Abstract
BackgroundCymbidium ensifolium is one of the most important ornamental flowers in China, with an elegant shape, beautiful appearance, and a fragrant aroma. Its unique flower shape has long attracted scientists. MicroRNAs (miRNAs) are critical regulators in plant development and physiology, including floral development. However, to date, few studies have examined miRNAs in C. ensifolium.ResultsIn this study, we employed Solexa technology to sequence four small RNA libraries from two flowering phases to identify miRNAs related to floral development. We identified 48 mature conserved miRNA and 71 precursors. These conserved miRNA belonged to 20 families. We also identified 45 novel miRNA which includes 21 putative novel miRNAs*, and 28 hairpin forming precursors. Two trans-acting small interfering RNAs (ta-siRNAs) were identified, one of which was homologous to TAS3a1. TAS3a1 belongs to the TAS3 family, which has been previously reported to target auxin response factors (ARF) and be involved in plant growth and floral development. Moreover, we built a C. ensifolium transctriptome database to identify genes targeted by miRNA, which resulted in 790 transcriptomic target unigenes. The target unigenes were annotated with information from the non-redundant (Nr), gene ontology database (GO), eukaryotic orthologous groups (KOGs) and Kyoto encyclopedia of genes and genomes (KEGG) database. The unigenes included MADS-box transcription factors targeted by miR156, miR172 and miR5179, and various hormone responding factors targeted by miR159. The MADS-box transcription factors are well known to determine the identity of flower organs and hormone responding factors involved in floral development. In expression analysis, three novel and four conserved miRNA were differentially expressed between two phases of flowering. The results were confirmed by RNA-seq and qRT-PCR. The differential expression of two miRNA, miR160 and miR396, targeted ARFs and growth regulating factor (GRF), respectively. However, most of these small RNA were clustered in the uncharacterized group, which suggests there may be many novel small non-coding RNAs yet to be discovered.ConclusionOur study provides a diverse set of miRNAs related to cymbidium floral development and serves as a useful resource for investigating miRNA-mediated regulatory mechanisms of floral development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1764-1) contains supplementary material, which is available to authorized users.
Highlights
Cymbidium ensifolium is one of the most important ornamental flowers in China, with an elegant shape, beautiful appearance, and a fragrant aroma
Ta-shortinterfering RNAs (siRNAs) were generated from TAS gene transcripts, which are cleaved by a miRNA, resulting in the production of small RNA fragments 21 nt in length that are in phase with the miRNA cleavage site [12]
Deep sequencing of the small RNA libraries generated the mean number of raw reads, 10,372,041 and 9,105,582, for phase 1 (PS1) and phase 4 (PS4), respectively (Table 1)
Summary
Cymbidium ensifolium is one of the most important ornamental flowers in China, with an elegant shape, beautiful appearance, and a fragrant aroma. Small, non-coding RNAs are grouped into two major classes: microRNAs (miRNAs) and shortinterfering RNAs (siRNAs). Both small RNA classes have the same chemical composition and mechanism of action. MiRNAs are endogenous non-coding RNAs of ~21 nucleotides (nt) in length, derived from single-stranded RNA hairpin precursors cleaved by a double-stranded-specific RNase (Dicer) in animals and Dicer-like (DCL1) in plants [6]. There exist non-conserved or species-specific small interfering RNA in plants, which are the transacting small interfering RNAs (ta-siRNAs). Ta-siRNAs were generated from TAS gene transcripts, which are cleaved by a miRNA, resulting in the production of small RNA fragments 21 nt in length that are in phase with the miRNA cleavage site [12]
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