**4. Discussion**

Using molecular markers, genetic differences can be quickly and easily identified at the DNA level without phenotyping. RAPD markers can easily identify polymorphisms by using short random primers, commonly 10 bp in length. In addition, it does not rely on knowing the target DNA sequence information, and RAPD is inexpensive, simple, quick, and easy to use [25–27]. Based on the RAPD results, small genetic differences between large genomes can be identified. Genetic relationships between the onion lines can also be confirmed through the unweighted pair group method with an arithmetic mean cluster dendrogram [15,16].

In this study, a phylogenetic analysis of gray mold disease-resistant and -susceptible lines was conducted using RAPD. The phylogenic analysis obtained from the RAPD showed that the resistant lines were closely related to other resistant lines, and the susceptible lines were closely related to other susceptible lines. S&P 7522, S&P 7521, S&P 7129, and S&P 7168 belong to the gray mold disease-resistant line group, and S&P 7483, S&P 7130, and S&P 7175 belong to the gray mold disease-susceptible line group. Additionally, a close genetic relationship appeared between the resistant S&P 7522 and S&P 7521 lines and between the resistant S&P 7129 and S&P 7168 lines. Similarly, susceptible lines S&P 7483, S&P 7130, and S&P 7175 showed close genetic relationships in the phylogenic analysis. These results were similar to the pedigree of the 'Seeds & People' Co. onion lines. According to the pedigrees, two gray mold disease-resistant lines, S&P 7129 and S&P 7168, were bred by crossing the resistant lines S&P 7522 and S&P 7521. The gray mold disease-susceptible lines S&P 7130 and S&P 7175 were bred through the crossing of the susceptible lines S&P 7406 and S&P 7405. A comparison of the results of the phylogenetic analysis obtained from the RAPD with the pedigree of the breeding lines revealed similar genetic relationships, thereby demonstrating the reliability of the RAPD results.

The SCAR-OPAN1 marker was developed using the polymorphic fragment in the RAPD analysis. The SCAR-OPAN1 marker sequence was designed to extend longer, including the 5 -ACT CCA CGT C-3 sequence of the OPAN-1 random primer. The primer set for the SCAR- OPAN1 marker was designed considering the annealing temperature, GC content, the possibility of the dimer formation, and the hairpin loops. A BLAST analysis was performed to analyze the products amplified by the SCAR marker. In the BLAST

analysis of the DNA sequence of the fragment amplified from the SCAR marker, there was no significant percentage of sequences matched to the other sequences from onions and other species; therefore, additional analysis is required. SCAR markers have been used to distinguish between various cultivars and species [28,29]. SCAR markers can be used to select individuals with specific traits and to identify disease-resistant individuals. Using SCAR markers, the selection of male infertility-dominant *Ms* and -recessive *ms* in onions [30], anthrax resistance in grapes [31], and Acokita blight resistance in lentils [32] have been reported. It was confirmed that the selection of gray mold disease-resistant onions was possible using this SCAR- OPAN1 marker in the onion breeding lines of 'Seeds & People' Co.

In addition, to validate the developed SCAR-OPAN1 marker, additional gray mold disease-resistant and -susceptible lines from the 'Asia Seed' Co. were analyzed. In the analysis of the gray mold disease-resistant and -susceptible 'Asia Seed' Co. lines, it was confirmed that a product of 2Kb was amplified only in the resistant lines. Therefore, it was confirmed that the SCAR-OPAN1 marker developed in this study not only facilitates the selection of gray mold-resistant onions but also facilitates easy selection in a considerably short time.

The HRM marker was developed to select gray mold disease-resistant onion lines by using the selected transcripts through a DEG analysis from RNA sequencing. After analyzing the RNA-seq, the selected 14 transcripts were analyzed using the Arabidopsis Information Resource (TAIR) ID derived from Arabidopsis. The matched transcripts were analyzed using GO annotation and were largely divided into cellular components, molecular functions, and biological processes. Among them, the transcripts related to disease resistance, classified as 'response to biotic stimulus' and 'response to stress', were selected.

