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

Abstract

Background: Orchids are one of the most diversified angiosperms, but few genomic resources are available for these non-model plants. In addition to the ecological significance, Phalaenopsis has been considered as an economically important floriculture industry worldwide. We aimed to use massively parallel 454 pyrosequencing for a global characterization of the Phalaenopsis transcriptome.

Results: To maximize sequence diversity, we pooled RNA from 10 samples of different tissues, various developmental stages, and biotic- or abiotic-stressed plants. We obtained 206,960 expressed sequence tags (ESTs) with an average read length of 228 bp. These reads were 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 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Among these annotations, over 780 unigenes encoding putative transcription factors were identified.

Conclusion: Pyrosequencing was effective in identifying a large set of unigenes from Phalaenopsis. The informative EST dataset we developed constitutes 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 study of model organisms with many genomic resources and species that are important for ecological and evolutionary studies.

Figure 1

Comparison of sequences generated by use of 454 sequencing and Phalaenopsis ESTs published in the NCBI EST database by BLASTN.

Figure 2

Comparison of percentage of hits among different sized contigs against Phalaenopsis ESTs published in the NCBI EST database by BLASTN (E-value < 10^-10).

Figure 3

The number of sequence hits against the NCBI non-redundant (NR) database by BLASTX.

Figure 4

Comparison of percentage of hits in the NR database for different sized ESTs with (black bars) and without (white bars) homology (E-value < 10^-7).

Figure 5

Representation of genome ontology assignments for Phalaenopsis ESTs derived from 454 sequencing. The GO Slim Classification for Plants developed at TAIR was used to characterize the ESTs functionally.

Figure 6

Graphic representation of terpenoid backbone biosynthesis pathways, including cytosolic mevolonate pathway and plastic methylerythritol phosphate pathway, for Phalaenopsis orchids. The red and blue numbers represent the ESTs generated by this study and derived from P. bellina [16], respectively, that are homologous to genes involved in the terpenoid backbone biosynthesis pathway. The first number in the bracket is the number of unigenes corresponding to the catalytic gene in the pathway, and the second number is the number of reads for the unigene.

Figure 7

Number of ESTs related to transcription factors in each transcription factor family. A total of 2,424 putative Oryza sativa subsp. japonica transcription factors were searched against the Phalaenopsis transcriptome, and target ESTs were classified into corresponding transcription factor families. Black bars are the number of reads in each family. Shaded bars are the number of unigenes in each family.