Genetic Diversity Analysis and Validation of Microsatellite Markers Linked with Tolerance to Powdery Mildew Disease in Mungbean [Vigna radiata (L.) Wilczek]

Greengram or Mungbean [Vigna radiata (L.) Wilczek] is an important pulse crop. It is a self-pollinated crop (2n=22) and genome size of 560 Mb. It is an important legume (Pulse) crop after Chickpea and Pigeon pea in India and widely cultivated in different parts of India as it is well adapted to multiple cropping systems in the drier and warmer climates. In India during 2019-20, about 31.15 lakh hectares area was covered under greengram. Among all the states Rajasthan had the highest area 18.30 lakh ha where Odisha covers only 1.63 lakh ha. In particular, the state of Odisha has recorded very low productivity (337 kg/ha) mainly due to inherent genotype failures and losses due to pests and diseases. On account of its dense crop canopy, photo insensitivity and short duration, it assumes special significance in crop diversification, intensification and conservation of natural resources and sustainability of production systems. The significant constraints behind the production of this crop arethe non-availability of high yielding and disease-resistant varieties. Among all Powdery mildew disease caused by Erisyphepolygoni is economically significant because it reduces photosynthetic activity and physiological changes which results in 20-40 per cent reduction in yield. So now it is a crucial time for achieving a gain in yield and for developing resistant and well adaptable genotypes through the breeding program. For this purpose scientific collection, characterization and evaluation of mungbean germplasm are highly essential. It also helps in the proper identification of genotypes with the desired characteristics.
       
Simple sequence repeats (SSRs) or microsatellites (as short tandemly repeated DNA sequences 2-5 bp in length) markers have been developed for major crop plants and this marker system is predicted to lead to even more rapid advances in both marker development and implementation in breeding programs. A potentially powerful technique for DNA fingerprinting and phylogenetic studies (Suman et al., 2019) is SSRs, which have long been known to be polymorphic and wide in plant genomes. SSR marker VrCSSR1 and one STS marker VrCSSTS1 which were reported as co-segregating with RFLP marker VrCS65 are found to be associated with PM resistance. These markers explained about 16.02% and 20.18% of the total phenotypic variance respectively. By composite-interval mapping, Kasettranan et al., (2010) reported two QTLs on two linkage groups, qPMR-1 and qPMR-2 which accounted for 20.10% and 57.81% of the total variation for plant response to powdery mildew disease. In this study, marker MB-SSR238, which was reported to be very closely linked to qPMR-2 locus and marker CEDG191, which was reported to be very closely linked to qPMR-1 were also used.

Experimental Details
 
The present investigation was carried out to evaluate thirty-one mungbean genotypes collected from the different parts of India. The genotypes were sown in an incomplete bock design augmented with four checks varieties in the month of January 2017 at KVK, Sakhigopal, Puri and November, 2018 in the Experimental Field station, EB-1, OUAT, Bhubaneswar. The accessions were accommodated in 3 blocks each having 13 plots and 13 treatments and each plot contains one treatment and seeds of each variety were sown in three lines in each plot which is 2m in length, along with four checks ‘Kamdev’, ‘OBGG-52’, ‘IPM-02-3’, ‘IPM-02-14’. The checks were randomly allocated along with the test genotypes within the blocks and replicated as many times as the number of blocks.
 
Phenotyping
Screening of Germplasm according to DUS Descriptor
 
The observations were taken as per the DUS Guidelines for the Conduct of Test for Distinctness, Uniformity and Stability on mungbean framed by the “Protection of Plant Varieties and Farmers’ Rights Authority (PPV and FRA)” (ACC, 2007). These included both quantitative and visual characters (Table 1).
 
Disease screening
 
Mungbean germplasm was evaluated in the field as per the DUS guidelines. The germplasm was screened using a 0 - 5 scale for Powdery mildew disease as recommended by Reddy et al., 1994 under field conditions. The disease scoring was carried out at 35 days and 55 days after sowing under natural conditions. Genotypes for powdery mildew disease were scored and were classified into five different classes. Observations were recorded on numbers of spots on each leaflet in a plant at different scales. Finally, the mean of each observation was calculated and the per cent disease index (PDI) was determined by using the following formula:
 
 
 
Statistical Analysis
 
The selected genotype was compared with the rest of the genotypes by DIST Coefficient Matrix. Higher the value of the coefficient, compared variety will be more similar to the selected variety. In addition to this, the value of coefficient also signifies about the extent of dissimilarity between the two varieties. DIST Coefficient Matrix was subjected to generate a dendrogram using software program NTSYS pc Ver 2.2. Exeter Software, N.Y. (Rohlf, 2000).
 
