Assessment of Genetic Diversity for Cercospora Leaf Spot (CLS) Resistance in Mung Bean [Vigna radiata (L.) Wilczek] using SSR Markers

Mung bean [Vigna radiata (L.) Wilczek] is a popular commercial species of the genus Vigna and its production is steadily increasing in South and Southeast Asia. It is a popular legume because of its short-life cycle (Chen et al., 2021). Mung bean has valuable nutritional and health benefits due to its high digestibility, Vitamin B and protein content (Chand et al., 2015). However, mung bean often succumbs to the spread of Cercospora canescens, which is the causal organism of Cercospora leaf spot disease, but requires special attention as it can affect plant growth and reduce seed yield (Priyadarshini et al., 2020). The incidence of mung bean CLS is widespread in India during the kharif season in warm and humid climatic conditions, causing considerable losses to mung bean growers. In the warm and wet seasons, yield losses of up to 47% under the northeast plain zone in India (Kumari et al., 2021), 61% in Australia and Okara, Pakistan (Rasool et al., 2021), 23-75% in Thailand (Somta et al., 2015) and 37.4% in South Korea (Bushra et al., 2013) have been documented world wide.
       
Despite the efforts of plant breeders during the past few decades, the CLS resistance varieties or lines of mung bean has not been developed substantially due to a lack of sufficient genetic diversity for desirable traits in the germplasm used for breeding (Kundu et al., 2022). It is imperative to further explore the genetic diversity available in this crop for better utilization of genetic resources for CLS resistance (Sahoo and Sharma, 2018). Various molecular markers linked to fungal disease resistance were also identified in mung bean (Lakhanpaul et al., 2000), black gram (Souframanien and Gopalakrishna, 2004) and cowpea (Bangar et al., 2021), including simple sequence repeat (SSR) markers for CLS disease resistance (Chankaew et al., 2011). However, limited work has been done so far for genetic diversity assessment in mung beans for CLS resistance. Therefore, the present study evaluated the genetic diversity among the mung bean germplasm using SSR markers under CLS stress.

Experimental details
 
CLS disease reaction of a ninety mung bean genotypes, including four suitable susceptible checks were evaluated under natural field condition with augmented block design without replication in the EB-2, at College of Agriculture, OUAT, Bhubaneswar, during the pre rabi 2018 and the pre  rabi 2019, respectively. Standard package and practices were followed for growing the mung bean crops except the use of fungicides. All the necessary measures were taken to ensure maximum CLS disease pressure, including maintaining optimum humidity and planting susceptible checks all along the borders and after every twenty test genotypes. At 20, 25 and 30 days after sowing (DAS), prepared spore suspension was artificially inoculated by spraying using a manual sprayer to all the genotypes. After inoculation, the crop was regularly monitored for the development of CLS disease. Infection on the leaves of each plant was then scored for CLS reaction at 40 DAS on a rating scale of 1-5 (Chankaew et al., 2011), where, 1: resistance (R); 2: moderately resistance (MR); 3: moderately susceptible (MS); 4: susceptible (S) and 5: highly susceptible (HS).
 
SSR marker genotyping
 
Plant genomic DNA was extracted from young leaf tissue for each of the ninety mung bean genotypes by following a standard protocol (Doyle and Doyle, 1987), with little modification. The quality of DNA extracted was checked by electrophoreting the samples using 0.8% agarose gel and quantified using Nanodrop® ND-1000 Spectrophotometer. Polymerase chain reactions for SSR analysis were carried out under standard conditions for all the sixty-six primer pairs (Wang et al., 2004; Somta et al., 2009; Isemura et al., 2012) using 1 U of Taq polymerase with 1× PCR buffer (100 mm Tris-HCl at pH 9.0, 500 mm KCl and 15 mm MgCl2), 2.5 mm dNTP, 3 mm MgCl2, 20 pM of each primer and 50 ng of DNA template with a final reaction volume of 25 μL. The PCR reactions were denatured at 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, required annealing temperature for 1 minute and extension at 72°C for 2 minutes. The final extension was done at 72°C for 7 minutes. The amplicons were run on a 3% agarose gel with a 100 bp DNA ladder and then visualized through a UV GELDOC unit to assess the results.
 
Genotyping data Scoring and Statistical analysis
 
The PCR fragments were scored for presence and absence (Fig 1). Spurious and missing data were repeated for verification. The genetic diversity parameters i.e., polymorphism information content (PIC) (Powell et al., 1996), the allele frequency distribution (Scitable-Nature, 2022), the percentage of polymorphic loci per population (Glsturgeon, 2022), the Shannon diversity index (Shrivastava et al., 2014), allelic patterns across the population (Bangar et al., 2021), the Jaccard’s similarity coefficient (Jaccard, 1908) and archaeopterx tree were analyzed using GenAlE × 6.51b2 and TASSEL 5.0, software program.