Plant cells synthesized reactive oxygen species (ROS) and pathogenesis-related proteins (PR proteins) after the detection of the presence of pathogens, along with a hypersensitive response (HR) to prevent the growth of pathogens. These resistance responses can induce a systemic acquired resistance (SAR) response, which is a resistance response of the whole plant. When SAR is activated by pathogens such as fungi, bacteria, and viruses, salicylic acid (SA) accumulates, and the accumulation of SA is essential for SAR expression [33,34].

In this study, the SAR gene was targeted by the SNP-10 transcript and showed DEGs and variants between the gray mold disease-resistant and -susceptible line groups (Table 2). After analyzing the relationship between SA accumulation and SAR in the early 1990s, SARs have been identified in *Arabidopsis thaliana*, tobacco, and cucumbers. The immune response by SAR is induced by PR proteins and has been studied to recognize SA as a signal [35–37]. When tobacco began to show resistance to *B. cinerea* and *Psequdomonas syringae*, it was confirmed that the resistance mechanism was initiated by the involvement of SAR, PR genes (PR-1 and PR-5), and SA before preparation in whole plants [38].

In contrast to SAR, the induced systemic resistance (ISR) with other signaling pathways induced by the plant hormones jasmonic acid (JA) and ethylene is also one of the resistance responses of plants. JA is a plant hormone that is related to disease resistance. The transcript targeted by SNP-1 was found to be the lipoxygenase (LOX) 3 gene (Table 2). The LOX gene is involved in JA biosynthesis. In particular, LOX3 was rapidly upregulated when the pathogen *B. cinerea* was inoculated in *Arabidopsis thaliana*. This LOX-upregulated response was shown in LOX3 and LOX4, which was presumably related to the early JA response of oligogalacturonides acting as damage-associated molecular patterns (DAMPs) [39].

In addition, the glutathione S-transferase (GST) gene, the transcript targeted by SNP-6, has been identified in many studies on disease resistance in plants (Table 2) [40–42]. GST plays a major role as an antioxidant and is resistant to plants in relation to a hypersensitive response to cell death. The transformed *Nicotiana benthamiana* was resistant to *Colletotrichum destructivum* and *C. orbiculare* by the GST gene [43].

In other selected transcripts, it was also confirmed that target genes have various functions related to plant resistance. Among them, the SNP-3 transcript developed as an HRM marker was confirmed to be an aldo-keto reductase gene (AKR) (Table 2). It was confirmed that the SNP-3 HRM marker related to the AKR gene can also select gray mold disease-resistant lines from 'Seeds & People' Co. and 'Asia Seed' Co. AKR gene expression was higher in the gray mold disease-resistant line group than in the -susceptible line group. AKR has been mentioned in previous studies and is known to increase the resistance at high expression levels [44,45].

Since the SNP-3 HRM marker was a gene-based marker, a qPCR analysis was conducted to confirm the expression level of the AKR gene. The expression level of AKR was higher in the resistant group than in the susceptible group. The qPCR results were compared with the in silico data. In the RNA-seq results, the expression level of AKR was higher in the resistant group than in the susceptible group, and S&P7522 showed the highest expression level. The similarity of the AKR gene expression levels between the qPCR analysis and in silico RNA-seq results showed that the gene was associated with resistance to gray mold disease, increasing the reliability of the in silico data used to develop the molecular markers.

It has also been identified that the AKR gene provides multiple stress tolerances in plants. In particular, there have been studies related to abiotic stresses such as herbicide resistance, heat stress tolerance, and biotic stress, such as mildew [41,46,47]. For biotic stress from pathogens such as microorganisms, the AKR gene group is mainly involved in plant secondary metabolic pathways, including flavonoid biosynthesis in plant-microbial interactions. Therefore, these AKRs primarily function as plant defense systems against biological stresses, such as a pathogen attack. However, despite reports identifying plant AKR as a potential target for developing abiotic and biotic stress-tolerant plant species, the importance of plant AKR has not yet been emphasized [46,48].

Various studies have revealed that AKR plays a stress-related role. This study also showed differences in the resistance and gene expression levels according to SNPs between the gray mold-resistant and -susceptible line groups. It was considered that the AKR gene affected the resistant group to show a resistance to gray mold disease. Therefore, further studies of AKR will be needed, and the SNP-3 HRM marker developed in this study could be used to select gray mold disease-resistant onion lines.
