BACKGROUND
conifers  have very large genomes , and this poses difficulties for both structural and functional genomic studies.
in addition, their generation times are long and their habitual out-breeding nature prevents the development of inbred strains useful for genetics research.
a further difficulty in conifer genomics is the large evolutionary distance between conifers and angiosperms , separated by  <dig> million years of evolution  <cit> , which severely restricts gene comparisons of conifers with angiosperms.
while there are several completely sequenced angiosperm genomes, as well as high-quality sequence-finished full-length cdna resources, for arabidopsis  <cit> , rice  <cit> , poplar , grapevine , and a moss , these basic genomics resources have not yet been developed for the conifer phyla or for any other gymnosperm.
in species with large genomes, a critical first step for genome characterization is to survey the expressed genes.
a common approach to characterize the expressed genome is to sequence cdna libraries and to assemble large collections of expressed sequence tags   <cit> .
in the absence of a conifer genome sequence, large and deep est collections are particularly useful.
sequencing of cdna libraries constructed from diverse tissues and developmental stages, and from materials subjected to diverse environmental conditions or treatments, enhances the diversity of genes captured in est populations.
in addition, normalization techniques reduce the frequency of highly expressed genes and increase the rate of rare gene discovery  <cit> , thus providing more comprehensive coverage of the expressed genome.
in conifers, gene discovery via est sequencing was first conducted in loblolly pine , the most economically important tree species in the southeastern usa.
the early emphasis in loblolly pine was on wood forming tissues  <cit> , but newer projects have involved treatments such as drought stress  <cit>  and embryogenesis  <cit> .
as of may  <dig>  the loblolly pine est collection contains more than  <dig>  sequences  <cit> .
recent est projects with species of spruce have used tissues related to shoot growth and xylem development in white spruce  <cit> , wound treatment in interior spruce  <cit> , root development in sitka spruce  <cit> , and xylem development and bud burst in norway spruce .
est resources have also been developed for a few other gymnosperm species outside of the pine family, such as cycas , ginkgo , japanese yew , japanese cedar  and hinoki cypress .
in addition to deep est sampling, other important components of a cdna sequence resource are the quality and length of sequence coverage for a given gene.
ideally, flcdna clones that capture the entire mature transcript of a gene should be identified and completely sequenced with high accuracy.
flcdna sequences should span not only the protein-coding open reading frame  region but also the non-coding 5' and 3' untranslated regions .
most importantly, true flcdna sequences should be derived from a single individual flcdna clone.
using individual clones prevents the assembly of chimeric flcdna sequences consisting of ests from multiple cdna clones representing closely related genes.
furthermore, allelic nucleotide polymorphisms and alternatively spliced variants of a gene are difficult to detect using in silico assembled sequence contigs from multiple clones.
to further discriminate among closely related genes, the authenticity of sequences should be verified by re-sequencing of the same clone .
compared to single-pass ests or in silico assembled sequence contigs originating from multiple clones, sequence-verified flcdna clones offer several advantages for comparative, structural, and functional genome analyses, in particular for conifers with their great evolutionary distance from angiosperms.
first, the complete protein-coding regions of flcdnas can be unambiguously identified.
an accurate prediction of full-length protein sequences aides in the correct identification of distant angiosperm homologues.
second, in anticipation of a future conifer genome sequence, flcdnas can be used to improve gene prediction from genomic sequences as demonstrated in arabidopsis  <cit>  and poplar  <cit> .
third, flcdna clones can be used for functional characterization of conifer genes using biochemical approaches  or for functional complementation of mutants in heterologous systems.
given the lack of knock-out mutants in conifers and the slow process of generating knock-down mutants in conifers, biochemical approaches and heterologous complementation that rely on flcdna clones are essential tools for functional genomics in conifers.
finally, flcdnas can be used to accurately identify peptides in large-scale conifer proteome analyses  <cit> .
despite their immense value, sequence-verified flcdna clones have not been generated in most plant species subjected to genome analysis.
only a few resources of large and sequence-verified flcdna data sets have been generated for angiosperm plant species; namely, for arabidopsis  <cit> , rice  <cit> , and poplar  <cit> .
in contrast, no substantial flcdna resource has been reported for a conifer or any other gymnosperm species.
the conifer forest health genomics project "treenomix"  <cit>  aims to develop genomic resources for spruce, characterize mechanisms of resistance against insect pests and adaptation to local environments, and identify genes for the formation of oleoresin-based terpenoid biomaterials  <cit> .
here, we report on a comprehensive spruce est and flcdna resource and discuss its utility for conifer genomics.
a total of  <dig>  ests were obtained by sequencing  <dig> standard, normalized or full-length cdna libraries derived from sitka spruce, white spruce, and interior spruce.
analysis of ests identified  <dig>  putative unique transcripts .
we describe advantages covered by the first large set of  <dig>  sequence-verified, high-quality flcdnas obtained from a single clonally propagated tree of sitka spruce.