Polymorphism information content (PIC) values
 
PIC measures the ability of a marker to detect polymorphisms and therefore has enormous importance in selecting markers for genetic studies (Serrote et al., 2020). A moderate molecular diversity was observed in the panel population. The marker CEDG118 estimated the maximum genetic diversity (PIC value = 0.74). Wide variation in alleles showed a range of 80 bp to 300 bp. The average PIC value was obtained to be 0.40 for all the employed SSR markers. However, PIC values for co-dominant markers generally range from 0 (monomorphic) to 1 (very highly informative) (Serrote et al., 2020). Results obtained from the present study revealed that, the PIC values of the markers were found between 0 to 1, which was similar to previous research finding in mung bean (Chen et al., 2021; Dash et al., 2022; Sahoo et al., 2020a). However, the present investigation on trait-based genetic diversity is almost similar to the earlier findings (Sushmita et al., 2021; Sahoo et al., 2020b; Sahoo et al., 2018) of moderate genetic diversity reported in mung bean for the single trait.
 
Allele frequency distribution
 
In the present investigation, the allelic frequency distribution across the codominant loci varied between 0 to 1 for all the population types, i.e. highly susceptible, moderately resistance, moderately susceptible, resistance and susceptible (Fig 2). However, Similar results were also observed in some previous studies (Moe et al., 2012; Sahoo et al., 2019) in mung bean germplasm, which suggests that, determining the allele frequency of candidate gene could have practical applications for research design, data interpretation, identifying genetic associations with particular diseases, estimating the number of individuals with disease susceptibility in a population and performing evolutionary studies.


 
Percentage of polymorphic loci
 
In the present study, the maximum percentage of polymorphic loci was found to be 75.76% in the resistance genotypes followed by 56.06% in moderately resistance genotypes. The least percentage of polymorphic loci was 6.06% in the highly susceptible genotypes. The mean and SE for the percentage of polymorphic loci were found to be 39.09% and 52.57%, respectively (Table 1). These findings are similar to the previous findings suggesting a range of 25-85% in mung bean by ISSR markers (Singh et al., 2013) and 95.6% in mung bean and also in black gram population panel by using ISSR markers (Tantasawat et al., 2010). However, the high degree of polymorphism is not due to a single species or population within a species, but rather polymorphic loci are spread evenly across all species and individual populations (Sahoo et al., 2021a).


 
Shannon information diversity by population
 
In the present study, the Shannon information diversity statistics showed that the Shannon information was 0.269 among the population and 0.336 within the population (Table 2). The diversity estimate among the population was 1.308 and within the population 1.399, with the estimated probability among the population 0.001 and within the population 1, respectively. The percentages of Shannon information among and within the population were 44.452% and 55.548%, respectively. The scaled diversity was found to be 0.347 and 0.595 and the scaled overlap was found to be 0.653 and 0.405 among and within the population respectively. These findings are similar to the previous studies (Chen et al., 2015; Sahoo et al., 2021b). However, the index was proven negatively biased at small sample sizes. Modifications to the original Shannon’s formula have been proposed to obtain an unbiased estimator (Sahoo et al., 2022b; Samal et al., 2021).
 


Allelic patterns distribution across the population
 
In the present study, the analysis of mean allelic patterns showed that the mean of expected heterozygosity was highest in the resistance population type (0.255), followed by the moderately resistance population type (0.210) (Fig 3). Results obtained from the analysis of allelic patterns for codominant data were similar to the results obtained in the previous studies on mung bean using SSR markers (Sarýkamýþ et al., 2009; Sahoo et al., 2022a) and in alfalfa using SSR markers (Diwan et al., 2000). However, genetic variation in a population is derived from a wide assortment of genes and alleles. The persistence of populations over time through changing environments depend on their adaptability to the shifting external conditions (Scitable-Nature, 2022).


 
Cluster diagram and distribution of genotypes in different clusters
 
The archaeopterx tree construction revealed two major clusters for all the ninety genotypes, i.e., I and II (Table 3), containing equal number (45 number) of accessions. Cluster I was again sub-divided into two major sub-clusters, i.e., IA and IB respectively, which were again subdivided into different sub-sub clusters. Cluster II was also sub-divided into two sub-clusters, i.e., IIA and IIB, respectively, which were again subdivided into different sub-sub clusters (Table 3). Therefore, it is concluded that the panel population used for the study possesses considerable genetic variation for CLS resistance.



Earlier researchers had also confirmed about the existence of genetic variation for CLS resistance in mung bean (Datta et al., 2012; Kundu et al., 2022). On the whole, the clustering results revealed by SSR closely reflected the previously understood relationship among these mung bean accessions.

The results obtained from the present investigation indicate that, SSR markers used in the present studies could be effectively used for molecular studies in mung bean, because these were highly influential in estimating mung bean’s genetic diversity. However, sufficient variability exists in the genotypes of current mung bean panel population, which could be useful in selecting suitable parents for breeding purposes and genetic map development for CLS disease resistance.

The authors are indebted to the Rashtriya Krishi Vikas Yojana (RKVY), Department of Agriculture and Farmers’ Empowerment, Government of Odisha, for providing support for undertaking this experiment.

The authors have no conflict of interest.