4.8. The Analysis of Differentially Expressed Genes

Raw reads generated in this study are available at the NCBI BioProject under the accession number PRJNA785089. The quality of the reads was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 13 November 2021). Reads with a q-score < 30 and rRNA-corresponding reads were filtered out using Trimmomatic and SortMeRNA, respectively [88,89]. Pseudo-alignment and quantification of filtered reads were carried out using Kallisto [90] with default parameters and reference transcript sequences of Secale cereale inbred line ‘Lo7′ [33]. The edgeR package [91] was used to reveal differentially expressed genes (DEGs). Genes that had TMM-normalized read counts per million (CPM) values ≥ 1 in all replicates within at least one of the experimental conditions were considered to be expressed in our study. Genes with |log₂FC| < 1 and FDR < 0.05 were considered to be DEGs. To interpret the RNA-Seq data in terms of general physiology, the reference rye transcript annotations were fused to our RNA-Seq data. Then, all transcripts were additionally annotated using eggNOG mapper [92] with default parameters, and BLAST+ against the reference proteomes of Arabidopsis thaliana, Oryza sativa, Triticum aestivum, and Hordeum vulgare obtained from the UniProt website (http://uniprot.org/, accessed on the 22 November 2021). The GO, KEGG, KOG, and CAZy mappings obtained from eggNOG were used to perform the automatic classification of DEGs into functional categories using R software. The merged classification based on the above-mentioned databases was created, manually checked, edited, and enriched by the “missed” genes based on the information from the UniProt database.

The functional DEG classification was hierarchically organized into 4 levels of categories. Categories of successive levels are nested, with the 1st level categories being the largest and the 4th level categories being the smallest. The number of up- and downregulated DEGs in each category were calculated. The classification markup and DEG numbers are given in Supplementary Table S2. The measures of the expression levels are given in Supplementary Table S3.