BACKGROUND
the family of orchidaceae is the largest family of flowering plants and the number of species may exceed  <dig>   <cit> .
like all other living organisms, present-day orchids have evolved from ancestral forms as a result of selection pressure and adaptation.
they show a wide diversity of epiphytic and terrestrial growth forms and have successfully colonized almost every habitat on earth.
factors promoting orchid species richness include specific interaction between the orchid flower and pollinator  <cit> , sequential and rapid interplay between drift and natural selection  <cit> , obligate interaction with mycorrhiza  <cit> , and epiphytism which is true for most of all orchids and probably two-thirds of the epiphytic flora of the world.
the radiation of the orchid family has probably taken place in a comparatively short period as compared with that of most flowering plant families, which had already started to diversify in the mid-cretaceous  <cit> .
the time of origin of orchids is in dispute, although dressler suggests that they originated  <dig> to  <dig> million years ago   <cit> .
recently, the origin of the orchidaceae was dated with a fossil orchid and its pollinator.
the authors showed that the most recent common ancestor of extant orchids lived in the late cretaceous   <cit> .
they also suggested that epidendroideae and orchidoideae, two of the largest orchid subfamilies, which together represent > 95% of living orchid species, began to diversify early in the tertiary   <cit> .
according to molecular phylogenetic studies, orchidaceae comprise  <dig> subfamilies: apostasioideae, cypripedioideae, vanilloideae, orchidoideae and epidendroideae.
the apostasioideae is considered the sister group to other orchids.
vanilloideae diverged just before cypripedioideae.
both subfamilies have relatively low numbers of genera and species.
most of the taxonomic diversity in orchids is in  <dig> recently expanded sister-subfamilies: orchidoideae and especially epidendroideae  <cit> .
orchids are known for their diversity of specialized reproductive and ecological strategies.
for successful reproduction, the production of labellum and gynostemium  to facilitate pollination is well documented and the co-evolution of orchid flowers and pollinators is well known  <cit> .
in addition, the especially successful evolutionary progress of orchids may be explained by mature pollen grains packaged as pollinia, pollination-regulated ovary/ovule development, synchronized timing of micro- and mega-gametogenesis for effective fertilization, and the release of thousands or millions of immature embryos  in a mature capsule  <cit> .
however, despite their unique developmental reproductive biology, as well as specialized pollination and ecological strategies, orchids remain under-represented in molecular studies relative to other species-rich plant families  <cit> .
the reasons may be associated with the large genome size, long life cycle, and inefficient transformation system of orchids.
the genomic sequence resources currently available for orchids are limited.
very recently, a sketch of the phalaenopsis orchid genome from sequencing the ends of  <dig> bacterial artificial chromosome libraries of p. equestris was reported  <cit> .
in addition, a number of studies have developed expressed sequence tags  resources for orchids by using sanger sequencing  <cit> .
fewer than  <dig>  ests, including  <dig>  from p. equestris,  <dig>  from p. bellina,  <dig>  from oncidium gower ramsey, and  <dig>  from vanda mimi palmer, have been deposited in public database.
these studies have highlighted the utility of cdna sequencing for discovering candidate genes for orchid floral development  <cit> , floral scent production  <cit>  or flowering time  <cit>  in the absence of a genomic sequence.
however, a comprehensive description of the full complement of gene expressed in orchids remains unavailable.
massively parallel  <dig> pyrosequencing has become feasible for increasing sequencing depth and coverage while reducing time, labour, and cost  <cit> .
this technology can be used to deeply explore the nature and complexity of a given transcriptional universe.
<dig> sequencing of transcriptomes for model organisms has confirmed that the relatively short reads produced by this technology can be effectively assembled and used for gene discovery  <cit> .
in addition, the superior performance of this technology has been demonstrated in several studies  <cit> , including those of mustard weed arabidopsis thaliana  <cit> , the model legume medicago truncatula  <cit> , maize zea may  <cit> , the tree eucalyptus grandis  <cit> , chestnut  <cit> , oil crop olea europaea  <cit> , oilseed rape brassica napus  <cit> , and the antimalarial plant artemisia annua  <cit> .
the genus phalaenopsis blume , a beautiful and one of the most popular ornamental flowers exported worldwide, comprises  <dig> species according to the latest classification by christenson  <cit> .
the species are found throughout tropical asia and the larger islands of the pacific ocean.
in taiwan,  <dig> of the native species, p. equestris and p. aphrodite subsp.
formosana, are usually used as parents for breeding.
p. equestris possesses several favorable commercial traits such as numerous spikes and branches and multitudinous and colorful flowers.
p. aphrodite subsp.
formosana has a perfect arrangement of flower positions at the spike and an elegant flower shape with extended longevity.
the flowers of both species are scentless.
many of the scent traits in the p. hybrids are mainly derived from p. bellina and/or p. violaceae, the native species in malaysia.
both p. equestris and p. aphrodite subsp.
formosana are diploid plants with  <dig> chromosomes , which are small and uniform in size .
the estimated haploid genome sizes are  <dig>  mb  and  <dig>  mb  for p. equestris and p. aphrodite subsp.
formosana, respectively, which are relatively small in genus of phalaenopsis  <cit> .
