BACKGROUND
melon  is an important horticultural crop grown in temperate, subtropical and tropical regions worldwide.
melon is among the most important fleshy fruits for fresh consumption, its total production in  <dig> exceeding  <dig> million metric tons, of which  <dig> % are produced in asia,  <dig> % in europe,  <dig> % in america and  <dig> % in africa, being a significant component of fresh fruit traded internationally  <cit> .
melon belongs to the cucurbitaceae family, which comprises up to  <dig> different species distributed in  <dig> genera.
species in this family include watermelon, cucumber, squash and marrow, all of them cultivated essentially because of their fruits, but this family also includes species of interest for other reasons, as, for example, their contents in potentially therapeutic compounds   <cit> .
melon is a diploid species, with a basic number of chromosomes x =  <dig>  and an estimated genome size of  <dig> to  <dig> mb  <cit> , similar in size to the rice genome   <cit>  and about three times the size of the arabidopsis genome   <cit> .
melon has been classified into two subspecies, c. melo ssp.
agrestis and c. melo ssp.
melo with india and africa being their centres of origin, respectively  <cit> .
melon has a great potential for becoming a model for understanding important traits in fruiting crops.
melon fruits have wide morphological, physiological and biochemical diversity  <cit>  which can be exploited to dissect biological processes of great technological importance, among them flavour development and textural changes that occur during fruit ripening.
the contemporary melon cultivars can be divided into two groups, climacteric and nonclimacteric, according to their ripening patterns  <cit> .
climacteric fruits are characterized by rapid and profound changes during ripening associated to increased levels of respiration and release of ethylene, whereas the nonclimacteric varieties do not produce ethylene and have long shelf-life.
analyses of climacteric and nonclimacteric melons have illustrated the process of aroma formation  <cit>  and the temporal sequence of cell wall disassembly  <cit> .
melon can be also a very useful experimental system to analyse other aspects of fundamental plant biology.
for example, melon and other cucurbits have been used to analyse the development of the plant vasculature and the transportation of macromolecules through it  <cit> , and different interactions between melon and pests and pathogens have been characterised with varying depths  <cit> .
important genetic tools have been described for melon, as for example linkage genetic maps  <cit>  and the development of a genomic library of near isogenic lines  from an exotic accession  <cit> ; also, biotechnology is feasible in melon  <cit> .
however, the great majority of genes involved in the aforementioned traits are yet to be identified in melon.
partial sequencing of cdna inserts of expressed sequence tags  have been used as an effective method for gene discovery.
by sequencing clones derived from rna from different sources, and/or by normalizing cdna libraries, the total set of genes sampled can be maximized.
bioinformatic analysis, annotation and clustering of sequences could yield databases which mining can be used to select candidate genes implicated in traits of interest.
est collections can also serve to construct microarrays useful for identifying sets of plant genes expressed during different developmental stages and/or responding to environmental stimuli  <cit> .
in addition, est collections are good sources of simple sequence repeats  and single-nucleotide polymorphisms  that can be used for creating saturated genetic maps  <cit> .
thus, est collections have been generated for many plant species, being the most comprehensive those of arabidopsis <cit>  and rice  <cit> .
fruit crops have been less extensively surveyed, but important collections are publicly available for several species, including tomato  <cit> , apple  <cit> , grape  <cit>  and citrus  <cit> .
despite the importance of the family cucurbitaceae, relatively little est information is currently available: only  <dig>  nucleotide sequences have been annotated from the whole cucurbitaceae family in the publicly accessible genbank database as of november 2006; out of these,  <dig>  correspond to the cucumis genus and  <dig>  to melon.
these numbers are in sharp contrast with the data available for families composed of other important food crops like solanaceae , fabaceae , brassicaceae , vitaceae  and rosaceae .
here we describe a public est sequencing project in melon.
we report the determination and analysis of  <dig>  high-quality melon ests, sequenced from eight normalized cdna libraries corresponding to different tissues in different physiological conditions.
we have classified the sequences into functional categories and described ssrs and snps of potential use in genetic maps and marker-assisted breeding programs.
a database which contains all est sequences, contig images and several tools for analysis and data mining has been created.
in addition, we have analyzed the est melon dataset to identify candidate genes potentially coding micrornas or involved in fruit maturation processes and pathogen defence.
the pattern of transcript accumulation in different physiological conditions has been characterised by real-time-qpcr for  <dig> of these candidate genes.
RESULTS
est sequencing and clustering
eight cdna libraries were constructed using material from "piel de sapo" spanish cultivars, the c- <dig> cantaloupe line  and the accession pat <dig> of c. melo l. ssp.
agrestis  pangalo.
the sources of rna to construct each library were fruits of  <dig> and  <dig> days after pollination , leaves, photosynthetic cotyledons inoculated with cucumber mosaic virus , healthy roots and monosporascus cannonballus pollack et uecker  infected roots .
approximately  <dig>  sequences were determined from each library by single-pass 5' sequencing, except for the library prepared from cmv infected cotyledons for which approximately  <dig>  sequences were determined, yielding a total of  <dig>  raw sequences.
processing to eliminate vector sequences, low quality chromatograms and sequences of less than  <dig> base pairs  gave rise to  <dig>  good quality expressed sequence tags   implying a cloning success of approximately 89%.
the average edited length was  <dig> bp, and only a  <dig> % of the sequences had less than  <dig> bp.
clustering of the sequences using default parameters of the est analysis pipeline est2uni  <cit>  yielded  <dig>  tentative consensus sequences  and  <dig>  unclustered sequences , with a total of  <dig>  non-redundant sequences or unigenes .
all good quality ests were used for clustering, independently of the melon genotype of origin, because single nucleotide polymorphisms  were expected among genotypes.
the number of ests per unigene was between  <dig> and  <dig> , with an average of  <dig>  ests per contig, as a high proportion of contigs  contained less than  <dig> ests and contigs with more than  <dig> ests were scarce .
therefore, redundancy values were notably low .
the unigene length varied between  <dig> bp and  <dig>  bp, averaging  <dig> bp .
library specific unigenes were about one third of the total for each library .
a second round of clustering yielded  <dig>  unigene clusters, referred to as superunigenes.
a web integrated database that contains all est sequences, contig images and several tools for analysis and data mining has been created and named melogen  <cit> .
codon usage was estimated using this est collection.
as expected, the codon usage of melon was very similar to that of arabidopsis and other dicots.
