some of the hormone-related genes whose sequence information is provided in the present paper  was further studied in fruits.
for auxin we selected genes encoding arf  proteins, which are transcription factors controlling the expression of auxin-induced genes  <cit> .
regarding ethylene, we studied genes encoding ethylene response transcription factors  that belong to the large ap2/erf family regulating ethylene-responsive genes  <cit> .
patterns of the expression of these genes in achenes and receptacle at three developmental stages are shown in figure  <dig>  values of expression by qrt-pcr are relative for each gene, therefore is not possible to have information on absolute values of expression of different genes.
however, it is apparent that each gene presents a tissue- and developmental-specific expression pattern with significant differences among samples .
thus, transcriptional activity of faarf <dig> is highest in red receptacle whereas for faarf <dig> the highest level of transcripts is detected in white receptacle.
for the faerf genes high and significant changes occur for faerf <dig> and faerf <dig>  having the first the highest value in green achenes and the second in green receptacle.
a more conspicuous case is brassinosteroids, whose involvement in fruit developmental processes has been studied in only a few species  <cit> ,  <cit> .
we have identified ests homologous to the receptor and two components of the signalling pathway  .
their expression also varies with the fruit part, achene or receptacle, and the developmental stage .
highest changes occur for the receptor fabri <dig> whose expression is higher in receptacle and clearly increases with ripening
transcript analysis in red receptacle of f. xananassa and f. vesca
utility of the est collection from f. xananassa was finally tested in transcriptomic studies.
one set of  <dig>  fragaria xananassa non redundant sequences here reported, and another set of  <dig>  fragaria vesca non redundant sequences available in the genbank, were used to design an oligo-based microarray for the expression studies.
fruit characteristics of the cultivated f. xananassa are very different from the f. vesca in terms of size, colour, softness and volatiles  <cit> .
these differences have their origin in the receptacle tissue and become more apparent at the ripe stage.
therefore, analysis of transcripts was performed in the receptacle of ripe fruits from cultivated strawberry f. xananassa  and f. vesca.
expression values are provided in the additional file  <dig>  prior to the analysis of the results, redundancy between the two sets of sequences was determined.
a blastn between both datasets with an e-value < 1e- <dig>  and a similarity percentage > 90% were used as discriminatory criteria.
global analysis was restricted to genes with very different expression level in the two species.
thus, figure  <dig> shows the results of the genes that were over 4-fold up-  and down-regulated  in f. xananassa relative to f. vesca, analyzed by go terms, when differences were statistically significant .
in general distribution of genes between categories of up- and down-regulated genes was similar between them, and also to the distribution in the f. xananassa est collection here analyzed .
however, there are two categories where differences, albeit minor, appear meaningful.
the category "response to stress" was more abundant among the genes up-regulated in f vesca  in comparison to those up-regulated in f. xananassa .
most of these genes correspond to heat shock proteins , which have been reported to play a role not only in thermo tolerance but also in plant development  <cit> ,  <cit> .
a second difference was observed in the category of "regulation of cellular processes" that was more highly represented among the genes up-regulated in f. xananassa.
detailed analysis of the genes reveals that most of them encode for proteins involved in signalling processes, some of them related to hormone action, especially auxin .
expression study was performed in a microarray system as described in the methods.
sequences were selected after establishing a 4-fold threshold and passing a t-test and fdr  test  for significance.
expression study was performed in a microarray system as described in the methods.
sequences were selected after establishing a 4-fold threshold and passing a t-test and fdr  test  for significance.
discussion
sequencing information has produced important data that is being used to investigate both basic and applied aspects of plant growth and development.
it is the first step towards a functional genomics, and a basic tool for molecular breeding.
however, this information has been mainly generated either in model species or species with great impact in global food supply.
fruits of cultivated strawberry  are appreciated both as fresh and as processed foods.
however, there have been only limited genetic and genomic resources developed in this species due to its growing characteristics and the inherent difficulty of working with an octoploid.
despite this, genetic and genomic information is slowly appearing and recently the first genetic map has been reported  <cit> .
in this work we analyzed more than  <dig>  ests from f. xananassa, assembled in more than  <dig>  unigenes.
half of these sequences proceeded from our own sequencing project; a second set of sequences was obtained from the genbank dbest database.
regarding the new sequences reported here it is worth emphasizing that they proceed from different fruit parts , at different developmental stages , and after hormone treatment .
in addition to the genetic characteristics, difficulties analyzing strawberry fruit growth and ripening arise from the fact that the commercial fruit is not a true fruit but includes an engrossed flower receptacle with the true fruits, the achenes, attached on its surface.