the  <dig> species could be considered model organisms for studying orchid biology because of their relative small genome size  <cit> , high performance of culture system and well applicable functional genomic tools such as genetic transformation  <cit>  and virus-induced gene silencing system  <cit> .
in this report, we provide the first comprehensive characterization of the transcriptome of phalaenopsis orchids by massively parallel  <dig> pyrosequencing.
to expansively cover the phalaenopsis orchid transcriptome and facilitate identifying sets of genes involved in a broad range of biological processes, we developed the est set from  <dig> samples derived from  <dig> different species of phalaenopisis .
based on data corresponding to a single run on the gs flx sequencer, almost  <dig> million bases were assembled into ~ <dig>  pieces of putative transcripts and the majority of these have been annotated and functionally classified.
all the sequences were deposited in orchidbase  and short read archive  division of the genbank repository .
these results can provide the means for future genome-wide orchid biology and biotechnology research in phalaenopsis.
RESULTS
sequencing and assembly of  <dig> pyrosequenced ests
in total,  <dig>  ests  averaging  <dig> bp were generated from one pyrosequencing run.
cleaning  of the raw sequences resulted in a total of  <dig>  high-quality reads with an average length of  <dig> bp nucleotides totaling  <dig> mb.
after assembly using gs flx gsassembler, these reads were assembled into  <dig>  contigs and  <dig>  singletons under the criteria of minimum  <dig> bases overlap with at least 95% identity.
the average length of contigs and singletons was  <dig> bp and  <dig> bp, respectively.
these contigs and singletons represented up to  <dig>  unigenes.
the length distribution of unigenes and their component reads are summarized in table  <dig> and additional file  <dig> and additional file  <dig>  our assembly also included a larger proportion of long contigs   than the 8% reported by novaes et al.
<cit> .
the unigene set was also compared to the ests derived from p. equestris  <cit>  and p. bellina  <cit> .
about  <dig> %  of the unigenes could be matched to the published phalaenopsis ests .
in addition, different sized contigs were selected to recalculate the blast hits.
result showed that the percentage of hits was proportion to contig size .
furthermore, the previously available ests derived from phalaenopsis orchids were blast against ests generated by this study.
results showed that among  <dig>  unigenes,  <dig>   matched the ests produced by  <dig> pyrosequencing.
the unigenes were searched against the ncbi non-redundant  protein database by use of the blastx algorithm.
among the  <dig>  unigenes,  <dig>   had at least one significant alignment to existing genes in the database  , and  <dig>  of these  had an open reading frame .
a majority  of the pyrosequencing assemblies did not match any known sequences in the database.
to analyze effects of fragment size on annotation efficiency, we compared the percentage of hits between different sized ests with and without homology at e-value < 10- <dig> .
the result showed that  <dig> % of genes without homology and 13% of those with homology were <  <dig> bp.
thus, short reads may have a low level of annotation.
in addition, it is also possible that unannotated reads may likely represent novel genes.
alternatively, these sequences might correspond to divergent 5' or 3' regions of genes.
functional annotation of novel transcripts
to determine the possible functions of genes tagged, we used the gene ontology  classification system for plants developed at tair  <cit>  .
the functions of genes identified cover various biological processes, molecular functions and cellular components .
in total,  <dig>  of the  <dig>  transcripts were assigned to biological processes.
the largest proportion of functionally assigned ests fell into protein metabolism  and transport  except of unknown biological processes  and other cellular processes .
among the molecular functions,  <dig>  out of a total  <dig>  transcripts were assigned in this category.
the molecular functions most represented were hydrolase activity , transferase activity  and nucleotide binding  except for other enzyme activity  and unknown molecular functions .
among the cellular components,  <dig>  out of a total  <dig>  transcripts were assigned in this category.
chloroplast  and plasma membrane  are the most represented categories in cellular components except for unknown cellular components .
these ests obtained from a non-normalized cdna library could be used to reveal global gene expression patterns as deduced from transcript abundance.
the transcripts highly expressed in phalaenopsis tissues are listed in table  <dig>  the most abundant ests coded for cytochrome p450-like protein precursor , followed by triple gene block  <dig>  and cytochrome p <dig> monooxygenase .
these ests probably correspond to the most highly expressed genes in the phalaenopsis.
plant systems utilize a diverse array of cytochrome p <dig> monooxygenases in their biosynthetic and detoxicative pathways.
high expression of the cytochrome p <dig> gene family suggests that these genes are important for orchid subsistence and adaptation to the environment.
the high expression of triple gene block  <dig> raised the interesting question of whether the sequence samples were infected by cymbidium mosaic virus  or if the gene is included in the phalaenopsis genome.
therefore, we screened other genes of the same cymmv in the est dataset.
in addition to triple gene block  <dig>  several genes of cymmv were found, including  <dig> unigenes of rna dependent rna polymerase,  <dig> of coat protein,  <dig> of triple gene block  <dig>  and  <dig> of triple gene block  <dig>  these transcripts showed significant expression in phalaenopsis, which indicates that some of the experimental materials used here may have been infected with virus prior to the sampling.
n.d.: not determined
analysis of a large number of ests has revealed ancient polyploidy throughout the major angiosperm lineages  <cit> .
