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Molecular Analysis of Hexaploid Wheat (triticum Aestivum L.) Cultivars by Rapd Markers

S. Mitra
Department of Biotechnology, Bangladesh Agricultural University (BAU), Mymensingh

K. M. Nasiruddin
Department of Biotechnology, Bangladesh Agricultural University (BAU), Mymensingh

E. H. Chowdhury
Department of Pathology, BAU, Mymensingh, Bangladesh

Abstract/ Executive Summary:

RAPD assay was conducted for molecular genetic analysis of six wheat cultivars, such as, Kanchan, Sourav, Gourab, Shatabdi, Pavon and BAW‐1006 to observe genetic variability and relatedness among these cultivars. Three out of 12 decamer random primers showed distinctly polymorphic bands when used to amplify genomic DNA. The primers yielded a total of 23 RAPD markers of which 14 were considered as polymorphic. The proportion of polymorphic loci and gene diversity (h) values were 34.78% and 0.153 for BAW‐1006, 30.43% and 0.124 for Kanchan, 26.09% and 0.127 for Shatabdi, 26.09% and 0.127 for Pavon, 26.09% and 0.111 for Gourab, 21.74% and 0.098 for Sourav, respectively. The coefficient of gene differentiation (Gst) and gene flow (Nm) values across all the loci were 0.50 and 0.50, respectively indicating genetic divergence among populations. The UPGMA dendrogram based on Nei’s genetic distance, grouped six cultivars into two main clusters: Kanchan, Sourav, Gourab and Shatabdi in cluster I; Pavon and BAW‐1006 in cluster II. The cluster I was further separated: Kanchan alone in sub‐cluster I and Sourav, Gourab, Shatabdi in subcluster II; furthermore, Sourab and Gourab grouped together in sub‐sub‐cluster I of sub‐cluster II with the lowest genetic distance of 0.035. Thus, RAPD offer a potentially simple, rapid and reliable method to evaluate genetic variation and relatedness among six wheat cultivars. Introduction

Keywords: RAPD, Genetic diversity, Polymorphic loci, Wheat

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Wheat

Objectives:

The goal of the study was inspired from mentioned research and emphasized on genetic variation and relatedness among the cultivars. To our knowledge, this was the first preliminary attempt to study the molecular analysis of wheat cultivars in Bangladesh

Methodology:

Six high yielding hexaploid wheat cultivars, namely Kanchan, Sourav, Gourab, Shatabdi, Pavon and BAW‐1006 were collected from the Wheat Research Center (WRC) of Bangladesh Agricultural Research Institute (BARI). Genomic DNA was extracted from fresh leaf samples of 15 day old seedlings and a total of 18 individuals (three from each cultivar) were taken randomly to carry out the RAPD analysis. Twelve primers of random sequence (Genei Pvt. Ltd., Banglore, India) were screened to evaluate their suitability for amplifying DNA sequences, which could be accurately scored. Primers were selected on the basis of band resolution intensity, presence of smearing, consistency within individuals and potential for population discrimination. Finally, three primers were selected for the analysis of the whole sample set of the six cultivars. PCR reactions were performed on each DNA sample in a 10 μl reaction mix containing 1 μl of 10x ampli taq polymerase buffer, 2.5 μl of 10 μM primer, 1 μl of 250 μM dNTPs, 1 unit of ampli taq DNA polymerase and 3 μl of genomic DNA and a suitable amount of sterile deionized water (Williams et al. 1990). DNA was amplified in a thermal cycler (Master Cycler Gradient, Eppendorf, Germany) and the reaction mix was performed at 94ºC for 3 min followed by 40 cycles of 1 min denaturation at 94ºC, 1 min annealing at 37ºC and elongation at 72ºC for 2 mins. After the last cycle, a final step of 7 min at 72ºC was added to allow complete extension of all amplified fragments. The amplified product from each sample was separated electrophoretically on 1.4% agarose gel containing ethidium bromide in 1xTBE buffer. A molecular weight DNA marker (Lambda DNA with Hind III digest and/or 100 bp DNA ladder) was loaded in the first lane of the gel. DNA bands were observed on UV‐transilluminator in the Image Documentation System and the image was viewed on the monitor and saved in a diskette, as well as printed on thermal paper.

Source of Information: Plant Tissue Cult. & Biotech. 19(1): 35-44, 2009 (June)

Article Link (Online Journal):

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Total Budget (Tk.) :

Achievement/Findings:

In conclusion, RAPD markers have been proved to be powerful tools for molecular genetic analysis of wheat cultivars for plant breeding program to assess available genetic diversity. This study indicated that BAW‐1006 line and Sourav variety contained the highest genetic variation and the lowest genetic variation, respectively which could be used as germplasm for future crossbreeding programs with the goal to improve wheat variety. Moreover, these markers allow for the production of new wheat varieties that are aimed at crop productivity improvement enabling them to withstand biotic and abiotic stresses.

Knowledge Management: Journal