Mango (Mangifera Indica L.): Morphological and Genetical Diversity in India

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Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 Available online at www.ijpab.com

DOI: http://dx.doi.org/10.18782/2320-7051.7425 ISSN: 2320 – 7051 Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) Research Article

Mango (Mangifera indica L.): Morphological and Genetical Diversity in India

Abhinav Kumar Singh1*, Neeraj Kumar Charmkar1 and Rajesh Singh2 1Centre for Biotechnology Studies, A.P.S. University, Rewa (M.P.) 2KVK, College of Agriculture, Rewa (M.P.) *Corresponding Author E-mail: [email protected] Received: 3.03.2019 | Revised: 7.04.2019 | Accepted: 20.04.2019

ABSTRACT Mango (Mangifera indica L.) is known as the 'ruler of natural products' for its rich taste, season, shading, creation volume and various end use. It has a place with plant family Anacardiaceae and has a little genome size of 439 Mb (2n = 40). Old writing shows inception of developed mango in India. Albeit wild types of sort Mangifera are circulated all through South and South- East Asia, recuperation of Paleocene mango leaf fossils close Damalgiri, West Garo Hills, Meghalaya point to the beginning of class in peninsular India before joining of the Indian and

Asian mainland plates. India delivers in excess of 50% of the world's mango and develops in

excess of thousand assortments. Notwithstanding its enormous financial criticalness genomic

assets for mango are restricted and hereditary qualities of helpful plant characteristics are

ineffectively comprehended. Here we present a short record of our ongoing endeavors to create

genomic assets for mango and its utilization in the examination of hereditary assorted variety

and populace structure of mango cultivars.

Sequencing of leaf RNA from mango cultivars 'Neelam', 'Dashehari' and their mixture 'Amrapali'

uncovered generously more elevated amount of heterozygosity in 'Amrapali' over its folks and created genic basic grouping rehash (SSR) and single nucleotide polymorphism (SNP) markers. Sequencing of twofold processed limitation site-related genomic DNA (ddRAD) of 84 assorted mango cultivars recognized 1.67 million top notch SNPs and two noteworthy sub-populaces. We have collected 323 Mb of the exceedingly heterozygous 'Amrapali' genome utilizing long succession peruses of PacBio single atom ongoing (SMRT) sequencing science and anticipated 43,247 protein coding qualities. Mangifera indica L. is the most prevalent and broadly developed natural product crop in the tropics. As indicated by verifiable information, mango trees are personally associated with Indian culture and old stories since 4000 years prior.

Key words: Mango (Mangifera indica L.), SSR, RAPD, SNP markers, Genetic diversity.

INTRODUCTION among the best fruits of the world. It is widely Mango (Mangifera indica L.) is the choicest grown in the tropical and subtropical regions fruit of India and occupies a prominent place world over.

Cite this article: Singh, A.K., Charmkar, N.K., Singh, R., Mango (Mangifera indica L.): Morphological

and Genetical Diversity in India, Int. J. Pure App. Biosci. 7(2): 382-395 (2019). doi: http://dx.doi.org/10.18782/2320-7051.7425

