AFLP analysis of Genetic diversity and Phylogenetic relationships of

Vigna radiata (L) Wilczek


Urmi Roy1, Ushri Roy2*

1Department of Botany, Vijaygarh Jyotish Ray College, 8/2, Bijoygarh, Jadavpur, Kolkata, West Bengal 700032.

2Department of Botany, Bhairab Ganguly College, Belgharia, 2, Feeder Rd, Beehive Garden, Belghoria, Kolkata, West Bengal 700056.

*Corresponding Author E-mail:



Among the important pulse-yielding plants Vigna radiata is important. In the Indian subcontinent, it is an essential source of dietary protein particularly in the vegetarian population. It contains abundant nutrients with biological activities. Soaked seeds are eaten as a good source of protein and mature seeds are cooked or can be sprouted for human consumption. Mung beans and its sprouts contain chemical constituents like flavonoids, phenolic acids, organic acids, amino acids, carbohydrates, and lipids. It is related to different biological activities like antioxidant, antimicrobial, antitumor, anti-inflammatory, anti-diabetic, and antihypertensive effects, etc. The seeds are a traditional source of cures for paralysis, rheumatism, coughs, fevers, and liver ailments. As a green manure or cover crop, the plant can be grown, enriching the soil with the nitrogen formed on its roots. The powerful Amplified Fragment Length Polymorphism (AFLP) is a molecular marker used for a variety of applications like assessing genetic diversity within species or among closely related species, inferring population-level phylogenies, generating genetic maps, and determining the relatedness among cultivars. AFLP has become extremely beneficial in the study of taxa including bacteria, fungi, and plants, where much is still unknown about the genomic makeup of various organisms. A study was made to assess the genetic diversity and phylogenetic relationships of a set of five varieties of Vigna radiata (L) Wilczek using the AFLP technique. Five different varieties of Vigna radiata viz. B1, TARM 2, PDM 84, TM 99, and TM 98 were subjected to AFLP analysis. A total of 471 fragments were scored across all the 12 AFLP primer sets used and the results were used to plot a dendrogram. It was observed that these five varieties formed three distinct groups among which the aromatic B1 variety formed a distinct group.


KEYWORDS: Mung bean, Vigna radiata, AFLP, genetic diversity, genetic relationships, phylogeny, medicinal plant.




Ever since ancient times, people looked for drugs in nature.  Plants have been a valuable source of natural products, for maintaining human health, with natural therapies. The use of phytochemicals for pharmaceutical purposes has gradually increased in many countries1,2,3. Mung beans (Vigna radiata) are green beans belonging to the legume (Fabaceae) family. It has been cultivated in India since ancient times. Mung beans are eaten in salads, soups, and in vegetarian diets.


They have high nutritional value and are believed to aid many ailments. Mung beans are the best plant-based sources of protein and are rich in essential amino acids like phenylalanine, leucine, isoleucine, lysine, arginine, etc. Along with high protein, it also contains a good amount of carbohydrates, fiber, folate (B9), manganese, magnesium, phosphorus, iron, copper, potassium, zinc, and vitamins (B1, B2, B3, B5, B6). Mung beans contain many healthy antioxidants, like phenolic acids, flavonoids, caffeic acid, cinnamic acid etc4. Vigna shows hepatoprotective activity5. It has a high level of antioxidants that helps to neutralize the potentially harmful molecules known as free radicals that interact with cellular components6. Mung bean antioxidants have been shown to lower blood LDL cholesterol and protect the LDL particles from interacting with unstable free radicals as proven by animal studies7. Vigna radiata plants have the capacity to cause the destruction of bladder stones (i.e. lithotriptic property). The crude aqueous extract of V. radiata exhibits antiurolithiatic activity and helps in the dissolution of kidney stone8.


