BACKGROUND
theobroma cacao is a diploid species  with a small genome size of  <dig> mbp  <cit> .
it is a tree fruit originating from the tropical rainforest of south america.
according to cheesman   <cit> , its center of origin is the lower eastern equatorial slopes of the andes.
t. cacao is now cultivated in all tropical lowlands of the world and its beans are used to produce chocolate and cocoa butter after a post harvest treatment including fermentation, drying and torrefaction steps.
t. cacao is one of the major cash crops for several tropical countries.
its economic importance is high and presently cocoa is the third most important internationally traded raw material after sugar and coffee.
cocoa is mainly produced on smallholdings.
it is estimated that approximately  <dig> million people around the world rely on cacao plantations for income.
t. cacao production is seriously affected by several fungal diseases and insect attacks.
oomycetes and especially phytophthora, spp.,  are responsible, worldwide, for 30% of losses.
several species are involved.
p. palmivora is present in the entire cacao growing area, whereas p. capsici and p. citrophthora are prevalent in south america.
p. megakarya is limited to some countries in west africa, however it is by far the most aggressive species causing losses of production up to 50% harvest losses due to phytophthora species were estimated to be  <dig>  tons  <cit> .
two basidiomycetes, moniliophthora roreri  and moniliophthora perniciosa  are also responsible for important harvest losses.
in brazil, m. perniciosa was responsible for a drastic yield loss with a fall in production from  <dig>  tons in  <dig> to less than  <dig>  tons in  <dig>  moniliophthora roreri causes a very destructive pod rot and has already had dramatic effects in some countries such as ecuador  <cit>  and costa rica  <cit> .
m. roreri was confined to several countries of central and northern south america, but is continuously spreading towards other central american countries like mexico or southward towards countries like peru.
several sources of disease resistance have been identified in different genetic backgrounds, and the search for a sustainable disease resistance, cumulating the different resistance genes is one of the major challenges of t. cacao genetic breeding programs  <cit> .
other traits of importance in t. cacao are quality traits.
food quality improvement, nutritional as well as organoleptic, is now a strong demand of consumers.
fundamental knowledge of the genetic basis of quality is an important challenge that can address this demand.
flavor is among the main criteria of quality for chocolate manufacturers, but these characteristics are largely understudied by the cocoa research and breeding community due to their complexity and a dramatic lack of fundamental knowledge about these traits.
flavour components depend strongly on conditions of post-harvest processing  <cit> .
after pod harvests, fresh seeds need to be fermented for  <dig> to  <dig> days, then dried and roasted to develope good cocoa aromas.
raw seeds, embedded in a pulp rich in sugar, undergo biochemical changes under the effect of various microorganisms present in the environment.
the initial anaerobic, low ph and high sugar conditions of the pulp favour yeast activity, converting sugars in the pulp to alcohol and carbon dioxide.
bacteria then start oxidising the alcohol into lactic acid and then into acetic acid as conditions become more aerobic.
these biochemical changes are accompanied by changes of amount and composition of several compounds having a major effect on cocoa flavor such as peptide aroma precursor formation, procyanidines or terpenes content.
however, it is now well recognized that the genetic origin is also a strong determinant of flavor, independent of the conditions of post-harvest processing  <cit> .
although some aromas are prominently defined by a single molecule, most aromas are composed of a bulk of volatile compounds responsible for aroma perception, and belonging to different classes of organic compounds.
interestingly, despite the vast number of chemical structures involved, the large majority of scent compounds are biosynthesized by a surprisingly small number of metabolic pathways.
parts of these metabolic pathways are ubiquitous, and have been developed by small but important modifications of ancestral genes and pathways  <cit> .
in t. cacao more than  <dig> volatile compounds have been detected.
however, only a small number are thought to play a key role in natural aroma variations.
cocoa is classified into two classes: the «standard quality cocoa» corresponding to 95% of the total market, and the «fine flavor cocoa» produced by t. cacao trees originated from two main varieties: criollo and nacional, which bring a higher price in the market.
an important class of volatile compounds, the terpenes, plays an important role in the aromatic flavor of these varieties.
for example, a high level of linalool, a monoterpene, has been observed in nacional varieties  <cit>  from ecuador, characterised by a floral taste, and could be at the origin of this specific flavor which represents an important economic «niche» for the country.
however, the modern and hybrid nacional varieties present a wide range of flavor variations due to introgressions of foreign and more vigorous varieties, leading to a dilution of this specific floral flavor, and recently a part of ecuador cocoa production was declassified from fine flavor to "bulk cocoa" with a lower price.
an increased knowledge of the metabolic pathways and expression of genes involved in terpene synthesis could help to improve the aromatic flavor of new "nacional" varieties.
independent to volatile compounds, some other biochemical compounds are known to interact with t. cacao organoleptic traits.
this is the case with polyphenols.
catechin, epicatechin and procyanidines are the main polyphenols present in t. cacao.
they have well known antioxidant biological activities and beneficial effects on the cardiovascular system  <cit> .
contributing to bitterness and astringency, polyphenols influence t. cacao organoleptic quality  <cit> .
they influence aromatic profiles of t. cacao in restricting maillard's reactions, which generates a majority of the aromatic compounds of t. cacao.
