**5. Conclusions**

Onions have a large genome size, and the information on the existing DNA sequences is insufficient. In this study, a molecular marker was developed to breed resistant onions against gray mold disease. A phylogenetic analysis between the gray mold disease-resistant and -susceptible lines was performed using a dendrogram derived from the RAPD analysis. The constructed dendrogram was compared with the pedigree of the gray mold diseaseresistant and -susceptible breeding lines provided by 'Seeds & People' Co. The results of the phylogenetic analysis were consistent with the genetic relationship of the pedigree of the breeding lines and showed the reliability of the RAPD results. Thereafter, a SCAR-OPAN1 marker was developed based on the RAPD results showing polymorphic fragments between the resistant and susceptible lines. The SCAR-OPAN1 marker only amplified the resistant lines of the specific 2-Kb product, and no product was amplified in the susceptible lines.

In addition, the RNA-seq of the gray mold disease-resistant and -susceptible onion lines was analyzed using NGS technology. Based on the RNA-seq results, DEG and GO analyses were performed to identify the variants, such as SNPs and indels. As a result, a selectable marker, SNP-3 HRM, was developed to select gray mold-resistant lines. The SNP-3 HRM marker for the selection of the resistant lines includes SNPs present in the AKR gene exhibiting high expression levels in these lines. Consequently, in this study, the SCAR-OPAN1 and SNP-3 HRM markers were developed for the selection of resistant onion lines in breeding programs to reduce the damage caused by gray mold disease. Thus, using these molecular markers, the breeding period of biennial onions can be shortened by selecting an onion line that is resistant to gray mold disease, thereby alleviating the economic loss of onions.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/horticulturae7110436/s1: Figure S1: The pedigree of the onion lines provided by 'Seeds & People' Co. Figure S2: RAPD analysis of the resistant and susceptible lines provided by 'Seeds & People' Co. by using the OPAN-1 random primer. Figure S3: MA plot to verify the significantly expressed genes in the groups of resistant and susceptible lines. Figure S4: Volcano plot of the transcripts that satisfied the following conditions: FDR < 0.05 and |log2 fold change| ≥ 2. Figure S5: GO annotation of 182 transcripts with increased expression levels. Table S1: Characteristics of the four resistant lines (S&P 7129, S&P 7168, S&P 7521, and S&P 7522) and three susceptible lines (S&P 7130, S&P 7175, and S&P 7483) provided by 'Seeds & People' Co., Ltd. (Yeonggwang, Korea). Table S2: Sequence information of 40 OPERON random primers used for the RAPD analysis. Table S3: Results of the raw data obtained by RNA-seq, trimming, and mapping.

**Author Contributions:** Conceptualization, S.-J.K., J.-S.P., and Y.-D.P.; investigation and writing original draft preparation, S.-J.K. and J.-S.P.; resources, T.P.; data curation, H.-M.L.; investigation, J.-R.C.; and conceptualization, supervision, writing—review and editing, and funding acquisition, Y.-D.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA).

**Data Availability Statement:** The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

**Acknowledgments:** This work was carried out with the support of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Golden Seed Project (213007-05-5-SBB20).

**Conflicts of Interest:** The authors declare that they have no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.
