Deep Sequencing-Based Analysis of the Cymbidium ensifolium Floral Transcriptome

Abstract

Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs), 41,690 into 58 gene ontology (GO) terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium.

De novo assembly and characterization of the root transcriptome and development of simple sequence repeat markers in Paphiopedilum concolor

“…Di-nucleotide motifs were the most frequent SSR motif type. This finding is consistent with the results that were reported for sugar beets, cabbage, soybeans, sunflowers, grapes, and sesame plants (Kumpatla and Mukhopadhyay, 2005;Wei et al, 2011), whereas tri-nucleotide motifs were the most abundant SSRs in rice, wheat, barley, radish, and Cymbidium ensifolium (La Rota et al, 2005;Wang et al, 2012;Li et al, 2013). Among the di-nucleotide repeats, AG/CT was the most abundant motif in our data ( Figure 5B).…”

“…This finding is consistent with the results that were reported for other plant species (Wei et al, 2011;Wang et al, 2012). Among the tri-nucleotide motifs, the most frequent motifs in our data were CCG/CGG, whereas AAG/CTT were the most frequent motifs in other plant species, such as radish, sesame, and C. ensifolium (Wei et al, 2011;Wang et al, 2012;Li et al, 2013).…”

“…Currently, nextgeneration sequencing technologies, such as Illumina Genome Analyzer, the Roche/454 Genome Sequencer FLX Instrument, and the ABI SOLiD System, have proven to be powerful and costeffective tools for advanced research in many areas, including de novo transcriptome sequencing, gene discovery, expression profiling analysis, and molecular marker development in non-model organisms (Hsiao et al, 2011;Wang et al, 2012;Li et al, 2013). Recently, the mature flower transcriptome of Paphiopedilum armeniacum has been sequenced using next-generation sequencing (Tsai et al, 2013).…”

De Novo Transcriptome Assembly from Inflorescence of Orchis italica: Analysis of Coding and Non-Coding Transcripts

“…The number of transcripts and unigenes assembled for the inflorescence of O. italica is higher than those assembled with the same deep sequencing approach for the inflorescence of the orchid Cymbidium ensifolium (101,423 transcripts and 51,696 unigenes) [34] and similar or slightly lower than those assembled for mixed vegetative and reproductive tissues of Cymbidium sinense [33] and Erycina pusilla [36]. The other orchid transcriptomes currently available ( Oncidium ‘Gower Ramsey’, Phalaenopsis aphrodite and Ophrys ) were obtained by applying combined approaches of different next generation sequencing (NGS) techniques [32], [35], [37], resulting in transcriptomes composed of both contigs and singletons.…”

“…Recently, next generation sequencing approaches have been applied to identify genes associated with flowering in some orchid species belonging to the Epidendroideae sub-family (genera Phalaenopsis , Cymbidium , Oncidium , Ericina ) [29], [32], [33], [34], [35], [36], whereas similar studies in the Orchidoideae sub-family have been limited to Ophrys [37]. Although all of these RNA-seq studies include comprehensive analyses of coding and/or small non-coding RNAs in orchid species, none of them focus on the long non-coding RNAs (lncRNAs).…”