Field Evaluation and Molecular Validation of Soybean Genotypes for Resistance to Yellow Mosaic Disease using SSR Markers

The soybean [Glycine max (L.) Merrill], also known as the golden bean, poor man’s meat, boneless meat, yellow jewel and the miracle crop of the 20^th century, is a highly significant legume crop worldwide (Jain et al., 2018, Sivabharathi et al., 2023, Barpanda et al., 2024). Soybean-based foods are trendy in eastern countries such as China and Japan. Soybean is rich in oil, protein, macronutrients and minerals (Banerjee et al., 2023). The central region of the country, comprising Madhya Pradesh (45%), Maharashtra (38%) and Rajasthan (8%), is a significant hub for soybean cultivation (Amrate et al., 2024; Mishra et al., 2024). In India, soybean cultivation has expanded to approximately 12.07 million hectares, resulting in a total production of about 13.98 million tons and an average productivity of roughly 1158 kg per hectare in the Kharif 2022-23 (Annual Report 2023, ICAR- Indian Institute of Soybean Research, Indore). Madhya Pradesh has significantly contributed to India’s soybean production, dedicating 5.51 million hectares of land to cultivation and achieving a yield of 5.39 million metric tonnes (Mishra et al., 2024).
       
Soybean yellow mosaic disease is one of the significant soybean diseases in Madhya Pradesh. Yellow Mosaic Disease is caused by the Mungbean yellow mosaic India virus (MYMIV) (Amrate et al., 2023) transmitted by white flies (Bemisia tabacci). The most distinctive symptom of Yellow Mosaic Disease is the presence of contrasting yellow-green patches (mottles) on the leaves. Under severe conditions, infected leaves may turn yellow, leaving the veins green (Amrate et al., 2020; Amrate, 2024). Screening soybean genotypes for YMV resistance is primarily impaired by the vectors’ non-arrival in the field, poor intensity, uneven distribution, environmental conditions, etc. Therefore, it is necessary to find some linked molecular markers for effective and efficient genotype screening (Kumar, 2013). Simple sequence repeats (SSRs) are one of the widely used markers for genotypic analysis and molecular mapping, cultivar identification, protecting germplasm, determination of hybrids, analysis of gene pool variation and as diagnostic markers for traits of economic value (Powell et al., 1996). In plant due to their species specificity and high reproducibility, simple sequence repeats markers were used in PCR amplification for molecular analysis of soybean. The present investigation was conducted to identify donor parents and SSR marker linked to YMV resistant in soybean.

The seventy soybean genotypes were planted on July 1^st, 2023, during the kharif season, using an Augmented Block Design with a spacing of 45 cm x 7 cm. Each genotype was grown in two rows, with each row being 3 m long, for field screening of the soybean genotype against yellow mosaic disease in the soybean research field (23^o12'45N, 79^o56'46E) at Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh. Molecular screening using SSR markers was conducted at the Molecular Biology Laboratory, Biotechnology Centre, Jawaharlal Nehru Agriculture University, Jabalpur (M.P).
       
The experimental area had uniform topography and fertility. The soil was clay loam with a good texture, medium NPK status and good water-holding capacity.
 
Experimental material
 
The study was conducted on seventy genotypes of soybean and collected from All India Coordinated Research Project on Soybean (Table 1).


 
Recording of coefficient of infection for YMD
 
The severity of the symptoms of YMD was evaluated based on a 0-5 rating scale 50 days after sowing (Table 2) and the coefficient of infection was calculated by multiplying the percentage incidence of YMD by the corresponding symptom severity grades. Genotypes were then assigned different reaction categories based on the coefficient of infection, as described by Amrate et al., (2020 and 2023).

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Isolation of DNA of soybean genotype
 
Leaf samples were collected from one month old seedling of soybean genotypes from research field on soybean. Usually fresh and young leaves are used in molecular work and DNA extraction.
 