moreover, the development pattern of these two parts of the commercial fruit is not synchronous in that the achenes reach their mature stage much earlier than receptacle  <cit>  thus, the sequence information provided in this report specific for achenes and receptacle libraries is highly valuable.
this is highlighted by the high number of ests encoding prunins in the achene library that is absent in the receptacle library.
similarly a large number of ests encoding metallothioneins were identified in the receptacle library with a low number of ests in the achene library.
prunins are known as the globulins of the genus prunus, which comprise the main family of storage proteins synthesized in seeds during embryogenesis  <cit> .
metallothioneins belong to a family of cysteine-rich, low molecular weight proteins that have the capacity to bind metals through the thiol group of the cysteine residues, which represent nearly 30% of their amino acidic residues.
these proteins have been shown to be involved in metal scavenging and detoxification  <cit> , as well as in biotic and abiotic plant responses  <cit> ,  <cit> .
their high abundance in green receptacle suggests their important role in this organ.
the gene index analysis of the sequences reflected the genetic proximity of strawberry with other species of rosaceae.
effectively, fragaria sp.
belongs to the rosaceae family that includes apple, peach and apricot, and to the rosaceae supertribe  <cit>  that includes rose.
the highest identity in the alignment was with f. vesca, from the same genus, followed by rosa hybrida from the same supertribe, and prunus and malus from the same family.
previous studies on genomic resources of fragaria and rosa have also shown a high level of genetic proximity  <cit> ,  <cit> .
there are more than  <dig>  ests available from the diploid f. vesca  <cit>  that has been proposed as a model plant for genomic studies.
recent studies have predicted approximately  <dig> mb for its genome size  <cit>  which might facilitate its complete sequencing.
however, cultivated strawberry is an octoploid species with at least two genomes involved in its origin; one is thought to be an ancestor of f. vesca or f. manchurica, and the other an ancestor of f. iinumae, or potentially other species  <cit> .
overall comparison between the f. vesca and f. xananassa has revealed that only  <dig> % of the diploid species had a corresponding putative orthologous gene in the octoploid.
a possible explanation for this low value would be that the f. vesca derived subgenome is silenced in f. xananassa, as it has been previously described for specific genes  <cit> , or even that the donor subgenome could be an ancestor or f. vesca.
however, these hypothesis needs further studies since it could also be just a consequence of the different precedence of the est sequences used in this comparison, mostly from plantlets in f. vesca and from fruits in f. xananassa.
in any case, cultivated strawberry still represents a great potential source of alleles that might be important for selected traits, since in other species it has been shown that polyploidization is accompanied by changes in the gene expression, and accordingly in phenotypic variation  <cit> .
in addition, the strawberry fruit produces some metabolites that are not found in other fruit models, such as tomato.
these aspects make the ests information provided here valuable since it might eventually be used to probe for specific genes in other species, some of them closely related as some berries of the rubus genus, like raspberry and blackberry that are classified in the same supertribe of rosoideae as fragaria  <cit> .
ssrs derived from ests have been used as functional markers in the generation of maps and in breeding programs.
in strawberry, we have previously used some of these markers to study genetic diversity within the species  <cit> .
based on the high level of identity found with corresponding genes of genetically close species, like those of the rosaceae family, we foresee their transferability to these species, as other authors have shown  <cit> ,  <cit> .
for this purpose, it is important to indicate that strawberry comparative map reveals a high level of co-linearity between diploid and octoploid fragaria species  <cit> .
for other species of the rosaceae family this transferability deserves to be evaluated.
the function played by hormones in the development of strawberry fruits is still an unresolved question.
considered as a non-climacteric fruit, the main role has been attributed to the auxin synthesized in the achenes  <cit> .
a search for genes involved in hormones response was performed.
auxin response factors  are transcription factors acting on the signalling pathway of this hormone  <cit> .
we have unequivocally identified two of them in the strawberry ests database, faarf <dig> and faarf <dig>  for the faarf <dig>  the highest homology corresponds to a gene expressed in tomato  <cit> , and to the arabidopsis arf <dig> gene  <cit> .
faarf <dig> has high homology to both arf <dig> genes from tomato and arabidopsis.
the strawberry gene faarf <dig> is mostly expressed in the receptacle at the white stage.
at this stage the content of auxin is decreasing but still high in comparison to red fruits  <cit> ,  <cit> , and cell expansion determines the final size of the receptacle.
the ethylene binding factors  constitute a family of transcription factors that were identified by their capacity to bind ethylene-responsive elements  present as cis-sequences in the ethylene-inducible genes.
further studies revealed that they act as transcriptional activators or repressors of gcc box-mediated gene expression  <cit> .
in tomato fruits it has been reported that some of them participate in the signalling pathway initiated by ethylene during the ripening of the fruits  <cit> .
