Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 778-787

ISSN: 2319-7706 Volume 5 Number 1(2016) pp. 778-787 Journal homepage: http://www.ijcmas.com

Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.501.079 Genetic Diversity and Relationships among Varieties using RAPD Molecular Markers

R. Pruthvish1 and B.K.Chikkaswamy2*

1Department of Biotechnology, Acharya Institute of Technology, Soladevanahalli Hesaraghatta Road, Bangalore, India 2Sigma BioScience Research Center, Indira Nagar Bangalore-560038, India

*Corresponding author

A B S T R A C T

K e y w o r d s Mango is the genus of tree & bushy tree in the family Mangnoliaceae. Most of the Genetic Mango varieties are dioceous and cross-pollinate to produce to produce fertile Diversity, hybrids suggesting a closer genetic relationship which is not expected at species Mango level. The traditional methods using morphological characters are not successful in Varieties, establishing the diversity and relationship among 19 different Mango varieties RAPD, because of the environmental influence.PCR based molecular marker method, Molecular RAPD was employed to study the genetic diversity and Inter-relationship among Markers 19 Mango varieties. On an average RAPD analysis generated 5-6 discrete bands/ varieties with 10 nucleotides primers. The size of the amplified products ranged Article Info from 100-3500 base pairs in length. With an average of 5-10 bands per primer. Of Accepted: 21 amplified fragments 50 were polymorphic (80%) with at least one pair wise 25 December 2015 comparison between 19 varieties. RAPD analysis identified varieties specific Available Online: amplification products which will be useful in germplasm classification and 10 January 2016 introgression studies. These results indicated that RAPD based markers are useful for genetic characterization of Mango /varieties/accessions.

Introduction

Genetic fingerprinting has been expense, and use of (32 P) for labeling accomplished traditionally through the use with RELPs and VNTRs. Polymerase Chain of isozymes, and more recently through Reaction (RAPD) uses arbitrary 10-base reactions fragment length polymorphisms primers to amplify randow portions of the (RELPs), variable number tandem repeats genome (Welsh and McCIelland 1990; (VNTRs), or a combination of these. While Williams et al. 1990).The fragments these methods have been very useful in produced are easily visualized on an cultivar identification, they have a number ethidium bromide stained gel, and of disadvantages, including a limited polymorphisms between genotypes reflect number of isozyme loci, and the time, heritable differences in the genome. Large

778 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 778-787 numbers of bands, or loci, can be generated Until recently, several promising species with relative ease. Due to the arbitrary released formally are characterized on the nature of the primers, RAPD markers, unlike basis of morphological data, mango content RELPs, VNTRs, and isozymes, represent a and yield potential. Worldwide demands on random sample from the entire genome. mango necessitate work on conservation of They are, however, inherited as dominant mango germplasm and their further genetic markers, requiring larger numbers of loci to improvement. Therefore precise be identified and screened to glean the same characterization of promising species and information as from RELPLs, VNTRs, and determination of genetic variation among isozymes. Amplification of bands in the those are felt necessary. progeny which were not amplified in either parent has also been reported (Riedy et al. Until recently, several promising species 1992). released formally are characterized on the basis of morphological data, mango content The use of RAPDs to determine genetic and yield potential. These characters differ relationships has been demonstrated in under varying environmental condition maize (Welsh et al. 1991), conifers (Carlson thereby posing problem identification of et al. 1991), caca (Wide et al. 1992), and species. Unlike morphological markers, various leguminous pecies (Charlmers et al. cytological (chromosome numbers, nuclear 1992, Echt et, al.1992). Within DNA content) and molecular markers (mango) species RAPDs have been used to (RAPD, AFLP, ISSR etc.) are not prone to determine phylogenetic relationships environmental influences and characterize (Schnell and Knight 1993). On the basis of the plants portraying the extent of genetic the classification of Kostermans and Bom. diversity among taxa (Bennett 1987,Bennett and Smith 1991,Waugh and Powell RAPD fingerprinting techniques have been 1992,Chalmers et al.1994, Das et al.1998, used for the identification of horticultural Rodriguez et al.1999, Das et al.2001). crop varieties, description of cultivars genotypes and for protecting breeder s rights Of the different markers, RAPD has been (Williams et al., 1990) enabling assay to be widely used in the last decade in species performed at any stage of plant identification programmed (Schnell et development. RAPD markers have been al.1995) and in assessing genetic variations extensively used to distinguish intraspecific among different taxa at DNA level because genetic variation in ornamental crops and of its cost effectiveness and simple operation detection of hybrids and clones (Collins et without requiring prior knowledge of al., 2003; Arus, 2000; Debener, 2001a). species DNA sequences (Williams et al.1990, Frankel et al.1995). RAPDs reveal Ranamukhaarachchi et al. (2001) showed similar patterns of genetic diversity when that RAPD markers had the ability to compared with other marker types and can identify pot-plant mango cultivars is crucial be performed more rapidly than most other to local breeders involved in hybridization methods (Morell et al.1995) and can provide programs for varietal development but is vital information for the development of currently lacking. In this study, RAPD genetic sampling, conservation and markers were used to evaluate the extent of improvement strategies (Waugh and Powell genetic variation among some mango cut 1992, Chalmers et al.1994). No report has flower cultivars. been published so far either on the genetic

