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RAPD Marker-assisted Identification of Genetic Diversity among Mango (Mangifera indica) Varieties in Mauritius.

Byline: ANUSHA DEVI RAMESSUR AND V.M. RANGHOO-SANMUKHIYA

ABSTRACT

Morphological and molecular techniques were used to evaluate the characteristics of 12 mango (Mangifera indica L.) varieties cultivated in Mauritius and to compare their genetic relatedness. Genomic DNA was extracted from leaf samples and genetic characterization was carried out using the RAPD technique making use of 30 decamer Operon primers. 77.1% polymorphism was obtained with Operon primer OPD-2 being the least discriminatory in contrast to OPA-9 and OPL-14, which were most polymorphic. An Un-weighted Pair Group Method with Arithmetic Mean dendogram of cophenetic ratio 0.9732 was constructed with distance values of 0.222 to 0.667. Two major clusters consisting of a mixture of foreign and native mango varieties were delimited with Overseer being the least related to the rest of the varieties. No clear distinction between the landraces and exotic varieties was observed. Phenotypically similar Jose and Eugenie varieties were also genetically close.

The 3 'pickle' varieties Dauphine, Sabre and Blanc were within the same cluster. Considerable genetic variation was observed between the studied mango varieties. The RAPD technique together with the analysis of morphological characters proved useful for assessing the diversity existing between the mango varieties. In conclusion, the mango varieties used in this study could be tapped in breeding programs aimed at cultivar improvement. (c) 2011 Friends Science Publishers

Key Words: Mango; Mangifera indica L; Mauritius; Genetic diversity; RAPD

INTRODUCTION

Mango (Mangifera indica L.), native to South-East Asia, comprises 62 species about 16 of which have an edible fruit. Mango was introduced in Mauritius two centuries ago by the Dutch and has been in cultivation ever since (Rouillard and Joseph, 1999). Prevailing favorable climatic conditions allow the tropical fruit to thrive particularly in the regions receiving 30 inches of rainfall annually (France Du Pavillon, 1991). Flowering starts in July over 5 months and mango yield peaks in summer months notably from October to February.Mango varieties of differing skin color, stone size, sweetness and composition are available on the market, some of which are native and others exotic ones introduced from Australia, Israel, Reunion, America and Europe by the Agricultural department in Mauritius to expand the local 'genetic pool' of the mango fruit (Seeruttun, 2001).

An important demand for mangoes exists in supermarkets and hotels for certain varieties like Jose. Mango is not currently exported but a market for export could be exploited for delicious endemic varieties. These varieties represent potential candidates for any future breeding work aimed at producing high yielding, disease-resistant hybrids adapted to the local microclimatic conditions. Genetic resources for potential crop improvement are invaluable, hence their collection, evaluation, characterization and documentation is important.

Traditionally, in Mauritius, mango varieties have been differentiated based particularly on fruit characteristics like size, shape and color, which being influenced by environmental parameters, are unreliable. Molecular markers, by virtue of their determinate number and protection against environmental influence, can solve problems posed by morphological markers. Identification of genotype using molecular markers can help in identification of trees for sale, minimizing the risk of mix-ups in orchards (Struss et al., 2001) and different markers have been used to study mango diversity namely Random Polymorphic DNA (RAPD) markers and Amplified Fragment Length Polymorphism (AFLP) and Inter Simple Sequence Repeat (ISSR). RAPD was considered practical to use over the other markers since the DNA purity required is lower, it is less costly, does not require blotting and data can be collected quickly (Welsh and McClelland, 1990; Williams et al., 1990).

Adato et al. (1995) used RFLP analysis based on minisatellite probes and came to the conclusion that Indian cultivars represented a varied group. Molecular markers are co-dominant, specific and highly variable, rendering them highly suitable to study diversity in supposedly related populations or cultivars (Duval and Bunel, 2005). Ravishankar et al. (2004) used dendogram analysis of RAPD and chloroplast DNA to group Indian cultivars into two clusters based on embryo type and geographical origin. In another study, Ravishankar et al. (2000) used RAPD analysis on 18 Indian cultivars and came to the conclusion that eastern, western and northern cultivars were genetically close, while southern cultivars formed a different genetic group. Recently, twenty-eight polymorphic microsatellite loci for the mango genome were developed and described (Duval and Bunel, 2005).

