Identification of known and novel miRNAs in B. rapa
We aligned the clean reads with known miRNAs in the miRBase
(https://www.mirbase.org/), allowing two mismatches using TopHat
(http://ccb.jhu.edu/software/tophat/index.shtml). The potential miRNAs
were mapped to B. rapa reference genome
(http://brassicadb.cn/#/) to extract the 600-bp flanking sequence. The
flanking sequence of miRNAs were also aligned with known pre-miRNAs to
confirm their identities of miRNA family. The flanking sequence of
unknown small RNAs was also used to predict secondary structures using
RNAfold software
(http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). To
identify novel miRNA, Small RNAs that showed less than 2 mismatches with
known miRNAs in miRbase were filtered. The potential miRNAs that had
fewer than 10 hits in the reference genome were retained. 600-bp
flanking sequences of small RNAs were used to predict their secondary
structures using RNAfold software with default parameters. Novel miRNAs
were identified according to the plant miRNA criteria (Axtell et al.,
2018, Yu et al., 2011). In brief, a key judgement was made by
determining whether the potential miRNAs and their miRNA* were on the
stem with fewer than four bulges, and the miRNA candidate were most
abundant among small RNAs generated from given miRNA precursors. After
Blast these novel miRNA precursors in NCBI, we found them were only
detected in Brassica, therefore, they were defined as Brassica-specific
miRNAs.