Generation, annotation, analysis and database integration of 16,500 white spruce EST clusters.
BACKGROUND
The sequencing and analysis of ESTs is for now the only practical approach for large-scale gene discovery and annotation in conifers because their very large genomes are unlikely to be sequenced in the near future. Our objective was to produce extensive collections of ESTs and cDNA clones to support manufacture of cDNA microarrays and gene discovery in white spruce (Picea glauca [Moench] Voss).


RESULTS
We produced 16 cDNA libraries from different tissues and a variety of treatments, and partially sequenced 50,000 cDNA clones. High quality 3' and 5' reads were assembled into 16,578 consensus sequences, 45% of which represented full length inserts. Consensus sequences derived from 5' and 3' reads of the same cDNA clone were linked to define 14,471 transcripts. A large proportion (84%) of the spruce sequences matched a pine sequence, but only 68% of the spruce transcripts had homologs in Arabidopsis or rice. Nearly all the sequences that matched the Populus trichocarpa genome (the only sequenced tree genome) also matched rice or Arabidopsis genomes. We used several sequence similarity search approaches for assignment of putative functions, including blast searches against general and specialized databases (transcription factors, cell wall related proteins), Gene Ontology term assignation and Hidden Markov Model searches against PFAM protein families and domains. In total, 70% of the spruce transcripts displayed matches to proteins of known or unknown function in the Uniref100 database (blastx e-value < 1e-10). We identified multigenic families that appeared larger in spruce than in the Arabidopsis or rice genomes. Detailed analysis of translationally controlled tumour proteins and S-adenosylmethionine synthetase families confirmed a twofold size difference. Sequences and annotations were organized in a dedicated database, SpruceDB. Several search tools were developed to mine the data either based on their occurrence in the cDNA libraries or on functional annotations.


CONCLUSIONS
This report illustrates specific approaches for large-scale gene discovery and annotation in an organism that is very distantly related to any of the fully sequenced genomes. The ArboreaSet sequences and cDNA clones represent a valuable resource for investigations ranging from plant comparative genomics to applied conifer genetics.
Pavy
Nathalie
N
Pavillon Charles-Eugène-Marchand, Université Laval, Ste.Foy, Québec G1K 7P4, Canada. nathalie.pavy@rsvs.ulaval.ca
Paule
Charles
C
Parsons
Lee
L
Crow
John A
JA
Morency
Marie-Josee
MJ
Cooke
Janice
J
Johnson
James E
JE
Noumen
Etienne
E
Guillet-Claude
Carine
C
Butterfield
Yaron
Y
Barber
Sarah
S
Yang
George
G
Liu
Jerry
J
Stott
Jeff
J
Kirkpatrick
Robert
R
Siddiqui
Asim
A
Holt
Robert
R
Marra
Marco
M
Seguin
Armand
A
Retzel
Ernest
E
Bousquet
Jean
J
MacKay
John
J
D017360:Arabidopsis / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D016000:Cluster Analysis; D020451:Contig Mapping; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D030541:Databases, Genetic; D020224:Expressed Sequence Tags; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017343:Genes, Plant; D018745:Genome, Plant; D023281:Genomics; D005810:Multigene Family; D012275:Oryza / Q000235:genetics; D028222:Picea / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012984:Software
D018076:DNA, Complementary; D012333:RNA, Messenger