In Mauritius, Seeruttun (2001) carried out morphological characterization of 16 important varieties on the basis of fruit, skin colour, stone, pulp firmness and sweetness. The current study adds to the previous one in providing more insight into the morphology and genetic diversity that exists among the mango varieties in the face of growing interest in the potential of the mango production sector. The aim of the study was to evaluate the genetic relatedness between different mango varieties native and exotic cultivated in Mauritius and to find the possible origin of Mauritian mango varieties. Morphological data was correlated with molecular data to validate their versatility to be used for characterisation of these mango varieties.

MATERIALS AND METHODS

Plant material:

The accessions used were both local and exotic ones from America, Australia and Reunion as follows: Dauphine, Sabre, Blanc, Maison Rouge, Figette and Overseer (local), Bowen (Australia), Irwin, Early Gold (America), Jose, Eugenie and Christian (Reunion). Leaf material (5 replicates of each variety) was collected in paper bags from mango varieties at different accession sites from the Ministry of Agroindustry and Fisheries (Mauritius) namely Barkly, Plaisance experimental stations and Roches Brunes Seed Production Center. The leaves were wrapped in moist tissue paper and were refrigerated at -50degC when not subjected to immediate use. All experiments were carried out from August 2007 to March 2008 in the molecular biology laboratory, Faculty of Agriculture, The University of Mauritius.

Morphological characterization: Mature mango leaves and ripe fruits of all 12 varieties were characterized based on the International Plant Genetic Resources Institute (IPGRI) descriptors for mango and the 1986 journal of the Californian Rare fruit growers. The 9 leaf traits described included base shape, apex shape, blade shape, margin, colour at maturity, fragrance, pubescence, angle of secondary veins to midrib and secondary vein curvature. The 6 fruit traits described include skin colour, fruit form, shoulder form, forms of the sinus and apex, beak form and base form.

DNA isolation protocol and purification:

Fresh leaf tissue (0.5 g) was ground in liquid Nitrogen to form a thin powder, which was transferred into 5 mL of freshly prepared, preheated (60*C) Cetyltrimethylammonium Bromide (CTAB) buffer in a tube followed by addition of 0.2% (v/v) b- mercaptoethanol and 2% (v/v) Polyvinylpyrrolidone (PVP). The tube was incubated in a water bath held at 60*C for 45 min with intermittent swirling. 2/3 volume of chloroform: isoamyl alcohol (24:1 v/v) was added to the tube, which was tilted several times and centrifuged for 10 min at 2208 g. This extraction step was repeated twice. 2/3 volume of ice-cold isopropanol was added to the supernatant and the tube was incubated at - 20degC overnight. The tube was centrifuged for 30 min at 5652 g (40degC). The supernatant was discarded and the DNA pellet was washed with 70% ethanol followed by centrifugation for 2 min at 10000 rpm to resettle the pellet. The alcohol was poured off and the DNA pellet was allowed to dry and thereafter it was dissolved in 1 mL of TE buffer.

The DNA was purified by incubation with RNase followed by a phenol treatment.

RAPD reaction:

RAPD reactions were carried out in a DNA Thermocycler (MJ Research Inc. USA). Negative controls comprised all PCR components excluding the template. Each 25 uL reaction volume comprised 100 ng of template DNA, 1X PCR Buffer (10 mM Tris Hcl pH 8.8; 50 mM KCl), 4 mM MgCl2, 0.2 mM dNTP, 0.5 uM of single primer,1 U of Taq DNA polymerase. The thermal profile set comprised a denaturation step of 5 min at 94degC, followed by 38 cycles of 30 s at 94degC, 30 s at 35degC, an extension at 72degC for 2 min and a final extension at 72degC for 10 min. 30 Operon primers were used to screen for polymorphism. PCR products were electrophoresed on 1.5% (w/v) agarose gels using 0.5x TBE Buffer at 115 V and stained with ethidium bromide (0.5 ug/mL) for visualization and photographing under UV light.

RAPD profile analysis:

The screened primers that gave bands were used to amplify the DNA of all the 12 mango varieties. Each genotype was characterized by its banding pattern (Figs. 1-3), using the DNA hyperladder 2 (Bioline) as basepair ladder. The RAPD markers as viewed from the gels after electrophoresis and staining were converted into a matrix of binary data, where the presence of the band corresponded to value 1 and the absence to value 0. The statistical software NTSYS-PC (Rohlf, 2005) and DARwin 5 software (Perrier and Jacquemoud-Collet, 1996) were used to construct a UPGMA dendrogram using hierarchical clustering. Using NTSYS software, a dissimilarity matrix was calculated utilising Jaccard's (1908) coefficient. The matrix was converted to a dissimilarity matrix corresponding to the complement (dissimilarity=1[?]similarity).