RESULTS
sequencing and assembly of spruce ests
we constructed  <dig> unidirectional standard, normalized or full-length enriched cdna libraries from various tissues, developmental stages, and stress treatments of sitka spruce, white spruce and interior spruce .
several libraries were made from trees subjected to insect feeding by white pine weevils  or spruce budworms , or to herbivory-simulation treatments such as mechanical wounding or methyl jasmonate application.
from these libraries, we obtained  <dig>  est sequences, consisting of  <dig>  3'-end est sequences and  <dig>  5'-end est sequences .
we initially focused on 3'-end sequencing.
subsequent sequence reads from 5'-ends were performed as paired end reads, primarily from clones derived from flcdna libraries, to support the identification of a non-redundant flcdna set for complete insert sequencing.
removing low-quality and vector sequences , as well as any obvious contaminant sequences, provided a database containing  <dig>  high-quality  3' ests  with an average read length of  <dig> bp .
when we analyzed the  <dig>  hq 3'-end ests using the cap <dig> program ,  <dig>  ests assembled into  <dig>  contigs and the remaining  <dig>  ests were classified as singletons, suggesting a combined total of  <dig>  puts across sitka spruce, white spruce and interior spruce .
on average, contigs contained six assembled est sequences.
only  <dig> contigs consisted of greater than  <dig> ests.
the five largest contigs contain  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  ests.
the proportion of est sequences from organelles was small.
known and putative mitochondrial and chloroplast sequences contribute only  <dig>  and  <dig>  ests to the entire data set, respectively.
in separate species-specific assemblies using ests from only white spruce or sitka spruce, we identified  <dig>  puts  and  <dig>  puts , respectively.
astandard cdna library; bnormalized cdna library; cfull-length cdna library; dfield site located at kalamalka research station in vernon, british columbia; eone- or two-year old trees grown in potted soil under greenhouse conditions at the university of british columbia
ahigh-quality  sequences only.
ba sequence is considered of hq if it is not derived from contaminant species and its vector-trimmed and poor-quality-trimmed phred  <dig> length is > <dig> bases.
ca contig  contains two or more ests; 3' sequences only.
dnumber of putative unique transcripts  among assembled 3' ests equals the number of contigs plus the number of singletons.
ethreshold for blastx significance versus the non-redundant  database of genbank is a score value >  <dig>
gene discovery in normalized and non-normalized cdna libraries
from each of the  <dig> cdna libraries, between  <dig>  and  <dig>  clones were 3'-end sequenced, with the rate of hq sequences ranging from  <dig> % to  <dig> % and an average est length of  <dig> bp to  <dig> bp in each library .
the rate of gene discovery for each library was assessed from:  the number of unique transcripts sequenced from each library;  the average number of est sequences forming contigs;  the percentage of ests with no similarity to protein sequences in the non-redundant  database of genbank using blastx;  the percentage of singleton ests; and  the percentage of library-specific transcripts.
based on these criteria, all but two of the normalized libraries  showed considerably higher rates of gene discovery, and hence higher complexity, than the corresponding non-normalized libraries .
for example, among the six successfully normalized est libraries, the percentage of unique transcripts identified within the first  <dig>  reads averaged  <dig> % , whereas among the seven corresponding standard est libraries made from the same rna samples, the average was only  <dig> % .
the diversity of starting biological materials combined with normalization resulted in low sequence redundancy demonstrated by the presence of only three puts  sequenced in all of the  <dig> cdna libraries .
these three transcripts were identified as translationally controlled tumor protein , eukaryotic translation initiation factor 5a  and s-adenosylmethionine synthase .
quality assessment of flcdnas
flcdnas are defined as individual cdna clones that contain the complete orf coding sequence as well as at least partial 5' and 3' utrs for a given transcript.
we prepared three flcdna libraries using the biotinylated cap trapper method  <cit> .
all flcdna libraries were made from insect-induced tissues of a single sitka spruce genotype .
from these libraries, we identified  <dig>  cdna candidate clones for complete insert sequencing, which resulted in  <dig>  hq sequence-verified flcdna clones .
analysis of the  <dig>  flcdna sequences using the cap <dig> program  identified  <dig>  flcdnas as singletons, with the remaining  <dig>  grouping into  <dig> contigs, suggesting a total of  <dig>  puts represented with finished flcdna sequences.
the high rate  of unique transcript discovery resulted from a successful strategy for selection of a low-redundancy flcdna clone set prior to sequence finishing .
all  <dig>  sequence-verified flcdnas achieved a minimum of phred <dig> sequence quality at every base .
the majority were of even higher quality with the minimum and average quality values exceeding phred <dig>  and phred <dig> , respectively .
we predicted the complete protein-coding orfs for all  <dig>  flcdnas .
the average sequenced flcdna length  was  <dig>  ±  <dig> bp , and ranged from  <dig> to  <dig>  bp, whereas the average predicted orf was  <dig> ±  <dig> bp and ranged from  <dig> to  <dig>  bp .
orfs could not be detected  for  <dig> flcdnas.
the 5' and 3' utrs averaged  <dig> ±  <dig> bp and  <dig> ±  <dig> bp, respectively .
to further assess the quality of the flcdnas, we performed reciprocal blast analysis using  <dig> known fl sequences from other conifer and gymnosperm species identified in previous entries in the nr database of genbank.
using a stringent similarity threshold  we identified  <dig> pairs of sitka spruce and other gymnosperm flcdnas.
of these pairs,  <dig>  agreed well with regard to their orf lengths  and positions of their starting methionine and stop codons .
for the remaining pairs, the predicted 5' and/or 3' orf ends did not match, suggesting alternative start or stop codons, splice variants, or the possibility that one of the pair members was truncated or had an incorrectly predicted orf.