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 India has rich mango varietal wealth and the fingerprints to identify individuals with country has the richest wealth of mango absolute certainty. Fingerprinting a vast germplasm in Southeast Asia. Reportedly, number of mango cultivars is a significant there are over 1,000 varieties of mango found contribution to mango cultivation, as presently in India. However, there is a lot of confusion several mango cultivars have many synonyms in nomenclature of the mango cultivars noted in different regions, which make identification that lack of systematic approach in naming of difficult and create confusion. Simple mango cultivars in the past has resulted in a Sequence Repeat (SSR) markers, also known great confusion in their nomenclature due to as microsatellites, are tandemly repeated many synonyms and duplication of names in motifs of 1-6 nucleotides found in all the absence of any rules governing prokaryotic and eukaryotic genomes nomenclature27,33. Microsatellites, with a polymorphism based on Indian mango breeders have realized different numbers of repeated motifs at a given this recent trend in mango breeding to catch locus, are becoming markers of choice in the emerging export market as well as to meet many fruit breeding programmes since they the demands of domestic consumers. A are multi-allelic and amenable to automation. notable feature of Indian mango indusry is the In addition to their usefulness in mapping and negligible presence of Indian exporters in breeding, have become the markers of choice world mango market. Subrahmanyam noted for fingerprinting purposes in most plant that although the international trade in mango species due to their high polymorphism, co- was increasing rapidly, India continued to lag dominancy and reproducibility14. behind in total mango exports as compared to History of Mango – ‘King of Fruits’: other exporting countries like Mexico, The history of Mango began thousands of Philippines and Venezuela. Though there is a years ago on the Indian sub-continent. The vast potential for export to western countries, Mango is the national fruit of India, Pakistan constraints like suitability of a few varieties and the Philippines. It is also the national tree for export, pests and disease problems have of Bangladesh. Not only is it one of the most restricted the expansion of the exports from highly prized fruits of South Asia, it is also India. Removal of some of the constraints will intimately connected with folklore and legends increase the potential for exports to USA and across many religions. There is consensus Japan. Research efforts are also needed for among the historians and horticulturists that prolonging the shelf life of fruit so that it could the cultivated mango has originated in India. be made available for longer period in the Vavilov has suggested Indio – Burma region international market. The existing scenario as the centre of origin of Mango based on the reminds us the importance of frontier sciences observed level of genetic diversity. like marker assisted breeding and their Mukherjee28 considered origin of genus integration with conventional fruit breeding Mangifera probably in the South – East Asia for obtaining desirable results with more but the origin of cultivated Mango in the precision. Assam – Burma region. Scientists of the Birbal The term DNA fingerprinting in its Sahni Institute of Palaeibitany, Lucknow, have original sense refers to the method developed traced the origin of genus Mangifera from 60 in 1985 by Sir Alec Jeffreys and his associates million years old fossil compressions of for the detection of highly variable DNA carbonized mango leaves in the Palaeocene fragments by hybridization of specific sediments near Damalgiri, west Garo Hills, multilocus probes to electrophoretically Meghalaya and named it Eomangiferop- separated restriction fragments. The DNA hyllum damalgiriensis. fingerprints, resembling barcodes, are unique History of Mangoes in India: to the individual and hence can be utilized in Mango is native to India and is one of the most much the same way as conventional important fruit crops world-wide. Its botanical Copyright © March-April, 2019; IJPAB 383

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 name is Mangifera indica L. and is the most Indonesia, Fiji Islands, Egypt, Israel, South important species of the genus Mangifera, Africa, Sudan, Somalia, Kenya, Uganda, which produces the most delicious fruit called Tanzania, Niger, Nigeria, Zaire, Madagascar, the mango. The genus Mangifera contains Mauritius, Venezuela, Mexico, West Indies about 49 species, of which 8 are of doubtful Islands, Cambodia, etc47. status and 41 are valid species. Global distribution of Mango: Morphologically the genus could be separated Distribution of Mango Presently, besides under two sections based on the character of India, it is being cultivated in Pakistan, the flower disc: the first, with 34 species, has Bangladesh, Burma, Sri Lanka, Thailand, flowers with well developed swollen disc, and Vietnam, Malaysia, the Philippines, Indonesia, the second, with 7 species, has obsolete or the Fiji Islands, Tropical Australia, Egypt, pedicellate disc. The cultivation of mango in Israel, Sudan, Somalia, Kenya, Uganda, India is as old as 4,000 to 6000 years. Hsüan- Tanzania, South Africa, Nigeria, Zaire, tsang appears to be the first person to bring the Madagascar, Mauritius, the USA (Florida, mango to the notice of people outside India. Hawaii, Puerto and Rico), Venezuela, Mexico, Geographic spread of mango was essentially Brazil, Australia, West Indies Islands and completed in the last half of the 19th Century Cambodia.98 The mango, which has been with its introduction to such far flung places as under cultivation in India for 4000 years or Florida, Hawaii, Fiji, Queensland and Natal. more, forms now a commercial crop in many The Portuguese are said to introduce countries. The account of the introduction and vegetative propagation methods in India for distribution of the mango in other countries of the first time to clone superior mono- the world is interesting. Perhaps Malaya was embryonic trees in the 15th Century. The most one of the earliest to obtain the material from important mango cultivars of India (Alphonso, India. The size of the seeds is too great to Dashehari, Langra etc.), are selections made at allow carriage by birds or other animals and the time of Akbar (1542–1605 AD) and have suggest dispersal by human agency. The been propagated by vegetative method for dissemination of Mango throughout the world several hundred years. Though, a tropical fruit, started with the commencement of trade it is now cultivated under subtropical between Asia and Europe. The Portuguese conditions in 89 countries of the world. The were the first to come to India, and they seized major mango growing countries are India, the opportunity of trading in spices and other Pakistan, Bangladesh, Myanmar, Sri Lanka, vegetable products of the East.100 It had Florida and Hawaii of USA, Australia, Brazil, become established in Somaliland on the Thailand, the Philippines, Malaysia, Vietnam, eastern coast of Africa before 1331.