in the ests collection we have identified three putative erfs  proceeding from the library prepared from the achenes, and this is consistent with the finding that achenes produced four to ten-fold more ethylene than fruit epidermal peels  <cit> .
both faerf <dig> and faerf <dig> have highest expression at the green stage and show high homology with slerf <dig>  <cit>  and mderf <dig>  <cit> , respectively, involved in tomato and apple fruit ripening.
the corresponding arabidopsis genes for faefr <dig> and faerf <dig> belong to the subfamily b- <dig>   <cit> .
in contrast, the arabidopsis gene homologous to faerf <dig>  which shows minor variation, is classified in the subfamily b- <dig>   <cit> .
the genes in group ix have often been linked in defensive gene expression in response to pathogen infection.
in strawberry there is no information on the content of active brassinosteroid in the ripening fruit.
the preferential expression of fabri <dig> in red receptacle suggests an increased concentration of this hormone in this tissue at later stages of ripening.
however, a relationship between fabri <dig> expression and an increased concentration is not direct since it is needed to know the expression of other important elements in the brassinosteroid signalling pathway such as bak <dig>  and bki <dig>   <cit> .
bzr <dig> is a transcription factor  <cit>  whose cell location depends on its phosphorylation status, mainly controlled by bin <dig>  <cit> .
when bzr <dig> is phosphorylated goes to the nucleus where induces the expression of brassinosteroid dependent genes.
expression of genes fabzr and fabin <dig> occurs in achenes and receptacle at all stages, but the expression ratio fabzr/fabin <dig> is higher in the white achene and lower in white receptacle.
these expression patterns must be interpreted under the light of the interaction of the encoded proteins as above indicated.
in summary, the functional relevance of all these expression studies in terms of the role of hormones in fruit ripening is limited.
however, they illustrate the possibility of using the sequence information here reported to initiate the molecular dissection the problem with gene-specific tools.
the database here reported allowed the comparison of the transcriptome in the ripe receptacle of f. xananassa  and the diploid f. vesca.
as expected, there are very specific changes in genes related to secondary metabolism .
however, global analysis revealed that differences in the transcriptomes being more quantitative than qualitative i.e.
supported by activation/depletion rather than by gain/loss of biological processes.
the two minor differences found in "response to stress", up-regulated in f. vesca, and "regulation of cellular processes", up-regulated in f. xananassa, are probably related to the domestication of the species.
natural environment of the wild f. vesca is more cold climate and high altitude than f. xananassa  <cit> , and it is probable that its cultivation under temperate conditions triggers the heat stress response.
on the other hand, is not surprising that hormone signalling pathways are more efficient in f. xananassa especially those related to auxin action since it has been reported that increasing auxin content in both f. xananassa and f. vesca has the effect of increasing weight and size of fruits  <cit> .
the relevance of these changes here reported deserves further investigation by a deep study of specific genes.
this is currently under progress.
CONCLUSIONS
we anticipate that the generation of this strawberry gene dataset will be important in further genomic studies of this species.
it doubled the number of ests available for this species and combined and analysed all the information presently available for the strawberry.
the analysis of the information reported and gathered in relation to the cultivated strawberry when compared with the available information on the wild strawberry, the diploid fragaria vesca, is valuable to establish their genetic relationship.
it is an essential source of information for the study of the expression of genes, either by qrt-pcr or by microarray.
it will also allow the establishment of few tools for the analysis of metabolic and hormone signalling pathways playing a role in the different developmental processes of this species.

