BACKGROUND
among starch producing crops, cassava  has a higher carbohydrate production than rice or maize under suboptimal conditions  <cit> ; more than  <dig> million tons are produced in the world each year and about 84% of them are used for direct human consumption and animal feed  <cit> .
cassava starch is used as a raw material for a wide range of food products and industrial goods, including paper, cardboard, textile, plywood, glue and alcohol  <cit> .
moreover, because starch production from cassava is cheap compared to other crops, it is gaining attention as a biomass source for fuel production  <cit> .
the growing interest in cassava as an energy crop is evidenced by a genome sequencing project  <cit>  and the increasing production and technical advancements in tropical countries; for instance, cassava fresh root production in thailand increased from  <dig>  to  <dig> million tons between  <dig> and  <dig>  <cit>  while a  <dig> % increase per year has been reported for the same period worldwide  <cit> .
by virtue of its remarkable tolerance to abiotic stresses, cassava is grown in marginal, low fertility acidic soils showing increased nutrient use efficiency  <cit> .
it is known to maintain a healthy appearance in drought-prone areas, remaining photosynthetically active though at a reduced rate  <cit> .
because cassava is very drought-resistant and the tubers can be left in the soil for a couple of years, it is considered an important reserve carbohydrate source to prevent or relieve famine  <cit> .
cassava has some unusual characteristics that make it highly productive in near optimum environments , these include elevated activities of the c <dig> phosphoenolpyruvate carboxylase enzyme, long leaf life and low photorespiration rates  <cit> ; it, however, is usually grown in marginal highly eroded soils with uncertain rainfall and almost no agrochemical input.
although cassava has some features that allow it to cope with stress better than other crops, e.g.
high stomatal sensitivity to environmental humidity  <cit> , deep rooting capacities and quick recovery after stress  <cit> , under these conditions productivity is sub-optimal and unstable  <cit> .
cassava productivity is also threatened by bacterial and viral diseases  <cit> , as well as arthropod pests  <cit> .
moreover, its high starch content is in contrast with its deficiency in proteins and key micronutrients , as well as the production of toxic hydrogen cyanide  <cit> .
to address these issues, traditional breeding methods have had some success, particularly in improving fresh root yield and dry matter content under non-stress conditions  <cit> , however, because of the crop's heterozygous genetic makeup and long growth cycle, progress with this approach is slow  <cit> .
the use of biotechnology to improve cassava cultivars is a more straightforward strategy that relies on the tools of molecular and cell biology to find genetic determinants of desirable phenotypes  <cit> .
the construction of genetic maps and the identification of quantitative trait loci have yielded some results in cassava response to biotic stress  <cit> , yet, the identification of candidate genes with this approach is a time consuming process involving the construction of bacterial artificial chromosome  libraries and anchoring of these clones to the genetic map  <cit> .
a reverse-genetics approach  <cit>  can be a more direct solution, relying on the accumulated knowledge of gene function in model species it is possible to assess the effects of selected genes through regulation of their expression.
as an example, silencing of p- <dig> cytochromes has allowed the production of cyanogen-free transgenic cassava plants  <cit> .
one tool that may assist both, the characterization of a plant expressed genes and the isolation of nucleotide sequences of genes with known function, are ests  <cit> .
these are a cost-effective gene discovery methodology that is also useful for the study of gene expression  <cit> .
despite its importance, large-scale sequence collections from cassava are scarce, there are  <dig> expressed cassava sequences in the dbest database  <cit>  as of april  <dig>  which is a small number compared to the number of ests of maize , rice , soybean , potato  or sugarcane .
this is likely to change with the release of a cassava draft genome sequence this year by the united states department of energy's joint genome institute.
although ests can aid the annotation of the cassava genome, the fact that most of them come from libraries made of random mrna fragments, make them insufficient to accurately and fully define gene models  <cit> , ests are not only derived from partial transcripts, but also they can confound alternatively spliced forms during the assembly process  <cit> .
moreover due to the fragmentary nature of ests, their use in gene functional analysis is limited  <cit> .
full-length cdna libraries, on the other hand, are built in such a way that one insert represents one transcription unit, providing information on complete molecules for the functional dissection of genes  <cit> .
we built a full-length enriched cdna library from cassava leaves and roots subject to drought, heat, and acidic conditions, as well as from roots subject to post-harvest physiological deterioration , a major obstacle for cassava commercialization  <cit> .
the aim of this library is to support research in cassava improvement for high yield under abiotic stress, providing full sequences of stress-responsive genes and expanding the gene catalog of this species.
the characterization of the transcripts captured in the library and the selection of non-redundant clones will certainly aid the annotation of genomic sequences  <cit>  and the construction of microarrays or other tools for functional genomics  <cit> .
in order to characterize the library and find the number and putative functions of the transcripts captured, nearly  <dig>  clones were sequenced from both ends, these ests, although unlikely to include the whole sequence of the inserts, are tagged with clone names.
because this information is considered during the assembly process, ests derived from a full-length library allow, in principle, a more accurate definition of transcript units than normal ests.
the annotation of the sequences acquired and the availability of the genome sequences of two species closely related to cassava such as castor bean  and poplar   <cit>  as well as the complete set of genes from arabidopsis thaliana, provide altogether an opportunity to study the evolution of the cassava genome by means of comparative genomics  <cit> ; if it is possible to define gene correspondences between these species and on that basis find sequences that are unique to cassava, a closer inspection of these genes could provide hints as to the mechanisms underlying cassava's unique features.
cassava is believed to be an allotetraploid that appeared by hybridization of wild manihot species  <cit> , it would then be interesting to see what genes within a highly heterozygous gene pool have remained functional during cassava domestication; for this we use a methodology for the detection of recent duplications that is based on the detection of groups of genes sharing similarity to single sequences in other genomes, hopefully the genes detected with this strategy will aid cassava research for the genetic improvement of an already outstanding crop.
