Open Access

SolEST database: a "one-stop shop" approach to the study of Solanaceae transcriptomes

  • Nunzio D'Agostino1,
  • Alessandra Traini1,
  • Luigi Frusciante1 and
  • Maria Luisa Chiusano1Email author
BMC Plant Biology20099:142

DOI: 10.1186/1471-2229-9-142

Received: 31 July 2009

Accepted: 30 November 2009

Published: 30 November 2009

Abstract

Background

Since no genome sequences of solanaceous plants have yet been completed, expressed sequence tag (EST) collections represent a reliable tool for broad sampling of Solanaceae transcriptomes, an attractive route for understanding Solanaceae genome functionality and a powerful reference for the structural annotation of emerging Solanaceae genome sequences.

Description

We describe the SolEST database http://biosrv.cab.unina.it/solestdb which integrates different EST datasets from both cultivated and wild Solanaceae species and from two species of the genus Coffea. Background as well as processed data contained in the database, extensively linked to external related resources, represent an invaluable source of information for these plant families. Two novel features differentiate SolEST from other resources: i) the option of accessing and then visualizing Solanaceae EST/TC alignments along the emerging tomato and potato genome sequences; ii) the opportunity to compare different Solanaceae assemblies generated by diverse research groups in the attempt to address a common complaint in the SOL community.

Conclusion

Different databases have been established worldwide for collecting Solanaceae ESTs and are related in concept, content and utility to the one presented herein. However, the SolEST database has several distinguishing features that make it appealing for the research community and facilitates a "one-stop shop" for the study of Solanaceae transcriptomes.

Background

Solanaceae represents one of the largest and most diverse plant families including vegetables (e.g. tomato, potato, capsicum, and eggplant), commercial (e.g. tobacco) and ornamental crops (e.g. petunia). Some Solanaceae plants are important model systems such as tomato for fruit ripening [1, 2], tobacco for plant defence [3], and petunia for the biology of anthocyanin pigments [4].

Since no full genome sequence of a member of the Solanaceae family is yet available, though genome sequencing efforts are at the moment ongoing for tomato [5], potato http://www.potatogenome.net/ and tobacco http://www.tobaccogenome.org/, much of the existing worldwide sequence data consists of Expressed Sequence Tags (ESTs). Because of the useful information these data bring to the genomics of Solanaceae plants, the availability of EST collections has dramatically increased in size, partly thanks to the start-up of sequencing initiatives [6]. EST collections are certainly no substitute for a whole genome scaffold. However, they represent the core foundation for understanding genome functionality, the most attractive route for broad sampling of Solanaceae transcriptomes and, finally, a valid contribution to comparative analysis at molecular level on the Solanaceae family members. ESTs are a versatile data source and have multiple applications which result from the specific analytical tools and methods accordingly used to process this type of sequence.

Therefore EST databases are useful not only to strictly serve as sequence repositories, but as powerful tools, albeit relatively under-exploited and far from complete.

Several web resources have been established for collecting ESTs and for improving and investigating their biological information content due to the growing interest in Solanaceae genomics research. Some of them, mainly focusing on individual species, address the needs of a particular research community by providing a catalogue of putative transcripts, describing their functional roles and enabling gene expression profiling [7, 8]. The remaining represent data-gathering centres or rather comprehensive resources aiming to meet the challenges raised by the management of multiple information from diverse sources worldwide [911].

The ultimate goal of these gene index providers is to represent a non-redundant view of all EST-defined genes. The unigene builds, which emerge, serve as the basis for a number of analyses comprising the detection of full-length transcripts and potential alternative splicing, expression pattern definition, association to array probes and, as a consequence, to microarray gene expression databases; association to metabolic and signalling pathways; development of simple sequence repeat (SSR) and conserved ortholog set (COS) markers etc.

We present the SolEST database, which integrates different EST datasets from both cultivated and wild Solanaceae species and also two EST collections from Rubiaceae (genus Coffea). SolEST is built on the basis of a preceding effort which was centred on the investigation of ESTs from multiple tomato species [12]. The main purpose is corroborating the existing transcriptomics data which are part of the multilevel computational environment ISOL@ [13]. In addition, the Solanaceae EST-based survey can considerably contribute to genome sequence annotation by highlighting compositional and functional features. Indeed, SolEST is a valuable resource for the ongoing genome sequencing projects of tomato (S. lycopersicum) [5] and potato (S. tuberosum; http://www.potatogenome.net/) and has the potential to significantly improve our understanding of Solanaceae genomes and address sequence-based synteny issues.

A common complaint in the SOL community concerns the different unique transcript sets generated for a given Solanaceae species by diverse research groups. These worldwide resources [912] are built starting from different primary data sets and by applying diverse methods and user-defined criteria for sequence analysis. Of course, there are advantages and disadvantages associated to each set, but to our knowledge, there is currently no easy way to compare them and, as a consequence, to provide the scientific community with a comprehensive overview. To this end, we also propose, as a novel feature of the SolEST database, a combined resource/interface dedicated to enabling the combination of different unigene collections for each Solanaceae species based on the UniProt Knowledgebase annotations.

The collection and integration of the whole public dataset of Solanaceae ESTs facilitate a "one-stop shop" for the study of Solanaceae transcriptomes.

Construction and content

Sequence retrieval

EST sequences are downloaded from dbEST http://www.ncbi.nlm.nih.gov/dbEST/ and from the Nucleotide/mRNA division of GenBank (release 011008).