 strawberry is one of the most important berry crops in the world; in its production was approximately million metric tons. the benefits that strawberry fruit consumption has on cardiovascular, neurodegenerative, and other diseases like aging, obesity, and cancer has been a subject of increased study over recent years. the strawberry belongs to the family rosaceae in the genus fragaria. there are four basic fertility groups in fragaria that are associated primarily with their ploidy level or chromosome number. the most common native species, f. vesca l., has chromosomes and it is considered to be a diploid and proposed as model for the genus. the most important cultivated strawberry is a perennial and herbaceous octoploid plant, with fifty six chromosomes, that stems from the cross of the octoploids f. virginiana duchesne from eastern north america, which was noted for its fine flavour, and f. chiloensis mill. from chile, noted for its large size. numerous varieties of strawberries have been developed in the temperate zones of the world by different breeding programs. strawberry has been considered a non-climacteric fruit, since there is no concomitant burst of respiration and production of the hormone ethylene that triggers the ripening process. the berry results from the development of the flower receptacle that consists of a pith at the centre, a fleshy cortex, an epidermis, and a ring of vascular bundles with branches leading to the achenes, the true fruits. each achene contains a single seed and a hard pericarp. the achenes are attached to the receptacle by vascular strands. when classifying the strawberry as non-climacteric, no distinction was made between the receptacle and the achenes. growth and ripening of strawberry fruits is an important field of research, which includes the role played by hormones, the synthesis of anthocyanins and flavour compounds, and the cell wall changes occurring during the late stages of ripening. it is reasonable to think that those changes that are important for fruit quality, like anthocyanins and flavour content, as well as fruit softening, mostly rest on the receptacle, whereas hormone control of the process might be supported by the achenes. therefore, the generation of tools to distinguish the functional roles of these two parts in the growth and ripening of the whole berry is important. the hormone auxin, which is supplied by the achenes, is considered as a key regulator of growth and ripening. removal the achenes from the receptacle has different effects depending on the developmental stage. in the early green stage it stops receptacle growth, whereas in the late green and white stages it accelerates ripening. interestingly, both effects are suppressed by the exogenous application of auxin restoring normal development,. therefore the role of ethylene in fruit ripening has been considered as negligible. recently, however, it has been reported that the achenes of red fruits produce ethylene at low concentrations, although its role in fruit ripening is unclear. genes related to biochemical processes and metabolites, such as the health promoting metabolites anthocyanin and vitamin c, with important roles in modulating fruit quality have been studied. the aroma, an important criterion defining strawberry quality is dependent on more than volatile compounds, many of them esters, whose synthesis is dependent on the strawberry alcohol acyltransferase activity encoded by the fasaat gene. of all the volatiles, furaneol is the main one responsible of the aroma of the strawberry fruit. the genes of two enzymes related to the biosynthesis of hdmf have been cloned,. due to the importance of the cell wall in the integrity of the strawberry fruit, genes encoding for cell wall modifying enzymes have been analysed, including expansins, cellulases, beta-galactosidase, pectate lyases,, and pectinmethylesterases,. despite all the previous molecular studies, including a recent report on metabolic changes during fruit growth and ripening, information on regulatory genes involved in the strawberry fruit development is still scarce. the development of genomic tools will, no doubt, constitute important input that will facilitate strawberry research. in recent years molecular markers for this species have been developed,, and microarray gene expression experiments during fruit ripening,, and in relation to fruit firmness have been reported. one of the most useful tools in the gene discovery, and further assignment of function, is the availability of expressed sequence tags. these sequences stem from cdna libraries constructed from different tissues and organs, under different environmental conditions and stages of development, so they represent a broad set of expressed genes. ests collections have been used in gene expression studies and to saturate genetic maps with simple sequence repeats or single nucleotide polymorphisms. they also allow the identification of mirna precursors and targets, and massive transcriptome analysis using microarrays,. at present there are more than million ests in the genbank database, a quarter of which are from plants. although fruit crops have been less studied than other plants like arabidopsis, rice, soybean, maize or pine, there is a significant number of ests obtained from fruits like tomato, grape, apple, citrus and melon. in this report we have analysed around ests from f. xananassa, of which originated from our own sequencing project, and are from the genbank database. these ests have been processed, clustered, annotated and classified into different functional categories. we have searched for ssrs and snps in the ests set in order to evaluate their potential in marker-assisted breeding programs. creation of a gene index and comparisons with other species enabled the conclusion that the highest average sequence identity was with the wild diploid relative f. vesca, up to a value of % between sequences of orthologous genes. expression studies of selected ests using qrt-pcr allowed investigating on the possible involvement of hormones like auxin, ethylene, and brassinosteroid in strawberry fruit ripening. in addition, the set of non-redundant sequences from f. xananassa jointly with an equivalent number of sequences from f. vesca has been used to design and perform a microarrays-based expression studies in ripe receptacle of these two species.  