Diversity in organisms is very interesting and important for understanding the evolutionary history of organisms. Different kinds of procedures are used to ascertain the relationship or diversities among the different variants or samples starting from simple morphological analysis to modern molecular markers that are used to relate the genetic diversities or relationships among organisms 9-13. For determining the genetic variation, organismal evolution, and phylogenetic relationship between individuals, molecular markers are the most efficient tools. Vos et al (1995) first described the AFLP technique14. Amplified Fragment Length Polymorphism (AFLP) is a polymerase chain reaction (PCR) based genetic fingerprinting technique for detecting polymorphisms in DNA. Hundreds of amplified DNA restriction fragments can be observed simultaneously which can be used through the AFLP technique, for identifying the genetic variation among closely related species or to determine minute differences within populations for assessing the degree of relatedness or variability among cultivars and also used in linkage studies to generate maps for QTL analysis. Marker-assisted selection (MAS) offers a form of genotype selection, which is not affected by environmental variation or by complexities of interaction affecting phenotypic selection. AFLP technique was used to study the genetic relationship among the five varieties of mung bean with twelve primer combinations.



Population selection and material collection:

Five varieties of Vigna seeds (namely B1, TM 99, TARM 2, PDM 84, and TM 98) have been collected from Baharampur Pulse and Oil Seed Research Centre, W. B. The collected seeds were germinated in the greenhouse. Sowing was carried out during the month of November and 6 to 8 seeds of each variety were sown in each pot filled with soil rite and soil mix (1:1) and were kept in a greenhouse where photoperiod was maintained for 16 h. The average day temperature was 250C with a night temperature maintained at 150C. The morphology of the plants was studied after 2 months of growing in the greenhouse.


Measurement of morphological traits:

The leaf traits were measured on the 4th or 5th healthy compound leaves from the top of the branches. Flower and fruit traits were measured at random during the full-blooming stage and the mature stage, respectively. Seven traits of plants were measured: plant height, length of internodes, leaf length, leaf width, inflorescence length, pod length, number of pods.



Vigna seeds were sterilized in 10% sodium hypochlorite solution (NaOCl, v/v) taken in a sterile conical flask in front of laminar airflow. Seeds were taken in conical flasks containing 20ml 10% bleach solution and vigorous stirring was done for 20 min for proper sterilization. The bleach was then washed off by rinsing with sterilized distilled water with a duration of 10 min of shaking for each wash and the rinsing was repeated five to six times.


DNA Extraction:

For DNA extraction seeds were germinated under aseptic condition. Seeds of different varieties were sterilized and imbibed (overnight) seeds were then allowed to germinate aseptically in agar (0.9%, w/v) and sucrose (3%, w/v) medium. Extra pure DNA was isolated from 12 days old young leaves of seedlings of five varieties of Vigna radiata using the Phenol-Chloroform extraction procedure15. Young, green leaves of Vigna radiata were cut and crushed in the extraction buffer (200mM Tris-HCl, pH 7.5, 250mM NaCl, 25mM EDTA, and 0.5% SDS). The supernatant was mixed thoroughly with 750μl Tris-EDTA saturated phenol-chloroform (pH 8.0). After centrifugation, the supernatant was collected and was again added to 680μl Tris-EDTA saturated phenol-chloroform. The clear upper supernatant was collected after centrifugation and was added to 600μl chloroform. The contents were mixed by inversion and then centrifuged. The upper clear supernatant was mixed with 500μl isopropyl alcohol and 50μl 3M ammonium acetate. Finally, the DNA was spooled out with the help of an autoclaved capillary tube. The spooled DNA was cleaned with 70% ethanol. Triple distilled water was added to it and was kept at 40C overnight. DNA isolated was treated with RNaseA (10mg/ml) to eliminate RNA and was transferred to -700C. 