EST/mRNA processing pipeline

The EST processing and annotation pipeline is described in [14] although it has been recently upgraded by updating the set of databases used in EST vector cleaning and repeat masking and in the annotation phase. In addition, the clustering tool was replaced with a more efficient novel method presented in [15]. This pipeline, divided into four consecutive steps, was used for processing EST data from 14 cultivated and wild Solanaceae species and from two species belonging to the genus Coffea (Table 1).
Table 1

The SolEST database statistics.

Source

# ESTs

EST length

# mRNA

# mRNA length

# Cluster

# TCs

TC length

# ESTs in TCs

# sESTs

SEST length

# Unique transcripts

SOLLC

259990

522.47 ± 156.39

5770

1377.23 ± 735.42

17001

20548

1019.43 ± 544.69

234297

30937

491.07 ± 246.90

51485

SOLPN

8346

460.38 ± 129.80

13

1854 ± 974.32

817

844

666.91 ± 265.85

5249

3110

470.86 ± 165.38

3954

SOLHA

8000

617.39 ± 165.42

30

864.07 ± 537.43

1119

1243

900.79 ± 342.79

5323

2707

561.14 ± 171.26

3950

SOLLP

1008

352.89 ± 133.45

-

-

103

109

478.06 ± 151.54

413

594

342.03 ± 136.70

703

RNA

231275

611.41 ± 205.52

1704

1144.24 ± 663.38

18590

23453

983.56 ± 429.02

184233

48630

627.26 ± 236.38

72083

SOLCH

7752

812.65 ± 152.46

60

1008.97 ± 533.89

632

637

845.18 ± 266.09

1513

6279

824.93 ± 154.44

6916

TOBAC

240440

601.54 ± 231.81

3605

795.75 ± 805.60

24274

28571

934.99 ± 423.1

158264

81247

565.44 ± 262.19

109818

NICBE

42566

611.86 ± 243.81

301

1260.82 ± 1379.73

4452

5006

984.41 ± 443.85

29051

13784

505.82 ± 299.38

18790

NICSY

8583

381.48 ± 168.21

94

1577.17 ± 1125.60

662

674

457.99 ± 437.09

1838

6831

400.25 ± 209.85

7505

NICAT

329

303.60 ± 152.31

94

1239.71 ± 726.51

32

32

949.5 ± 775.84

68

352

461.88 ± 463.58

384

NICLS

12448

492.11 ± 205.98

95

831.58 ± 456.42

1268

1379

651.89 ± 252.34

7570

4969

467.14 ± 215.44

6348

CAPAN

33311

466.73 ± 154.98

564

974.95 ± 587.75

4082

4293

760.45 ± 331.46

22144

11714

460.65 ± 194.94

16007

CAPCH

372

464.35 ± 228.64

105

1072.8 ± 642.35

32

34

901.97 ± 490.54

86

389

572.65 ± 446.71

423

PETHY

14017

500.50 ± 185.75

323

1254.12 ± 724.09

1627

1738

704.4 ± 308.88

6642

7612

520.40 ± 268.87

9350

COFCA

55694

613.87 ± 174.08

100

1158.32 ± 643.76

6141

6620

863.97 ± 325.59

42873

12732

548.20 ± 181

19352

COFAR

1577

413.29 ± 149.78

150

725.25 ± 534.10

129

137

644.16 ± 333.3

455

1271

421.38 ± 207.41

1408

 

925708

 

13008

 

80961

925708

 

700019

233158

 

328476

Source: SOLLC: S. lycopersicum; SOLPN: S. pennellii; SOLHA: S. habrochaites; SOLLP: S. lycopersicum × S. pimpinellifolium; SOLTU: S. tuberosum; SOLCH: S. chacoense; TOBAC: N. tabacum; NICBE: N. benthamiana; NICSY: N. sylvestris; NICAT: N. attenuata; NICLS: N. langsdorffii × N. sanderae; CAPAN: C. annuum; CAPCH: C. chinense; PETHY: Petunia × hybrida; COFCA: C. canephora; COFAR: C. arabica. #ESTs: number of raw ESTs from dbEST; EST length: average length and standard deviation; #mRNA: number of mRNA from GenBank;mRNA length: average length and standard deviation; #cluster: number of clusters created by grouping overlapping EST sequences; #TCs: number of tentative consensuses which are generated from multiple sequence alignments of ESTs (assembling process); TC length: average length and standard deviation; #ESTs in TCs: number of ESTs assembled to generate TCs; #sESTs:number of singleton ESTs; sESTs length: average length and standard deviation;#Unique transcripts: number of total transcripts obtained adding the sESTs to the TCs.

(1) Vector cleaning

RepeatMasker http://repeatmasker.org is used to identify and mask vector sequences by using the NCBI's Vector database (ftp://ftp.ncbi.nih.gov/blast/db/FASTA/vector.gz; update October 2008). The masked regions are removed with an in-house developed trimming tool.

(2) Repeat masking

EST sequences are masked using the RepeatMasker program with the RepBase.13.06 http://www.girinst.org/ as selected repeat database. Targets for masking include low-complexity regions, simple sequence repeats (SSR, also referred to as microsatellites) and other DNA repeats (e.g. transposable elements).

(3) Clustering and assembling

For each collection the rate of sequence redundancy was evaluated by first clustering, then assembling EST reads to produce tentative consensus sequences (TCs) and singletons (sESTs; see Table 1). The wcd tool [15] was used with its default parameters for the clustering process. The CAP3 assembler [16] with overlap length cutoff (= 60) and an overlap percent identity cutoff (>85) was run to assemble each wcd cluster into one or more assembled sets of sequences (i.e. TCs). Indeed, when sequences in a cluster cannot always be all reconciled into a solid and reliable multiple alignment during the assembly process, they are divided into multiple assemblies/TCs. Possible interpretations are: (i) alternative transcription, (ii) paralogy or (iii) protein domain sharing. All the ESTs that did not meet the match criteria to be clustered/assembled with any other EST in the collection were defined as singleton ESTs.