it would be interesting to analyze how many subfamilies exist in very high number of ests to evaluate the possibility of gene duplication in phalaenopsis orchids.
we set more stringent criteria for assembly  and found a greater number of unigenes , suggesting that some genes may have been undergone gene duplication.
however, more evidence is needed to solve the causes that lead to the formation of paralogous genes, such as whole-genome duplication, tandem gene duplication or segmental duplication.
gene families and pathways
to evaluate the effectiveness of the orchid transcriptome library, we categorized the assembled unigenes through the use of arabidopsis proteome as targets.
in total,  <dig>  unigenes were classified into  <dig> gene families representing  <dig> %  of arabidopsis gene families .
we also mapped assembled unigenes into the kegg pathways including metabolism, genetic information processing, environmental information processing, cellular processes, and organism systems .
a total of  <dig>  unigenes were mapped onto kegg pathways .
among these,  <dig>  unigenes were related to metabolism,  <dig>  unigenes corresponded to genetic information processing,  <dig> unigenes mapped to environmental information processing,  <dig> unigenes were classified as cellular processes, and  <dig> unigenes belonged to organism systems .
the presence genes for these essential cellular processes suggest that these sequences account for most of the comprehensive phalaenopsis transcriptome.
previously, we identified the major compounds emitted from p. bellina flowers to be monoterpenes, including linalool and geraniol  <cit> .
identification of the potential genes involved in the biosynthetic pathways for terpenoid precursors will be important for understanding the regulation of scent biosynthesis in orchids.
in our  <dig> sequence dataset,  <dig> unigenes assembled from  <dig> reads were found potentially related to the methylerythritol phosphate pathway  and mevalonate pathway  .
analysis of previous ests from p. bellina showed  <dig> unigenes assembled from  <dig> reads were potentially related to the mep and mva pathways .
the use of  <dig> pyrosequencing identified more genes and more ests per gene constituted in the terpenoid backbone biosynthesis than did sanger sequencing .
at least one unigene was found to correspond to the enzymes participating in the two pathways, except the third step of the mep pathway .
thus, the large amount of transcriptomic information provided by  <dig> pyrosequencing may accelerate the progress for the study of scent production and regulation in orchids.
transcription factors
because transcription factors control the expression of a genome and play important roles in all aspects of the life cycle of higher plants, we characterized the transcription factor-associated ests from the transcriptome of phalaenopsis by using rice transcription factor sequences downloaded from the plant transcription factor database  as queries.
in the phalaenopsis transcriptome, we identified  <dig> unigenes consisted of  <dig>  reads encoding putative transcription factors, occupying  <dig> %  of the unigenes of phalaenopsis transcriptome.
compared to the  <dig> % of plant genes that have been shown to be transcription factor genes  <cit> ,  <dig> % of genes related to transcription factors in phalaenopsis is low.
to analyze the underestimation, we mapped all unigenes to  <dig> full-length cdnas  encoding mads-box proteins  derived from p. equestris.
in total,  <dig> unigenes could be mapped to the  <dig> fl-cdnas.
among the  <dig> unigenes,  <dig> are located in untranslated regions, and the other two are short fragments  located within coding regions.
however, these seven unigenes were not identified as transcription factor genes.
these results suggest that one of the reasons for an underestimation of transcription factor genes in phalaenopsis can be explained by sequences corresponding to divergent 5' or 3' regions of genes and/or they are short reads per se.
the most abundantly expressed transcription factor gene families, c3h and ap2/erf, accounted for a full  <dig> %  of the overall transcription factor expression .
in addition, bhlh , myb  and nac  families were found in turn .
these five families occupied approximately  <dig> % of expressed transcription factors.
however, five families of transcription factors including lfy, m-type, stat, voz, and wox were not detected.
the few families that could not be found might result from inadequate sampling of the transcriptome or genes that are truly rarely expressed.
large-scale efforts in deeper sampling and sequencing of the transcriptome will help completely identify genes related to of transcription factor families in orchids.
discussion
as the most species rich and diversified family, orchidaceae has mesmerized botanists for centuries.
as for most other non-model plant species, we lack genetic and genomic resources for molecular biological study.
although a precise estimate of transcriptome coverage is unattainable without the full genomic sequence, the massively parallel pyrosequencing characterization can be considered an initial step for functional genomics studies in phalaenopisis.
from  <dig>  sequence reads, we assembled data for  <dig>  unigenes consisting of  <dig>  contigs and  <dig>  singletons from phalaenopsis.
although a high number of transcripts are short-length reads which may result in several assembled contigs and singletons for each gene, the dataset we report here still provides a plentiful dataset of different genes representing a substantial part of the transcriptome of orchids, which in turn reflects these plants' sophisticated designs for successful pollination, reproduction and adaptation to the environment.
homology searches showed that  <dig> % of the ests have no significant similarities to any other protein sequences in public databases.
about  <dig> % of these ests are <  <dig> bp, indicating that the short size has a negative effect on successful annotation.
however, these genes may perform specific roles in orchids and be quite divergent from those of other plant species.
the orchids, indeed, have diverse specialized reproductive and ecological strategies for adaptive radiation.
