Gene discovery using next-generation pyrosequencing to develop ESTs for Phalaenopsis orchids

Background: Orchids represent one of the most diversified angiosperms but few genomic resources are available in these non-model plants. In addition to the ecological significance, Phalaenopsis also has been considered as an economically important floriculture industry worldwide. We applied massively parallel pyrosequencing technique to explore the transcriptional complexity for Phalaenopsis. Results: We present a global characterization of Phalaenopsis orchids transcriptome using 454 pyrosequencing. To maximize sequence diversity, we pooled RNA extracted from ten different tissues, developmental stage, and biotic or abiotic stressed Phalaenopsis plants. A total of 206,960 ESTs with an average read length of 228 bp was obtained. These reads assembled into 8,233 contigs and 34,630 singletons. The unigenes were searched against the NCBI non-redundant (NR) protein database. Based on sequence similarity with known proteins, these analyses identified 22,234 different genes (E-value cutoff, e-7). Assembled sequences were annotated with Gene Ontology, Gene Family and KEGG pathways. In addition, we identified over 650 unigenes encoding putative transcription factors. Conclusion: Pyrosequencing is an effective approach to identify a large set of unigenes from Phalaenopsis. The informative EST dataset developed from this study constitute a much-needed resource for discovery of genes involved in various biological processes in Phalaenopsis and other orchid species. These transcribed sequences will narrow the gap between approaches based on model organisms with plenty genomic resources and species that are important for ecological and evolutionary studies.