(4) Annotation

Functional annotation, which is performed both on EST sequences and TCs, is based on the detection of similarities (E-value ≤ 0.001) with proteins by BLAST searches versus the UniProtKB/Swiss-prot (Release 14.3) database. BLAST annotation is detailed including fine-grained gene ontology terms http://www.geneontology.org/ and Enzyme Commission numbers http://www.expasy.ch/enzyme/. A back-end tool to align on-the-fly the unique transcripts against the annotated KEGG-based metabolic pathways http://www.genome.jp/kegg/ was also implemented.

Database content and web interface

Raw input ESTs, intermediate data (from the pre-processing analysis) as well as transcript assembly data and annotation information were stored in a MySQL relational database whose structure reproduces the one described in [12]. Web interfaces were implemented in dynamic PHP pages and include Java tree-views for easy object navigation (http://biosrv.cab.unina.it/solestdb/; Figure 1). In addition to well-established access to the EST-based resources via web interfaces, all sequence datasets are available for bulk download in FASTA format in a typical web-based data exchange scenario on the web http://biosrv.cab.unina.it/solestdb/download.php.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-9-142/MediaObjects/12870_2009_Article_484_Fig1_HTML.jpg
Figure 1

Snapshots of the SolEST database web interface. A: TC structure and functional annotation. B: BLASTx alignment to protein. C1: Data classification by ENZYME scheme. C2: Data classification by KEGG metabolic pathways. D: Transcript association to KEGG metabolic maps.

Utility

Simple sequence repeat (SSR) characterization

EST and mRNA sequences were explored for the existence of microsatellite repeat motifs since they are potential resources for SSR marker discovery [17, 18]. Our research focused on trimeric, tetrameric, pentameric and hexameric repeat motifs. In the entire collection we found 10 trimeric, 28 tetrameric, 88 pentameric and 16 hexameric motifs. SSR summary statistics are reported in Table 2, while the frequency of different types of SSR motifs, which were identified species by species, can be found in Additional file 1. In Figure 2, we report the average repeat length and the standard deviation for each SSR motif.
Table 2

Simple Sequence Repeats (SSR) summary statistics.

 

# sequences analysed

#SSRs identified

# SSR-containing sequences

#sequences containing >1 SSR

SOLLC

265760

9636

8758

698

SOLPN

8359

360

349

10

SOLHA

8030

400

367

27

SOLLP

1008

17

15

2

SOLTU

232979

12364

10591

1551

SOLCH

7812

362

321

30

TOBAC

244045

9875

8434

958

NICBE

42867

2109

1880

197

NICSY

8677

0

0

0

NICAT

423

11

11

0

NICLS

12543

278

265

10

CAPAN

33875

1386

1271

108

CAPCH

477

14

13

1

PETHY

14340

454

420

27

COFCA

55794

3173

2936

200

COFAR

1727

142

121

15

Σ

938716

40581

35752

3834

Source: SOLLC: S. lycopersicum; SOLPN: S. pennellii; SOLHA: S. habrochaites; SOLLP: S. lycopersicum × S. pimpinellifolium; SOLTU: S. tuberosum; SOLCH: S. chacoense; TOBAC: N. tabacum; NICBE: N. benthamiana; NICSY: N. sylvestris; NICAT: N. attenuata; NICLS: N. langsdorffii × N. sanderae; CAPAN: C. annuum; CAPCH: C. chinense; PETHY: Petunia × hybrida; COFCA: C. canephora; COFAR: C. arabica.

For each species we show the number of the sequences analysed, the number of the microsatellites identified, the total of the SSR-containing sequences and the amount of sequences containing more than one SSR.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-9-142/MediaObjects/12870_2009_Article_484_Fig2_HTML.jpg
Figure 2

SSR motif average length.

Comparison of different Solanaceaeunique transcript sets

We considered the most accessed and referenced Solanaceae unigene collections freely available on the web [911] in an effort to enable comparisons of different unigene projects for a given species by a comprehensive approach.

Different Solanaceae and Rubiaceae (genus Coffea) expressed unique transcript sets from the DFCI Gene Index Project (DFCI; http://compbio.dfci.harvard.edu/tgi/plant.html), the plantGDB (PGDB; http://www.plantgdb.org/download/download.php?dir=/Sequence/ESTcontig) and the Solanaceae Genome Network (SGN; ftp://ftp.sgn.cornell.edu/unigene_builds/) were downloaded.

In Table 3 the number of collected sequences per species is reported for each of the resources taken into account. Each dataset was compared versus the UniProtKB/Swiss-prot (Release 14.3) database using BLASTX (e-value = 0.001) and the corresponding results are summarized in Table 4. A total of 29,463 distinct proteins were matched corresponding to ~7.35% of the whole protein collection made up of 400,771 sequences. When considering annotations with respect to the origin of the protein data source, the bulk of the identifications concerned proteins of plant and vertebrata origin (35% and 34%, respectively), while protein from bacteria and fungi represent 12% and 9% as reported in figure 3. We built a web tool dedicated to enable the association of different unigene collections for a given Solanaceae species based on the UniProt Knowledgebase annotations. Data can be accessed by specifying the UniProt accession number, the UniProt entry name or keywords; the latter may be searched in the protein description lines http://biosrv.cab.unina.it/solestdb/solcomp.php.
Table 3

Number of sequences per species collected from different web sources.