on the other hand, we could not reliably annotate a high proportion of unigenes lacking assignment of a putative function because they did not cover the full length of the transcript or because they represent untranslated regions.
comparing the distributions of the functional categories among ests provides support for the expression levels of the different gene classes.
the transcripts with the first and third highest expression we found for phalaenopsis were homologous to the members of cytochrome p <dig> .
plant cytochromes p <dig> catalyze a wide variety of monooxygenation/hydroxylation reactions in primary and secondary metabolism.
genomic sequencing projects have revealed that cytochromes p <dig> genes represent approximately 1% of the total gene annotations for each plant species  <cit> .
in addition to revealing the highest transcript expression in phalaenopsis, the next-generation transcriptome sequencing also generated  <dig> members of the cytochrome p <dig> family.
the extraordinary expression level and remarkable diversification of this gene family may have led to the phalaenopsis orchid survival ability.
the transcript with the second highest level of expression was homologous to triple gene block  <dig>  of cymmv.
we also found genes with significant expression that were homologous to rna dependent rna polymerase, coat protein, triple gene block  <dig> and triple gene block  <dig> of cymmv.
even though we found no substantial virus-infected symptoms in our samples, some of the experimental materials have been infected with virus prior to the sampling.
we also found transcripts with high expression  that were homologous to lla- <dig> derived from lily .
this transcript is found in immature anther, tepal, pistil, stem and leaf in lily  <cit> , however it has an as yet unknown function.
dissection of function of these genes might be a useful direction for further study of orchid biology.
the fact that whole-genome duplication often gives rise to species-rich groups of organisms, such as >  <dig>  species of asteraceae and >  <dig>   <dig> species of fabaceae, highlights that polyploidy can facilitate diversification and speciation of organisms  <cit> .
the orchidaceae contains more than  <dig>  species and has successfully colonized almost every habitat on earth.
whole-genome duplication may also have occurred in the orchid genome.
based on the results from analyzing how many subfamilies exist with very high number of ests, we suggested gene duplication probably have occurred on these genes.
however, these gene duplication events may be caused by whole-genome duplication, tandem gene duplication or segmental duplication.
only after completeness of whole-genome sequencing of phalaenopsis has been performed, it will be possible to differentiate whole-genome duplications from segmental and tandem duplications by mapping chromosome locations of the duplicated genes or blocks of genes.
to evaluate whether the sequences annotated in this study include all genes expressed in these tissues, developmental stages, and treatments, we searched for a number of genes involved in metabolic pathways and homologous to members characterized in arabidopsis gene families.
the genes associated with metabolic pathways were based on the kegg database of pathways and those for gene family on the tair database of arabidopsis proteome.
the rationale for these searches was that those essential genes must be expressed to maintain cellular functions, so failure to find these sequences in the transcriptome would reflect either inadequate sequencing depth or ineffective annotation.
for the pathways considered here, essentially all genes involved in the pathways were found except those involved in anthocyanin biosynthesis.
for the  <dig> gene families in arabidopsis, total  <dig>  unigenes were classified into  <dig> gene families occupying more than 70%  of arabidopsis gene families.
a caution for these finding is that high levels of expression might be expected for some essential house-keeping genes, leading them to be well represented in even an incomplete transcriptome sequencing effort.
to account for this possibility, we searched for genes associated with transcription factors.
because of their more restricted spatial and temporal expression profiles, transcription factor genes are not expected to be as highly expressed as essential house-keeping genes in whole-organism libraries.
we successfully identified genes from nearly all the transcription factor families considered .
the few genes that could not be found might result from incomplete annotation or inadequate sampling of the transcriptome or they may be truly not expressed.
overall, these searches support that the collection of annotated sequences we produced represents a reasonably broad description of the phalaenopsis transcriptome.
these sequences of expressed transcripts will be very useful for genome annotation of phalaenopsis genome in the future.
analysis of expression profiles of transcription factors in the transcriptome of phalaenopsis is meaningful because these are master-control proteins in all living cells.
surprisingly, we found that c3h and ap2/erf families together represented more than 30% of expression of phalaenopsis transcription factors.
the c3h family has been reported to be involved in arabidopsis embryogenesis  <cit> , shoot apical meristem maintenance  <cit> , drought tolerance  <cit> , and response to abscisic acid in craterostigma plantagineum  <cit> .
the ap2/erf superfamily is defined by the ap2/erf domain, of about  <dig> to  <dig> amino acids, and is involved in dna binding.
a combination of genetic and molecular approaches has been used to characterize a series of regulatory genes of the ap2/erf family.
the members of this family are involved in regulating various biological processes related to growth and development, as well as various responses to environmental stimuli.
this family includes genes related to drought  <cit> , high salt concentration  <cit> , low temperature  <cit> , diseases  <cit> , and the control of ovule development and flower organ growth  <cit> .
understanding the functions of these genes will advance our understanding of the great morphological diversity and successful adaptation of orchids.
however, we did not find the transcription factor families lfy, m-type, stat, voz, and wox, in the phalaenopsis transcriptome.
these families might either be rarely expressed, or might not have appeared in our cdna sampling.
