MicroRNA166 (miR166) is highly conserved and has diverse functions across plant species. The highbush blueberry (Vaccinium corymbosum) genome is thought to harbor 10 miRNA166 loci (Vco-miR166), but the extent of their evolutionary conservation or functional diversification remains unknown. In this study, we identified six additional Vco-miR166 loci based on conserved features of the miR166 family. Phylogenetic analyses showed that mature Vco-miR166s and their precursor cluster in several clades are evolutionary conserved with diverse species. The cis-regulatory elements in the Vco-miR166 promoters indicated functions related to different phytohormones and defense responses. We also identified putative targets of vco-miR166s, which targeted the same gene families, suggesting the functional conservation and diversification of Vco-miR166 family members. Furthermore, we examined the accumulation patterns of six mature Vco-miR166s in response to abiotic stresses by stem-loop reverse RT-qPCR, which revealed their upregulation under freezing, cold, and heat stress, while they were downregulated by drought compared to control growth conditions. However, Vco-miR166 members showed different expression patterns when exposed to salt stress. These results showed that conserved Vco-miR166 family members display functional diversification but also coordinately influence plant responses to abiotic stress.
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