 

TOTAL UNIQUE SEQUENCES

SOURCE

DFCI

PlantGDB

SGN

SOLLC

46849

48945

34829

SOLPN

 

3718

 

SOLHA

 

4024

 

SOLTU

?

70344

31072

SOLCH

 

7110

 

TOBAC

83083

114188

84602

NICBE

16127

18037

16024

NICSY

 

7612

6300

NICLS

 

6791

 

CAPAN

14249

15278

9554

PETHY

8729

9884

5135

COFCA

17632

20168

15721

COFAR

 

1093

 

Source: SOLLC: S. lycopersicum; SOLPN: S. pennellii; SOLHA: S. habrochaites; SOLLP: S. lycopersicum × S. pimpinellifolium; SOLTU: S. tuberosum; SOLCH: S. chacoense; TOBAC: N. tabacum; NICBE: N. benthamiana; NICSY: N. sylvestris; NICAT: N. attenuata; NICLS: N. langsdorffii × N. sanderae; CAPAN: C. annuum; CAPCH: C. chinense; PETHY: Petunia × hybrida; COFCA: C. canephora; COFAR: C. arabica.CAB: Computer Aided Bioscience group http://cab.unina.it collection; DFCI: The DFCI Gene Index Project http://compbio.dfci.harvard.edu/tgi/; PGDB: PlantGDB http://www.plantgdb.org/; SGN: The unigene collection at Solanaceae Genomics Network http://www.sgn.cornell.edu/. '?' indicates that the corresponding sequence file was corrupted at the time of the analysis.

Table 4

Statistics on UniProtKB-based annotations.

Unique transcripts with matches in UniProt

SOURCE

CAB

DFCI

PGDB

SGN

SOLLC

28737 (55.82%)

27240 (58.1%)

27763 (56.7%)

20720 (59.4%)

SOLPN

2319 (58.65%)

-

2169 (58.3%)

-

SOLHA

2652 (67.14%)

-

2690 (66.8%)

-

SOLLP

483 (68.71%)

-

-

-

SOLTU

39201 (54.38%)

-

37920 (53.9%)

17122 (55.1%)

SOLCH

4163 (60.19%)

-

4062 (57.1%)

 

TOBAC

45647 (41.5%)

30958 (37.26%)

46618 (40.83%)

33415 (39.5%)

NICBE

8108 (43.15%)

7631 (47.32%)

8564 (47.48%)

7239 (45.18%)

NICSY

3930 (52.3%)

-

4030 (52.9%)

3512 (55.7%)

NICAT

177 (46.09%)

-

-

-

NICLS

3116 (49.09%)

 

3391 (49.9%)

-

CAPAN

8457 (52.83%)

7947 (55.7%)

8454 (55.3)%

5723 (59.90%)

CAPCH

311 (73.52%)

-

-

-

PETHY

5041 (53.9%)

4999 (57.27%)

5474 (55.3%)

2967 (57.7%)

COFCA

9896 (51.14%)

9464 (53.6%)

10697 (53.0%)

8316 (52.9%)

COFAR

701 (49.79%)

-

530 (48.49%)

-

The table shows the number of sequences with significant matches to the UniProtKB/Swiss-prot database and, in brackets, the corresponding percentage on the total.

Source: SOLLC: S. lycopersicum; SOLPN: S. pennellii; SOLHA: S. habrochaites; SOLLP: S. lycopersicum × S. pimpinellifolium; SOLTU: S. tuberosum; SOLCH: S. chacoense; TOBAC: N. tabacum; NICBE: N. benthamiana; NICSY: N. sylvestris; NICAT: N. attenuata; NICLS: N. langsdorffii × N. sanderae; CAPAN: C. annuum; CAPCH: C. chinense; PETHY: Petunia × hybrida; COFCA: C. canephora; COFAR: C. arabica.CAB: Computer Aided Bioscience group http://cab.unina.it collection; DFCI: The DFGI Gene Index Project http://compbio.dfci.harvard.edu/tgi/; PGDB: Plant Genome Database http://www.plantgdb.org/; SGN: The unigene collection at Solanaceae Genomics Network http://www.sgn.cornell.edu/).

https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-9-142/MediaObjects/12870_2009_Article_484_Fig3_HTML.jpg
Figure 3

Pie chart representing protein annotations with respect to the origin of the protein data source.

The results of a query are displayed in matrix format where each row represents a protein and each column refers to a single species for each web resource. The (i, j)th entry of the matrix identifies the number of unique transcripts matching a protein sequence (Figure 4A). By clicking on a single matrix cell the user can access the list of source-specific sequence identifiers (Figure 4B), each of which is, in turn, used to generate a cross-reference to the SolEST database itself as well as to the corresponding external database.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-9-142/MediaObjects/12870_2009_Article_484_Fig4_HTML.jpg
Figure 4

Screenshot of the web tool for comparing different unigene collections for a given Solanaceae species. Panel A shows results from a query in matrix format where each row represents a protein from the UniProt Knowledgebase database and each column refers to a single Solanaceae species and unigene collection. Each matrix cell defines the number of unique transcripts matching a protein sequence. By clicking on a single matrix cell the user can access the list of source-specific sequence identifiers (Panel B).

Exploiting SolEST for Solanaceaegenome sequencing

EST-based collections represent a much-needed reference for the structural annotation of the emerging Solanaceae genome sequences and for addressing sequence-based synteny studies. In addition, they can support technical issues arising while sequencing efforts are ongoing.

