AGRICULTURAL RESEARCH COMMUNICATION CENTREwww.arccjournals.com/www.legumeresearch.in

*Corresponding author’s e-mail: swapankumartripathy@gmail.com.

Legume Research. 38 (3) 2015: 279-288Print ISSN:0250-5371 / Online ISSN:0976-0571

RAPD markers for genetic characterization in mungbean[Vigna radiata (L.) Wilczek]Swapan K. Tripathy*, Suchinnata S. Sardar, Sasmita Dash, Shovina Pal, Tanuja Acharya, Bhumika Ray Mohapatra,Anath B. Das and Gyana R. Rout

Sinha Molecular Breeding Laboratory, Department of Plant Breeding & Genetics,College of Agriculture, OUAT, Bhubaneswar-751 003, India.Received: 17-09-2013 Accepted: 25-03-2014 DOI: 10.5958/0976-0571.2015.00023.5

ABSTRACTInformation on genetic variability and relatedness among 20 selected high yielding Vigna genotypes were assessed usingRAPD profiles. Ten RAPD primers generated 81 amplicons resulting 98.7% polymorphism. 5-12 bands were produced perprimer with an average of 8.1 bands and the maximum being resolved using primer OPD 08. All primers revealed 100%polymorphic loci except the primer OPD 15 which produced one monomorphic amplicon (500bp). Jaccard’s similaritycoefficient values indicated >80% genomic homology between the T-series mutants of Dhauli (parent variety). T 2-1 hadshown two specific bands of amplicon size 750bp and 920bp produced by primer OPD 18. Similarly, a 900bp band amplifiedby primer OPA-8 was specific to Purusattam local whereas, it was absent in all other local land races. RAPD analysisconfirmed that wild accession of mungbean (Vigna radiata var. Sublobata) is more close to mungbean particularly localland races than urdbean. The UPGMA cluster analysis distinguished each single variety resulting single variety cluster ateven 0.86 similarity index. Further, the RAPD analysis revealed high divergence of UG 218, LGG460 and TCR 20 fromAB 2557, ML 267 and Pusa 172. These genotypes could serve as valuable genetic materials in recombination breeding.

Key words: Genetic diversity, Mungbean, Molecular characterization, RAPD markers.

INTRODUCTIONMungbean is an important short duration legume

crop in Asian countries and India leads the list of mungbeangrowing countries in terms of area(2.98 mill. ha) andproduction (1.29 mill. Tones (Ali and Kumar 2006). It is aninexpensive source of easily digestible protein, essentialamino acids, vitamins and minerals in vegetarian dietparticularly in the developing countries. Protein content ofseeds varies from 17.2 to 29.9% among varieties with anaverage of 22.4%. Dry seeds are rich in Vit A (114mg) andVit B (thiamine- 0.621mg and riboflavin- 0.233mg) whilesprouted seeds synthesize an appreciable amount of VitC(ascorbic acid- 4.8mg). But, mungbean continues to be aslow runner in productivity (332kg/ha in India) owing to usualpractice of minimal cultivation in marginal and sub-marginallands, poor seed replacement rate, vulnerability to biotic(YMV, Cercospora leaf spots, powdery mildew, root rot, fruitfly etc) and abiotic stresses (drought, salinity and waterlogging) and insufficient irrigation facility.

Germplasm including exotic backgrounds is a vitalsource of valuable genes which help to augment cropproduction and improve quality of the produce. Therefore,

genotypic characterization is a prerequisite to study geneticdiversity, and breeders often rely upon diverse parents inhybridization programme to assemble favourable genes in asingle genotype. Besides, practical application of genotypiccharacterization lies with testing of genotype purity, cultivaridentification leading to speed up DUS test, elimination ofduplicates in the germplasm and designing mappingpopulation for genome mapping. Morphologicalcharacterization of germplasm collections is an important stepin this regard. Many often biochemical and molecular markersare sought owing to paucity of specific morphological traitsor descriptors for categorization of genotypes. In this regard,restriction fragment length polymorphism (RFLP), DNAamplification fingerprinting (DAF), amplified fragment lengthpolymorphism (AFLP), Inter simple sequence repeats (ISSR),simple sequence repeats (SSR), sequence tagged sites (STS)and random amplified polymorphic DNA (RAPD) serve asexcellent tools for characterization of germplasm lines.Among these, RAPD has a vast scope for preliminaryassessment of the extent of genetic variability and isolationof divergent genotypes in a set of germplasm lines owing toits technical simplicity, low cost and speed of experimentation

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(Newburry and Lioyd 1993). Therefore, the presentinvestigation was attempted to verify the efficacy of RAPDmarker system for study of genetic diversity in mungbean.