Genotyping
DNA Isolation and purification
 
The leaves were first washed, air-dried and then crushed on to a sterilised pestle and mortar in liquid nitrogen to obtain a fine powder. Care was taken to avoid thawing of the material. Extraction of total genomic DNA was carried out as described by Doyle and Doyle, (1987) with minor modifications to suit the materials under consideration. Then the DNA was dissolved in 200 µl of TE (10:1) buffer (pH 8.0) and kept overnight for complete dissolution. RNase solution (10mg/ml) was added @ 15µl/100 µl to the tube containing dissolved DNA and incubated at 37°C for 1 hour. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added and mixed properly for 2 min and spun 5 min and then purification steps were followed to remove the contaminants. Then the DNA was dried under vacuum, dissolved in TE (10:1) buffer at room temperature and then stored at -20°C for further use.
 
Standardisation of PCR for Gene Specific primer
 
Optimisation of primers is essential to make the assay perfect. Poor optimisation leads to lack of reproducibility between replicates as well as inefficient and insensitive assays, hence to pass up this intact problem prior to PCR amplification a gradient was set up such that it spans the calculated annealing temperature of the primers ± 2°C. This range can then be further customised to polish up the assay. This step can be combined with various primer concentration combinations to optimise both annealing temperature and primer concentration simultaneously. In this study, all the primers were optimised by the setting of a gradient temperature. Once the experiment has ended, the samples are run and visualised on an agarose gel (1.5%) to assess the results. The optimal annealing temperature and primer combination give the band with the highest intensity (yield) with no non-specific products.
 
Microsatellite marker Analysis
 
Seven Microsatellite markers tested for PCR amplification according to annealing temperature for 31 genotypes. The experiment was performed in eppendorf Thermal Cycler programmed under standard conditions for all the primer pairs using 1 U of Taq polymerase with 1X polymerase chain reaction buffer (100mMTris-HCl at pH 9, 500mMKCl and 15mM MgCl₂), 2.5mMdNTP, 3mM MgCl₂, 20pM 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 72°C for 7 minutes. The amplicons were run on an agarose gel (1.8%) with 100bp DNA ladder, which was then visualised through UV GELDOC unit to assess the results.

Morphological Characterization
 
Analysis of data of thirty-one genotypes on the basis of 24 visual characters and various categorical descriptors revealed the occurrence of 62 numbers of variants. Eighteen variants (erect plant growth, purple stem colour, absent in leaflet lobes, deltoid leaf shape, lanceolate leaf shape, cuneate leaf shape, green leaf colour, green leaf vein colour, purple leaf vein colour, green petiole colour, purple petiole colour, large leaf size, yellow flower colour of petiole colour, absent pod pubescence, medium and long plant height, long pod length of the mature pod, large seed size) out of possible 62 variants could not be observed in any of the genotypes. Only one limited number of categorical descriptors (yellow seed colour) that have contributed to the distinctiveness of specific mungbean genotypes had been observed. Yield assessment was analyzed for each mungbean genotypes under field conditions and calculated the yield (kg/ha) given in the (Table 4). Majority of genotypes showed good yield viz. ‘OBGG-52’ (870kg/ha), ‘Kamadev’ (830kg/ha), ‘KalahandiLocal-1’ (846kg/ha), ‘BerhempurLocal-1’ (920kg/ha), ‘Banpur Local’ (952kg/ha), ‘IPM-02-03’ (895kg/ha) and while rest of the genotypes showed low yield viz. ‘DigapahandiLocal’ (275kg/ha), ‘Sambalpur Local’ (290kg/ha), ‘Nadika Local’ (220kg/ha), ‘NayagarhLocal-6’(380kg/ha).
 