1,215 BAC sequences from S. lycopersicum and 708 from S. tuberosum were retrieved from GenBank on July 2009. ESTs and TC sequences from tomato and potato were spliced-aligned along BAC sequences using GenomeThreader [19]. Alignments with a minimum score identity of 90% and a minimum sequence coverage of 80% were filtered out.

Table 5 shows the number of ESTs and TCs per species successfully mapped along the available BAC sequences from tomato and potato (see Methods).
Table 5

Counting of ESTs/TCs mapped along tomato and potato genomic sequences.

  

mapped on TOMATO

mapped on POTATO

mapped on TOMATO and/or POTATO

SOURCE

TOTAL (ESTs/TCs)

# total (ESTs/TCs)

# multiple matches (ESTs/TCs)

# single matches (ESTs/TCs)

# total (ESTs/TCs)

# multiple matches (ESTs/TCs)

# single matches (ESTs/TCs)

only TOMATO (ESTs/TCs)

only POTATO (ESTs/TCs)

TOMATO & POTATO (ESTs/TCs)

CAPAN

33311/4293

3015/365

805/89

2210/276

1051/117

258/24

793/93

2585/307

621/59

430/58

CAPCH

372/34

41/3

20/3

21/0

23/2

3/1

20/1

35/3

17/2

6/

COFAR

1577/137

30/3

10/1

20/2

30/3

8/0

22/3

1/3

1/

29/3

COFCA

55694/6620

73/7

51/6

22/1

58/7

42/5

16/2

22/1

7/1

51/6

NICAT

329/32

16/0

8/0

8/0

14/0

4/0

10/0

7/0

5/0

9/0

NICBE

42566/5006

1016/80

363/20

653/60

499/37

102/11

397/26

764/60

247/17

252/20

NICLS

12448/1379

207/27

64/7

143/20

106/10

37/1

69/9

163/23

62/6

44/4

NICSY

8583/674

546/52

140/17

406/35

215/19

49/5

166/14

464/46

133/13

82/6

PETHY

14017/1738

37/278

12/64

25/214

12/119

1/22

11/97

33/227

8/68

4/51

SOLCH

7752/637

1068/117

262/29

806/88

469/58

107/10

362/48

925/103

326/44

143/14

SOLHA

8000/1243

1996/346

658/99

1338/247

600/99

194/15

406/84

1786/306

390/59

210/40

SOLLC

259990/20548

86547/6184

24129/1576

62418/4608

22126/1409

4985/299

17141/1110

76161/5485

11740/710

10386/699

SOLLP

1008/109

42/0

13/0

29/0

9/0

2/0

7/0

39/0

6/0

3/0

SOLPN

8346/844

2731/269

766/77

1965/192

772/66

100/14

672/52

2425/240

466/37

306/29

SOLTU

231275/23453

48264/4314

12936/1096

35328/3218

22189/1974

5454/458

16735/1516

41371/3693

15296/1353

6893/621

TOBAC

240440/28571

8171/499

2310/123

5861/376

3686/247

977/58

2709/189

6516/392

2031/140

1655/107

The total number of ESTs/TCs for each Solanaceae species collected in the SolEST database is shown in the first two columns. The number of ESTs/TCs splice-aligned along BACs, the number of ESTs/TCs mapped more than once and the number of EST/TC single matches is reported for the tomato and potato genomes, respectively. In addition, the table lists the number of ESTs/TCs exclusively mapped onto the tomato or potato genome and, finally, the number of ESTs/TCs splice-aligned along both the genomes.

We estimated the level of coverage of the Solanaceae transcriptome by counting the number of ESTs/TCs mapped with respect to the total number of the sequences collected in SolEST. The different transcriptome coverage per species is informative per se of the similarity level of the Solanaceae transcriptomes. For example, the EST/TC dataset from tobacco (Nicotiana tabacum), even if it is solid in number, proved poorly mapped on both tomato and potato BACs, showing a transcriptome distance with respect to S. lycopersicum, S. tuberosum or C. annuum.

Columns 4 and 7 in Table 5 report the number of ESTs/TCs with multiple matches along tomato as well as potato BACs. This is expected since sequencing proceeds on a BAC-by-BAC basis, aiming at a minimal tiling path of BACs. In other words, it is evident that several transcripts are aligned along different BACs of the same chromosome because of BAC overlaps. As an alternative, transcripts with multiple matches can be identified with repetitive sequences in the genomes.

Table 6 shows that concurrent mapping of Solanaceae ESTs/TCs along the tomato and potato BAC sequences is informative not only for investigating genome co-linearity between the two species but also for supporting genome sequencing and assignment of BACs to the corresponding chromosomes.
Table 6

Examples of S. lycopersicum TCs that are independently splice-aligned along tomato and potato BACs.

   

S.lycopersicum BACs

S.tuberosum BACs

 

TC ID

UniProtKB Annotation

# chr

BAC ID

start

stop

# chr

BAC ID

start

stop

A

SOLLC004853:Contig2

 

1

AC171727.1

13505

14336

-

-

-

-

 

SOLLC004853:Contig1

 

1

AC171727.1

13540

14538

-

-

-

-

 

SOLLC005165:Contig75

Q8L9T5 | ATL3F_ARATH | RING-H2 finger protein ATL3F OS = Arabidopsis thaliana

1

AC171727.1

36486

37772

-

-

-

-

 

SOLLC007669:Contig1

Q43043 | PME_PETIN | Pectinesterase OS = Petunia integrifolia

1

AC171727.1

      
 

SOLLC021190:Contig1

Q43043 | PME_PETIN | Pectinesterase OS = Petunia integrifolia

1

AC171727.1

109415

109926

-

-

-

-

 

