BACKGROUND
in non-model species with large genome size, est  sequencing and their annotation can provide the first step towards understanding the transcriptome and expression patterns of specific genes, which can complement the whole genome sequencing, and can assist with genome sequence annotation.
traditionally, est sequencing was conducted with the sanger sequencing system  <cit> .
more recently, next-generation sequencing  platforms have been used to generate enormous amounts of genome and transcriptome sequences  <cit> .
ngs methods facilitate whole transcriptome sequencing at a fraction of the time and cost previously required for the sanger method  <cit> .
however, commonly used ngs platforms produce shorter reads and/or reduce the quality per base call  <cit> .
the improved length and accuracy of reads obtained from sanger sequencing can complement ngs workflows.
this technology can assist in validating the ngs platform sequences by serving as a reference by which short reads can be aligned and corrected  <cit> .
therefore, est sequences derived from the sanger method are still a valuable resource in the ngs era.
conifers have a large genome size  and are ecologically and economically important, long-lived plants.
they form a major part of the northern boreal and temperate forests, which constitute the major biome of the world.
the genus pinus  and picea  are two important genera among conifers.
black spruce  b.s.p.)
is a widely-distributed transcontinental species of the north american boreal and temperate forests with high ecological and economic importance  <cit> .
black spruce is one of the most important softwood species for the production of pulp and paper in canada  <cit> .
it is an early successional species and has a corresponding suite of species-specific life history, growth, eco-physiological and adaptive traits  <cit> .
the estimated haploid genome size of black spruce is about  <dig>  gbp, with 1c contents of  <dig>  pg  <cit> .
as of, january 1st,  <dig> dbest release , there were approximately  <dig>  million ests from  <dig>  species available in the genbank at the national centre for biotechnology information   <cit> .
in conifers, the major est contributing species are loblolly pine - pinus taeda , followed by white spruce - picea glauca  and sitka spruce - picea sitchensis .
among spruce species, white spruce has the maximum number of ests, followed by p. sitchensis and p. engelmannii x p. sitchensis.
recently  <dig>  unique cdna clusters  have also been reported for p. glauca <cit> .
also, very recently, draft genomes of norway spruce  and white spruce have been published  <cit> .
however, the black spruce transcriptome is not yet fully characterized and only  <dig> ests and  <dig> cdna sequences are reported within the ncbi’s dbest .
due to a number of life history, morphological, adaptive, eco-physiological, and insect resistance traits and phylogenetic differences of black spruce from white spruce and sitka spruce  <cit> , we expect some unique genes in the black spruce transcriptome.
black spruce is an early successional shade-intolerant species whereas white spruce and sitka spruce are late successional shade-tolerant species.
black spruce can grow in poor conditions, such as bogs, whereas white spruce grows on well-drained soils.
these species-specific traits affirm a need to sequence and characterize the black spruce transcriptome.
much of the est sequencing in conifers has been performed using wood forming tissues and secondary xylem due to the economic importance of wood  <cit> .
there are a number of studies that have sequenced transcripts from needle tissues, including: lodgepole pine   <cit> , sugar pine   <cit> , loblolly pine  <cit> , maritime pine   <cit>  and norway spruce  <cit> .
these studies have provided some basis for the genes expressed in needle tissue; however, more comparative work is needed to understand their role in important metabolic pathways, such as photosynthesis.
the objective of our study was to develop a black spruce transcript resource, and thus, facilitate structural and functional spruce genomics projects related to growth and adaptation.
here, we report the results of the first est sequencing project from black spruce in which cdna clones were isolated and sequenced from a standard cdna library constructed from needle tissues in  <dig>  we conducted bidirectional sanger sequencing of ests to produce high quality, long reads to assist with the identification of full-length genes.
we assembled ests into contigs and singletons, and subsequently performed comparative protein annotations with the non-redundant  protein database and unigene clusters available at ncbi for model plant species.
we further conducted nucleotide similarity analysis with est sequences available from all major plant species , as well as species-specific sequences from various gymnosperms and angiosperms.
gene ontology terms were assigned and the ests were manually evaluated for specific categories, including transcription factors and photosynthetic genes.
finally we used black spruce est data for the detection of simple sequence repeats .
methods
plant material and cdna library construction
total rna was extracted from 2 g of freshly growing needles of three different black spruce seedlings established in the greenhouse at dalhousie university, following the protocol described in  <cit> .
quality and quantity of the isolated rna were determined using a spectrophotometer  and extracted rna was found to be of high quality .
the quantity of isolated rna was approximately 120 μg per g of the needle tissue used.
the polya rna was purified using rneasy mini kit  following the manufacturer’s instructions.
the cdna library was constructed using creator smart cdna library construction kit .
the oligo dt primed cdna inserts were directionally cloned in pdnr-lib vector and transformed using xl- <dig> gold ultra-competent cells of escherichia coli.
plasmid dna was isolated from the transformed white colonies selected from the overnight grown cells on luria broth agar plates containing chloramphenicol  using qiaprep spin miniprep kit .
the quality and quantity of the isolated plasmid dna was confirmed on  <dig> % agarose gels with known amount of lambda dna before sequencing.
cdna sequencing
sequencing reactions were performed in a ptc- <dig> thermal cycler  using the thermosequenase fluorescent labeling primer cycle sequencing kit with 7-deaza dgtp  according to the manufacturer’s instructions.
the sequencing products were resolved on a li-cor 4200 l sequencing system .
a total of  <dig>  cdna clones were sequenced in both directions using ird labeled m13f  and m13r  primers.
preliminary sequence processing
processing of raw trace files was performed through the customized treegenes est pipeline  <cit> .
base-calling and quality-assignment of the sequences were conducted with phred   <cit> .
low quality bases below phred <dig> were masked and vector sequences were trimmed from the ends.
the cross_match program was used for this purpose with minmatch  <dig> and minscore  <dig>  <cit> .
sequences with less than  <dig> high-quality bases  after trimming and sequences with polya tails of ≥  <dig> bases were removed from the analysis.
the resulting sequence set was compared against the non-redundant  protein database  <cit>  and top ranked blast matches to species other than plants with score values >  <dig> were flagged as contaminants; no such sequences were found in our sequence dataset.
the processed sequences were assembled into contigs and singletons using usearch v <dig>   <cit>  with 95% identity.
est and contig redundancy was calculated as described in kirst et al.