the preferred stop codon was uga occurring in the 48% of the sequences.
suppression of the cg dinucleotide in the last two codon positions is very frequent in dicots, possibly as a consequence of methylation of c in the cg dinucleotide, resulting in an increased mutation rate  <cit> ; in agreement with these data, the ratio xcg/xcc for melon was  <dig> , very similar to the corresponding figure for tomato , pea , potato  and other dicots  <cit> .
libraries obtained from tissues inoculated with m. cannonballus were expected to contain sequences from the fungus.
to estimate the proportion of sequences of fungal origin in these libraries, blast analyses against a database with plant and fungal sequences were carried out  <cit> .
only  <dig> sequences from these libraries were found to have a more significant similarity with fungal sequences than with plant sequences .
consequently, these sequences were considered of fungal origin  <cit> .
ssrs and snps
we have analysed the nature and frequency of microsatellites or simple sequence repeats  in the melon sequence dataset.
a search for repeats of two, three or four nucleotides in the dataset yielded  <dig>  potential ssrs.
approximately, 6% of the unigenes contained at least one of the considered ssrs motifs, with repeats of three nucleotides being prevalent .
the maximum and minimum lengths of the repeats were  <dig> and  <dig> nucleotides, respectively, and the average length was  <dig> nucleotides.
the most common repeat among dinucleotides was, by far, the ag repeat, constituting the 83% .
repeats of at and ac dinucleotides followed, with approximately 9% and 7%, respectively.
among the trinucleotide repeats, the most frequent was aag , and the least frequent was act .
among tetranucleotide repeats, the most frequent was aaag .
a high proportion of ssrs  were found in open reading frames , though an analysis of the localization of di-, tri- and tetranucleotides separately showed that di- and tetranucleotides localised preferentially in untranslated regions , whereas trinucleotides localised in both, utrs and orfs .
*the number of di-, tri- and tetranucleotide repeats identified in the melon database is shown for the complete set of putative ssrs .
*only full length melon unigenes were used for this analysis.
full length unigenes were automatically selected by checking the presence of the start codon by comparison of the 5'-terminal region of the unigene with the corresponding arabidopsis gene used for annotation.
†imprecise localization of the ssr with respect to putative initiation or termination codons.
single nucleotide polymorphisms  are the most abundant variations in genomes and, therefore, constitute a powerful tool for mapping and marker-assisted breeding.
we initially identified in the melon sequence dataset  <dig>  single nucleotide sequence variations and therefore potential snps  distributed in  <dig>  contigs; however, these variations would include high-quality snps  but also sequencing errors and mutations introduced during the cdna synthesis step.
using more stringent criteria, these figures were substantially reduced: putative snps were annotated only when the least represented allele was present in at least two est sequences from the same genotype in a given contig and showing the same base change.
two accessions of the same cultivar  represented  <dig> % of the sequences, but more than one half of the sequences were from two other more distant genotypes, the c- <dig> cantaloupe accession  and the pat <dig> agrestis accession .
thus, a total of  <dig> high-quality snps were found in  <dig> contigs, averaging  <dig>  snps per contig.
transitions were much more common than transversions.
there were  <dig> ag and  <dig> ct transitions compared with  <dig> ac,  <dig> at and  <dig> gt transversions .
cg transversions were not detected.
the melogen database  <cit>  includes a tool for designing oligonucleotide primers to amplify the region containing the polymorphism to generate the corresponding molecular marker.
*type and number of transition and transversions are shown for putative single nucleotide variations in sequence  and for putative high-quality single nucleotide polymorphism  identified in the melon database.
functional annotation
in order to identify melon unigenes potentially encoding proteins with known function, we carried out a blastx analysis  <cit>  of the sequence dataset against the databases listed in table  <dig>  out of the  <dig>  unigenes with a hit with proteins in databases,  <dig>   unigenes showing an e value of ≤ 1e- <dig> were annotated.
on the other hand,  <dig> % of the unigenes did not show significant similarity to any protein in the databases and, therefore, were not annotated.
* databases searched were: arabidopsis  <cit> ; uniref <dig>  <cit> ; cucurbitaceae: all cucurbitaceae sequences available in the national center for biotechnology information ; any database: results using arabidopsis, cucurbitaceae and uniref <dig> databases all together; pfam  <cit> .
additionally, we performed a functional classification of the unigenes following the gene ontology scheme.
gene ontology provides a structured and controlled vocabulary to describe gene products according to three ontologies: molecular function, biological process and cellular component  <cit> .
to do that, we added go terms based on the automated annotation of each unigene using the arabidopsis database  <cit> .
a summary of the results with the percentage of unigenes annotated in representative categories corresponding to the go slim terms  <cit>  is shown, as well as a comparison of the distribution of melon and arabidopsis unigenes .
the distributions of melon and arabidopsis unigenes follow similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome.
in total,  <dig>  unigenes could be mapped to one or more ontologies, with multiple assignments possible for a given protein within a single ontology.
a high percentage of unigenes in both species was classified as "unknown function".
out of the  <dig>  assignments made to the cellular component category,  <dig> % corresponded to membrane proteins and  <dig> % to plastidial proteins .
under the molecular function category, assignments were mainly to catalytic activity  and to hydrolase activity  .
the distribution of unigenes under the biological process category was more uniform, with  <dig> % of assignments to cellular process and  <dig> % to biosynthesis .
we have also identified  <dig>   melon unigenes with an ortholog in the arabidopsis database, and a hmmer motif has been assigned to  <dig>   unigenes by comparisons with the pfam database  <cit>  .
all these results are compiled in the melogen database, which also contains direct links to the databases used to carry out analyses.
genes potentially encoding micrornas
central to rna silencing are small rna molecules  that can arise from endogenous or exogenous sources from precursors with double-stranded rna  pairing.
one class of such srnas are micrornas , which originate from endogenous long self-complementary precursors that mature in a multi-step process involving many enzymes  <cit> .
recently, a comprehensive strategy to identify new mirna homologs in est databases has been developed  <cit> .
we have followed this strategy to identify potential melon mirnas.
a total of  <dig> ests that contained homologs to mirnas in the microrna registry database  <cit>  were identified and grouped into  <dig> contigs and, after manual inspection of secondary foldback hairpin structure,  <dig> unigenes were selected .
contig sequences varied between  <dig> and  <dig> nucleotides long, and had negative folding free energies of - <dig>  to - <dig>  kcal mol- <dig>  according to mfold  <cit> , which are in the range of the computational values of arabidopsis mirna precursors  <cit> .
their predicted secondary structures showed that there were at least  <dig> nucleotides paired between the sequence of the potential mature mirna and its opposite arm  in the corresponding hairpin structure .
the location of the potential mirnas varied among ests,  <dig> were found in the sense orientation of the est,  <dig> was found in the antisense orientation.
we have also searched for potential targets of the potential mirnas in the melon est dataset, identifying  <dig> of them .
however, minimal folding free energy indexes   <cit>  were below the - <dig>  cut-off value proposed by zhang et al.