Cluster analysis based on the dissimilarity matrix, was performed using un-weighted pair-group method arithmetic averages (UPGMA) (Sneath and Sokal, 1973) of the NTSYS-PC version 2.2 (Rohlf, 2005).

RESULT

Morphological characterization: A high degree of similarity among varieties in some of the leaf traits e.g., curvature of secondary veins, angle of secondary veins to midrib and leaf pubescence was observed (Table I).

Table I: Morphological leaf traits of the different mango varieties

Variety###Eugenie###Jose###Irwin###Bowen###Early###Christian Maison###Figette###Overseer Sabre###Dauphine###Blanc

###Gold###Rouge###Barkly

###1.Acute###X###X###X###X###X###X###X###X###X###

1. Leaf base shape###2.Obtuse###X###X###X###

###1.Acute###X###X###X###X###X###X###

2. Leaf apex shape###2.Acuminate###X###X###X###X###X###X###

###1.Lanceolate###X###X###X

3. Leaf blade shape###2.Elliptic###X###X###X###X###X###X

###3.Oblong###X###X###X###

###1.Wavy###X###X###X###X###X###X###X###X###X###X###

4. Leaf margin###2.Entire###X###X###

###1.Dark green###X###X###X###X###X###X###X

5. Colour of fully developed leaf2.Green###X###X###X###X###X###

###1.Mild###X###X###X###X###X###X###X###X###

6. Leaf fragrance###2.strong###X###X###X###X

###3.Absent###

###1.Absent###X###X###X###X###X###X###X###X###X###X###X###X

###2.Present

7. Leaf pubescence###2.Medium (45-60deg)###X###X###X###X###X###X###X###X###X###X###X###X

###3.Narrow (45deg)###

8. Secondary vein curvature###1.Present###X###X###X###X###X###X###X###X###X###X###X###X

###2.Absent

Table II: Morphological fruit traits of the different mango varieties

Variety###Eugenie###Jose###Irwin###Bowen###Early###Christian Maison###Figette###Overseer Sabre###Dauphine###Blanc

###Gold###Rouge###Barkly

###1.Green###X###

###2.Yellow###X###X###X###X###X###

1.Skin colour###3.Orange###X###

###4.Purple###X###X###X###X###X###X###

###5.Red###

###1.Roundish###X###X###X###X###

###2.Oval###X###X###X###

###3.Cordate###X###

2.Fruit form###4.Peento###

###5.Oblong###X###

###6.Ovate oblique###X###

###7.Oblong reniform###X###

###8.Ovate reniform###X###

###1.Broadly rounded###X###X###X###

###2.Rising then rounded###X###X###

3.Shoulder form###3.Ending in long curve###X###X###X###

###4.Sloping###X###X###X###

###5.Falling abruptly###X###

###1.Sinus absent###X###X###X###X###X###

###2.Slight sinus###X###X###

4.Forms of Sinus###3.Broadly rounded apex###

and apex###4.Broadly pointed apex###X###

###5.Deep sinus###

###6.Rounded apex###X

###1.Point absent###X###X###X###X###X###X###X###

###2.Small point###X###

5.Form of the beak###3.Mammi form###X###

###4.Curved beak###X###X###

###5.Slightly prominent###X###

###1.Flattened###X###

###2.Slightly flattened###X###X###X###X###

6.Base form###3.Rounded###X###X###X###

###4.Extended###X###

###5.Obliquely flattened###X###X###

###6.Necked###X

Table III: RAPD markers used for the generation of polymorphism in local and imported mango varieties

Primer###Primer sequence###Number of###Number of###%

###(5' - 3')###markers###polymorphic###polymorphism

###(Total)###markers###

OPD-2###GGACCCAACC###6###0###0.0

OPD-10###TGTCTGGGTG###10###9###90.0

OPL-14###GTGACAGGCT###9###9###100.0

OPJ-13###CCACACTACC###11###8###72.7

OPW-4###CAGAAGCGGA###13###7###53.8

OPA-9###GGGTAACGCC###21###21###100.0

Total###70###54###77.1

In contrast, there appeared to be greater variation in the fruit traits. Each fruit character (Table II) had a large number of character states with many intermediate gradations between extremes, while leaf character states were two to threefold.

