Genome-wide identification of microRNAs associated with the somatic embryogenesis in Eucalyptus

Background: MicroRNAs (miRNAs) are a class of small noncoding RNAs with 18-24 nucleotides in length and function in many biological processes in plant. Although Eucalyptus trees are widely planted across the world, our understanding of the miRNA regulation in the somatic embryogenesis of Eucalyptus is still poor. Here we reported for the first time the miRNA profiles of differentiated and dedifferentiated tissues of two Eucalyptus cultivars and identified miRNAs involved in the somatic embryogenesis of Eucalyptus.Results: Stem and tissue-culture induced callus were obtained from the subculture seedlings of E. camaldulensis and E. grandis x urophylla, and were used as differentiated and dedifferentiated samples, respectively. We generated 346.4 million reads for 12 samples (n=3) and identified 888 miRNA precursors (197 known and 691 novel) which can produce 1,067 mature miRNAs. These miRNAs were mainly distributed in chromosomes Chr03, Chr05 and Chr08, and can produce 46 miRNA clusters. In these samples we detected 998 miRNAs with TPM (transcripts per million reads) > 5 and found that highly expressed miRNAs varied across samples. We identified 327 and 343 differentially expressed miRNAs in the dedifferentiation process of E. camaldulensis and E. grandis x urophylla, respectively. Dysregulated miRNAs shared by the two cultivars might be involved in the development of embryonic callus of Eucalyptus, such as MIR156, MIR159, MIR160, MIR164, MIR166, MIR169, MIR171, MIR399 and MIR482. We also identified 81 up-regulated (e.g., miR159c-3p, miR167a-5p, miR397a-3p, miR397c-5p, miR397d-3p, miR397d-5p, N-miR1-5p and N-miR5-5p) and 67 down-regulated (e.g., miR482b-3p, N-miR3-3p, miR156a-3p, N-miR40-3p and N-miR18-5p) miRNAs specific to E. camaldulensis. Target prediction and functional analysis showed they might be involved in longevity regulating and plant hormone signal transduction pathways. Then, the expression patterns of these miRNAs were confirmed by qRT-PCR. Conclusions: This is the first time to study the miRNAs profiles in the dedifferentiation process of Eucalyptus and it will provide a valuable resource for future studies. More importantly, our findings will improve our understanding of miRNA regulation and molecular mechanisms during the somatic embryogenesis of Eucalyptus, and the output of this study will benefit the Eucalyptus breeding program.