<cit>  only for m <dig> .
potential melon mirna m <dig> has a precursor of  <dig> nt in length and codes for a melon ortholog of the arabidopsis mir <dig>  mir <dig> targets a transcription factor of the tcp family  <cit> ; in the melon dataset, an ortholog of this arabidopsis gene has been found in a unigene annotated as a tcp transcription factor.
in this case, the melon mirna and its potential target have a pattern of paired/non-paired bases between the target and the mirna identical to the corresponding target-mirna pattern in arabidopsis .
*nucleotide sequences correspond to potential mature mirna sequences as deduced from blast searches using known plant mirna sequences  <cit>  and analysis of secondary structure predictions  <cit> .
†accession numbers  of potential precursors of melon mirnas are given.
§computational values of folding free energies have been calculated using mfold  <dig>   <cit> .
Δminimal folding free energy indexes  have been calculated as described by zhang et al.
<cit> .
genes potentially encoding pathogen resistance and fruit quality traits
pathogens affect severely the productivity of melon crops.
three of the cdna libraries sequenced here correspond to pathogen-infected tissues and, thus, should contain transcripts from genes whose expression is induced in response infection.
we have carried out a bioinformatics search for homologs of genes involved in pathogen resistance response  and virus susceptibility  <cit> , finding among them at least one melon ortholog to the arabidopsis fls <dig> receptor  <cit> , several unigenes potentially encoding disease resistance proteins as well as mitogen-activated protein kinases, homologs to translation initiation factors constituting potential virus susceptibility factors, etc.
.
fruit development and ripening are the most important processes determining the fruit quality traits of fleshy fruits like melon.
at present most of the molecular and genetic data available about fruit development and ripening come from tomato  <cit>  and arabidopsis  <cit> .
in recent years, several genes and quantitative trait loci controlling fruit quality traits have been described in melon  <cit> .
as for developmental processes, homologs to genes involved in melon fruit development, ripening and quality have been found in the melon dataset.
these include several mads-box genes, homologs to the fw <dig>  and ovate qtls  <cit> , several homologs to members of the sbp-box gene family to which the major tomato ripening gene colorless non-ripening belongs  <cit> , several acc synthase and acc oxidase genes, unigenes from several cell wall-metabolism enzymes, etc .
expression analysis of selected ests by real-time-qpcr
the accumulation of transcripts for  <dig> selected genes was analyzed by reverse transcription real-time-qpcr.
ests for this analysis were preferentially chosen among those showing significant similarity with genes related to response to infection and fruit quality characteristics in melon and other species, and included ctl <dig>  eif4a- <dig>  eif4e, ein <dig>  ga2ox <dig>  hsp <dig>  hsp <dig>  iaa <dig>  lsm <dig>  lut <dig>  ncbp, svp, hir, tch <dig>  tip <dig>  tom <dig>  tom2a, tom <dig>  uge <dig> and wrky <dig> .
preliminary experiments were carried out to choose between gapdh and cyclophilin  rnas as endogenous controls; results showed that the cyp <dig> rna levels varied the least among treatments  and, therefore, transcript accumulation levels were expressed relative to cyp <dig> rna levels.
the response of selected genes in roots inoculated with m. cannonballus was analysed in melon genotypes known to be susceptible  and partially resistant  to the infection by this fungus.
the patterns of transcript accumulation resulted clearly different for both genotypes.
for pat <dig> , transcription factors wrky <dig> and svp increased their expression between  <dig> and  <dig> times after inoculation; other stress-inducible genes  showed only a moderate increase .
for "piel de sapo" , accumulation of wrky <dig> and svp transcripts only increased about  <dig>  times after inoculation whereas the expression of hsp <dig> showed a marked increase .
it is also worth noting the differential response of the ga2ox <dig> gene in the two genotypes.
expression of ga2ox <dig> increased about  <dig>  times in pat <dig> roots after the m. cannonballus attack, whereas it decreased in "piel de sapo" roots after fungal infection .
comparison of patterns of transcript accumulation at two stages of fruit development showed increased levels of gene expression for  <dig> of the analysed genes.
this was particularly evident for hsp <dig>  tom2a, tom <dig>  ein <dig> and iaa <dig>  in contrast, decreased levels of transcript accumulation were observed for the other  <dig> genes.
discussion
in this paper we provide an initial platform for functional genomics of melon by the identification of more than  <dig>  unigenes assembled from almost  <dig>  ests sequenced from  <dig> melon cdna libraries.
it is probably premature to estimate the proportion of melon genes represented in this dataset, but based on available data for other plant species , it is likely that the melon unigene set characterised here represents approximately between half and one-third of the number of expressed, protein coding genes of melon.
libraries were constructed from various tissue types, but with a bias towards fruit development and pathogen-infected tissues.
data from these libraries will become a useful resource of genes for experiments aimed at understanding important processes involved in fruit development and resistance to viral and fungal pathogens.
also, data presented here provide an important tool for generating markers to saturate melon genetic maps.
in contrast to typical est gene-sampling strategies reported previously, we have found a low degree of redundancy in the sequences determined.
the process of clustering reduced the number of sequences to 56%, from  <dig>  good quality ests to  <dig>  contigs and  <dig>  singletons.
contigs with more than  <dig> ests were scarce, the majority of them being formed by  <dig> or  <dig> ests.
redundancy of the sequences derived from each library ranged from 13% to 20%, with singletons constituting approximately one third of the unigenes determined per library.
this low redundancy is probably due to the success of the normalization process, responsible for the suppression of superabundant transcripts specific for a given tissue or condition.
normalization precludes in silico analysis of gene expression, but greatly increases the number of unigenes that can be determined by reducing redundancy  <cit> .
here we have used a recently described normalization protocol which is based on the cleavage of dna or dna-rna duplexes by a specific dnase  <cit> ; this process, in our hands, has proven simple, reproducible and efficient.
