Development and functional annotation of an 11,303-EST collection from Eucalyptus for studies of cold tolerance

A cDNA library was constructed from leaves of a cold-acclimated Eucalyptus gunnii elite clone and 13,056 ESTs were sequenced. When the 5,457 unique sequences were compared with Arabidopsis , rice, and poplar datasets, more than 38% of no-hits were found and the best annotation was provided by the woody perennial poplar database. However, among the 62% of sequences that could be annotated, the vast majority (87.5%) was found to be common to the four compared plant species, which shows how highly conserved this gene pool is across the plant kingdom. When the distribution of the annotated sequences was studied according to the Gene Ontology classification, some typical features of the dehydrative stress transcriptome were observed. In particular, genes from sugar metabolism and, above all, those induced by stress or external stimuli were well represented (6% in total). In addition, the library was found to contain a substantial number of ESTs encoding putative transcription factors including CBF (CRT-Binding Factor). The enrichment of this library with stress-related genes was strongly suggested by the high redundancy (up to 300 ESTs) of several genes known to be involved in cell protection such as the PCP (Pollen Coat Protein), Lti6b (Low temperature induced) or metallothionein. It was further confirmed by demonstrating the cold induction of a set of representative genes through Real Time-PCR. All these characteristics make this Eucalyptus EST collection very suitable for investigating the molecular basis of cold tolerance in a woody perennial and for isolating important or new candidate frost tolerance genes.