CONCLUSIONS
thanks to recent advances in next-generation sequencing technology, we have applied rna-seq to facilitate transcriptome analysis of orchids which present important biological questions but lack a fully sequenced genome.
our findings represent substantial contributions to the publicly accessible expressed sequences for the orchidaceae family.
with the whole genome sequencing of p. equestris in progress, this collection of ests is a valuable resource that will be immediately useful for researchers, allowing for correction of assemblies, annotation, and construction of gene models to establish accurate exon-intron boundaries.
application of these resources through the common language of nucleotide sequences will greatly enhance the insights into the reproductive success of orchids.

 the family of orchidaceae is the largest family of flowering plants and the number of species may exceed. like all other living organisms, present-day orchids have evolved from ancestral forms as a result of selection pressure and adaptation. they show a wide diversity of epiphytic and terrestrial growth forms and have successfully colonized almost every habitat on earth. factors promoting orchid species richness include specific interaction between the orchid flower and pollinator, sequential and rapid interplay between drift and natural selection, obligate interaction with mycorrhiza, and epiphytism which is true for most of all orchids and probably two-thirds of the epiphytic flora of the world. the radiation of the orchid family has probably taken place in a comparatively short period as compared with that of most flowering plant families, which had already started to diversify in the mid-cretaceous. the time of origin of orchids is in dispute, although dressler suggests that they originated to million years ago. recently, the origin of the orchidaceae was dated with a fossil orchid and its pollinator. the authors showed that the most recent common ancestor of extant orchids lived in the late cretaceous. they also suggested that epidendroideae and orchidoideae, two of the largest orchid subfamilies, which together represent > 95% of living orchid species, began to diversify early in the tertiary. according to molecular phylogenetic studies, orchidaceae comprise subfamilies: apostasioideae, cypripedioideae, vanilloideae, orchidoideae and epidendroideae. the apostasioideae is considered the sister group to other orchids. vanilloideae diverged just before cypripedioideae. both subfamilies have relatively low numbers of genera and species. most of the taxonomic diversity in orchids is in recently expanded sister-subfamilies: orchidoideae and especially epidendroideae. orchids are known for their diversity of specialized reproductive and ecological strategies. for successful reproduction, the production of labellum and gynostemium to facilitate pollination is well documented and the co-evolution of orchid flowers and pollinators is well known. in addition, the especially successful evolutionary progress of orchids may be explained by mature pollen grains packaged as pollinia, pollination-regulated ovary/ovule development, synchronized timing of micro- and mega-gametogenesis for effective fertilization, and the release of thousands or millions of immature embryos in a mature capsule. however, despite their unique developmental reproductive biology, as well as specialized pollination and ecological strategies, orchids remain under-represented in molecular studies relative to other species-rich plant families. the reasons may be associated with the large genome size, long life cycle, and inefficient transformation system of orchids. the genomic sequence resources currently available for orchids are limited. very recently, a sketch of the phalaenopsis orchid genome from sequencing the ends of bacterial artificial chromosome libraries of p. equestris was reported. in addition, a number of studies have developed expressed sequence tags resources for orchids by using sanger sequencing. fewer than ests, including from p. equestris, from p. bellina, from oncidium gower ramsey, and from vanda mimi palmer, have been deposited in public database. these studies have highlighted the utility of cdna sequencing for discovering candidate genes for orchid floral development, floral scent production or flowering time in the absence of a genomic sequence. however, a comprehensive description of the full complement of gene expressed in orchids remains unavailable. massively parallel pyrosequencing has become feasible for increasing sequencing depth and coverage while reducing time, labour, and cost. this technology can be used to deeply explore the nature and complexity of a given transcriptional universe. sequencing of transcriptomes for model organisms has confirmed that the relatively short reads produced by this technology can be effectively assembled and used for gene discovery. in addition, the superior performance of this technology has been demonstrated in several studies, including those of mustard weed arabidopsis thaliana, the model legume medicago truncatula, maize zea may, the tree eucalyptus grandis, chestnut, oil crop olea europaea, oilseed rape brassica napus, and the antimalarial plant artemisia annua. the genus phalaenopsis blume, a beautiful and one of the most popular ornamental flowers exported worldwide, comprises species according to the latest classification by christenson. the species are found throughout tropical asia and the larger islands of the pacific ocean. in taiwan, of the native species, p. equestris and p. aphrodite subsp. formosana, are usually used as parents for breeding. p. equestris possesses several favorable commercial traits such as numerous spikes and branches and multitudinous and colorful flowers. p. aphrodite subsp. formosana has a perfect arrangement of flower positions at the spike and an elegant flower shape with extended longevity. the flowers of both species are scentless. many of the scent traits in the p. hybrids are mainly derived from p. bellina and/or p. violaceae, the native species in malaysia. both p. equestris and p. aphrodite subsp. formosana are diploid plants with chromosomes, which are small and uniform in size. the estimated haploid genome sizes are mb and mb for p. equestris and p. aphrodite subsp. formosana, respectively, which are relatively small in genus of phalaenopsis. the species could be considered model organisms for studying orchid biology because of their relative small genome size, high performance of culture system and well applicable functional genomic tools such as genetic transformation and virus-induced gene silencing system. in this report, we provide the first comprehensive characterization of the transcriptome of phalaenopsis orchids by massively parallel pyrosequencing. to expansively cover the phalaenopsis orchid transcriptome and facilitate identifying sets of genes involved in a broad range of biological processes, we developed the est set from samples derived from different species of phalaenopisis. based on data corresponding to a single run on the gs flx sequencer, almost million bases were assembled into ~ pieces of putative transcripts and the majority of these have been annotated and functionally classified. all the sequences were deposited in orchidbase and short read archive division of the genbank repository. these results can provide the means for future genome-wide orchid biology and biotechnology research in phalaenopsis.  