despite the relatively small number of other gymnosperm fl sequences available for pairwise comparison, the high sequence similarity within this dataset indicates that most of the  <dig>  flcdnas represent true fl transcripts with complete orfs and correctly annotated start and stop codons.
most spruce ests have low similarity with angiosperm sequences
since conifers and other gymnosperms are difficult experimental systems with few functionally characterized proteins, in silico annotation of spruce ests was performed against predicted peptides from sequenced genomes of four angiosperms  and the moss physcomitrella patens, together with all protein sequences in the nr database of genbank.
among hq 3'-end ests >  <dig> bases in length , between  <dig> % and  <dig> % have matches against each of the five plant genomes with a low stringency blastx score of >  <dig> .
using a more stringent threshold of score >  <dig>  between  <dig> % and  <dig> % of spruce 3'-end ests match peptides from each of the five plant genomes of this comparison.
blastx matches with hq 3'-end ests were slightly higher  when compared to the more comprehensive collection of proteins in the nr database .
similar results were obtained using the assembled contig set of  <dig>  spruce puts derived from 3'-end ests .
among hq 5'-end ests >  <dig> bases in length , sequence similarity with proteins predicted from the five plant genome sequences was higher compared to 3' ests and puts, with between  <dig> % and  <dig> %  and  <dig> % and  <dig> %  of 5'-end ests matching each of the plant genomes .
as observed with 3'-end ests and puts, an even higher proportion of 5'-end ests had blastx matches against the nr database .
these results illustrate the challenge of in silico annotation of conifer ests, even with hq sequences averaging >  <dig> bases in length.
we also compared the spruce ests and puts against ests from all gymnosperm species combined  using blastn.
as expected, sequence similarity between the spruce ests and published gymnosperm ests was high .
among puts , hq 3'-end and 5'-end ests >  <dig> bases in length,  <dig> %,  <dig> % and  <dig> %, respectively, have matches with scores >  <dig>  at higher blastn stringency levels , sequence matches for puts, 3'-end and 5'-end ests remain consistently high.
among those puts, 3'-end and 5'-end ests >  <dig> bases in length and with no obvious similarity to proteins from the five sequenced plant genomes ,  <dig> %,  <dig> %, and  <dig> %, respectively, have blastn scores >  <dig> versus published gymnosperm ests .
when the spruce ests are compared against published ests from white spruce and loblolly pine, the two gymnosperm species with the most substantial est collections, a higher proportion of puts, and 3'-end and 5'-end ests show sequence similarity to white spruce compared to loblolly pine, especially at the highest blastn threshold .
utility of spruce flcdnas for comparative sequence annotation
as might be expected, sequence similarity between the  <dig>  sitka spruce flcdnas and other gymnosperm ests is very high, with  <dig> %,  <dig> % and  <dig> % of flcdnas matching published gymnosperm ests at low, medium, and high sequence similarity thresholds, respectively .
as observed with spruce ests, sequence similarity was highest between spruce flcdnas and white spruce ests, with lower similarity observed with loblolly pine ests .
next, the spruce flcdnas were compared against predicted proteins from five plant genome sequences and protein sequences in the complete nr database of genbank.
at a low sequence similarity threshold of score >  <dig>  between  <dig> % and  <dig> % of flcdnas matched proteins from each of the plant genomes of this comparison, whereas at a higher threshold of score >  <dig> the percentages of flcdnas with matches in the plant genome sequences ranged from  <dig> % to  <dig> % .
overall, the sitka spruce flcdnas show greater similarity to predicted proteins from sequenced plant genomes compared to the spruce ests.
the proportion of spruce flcdnas with similarity to proteins in the nr database was also higher than spruce ests at  <dig> % and  <dig> % at score >  <dig> and score >  <dig>  respectively .
these results show that flcdnas provide a clear advantage over ests for large scale in silico annotation of spruce sequences.
nevertheless, when using high stringency criteria relevant for in silico functional annotation , the comparison of spruce flcdnas against the five plant genomes, as well as all plant species in the nr database, still identifies a substantial number of sequences that only show significant matches with other gymnosperms, as opposed to angiosperms.
among the  <dig>  spruce flcdnas, we found  <dig>  without a reliable match to angiosperm sequences at a low stringency , of which  <dig>  match with high sequence similarity  to a published gymnosperm est sequence .
a very small number of spruce flcdnas lack sequence similarity to angiosperm or gymnosperm sequences  and display a best match with non-plant species in the nr database of genbank;  <dig> % at score >  <dig> and  <dig> % at score >  <dig> .
in these cases, the best match is often an insect sequence suggesting small amounts of contaminants in the cdna libraries.
comparing the entire spruce flcdna dataset against sequences from all species identified that  <dig> %  or  <dig> %  have matches in all seven datasets  .
it is notable that at the higher threshold of score >  <dig>   <dig> % of spruce flcdnas match only to a single database, and in the vast majority of cases this is a gymnosperm sequence .
another  <dig> %  or  <dig> %  of spruce flcdna sequences do not align to any sequences in available databases.
these sequences could represent genes from spruce  that have not been sequenced before in any source.
discussion
spruce ests and flcdnas enhance conifer genomics resources
genomics research on conifers has been limited by the lack of a relevant gymnosperm reference genome sequence.
