A total of 16892 pre-miRNA sequences were obtained from  PMRD   and   miRNEST2.0    on January 31, 2014, as well as from in-house mining of pre-miRNA sequences from fungi and citrus EST databases. Pre-miRNA sequences for the fungi and citrus species were predicted from the EST sequences. miRNA database version 21 used for homology search against fungi and citrus EST collection, then best hits with a minimum length of 18 nucleotides and a maximum miRNA length cover up to 26 nucleotides with not more than 3 mismatches were considered for putative candidate miRNA selection. Further, BLASTx was applied to eliminate protein-coding transcripts. After that, Mfold uses to predict stem-loop structure. Then potential pre-miRNA sequences were selected for this study following the criteria: (a) mature miRNA must be located on the arm of the hairpin; (b) minimum paired and unpaired residues in miRNA are 14 and ≤ 5, respectively; (c) the maximum number of G-U pairs in miRNA is 5, (d) maximum bulge size is 3nt, (e) the negative minimal folding free energy (MFE) is low (≤ -18 kcal/mol), and (f) minimal folding free energy index (MFEI = [(MFE/length of the RNA sequence) * 100]/(G+C)%) is high (>0.85).