Open Access

Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding

  • Steven G Ralph1, 6,
  • Hye Jung E Chun2,
  • Dawn Cooper1,
  • Robert Kirkpatrick2,
  • Natalia Kolosova1, 3,
  • Lee Gunter4,
  • Gerald A Tuskan4,
  • Carl J Douglas3,
  • Robert A Holt2,
  • Steven JM Jones2,
  • Marco A Marra2 and
  • Jörg Bohlmann1, 3, 5Email author
Contributed equally
BMC Genomics20089:57

DOI: 10.1186/1471-2164-9-57

Received: 06 November 2007

Accepted: 29 January 2008

Published: 29 January 2008

Abstract

Background

The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions.

Results

As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in poplar leaves attacked by forest tent caterpillars.

Conclusion

This study has generated a high-quality FLcDNA resource for poplar and the third largest FLcDNA collection published to date for any plant species. We successfully used the FLcDNA sequences to reassess gene prediction in the poplar genome sequence, perform comparative sequence annotation, and identify differentially expressed transcripts associated with defense against insects. The FLcDNA sequences will be essential to the ongoing curation and annotation of the poplar genome, in particular for targeting gaps in the current genome assembly and further improvement of gene predictions. The physical FLcDNA clones will serve as useful reagents for functional genomics research in areas such as analysis of gene functions in defense against insects and perennial growth. Sequences from this study have been deposited in NCBI GenBank under the accession numbers EF144175 to EF148838.

Background

Poplars are keystone tree species in several temperate forest ecosystems in the Northern Hemisphere. Poplars are also intensively cultivated in plantation forestry for the production of wood, pulp, and paper. Fast growing poplars can serve functions in phytoremediation, as a sink for carbon sequestration, and as a feedstock for biofuel production. Poplar has also been firmly established as a model research system for long-lived woody perennials (reviewed in [1]). Advances in functional genomics of poplar have been greatly enhanced by the availability of a high-quality genome sequence from P. trichocarpa (Nisqually-1; [2]), combined with comprehensive genetic [36] and physical genome [7] maps, as well as the availability of several platforms for transcriptome analysis [811] and genetic transformation. Large collections of expressed sequence tags (ESTs) have also been developed from a variety of poplar species and hybrids focussing on gene discovery in wood formation, dormancy, floral development and stress response [9, 1120]. These short, single-pass EST reads have been a critical resource for gene discovery, genome annotation, and the construction of microarray platforms.

High-accuracy, sequence-verified FLcDNA sequences that span the entire protein-coding region of a given gene can advance comparative, functional, and structural genome analysis. For example, the accuracy of ab initio prediction of protein-coding regions in genome sequences is limited by the difficulty of finding islands of coding sequences within an ocean of non-coding DNA, and by the complexity of individual genes that may code for multiple peptides through alternative splicing. More robust approaches that unambiguously identify protein-coding regions in a genome sequence have used FLcDNA data, as demonstrated for example in Arabidopsis thaliana [2123]. Despite their immense value, sequence-verified FLcDNA clones, where multiple passes verify the authenticity of reads, have not been generated in most plant species subjected to genomic analysis. Only a few large FLcDNA data sets have been generated for plants; namely for rice [24], Arabidopsis [25], and maize [26, 27]. In contrast, as of September 2007, there were only 1,409 complete sequences from individual poplar FLcDNA clones in the non-redundant (NR) division of GenBank, in addition to a larger number of putative full-length sequences assembled from EST reads of multiple cDNA clones.

Our poplar FLcDNA program in the areas of forest health genomics and wood formation has focused on mechanisms of defense and resistance against insects and genes associated with xylem development. The forest tent caterpillar (Malacosoma disstria; FTC; [28]) is a major insect pest that threatens the productivity of natural and plantation forests. Poplars deploy an array of combined defense strategies against herbivores that can be grouped as chemical and physical defenses, direct and indirect defenses, constitutive and induced defenses, as well as local and systemic defenses (reviewed in [29]). Several recent studies have been conducted on the molecular mechanisms underlying inducible defenses against herbivores in poplar [11, 18, 3037].

In this paper, we report on the development of four FLcDNA libraries from poplar that served as the starting template for creating a substantial genomic resource of 4,664 sequence-verified FLcDNAs. We describe the overall structural features of these FLcDNA clones, annotation based on comparisons with other species, and the identification of 536 putative poplar-specific transcripts. Mapping the FLcDNA collection to the poplar genome sequence confirmed the overall high quality of the assembled genome sequence as well as the high quality of the FLcDNA resource, while also identifying 39 expressed poplar transcripts that appear to be derived from gap regions of the current genome sequence assembly and 173 new poplar genes that have not previously been identified in the genome assembly. By mapping 3,854 FLcDNAs to a poplar 15.5 K cDNA microarray platform and performing a comparison with existing transcriptome data, we identified 153 FLcDNAs that match transcripts differentially expressed following insect attack by FTC on poplar leaves.

Results

Selection and sequence finishing of FLcDNAs

FLcDNAs are defined as individual cDNA clones that contain the complete protein-coding sequence and at least partial 5' and 3' untranslated regions (UTRs) for a given transcript. This definition distinguishes bona fide FLcDNAs from in silico assembled EST sequences derived from multiple cDNA clones. In the latter case, it is possible that multiple, closely related genes or allelic variants of the same gene are assembled into a single consensus sequence. This problem is avoided when only sequences derived from the same physical FLcDNA clone are assembled. We prepared four FLcDNA libraries using the biotinylated CAP trapper method [38]. Three libraries constructed from xylem, phloem and cambium, and green shoot tips and leaves were derived from the P. trichocarpa Nisqually-1 genotype, for which the genome sequence has been reported [2]. An additional library was developed from the P. trichocarpa × P. deltoides hybrid H11–11 genotype using leaves subjected to FTC herbivory (Table 1).
Table 1

Libraries, tissue sources and species for sequences described in this study

cDNA Library

Tissue/Developmental Stage

Species (genotype)

PT-X-FL-A-1

Outer xylema.

Populus trichocarpa (Nisqually-1)

PT-P-FL-A-2

Phloem and cambiuma.

P. trichocarpa (Nisqually-1)

PT-GT-FL-A-3

Young and mature leaves, along with green shoot tipsa.

P. trichocarpa (Nisqually-1)

PTxD-IL-FL-A-4

Local and systemic (above region of feeding) mature leaves harvested after continuous feeding by forest tent caterpillars, Malacosoma disstria. Local tissue was collected 4, 8 and 24 h post-treatment and systemic tissue 4, 12 and 48 h post-treatmentb.

P. trichocarpa × deltoides (H11–11)

aHarvested May 15th, 2001 from eight year old trees within the Boise Cascade region of Washington state.

bOne or two year old saplings grown in potted soil under greenhouse conditions at the University of British Columbia.

To select candidate FLcDNAs for complete insert sequencing, we used a previously described bioinformatic pipeline for EST processing [11]. An initial set of 26,112 3' ESTs derived from FLcDNA libraries was combined with 81,407 3' ESTs from standard EST libraries [11] to generate a starting set of 107,519 3'-end ESTs, which resulted in 90,368 high-quality ESTs after filtering to remove sequences of low quality and contaminant sequences from yeast, bacteria and fungi. These sequences were then clustered using the CAP3 assembly program ([39]; assembly criteria: 95% identity, 40 bp window) to identify a set of 35,011 putative unique transcripts (PUTs; Figure 1). To maximize the capture of complete open reading frames (ORFs) and UTRs, only clones from full-length libraries were considered further. Using this strategy, we identified 5,926 cDNA candidate clones for full insert sequencing, which resulted in 4,664 sequence-verified poplar FLcDNA clones (see Additional file 1 and Figure 2). Inserts of 2,672 clones were completely sequenced using end reads only, with an average sequenced insert size of 735 ± 434 bp (average ± SD) and required an average of 4.5 ± 1.3 end reads to finish to high sequence quality. Using a combination of end reads and primer walking, inserts of an additional 1,992 clones were completely sequenced, with an average insert size of 1,308 ± 567 bp requiring 5.9 ± 2.8 end reads and 3.4 ± 1.8 internal primer reads per clone.
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Figure 1

Schematic of clone selection and complete insert sequencing of 4,664 FLcDNAs. CAP3 assembly of 90,368 high-quality 3'-end ESTs identified 35,011 putative unique transcripts (PUTs) for the identification of candidate FLcDNAs. Only those PUTs containing at least one clone from a FLcDNA library were considered further. To maximize the number of FLcDNAs captured, candidate clones were excluded from further analysis if: (1) the 5' second strand primer adaptor (SSPA) was absent; (2) a polyA tail was absent; (3) 5'- and/or 3'-end ESTs had a Phred20 quality length (Q20) of < 100 nt; or (4) BLASTN (E < 1e-80) versus poplar ESTs in the public domain identified a candidate as potentially truncated (i.e., > 100 nt shorter) at the 5' end of the transcript relative to a matching EST. Among the 5,926 candidates selected for sequencing, only 483 (8%) were aborted at various stages of the sequence finishing pipeline due to: (1) missing cloning structures; (2) errors in re-array of glycerol stocks; (3) problematic sequencing such as hard stops; or (4) problematic clone features such as chimeric sequences. Through a combination of end reads and gap closing using primer walking, 4,664 (79%) sequence-verified FLcDNAs were completed. An additional 779 clones (13%) from the starting set of 5,926 will be finished in future work.