RESULTS
sequencing and assembly of both-end, single-pass sequences
a full-length cdna library was constructed from leaves and roots of cassava plants under various environmental conditions ,  <dig> clones  were sequenced from both ends; the clones are available at riken bioresource center  <cit>  and the sequences can be obtained from the dna databank of japan  under accession numbers db920056-db <dig>
sequence reads were trimmed for low quality and vector contamination;  <dig> sequences belonging to  <dig> clones were obtained after this process.
for the clones with 5' and 3' sequence data showing significant sequence similarity to known proteins, the calculated full-length ratio was  <dig> , meaning that roughly 85% of the clones contain the complete coding sequence  of their inserts.
the sequences were assembled into  <dig> contigs and  <dig> singletons using cap3; however, given that all sequences were tagged with their respective clone ids, we were able to further cluster the results of the cap <dig> assembly to build  <dig> scaffolds representing distinct transcripts.
of these,  <dig>  contained, in a single contig, both ends of the respective clones and were thus considered full-length sequences.
alternative splicing variants had to be detected in order to estimate the number of different genes in the library; using the approach described in the methods section, we identified  <dig> transcripts of just  <dig> genes.
we determined that the full-length library includes transcripts of a total of  <dig> distinct genes, with alternative transcripts of about 26% of them.
to find the number of new transcripts captured in this library, relative to the number of expressed sequences from cassava already present in genbank, we conducted a blastn search of the sequences in our assembly against the  <dig> est sequences in dbest as of april  <dig>  any sequence with no hit to the database or with an e-value > 1e- <dig> and a percent identity <  <dig> % was considered to be a new cassava transcript.
in this way we found  <dig> new cassava sequences in our set.
furthermore, by running blastx against a uniprot – trembl database of plant proteins, we found  <dig> transcripts with no similarity  to known proteins in other plant species .
the information in the cap <dig> assembly and the names of the sequenced clones were used to build a cluster profile representing the number of clones per assembled scaffold ; this was done in order to provide an approximation of the total number of cassava transcripts using the compound poisson process model implemented in the eststat package  <cit> .
we obtained a number of  <dig> transcripts, which is in the range of the number of transcripts estimated in poplar, arabidopsis and rice .
sequence functional annotation
the  <dig> different transcripts defined upon the assembly were annotated with gene function using the gomp package .
sequences were thus assigned gene ontology  terms  <cit>  and mapped to the kyoto encyclopedia of genes and genomes  metabolic pathways  <cit>  based on sequence similarity.
of the  <dig> sequences,  <dig>  were annotated with terms of either of these controlled vocabularies, while  <dig>  had no function assigned.
the use of the kegg orthology  system  <cit>  to annotate sequences allowed us to draw pathway maps of the transcripts in our library using arabidopsis graphs as templates .
we assigned cassava sequences to  <dig> of the  <dig> a. thaliana pathways, and according to the electronic annotation we may have captured about 60%  of the enzymatic activities  reported for arabidopsis .
for some pathways we captured the full-length transcript of genes homologous to more than 70% of the enzymes involved according to the arabidopsis annotation, these almost-complete pathways include: 'glycolysis/gluconeogenesis' , 'starch and sucrose metabolism' , 'proteasome' , 'carbon fixation' , 'pyruvate metabolism' , 'biosynthesis of steroids' , 'pentose phosphate pathway'  and 'stilbene, coumarine and lignin biosynthesis'  among others.
the metabolic pathway of starch metabolism is of special interest in the case of cassava; the synthesis of this biopolymer is a relatively simple process that relies on the activities of three major enzymes: adp glucose pyrophosphorylase , starch synthase  and starch branching enzyme   <cit> ; as shown in figure  <dig>  we captured the full-length sequence of adpgpase and ss, the pathway visualization also indicates that the sbe was not found in the library.
three cassava transcripts of adpgpase were identified; these included one sequence of the small subunit of this enzyme and two alternative splicing variants of the large subunit.
for the ss enzyme we found five sequences, these appear to be alternative transcripts of two enzyme isoforms.
molecular markers are an important tool for crop improvement.
using the ssrfinder set of perl scripts  <cit>  and the autosnp package  <cit> , we designed  <dig> simple sequence repeats  and  <dig> single nucleotide polymorphism  markers for  <dig> of the  <dig> captured transcripts; these markers were stored in a relational database where they were linked to the functional annotation of the sequences.
after this process we got either a snp or a ssr marker for  <dig> of the  <dig> cassava transcripts identified as enzymes in the starch and sucrose metabolism pathway, these enzymes include ss, starch phosphorylase , sucrose phosphate synthase  and udp-glucose 6-dehydrogenase , which are enzymes known to have an effect on starch production  <cit> .
of the remaining  <dig> genes associated with molecular markers,  <dig> were inside genes included in  <dig> different pathways.
to recognize stress inducible genes in this remarkably tolerant crop, we compared our sequences to the collection of drought and cold induced genes identified with the riken arabidopsis full length  cdna microarray  <cit> .
table  <dig> shows genes from that experiment with significant hits in our library; for  <dig> stress-induced genes in arabidopsis, we captured  <dig> cassava transcripts showing significant sequence similarity  to  <dig> of them.
those genes for which we found more cassava transcripts include enzymes in the following categories: aquaporins, endoxyloglucan transferases, beta-glucosidases, thiol proteases, heat shock proteins , ascorbate peroxidases, thioredoxins, ethylene responsive element binding /ap2-like proteins and catalases.
gene correspondence and in-paralog  detection
in the following sections the term ortholog will be used to designate sequences that are derived from a single ancestral gene in the last common ancestor of the species that are being compared  <cit> .
this definition allows for cases were a single copy of a gene exists in each of these genomes  and cases where recent gene duplication has occurred and two or more genes in one species are orthologs with a single gene in another.
in the later case, genes produced by gene duplication after a speciation event are called in-paralogs and they are co-orthologs of the corresponding gene in other species.
it is not our objective to provide a full classification of the transcripts captured in the full-length library into orthologs and paralogs, but to make use of the methods available to describe some interesting features of this collection of genes.
first we use blast to designate pairs of genes that are reciprocal best hits  when cassava transcripts are compared to those of other species.
with this approach, rbhs are interpreted as potential one-to-one orthologs whereas co-orthologs are ignored; this way we are able to look for go terms overrepresented in the set of sequences that remain unpaired  as a means to recognize functional categories that are particularly frequent in the annotation of cassava transcripts.
second, we use blast to identify putative in-paralogs from a set of sequences from which alternative transcripts have been removed; this way we can produce a list of potential recent gene duplications for further analysis.
the rbh criterion was used to define one-to-one orthologous pairs of genes between cassava and three other species: r. communis, p. trichocarpa, and a. thaliana.
we found  <dig>   <dig> and  <dig> shared sequences respectively.