est sequencing and clustering more than clones were sequenced from six cdna libraries prepared from fruits of several varieties of the cultivated strawberry. because we are interested in fruit ripening, transcripts were extracted from two ripening stages, green and red, and two different fruit parts: achenes and receptacle. in addition, transcripts from red fruits were favoured vs. transcripts from green fruits using two different subtraction procedures. also, sequences were obtained from transcripts corresponding to genes differentially expressed in ethylene-treated ripe fruits. a web-accessible database containing all the est sequences, contigs, and bioinformatic tools for their analysis and data mining has been created and named frest http://fresa.uco.uma.es/srs the set was completed with the dbest genbank sequences of f. xananassa. in total, sequences were analyzed in the present study. * these libraries were pooled for their analysis and named here after as sgbl clustering and determination of the consensus sequences were performed through the tgicl pipeline as described in the methods section. the raw sequences were processed to remove vector and adaptor sequences and to discard sequences with either more than 3% of n or being less than bp in length. the mean length ranged from to bp, and the accuracy was evaluated by the frequency of appearance of an undetermined nucleotide, and changed from an average of once every to bp. all ests that passed the quality control were used for clustering. a total of singletons and contigs/tentative consensuses were obtained, resulting in unigenes/non redundant sequences. some genes were represented by multiple ests as shown in the table that includes the contigs with more than ests. in the case of contigs corresponding to metallothionein-like and prunin, with more than ests for each one, it is notable that they are overrepresented in the m and m libraries, respectively. this is related to the high expression level of these genes in these fruit parts, i.e. the receptacle and achenes, at this developmental stage. abundancy abundancy functional annotation a summary of the different parameters studied in the annotation of the complete set of unigenes from f. xananassa is shown in table the number of chimeras was very low based on the blastx sequence searches against the arabidopsis tair database. annotation included not only sequence homology comparisons in the genbank at two e-value cutoff, but also search for domains using interproscan, signal peptides using signalp tool, association to gene ontology terms, and numbers of the enzyme commission. in total, a % unigenes were annotated in at least one of the categories of table this means that there is still a % of the unigenes that remained unknown for any putative function. further, a global analysis by gene ontology groups was performed with the blast2go software. blast2go uses different tools as blastx and interproscan to annotate sequences. figure shows the result of this analysis. metabolic processes account for almost 60% of the annotated sequences, including primary, macromolecule, and cellular metabolic processes. remarkable is the dominance of biosynthetic processes over catabolic processes. proteins involved in transport are represented by %, and other groups of proteins encoded by est correspond to a wide variety of biological processes. sequence analyses allowed the identification of genes involved in metabolic and regulatory processes of fruit ripening we were interested in the fruit ripening process; therefore the rna used in the preparation of the cdna libraries of the present publication was extracted from two parts of the berry at two developmental stages, as it was ripening-enriched by subtraction. there has been previous sequencing project in strawberry focused on the fruit ripening and we now complete this previous information. we have performed manual assignment of f. xananassa unigenes to specific metabolic and signalling pathways providing an exhaustive catalogue of f. xananassa sequences to be further used in specific research projects. we summarize in table the contribution of the new sequences to the information previously available on strawberry genes relevant for the fruit ripening process. in of the metabolic pathways the new genes account for more than percent of the total number of genes known. more interestingly, in hormone signalling the information on new genes is very significant, being over percent in of the pathways. in some cases, like brassinosteroid, gibberellins and abscisic acid, there is not previous information on gene sequences of the corresponding signalling pathways. overall comparison of f. xananassa sequences with other species. gene index we determined the f. xananassa gene index and related it to different plant species using the dfci gene index database, and "ad hoc" gene indices created from the genbank dbest. the homology search was performed using the blastn tool against non-redundant sequences and true orthologues were considered as having e values of ≤ 1e- results of this analysis are shown in table the analysis of these orthologous groups was made from three different perspectives. percentage of orthologous unigenes of each species relative to f. xananassa unigenes, percentage of f. xananassa orthologous unigenes relative to each species unigenes, and the average identity, after the alignments, of unigenes from each species with the corresponding f. xananassa orthologous unigene. values for the first two comparisons are highly dependent on both the number and the length of the available sequences for the species compared. we focus on the values obtained for f. vesca, the wild diploid species of the same genus of f. xananassa. it is noteworthy that there is a percent of unigenes from cultivated strawberry that show no putative orthologues to f. vesca unigenes. thus, although the number of ests available for f. vesca is more than four-fold the number of ests for f. xananassa there are a high number of sequences of cultivated strawberry that have not been revealed in the f. vesca sequencing projects. high homology to strawberry high homology to x species the alignments the average identity was calculated after the alignments of these putative orthologous sequences from different species with the f. xananassa sequences. as expected, the highest value was for f. vesca reaching the percent, as the genome of this species probably shares a common ancestor with f. xananassa. the order of the species in this column reflects the taxonomic proximity with close relatives, having rosa hybrid, prunus and malus the highest values. however, this is not an analysis of phylogeny, but the result of the multiple alignments of sequences available in the databases for the different species. therefore, it is not possible to gain taxonomic information from the results here presented on species out of the rosaceae family actual polymorphisms evaluation inside