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Figure 2

Distribution of open reading frame (ORF) and 5' and 3' untranslated region (UTR) sizes among the finished 4,664 FLcDNAs (A), and the mean ORF and UTR length (± standard deviation) (B). Each finished FLcDNA sequence was examined for the presence of ORFs using either the EMBOSS getorf program (version 2.5.0; [55]) or an in-house BLAST-aided program. The getorf program identifies the longest stretch of uninterrupted sequence between a start (ATG) and stop codon (TGA, TAG, TAA) in the 5' to 3' direction for the predicted ORF. The BLAST-aided program detects ORFs by finding the starting methionine and stop codon in a poplar FLcDNA sequence relative to the same features in the most closely related Arabidopsis protein identified by BLASTX (E values < 1e-20). For this study, ORFs identified by the BLAST-aided method were utilized except in cases where the FLcDNA sequence did not show high similarity to an Arabidopsis protein, in which case the ORF identified by the getorf program was chosen. The presence and coordinates of the 5' second strand primer adaptor sequence (SSPA) and polyA tail were also noted. The regions between the 5'SSPA and the predicted ORF start and between the predicted ORF stop and the polyA tail were taken to be the 5' and 3' UTRs, respectively. The 5' SSPA and 3' polyA tail lengths were not included when determining UTR length.

Analysis of the 4,664 FLcDNA sequences using the CAP3 clustering and assembly program ([39]; assembly criteria: 95% identity, 40 bp window) identified 3,505 FLcDNAs as unique singletons, with the remaining 1,159 grouping into 485 contigs, suggesting a total of 3,990 unique genes represented with finished FLcDNA sequences. The high percentage of unique transcripts (85.5%) within this set confirms the successful clone selection strategy (Figure 1) for establishing a low-redundancy clone set prior to sequence finishing.

Sequence quality and "full-length" assessment of poplar FLcDNAs

All 4,664 finished FLcDNAs achieved a minimum of Phred30 (i.e., one error in 103 bases) sequence quality at every base. The majority of FLcDNAs were of even higher quality with the minimum and average Phred values exceeding Phred45 (i.e., one error in 3 × 104 bases) and Phred80 (i.e., one error in 108 bases), respectively (Figure 3). We predicted the complete protein-coding ORFs for all 4,664 FLcDNAs. The distribution of 5' UTR, ORF and 3' UTR lengths is illustrated in Figure 2 [also see Additional file 1]. The average sequenced FLcDNA length (from the beginning of the 5' UTR to the end of the polyA tail) was 1,045 ± 475 bp (mean ± SD), and ranged from 147 to 3,342 bp, whereas the average predicted ORF was 649 ± 429 bp and ranged from 33 to 2,935 bp. ORFs could not be detected (i.e., 30 bp or less) for 96 FLcDNAs. The 5' and 3' UTRs averaged 109 ± 138 bp and 228 ± 152 bp, respectively. These results are comparable to CAP trapper FLcDNA collections from other plant species including maize (cDNA insert 799 bp, 5' UTR 99 bp, 3' UTR 206 bp; [27]), Arabidopsis (cDNA insert ca. 1.2 kb; [40]) and rice (5' UTR 259 bp, 3' UTR 398 bp; [24]). Similarly, the average transcript length of the 45,555 poplar reference genes predicted ab initio from the genome sequence was 1,079 bp and 5' and 3' UTRs averaged 92 bp [2], in close agreement with our results obtained with FLcDNAs.
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Figure 3

Validation of sequence quality of FLcDNAs. Sequence accuracy was measured as the percentage of the 4,664 FLcDNAs which, with 100%, 95.0–99.9%, 90.0–94.9% or < 90.0% of their sequence length, exceeded Phred30, Phred40, Phred50 or Phred60 sequence quality thresholds. All 4,664 FLcDNAs exceeded the Phred30 quality thresholds (calculated as less than 1 error in 103 sequenced nucleotides) over 100% of their sequence length. Even at the threshold level of Phred60 (calculated as less than 1 error in 106 sequenced nucleotides) the majority (61.2%) of the FLcDNA sequences met this very high sequence quality score over > 90.0% of their length.

To further assess the quality of the 4,664 poplar FLcDNAs, we performed reciprocal BLAST analysis against peptide sequences in The Arabidopsis Information Resource (TAIR) and against a set of 1,409 poplar sequences previously identified to be full-length (collected from the NR division of GenBank). Reciprocal BLAST analysis was performed with a stringent similarity threshold [% identity ≥ 50%; expect (E) value ≤ 1e-20] and identified 2,774 and 288 pairs, respectively, with Arabidopsis and previously published poplar FLcDNAs (Figure 4). Of the 288 homologous poplar transcript pairs (i.e., previously published poplar sequences with high sequence similarity to FLcDNAs reported in this study), 228 (79.2%) agreed well with regard to their ORF lengths and position of their start and stop codons (± ten amino acids; Figure 4). 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 either truncated or had an incorrectly predicted ORF. When comparing the poplar FLcDNA collection to reciprocal matches from TAIR Arabidopsis peptides, we observed a similar number of 2,151 (77.5%) pairs with similar ORF lengths and positions of their starting methionine and stop codons (± ten amino acids; Figure 4). These results indicate the majority of the 4,664 poplar FLcDNAs represent true full-length transcripts with complete ORFs and correctly annotated start and stop codons.
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Figure 4

Validation of poplar FLcDNAs by comparison to reciprocal BLAST matches against Arabidopsis peptides and previously published poplar FLcDNAs. The set of 4,664 poplar FLcDNAs were compared using BLASTX to both The Arabidopsis Information Resource (TAIR) non-redundant Arabidopsis peptide set (28,952 sequences [56]) and a collection of 1,409 previously published poplar sequences from the non-redundant (NR) division of GenBank ([57], the NR release of December 19th, 2006) annotated as full-length (excluding predicted proteins derived from genomic DNA). FLcDNAs were excluded from the analysis when the in-house BLAST-aided ORF detection software identified a FLcDNA as problematic according to the following categories: truncation at the 5'-end (319), truncation at the 3'-end (50), frameshift (12), stop codon in the middle of an ORF (9), or inverted insert (3) [see Additional file 1]. No problematic features were identified in the remaining 4,271 FLcDNAs. This comparison identified 2,774 homologous Arabidopsis-poplar pairs and 288 homologous poplar transcript pairs. A FLcDNA pair was considered homologous if (1) the top BLASTX match exceeded a stringent threshold (% identity ≥ 50%; expect value ≤ 1e-20) and (2) the reciprocal TBLASTN analysis identified the same poplar FLcDNA with a score value equal to or within 10% of the top match. ORF lengths for Arabidopsis and public poplar sequences were extracted from the TAIR and NR records, respectively, and poplar ORF lengths from this study were predicted using either the EMBOSS getorf or in-house BLAST-aided programs (see Figure 2 legend). The greyscale shading of each hexagon represents poplar FLcDNA abundance. ORF lengths for three Arabidopsis-poplar pairs and eight homologous poplar transcript pairs differed by more than 500 aa and are not included in the figure.