<cit> .
simple sequence repeats  present in the est sequences were identified and analyzed using the simple sequence repeat identification tool   <cit> .
the parameters were set for detection of perfect di-, tri-, tetra-, and pentanucleotide motifs with a minimum of  <dig>   <dig>   <dig>  and  <dig> repeats, respectively.
comparative sequence analysis
the following databases were used to perform blastx <cit>  and blastn  <cit>  analyses for annotation of the est singletons and contigs: 1) arabidopsis thaliana, unigene build # <dig>   <dig>  clusters; 2) populus unigene build# <dig>   <dig>  clusters; 3) oryza sativa, unigene build # <dig>   <dig>  clusters; 4) vitis vinifera, unigene build # <dig>   <dig>  clusters; 5) physcomitrella patens, unigene build # <dig>   <dig>  clusters; 6) pinus and picea, unigene build # <dig>   <dig>  clusters; 7) nr database of genbank, ncbi release  <dig>  release date october  <dig>  2012; 8) est_others in ncbi download date october  <dig>  2012; 9) uniprot plant protein databank in ncbi download date october  <dig>   <dig>  all blast searches were subject to an e-value cut-off of 1e -  <dig>  in reporting blast results, the blast score was used which incorporates both the similarity metric and the e-value to provide a representation of the hit’s uniqueness and overall similarity to the query sequence.
blastx searches were targeted against model species while blastn searches focused on comparisons against conifer species with public sequence resources.
in addition to blast annotations, the pipeline-directed gene ontology  assignments were conducted from applicable results in the categories of molecular function and biological process.
the hierarchical go structure was stored locally to resolve consistent levels of annotation.
in order to classify sequences into comparable categories, interpro scan wrappers were applied to generate brenda enzyme, signalp, tmhmm, and pfam protein domain results.
full-length unique ests were identified from blastx sequence similarity searches.
to be considered full-length, sequence were required to have greater than 80% identity and include the start codon for the translated protein.
RESULTS
est sequence quality, contigs and unique sequences
a sequence read length of >100 bp from either one or both directions was obtained for  <dig>  cdna clones.
after sequence processing and removing vector and low quality sequences, a total of  <dig>  high quality reads  were obtained .
the average read-length of ests used for sequence analysis was 532 bp.
the est length was distributed from  <dig> to 500 bp ,  <dig> to 800 bp  and  <dig> to 1242 bp .
sequence length of greater than 800 bp was obtained for one-eighth of our ests.
we identified a total of  <dig>  unique sequences, consisting of  <dig>  singletons and  <dig> contigs from the assembly of  <dig>  overlapping and contiguous quality reads .
the average length of singletons, and contigs, was 526 bp, and 560 bp, respectively.
the average and maximum number of ests in a contig was  <dig> , and  <dig>  respectively .
the average gc content of the unique sequences set is  <dig> %.
all of the  <dig>  high quality est sequences have been deposited into genbank under the accession numbers dbest jz <dig> - jz <dig>  they have also been submitted to the treegenes database  <cit> .
est redundancy in the present study was 51%, which is lower than estimates of  <dig> to 85% reported in other representative conifer studies , even though our ests were sequenced from a standard non-normalized cdna library versus normalized cdna libraries used in most of the other studies.
the lower est redundancy could be due to the fact that relatively small number of sequences from the cdna library in this study.
as the number of ests sequenced from a single cdna library increases, the percent est redundancy also increases.
inevitable causes of redundancy in cdna libraries are non-uniform abundance of mrnas from different genes  <cit> , presence of paralogous genes, and members of multigene families  <cit> .
on the other hand, the redundancy inherent in standard cdna libraries improves the assemblies as overlapping ests from a single gene can be aligned to generate a single contig  <cit> .
the contig redundancy of  <dig> % in our study was similar to that observed in other representative conifer tree species .
thus, the quality of our est sequences appears to be quite high.
it should be noted that it is difficult to make comparisons between numbers of transcripts among different projects and genera as they are influenced by the genome and/or transcriptome size of the study species.
in addition, these comparisons are influenced by the assembly method  <cit>  and the number of input reads.
picea
and
pinus
studies
picea mariana
picea glauca
picea sitchensis
pinus taeda
pinus contorta
functional gene annotations for unique transcript sequences and gene discovery
translated nucleotide to protein comparisons were made for the  <dig>  p. mariana unique sequences  against the non-redundant  protein database.
<dig>   of  <dig>  singletons and  <dig>  of  <dig> contigs, had significant blastx hits to known proteins, yielding annotations for  <dig>   black spruce unique sequences .
as expected, the percentage of contigs  showing significant similarity with the nr database was higher than singletons .
this may be due to greater sequence lengths of the contigs in comparison to shorter singletons.
of the  <dig>  annotated unique sequences,  <dig>   represented sequences with known gene functions.
in all cases, the most significant, informative annotation was selected.
the remaining  <dig> annotated sequences had annotations that were predicted, hypothetical, or unknown .
no contaminants were found after analysis of blastx results as the cdna library was developed from fresh needles of green-house grown seedlings.
a total of  <dig>   sequences had no significant blastx hits with the nr protein database.
the sequence divergence among gymnosperms and angiosperms is a limiting factor for gene annotation in conifers.
similar statistics were obtained for blastx similarity analysis of ests against publically available databases for white spruce  <cit>  sitka spruce  <cit> , and norway spruce  <cit> , which reported no annotations for 15-30% of the transcripts.
these results demonstrate that available datasets are not sufficient for annotation of conifer transcripts.
in theory, these un-annotated sequences  could be p. mariana specific transcripts or short segments of genes that would be recognized as homologs if more substantial sequences sets were available.
perhaps, these sequences represent regions of proteins that have diverged too much and escaped our similarity search criteria.
finally, these un-annotated sequences could represent partial transcripts with mostly utrs which, in general, show lower degree of conservation among species.
following the initial analysis of ests with the nr database, sequences were annotated against the highly curated plant protein uniprot databank and produced a total of  <dig>  significant annotations with known functions .
the gene annotations from ests in this study represent only a portion of gene repertoire in p. mariana, more transcriptome sequencing is needed to identify the needle tissue transcriptome.
predicted proteins from the first whole genome sequence of norway spruce have become available  <cit> .
however, the norway spruce genome assembly and protein predictions are at the very first stage, whereas we have used highly curated and reliable plant protein and nr protein databases for annotation of our black spruce unique contigs and singletons.
thus, the functional annotations reported here, although conservative, should be quite reliable.
also, the first draft genome of white spruce is published  <cit>  but there is no information available on its predicted proteins.
in future, with the availability of advance generations of norway spruce genome assembly and identification of functional proteins, the black spruce unique transcriptome sequences should be analyzed against the norway spruce and other conifers  functionally analyzed and predicted proteins.
this may provide information if an est is a member of a longer protein that is actually or predictably expressed.
the blastx comparisons of p. mariana 3' singletons, 5' singletons, and contigs were also conducted against the protein sequences of five sequenced plant genomes  and moss  .
in all species, contigs and 5' singletons showed higher similarity  with the species-specific peptides than 3' singletons  .