Extraction of genomic DNA and purification
 
Genomic DNA was isolated using the Sahai-Marof et al. (1984) method with suitable modification. The quality of isolated DNA was checked by horizontal submarine gel electrophoresis on 0.8% agarose gel. The purity of DNA was checked by taking the ratio of optical density (OD) using a spectrophotometer at 260 nm to that of 280 nm. The samples with an OD ratio between 1.7 and 1.9 were used in subsequent experiments. If DNA samples show a value beyond this range, they must be repurified.
 
PCR amplification
 
The PCR reaction was carried out in a ten μl reaction volume with various components. The reaction mixture comprised 1.0 μl DNA, 5.7 μl nuclease-free water, 0.7 μl MgCl‚ , 0.4 μl dNTPs, 1.0 μl primers, 0.2 μl Taq DNA polymerase and 1.0 μl 10x buffer. The reaction mixture underwent a brief spin to ensure thorough mixing. PCR samples were stored at 4oC for short-term use and -20^oC for long-term preservation.
       
The PCR thermal cycling conditions for SSR markers were fine-tuned with various parameters, including initial denaturation, denaturation, annealing, elongation and final elongation, as outlined in Table 3.

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Agarose gel electrophoresis of SSR-PCR products
 
Amplified DNA fragments were loaded in submerged horizontal agarose gel electrophoresis in 2.5 percentage (w/v) agarose gel and visualized by staining with Ethidium bromide. PCR product mixed with loaded dye and loaded into gel wells. The gel was run at a voltage of 80 volts for 1 hour (Fig 1 and 2). The result was recorded in the gel documentation system.




 
Data analysis
 
NTSYS-PC version 2.02 software was used to observe data analysis. Using the data produced by the cluster analysis, a UPGMA dendrogram was also created (Fig 3 and 4).




 
Data scoring
 
The polymorphism in SSR was analyzed based on the presence or absence of the SSR bands. The genetic associations among varieties were analyzed by calculating the similarity coefficient (Jaccard, 1908) for pair-wise comparisons based on the proportions of shared bands produced by primers.The polymorphism information content (PIC) of the SSR markers was calculated according to Anderson et al., (1993) (Table 4).

The current study, 70 soybean genotypes were assessed for their resistance to yellow mosaic disease under field conditions. The susceptible varieties (JS 93-05, JS 20-34) were kept in check. The experimental site was one of the hot spot locations for yellow mosaic in soybeans (Amrate et al., 2018; Pancheshwar et al., 2016; Amrate et al., 2020; Amrate et al., 2023,).
       
Among the seventy genotypes, eighteen, including JS 24-25, JS 20-29, JS 25-01, DS 1510, NRC 189, JS 24-31, NRC 186, JS 20-89, PS 1660, JS 24-26, JS 24-78, JS 24-32, JS 20-69, JS 22-08, RSC 11-48, JS 20-79, KDS 1009 and JS 22-24, they demonstrated high resistance (Table 5). Additionally, twenty-seven genotypes were moderately resistant and eighteen were susceptible. Six genotypes exhibited susceptibility, including JS 95-60 check, JSM 230, JS 20-34, KDS 10-73, RVS 13-7 and AMS 243, while only one genotype, JS 93-05 (check), was highly susceptible. The present investigation was based on recently evolved soybean genotypes. Similar results were found by Amrate et al., (2023), who observed that JS 20-94 showed moderate resistance to Yellow Mosaic Disease. Soybean yellow mosaic disease is one of the significant soybean diseases in Madhya Pradesh. Yellow Mosaic Disease is caused by the Mungbean yellow mosaic India virus (MYMIV) (Amrate et al., 2023) transmitted by white flies (Bemisia tabacci). The most distinctive symptom of Yellow Mosaic Disease is the presence of contrasting yellow-green patches (mottles) on the leaves. Under severe conditions, infected leaves may turn yellow, leaving the veins green (Amrate et al., 2020; Amrate, 2024).
       
The genotypes that showed susceptibility to yellow mosaic were also found susceptible in previous studies (Rani et al., 2016; Amrate et al., 2023). A similar kind of moderate resistance reaction for JS 20-94 and JS 20-98 was also reported by Amrate et al., (2023) and Mishra et al., (2022).
       
