**3. Gene expression regulation by miRNA**

For controlling various function of plant biology, specifically process control by transcription actors, miRNA diversity is significant [15]. miRNAs act as a significant controllers of gene expression and investigation on this aspect increasing now a days [2, 13]. Gene expression regulated by miRNA through high sequence similarity at the post-transcriptional level. Proper pairing between miRNA and targeted mRNA promotes the corresponding mRNA degradation and improper pairing between miRNA and target mRNA leads translation inhibition [13]. Poly(A) tail removal induced by mRNA which further promotes the destabilization and degradation of the target mRNAs [16]. Along with post-transcriptional gene expression regulation, miRNAs also decrease arbitrary fluctuation in transcript copy number and promote different metabolic pathways by transcription inhibition. miRNA of different length produces by different gene, as well as varied length miRNAs originated from the same gene. DCL1 enzyme mainly processed plant pre-miRNAs and produce 21 nucleotide base pair long mature miRNAs, but few other DCLs *i.e.* 2–4 can also be involved to produce miRNA of various lengths [17]. Diversity miRNA pool expanded by such miRNA heterogeneity and can efficiently enhance their monitoring possibilities. Additionally, miRNA diversity has practical used for the production of miRNA precursor-based expression casettes designed to produce artificial and sequence specific miRNAs [18].

## **4. Molecular techniques used for miRNA study**

#### **4.1 Techniques used for miRNA isolation, identification, and characterization**

For miRNA identification first crucial step is to recognize their roles. Direct cloning, sequencing, genetic screening and some bioinformatic approaches commonly used for miRNAs identification [19]. In *Arabidopsis* first plant miRNAs identified such as miR156, miR159, miR164 and miR171 by isolating, cloning and sequencing of small RNA populations [20]. Small number of miRNAs have been

recognized by genetic screening, mainly due to the redundancy and sequence similarity with other miRNA-coding genes. Genetic screening and activation-tagging approach used for miRNA identification and significant to separate prominent miRNA mutants like miR172a-2 [21]. JAW (jagged and wavy) loci also identified by genetic screening, which generate a microRNA (miR-JAW) that can leads the degradation of mRNAs of TCP genes which control leaf development [22]. Mutations like deletions/insertions/further promotes the loss/gain of miRNA binding sites during co-evolution of miRNAs and their target sites [23]. Mutation also contributes in identification for specific defect during development. In the earlier decade, thousands of plant miRNAs identified by both experimental and computational approaches. The main computational method used for miRNAs identification on the basis of sequence similarity against DNA/genome sequence of some important crop plants is BLAST (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/ blast/) [24–27].

Direct cloning and genetic screening are experimentally old methods used for identification and functional characterization of miRNAs [20]. Sanger sequencing technique used after direct cloning for identification of sequence of base pair. But now a days next generation sequencing technology evolved as a powerful tool for discovery of novel miRNA and target identification in crop plants [9, 28]. Real time PCR and Northern bloting technique used for validation of identified miRNAs expression [29, 30]. Along with this, miRNA identification at protein level possible by using some other methods like mass spectrometry, proteins chromatography, protein foot printing, Western blotting, *etc*. at the protein level. Outcomes from these approaches showed that miRNAs act as rheostats to make fine-scale alterations in protein output. Further, miRNA identification promotes the development of different database which contains searchable evidence on the miRNAs. miRbase (http://www.mirbase.org) is the most significant and crucial bioinformatics tool used for miRNA research which is a searchable and comprehensive miRNA database mainly based on miRNA name, annotation, references and keyword [30]. A another bioinformatic database like The Plant MicroRNA Database (PMRD; http://mirnablog.com/ plant-micrornadatabase-goes-online/) also contains information about plant miRNAs, like miRNA and their target(s), expression profiling, genome browser and secondary structure [31]. Several computational tools such as AthaMap (http://www.athamap.de/), CLC Genomic workbench 6 software (CLC Bio, Cambridge, MA, USA) and Next-Gen sequence databases also enhance the NGS performance along with the knowledge about miRNA and their role in Plants (https://mpss.udel.edu/index.php) [32].

## **4.2 Approaches for miRNA target screening and prediction**

Several bioinformatics approaches and tools used for identification of miRNA target gene [33]. Sequence similarity scoring and secondary structure investigation are main bioinformatics criteria used for miRNA and target identification are: miRTarBase, miRTour (http://mirnablog.com/mirtour-plantmirna-and-target-prediction-tool/, psRNATarget (http://plantgrn. noble.org/psRNATarget/) and TAPIR (http://bioinformatics.psb.ugent.be/webtools/tapir/) [30]. Relationship between microRNA and its target confirmed by micRTarBase database (http://mirtarbase. mbc.nctu.edu.tw/) [34]. While the target mRNA expression levels can be observed by real-time PCR, for mapping the target site 5′-Rapid Amplification of cDNA Ends (RACE) used. Now a days, the degradome sequencing technique was developed for identification of comparative profusion of cleaved targets [30].
