*2.4. RNA Sequencing and Variant Analysis*

For RNA sequencing, the leaves of four resistant and three susceptible lines were ground, and the total RNA was extracted using the RNease® kit (QIAGEN, Hilden, Germany). Before analyzing the RNA sequence, the OD values were measured using Dropsense96 (Trinean, Gentbrugge, Belgium), and the total RNA quality was checked using a Bioanalyzer RNA Chip (Agilent Technologies, Santa Clara, CA, USA). The library was constructed using the TruSeq Stranded mRNA kit (Illumina, San Diego, CA, USA), and RNA-seq was performed using HiseqX (Illumina, San Diego, CA, USA) by the DNAcare Company (Seoul, Korea). To remove the low-quality base and Illumina adapters, the Trimmomatic program (USADELLAB, Aachen, Germany) was used on the RNA-seq raw data of each line. After trimming, only paired reads containing at least 50 nucleotides were used for the analysis. In addition, quality trimming was performed by applying options such as a sliding window, average quality, and minimum read size. Thereafter, the generated trimmed data were used for the de novo assembly of resistant and susceptible lines by using the Trinity program. As a reference for mapping, the onion reference transcript (National Agricultural Biotechnology Information Center (NABIC), Rural Development

Administration (RDA), JeonJu, Korea) and the data obtained through the de novo assembly were used. Read mapping was performed using the BWA-Mem algorithm. To remove the duplicated PCR reads from the produced BAM file, the Picard program (Broad Institute, Cambridge, MA, USA) was used. Thereafter, the variant information of seven lines was produced using haplotypeCaller of the Genome Analysis Tool Kit (GATK, Broad Institute, Cambridge, MA, USA), and the final raw variant call file (vcf) was generated by integrating the variant files of each line. Variant filtering was performed using Vcftools (VCFtools, 1000 Genomes Project Analysis Group, http://vcftools.sourceforge.net/ accessed on 1 October 2021) to remove the low-quality genotypes, target missing levels of depth coverage (DP), genotype quality (GQ), and genotype data. The filtering conditions were set to min DP = 5, max DP = 100, and min GQ = 220, and the missing was set to 20%. Various information was used to select the variants showing polymorphisms between the resistant and susceptible lines. For the selection condition for the variants, those showing the same genotype in each resistant and susceptible group and showing polymorphisms between these groups were selected.
