Identification and confirmation of new common miRNAs in bread wheat and different Aegilops species by bioinformatics approach and Real time PCR

Bread wheat is one of the most important cereals widely cultivated in the world. miRNAs are a type of small regulatory RNAs that have about 18-22 nucleotides long and can use as a novel breeding tool for plant genetic improvement. Because miRNAs are evolutionarily conserved between plant and animal kingdoms, the use of comparative genomics and computational methods to identify miRNAs is interesting. In this study, the goal was to identify potential new miRNAs in bread wheat by EST-based similarity search method. For this purpose, known plant miRNAs were used as reference against wheat ESTs and BLASTn was performed. Then, protein coding sequences were identified and deleted by BLASTx. The secondary structure was examined and then the candidate miRNAs were analyzed in the Rfam database. Finally, three new miRNAs were identified from this method, which were sequentially similar to the miRNAs known in Ae. tauschii. In order to confirm their presence in wheat and Agileops species containing D genome including tauschii, cylindrica and crossa, Real time PCR reaction was performed and finally confirmed the presence of myseq2 sequence in the shoots of studied plants. Identification of target genes of the confirmed miRNA revealed that most of them such as RGA, PIK and NB-ARC had an impact on resistance to various diseases and some of them played a role in resistance to abiotic stresses, such as drought and salt stress. The results showed that computational methods can be a suitable tool for predicting candidate miRNAs, which saves time and cost in identifying miRNAs.