MATERIALS AND METHODSExperimental materials: The experimental materials usedin the present investigation consisted of 20 genetically puregenotypes including 13 promising improved varieties, fourpopular locally adapted mungbean land races of Odisha(India), one wild cultivar of mungbean (TCR 20), onegenotype of its closely related species Vigna mungo, cv. UG218 (urdbean) and a popular mungbean variety LGG 460 asstandard check.. These genotypes were purposefully selectedfrom a set of 314 germplasm lines based on significant higherseed yield than the best standard checks. The details of thesegenotypes representing a core germplasm are given inTable 1. The test genotypes were grown in the field followingrandomized block design with three replications to assessyield and ancillary traits including protein content. The presentinvestigation was carried out at Dept. of Plant Breeding andGenetics, College of Agril., Orissa Univ. of Agril. andTech.(OUAT) during the year 2010-12.

Isolation of genomic DNA: Genomic DNA was isolated fromtwo gm. tender young leaves of aseptically grown seedlingson the same day of collection using standard CTAB (cetyltrimethyl ammonium bromide) method (Doyle and Doyle1990) with minor modification. The contaminating RNA wasremoved by treatment with 3µl RNase (10mg/ml) for eachml of DNA extract followed by incubation in water bath at370C for 1hr and then addition of equal volume of chloroform:isoamyl alcohol(24:1). The content was centrifuged for 10min

at 10,000 rpm and the upper acquous phase was pipette out.Starting from the addition of chloroform- isoamyl alcohol,the entire process was repeated twice. After finalcentrifugation, 1/10th volume of 3M sodium acetate, pH 4.8was mixed to the upper acquous phase separated to a freshcentrifuge tube. DNA was precipitated by adding 2.5 volumeof chilled absolute ethanol and pellated by spinning. The DNApellet was washed twice with 70% ethanol, carefully and driedunder vacuum. The Dried DNA was dissolved in TE buffer(10mM Tris-HCl, pH 8.0 and 1mM EDTA) and stored inrefrigerator for future use.

Test for quality and quantity of the purified DNA: Theamount of DNA was determined through UV-VISspectrophotometer. The absorbance at 260nm wave lengthgave the quantity of the total DNA, and the ratio of theabsorbance at 260nm and 280nm indicated the quality of thepurified DNA. The quantification was done in comparisonwith the known standard. After quantification, the DNA wasdiluted in TE buffer to a working concentration of 10ng/µlfor PCR analysis. The DNA was also loaded in 0.8% agarosegel alongside diluted uncut lambda DNA as standard torecheck the quality and quantity of DNA.

DNA amplification and agarose gel electrophoresis: Tendecamer primers were used for amplification of genomicDNA. The amplification was performed in a reaction volume25µl containing 1X reaction buffer (10mM Tris HCl, pH9.0, 1.5mM MgCl2, 50mM KCl, 0.01% gelatin), 2.5mM eachof dNTPs, 10ng of single random primer, 20ng of genomicDNA and 1 unit of Taq polymerase (Genei, Bangalore). DNAamplification was performed in the Gene-Pro Thermocycler

TABLE 1: List of mungbean and allied test genotypes.

Genotypes Place of origin Seed coat colour Seed sizeAB 2557 PKV, Akola Green NormalML 267 PAU, Ludhiana Green NormalPMB 27 IIPR, Kanpur Green NormalDhauli IARI, New Delhi Green NormalPusa 172 -do- Green NormalRCM 12 OUAT, (Orissa) Green BoldTM 98-2 BARC, Trombey Green NormalT 2-1 OUAT, (Orissa) Green NormalT 7-1 -do- Green NormalT 7-3 -do- Green NormalT 7-7 -do- Brown NormalT 7-10 -do- Shiny black NormalT 12-2 -do- Shiny black NormalHinjli local- B Local land race, Orissa Reddish brown NormalNandika local -do- Dull green NormalPurusattam local -do- Shiny green BoldKhadabhanga local -do- Dull black BoldTCR 20 (Wild Accession) NBPGR, New Delhi Reddish brown NormalUG 218 (Urdbean ) - Dull black boldLGG 460 ANGRAU, AP Green Normal

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(Bioer Tech. Co., Ltd, Japan), programmed for 5min at 940C,40 cycles of 1min at 940C, 1min at 350C and 2min at 720Cand final extension for 4 min at 720C followed by storing at4oC till loading to the agarose gel. The amplified productswere loaded in 1.6% agarose gel containing 1µg/ml ethidiumbromide and electrophoresed in a constant voltage at 50V.The amplifications were checked for their reproducibility.