genomic research provides new tools to study the genetic and molecular bases of important trait variations: est sequencing projects carried out on other plant models have allowed the characterization of the transcriptome and facilitated the gene discovery of important trait variations  <cit> .
in tree crops, except for poplar whose genome has been recently sequenced  <cit> , genomic resources are generally limited, and few large est collections have been produced.
recently, a citrus est collection comprising  <dig>  putative transcription units  <cit>  has been produced, allowing the identification of clusters associated with fruit quality, production and salinity tolerance.
a cotton study identified  <dig>  unigenes from a global assembly of  <dig>  cotton ests,  <cit>  providing a framework for future investigation of cotton genomics.
the same approach was used to characterize the grape transcriptome during berry development by the analysis and annotation of  <dig>  unigenes from  <dig>  ests  <cit> .
in t. cacao, only small collections of ests have been produced so far and used to study gene expression related to stress or disease resistance and defense  <cit>
the objective of this study was to produce a large t. cacao est collection from a wide range of organs, providing a good representation of t. cacao genes expressed during t. cacao development and suitable for further analysis of all kind of traits in t. cacao.
moreover, we emphasized the production of tools to further study t. cacao diseases, a major constraint for cocoa production, and quality features.
therefore, we also produced cdna libraries relevant to disease resistance and quality traits.
ests were produced from t. cacao tissues interacting with various pest and fungal diseases, from seeds at different stages of development and during the fermentation steps.
this large est collection will provide valuable tools to carry out functional genomic studies and discover genes essential to important agronomic and quality trait variation in t. cacao, aiming to accelerate t. cacao improvement.
a multidisciplinary approach combining functional genomic and quantitative genetic approaches could lead to a better understanding of gene function involved in disease resistance mechanisms or quality trait variations.
t. cacao's phylogenetic proximity to the model plant arabidopsis will facilitate our understanding of most metabolic pathways.
however, t. cacao is a tree, and expresses traits not found in arabidopsis, thus we hypothesize that genes not found in arabidopsis play important roles in cacao development.
RESULTS
library construction
fifty-six libraries were constructed from two main genotypes representing three contrasting genetic origins: ics <dig>  a hybrid between criollo and forastero from lower amazonia of brazil, and scavina  <dig>  a forastero from upper amazonia of peru.
a few other genotypes characterized by specific resistance or quality traits and belonging to various genetic origins were also used.
the plant materials were provided from a various panel of different t. cacao l. organs .
among them,  <dig> libraries corresponded to t. cacao tissues introduced to different biotic stresses: pods inoculated by phytophthora palmivora, phytophthora megakarya, moniliophthora perniciosa and moniliophthora roreri, leaves inoculated by phytophthora palmivora and phytophthora megakarya, stems inoculated by moniliophthora perniciosa and ceratocystis fimbriata, and stems attacked by sahlbergella singularis .
among these libraries,  <dig> are suppressive subtractive hybridization  libraries.
finally, two libraries corresponded to t. cacao tissues introduced to drought stresses and  <dig> corresponded to seed development and fermentation stages.
est sequencing and assembly
from the  <dig> libraries,  <dig> clones were first sequenced on both strands using forward and reverse primers, to have an overview of the quality of the libraries, and then  <dig>  clones were single-pass sequenced from 5' or from 3' end .
this represented a total number of  <dig>  chromatograms that were used in this analysis.
after low quality, vector and adapters trimming,  <dig>  sequences longer than  <dig> bp remained as good quality sequences.
the average sequence length was  <dig> bp and 62% were longer than  <dig> bp.
these individual ests  were assembled using the tigr gene indices clustering tools   <cit> .
the assembly process produced  <dig>  contigs and  <dig>  singletons that represented a total of  <dig>  mb of transcripted sequences.
the combined set of contigs and singletons resulted in  <dig>  unigenes which might correspond to different putative transcripts or different parts of the same transcript found in the theobroma cacao transcriptome.
the average length of this t. cacao non redundant sequences dataset was  <dig> bp.
an assembly of ests has already been published for theobroma cacao but has been limited to  <dig> unigenes  from two leaf and bean cacao libraries  <cit> , to the isolation of  <dig> unigenes  from cacao leaves treated with inducers of defense response  <cit>  and to  <dig> non redundant sequences from libraries of cacao meristems inoculated by moniliophthora perniciosa  <cit> .
the results of this study are more comparable to a cotton est project  <cit> , involving  <dig> cdna libraries.
this analysis detected  <dig>  unigenes in approximately  <dig>  gossypium ests.
analysis of est abundance in a contig can provide insights to gene expression levels, although this information must be taken with caution due to cloning and replication bias resulting form library construction and propagation steps.
the number of ests in the t. cacao contigs ranged from  <dig> to  <dig>  and  <dig> % were composed of  <dig> or less ests.
98% of the contigs contained less than  <dig> ests.
we evaluated the redundancy of transcripts in each library and among all libraries by studying the distribution of ests in contigs across multiple libraries.