sequencing and assembly of pyrosequenced ests in total, ests averaging bp were generated from one pyrosequencing run. cleaning of the raw sequences resulted in a total of high-quality reads with an average length of bp nucleotides totaling mb. after assembly using gs flx gsassembler, these reads were assembled into contigs and singletons under the criteria of minimum bases overlap with at least 95% identity. the average length of contigs and singletons was bp and bp, respectively. these contigs and singletons represented up to unigenes. the length distribution of unigenes and their component reads are summarized in table and additional file and additional file our assembly also included a larger proportion of long contigs than the 8% reported by novaes et al.. the unigene set was also compared to the ests derived from p. equestris and p. bellina. about % of the unigenes could be matched to the published phalaenopsis ests. in addition, different sized contigs were selected to recalculate the blast hits. result showed that the percentage of hits was proportion to contig size. furthermore, the previously available ests derived from phalaenopsis orchids were blast against ests generated by this study. results showed that among unigenes, matched the ests produced by pyrosequencing. the unigenes were searched against the ncbi non-redundant protein database by use of the blastx algorithm. among the unigenes, had at least one significant alignment to existing genes in the database, and of these had an open reading frame. a majority of the pyrosequencing assemblies did not match any known sequences in the database. to analyze effects of fragment size on annotation efficiency, we compared the percentage of hits between different sized ests with and without homology at e-value < 10-. the result showed that % of genes without homology and 13% of those with homology were < bp. thus, short reads may have a low level of annotation. in addition, it is also possible that unannotated reads may likely represent novel genes. alternatively, these sequences might correspond to divergent 5' or 3' regions of genes. functional annotation of novel transcripts to determine the possible functions of genes tagged, we used the gene ontology classification system for plants developed at tair. the functions of genes identified cover various biological processes, molecular functions and cellular components. in total, of the transcripts were assigned to biological processes. the largest proportion of functionally assigned ests fell into protein metabolism and transport except of unknown biological processes and other cellular processes. among the molecular functions, out of a total transcripts were assigned in this category. the molecular functions most represented were hydrolase activity, transferase activity and nucleotide binding except for other enzyme activity and unknown molecular functions. among the cellular components, out of a total transcripts were assigned in this category. chloroplast and plasma membrane are the most represented categories in cellular components except for unknown cellular components. these ests obtained from a non-normalized cdna library could be used to reveal global gene expression patterns as deduced from transcript abundance. the transcripts highly expressed in phalaenopsis tissues are listed in table the most abundant ests coded for cytochrome p450-like protein precursor, followed by triple gene block and cytochrome p monooxygenase. these ests probably correspond to the most highly expressed genes in the phalaenopsis. plant systems utilize a diverse array of cytochrome p monooxygenases in their biosynthetic and detoxicative pathways. high expression of the cytochrome p gene family suggests that these genes are important for orchid subsistence and adaptation to the environment. the high expression of triple gene block raised the interesting question of whether the sequence samples were infected by cymbidium mosaic virus or if the gene is included in the phalaenopsis genome. therefore, we screened other genes of the same cymmv in the est dataset. in addition to triple gene block several genes of cymmv were found, including unigenes of rna dependent rna polymerase, of coat protein, of triple gene block and of triple gene block these transcripts showed significant expression in phalaenopsis, which indicates that some of the experimental materials used here may have been infected with virus prior to the sampling. n.d.: not determined analysis of a large number of ests has revealed ancient polyploidy throughout the major angiosperm lineages. it would be interesting to analyze how many subfamilies exist in very high number of ests to evaluate the possibility of gene duplication in phalaenopsis orchids. we set more stringent criteria for assembly and found a greater number of unigenes, suggesting that some genes may have been undergone gene duplication. however, more evidence is needed to solve the causes that lead to the formation of paralogous genes, such as whole-genome duplication, tandem gene duplication or segmental duplication. gene families and pathways to evaluate the effectiveness of the orchid transcriptome library, we categorized the assembled unigenes through the use of arabidopsis proteome as targets. in total, unigenes were classified into gene families representing % of arabidopsis gene families. we also mapped assembled unigenes into the kegg pathways including metabolism, genetic information processing, environmental information processing, cellular processes, and organism systems. a total of unigenes were mapped onto kegg pathways. among these, unigenes were related to metabolism, unigenes corresponded to genetic information processing, unigenes mapped to environmental information processing, unigenes were classified as cellular processes, and unigenes belonged to organism systems. the presence genes for these essential cellular processes suggest that these sequences account for most of the comprehensive phalaenopsis transcriptome. previously, we identified the major compounds emitted from p. bellina flowers to be monoterpenes, including linalool and geraniol. identification of the potential genes involved in the biosynthetic pathways for terpenoid precursors