another factor that has contributed to the low redundancy values obtained has been the sequencing of libraries from very distinct tissues.
thus, the number of library specific unigenes was about one half of the total number of unigenes contributed by each library, suggesting that further sequencing of the libraries still has the potential to provide a good number of new, non-redundant sequences.
cdna sequences are a useful source of ssrs, which are excellent molecular markers due to their high degree of polymorphism.
a common feature of cdna sequences obtained from plants is the high frequency of ssrs that they contain  <cit> .
we have identified more than  <dig>  potential ssrs in the melon dataset, with approximately 6% of the melon unigenes containing di-, tri- or tetranucleotide repeats.
a clear bias toward ag and aag repeats existed, that account for 67% of the ssrs.
in contrast, the gc repeat was not found in the melon dataset.
a similar bias toward ag and against cg repeats has been identified in arabidopsis and other plant species  <cit> .
as proposed at least in one other instance  <cit> , this may be due to the tendency of cpg sequences to be methylated  <cit> , which potentially might inhibit transcription.
another interesting feature of melon ssrs relates to their pattern of localization with respect to putative initiation and termination codons.
it is known that the utrs of transcribed sequences are richer in ssrs than coding regions, particularly at the 5'-utrs  <cit> .
however, in the melon dataset, a high proportion of ssrs  were found in orfs.
an analysis of the localization of di-, tri- and tetranucleotide repeats separately showed that di- and tetranucleotides were preferentially located in utrs, whereas trinucleotides localised in both, utrs and orfs, consistently with maintenance of the orfs coding capacity.
thus, the prevalence of trinucleotide repeats in the melon dataset  explains this result.
we identified in the melon sequence dataset  <dig> high-quality snps.
since non-redundant sequences analysed here encompassed  <dig>  mb, one snp was found every  <dig>  pb of sequence.
this small figure is probably due to the limited number of melon genotypes used and the low redundancy found among libraries.
in fact, when the frequency of snps is computed in relation to the length and number of contigs containing snps, the corresponding value  is of the same order of magnitude as values previously calculated for melon  and other plant species  <cit> .
with the advent of high-throughput detection systems, the ssrs and snps identified here will constitute an important resource for mapping and marker-assisted breeding in melon and closely related crops.
as an approach to the function of melon unigenes, we carried out a bioinformatics analysis based on blastx and matches with the pfam database  <cit> .
the proportion of melon unigenes with no similar sequences in databases was quite high, suggesting that the melon dataset may encompass an important number of melon-specific sequences.
however, the proportion specific sequences might be overestimated because blasting has been made with unigene sequences, which in many cases do not cover the complete length of the transcript.
we performed a functional classification of the unigenes following the gene ontology scheme, which is one of the more versatile and complete systems for functional classification  <cit> .
a comparison of the distributions of melon and arabidopsis unigenes in go categories showed that both followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome.
this is remarkable, as the number of different libraries sequenced has been relatively small; again, this is probably due to the success of the normalization process.
we have also carried out specific searches for genes involved in pathways of particular relevance in melon, such are resistance response and fruit development, identifying a remarkable number of melon candidates.
for example, an ortholog of the flagellin receptor fls <dig> from arabidopsis  <cit>  has been identified, together with  <dig> candidate rlks that may have critical roles in pathogen recognition or diverse signalling processes.
similarly, up to  <dig> mads-box gene homologs with potential roles in development have been found in the melon dataset.
moreover, a bioinformatics approach  <cit>  allowed the identification of potential precursors of melon mirnas together with several potential targets in the melon dataset.
this finding opens the door to biotechnology approaches based on the use of artificial mirnas to specifically silence melon genes  <cit> .
the transcript accumulation analysis for the  <dig> selected genes revealed important changes in gene expression associated with pathogen infection and fruit development.
for virus infection, the accumulation of transcripts remained unaltered for  <dig> genes, but showed a significant increase for  <dig> genes and a decrease for  <dig>  among the set of genes analysed, tom2a and eif4e were known to code for virus susceptibility factors  <cit> ; the expression of tom2a was increased after cmv infection, consistently with its requirement by the virus, but this was not the case for eif4e.
different hypotheses can explain this result: since eif4e is an abundant, housekeeping protein, increased expression may not be essential for virus multiplication; alternatively, cmv may not use eif4e in melon or may use a factor coded by a different member of the 4e family; it may also be that timing of the sampling for this experiment was not appropriate to detect such an effect, as requirement of eif4e might occur very early during virus multiplication.
in the cases of infection of susceptible  and resistant  melons by m. cannonballus, more extensive alterations in gene expression seemed to occur in the susceptible than in the resistant accession.
significantly, for the susceptible accession, stress responsive genes  appeared to be maximally induced, whereas for the resistant accession, a gene encoding a wrky <dig> transcription factor, potentially involved in resistance response, was induced to high levels.
significantly, expression of ga2ox <dig> increased about  <dig>  times in pat <dig> after the m. cannonballus attack, whereas it decreased in "piel de sapo".
ga2ox is a major gibberellin  catabolic enzyme, with an important role in controlling ga levels in plants.
hormones control many plant developmental processes, and strong evidence indicates that hormone signalling is involved in the regulation of root growth and architecture  <cit> .
the differential response of the ga2ox <dig> gene in the two melon genotypes is consistent with an enhanced root growth in pat <dig> after infection  <cit> .
notably, other genes involved in hormone-mediated signalling pathways, such as the iaa <dig> gene, did not show such differential response to m. cannonballus infection in both genotypes.
in the case of fruit development, differences in the expression of selected genes between immature and ripening fruits appeared to be even sharper than in the cases of healthy and pathogen-infected tissues.
specific roles during fruit development for hsp <dig>  tom2a and tom <dig> have not been identified, though an increased expression has been shown at least in the case of tom2a in tomato  <cit> .
the ethylene receptor gene ein <dig> showed a two-fold increase in expression.
ein <dig> is the ortholog of arabidopsis ein <dig> and tomato leetr <dig>  <cit> .
in tomato, leetr <dig> is also highly expressed in ripening fruit, suggesting that it responds by modulating ethylene signalling during ripening  <cit> .
the mads-box gene  showed about a four-fold decrease in expression.
this gene is the ortholog of tomato jointless, which specifies the abscission zone in tomato.
in tomato fruit microarray hybridizations, the expression of jointless also decreased from  <dig> to  <dig> dap  <cit> , in agreement with our data for melon.
the lycopene epsilon cyclase  and xyloglucan endotransglycosylase  genes showed an approximately four-fold decrease in expression during melon fruit development.
these findings fit with the patterns of expression of these genes in tomato, where their transcript levels decrease to a non-detectable level in the ripe fruits  <cit> .