Cluster Analysis based on Morphology
 
The genetic dissimilarity matrix was analysed using UPGMA clustering algorithms for the construction of dendrogram that depicted the pattern of genetic relationships among mungbean genotypes (Table 3). The analysis revealed four main clusters at 8.75 dissimilarity coefficients with best possible discrimination between groups. The genotype pairs ‘Badahana Local’ and ‘Hinjili Local’ showed the maximum dis-similarity (2.67) followed by dis-similarity between ‘OBGG-52’ and ‘Berhampur Local 1’ among the thirty-one genotypes (Table 2), whereas, ‘Nayagarh Local’ and ‘Nayagarh Local 6’ showed lowest dis-similarity (0.01) followed by dis-similarity between ‘Kukudakhandi Local’ and ‘Banpur Local’(0.20). Fourteen genotypes viz., ‘Banpur Local’, ‘Kukudakhandi Local’, ‘PuriLocal- 4’, ‘Kalahandi Local 1’, ‘Kalahandi Local 1’, ‘Balangir Local’, ‘Nadika Local’, ‘NayagarhLocal-6’, ‘Nayagarh Local’, ‘Bhapur Local’, ‘Bolangiri local-1’, ‘Bhapur Black Local’, ‘Kamdev’ and ‘KVK Puri-2’ were included in Cluster I. But cluster-II was represented by only one genotype (Sambalpur Local 2'. Ten genotypes viz., ‘Nuagaon Brown Local’, ‘Kaniapada Local’, ‘SundergarhLocal 11-1’, ‘IPM-02-14’, ‘Behrampur Local 2’ , ‘KantapadaLocal’, ‘Berhampur Local 1’, ‘IPM-02-3’, ‘Kendrapada Local 1’ and  ‘Purushottamapur Local’ were represented in the Cluster III and cluster IV had six genotypes viz., ‘Badahana Local’, ‘Andapur Local’, ‘Hinjili Local’, ‘Seragada Local’, ‘OBGG-52’ and ‘Digapahandi Local’.
       
At 8.07 dis-similarity coefficient, Cluster-I was further sub-divided into two sub-clusters. The sub-cluster I A was represented by two genotypes viz., ‘Kamdev’ and ‘KVK Puri-2’. whereas Cluster IB had 12 genotypes viz., ‘Banpur Local’, ‘Kukudakhandi Local’, ‘PuriLocal-4’, ‘Kalahandi Local 1’, ‘Kalahandi Local 1’, ‘Balangir Local’, ‘Nadika Local’, ‘NayagarhLocal-6’, ‘Nayagarh Local’, ‘Bhapur Local’, ‘Bolangiri local -1’ and ‘Bhapur Black Local’.
 
Variability for disease reaction
 
Based on disease scale (0-5) given by Reddy et al., 1994, observations were carried out mainly on the basis of weekly intervals and disease incidence was recorded from the field given in the Plate 2 and 3. Majority of genotypes (22.58%) showed highly resistant for powdery mildew and 16.13% were resistant to powdery mildew. In comparison, 19.35% of genotypes exhibited were highly susceptible, with moderate resistant and susceptible. Among all genotypes, 3.23% of genotypes were exhibited susceptible to powdery mildew.
 
Molecular Characterisation
 
The study was aimed to validate the powdery mildew associated marker (Table 4) among mungbean genotypes. Seven primers of specific microsatellite loci were used to amplify the repeated regions in the mungbean samples. For all loci, 25µl reaction mixture containing genomic DNA concentration of 25-50 ng, primer concentration of 1 pM and MgCl2 concentration of 1.5mM, dNTPs 2.5mM, 10X buffer and 1 unit of Taq polymerase were given. In the present study primers were found to be polymorphic. These polymorphic loci amplified were in the mungbean genotypes given in the Fig 2 approximately of size viz. VrCSSTS1 (300bp), VrCSSTS2 (300bp), VrCSSSR3 (200-300bp), MB-SSR238 (200-300bp), CEDG191 (100-150bp), CEDG166 (220-300bp) and CEDG282(100bp) which are associated with powdery mildew disease recorded during field evaluation.
 
Relationship Between Genetic and Phenotyping Profiling
 
The results of this study support the existence of a relationship between genetic and phenotypic character among the mungbean varieties. This finding is of specific interest in diversity analysis and disease variability reaction with validation of microsatellites marker for mildew resistant make inferences about the genetics and phenotypic correlations among the genotypes. The phenotypic profiling (Table 1) showing that some genotypes are superior as compared to other and the disease variability reaction also showed that these superior genotypes are showing highly resistant, resistant and moderately resistant with better yield.  From the molecular profiling analysis (Table 6) of microsatellites marker also revealing similar result and validating that these markers are linked to the powdery mildew disease.
       
