Deep sequencing identified potential miRNAs involved in defence response, stress and plant growth characteristics of wild genotypes of cardamom.

Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses. In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer. We identified 161 potential miRNAs representing 42 families, including monocot/tissue-specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars. Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT-PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA-mRNA target pairing using RNA ligase-mediated 5' Rapid Amplification of cDNA Ends (5'RLM-RACE) PCR.