CONCLUSIONS
in summary, this collection of ests represents a substantial increase on the information available for melon.
the dataset contains ssr and snp markers that can be used for breeding, as well as a significant number of candidate genes that can be experimentally tested for their roles in various important processes.
this set of genes constitutes also the basis for a microarray for melon that is being used in experiments to further analyse fruit development and maturation and responses to pathogen infections.

 melon is an important horticultural crop grown in temperate, subtropical and tropical regions worldwide. melon is among the most important fleshy fruits for fresh consumption, its total production in exceeding million metric tons, of which % are produced in asia, % in europe, % in america and % in africa, being a significant component of fresh fruit traded internationally. melon belongs to the cucurbitaceae family, which comprises up to different species distributed in genera. species in this family include watermelon, cucumber, squash and marrow, all of them cultivated essentially because of their fruits, but this family also includes species of interest for other reasons, as, for example, their contents in potentially therapeutic compounds. melon is a diploid species, with a basic number of chromosomes x = and an estimated genome size of to mb, similar in size to the rice genome and about three times the size of the arabidopsis genome. melon has been classified into two subspecies, c. melo ssp. agrestis and c. melo ssp. melo with india and africa being their centres of origin, respectively. melon has a great potential for becoming a model for understanding important traits in fruiting crops. melon fruits have wide morphological, physiological and biochemical diversity which can be exploited to dissect biological processes of great technological importance, among them flavour development and textural changes that occur during fruit ripening. the contemporary melon cultivars can be divided into two groups, climacteric and nonclimacteric, according to their ripening patterns. climacteric fruits are characterized by rapid and profound changes during ripening associated to increased levels of respiration and release of ethylene, whereas the nonclimacteric varieties do not produce ethylene and have long shelf-life. analyses of climacteric and nonclimacteric melons have illustrated the process of aroma formation and the temporal sequence of cell wall disassembly. melon can be also a very useful experimental system to analyse other aspects of fundamental plant biology. for example, melon and other cucurbits have been used to analyse the development of the plant vasculature and the transportation of macromolecules through it, and different interactions between melon and pests and pathogens have been characterised with varying depths. important genetic tools have been described for melon, as for example linkage genetic maps and the development of a genomic library of near isogenic lines from an exotic accession; also, biotechnology is feasible in melon. however, the great majority of genes involved in the aforementioned traits are yet to be identified in melon. partial sequencing of cdna inserts of expressed sequence tags have been used as an effective method for gene discovery. by sequencing clones derived from rna from different sources, and/or by normalizing cdna libraries, the total set of genes sampled can be maximized. bioinformatic analysis, annotation and clustering of sequences could yield databases which mining can be used to select candidate genes implicated in traits of interest. est collections can also serve to construct microarrays useful for identifying sets of plant genes expressed during different developmental stages and/or responding to environmental stimuli. in addition, est collections are good sources of simple sequence repeats and single-nucleotide polymorphisms that can be used for creating saturated genetic maps. thus, est collections have been generated for many plant species, being the most comprehensive those of arabidopsis and rice. fruit crops have been less extensively surveyed, but important collections are publicly available for several species, including tomato, apple, grape and citrus. despite the importance of the family cucurbitaceae, relatively little est information is currently available: only nucleotide sequences have been annotated from the whole cucurbitaceae family in the publicly accessible genbank database as of november 2006; out of these, correspond to the cucumis genus and to melon. these numbers are in sharp contrast with the data available for families composed of other important food crops like solanaceae, fabaceae, brassicaceae, vitaceae and rosaceae. here we describe a public est sequencing project in melon. we report the determination and analysis of high-quality melon ests, sequenced from eight normalized cdna libraries corresponding to different tissues in different physiological conditions. we have classified the sequences into functional categories and described ssrs and snps of potential use in genetic maps and marker-assisted breeding programs. a database which contains all est sequences, contig images and several tools for analysis and data mining has been created. in addition, we have analyzed the est melon dataset to identify candidate genes potentially coding micrornas or involved in fruit maturation processes and pathogen defence. the pattern of transcript accumulation in different physiological conditions has been characterised by real-time-qpcr for of these candidate genes.  est sequencing and clustering eight cdna libraries were constructed using material from "piel de sapo" spanish cultivars, the c- cantaloupe line and the accession pat of c. melo l. ssp. agrestis pangalo. the sources of rna to construct each library were fruits of and days after pollination, leaves, photosynthetic cotyledons inoculated with cucumber mosaic virus, healthy roots and monosporascus cannonballus pollack et uecker infected roots. approximately sequences were determined from each library by single-pass 5' sequencing, except for the library prepared from cmv infected cotyledons for which approximately sequences were determined, yielding a total of raw sequences. processing to eliminate vector sequences, low quality chromatograms and sequences of less than base pairs gave rise to good quality expressed sequence tags implying a cloning success of approximately 89%. the average edited length was bp, and only a % of the sequences had less than bp. clustering of the sequences using default parameters of the est analysis pipeline est2uni yielded tentative consensus sequences and unclustered sequences, with a total of non-redundant sequences or unigenes. all good quality ests were used for clustering, independently of the melon genotype of origin, because single nucleotide polymorphisms were expected among genotypes. the number of ests per unigene was between and, with an average of ests per contig, as a high proportion of contigs contained less than ests and contigs with more than ests were scarce. therefore, redundancy values were notably low. the unigene length varied between bp and bp, averaging bp. library specific unigenes were about one third of the total for each library. a second round of clustering yielded unigene clusters, referred to as superunigenes. a web integrated database that contains all est sequences, contig images and several tools for analysis and data mining has been created and named melogen. codon usage was estimated using this est collection. as expected, the codon usage of melon was very similar to that of arabidopsis and other dicots. the preferred stop codon was uga occurring in the 48% of the sequences. suppression of the cg dinucleotide in the last two codon positions is very frequent in dicots, possibly as a consequence of methylation of c in the cg dinucleotide, resulting in an increased mutation rate; in agreement with these data, the ratio xcg/xcc for melon was, very similar to the corresponding figure for tomato, pea, potato and other dicots. libraries obtained from tissues inoculated with m. cannonballus were expected to contain sequences from the fungus. to estimate the proportion of sequences of fungal origin in these libraries, blast analyses against a database with plant and fungal sequences were carried out. only sequences from these libraries were found to have a more significant similarity