the est collection microsatellites, or simple sequence repeats, are stretches of dna consisting of tandem repeated short units of 1- base pairs in length. the uniqueness and the value of microsatellites as molecular markers arise from their multiallelic nature, co dominant inheritance, relative abundance, extensive genome coverage and simple detection by pcr. three hundred eighty three ssrs were identified in of the unigenes. fifty sequences contained more than ssr and of them with less than bp between consecutive ssrs. the frequency of ssr was one every kb of the sequence. as shown in table dinucleotides are the most frequent motifs, followed by trinucleotides. other nucleotide combinations are poorly represented. most of the ssrs found were on the 5' non-coding regions upstream of putative orfs, close to the initial atg. a total of ssrs have been amplified and have already been used for studies of f. xananassa varieties and fragaria species. the number of di-, tri-, tetra and pentanucleotides repeats identified in the strawberry database is shown for the complete set of putative ssrs of the contigs generated in the present study, contained a minimum of two alleles, of them with potential snps. in these contigs the changes corresponded to potential snps and indels. the final number of good quality true-snps was of them were transitions, were transversions, were tri-allelic polymorphisms, and were indels. the frequency of snp was one every bp, and a mean value of snps per contig. expression analysis of selected genes during fruit ripening a detailed catalogue of strawberry sequences of genes related to hormone biosynthesis and signalling pathways is shown in table the expression of some of the hormone-related genes whose sequence information is provided in the present paper was further studied in fruits. for auxin we selected genes encoding arf proteins, which are transcription factors controlling the expression of auxin-induced genes. regarding ethylene, we studied genes encoding ethylene response transcription factors that belong to the large ap2/erf family regulating ethylene-responsive genes. patterns of the expression of these genes in achenes and receptacle at three developmental stages are shown in figure values of expression by qrt-pcr are relative for each gene, therefore is not possible to have information on absolute values of expression of different genes. however, it is apparent that each gene presents a tissue- and developmental-specific expression pattern with significant differences among samples. thus, transcriptional activity of faarf is highest in red receptacle whereas for faarf the highest level of transcripts is detected in white receptacle. for the faerf genes high and significant changes occur for faerf and faerf having the first the highest value in green achenes and the second in green receptacle. a more conspicuous case is brassinosteroids, whose involvement in fruit developmental processes has been studied in only a few species,. we have identified ests homologous to the receptor and two components of the signalling pathway. their expression also varies with the fruit part, achene or receptacle, and the developmental stage. highest changes occur for the receptor fabri whose expression is higher in receptacle and clearly increases with ripening transcript analysis in red receptacle of f. xananassa and f. vesca utility of the est collection from f. xananassa was finally tested in transcriptomic studies. one set of fragaria xananassa non redundant sequences here reported, and another set of fragaria vesca non redundant sequences available in the genbank, were used to design an oligo-based microarray for the expression studies. fruit characteristics of the cultivated f. xananassa are very different from the f. vesca in terms of size, colour, softness and volatiles. these differences have their origin in the receptacle tissue and become more apparent at the ripe stage. therefore, analysis of transcripts was performed in the receptacle of ripe fruits from cultivated strawberry f. xananassa and f. vesca. expression values are provided in the additional file prior to the analysis of the results, redundancy between the two sets of sequences was determined. a blastn between both datasets with an e-value < 1e- and a similarity percentage > 90% were used as discriminatory criteria. global analysis was restricted to genes with very different expression level in the two species. thus, figure shows the results of the genes that were over 4-fold up- and down-regulated in f. xananassa relative to f. vesca, analyzed by go terms, when differences were statistically significant. in general distribution of genes between categories of up- and down-regulated genes was similar between them, and also to the distribution in the f. xananassa est collection here analyzed. however, there are two categories where differences, albeit minor, appear meaningful. the category "response to stress" was more abundant among the genes up-regulated in f vesca in comparison to those up-regulated in f. xananassa. most of these genes correspond to heat shock proteins, which have been reported to play a role not only in thermo tolerance but also in plant development,. a second difference was observed in the category of "regulation of cellular processes" that was more highly represented among the genes up-regulated in f. xananassa. detailed analysis of the genes reveals that most of them encode for proteins involved in signalling processes, some of them related to hormone action, especially auxin. expression study was performed in a microarray system as described in the methods. sequences were selected after establishing a 4-fold threshold and passing a t-test and fdr test for significance. expression study was performed in a microarray system as described in the methods. sequences were selected after establishing a 4-fold threshold and passing a t-test and fdr test for significance. discussion sequencing information has produced important data that is being used to investigate both basic and applied aspects of plant growth and development. it is the first step towards a functional genomics, and a basic tool for molecular breeding. however, this information has been mainly generated either in model species or species with great impact in global food supply. fruits of cultivated strawberry are appreciated both as fresh and as processed foods. however, there have been only limited genetic and genomic resources developed in this species due to its growing characteristics and the inherent difficulty of working with an octoploid. despite this, genetic and genomic information is slowly appearing and recently