Unveiling Genetic Variation in Garlic Genotypes in Response to Rust Disease Using RAPD Markers

Somayeh Alimoradi Askari ¹, Mehdi Nasr Esfahani ^{2, *} , Kowsar Shirazi ³, Arman Nasr Esfahani ⁴, Hossein Zeinalzadeh-Tabrizi ⁵, Mojtaba Mohammadi ⁶

1. Department of Biotechnology, Payame Noor University, Alborz, Iran

2. Plant Protection Research Department, Esfahan Agriculture and Natural Resource Research and Education Center, Esfahan, AREEO, Iran

3. Department of Plant Protection, Faculty of Agriculture, University of Zabol, Zabol, Iran

4. Department of Mycology and Parasitology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

5. Department of Horticulture and Agronomy, Faculty of Agriculture, Kyrgyz-Turkish Manas University, Bishkek, Kyrgyzstan

6. USDA-APHIS-PPQ, 1724 20 th St. E., Palmetto, FL 34221, USA

* Correspondence: Mehdi Nasr Esfahani

Academic Editor: Yuri Shavrukov

Special Issue: Molecular Markers in Plant Biology

Received: January 23, 2024 | Accepted: April 24, 2024 | Published: April 28, 2024

OBM Genetics 2024, Volume 8, Issue 2, doi:10.21926/obm.genet.2402231

Recommended citation: Askari SA, Esfahani MN, Shirazi K, Esfahani AN, Zeinalzadeh-Tabrizi H, Mohammadi M. Unveiling Genetic Variation in Garlic Genotypes in Response to Rust Disease Using RAPD Markers. OBM Genetics 2024; 8(2): 231; doi:10.21926/obm.genet.2402231.

© 2024 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

1. Introduction

Garlic (Allium sativum) is the second most significant crop in Allium. This bulb vegetable crop is next to onion as an essential nutritional crop grown in many countries around the globe [1]. Garlic production worldwide is more than 28 million tons per year, which has grown significantly in the last decade, with China alone accounting for 74% of the total world production [2]. Despite the agricultural potential in Iran, a scarcity of garlic persists in the market, primarily attributed to the widespread occurrence of plant diseases. The incidence of garlic diseases has reached 100% in some regions of cultivation in Iran [3].

Plant diseases are one of the significant constraints on crop production. Of various plant pathogens, fungi are of particular importance [4,5,6,7]. Despite using protective fungicides, garlic, like many other crops, is infected by some pathogens, particularly fungi [8,9]. One of the most devastating diseases of garlic is rust disease, caused by the fungus Puccinia Alli. It significantly threatens garlic plants during the growing season [10]. Early rust symptoms appear as tiny, white specks on the leaves and stem that develop into a circular or elongated posture. In severe cases when conditions are conducive, leaves turn yellow, wither, and die, leading to substantial yield losses. The disease spreads in the field by wind-blown spores [11].

Resistant genotypes are valuable genetic resources that can improve crop yield [12,13,14]. Identifying and using resistant genotypes is one of the essential ways to manage and control disease epidemics and reduce crop damage. In this case, pesticide application is reduced, and the environment and human health are preserved [15].

The growing significance of garlic in commercial, medicinal, and industrial contexts has led to a surging interest in enhancing production, performance, and the development of novel genotypes. Molecular markers can screen and identify resistant and susceptible genotypes even at early growth stages [16]. Selection for resistant genotypes based on phenotype is a time-consuming process in plant breeding, whereas molecular markers allow for the direct selection of favorable genotypes [17]. The use of molecular markers and the concept of polymorphism in systematic studies to determine the relationships between organisms, geographic distribution, speciation studies, genetic drift, genetic diversity, and the interpretation of population structure have received much attention in recent decades [18]. This is because the accuracy of phylogenetic information can play a significant role in the successful advancement of ecological research, preparation, protection, and maintenance of gene banks and breeding programs [19].