SOLLC020772:Contig1

Q43043 | PME_PETIN | Pectinesterase OS = Petunia integrifolia

1

AC171727.1

121192

121569

-

-

-

-

 

SOLLC015580:Contig1

Q43043 | PME_PETIN | Pectinesterase OS = Petunia integrifolia

1

AC171727.1

121518

123321

-

-

-

-

B

SOLLC004826:Contig1

Q766C2 | NEP2_NEPGR | Aspartic proteinase nepenthesin-2 OS = Nepenthes gracilis

-

-

-

-

5

AC233494.1

5393

6182

 

SOLLC021577:Contig1

Q766C3 | NEP1_NEPGR | Aspartic proteinase nepenthesin-1 OS = Nepenthes gracilis

-

-

-

-

5

AC233494.1

6647

7214

 

SOLLC005426:Contig1

 

-

-

-

-

5

AC233494.1

10450

13585

 

SOLLC007282:Contig1

Q9FT81 | TT8_ARATH | Protein TRANSPARENT TESTA 8 OS = Arabidopsis thaliana

-

-

-

-

5

AC233494.1

34176

35673

 

SOLLC012069:Contig1

Q94HW3 | DRL11_ARATH | Probable disease resistance protein RDL6/RF9 OS = Arabidopsis thaliana

-

-

-

-

5

AC233494.1

48246

53485

 

SOLLC024651:Contig1

Q6L400 | R1B16_SOLDE | Putative late blight resistance protein homolog R1B-16 OS = Solanum demissum

-

-

-

-

5

AC233494.1

51418

52370

 

SOLLC010379:Contig1

Q38950 | 2AAB_ARATH | Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform OS = Arabidopsis thaliana

-

-

-

-

5

AC233494.1

71687

79002

 

SOLLC024982:Contig1

 

-

-

-

-

5

AC233494.1

84414

87845

C

SOLLC010896:Contig1

 

11

AC212431.2

128060

132631

0

AC232103.1

40703

46333

 

SOLLC005049:Contig1

Q3ZAF9 | KGUA_DEHE1 | Guanylate kinase OS = Dehalococcoides ethenogenes

11

AC212431.2

137482

138460

0

AC232103.1

33385

34367

 

SOLLC032933:Contig1

P54677 | PI4K_DICDI | Phosphatidylinositol 4-kinase OS = Dictyostelium discoideum

11

AC212431.2

159306

163292

0

AC232103.1

13520

17677

D

SOLLC001795:Contig1

acc = P43394 entry_name = K502_ACTDE desc = Fruit protein pKIWI502 OS = Actinidia deliciosa

0

CU914756.3

126304

131068

5

AC233527.1

81221

85811

 

SOLLC001995:Contig1

acc = P17614 entry_name = ATPBM_NICPL desc = ATP synthase subunit beta, mitochondrial OS = Nicotiana plumbaginifolia

0

CU914756.3

121815

125827

5

AC233527.1

75747

79701

 

SOLLC002780:Contig1

 

0

CU914756.3

132958

137854

5

AC233527.1

87980

93448

 

SOLLC002780:Contig2

 

0

CU914756.3

132996

137857

5

AC233527.1

88066

93451

 

SOLLC011112:Contig1

acc = P54086 entry_name = Y194_SYNY3 desc = Uncharacterized protein sll0194 OS = Synechocystis

0

CU914756.3

68182

73146

5

AC233527.1

56111

61611

 

SOLLC014928:Contig1

acc = P34552 entry_name = ALX1_CAEEL desc = Apoptosis-linked gene 2-interacting protein X 1 OS = Caenorhabditis elegans

0

CU914756.3

141248

142048

5

AC233527.1

96451

97251

E

SOLLC013836:Contig1

 

11

AC171736.1

46790

49549

11

AC231674.1

1

2996

 

SOLLC033755:Contig1

Q5T9S5 | CCD18_HUMAN | Coiled-coil domain-containing protein 18 OS = Homo sapiens

11

AC171736.1

61542

62195

11

AC231674.1

19771

20423

 

SOLLC011109:Contig2

Q9FIH9 | CML37_ARATH | Calcium-binding protein CML37 OS = Arabidopsis thaliana

11

AC171736.1

67251

68067

11

AC231674.1

29350

30116

 

SOLLC027329:Contig1

Q7Z2Z2 | ETUD1_HUMAN | Elongation factor Tu GTP-binding domain-containing protein 1 OS = Homo sapiens

11

AC171736.1

71740

72558

11

AC231674.1

34634

35449

 

SOLLC011285:Contig1

 

11

AC171736.1

78845

82124

11

AC231674.1

46882

50643

 

SOLLC000854:Contig1

Q9D7N9 | APMAP_MOUSE | Adipocyte plasma membrane-associated protein OS = Mus musculus

11

AC171736.1

82643

87091

11

AC231674.1

50695

55627

 

SOLLC016421:Contig1

Q6R2K2 | SRF4_ARATH | Protein STRUBBELIG-RECEPTOR FAMILY 4 OS = Arabidopsis thaliana

11

AC171736.1

101708

106816

11

AC231674.1

72838

78011

 

SOLLC004514:Contig1

P42824 | DNJH2_ALLPO | DnaJ protein homolog 2 OS = Allium porrum

11

AC171736.1

107145

110458

11

AC231674.1

78559

81871

 

SOLLC014445:Contig1

Q9SF32 | IQD1_ARATH | Protein IQ-DOMAIN 1 OS = Arabidopsis thaliana

11

AC171736.1

119739

122235

11

AC231674.1

97126

101323

 

SOLLC026

 

11

AC17

12536

12605

11

AC23

10606

10674

 