then, to assess the function of the sequences that under these terms were found only in cassava, we compared the go annotation of the sequences that were assigned to an orthologous pair and the annotation of those that were not.
as a result , the go terms enriched with cassava sequences  that were not assigned to a one-to-one pair included: 'protein biosynthesis', 'cellular protein catabolism', 'hormone mediated signaling', 'aminoacid biosynthesis', 'response to pest, pathogen or parasite' and 'lignin biosynthesis' among others.
on the other hand, go terms enriched with sequences assigned to an orthologous pair included: 'dna repair', 'regulation of transcription' and 'rna processing'.
besides go terms that are immediately associated to stress response like 'response to high light intensity,' 'response to heat' or 'response to oxidative stress,' sequences without a reciprocal best hit were frequently annotated with terms related to the synthesis of stress-responsive molecules like 'phenylpropanoid biosynthesis'  <cit> ; also they were annotated with terms describing cellular processes that are enhanced during stress such as 'ubiquitin-dependent protein catabolism'  <cit>  and 'abscisic acid mediated signaling'  <cit> ; or, as a third example, with terms like 'photosynthesis, light harvesting', which we found to include mainly homologues of chlorophyll binding proteins, that might help protect the photosystems during high-light stress  <cit> .
given that many of the sequences without assigned orthologs were somehow involved in response to stress, we wanted to see if those unmatched sequences corresponded to recent gene duplications of stress-related genes instead of alternatively spliced forms or assembly errors of single genes.
for this we excluded from our set of sequences the scaffolds that were identified as alternative splicing variants of other sequences.
then, we defined in-paralogs as sequences that were similar to each other and shared the same best hit in another genome .
using this approach and the additional restrictions mentioned in the methods section, we found  <dig> possible gene duplications; the go annotation of these sequences is presented in figure  <dig>  most of them are homologous to enzymes involved in primary metabolism and macromolecule modification, however, there are several of these duplications in the 'response to stimulus' category.
a closer look at this sequences revealed that enzymes such as monodehydroascorbate reductase , glutaredoxin , glutathione reductase , glutamate cysteine ligase , ferredoxin nadp+ reductase  and nadph thioredoxin reductase , seem to be duplicated; as shown in figure  <dig>  these enzymes catalyze important steps in reactive oxygen species  scavenging pathways, moreover enzymes like a mitogen activated protein kinase kinase  and heat shock protein  that were also duplicated, are known to play important roles in stress response  <cit> .
multiple sequence alignments and the construction of parsimony trees for the sequenced regions of these genes support the idea of lineage specific expansions in cassava .
discussion
value of the cassava full-length cdna library
we built the first est characterized full-length cdna library of cassava, providing nearly the same number of sequences previously available in est databases of this species.
the high number of novel sequences captured in this library can be taken as an indication of how poorly characterized the cassava transcriptome is; our library was not normalized, however, the fact that we extracted mrna from leaves and roots of cassava plants under different environmental conditions, resulted in a low-redundancy set with more than  <dig> distinct sequences represented by just one clone .
this low redundancy could be the outcome of different gene expression patterns in response to the varying conditions used to build the library, also, the small overlap between our set of ests and those of previous efforts that focused on cassava traits like starch content and response to pathogens  <cit> , could be an indication of the presence in our library of many genes specific to the abiotic stresses used in this study.
full-length cdnas are useful for the detailed annotation of sequence features in coding sequences and untranslated regions   <cit> .
while the analysis of the first can sometimes render valuable information about protein structure and function through the annotation of amino acid motifs or protein domains  <cit> , utr sequences can be useful for the analysis of gene expression by means of the identification of transcription factor binding motifs  <cit> , polyadenylation signals  <cit>  and other structural features.
given the above, the importance of our effort is not only measured in terms of the amount of sequences captured, but also in terms of the quality and relevance of the genes represented in the library.
we found that approximately 85% of the clones in our library contain full-length inserts; although this means that some of the cloned fragments are incomplete, the functional characterization of partial cdnas in the library still allows the retrieval of sequence data for further experiment design and for the isolation of the full-length cdna of specific genes.
moreover, from the est information alone, we were able to determine the 5'utrs of  <dig> sequences and the 3'utrs of  <dig> sequences, as well as the complete coding sequence of  <dig> genes by running blastx against a set of known proteins, this information can be valuable to look for functional features such as micro rna binding sites  <cit> .
we tried to minimize annotation errors by using curated databases of protein function to retrieve go and ko terms .
although this can prevent the propagation of such errors, sequence similarity does not always guarantee functional relationship, especially when identity is low  <cit> .
in our dataset, only  <dig> percent of the alignments that were used to retrieve functional annotations had a percent identity below  <dig> % and more than  <dig> % of the times the e-value was less than 10-30; as shown by joshi and xu  <cit> , this level of sequence similarity can be expected to provide a  <dig> to  <dig> percent probability that two proteins will have similar functions, even for the most specific go terms.
wilson and collaborators  <cit>  have also showed that precise function is generally well conserved when sequence identity is above 40%.
we trust that the overall representation of functional categories of the sequenced transcripts should not be very different from what we presented, however, at the more specific levels, one should be very careful in verifying the functional significance of sequence similarity.
putative functions were assigned to 78% of the sequenced clones, this is in contrast with previous cassava est collections for which as much as 63% of the sequences showed no significant similarity to known proteins  <cit> , the high number of annotated sequences in our library may be due to an increase in the number of annotations in go.
compared to similar reports in other species, we assigned a function to more sequences than those reported for maize  <cit>  or wheat  <cit>  full-length libraries, in these cases the amount of sequences with no function assigned were  <dig> and 44% respectively.
the fact that a large portion of the sequences in our library has been assigned a function through sequence similarity aids the detection and isolation of particular genes known to participate in relevant biological processes, or at least of genes with features such as protein motifs that would make them interesting targets for research.
while most of the clones were linked to a molecular function or biological process using go, the use of kegg pathways to visualize functional assignments allows a much easier assessment of the enzymatic activities and metabolic processes for which we have transcripts.
we mapped our cassava sequences to almost all of the pathway graphs of arabidopsis; it is noteworthy that with only  <dig> distinct transcripts, the equivalent to more than half of the pathway knowledge represented in kegg for arabidopsis been inferred from electronic annotation in cassava.