Mapping FLcDNAs to the poplar genome sequence to reassess gene prediction and to identify possible gaps in the genome assembly

As part of the poplar genome sequencing project [2], the poplar FLcDNAs were used to train a series of gene prediction algorithms to identify coding regions in the genome sequence. To reassess the effectiveness of gene prediction in the current genome assembly and to search for possible genome sequence gaps, we took two approaches: 1) BLAT [41] was utilized to map FLcDNAs to the assembled genome sequence, and 2) BLASTN was applied to align FLcDNAs with the 45,555 protein-coding gene loci predicted from the poplar genome sequence. Using BLAT, we mapped 4,642 poplar FLcDNAs (99.5%) to the genome at a minimum threshold (tile match length ≥ 11 bp, score ≥ 30, sequence identity ≥ 90%; Figure 5). From this set, 3,847 (82.9%) mapped to the 19 linkage groups (i.e., chromosomes) whereas the remainder mapped to scaffold segments that were not incorporated into the poplar genome sequence assembly. Examination of the linkage group location of FLcDNAs suggests a pattern of random distribution when grouped by cDNA library/tissue of origin, with an approximately even distribution of FLcDNAs throughout the genome (Figure 5). When we applied a more stringent similarity threshold (sequence identity ≥ 95%, alignment coverage ≥ 95%), the number of poplar FLcDNAs matching to the genome was only slightly reduced to 4,487 (96.2%).
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Figure 5

Mapping FLcDNAs to the poplar genome. 4,664 poplar FLcDNAs were aligned to the genome using BLAT with default parameters (match length ≥ 11 bp, BLAT score ≥ 30, sequence identity ≥ 90%). Prior to alignment, the 5' second strand primer adaptor sequences (SSPA) and polyA tails were removed. Among 4,642 poplar FLcDNAs that exceeded the minimal criteria for a match to the genome, 3,847 mapped to chromosomes whereas the remainder mapped to scaffold segments. Colored bars indicate the cDNA library of origin for those FLcDNAs mapping to one of the 19 poplar chromosomes. Applying a higher stringency threshold (sequence identity ≥ 95%, alignment coverage ≥ 95%), 4,487 or 96.2% of poplar FLcDNAs could be mapped to the genome.

In addition to BLAT analysis, we also compared the FLcDNAs with the 45,555 predicted protein-coding gene loci identified in the genome sequence using BLASTN and observed 4,452 (95.5%) matched at an E value < 1e-50 (see Additional file 1). In order to identify possible sequence gaps in the 7.5× coverage genome, we searched for FLcDNAs lacking a stringent BLAT to the genome match and a BLASTN match (E value ≥ 1e-50) to the predicted gene models. This approach identified only 39 candidates, of which 20 (0.4%) FLcDNAs also had a strong match by BLASTN (E value < 1e-50) to one or more poplar ESTs in the public domain, excluding ESTs reported in this study (Table 2 and see Additional file 1), suggesting that these FLcDNAs represent expressed poplar genes that likely map to gap regions within the current genome draft. We cannot exclude the possibility that the remaining 19 FLcDNAs represent sequences from bacterial, fungal or insect species present on poplar tissues harvested for cDNA library construction, which were not filtered as contaminant sequences in our EST and FLcDNA processing procedures.
Table 2

Expressed FLcDNAs that identify possible gaps in the genome sequence assembly

Clone ID

GenBank ID

FLcDNA length (bp)

FL status/ORF size (aa)

NR BLASTP best match

dbEST BLASTN best match

    

GenBank accession, gene name, species

BLAST Score

GenBank accession, species

BLAST Score

WS0138_J20

EF148816

1444

FL/340

AAB39877.1, NMT1 protein, Uromyces fabae

1572

DN493922.1, Populus tremula

770

WS01313_D10

EF148323

1439

FL/363

At3g20790, oxidoreductase, Arabidopsis thaliana

1233

DN501083, P. trichocarpa

1318

WS0127_P01

EF148143

1237

FL/299

AAD01907, methenyltetrahydrofolate dehydrogenase, Pisum sativum

1213

CV131075.1, P. deltoides

1511

WS01231_K20

EF147482

1207

FL/256

At5g20060, phospholipase/carboxylesterase family, A. thaliana

1026

DV464443.2, P. fremontii × P. angustifolia

1479

WS0135_G15

EF148633

992

n.a.

No matches

n.a.

BU891205, P. tremula

240

WS01312_F21

EF148269

946

n.a.

No matches

n.a.

BI122644.1, P. tremula × P. tremuloides

729

WS01315_I11

EF148467

836

n.a.

No matches

n.a.

BU824948.1, P. tremula × P. tremuloides

339

WS01312_H02

EF148274

835

n.a.

No matches

n.a.

BU791223.1, P. trichocarpa × P. deltoides

779

WS01212_B01

EF146690

821

FL/88

BAB68268.1, drought-inducible protein, Saccharum officinarum

147

BU879805.1, P. trichocarpa

595

WS0122_E05

EF147284

739

FL/131

CAB80775.1, proline-rich protein, A. thaliana

340

BU866461.1, P. tremula

890

WS0122_O15

EF147357

736

FL/162

At4g10300, hypothetical protein, A. thaliana

444

CX181869.1, Populus × canadensis

1215

WS0113_C11

EF145750

722

FL/136

At3g12260, complex 1/LVR family protein, A. thaliana

426

BU879375.1, P. trichocarpa

1223

WS0125_P18

EF147919

596

3' trunc./70

AAF71823.1, pumilio domain protein, P. tremula × P. tremuloides

167

CX187487.1, Populus × canadensis

722

WS01123_K15

EF145357

483

n.a.

No matches

n.a.

CK319617.1, P. deltoides

268

WS01231_G04

EF147458

416

5' trunc./62

At3g18790, hypothetical protein, A. thaliana

200

CX184264.1, Populus × canadensis

543

WS0124_L22

EF147751

360

n.a.

No matches

n.a.

BI128250.1, P. tremula × P. tremuloides

494

WS0126_O09

EF148027

342

n.a.

No matches

n.a.

CF228572.1, P. tremula × P. alba

410

WS01118_P04

EF144846

300

n.a.

No matches

n.a.

CX184524.1, Populus × canadensis

242

WS0136_N09

EF148717

278

n.a.

No matches

n.a.

CX179364.1, Populus × canadensis

458

WS0138_I14

EF148811

231

n.a.

No matches

n.a.

CX170421.1, P. deltoides

228

To identify expressed genes that were not predicted in the original genome annotation [2], we searched among the set of 4,487 FLcDNAs with a stringent BLAT match to the genome that did not match to any of the 45,555 predicted gene models (E value ≥ 1e-50). This analysis revealed 173 FLcDNAs, 79 of which also showed strong similarity (E value < 1e-50) to one or more poplar ESTs in the public domain (see Additional file 1), suggesting that these 79 FLcDNAs represent expressed genes and possibly non-coding RNAs, that were missed by gene prediction software during the annotation of the poplar genome. The fact that these poplar transcripts had been missed could be due in part to the relatively short lengths of these 79 FLcDNAs (average FLcDNA and predicted ORF length of 555 bp and 67 bp, respectively; see Additional file 1).

Comparative sequence annotation of poplar FLcDNAs against Arabidopsis and other plants identifies proteins unique to poplar

Despite the growing research interest in poplar as a model angiosperm tree species and the recent completion of the poplar genome sequence, poplar still represents a difficult experimental system with relatively few functionally characterized proteins, compared to other established model systems such as Arabidopsis. Therefore, our effort of in silico annotation of poplar FLcDNAs was largely based on comparison with Arabidopsis together with the NR database of GenBank containing sequences from all plants, among other species. Using BLASTX, we found that the proportion of FLcDNAs with similarity to TAIR Arabidopsis proteins was 87.5% (4,081) at E value < 1e-05 and 55.5% (2,590) at E value < 1e-50 (Figure 6A). Similar values were obtained when using BLASTX to compare against peptides from other species in the NR division of GenBank (88.0% matches at E value < 1e-05 and 56.9% matches at E value < 1e-50) (Figure 6A). As expected, the proportion of poplar FLcDNAs with sequence similarity to previously published poplar ESTs (i.e., ESTs available in the dbEST division of GenBank, excluding ESTs from this study) by BLASTN was very high, with 96.3% (4,496) and 94.3% (4,401) of FLcDNAs having matches with E values < 1e-05 and < 1e-50, respectively (Figure 6A).
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Figure 6

Sequence annotation of 4,664 high-quality poplar FLcDNAs against published databases. Panel A shows the percentage of FLcDNAs with similarity to entries in three databases using expect (E) value thresholds of < 1e-05 and < 1e-50: matches to previously published poplar ESTs (i.e., ESTs available in GenBank, excluding ESTs from this study) identified by BLASTN; amino acid sequences in the non-redundant (NR) division of GenBank identified by BLASTX; and The Arabidopsis Information Resource (TAIR) non-redundant Arabidopsis peptide matches identified by BLASTX. Panel B shows a Venn diagram of distinct and overlapping patterns of sequence similarity against the three databases (public poplar ESTs, TAIR, NR) at a BLAST E value threshold of < 1e-05. At this threshold, 95 poplar FLcDNAs had no similarity to sequences in any of the databases examined.

