## 3 Discussion  
The miRNAs are important for plant development and responses to biotic and abiotic stresses. The development of high-throughput sequencing technology has led to the identification of miRNAs and their targets in many plant species. However, there is very limited information available regarding miRNAs and their targets in blueberry, which is an economically important fruit crop. Thus, we completed high-throughput sequencing and degradome analyses to comprehensively characterize the regulation of miRNAs during the development of blueberry flowers and fruits. Sequencing the sRNA libraries for _V. ashei_ flowers, young fruits, and rip fruits resulted in 11.9, 12.1, and 10.8 million clean reads, respectively. Read lengths for the three samples exhibited a similar distribution pattern, and ranged from 18 to 30 nt. The majority of unique sRNAs were 21-24 nt long (Figure 3a), corresponding to the typical size for dicer-derived products. The most abundant sRNAs were 24 nt long. The result was consistent with those of previous studies involving potato [21], citrus trifoliata [22],durum wheat [23], and rice [24]. However, it was in contrast to the data obtained  
Figure 2: Blueberry miRNA–target gene regulatory networks involved in anthocyanin biosynthesis (**a**) and cytokin signal transduction (**b**). The right of each miRNA showed the corresponding miRNA:target alignment and the expression levels of miRNA and its target gene in flowers, young fruits, and ripe fruits. The arrow on the target transcript indicates the cleavage site and the number above the alignment indicates the number of RNA degradation tags with the 5′-end overlapped to the cleavage site. for grapevine [12] and tomato [25], in which the predominant sRNAs were 21 nt long. An analysis of the distribution of clean read lengths also revealed the most common length was 24 nt (Figure 3b). These observations suggested 24-nt sRNAs may be particularly important in blueberry flowers and fruits. Our results were also indicative of the complexity of the blueberry genome as the 24 nt sRNAs were mainly siRNAs associated with repressive chromatin [1]. Cross-species comparisons of miRNAs have identified 29 conserved miRNA families in fruit trees [26]. These families can be detected in at least three of the following four fruit tree species: peach, apple, citrus, and grapevine. We detected 21 of the 29 families in the current study. This indicates these miRNAs are also conserved in blueberry. Further compared with the two datasets of blueberry miRNAs published respectively by [PERSON] [6] and [PERSON] [7], a total of 61 miRNAs were shared with one of [PERSON]'s and [PERSON]'s in our miRNAs (Table S5). Excepting these, there are still about 73% new miRNAs reported. This implicated, the number of blueberry miRNAs might be not close to saturation to date, on the one hand. On the other hand, it also implicated that there might be false positive miRNAs in published data. The study of blueberry miRNAs still has a long way to go. Our degradome analysis has resulted in the identification of 141 target genes for 44 known and 19 novel miRNAs. GO analysis revealed that genes related to the metabolic process, catalytic activity, cellular process categories, response to stimulus, transcription factor activity, etc. were the main miRNA targets in blueberry flowers and fruits. Earlier studies examining the blueberry transcriptome or expressed sequence tags also concluded that genes associated with metabolic processes, catalytic activities, and cellular processes were actively expressed [6; 10; 27]. These results provide clues to further study functions of miRNA-target regulatory pathway. Blueberry fruits are a rich source of anthocyanins. The TT2-type MYBs can promote anthocyanin accumulation in diverse fruits by binding to the promoters of structural genes to regulate expression [28; 29]. In apple, a gene encoding MdMYB9 is a predicted target of mdm-miR858, and the MdMYB9 and miR858 expression levels reportedly exhibit the opposite trends [30].