Identification of B. rapa miRNAs responsive to cold stress
To identify known miRNAs in our sequence libraries, we aligned the small RNA sequences with mature miRNAs in miRBase and the B. rapareference genome, allowing no more than two mismatches. In total, 77 mature miRNAs related to 184 conserved miRNA precursors were found in our libraries. Among them, 52 miRNAs were conserved with those inArabidopsis , while 16 miRNAs were Brassica -specific miRNAs (Table S2). To further identify novel miRNA candidates, the flanking sequences of unknown small RNAs in genome were extracted for RNA secondary structure prediction using RNAfold. Based on the plant miRNA criteria (Axtell et al., 2018, see the detail in the method), we identified 11 highly credible novel miRNAs (Table S3). Among them, novel-miR1 and novel-miR11 were identified with more than 100 reads.
Among the 77 known miRNAs, 16 miRNAs were differentially expressed between the sample under cold treatment and the control (>1.5-fold) (Table 1). Among these mature miRNAs, bra-miR157a~c, bra-miR161, bra-miR393f, bra-miR396b, bra-miR319c, bra-miR156k, bra-miR1140, bra-miR158a and bra-miR156a~f were down-regulated; and bra-miR168d, bra-miR403, bra-miR390a~b, bra-miR398b~c, bra-miR408, and bra-miR1885 were up-regulated under cold treatment. Among them, theBrassica- specific bra-miR1885 was induced more than four-fold by cold stress. Among the novel miRNAs, only novel-miR11 was cold-responsive. Taken together, the small RNA profiling provided potential candidates of cold-responsive miRNAs in B. rapa .