the 3' singletons obtained the most annotations with a blast score range from 50– <dig>  between 49% and 64% of the 3' singletons contained an annotation with arabidopsis, populus, oryza sativa, vitis vinifera, and physcomitrella patens at score range of 50– <dig> .
however, 5' singletons had a larger percentage of blast hits with a score range of 100– <dig> .
contigs and 5' singletons, had the best alignments with scores > <dig> in all model plant species queried .
the greatest number of black spruce contig annotations, with a score range of 50– <dig> , were obtained from the comparisons with the populus and oryza sativa proteins.
a previous study involving 5' singletons, 3' singletons, and contigs for white spruce and sitka spruce, showed a higher percentage of blastx similarity with the same angiosperm species as in our study  <cit> .
the percentage of white spruce and sitka spruce 5' singletons  showing blastx similarity was higher than 3' singletons   <cit> .
in contrast, only  <dig> % of loblolly pine ests showed significant blastx hits with arabidopsis peptides  <cit> .
based on the functional gene annotation analysis, p. mariana transcripts showed higher annotation rates with angiosperms peptides used in the present study than with moss peptides.
it may be due to the fact that conifers are phylogenetically closer to angiosperms than to mosses  <cit> .
black spruce transcripts are expected to show the highest annotations with highly curated spruce and pine peptide database when it becomes publicly available.
gene ontology classification, full-length genes, and gene families
gene ontology  results were assigned to describe the functional distribution within the est unique sequences derived from p. mariana needle tissue .
a total of  <dig> unique sequences were associated with at least one molecular function term and  <dig> sequences were associated with at least one biological process term .
molecular function assignment revealed that oxidoreductase activity, transferase activity, ion binding and nucleic acid/nucleotide binding accounted for the largest portion of the p. mariana unique genes identified .
among the ion binding proteins, the majority were small metal ion binding proteins such as metallotheonins, zinc and calcium binding proteins.
these genes are involved in stress response  <cit>  and protect cells from toxic metal and assist in metal transport  <cit> .
metallothioneins are able to sequester excess amounts of metal ions, and participate in homeostasis and antioxidant functions  <cit> .
various nucleic acid binding proteins were also targeted with high frequency as they are important components of transcriptional machinery of cells  <cit> .
previous study involving quercus spp.
contigs also reported nucleotide binding as abundant go term in terms of molecular function  <cit> .
other significant categories included hydrolase activity, small molecule binding and organic cyclic compound binding  .
it is worth noting that although the est number in our study is relatively small, genes involved in  <dig> molecular functions could be identified.
biological process describes the major biochemical pathways that the sequences may be involved in .
the primary categories in our study include cellular metabolic process , primary metabolic process , macromolecule metabolic process , and biosynthetic process  .
the results from the functional distribution highlight that transcripts from diverse categories are represented in p. mariana unique sequences.
the molecular functions and biological processes assigned for the black spruce unique contigs and singletons are consistent with the metabolic pathways active during vigorously-growing black spruce seedlings in the greenhouse conditions from which samples for rna preparation were collected.
the molecular function and biological process gene ontology terms are also consistent with the similar results reported in other studies that used needles for est or transcriptome sequencing  <cit> .
a combination of blast annotations, go terms, and supplemental interproscan data was used to generate lists of gene families of interest.
two of the groups highlighted here include transcription factors  and genes involved in the photosynthetic pathway .
transcription factors are dna-binding sequence-specific proteins that interact with the promoter sequences of target genes and modulate the gene expression  <cit> .
a total of  <dig> putative transcription factor sequences were identified based on the go term assignment of p. mariana unique sequences.
majority of the transcription factors were represented by myc, myb, and wrky domains.
these transcription factors families have also been reported in the white spruce est collection  <cit> .
they are key regulators of various biological processes involved in growth and development  <cit> .
also, they are expressed in response to various biotic and environmental stresses encountered by needles during their life cycle  <cit> .
some wrky proteins have been found to be involved in the signal transduction pathway  <cit> .
since the cdna library was constructed from needle tissues, genes related to photosynthesis were expected to be abundantly expressed.
a total of  <dig> p. mariana unique transcripts related to photosynthetic pathway were identified .
this group included chlorophyll a/b binding protein, light-harvesting complex proteins, photosystem i and ii reaction center proteins, ribulose bisphosphate carboxylase , oxygen-evolving enhancer protein, granule-bound starch synthase and nucleoside diphosphate.
in plants, rubisco is more abundant during the day when it is transcriptionally regulated by the light receptor phytochrome, thus fixing carbon dioxide in photosynthesis  <cit> .
we collected needle samples for rna extraction during daytime.
blast results against individual species as well as the nr repository provided details on the  <dig> full-length ests identified .
of these, 32% had annotations in three of the five model plant species .
the  <dig> full-length gene sequences were represented by  <dig> contigs,  <dig> 5' singletons, and  <dig> 3' singletons.
the full-length cdna sequences are an important resource for characterization of individual genes and members of multigene families, as well as for functional and comparative genomics.
additionally, they are essential resources for downstream annotation of related conifer genomic sequences.
gene expression and highly abundant transcripts
a preliminary estimation of gene expression can be given by the frequency distribution of the ests representing a gene in the library.
among the annotated sequences,  <dig> contained at least  <dig> supporting ests .
a family of lipid-transfer protein  was most abundant.
they are known to be involved in biotic and abiotic stress responses  <cit> .
additionally, transcripts related to bet v i allergen family, antimicrobial protein, germin-like protein, and many photosynthetic pathway genes were also highly abundant.
the supporting ests represent between  <dig> and  <dig> unique contigs .
the supplemental additional file 5: table s <dig> provides details on  <dig> annotated sequences with supporting ests.
the examination of the complete set shows that several growth factors, disease responsive genes, transcription factors, and photosynthetic mechanism proteins are represented by multiple ests .
these may be the result of multigene families, in addition to the inherent redundancy of ests in non-normalized cdna libraries  <cit> .
high abundance of genes involved in photosynthesis, growth and transcription factors is quite expected because the cdna library was constructed from needles of actively growing black spruce seedlings in the greenhouse conditions.
abundance of stress and disease responsive genes expressed in black spruce seedlings growing under optimal greenhouse conditions suggest that these genes are also involved in plant functions other than their response to abiotic and biotic stress.
nevertheless, the stress and disease responsive genes identified in our study provide very valuable transcriptomic resource for structural and functional genomics studies in black spruce.
sequence similarities, life history and ecological traits and evolutionary relationships
unique sequences were also compared with ests from major plant species combined  using blastn.
as expected, sequence similarity between the p. mariana sequences and published gymnosperm ests was high .
results were examined first by genus  and subsequently by specific species within picea and pinus .
a total of  <dig> sequences did not have a significant blastn hit and are therefore considered to be novel sequences.