will be important for understanding the regulation of scent biosynthesis in orchids. in our sequence dataset, unigenes assembled from reads were found potentially related to the methylerythritol phosphate pathway and mevalonate pathway. analysis of previous ests from p. bellina showed unigenes assembled from reads were potentially related to the mep and mva pathways. the use of pyrosequencing identified more genes and more ests per gene constituted in the terpenoid backbone biosynthesis than did sanger sequencing. at least one unigene was found to correspond to the enzymes participating in the two pathways, except the third step of the mep pathway. thus, the large amount of transcriptomic information provided by pyrosequencing may accelerate the progress for the study of scent production and regulation in orchids. transcription factors because transcription factors control the expression of a genome and play important roles in all aspects of the life cycle of higher plants, we characterized the transcription factor-associated ests from the transcriptome of phalaenopsis by using rice transcription factor sequences downloaded from the plant transcription factor database as queries. in the phalaenopsis transcriptome, we identified unigenes consisted of reads encoding putative transcription factors, occupying % of the unigenes of phalaenopsis transcriptome. compared to the % of plant genes that have been shown to be transcription factor genes, % of genes related to transcription factors in phalaenopsis is low. to analyze the underestimation, we mapped all unigenes to full-length cdnas encoding mads-box proteins derived from p. equestris. in total, unigenes could be mapped to the fl-cdnas. among the unigenes, are located in untranslated regions, and the other two are short fragments located within coding regions. however, these seven unigenes were not identified as transcription factor genes. these results suggest that one of the reasons for an underestimation of transcription factor genes in phalaenopsis can be explained by sequences corresponding to divergent 5' or 3' regions of genes and/or they are short reads per se. the most abundantly expressed transcription factor gene families, c3h and ap2/erf, accounted for a full % of the overall transcription factor expression. in addition, bhlh, myb and nac families were found in turn. these five families occupied approximately % of expressed transcription factors. however, five families of transcription factors including lfy, m-type, stat, voz, and wox were not detected. the few families that could not be found might result from inadequate sampling of the transcriptome or genes that are truly rarely expressed. large-scale efforts in deeper sampling and sequencing of the transcriptome will help completely identify genes related to of transcription factor families in orchids. discussion as the most species rich and diversified family, orchidaceae has mesmerized botanists for centuries. as for most other non-model plant species, we lack genetic and genomic resources for molecular biological study. although a precise estimate of transcriptome coverage is unattainable without the full genomic sequence, the massively parallel pyrosequencing characterization can be considered an initial step for functional genomics studies in phalaenopisis. from sequence reads, we assembled data for unigenes consisting of contigs and singletons from phalaenopsis. although a high number of transcripts are short-length reads which may result in several assembled contigs and singletons for each gene, the dataset we report here still provides a plentiful dataset of different genes representing a substantial part of the transcriptome of orchids, which in turn reflects these plants' sophisticated designs for successful pollination, reproduction and adaptation to the environment. homology searches showed that % of the ests have no significant similarities to any other protein sequences in public databases. about % of these ests are < bp, indicating that the short size has a negative effect on successful annotation. however, these genes may perform specific roles in orchids and be quite divergent from those of other plant species. the orchids, indeed, have diverse specialized reproductive and ecological strategies for adaptive radiation. on the other hand, we could not reliably annotate a high proportion of unigenes lacking assignment of a putative function because they did not cover the full length of the transcript or because they represent untranslated regions. comparing the distributions of the functional categories among ests provides support for the expression levels of the different gene classes. the transcripts with the first and third highest expression we found for phalaenopsis were homologous to the members of cytochrome p. plant cytochromes p catalyze a wide variety of monooxygenation/hydroxylation reactions in primary and secondary metabolism. genomic sequencing projects have revealed that cytochromes p genes represent approximately 1% of the total gene annotations for each plant species. in addition to revealing the highest transcript expression in phalaenopsis, the next-generation transcriptome sequencing also generated members of the cytochrome p family. the extraordinary expression level and remarkable diversification of this gene family may have led to the phalaenopsis orchid survival ability. the transcript with the second highest level of expression was homologous to triple gene block of cymmv. we also found genes with significant expression that were homologous to rna dependent rna polymerase, coat protein, triple gene block and triple gene block of cymmv. even though we found no substantial virus-infected symptoms in our samples, some of the experimental materials have been infected with virus prior to the sampling. we also found transcripts with high expression that were homologous to lla- derived from lily. this transcript is found in immature anther, tepal, pistil, stem and leaf in lily, however it has an as yet unknown function. dissection of function of these genes might be a useful direction for further study of orchid biology. the fact that whole-genome duplication often gives rise to species-rich groups of organisms, such as > species of asteraceae and > species of fabaceae, highlights that polyploidy can facilitate diversification and speciation of organisms. the orchidaceae contains more than species and has successfully colonized almost every habitat on earth. whole-genome duplication may also have occurred in the orchid genome. based on the results from analyzing how many subfamilies exist with very high number of ests, we suggested gene duplication probably have occurred on these genes. however, these gene duplication events may be caused by whole-genome duplication, tandem gene duplication or segmental duplication. only after completeness