Flower development plays vital role in horticultural plants. Post-transcriptional regulation via small RNAs is important for plant flower development. To uncover post-transcriptional regulatory networks during the flower development in Dimocarpus longan Lour. ‘Shixia’, an economically important fruit crop in subtropical regions, we collected and analyzed sRNA deep-sequencing datasets and degradome libraries Apart from identifying miRNAs and phased siRNA generating loci (PHAS loci), 120 hairpin loci, producing abundant sRNAs, were identified by in-house protocols. Our results suggested that 56 miRNA-target pairs, 2221-nt-PHAS loci, and 111 hairpin loci are involved in post-transcriptional gene silencing during longan reproductive development. Lineage-specific or species-specific post-transcriptional regulatory modules have been unveiled, including miR482-PHAS and miRN15. miR482-PHAS might be involved in longan flower development beyond their conserved roles in plant defense, and miRN15 is a novel miRNA likely associated with a hairpin locus (HPL-056) to regulate strigolactone receptor gene DWARF14 (D14) and the biogenesis of phasiRNAs from D14. These small RNAs are enriched in flower buds, suggesting they are likely involved in post-transcriptional regulatory networks essential for longan flower development via the strigolactone signaling pathway.

Small RNAs (sRNAs) are vital regulators of gene expression and involved in various biological processes. Among them, microRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) have been well defined and studied in the past decades. A bunch of scripts or pipelines were developed to annotate miRNAs and phasiRNAs. However, some computational annotations are rough and without careful manual check, resulting in low quality annotation. In this study, 19 public strawberry (Fragaria vesca) sRNA sequencing data from nine different tissues were collected to annotate miRNAs and PHAS loci in F. vesca. After bioinformatics analysis and careful manual checking, 167 known miRNAs, 27 miRNA*s with notable abundance, 54 novel miRNAs were accurately annotated. The terms of two miRNAs were corrected from miR477b and miR5225 using miRN47 and miR3627h, respectively. Besides 21 nucleotides (nt) miR390, eleven miRNAs with a length of 22-nt are in charge of triggering the biogenesis of 21-nt phasiRNAs from 110 PHAS loci in strawberry. In particular, we found several PHAS loci were targeted by two different miRNAs (similar to the “two-hit” model) and the phasiRNA generating region located between two target sites. We speculate that one target site is in control of triggering phasiRNA biogenesis and the other target site define the boundary of the region of phasiRNA biogenesis, which likely provide an accurate way for phasiRNA generation. Overall, we provided a comprehensive and accurate annotation of miRNAs and PHAS loci in the F. vesca genome.