kegg pathways consist of reference diagrams on top of which species-specific enzymes can be drawn, since not all the metabolic pathways are as conserved as to allow the construction of a reference diagram, most of the kegg pathway graphs are of intermediary metabolism processes, and only a few regulatory pathways for a particular species like a. thaliana are available  <cit> .
nonetheless, traits of agronomical value such as starch content and quality  <cit> , carotene production  <cit> , photosynthesis  <cit>  and lignin biosynthesis  <cit>  that are important targets for cassava improvement are easily related to some of the kegg pathway maps ; identification of cassava genes participating in these as well as other processes would allow a rapid selection, isolation and characterization of key enzymes for the improvement of the crop, i.e.
rate limiting enzymes or catalytic elements missing from a biosynthetic pathway.
as an example, adpgpase has been shown to be a rate limiting enzyme in starch biosynthesis whose over-expression in cassava leads to increased root biomass  <cit> , the characterization of the molecular diversity of this as well as other enzymes could lead to the development of higher yielding crops.
although our library was oriented towards stress genes, the fact that we captured the full-length transcript of important enzymes in processes such as starch biosynthesis shows that we have a valuable resource for several aspects of cassava research.
furthermore, since we were able to design molecular markers for genes that directly or indirectly affect the poising between starch production and the synthesis of other molecules like sucrose  <cit>  or glucuronate; it is possible that the information provided by this library will provide elements for marker assisted selection in this as well as other processes, i.e.
protein biosynthesis, carotene accumulation, disease resistance, etc.
once a candidate gene in cassava is selected, the complete cdna sequence can be easily isolated from the corresponding clones.
this sequence can then be used to screen the molecular diversity of the studied loci to find gene variants suited for molecular marker development in a breeding program; with this in mind, we used the ests to design ssr and snp molecular markers; if these markers are within interesting genes, then they could be valuable for the detection of quantitative trait loci .
moreover, once the cassava genome is revealed, they could serve as a tool for pseudomolecule assembly  <cit> , which is an addition to the central role of the full-length sequences in gene annotation.
we found that our library contains transcripts of  <dig> different genes, this is a small number compared to the expected number of genes for a higher plant  <cit> ; the calculated number of  <dig> transcripts in cassava is a guide for the future efforts required for the completion of the gene catalog for this species.
this figure however may well be an overestimation of the true number of transcripts, mainly because our library is believed to be rich in rare transcripts that result from specific stress conditions  <cit> .
transcripts of stress-related genes
rna from different tissues of plants under normal conditions was pooled with that of plants under ppd, drought, heat and acidic soil stresses, we therefore suppose that our library should be enriched with transcripts of genes induced by these conditions.
if this is the case, one of the objectives of the analysis should be the identification of the types of stress-induced genes in the library.
as stated before, the abundance of novel cassava sequences in our set of transcripts could be an indication of the specificity of the genes captured according to the different treatments; this assumption is supported by the fact that we captured most of the stress-induced genes detected by the rafl microarray  <cit>  and that most of the sequences without assigned one-to-one orthologs in other species are in go categories linked directly or indirectly to stress response.
the comparison of the annotation of sequences with and without rbhs that we made for the detection of stress-induced genes is based on the following assumption: if there is more than a spliced form, allele or copy of a certain gene in a set of sequences, the use of the rbh criterion to define orthologs in a second set would assign just one of them to the corresponding sequence in the second group, this would leave an amount of unpaired sequences that would enrich the corresponding go category in the group of sequences without orthologs.
if our library includes many transcripts of stress related genes, then it is more likely to find alternative splicing variants or even more assembly errors of these sequences; this would lead to an overrepresentation of genes annotated with go terms corresponding to the functional categories of these sequences.
one possible explanation for the fact that many of the sequences without one-to-one orthologs were associated with stress-related genes could be the existence of recent gene duplications of these sequences in the cassava lineage.
we applied a very conservative methodology to detect some of these duplications ; with this method, two or more cassava sequences sharing their best hit in at least two other genomes are considered as potential in-paralogs.
in order to create a method to establish gene correspondences across genomes while dealing with recent and ancient gene duplications, kellis and collaborators have defined a best unambiguous subset as a group of genes such that all best hits of any gene within the set are contained within the set and no best hit of a gene outside the set is contained within the set  <cit> ; accordingly, we defined potential gene duplications in cassava by finding pairs of potential in-paralogs in which only one of the sequences was a best hit of genes in a second genome; in this way we avoid the report of false positives in which orthologs exists for the candidate in-paralogs, but both best blast hits point to just one of them  <cit> , in many cases as a result of different sequence lengths.
as shown in figure  <dig>  almost 5% of these potential duplications are annotated in the 'response to stimulus' go category; although this number may seem small, we have already seen that not all of the sequences related to stress response are annotated in that category.
for instance, many sequences involved in ubiquitin-mediated proteolysis correspond to a large portion of the duplications in the 'macromolecule metabolism' category.
a detailed inspection of the  <dig> possible gene duplications that we found, revealed that they were homologous to several enzymes related to ros metabolism; since heat  <cit> , drought  <cit> , acidic soils  <cit>  and ppd  <cit>  have been reported to induce ros production, we think that these potential duplications are a good example of how the full-length library can provide hints as to the mechanisms underlying cassava stress response features.
most ros in plants are produced by dismutation of superoxide generated by electron transfer to molecular oxygen in the mehler reactions of the chloroplast  <cit> .
during stress response, h2o <dig> production can be increased  <cit>  in a process that sometimes involves abscisic acid mediated stomatal closure and reduction of co <dig> levels for photosynthesis  <cit> .
high dosage of hydrogen peroxide results in hypersensitive cell-death while low quantities of this molecule can trigger a protective function against different stress conditions  <cit> , this ros-mediated activation of stress responses is dependent on a network of many genes that balance the outcome of ros-scavenging and ros-producing proteins  <cit> .
what we found in our sample of the cassava transcriptome is that many important enzymes in the main pathways of h2o <dig> scavenging seem to be duplicated ; we propose that these duplications may account, at least in part, for the stress tolerance characteristics of cassava, and that this could be due to a tighter control of some of the ros signaling mechanisms already described for other plants.
we base our hypothesis on the extensive literature regarding the importance of molecules like mdar  <cit> , fnr  <cit> , ntr  <cit> , glr  <cit> , thioredoxin  <cit>  and glutathione  <cit>  in plant sensitivity to environmental stress.
it is believed that the genus manihot emerged by recent allopolyploidization, this event is considered responsible for both, rapid speciation and weak interespecific barriers leading to hybridization  <cit> .
we hypothesize that potential duplications of stress-responsive genes could have originated in this polyploidization event; since it has been shown that most genes are rapidly silenced after these episodes unless they diversify in function  <cit> , it would be interesting to see if some of the detected duplications show evidence of subfunctionalization.
a first step to do this would be the evaluation of the expression profiles in different organs and under various conditions of the plausible gene duplications detected with this library, this could readily be done through microarray construction, for which once again the full-length library would be an invaluable resource  <cit> .
