Identification of miRNAs in Response to Cold Stress in ‘Chaling’ Common Wild Rice (Oryza rufipogon Griff.)

(1) Background: 'Chaling' common wild rice (CLWR), one of the two wild rice populations with the northernmost natural distribution worldwide, exhibits excellent cold tolerance. The role of microRNA (miRNA) in regulating cold tolerance in plants has been reported in some species. However, the miRNA landscape in CLWR remains unexplored. (2) Methods: We assessed cold tolerance in CLWR and the conventional rice variety 9311 at 4 °C, and conducted small RNA sequencing and analysis on eight samples from both CLWR and 9311, before and after cold treatment. (3) Results: All seedlings of CLWR survived after cold treatment and recovery, while all seedlings of 9311 died. After quality control and classification analysis of the small RNA sequences, numerous known and novel microRNAs (miRNAs) were identified. The expression analysis showed 59 differentially expressed miRNAs in CLWR before and after cold treatment, and 19 in 9311, with eight overlapping differentially expressed miRNAs between the two varieties. Target gene prediction for these miRNAs indicated that some predicted target genes, such as <i>CTB4a</i> and <i>GRF4</i>, are key genes involved in regulating cold tolerance in rice. Additionally, CLWR actively mobilizes more miRNAs and their target genes to resist cold stress than 9311. (4) Conclusions: This study offers new insights into the regulatory mechanisms of cold tolerance in CLWR at the miRNA level, providing a wealth of gene (miRNA) resources for genetic breeding research in rice aimed at enhancing cold tolerance.