Fig. 1: Worldwide distribution of mango21

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 Indian distribution of Mango: Butty, Natty and Gaddemar. The name of the Varieties of Mango for successful mango mango variety is varies from region to region. growing, it is necessary that the varieties The same variety may be known by a different planted in a commercial orchard are name in a different location. For example, productive, of good quality and adaptable to Himsagar of South Bengal area is known as the climate of the tract. Different varieties are Khirsapati in Malda. There are nearly 1000 suitable for growing in different climatic mango varieties in India. However, only the conditions. The number of varieties of mango following varieties are grown on a commercial found in India is great. In fact, there are far too scale in different States: Alponso Bangalora many varieties. The number of commercial Banganapalli Bambai Bambay Green varieties is estimated at over 1000104 each Dashehari Fajri Fernandiein Himsagar differing in size, shape, colour, texture and Kasar Kishenbhug Langra Mankurad taste. The popular varieties grown in Tamil Mulgao Neelum Sambarbehisht Chausa Nadu are Neelum, Bangalara, Banganapalli, Rumani and Raspuri3. Rumani and Mulgoa. Pickle varieties are

Fig. 2: Indian distribution of mango3

Morphological Diversity: cultivars identified two main sub-populations Molecular diversity analysis and fingerprinting representing mango cultivars from the North- of mango cultivars has been carried out using East and South- West regions of India. We different types of DNA markers, including identified 1.67 million high quality SNPs by random amplified polymorphic DNA (RAPD) sequencing double digested restriction site by Schnell et al., However, most of these associated DNA (ddRAD) from 84 diverse studies were carried it on small sets of mango cultivars from different regions of India genotypes. Recently, Ravishankar et al., using and abroad and a database of SNPs was 14 carefully selected SSR markers on a created. comprehensive set of 367 Indian mango

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051

Fig. 3: A sample of the diversity in the color, size and shape of mature mango fruits of Indian mango cultivars

Varieties : Though there are nearly 1000 regular bearing habit. Fruits are medium sized varieties of mango in India, only following with light yellow coloured skin, firm and varieties are grown in different states : fibreless flesh and sweet to taste. Alphonso, Bangalora, Banganpalli, Bombai, Ratna : It is a cross between Neelam and Bombay Green, Dashehari, Fazli, Fernandin, Alphonso. It is a regular bearer and free from Himsagar, Kesar, Kishen Bhog,Langra, spongy tissue. Fruits are medium sized with Mankhurd, Mulgoa, Neelam, Samarbehist, excellent quality. Flesh is firm and fibreless, Chausa, Suvarnarekha, Vanaraj and Zardalu. deep orange in colour with high TSS (19-21 Recently some mango hybrids have been Brix). released for cultivation by different institutes / Arka Aruna : It is a hybrid between universities. A brief introduction to such Banganapalli and Alphonso with regular varieties is presented below : bearing habit and dwarf in stature. About 400 Mallika - It is a cross between Neelam and plants can be accommodated per hectare. Dashehari. Fruits are medium sized cadmium Fruits are large sized (500- 700 gm) with coloured with good quality, reported to be a attractive skin colour. Pulp is fibreless, sweet regular bearer. to taste (20-22 Brix). Pulp percentage is 73 Amrapali - It is a cross between Dashehari and and the fruits are free from spongy tissue. Neelam. It is a dwarf vigorous type with Arka Puneet : It is a regular and prolific regular and late bearing variety. It yields on an bearing hybrid of the cross between Alphonso average 16 t/ha and about 1600 plants can be and the Banganapalli. Fruits are medium sized accommodated in one hectare. (220-250 gm) with attractive skin colour, Mangeera : It is a cross between Rumani and having red blush. Pulp is free from fibre, pulp Neelam. It is a semi vigorous type with a percentage being 70 percent. Fruits are sweet Copyright © March-April, 2019; IJPAB 386