779 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 778-787 characterization or on the extent of genetic out on RAPD molecular marker studies in variations existing among promising species varieties of mango plants with the following of mango. objectives. To develop protocol for isolation of DNA from different 19 varieties of The present study deals with the in situ mango plants. To develop PCR protocol and DNA estimation and RAPD analysis of 19 to identify RAPD markers to score in promising species of Mango to identify and various mango plants.To assess the genetic evaluate extent of genetic variation existing diversity and relationships among mango among these. plants using RAPD molecular markers belonging to family magnoliace. As the efficiency of selection scheme or genetic analysis based on phenotype is a Matias Kirst et.al.,(2004) studied on DNA function of heritability of the trait, factors marker RAPD,RFLPs and AFLP etc in like environment, traits of mutagenic and forest tree species and in particular its quantitative inheritance or partial and application to tree breeding and tree complete dominance often confound the genomics. expression of genetic traits. Many of these complications of a phenotype-based assay Million and Chinnappa (2000) assessed the can be overcome through direct genetic divergence in forty seven genotypes identification of species with DNA based of diagnostic assay. For this reason DNA based genetic markers are being integrated into Stellaria longipes (Caryophyllaceae) using several plant systems under expected to play RAPD analysis. Datta and Mitrick an important role in the future plant et.al.,(1997) studied the classification of improvement programmes. common bean Phaseolus vulgaris from Chile using RAPD data.The study reveals Advent of Polymerase chain reaction (PCR) that 95 bean accessions analysed using 25 technology as lead to the development of primers that generated 106 polymorphic several novel genetic assays based on bands of RAPD collected from different selective DNA amplification Krawetz locations. (1989) and Innis et al (1990). RAPD assay detects nucleotides sequence of Nirupama et.al., (2003). Reported genetic polymorphism in DNA using only a single diversity and relationship of 51 accessions primer of arbitrary nucleotide sequence. The of Vetiver using RAPD/AFLP analysis.Total protocol is also relatively quick and easy to of 20 primers were used to generate 5 perform and uses fluorescence instead of clusters using UPGMA cluster analysis radioactivity. Because the RAPD technique where as for AFLP analysis nine primers are is amplification- based assay, only used to generate 383 unique bands specific nanogram quantity of DNA is required. One to different accessions compared 81 of the strengths of these new assays is that monomorphic bands.In contrast sufficient they are more amenable to automation than diversity existing with in the wild and conventional techniques. It is simple to cultivated.Indian germplasm. Raghvendra perform and is preferable to experiment; saxena and Amaresh Sanghamitra Nayak their species of large number of individuals et.al. (2006) studied the 4C nuclear DNA are to be determined at a few genetic content and RAPD analysis of 17 promising loci.The present investigation will be carried cultivars of turmeric Curcuma longa of

