Sequencing-Based Approaches Reveal Low Ambient Temperature-Responsive and Tissue-Specific MicroRNAs in Phalaenopsis Orchid

Plant small RNAs (smRNAs) are short, non-coding RNA molecules that mediate RNA silencing and regulate a group of genes involved in plant development and responses to environmental stimuli. Low temperature is necessary to initiate stalk development in the orchid Phalaenopsis aphrodite subsp. formosana. To identify smRNAs in Phalaenopsis responding to low temperatures, a smRNA profiling analysis using high-throughput sequencing technology was performed. Subsequent bioinformatic analysis was applied to categorize the miRNAs identified. A total of 37,533,509 smRNA reads yielded 11,129 independent orchid miRNA sequences, representing 329 known miRNA families identified in other plant species. Because the genomic resources available for Phalaenopsis are limited, a transcriptomic database was established using deep sequencing data sets to identify miRNAs precursors and their target transcripts. Comparing small RNAs and the transcriptomic database, 14 putative miRNA precursors of 10 miRNA families were identified, as were hundreds of putative targets. Comparing sequencing data and smRNA northern hybridization results identified miR156, miR162, miR528 and miR535 as low temperature-induced miRNAs. In addition, tissue-specific expression of these miRNAs was investigated. It was concluded that miR156 and miR172 may be components of a regulatory pathway mediating transition from the vegetative to the reproductive phase in Phalaenopsis. The smRNA and transcriptomic databases could be the foundations for further research aimed at elucidating the control of the flowering time in orchids.