these novel sequences may represent transcripts specific to p. mariana related to its species-specific traits.
as noted earlier, p. mariana differs from p. glauca, p. sitchensis and p. engelmannii for many life history, growth, morphological, ecophysiological, adaptive and insect resistance traits  <cit> .
black spruce is an early successional pioneering species, whereas white and sitka spruces are late-successional and climax species.
it is a slower growing species than white and sitka spruce.
black spruce unlike white and sitka spruce can grow on poorly drained, wet organic, loamy clay nutrient-poor soils, with low soil temperatures and high moisture contents  <cit> .
black spruce is less susceptible to some insect diseases, such as spruce budworm , than white spruce  <cit> .
white spruce is very closely related to engelmann spruce and sitka spruce, and hybridizes naturally with these species in the zone of overlap  <cit> .
phylogenetically, black spruce is distinct from white, sitka and engelmann spruces  <cit> .
occurrence of some species-specific genes in p. mariana is therefore expected.
it is noteworthy that despite the relatively smaller p. mariana est dataset as compared to p. glauca, and p. sitchensis, a significant number of novel transcripts could be detected in our study.
this number may be an over-estimate as some of these novel transcripts may be gene segments or regions from 5' or 3' ends of genes sufficiently diverged to escape our similarity criteria.
among the remaining,  <dig>  sequences, 96% had a hit to a member of the picea genus and 6% had significant similarities to a member of the pinus genus .
when viewing the results by species, picea glauca and picea sitchensis had the majority of significant matches, with more than 65% of the sequences generating a blast score >  <dig> .
these similarity results suggest that the majority of p. mariana genes discovered are homologues  of other picea species genes and may have originated from a common ancestor.
the significant similarities with p. glauca ests is not surprising as both species are sympatric transcontinental boreal species which can hybridize naturally, although rarely  <cit> .
within pinus, the greatest number of hits was observed with pinus taeda  as expected since the est resource generated for that species is very large .
pinus contorta followed with  <dig> unique sequence similarities with scores >  <dig> .
only  <dig>  sequences had significant similarity to another plant species outside of the picea and pinus families.
these  <dig> % blast hits to distant species may represent sequences not well characterized in closely related conifer species.
simple sequence repeats
a total of  <dig> different di-, tri-, tetra-, and penta-nucleotide repeats were identified among the p. mariana ests .
these were represented by  <dig> dinucleotide,  <dig> trinucleotide, three tetranucleotide, and three pentanucleotide repeats.
dinucleotide motifs were the most frequent , followed by trinucleotide motifs, which constitute 26% of total number of ssrs.
among the dinucleotide and trinucleotide repeats at, and cag motifs, respectively, were the most abundant.
ssr markers have been developed from some of these ssr-containing sequences and mapped on a black spruce genetic map  <cit> .
the ssr markers developed and other that can be developed in the future from the black spruce est sequences reported here provide a highly valuable resource for various population and conservation genetic studies in black spruce and other conifers.
CONCLUSIONS
we report here the first est resource of high quality for a widely-distributed, ecologically and economically important boreal conifer, black spruce.
despite the relatively small number of est sequences compared to picea glauca and p. sitchensis, our study identified  <dig> novel transcripts with no nucleotide similarity with dbest, and therefore, represent important addition to dbest.
we have identified genes involved in  <dig> molecular functions and  <dig> biological processes.
genes involved in stress response, photosynthetic pathway and growth were most abundant in the ests.
we have identified  <dig> full-length genes, ranging from  <dig> to  <dig> amino acids in length.
the sequences showed the greatest similarities to ests from the congeneric and sympatric species, picea glauca.
black spruce ests containing  <dig> different di-, tri-, tetra-, and penta-nucleotide repeats were identified.
these sequences could be used for the development of microsatellite dna markers.
the ests, and their annotations provide a valuable genomics resource to the forest tree genomics community in specific and plant genomics community in general.
markers developed from some of the est sequences have already been mapped on a black spruce genetic linkage map  <cit> .
the ests reported will provide an excellent resource for future assembly and annotation of transcriptome sequences from the ngs platforms, as well as for annotation of the spruce whole genome sequences.
a comparison of  <dig> and sanger reads showed that sanger reads can improve the assembly and annotation of the  <dig> datasets  <cit> .
availability of supporting data
all of the  <dig>  high quality est sequences have been deposited into genbank under the accession numbers dbest jz <dig> - jz <dig>  they have also been submitted to the treegenes database admits multiple queries on the occurrence of ests in the library and their functional annotation.
abbreviations
ests: expressed sequence tags; nr: non-redundant; go: gene ontology; dbest: est database; ssrs: simple sequence repeats; ngs: next-generation sequencing.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ikm was partly involved in library construction, sanger sequencing, quality assurance, sequence analysis and processing, and prepared the first draft of the manuscript.
jw carried out sequence assembly, annotation, submitted sequences to public databases, and contributed to manuscript preparation; opr is the principal investigator of the spruce genomics project and contributed to the project conception, secured project funding, provided overall project direction and guidance, and contributed to the manuscript preparation and revision.
all authors read and approved the final manuscript.
supplementary material
additional file 1: table s1
detailed annotation of complete set of black spruce singletons and contigs.
click here for file
additional file 2: table s2
blastx similarity results against peptides from five model species: arabidopsis, populus, oryza sativa, and vitis vinifera, and physcomitrella patens.
click here for file
additional file 3: table s3
est singletons and contigs annotated as putative photosynthetic pathway genes.
click here for file
additional file 4: table s4
predicted annotation, genbank accession numbers for the black spruce predicted full length genes.
click here for file
additional file 5: table s5
estimation of gene expression: unique est sequences with 1– <dig> ests.
click here for file
additional file 6: table s6
sequence similarity results for back spruce unique sequences against dbest.
click here for file
acknowledgements
we thank john major for providing black spruce seedlings, dr daoquian xiang for assistance with cdna library construction and est sequencing, taralynn cluney, brent higgins and waleed abousamak for help in running licor sequencing gels, and minyoung choi and ben figueroa for assistance with bioinformatics analysis.
the research was funded by a natural sciences and engineering research council of canada  strategic project grant  to o.p.
rajora.
i.k.
mann was financially supported by nserc strategic grants funds  to o.p.
rajora and dalhousie university graduate scholarship.
o.p.
rajora held the stora enso senior chair in forest genetics and biotechnology at dalhousie university, which was supported by stora enso port hawkesbury ltd., and the senior canada research chair in forest and conservation genomics and biotechnology at unb, which was supported by the canada research chair program .

