Due to the disruptions by Covid-19 pandemic and termination ahead of schedule, we only completed parts of the planned objectives:
1. Developed a SHAPE-MaP procedure applied to RNA structure probing in wheat seedlings.
2. Performed SHAPE-MaP in wheat seedlings under different temperatures.
3. Generated RNA structure probing data for wheat seedlings under different temperature.
4. Validated the accuracy and sensitivity of SHAPE-MaP method in RNA structure probing in wheat seedlings.
We performed SHAPE-MaP RNA structure probing in wheat seedlings subject to high temperature (38℃) stresses. Totally, ~500 million high through-put sequencing reads were obtained (Figure 1). About 60-70% of the reads can be aligned to wheat reference genome sequence (Chinese Spring) (Figure 2). We then checked the reads coverage along transcripts and found that sequenced reads were nearly equally covered from transcript 5’ end to 3’ end, which indicated the high quality of our sequencing libraries (Figure 3).
For SHAPE-MaP method, RNA structure information is reflected by mutation rate calculated from sequenced reads. We calculated mutation rate for all highly covered positions in transcripts and found that more than 35% of the nucleotides have mutation rate >=0.5%, which is high enough to be detected (Figure 3). No apparent difference was observed between normal and high temperature, which needs further analysis (Figure 3). To check the accuracy of SHAPE-MaP method in wheat, we mapped the mutation rate difference to 18S ribosome RNA secondary structure and found that our SHAPE-MaP mutation rate can properly reflect the phylogenetical structure of 18S rRNA (Figure 4).
Only positions with >1000x coverage were counted. For both normal and high temperature conditions, about 35% of the nucleotides have mutation rate >=0.5%, which is high enough to be detected. No apparent difference was observed between normal and high temperature.