AFLP Analysis:

For AFLP, extra pure plant DNA was used with the help of the AFLP ® analysis system I AFLP ® starter primer kit (Invitrogen). 250 ng genomic DNA was digested with EcoRI and MSeI enzymes. 25µl Digested DNA solution was added to the adapter ligation solution, according to the kit manual. Pre-amplification reaction, primer labeling, selective amplification were performed according to the protocol. Selective amplification was performed with 12 pairs of primers using E-ACG, E-AGG, M-CAC, M-CAA, M-CAT, M-CAG, M-CTA, M-CTT, M-CTG, and M-CTC in combination. PCR amplification was carried out with radioactive forward primers (labeled with γ-32P). The PCR reaction was terminated at different positions with respective reverse primers to produce amplified products of different lengths. The selective amplified products were separated by polyacrylamide gel electrophoresis (PAGE).


Data Interpretation and Analysis:

Each selective primer pair produced a specific banding pattern depending upon the genomic DNA samples used in the PCR reaction. Easily visible and scorable bands were counted (as “1” for the presence and “0” for the absence of a band) across the lanes for each of the primer combinations in the autoradiogram and transferred to the binary matrix for the cluster analysis using the Software Package SPSS-version 9.0. Jaccard’s coefficient (Proximity matrix) was used to calculate the similarity between varieties. Cluster analysis was performed by the proximity matrix using the complete linkages that generate a dendrogram.


The distance migrated by molecular weight markers (in bp) from a particular position of autoradiogram was measured and was plotted on a millimeter graph. A calibration curve was prepared from that graph. The corresponding weight (in bp) of AFLP bands of different accessions was determined by measuring the distance migrated in the autoradiogram and comparing it with the calibration curve.



Phenotypic differentiation based on morphological traits:

Five different varieties of Vigna radiata (viz. B1, TM99, PDB84, TARM2, and TM98) were grown in the greenhouse. The plants were highly branched with trifoliate leaves. The upright part of the plants varied in height (Figure 1a) i.e. from 20cm and 35cm as well as the distance of the internodes (Figure 1b) varied in different species. The leaves were oval with a sharp blade tip. The leaf length (Figure 1c) varied from 3 and 5cm and the width (Figure 1d) varied from 2cm and 3cm.  The leaves and stems were very furry.


The flowers were pale yellow in colour, borne in the inflorescence; a cluster of 12 to 15 flowers near the top of the plant (Figure 1e). The mature pods were variable in color (yellowish-brown to black) and length. Pods were linear to cylindrical and 3 to 10cm long with a diameter of 0.5cm.  Pods were hairy, dark brown to blackish in colour, and died after maturity. The length of pods (Figure 1f) and the number of seeds (Figure 1g) varied from 7 to 13 per pod. The beans were round in shape.  



Figure1.  The mean value of seven quantitative traits (plant height, length of internode, length and width of leaves, length of inflorescence, length of pods, and numbers of seeds) of five varieties of Vigna radiata (viz. B1, TM-99, PDM- 84, TM 98, TARM-2) represented in chart form.

Genetic differentiation based on AFLP markers:

The agarose gel electrophoresis of DNA isolated from Vigna radiata (Figure 2) showed a clear genomic DNA band without RNA contamination and smear formation.  In the AFLP study, 12 primer pairs were used. Among the 12 primer pairs used, all the primers did not show similar binding efficiency with Vigna DNA samples. Some primers bound to all varieties of Vigna DNA, but few primers were unable to bind with specific varieties of DNA.


Fig. 2:  The genomic DNA band isolated from Vigna radiata            


Figure 3: AFLP Profile with V. radiata (EACG-MCAG Primer Pairs)


