**1. Introduction**

Onions (*Allium cepa* L.) are one of the most economically and nutritionally important crops worldwide. They are also one of the oldest cultivated crops and are used as an ingredient in various foods and sauces to enhance flavor and promote health, such as for lowering cholesterol levels [1–3]. Therefore, it is important to breed and produce higherquality onions to improve their competitive advantage in the market. Onion breeding is performed for various purposes, such as to improve the onion yield; for qualities like size, taste, or color; for male sterility; and for a resistance against biotic and abiotic stresses [3–5].

Onions are susceptible to many pathogens and insects [3]; therefore, breeding for resistant onions has been extensively studied to reduce the damage caused by various diseases, many of which are caused by the genus *Botrytis*. Onion botrytis leaf blight is

**Citation:** Kim, S.-J.; Park, J.-S.; Park, T.; Lee, H.-M.; Choi, J.-R.; Park, Y.-D. Development of Molecular Markers Associated with Resistance to Gray Mold Disease in Onion (*Allium cepa* L.) through RAPD-PCR and Transcriptome Analysis. *Horticulturae* **2021**, *7*, 436. https://doi.org/ 10.3390/horticulturae7110436

Academic Editor: Yuyang Zhang

Received: 25 September 2021 Accepted: 20 October 2021 Published: 25 October 2021

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caused by *B. squamosa*, and onion neck rot is caused by *B. aclada*, *B. alli*, *B. squamosa,* and *B. porri* [6]. In particular, *B. cinerea* is a necrotrophic pathogen with more than 200 host crop species, causing severe damage to onions [7]. Gray mold, caused by *B. cinerea* and *B. squamosa*, reduces the yield and storage capacity. Gray mold disease affecting the onion bulb is caused by *B. squamosa* during bulb formation and bulb filling and by *B. cinerea* during the later cultivation and storage periods [8,9]. Previous studies have attempted chemical and biological controls to prevent damage to onions by gray mold. To prevent this disease, onions should be kept dry, infected onions should be rapidly removed, and crops must be rotated every 3 to 4 years. In addition, many fungicides are used to control gray mold, with approximately 10% of the global fungicide market focused on controlling *B. cinerea* [10]. However, despite these efforts, it is difficult to control gray mold disease. Furthermore, synthetic fungicides can cause problems, such as residue concerns and a negative impact on human health, the emergence and increase of resistant pathogen populations, and environmental pollution [11–14]. Therefore, breeding disease-resistant onions can reduce the damage caused by diseases, such as gray mold disease, increase production, and reduce labor and environmental pollution.

Disease-resistant onions have a long breeding period, as onions are a biennial plant; therefore, a complete generation of onions requires two years. Marker assistant selection (MAS) can be used to shorten the breeding period of onions. In addition, a genome analysis of onions is difficult, because onions have a large genome size (16Gb), which is 100 times larger than that of Arabidopsis genomes [3,4]. Molecular markers such as random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) have been developed for various purposes. These markers are used for MAS, facilitating selection in breeding and shortening the breeding periods. RAPD is a PCR-based marker using short random primers; therefore, a RAPD analysis can reveal small genetic polymorphisms between large genomes, such as that of onions [15,16]. The disadvantage of RAPD markers is their low reproducibility. Therefore, in this study, we developed a SCAR marker by using a more specific primer than RAPD from the results of polymorphism studies between resistant and susceptible lines of onion.

In addition, NGS technologies such as RNA sequencing have enabled large-scale transcriptome data analysis, which has improved the efficiency of gene discovery despite no prior knowledge of reference genome sequences [4,17,18]. In this study, RNA-seq was performed to develop molecular markers for breeding gray mold-resistant onions. RNAseq was used for a DEG analysis and the analysis of variants such as SNPs and insertions and deletions (InDels) between the resistant and susceptible groups. The HRM markers confirm the fluorescence of the PCR product's melting curve when the double-stranded DNA becomes single-stranded DNA. The sequence region of interest was amplified with a fluorescent dsDNA-binding dye, and the PCR melting curve was measured when the product was gradually melted. The melting curve varies depending on the sequence, such as the presence of SNPs, GC content, length, and heterozygosity [19,20]. In this study, the HRM marker was also developed through selected transcripts from gene ontology (GO) and variant analysis using RNA-seq data to breed gray mold-resistant onions.
