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The roles of microRNAs (miRNAs) related to ethylene response in banana fruits remain unknown because many miRNAs are differentially expressed as the fruit ripens, making the identification of ethylene-responsive miRNAs difficult. Using newly harvested banana fruits (within 5 h after harvest) as material, we found that these fruit did not ripen when treated with 5 μL/L of ethylene for 12 h at 22 °C. Two miRNA libraries were generated from newly harvested banana fruits with and without ethylene treatment and sequenced. In total, 128 known miRNAs belonging to 42 miRNA families were obtained, and 12 novel miRNAs were identified. Among them, 22 were differentially expressed in response to ethylene treatment, among which 6 known miRNAs and their putative targets were validated using qRT-PCR. These putative targets encoded proteins including GATA, ARF, DLC, and AGO, etc. KEGG and GO analyses showed that miRNAs differentially expressed in response to ethylene mainly function in the molecular and biological processes.

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