Among the primer pair used, E-ACG/M-CAT, E-ACG/ M-CTT, E-ACG/M-CTG, E-ACG/M-CTC, E-AGG/M-CAA, E-AGG/M-CAA, E-AGG/M-CAT, E-AGG/M-CAG were proved to be very good primer pairs in Vigna where amplified products were found in all five varieties studied through AFLP technique (Figure 3). E-ACG/M-CAG and E-AGG/ M-CAC were also good primer pairs producing bands in all varieties except one variety (TM 98 and PDM 84 respectively). Among the primer pairs used, E-ACG / M-CTA was unable to bind with DNA of any varieties of Vigna, and no product was amplified. Different numbers of bands were found in different primer pairs. Among all primer pairs used, E-ACG / M-CAT was able to produce a maximum number of amplified products, producing 170 total amplified fragments in all five varieties. In this primer pair, TM 98 and TM 99 variety showed more than 50 amplified products. E-AGG / M-CAC showed poor binding with Vigna DNA producing 50 amplified products. Among the 12 primer pairs used E-ACG / M-CTA was unable to bind to DNA of any varieties.


The size of the bands showed that maximum primer pairs produced smaller fragments (50 base pair to 600 base pair) and few showed larger fragments in PAGE. E-ACG/M-CAT and E-ACG/ M-CTC were two primer pairs which produced fragments above 100 base pair in all varieties. Some amplified products were monomorphic across all the varieties while some fragments were variety specific. A band produced by a specific primer pair representing only one variety will be designated as a molecular marker. Molecular markers specific for each variety were counted and represented in chart form (Figure 4).



Figure 4: Number of unique molecular marker fragments specific for each variety of Vigna radiata generated by three primer pair combinations.


The relativity of the banding patterns among five different varieties was constructed and was transferred to Proximity Matrix (Complete Linkage). The Jaccard’s coefficient values calculated (table 1) from the data obtained from the selected primer pairs ranged from 0.078 to 0.227 (min to max) in the proximity matrix.


Table 1:  Proximity Matrix of Jaccard’s Coefficient


Matrix Input


TM 99


PDM 84

TM 98







TM 99












PDM 84






TM 98







The data obtained by the proximity matrix were used to construct a dendrogram among the five varieties studied (Figure 5).


Figure 5: Dendrogram of five varieties of Vigna radiata


The AFLP analysis was proved to be an efficient tool for the separation of five varieties of mung bean studied. The resultant dendrogram comprised mainly of three clusters (A, B, and C) of five varieties. Cluster A and C were further divided into two subclusters. Variety TM 99 and TM 98 were clustered together forming a single group (A). TARM 2 produced a separate cluster. B1 and PDM 84 varieties were grouped together representing a single cluster (C).



Plants are used for curing different health problems and for this reason many herbal treatments are coming up with no harmful side effects and cost-effectiveness as compared to other forms of treatment16,17,18. Plant members belonging to the family Fabaceae are economically important. The seeds for many members are rich in protein and slow antioxidants and anti-inflammatory activity19. Different types of molecular techniques are used to identify, predict, or characterize plants at inter or intraspecific labels, among which RAPD is less expensive and low time consuming20.  AFLP analysis generated a large number of markers that were linked to targeting genes and revealed high polymorphism. AFLP analysis was used to study the basic diversity and genetic variation among different cultivars of Vigna radiata.


The intra and interspecific variations in Vigna subgenus Ceratotropis sp were determined through AFLP technique21. A similar kind of experiment was also performed to find the genetic relationship among 27 cultivars in Indian mung bean22. In 2002, a linkage map of Vigna radiata was constructed to identify drought-resistant varieties using the AFLP technique23. The diversity and the relationship at the genetic level among domesticated and wild varieties of cowpea (Vigna unguiculata) were evaluated24. Wild cowpea varieties were more diverse than domesticated cowpea varieties. Different landraces of Bambara groundnut (Vigna subterranea) were analyzed through AFLP technique25. A genetic linkage map of cowpea was constructed which included 412 markers (AFLP based) that were linked to resistance genes or traits. The genetic relationship in the different species of Vigna minima complex showed a high degree of intra-specific polymorphism depending upon the AFLP methodology26. The genetic diversity among different accessions of the Bambara groundnut was assessed through AFLP analysis27. The AFLP technique along with the RAPD procedure were used to draw genetic relationships among different accessions of Vigna angularis (Azuki)28. AFLP marker proved to be very informative to monitor the genetic diversity of Albizia species29.


