Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak
1 INRA, UMR 1202 BIOGECO, 69 route d'Arcachon, F-33612 Cestas, France
2 Forestry and Forest Products Research Institute, Department of Forest Genetics, Tree Genetics Laboratory, 1 Matsunosato, Tsukuba, Ibaraki, 305-8687, Japan
3 Plateforme bioinformatique Genotoul, UR875 Biométrie et Intelligence Artificielle, INRA, 31326 Castanet-Tolosan, France
4 INRA/UBP UMR 1095, Laboratoire Génétique, Diversité et Ecophysiologie des Céréales, 234 avenue du Brézet, 63100 Clermont Ferrand, France
5 INRA, UMR1137 EEF "Ecologie et Ecophysiologie Forestières", F 54280 Champenoux, France
6 Université de Bordeaux, Centre de Bioinformatique de Bordeaux, Bordeaux, France
7 CNRS, UMR 5800, Laboratoire Bordelais de Recherche en Informatique, Talence, France
8 CEA, DSV, Genoscope, Centre National de Séquençage, 2 rue Gaston Crémieux CP5706 91057 Evry cedex, France
BMC Genomics 2010, 11:650 doi:10.1186/1471-2164-11-650Published: 23 November 2010
The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.
We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html webcite.
This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.