 in non-model species with large genome size, est sequencing and their annotation can provide the first step towards understanding the transcriptome and expression patterns of specific genes, which can complement the whole genome sequencing, and can assist with genome sequence annotation. traditionally, est sequencing was conducted with the sanger sequencing system. more recently, next-generation sequencing platforms have been used to generate enormous amounts of genome and transcriptome sequences. ngs methods facilitate whole transcriptome sequencing at a fraction of the time and cost previously required for the sanger method. however, commonly used ngs platforms produce shorter reads and/or reduce the quality per base call. the improved length and accuracy of reads obtained from sanger sequencing can complement ngs workflows. this technology can assist in validating the ngs platform sequences by serving as a reference by which short reads can be aligned and corrected. therefore, est sequences derived from the sanger method are still a valuable resource in the ngs era. conifers have a large genome size and are ecologically and economically important, long-lived plants. they form a major part of the northern boreal and temperate forests, which constitute the major biome of the world. the genus pinus and picea are two important genera among conifers. black spruce b.s.p.) is a widely-distributed transcontinental species of the north american boreal and temperate forests with high ecological and economic importance. black spruce is one of the most important softwood species for the production of pulp and paper in canada. it is an early successional species and has a corresponding suite of species-specific life history, growth, eco-physiological and adaptive traits. the estimated haploid genome size of black spruce is about gbp, with 1c contents of  pg. as of, january 1st, dbest release, there were approximately million ests from species available in the genbank at the national centre for biotechnology information. in conifers, the major est contributing species are loblolly pine - pinus taeda, followed by white spruce - picea glauca and sitka spruce - picea sitchensis. among spruce species, white spruce has the maximum number of ests, followed by p. sitchensis and p. engelmannii x p. sitchensis. recently unique cdna clusters have also been reported for p. glauca. also, very recently, draft genomes of norway spruce and white spruce have been published. however, the black spruce transcriptome is not yet fully characterized and only ests and cdna sequences are reported within the ncbi’s dbest. due to a number of life history, morphological, adaptive, eco-physiological, and insect resistance traits and phylogenetic differences of black spruce from white spruce and sitka spruce, we expect some unique genes in the black spruce transcriptome. black spruce is an early successional shade-intolerant species whereas white spruce and sitka spruce are late successional shade-tolerant species. black spruce can grow in poor conditions, such as bogs, whereas white spruce grows on well-drained soils. these species-specific traits affirm a need to sequence and characterize the black spruce transcriptome. much of the est sequencing in conifers has been performed using wood forming tissues and secondary xylem due to the economic importance of wood. there are a number of studies that have sequenced transcripts from needle tissues, including: lodgepole pine, sugar pine, loblolly pine, maritime pine and norway spruce. these studies have provided some basis for the genes expressed in needle tissue; however, more comparative work is needed to understand their role in important metabolic pathways, such as photosynthesis. the objective of our study was to develop a black spruce transcript resource, and thus, facilitate structural and functional spruce genomics projects related to growth and adaptation. here, we report the results of the first est sequencing project from black spruce in which cdna clones were isolated and sequenced from a standard cdna library constructed from needle tissues in we conducted bidirectional sanger sequencing of ests to produce high quality, long reads to assist with the identification of full-length genes. we assembled ests into contigs and singletons, and subsequently performed comparative protein annotations with the non-redundant protein database and unigene clusters available at ncbi for model plant species. we further conducted nucleotide similarity analysis with est sequences available from all major plant species, as well as species-specific sequences from various gymnosperms and angiosperms. gene ontology terms were assigned and the ests were manually evaluated for specific categories, including transcription factors and photosynthetic genes. finally we used black spruce est data for the detection of simple sequence repeats. methods plant material and cdna library construction total rna was extracted from 2 g of freshly growing needles of three different black spruce seedlings established in the greenhouse at dalhousie university, following the protocol described in. quality and quantity of the isolated rna were determined using a spectrophotometer and extracted rna was found to be of high quality. the quantity of isolated rna was approximately 120 μg per g of the needle tissue used. the polya rna was purified using rneasy mini kit following the manufacturer’s instructions. the cdna library was constructed using creator smart cdna library construction kit. the oligo dt primed cdna inserts were directionally cloned in pdnr-lib vector and transformed using xl- gold ultra-competent cells of escherichia coli. plasmid dna was isolated from the transformed white colonies selected from the overnight grown cells on luria broth agar plates containing chloramphenicol using qiaprep spin miniprep kit. the quality and quantity of the isolated plasmid dna was confirmed on % agarose gels with known amount of lambda dna before sequencing. cdna sequencing sequencing reactions were performed in a ptc- thermal cycler using the thermosequenase fluorescent labeling primer cycle sequencing kit with 7-deaza dgtp according to the manufacturer’s instructions. the sequencing products were resolved on a li-cor 4200 l sequencing system. a total of cdna clones were sequenced in both directions using ird labeled m13f and m13r primers. preliminary sequence processing processing of raw trace files was performed through the customized treegenes est pipeline. base-calling and quality-assignment of the sequences were conducted with phred. low quality bases below phred were masked and vector sequences were trimmed from the ends. the cross_match program was used for this purpose with minmatch and minscore. sequences with less than high-quality bases after trimming and sequences with polya tails of ≥  bases were removed from the analysis. the resulting sequence set was compared against the non-redundant protein database and top ranked blast matches to species other than plants with score values >  were flagged as contaminants; no such sequences were found in our sequence dataset. the processed sequences were assembled into contigs and singletons using usearch v with 95% identity. est and contig redundancy was calculated as described in kirst et al.. simple sequence repeats present in the est sequences were identified and analyzed using the simple sequence repeat identification tool. the parameters were set for detection of perfect di-, tri-, tetra-, and pentanucleotide motifs with a minimum of and repeats, respectively. comparative sequence analysis the following databases were used to perform blastx and blastn analyses for annotation of the est singletons and contigs: 1) arabidopsis thaliana, unigene build # clusters; 2) populus unigene build# clusters; 3) oryza sativa, unigene build # clusters; 4) vitis vinifera, unigene build # clusters; 5) physcomitrella patens, unigene build # clusters; 6) pinus and picea, unigene build # clusters; 7) nr database of genbank, ncbi release release date october 2012; 8) est_others in ncbi download date october 2012; 9) uniprot plant protein databank in ncbi download date october all blast searches were subject to an e-value cut-off of 1e -  in reporting blast results, the blast score was used which incorporates both the similarity metric and the e-value to provide a representation of the hit’s uniqueness and overall similarity to the query sequence. blastx searches were targeted against model species while blastn searches focused on comparisons against conifer species with public sequence resources. in addition to blast annotations, the pipeline-directed gene ontology assignments were conducted from applicable results in the categories of molecular function and biological process. the hierarchical go structure was stored locally to resolve consistent levels of annotation. in order to classify sequences into comparable categories, interpro scan wrappers were applied to generate brenda enzyme, signalp, tmhmm, and pfam protein domain results. full-length unique ests were identified from blastx sequence similarity searches. to be considered full-length, sequence were required to have greater than 80% identity and include the start codon for the translated protein.  