## 2 Results
### Identification of miRNA Targets Based on a Degradation Analysis  
To characterize the regulatory roles of blueberry miRNAs, the target genes of all detected miRNAs were identified by analyzing the degradome sequencing data. A total of 20.6, 18.7, and 17.6 million tags were obtained for flowers, young fruits, and ripe fruits, respectively. After removing the adaptor tags, low-quality tags, rRNA, snRNA, snRNA, and tRNA, approximately 20.3, 18.4, and 17.4 million clean tags remained for flowers, young fruits, and ripe fruits, respectively. Mapping degradome tags to the _V. ashei_ unigenes and a subsequent analysis using the CleaveLand pipeline resulted in the identification of 141 target genes for 42 known and 19 novel miRNAs (Table S4). Of these targets, 53, 53, and 65 were for flowers, young fruits, and ripe fruits. Additionally, 11 targets were common to 2 tissues, while 9 were common to all 3 tissues. To further gain insights into the common biological functions, we conducted a GO analysis on all miRNA target genes, which resulted in the annotation of 50 miRNA target genes with GO terms. The annotations covered a wide range of biological activities (Figure 1). These included 41 genes involved in 13 biological processes, 18 genes related to 9 cellular components, and 39 genes involved in 5 molecular functions. Metabolic process (GO: 0008152), catalytic activity (GO: 0003824), and cellular process (GO: 0009987) were the top three terms, with 29, 24, and 23 target genes, respectively. Their \(p\) values of enrichment, calculated on basis of hypergeometric distribution by using whole genome as background, were 5.1 \(\times\) 10\({}^{-3}\), 2.7 \(\times\) 10\({}^{-2}\), and 4.5 \(\times\) 10\({}^{-2}\), respectively, indicating that cellular metabolic processes, biologically catalyzed reactions, and cell physiological processes are the typical activities regulated by miRNAs in _V. ashei_ flowers and fruits. Additionally, some of target genes were also significantly enriched in function categories, response to stimulus(GO:0050896) and transcription factor activity(GO:0000988). Although their numbers were the highest, those results provide clues to further study functions of miRNA-target regulatory pathway. To further elucidate the miRNA-target gene regulatory networks, we mapped miRNA target genes to KEGG (kyoto encyclopedia of genes and genomes) pathways. Interestingly, several important pathways related to floral and fruit development or fruit quality were included. The first is the anthocyanin biosynthesis pathway (Figure 2a). Three miRNAs and their target genes were identified in the present study. The first one was vas-miR-21. Its expression levels were 0.90 and 3.08 RPM in young and ripe fruits, respectively. Its target was a gene encoding chalcogen isomerase (CHI), which was expressed at 365.60 and 191.28 FPKM (fragments per kilo base of transcript per million fragments mapped) in young and ripe fruits, respectively. Our degradome analysis revealed that the CHI transcript was cleaved by vas-miR-21 in ripe fruits, but not in young fruits. The second identified miRNA was vas-miR67a. Our degradome analysis confirmed that vas-miR67a cleaved the transcript of flavanone 3-hydroxylase(F3H) in young fruits. Its expression levels were 1180.38 and 369.56 RPM in young and ripe fruits respectively, while F3H transcripts accumulated to 469.73 and 286.66 FPKM in young and ripe fruits respectively. It indicated F3H expression might also be regulated by another unknown mechanism. The third miRNA was vas-miR858b, which was expressed at 217.02 and 1017.67 RPM in young and ripe fruits, respectively. Its target was a transcription factor gene encoding a TT2 (transparent testa 2)-type MYB (v-myb avian myeloblastosis viral oncogene homolog). Our degradome analysis indicated the transcript was cleaved by vas-miR858b in young and ripe fruits. Moreover, the transcript encoding the TT2-type MYB accumulated to 5.35 and 0.55 FPKM in young and ripe fruits, respectively. The second is cytokinin signal transduction pathway.