122:Contig1

  

1736.1

1

5

 

1674.1

6

4

 

SOLLC013

P16577 | UBC4_WHEAT |

11

AC17

13590

13989

11

AC23

11693

12091

 

099:Contig4

Ubiquitin-conjugating enzyme E2-23 kDa OS = Triticum aestivum

 

1736.1

0

2

 

1674.1

4

9

F

SOLLC014596:Contig1

 

4

CU914524.3

11054

11642

1

AC233501.1

124622

125217

 

SOLLC004364:Contig1

Q9SZA7 | DRL29_ARATH | Probable disease resistance protein At4g33300 OS = Arabidopsis thaliana

4

CU914524.3

19738

21366

1

AC233501.1

115958

117638

 

SOLLC003401:Contig2

Q9SZA7 | DRL29_ARATH | Probable disease resistance protein At4g33300 OS = Arabidopsis thaliana

4

CU914524.3

21414

22905

1

AC233501.1

114425

115910

 

SOLLC003654:Contig1

Q8GZD4 | NAT3_ARATH | Nucleobase-ascorbate transporter 3 OS = Arabidopsis thaliana

4

CU914524.3

24161

31350

1

AC233501.1

105840

113085

 

SOLLC009218:Contig1

Q9S7T8 | SPZX_ARATH | Serpin-ZX OS = Arabidopsis thaliana

4

CU914524.3

34754

37499

1

AC233501.1

100513

103214

 

SOLLC010320:Contig1

Q9S7T8 | SPZX_ARATH | Serpin-ZX OS = Arabidopsis thaliana

4

CU914524.3

39986

41937

1

AC233501.1

79650

81701

 

SOLLC005339:Contig2

Q9S7T8 | SPZX_ARATH | Serpin-ZX OS = Arabidopsis thaliana

4

CU914524.3

43123

43880

1

AC233501.1

77665

78422

 

SOLLC007010:Contig1

Q05085 | CHL1_ARATH | Nitrate/chlorate transporter OS = Arabidopsis thaliana

4

CU914524.3

52979

54985

1

AC233501.1

40918

42609

 

SOLLC005747:Contig1

Q05085 | CHL1_ARATH | Nitrate/chlorate transporter OS = Arabidopsis thaliana

4

CU914524.3

56811

57920

1

AC233501.1

41189

42544

 

SOLLC008759:Contig1

 

4

CU914524.3

60617

63084

1

AC233501.1

30324

32692

 

SOLLC002208:Contig1

O04348 | TPP1_ARATH | Thylakoidal processing peptidase 1, chloroplastic OS = Arabidopsis thaliana

4

CU914524.3

63803

67165

1

AC233501.1

25016

29581

 

SOLLC004022:Contig1

 

4

CU914524.3

74867

75517

1

AC233501.1

12497

13142

Each selected TC is identified by its ID (TC ID), and a putative functional annotation is associated to it (UniProtKB Annotation). In case of TCs aligned along S. lycopersicum and/or S. tuberosum BACs, the chromosome number (# chr), the BAC accession number from GenBank (BAC ID), and the BAC start and stop coordinates are reported.

First of all, panels A and B in table 6 report instances of S. lycopersicum TCs solely mapped on BACs from a unique species. In particular, 5,904 S. lycopersicum TCs mapped exclusively on tomato genome sequences, while 488 were successfully aligned only along potato BACs, suggesting that the potato sequencing project, although started later, is providing a complementary contribution to that of tomato.

Tomato as well as potato BACs with ambiguous positioning on chromosomes, which have been assigned to the arbitrary-defined chromosome 0, can be correctly associated to the corresponding chromosomes by exploiting (Table 6, panels C and D) evidence from the potato or tomato counterpart, respectively.

In most cases, it is useful to refer to a comparison of BAC sequences, while they are released, in an attempt to find clear genome co-linearity with tomato/potato (Table 6 panels E) or to highlight neighboring genetic loci which retain their relative positions and orders on different chromosomes of the two species (Table 6 panel F).

Figure 5 shows an example that points to the power of a comparative approach based on different transcriptome and genome collections integrated in a single platform. Transcripts from S. lycopersicum and S. tuberosum were mapped onto the BAC CU914524.3 from tomato and the BAC AC233501.1 from potato. The two BACs are present schematically at the center of the figure and were selected because they share a remarkable number of TCs (20 TCs) which are represented as colored bars (the same colors identified the same TCs). Clearly, all the TCs successfully aligned along the BAC CU914524.3 are mapped onto the potato BAC AC233501.1 maintaining their relative positions and orders. It can be easily assumed that the two genomic regions taken into account are co-linear. However, they differ in size, the potato genomic region being 120 kb and that of tomato 70 kb. This is due to insertions in the potato BAC. In these inserted regions TCs from both the species are present (black bars). The region which we are describing is highlighted in yellow and is "zoomed-in" in order to display details on the TC splice-alignments.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2229-9-142/MediaObjects/12870_2009_Article_484_Fig5_HTML.jpg
Figure 5

Representation of the co-linearity between the tomato BAC CU914524.3 and the potato BAC AC233501.1. The BAC CU914524.3 from tomato and the BAC AC233501.1 from potato are present schematically at the center of the figure. Transcripts from S. lycopersicum and S. tuberosum mapped onto them are represented as colored bars (same colors identified same TCs). A particular genomic region is emphasized in yellow and is "zoomed-in" in order to display details on the TC splice-alignments.

SolEST currently provides information on the mapping of both EST and TC datasets on the draft sequences of the tomato and potato genome in the framework of platform ISOL@ [13].