780 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 778-787 zingiberaceae is an important spice and isolation of DNA from mature strawberry anticancer properties and also showing leaf tissue that produces consistent results differential genetic variation among for a variety of different species, both cultivars. The polymorphism ranged from octoploid and diploid, and is both stable and 35.6% to 98.6%. PCR amplifiable before and after extended storage. Schnell et.al.,(1995) studied the identification of cultivars and genetic Quantification of DNA relationships in using RAPD technique. 25 accessions of mango DNA quantification can be done by were examined using 80 primers.Of the 80 flurometry, spectrometry and agarose gel primers 33 did not get amplified 19 were electrophoresis with standard DNA monomorphic and 28 gave concentrations (Boiteux et al., 1999). The quality can be assessed by restriction Materials and Methods digestion with restriction endonucleases (EcoRI, HindIII etc.,) electroporation and The present study selected leaf samples of spectral properties. Quality is that to what the 19 mango plants plants that were extent the DNA is pure of secondary collected from the conservatory of metabolites and other substances, which Biotechnology Centre, Hulimavu, hinder further use of DNA in molecular Department of Horticulture, Bangalore, techniques. A good DNA preparation Karnataka, India. Which may represent the generally exhibits the following spectral wide variation prevalent in the genome The properties. It will have A230, A230/A260, recently matured leaves were collected and A280/A260 or A260/A280 rations of less than used for DNA extraction. 0.10, less than 0.45, less than 1.65 or more than 1.80, respectively (Shantha et al., Porbeski et al. (1997) described a relatively 1998). If a DNA preparation exhibits quick, inexpensive and consistent protocol A260/A280 more than 1.80, it shows the for extraction of DNA from expanded leaf presence of RNA and if it is less than 1.65 material containing large quantities of or less indicates protein contamination polyphenols, tannins and polysaccharides. (Sambrook et al., 1989). Mature strawberry leaves, which contain high levels of the secondary components, Results and Discussion were used as a study group. The method involved a modified CTAB extraction, The data obtained in the present study employing high salt concentrations to regarding RAPD Molecular Marker studies remove polysaccharides, the use of in 19 Mango varieties The present study polyvinyl pyrrolidone (PVP) to remove reveals that RAPDmarkers are good choice phenols, an extended RNAse treatment and for assessing the genetic diversityand phenol-chloroform extraction. Average relationship in Mango varieties with yields ranged from 20-84µg/g, mature leaf polymorphism levels enough to establish tissue for both wild and cultivated octoploid informative fingerprints with a few markers. and diploid Mango. Results from 19 plants The information obtained could be of were examined and were consistently practical use for mapping the mulberry amplifiable in the RAPD reaction with as genome as well as for classical breeding. little as 0.5ng DNA per 25µ reaction. The study also provides a closer basis for Presently this is the first procedure for the 781 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 778-787 mango breeders to make informative choice available abundantly with in 19 Mango on selection of parental materials based on species even though a few species of Mango genetic diversity and overcome the problem occur naturally in India and Karnataka many usually associated with mango crop cultivated species of Mango varieties do not improvement. The important primers find their origin outside the country of some identified will be useful for molecular of the cultivated Mango accessions which characterization of gene bank accessions. are classified as different species, cross pollinate with each other and produced 19 collections of Mango varieties fertile offspring showing no signs of sexual (, Bangalora, Mulgoa, Neelum, incompatibility characteristics of species. Pairi, Banganapalli, , Bombay This fact suggests a close genetic Green, Chausa, Dashehari, , relationship among the cultivated Mango Fernandian, , Kesar, Kishen Bhog, species. The present investigation involving , , Mankurad, Totapuri) 19 Mango varieties and its RAPD analysis which are distributed in different regions further supports this view. Bangalore, are from diverse origin and possibly represent the genetic diversity The high level of polymorphism (80%) in existing in the species table 1 OPD 1-12. Mango varieties reflects out crossing nature on par with results obtained with RAPD in The RAPD analysis: The random primers other fruits and nut tree species such as amplified with genomic DNA of different Pistachio (Hormaza 1995),olive (Fabbri Mango varieties generated 55 RAPD bands et.al.,1995),walnut (Nieise et.al.,1998) and in the size ranged from 1000 base pairs in vegetatively propagated crop such as banana length. The number of bands obtained per (Krammer et.al.,1992) and apple (Koller primer ranged from 5-6 with an average of et.al.,1993).Similar results was reported by 11 with exception in some of the lanes Schnell et.al., (1995) Karinaloo et.al., (2003) where no amplification and bands formation & Maria cristina et.al., (2006).The has not taken place was observed. A total of molecular analysis using RAPD markers did 80% bands was polymorphic. The complete not show corelation between the ploidy and amplification details are presented in the number of polymorphic bands suggesting table 2. DNA profiles generated by OPD 1- the redundancy of genome complements in 12 are shown in fig 1 these species. The ploidy level of a plant does not appear to influence the number of The genetic analysis of RAPD markers fragments per primer (Wolf and Peter based on Ward s method of Euclidean Vanrijn 1993).In Mango varieties distance showed genetic similarity among percentage of similarity based on F value the collection of Mango varieties Fig 2.The ranged 80 to 30 most of the Mango pairs had collection of Alphonso & Bangalora was shown similarity value some of the closest with the maximum similarity (95%) morphological variation and improvement in whereas Mulgoa and Neelum forms separate agronomic traits, leaf yield have been cluster than other varieties. Euclidean achieved through selective cultivation and Ward s method clustering based on RAPD breeding programmes, there appears to be data showed clear separation of collections effort has been made to utilise the useful fig 2. Some of the Mango varieties are trait found in Mango varieties.

