Legume Research

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Legume Research, volume 44 issue 7 (july 2021) : 743-750

Assessment of the Genetic Diversity of Black Gram [Vigna Mungo (L.) Hepper] Collections for Yellow Mosaic Virus Resistance Using Simple Sequence Repeat Markers 

N. Sathees1, D. Shoba1, S. Saravanan1, S. Merina Prem Kumari1, M. Arumugam Pillai1,*
1Department of Plant Breeding and Genetics, Agricultural College and Research Institute, Killikulam, Vallanad, Tuticorin-628 252, Tamil Nadu, India.
  • Submitted16-04-2019|

  • Accepted11-09-2019|

  • First Online 03-12-2019|

  • doi 10.18805/LR-4155

Cite article:- Sathees N., Shoba D., Saravanan S., Kumari Prem Merina S., Pillai Arumugam M. (2019). Assessment of the Genetic Diversity of Black Gram [Vigna Mungo (L.) Hepper] Collections for Yellow Mosaic Virus Resistance Using Simple Sequence Repeat Markers . Legume Research. 44(7): 743-750. doi: 10.18805/LR-4155.
A total of nine black gram genotypes were tested for yellow mosaic virus (YMV) resistance along with other nine biometrical traits. The genotypes VBN 4, KKB-14-015, KKB-14-45 and KKB-14-022 exhibited complete resistance to YMV in field screening.  From the molecular characterization, out of 42 SSR markers studied, 15 SSR markers expressed polymorphism. The PIC values ranged from 0.37 (for SSR marker CEDG 024) to 0.79 (CEDG 154) with an average of 0.63. From the cluster analysis, YMV resistant genotypes KKB-14-045 and KKB-14-015 were located on clusters 6 and 7 and the susceptible but agronomic desirable genotypes IC 343943 and IC 436656 were located on cluster 3 which showed the diverse nature of these genotypes. Hence combinations viz., IC 343943 x KKB-14-015, IC 343943 x KKB-14-045, IC 436656 x KKB-14-015 and IC 436656 x KKB-14-045 would be ideal to produce desirable recombinants for YMV resistance breeding in black gram.
Pulses also called as the poor man’s meat and rich man’s vegetables are the main basis of protein to vegetarian diet. Among the pulses, black gram [Vigna mungo (L.) Hepper] is a diploid (2n=2x=22), short duration and cleistogamous crop belonging to the family leguminosae (Naik et al., 2017). It is grown in 21-22 million hectares globally with an annual production of 12-16 million tonnes (Jeevitha et al., 2018). In India, black gram productivity is 841 Kg/ha (Pulses Revolution- from Food to Nutritional Security, 2018) and the poor productivity of pulses in India is credited primarily to poor spread of improved varieties and technologies, untimely and inadequate availability of quality seed of improved varieties and other inputs, water-stress due to dependence on rainfall, low and high temperature stress, vulnerability to pests and diseases and cultivation on marginal and sub-marginal land (Rana et al., 2016). Among the biotic stresses, yellow mosaic virus (YMV) disease is a serious problem in black gram production and yield reduction up to 100 per cent has been recorded (Nene, 1972 and Rathi, 2002). Hence identification of YMV resistant lines in black gram is very essential for breeding programs.
Genetic diversity is a basic requirement for effective genetic improvement program in crop plants (Jeevitha et al., 2018). The significant decline in the black gram production in India is due to lack of suitable varieties and genotypes and the absence of distinct morphological and genetic characterization of diverse genotypes. Documentation of diversity of genotypes is needed for genetic improvement of legume species (Sivaprakash et al., 2004 and Palaniappan and Murugaiah, 2012).
Molecular markers are widely used in diversity studies, since they are not influenced by environmental factors. Molecular markers differentiate DNA sequences of individual genotypes and detect more polymorphism than morphological and isozyme markers (Tanksley et al., 1989 and Shoba et al., 2010). Among the molecular markers, simple sequence repeat (SSR) markers comprise immense potential due to their co-dominant, multi allelic nature, reproducibility and good genetic coverage (Naik et al., 2017). SSR markers are short tandem repetitive DNA sequences with repeat length of few (1 to 5) base pairs and are used in wide range of applications such as genetic mapping, genome analysis, genotype identification, seed purity evaluation, diversity studies, paternity determination, pedigree analysis, QTL analysis and marker assisted breeding (Archana and Jawali, 2007 and Palaniappan and Murugaiah, 2012). SSR primers amplify regions between the microsatellites and provide larger number of fragments per primer than RAPD (Bajpai et al., 2008 and Shoba et al., 2010). The occurrence of variation among the genotypes based on morphological and molecular characterization helps to choose the diverse parents for hybridization programs (Hari et al., 2017). Efforts were made continuously to identify the YMV resistance lines in black gram (Basamma et al., 2015, Vanniarajan et al., 2017 and Priya et al., 2018). Marker Assisted Selection (MAS) is one of the key methods for improving the traditional breeding practices (Prasanthi et al., 2011). Keeping the above points in view, the present investigation was concentrated on assessing the genetic diversity among nine black gram genotypes using SSR markers to identify the diverse parents. YMV resistance was assessed using field screening techniques to identify the YMV resistant genotypes for making desirable cross combinations in recombination breeding programs.
Plant materials
Field experiment was carried out at Department of Plant Breeding and Genetics, Agricultural College and Research Institute, Killikulam, Tamil Nadu during 2017-2018. A total of nine black gram genotypes collected from different sources were taken for YMV screening. The pedigree details of the genotypes are presented in Table 1.