with fungal sequences than with plant sequences. consequently, these sequences were considered of fungal origin. ssrs and snps we have analysed the nature and frequency of microsatellites or simple sequence repeats in the melon sequence dataset. a search for repeats of two, three or four nucleotides in the dataset yielded potential ssrs. approximately, 6% of the unigenes contained at least one of the considered ssrs motifs, with repeats of three nucleotides being prevalent. the maximum and minimum lengths of the repeats were and nucleotides, respectively, and the average length was nucleotides. the most common repeat among dinucleotides was, by far, the ag repeat, constituting the 83%. repeats of at and ac dinucleotides followed, with approximately 9% and 7%, respectively. among the trinucleotide repeats, the most frequent was aag, and the least frequent was act. among tetranucleotide repeats, the most frequent was aaag. a high proportion of ssrs were found in open reading frames, though an analysis of the localization of di-, tri- and tetranucleotides separately showed that di- and tetranucleotides localised preferentially in untranslated regions, whereas trinucleotides localised in both, utrs and orfs. *the number of di-, tri- and tetranucleotide repeats identified in the melon database is shown for the complete set of putative ssrs. *only full length melon unigenes were used for this analysis. full length unigenes were automatically selected by checking the presence of the start codon by comparison of the 5'-terminal region of the unigene with the corresponding arabidopsis gene used for annotation. †imprecise localization of the ssr with respect to putative initiation or termination codons. single nucleotide polymorphisms are the most abundant variations in genomes and, therefore, constitute a powerful tool for mapping and marker-assisted breeding. we initially identified in the melon sequence dataset single nucleotide sequence variations and therefore potential snps distributed in contigs; however, these variations would include high-quality snps but also sequencing errors and mutations introduced during the cdna synthesis step. using more stringent criteria, these figures were substantially reduced: putative snps were annotated only when the least represented allele was present in at least two est sequences from the same genotype in a given contig and showing the same base change. two accessions of the same cultivar represented % of the sequences, but more than one half of the sequences were from two other more distant genotypes, the c- cantaloupe accession and the pat agrestis accession. thus, a total of high-quality snps were found in contigs, averaging snps per contig. transitions were much more common than transversions. there were ag and ct transitions compared with ac, at and gt transversions. cg transversions were not detected. the melogen database includes a tool for designing oligonucleotide primers to amplify the region containing the polymorphism to generate the corresponding molecular marker. *type and number of transition and transversions are shown for putative single nucleotide variations in sequence and for putative high-quality single nucleotide polymorphism identified in the melon database. functional annotation in order to identify melon unigenes potentially encoding proteins with known function, we carried out a blastx analysis of the sequence dataset against the databases listed in table out of the unigenes with a hit with proteins in databases, unigenes showing an e value of ≤ 1e- were annotated. on the other hand, % of the unigenes did not show significant similarity to any protein in the databases and, therefore, were not annotated. * databases searched were: arabidopsis; uniref; cucurbitaceae: all cucurbitaceae sequences available in the national center for biotechnology information; any database: results using arabidopsis, cucurbitaceae and uniref databases all together; pfam. additionally, we performed a functional classification of the unigenes following the gene ontology scheme. gene ontology provides a structured and controlled vocabulary to describe gene products according to three ontologies: molecular function, biological process and cellular component. to do that, we added go terms based on the automated annotation of each unigene using the arabidopsis database. a summary of the results with the percentage of unigenes annotated in representative categories corresponding to the go slim terms is shown, as well as a comparison of the distribution of melon and arabidopsis unigenes. the distributions of melon and arabidopsis unigenes follow similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. in total, unigenes could be mapped to one or more ontologies, with multiple assignments possible for a given protein within a single ontology. a high percentage of unigenes in both species was classified as "unknown function". out of the assignments made to the cellular component category, % corresponded to membrane proteins and % to plastidial proteins. under the molecular function category, assignments were mainly to catalytic activity and to hydrolase activity. the distribution of unigenes under the biological process category was more uniform, with % of assignments to cellular process and % to biosynthesis. we have also identified melon unigenes with an ortholog in the arabidopsis database, and a hmmer motif has been assigned to unigenes by comparisons with the pfam database. all these results are compiled in the melogen database, which also contains direct links to the databases used to carry out analyses. genes potentially encoding micrornas central to rna silencing are small rna molecules that can arise from endogenous or exogenous sources from precursors with double-stranded rna pairing. one class of such srnas are micrornas, which originate from endogenous long self-complementary precursors that mature in a multi-step process involving many enzymes. recently, a comprehensive strategy to identify new mirna homologs in est databases has been developed. we have followed this strategy to identify potential melon mirnas. a total of ests that contained homologs to mirnas in the microrna registry database were identified and grouped into contigs and, after manual inspection of secondary foldback hairpin structure, unigenes were selected. contig sequences varied between and nucleotides long, and had negative folding free energies of - to - kcal mol- according to mfold, which are in the range of the computational values of arabidopsis mirna precursors. their predicted secondary structures showed that there were at least nucleotides paired between the sequence of the potential mature mirna and its opposite arm in the corresponding hairpin structure. the location of the potential mirnas varied among ests, were found in the sense orientation of the est, was found in the antisense orientation. we have also searched for potential targets of the potential mirnas in the melon est dataset, identifying of them. however, minimal folding free energy indexes were below the - cut-off value proposed by zhang et al. only for m. potential melon mirna m has a precursor of nt in length and codes for a melon ortholog of the arabidopsis mir mir targets a transcription factor of the tcp family; in the melon dataset, an ortholog of this arabidopsis gene has been found in a unigene annotated as a tcp transcription factor. in this case, the melon mirna and its potential target have a pattern of paired/non-paired bases between the target and the mirna identical to the corresponding target-mirna pattern in arabidopsis. *nucleotide sequences correspond to potential mature mirna sequences as deduced from blast searches using known plant mirna sequences and analysis of secondary structure predictions. †accession numbers of potential precursors of melon mirnas are given. §computational values of folding free energies have been calculated using mfold. Δminimal folding free energy indexes have been calculated as described by zhang et al.. genes potentially encoding pathogen resistance and fruit quality traits pathogens affect severely the productivity of melon crops. three of the cdna libraries sequenced here correspond to pathogen-infected tissues and, thus, should contain transcripts from genes whose expression is induced in response infection. we have carried out a bioinformatics search for homologs of genes involved in pathogen resistance response and virus susceptibility, finding