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Fig.1 Gel Diagram of Mango Using Opd 1-12 for 19 Mango Varieties

Tree Diagram for 19 Variables Single Linkage Euclidean distances

VAR1 VAR2 VAR9 NEWVAR11 NEWVAR12 VAR7 VAR10 NEWVAR18 NEWVAR15 NEWVAR16 VAR6 NEWVAR19 VAR5 VAR4 VAR3 VAR8 NEWVAR13 NEWVAR14 NEWVAR17

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Linkage Distance

The present study has made an attempt to improvement and conservation programme assess the genetic diversity among 10 in mulberry (Sharma et al., 2000). collection from different region of Bangalore. Morphological traits are greatly The present results also showed number of influenced by the environment. It has always RAPD bands such as 2,2,0,20,4,4,2,2,46 been difficult to accurately assess the respectively in Mango varieties, Mango 1 diversity and interrelationship among and Mango 2 further Ward s method of Mango varieties similar report was reported genetic distance bu Squared Eucleidean by (Awasthi et al., 2004) on mulberry plant. distances dendrogram revealed maximum DNA marker such as RAPD and ISSR have similarity between Alphonso & Bangalora become a handy tool for quick and reliable Whereas Mulgoa and Neelum forms estimation of diversity for crop separate cluster than other varieties. 783 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 778-787

The mean genetic similarity was less in the Theor.Appl.Genet., 94 61-67. case of Mango officinalis and Mango Akagi, H.; Yokozeki, Y.; Inagaki, A.; malabaricum indicating a diverse genetic Nakumura, A.and Fujimura, T. (1996) background of the former. This conclusion A codominant DNA marker closely is also supported by the large variabilty linked to the rice nuclear restorer gene, exhibited in phenotypic traits reported by RF-1, identified with inter SSR Ravindran et al., 1997 in mulberry plants. fingerprinting.Genome, 39:1205-1209. The RAPD data separated the varieties in to Akito Kaga,Norihiko Tomooka,Yoshinobu three distinct group confirming their Egawa, Kazuyoshi Hosaka & Osamu taxonomic status unambiguously which are Kamijima.Species relationships in the geographically isolated and thriving in subgenus Ceratotropis (genus Vigna) as comparatively different habit. Due to high revealed by RAPD analysis. Euphytica heterozygosity of the species it is not 88:17-24.1996. possible to conserve the seeds unresisting Akkaya, M.S., Shoemaker, R.C., Specht, condition and is maintained in the vegetative J.E., Bhagwat, A.A., and Cregan, P.B. form in the field gene bank. Most of the (1995).Integration of simple sequence primers used for DNA profiling of the repeat DNA markers into a soybean Jamine collections generated linkage map. Crop Sci.35:1439-1445. polymorphisms. Alstrom-Rapaport, C., Lascoux, M.,-Wang, Y.C., Roberts, G.and Tuskan, G.A, The present study gives an instinct into the (1998), Identification of RAPD marker broad genetic structure of this varieties. The linked to sex determination in basket information generated from the study will be willow (salix viminalis L.). J. Heredity, useful for further in depth analysis of these 89 (1):44-49. varieties towards utilization in Mango Antoni rafalski, scott tingey and john improvement and conservation programmed. g.k.williams.1994.Random amplified polymorphic DNA (RAPD) References markers.Plant Molecular Biology Manual.H4:1-8. Ajibade S.R., Weeden.N.F & Chite S.M., Armour. R.P. (1959), (Investigations on Inter simple sequence repeat analysis of Simarouba glauca DC in E1 Salvador.) genetic relationship in the genus Eco. Bot., 13:41-66 Vigna.Euphytica 111:47-55. (2000). Arumuganathan, and Earle, D, (1991). Ajit K.Shasany, Alka Srivastava, Janak Nuclear DNA content of some R.Genetic diversity assessment of important plant species Pant.Mol.Bio Mentha spicata L.germplasm through Rep., 9(3):208-218. RAPD analysis fifteen elite accessions Arus P,Shields C.R,Orton J J (1985) in the national gene bank of Mentha Application of isozyme electrophoresis spicta L.IPGRI Plant Genetic Resources for purity testing and cultivar Newsletter Portal.130, 1-5, (2002). identification of F1 hybrids in Brassica Akagi, H.; Yokozeki, Y.; INagaki, A and oleracea. Euphytica 34:651-657. Fugimura, T. (1997) highly Atal.C.K., (1959), (Sex reversal in hemp by polymorphic microsatellites of rice application of gibberelin.) consist of AT repeats and a Curr.Sci.28:408-409 classification of closely related cultivars Ausubel.M.F, Brent.R.Kinton.E.R. Moore. with these microsatellite loci. D. D. Smith.A.S.and Struhl.K, (1989),

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How to cite this article:

Pruthvish, R., and Chikkaswamy, B.K.2016. Genetic Diversity and Relationships among Mango Varieties using RAPD Molecular Markers. Int.J.Curr.Microbiol.App.Sci. 5(1): 778-787 http://dx.doi.org/10.20546/ijcmas.2016.501.079

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