Senecio ovatus subsp. ovatus and S. germanicus subsp. germanicus form distinct entities through AFLP fingerprint data30. The genetic diversity of 20 garlic cultivars was evaluated by combining morphological characters and AFLP molecular marker data31. AFLP markers were used in gene mapping and in the early selection of medicinally important orchid Dendrobium thyrsiflorum32.  AFLP is highly suitable for the evaluation of within and between accession diversity in gene banks33. Morphological traits were used in clustering 6-natural populations of Rosa platyacantha and the data were analyzed with AFLP based markers34. The populations were mainly grouped with similar altitudes and geographic distances. When genetic diversity among mung bean, lentil, and faba bean genotypes were studied, it was found that amplified fragments length polymorphism (AFLP) markers exhibited a considerable genetic diversity35. Two distinct ancestral clusters were obtained from 25 Salvia officinalis populations using the AFLP marker36. Two genetic groups were constructed from 11 Prunus padus populations through AFLP makers37. AFLP fingerprints were proved to be useful to assess the degree of intraspecific variability among Sporothrix species38.


The genetic relationship and the genetic diversity of the five varieties of mung bean were established using the AFLP technique. The AFLP tool was proved to be useful to draw relationships among these five varieties studied. All primer pairs used were able to amplify Vigna DNA except the E-ACG/M-CTA primer pair. Among all the primer pairs those were able to amplify DNA fragments with all the five varieties E-ACG/M-CAT showed the best result producing the maximum number of amplified products (total 170 amplified fragments). The size range of amplified products varied from 40 bp to 600 bp with different samples with different primer pairs. The 11 primer pairs were able to produce polymorphic bands, which were variety-specific. E-ACG/M-CAG, E-ACG/ M-CTG, and E-AGG/M-CAG primer pairs were the choice of primer pairs for use of determining unique molecular marker fragments in Vigna radiata varieties among which E-AGG/M-CAG was the best primer pair producing total 42 polymorphic bands in the five varieties studied. 


From the above study, E-ACG/M-CTG and E-AGG/M-CAG primer pairs could be recommended for the detection of the variety-specific fragment in Vigna radiata. The proximity matrix ranged from min 0.078 to max 0.227 in Jaccard’s coefficient matrix. From dendrogram analysis, it was observed that TM98 and TM 99 formed a single group forming a close cluster. These proved that they might have originated from a similar ancestor in their origin and have maximum similarity at the genetic level. TARM 2 was proved to be distantly related thus forming a separate group in the dendrogram. The B1 variety was also representing different clusters showing the difference in AFLP banding patterns with other varieties studied. PDM84 variety formed an outgroup from B1 variety showing similarity to some extent with this variety. It may be concluded that intervarietal genetic variation clearly distinguished the aromatic B1 variety from the rest of the high yielding varieties.



Assessing the genetic relationships among different varieties of Vigna radiata through AFLP analysis is very useful. The ability to determine genetic variation among the varieties at the molecular level is directly related to the number of polymorphisms detected and their reproducibility. AFLP markers were used to study the relationship among individuals, populations, and independently evolving lineages, such as species. The relationship and distance of the five varieties (B1, TM 99, TARM 2, PDM 84, and TM 98) of mung bean were analyzed through the AFLP technique. Twelve primer pairs were used to amplify the DNA of these five varieties. Among all the primer pairs used, a maximum number of amplified products were produced by E-ACG/ M-CAG. E-AGG/ M-AAG was the best primer pair to produce maximum polymorphic bands or molecular markers for a specific variety.


The above experimental data and examples have proved that AFLP markers are useful for assessing genetic differences and were also useful to determine the genetic relationship among cultivars of Vigna radiata.



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Received on 11.04.2020           Modified on 22.09.2020

Accepted on 12.11.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(8):4122-4128.

DOI: 10.52711/0974-360X.2021.00714