<dig>  had members from more than one library  and  <dig> contigs were specific from one library.
no contigs had members from all  <dig> libraries.
two contigs were found in  <dig> libraries: the contig cl1contig <dig> was similar to the mitochondrial large subunit ribosomal rna gene and the contig cl1contig <dig> to the 18s ribosomal rna gene.
the contig cl18contig <dig>  cl2contig <dig> and cl15contig <dig>  similar to an atp synthase beta subunit, a metallothionein-like protein and a photosystem ii d <dig> protein respectively, were found in  <dig> libraries.
unigene set annotation
blastn against cacao ests
the unigene dataset was used to detect how many cacao sequences had not been already described in public databases.
to answer this question, we collected all  <dig> t. cacao unique sequences already published by the dana farber cancer institute  gene index  <cit>  and we did a blastn search against our unigenes.
an e-value cutoff of 1e- <dig> was used to ensure that only highly similar sequences were detected.
a total of  <dig> unigenes produced a significant hit with  <dig> unique sequences from the dfci gene index, therefore these sequences may correspond to t. cacao sequences already published or may match different parts of the gene index sequences.
they may be also produced by closely related genes .
finally,  <dig>  unique sequences did not produce a significant hit, therefore these sequences may be new.
blastx and blastn annotation
the unigenes were first translated into amino-acid sequences and then searched for similar protein with the blastx program using an e-value cutoff of 1e- <dig> against the non-redundant protein sequence database  with entries from genpept, swissprot, pir, pdf, pdb and ncbi refseq.
the  <dig> best hits were retained for the annotation, providing an annotation for  <dig>  cacao sequences .
the  <dig> % of the unigenes that did not have any match were searched for similar nucleotide sequences from the genbank nucleotide collection nt with the blastn program.
an e-value cutoff of 1e- <dig> was also used and the  <dig> best hits were used for the annotation.
<dig> unigenes exhibited a significant similarity with nucleotide sequences providing a blastx or blastn annotation for  <dig>  unigenes.
the  <dig> blastx hits were used to classify the unigenes according to the species associated with the annotation .
a total of  <dig>  hits  involved proteins from vitis vinifera, arabidopsis thaliana or oryza sativa, while  <dig> hits involved proteins from gossypium hirsutum, a closely related species from the malvaceae family  <cit> .
although fewer protein sequences from vitis vinifera than from arabidopsis thaliana  were present in the non redundant database we used for blastx, and although the evolutionary distance between vitis vinifera and theobroma cacao is higher than the distance between arabidopsis thaliana and theobroma cacao  <cit> , we found more similarities with vitis vinifera  than with arabidopsis thaliana .
to further investigate this unexpected result we compared with the blastx program the cacao unigenes dataset against the two proteomes of arabidopsis thaliana and vitis vinifera .
for each blast result, we selected the species found in the first hit having an expected value lower than 1e- <dig> to detect similar sequence.
a total of  <dig>  theobroma cacao sequences  presented at least a significant hit with an arabidopsis thaliana or vitis vinifera protein.
the results showed that  <dig>  theobroma cacao sequences presented a higher similarity to the vitis vinifera proteome whereas only  <dig> theobroma cacao sequences presented a first blast hit similar to the arabidopsis thaliana proteome .
moreover, it was determined that these first significant hits involved  <dig> vitis vinifera proteins  and  <dig> arabidopsis thaliana proteins  .
these surprising results suggest that the genes expressed in theobroma cacao are more similar to vitis vinifera proteins than to those of arabidopsis thaliana.
these findings could be explained by the fact that theobroma cacao and vitis vinifera are both fruit trees.
this idea could be supported by the large amount of blast hits found with other tree crops such as populus trichocarpa , despite a small number of non redundant proteins in the databases for this species.
gene ontology annotation
we used blast2go  <cit> , a program that retrieves go terms based on blast definition, to assign gene ontology  annotation  <cit>  to the unigene dataset.
to best exploit go results, we built a local amigo browser  <cit> .
a total number of  <dig>  annotations were found and  <dig>  unigenes were characterized by at least one annotation.
these annotations were distributed among the main go categories into  <dig>  biological process ,  <dig>  cellular component  and  <dig>  molecular function  .
the most abundant high-level direct go counts within these categories were c: mitochondrion , c: membrane , c: plastid , f: atp binding  and c: chloroplast  .
genes involved in defense and resistance mechanisms
some of the libraries provide an important resource to study plant/pathogens interactions.
using the annotations provided by blast and gene ontology, we specifically focussed on genes known to play a crucial role in plant pathogen resistance and defense mechanisms  <cit> .
using the amigo browser, we identified  <dig> gene product associations to "response to stress" .
both searches with blast result and gene ontology annotation resulted in the identification of unigenes similar to known proteins involved in resistance or defense mechanisms such as lrr-nbs  <cit>  , chitinase  <cit>  , 1– <dig> beta glucanase  <cit>   or pathogenesis-related protein .
other genes related to resistance/defense mechanisms were also found more specifically in libraries produced from pathogen infected tissues, such as those involved in regulation of pathogen-induced genes like transcription factors , in signal transduction  or in the cell death program.
the identification of a unigene set gathering sequences from all genes known to be involved in plant resistance and defense mechanisms, and the construction of a corresponding microarray could constitute a valuable tool to progress in the understanding of plant/pathogens interactions.
genes involved in particular metabolic pathways or biological activities
to check the representativeness of our est collection, we looked for ests encoding proteins known to be involved in the flavonoid and the terpene pathways, already studied in other plant species, and at the basis of important traits of interest in t. cacao.
generally, polyphenols play a major role in chocolate quality, acting as colour precursors or taste agents  <cit> .