the very large size of conifer genomes , dominated by repetitive dna, has been a roadblock to a conifer genome sequence project.
furthermore, the phylogenetic distance between conifers and the well-studied angiosperms is more than  <dig> million years  <cit> , limiting the utility of angiosperm genome information for research in conifers.
to overcome these obstacles to conifer genome research, we have developed two new valuable components for the "conifer genomics toolbox".
first, we have assembled a large collection of high-quality, sequence-verified flcdna clones from sitka spruce, along with a corresponding database of in silico annotations .
these flcdnas are of very low redundancy.
they represent the third largest sequence-verified flcdna resource for any plant species, behind only rice  <cit>  and arabidopsis  <cit> , and are the only substantial flcdna resource for a conifer or any other gymnosperm.
second, we have added a large number of new est sequences to the public spruce est collection in genbank, along with corresponding databases of in silico annotations .
this resource, which was developed from sitka, white and interior spruce , substantially improves the size and quality of the previously described spruce est collections  <cit> .
the spruce est collection, along with the ests from loblolly pine  <cit> , is now one of the two largest est resources for any conifer species.
to enhance gene discovery, we strategically employed library normalization, which had previously not been applied to a conifer est program.
also, we have added sequences from an until now poorly represented class of tissues representing a biologically important component of conifer defense: insect-, wound- or elicitor-induced tissues.
we identified  <dig>  puts  in the three species groups surveyed here; sitka spruce, white spruce, and interior spruce.
the rates of put discovery for all species combined , white spruce only  and sitka spruce only  are comparable, as are the ratios of singletons to contigs in each collection.
among contigs from the combined analysis of white and sitka spruce ests,  <dig> % contained ests from both species, suggesting that ests derived from different spruce species representing the same spruce gene often cluster together.
the puts identified here may represent a substantial portion of the expressed gene catalogue for species of spruce, but a complete genome sequence is needed for assessment of true gene numbers in conifers.
the spruce ests described here have already provided the foundation for functional and comparative genomics research on conifer defense against insects, adaptation to the environment, somatic embryogenesis and wood formation, via both transcriptome and proteome analyses  <cit> .
they have also allowed development of three types of genetic markers: microsatellites  <cit> , single nucleotide polymorphisms  and conserved orthologous sequences   <cit> .
the flcdna sequences enable rigorous large-scale comparisons of evolutionary patterns at large evolutionary scales .
utility of spruce flcdnas for functional characterization of gene families including nearly identical paralogous genes
prior to this work, only a few dozen complete spruce protein sequences were available in the swissprot database, and no substantial flcdna resource was available for any gymnosperm.
using flcdnas, detailed pathway annotation, gene expression analysis, and biochemical functional characterization of individual genes and gene families are now possible .
the sitka spruce flcdnas have already advanced the discovery and the characterization of conifer defense genes  <cit> .
importantly, sitka spruce flcdnas allow for accurate analysis of closely related members of gene families such as cytochrome p450-dependent monooxygenases or terpenoid synthases  involved in defense against insects or pathogens  <cit> .
for example, tpss represent a gene family containing many pairs or groups of nearly identical paralogous genes each with a potentially different biochemical function  <cit> .
our recent mutational analysis of two closely related paralogous norway spruce di-tps illustrated that a single amino acid mutation in a background of more than  <dig> amino acids completely alters biochemical product profiles  <cit> .
similarly, in rice, the functional divergence of two distinct tps of primary and secondary metabolism was due to a single amino acid substitution  <cit> .
these examples illustrate the utility of true flcdnas for discovery of nearly identical paralogous genes and for functional assessment of gene evolution that is now possible in sitka spruce.
utility of flcdnas for conifer proteome and genome characterization
beyond their importance for functional characterization of individual genes and the analysis of gene families, on an even larger scale, flcdnas are also superior to ests for overall proteome and genome characterization in a conifer.
because the sitka spruce flcdnas allow for a much more reliable prediction of the complete protein-coding orf than ests, they have been invaluable for proteome predictions and practical proteome analyses  <cit> .
in expectation of future efforts to sequence a conifer genome, flcdnas and their orfs will be essential for the development and training of gene prediction software, as has recently been demonstrated for poplar  <cit> .
spruce flcdnas from insect-induced libraries reveal genes not detected in angiosperms
comparison of sitka spruce sequences against angiosperm plants suggests that there are likely a substantial number of genes in the collection of  <dig>  flcdnas that are either absent in other species, or lack significant sequence similarity for unambiguous identification.
in earlier work, kirst et al.
<cit>  suggested that less than 10% of loblolly pine transcripts lack a related gene in arabidopsis .
when we analyzed the spruce flcdnas, we found that approximately 14% had no similarity to any angiosperm at a blastx stringency of score  <dig> , based on comparisons to four sequenced angiosperm genomes and all angiosperm sequences in the nr database.
this slightly higher rate may be the result of sequencing libraries made from tissues induced by insect attack, which may disproportionally represent genes with specialized functions in conifer defense that are subject to high levels of natural selection due to biotic interaction.
by contrast, genes involved in xylem development and wood formation appear to be well conserved in angiosperms and conifers  <cit> .