To identify genes that are potentially unique to poplar, we next examined the relationship of sequence similarity among the poplar FLcDNAs and best matching sequences in the TAIR Arabidopsis proteins, other NR database proteins (which includes all plant species), and previously published poplar EST datasets. Of the 4,664 poplar FLcDNAs, 3,994 (85.6%) had at least low sequence similarity to sequences in all three databases (E values < 1e-05; Figure 6B). Only 95 FLcDNAs had no similarity (E values ≥ 1e-05) to sequences in any of these databases; however, 87 of these strongly matched to the poplar genome using BLAT (sequence identity ≥ 95%, alignment coverage ≥ 95%). Our results suggest that these 87 genes that are represented with FLcDNAs and with poplar genomic sequences are new genes that have not previously been identified in other poplar EST collections or among genes in Arabidopsis and other plant species (see Additional file 1).

In addition, we also identified 536 poplar FLcDNAs (including the 95 FLcDNAs with no similarity to sequences in the three databases examined) with no similarity to Arabidopsis or NR proteins (E values ≥ 1e-05), of which 346 FLcDNAs matched with high similarity to both the poplar genome by BLAT and to previously published poplar ESTs by BLASTN (E values < 1e-50; Figure 6B and see Additional file 1). These poplar FLcDNAs could represent genes that were gained and then rapidly diverged in sequence since the recent whole genome duplication in poplar, or they may also represent non-coding RNAs or small peptides in poplar that share limited sequence similarity with other plants. The fact that these putative poplar-specific FLcDNAs do not share similarity with existing plant sequence data may also reflect the limited availability of sequence data from Salicaceae species closely related to poplar in the current NR database. To test these putatively poplar-specific FLcDNAs for known functional domains, we performed a search of the Pfam database [42]. At a threshold of E values < 1e-05, we identified 2,908 (62.3%) poplar FLcDNAs with similarity to a Pfam domain; however, among the collection of 346 putatively poplar-specific genes only 8 FLcDNAs in this set matched a Pfam domain (see Additional file 1). Domain matches included PF05162.3/ribosomal protein L41 (WS0112_A21, WS0116_F12, WS0124_J06, WS01230_B01, and W01118_I11), PF05160.3/DSS1/SEM1 family (WS0123_P21), PF06376.2/unknown function (WS0112_B13), and PF04689.3/DNA binding protein S1FA (WS01110_K04).

Annotation of poplar FLcDNA transcripts affected by FTC herbivory

A major emphasis of the program that motivated the development and analysis of poplar FLcDNAs is the discovery of genes affected by insect attack. To identify herbivore-responsive genes among the poplar FLcDNAs, we first mapped the FLcDNA set onto a poplar 15.5 K microarray based on BLASTN comparison to ESTs spotted on the array. This microarray platform was previously used for profiling of the poplar leaf transcriptome affected by FTC larvae feeding [11]. Using a stringent similarity threshold of ≥ 95% identity over ≥ 95% alignment coverage, we identified 3,854 FLcDNAs that matched with 3,974 EST elements on the array (see Additional file 2). Although we did observe some cases of individual FLcDNAs mapping to multiple array elements, as well as multiple FLcDNAs mapping to the same array element, it should be noted that the in silico match stringency applied here is likely higher than the capability of cDNA microarrays to discriminate among highly similar transcripts by actual DNA hybridization. Next, we identified poplar FLcDNAs with a role in the response to insect attack by screening the 3,854 FLcDNAs against existing transcriptome data of differentially expressed (DE) genes in leaves that were exposed for 24 hours to FTC feeding [11]. This approach resulted in the identification of 129 and 24 FLcDNAs that were induced or repressed, respectively, in FTC-treated leaves compared to untreated control leaves (Tables 3 and 4) using the DE criteria of fold-change ≥ 2.0-fold, P value < 0.05 and Q value < 0.05. A complete list of expression data is provided [see Additional file 2]. Each of the 153 FLcDNAs was translated and evaluated for the presence of ORFs, and annotation was assigned based on manual examination of the highest scoring and most informative BLASTX matches in NR.
Table 3

FLcDNAs corresponding to transcripts most strongly induced by forest tent caterpillar (FTC) feeding [fold-change (FC) ≥ 2.0, P value < 0.05, Q value < 0.05]

    

NR BLASTP best match

FTC feeding @ 24 h

15.5 K Array ID

Matching FLcDNA ID

GenBank ID

FL status/ORF size (aa)

GenBank accession, gene name, species

BLAST score

FC

P

Q

WS0151_M13

WS0131_K04a

EF148503

FL/202

BAB85998.1, Kunitz trypsin inhibitor, Populus nigra

396

60.4

<0.001

<0.001

WS0132_F23

WS0133_O14a

EF148554

FL/202

BAB85997.1, Kunitz trypsin inhibitor, P. nigra

380

50.2

<0.001

<0.001

WS0134_B13

WS0134_B13

EF148557

FL/212

AAQ84217.1, Kunitz trypsin inhibitor, Populus trichocarpa × deltoides

387

46.2

<0.001

<0.001

WS0133_N23

WS0133_N23

EF148553

FL/197

CAJ21341.1, Kunitz trypsin inhibitor, P. nigra

383

38.8

<0.001

<0.001

WS0124_G12

WS0124_G12

EF147703

FL/159

AAQ08196.1, translation initiation factor 5A, Hevea brasiliensis

316

29.0

<0.001

<0.001

WS01223_D01

WS01223_D01

EF146918

FL/359

At1g74320, choline kinase, Arabidopsis thaliana

537

28.4

<0.001

<0.001

WS0134_E16

WS0134_E16

EF148571

5' trunc./124

AAA16342.1, vegetative storage protein, P. trichocarpa × deltoides

239

27.4

<0.001

<0.001

WS01120_O24

WS01120_O24

EF145143

3' trunc./56

At4g07960, putative glucosyltransferase, A. thaliana

72

26.4

<0.001

<0.001

WS01211_H19

WS01211_H19

EF146657

FL/337

CAN72815, hypothetical protein, Vitis vinifera

253

26.0

<0.001

<0.001

WS0121_J16

WS0122_N13

EF147347

FL/339

AAK01124.1, vegetative storage protein, P. trichocarpa × deltoides

509

25.4

<0.001

<0.001

WS0141_P05

WS0132_K10a

EF148516

FL/202

AAQ84216.1, Kunitz trypsin inhibitor, Populus trichocarpa × deltoides

386

22.7

<0.001

<0.001

WS01118_D16

WS01118_D16

EF144781

n.a.

No protein matches

n.a.

16.8

<0.001

<0.001

WS0168_C17

WS01119_J20

EF144899

FL/285

AAY43790.1, hypothetical protein, Gossypium hirsutum

77

16.0

<0.001

<0.001

WS01119_E18

WS01119_E18

EF144877

3' trunc./67

At5g61770, brix domain-containing protein, A. thaliana

85

15.7

<0.001

<0.001

WS0133_B24

WS0133_K20a

EF148543

FL/202

CAH59150.1, Kunitz trypsin inhibitor, Populus tremula

351

15.5

<0.001

<0.001

WS0155_D02

WS0138_H02a

EF148810

FL/251

BAB21610.2, mangrin/allene oxide cyclase, Bruguiera sexangula

336

14.4

<0.001

<0.001

WS0152_M24

WS0128_J15

EF148194

FL/91

At5g24165, hypothetical protein, A. thaliana

72

13.7

<0.001

<0.001

WS01118_N14

WS01118_N14

EF144837

frameshift/47

At4g27960, ubiquitin conjugating enzyme 9, A. thaliana

96

13.2

<0.001

<0.001

WS01212_M19

WS0128_D22

EF148166

FL/509

ABA01477.1, cytochrome P450, Gossypium hirsutum

726

12.3

<0.001

0.002

WS01211_N06

WS0118_O23a

EF146529

FL/225

ABS12347.1, dehydrin, P. nigra

167

11.8

<0.001

<0.001

WS0132_A15

WS01313_N19

EF148368

FL/396

At4g18550, lipase class 3 family protein, A. thaliana

385

11.6

<0.001

0.001

WS01212_B20

WS0128_L03

EF148205

FL/318

CAA73220.1, isoflavone reductase, Citrus × paradise

469

10.4

<0.001

<0.001

WS0122_C03

WS0122_C03

EF147271

FL/133

CAN82925.1, hypothetical protein, V. vinifera

114

9.2

<0.001

0.001

WS0113_H20

WS0113_H20

EF145803

n.a.

No protein matches

n.a.

8.8

<0.001

<0.001

WS0134_J14

WS0134_J14a

EF148597

FL/202

AAQ84216.1, Kunitz trypsin inhibitor, P. trichocarpa × deltoides

380

7.9

<0.001

<0.001

WS01120_N21

WS01120_N21

EF145138

n.a.