CONCLUSIONS
the cassava research community will certainly benefit from the full-length library here presented.
the analysis of the sequenced clones already suggests tempting research directions for the improvement of this crop.
an in-depth analysis of gene features and gene families in cassava, as well as the fine-tuning of their assigned functions, could provide the necessary elements for the enhancement of production under stress environments, moreover, what we learn from this crop should lead to important achievements in other important but not so tolerant plant species.

 among starch producing crops, cassava has a higher carbohydrate production than rice or maize under suboptimal conditions; more than million tons are produced in the world each year and about 84% of them are used for direct human consumption and animal feed. cassava starch is used as a raw material for a wide range of food products and industrial goods, including paper, cardboard, textile, plywood, glue and alcohol. moreover, because starch production from cassava is cheap compared to other crops, it is gaining attention as a biomass source for fuel production. the growing interest in cassava as an energy crop is evidenced by a genome sequencing project and the increasing production and technical advancements in tropical countries; for instance, cassava fresh root production in thailand increased from to million tons between and while a % increase per year has been reported for the same period worldwide. by virtue of its remarkable tolerance to abiotic stresses, cassava is grown in marginal, low fertility acidic soils showing increased nutrient use efficiency. it is known to maintain a healthy appearance in drought-prone areas, remaining photosynthetically active though at a reduced rate. because cassava is very drought-resistant and the tubers can be left in the soil for a couple of years, it is considered an important reserve carbohydrate source to prevent or relieve famine. cassava has some unusual characteristics that make it highly productive in near optimum environments, these include elevated activities of the c phosphoenolpyruvate carboxylase enzyme, long leaf life and low photorespiration rates; it, however, is usually grown in marginal highly eroded soils with uncertain rainfall and almost no agrochemical input. although cassava has some features that allow it to cope with stress better than other crops, e.g. high stomatal sensitivity to environmental humidity, deep rooting capacities and quick recovery after stress, under these conditions productivity is sub-optimal and unstable. cassava productivity is also threatened by bacterial and viral diseases, as well as arthropod pests. moreover, its high starch content is in contrast with its deficiency in proteins and key micronutrients, as well as the production of toxic hydrogen cyanide. to address these issues, traditional breeding methods have had some success, particularly in improving fresh root yield and dry matter content under non-stress conditions, however, because of the crop's heterozygous genetic makeup and long growth cycle, progress with this approach is slow. the use of biotechnology to improve cassava cultivars is a more straightforward strategy that relies on the tools of molecular and cell biology to find genetic determinants of desirable phenotypes. the construction of genetic maps and the identification of quantitative trait loci have yielded some results in cassava response to biotic stress, yet, the identification of candidate genes with this approach is a time consuming process involving the construction of bacterial artificial chromosome libraries and anchoring of these clones to the genetic map. a reverse-genetics approach can be a more direct solution, relying on the accumulated knowledge of gene function in model species it is possible to assess the effects of selected genes through regulation of their expression. as an example, silencing of p- cytochromes has allowed the production of cyanogen-free transgenic cassava plants. one tool that may assist both, the characterization of a plant expressed genes and the isolation of nucleotide sequences of genes with known function, are ests. these are a cost-effective gene discovery methodology that is also useful for the study of gene expression. despite its importance, large-scale sequence collections from cassava are scarce, there are expressed cassava sequences in the dbest database as of april which is a small number compared to the number of ests of maize, rice, soybean, potato or sugarcane. this is likely to change with the release of a cassava draft genome sequence this year by the united states department of energy's joint genome institute. although ests can aid the annotation of the cassava genome, the fact that most of them come from libraries made of random mrna fragments, make them insufficient to accurately and fully define gene models, ests are not only derived from partial transcripts, but also they can confound alternatively spliced forms during the assembly process. moreover due to the fragmentary nature of ests, their use in gene functional analysis is limited. full-length cdna libraries, on the other hand, are built in such a way that one insert represents one transcription unit, providing information on complete molecules for the functional dissection of genes. we built a full-length enriched cdna library from cassava leaves and roots subject to drought, heat, and acidic conditions, as well as from roots subject to post-harvest physiological deterioration, a major obstacle for cassava commercialization. the aim of this library is to support research in cassava improvement for high yield under abiotic stress, providing full sequences of stress-responsive genes and expanding the gene catalog of this species. the characterization of the transcripts captured in the library and the selection of non-redundant clones will certainly aid the annotation of genomic sequences and the construction of microarrays or other tools for functional genomics. in order to characterize the library and find the number and putative functions of the transcripts captured, nearly clones were sequenced from both ends, these ests, although unlikely to include the whole sequence of the inserts, are tagged with clone names. because this information is considered during the assembly process, ests derived from a full-length library allow, in principle, a more accurate definition of transcript units than normal ests. the annotation of the sequences acquired and the availability of the genome sequences of two species closely related to cassava such as castor bean and poplar as well as the complete set of genes from arabidopsis thaliana, provide altogether an opportunity to study the evolution of the cassava genome by means of comparative genomics; if it is possible to define gene correspondences between these species and on that basis find sequences that are unique to cassava, a closer inspection of these genes could provide hints as to the mechanisms underlying cassava's unique features. cassava is believed to be an allotetraploid that appeared by hybridization of wild manihot species, it would then be interesting to see what genes within a highly heterozygous gene pool have remained functional during cassava domestication; for this we use a methodology for the detection of recent duplications that is based on the detection of groups of genes sharing similarity to single sequences in other genomes, hopefully the genes detected with this strategy will aid cassava research for the genetic improvement of an already outstanding crop.  sequencing and assembly of both-end, single-pass sequences a full-length cdna library was constructed from leaves and roots of cassava plants under various environmental conditions, clones were sequenced from both ends; the clones are available at riken bioresource center and the sequences can be obtained from the dna databank of japan under accession numbers db920056-db sequence reads were trimmed for low quality and vector contamination; sequences belonging to clones were obtained after this process. for the clones with 5' and 3' sequence data showing significant sequence similarity to known proteins, the calculated full-length ratio was, meaning that roughly 85% of the clones contain the complete coding sequence of their inserts. the sequences were assembled into contigs and singletons using cap3; however, given that all sequences were tagged with their respective clone ids, we were able to further cluster the results of the cap assembly to build scaffolds representing