Identification of Novel MicroRNAs and their Target Prediction in Stevia rebaudiana


miRNAs are a class of endogenous, small, noncoding, ~22 nucleotides single-stranded RNAs that act as posttranscriptional regulators in eukaryotes . They have reported to be located mostly within noncoding regions of genomes, and usually transcribed from RNA polymerase II promoters . The generation of mature miRNA is a complicated enzyme-catalyzed process, from the initial transcript pri-miRNA to the precursor (pre-miRNA) with a characteristic hairpin structure, then a miRNA duplex (miRNA:miRNA* ). In the end, the mature miRNAs function within large complexes to negatively regulate specific target mRNAs. Perfect complementarity between miRNA: mRNA strand generally results in cleavage, such as in plants, whereas imperfect base-pairing leads to translational repression. MiRNA genes represent about 1%-2% of the known eukaryotic genomes and constitute an important class of fine-tuning regulators that are involved in several physiological or disease-associated cellular processes. Plant miRNAs target a large number of genes with functions in a range of development processes, including meristem cell identity, leaf organ morphogenesis and polarity and floral differentiation and development . miRNAs are also reported to be involved in plant responses to biotic and environmental stresses.Increasing evidences showed that miRNA repertoire of plant or animal species includes a set of conserved (ancient, abundant) and non-conserved or novel (species specific, recently evolved) miRNAs . However, the biggest challenge in plants is to identify novel miRNAs and to understand their mode of action, role in various metabolic processes. The availability of high throughput next generation sequencing (NGS) technologies such as 454 and Illumina have further revolutionized sRNA discovery. Whereas, transcribed sequences such as ESTs has led to the identification of only conserved (abundant) miRNAs or miRNAs previously identified in other species, on the other hand, NGS provides high throughput tools to make new discoveries of additional species specific or lowly expressed miRNAs in plants irrespective of whether their genome is sequenced or not, e.g. Arabidopsis, Oryza sativa, Populus trichocarpa, Triticum aestivum, Brachypodium distachyon, Vitis vinifera, Arachis hypogaea, Citrus trifoliate, Carthamus tinctorius and many more. NGS strategy for discovery of miRNAs may be successful not only for these plant species of full genomic and sufficient EST database available, but as well as for those with incomplete genomic information but with sufficient EST sequences available that is the case of our model plant Stevia rebaudiana. The latest miRBase release (v20, June 2013) contains 24,521 microRNA loci from 206 species, processed to produce 30,424 mature microRNA products. 

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