No protein matches

n.a.

6.9

<0.001

<0.001

WS0114_H12

WS0114_H12

EF145947

FL/252

At4g01470, major intrinsic family protein, A. thaliana

364

6.3

<0.001

<0.001

WS0126_E15

WS0126_E15

EF147963

FL/325

At1g30910, molybdenum cofactor sulfurase family protein, A. thaliana

444

6.2

<0.001

<0.001

WS0168_F14

WS01123_O20

EF145380

FL/217

At3g18030, phosphopantothenoyl cysteine decarboxylase, A. thaliana

350

6.2

<0.001

<0.001

PX0019_C05

PX0019_C05

EF144379

FL/214

AAF64453.1, heat-shock protein 90, Euphorbia esula

330

5.7

<0.001

<0.001

WS0205_K16

WS01214_G11

EF146815

FL/387

CAN71454.1, hypothetical protein, V. vinifera

682

5.6

<0.001

<0.001

WS0152_N17

WS0114_F10a

EF145928

FL/70

BAA03527.1, ATP synthase epsilon subunit, Ipomoea batatas

120

5.6

<0.001

0.001

WS01118_A11

WS0113_M04

EF145848

FL/97

At1g77710, ubiquitin-fold modifier precursor, A. thaliana

150

5.5

<0.001

<0.001

WS0132_L23

WS0132_L23

EF148518

FL/372

AAP87281.1, beta-1,3-glucanase, Hevea brasiliensis

540

5.4

<0.001

0.002

WS0124_C22

WS0124_C22

EF147658

5' trunc./142

CAA42660.1, luminal binding protein, Nicotiana tabacum

213

5.4

<0.001

<0.001

WS01116_C06

WS01123_N20

EF145376

FL/250

At4g38210, expansin A20 precursor, A. thaliana

351

5.2

<0.001

<0.001

WS0114_D04

WS01211_M02a

EF146676

FL/414

AAB71419.1, calreticulin, Ricinus communis

556

5.0

<0.001

<0.001

WS01117_O15

WS01117_O15

EF144759

FL/230

At4g11150, Vacuolar ATP synthase subunit E1, A. thaliana

295

4.7

<0.001

<0.001

WS0133_J24

WS0133_J24

EF148541

FL/177

At1g01250, AP2 transcription factor, A. thaliana

303

4.6

0.001

0.004

WS0148_P02

WS0127_F13

EF148073

5' trunc./283

At1g64660, methionine gamma-lyase, A. thaliana

424

4.5

<0.001

0.001

WS02010_D02

WS0126_C10a

EF147943

FL/68

NP_001066879.1, hypothetical protein, Oryza sativa

175

4.4

<0.001

<0.001

WS0155_H06

WS0125_E23

EF147828

FL/215

CAN69111.1, glutathione-S-transferase, V. vinifera

415

4.3

<0.001

<0.001

WS01119_L18

WS01119_L18

EF144906

FL/56

NP_001068325.1, 40S ribosomal protein, O. sativa

182

4.3

<0.001

<0.001

WS0134_F23

WS0134_F23

EF148579

FL/312

CAN79077.1, annexin, V. vinifera

575

4.2

<0.001

<0.001

WS0117_C05

WS0124_M24

EF147756

FL/538

AAA80588.1, calnexin, Glycine max

1231

4.1

<0.001

<0.001

WS0175_A23

WS01125_H02a

EF145504

FL/181

AAT08648.1, ADP-ribosylation factor, Hyacinthus orientalis

587

4.0

0.004

0.014

WS0153_O15

WS0135_A12

EF148616

FL/388

At4g24220, vein patterning 1, A. thaliana

711

4.0

<0.001

<0.001

WS0141_G12

WS01312_A02

EF148234

FL/273

At1g19180, hypothetical protein, A. thaliana

160

4.0

<0.001

0.003

WS0168_D23

WS01230_E07

EF147385

FL/420

ABD32854.1, hypothetical protein, Medicago truncatula

670

4.0

<0.001

0.001

WS0154_B02

WS01228_N21

EF147184

5' trunc./186

At5g07340, calnexin, A. thaliana

251

3.9

<0.001

<0.001

WS01116_D23

WS01116_D23

EF144634

FL/84

At3g60540, sec61beta family protein, A. thaliana

92

3.8

<0.001

<0.001

WS0117_O22

WS0117_O22a

EF146403

FL/68

At1g27330, hypothetical protein, A. thaliana

103

3.5

<0.001

<0.001

WS0122_A01

WS01227_N20

EF147117

FL/399

At1g74210, glycerophosphodiester phosphodiesterase, A. thaliana

606

3.5

<0.001

<0.001

WS0144_K08

WS01119_H21

EF144889

FL/358

ABQ10199.1, cysteine protease, Actinidia deliciosa

594

3.5

<0.001

<0.001

WS0147_I02

WS0125_D08

EF147814

FL/444

AAS79603.1, prephenate dehydratase, Ipomoea trifida

653

3.3

<0.001

0.001

WS0111_C18

WS0125_B22a

EF147800

FL/395

P47916, S-adenosyl methionine synthetase, P. deltoides

785

3.3

<0.001

0.001

WS0151_N14

WS0127_M05

EF148121

FL/485

Q01781, S-adenosylhomocysteine hydrolase, Petroselinum crispum

939

3.3

<0.001

<0.001

WS01212_P09

WS01212_P09

EF146734

FL/161

ABC47922.1, pathogenesis-related protein 1, Malus × domestica

236

3.2

0.005

0.016

PX0015_M10

PX0015_M10

EF144335

n.a.

No protein matches

n.a.