the first genetic map has been reported. in this work we analyzed more than ests from f. xananassa, assembled in more than unigenes. half of these sequences proceeded from our own sequencing project; a second set of sequences was obtained from the genbank dbest database. regarding the new sequences reported here it is worth emphasizing that they proceed from different fruit parts, at different developmental stages, and after hormone treatment. in addition to the genetic characteristics, difficulties analyzing strawberry fruit growth and ripening arise from the fact that the commercial fruit is not a true fruit but includes an engrossed flower receptacle with the true fruits, the achenes, attached on its surface. moreover, the development pattern of these two parts of the commercial fruit is not synchronous in that the achenes reach their mature stage much earlier than receptacle thus, the sequence information provided in this report specific for achenes and receptacle libraries is highly valuable. this is highlighted by the high number of ests encoding prunins in the achene library that is absent in the receptacle library. similarly a large number of ests encoding metallothioneins were identified in the receptacle library with a low number of ests in the achene library. prunins are known as the globulins of the genus prunus, which comprise the main family of storage proteins synthesized in seeds during embryogenesis. metallothioneins belong to a family of cysteine-rich, low molecular weight proteins that have the capacity to bind metals through the thiol group of the cysteine residues, which represent nearly 30% of their amino acidic residues. these proteins have been shown to be involved in metal scavenging and detoxification, as well as in biotic and abiotic plant responses,. their high abundance in green receptacle suggests their important role in this organ. the gene index analysis of the sequences reflected the genetic proximity of strawberry with other species of rosaceae. effectively, fragaria sp. belongs to the rosaceae family that includes apple, peach and apricot, and to the rosaceae supertribe that includes rose. the highest identity in the alignment was with f. vesca, from the same genus, followed by rosa hybrida from the same supertribe, and prunus and malus from the same family. previous studies on genomic resources of fragaria and rosa have also shown a high level of genetic proximity,. there are more than ests available from the diploid f. vesca that has been proposed as a model plant for genomic studies. recent studies have predicted approximately mb for its genome size which might facilitate its complete sequencing. however, cultivated strawberry is an octoploid species with at least two genomes involved in its origin; one is thought to be an ancestor of f. vesca or f. manchurica, and the other an ancestor of f. iinumae, or potentially other species. overall comparison between the f. vesca and f. xananassa has revealed that only % of the diploid species had a corresponding putative orthologous gene in the octoploid. a possible explanation for this low value would be that the f. vesca derived subgenome is silenced in f. xananassa, as it has been previously described for specific genes, or even that the donor subgenome could be an ancestor or f. vesca. however, these hypothesis needs further studies since it could also be just a consequence of the different precedence of the est sequences used in this comparison, mostly from plantlets in f. vesca and from fruits in f. xananassa. in any case, cultivated strawberry still represents a great potential source of alleles that might be important for selected traits, since in other species it has been shown that polyploidization is accompanied by changes in the gene expression, and accordingly in phenotypic variation. in addition, the strawberry fruit produces some metabolites that are not found in other fruit models, such as tomato. these aspects make the ests information provided here valuable since it might eventually be used to probe for specific genes in other species, some of them closely related as some berries of the rubus genus, like raspberry and blackberry that are classified in the same supertribe of rosoideae as fragaria. ssrs derived from ests have been used as functional markers in the generation of maps and in breeding programs. in strawberry, we have previously used some of these markers to study genetic diversity within the species. based on the high level of identity found with corresponding genes of genetically close species, like those of the rosaceae family, we foresee their transferability to these species, as other authors have shown,. for this purpose, it is important to indicate that strawberry comparative map reveals a high level of co-linearity between diploid and octoploid fragaria species. for other species of the rosaceae family this transferability deserves to be evaluated. the function played by hormones in the development of strawberry fruits is still an unresolved question. considered as a non-climacteric fruit, the main role has been attributed to the auxin synthesized in the achenes. a search for genes involved in hormones response was performed. auxin response factors are transcription factors acting on the signalling pathway of this hormone. we have unequivocally identified two of them in the strawberry ests database, faarf and faarf for the faarf the highest homology corresponds to a gene expressed in tomato, and to the arabidopsis arf gene. faarf has high homology to both arf genes from tomato and arabidopsis. the strawberry gene faarf is mostly expressed in the receptacle at the white stage. at this stage the content of auxin is decreasing but still high in comparison to red fruits,, and cell expansion determines the final size of the receptacle. the ethylene binding factors constitute a family of transcription factors that were identified by their capacity to bind ethylene-responsive elements present as cis-sequences in the ethylene-inducible genes. further studies revealed that they act as transcriptional activators or repressors of gcc box-mediated gene expression. in tomato fruits it has been reported that some of them participate in the signalling pathway initiated by ethylene during the ripening of the fruits. in the ests collection we have identified three putative erfs proceeding from the library prepared from the achenes, and this is consistent with the finding that achenes produced four to ten-fold more ethylene than fruit epidermal peels. both faerf and faerf have highest expression at the green stage and show high homology with slerf and mderf, respectively, involved in tomato