Table 1: Pedigree details of black gram genotypes used in the study.

Field screening for YMV resistance
Each black gram genotype was sown in 3 meter row to accommodate 20 plants per row. The experiment was conducted by adopting a randomized block design (RBD) replicated twice. Spacing was adopted with 30 x 10 cm which denoted the distance between rows and distance between plants respectively. On tenth day after sowing, the crop was thinned out, leaving one healthy seedling per hill. Recommended agronomic practices were adopted. Observation on the YMV disease incidence was recorded on a 1-9 arbitrary scale (Alice and Nadarajan, 2007). YMV disease reaction scale was classified as 1&2 - resistant; 2.1&4- moderately resistant; 4.1&5- moderately susceptible; 5.1&7- susceptible; 7.1&9- highly susceptible and the detail of scales is given in Table 2.

Table 2: Rating scale for scoring YMV disease severity in black gram (Alice and Nadarajan, 2007).

Data collection
Biometrical observations on days to 50% flowering, plant height (cm), number of primary branches per plant, number of clusters per plant, number of pods per plant, number of seeds per pod, pod length (cm), hundred seed weight (g) and single plant yield (g) were recorded at maturity stage. From each replication, five random plants were tagged for observing yield and other quantitative characters. The mean value of the five plants was computed and taken for analysis.
Molecular analysis
Extraction of genomic DNA and PCR amplification
DNA was extracted from the leaf samples of the germplasm lines following CTAB method developed by Saghai-Maroof et al., (1984). PCR reaction was performed with a total of 43 SSR markers. The list of SSR markers used for surveying genotypes is furnished in Table 3. PCR reaction was performed with a total volume of 10 μl. The thermal cycler programme as follows, step 1-Initial denaturation (94°C for 5 minutes), step 2-Denaturation (94°C for 30 seconds), step 3-Annealing (55°C for 30 seconds), step 4-Extension (72°C for 1 minute), steps 2-4 (40 cycles), step 5-final extension (72°C for 10 minutes) and step 6 (Final Hold-4°C until sample retrieval).

Table 3: Sequence details of the SSR markers.

Polymorphism information content (PIC)
SSR markers were used to identify the polymorphism in black gram germplasm to observe the variation at DNA level. Agarose gel electrophoresis was performed to see the amplified DNA products. Clearly resolved, unambiguous polymorphic bands were scored using the molecular weight standard 100 bp ladder. The banding pattern was scored in binary digits as absence of band was indicated as ‘0’ and presence of band was indicated as ‘1’. Polymorphic Information Content (PIC) was estimated using the formula 1-Σ (Pi)2, whereas Pi depicts the proportion of samples carrying the ith allele.
Molecular diversity analysis
Genetic similarity index was used to construct the dendrogram which illustrated the genetic relationship among the genotypes of black gram. Allele number was given and scored according to its presence or absence, based on difference in molecular weight. Only the clear and unambiguous bands were scored. Polymorphic markers were scored for the presence (1) and absence (0) of the corresponding band among the genotypes. Consequently, a data matrix comprising ‘1’ and ‘0’ was formed and subjected to further analysis. Further processing of data was done by carrying out sequential agglomerative hierarchical non overlapping clustering (SAHN), on squared Euclidean distance matrix. Similarity matrix was done using Jaccard’s coefficient in which similarity matrices were utilized to construct the UPGMA (Unweighted Pair Group Method with Arithmetic average) dendrogram. Data analysis was done using NTSYs PC (Hari et al., 2017). 
Field screening for YMV resistance and mean performance on agronomic different traits
The nine black gram genotypes were scored for YMV resistance and the results are given in Table 4. The genotype IC 436656 was highly susceptible to YMV whereas the genotypes viz., VBN 4, KKB 14-022, KKB 14-045 and KKB 14-015 were resistant to YMV. The mean performance of black gram genotypes for agronomic different traits is presented in Table 5. The genotypes IC436656, IC343943, KKM1, ADT3, KKB-14-022, KKB-14-052, KKB-14-045 and VBN 4 recorded significantly superior mean performance for days to 50% flowering viz., 33, 35, 35, 34, 35, 32, 32.50 and 32.50 days respectively. The genotypes KKM 1, KKB-14-022 and KKB-14-045 recorded desirable mean performance for plant height viz., 59cm, 58.40cm and 61.65cm respectively. The genotype KKB-14-045 had shown significantly superior mean performance for number of primary branches per plant (5.65) whereas the genotypes KKB-14-022, KKB-14-052 and KKB-14-045 recorded significantly more number of clusters per plant viz., 24.80, 40.15 and 30.65 respectively.  The genotypes viz., KKB-14-052 and KKB-14-045 exhibited significantly superior mean performance for higher number of pods per plant viz., 77.50 and 66.65 respectively and the genotypes viz., IC436656, KKB-14-052 and VBN 4 recorded desirable performance for pod length viz., 5.20cm, 5.15cm and 5.05cm respectively. The genotype KKB-14-045 had shown superior performance for the trait 100 seed weight (5.15g) and the genotypes IC343943, KKB-14-045 and KKB-14015 significantly well performed for single plant yield viz., 28.10g, 31g and 31.85g respectively.

