*3.7. Transcripts Selection to Develop SNP Markers and a HRM Analysis*

Based on variant analyses, including the DEG and GO analyses, the transcripts were selected to develop SNP markers for the screening of gray mold-resistant onions. A total of 118 transcripts with 233 variants, such as SNPs and indels, between the resistant and susceptible groups were selected by variant analyses. Thereafter, 29 transcripts that were found to be related to disease resistance through the GO analysis and showed higher gene expression levels in the resistant group than in the susceptible group in the DEG analysis were selected. Finally, a total of 14 transcripts with SNPs were selected to develop SNP markers, and HRM primers were designed from these 14 selected transcripts with SNPs

that existed between the resistant and susceptible lines (Table 1). These selected transcripts were associated with genes with functions related to 'response to biotic stimulus' and 'response to stress'. Plants are known to show resistance to pathogens in various ways. Although it prevents infection by pathogens structurally, such as through cell walls, it has several defense systems that show resistance to invading pathogens. The gene functions of the selected transcripts were 'lipoxygenase 3', 'Glutathione S-transferase', and 'systemic acquired resistance', which were genes related to plant resistance (Table 2).

**Table 2.** Gene functions of 14 selected transcripts with SNPs that exist between the resistant and susceptible lines.


<sup>z</sup> The Arabidopsis Information Resource ID.

Each HRM amplicon obtained from the 14 HRM primers was confirmed to match the expected size by electrophoresis. In addition, the sequence analysis confirmed that SNPs existed between the resistant and susceptible lines and revealed the amino acid sequences that were altered by the SNP (Figure 5). The SNPs targeted by the HRM primers are shown in green, and the nonsynonymous mutations are shown in red. Synonymous mutations are shown in blue and were present in SNP-1, SNP-2, SNP-4, SNP-12, and SNP 14. Nonsynonymous mutations were identified in SNP-3, SNP-5, SNP-6, SNP-7, SNP-8, SNP-9, SNP-10, SNP-11, and SNP-13. The melting peak values obtained from the HRM analysis were confirmed by ANOVA grouping. As a result, SNP-3 primers targeting the AKR gene transcript capable of distinguishing resistance and susceptibility were selected. The expected amplicon size of the SNP-3 primer was 134 bp. The HRM amplicon sizes of all seven onion lines were identified as the same. In addition, the sequences of the four resistant and three susceptible lines targeted by the SNP-3 primer were analyzed (Figure 6). Two SNPs were identified within the sequence. One SNP showed the 'T' allele in the resistant lines and the 'C' allele in the susceptible lines, while the other SNP showed the 'C' allele in the resistant lines and the 'G' allele in the susceptible ones.


**Figure 5.** SNPs and amino acid sequences targeted by the HRM primers in the resistant and susceptible lines. The SNPs targeted by HRM primers are shown in green. The synonymous mutations are shown in blue, and the nonsynonymous mutations between the resistant and susceptible groups are shown in red.


**Figure 6.** Sequences of the products amplified with the SNP-3 HRM primer in four resistant and three susceptible onion lines. Two SNPs were identified within the sequence. One SNP showed the 'T' allele in the resistant lines and the 'C' allele in the susceptible lines, while the other SNP showed the 'C' allele in the resistant lines and the 'G' allele in the susceptible ones. The forward (F) and reverse (R) primers for the SNP-3 HRM marker are underlined with the arrow.

> In the results of the HRM analysis, which was repeated six times using the SNP-3 primer, the melting values of the resistant strains were 79.642–79.838 and those of the susceptible strains were 80.353–80.472. Upon statistically grouping these values by the Duncan method using ANOVA, significant results were obtained by grouping four resistant lines into one group (A) and three susceptibility lines into another group (B) (Table 3a). In the HRM result graph, the resistant lines (red) show a distinct curve from the susceptible lines (blue) (Figure 7a). Compared with the sequence results, it was confirmed that the resistant group that showed 'T' and 'C' at the SNP position had a lower melting temperature than the susceptible group that had 'C' and 'G'. In addition, for validation, the 'Asia Seed' Co. onion lines were analyzed using the SNP-3 HRM marker. As a result of ANOVA grouping, the resistant lines (Asia-12, Asia-48, and Asia-53) were grouped into one group (A), and the susceptible lines (Asia-30, Asia-35, Asia-45, and Asia-50) were also grouped together (B) (Table 3b). Similar to the HRM results of the 'Seeds & People' Co. onion lines, the HRM curve graph of the 'Asia Seed' Co. onion lines showed that the resistant lines were melted at a lower temperature than the susceptible lines (Figure 7b). Based on this result, it was confirmed that the SNP-3 HRM marker is versatile and can be used to select resistant onions.


**Table 3.** HRM analysis with the SNP-3 primers and statistically grouped by using ANOVA.

(A) HRM analysis results of the 'Seeds & People' Co. onion lines.

\* In each column, means with the same letter are not significantly different.

**Figure 7.** HRM analysis of the 'Seeds & People' Co. and 'Asia Seed' Co. onion lines. (**A**) HRM curve of the 'Seeds & People' Co. onion lines. Red: resistant lines (S&P 7522, S&P 7521, S&P 7129, and S&P 7168) and blue: susceptible lines (S&P 7130, S&P 7175, and S&P 7483). (**B**) HRM curve of the 'Asia Seed' Co. onion lines. Red: resistant lines (Asia-12, Asia-42, and Asia-53) and, blue: susceptible lines (Asia-30, Asia-35, Asia-45, and Asia-50).