CONCLUSIONS
the  <dig>  ests and  <dig>  flcdnas and the corresponding in silico annotated sequence databases provide a new and valuable genomics resource for species of spruce, as well as for gymnosperms in general.
our emphasis on flcdnas and ests from cdna libraries constructed from herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, gives a rich conifer est resource which also apparently contains a substantial number of transcripts with no obvious sequence similarity to known angiosperm sequences.
recent research has begun to fully realize the application of these est and flcdna sequences, and flcdna clones.

 conifers have very large genomes, and this poses difficulties for both structural and functional genomic studies. in addition, their generation times are long and their habitual out-breeding nature prevents the development of inbred strains useful for genetics research. a further difficulty in conifer genomics is the large evolutionary distance between conifers and angiosperms, separated by million years of evolution, which severely restricts gene comparisons of conifers with angiosperms. while there are several completely sequenced angiosperm genomes, as well as high-quality sequence-finished full-length cdna resources, for arabidopsis, rice, poplar, grapevine, and a moss, these basic genomics resources have not yet been developed for the conifer phyla or for any other gymnosperm. in species with large genomes, a critical first step for genome characterization is to survey the expressed genes. a common approach to characterize the expressed genome is to sequence cdna libraries and to assemble large collections of expressed sequence tags. in the absence of a conifer genome sequence, large and deep est collections are particularly useful. sequencing of cdna libraries constructed from diverse tissues and developmental stages, and from materials subjected to diverse environmental conditions or treatments, enhances the diversity of genes captured in est populations. in addition, normalization techniques reduce the frequency of highly expressed genes and increase the rate of rare gene discovery, thus providing more comprehensive coverage of the expressed genome. in conifers, gene discovery via est sequencing was first conducted in loblolly pine, the most economically important tree species in the southeastern usa. the early emphasis in loblolly pine was on wood forming tissues, but newer projects have involved treatments such as drought stress and embryogenesis. as of may the loblolly pine est collection contains more than sequences. recent est projects with species of spruce have used tissues related to shoot growth and xylem development in white spruce, wound treatment in interior spruce, root development in sitka spruce, and xylem development and bud burst in norway spruce. est resources have also been developed for a few other gymnosperm species outside of the pine family, such as cycas, ginkgo, japanese yew, japanese cedar and hinoki cypress. in addition to deep est sampling, other important components of a cdna sequence resource are the quality and length of sequence coverage for a given gene. ideally, flcdna clones that capture the entire mature transcript of a gene should be identified and completely sequenced with high accuracy. flcdna sequences should span not only the protein-coding open reading frame region but also the non-coding 5' and 3' untranslated regions. most importantly, true flcdna sequences should be derived from a single individual flcdna clone. using individual clones prevents the assembly of chimeric flcdna sequences consisting of ests from multiple cdna clones representing closely related genes. furthermore, allelic nucleotide polymorphisms and alternatively spliced variants of a gene are difficult to detect using in silico assembled sequence contigs from multiple clones. to further discriminate among closely related genes, the authenticity of sequences should be verified by re-sequencing of the same clone. compared to single-pass ests or in silico assembled sequence contigs originating from multiple clones, sequence-verified flcdna clones offer several advantages for comparative, structural, and functional genome analyses, in particular for conifers with their great evolutionary distance from angiosperms. first, the complete protein-coding regions of flcdnas can be unambiguously identified. an accurate prediction of full-length protein sequences aides in the correct identification of distant angiosperm homologues. second, in anticipation of a future conifer genome sequence, flcdnas can be used to improve gene prediction from genomic sequences as demonstrated in arabidopsis and poplar. third, flcdna clones can be used for functional characterization of conifer genes using biochemical approaches or for functional complementation of mutants in heterologous systems. given the lack of knock-out mutants in conifers and the slow process of generating knock-down mutants in conifers, biochemical approaches and heterologous complementation that rely on flcdna clones are essential tools for functional genomics in conifers. finally, flcdnas can be used to accurately identify peptides in large-scale conifer proteome analyses. despite their immense value, sequence-verified flcdna clones have not been generated in most plant species subjected to genome analysis. only a few resources of large and sequence-verified flcdna data sets have been generated for angiosperm plant species; namely, for arabidopsis, rice, and poplar. in contrast, no substantial flcdna resource has been reported for a conifer or any other gymnosperm species. the conifer forest health genomics project "treenomix" aims to develop genomic resources for spruce, characterize mechanisms of resistance against insect pests and adaptation to local environments, and identify genes for the formation of oleoresin-based terpenoid biomaterials. here, we report on a comprehensive spruce est and flcdna resource and discuss its utility for conifer genomics. a total of ests were obtained by sequencing standard, normalized or full-length cdna libraries derived from sitka spruce, white spruce, and interior spruce. analysis of ests identified putative unique transcripts. we describe advantages covered by the first large set of sequence-verified, high-quality flcdnas obtained from a single clonally propagated tree of sitka spruce.  