moreover, they are strongly implicated in health benefits associated with chocolate consumption  <cit> .
the flavonoid pathway
the flavonoid pathway has been already studied in several plants  <cit> .
in t. cacao, this pathway is the source of numerous essential components for human health benefits of chocolate  <cit>  and resistance against pathogens  <cit> .
gene ontology analysis highlighted  <dig> est sequences implicated in "phenylpropanoid biosynthetic process" , most of them implicated in flavonoid biosynthesis.
for example, the go analysis, together with keyword esttik database searching  into blast results allowed us to find sequences encoding phenylalanine ammonia lyase , cinnamate-4-hydroxylase , the 4-coumarate-coa ligase , chalcone synthase  and chalcone isomerase , all major enzymes of the general flavonoid pathway .
most specific enzymes, implicated in anthocyanin biosynthesis  were also represented in this t. cacao est resource.
the terpene pathway
terpenoid compounds, synthesized in the isoprenoid pathway , are compounds of importance for specific scent and aromatic qualities of chocolates classified as "fine and flavor".
for example, linalool, a monoterpenol, is found in high quantity in arriba nacional varieties from ecuador and in some criollo clones from venezuela  <cit> .
linalool, together with other volatiles, could be responsible for the typical floral aroma  <cit>  of these chocolates.
one of our goals was to identify enzymes involved in the terpenoid pathway that could be responsible for linalool content variations among nacional clones.
as a first step we identified sequences encoding isoprenoid pathway enzymes .
the final step enzyme for linalool synthesis, linalool synthase, was represented by  <dig> contigs and  <dig> singletons.
nearly all enzymes reported to be involved in this biochemical pathway were present in our esttik database, allowing the analysis of the t. cacao terpene pathway based on oligonucleotide microarrays derived from these ests.
the fact that nearly all of the genes involved in these two pathways as described in other plant species were identified in ests from our collection demonstrates the high level of representation of this resource and suggests that the majority of cacao genes have been sampled.
thus, this est collection offers a comprehensive resource to search for candidate genes involved in quality traits and other important agronomical traits variation.
production of ssr and snp markers
molecular markers derived from ests are part of, or adjacent to genes, and therefore they provide an efficient means of gene mapping.
simple sequence repeats  were identified in the unigene dataset with the misa pipeline  <cit> .
in this study, ssrs were defined as dimers with at least  <dig> repetitions and trimers, tetramers, pentamers and hexamers with at least  <dig> repeats.
microsatellites were considered compound when two ssrs were not separated by more than  <dig> bp.
a total of  <dig> ssrs were identified as  <dig> unigenes, and  <dig> unigenes had more than  <dig> ssr.
dimers and trimers were the most common types  and represented  <dig> % of ssrs found in unigenes.
the distribution of all possible dimer and trimer motifs found in the unigenes is listed in table  <dig>  the polyn and polyn groups were the most abundant motifs in t. cacao unigenes.
for each ssr identified, if possible,  <dig> couples of primers were defined using primer <dig>  <cit> .
a total of  <dig> flanking sequences were designed and it was possible to define at least one couple of primers for  <dig> ssrs.
the exploration of redundant ests in contigs was shown to be a valuable resource of single nucleotide polymorphisms   <cit> .
snps were detected using qualitysnp  <cit>  pipeline from unigene contigs.
we assumed that contigs with at least  <dig> members contained paralogous sequences  <cit>  therefore we selected  <dig> contigs that contained at least  <dig> sequences but no more than  <dig> sequences.
a preliminary study assembled  <dig> snps into  <dig> contigs.
transitions  represented  <dig> % of the snps found, transversions  <dig> % and indels  <dig> %.
CONCLUSIONS
the present assembly of  <dig>  t. cacao ests produced from  <dig> cdna libraries constructed from different organs and environmental conditions is the largest transcriptome dataset produced so far for t. cacao, and among the largest ones generated for any tree fruit crop.
it provides a major resource for cacao genetic and functional genomic analyses of important t. cacao traits, with the identification and annotation of  <dig>  different putative transcripts.
the improved knowledge of the t. cacao transcriptome will enhance our understanding of main disease resistance mechanisms and will be useful to improve new varieties and establish a sustainable t. cacao resistance to pests and diseases.
towards this goal, a large number of cdna libraries have been produced from t. cacao/pathogens or pest interactions, and an important set of unique transcripts homologous to genes known in other species involved in defense and resistance mechanisms have been identified in the whole est collection using keywords and gene ontology tools.
it provides a cdna resource available for the broad scientific community and suitable for cdna-based microarray analyses.
this collection of ests also provides a valuable framework for the discovery of candidate genes involved in chocolate quality traits.
tested for two distinct metabolic pathways, this collection displays a good representation of the t. cacao transcriptome involved in quality trait elaboration and will allow the comparative analysis of contrasting genotypes for t. cacao qualities to better understand the genetic basis of quality.
this est collection also will provide a large number of genetic tools, such as ssr and snp markers, which will be used to construct high density gene maps, facilitating the integration of genetic and genomic approaches to discover the genes that effect trait variations, and also facilitating the sequence assembly in further activities of whole t. cacao genome sequencing.
finally, the assembly and annotation associated will also provide a valuable resource for future investigation of t. cacao evolutionary genomics with related species such as gossypium hirsutum or arabidopsis thaliana.