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 to taste (20-22 Brix) with good keeping quality oblong leaf blade shape, semi-erect to and free from spongy tissue. It is a good horizontal leaf attitude in relation to branch, a variety for processing also. medium category in the angle of secondary Arka Anmol : It is a semi-vigorous plant type veins to the midrib, acuminate apex, acute from the cross between Alphonso and base shape, and mild leaf fragrance were Janardhan Pasand. It is also a regular bearing observed in the majority of the cultivars. These and free from spongy tissues. Fruits ripen to characters are among the important attributes uniform yellow colour. Keeping quality of the that could be utilized for classification of the fruit is very good and it is suitable for export. cultivars. It has got excellent sugar and acid blend and Fruit, stone and seed characters fruits weigh on an average about 300 g Pulp is The predominant fruit shape of the cultivars orange in colour. was oblong followed by roundish, obtuse fruit Morphological marker: apex, absent fruit stalk cavity, absent to Commercially grown mango cultivars have slightly neck prominence and perceptible beak been identified on the basis of vegetative and type. The majority of the cultivars had orange, reproductive parameters such as leaf size, leaf greenish yellow to yellow skin color and shape, shoot length, panicle length, fruit size, orange to yellow pulp color when ripe. The fruit shape, peel colour, stone size and stone cultivars fruit attractiveness was from average weight. These parameters, based on to good though there were excellent attractive morphological traits, constitute the cultivars (26.1%). Most had low to morphological markers, which are the oldest intermediate fiber in fruit pulp, very juicy, and most widely used genetic markers for the intermediate aroma, and very good to excellent germplasm characterization and cultivar eating quality. This indicated the potential of identification. Extensive studies have been cultivars for the table as well as processing carried out on the morphological diversity of purpose if further studied. Most cultivars in Indian mangoes and scores of morphological Shendi, Sudan also reported oblong fruit shape markers have been reported in mango by followed by round and obtuse fruit apex. several workers for the purpose of evaluation Alphonso (Happus) Fruit is medium in of cultivars and hybrids40. size, ovate oblique in shape, orange yellow in Variation of mango cultivars for qualitative colour. Juice is moderate-abundant, excellent raits keeping quality, good for pulping and canning. Tree and leaf characters Mainly exported as fresh fruit. Flesh develops The cultivars were largely non-grafted spongy tissue. Bangalora (Totapuri) The fruit seedlings, irregular (alternate) bearing is medium-large, oblong shaped with pointed behavior, tree height range from medium to base with golden yellow colour. Good keeping very tall group, broadly pyramidal to semi- quality; used for processing; heavy and regular circular crown shape, and spreading growth bears variety, Susceptible to bacterial spot. habit. The alternate bearing which is Banganapalli (Baneshan, Safeda) Fruit is large dependent on agronomic practices sized, obliquely oval in shape, golden yellow environmental conditions and genetic makeup in colour, good keeping quality, and good for is a common phenomenon of mango. Most canning, biennial in habit. Variety suited for cultivated mango trees are between 3 and 10 m dry areas Bombai (Malda) Variety is alternate in height when fully matured depending on the bearer. Fruit is medium, ovate and yellow in way of pruning. However, mango trees can colour. Keeping quality is medium. Bombay reach a height of 40 m or more while grafted Green Fruit size is medium, shape ovate ones are usually shorter Tree canopies vary in oblong with spinach green colour. Keeping genotypes, propagation method, the density of quality is medium. Early seasoning variety. plantation, and prevailing agro-climatic Biennial in habit highly susceptible to both conditions Intermediate foliage density, vegetative and floral malformation. Dashehari Copyright © March-April, 2019; IJPAB 387