It is estimated that a 10% error in phylogenetics classification can reduce the genetic progress of a breeding program by three to four percent [20]. A group of diagnostic markers that can distinguish homogenous species from each other can be found among the repetitive sequences of genomes. Among conventional markers, random amplified polymorphic DNA (RAPD) is one of the most widely used markers due to its uniform distribution in the whole genome and its generality among different organisms [21]. In addition, the RAPD procedure is simple, requires a meager amount of DNA for analysis, costs less, and is faster and more accessible technology than amplified fragment length polymorphism (AFLP) or simple sequence repeat (SSR) analysis. RAPD markers are recommended for selecting resistant cultivars [16,22].

The comprehensive evaluation of garlic genotypes in this study suggests that the selected genotypes have high genetic variability in rust disease resistance. RAPD markers are expected to reveal unique genetic profiles in genotypes, especially resistant ones. Additionally, using RAPD markers to characterize these garlic genotypes may help manage and control rust disease in garlic fields by selecting resistant cultivars more efficiently and precisely. These molecular markers should also speed up breeding and help create rust-resistant garlic varieties.

This study aimed to elucidate the genetic basis of rust resistance in garlic breeding programs to improve garlic production and reduce dependence on protective fungicides in disease management.

2. Materials and Methods

2.1 Plant Materials

Eleven local Iranian garlic genotypes (Gandagh, Barfgin, Toein, Aliabad, Maryang, Moein, Bahar, Solan, Toyserkan, Hydare, Shorin) and one Chinese garlic genotype were obtained from the Plant Pathology Collection of the Agriculture and Natural Resources Research Center, Esfahan, Iran. Garlic is cultured as a winter crop between September and October and harvested from April to May the following year. A greenhouse experiment was laid out at the Agriculture and Natural Resources Research Center, Esfahan, Iran (51°34' E; 32°36' N) at 1500 meters above sea level during the 2020 growing season. Garlic genotypes were planted in pots containing pasteurized soil and maintained under greenhouse conditions. Bulbs were harvested at physiological maturity when the leaves fell and 70% dried. Each treatment was replicated five times in a randomized complete block design.

3. Disease Assessment

3.1 Frequency of Infection

The number of healthy and infected plants was meticulously counted independently to evaluate the frequency of rust fungus infection among the genotypes Negash et al. described [23]. The ratio of the total number of infected plants to the total number of samples was calculated as the percentage of plants infected with rust disease. The frequency of infection was calculated using the following formula:

$\mathrm{DF}\left(\%\right)=\frac{\sum_{i=2}^{N} R_{i}}{N}\times100$

Where R: Total number of infected plants; N: Total number of plants.

3.2 Disease Severity

Disease severity was recorded on 10 bulbs from each phenotype after the appearance of the disease symptom. Symptoms (rust postulates) in each bulb sample were scored based on disease categories (0, 5, 10, 25, 50, 75, and 100), which corresponded to the estimated percentage of lesions in the entire disease area (0, 1, 2, 4, 8, 16 and above 32), respectively [24]. A score of zero represents the most resistant phenotype, whereas a score of 100 represents the most susceptible one. The scores were converted to a susceptibility index:

SI = [(Sum of grade value × Number of bulbs in that grade)/(Total bulb number × Highest grade value)] × 100.

$\mathrm{D}\mathrm{S}=\frac{\sum _{i=2}^N R_i.S_i}{N}\times 100$

The average SI across the two-year study was used as the final SI for each genotype. Based on this, the resistance level of each genotype was classified into five classes based on its SI: Immune (IS), SI = 0, class 1; Highly resistant (HR), SI = 0.1-0.5, class 2; Resistant (R), SI = 5.1-25, class 3; Susceptible (S), SI = 25-50, class 4; Highly susceptible (HS), SI = 50-100, class 5 [25,26].