 theobroma cacao is a diploid species with a small genome size of mbp. it is a tree fruit originating from the tropical rainforest of south america. according to cheesman, its center of origin is the lower eastern equatorial slopes of the andes. t. cacao is now cultivated in all tropical lowlands of the world and its beans are used to produce chocolate and cocoa butter after a post harvest treatment including fermentation, drying and torrefaction steps. t. cacao is one of the major cash crops for several tropical countries. its economic importance is high and presently cocoa is the third most important internationally traded raw material after sugar and coffee. cocoa is mainly produced on smallholdings. it is estimated that approximately million people around the world rely on cacao plantations for income. t. cacao production is seriously affected by several fungal diseases and insect attacks. oomycetes and especially phytophthora, spp., are responsible, worldwide, for 30% of losses. several species are involved. p. palmivora is present in the entire cacao growing area, whereas p. capsici and p. citrophthora are prevalent in south america. p. megakarya is limited to some countries in west africa, however it is by far the most aggressive species causing losses of production up to 50% harvest losses due to phytophthora species were estimated to be tons. two basidiomycetes, moniliophthora roreri and moniliophthora perniciosa are also responsible for important harvest losses. in brazil, m. perniciosa was responsible for a drastic yield loss with a fall in production from tons in to less than tons in moniliophthora roreri causes a very destructive pod rot and has already had dramatic effects in some countries such as ecuador and costa rica. m. roreri was confined to several countries of central and northern south america, but is continuously spreading towards other central american countries like mexico or southward towards countries like peru. several sources of disease resistance have been identified in different genetic backgrounds, and the search for a sustainable disease resistance, cumulating the different resistance genes is one of the major challenges of t. cacao genetic breeding programs. other traits of importance in t. cacao are quality traits. food quality improvement, nutritional as well as organoleptic, is now a strong demand of consumers. fundamental knowledge of the genetic basis of quality is an important challenge that can address this demand. flavor is among the main criteria of quality for chocolate manufacturers, but these characteristics are largely understudied by the cocoa research and breeding community due to their complexity and a dramatic lack of fundamental knowledge about these traits. flavour components depend strongly on conditions of post-harvest processing. after pod harvests, fresh seeds need to be fermented for to days, then dried and roasted to develope good cocoa aromas. raw seeds, embedded in a pulp rich in sugar, undergo biochemical changes under the effect of various microorganisms present in the environment. the initial anaerobic, low ph and high sugar conditions of the pulp favour yeast activity, converting sugars in the pulp to alcohol and carbon dioxide. bacteria then start oxidising the alcohol into lactic acid and then into acetic acid as conditions become more aerobic. these biochemical changes are accompanied by changes of amount and composition of several compounds having a major effect on cocoa flavor such as peptide aroma precursor formation, procyanidines or terpenes content. however, it is now well recognized that the genetic origin is also a strong determinant of flavor, independent of the conditions of post-harvest processing. although some aromas are prominently defined by a single molecule, most aromas are composed of a bulk of volatile compounds responsible for aroma perception, and belonging to different classes of organic compounds. interestingly, despite the vast number of chemical structures involved, the large majority of scent compounds are biosynthesized by a surprisingly small number of metabolic pathways. parts of these metabolic pathways are ubiquitous, and have been developed by small but important modifications of ancestral genes and pathways. in t. cacao more than volatile compounds have been detected. however, only a small number are thought to play a key role in natural aroma variations. cocoa is classified into two classes: the «standard quality cocoa» corresponding to 95% of the total market, and the «fine flavor cocoa» produced by t. cacao trees originated from two main varieties: criollo and nacional, which bring a higher price in the market. an important class of volatile compounds, the terpenes, plays an important role in the aromatic flavor of these varieties. for example, a high level of linalool, a monoterpene, has been observed in nacional varieties from ecuador, characterised by a floral taste, and could be at the origin of this specific flavor which represents an important economic «niche» for the country. however, the modern and hybrid nacional varieties present a wide range of flavor variations due to introgressions of foreign and more vigorous varieties, leading to a dilution of this specific floral flavor, and recently a part of ecuador cocoa production was declassified from fine flavor to "bulk cocoa" with a lower price. an increased knowledge of the metabolic pathways and expression of genes involved in terpene synthesis could help to improve the aromatic flavor of new "nacional" varieties. independent to volatile compounds, some other biochemical compounds are known to interact with t. cacao organoleptic traits. this is the case with polyphenols. catechin, epicatechin and procyanidines are the main polyphenols present in t. cacao. they have well known antioxidant biological activities and beneficial effects on the cardiovascular system. contributing to bitterness and astringency, polyphenols influence t. cacao organoleptic quality. they influence aromatic profiles of t. cacao in restricting maillard's reactions, which generates a majority of the aromatic compounds of t. cacao. genomic research provides new tools to study the genetic and molecular bases of important trait variations: est sequencing projects carried out on other plant models have allowed the characterization of the transcriptome and facilitated the gene discovery of important trait variations. in tree crops, except for poplar whose genome has been recently sequenced, genomic resources are generally limited, and few large est collections have been produced. recently, a citrus est collection comprising putative transcription units has been produced, allowing the identification of clusters associated with fruit quality, production and salinity tolerance. a cotton study identified unigenes from a global assembly of cotton ests, providing a framework for future investigation of cotton genomics. the