In Silico Identification and Validation of Potential microRNAs in Kinnow Mandarin (Citrus reticulata Blanco)

MicroRNAs (miRNAs) are a large family of 19-25 nucleotides, regulatory, non-coding RNA molecules that control gene expression by cleaving or inhibiting the translation of target gene transcripts in animals and plants. Despite the important functions of miRNAs related to regulation of plant growth and development processes, metabolism, and abiotic and biotic stresses, little is known about the disease-related miRNA. Here, we present a new pipeline for miRNA analysis using expressed sequence tags (ESTs)-based bioinformatics approach in Kinnow mandarin, a commercially important citrus fruit crop. For this, 56,041 raw EST sequences of Citrus reticulata Blanco were retrieved from EST database in NCBI through step-by-step filtering and processing methods and 130 miRNAs were predicted. Upon blast with Citrus sinensis transcriptome data, these produced potential targets related to disease resistance proteins, pectin lyase-like superfamily proteins, lateral organ boundaries (LOB) domain-containing proteins 11, and protein phosphatase 2C family proteins, protein kinases, dehydrogenases, and methyltransferases. Majority of the predicted miRNAs were of 22, 23, and 24 nucleotides in length. To validate these computationally predicted miRNA, poly(A)-tailed Reverse Transcription-PCR was applied to detect the expression of seven miRNA which showed disease-related potential targets, in citrus greening diseased leaf tissues in comparison to the healthy tissues of Kinnow mandarin. Our study provides information on regulatory roles of these potential miRNAs for the citrus greening disease development, miRNA targets, and would be helpful for future research of miRNA function in citrus.