Discussion

Different databases worldwide are related in concept, content and utility to the one presented herein. All of them aim to partition EST sequences into a non-redundant set of gene-oriented clusters and to provide sequences with related information such as biological function and the tissue types in which the gene is expressed. Of course, they differ in their database update policy, in data quality standards and finally in the level of detail with which the database is endowed, which supports investigations on structural and functional information and on expression patterns to different extents.

The SolEST database presents several features making it appealing for the SOL research community and for those interested in EST data management. The Solanaceae EST collection is endowed with both immediate graphical interfaces and details on the organization of multiple alignments and consensus sequence structure to permit a user friendly interpretation of the results as well as easy access to accessory information. SolEST can be accessed through different access points which are briefly summarized to describe the main features of the database that were, however, inherited by TomatEST [12]. The 'Unique Transcript' access point allows the list of singletons and tentative consensus sequences to be associated to the enzymes they encode and, as a consequence, to be mapped 'on the fly' on the KEGG-based metabolic pathways. Singleton ESTs as well as ESTs which were assembled generating the corresponding TC can be independently browsed through the 'ESTs' access point. The maintenance of the single ESTs as well as of the background information related to each of them (presence of contamination and of repeat subsequences, functional annotation), makes SolEST suitable for accessing raw data also in the event of updating the database. This represents an attractive feature of TomatEST [12] which was saved in SolEST. Finally, the 'cluster' access point allows those clusters which have been split into multiple assemblies to be browsed. It can be exploited for a priori identification of putative alternative transcripts or allele-specific transcript isoforms and for investigation of heterozygosity and on the level of ploidy of many of the included species

Being in possession of the entire publicly available EST collection for Solanaceae permitted identification of SSR and the building of a comprehensive EST-derived SSR catalogue for Solanaceae which is accessible to users. This catalogue can be used to develop genetic markers, opening up additional paths into Solanaceae phylogenetic and evolutionary analysis and genetic mapping.

Another novel feature of the SolEST database is aimed at resolving a common complaint in the SOL community as to whether different Solanaceae assemblies generated by diverse research groups should be compared. Various clustering and assembly programs or parameters result in differences among the unique transcript sets provided by different reference databases. In addition, given that each set can be built starting from non-homogeneous primary data sources (e.g. dbEST, RefSeq, genomic or unfinished high-throughput cDNA sequencing (HTC) entries), differences can further become larger. The different major collections now available for the Solanaceae transcriptomes are however equivalently used by the entire community, as they represent the basic collection for specific expression arrays (e.g. a list of microarray resources for tomato is available in [20] Table 2), for COS marker definition [11], for genome annotation [12]. In order to overcome such differences, we decided to compare each unigene collection with the UniProt Knowledgebase. The use of a protein reference database may represent a useful tool to cross-link the different collections through a specific service and, more interestingly, it is an immediate approach to compare the different EST-based available resources.

One of the most novel features in SolEST, when compared to other resources, is the option of accessing and then visualizing Solanaceae EST/TC alignments along the tomato and the potato genomes. The mapping of Solanaceae ESTs certainly provides insights into the location of potential candidate genes and facilitates EST-driven gene annotation. This represents the first attempt to provide a unique view of the data from both the sequencing efforts, which we believe will be appreciated by the SOL community. In addition, having ESTs from Solanaceae and Rubiaceae mapped along the genomes of two of the major representatives of the family will support comparative genomics approaches aimed at addressing the most fundamental issues such as diversity and adaptation within the Solanaceae family and heterogeneity in gene expression patterns. Finally, the TCs we defined will provide support for solving technical issues arising from BAC-by-BAC genome sequencing and will undoubtedly provide a reference for forthcoming WGS (whole genome shotgun) efforts in both tomato and potato.

Conclusion

To our knowledge, no similar work has yet been carried out on the construction of an EST database offering a broad overview of Solanaceae as well as Coffea transcriptomes. Multiple sequence analysis results from the database (e.g. developing a unigene set, annotation with putative function and identification of SSRs) extensively linked to external related resources, represent a major source of information for these plant families, opening up novel vistas conducive to comparative evolutionary studies. We think the SolEST database will represent an invaluable resource for supporting the structural annotation of the emerging Solanaceae genome sequences and addressing technical issues arising while sequencing efforts are being made. Finally, the SolEST database meets the challenge of connecting the different EST-centered collections worldwide generated by applying various methods and starting from disparate primary data sources.

Availability and requirements

The SolEST database is available with no restrictions at the following URL: http://biosrv.cab.unina.it/solestdb/.

The SolEST update is scheduled at the end of each year and comprises the retrieval of primary data sources (i.e. EST/mRNA sequences) and the generation of novel unigenes/TCs as well as their annotation. Therefore, at each release the update of the satellite databases (i.e. UniVec, RepBase, UniProtKB/Swiss-prot, Gene Ontology, Enzyme, KEGG) used in the cleaning, repeat masking and annotation phases is also performed.

The retrieval of new S. lycopersicum and S. tuberosum BAC sequences from the GenBank repository is ensured daily by an automated pipeline [13]. The switch to genome contigs will beensured as the sequencing status will evolve.

Declarations

Acknowledgements

The authors thank Mark Walters for editing the manuscript.

This work is supported by the EU-SOL project (European Union) (Contract no. PL 016214-2) and by the GenoPom Project (MIUR, Italy).

This is the contribution DISSPAPA no. 2000.

Authors’ Affiliations

(1)
University of Naples 'Federico II', Dept of Soil, Plant, Environmental and Animal Production Sciences

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Copyright

© D'Agostino et al; licensee BioMed Central Ltd. 2009

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.