same approach was used to characterize the grape transcriptome during berry development by the analysis and annotation of unigenes from ests. in t. cacao, only small collections of ests have been produced so far and used to study gene expression related to stress or disease resistance and defense the objective of this study was to produce a large t. cacao est collection from a wide range of organs, providing a good representation of t. cacao genes expressed during t. cacao development and suitable for further analysis of all kind of traits in t. cacao. moreover, we emphasized the production of tools to further study t. cacao diseases, a major constraint for cocoa production, and quality features. therefore, we also produced cdna libraries relevant to disease resistance and quality traits. ests were produced from t. cacao tissues interacting with various pest and fungal diseases, from seeds at different stages of development and during the fermentation steps. this large est collection will provide valuable tools to carry out functional genomic studies and discover genes essential to important agronomic and quality trait variation in t. cacao, aiming to accelerate t. cacao improvement. a multidisciplinary approach combining functional genomic and quantitative genetic approaches could lead to a better understanding of gene function involved in disease resistance mechanisms or quality trait variations. t. cacao's phylogenetic proximity to the model plant arabidopsis will facilitate our understanding of most metabolic pathways. however, t. cacao is a tree, and expresses traits not found in arabidopsis, thus we hypothesize that genes not found in arabidopsis play important roles in cacao development.  library construction fifty-six libraries were constructed from two main genotypes representing three contrasting genetic origins: ics a hybrid between criollo and forastero from lower amazonia of brazil, and scavina a forastero from upper amazonia of peru. a few other genotypes characterized by specific resistance or quality traits and belonging to various genetic origins were also used. the plant materials were provided from a various panel of different t. cacao l. organs. among them, libraries corresponded to t. cacao tissues introduced to different biotic stresses: pods inoculated by phytophthora palmivora, phytophthora megakarya, moniliophthora perniciosa and moniliophthora roreri, leaves inoculated by phytophthora palmivora and phytophthora megakarya, stems inoculated by moniliophthora perniciosa and ceratocystis fimbriata, and stems attacked by sahlbergella singularis. among these libraries, are suppressive subtractive hybridization libraries. finally, two libraries corresponded to t. cacao tissues introduced to drought stresses and corresponded to seed development and fermentation stages. est sequencing and assembly from the libraries, clones were first sequenced on both strands using forward and reverse primers, to have an overview of the quality of the libraries, and then clones were single-pass sequenced from 5' or from 3' end. this represented a total number of chromatograms that were used in this analysis. after low quality, vector and adapters trimming, sequences longer than bp remained as good quality sequences. the average sequence length was bp and 62% were longer than bp. these individual ests were assembled using the tigr gene indices clustering tools. the assembly process produced contigs and singletons that represented a total of mb of transcripted sequences. the combined set of contigs and singletons resulted in unigenes which might correspond to different putative transcripts or different parts of the same transcript found in the theobroma cacao transcriptome. the average length of this t. cacao non redundant sequences dataset was bp. an assembly of ests has already been published for theobroma cacao but has been limited to unigenes from two leaf and bean cacao libraries, to the isolation of unigenes from cacao leaves treated with inducers of defense response and to non redundant sequences from libraries of cacao meristems inoculated by moniliophthora perniciosa. the results of this study are more comparable to a cotton est project, involving cdna libraries. this analysis detected unigenes in approximately gossypium ests. analysis of est abundance in a contig can provide insights to gene expression levels, although this information must be taken with caution due to cloning and replication bias resulting form library construction and propagation steps. the number of ests in the t. cacao contigs ranged from to and % were composed of or less ests. 98% of the contigs contained less than ests. we evaluated the redundancy of transcripts in each library and among all libraries by studying the distribution of ests in contigs across multiple libraries. had members from more than one library and contigs were specific from one library. no contigs had members from all libraries. two contigs were found in libraries: the contig cl1contig was similar to the mitochondrial large subunit ribosomal rna gene and the contig cl1contig to the 18s ribosomal rna gene. the contig cl18contig cl2contig and cl15contig similar to an atp synthase beta subunit, a metallothionein-like protein and a photosystem ii d protein respectively, were found in libraries. unigene set annotation blastn against cacao ests the unigene dataset was used to detect how many cacao sequences had not been already described in public databases. to answer this question, we collected all t. cacao unique sequences already published by the dana farber cancer institute gene index and we did a blastn search against our unigenes. an e-value cutoff of 1e- was used to ensure that only highly similar sequences were detected. a total of unigenes produced a significant hit with unique sequences from the dfci gene index, therefore these sequences may correspond to t. cacao sequences already published or may match different parts of the gene index sequences. they may be also produced by closely related genes. finally, unique sequences did not produce a significant hit, therefore these sequences may be new. blastx and blastn annotation the unigenes were first translated into amino-acid sequences and then searched for similar protein with the blastx program using an e-value cutoff of 1e- against the non-redundant protein sequence database with entries from genpept, swissprot, pir, pdf, pdb and ncbi refseq. the best hits were retained for the annotation, providing an annotation for cacao sequences. the % of the unigenes that did not have any match were searched for similar nucleotide sequences from the genbank nucleotide collection nt with the blastn program. an e-value cutoff of 1e- was also used and the best hits were used for the annotation. unigenes exhibited a significant similarity with nucleotide sequences providing a blastx or blastn annotation for unigenes. the blastx hits were used