Small RNA and mRNA Sequencing Reveal the Roles of microRNAs Involved in Pomegranate Female Sterility

Female sterility is a key factor restricting plant reproduction. Our previous studies have revealed that pomegranate female sterility mainly arose from the abnormality of ovule development. MicroRNAs (miRNAs) play important roles in ovule development. However, little is known about the roles of miRNAs in female sterility. In this study, a combined high-throughput sequencing approach was used to investigate the miRNAs and their targeted transcripts involved in female development. A total of 103 conserved and 58 novel miRNAs were identified. Comparative profiling indicated that the expression of 43 known miRNAs and 14 novel miRNAs were differentially expressed between functional male flowers (FMFs) and bisexual flowers (BFs), 30 known miRNAs and nine novel miRNAs showed significant differences among different stages of BFs, and 20 known miRNAs and 18 novel miRNAs exhibited remarkable expression differences among different stages of FMFs. Gene ontology (GO) analyses of 144 predicted targets of differentially expressed miRNAs indicated that the “reproduction process” and “floral whorl development” processes were significantly enriched. The miRNA–mRNA interaction analyses revealed six pairs of candidate miRNAs and their targets associated with female sterility. Interestingly, pg-miR166a-3p was accumulated, whereas its predicted targets (Gglean012177.1 and Gglean013966.1) were repressed in functional male flowers (FMFs), and the interaction between pg-miR166a-3p and its targets (Gglean012177.1 and Gglean013966.1) were confirmed by transient assay. A. thaliana transformed with 35S-pre-pg-miR166a-3p verified the role of pg-miR166a-3p in ovule development, which indicated pg-miR166a-3p’s potential role in pomegranate female sterility. The results provide new insights into molecular mechanisms underlying the female sterility at the miRNA level.