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 Best varieties of the country. Mankur The roundish-oblique in shape and yellow in variety develops black spots on the skin in colour. High fruit quality and good keeping rainy season. Fruit is medium ovate and quality. Neelum Fruit is medium ovate-oblique yellow in colour. Fruit quality is very good but in shape and saffron yellow in colour. Good keeping quality is poor. Mulgoa Fruit is large keeping quality

Table 1: Studies on fruit morphological characteristics of mango varieties

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 Table 2: Studies on fruit weight and size of different mango varieties

Table 3: Studies on fruit physical parameters of different mango varieties

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 Genetic Diversity: Random Amplified Polymorphic DNA DNA markers (RAPD) The importance of genetic variations in The potential of RAPD markers to establish facilitating plant breeding and/or conservation genetic relationships among Mangifera strategies has long been recognized Molecular species, in the identification of Mangifera markers are useful tools for assaying genetic indica L. cultivars and in the validation of variation and provide an efficient means to genetic relationships among them has been link phenotypic and genotypic variation. In demonstrated by many authors1,35. recent years, the progress made in the Variable Number Tandem Repeat Sequence development of DNA based marker systems (VNTRS) has advanced our understanding of genetic Adato et al., analyzed DNA fingerprint resources. These molecular markers are information of some mango genotypes using classified as: (i) hybridization based markers, minisatellite multilocus probes. Genomic i.e., restriction fragment length polymorphisms DNA of 26 mango cultivars and 14 mango (RFLPs), (ii) PCR-based markers i.e. random rootstocks probed with 33.6, R18.1, 22.3, amplification of polymorphic DNAs (RAPDs), (GGAT)4, (GTG)5, and (GATA)4 revealed amplified fragment length polymorphisms resolvable and complex band pattern. DNA (AFLPs), inter simple sequence repeats fingerprinting pattern of mango cultivar and (ISSRs) and microsatellites or simple sequence rootstocks showed high polymorphism repeats (SSRs), and (iii) sequence based between unrelated cultivars. Each cultivar or markers, i.e., single nucleotide polymorphisms rootstock can be identified by its DSP pattern. (SNPs). Majority of these molecular markers The probability of two unrelated cultivars and have been developed either from genomic rootstocks having the same pattern is 8 x 10-6 DNA library (e.g. RFLPs or SSRs) or from and 1.2 x 10-5, respectively. The presence of random PCR amplification of genomic DNA many specific bands and low levels of band (e.g. RAPDs) or both (e.g. AFLPs) sharing between cultivars reflects the high Availability of an array of molecular marker level of informativeness of probe 33.6. The techiques and their modifications led to loci detected by other microsatellites were of comparative studies among them in many low polymorphism. Based on DNA fingerprint crops including fruit crops Molecular markers information, genetic distances between 20 provide an attractive and more reliable mango cultivars were evaluated and an alternative to morphological and biochemical evolutionary tree was established. Analysis of markers. In mango, phenotypic markers have DNA fingerprint band patterns of 12 progeny been the commonly used method of describing resulting from a cross between Tommy Atkins cultivars. Although this approach is useful to and Keitt mango revealed neither linked or nor distinguish distantly related cultivars, its allelic bands. The results suggested that DNA reliable application has proven more difficult fingerprints in mango are likely to be useful when it comes to differentiating closely related for identification and breeding purposes lines or off types of a particular cultivar. This Restriction Fragment Length is because phenotypically indistinguishable Polymorphism (RFLP) trees can be genotypically different and Description of Restriction Fragment Length therefore variants of a given cultivar cannot be Polymorphism (RFLP) as a DNA profiling easily detected by phenotypic assessment. technique and elucidation of its potential use Consequently, a more refined technique, such in varietal identification by Botstein et al., as molecular markers that are highly opened new avenues in genetic studies and polymorphic is required. DNA-based markers varietal improvement programmes. Capote et can be gainfully used in mango breeding for al., and Ravishankar et al.35, have employed marker assisted selection (MAS) and for the RFLP technique for genetic studies in cultivar identification. mango. Copyright © March-April, 2019; IJPAB 390

