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Assessment of Genetic Diversity in 13 Local Banana (musa Spp.) Cultivars Using Simple Sequence Repeat (ssr) Markers

M H Kabir
Institute of Food and Radiation Biology, Atomic Energy Research Establishment G. P. O. Box 3787, Dhaka Bangladesh

A N K Mamun
Institute of Food and Radiation Biology, Atomic Energy Research Establishment G. P. O. Box 3787, Dhaka Bangladesh

H Fatema
Department of Biotechnology and Genetic Engineering, Islamic University, Kustia, Bangladesh

R Amin
National Mushroom Development and Extension Centre, Sobhanbag, Savar, Dhaka, Bangladesh

Abstract/ Executive Summary:

A study was conducted to investigate the genetic variability among 13 local banana cultivars using 3 SSR primers of Mb1-69, Mb1-113 and Mb1-134. All the primer pairs amplified a total of 29 different marker bands with an average of 9.6 bands per primer. Among the 29 bands only 4 bands were monomorphic and the rest 25 bands were polymorphic. The sizes of the amplified DNA bands in 13 local banana cultivars varied from 200 bp to 600 bp. The primer Mb1-113 amplified the highest (14) number of DNA bands and the primer Mb1-69 amplified the lowest (7) number of DNA bands whilst primer Mb1-134 amplified 8 DNA bands. The values of pairwise genetic distances ranged from 1.00 to 9.00 indicating the presence of wide genetic diversity. The dendogram constructed based on phylogenetic relationship analysis revealed that the highest genetic diversity (9.00) found between the cultivars champa and jawayta and also the cultivars champa and jahazy whilst the lowest (1.00) between the cultivars doubled haploid and kathaly, doubled haploid and sorishafruity, doubled haploid and amritsagor and doubled haploid and ganasundory. The UPGMA dendogram has segregated the 13 local banana cultivars into two major clusters. Agnishwar and champa formed in cluster 1 and the rest of the cultivars like sobri jesore, sobri, anazy, kathaly, jawayta, sorishafruity, amritsagor, jahazy, bangle, ganasundory and doubled haploid have constituted the cluster 2.

Keywords: Genetic diversity, SSR markers, Banana.

Location: Institute of Food and Radiation Biology, Atomic Energy Research Establishment G. P. O. Box 3787, Dhaka Bangladesh

Start Date:

End Date:

Program Area: Conservation and Biodiversity

Commodity: Diversity, Banana

Objectives:

The present study was undertaken to investigate the potentiality of SSR markers for distinguishing individual accessions to rule out duplications among cultivars and to develop a standardized procedure for Musa genotyping which could serve as a basis for molecular characterization of newly introduced global Musa gene bank as well as Musa research and breeding community.

Methodology:

Thirteen local banana cultivars namely sobri jessore (large size), sobri (small size), anazy (cooking, large), kathaly (cooking, small), jawayta (seeded, large), sorishafruity (seeded, small), amritsagor, jahazy, agnishwar, champa, ganasundory, bangle and doubled haploid (anther derived plant) were used for molecular characterization through SSR analysis. The cultivars were collected from the experimental garden of Plant Biotechnology and Genetic Engineering Division, Atomic Energy Research Establishment, Savar, Dhaka. Young and fresh leaf were used for DNA extraction. The harvested leaf of 0.25g was cut down into small pieces and isolated DNA using modified CTAB (Abdullah et al., 2012). The concentration of DNA sample was determined using UV spectrophotometer (PG Instruments Ltd. Korea) at 260/280 nm readings were 1.6 to 1.8. The PCR reaction mixture for 10 μl containing template DNA (50 ng) 1 μl, deionized distilled water 1.8 μl, Taq buffer A 10X (15mM Tris-Hcl with Mgcl2) 1 μl, primer (2 μM) 5 μl, dNTPs (2.5 mM) 1 μl and Taq DNA polymerase (5u/ μl) 0.2 μl. The reaction with each primer was replicated thrice to check the reproducibility of DNA. DNA amplification was carried out in an oil-free DNA thermal cycler (Finnzymes Instruments, EU) at the following thermal profile: initial denaturation for 5 minutes at 94ºC followed by denaturation for 60 seconds at 94ºC, annealing at 55ºC for 60 seconds and extension at 72ºC for 120 seconds. A final extension at 72ºC for 7 minutes was allowed for complete extension of all amplified fragments. Amplified fragments were separated on a 1.4% agarose gel containing 0.5 μg/ml ethidium bromide in 1X TBE buffer for 1 hour at 100 volts. The gel was visualized by UVtransilluminator and photographed by gel documentation system (Major Science, USA). The amplified bands were visually scored as present (1) and absent (0) of bands, size of bands and overall polymorphism of bands. The scores obtained using all primers in the SSR analysis were then pooled for constructing a single data matrix. This was used to estimate polymorphic loci, genetic diversity, genetic distance (Nei, 1972) and a UPGMA (Unweighted Pair Group Method with Arithmatic Means) dendrogram using computer program ‘Statistica’.

Source of Information: International Journal of Recent Research in Life Sciences (IJRRLS) Vol. 2, Issue 1, pp: (65-69), Month: January - March 2015,

Article Link (Online Journal): Available at: www.paperpublications.org

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The banding patterns of different banana cultivars using 3 primers are shown. Band size ranging from 200 to 600 bp in PCR amplification products scored for primers. Strong and weak bands were produced in the PCR reactions. Weak bands produced from low homology between the primer and the pairing site on the DNA strand (Thormann et al., 1994). This study shows the genetic diversity within Musa germplasm collection in Atomic Energy Research Establishment. There are several limitations inbreeding and genetic improvement in Musa genotypes. Therefore, selecting the most suitable parents that would result in higher diversity is crucial for significant genetic progress.

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