Visualization, scoring of bands and statistical analysis:The gels were documented by gel doc system (Fire Reader-Uvtec, Cambridge, UK) for scoring the bands. The size ofthe amplicons were determined by comparing with the lambdaDNA ladder (100bp) with known mol. wt. fragments. Gelswere scored for the presence (1) or absence (0) of bands andthe binary data matrix of RAPD score was analysed toestimate Jaccard’s similarity coefficient (Jaccard 1908)values. The dendrogram was constructed using UnweightedPaired Group method with Arithmetic Averages (UPGMA)employing Sequential agglomerative Hierarchic and Non-overlapping Clustering (SAHN).

RESULTS AND DISCUSSIONRAPD has been standardized and employed

successfully by different workers (Tomooka et al. 1995, Kagaet al. 1996, Xu et al. 2000, Samarajeewa et al. 2002) toanalyse samples of Vigna species. The success in generatingwide range of polymorphic loci depends on proper choice ofprimers for DNA amplification. The optimum number ofprimers required to differentiate two or more cultivars mayvary with the test materials used. When the variation in thecultivars is high the use of few primers can serve the purposeof generating useful information. Dhanaraj et al. (2002) usedonly seven primers, while only 2-3 primers were sufficient todistinguish between cultivars of broccoli (Hu and Quaros1991). However, it is in vogue to use more number of primersfor study of more closely related cultivars. For instance,Mackill (1995) could not differentiate a few number ofjaponica rice cultivars even using 21 RAPD primers due tolow germplasm diversity. In the present investigation, initiallyfifteen random primers (Operon technologies Inc., USA) wereexamined out of which two primers did not produce anyamplified products of DNA and three primers gave smearedbackground without distinct fragments. As a result, ten primerswere used for genomic DNA amplification of 20 testgenotypes. The list of such informative primers used in thisinvestigation is presented in Table 2. The number of bandsper primer ranged from 5-12 with an average of 8.1 bands.The PCR amplification with ten RAPD primers resulted total81 amplicons. Fig. 1a showed nine polymorphic bands incase of primer OPA 8. All primers produced 100%polymorphic loci except the primer OPD 15 which produced

one monomorphic (500bp) and eight polymorphic amplicons(Fig. 1b). Thus, the present set of materials revealed extremelyhigher level of polymorphism (98.7%) of RAPD markers.The level of polymorphism obtained in the presentinvestigation may be ascribed to higher genetic variation inthe selected 20 test genotypes. Lakhanpaul et al. (2000) andChattopadhyay et al.(2005) reported moderate level (64-70%) of polymorphism. Saini et al. (2004) observed only39.08% polymorphism in a set of 46 mungbean cultigens,but an another group of workers (Saini et al. 2010) revealedpolymorphism of RAPD products as high as 92.9% using 30primers over 39 mungbean genotypes. The inconsistency ofvarious studies may be attributed to different set of genotypesand primers used. Primer OPD 08 and OPN 08 gavemaximum number of polymorphic bands i.e. 12 and 10 bandswithin the range of fragment size 350-2200 and 300-2300bprespectively (Table 2).

In the present investigation, bands with similarmobility to those detected in the negative control if any, werenot scored. The RAPD profiles (pooled over primers) revealed39 distinct dense electrophoretic bands out of total 81 bandsamong which an erstwhile mentioned amplicon of fragmentsize 500bp was monomorphic and rest of the bands had shownpolymorphism. These 39 bands (including the monomorphicband) were considered for study of genetic relationship amongthe test genotypes. As a whole, the resulting data matrixrevealed a total of 505 distinct bands over the genotypes andprimers out of which 485 bands are polymorphic. Thisindicates that 96.04% of the bands generated werepolymorphic. Such a high level of polymorphism with RAPDmarkers depicts diverse genetic base of the present set ofmaterials.

Similarity matrix generated by Jaccard’s coefficientvalues (Table 3) revealed extent of relationship between thetest genotypes. Higher the inter-distance between thegenotypes, the better is the scope of using them inhybridization to achieve transgressive segregants.

