Deciphering microRNAs and Their Associated Hairpin Precursors in a Non-Model Plant, Abelmoschus esculentus

MicroRNAs (miRNAs) are crucial regulatory RNAs, originated from hairpin precursors. For the past decade, researchers have been focusing extensively on miRNA profiles in various plants. However, there have been few studies on the global profiling of precursor miRNAs (pre-miRNAs), even in model plants. Here, for the first time in a non-model plant-Abelmoschus esculentus with negligible genome information-we are reporting the global profiling to characterize the miRNAs and their associated pre-miRNAs by applying a next generation sequencing approach. Preliminarily, we performed small RNA (sRNA) sequencing with five biological replicates of leaf samples to attain 207,285,863 reads; data analysis using miRPlant revealed 128 known and 845 novel miRNA candidates. With the objective of seizing their associated hairpin precursors, we accomplished pre-miRNA sequencing to attain 83,269,844 reads. The paired end reads are merged and adaptor trimmed, and the resulting 40-241 nt (nucleotide) sequences were picked out for analysis by using perl scripts from the miRGrep tool and an in-house built shell script for Minimum Fold Energy Index (MFEI) calculation. Applying the stringent criteria of the Dicer cleavage pattern and the perfect stem loop structure, precursors for 57 known miRNAs of 15 families and 18 novel miRNAs were revealed. Quantitative Real Time (qRT) PCR was performed to determine the expression of selected miRNAs.