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 Amplified Fragment length Polymorphism observations confirmed by molecular pattern (AFLP) comparisons with the original Floridian Amplified fragment length polymorphism varieties. Galvez-Lopez et al., investigated (AFLP) is a PCR-based technique which genetic relationships among 112 mango allows inspection of polymorphism at fairly a (Mangifera indica) plants native to 16 states of large number of loci within a very short span Mexico and four controls [three mango of time and at the same time requires a very cultivars (Ataulfo, Manila and Tommy Atkins) small amount of DNA. The usefulness of and one accession of Mangifera odorata] AFLP marker system in genetic diversity based on simple sequence repeat (SSR) analysis and fingerprinting of mango markers. cultivars/genotypes has been reported by Despite the low degree of genetic Eiadthong et al10,. differentiation among accessions, Myanmar’s Inter Simple Sequence Repeat markers accessions were distinguishable from mango (ISSR) accessions from Florida, India, and Southeast Amplification of inter-simple-sequence- Asia in a principal coordinates plot. Genetic repeats (ISSRs) is a relatively novel technique differentiation of the Myanmar accessions and has proven to be a powerful, rapid, simple, from other groups was also observed in a reproducible and inexpensive way to assess Bayesian cluster analysis. No population genetic diversity or to identify closely related structure among Myanmar accessions was cultivars in many species, including fruit revealed by a neighbor-joining tree. The trees12. A number of reports are available results revealed a broad genetic background regarding the potential application of ISSR and genetic distinctiveness of mango in markers in genetic diversity estimation and Myanmar. Olano et al., used 25 SSR markers fingerprinting in mango10,12,17,34,38,40. to evaluate the genetic background of 63 Simple Sequence Repeat (SSR) markers Florida mango varieties, as well as cultivars Among the molecular markers available for from India, Asia and other locations. The genetic studies, SSR markers have gained parentage analysis, performed in a multistage considerable importance owing to many process resulted in four parent-offspring sets. desirable attributes including hypervariability, The results indicated that as few as four Indian multiallelic nature, codominant inheritance, cultivars, and the ‘Turpentine’ land race were reproducibility, relative abundance, extensive involved in the early cultivar selections. Sixty- genome coverage (including organellar three of the 85 parents identified across the genomes), chromosome specific location, four generations were other Florida cultivars. amenability to automation and high throughput Results of the study indicated that the Florida genotyping. In contrast, RAPD assays are not types are more closely related to Indian than to sufficiently reproducible, whereas RFLPs are Southeast Asian types. It was also confirmed not readily adaptable to high throughput that the Florida group is not more diverse than sampling. AFLP is complicated as individual either of the parental groups. Schnell et al., bands are often composed of multiple reported the development and characterization fragments mainly in large genome templates. of 15 microsatellite loci isolated from SSRs have been succesfully used for genetic Mangifera indica L. diversity estimation and DNA fingerprinting in SSR Amplification Polymerase chain mango44. reaction (PCR) amplification was performed in A dendrogram was constructed from a Perkin Elmer Thermocycler (PCR-Gene the 304 accessions. These accessions displayed Amp PCR System 9700) as per the protocol 207 different genotypes. Some suspected suggested by Williams et al., using 109 identification errors in the germplasm bank mango-specific microsatellite markers (Table were confirmed. Eight accession identities 2). Amplified products were separated by could be corrected according to morphological electrophoresis in a 3% metaphor-agarose gel Copyright © March-April, 2019; IJPAB 391

Singh et al Int. J. Pure App. Biosci. 7 (2): 382-395 (2019) ISSN: 2320 – 7051 using Tris-acetate EDTA Tris-acetate EDTA Cambridge, United Kingdom). 198 alleles (TAE) buffer at pH 8.0. The amplified were chosen for their clear pattern and high fragments were observed and photographed allele numbers to study diversity within the under UV light in Gel Doc System (Syngene, entire sample.

Table 4:

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