Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak

Abstract

BackgroundThe 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.ResultsWe 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.ConclusionsThis 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.

Transcriptome analysis reveals positive selection on the divergent between topmouth culter and zebrafish

“…3). Similar results have been found in oak and Nothofagus nervosa (Ueno et al, 2010). Then, the 384 SSRs (12.3%) had sufficient flanking sequences to allow the design of appropriate unique primers.…”

A White Spruce Gene Catalog for Conifer Genome Analyses

“…Recent studies involving uncharacterized genomes such as P. contorta (Parchman et al, 2010) and European oaks (Quercus petraea and Quercus robur; Ueno et al, 2010) have shown that GS-FLX is efficient for transcriptome sequencing, but clustering methods are prone to generate exceedingly large numbers of unique sequences upon assembly, particularly when multiple individuals are pooled for sequencing. Even when integrating Sanger and GS-FLX reads in a hybrid analysis, the assembly of oak sequences yielded a minimum of 116,000 unique sequence elements (Ueno et al, 2010). Assembly of GS-FLX data alone generated short contigs averaging 353 bp in Eucalyptus (Novaes et al, 2008; de novo assembly) and 500 bp in pine (Parchman et al, 2010; combined de novo and reference assembly).…”

Identification of SNPs in candidate genes potentially involved in bud burst in European beech (Fagus sylvatica L.)

“…The ten selected genes ( Table 2) are expected to have an impact on bud burst in oak (DERORY et al, 2006;UENO et al, 2010), a genus of the Fagaceae family related to beech. The Evoltree EST database (http:// www.evoltree.eu) and the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) were used to find corresponding F. sylvatica sequences.…”