distinct transcripts. of these, contained, in a single contig, both ends of the respective clones and were thus considered full-length sequences. alternative splicing variants had to be detected in order to estimate the number of different genes in the library; using the approach described in the methods section, we identified transcripts of just genes. we determined that the full-length library includes transcripts of a total of distinct genes, with alternative transcripts of about 26% of them. to find the number of new transcripts captured in this library, relative to the number of expressed sequences from cassava already present in genbank, we conducted a blastn search of the sequences in our assembly against the est sequences in dbest as of april any sequence with no hit to the database or with an e-value > 1e- and a percent identity < % was considered to be a new cassava transcript. in this way we found new cassava sequences in our set. furthermore, by running blastx against a uniprot – trembl database of plant proteins, we found transcripts with no similarity to known proteins in other plant species. the information in the cap assembly and the names of the sequenced clones were used to build a cluster profile representing the number of clones per assembled scaffold; this was done in order to provide an approximation of the total number of cassava transcripts using the compound poisson process model implemented in the eststat package. we obtained a number of transcripts, which is in the range of the number of transcripts estimated in poplar, arabidopsis and rice. sequence functional annotation the different transcripts defined upon the assembly were annotated with gene function using the gomp package. sequences were thus assigned gene ontology terms and mapped to the kyoto encyclopedia of genes and genomes metabolic pathways based on sequence similarity. of the sequences, were annotated with terms of either of these controlled vocabularies, while had no function assigned. the use of the kegg orthology system to annotate sequences allowed us to draw pathway maps of the transcripts in our library using arabidopsis graphs as templates. we assigned cassava sequences to of the a. thaliana pathways, and according to the electronic annotation we may have captured about 60% of the enzymatic activities reported for arabidopsis. for some pathways we captured the full-length transcript of genes homologous to more than 70% of the enzymes involved according to the arabidopsis annotation, these almost-complete pathways include:'glycolysis/gluconeogenesis','starch and sucrose metabolism','proteasome','carbon fixation','pyruvate metabolism','biosynthesis of steroids','pentose phosphate pathway' and'stilbene, coumarine and lignin biosynthesis' among others. the metabolic pathway of starch metabolism is of special interest in the case of cassava; the synthesis of this biopolymer is a relatively simple process that relies on the activities of three major enzymes: adp glucose pyrophosphorylase, starch synthase and starch branching enzyme; as shown in figure we captured the full-length sequence of adpgpase and ss, the pathway visualization also indicates that the sbe was not found in the library. three cassava transcripts of adpgpase were identified; these included one sequence of the small subunit of this enzyme and two alternative splicing variants of the large subunit. for the ss enzyme we found five sequences, these appear to be alternative transcripts of two enzyme isoforms. molecular markers are an important tool for crop improvement. using the ssrfinder set of perl scripts and the autosnp package, we designed simple sequence repeats and single nucleotide polymorphism markers for of the captured transcripts; these markers were stored in a relational database where they were linked to the functional annotation of the sequences. after this process we got either a snp or a ssr marker for of the cassava transcripts identified as enzymes in the starch and sucrose metabolism pathway, these enzymes include ss, starch phosphorylase, sucrose phosphate synthase and udp-glucose 6-dehydrogenase, which are enzymes known to have an effect on starch production. of the remaining genes associated with molecular markers, were inside genes included in different pathways. to recognize stress inducible genes in this remarkably tolerant crop, we compared our sequences to the collection of drought and cold induced genes identified with the riken arabidopsis full length cdna microarray. table shows genes from that experiment with significant hits in our library; for stress-induced genes in arabidopsis, we captured cassava transcripts showing significant sequence similarity to of them. those genes for which we found more cassava transcripts include enzymes in the following categories: aquaporins, endoxyloglucan transferases, beta-glucosidases, thiol proteases, heat shock proteins, ascorbate peroxidases, thioredoxins, ethylene responsive element binding /ap2-like proteins and catalases. gene correspondence and in-paralog detection in the following sections the term ortholog will be used to designate sequences that are derived from a single ancestral gene in the last common ancestor of the species that are being compared. this definition allows for cases were a single copy of a gene exists in each of these genomes and cases where recent gene duplication has occurred and two or more genes in one species are orthologs with a single gene in another. in the later case, genes produced by gene duplication after a speciation event are called in-paralogs and they are co-orthologs of the corresponding gene in other species. it is not our objective to provide a full classification of the transcripts captured in the full-length library into orthologs and paralogs, but to make use of the methods available to describe some interesting features of this collection of genes. first we use blast to designate pairs of genes that are reciprocal best hits when cassava transcripts are compared to those of other species. with this approach, rbhs are interpreted as potential one-to-one orthologs whereas co-orthologs are ignored; this way we are able to look for go terms overrepresented in the set of sequences that remain unpaired as a means to recognize functional categories that are particularly frequent in the annotation of cassava transcripts. second, we use blast to identify putative in-paralogs from a set of sequences from which alternative transcripts have been removed; this way we can produce a list of potential recent gene duplications for further analysis. the rbh criterion was used to define one-to-one orthologous pairs of genes between cassava and three other species: r. communis, p. trichocarpa, and a. thaliana. we found and shared sequences respectively. then, to assess the function of the sequences that under these terms were found only in cassava, we compared the go annotation of the sequences that were assigned to an orthologous pair and the annotation of those that were not. as a result, the go terms enriched with cassava sequences that were not assigned to a one-to-one pair included:'protein biosynthesis','cellular protein catabolism','hormone mediated signaling','aminoacid biosynthesis','response to pest, pathogen or parasite' and'lignin biosynthesis' among others. on the other hand, go terms enriched with sequences assigned to an orthologous pair included:'dna repair','regulation of transcription' and'rna processing'. besides go terms that are immediately associated to stress response like'response to high light intensity,''response to heat' or'response to oxidative stress,' sequences without a reciprocal best hit were frequently annotated with terms related to the synthesis of stress-responsive molecules like'phenylpropanoid biosynthesis'; also they were annotated with terms describing cellular processes that are enhanced during stress such as'ubiquitin-dependent protein catabolism' and'abscisic acid mediated signaling'; or, as a third example, with terms like'photosynthesis, light harvesting', which we found to include mainly homologues of chlorophyll binding proteins, that might help protect the photosystems during high-light stress. given that many of the sequences without assigned orthologs were somehow involved in response to stress, we wanted to see if those unmatched sequences corresponded to recent gene duplications of stress-related genes instead of alternatively spliced forms or assembly errors of single genes. for this we excluded from our set of sequences the scaffolds that were identified as alternative