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Molecular Characterization of 25 Mango Germplasm Using Rapd Markers Available in the South-western Region of Bangladesh

M. A. Hossain*
Agrotechnology Discipline, Khulna University, Khulna (9208), Bangladesh

M. M. Islam
Agrotechnology Discipline, Khulna University, Khulna (9208), Bangladesh

M. A. Mannan
Agrotechnology Discipline, Khulna University, Khulna (9208), Bangladesh

S. K. Roy
Agri Studies, Khulna Public College, Boyra, Khulna (9000), Bangladesh

P. Shil
Agri. Studies, Khulna Model College, Boyra, Khulna (9000), Bangladesh

Abstract/ Executive Summary:

RAPD (Randomly amplified polymorphic DNA) markers were used for the genetic variation and relationship analysis among 25 Mango (Mangifera indica L.) germplasm (Gp). PCR amplification with seven primers generated 48 reproducible, clear and distinct bands, out of which 46(95.92%) bands are considered polymorphic and the remaining 2 fragments (4.08%) monomorphic.RAPD matrix of the 25 germplasm of mango using seven primer were assembled for statistical analysis the sizes of the fragments were estimated using DNA 1 kb and 100 bp ladder by electrophoresis with the PCR products.The average polymorphism in all the 25 cultivars using the seven primers was found to be 95.92%. Among all the primers AL 07, OPO 07, OPK 14, OPG 17 and OPA 06 have shown 100% polymorphism while OPH 15 and OPF 08 were found to be least polymorphism (85.71%). The UPGMA (Unweighted Pair Group Method of Arithmetic Mean) dendrogram based on Jaccard’s similarity coefficient segregated the 25 mango germplasm into two clusters. The dendrogram shows that all the germplasm were grouped into two major clusters 16 mango germplasm in one groups and rest 9 mango germplasm is another group.The values of pair-wise comparisons of Nei’s (1979) genetic distance among 25 mango genotypes were computed from 0.043 to 0.510. Comparatively the highest genetic distance 0.510 was found between Gp no. 52 (Langra) vs. Gp73 (Hybrid-10). The lowest genetic distance was found between Gp no. 50 (0.043) (Bhut Bomby) and Gp no. 52 (Langra). Results from the given study would be useful breeders for further improvement of mango varieties.

Keywords: Mango germplasm, Molecular characterization, Genetic diversity

Location: Molecular Horticulture laboratory, Agrotechnology Discipline, Khulna University, Khulna-9208, Bangladesh.

Start Date: 00-08-2013

End Date: 00-06-2014

Program Area: Variety and Species

Commodity: Mango

Objectives:

The present study was conducted to assess genetic diversity and genetic relationships among mango germplasm/genotypes.

Methodology:

2.1. Experimental location, sample collection and preparation Random Amplified Polymorphic DNA analysis (RAPD) of mango Germplasm was carried out during the period of August 2013 to June 2014 in the Molecular Horticulture laboratory, Agrotechnology Discipline, Khulna University, Khulna-9208, Bangladesh. In the present study, 25 mango genotypes were obtained from the south-western part of Bangladesh. Approximately 5 g of recently matured and tender leaves (7−10 days old) was collected, washed using distilled water, wiped with 70% (v/v) ethanol and then air dried, of which 1 g was weighed out and stored in sealed plastic bags at -80 °C for further use. 2.2. DNA extraction and purification The DNAzol protocol which is based on the use of a novel guanidine detergent lysing solution that allows the selective precipitation of DNA from the lysate (Chomczynski et al., 1998) is fast and permits efficient isolation of genomic DNA from a variety of plant tissues that why we followed the methods of Chomczynski et al. (1998). 0.5 g to 1 g leaf samples were ground into powder with mortar and pestle and 1−1.5 ml of DNAzol (Invitrogen, USA) was added and then homogenized properly. The mixture was centrifuged at 12,000 rpm for 10 minutes and transferred the supernatant to another 1.5 ml centrifuge tube. DNA was precipitated from the lysate/homogenate by the addition of 0.5−0.75 ml of 100% ethanol and then mixed gently by inverting tubes 3−5 times and stored at room temperature for 2−3 minutes to form a well mixed homogenous solution. As DNA became visible as a cloudy precipitate and then the mixer was centrifuged at 12,000 rpm for 10 minutes again. The DNA precipitated was found and needed to discard supernatant and there after precipitated DNA was washed with 75% of ethanol about 2−3 times. After addition of ethanol, inverting the tubes 8−10 times and were stored the tubes vertically for 0.5–1 minutes to allow the DNA to settle to the bottom of the tubes and were removed ethanol through decanting and pepeting. Finally air dried the DNA pellet. 2.3. DNA solubilization and quantification The air dried pellet was dissolved in 300 μl of 8 mM NaOH was stored at -20 °C. The DNA was checked in 1 percent agarose gel electrophoresis and quantified using UV spectrophotometer (T 60 UV-Visible, PG instrument Ltd). 2 μl of isolated DNA sample from each germplasm of mangoes were taken and made up to 2000 μl or 2 ml with 8 mM NaOH. The entire isolated DNA samples were quantified using UV spectrophotometer at 260 nm and 280 nm. The ratio OD 260/280 should be determined to assess the purity of the sample. The concentrations of absorbance were calculated by using the following formula: DNA concentration (µg ml-1)=(OD 260)×(dilution factor)×(50 µg DNA ml-1)/(100 OD260 unit). The DNA solution was expressed in ng mL-1 (Hoisington et al., 1994). The final concentrations of mango DNA stock solutions were adjusted to 100 ng mL-1 for PCR reaction. 2.4. Selection of primer Primers were selected mainly based on GC content, intensity of bands, presence of smearing, consistency within individuals and potential for population discrimination. Primers that have GC content more than 60% are suitable for RAPD analysis. We selected seven subsets of primers supplied by Bioneer, South Korea among them seven (AL07, OPA06, OPF08, OPG17, OPO07, OPH15 and OPK14) were exhibited good quality banding patterns and sufficient variability. Annealing temperature was calculated by the following formula: 0.3× Tm (primer)+0.7×Tm (product) -25.

Source of Information: International Journal of Bio-resource and Stress Management 2016, 7(4):807-813

Article Link (Online Journal): DOI: HTTPS://DOI.ORG/10.23910/IJBSM/2016.7.4.1629a

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

RAPD analysis is efficient and accurate for the investigation of distribution of commercial mango or local mango. The RAPD analysis is useful in the fingerprinting of each mango sample. The geographical locations, growth altitude, and climates may contribute the polymorphic RAPD of mango trees in Bangladesh. This result is beneficial for further research on the mango functionality.Cultivars from Southwest region of Bangladesh unveiled maximum diversity and indicated the potential of RAPD markers for the identification of management of mango germplasm for breeding purposes.

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