T-series mutants exhibited more than 30 bands andmaximum up to 35 bands being in T12-2. In contrast, theurdbean accession UG 218 and the best standard check LGG460 scored only seven and eight bands respectively. Fourbands were found common between UG218 and LGG 460.Besides, two specific bands (700bp and 900bp) produced byprimer OPA 4 were absent in urdbean as well as LGG 460,but present in all other genotypes including the wild accessionTCR20. It evidences that LGG 460 is more proximal tourdbean. This was also revealed from similarity value as highas 36.4% of UG 218 with LGG 460 than rest of the testgenotypes (Table 3). In addition, other test genotypes had

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TABLE 2: Amplified products with different primers in twenty test genotypes.

RAPDPrimer Sequences(5'-3') No of Polymor- No. of monomorphic Total Range ofphicbands bands bands fragmentsize ( bp)

OPA-02 TGCCGAGCGT 8 - 8 400-1800OPA-04 AATCGGGCTG 7 - 7 500-2000OPA-08 GTGACGTAGG 9 - 9 200-1750OPA-12 TCGGCGATAG 9 - 9 230-2000OPD-08 GTGTGCCCCA 12 - 12 350-2200OPD-15 CATCCGTGCT 8 1 9 350-2000OPD-18 GAGAGCCAAC 6 - 6 200-1950OPN-08 ACCTCAGCTC 10 - 10 300-2300OPN-12 CACAGACACC 6 - 6 245-2000OPN-16 AAGCGACCTG 5 - 5 180-1900

very low similarity coefficient value with these two genotypes.Hence, UG 218 and LGG460 could be considered highlydivergent from rest of the genotypes. Among the testgenotypes, the urdbean accession UG 218 was most divergentfrom T2-1, T7-7 and T-7-10; while LGG460 exhibited veryhigh genetic dissimilarity with Khadabhanga local andPurusattam local as revealed from their inter se similaritycoefficient values (0.142- 0.161).

Fourteen bands were common in TCR20 and localland races, whereas three other bands were commonly absentin all such genotypes which suggested comparative proximityof TCR 20 with the local varieties than the improved testgenotypes. Besides, out of 24 bands scored in TCR 20, fivebands are common with urdbean and rest of the bands areshared by mungbean genotypes.. Thus, the wild accession isconsidered to be more genetically close to mungbean(particularly land races) than urdbean. This was also viewedsimilarly by Samarajeewa et al. (2002) while studying wildand cultivated Vigna species. Intra-specific diversity usingDNA markers has been also studied in many legume cropse.g., mungbean (Bhat et al.2005), urdbean (Souframanienand Gopalakrishna 2004), rice bean (Muthusamy et al.2008),common bean(Galvan et al.2003), cowpea(Fang et al.2007),soybean (Caetano-Anolles and Gresshof 1994), andChickpea (Winter et al. 2000, Benko-Iseppon et al. 2003,Rakshit et al. 2003).

Among the T-series mutants of Dhauli, 20 bandswere found monomorphic which evidences greaterhomology among such genotypes. This resulted highsimilarity value between the T-series mutants and it wasmaximum as high as >80% in case of T7-7 with T7-3,T7-10 and T12-2 and in case of T2-1 with T7-3 and T7-10.However, the mutant T 12-2 was distinguished from rest ofthe T-series mutants and its parent Dhauli by presence oftwo unique bands of fragment size 1020bp and 1250bpproduced by primer OPD 8. Similarly, T 2-1 had showntwo specific bands of amplicon size 750bp and 920bp

produced by primer OPD 18. This indicates that mutationsin some mungbean genotypes have affected the genome toa large extent.

Besides, all local varieties had seventeen commonbands which indicates similar lineage in ancestral hierarchy.Hinjli local had shown absence of six bands which werepresent in all other local land races. Similarly, Khadabhangalocal as well as Hinjli local each exhibited absence of a fewunique bands which were present in all other local land races.Similarity coefficient values between local land races rangedfrom 0.588 (Hinjli local vs Purusattam local) to as high as0.839 (Khadabhanga local vs Purusattam local). A 900bpband amplified by primer OPA-8 was specific to Purusattamlocal whereas, it was absent in all other local landraces(Fig.1a). The above information will be of immensevalue for varietal identification and elimination of duplicates.RAPD based DNA finger printing has been also reported todifferentiate genotypes with a great success in many cerealcrops including rice (Ko et al. 1994), barley (Fernandezet al. 2002) and pearl millet (Chowdhary et al. 1998). In

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TM 98-2, T 2-1, T 7-1, T 7-3, T 7-7, T 7-10, T 12-2, Hinjli local-B, Nandika local, Purusattam local, Khadabhanga local, TCR

20(WA), UG 218(urdbean), LGG 460(Check).

