Multiple microRNAs Regulate the Floral Development and Sex Differentiation in the Dioecious Cucurbit Coccinia grandis (L.) Voigt

Abstract

Despite having a small genome and heteromorphic sex chromosomes, the molecular basis of sex differentiation in the dioecious cucurbit, Coccinia grandis, largely remains unclear. Increasing evidences indicate a vital regulatory role of microRNAs in the reproductive development of plants. In this study, we used a combination of high-throughput small RNA sequencing and computational analysis to identify sex-specific miRNAomes from male and female buds of C. grandis. Ninety-eight conserved miRNAs from 22 families and 44 novel miRNAs specific to C. grandis were detected. Comparative profiling together with Northern blot and qRT-PCR analysis revealed 41 significantly differentially expressed (DE) miRNAs, of which 16 could be fundamental to the regulation of sexual dimorphism. One hundred six target genes were predicted for 35 DE miRNAs that were significantly involved in flower organogenesis, phytohormone signaling, metabolism, transcription regulation, and DNA methylation. Temporal analysis of a set of 16 target genes at three stages of the bud development revealed a reciprocal alteration in their expression pattern with the complementary miRNAs. Further, the miR167a-1, miR393a, miR398b, and miRn9 mediated down-regulation of four predicted targets associated with reproductive organ development in C. grandis was confirmed through transient co-expression in Nicotiana benthaminana. Taken together, the present study represents the first report suggesting that multiple miRNA-mediated gene silencing cascade could be involved in regulating the molecular basis of sex differentiation in C. grandis.