3.3 DNA Extraction

Total genomic DNA was extracted from dried leaf tissue following the standard CTAB (cetyltrimethylammonium bromide) method described by Doyle and Doyle [27]. Leaves (100 mg) were pulverized in liquid nitrogen, then homogenized in 25 ml of extraction buffer (2% CTAB, 20 mM EDTA, 2% PVP, 1.4 M NaCl, 100 mM Tris-HCl, pH 8.0, and 1% β-mercaptoethanol) containing 4 μl RNAse and incubated at 65°C for 1 h. The supernatant was extracted twice with chloroform: isoamyl alcohol (24:1 v/v). RNase was again added, and the mixture was incubated at room temperature for 30 minutes. The DNA was pelleted with chilled isopropanol, washed twice with 70% ethanol, air-dried, dissolved in 500 mL TE buffer, and stored at -20°C. DNA quality and quantity were evaluated using a NanoDrop spectrometer and 2% (w/v) agarose gel electrophoresis [28].

3.4 RAPD Amplification

The genomic DNA was subjected to polymerase chain reaction (PCR) amplification using RAPD markers. The PCR was run with 20 RAPD primers in a 25 μL total volume, containing 500 mM KCl, 100 mM Tris-HCl, pH 9.0, 1% Triton X-100, 1.5 mM MgCl₂, 0.2 mM each dNTP, 0.15 μM primer, 45 ng genomic DNA, and 1 U Taq DNA polymerase (Bioneer Company, Seoul, South Korea). The amplification was performed in a DNA thermal cycler with pre-denaturation set at 95°C for 5 min followed by 45 cycles of 1 min at 95°C, 1 min at 36°C, and 2 min at 72°C, with a final extension step at 72°C for 7 min [29]. The amplification products were resolved by electrophoresis on 2% (w/v) agarose gel containing 0.5 μg/mL ethidium bromide in 1× TAE buffer (40 mM Tris-acetate, pH 8.0, 1 mM EDTA) at 120 V for 1 h. In all cases, 1 kb DNA ladder was utilized as the size marker. The gel was examined and photographed on a UV transilluminator. All reactions were repeated at least three times [30,31].

3.5 Statistical Analysis

Each treatment was prepared in three replicates, and the experiment was repeated twice. All statistical analysis was performed using SAS 9.1 software. Data were analyzed using the SIMQUAL route to generate Jaccard's similarity coefficient using NTSYS-pc version 2.02 (Numerical Taxonomy System), and significant differences were identified by one-way analysis of variance (ANOVA) followed by Turkey's HSD (Honestly Significant Difference) test. Mean comparisons of every treatment were carried out using Duncan's multiple-range test. Differences were considered to be significant at p ≤ 0.01. Clustering of genotypes was done based on UPGMA using NTSYS version 2.02 software [32,33].

4. Results

Rust postulates initially appeared on the lower leaf surface of garlic plants, as shown in Figure 1. Yellow spots turned orange and gradually spread to the other leaves. Microscopic examination revealed the presence of uredospores characterized as spherical or oval structures measuring 20-24 ×23-29 µm (Figure 1). Based on symptom development, morphology, and microscopic analysis, the causal agent was identified as Puccinia allii.

Figure 1 Garlic rust disease: A) Infected garlic plant with rust disease; B) Yellow spots on garlic leaves; and C, D) Uredospores. (Scales bars = 20 µm).

4.1 Evaluation of Rust Disease Resistance Performance in Garlic Genotypes

Results from the two-year combined analysis of variance showed significant differences in resistance to the rust fungus among garlic genotypes at 1% level (Table 1 and Table 2). During the initial growth stage, the genotypes Shorin, Toein, and Toyserkan exhibited the highest percentages of infection at 55%, 51.52%, and 46.25%, respectively (Table 3), while the Solan genotype recorded the lowest at 75.28% (Table 3). Moving to the second stage, the genotypes Moein, Toein, Hydare, and Aliabad displayed the highest infection percentages at 83.75%, 76.25%, 75%, and 73.75%, respectively (Table 4). Further, the data indicated that during the first stage, the genotype Toein exhibited the highest disease severity at 8.85%, followed by Shorin at 8.5%, and Toyserkan at 8.43%, placing them in a statistically significant group compared to others (Table 3).

