Genome-wide screening and characterization of long non-coding RNAs involved in flowering development of trifoliate orange (Poncirus trifoliata L. Raf.)

Long non-coding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in post-transcriptional and transcriptional regulation in Arabidopsis. However, lncRNAs and their functional roles remain poorly characterized in woody plants, including citrus. To identify lncRNAs and investigate their role in citrus flowering, paired-end strand-specific RNA sequencing was performed for precocious trifoliate orange and its wild-type counterpart. A total of 6,584 potential lncRNAs were identified, 51.6% of which were from intergenic regions. Additionally, 555 lncRNAs were significantly up-regulated and 276 lncRNAs were down-regulated in precocious trifoliate orange, indicating that lncRNAs could be involved in the regulation of trifoliate orange flowering. Comparisons between lncRNAs and coding genes indicated that lncRNAs tend to have shorter transcripts and lower expression levels and that they display significant expression specificity. More importantly, 59 and 7 lncRNAs were identified as putative targets and target mimics of citrus miRNAs, respectively. In addition, the targets of Pt-miR156 and Pt-miR396 were confirmed using the regional amplification reverse-transcription polymerase chain reaction method. Furthermore, overexpression of Pt-miR156a1 and Pt-miR156a1 in Arabidopsis resulted in an extended juvenile phase, short siliques, and smaller leaves in transgenic plants compared with control plants. These findings provide important insight regarding citrus lncRNAs, thus enabling in-depth functional analyses.