## 3 Discussion
Similarly, we identified a gene encoding a TT2-type MYB as the target of vas-miR858b. Moreover, vas-miR858b and the TT2-type MYB gene were differentially expressed. Specifically, vas-miR858b expression levels were 217.02 and 1017.67 RPM in young and ripe fruits, respectively, while the TT2-type MYB transcript levels were 5.35 and 0.55 FPKM in young and ripe fruits, respectively. This further confirms that vas-miR858b negatively regulates the accumulation of anthocyanin in blueberry fruits, which implicated that anthocyanin biosynthesis might be very week in fully mature fruit. Interestingly, we identified the CHI and F3H structural genes as the targets of vas-miR8-21 and vas-miR867a, respectively. A previous study revealed that miR167a targets an IAA amidohydrolase gene, IAR3, as part of a drought tolerance mechanism [31]. The higher accumulation of transcripts in young fruits suggested CHI was more actively expressed in developing fruits than in ripe fruits. Our degradome analysis revealed that the CHI transcript was cleaved by vas-miR-21 in ripe fruits, but not in young fruits. This further indicated CHI expression was repressed in ripe fruits, which was consistent with  
Figure 3: Length distribution of unique sRNAs (**a**) and clean reads (**b**) for _V. ashei_ flowers, young fruits, and ripe fruits. the lower accumulation of CHI in ripe fruits than in young fruits. While positive correlation between expression levels of F3H and vas-miRR167a implicated that F3H expression might also be regulated by another unknown mechanism. Cytokinin is an essential hormone for nearly all aspects of plant growth and development. Cytokin activities progressively decline as fruits mature [32], implying there exists a mechanism that gradually inactivates cytokinin signal transduction. We identified a gene encoding a histidine kinase as a target of vas-miR5021 in ripe fruits. Histidine kinase is a cytokinin receptor that can transduce cytokinin signals across the plasma membrane. Our sequencing results confirmed that the transcript abundance for the histidine kinase gene decreased as young fruits ripened, while vas-miR5021 expression significantly increased during the same period. This further supports the likelihood of a mechanism that inactivates cytokinin signal transduction in ripening fruits. Abscisic acid is a central regulator of fruit ripening. Its content increases markedly to initiate blueberry fruit ripening, and peak levels occur at the \(\sim\)75% blue stage [27]. In our study, PP2C expression exhibited a similar pattern, with the corresponding transcript levels reaching 40.61 and 290.06 FPKM in young and ripe fruits, respectively. An earlier study involving beechnut indicated that PP2C expression levels in seeds and young seedlings were higher expressed by exogenously applied ABA. However, the over-expression of PP2C in transgenic _A. thaliana_ decreased ABA sensitivity in germinating seeds and in response to different abiotic stresses [33]. Additionally, PP2C has been confirmed as a negative regulator of ABA signaling. In the present study, PP2C was identified as a target of vas-miR-74 in ripe blueberry fruits. The vas-miR-74 expression levels were 0 and 0.81 RPM in young and ripe fruits, respectively, which is consistent with the PP2C expression levels. In a previous study, PP2C was identified as a target of cand_mir_322-3p in the ABA signaling network [34], a target of ttu-miR008 and tae-miRR408 in wheat [23; 35], and a target of five miRNAs--including sy-miRR159b-3p, sy-miRR393a, sy-miRR268, sy-miRR6020a-5p, and sy-miRR7696a-3p--in tomato [36].