During the present study, the genomic DNA of 40 randomly selected soybean genotypes were characterized for genetic diversity using 15 SSR markers to achieve the research goal. The polymorphism information content, or PIC, measures the variation at a specific gene position (Kumar et al., 2023). A large number suggests that the plants were most likely significantly different. These PIC values varied from 0 (Sct_199) to 0.84 (Satt 267). Each marker indicates that these plants’ genes differ significantly at these specific sites, with an average PIC value of 0.43.  Alleles ranged between 1-3 with an average value of 2.1 and allele size ranged from 60 to 260 base pairs.
       
Similarly, Kumar et al., (2015) also reported two markers (Satt 301 and GMSHP 179) with possible association with yellow mosaic disease in soybeans. Similar results were found by Mishra  et al. (2022), who observed that JS 335, JS 95-72 and RVS 2001-4 showed highly susceptible expression to yellow mosaic disease. Similarly, Rani et al., (2016) identified the genetic basis of 41 soybean genotypes varying in resistance against yellow mosaic virus using 58 simple sequence repeat primers. An average of 2.41 alleles per locus was detected in their study. Kumar et al., (2014) found that JS 335 was highly susceptible to yellow mosaic disease at the molecular and phenotypic levels (Table 5 and 6).



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UPGMA cluster analysis generated a dendrogram using genetic distance data. The genotypes of soybeans were divided into two groups, A and B.  Group A was subdivided into A1 and A2. Subgroup A1a contained thirteen genotypes. This group had the most high resistance and moderate resistance. Subgroup A1b contained three genotypes. The A2 subgroup contained four genotypes. Group B is also split into B1 and B2. There are seven genotypes in subgroup B1. B2a and B2b were the divisions of group B. There were eleven genotypes in subgroup B2a and two in subgroup B2b. Group B2 had the most genotypes that showed the most susceptibility to yellow mosaic disease. Similarly, Koutu et al., (2019) generated a dendrogram for the clustering of soybean varieties, i.e. JS 93-05, JS 20-69, JS 20-29, JS 97-52, JS 95-60, JS 20-93, JS 20-34 and JS 335, which is based on SSR marker analysis. Similar results in soybeans were also reported by Kumar et al., (2014), Mishra et al., (2022), Sahu et al., (2024) and Tiwari et al., (2019).
       
The values of Jaccard’s similarity coefficient have been used to establish a genetic relationship between several genotypes of soybeans. The RVS 13-7 and JS 20-116 showed the maximum similarity (0.94), whereas NRC 192 and RVSM 2012-4 showed the lowest similarity (0.41). Seven genotypes had the same similarity (0.91) coefficient among them: JS 22-08, JS 93-05; KDS 10-73, AMS 243; JS 20-98, JS 20-69; JS 21-72, JS 20-69; NRC 189, JS 20-69; NRC 189, JS 20-29; JS 20-69, JS 20-29. Among the seven genotypes, most entries were resistant to yellow mosaic disease in soybeans. Genotypes RVS 13-7 and JS 20-116 showed the maximum similarity because they share the same parentage.

Seventy soybean genotypes were screened in field conditions. Eighteen genotypes were found to be highly resistant, twenty-seven genotypes showed moderate resistance, eighteen soybean genotypes were screened as moderately susceptible, six genotypes were found susceptible and only one genotype showed high susceptibility to yellow mosaic virus. The high Polymorphic Information Content (PIC) value indicates significant variability, demonstrating that the selected markers effectively differentiate between genotypes. Our results showed that closely related genotypes with a common similarity coefficient exhibited similar or dissimilar reactions. To validate the field screening data more accurately, it is recommended to conduct field screening over multiple seasons and locations and to utilize a greater number of SSR primers.

The authors would like to extent their gratitude to Department of Genetics and Plant Breeding and Biotechnology Centre, College of Agriculture JNKVV, Jabalpur (M.P.) for their support by allowing us to use the tools, materials, supplies, offices and places to conduct this study.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.