sequencing and assembly of spruce ests we constructed unidirectional standard, normalized or full-length enriched cdna libraries from various tissues, developmental stages, and stress treatments of sitka spruce, white spruce and interior spruce. several libraries were made from trees subjected to insect feeding by white pine weevils or spruce budworms, or to herbivory-simulation treatments such as mechanical wounding or methyl jasmonate application. from these libraries, we obtained est sequences, consisting of 3'-end est sequences and 5'-end est sequences. we initially focused on 3'-end sequencing. subsequent sequence reads from 5'-ends were performed as paired end reads, primarily from clones derived from flcdna libraries, to support the identification of a non-redundant flcdna set for complete insert sequencing. removing low-quality and vector sequences, as well as any obvious contaminant sequences, provided a database containing high-quality 3' ests with an average read length of bp. when we analyzed the hq 3'-end ests using the cap program, ests assembled into contigs and the remaining ests were classified as singletons, suggesting a combined total of puts across sitka spruce, white spruce and interior spruce. on average, contigs contained six assembled est sequences. only contigs consisted of greater than ests. the five largest contigs contain,,, and ests. the proportion of est sequences from organelles was small. known and putative mitochondrial and chloroplast sequences contribute only and ests to the entire data set, respectively. in separate species-specific assemblies using ests from only white spruce or sitka spruce, we identified puts and puts, respectively. astandard cdna library; bnormalized cdna library; cfull-length cdna library; dfield site located at kalamalka research station in vernon, british columbia; eone- or two-year old trees grown in potted soil under greenhouse conditions at the university of british columbia ahigh-quality sequences only. ba sequence is considered of hq if it is not derived from contaminant species and its vector-trimmed and poor-quality-trimmed phred length is > bases. ca contig contains two or more ests; 3' sequences only. dnumber of putative unique transcripts among assembled 3' ests equals the number of contigs plus the number of singletons. ethreshold for blastx significance versus the non-redundant database of genbank is a score value > gene discovery in normalized and non-normalized cdna libraries from each of the cdna libraries, between and clones were 3'-end sequenced, with the rate of hq sequences ranging from % to % and an average est length of bp to bp in each library. the rate of gene discovery for each library was assessed from: the number of unique transcripts sequenced from each library; the average number of est sequences forming contigs; the percentage of ests with no similarity to protein sequences in the non-redundant database of genbank using blastx; the percentage of singleton ests; and the percentage of library-specific transcripts. based on these criteria, all but two of the normalized libraries showed considerably higher rates of gene discovery, and hence higher complexity, than the corresponding non-normalized libraries. for example, among the six successfully normalized est libraries, the percentage of unique transcripts identified within the first reads averaged %, whereas among the seven corresponding standard est libraries made from the same rna samples, the average was only %. the diversity of starting biological materials combined with normalization resulted in low sequence redundancy demonstrated by the presence of only three puts sequenced in all of the cdna libraries. these three transcripts were identified as translationally controlled tumor protein, eukaryotic translation initiation factor 5a and s-adenosylmethionine synthase. quality assessment of flcdnas flcdnas are defined as individual cdna clones that contain the complete orf coding sequence as well as at least partial 5' and 3' utrs for a given transcript. we prepared three flcdna libraries using the biotinylated cap trapper method. all flcdna libraries were made from insect-induced tissues of a single sitka spruce genotype. from these libraries, we identified cdna candidate clones for complete insert sequencing, which resulted in hq sequence-verified flcdna clones. analysis of the flcdna sequences using the cap program identified flcdnas as singletons, with the remaining grouping into contigs, suggesting a total of puts represented with finished flcdna sequences. the high rate of unique transcript discovery resulted from a successful strategy for selection of a low-redundancy flcdna clone set prior to sequence finishing. all sequence-verified flcdnas achieved a minimum of phred sequence quality at every base. the majority were of even higher quality with the minimum and average quality values exceeding phred and phred, respectively. we predicted the complete protein-coding orfs for all flcdnas. the average sequenced flcdna length was ± bp, and ranged from to bp, whereas the average predicted orf was ± bp and ranged from to bp. orfs could not be detected for flcdnas. the 5' and 3' utrs averaged ± bp and ± bp, respectively. to further assess the quality of the flcdnas, we performed reciprocal blast analysis using known fl sequences from other conifer and gymnosperm species identified in previous entries in the nr database of genbank. using a stringent similarity threshold we identified pairs of sitka spruce and other gymnosperm flcdnas. of these pairs, agreed well with regard to their orf lengths and positions of their starting methionine and stop codons. for the remaining pairs, the predicted 5' and/or 3' orf ends did not match, suggesting alternative start or stop codons, splice variants, or the possibility that one of the pair members was truncated or had an incorrectly predicted orf. despite the relatively small number of other gymnosperm fl sequences available for pairwise comparison, the high sequence similarity within this dataset indicates that most of the flcdnas represent true fl transcripts with complete orfs and correctly annotated start and stop codons. most spruce ests have low similarity with angiosperm sequences since conifers and other gymnosperms are difficult experimental systems with few functionally characterized proteins, in silico annotation of spruce ests was performed against predicted peptides from sequenced genomes of four angiosperms and the moss physcomitrella patens, together with all protein sequences in the nr database of genbank. among hq 3'-end ests > bases in length, between % and % have matches against each of the five plant genomes with a low stringency blastx score of >. using a more stringent threshold of score > between % and % of spruce 3'-end ests match peptides from each of the five plant genomes of this comparison. blastx matches with hq 3'-end ests were slightly higher when compared to the more comprehensive collection of proteins in the nr database. similar results were obtained using the assembled contig set of spruce puts derived from 3'-end ests. among hq 5'-end ests > bases in length, sequence similarity with proteins predicted from the five plant genome sequences was higher compared to 3' ests and puts, with between % and % and % and % of 5'-end ests matching each of the plant genomes. as observed with 3'-end ests and puts, an even higher proportion of 5'-end ests had blastx matches against the nr database. these results illustrate the challenge