to classify the unigenes according to the species associated with the annotation. a total of hits involved proteins from vitis vinifera, arabidopsis thaliana or oryza sativa, while hits involved proteins from gossypium hirsutum, a closely related species from the malvaceae family. although fewer protein sequences from vitis vinifera than from arabidopsis thaliana were present in the non redundant database we used for blastx, and although the evolutionary distance between vitis vinifera and theobroma cacao is higher than the distance between arabidopsis thaliana and theobroma cacao, we found more similarities with vitis vinifera than with arabidopsis thaliana. to further investigate this unexpected result we compared with the blastx program the cacao unigenes dataset against the two proteomes of arabidopsis thaliana and vitis vinifera. for each blast result, we selected the species found in the first hit having an expected value lower than 1e- to detect similar sequence. a total of theobroma cacao sequences presented at least a significant hit with an arabidopsis thaliana or vitis vinifera protein. the results showed that theobroma cacao sequences presented a higher similarity to the vitis vinifera proteome whereas only theobroma cacao sequences presented a first blast hit similar to the arabidopsis thaliana proteome. moreover, it was determined that these first significant hits involved vitis vinifera proteins and arabidopsis thaliana proteins. these surprising results suggest that the genes expressed in theobroma cacao are more similar to vitis vinifera proteins than to those of arabidopsis thaliana. these findings could be explained by the fact that theobroma cacao and vitis vinifera are both fruit trees. this idea could be supported by the large amount of blast hits found with other tree crops such as populus trichocarpa, despite a small number of non redundant proteins in the databases for this species. gene ontology annotation we used blast2go, a program that retrieves go terms based on blast definition, to assign gene ontology annotation to the unigene dataset. to best exploit go results, we built a local amigo browser. a total number of annotations were found and unigenes were characterized by at least one annotation. these annotations were distributed among the main go categories into biological process, cellular component and molecular function. the most abundant high-level direct go counts within these categories were c: mitochondrion, c: membrane, c: plastid, f: atp binding and c: chloroplast. genes involved in defense and resistance mechanisms some of the libraries provide an important resource to study plant/pathogens interactions. using the annotations provided by blast and gene ontology, we specifically focussed on genes known to play a crucial role in plant pathogen resistance and defense mechanisms. using the amigo browser, we identified gene product associations to "response to stress". both searches with blast result and gene ontology annotation resulted in the identification of unigenes similar to known proteins involved in resistance or defense mechanisms such as lrr-nbs, chitinase, 1– beta glucanase or pathogenesis-related protein. other genes related to resistance/defense mechanisms were also found more specifically in libraries produced from pathogen infected tissues, such as those involved in regulation of pathogen-induced genes like transcription factors, in signal transduction or in the cell death program. the identification of a unigene set gathering sequences from all genes known to be involved in plant resistance and defense mechanisms, and the construction of a corresponding microarray could constitute a valuable tool to progress in the understanding of plant/pathogens interactions. genes involved in particular metabolic pathways or biological activities to check the representativeness of our est collection, we looked for ests encoding proteins known to be involved in the flavonoid and the terpene pathways, already studied in other plant species, and at the basis of important traits of interest in t. cacao. generally, polyphenols play a major role in chocolate quality, acting as colour precursors or taste agents. moreover, they are strongly implicated in health benefits associated with chocolate consumption. the flavonoid pathway the flavonoid pathway has been already studied in several plants. in t. cacao, this pathway is the source of numerous essential components for human health benefits of chocolate and resistance against pathogens. gene ontology analysis highlighted est sequences implicated in "phenylpropanoid biosynthetic process", most of them implicated in flavonoid biosynthesis. for example, the go analysis, together with keyword esttik database searching into blast results allowed us to find sequences encoding phenylalanine ammonia lyase, cinnamate-4-hydroxylase, the 4-coumarate-coa ligase, chalcone synthase and chalcone isomerase, all major enzymes of the general flavonoid pathway. most specific enzymes, implicated in anthocyanin biosynthesis were also represented in this t. cacao est resource. the terpene pathway terpenoid compounds, synthesized in the isoprenoid pathway, are compounds of importance for specific scent and aromatic qualities of chocolates classified as "fine and flavor". for example, linalool, a monoterpenol, is found in high quantity in arriba nacional varieties from ecuador and in some criollo clones from venezuela. linalool, together with other volatiles, could be responsible for the typical floral aroma of these chocolates. one of our goals was to identify enzymes involved in the terpenoid pathway that could be responsible for linalool content variations among nacional clones. as a first step we identified sequences encoding isoprenoid pathway enzymes. the final step enzyme for linalool synthesis, linalool synthase, was represented by contigs and singletons. nearly all enzymes reported to be involved in this biochemical pathway were present in our esttik database, allowing the analysis of the t. cacao terpene pathway based on oligonucleotide microarrays derived from these ests. the fact that nearly all of the genes involved in these two pathways as described in other plant species were identified in ests from our collection demonstrates the high level of representation of this resource and suggests that the majority of cacao genes have been sampled. thus, this est collection offers a comprehensive resource to search for candidate genes involved in quality traits and other important agronomical traits variation. production of ssr and snp markers molecular markers derived from ests are part of, or adjacent to genes, and therefore they provide an efficient means of gene mapping. simple sequence repeats were identified in the unigene dataset with the misa pipeline. in this study, ssrs were defined as dimers with at least repetitions and trimers, tetramers, pentamers and hexamers with