RNA-seq based expression of DEGs in roots and leaves of bread wheat

RNA from roots and leaves of twenty eight bread wheat genotypes was extracted, purified, quantified and sent to Beijing Genomics Institute (BGI) for sequencing. Briefly, mRNAs were isolated and fragmented from total RNA using the oligo (dT) method for cDNA synthesis. The 75-bp single-end sequencing libraries were constructed, and sequencing was performed on HiSeq X Ten (Illumina, San Diego, USA) using standard protocols. Adapters with unknown bases (N's > 5%) and low quality were removed from raw reads to produce 'clean data' as FastQ data files using a quality control software, SOAPnuke version 2.1.6. High quality single-end reads were mapped to the bread wheat reference genome (IWGSC, INSDC Assembly GCA_900519105.1) using HISAT2 (Hierarchical Indexing for Spliced Alignment of Transcripts) software version 2.2.1. Alignment of the reference sequence with reads were performed using Bowtie. Quantification of the reads was performed using featureCounts software program. Differentially expressed genes (DEGs) were identified using DeSEQ2 in R version  4.1.1. DEGs were then filtered based on their adjusted p-value. A total of 120744 DEGs were identified with varying expression in roots and leaves. The threshold value for filtering was set at 0.1.