Table 1 Results of combined analysis of variance for resistance to rust in garlic genotypes at the first growth stage.

Table 2 Results of combined analysis of variance for resistance to rust in garlic genotypes at the second growth stage.

Table 3 Mean comparison of resistance to rust in garlic genotypes at the first growth stage.

Table 4 Mean comparison of resistance to rust in garlic genotypes at the second growth stage.

The combined analysis of the disease index demonstrated a significant variation among the genotypes examined (Table 1). The highest indices were associated with the Toein, Shorin, and Toyserkan genotypes at 0.76, 0.75, and 0.72, respectively, thus placing them in a distinct statistical group different from other genotypes. On the other hand, the lowest disease indices were observed in the Solan, Aliabad, and Bahar genotypes at 0.3, 0.45, and 0.5, respectively. These genotypes occupied a distinctive statistical position, showing a significant impact compared to others, while the remaining genotypes fell within the intermediate range between these two groups (Table 3).

Analysis of variance revealed a significant variation among garlic genotypes about disease severity over the two years during the second growth stage (Tables 2-4). It significantly increased in the second growth stage compared to the first. Notably, the Aliabad genotype displayed the highest disease severity at 25.3% in the second growth stage compared to the first. Thus, this genotype seems the most susceptible among other genotypes tested (Table 4).

Disease index based on the two-year dataset at the first growth stage correlated with disease severity. More specifically, the Aliabad genotype showed the highest disease index at 2.83, thus placing it in a distinct statistical group with a significant impact (Table 2). On the other hand, both Shorin and Toyserkan genotypes exhibited the lowest disease index of 1.11, forming a separate statistical group that differed significantly from other genotypes (Table 4). This divergence in disease indices suggests that garlic genotypes exhibited distinct behaviors across growth stages, indicative of varied genetic factors influencing their resistance to garlic rust fungus. The outcome of this study highlights the diverse responses of garlic genotypes, classifying them into resistant, partially resistant, partially susceptible, and susceptible categories.

4.2 Identification of RAPD Markers Associated with the Rust Disease

Genomic DNA extracted from various garlic genotypes was subjected to RAPD-PCR analysis utilizing a panel of 20 RAPD primers. In the initial experiments, 10 of the 20 tested primers yielded distinct and reproducible polymorphic banding profiles. These selected primers, namely OPA-07, OPA-09, OPA-10, OPA-16, OPA-19, OPAB-04, OPC-06, OPC-09, OPD-06, and OPG-13, generated polymorphic bands across all garlic genotypes as shown in Figure 2.

Figure 2 RAPD patterns on 2% agarose gel of amplified fragments generated from garlic genotypes with primers OPA-16 and OPC-06.

The percentage of polymorphism varied from 42.86% (OPA-10) to 84.62% (OPAB-04), with an average polymorphism rate of 67.66% (Table 5). A total of 95 fragments were detected, spanning the range of 300 to 1500 bp. Of the total, 66 fragments exhibited polymorphism ranging from 5 to 15 pieces per primer and averaging 9.5 fragments per primer. The OPC-06 primer generated the highest number of bands, whereas the OPC-03 and OPC-19 primers exhibited the lowest. Notably, the OPAB-04 primer demonstrated the highest polymorphism, featuring 11 bands, whereas the OPA-19, OPA-10, and OPD-03 primers displayed the lowest polymorphism with 3 bands each (Table 5). Our data show that RAPD markers can be a convenient and reliable tool for distinguishing garlic genotypes. It further demonstrates a significant genetic variation among these genotypes.

Table 5 RAPD primers amplified specific bands (bp) for resistant and susceptible garlic genotypes.