splicing variants of other sequences. then, we defined in-paralogs as sequences that were similar to each other and shared the same best hit in another genome. using this approach and the additional restrictions mentioned in the methods section, we found possible gene duplications; the go annotation of these sequences is presented in figure most of them are homologous to enzymes involved in primary metabolism and macromolecule modification, however, there are several of these duplications in the'response to stimulus' category. a closer look at this sequences revealed that enzymes such as monodehydroascorbate reductase, glutaredoxin, glutathione reductase, glutamate cysteine ligase, ferredoxin nadp+ reductase and nadph thioredoxin reductase, seem to be duplicated; as shown in figure these enzymes catalyze important steps in reactive oxygen species scavenging pathways, moreover enzymes like a mitogen activated protein kinase kinase and heat shock protein that were also duplicated, are known to play important roles in stress response. multiple sequence alignments and the construction of parsimony trees for the sequenced regions of these genes support the idea of lineage specific expansions in cassava. discussion value of the cassava full-length cdna library we built the first est characterized full-length cdna library of cassava, providing nearly the same number of sequences previously available in est databases of this species. the high number of novel sequences captured in this library can be taken as an indication of how poorly characterized the cassava transcriptome is; our library was not normalized, however, the fact that we extracted mrna from leaves and roots of cassava plants under different environmental conditions, resulted in a low-redundancy set with more than distinct sequences represented by just one clone. this low redundancy could be the outcome of different gene expression patterns in response to the varying conditions used to build the library, also, the small overlap between our set of ests and those of previous efforts that focused on cassava traits like starch content and response to pathogens, could be an indication of the presence in our library of many genes specific to the abiotic stresses used in this study. full-length cdnas are useful for the detailed annotation of sequence features in coding sequences and untranslated regions. while the analysis of the first can sometimes render valuable information about protein structure and function through the annotation of amino acid motifs or protein domains, utr sequences can be useful for the analysis of gene expression by means of the identification of transcription factor binding motifs, polyadenylation signals and other structural features. given the above, the importance of our effort is not only measured in terms of the amount of sequences captured, but also in terms of the quality and relevance of the genes represented in the library. we found that approximately 85% of the clones in our library contain full-length inserts; although this means that some of the cloned fragments are incomplete, the functional characterization of partial cdnas in the library still allows the retrieval of sequence data for further experiment design and for the isolation of the full-length cdna of specific genes. moreover, from the est information alone, we were able to determine the 5'utrs of sequences and the 3'utrs of sequences, as well as the complete coding sequence of genes by running blastx against a set of known proteins, this information can be valuable to look for functional features such as micro rna binding sites. we tried to minimize annotation errors by using curated databases of protein function to retrieve go and ko terms. although this can prevent the propagation of such errors, sequence similarity does not always guarantee functional relationship, especially when identity is low. in our dataset, only percent of the alignments that were used to retrieve functional annotations had a percent identity below % and more than % of the times the e-value was less than 10-30; as shown by joshi and xu, this level of sequence similarity can be expected to provide a to percent probability that two proteins will have similar functions, even for the most specific go terms. wilson and collaborators have also showed that precise function is generally well conserved when sequence identity is above 40%. we trust that the overall representation of functional categories of the sequenced transcripts should not be very different from what we presented, however, at the more specific levels, one should be very careful in verifying the functional significance of sequence similarity. putative functions were assigned to 78% of the sequenced clones, this is in contrast with previous cassava est collections for which as much as 63% of the sequences showed no significant similarity to known proteins, the high number of annotated sequences in our library may be due to an increase in the number of annotations in go. compared to similar reports in other species, we assigned a function to more sequences than those reported for maize or wheat full-length libraries, in these cases the amount of sequences with no function assigned were and 44% respectively. the fact that a large portion of the sequences in our library has been assigned a function through sequence similarity aids the detection and isolation of particular genes known to participate in relevant biological processes, or at least of genes with features such as protein motifs that would make them interesting targets for research. while most of the clones were linked to a molecular function or biological process using go, the use of kegg pathways to visualize functional assignments allows a much easier assessment of the enzymatic activities and metabolic processes for which we have transcripts. we mapped our cassava sequences to almost all of the pathway graphs of arabidopsis; it is noteworthy that with only distinct transcripts, the equivalent to more than half of the pathway knowledge represented in kegg for arabidopsis been inferred from electronic annotation in cassava. kegg pathways consist of reference diagrams on top of which species-specific enzymes can be drawn, since not all the metabolic pathways are as conserved as to allow the construction of a reference diagram, most of the kegg pathway graphs are of intermediary metabolism processes, and only a few regulatory pathways for a particular species like a. thaliana are available. nonetheless, traits of agronomical value such as starch content and quality, carotene production, photosynthesis and lignin biosynthesis that are important targets for cassava improvement are easily related to some of the kegg pathway maps; identification of cassava genes participating in these as well as other processes would allow a rapid selection, isolation and characterization of key enzymes for the improvement of the crop, i.e. rate limiting enzymes or catalytic elements missing from a biosynthetic pathway. as an example, adpgpase has been shown to be a rate limiting enzyme in starch biosynthesis whose over-expression in cassava leads to increased root biomass, the characterization of the molecular diversity of this as well as other enzymes could lead to the development of higher yielding crops. although our library was oriented towards stress genes, the fact that we captured the full-length transcript of important enzymes in processes such as starch biosynthesis shows that we have a valuable resource for several aspects of cassava research. furthermore, since we were able to design molecular markers for genes that directly or indirectly affect the poising between starch production and the synthesis of other molecules like sucrose or glucuronate; it is possible that the information provided by this library will provide elements for marker assisted selection in this as well as other processes, i.e. protein biosynthesis, carotene accumulation, disease resistance, etc. once a candidate gene in cassava is selected, the complete cdna sequence can be easily isolated from the corresponding clones. this sequence can then be used to screen the molecular diversity of the studied loci to find gene variants suited for molecular marker development in a breeding program; with this in mind, we used the ests to design ssr and snp molecular markers; if these markers are within interesting genes, then they could be valuable for the detection of quantitative trait loci. moreover, once the cassava genome is revealed, they could serve as a tool for pseudomolecule assembly, which