Identification and expression profiling of miRNAs in two color variants of carrot (Daucus carota L.) using deep sequencing

Abstract

microRNAs represent small endogenous RNAs which are known to play a crucial role in various plant metabolic processes. Carrot being an important vegetable crop, represents one of the richest sources of carotenoids and anthocyanins. Most of the studies on microRNAs have been conducted in the aerial parts of the plants. However, carrot has the rare distinction of storing these compounds in roots. Therefore, carrot represents a good model system to unveil the regulatory roles of miRNAs in the underground edible part of the plant. For the first time, we report the genome wide identification and expression profiling of miRNAs in two contrasting color variants of carrot namely Orange Red and Purple Black using RNA-seq. Illumina sequencing resulted in the generation of 25.5M and 18.9M reads in Orange Red and Purple Black libraries, respectively. In total, 144 and 98 (read count >10), conserved microRNAs and 36 and 66 novel microRNAs were identified in Orange Red and Purple Black, respectively. Functional categorization and differential gene expression revealed the presence of several miRNA genes targeting various secondary metabolic pathways including carotenoid and anthocyanin biosynthetic pathways in the two libraries. 11 known and 2 novel microRNAs were further validated using Stem-Loop PCR and qRT-PCR. Also, target validation was performed for selected miRNA genes using RLM-RACE approach. The present work has laid a foundation towards understanding of various metabolic processes, particularly the color development in carrot. This information can be further employed in targeted gene expression for increasing the carotenoid and anthocyanin content in crop plants.