The UPGMA (Unweighted Pair Group Method with Arithmetic Average) dendrogram derived from the cluster analysis of RAPD data is shown in Figure 3. It categorizes the garlic genotypes into six distinct clusters, making up 76% of the observed variation, with Jaccard's similarity coefficient falling within the specified range. Specifically, the first cluster only included the genotype Gandagh; the second cluster comprised genotypes Barfgin, Toein, and Aliabad; the third cluster involved genotypes Mariyang and Moein; the fourth cluster constituted genotypes Bahar, Solan, Toyserkan, and Hydare; the fifth cluster featured genotype Shorin whereas the sixth cluster included the Chinese genotype.

Figure 3 UPGMA dendrogram of the accessions studied, based on the RAPD data using Jaccard's similarity coefficients.

The highest genetic similarity was observed between the Mariyang and Moein genotypes, with a similarity coefficient 0.826 (Table 6). This suggests a common ancestral origin, or they were initially the same genotype, albeit assigned different names over time. On the other hand, the lowest similarity coefficient of 0.520 was obtained between the Bahar and Chinese genotypes and between the Solan and Chinese genotypes at 0.529. Notably, the Chinese genotype exhibited the slightest similarity with the other genotypes under investigation. Within the native genotypes, the Shurin genotype displayed the lowest similarity with others, forming a distinct cluster. The genetic similarity coefficients among genotypes ranged from 0.520 to 0.826, with an average estimated at 0.70 (Table 6).

Table 6 Jaccard similarity coefficient of garlic genotypes accessions based on RAPD data analysis.

5. Discussion

Molecular markers are crucial in assessing the genetic diversity among different genotypes. The RAPD markers, in particular, are favored over alternative molecular markers for genetic diversity analysis due to their cost-effectiveness, rapid assessment of DNA-level variability, simplicity, ease of use, dominant nature, and the fact that it does not necessitate specific knowledge of the DNA sequence. These attributes make the RAPD markers a practical and efficient tool for exploring genetic diversity in various genotypes [12]. Phylogenetic analysis of RAPD profiles further reveals intergenic and intraspecies variation. Despite its many advantages, there are certain limitations to RAPD [34]. RAPD markers predominate, and amplification may or may not occur at a locus, leading to scores based on the presence or absence of bands. This means that homozygotes cannot be distinguished from heterozygotes [35]. Furthermore, the absence of a band due to the lack of a target sequence cannot be distinguished from DNA non-amplification for technical reasons (poor quality DNA), thus contributing to the ambiguity in interpreting the results [12].

Patel et al. [36] reported that morphological propensity, environmental factors, variation, and inter-hybridization may cause misidentification of genotypes. Polymorphism occurs when two or more different phenotypes exist in a population of the same species or when more than one morph or form exists [37]. Polymorphism is common in nature and is related to biodiversity, genetic diversity, and adaptation. It usually acts to maintain different forms in a population living in a diverse environment [38]. Polymorphism results from evolutionary processes, as does any aspect of a species. It is inherited and modified by natural selection. The ability of a species to respond adaptively to environmental changes depends on its level of genetic diversity [39]. Extensive studies on evaluating the genetic diversity of a diverse plant species using molecular markers have proven the relationship between geographic distance and genetic similarity between individuals [12]. Likewise, many studies have evaluated the establishment of genetic relationships in various cultivars and plant species [36].