est sequence quality, contigs and unique sequences a sequence read length of >100 bp from either one or both directions was obtained for cdna clones. after sequence processing and removing vector and low quality sequences, a total of high quality reads were obtained. the average read-length of ests used for sequence analysis was 532 bp. the est length was distributed from to 500 bp, to 800 bp and to 1242 bp. sequence length of greater than 800 bp was obtained for one-eighth of our ests. we identified a total of unique sequences, consisting of singletons and contigs from the assembly of overlapping and contiguous quality reads. the average length of singletons, and contigs, was 526 bp, and 560 bp, respectively. the average and maximum number of ests in a contig was, and respectively. the average gc content of the unique sequences set is %. all of the high quality est sequences have been deposited into genbank under the accession numbers dbest jz - jz they have also been submitted to the treegenes database. est redundancy in the present study was 51%, which is lower than estimates of to 85% reported in other representative conifer studies, even though our ests were sequenced from a standard non-normalized cdna library versus normalized cdna libraries used in most of the other studies. the lower est redundancy could be due to the fact that relatively small number of sequences from the cdna library in this study. as the number of ests sequenced from a single cdna library increases, the percent est redundancy also increases. inevitable causes of redundancy in cdna libraries are non-uniform abundance of mrnas from different genes, presence of paralogous genes, and members of multigene families. on the other hand, the redundancy inherent in standard cdna libraries improves the assemblies as overlapping ests from a single gene can be aligned to generate a single contig. the contig redundancy of % in our study was similar to that observed in other representative conifer tree species. thus, the quality of our est sequences appears to be quite high. it should be noted that it is difficult to make comparisons between numbers of transcripts among different projects and genera as they are influenced by the genome and/or transcriptome size of the study species. in addition, these comparisons are influenced by the assembly method and the number of input reads. picea and pinus studies picea mariana picea glauca picea sitchensis pinus taeda pinus contorta functional gene annotations for unique transcript sequences and gene discovery translated nucleotide to protein comparisons were made for the p. mariana unique sequences against the non-redundant protein database. of singletons and of contigs, had significant blastx hits to known proteins, yielding annotations for black spruce unique sequences. as expected, the percentage of contigs showing significant similarity with the nr database was higher than singletons. this may be due to greater sequence lengths of the contigs in comparison to shorter singletons. of the annotated unique sequences, represented sequences with known gene functions. in all cases, the most significant, informative annotation was selected. the remaining annotated sequences had annotations that were predicted, hypothetical, or unknown. no contaminants were found after analysis of blastx results as the cdna library was developed from fresh needles of green-house grown seedlings. a total of sequences had no significant blastx hits with the nr protein database. the sequence divergence among gymnosperms and angiosperms is a limiting factor for gene annotation in conifers. similar statistics were obtained for blastx similarity analysis of ests against publically available databases for white spruce sitka spruce, and norway spruce, which reported no annotations for 15-30% of the transcripts. these results demonstrate that available datasets are not sufficient for annotation of conifer transcripts. in theory, these un-annotated sequences could be p. mariana specific transcripts or short segments of genes that would be recognized as homologs if more substantial sequences sets were available. perhaps, these sequences represent regions of proteins that have diverged too much and escaped our similarity search criteria. finally, these un-annotated sequences could represent partial transcripts with mostly utrs which, in general, show lower degree of conservation among species. following the initial analysis of ests with the nr database, sequences were annotated against the highly curated plant protein uniprot databank and produced a total of significant annotations with known functions. the gene annotations from ests in this study represent only a portion of gene repertoire in p. mariana, more transcriptome sequencing is needed to identify the needle tissue transcriptome. predicted proteins from the first whole genome sequence of norway spruce have become available. however, the norway spruce genome assembly and protein predictions are at the very first stage, whereas we have used highly curated and reliable plant protein and nr protein databases for annotation of our black spruce unique contigs and singletons. thus, the functional annotations reported here, although conservative, should be quite reliable. also, the first draft genome of white spruce is published but there is no information available on its predicted proteins. in future, with the availability of advance generations of norway spruce genome assembly and identification of functional proteins, the black spruce unique transcriptome sequences should be analyzed against the norway spruce and other conifers functionally analyzed and predicted proteins. this may provide information if an est is a member of a longer protein that is actually or predictably expressed. the blastx comparisons of p. mariana 3' singletons, 5' singletons, and contigs were also conducted against the protein sequences of five sequenced plant genomes and moss. in all species, contigs and 5' singletons showed higher similarity with the species-specific peptides than 3' singletons. the 3' singletons obtained the most annotations with a blast score range from 50– between 49% and 64% of the 3' singletons contained an annotation with arabidopsis, populus, oryza sativa, vitis vinifera, and physcomitrella patens at score range of 50–. however, 5' singletons had a larger percentage of blast hits with a score range of 100–. contigs and 5' singletons, had the best alignments with scores > in all model plant species queried. the greatest number of black spruce contig annotations, with a score range of 50–, were obtained from the comparisons with the populus and oryza sativa proteins. a previous study involving 5' singletons, 3' singletons, and contigs for white spruce and sitka spruce, showed a higher percentage of blastx similarity with the same angiosperm species as in our study. the percentage of white spruce and sitka spruce 5' singletons showing blastx similarity was higher than 3' singletons. in contrast, only % of loblolly pine ests showed significant blastx hits with arabidopsis peptides. based on the functional gene annotation analysis, p. mariana transcripts showed higher annotation rates with angiosperms peptides used in the present study than with moss peptides. it may be due to the fact that conifers are phylogenetically closer to angiosperms than to mosses. black spruce transcripts are expected to show the highest annotations with highly curated spruce and pine peptide database when it becomes publicly available. gene ontology classification, full-length genes, and gene families gene ontology results were assigned to describe the functional distribution within the est unique sequences derived from p. mariana needle tissue. a total of unique sequences were associated with at least one molecular function term and sequences were associated with at least one biological process term. molecular function assignment revealed that oxidoreductase activity, transferase activity, ion binding and nucleic acid/nucleotide binding accounted for the largest portion of the p. mariana unique genes identified. among the ion binding proteins, the majority were small metal ion binding proteins such as metallotheonins, zinc and calcium binding proteins. these genes are involved in stress response and protect cells from toxic metal and assist in metal transport. metallothioneins are able to sequester excess amounts of metal ions, and participate in homeostasis and antioxidant functions. various nucleic acid binding proteins were also targeted with high frequency as they are important components of transcriptional machinery of cells. previous study involving quercus spp. contigs also reported nucleotide binding as abundant go term in terms of molecular function. other significant categories included hydrolase activity, small molecule binding and organic