3.2

<0.001

<0.001

WS0111_A20

WS0111_A20

EF144935

FL/360

CAN67616.1, cupin family protein, V. vinifera

474

3.2

<0.001

<0.001

WS0117_P18

WS0117_P18

EF146411

FL/93

NP_001047293.1, hypoxia-responsive family protein, O. sativa

122

3.2

<0.001

<0.001

WS0131_J08

WS0131_J08

EF148502

FL/452

AAA70334.1, omega-3 fatty acid desaturase, Sesamum indicum

708

3.1

<0.001

<0.001

WS0173_J22

WS01229_P15

EF147254

frameshift/441

CAH05011.1, alpha-dioxygenase, Pisum sativum

679

3.1

<0.001

0.002

WS0151_H21

WS01314_F07a

EF148393

FL/505

AAB05641.1, protein disulphide isomerase, R. communis

786

3.1

<0.001

<0.001

WS0141_E06

WS0128_M17

EF148216

FL/338

CAN79663.1, hypothetical protein, V. vinifera

284

3.0

<0.001

<0.001

WS01211_D15

WS01211_D15

EF146643

FL/258

NP_001061550.1, 60S ribosomal protein L7A, O. sativa

398

3.0

0.004

0.012

WS01110_A05

WS01110_A05

EF144530

5' trunc./46

AAT45244.1, EPSP synthase, Conyza canadensis

87

3.0

<0.001

<0.001

WS0122_A21

WS0122_A21

EF147261

FL/349

At3g62600, DNAJ heat shock family protein, A. thaliana

542

3.0

<0.001

<0.001

WS0154_D16

PX0019_K19

EF144475

FL/172

ABL67655.1, cyclophilin, Citrus cv. Shiranuhi

303

3.0

<0.001

<0.001

WS0114_N12

WS0114_N12

EF146003

5' trunc./243

AAU08208.1, chloroplast ferritin precursor, Vigna angularis

357

3.0

0.001

0.007

WS0153_O16

WS0136_K07a

EF148708

FL/113

CAA40072.1, hypothetical protein, P. trichocarpa × deltoides

225

2.9

<0.001

<0.001

WS01117_D04

WS01117_D04

EF144703

FL/137

CAN73155.1, hypothetical protein, V. vinifera

110

2.9

<0.001

<0.001

WS01120_A02

WS01120_A02

EF145080

5' trunc./105

At1g03010, phototropic-responsive NPH3 family protein, A. thaliana

177

2.8

<0.001

0.001

WS0178_L06

WS01211_M01

EF146675

FL/415

NP_001064428.1, no apical meristem transcription factor, O. sativa

98

2.8

<0.001

0.001

WS0143_C23

WS01228_M23a

EF147179

FL/212

ABB89210.1, dehydroascorbate reductase, S. indicum

343

2.7

<0.001

<0.001

WS0127_I09

WS0127_I09

EF148095

FL/235

CAB77025.1, Rho GDP dissociation inhibitor, N. tabacum

294

2.7

0.003

0.012

PX0015_K10

PX0015_K10

EF144326

3' trunc./65

At2g15590, hypothetical protein, A. thaliana

39

2.7

0.001

0.004

WS0152_M05

WS01111_A23

EF144570

FL/125

At1g69230, nitrilase-associated protein, A. thaliana

80

2.7

0.001

0.006

WS0134_H19

WS0134_H19

EF148589

FL/461

At5g28237. tryptophan synthase, A. thaliana

579

2.7

<0.001

0.001

WS0122_P22

WS0122_P22

EF147367

5' trunc./46

AAS89832.1, flavonoid 3-O-glucosyltransferase, Fragaria × ananassa

47

2.6

0.009

0.023

WS0113_E03

WS0113_E03

EF145764

5' trunc./130

At1g73600, phosphoethanolamine N-methyltransferase, A. thaliana

198

2.6

<0.001

0.001

WS02012_L20

WS01212_L02a

EF146720

FL/440

AAV50009.1, N-hydroxycinnamoyl/benzoyltransferase, Malus × domestica

451

2.5

<0.001

0.001

WS0116_I22

WS01119_O01a

EF144919

FL/212

ABB89210.1, dehydroascorbate reductase, S. indicum

360

2.5

<0.001

0.001

WS0128_C01

WS0128_C01

EF148156

FL/205

CAC85245.1, salt tolerance protein, Beta vulgaris

246

2.5

0.001

0.005

PX0011_E19

PX0011_C19

EF144204

FL/341

At1g10840, eukaryotic translation initiation factor subunit 3, A. thaliana

573

2.5

<0.001

0.002

WS0128_M01

WS0128_M01

EF148209

5' trunc./197

ABN08481.1, homeodomain-related, M. truncatula

103

2.4

<0.001

0.003

WS01126_B13

WS01126_B13

EF145551

3' trunc./136

CAN77060.1, ubiquitin activating enzyme, V. vinifera

239

2.4

0.017

0.035

WS01125_E14

WS01125_E14a

EF145493

FL/207

NP_001058535.1, cyclophilin, O. sativa

340

2.4

<0.001

0.001

WS01218_P22

WS01120_G07a

EF145102

FL/170

NP_001050870.1, glycine-rich RNA-binding protein, O. sativa

144

2.4

0.004

0.013

WS01117_L06

WS01117_L06

EF144744

frameshift/136

NP_001046690.1, ribosomal protein L10A, O. sativa

171

2.4

<0.001

<0.001

WS01117_E15

WS01117_E15

EF144711

n.a.

No protein matches

n.a.

2.4

<0.001

0.001

WS01110_A14

WS0122_K19

EF147330

FL/476

AAF18411.1, integral membrane protein, Phaseolus vulgaris

897

2.4

<0.001

<0.001

WS0156_A21

WS0127_G12a

EF148080

n.a.

No protein matches

n.a.

2.4

0.017

0.035

WS0127_G19

WS0127_G19

EF148082

frameshift/251

At4g11640, serine racemase, A. thaliana

354

2.4

<0.001

0.002

WS0112_O04

WS0112_O04

EF145713

5' trunc./566

ABS01352.1, methionine synthase, Carica papaya

1073

2.4

<0.001

0.001

WS0155_E17

WS01212_I06a

EF146705

FL/363

ABM67589.1, flavanone 3-hydroxylase, V. vinifera

645

2.4

0.003

0.012

WS0168_M07

WS0137_H13a

EF148760

FL/62

ABF98145.1, hypothetical protein, O. sativa

57

2.4

<0.001

0.003

WS0119_H18

WS0117_P08

EF146405

5' trunc./188

CAN83141.1, hypothetical protein, V. vinifera

218

2.3

<0.001

0.003

WS0157_L22

WS0128_B17

EF148154

5' trunc./388

CAN76057.1, glucosyltransferase, V. vinifera

411

2.3

0.002

0.008

WS0185_E12

WS0124_A18

EF147646

FL/285

CAH60723.1, aquaporin, P. tremula × tremuloides

488

2.3

0.001

0.007

WS0125_I01

WS0125_I01

EF147858

FL/477

BAA36972.1, flavonoid 3-O-galactosyl transferase, Vigna mungo

442

2.3

0.003

0.011

PX0019_C07

PX0019_C07

EF144380

5' trunc./222

CAN74465.1, hypothetical protein, V. vinifera

369

2.3

0.015

0.033

WS01111_E24

WS0113_P06

EF145877

FL/290

AAN32641.1, short-chain alcohol dehydrogenase, Solanum tuberosum

399

2.3

<0.001

0.003

WS01212_B14

WS01214_D06a

EF146806

FL/363

ABM67589.1, flavanone 3-hydroxylase, V. vinifera

644

2.3

0.003

0.011

WS0181_A04

WS01312_M14

EF148294

frameshift/232

CAN74806, bZIP transcription factor, V. vinifera

152

2.3

0.002

0.009

WS0116_F22

WS0116_F22

EF146228

frameshift/239

At3g05290, mitochondrial substrate carrier protein, A. thaliana

283

2.3

0.004

0.013

WS01121_C12

WS01121_C12

EF145159

FL/216

At2g25110, MIR domain-containing protein, A. thaliana

349

2.3

<0.001

<0.001

WS01214_P11

WS01214_P11

EF146849

FL/219

ABL84692, glutathione S-transferase, V. vinifera

345

2.3

0.002

0.009

WS0128_G16

WS01228_N10

EF147182

FL/207

AAN03471.1, hypothetical protein, G. max

99

2.2

<0.001

<0.001

WS0209_J01

WS0135_O22

EF148667

FL/318

AAG23965.1, endochitinase, Vigna sesquipedalis

461

2.2

0.001

0.004

WS01119_M12

WS01110_H18

EF144553

FL/118

At5g04750, F1F0-ATPase inhibitor protein, A. thaliana

52

2.2

<0.001

<0.001

WS0205_L05

WS01228_D08

EF147142

frameshift/233

AAX85981.1, NAC4 protein, G. max

362

2.2

0.019

0.038

WS0123_D13

WS0137_E08

EF148737

FL/533

At5g58270, STARK1 ATPase, half ABC transporter, A. thaliana

642

2.2

<0.001

<0.001

WS0112_P02

WS0116_L21

EF146273

FL/145

At5g27670, histone 2A, A. thaliana

196

2.2

<0.001

0.002

WS01214_A14

WS01225_E15

EF146945

FL/330

At5g07010, sulfotransferase family protein, A. thaliana

394

2.2

0.002

0.009

WS01211_G15

WS01211_G15

EF146653

FL/507

AAL24049.1, cytochrome P450, Citrus sinensis

677

2.2

<0.001

0.002

WS0123_E09

WS0123_E09

EF147535

FL/210

ABB89210.1, dehydroascorbate reductase, S. indicum

332

2.2

<0.001

<0.001

WS0114_N11

WS0114_N11

EF146002

5' trunc./313

AAF73006.1, NADP-dependent malic enzyme, R. communis

450

2.1

<0.001

<0.001

WS0154_G22

WS0122_L10

EF147335

5' trunc./381

CAN74204.1, hypothetical protein, V. vinifera

535

2.1

0.001

0.005

WS0181_N15

WS0133_H05

EF148536

FL/283

ABG73415.1, chloroplast pigment-binding protein, N. tabacum

496

2.1

<0.001

0.001

WS0131_L08

WS0137_P12a

EF148792

FL/214

NP_001060368.1, emp24/gp25L/p24 transmembrane protein, O. sativa

288

2.1

<0.001

<0.001

WS0124_N24

WS0124_N24

EF147765

FL/584

NP_001048852.1, acyl-activating enzyme 11, O. sativa

750

2.1

0.017

0.036

WS0116_E14

WS0116_E14

EF146213

n.a.

No protein matches

n.a.

2.1

0.001

0.004

WS0128_N06

WS0128_N06

EF148221

FL/257

At4g18260, cytochrome b-561, A. thaliana

294

2.1

0.005

0.016

WS01122_N10

WS01122_N10

EF145286

FL/91

At1g62440, leucine-rich repeat extensin, A. thaliana

107

2.0

0.010

0.025

WS01214_M13

WS01214_M13

EF146841

FL/378

At5g45670, GDSL-motif/hydrolase family protein, A. thaliana

298

2.0

<0.001

0.001

WS01213_H17

WS01213_H17

EF146756

FL/597

At4g34200, phosphoglycerate dehydrogenase, A. thaliana

884

2.0

<0.001

0.003

WS01122_N02

WS01231_J04a

EF147472

FL/196

XP_001334748.1, hypothetical protein, Danio rerio

59

2.0

0.003

0.010

WS0156_F12

WS0118_O10

EF146525

FL/102

At2g18400, ribosomal protein L6, A. thaliana

165

2.0

<0.001

<0.001

aMultiple FLcDNAs match to the same microarray EST, a complete list of matching FLcDNAs is provided elsewhere [see Additional file 2].