The present study, 20 RAPD primers were used to achieve diversity within and between 11 Iranian garlic genotypes and one Chinese garlic genotype. 10 RAPD primers scored 66 bands in eleven Iranian and one Chinese garlic genotype. In a study on the genetic diversity of Iranian garlic genotypes using AFLP as a molecular marker, Vafaei et al. [40] classified Iranian garlic genotypes into 6 groups. The results showed that garlic genotypes react differently to rust, indicating the existence of genetic diversity among garlic genotypes. Garlic rust disease spreads in all genotypes with differences in infection percentage, severity, and disease index in the first and second growth stages. These results are supported by those reported by Periyannan et al. [41] regarding the genetics of resistance against rusts. In another study, Anjomshoaa et al. [12] showed that Iranian garlic genotypes reacted differently to the rust fungus (Puccinia allii) and were separated into resistant, partially resistant, partially susceptible, and fully susceptible genotypes. A study in Pakistan on garlic rust resistance showed 0.8% rust severity recorded on the Hazero variety [42]. Another study in California reported that the early and late maturing garlic genotypes, including Spanish and Chinese cultivars, were relatively tolerant to rust disease [43]. A California breeding program evaluating garlic rust resistance revealed that most genotypes were infected, and only about 1% had less than 26% infection [44]. Merga et al. [45] reported that using resistant or tolerant garlic cultivars is economically, sustainably effective, and environmentally friendly for managing rust disease.

Various researchers have utilized diverse markers to identify the genetic diversity of resistance genes in garlic and other crops. For example, the utilization of RAPD markers to uncover genetic diversity and resistance genes is further exemplified in the research by Poulsen et al. [46], where bulk segregant analysis led to the identification of an RAPD marker linked to leaf rust resistance in barley. This approach demonstrates the potential of molecular markers in breeding programs to enhance resistance against rust pathogens [47].

A study by Fernández-Aparicio et al. [48] delved into identifying and characterizing resistance against Puccinia allii in garlic germplasm, shedding light on the defense mechanisms impeding fungal development at various stages.

Additionally, Junghans et al. [49] identified a major gene associated with resistance to Puccinia psidii Winter in Eucalyptus, highlighting the involvement of a significant gene in a non-coevolved pathosystem. The work by Becerra et al. [50] explored the high genetic diversity in Chilean wheat yellow rust (Puccinia striiformis) populations, emphasizing the need to consider multiple significant genes in cultivars. These studies underscore the significance of genetic markers and diversity in understanding rust diseases caused by Puccinia species. These findings emphasize the importance of leveraging genetic diversity to develop cultivars with durable resistance to rust pathogens [51].

Applying RAPD markers in this study showed a significant genetic variation among garlic genotypes. As demonstrated in our research, this technique is proper in breeding programs, integrating with marker-assisted selection and comparative genomics studies. Further investigation may shed some light on garlic genes involved in resistance against rust pathogen and the resistance mechanism involved.

6. Conclusion

The OPAB-04 primer exhibited the highest level of polymorphism, revealing 11 bands in the study. Notably, genotype Solan displayed the highest susceptibility to rust disease, with a severity rate of 30.81%, while genotypes Aliabad and Bahar demonstrated resistance, with infection rates as low as 13% and 16.37%, respectively. Additionally, genetic diversity was evaluated using random-amplified polymorphic DNA (RAPD) markers, generating 95 scorable bands, of which 66 (69.48%) were polymorphic. A dendrogram was constructed using the UPGMA method based on Jaccard's coefficient of similarity, revealing six distinct clusters among the genotypes. Garlic rust, attributed to Puccinia alli, poses a significant threat to garlic crops globally, leading to economic constraints. To combat this challenge, various disease management strategies are employed. Utilizing resistant or tolerant garlic cultivars has emerged as a cost-effective, sustainable, and environmentally friendly approach. RAPD markers are a robust tool for identifying resistant and susceptible garlic genotypes against rust disease, offering valuable insights for breeding programs and disease management strategies.

Acknowledgments

The authors express their sincere gratitude to the Agriculture and Natural Resources Research and Education Center (AREEO) in Isfahan, Iran, for generously providing the necessary facilities that enabled the successful execution of this project.

Author Contributions

SAA: Running the project, Resources, Writing - original draft. MNE: Supervision, Conceptualization, Methodology, Data curtain, review and editing. KS, ANE, HZT and MM: Data curtain and analysis, review and editing.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing Interests

The authors stated that, this research was conducted in the absence of any commercial and or financial relationships that could be construed as a potential conflict of interest.