Transcriptome-wide characterization of miRNA-directed and non-miRNA-directed endonucleolytic cleavage using Degradome...

Plant microRNAs (miRNAs) regulate gene expression through post-transcriptional gene silencing. Phalaenopsis aphrodite subsp. formosana is an orchid species native to Taiwan, which has high economic value and a high frequency of floral polymorphism. To date, few studies have focused on the regulatory roles of miRNAs and functional small RNAs (sRNAs) in orchids although understanding the regulation of flower development and flowering time is potentially important. Here, we combined analyses of the transcriptome, sRNAs and the degradome to identify sRNA-directed transcript cleavages in Phalaenopsis. Degradome analysis provided large-scale evidence of conserved and novel miRNA-directed cleavage of target transcripts, and 46 abundant sRNA groups and their target transcripts were identified. Low temperature-responsive sRNAs were validated with normalized reads from an sRNA library and quantitative stem-loop reverse transcription-PCR (RT-PCR) analysis. According to gene ontology (GO) categorization, target transcripts of the novel miRNAs and sRNAs are functionally involved in metabolic processes or responses to stress. One particular homologous gene, Allcontig28452, which encodes digalactosyldiacylglycerol synthase 2 (DGD2), was found to be targeted by natural antisense transcripts (NATs) unique to Phalaenopsis. In summary, comprehensive analyses of the transcriptome, sRNAs and degradome using deep sequencing technology provided a useful platform for investigating miRNA-directed and non-miRNA-directed endonucleolytic cleavage in a non-model plant, the orchid Phalaenopsis.