Table 4

FLcDNAs corresponding to transcripts most strongly repressed by forest tent caterpillar (FTC) feeding [fold-change (FC) ≥ 2.0, P value < 0.05, Q value < 0.05]

    

NR BLASTP best match

FTC feeding @ 24 h

15.5 K Array ID

Matching FLcDNA ID

GenBank ID

FL status/ORF size (aa)

GenBank accession, gene name, species

BLAST score

FC

P

Q

WS0162_B18

WS01227_D07

EF147075

FL/465

AAX84673.1, cysteine protease, Manihot esculenta

782

0.33

<0.001

<0.001

WS0112_D20

WS0112_D20

EF145637

FL/99

At1g67910, hypothetical protein, Arabidopsis thaliana

69

0.34

<0.001

0.001

WS0126_C06

WS0126_C06

EF147942

FL/121

At2g45180, protease inhibitor/lipid transfer protein, A. thaliana

108

0.34

0.018

0.038

WS0131_P03

WS0131_P03a

EF148510

FL/303

CAN63090.1, zinc finger transcription factor, Vitis vinifera

135

0.36

<0.001

0.001

WS0178_F11

WS01228_M08

EF147174

5' trunc./106

At1g22770, gigantea protein, A. thaliana

150

0.38

<0.001

0.002

WS0127_F15

WS0127_F15

EF148074

FL/173

CAN68427.1, hypothetical protein, V. vinifera

207

0.40

<0.001

0.001

WS0121_B24

WS0128_M21

EF148217

FL/139

AAU03358.1, acyl carrier protein, Lycopersicon esculentum

119

0.41

<0.001

<0.001

WS0147_J04

WS0134_M10

EF148605

n.a.

No protein matches

n.a.

0.41

0.004

0.014

WS0158_G10

WS0128_E13

EF148173

5' trunc./628

At1g56070, elongation factor, A. thaliana

1239

0.41

0.001

0.005

WS0152_E14

WS0112_O08a

EF145715

FL/252

ABH09330.1, aquaporin, V. vinifera

375

0.42

<0.001

0.003

WS0143_B24

WS01227_O15

EF147121

FL/267

At1g06460, small heat shock protein, A. thaliana

146

0.42

<0.001

0.001

WS0127_G18

WS0127_G18

EF148081

n.a.

No protein matches

n.a.

0.43

<0.001

<0.001

WS0182_D02

WS01226_N23

EF147055

FL/335

CAN75691.1, methyltransferase, V. vinifera

534

0.43

0.001

0.005

WS0124_D16

WS0124_D16

EF147668

FL/164

At3g62550, universal stress protein, A. thaliana

188

0.44

<0.001

0.001

WS0163_G24

WS0115_E02

EF146059

FL/341

AAD56659.1, malate dehydrogenase, Glycine max

566

0.45

0.003

0.010

WS0175_O14

WS01313_J01a

EF148349

FL/239

CAN63226.1, hypothetical protein, V. vinifera

313

0.45

<0.001

0.001

WS0178_N22

WS01111_H24

EF144589

FL/161

ABG27020.1, SKP1-like ubiquitin-protein ligase, Medicago truncatula

219

0.46

<0.001

<0.001

WS0121_H19

WS0121_H19

EF146882

FL/350

AAW66657.1, thiamine biosynthetic enzyme, Picrorhiza kurrooa

539

0.48

0.005

0.016

WS0206_B21

WS0131_B11

EF148494

FL/133

CAA59409.1, photosystem II reaction center protein, Spinacia oleracea

140

0.48

0.001

0.006

WS0155_M12

WS0136_E20

EF148683

FL/234

CAN60736.1, hypothetical protein, V. vinifera

313

0.48

0.001

0.007

WS0152_F02

WS01117_K24

EF144742

FL/384

CAN83255.1, CCCH-type zinc finger protein, V. vinifera

432

0.49

<0.001

0.002

WS01224_P10

WS0124_L08a

EF147742

FL/137

CAA28450.1, photosystem II 10 kDa polypeptide, Solanum tuberosum

191

0.49

<0.001

0.003

WS0115_N05

WS0115_N05

EF146146

FL/250

AAM21317.1, auxin-regulated protein, Populus tremula × tremuloides

449

0.50

0.005

0.016

WS0125_F02

WS0125_F02

EF147829

FL/516

At1g60590, polygalacturonase, A. thaliana

715

0.50

0.001

0.005

aMultiple FLcDNAs match to the same microarray EST, a complete list of matching FLcDNAs is provided elsewhere [see Additional file 2].

Among FTC-induced transcripts represented with FLcDNAs, we identified a large number of defense-related and stress response proteins such as chitinases, Kunitz protease inhibitors, dehydrins, beta-1,3-glucanases, pathogenesis related protein PR-1, and glutathione-S-transferase (Table 3). Several classes of transcription factors (TFs) were also strongly affected by FTC feeding such as bZIP domain TFs, NAC domain TFs, NAM domain TFs and ethylene response factor TFs. A number of genes associated with signaling were also strongly affected by FTC feeding, including allene oxide cyclase involved in jasmonate formation and calreticulin associated with calcium signaling. We also observed a substantial number of FLcDNAs annotated as involved in phenolic metabolism, particularly flavonoid biosynthesis, including isoflavone reductase, EPSP synthase, flavonoid 3-O-glycosyl transferase and flavanone 3-hydroxylase, along with several cytochrome P450s of unknown function (Table 3). Among the FTC-repressed transcripts represented with FLcDNAs, we observed photosystem II proteins associated with photosynthesis, malate dehydrogenase and thiamine biosynthesis enzyme associated with primary metabolism, several zinc finger TFs, and stress-responsive proteins such as small heat shock and universal stress proteins (Table 4). Twenty two of the 153 FTC-responsive genes represented with FLcDNAs matched to hypothetical proteins of unknown function and nine have no obvious similarity to any proteins in the NR database.

Discussion

Previous studies using the biotinylated CAP trapper method for FLcDNA library construction have demonstrated this technique to be highly effective for capturing predominantly true full-length clones in large-scale projects [24, 25, 27]. In this study, we generated a set of 4,664 FLcDNAs, which represents the third largest plant FLcDNA resource published to date, behind only Arabidopsis and rice. CAP3 clustering and assembly indicates that more than 85% of the FLcDNAs are non-redundant within this collection. The average sequence length, ORF and UTR sizes of the poplar FLcDNAs were comparable to those observed with the CAP trapper-derived FLcDNA collections for maize [27], Arabidopsis [40] and rice [24], and were also very similar to the ab initio predicted reference genes in the poplar genome sequence [2]. Applying a reciprocal BLAST strategy, we demonstrated that among FLcDNAs with high sequence similarity to known Arabidopsis peptides and/or previously published poplar FLcDNAs, nearly 80% had similar ORF lengths and starting methionine and stop codon positions. Collectively, these data show that the poplar FLcDNA libraries are of high quality and that our clone selection strategy combined with the CAP trapper method was effective in capturing bona fide FLcDNAs from poplar.

Comparison of poplar FLcDNAs and the poplar genome sequence assembly confirmed both the overall high accuracy of the current genome assembly, as well as the quality of the FLcDNA resource described here. However, as has been previously demonstrated with efforts to identify the complete catalogue of genes in Arabidopsis and rice, gene prediction and genome assembly is an iterative process. The results reported here for the mapping of FLcDNAs to the poplar genome sequence reveal opportunities for improvement of the genome sequence assembly (i.e., targeting apparent gaps for re-sequencing), as well as opportunities to further improve tools for the in silico prediction of genes. To address the discovery of apparent gaps in the genome assembly, the availability of 39 FLcDNAs that are not covered in the current assembly could be used to target BAC clones for re-sequencing and filling of gap regions. Similarly, the discovery of 173 FLcDNAs that do not have corresponding gene predictions in the current genome annotation may provide an opportunity to further improve gene prediction tools for poplar. Algorithms used for gene prediction in the poplar genome sequence assembly could be tested with these 173 FLcDNAs to find out why they may have initially been missed. If this leads to an improvement of prediction tools, the assembled genome sequence could be tested with the modified tools to identify additional genes.