On the basis of seed yield per plant, number of pods per plant, number of branches per plant, number of seeds per pod and 100 seed weight some varieties are considered as superior genotypes for the further breeding program which has shown similar results with  Gokulakrishnan et al., 2012.; Behera et al., 2020.Thirty-one genotypes of mungbean were screened under field conditions and the incidence of disease was present in almost all the genotypes from 35 DAS to 55 DAS (Days After Sowing). Among them, six genotypes showed a moderately resistant and moderately susceptible reaction; five genotypes showed resistance reaction, seven genotypes showed highly resistance reaction, six genotypes showed highly susceptible reaction and only one showed a susceptible reaction. These results are in line with Khare and Lankpale (1998) who carried out mungbean trials during Rabi season over three years against powdery mildew and only seven entries were found to be resistant to powdery mildew over the years. A previous study by Thakur and Agarwal (1995) suggested that environmental factors have a large impact on the extent and severity of powdery mildew. They reported a high incidence of powdery mildew during Kharif than Rabi (Mishra et al., 2019). In the present study, high incidence of powdery mildew on mungbean varieties was observed during Kharif season under field condition. This indicated that powdery mildew development is largely depended on favourable environmental condition and cool, dry season favours their development andinfection.
       
Among the different DNA markers, microsatellite or Simple Sequence Repeats (SSRs) are the marker of choice for various genetic studies because, which detect sequence variation in the hyper-variable region of tandem repeats of 2-4bp, are a powerful tool for genome analysis because of their co-dominant nature, loci specificity and high reproducibility (Tautz and Renz, 1984). Microsatellite markers have been used for genome mapping and genetic diversity studies in many crop plants (Ford et al., 2002; Blair et al., 2006; Sangiri et al., 2007; Gupta et al., 2013; Singh et al., 2014 ; Behera et al., 2020). In addition to being able to be readily assayed, they are highly informative, transferable, widely distributed in most plant species and highly polymorphic between genotypes. The present molecular data are showing amplification of VrCSSSR3 at 200-300 bp regions, VrCSSTS1 and VrCSSTS2 at 300bp region, which is in congruent with the result reported by Zhang et al., (2008) who developed new SSR and STS markers co-segregated with RFLP marker VrCS65 by BAC cloning. In his study, SSR marker VrCSSR1 and one STS marker VrCSSTS1 which were reported as co-segregating with RFLP marker VrCS65 are found to be associated with PM resistance. These markers explained about 16.02% and 20.18% of total phenotypic variance respectively. CEDG191amplified at100-150bp region, CEDG166 amplified at 220-300bp region andCEDG282 at100bp region are considering associated with powdery mildew disease recorded during field evaluation. Multiple-interval mapping by Chankaew et al., (2013) also shows the similar types of result with little divergence. He reported that two markers from LG 6 (CEDG121 and CEDG169) were associated with qPMRUM2 locus and two markers from LG9 (VRSSR010 and DMBSSR130) were associated with qPMV4718-3 locus and he reported that Z test indicated that marker DMBSSR130 and CEDG121 showed significant association with powdery mildew resistance. The MB-SSR238 marker exhibits amplification band within 200-300 bp regions showing the association with powdery mildew. The composite-interval mapping by Kasettranan et al., (2010) reported two QTLs on two linkage groups, qPMR-1 and qPMR-2 which accounted for 20.10% and 57.81% of the total variation for plant response to powdery mildew disease. In this study, marker MB-SSR238, which was reported to be very closely linked to qPMR-2 locus and marker CEDG191, which was reported to be very closely linked to qPMR-1 were also used.

Based on the present study, significant differences among mungbean genotypes were observed for most of the yield attributing traits. The resistant sources obtained in the present study can be utilised in the breeding program for evolving varieties resistant to powdery mildew. The powdery mildew linked validated markers (VrCSSTS1, VrCSSTS2, CEDG191, CEDG166, CEDG238 and MB-SSR238) could be utilised for the breeding programme for the development of powdery mildew resistant mungbean genotypes. The genotypes identified for different characters serve as a source for breeders to select them and use them for constructing mapping population. The germplasm identified with different characters serves as genepools which provide sufficient scope for a breeder to select within the genotypes. The promising germplasm lines could be used in various breeding programs.

The authors wish to acknowledge the Department of Biotechnology, Government of India for providing student research grant under PG-HRD course program. The authors also wish to acknowledge Rashtriya Krishi Vikas Yojana (RKVY), Govt. of Odisha for the generous support. The authors are also thankful to Krishi Vigyan Kendra, Puri and Centre for Pulse Research, OUAT, Berhampur, Odisha for their support for conducting field trials.

All authors declare that they have no conflicts of interest.