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Genetic Diversity of Fruit Borer, Helicoverpa Armigera (lepidoptera: Noctuidae) Based on Random Amplified Polymorphic DNA- Polymerase Chain Reaction

A. K. M. Z. RAHMAN
Entomology Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701

M. A. HAQUE
Department of Entomology, Bangladesh Agricultural University (BAU), Mymensingh,Bangladesh,

S. N. ALAM
Entomology Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701

P. YASODHA
4Assistant Professor
(Agril. Entomology), Department of Agril. Processing& Basic Sciences, Agricultural Engineering College and Research Institute, Kumulur,

V. BALASUBRAMANI
Assistant Professor
(Agril. Entomology), Department of Agril. Processing& Basic Sciences, Agricultural Engineering College and Research Institute, Kumulur,

Abstract/ Executive Summary:

The genetic variability of Helicoverpa armigera (Hübner) at different agroecological zones of Bangladesh in comparison with Indian population was conducted in India during September 2008 to February 2009. A total of 12 H. armigera populations of which 10 populations collected from different agroecological zones of Bangladesh and two populations from India were tested for their genetic variability. Eight out of the ten primers produced scorable PCR products by amplifying the template DNA with taq polymerase and were subjected for analysis. Those eight primers got amplified to a total of 138 markers which produced polymorphic markers. The similarity coefficient based on 138 RAPD markers ranged from 0.000 to 0.777 of the pair-wise combination among twelve samples of H. armigera. An UPGMA dendrogram based on Jaccard’s similarity coefficient was constructed for the 12 samples of H. armigera. The dendrogram showed that H. armigera population from Bangladesh had 25 to 45 percent similarity, and in its Indian population the similarity remained within this range.

Keywords: Helicoverpa armigera, Genetic diversity, RAPD-PCR, Primers.

Location: IPM laboratory, Entomology Division, BARI

Start Date: 00-09-2008

End Date: 00-02-2009

Program Area: Pest Management

Commodity: Insects

Objectives:

An attempt was made to study the genetic variation of H. armigera using RAPD markers.

Methodology:

This study was conducted in the Department of Plant Molecular Biology and Biotechnology, Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore, India during September 2008 to February 2009. Sampling of insect: H. armigera larvae collected from different locations of Bangladesh and were reared at IPM laboratory, Entomology Division, BARI separately using their respective hosts. Indian H. armigera populations were also reared separately in their respective hosts. The emerged female from each locality was preserved immediately in vials containing 70 percent alcohol. The samples were maintained at -20°C until DNA extraction. A total of 10 samples of H. armigera were collected from 10 different localities of Bangladesh on different hosts like tomato, chickpea, chili, and mungbean during March to May of 2008. For comparison, two populations of H. armigera were collected from two locations of Coimbatore, Tamil Nadu, India during October 2008. Hence, a total of 12 samples were collected from 12 different localities depicting a garden-land ecosystem with field and horticultural crops that are being grown round the year. DNA extraction Total nucleic acids were extracted from individual female of H. armigera using C-TAB (Hexadecyl trimethyl ammonium bromide) method (Doyle and Doyle, 1987) with some modifications. Modifications were made as follows: 1. Second wash of crude DNA with Chloroform and iso amyl alcohol is prevented. 2. Washing of crude DNA with sodium acetate 3. Washing with 70% ethanol for several times is limited based on the final crude DNA pellet obtained. Single washing of crude DNA with ethanol was maximum followed. Quantity of the isolated DNA was measured in NanoDrop® ND-1000 spectrophotometer and the quality were checked in 0.8% Agarose gel electrophoresis before using it as the template for polymerase chain reactions (PCR). The reagents were purchased from Bangalore Genei Ltd., Bangalore, India. RAPD assays: Amplification reactions were performed in a 20 µL reaction mix, containing a final concentration of 2.5 mM dNTPs, 25 mM MgCl2, dimethyl sulfoxide, Taq polymerase 3U/µL, 10x Taq buffer, sterile water, primer 100 nmol and DNA 20- 25 ng/uL. The RAPD analysis was performed with seventeen decamers supplied by Operon Technologies Inc., California, USA. Amplification was performed in thermocycler (BioRad, USA) programmed as one cycle of initial denaturation at 95°C for 2 min; one cycle of denaturation at 95°C for 1 min; 30 cycles each of 95°C for 1 min., annealing at 40°C for 1 min., extension at 72°C for 1 min. and final extension at 72°C for 7 min. The PCR products were separated in 1.5 % agarose gel electrophoresis. Analysis of PCR amplification profiles: Data obtained by scoring the RAPD profiles of the eight primers individually were subjected to cluster analyses. PCR amplification products of the 12 samples were scored as presence (1) or absence (0) of bands. The data matrix was used to calculate Jaccard’s similarity coefficient (Sneath and Sokal, 1973), which does not consider the joint absence of a marker as an indication of similarity. The similarity values were used for cluster analyses. Sequencial agglomerative hierarchial non-overlapping (SAHN) clustering was done using Unweighted PairGroup method with arithmetic averages (UPGMA). This analysis was performed using NTSYS-PC software, version 2.0 (Rohlf, 1998).

Source of Information: Bangladesh J. Agril. Res. 39(2): 263-271, June 2014 ISSN 0258-7122

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Those eight primers got amplified to a total of 138 markers which produced polymorphic markers. The similarity coefficient based on 138 RAPD markers ranged from 0.000 to 0.777 of the pair-wise combination among twelve samples of H. armigera. An UPGMA dendrogram based on Jaccard’s similarity coefficient was constructed for the 12 samples of H. armigera. The dendrogram showed that H. armigera population from Bangladesh had 25 to 45 percent similarity, and in its Indian population the similarity remained within this range.

Knowledge Management: Journal