Genome-wide analysis of small RNAs responsive to cold stress inB. rapa
To study the cold response of B. rapa , the degree of cold tolerance was estimated by evaluating morphological traits under cold stress. After 7 days of cold treatment (4 °C), the plants stopped growing and their leaves drooped (Figure 1A). Therefore, to identify genome-wide cold-responsive miRNAs, we performed cold treatment for 7 days on 3-week-old seedlings of B. rapa in a growth chamber, and plants grown at 22 °C was used as control for small RNA sequencing.
Totally, 17.96 million and 17.17 million raw reads were obtained from these two small RNA sequencing samples treated with 22 °C and 4 °C, respectively (Figure 1B). Among them, most of small RNA sequences were shared between these two libraries, while 2.82 million small RNA sequences were specific in the seedling under normal temperature, and 2.23 million small RNA sequences were specific in the seedling under 4 °C (Figure 1B). Small RNA length distribution analysis showed that 24-nt small RNAs were most abundant, and 21-nt small RNAs were second most abundant (Figure 1C).
To learn the genomic loci that produced these small RNAs, 11.46 million (22 °C) and 11.36 million (4 °C) clean reads were mapped to the B. rapa reference genome (http://brassicadb.cn/#/). We found that the small RNAs were generated from exons, introns, miRNAs, rRNAs, snRNAs, snoRNAs, tRNAs, and repeat regions (Table S1). Notably, focusing on small RNAs from non-coding gene regions, most abundant small RNAs were produced from ribosomal RNAs (rRNAs) (22 °C, 44.67% and 4 °C, 48.10%), unknown RNAs (22 °C, 27.65% and 4 °C, 25.22%), and miRNA precursors (22 °C, 8.50% and 4 °C, 8.71%) (Figure S1).