at least repeats. microsatellites were considered compound when two ssrs were not separated by more than bp. a total of ssrs were identified as unigenes, and unigenes had more than ssr. dimers and trimers were the most common types and represented % of ssrs found in unigenes. the distribution of all possible dimer and trimer motifs found in the unigenes is listed in table the polyn and polyn groups were the most abundant motifs in t. cacao unigenes. for each ssr identified, if possible, couples of primers were defined using primer. a total of flanking sequences were designed and it was possible to define at least one couple of primers for ssrs. the exploration of redundant ests in contigs was shown to be a valuable resource of single nucleotide polymorphisms. snps were detected using qualitysnp pipeline from unigene contigs. we assumed that contigs with at least members contained paralogous sequences therefore we selected contigs that contained at least sequences but no more than sequences. a preliminary study assembled snps into contigs. transitions represented % of the snps found, transversions % and indels %.  the present assembly of t. cacao ests produced from cdna libraries constructed from different organs and environmental conditions is the largest transcriptome dataset produced so far for t. cacao, and among the largest ones generated for any tree fruit crop. it provides a major resource for cacao genetic and functional genomic analyses of important t. cacao traits, with the identification and annotation of different putative transcripts. the improved knowledge of the t. cacao transcriptome will enhance our understanding of main disease resistance mechanisms and will be useful to improve new varieties and establish a sustainable t. cacao resistance to pests and diseases. towards this goal, a large number of cdna libraries have been produced from t. cacao/pathogens or pest interactions, and an important set of unique transcripts homologous to genes known in other species involved in defense and resistance mechanisms have been identified in the whole est collection using keywords and gene ontology tools. it provides a cdna resource available for the broad scientific community and suitable for cdna-based microarray analyses. this collection of ests also provides a valuable framework for the discovery of candidate genes involved in chocolate quality traits. tested for two distinct metabolic pathways, this collection displays a good representation of the t. cacao transcriptome involved in quality trait elaboration and will allow the comparative analysis of contrasting genotypes for t. cacao qualities to better understand the genetic basis of quality. this est collection also will provide a large number of genetic tools, such as ssr and snp markers, which will be used to construct high density gene maps, facilitating the integration of genetic and genomic approaches to discover the genes that effect trait variations, and also facilitating the sequence assembly in further activities of whole t. cacao genome sequencing. finally, the assembly and annotation associated will also provide a valuable resource for future investigation of t. cacao evolutionary genomics with related species such as gossypium hirsutum or arabidopsis thaliana. 
a specific information system was constructed to process, store and manage this est collection allowing the user to query a database. to check the representativeness of our est collection, we looked for the genes known to be involved in two different metabolic pathways extensively studied in other plant species and important for t. cacao qualities: the flavonoid and the terpene pathways. most of the enzymes described in other crops for these two metabolic pathways were found in our est collection. this est collection displays a good representation of the t. cacao transcriptome, suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from these ests. this est collection represents a unique and important molecular resource for t. cacao study and improvement, facilitating the discovery of candidate genes for important t. cacao trait variation. fifty-six cdna libraries were constructed from different organs, different genotypes and different environmental conditions. theobroma cacao l., is a tree originated from the tropical rainforest of south america. it is one of the major cash crops for many tropical countries. t. cacao is mainly produced on smallholdings, providing resources for million farmers. disease resistance and t. cacao quality improvement are two important challenges for all actors of cocoa and chocolate production. t. cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses and quality improvement, nutritional and organoleptic, is also important for consumers. it will provide numerous genetic markers that will allow the construction of a high density gene map of t. cacao.
 theobroma cacao l., is a tree originated from the tropical rainforest of south america. it is one of the major cash crops for many tropical countries. t. cacao is mainly produced on smallholdings, providing resources for million farmers. disease resistance and t. cacao quality improvement are two important challenges for all actors of cocoa and chocolate production. t. cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses and quality improvement, nutritional and organoleptic, is also important for consumers. an international collaboration was formed to develop an est genomic resource database for cacao.  fifty-six cdna libraries were constructed from different organs, different genotypes and different environmental conditions. a total of valid est sequences were generated corresponding to unigenes, contigs and singletons. a total of unigenes shared significant homology with public sequences from other species. gene ontology annotation was applied to distribute the ests among the main go categories. a specific information system was constructed to process, store and manage this est collection allowing the user to query a database. to check the representativeness of our est collection, we looked for the genes known to be involved in two different metabolic pathways extensively studied in other plant species and important for t. cacao qualities: the flavonoid and the terpene pathways. most of the enzymes described in other crops for these two metabolic pathways were found in our est collection. a large collection of new genetic markers was provided by this ests collection.  this est collection displays a good representation of the t. cacao transcriptome, suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from these ests. it will provide numerous genetic markers that will allow the construction of a high density gene map of t. cacao. this est collection represents a unique and important molecular resource for t. cacao study and improvement, facilitating the discovery of candidate genes for important t. cacao trait variation. 