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Analysis of Genetic Diversity in Mango (mangifera Indica L.) Using Isozymetic Polymorphism

Majumder, D. A. N.*
Plant Biotechnology laboratory, BRAC Agricultural Research and Development Centre, Bangladesh

Hassan, L.
Department of Genetics and Plant Breeding, Bangladesh Agricultural University (BAU), Bangladesh

Rahim M. A.
Department of Horticulture, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

Kabir, M. A.
Department of Horticulture, Hajee Mohammad Danesh Science and Technology University, Dinaupur-5200, Bangladesh.

Abstract/ Executive Summary:

The isozymetic study was designed for assessing the genetic diversity among the selected mango cultivars/genotype available in Bangladesh. All the isozymes, used in the present study showed polymorphism for mango. A total of 25 different electrophoretic zymotypes were observed for three isozymes studied. Glutamate oxaloacetate transaminase, malate dehydrogenase and peroxidase analysis possessed 8, 10 and 7 zymotypes, respectively, and genotypes were grouped into different electrophoretic zymotypes which indicated higher level of genetic diversity of mango. For glutamate oxaloacetate transminase, G6 was the most common zymotypes whereas G3, G5, and G8 were found in few cases. Similarly, M8 and M6 in malate dehydrogenase as well as P6 and P5 in peroxidase were found more frequent while other zymotypes in both isozymes were less frequent. Frequency was very few for one zymotype in all cases of three isozymes such as, G8, M10 and P7. Cluster analysis through UPGMA dendogram using isozymes electrophoretic pattern provided strong information about existence of variability among the genotypes of mango. Based on Euclidean distance, a dendogram was constructed using banding pattern of 60 mango genotypes developed through three isozymes activities. The dendogram showed eight major clusters designated as I, II, III, IV, V, VI, VII and VIII. The results generated with isoenzyme will be helpful in improvement as well as may guide us in designing strategies that maximize the utility of genetic resources.

Keywords: Polymorphism, Isozyme, Mango, Genetic diversity, Variety.

Location: Molecular Biology Laboratory, Department of Genetics and Plant Breeding, Bangladesh Agricultural University

Start Date: 00-08-2007

End Date: 00-02-2008

Program Area: Conservation and Biodiversity

Commodity: Mango

Objectives:

The objective of the present investigation was aimed at elucidating the phylogenetic relationship among 60 genotypes of mango, based on the polymorphism of three enzymes. Moreover, it helps to obtain genetic information in large collection of mango being maintained at the Germplasm Repository of Bangladesh Agricultural University (BAU) from isozyme analysis.

Methodology:

The study was conducted with 60 mango genotypes in polyacrylamide gel electrophoresis (PAGE) techniques (Staub et al., 1997). Three isozymes viz., GOT, MDH and PER were used in this experiment. Isozyme analysis was done in the Molecular Biology Laboratory, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, during the period from August 2007 to February 2008. Extraction of enzyme sample Mature leaf samples were collected, cleaned and sealed in brown paper covers from the orchard of Germplasm Repository of Bangladesh Agricultural University (BAU) and brought to the laboratory. Approximately, 0.4 g of leaf material was crushed with acid washed sand and 400 ml extraction buffer. Extraction buffer consisted of 0.1 M Tris-HCl (pH 7.5) containing 20% sucrose. Preparation of gel for electrophoresis Gels of different concentration viz., staking gel 4.5% and separation gel 9% were prepared by using the stack solutions. Ammonium per sulphate (APS) solution was prepared immediately just before use. The gel dimensions were 14 × 11 × 0.1 cm. The stock solutions for separation gel except APS and tetramethylene diamine (TEMED) were gently mixed in a 100 ml beaker. Gel solution was poured into glass plate to the level of about 5 mm lower from the comb to be placed. Electrophoresis Electrophoresis means migration of suspended electrically charged particles such as protein macromolecules under the influence of an electric field. Vertical polyacrylamide slab gel electrophoresis (PAGE) was used for isozyme analysis. About 500 ml of the electrode buffer (Tris 0.025 M and Glycine 0.190 M) was poured into the electrophoresis chamber and 300 ml of buffer was poured on to the upper portion of the electrode. The electrode assembly was connected with the power supply unit (120 volts) at constant voltages and electrophoresis was carried out for 3.5 to 4 h. Staining of isozyme The electrophoresis of the proteins of leaf samples was carried out using PAGE technique and the gels were stained for GOT, MDH and PER were used for staining gel. Staining solution of GOT was prepared by aspartate aminotransferase (AAT) substrate solution (pH 7.4) α-Ketogluteric acid 292 mg, L- aspartic acid 1.07 g, PVP-40 (Polyvinylpyrolidine) 4.0 g, ethylenediaminetetraacetic acid (EDTA) 400 mg and sodium phosphate, dibasic 11.36 g were mixed with 800 ml distilled water. Then, 50 mg of fast blue BB salt was added to the AAT substrate solution and poured over the gel. For malate dehydrogenase, staining solution was prepared by mixing 50 mN Tris-HCl (pH 8.5) 50 ml, nicotinamide adenine dinucliotide (NAD) 10 mg, malic acid 1 ml (after neutralized with NaOH), nitro blue tetrazolium chloride (NBT) 10 mg and phenozine methosulphate (PMS) 2 mg. In case of peroxidase, 10 ml of POD-1 (500 ml of acetone + 1.05 g of 3-amino-9-ethylcarbazole and + 0.725 g of B-napthol) solution was added to 40 ml of POD-B (1.51 g of Tris buffer + 1.62 ml of glacial acetic acid) solution and mixed gently. About 50 μl of 30 % H2O2 was added and the gel was then incubated at room temperature in the dark and continuously shaken until the bands appeared. The formation of bands was completed within an hour. Identification of band Following Mouemar and Gasquez (1983), isozyme banding patterns were recorded on the basis of number and the relative front (Rf) values of the bands. The Rf value of each respective band on schematic isozyme patterns was determined to allow precise comparisons among the various genotypes. The Rf value is the mobility of each isozyme band that traveled from the origin divided by the distance traveled by the front tracking dye. The presence or absence of a certain isozymatic band was considered as a differentiating feature. Zymograms were drawn to scale and relative mobility values were calculated for each band. Analysis of similarity coefficient Similarity coefficients of two zymotypes from electrophoretic banding pattern were calculated using Nei and Li’s (1979) index of genetic similarity comparisons (Sxy), which are: Sxy = 2nxy/(nx+ny); Where, nxy = Number of shared bands; nx = number of bands in electrophoretic patterns of x zymotype; and ny = number of bands and electrophoretic patterns of y zymotype.

Source of Information: African Journal of Biotechnology Vol. 11(87), pp. 15310-15323, 30 October, 2012

Article Link (Online Journal): DOI: 10.5897/AJB12.2217

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The isozymetic study is suitable for investigating mango diversity and rendered huge information for further studies. It would be interesting to introduce more accessions from the land races and diversity to infer the relationships and to evaluate the genetic difference that has occurred in Bangladesh. Isozymetic study revealed a high degree of genetic diversity among the cultivars examined in the study, which can contribute to the improvement of fruit crops like mango. Furthermore, these electrophoretic zymotypes should supplement conventional breeding programmes aimed at developing elite mango cultivars possessing larger fruit size, smaller stone size and greater yield stability over biotic and aboitic tress. This study would also help for choosing diverse parents in mango breeding programme and would help to confirm the genetic purity of elite mango cultivars. To the best of my knowledge, this is the first reports on isozymatic clustering on mango of Bangladesh.

Knowledge Management: Journal