Molecular characterization:

The CTAB extraction protocol successfully yielded DNA (265-600 ng/uL) from all the mango varieties with a A260/A280 ratio of 1.600- 1.900. Out of 30 primers screened most polymorphism were obtained using OPA-16, OPJ-13, OPL-14, OPD-02, OPW-04, OPD-10 and OPA-09 (Table III). Primer OPA-9 gave the greatest number of bands and together with OPL-14 were the most polymorphic and hence most discriminatory between varieties in stark contrast to primer OPD-2, which was not polymorphic at all (Table III).

Two major clusters, A and B, could be delimited from the UPGMA dendogram generated (Fig. 4). Cluster B comprised Dauphine, Sabre, Bowen and Blanc, while the other encompassed Maison Rouge, Figette, Irwin, Early Gold, Christian, Eugenie and Jose. In cluster B, Blanc clustered out separately. The Reunion varieties Jose and Eugenie separated out as an independent clade in cluster A. The other Mauritian varieties, Maison Rouge, Figette, Irwin, Early Gold and Christian were found in another clade within the same cluster A. Within this cluster, Early Gold was the most different from the rest of the varieties of the group. Dauphine was particularly genetically very close to Sabre as were Maison Rouge to Irwin and Jose to Eugenie. Local varieties clustered with the American and Reunion ones in cluster A and with the Australian one in cluster B. The local variety Overseer was distantly related to the rest of the mango varieties.

Dissimilarity values ranged from 0.222 to 0.667; Dauphine and Overseer were the two varieties with most distantly related genotypes as inferred from the dissimilarity matrix in contrast to the two closest varieties Dauphine and Sabre (Table IV). The dendogram had a high goodness of fit and was robust as indicated by the cophenetic ratio of 0.9732 and was hence reliable for the analysis of the genetic relatedness among the varieties.

DISCUSSION

Each mango variety possesses a unique combination of fruit and leaf traits setting it apart from the other varieties. These sets of distinguishing morphological characteristics constitute a basis for separating varieties. Certain characters are uninformative for differentiating the varieties notably 'curvature of secondary veins', 'angle of secondary vein to midrib' and 'leaf pubescence'. Some leaf traits were similar in the overwhelming majority of varieties e.g., the wavy leaf margin, which were not useful in distinguishing varieties. On average, the leaf traits between the varieties did not show as much diversity as the fruit traits, suggesting the superiority of the latter in easy differentiation of varieties. These observations are congruent with the statement of Singh (1960) statement that mango fruit characters form the main basis for identification and classification of varieties of which primary characters e.g., fruit and beak form are the most useful.

Out of all the fruit traits, fruit form was observed to be more variable among the varieties. A strikingly close phenotypic similarity between the varieties

Table IV: Dissimilarity matrix based on the proportion of shared RAPD fragments among different mango varieties

###Blanc###Dauhine###Sabre###Bowen###Jose###Eugenie Maison. Rouge###Irwin###Figette###Early Gold###Christian

Dauphine###0.350###

Sabre###0.350###0.222###

Bowen###0.381###0.308###0.390###

Jose###0.442###0.451###0.420###0.380###

Eugenie###0.447###0.489###0.489###0.540###0.377###

Maison Rouge###0.463###0.527###0.442###0.463###0.373###0.429###

Irwin###0.527###0.481###0.481###0.500###0.407###0.436###0.222###

Figette###0.420###0.490###0.490###0.537###0.441###0.444###0.291###0.327###

Early Gold###0.447###0.386###0.422###0.447###0.436###0.440###0.429###0.377###0.444###

Christian###0.370###0.447###0.378###0.500###0.400###0.431###0.333###0.340###0.314###0.333###

Overseer###0.630###0.667###0.615###0.630###0.525###0.589###0.466###0.418###0.455###0.564###0.472

Jose and Eugenie with regard to fruit traits exists, understandably giving rise to confusion in orchards. The resemblance suggests that they may be closely related to each other although it was found that few of the studied leaf traits are shared between the two varieties.Primers OPD-10, OPL-14 and OPA-9 were most discriminatory in assessing genetic diversity between mango varieties. The Reunion varieties Jose and Eugenie, which were phenotypically similar in fruit traits clustered together in the dendogram obtained following analysis of the RAPD data, confirming similarity at genetic level as well. Primer OPW-4 was most suitable in differentiating these two varieties since many bands present in one variety were absent in the other. These could hypothetically be associated with the differential characteristics setting Jose apart from Eugenie e.g., 'acuminate leaf apex' and 'elliptic leaf blade'. Local varieties Dauphine and Sabre clustered together although morphologically no such closeness was observed.