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Fig 2: Dendrogram showing genetic diversity of genotypesbased on DNA fingerprinting using RAPD markers.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 203000

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Fig 1b: RAPD profile of different varieties of mungbeanamplified with OPD-15 primer. M=DNA molecular marker, Lane1-20: AB 2557, ML 267, PMB 27, Pusa 105, Pusa 172, RCM 12,TM 98-2, T 2-1, T 7-1, T 7-3, T 7-7, T 7-10, T 12-2, Hinjli local-

B, Nandika local, Purusattam local, Khadabhanga local, TCR20(WA), UG 218(urdbean), LGG 460(Check).

addition, RAPD is suggested to be a potent technique foridentification of hybrids and use in marker assisted selection.

Clustering based on RAPD profile: The whole range often primer based RAPD profile of twenty selected testgenotypes revealed five clusters at even 56% phenon level(Fig. 2). RAPD profile brought about initial discriminationof UG 218 (urdbean) and LGG 460 forming single varietyCluster I and Cluster II respectively at even 37% phenonlevel followed by TCR 20 (Cluster III at 56% phenon level).Thus, RAPD analysis confirmed that wild accession ofmungbean (Vigna radiata var. Sublobata) is more close tomungbean than urdbean. Samarajeewa et al. (2002) carriedout cluster analysis based on RAPD markers and alsoestablished that Vigna radiata var. Sublobata is mostclosely related to mungbean. Cluster-IV was the largestcluster which included twelve genotypes. At bit higher phenonlevel, these genotypes were separated into two distinct sub-clusters (IVA and IVB). Six T-series mutants and all localland races except Hinjli local showed a multi-varietyclustering pattern and all these were grouped in sub-cluster-IVA. In the present investigation at DNA level, Hinjli localwas observed to be more closer to TM 98-2 and RCM 12than other local land races. These three test genotypes formedSub-cluster IVB. At still higher phenon level, the sub-cluster-IVA was again sub-divided into Cluster-IVA1 which includedtwo local land races (Purusattam local and Khadabhangalocal); one single variety Cluster IVA2 containing Nandikalocal; and a multivariety Cluster IVA3 which contained all T-series mutants of mungbean. Rest five improved varietieswere clubbed together in Cluster V. Subsequently at 60%phenon level, Cluster V was divided into two sub-clusterscontaining “Dhauli” and PMB 27 in Sub-cluster VA ; andPusa 172, ML 267 and AB2557 in Sub-cluster-VB. ML 267and Pusa 172 were found to have some degree of homology

with AB 2557. Dhauli and PMB 27, had also maintained closerelationship at DNA level. In the present investigation,RAPD profile reflected very high divergence of UG 218(urdbean) and LGG 460 from AB 2557, ML 267 and Pusa172, while T-series mutants derived through chemicalmutagenesis and local land races revealed less divergencefrom the above test genotypes. Kaga et al.(1996) studiedgenetic variation among 23 accessions of five Vigna spp. ofsub-genus Ceratotropis using 24 RAPD primers. Theyrevealed largest variation in V. radiata in which the wild formsshowed more differences than the cultivated forms.Lakhanpaul et al. (2000) observed moderate to low level ofpolymorphism among some selected varieties of mungbeanusing RAPD markers. Afzal et al. (2004) used 34 RAPDmarkers to study genetic diversity in mungbean, but foundnarrow genetic base with moderate to high similarity co-efficients. Low genetic diversity was also detected by Bhatet al. (2005) in cowpea using RAPD marker system. But,India being the proposed centre of diversity (de Condolle1886, Vavilov 1926, and Zukovskii 1962), it shows thatmungbean does not lack variability in the germplasm materialavailable in India.