among them at least one melon ortholog to the arabidopsis fls receptor, several unigenes potentially encoding disease resistance proteins as well as mitogen-activated protein kinases, homologs to translation initiation factors constituting potential virus susceptibility factors, etc.. fruit development and ripening are the most important processes determining the fruit quality traits of fleshy fruits like melon. at present most of the molecular and genetic data available about fruit development and ripening come from tomato and arabidopsis. in recent years, several genes and quantitative trait loci controlling fruit quality traits have been described in melon. as for developmental processes, homologs to genes involved in melon fruit development, ripening and quality have been found in the melon dataset. these include several mads-box genes, homologs to the fw and ovate qtls, several homologs to members of the sbp-box gene family to which the major tomato ripening gene colorless non-ripening belongs, several acc synthase and acc oxidase genes, unigenes from several cell wall-metabolism enzymes, etc. expression analysis of selected ests by real-time-qpcr the accumulation of transcripts for selected genes was analyzed by reverse transcription real-time-qpcr. ests for this analysis were preferentially chosen among those showing significant similarity with genes related to response to infection and fruit quality characteristics in melon and other species, and included ctl eif4a- eif4e, ein ga2ox hsp hsp iaa lsm lut ncbp, svp, hir, tch tip tom tom2a, tom uge and wrky. preliminary experiments were carried out to choose between gapdh and cyclophilin rnas as endogenous controls; results showed that the cyp rna levels varied the least among treatments and, therefore, transcript accumulation levels were expressed relative to cyp rna levels. the response of selected genes in roots inoculated with m. cannonballus was analysed in melon genotypes known to be susceptible and partially resistant to the infection by this fungus. the patterns of transcript accumulation resulted clearly different for both genotypes. for pat, transcription factors wrky and svp increased their expression between and times after inoculation; other stress-inducible genes showed only a moderate increase. for "piel de sapo", accumulation of wrky and svp transcripts only increased about times after inoculation whereas the expression of hsp showed a marked increase. it is also worth noting the differential response of the ga2ox gene in the two genotypes. expression of ga2ox increased about times in pat roots after the m. cannonballus attack, whereas it decreased in "piel de sapo" roots after fungal infection. comparison of patterns of transcript accumulation at two stages of fruit development showed increased levels of gene expression for of the analysed genes. this was particularly evident for hsp tom2a, tom ein and iaa in contrast, decreased levels of transcript accumulation were observed for the other genes. discussion in this paper we provide an initial platform for functional genomics of melon by the identification of more than unigenes assembled from almost ests sequenced from melon cdna libraries. it is probably premature to estimate the proportion of melon genes represented in this dataset, but based on available data for other plant species, it is likely that the melon unigene set characterised here represents approximately between half and one-third of the number of expressed, protein coding genes of melon. libraries were constructed from various tissue types, but with a bias towards fruit development and pathogen-infected tissues. data from these libraries will become a useful resource of genes for experiments aimed at understanding important processes involved in fruit development and resistance to viral and fungal pathogens. also, data presented here provide an important tool for generating markers to saturate melon genetic maps. in contrast to typical est gene-sampling strategies reported previously, we have found a low degree of redundancy in the sequences determined. the process of clustering reduced the number of sequences to 56%, from good quality ests to contigs and singletons. contigs with more than ests were scarce, the majority of them being formed by or ests. redundancy of the sequences derived from each library ranged from 13% to 20%, with singletons constituting approximately one third of the unigenes determined per library. this low redundancy is probably due to the success of the normalization process, responsible for the suppression of superabundant transcripts specific for a given tissue or condition. normalization precludes in silico analysis of gene expression, but greatly increases the number of unigenes that can be determined by reducing redundancy. here we have used a recently described normalization protocol which is based on the cleavage of dna or dna-rna duplexes by a specific dnase; this process, in our hands, has proven simple, reproducible and efficient. another factor that has contributed to the low redundancy values obtained has been the sequencing of libraries from very distinct tissues. thus, the number of library specific unigenes was about one half of the total number of unigenes contributed by each library, suggesting that further sequencing of the libraries still has the potential to provide a good number of new, non-redundant sequences. cdna sequences are a useful source of ssrs, which are excellent molecular markers due to their high degree of polymorphism. a common feature of cdna sequences obtained from plants is the high frequency of ssrs that they contain. we have identified more than potential ssrs in the melon dataset, with approximately 6% of the melon unigenes containing di-, tri- or tetranucleotide repeats. a clear bias toward ag and aag repeats existed, that account for 67% of the ssrs. in contrast, the gc repeat was not found in the melon dataset. a similar bias toward ag and against cg repeats has been identified in arabidopsis and other plant species. as proposed at least in one other instance, this may be due to the tendency of cpg sequences to be methylated, which potentially might inhibit transcription. another interesting feature of melon ssrs relates to their pattern of localization with respect to putative initiation and termination codons. it is known that the utrs of transcribed sequences are richer in ssrs than coding regions, particularly at the 5'-utrs. however, in the melon dataset, a high proportion of ssrs were found in orfs. an analysis of the localization of di-, tri- and tetranucleotide repeats separately showed that di- and tetranucleotides were preferentially located in utrs, whereas trinucleotides localised in both, utrs and orfs, consistently with maintenance of the orfs coding capacity. thus, the prevalence of trinucleotide repeats in the melon dataset explains this result. we identified in the melon sequence dataset high-quality snps. since non-redundant sequences analysed here encompassed mb, one snp was found every pb of sequence. this small figure is probably due to the limited number of melon genotypes used and the low redundancy found among libraries. in fact, when the frequency of snps is computed in relation to the length and number of contigs containing snps, the corresponding value is of the same order of magnitude as values previously calculated for melon and other plant species. with the advent of high-throughput detection systems, the ssrs and snps identified here will constitute an important resource for mapping and marker-assisted breeding in melon and closely related crops. as an approach to the function of melon unigenes, we carried out a bioinformatics analysis based on blastx and matches with the pfam database. the proportion of melon unigenes with no similar sequences in databases was quite high, suggesting that the melon dataset may encompass an important number of melon-specific sequences. however, the proportion specific sequences might be overestimated because blasting has been made with unigene sequences, which in many cases do not cover the complete length of the transcript. we performed a functional classification of the unigenes following the gene ontology scheme, which is one of the more versatile and complete systems for functional classification. a comparison of the distributions of melon and arabidopsis unigenes in go categories showed that both followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. this