Table 4: Disease severity scoring scale of black gram genotypes for YMV resistance.


Table 5: Mean Performance of black gram genotypes for agronomic different traits.

Studies on polymorphism
Genetic diversity analysis was carried out in nine black gram genotypes using 42 SSR markers. Out of 42 SSR markers, 15 exhibited 35.7 per cent polymorphism. The pictorial representation of black gram genotypes for the SSR markers CEDG 141 and CEDG 008 is depicted in Fig 1. The informativeness of the SSR markers is revealed by calculating the Polymorphic Information Content (PIC) value. The PIC value of 15 SSR markers is presented in Table 6 and the values ranged from 0.37 (CEDG 024) to 0.79 (CEDG 154) with an average of 0.63. Pyngrope et al., (2015) screened 30 black gram genotypes using 12 SSR markers and from the study, PIC value ranged from 0.22 to 0.68 and three polymorphic markers CEDG 180, CEDG 139 and CEDG 279 were identified. Tamilzharasi et al., (2018) genotyped 48 black gram lines for YMV resistance and tagged the molecular markers CEDG180, ISSR8111357 and YMV1 FR. In the present investigation SSR marker CEDG 139 had shown the PIC value of 0.59 (Table 6).

Table 6: Polymorphism information content (PIC) value of the simple sequence repeat (SSR) markers for genetic diversity analysis in black gram.


Fig 1: The pictorial representation of black gram genotypes for the SSR markers CEDG 141 and CEDG 008.

Cluster analysis
Genetic similarity co-efficient estimated for nine black gram genotypes ranged from 0.48 to 0.82 (Table 7). Based on similarity values, genotypes KKB-14-045 and ADT3 had less similarity (0.48) followed by KKB-14-045 and IC 343943(0.50), VBN 4 and KKM 1(0.51) and VBN 4 and KKB-14-022 (0.51). Hence these genotypes possessed high genetic variation and hybridization of these genotypes may result desirable recombinants. The genotypes KKM-1 and IC 343943 had high similarity value (0.82) and combination of these genotypes would not be advantageous for breeding programmes. Hari et al., (2017) studied the genetic diversity analysis among YMV resistant and susceptible varieties in mung bean using SSR markers. From their studies, the mung bean variety WGG-2 was appeared to be more divergent with 42.1% similarity, while high similarity of 81.7% was recorded between two susceptible varieties KM 14-34 and KM 14-61.

Table 7: Genetic similarity co-efficient for diversity analysis in black gram collections.

In the present study, nine black gram genotypes were grouped into seven clusters at 3.80 coefficient in the dendrogram (Fig 2). Among the seven clusters, genotypes VBN 4, KKB 14-052, KKB 14-022, KKM 1, KKB 14-045 and KKB 14-015 were present separately in clusters viz., I, II, IV,V,VI and VII respectively. The genotypes viz., VBN 4, KKB 14-022, KKB 14-045 and KKB 14-015 were resistance to YMV and genotypes KKB 14-052 and KKM-1 were moderately resistance to YMV (Table 4). Susceptible genotypes for YMV viz., IC 436656 (highly susceptible), IC 343943 (moderately susceptible) and ADT-3(susceptible) were grouped together in single cluster (cluster III). Gupta et al., (2015) reported that 10 molecular markers (includes ISSR and microsatellite markers) were linked to YMV resistance in black gram and mung bean. From their studies, three molecular markers viz., ISSR8111357, YMV1-FR and CEDG180 differentiated YMV resistant and susceptible genotypes in black gram. Jeevitha et al., (2018) projected molecular genetic diversity analysis in black gram under an YMV hotspot regime. They analyzed 26 black gram genotypes using 19 SSR markers and the dendrogram showed four apparent clusters based on marker allele distribution and also considerable divergence was noted between the dissimilarity matrices.

Fig 2: Dendrogram of black gram collections (denoted in Table 1) based on SSR markers polymorphism.

In the present study, moderately susceptible genotype IC343943 and highly susceptible genotype IC 436656 were agronomically superior based on morphological characterization. Hence these genotypes may be used as female parents in hybridization programmes to improve the YMV resistance. The YMV resistant genotypes KKB-14-015 and KKB-14-045 could be utilized for male parents since they were located away from susceptible genotypes in the dendrogram. Hence, the combinations such as IC343943x KKB-14-015, IC343943 x KKB-14-045, IC 436656 x KKB-14-015 and IC 436656 x KKB-14-045 would be fruitful to yield desirable recombinants for YMV resistance breeding in black gram.

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