cyclic compound binding. it is worth noting that although the est number in our study is relatively small, genes involved in molecular functions could be identified. biological process describes the major biochemical pathways that the sequences may be involved in. the primary categories in our study include cellular metabolic process, primary metabolic process, macromolecule metabolic process, and biosynthetic process. the results from the functional distribution highlight that transcripts from diverse categories are represented in p. mariana unique sequences. the molecular functions and biological processes assigned for the black spruce unique contigs and singletons are consistent with the metabolic pathways active during vigorously-growing black spruce seedlings in the greenhouse conditions from which samples for rna preparation were collected. the molecular function and biological process gene ontology terms are also consistent with the similar results reported in other studies that used needles for est or transcriptome sequencing. a combination of blast annotations, go terms, and supplemental interproscan data was used to generate lists of gene families of interest. two of the groups highlighted here include transcription factors and genes involved in the photosynthetic pathway. transcription factors are dna-binding sequence-specific proteins that interact with the promoter sequences of target genes and modulate the gene expression. a total of putative transcription factor sequences were identified based on the go term assignment of p. mariana unique sequences. majority of the transcription factors were represented by myc, myb, and wrky domains. these transcription factors families have also been reported in the white spruce est collection. they are key regulators of various biological processes involved in growth and development. also, they are expressed in response to various biotic and environmental stresses encountered by needles during their life cycle. some wrky proteins have been found to be involved in the signal transduction pathway. since the cdna library was constructed from needle tissues, genes related to photosynthesis were expected to be abundantly expressed. a total of p. mariana unique transcripts related to photosynthetic pathway were identified. this group included chlorophyll a/b binding protein, light-harvesting complex proteins, photosystem i and ii reaction center proteins, ribulose bisphosphate carboxylase, oxygen-evolving enhancer protein, granule-bound starch synthase and nucleoside diphosphate. in plants, rubisco is more abundant during the day when it is transcriptionally regulated by the light receptor phytochrome, thus fixing carbon dioxide in photosynthesis. we collected needle samples for rna extraction during daytime. blast results against individual species as well as the nr repository provided details on the full-length ests identified. of these, 32% had annotations in three of the five model plant species. the full-length gene sequences were represented by contigs, 5' singletons, and 3' singletons. the full-length cdna sequences are an important resource for characterization of individual genes and members of multigene families, as well as for functional and comparative genomics. additionally, they are essential resources for downstream annotation of related conifer genomic sequences. gene expression and highly abundant transcripts a preliminary estimation of gene expression can be given by the frequency distribution of the ests representing a gene in the library. among the annotated sequences, contained at least supporting ests. a family of lipid-transfer protein was most abundant. they are known to be involved in biotic and abiotic stress responses. additionally, transcripts related to bet v i allergen family, antimicrobial protein, germin-like protein, and many photosynthetic pathway genes were also highly abundant. the supporting ests represent between and unique contigs. the supplemental additional file 5: table s provides details on annotated sequences with supporting ests. the examination of the complete set shows that several growth factors, disease responsive genes, transcription factors, and photosynthetic mechanism proteins are represented by multiple ests. these may be the result of multigene families, in addition to the inherent redundancy of ests in non-normalized cdna libraries. high abundance of genes involved in photosynthesis, growth and transcription factors is quite expected because the cdna library was constructed from needles of actively growing black spruce seedlings in the greenhouse conditions. abundance of stress and disease responsive genes expressed in black spruce seedlings growing under optimal greenhouse conditions suggest that these genes are also involved in plant functions other than their response to abiotic and biotic stress. nevertheless, the stress and disease responsive genes identified in our study provide very valuable transcriptomic resource for structural and functional genomics studies in black spruce. sequence similarities, life history and ecological traits and evolutionary relationships unique sequences were also compared with ests from major plant species combined using blastn. as expected, sequence similarity between the p. mariana sequences and published gymnosperm ests was high. results were examined first by genus and subsequently by specific species within picea and pinus. a total of sequences did not have a significant blastn hit and are therefore considered to be novel sequences. these novel sequences may represent transcripts specific to p. mariana related to its species-specific traits. as noted earlier, p. mariana differs from p. glauca, p. sitchensis and p. engelmannii for many life history, growth, morphological, ecophysiological, adaptive and insect resistance traits. black spruce is an early successional pioneering species, whereas white and sitka spruces are late-successional and climax species. it is a slower growing species than white and sitka spruce. black spruce unlike white and sitka spruce can grow on poorly drained, wet organic, loamy clay nutrient-poor soils, with low soil temperatures and high moisture contents. black spruce is less susceptible to some insect diseases, such as spruce budworm, than white spruce. white spruce is very closely related to engelmann spruce and sitka spruce, and hybridizes naturally with these species in the zone of overlap. phylogenetically, black spruce is distinct from white, sitka and engelmann spruces. occurrence of some species-specific genes in p. mariana is therefore expected. it is noteworthy that despite the relatively smaller p. mariana est dataset as compared to p. glauca, and p. sitchensis, a significant number of novel transcripts could be detected in our study. this number may be an over-estimate as some of these novel transcripts may be gene segments or regions from 5' or 3' ends of genes sufficiently diverged to escape our similarity criteria. among the remaining, sequences, 96% had a hit to a member of the picea genus and 6% had significant similarities to a member of the pinus genus. when viewing the results by species, picea glauca and picea sitchensis had the majority of significant matches, with more than 65% of the sequences generating a blast score > . these similarity results suggest that the majority of p. mariana genes discovered are homologues of other picea species genes and may have originated from a common ancestor. the significant similarities with p. glauca ests is not surprising as both species are sympatric transcontinental boreal species which can hybridize naturally, although rarely. within pinus, the greatest number of hits was observed with pinus taeda as expected since the est resource generated for that species is very large. pinus contorta followed with unique sequence similarities with scores > . only sequences had significant similarity to another plant species outside of the picea and pinus families. these % blast hits to distant species may represent sequences not well characterized in closely related conifer species. simple sequence repeats a total of different di-, tri-, tetra-, and penta-nucleotide repeats were identified among the p. mariana ests. these were represented by dinucleotide, trinucleotide, three tetranucleotide, and three pentanucleotide repeats. dinucleotide motifs were the most frequent, followed by trinucleotide motifs, which constitute 26% of total number of ssrs. among the dinucleotide and trinucleotide repeats at, and cag motifs, respectively, were the most abundant. ssr markers have been developed from some of these ssr-containing sequences and mapped on a black spruce genetic map. the ssr markers developed and other that can be developed in the future from the black spruce est sequences reported here provide a highly valuable resource for various population and conservation genetic studies in black spruce and other conifers.  