of in silico annotation of conifer ests, even with hq sequences averaging > bases in length. we also compared the spruce ests and puts against ests from all gymnosperm species combined using blastn. as expected, sequence similarity between the spruce ests and published gymnosperm ests was high. among puts, hq 3'-end and 5'-end ests > bases in length, %, % and %, respectively, have matches with scores > at higher blastn stringency levels, sequence matches for puts, 3'-end and 5'-end ests remain consistently high. among those puts, 3'-end and 5'-end ests > bases in length and with no obvious similarity to proteins from the five sequenced plant genomes, %, %, and %, respectively, have blastn scores > versus published gymnosperm ests. when the spruce ests are compared against published ests from white spruce and loblolly pine, the two gymnosperm species with the most substantial est collections, a higher proportion of puts, and 3'-end and 5'-end ests show sequence similarity to white spruce compared to loblolly pine, especially at the highest blastn threshold. utility of spruce flcdnas for comparative sequence annotation as might be expected, sequence similarity between the sitka spruce flcdnas and other gymnosperm ests is very high, with %, % and % of flcdnas matching published gymnosperm ests at low, medium, and high sequence similarity thresholds, respectively. as observed with spruce ests, sequence similarity was highest between spruce flcdnas and white spruce ests, with lower similarity observed with loblolly pine ests. next, the spruce flcdnas were compared against predicted proteins from five plant genome sequences and protein sequences in the complete nr database of genbank. at a low sequence similarity threshold of score > between % and % of flcdnas matched proteins from each of the plant genomes of this comparison, whereas at a higher threshold of score > the percentages of flcdnas with matches in the plant genome sequences ranged from % to %. overall, the sitka spruce flcdnas show greater similarity to predicted proteins from sequenced plant genomes compared to the spruce ests. the proportion of spruce flcdnas with similarity to proteins in the nr database was also higher than spruce ests at % and % at score > and score > respectively. these results show that flcdnas provide a clear advantage over ests for large scale in silico annotation of spruce sequences. nevertheless, when using high stringency criteria relevant for in silico functional annotation, the comparison of spruce flcdnas against the five plant genomes, as well as all plant species in the nr database, still identifies a substantial number of sequences that only show significant matches with other gymnosperms, as opposed to angiosperms. among the spruce flcdnas, we found without a reliable match to angiosperm sequences at a low stringency, of which match with high sequence similarity to a published gymnosperm est sequence. a very small number of spruce flcdnas lack sequence similarity to angiosperm or gymnosperm sequences and display a best match with non-plant species in the nr database of genbank; % at score > and % at score >. in these cases, the best match is often an insect sequence suggesting small amounts of contaminants in the cdna libraries. comparing the entire spruce flcdna dataset against sequences from all species identified that % or % have matches in all seven datasets. it is notable that at the higher threshold of score > % of spruce flcdnas match only to a single database, and in the vast majority of cases this is a gymnosperm sequence. another % or % of spruce flcdna sequences do not align to any sequences in available databases. these sequences could represent genes from spruce that have not been sequenced before in any source. discussion spruce ests and flcdnas enhance conifer genomics resources genomics research on conifers has been limited by the lack of a relevant gymnosperm reference genome sequence. the very large size of conifer genomes, dominated by repetitive dna, has been a roadblock to a conifer genome sequence project. furthermore, the phylogenetic distance between conifers and the well-studied angiosperms is more than million years, limiting the utility of angiosperm genome information for research in conifers. to overcome these obstacles to conifer genome research, we have developed two new valuable components for the "conifer genomics toolbox". first, we have assembled a large collection of high-quality, sequence-verified flcdna clones from sitka spruce, along with a corresponding database of in silico annotations. these flcdnas are of very low redundancy. they represent the third largest sequence-verified flcdna resource for any plant species, behind only rice and arabidopsis, and are the only substantial flcdna resource for a conifer or any other gymnosperm. second, we have added a large number of new est sequences to the public spruce est collection in genbank, along with corresponding databases of in silico annotations. this resource, which was developed from sitka, white and interior spruce, substantially improves the size and quality of the previously described spruce est collections. the spruce est collection, along with the ests from loblolly pine, is now one of the two largest est resources for any conifer species. to enhance gene discovery, we strategically employed library normalization, which had previously not been applied to a conifer est program. also, we have added sequences from an until now poorly represented class of tissues representing a biologically important component of conifer defense: insect-, wound- or elicitor-induced tissues. we identified puts in the three species groups surveyed here; sitka spruce, white spruce, and interior spruce. the rates of put discovery for all species combined, white spruce only and sitka spruce only are comparable, as are the ratios of singletons to contigs in each collection. among contigs from the combined analysis of white and sitka spruce ests, % contained ests from both species, suggesting that ests derived from different spruce species representing the same spruce gene often cluster together. the puts identified here may represent a substantial portion of the expressed gene catalogue for species of spruce, but a complete genome sequence is needed for assessment of true gene numbers in conifers. the spruce ests described here have already provided the foundation for functional and comparative genomics research on conifer defense against insects, adaptation to the environment, somatic embryogenesis and wood formation, via both transcriptome and proteome analyses. they have also allowed development of three types of genetic markers: microsatellites, single nucleotide polymorphisms and conserved orthologous sequences. the flcdna sequences enable rigorous large-scale comparisons of evolutionary patterns at large evolutionary scales. utility of spruce flcdnas for functional characterization of gene families