JAHAN, S. M. H.
Professor
Department of Entomology, Patuakhali Science and Technology University,
Dumki, Patuakhali-8602
LEE, K-Y.
Professor
Department of Agricultural Biology, Kyungpook National University, Daegu 702-701, Korea,
HOWLADER, M. I. A.
Senior Scientific Officer
On-Farm Research Division, Bangladesh Agricultural Research Institute, Patuakhali-8600, Bangladesh.
Genotypic clusters, Bemisia tabaci
Molecular Physiology Laboratory at Kyungpook National University in South Korea
Pest Management
Samples of adult B. tabaci were collected from bean, eggplant and okra of different places of eastern part of Bangladesh such as, Chittagong and Cox’s Bazar in 2012 and were immediately preserved in 99% ethanol (alcohol) and stored at -20 °C. Adults of B. tabaci B and Q biotypes were collected from whitefly rearing house in Insect Molecular Physiology Laboratory at Kyungpook National University in South Korea on cucumber and tomato plants in 2011 for morphological and genetic sequences comparison of Bangladesh whiteflies.
Total genomic DNA was extracted from individual B. tabaci according to protocol supplied by Invitrogen Purelink Genomic DNA mini kit. After removing from ethanol the sample had been washed with double-distilled water to remove alcohol, individual whiteflies were homogenized in 180 µl genomic digestion buffer using a 1.5 ml microcentrifuge tube and micropestle (homogenizer). Then added 200 µl genomic lysis/ binding buffer (1% SDS, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 25 mM NaCl, Proteinase K 200 mg/ml) and after that immediately added 200 µl absolute ethanol. Subsequently added wash buffer into the genomic column and finally added 20 µl genomic elusion buffer (Invitrogen Purelink, Carisbad, CA, USA). After 1 min incubation at room temperature, samples were centrifuged at about 12000 rpm for 1 min, and the supernatants/pellets were directly used for PCR detection of the secondary endosymbionts or were stored at -20 °C for later use. These procedures were followed by Dellaporta et al., 1983 and Jahan et al., 2011.
Bemisia tabaci was determined using the genomic DNA which was collected from Bangladesh with the primers listed, using Polymerase Chain Reaction (PCR). All PCR reaction mixture performed in 20 µl volume that included 1 µl of each primer (Forward and Reverse), 1 µl of DNA template and 17 µl smart buffer which were supplied by the manufacturer (Smart taq pre-mix). All PCR reactions were carried out on the PTC-200 DNA engine thermal cycler (MJ Research PTC-200 DNA Engine Thermal Cycler PCR). The mixtures with the cytochrome oxidase sub-unit1 (CO1) primer C1-J-2195(F) and L2-N-3014(R) were amplified in a PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) with a 1 minute initial denaturation at 95 ° C, 35 cycles (1 min at 94 ° C, 30 sec at 52 ° C, 2 min at 72 ° C), and finally by a 5 min extension at 72 ° C. For 16S rRNA primers LR-J-12887 (F) and LR-N-13398 (R) were also amplified with a 2 min initial denaturation at 94 ° C, 35 cycles (30 sec at 94 ° C, 1 min at 55 ° C, 1 min at 72 ° C), and finally by a 10 min extension at 72 °.
DNA sequences were aligned using CLUSTAL W. The aligned sequences were checked and compared the sequences similarity with the online published sequences using BLAST in the National Centre for Biotechnology Information (NCBI). Sequences were aligned and arranged using the Clustal W2 multiple alignments in BioEdit (version 7.0). The sequences divergences calculated by Molecular Evolutionary Genetics Aanlysis (MEGA) among intraspecific, interspecific species, based on Kimura-2-parameter (K2P) distances. Phylogenetic relationships were inferred by MEGA Software Version 4.0 using neibour-joining method (NJ). Bootstrap values were obtained from 1000 replicates. The sequences are deposited in the GenBank database.
ISSN 0258-7122 Bangladesh J. Agril. Res. 40(1): 1-16, March 2015
Journal