we report here the first est resource of high quality for a widely-distributed, ecologically and economically important boreal conifer, black spruce. despite the relatively small number of est sequences compared to picea glauca and p. sitchensis, our study identified novel transcripts with no nucleotide similarity with dbest, and therefore, represent important addition to dbest. we have identified genes involved in molecular functions and biological processes. genes involved in stress response, photosynthetic pathway and growth were most abundant in the ests. we have identified full-length genes, ranging from to amino acids in length. the sequences showed the greatest similarities to ests from the congeneric and sympatric species, picea glauca. black spruce ests containing different di-, tri-, tetra-, and penta-nucleotide repeats were identified. these sequences could be used for the development of microsatellite dna markers. the ests, and their annotations provide a valuable genomics resource to the forest tree genomics community in specific and plant genomics community in general. markers developed from some of the est sequences have already been mapped on a black spruce genetic linkage map. the ests reported will provide an excellent resource for future assembly and annotation of transcriptome sequences from the ngs platforms, as well as for annotation of the spruce whole genome sequences. a comparison of and sanger reads showed that sanger reads can improve the assembly and annotation of the datasets. availability of supporting data all of the high quality est sequences have been deposited into genbank under the accession numbers dbest jz - jz they have also been submitted to the treegenes database admits multiple queries on the occurrence of ests in the library and their functional annotation. abbreviations ests: expressed sequence tags; nr: non-redundant; go: gene ontology; dbest: est database; ssrs: simple sequence repeats; ngs: next-generation sequencing. competing interests the authors declare that they have no competing interests. authors’ contributions ikm was partly involved in library construction, sanger sequencing, quality assurance, sequence analysis and processing, and prepared the first draft of the manuscript. jw carried out sequence assembly, annotation, submitted sequences to public databases, and contributed to manuscript preparation; opr is the principal investigator of the spruce genomics project and contributed to the project conception, secured project funding, provided overall project direction and guidance, and contributed to the manuscript preparation and revision. all authors read and approved the final manuscript. supplementary material additional file 1: table s1 detailed annotation of complete set of black spruce singletons and contigs. click here for file additional file 2: table s2 blastx similarity results against peptides from five model species: arabidopsis, populus, oryza sativa, and vitis vinifera, and physcomitrella patens. click here for file additional file 3: table s3 est singletons and contigs annotated as putative photosynthetic pathway genes. click here for file additional file 4: table s4 predicted annotation, genbank accession numbers for the black spruce predicted full length genes. click here for file additional file 5: table s5 estimation of gene expression: unique est sequences with 1– ests. click here for file additional file 6: table s6 sequence similarity results for back spruce unique sequences against dbest. click here for file acknowledgements we thank john major for providing black spruce seedlings, dr daoquian xiang for assistance with cdna library construction and est sequencing, taralynn cluney, brent higgins and waleed abousamak for help in running licor sequencing gels, and minyoung choi and ben figueroa for assistance with bioinformatics analysis. the research was funded by a natural sciences and engineering research council of canada strategic project grant to o.p. rajora. i.k. mann was financially supported by nserc strategic grants funds to o.p. rajora and dalhousie university graduate scholarship. o.p. rajora held the stora enso senior chair in forest genetics and biotechnology at dalhousie university, which was supported by stora enso port hawkesbury ltd., and the senior canada research chair in forest and conservation genomics and biotechnology at unb, which was supported by the canada research chair program.
most abundantly expressed transcripts were associated with photosynthesis, growth factors, stress and disease response, and transcription factors. about 18% of the transcripts were novel, representing an important addition to the genbank est database. est sequences and their annotation provide a highly valuable resource for gene discovery, genome sequence annotation, and other genomics studies that can be applied in genetics, breeding and conservation programs for non-model organisms. based on est sequence similarities, black spruce showed close evolutionary relationships with congeneric picea glauca and picea sitchensis compared to other pinaceae members and angiosperms. a total of full-length genes were identified. we have also identified full-length genes and microsatellite-containing ests. we obtained high quality sequence reads, with an average read-length of 532 bp. black spruce, is a transcontinental species of the north american boreal and temperate forests. the est sequences reported here provide an important resource for genome annotation, functional and comparative genomics, molecular breeding, conservation and management studies and applications in black spruce and related conifer species. fifty-seven di-, tri-, tetra- and penta-nucleotide simple sequence repeats were identified. we conducted bidirectional sequencing of cdna clones from a standard cdna library constructed from black spruce needle tissues. conifers are long-lived plants that are ecologically and economically important globally, and have a large genome size. we have developed the first high quality est resource for black spruce and identified novel transcripts, which may be species-specific related to life history and ecological traits. the primary objective of our study was to develop a black spruce transcriptomic resource to facilitate on-going functional genomics projects related to growth and adaptation to climate change. genes involved in molecular functions and biological processes were discovered, including putative transcription factors and genes involved in photosynthesis. clustering and assembly of ests resulted in unique sequences, consisting of singletons and contigs. approximately two-thirds of unique sequences were functionally annotated. picea marianaexpressed sequence taggene discoverygene expressiongene ontologymicrosatellites
 est sequences and their annotation provide a highly valuable resource for gene discovery, genome sequence annotation, and other genomics studies that can be applied in genetics, breeding and conservation programs for non-model organisms. conifers are long-lived plants that are ecologically and economically important globally, and have a large genome size. black spruce, is a transcontinental species of the north american boreal and temperate forests. however, there are limited transcriptomic and genomic resources for this species. the primary objective of our study was to develop a black spruce transcriptomic resource to facilitate on-going functional genomics projects related to growth and adaptation to climate change.  we conducted bidirectional sequencing of cdna clones from a standard cdna library constructed from black spruce needle tissues. we obtained high quality sequence reads, with an average read-length of 532 bp. clustering and assembly of ests resulted in unique sequences, consisting of singletons and contigs. approximately two-thirds of unique sequences were functionally annotated. genes involved in molecular functions and biological processes were discovered, including putative transcription factors and genes involved in photosynthesis. most abundantly expressed transcripts were associated with photosynthesis, growth factors, stress and disease response, and transcription factors. a total of full-length genes were identified. about 18% of the transcripts were novel, representing an important addition to the genbank est database. fifty-seven di-, tri-, tetra- and penta-nucleotide simple sequence repeats were identified.  we have developed the first high quality est resource for black spruce and identified novel transcripts, which may be species-specific related to life history and ecological traits. we have also identified full-length genes and microsatellite-containing ests. based on est sequence similarities, black spruce showed close evolutionary relationships with congeneric picea glauca and picea sitchensis compared to other pinaceae members and angiosperms. the est sequences reported here provide an important resource for genome annotation, functional and comparative genomics, molecular breeding, conservation and management studies and applications in black spruce and related conifer species. picea marianaexpressed sequence taggene discoverygene expressiongene ontologymicrosatellites 