and apple fruit ripening. the corresponding arabidopsis genes for faefr and faerf belong to the subfamily b-. in contrast, the arabidopsis gene homologous to faerf which shows minor variation, is classified in the subfamily b-. the genes in group ix have often been linked in defensive gene expression in response to pathogen infection. in strawberry there is no information on the content of active brassinosteroid in the ripening fruit. the preferential expression of fabri in red receptacle suggests an increased concentration of this hormone in this tissue at later stages of ripening. however, a relationship between fabri expression and an increased concentration is not direct since it is needed to know the expression of other important elements in the brassinosteroid signalling pathway such as bak and bki. bzr is a transcription factor whose cell location depends on its phosphorylation status, mainly controlled by bin. when bzr is phosphorylated goes to the nucleus where induces the expression of brassinosteroid dependent genes. expression of genes fabzr and fabin occurs in achenes and receptacle at all stages, but the expression ratio fabzr/fabin is higher in the white achene and lower in white receptacle. these expression patterns must be interpreted under the light of the interaction of the encoded proteins as above indicated. in summary, the functional relevance of all these expression studies in terms of the role of hormones in fruit ripening is limited. however, they illustrate the possibility of using the sequence information here reported to initiate the molecular dissection the problem with gene-specific tools. the database here reported allowed the comparison of the transcriptome in the ripe receptacle of f. xananassa and the diploid f. vesca. as expected, there are very specific changes in genes related to secondary metabolism. however, global analysis revealed that differences in the transcriptomes being more quantitative than qualitative i.e. supported by activation/depletion rather than by gain/loss of biological processes. the two minor differences found in "response to stress", up-regulated in f. vesca, and "regulation of cellular processes", up-regulated in f. xananassa, are probably related to the domestication of the species. natural environment of the wild f. vesca is more cold climate and high altitude than f. xananassa, and it is probable that its cultivation under temperate conditions triggers the heat stress response. on the other hand, is not surprising that hormone signalling pathways are more efficient in f. xananassa especially those related to auxin action since it has been reported that increasing auxin content in both f. xananassa and f. vesca has the effect of increasing weight and size of fruits. the relevance of these changes here reported deserves further investigation by a deep study of specific genes. this is currently under progress.  we anticipate that the generation of this strawberry gene dataset will be important in further genomic studies of this species. it doubled the number of ests available for this species and combined and analysed all the information presently available for the strawberry. the analysis of the information reported and gathered in relation to the cultivated strawberry when compared with the available information on the wild strawberry, the diploid fragaria vesca, is valuable to establish their genetic relationship. it is an essential source of information for the study of the expression of genes, either by qrt-pcr or by microarray. it will also allow the establishment of few tools for the analysis of metabolic and hormone signalling pathways playing a role in the different developmental processes of this species. 
the collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. however, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative. the availability of libraries prepared from green growing fruits has allowed the cloning of cdnas encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. the study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith. in addition, the sequence information generated in the project, jointly with previous information on sequences from both f. xananassa and f. vesca, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. cultivated strawberry is a hybrid octoploid species whose fruit is highly appreciated due to its organoleptic properties and health benefits. we have produced a collection of expressed sequence tags from different cdna libraries prepared from different fruit parts and developmental stages. we then assembled this information together with that available from f. xananassa resulting a total of unigenes. the identification of ssrs and snps in many of the ests allowed their conversion into functional molecular markers. the sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in qtl analysis. further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative fragaria vesca. despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes.
 cultivated strawberry is a hybrid octoploid species whose fruit is highly appreciated due to its organoleptic properties and health benefits. despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative fragaria vesca. however, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. we have produced a collection of expressed sequence tags from different cdna libraries prepared from different fruit parts and developmental stages. the collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. the study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith.  we have sequenced over ests from fragaria xananassa, thus doubling the number of ests available in the genbank of this species. we then assembled this information together with that available from f. xananassa resulting a total of unigenes. the identification of ssrs and snps in many of the ests allowed their conversion into functional molecular markers. the availability of libraries prepared from green growing fruits has allowed the cloning of cdnas encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. in addition, the sequence information generated in the project, jointly with previous information on sequences from both f. xananassa and f. vesca, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative.  the present study generates essential knowledge and molecular tools that will be useful in improving investigations at the molecular level in cultivated strawberry. this knowledge is likely to provide useful resources in the ongoing breeding programs. the sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in qtl analysis. massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes. 