is remarkable, as the number of different libraries sequenced has been relatively small; again, this is probably due to the success of the normalization process. we have also carried out specific searches for genes involved in pathways of particular relevance in melon, such are resistance response and fruit development, identifying a remarkable number of melon candidates. for example, an ortholog of the flagellin receptor fls from arabidopsis has been identified, together with candidate rlks that may have critical roles in pathogen recognition or diverse signalling processes. similarly, up to mads-box gene homologs with potential roles in development have been found in the melon dataset. moreover, a bioinformatics approach allowed the identification of potential precursors of melon mirnas together with several potential targets in the melon dataset. this finding opens the door to biotechnology approaches based on the use of artificial mirnas to specifically silence melon genes. the transcript accumulation analysis for the selected genes revealed important changes in gene expression associated with pathogen infection and fruit development. for virus infection, the accumulation of transcripts remained unaltered for genes, but showed a significant increase for genes and a decrease for among the set of genes analysed, tom2a and eif4e were known to code for virus susceptibility factors; the expression of tom2a was increased after cmv infection, consistently with its requirement by the virus, but this was not the case for eif4e. different hypotheses can explain this result: since eif4e is an abundant, housekeeping protein, increased expression may not be essential for virus multiplication; alternatively, cmv may not use eif4e in melon or may use a factor coded by a different member of the 4e family; it may also be that timing of the sampling for this experiment was not appropriate to detect such an effect, as requirement of eif4e might occur very early during virus multiplication. in the cases of infection of susceptible and resistant melons by m. cannonballus, more extensive alterations in gene expression seemed to occur in the susceptible than in the resistant accession. significantly, for the susceptible accession, stress responsive genes appeared to be maximally induced, whereas for the resistant accession, a gene encoding a wrky transcription factor, potentially involved in resistance response, was induced to high levels. significantly, expression of ga2ox increased about times in pat after the m. cannonballus attack, whereas it decreased in "piel de sapo". ga2ox is a major gibberellin catabolic enzyme, with an important role in controlling ga levels in plants. hormones control many plant developmental processes, and strong evidence indicates that hormone signalling is involved in the regulation of root growth and architecture. the differential response of the ga2ox gene in the two melon genotypes is consistent with an enhanced root growth in pat after infection. notably, other genes involved in hormone-mediated signalling pathways, such as the iaa gene, did not show such differential response to m. cannonballus infection in both genotypes. in the case of fruit development, differences in the expression of selected genes between immature and ripening fruits appeared to be even sharper than in the cases of healthy and pathogen-infected tissues. specific roles during fruit development for hsp tom2a and tom have not been identified, though an increased expression has been shown at least in the case of tom2a in tomato. the ethylene receptor gene ein showed a two-fold increase in expression. ein is the ortholog of arabidopsis ein and tomato leetr. in tomato, leetr is also highly expressed in ripening fruit, suggesting that it responds by modulating ethylene signalling during ripening. the mads-box gene showed about a four-fold decrease in expression. this gene is the ortholog of tomato jointless, which specifies the abscission zone in tomato. in tomato fruit microarray hybridizations, the expression of jointless also decreased from to dap, in agreement with our data for melon. the lycopene epsilon cyclase and xyloglucan endotransglycosylase genes showed an approximately four-fold decrease in expression during melon fruit development. these findings fit with the patterns of expression of these genes in tomato, where their transcript levels decrease to a non-detectable level in the ripe fruits.  in summary, this collection of ests represents a substantial increase on the information available for melon. the dataset contains ssr and snp markers that can be used for breeding, as well as a significant number of candidate genes that can be experimentally tested for their roles in various important processes. this set of genes constitutes also the basis for a microarray for melon that is being used in experiments to further analyse fruit development and maturation and responses to pathogen infections. 
many potential molecular markers were identified in the melon dataset: potential simple sequence repeats and single nucleotide polymorphisms were found. remarkably, the distributions of melon and arabidopsis unigenes followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. bioinformatic analyses primarily focused on potential precursors of melon micro rnas in the melon dataset, but many other genes potentially controlling disease resistance and fruit quality traits were also identified. melon is one of the most important fleshy fruits for fresh consumption. to facilitate the discovery of genes involved in essential traits, such as fruit development, fruit maturation and disease resistance, and to speed up the process of breeding new and better adapted melon varieties, we have produced a large collection of expressed sequence tags from eight normalized cdna libraries from different tissues in different physiological conditions. sixty-nine percent of the melon unigenes showed a significant similarity with proteins in databases. functional classification of the unigenes was carried out following the gene ontology scheme. we determined over ests that were clustered into non-redundant sequences or unigenes, comprising tentative consensus sequences and unclustered sequences. a database which contains all est sequences, contig images and several tools for analysis and data mining has been created. patterns of transcript accumulation were characterised by real-time-qpcr for of these genes. the collection of ests characterised here represents a substantial increase on the genetic information available for melon.
 melon is one of the most important fleshy fruits for fresh consumption. despite this, few genomic resources exist for this species. to facilitate the discovery of genes involved in essential traits, such as fruit development, fruit maturation and disease resistance, and to speed up the process of breeding new and better adapted melon varieties, we have produced a large collection of expressed sequence tags from eight normalized cdna libraries from different tissues in different physiological conditions.  we determined over ests that were clustered into non-redundant sequences or unigenes, comprising tentative consensus sequences and unclustered sequences. many potential molecular markers were identified in the melon dataset: potential simple sequence repeats and single nucleotide polymorphisms were found. sixty-nine percent of the melon unigenes showed a significant similarity with proteins in databases. functional classification of the unigenes was carried out following the gene ontology scheme. in total, unigenes were mapped to one or more ontology. remarkably, the distributions of melon and arabidopsis unigenes followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. bioinformatic analyses primarily focused on potential precursors of melon micro rnas in the melon dataset, but many other genes potentially controlling disease resistance and fruit quality traits were also identified. patterns of transcript accumulation were characterised by real-time-qpcr for of these genes.  the collection of ests characterised here represents a substantial increase on the genetic information available for melon. a database which contains all est sequences, contig images and several tools for analysis and data mining has been created. this set of sequences constitutes also the basis for an oligo-based microarray for melon that is being used in experiments to further analyse the melon transcriptome. 