*2.6. HRM Primer Designs from Selected Transcripts and HRM Analysis*

HRM primers were designed from 14 selected transcripts with SNPs that exist between the resistant and susceptible lines. The conditions of the designed primers were as follows: the amplified product size was between 80 and 200 bp, the variant region was inside the PCR product, and the annealing temperature was approximately 60 ± 1 ◦C. The sequences and annealing temperature information of the HRM primers are listed in Table 1. HRM was performed using a total 10-μL reaction mixture containing the BioFactTM 2X Real-Time PCR

Master Mix (BIOFACT, Daejeon, Korea) with a 10-pmol primer and 50 ng of gDNA. The reaction conditions were: pre-denaturation at 95 ◦C for 15 min, 40 cycles of denaturation at 95 ◦C for 20 s, annealing at 60 ◦C for 30 s, and extension at 72 ◦C for 20 s. Thereafter, the temperature was sequentially increased from 65 ◦C to 95 ◦C, and the melt curve and peak value were measured. At least six repetitions were performed for each line. The HRM results were statistically grouped using the ANOVA of the XLSTAT program. In addition, to confirm the versatility of the selected HRM marker, which was able to distinguish between resistant and susceptible onion lines, an additional analysis was performed using 'Asia Seed' Co. onion lines.


**Table 1.** HRM primers designed from 14 selected transcripts with SNPs that exist between the resistant and susceptible lines.

## *2.7. Quantitative Real-Time PCR (qPCR) to Identify Expression Level of Transcripts*

The total RNA was extracted from the leaves of the resistant and susceptible lines by using the Plant RNA Extraction Kit (Takara, Shiga, Japan). The concentration and purity of the RNA were measured using a Nanodrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). Thereafter, cDNA was synthesized from 500 ng of RNA extracted from each line by using the HiSenScript™ RH[-] RT PreMix Kit (iNtRON, Seongnam, Korea). PCR for cDNA synthesis was performed using the following cycle: reverse transcription step at 42 ◦C for 1 h and RTase inactivation extension step at 85 ◦C for 10 min. The synthesized cDNA was stored at −20 ◦C. Quantitative real-time PCR (qPCR) was performed to compare the expression levels of the SNP-3 transcript related to the aldo-keto reductase (AKR) gene with in silico data, which showed significant HRM results in 14 transcripts. The qPCR primer conditions were as follows: the amplified product size was less than 200 bp, and the annealing temperature was approximately 60 ± 1 ◦C. The forward primer was 5 -CGT TAG CTC AAG TGG GTT TGA GGT G-3 , and the reverse primer was 5 -CTC CAG CAC ACG CCC TCC A-3 . Before the qPCR analysis, it was confirmed that the 171-bp band targeted by the qPCR primer set was amplified by RT-PCR

using the Maxime™ PCR PreMix Kit (iNtRON, Seongnam, Korea). The reaction cycle was as follows: 10 min of initial denaturation at 95 ◦C, followed by 40 cycles of 95 ◦C for 30 s, 60 ◦C for 30 s, 72 ◦C for 40 s, and a final extension at 72 ◦C for 10 min. The amplified product was electrophoresed using 1% agarose gel.

The qPCR analysis was performed with the TransStart Top Green qPCR Super Mix (TransGen, Beijing, China) containing 1 pmol of qPCR primer and 500 ng of cDNA by using Roter-GeneTM6000 (Corbett, Melbourne, Vic, Australia), and the qPCR program proceeded at 95 ◦C for 10 s, followed by 40 cycles consisting of 2 steps: 95 ◦C for 10 s and 60 ◦C for 30 s. After amplification, a fluorescence melting curve was obtained by heating the samples from 60 ◦C to 95 ◦C. AKR gene expression was identified using the Ct value. To compare the differences in the gene expression levels between the resistant and susceptible lines, the actin gene, the housekeeping gene, was used as a control. After obtaining the Ct value using Roter-Gene Q Series Software, the ΔCt value ((Ct value of target gene)−(Ct value of actin gene)) was calculated. To compare the expression levels based on the susceptible S&P 7483 lines, the ΔΔCt value ((ΔCt of each onion line)−(ΔCt of S&P 7483)) was obtained. The difference in the expression levels was confirmed by calculating the 2ΔΔCt. The expression level of each line was calculated from the results of six repetitions. Data were presented as the means with standard errors, and the means were compared using Duncan's multiple comparison test (*p* ≤ 0.05).
