Analysis of microRNAs in response to cycloastragenol treatment by small RNA sequencing in Arabidopsis thaliana

Purpose:we aimed to demonstrate the effects of cycloastragenol on the different plant signaling mechanisms and analyze microRNAomic responses in order to demonstrate its potential as a new key molecule to help plants overcome different environmental stresses. Methods: smallRNA-seq was employed to give a quantitative profile of microRNA expression and to identify new micro RNAs and in treated and non-treated A. thaliana calli. We sequenced two cDNA libraries developed from A. thaliana (wild type Col-0) calli, one non-treated and the other is treated with 1µM cycloatragenol. Reads were filtered, mapped, aligned and then compared to the reference annotation (miRBase database). Clean data was analyzed using different software in order to identify differentilly expressed miRNAs and their target genes. Results were validated by qRT-PCR using TaqMan and SYBER green assays. Results: We mapped more than 31 and 30 million tags from, respectively, control and CAG-treated samples. After filtration and mapping, a total of 273 known micro RNAs, 298 novels expressed miRNAs and 6160 target genes were identified, among which a total of 119 miRNAs and 961 target genes showed differential expression between control and treated sample with a p value < 0.05. Nine miRNAs, which have been chosen randomly, were validated with qRT-PCR. SmallRNA-seq data had a linear relationship with qRT-PCR for a goodness of fit (R2) of 0.938. Small RNAs response to cycloastragenol of two samples (Control and 1µM) and an elucidation of the differentially expressed sRNAs in Arabidopsis calli (developed in three biological repetitions for each sample)