Similar disparities between molecular and morphological data were made for Maison Rouge and Irwin. The use of morphological traits was not always the best way to evaluate genetic distance, since the degree of divergence between genotypes at the phenotypic level was not necessarily correlated with a similar degree of genetic difference. Similar discrepancies between RAPD data and morphologically based groupings were reported recently in cashew (Damodaran et al., 2007). Maison Rouge and Irwin shared a similar profile with most RAPD primers particularly OPD-10, while on the other hand they could be differentiated by OPL-14, OPJ-13, OPW-4 and OPA-9. Interestingly, the three local varieties Dauphine, Sabre and Blanc, which are known to be less sweet with high fibre content and hence suitable for pickling were within the same cluster, indicating a close relationship between them.

Most probably their attributes that make them more suitable for pickling than consumption as table fruits were inherited from a common parent having its roots in Mauritius. With OPD-10 for instance 80% of the markers were shared among the three varieties. Dauphine and Sabre could be discriminated by OPA-9, which yielded a 600 and 650 bp marker in Dauphine and a 1200 bp one in Sabre.

Analysis of RAPD data showed that the mango varieties did not cluster according to their country of origin. One possible explanation regarding this association between the supposedly distantly located genotypes could be attributed to the unique and broad genetic base of the mango species in general. Another could be that agro-climatic conditions prevailing in the island could have triggered adaptive changes in the introduced foreign varieties. This possibility is acknowledged by Padmesh et al. (1998), according to whom genotypes from different geographical regions can be genetically similar to genotypes with similar spatial relationships. Furthermore, compatibility between the varieties being high, inter-varietal hybridization in nature between the local and exotic varieties could have given birth to new varieties.

Karihaloo et al. (2003) reported a high diversity within regions in India and confirmed that this is not surprising given that mango is a cross-pollinated plant and selecting superior strains according to taste among naturally produced seedlings has given birth to the commercial cultivars and the observed appreciable range of variation (Mukherjee, 1950). Mango was introduced two centuries back into Mauritius and there are instances of typical local Mauritian varieties being introduced in other countries like Jamaica (Duval and Bunel, 2005). The local variety Overseer was most distantly related to the rest of the varieties (local or exotic). This is particularly evident from its distinct unique shape thus sharing little similarity at genetic level with the other varieties. Morphological traits like 'leaf pubescence absent' and DNA bands e.g., the 1200 bp amplified by OPD-10 were common to all the twelve varieties possibly being ancestral in origin and characteristic of mango.

Originally, mangoes had no names associated with them and monkeys were responsible for their propagation. They hybridized, conserving their ancestral characteristics (Rouillard and Joseph, 1999), which could explain their similar genetic profile.

The study highlights the diversity of mango fruit forms that exists in Mauritius, which has also been observed in wild and cultivated Indian mango (Karihaloo et al., 2003). Both molecular and morphological methods were effective at portraying the variation. The dendogram clusters are not in agreement with the geographical origin of the mango cultivars. It is conceivable that, keeping in mind the geographical proximity of Australia and Reunion to Mauritius, foreign varieties could have originated from local Mauritian varieties for instance, the Reunion variety Christian and the landrace Figette - explaining the observed relatedness. Similarly, Ravishankar et al. (2000) concluded that cultivars in India might have arisen from germplasm in a particular geographical area.

There are documented cases of an association of genotypes of different geographical location as in the study on the genetic diversity of Andrographis paniculata accessions from India, Thailand, Malaysia and Indonesia, where Padmesh et al. (1998) ascribed the observation to seed movement and gene flow. Schnell et al. (1995) in their study of 25 accessions also obtained a wide distribution of Indian cultivars in RAPD based dendogram, which is indicating how cultivars may vary in a specific region.

This study reports for the first time the genetic diversity of mango varieties cultivated in Mauritius and will pave the way for future studies aimed at better understanding their genetic variation. These molecular studies will help in identifying superior genotypes for cultivar upgrading or for generating new cultivars. Better understanding the distribution of genetic variation could help in identifying superior genotypes for cultivar upgrading or for use in generating new cultivars. These could give a boost to the local mango 'gene pool' by bringing together the greater adaptability of the landraces to the local agro-climatic conditions and the commercial characters of value from foreign varieties to give birth to appealing varieties from the point of view of consumers and marketing to capture the interest of export markets.

Developing SCARS (Sequence Characterized Amplified Region) primers based on unique polymorphic bands obtained among varieties could help in distinguishing morphologically similar cultivars as in the case of Jose and Eugenie.

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Author:Ramessur, Anusha Devi; Ranghoo-Sanmukhiya, V.M.
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:6MAUI
Date:Apr 30, 2011
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