A chromosome single segment substitution line (CSSL) DC90, which was generated by introgressing CTS-12, a locus derived from common wild rice (Oryza rufipogon Griff.), into the 9311 (Oryza sativa L. ssp. indica) background, exhibits a chilling tolerance phenotype under chilling stress. Here, an integration of microRNA (miRNA) deep sequencing and transcriptomic sequencing analysis was performed to explore the expression profiles of miRNAs and their target genes mediated by CTS-12 under chilling stress, and to reveal the possible regulatory mechanisms of miRNAs that are involved in chilling tolerance. Integration analysis revealed that a number of differentially expressed miRNAs (DEMs) and putative target genes with different expression patterns and levels were identified in 9311 and DC90 under chilling stress. KEGG enrichment analysis revealed that the target genes that are regulated by chilling-induced miRNAs are involved in the regulation of various biological processes/pathways, including protein biosynthesis, redox process, photosynthetic process, and chloroplast development in two genotypes. CRISPR/Cas9 editing of the target genes of the key DEMs in a chilling tolerant rice variety Zhonghua 11 (ZH11) found that LOC_Os11g48020 (OsGL1-11), one of the putative target genes of osa-miR1846a/b-5p and encoding a wax synthesis protein, is correlated with a chilling stress tolerance phenotype, implying osa-miR1846a/b-5p/OsGL1-11 plays an important role in CTS-12-mediated chilling stress tolerance regulatory pathway(s). Therefore, we speculate that the CTS-12 may regulate the key miRNA target genes in response to chilling stress by differential regulation of miRNAs in wild rice, thereby resulting in the variation of chilling tolerance phenotype between 9311 and DC90.

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