| Summary: | With versatile biological functions, microRNAs (miRNAs) participate in the regulation of post-transcriptional gene expression in plants. <i>Tulipa thianschanica</i> Regel is a key wild tulip resource of the Liliaceae; however, it reproduces poorly under natural conditions, and the spreading and expansion of its population rely on the release of its seeds. In this study, <i>T. thianschanica</i> seeds were subjected to stratification at 4 °C for three different durations: 1 d (physiological dormancy), 20 d (dormancy release), and 40 d (non-dormancy). Nine samples were selected, and miRNA databases were established and annotated. This research revealed information on 34 miRNA families, including 14 newly discovered families and 20 families that were previously known. <i>TpmiRNA167</i> and <i>TpmiRNA395</i> were observed to be upregulated. Conversely, <i>TpmiRNA166</i>, which targets the key enzymes involved in antioxidant reactions, was downregulated. Notably, <i>PC-5p-84014</i> and <i>TpmiRNA159</i> were found to target <i>TpPYL</i> and <i>TpDELLA</i>, respectively, which influenced the abscisic acid/gibberellin (ABA/GA) ratio. Additionally, <i>TpmiRNA160</i> and <i>TpmiRNA164</i> were found to participate in the indole-3-acetic acid signal transduction pathway and interact with ABA, thereby contributing to the release of dormancy. In conclusion, this study provides a comprehensive understanding of the mechanism used to release dormancy in <i>T. thianschanica</i> seeds at the molecular level.
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