The comparative sequence annotation of poplar FLcDNAs against Arabidopsis, the NR database, and previously published poplar ESTs revealed that ca. 88% of poplar FLcDNAs showed similarity to sequences in Arabidopsis or other plants. Many of the ca. 11.5% of poplar FLcDNAs without significant sequence similarity in Arabidopsis or other plants are supported with evidence of gene expression in the form of previously published poplar ESTs and matching the poplar genome sequence, thus excluding the possibility that they are artifacts of cDNA library construction. The discovery of poplar FLcDNAs without matches in other plant species is also in agreement with previous analysis of the poplar genome sequence where 11% of predicted proteins had no similarity to proteins in the NR database and 12% had no similarity to Arabidopsis proteins [2]. For comparison, only 64% of the 28,444 ORFs derived from rice FLcDNAs showed significant similarity to coding sequences predicted from the Arabidopsis genome and conversely, only 75% of Arabidopsis coding sequences had similarity to rice FLcDNAs [24]. These findings suggest that a substantial proportion of protein-coding sequences are not conserved among all plant species. The putative poplar-specific genes could be the product of past local or whole genome duplications in the lineage that led to extant poplar species [2, 43] followed by sequence divergence [44, 45]. Furthermore, ca. 2% of poplar FLcDNAs did not contain a predicted ORF suggesting these putative poplar-specific genes likely encode non-coding RNAs (i.e., rRNAs, tRNAs, snoRNAs etc.).

Conclusion

We developed a large FLcDNA resource of high sequence quality and low-level redundancy that facilitated the discovery of a substantial number of genes not present among the published sequences of other plant species, and that also facilitated the discovery of several hundred insect-affected genes in the poplar leaf transcriptome that were represented by FLcDNAs. The newly established poplar FLcDNA resource will be valuable for further improvement of the poplar genome assembly, annotation of protein-coding regions, and for functional and comparative analysis of poplar genes. Specifically, the identification of FLcDNAs that are not covered in the current genome assembly or that were not predicted during the genome annotation provides opportunities to further refine the current genome assembly. The availability of a large collection of FLcDNAs that show altered gene expression following insect herbivory affords more rapid characterization of the role of these genes in poplar biotic interactions.

Methods

Full-length cDNA libraries

Plant materials used in the construction of cDNA libraries are described in Table 1. Isolation of total and poly(A)+ RNA are described elsewhere (see Additional file 3). FLcDNA libraries were directionally constructed (5' Sst I and 3' Xho I) according to published methods [46, 47], with modifications described in detail elsewhere (see Additional file 3).

DNA sequencing and sequence filtering

Details of bacterial transformation with plasmids, clone handling, DNA purification and evaluation, and DNA sequencing are provided elsewhere (see Additional file 3). Sequences from each cDNA library were closely monitored to assess library complexity and sequence quality. DNA sequence chromatograms were processed using the PHRED software (versions 0.000925.c and 0.020425.c) [48, 49]. Sequences were quality-trimmed according to the high-quality (hq) contiguous region determined by PHRED and vector-trimmed using CROSS_MATCH software [50]. Sequences with less than 100 quality bases (Phred 20 or better) after trimming and sequences having polyA tails of ≥ 100 bases were removed from analysis. Also removed were sequences representing bacterial, yeast or fungal contaminations identified by BLAST searches [51, 52] against E. coli K12 DNA sequence (GI: 6626251), Saccharomyces cerevisiae [53], Aspergillus nidulans (TIGR ANGI.060302), and Agrobacterium tumefaciens (custom database generated using SRS, Lion Biosciences). Sequences were also compared to the GenBank NR database using BLASTX. Top ranked BLAST hits involving other non-plant species and with E values < 1e-10 were classified as contaminants and removed prior to EST assembly.

Selection of candidate FLcDNA clones and sequencing strategy

All 3'-end ESTs remaining after filtering were clustered and assembled using CAP3 [39] (assembly criteria: 95% identity, 40 bp window). The resulting contigs and singletons were defined as the PUT set. PUTs with a cDNA clone from a FLcDNA library were selected as candidates for complete insert sequencing (Figure 1). Candidate clones from FLcDNA libraries were single-pass sequenced from both 3'- and 5'-ends and both sequences were used for subsequent clone selection. Next, clones were screened for the presence of a polyA tail (3'-end EST) and the second-strand primer adaptor (SSPA; 5'-ACTAGTTTAATTAAATTAATCCCCCCCCCCC-3'; 5'-end EST). Clones lacking either of these features were eliminated. A polyA tail was defined as at least 12 consecutive, or 14 of 15 "A" residues within the last 30 nt of the 3'-end EST (5' to 3'). The presence of the SSPA was detected using the Needleman-Wunsch algorithm limiting the search to the first 30 nt of the 5'-end EST (5' to 3'). The SSPA was defined as eight consecutive "C" residues and a > 80% match to the remaining sequence (5'-ACTAGTTTAATTAAATTAAT-3'). In each case, the algorithms used to detect the 5' and 3' clone features were set to produce maximal sensitivity while maintaining a 0% false positive rate, as determined using test data sets. Candidate clones for which either of the initial 5'-end or 3'-end EST reads had a Phred20 quality length of < 100 nt were also excluded. Finally, candidate clones were compared to poplar ESTs in the public domain (excluding ESTs from this collection; BLASTN match E < 1e-80) to identify candidate FLcDNAs potentially truncated at the 5' end of the transcript relative to a matching EST. Any clone with a 5' end that was > 100 nt shorter than the matching public EST was excluded. For each PUT represented by multiple candidate clones after filtering, the clone with the longest 5' sequence was selected for complete insert sequencing. Insert sizing performed on 4,848 of 5,926 candidate clones using colony PCR with vector primers and standard gel electrophoresis revealed an average insert size of ca. 1,085 bp. Based on this information, a sequencing strategy emphasizing the use of end reads was chosen.

Sequence finishing of FLcDNA clones

FLcDNA clones selected for complete sequence finishing were rearrayed into 384-well plates, followed by an additional round of 5'-end and 3'-end sequencing using vector primers. All end reads from an individual clone were then assembled using PHRAP (version 0990329) [4850]. To meet our sequence quality criteria, the resulting clone consensus sequence was required to achieve a minimum average score of Phred35, with each base position having a minimum score of Phred30. Each base position also required at least two sequence reads, of minimum Phred20, that were in agreement with the consensus sequence (i.e., no high-quality discrepancies). Clones that did not meet these finishing criteria after two rounds of end read sequencing were then subjected to successive rounds of sequencing using custom primers designed using the Consed graphical tool version 14 [54] until the required quality levels were achieved. Regardless of the finishing strategy, all clones that did not meet the minimum finishing criteria according to an automated pipeline were flagged for manual examination. Clones were aborted if they were manually verified to lack the minimum finishing criteria after three rounds of custom primer design, were identified as chimeric sequences, or were refractory to sequence finishing due to the presence of a "hard-stop". FLcDNA sequences have been deposited in the NR division of GenBank [EF144175 to EF148838].

Gene expression meta-analysis of FLcDNAs

Poplar FLcDNA sequences were mapped to a cDNA microarray containing 15,496 poplar ESTs [[11]; Gene Expression Omnibus (GEO) platform number GPL5921] using BLASTN with a stringent threshold of ≥ 95% identity over ≥ 95% of alignment coverage. To identify FLcDNAs that were DE following FTC feeding, FLcDNAs mapping to the microarray were matched to an existing microarray dataset that examined gene expression in hybrid poplar leaves 24 hours after continuous FTC feeding ([11]; GEO series number GSE9522).

Notes

Declarations

Acknowledgements

We thank Diana Palmquist, Brian Wynhoven, Jerry Liu, Yaron Butterfield and Asim Siddiqui of the Genome Sciences Centre for assistance with bioinformatic analyses; Jeff Stott, George Yang and many other staff at the Genome Sciences Centre for assistance with DNA sequencing; Claire Oddy and Sharon Jancsik of the University of British Columbia for assistance with clone insert sizing; Bob McCron from the Canadian Forest Service for access to forest tent caterpillars; and David Kaplan for greenhouse support. The work was supported by Genome British Columbia, Genome Canada and the Province of British Columbia (Treenomix Conifer Forest Health grant to J.B., and Treenomix grant to J.B. and C.J.D.), and by the Natural Science and Engineering Research Council of Canada (NSERC, grant to J.B.). Salary support for J.B. has been provided, in part, by the UBC Distinguished University Scholar Program and an NSERC Steacie Memorial Fellowship.

Authors’ Affiliations

(1)
Michael Smith Laboratories, University of British Columbia
(2)
British Columbia Cancer Agency Genome Sciences Centre
(3)
Department of Botany, University of British Columbia
(4)
Environmental Sciences Division, Oak Ridge National Laboratory
(5)
Department of Forest Sciences, University of British Columbia
(6)
Department of Biology, University of North Dakota

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