including nearly identical paralogous genes prior to this work, only a few dozen complete spruce protein sequences were available in the swissprot database, and no substantial flcdna resource was available for any gymnosperm. using flcdnas, detailed pathway annotation, gene expression analysis, and biochemical functional characterization of individual genes and gene families are now possible. the sitka spruce flcdnas have already advanced the discovery and the characterization of conifer defense genes. importantly, sitka spruce flcdnas allow for accurate analysis of closely related members of gene families such as cytochrome p450-dependent monooxygenases or terpenoid synthases involved in defense against insects or pathogens. for example, tpss represent a gene family containing many pairs or groups of nearly identical paralogous genes each with a potentially different biochemical function. our recent mutational analysis of two closely related paralogous norway spruce di-tps illustrated that a single amino acid mutation in a background of more than amino acids completely alters biochemical product profiles. similarly, in rice, the functional divergence of two distinct tps of primary and secondary metabolism was due to a single amino acid substitution. these examples illustrate the utility of true flcdnas for discovery of nearly identical paralogous genes and for functional assessment of gene evolution that is now possible in sitka spruce. utility of flcdnas for conifer proteome and genome characterization beyond their importance for functional characterization of individual genes and the analysis of gene families, on an even larger scale, flcdnas are also superior to ests for overall proteome and genome characterization in a conifer. because the sitka spruce flcdnas allow for a much more reliable prediction of the complete protein-coding orf than ests, they have been invaluable for proteome predictions and practical proteome analyses. in expectation of future efforts to sequence a conifer genome, flcdnas and their orfs will be essential for the development and training of gene prediction software, as has recently been demonstrated for poplar. spruce flcdnas from insect-induced libraries reveal genes not detected in angiosperms comparison of sitka spruce sequences against angiosperm plants suggests that there are likely a substantial number of genes in the collection of flcdnas that are either absent in other species, or lack significant sequence similarity for unambiguous identification. in earlier work, kirst et al. suggested that less than 10% of loblolly pine transcripts lack a related gene in arabidopsis. when we analyzed the spruce flcdnas, we found that approximately 14% had no similarity to any angiosperm at a blastx stringency of score, based on comparisons to four sequenced angiosperm genomes and all angiosperm sequences in the nr database. this slightly higher rate may be the result of sequencing libraries made from tissues induced by insect attack, which may disproportionally represent genes with specialized functions in conifer defense that are subject to high levels of natural selection due to biotic interaction. by contrast, genes involved in xylem development and wood formation appear to be well conserved in angiosperms and conifers.  the ests and flcdnas and the corresponding in silico annotated sequence databases provide a new and valuable genomics resource for species of spruce, as well as for gymnosperms in general. our emphasis on flcdnas and ests from cdna libraries constructed from herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, gives a rich conifer est resource which also apparently contains a substantial number of transcripts with no obvious sequence similarity to known angiosperm sequences. recent research has begun to fully realize the application of these est and flcdna sequences, and flcdna clones. 
sequence-finished full-length cdnas and large collections of expressed sequence tags are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation. our emphasis on capturing flcdnas and ests from cdna libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed. as part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed standard, normalized or full-length enriched cdna libraries from sitka spruce, white spruce, and interior spruce. the sitka spruce flcdnas represent the third largest sequence-verified flcdna resource for any plant species, behind only rice and arabidopsis, and the only substantial flcdna resource for a gymnosperm. members of the pine family, especially species of spruce and pine, dominate many of the world's temperate and boreal forests. in contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. sequence comparisons against five plant genomes and the non-redundant genbank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. we sequenced and analyzed 3'- or 5'-end ests from these libraries, and developed a resource of high-quality sequence-finished flcdnas from sitka spruce. this paper provides detailed annotation and quality assessment of a large est and flcdna resource for spruce. the flcdnas were all obtained from a single sitka spruce genotype and represent puts. clustering and assembly of 3'-end ests resulted in contigs and singletons, representing putative unique transcripts.
 members of the pine family, especially species of spruce and pine, dominate many of the world's temperate and boreal forests. these conifer forests are of critical importance for global ecosystem stability and biodiversity. they also provide the majority of the world's wood and fiber supply and serve as a renewable resource for other industrial biomaterials. in contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. sequence-finished full-length cdnas and large collections of expressed sequence tags are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation.  as part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed standard, normalized or full-length enriched cdna libraries from sitka spruce, white spruce, and interior spruce. we sequenced and analyzed 3'- or 5'-end ests from these libraries, and developed a resource of high-quality sequence-finished flcdnas from sitka spruce. clustering and assembly of 3'-end ests resulted in contigs and singletons, representing putative unique transcripts. the flcdnas were all obtained from a single sitka spruce genotype and represent puts.  this paper provides detailed annotation and quality assessment of a large est and flcdna resource for spruce. the sitka spruce flcdnas represent the third largest sequence-verified flcdna resource for any plant species, behind only rice and arabidopsis, and the only substantial flcdna resource for a gymnosperm. our emphasis on capturing flcdnas and ests from cdna libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. sequence comparisons against five plant genomes and the non-redundant genbank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed. 