of whole-genome sequencing of phalaenopsis has been performed, it will be possible to differentiate whole-genome duplications from segmental and tandem duplications by mapping chromosome locations of the duplicated genes or blocks of genes. to evaluate whether the sequences annotated in this study include all genes expressed in these tissues, developmental stages, and treatments, we searched for a number of genes involved in metabolic pathways and homologous to members characterized in arabidopsis gene families. the genes associated with metabolic pathways were based on the kegg database of pathways and those for gene family on the tair database of arabidopsis proteome. the rationale for these searches was that those essential genes must be expressed to maintain cellular functions, so failure to find these sequences in the transcriptome would reflect either inadequate sequencing depth or ineffective annotation. for the pathways considered here, essentially all genes involved in the pathways were found except those involved in anthocyanin biosynthesis. for the gene families in arabidopsis, total unigenes were classified into gene families occupying more than 70% of arabidopsis gene families. a caution for these finding is that high levels of expression might be expected for some essential house-keeping genes, leading them to be well represented in even an incomplete transcriptome sequencing effort. to account for this possibility, we searched for genes associated with transcription factors. because of their more restricted spatial and temporal expression profiles, transcription factor genes are not expected to be as highly expressed as essential house-keeping genes in whole-organism libraries. we successfully identified genes from nearly all the transcription factor families considered. the few genes that could not be found might result from incomplete annotation or inadequate sampling of the transcriptome or they may be truly not expressed. overall, these searches support that the collection of annotated sequences we produced represents a reasonably broad description of the phalaenopsis transcriptome. these sequences of expressed transcripts will be very useful for genome annotation of phalaenopsis genome in the future. analysis of expression profiles of transcription factors in the transcriptome of phalaenopsis is meaningful because these are master-control proteins in all living cells. surprisingly, we found that c3h and ap2/erf families together represented more than 30% of expression of phalaenopsis transcription factors. the c3h family has been reported to be involved in arabidopsis embryogenesis, shoot apical meristem maintenance, drought tolerance, and response to abscisic acid in craterostigma plantagineum. the ap2/erf superfamily is defined by the ap2/erf domain, of about to amino acids, and is involved in dna binding. a combination of genetic and molecular approaches has been used to characterize a series of regulatory genes of the ap2/erf family. the members of this family are involved in regulating various biological processes related to growth and development, as well as various responses to environmental stimuli. this family includes genes related to drought, high salt concentration, low temperature, diseases, and the control of ovule development and flower organ growth. understanding the functions of these genes will advance our understanding of the great morphological diversity and successful adaptation of orchids. however, we did not find the transcription factor families lfy, m-type, stat, voz, and wox, in the phalaenopsis transcriptome. these families might either be rarely expressed, or might not have appeared in our cdna sampling.  thanks to recent advances in next-generation sequencing technology, we have applied rna-seq to facilitate transcriptome analysis of orchids which present important biological questions but lack a fully sequenced genome. our findings represent substantial contributions to the publicly accessible expressed sequences for the orchidaceae family. with the whole genome sequencing of p. equestris in progress, this collection of ests is a valuable resource that will be immediately useful for researchers, allowing for correction of assemblies, annotation, and construction of gene models to establish accurate exon-intron boundaries. application of these resources through the common language of nucleotide sequences will greatly enhance the insights into the reproductive success of orchids. 
the informative est dataset we developed constitutes a much-needed resource for discovery of genes involved in various biological processes in phalaenopsis and other orchid species. these transcribed sequences will narrow the gap between study of model organisms with many genomic resources and species that are important for ecological and evolutionary studies. orchids are one of the most diversified angiosperms, but few genomic resources are available for these non-model plants. among these annotations, over unigenes encoding putative transcription factors were identified. based on sequence similarity with known proteins, these analyses identified different genes. we aimed to use massively parallel pyrosequencing for a global characterization of the phalaenopsis transcriptome. assembled sequences were annotated with gene ontology, gene family and kyoto encyclopedia of genes and genomes pathways.
 orchids are one of the most diversified angiosperms, but few genomic resources are available for these non-model plants. in addition to the ecological significance, phalaenopsis has been considered as an economically important floriculture industry worldwide. we aimed to use massively parallel pyrosequencing for a global characterization of the phalaenopsis transcriptome.  to maximize sequence diversity, we pooled rna from samples of different tissues, various developmental stages, and biotic- or abiotic-stressed plants. we obtained expressed sequence tags with an average read length of bp. these reads were assembled into contigs and singletons. the unigenes were searched against the ncbi non-redundant protein database. based on sequence similarity with known proteins, these analyses identified different genes. assembled sequences were annotated with gene ontology, gene family and kyoto encyclopedia of genes and genomes pathways. among these annotations, over unigenes encoding putative transcription factors were identified.  pyrosequencing was effective in identifying a large set of unigenes from phalaenopsis. the informative est dataset we developed constitutes a much-needed resource for discovery of genes involved in various biological processes in phalaenopsis and other orchid species. these transcribed sequences will narrow the gap between study of model organisms with many genomic resources and species that are important for ecological and evolutionary studies. 