Cluster composition and comparative meanperformance of clusters and test genotypes: An effortwas made to note agronomic performance of the testgenotypes in relation to their distr ibution in thedendrogram clusters using RAPD markers. The order ofoccurrence of the genotypes in the above RAPD basedclusters was used as a reference for arranging performancevalues of the accessions for 14 agro-morphological traits

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that were observed in the field over two consecutiveseasons laid out in RBD. The average values of these traitsalong with protein content are given in Table 4. The tablevalues arranged cluster -wise allow a simultaneouscomparison of several quantitative traits in the testgenotypes with reference to the position of each genotypein the dendrogram. For better clarity, the significantlylower performance values for days to maturity, days to50% flowering and plant height; and for rest of themorpho-economic traits ( including yield) havingsignificantly higher mean values compared to theexperimental grand mean are marked astrick (*). It isobserved that the genotypes in the same cluster based onDNA profiling have some common desirable phenotypicperformance and such clusters with unique phenotypicperformance could be identified for genetic improvement.

For genetic improvement, dwarf plant types withearly maturity and early flowering habit are of specialconsideration. All T-series mutants (Cluster IVA3)exhibited significantly lower values for maturity durationthan overall grand mean. While genotypes under cluster -V flowered significantly earlier than rest of the testgenotypes. Genotypes under Cluster -II and Cluster-IVBincluded dwarf plant types. RAPD based clusteringreflected differentiation of four local land races, wildaccession as well as urdbean genotype into separateclusters. These genotypes have revealed significantlyabove average values for number of branches per plant.All T-series mutants except T 7-1 exhibited higher numberof pods/ cluster (NP/C). Cluster I (urdbean), ClusterIII(Wild accession) and most of the T-series mutantsincluded in Cluster IVA3 had shown to have significantlyhigher number of pods per plant. Three single varietyclusters e.g., Cluster II(LGG 460), Cluster III and ClusterIVA2 (Nandika local) were unique in significantly higherpod length as well as number of seeds per pod. Among allclusters, the cluster -IVA3 (T –series mutants) exhibitedsignificantly higher harvest index which has direct bearingon productivity. Cluster mean for protein content wasabove average in cluster-I, Cluster-II, Cluster-III andCluster-IVA1 which included UG 218 (urdbean), LGG460, wild accession (TCR 20), khadabhanga local,Purusattam local and Nandika local. Cluster-I, Cluster-IIIand Cluster IVA3 had shown significantly above averageproductivity. Phylogenetic tree based on RAPD markerswhich correlated with morphological characters wasdeveloped by Betal et al. (2004). Lavanya et al (2008)reported typical agronomic performance of genotypes

included in each of the clusters based on RAPD inmungbean germplasm. Such a distribution of clusters basedon agronomic performance and supported by RAPD profileanalyses are a very good starting point for further breedingefforts involving contrasting parental line, and will furtherenable tagging of genes and identification of QTLs forthese traits with molecular markers. Ghafoor et al. (2005)identified few QTLs for days to flowering, days to maturity,branches per plant, seeds per pod, 100-seed weight,biological yield and seed yield based on significantcorrelation of some polypeptide bands with the quantitativetraits in urdbean. Ghallab et al. (2007) correlatedsuperiority of two mungbean genotypes (L 2520 and L1720) in seed yield with absence of two bands at around94.6kd and a presence of a polypeptide band withmolecular weight 12.1kd under drought stress condition.However, such an interpretation needs critical examinationparticularly in the context of DNA markers.

From above discussion, it is understood that eachof the clusters based on molecular fingerprint (RAPD) hassome characteristic features for performance of morpho-economic traits. This could be due to the fact that some ofthe genotypes may have been descended from commonancestry and may have been subjected to similar selectionfor agro-economic traits during breeding programme.Besides, the test genotypes were distinguishable from eachother by their RAPD profiles. Cluster-IVA3 contained allT-series mutants of mungbean and these genotypesmaintained genomic homology and clustered together tillabout 71% phenon level; and beyond that these wereseparated gradually and fully discriminated from eachother at a bit less than 86% phenon level. This wasexpected as all the T-series mutants were derived followingmutagenesis of the same parent Dhauli. Similarly, all thelocal varieties except Hinjli local grouped together andoccupied a specific position in the dendrogram. These localland races might have same ancestry and possiblyaccumulated minor mutational changes during the courseof evolution leading to identification of separate geneticentities or genotypes.

Thus, the RAPD emerge as a potent technique togenerate wide array of polymorphism and as such, it couldserve valuable information for varietal identification andextent of genetic diversity. Further, the perspective of such amolecular approach could be more successful in conjunctionwith morpho-economic traits for genotype sorting and tosupport mungbean breeding programme.

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Volume 38 Issue 3, 2015 287

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