is an addition to the central role of the full-length sequences in gene annotation. we found that our library contains transcripts of different genes, this is a small number compared to the expected number of genes for a higher plant; the calculated number of transcripts in cassava is a guide for the future efforts required for the completion of the gene catalog for this species. this figure however may well be an overestimation of the true number of transcripts, mainly because our library is believed to be rich in rare transcripts that result from specific stress conditions. transcripts of stress-related genes rna from different tissues of plants under normal conditions was pooled with that of plants under ppd, drought, heat and acidic soil stresses, we therefore suppose that our library should be enriched with transcripts of genes induced by these conditions. if this is the case, one of the objectives of the analysis should be the identification of the types of stress-induced genes in the library. as stated before, the abundance of novel cassava sequences in our set of transcripts could be an indication of the specificity of the genes captured according to the different treatments; this assumption is supported by the fact that we captured most of the stress-induced genes detected by the rafl microarray and that most of the sequences without assigned one-to-one orthologs in other species are in go categories linked directly or indirectly to stress response. the comparison of the annotation of sequences with and without rbhs that we made for the detection of stress-induced genes is based on the following assumption: if there is more than a spliced form, allele or copy of a certain gene in a set of sequences, the use of the rbh criterion to define orthologs in a second set would assign just one of them to the corresponding sequence in the second group, this would leave an amount of unpaired sequences that would enrich the corresponding go category in the group of sequences without orthologs. if our library includes many transcripts of stress related genes, then it is more likely to find alternative splicing variants or even more assembly errors of these sequences; this would lead to an overrepresentation of genes annotated with go terms corresponding to the functional categories of these sequences. one possible explanation for the fact that many of the sequences without one-to-one orthologs were associated with stress-related genes could be the existence of recent gene duplications of these sequences in the cassava lineage. we applied a very conservative methodology to detect some of these duplications; with this method, two or more cassava sequences sharing their best hit in at least two other genomes are considered as potential in-paralogs. in order to create a method to establish gene correspondences across genomes while dealing with recent and ancient gene duplications, kellis and collaborators have defined a best unambiguous subset as a group of genes such that all best hits of any gene within the set are contained within the set and no best hit of a gene outside the set is contained within the set; accordingly, we defined potential gene duplications in cassava by finding pairs of potential in-paralogs in which only one of the sequences was a best hit of genes in a second genome; in this way we avoid the report of false positives in which orthologs exists for the candidate in-paralogs, but both best blast hits point to just one of them, in many cases as a result of different sequence lengths. as shown in figure almost 5% of these potential duplications are annotated in the'response to stimulus' go category; although this number may seem small, we have already seen that not all of the sequences related to stress response are annotated in that category. for instance, many sequences involved in ubiquitin-mediated proteolysis correspond to a large portion of the duplications in the'macromolecule metabolism' category. a detailed inspection of the possible gene duplications that we found, revealed that they were homologous to several enzymes related to ros metabolism; since heat, drought, acidic soils and ppd have been reported to induce ros production, we think that these potential duplications are a good example of how the full-length library can provide hints as to the mechanisms underlying cassava stress response features. most ros in plants are produced by dismutation of superoxide generated by electron transfer to molecular oxygen in the mehler reactions of the chloroplast. during stress response, h2o production can be increased in a process that sometimes involves abscisic acid mediated stomatal closure and reduction of co levels for photosynthesis. high dosage of hydrogen peroxide results in hypersensitive cell-death while low quantities of this molecule can trigger a protective function against different stress conditions, this ros-mediated activation of stress responses is dependent on a network of many genes that balance the outcome of ros-scavenging and ros-producing proteins. what we found in our sample of the cassava transcriptome is that many important enzymes in the main pathways of h2o scavenging seem to be duplicated; we propose that these duplications may account, at least in part, for the stress tolerance characteristics of cassava, and that this could be due to a tighter control of some of the ros signaling mechanisms already described for other plants. we base our hypothesis on the extensive literature regarding the importance of molecules like mdar, fnr, ntr, glr, thioredoxin and glutathione in plant sensitivity to environmental stress. it is believed that the genus manihot emerged by recent allopolyploidization, this event is considered responsible for both, rapid speciation and weak interespecific barriers leading to hybridization. we hypothesize that potential duplications of stress-responsive genes could have originated in this polyploidization event; since it has been shown that most genes are rapidly silenced after these episodes unless they diversify in function, it would be interesting to see if some of the detected duplications show evidence of subfunctionalization. a first step to do this would be the evaluation of the expression profiles in different organs and under various conditions of the plausible gene duplications detected with this library, this could readily be done through microarray construction, for which once again the full-length library would be an invaluable resource.  the cassava research community will certainly benefit from the full-length library here presented. the analysis of the sequenced clones already suggests tempting research directions for the improvement of this crop. an in-depth analysis of gene features and gene families in cassava, as well as the fine-tuning of their assigned functions, could provide the necessary elements for the enhancement of production under stress environments, moreover, what we learn from this crop should lead to important achievements in other important but not so tolerant plant species. 
a full-length cdna library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; clones were sequence-characterized using expressed sequence tags. the cassava full-length cdna library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. this library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. finally, we detected putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. the ests were assembled into contigs and singletons that were further grouped into scaffolds; we found new cassava sequences and sequences with no significant similarity to plant protein databases. transcripts of distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in arabidopsis. the annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes.
 cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. a full-length cdna library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; clones were sequence-characterized using expressed sequence tags.  the ests were assembled into contigs and singletons that were further grouped into scaffolds; we found new cassava sequences and sequences with no significant similarity to plant protein databases. transcripts of distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in arabidopsis. the annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. finally, we detected putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features.  the cassava full-length cdna library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. this library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. 