Rawnak Laila
Graduate Student
Deparment of Plant Pathology, Bangladesh Agricultural University, Mymensingh-2202
Dr. Md. Bahadur Meah
Professor
Deparment of Plant Pathology, Bangladesh Agricultural University, Mymensingh-2202
Mahbuba Khatun Siddiqua
Professor
Deparment of Horticulture, Bangladesh Agricultural University, Mymensingh-2202
Md. Ibrahim Khalil
Senior Scientific Officer
Plant Pathology Division, Bangladesh Institute of Nuclear Agriculture, BAU Campus, Mymensingh-2202.
Molecular characterization, Eggplant, RAPD, Collar rot
Bangladesh Agricultural University and Bangladesh Institute of Nuclear Agriculture, Mymensingh
Variety and Species
Genomic DNA Extraction and Quantification
Five eggplant varieties, BAU Begun 1, BAU Begun 2, Laffa S, ISD 006 and Dohazari G were used in the study. Seeds were collected from the IPM Laboratory of Bangladesh Agricultural University, Mymensingh and sown in earthen pot soil covering with the light soil uniformly for proper germination. As a source of genomic DNA youngest healthy leaf samples-collected from the 15-days old seedling were used. The experiment was carried out in the Biotechnology Laboratory of Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Modified CTAB mini-prep method was followed to extract DNA from leaf samples (Kabir, 2007). The concentration of DNA in the samples were determined using a UV Spectrophotometer at 260nm. The quality of the DNA was verified by electrophoreses on a 0.8% agarose gel in TBE (Tris-boric acid-EDTA) buffer.
PCR amplification and Electrophoresis
RAPD amplification reactions were maintained essentially following William et al. (1990) with some modifications. The screening was done with fifteen arbitrary decamer primers (Bengalore Genei, India) using DNA from parental cultivars. Three primers resulting scorable and reproducible bands were selected for subsequent RAPD analysis of eggplant germplasms. PCR reactions were performed on each DNA sample in a 10 μl reaction mixture containing 1x PCR buffer (10 mM Tris HCl pH 8.5, 50mM KCl and 15 mM MgCl2), 10 mM each dNTPs, 5 pmols primer, 2 U of Taq DNA polymerase (Bengalore Genei, India), 100 ng of genomic DNA and rest amount of sterile deionized water. DNA amplification was carried out in a DNA thermocycler (Biometra, Germany) as the following thermal profile: initial denaturation for 3 min at 94°C followed by 41 cycles of 1 min denaturation at 94°C, 1 min annealing at 35°C and extension at 72°C for 2 min. A final extension step at 72°C for 7 min was allowed for complete extension of all amplified fragments. The PCR products were kept at 4°C until electrophoresis. Reaction mixtures were mixed with 2.0 ml 6X loading dye. Amplified fragments were separated on a 1.5% agarose (Bengalore Genei, India) gel in 0.5 X TBE buffer along with 20 bp DNA weight marker (Bengalore Genei, India) for 2 hours at 100V. Gel was stained with Ethidium bromide solution (0.1 mg ml-1) for 15 min. Finally fragments were visualized under UV-transilluminator and photographed by Gel Documentation System (Biometra, Germany).
Scoring and Data analysis
It is assumed that each band represented the phenotype at a single locus because all RAPD markers are nearly dominant (Williams et al., 1990). The amplified bands were visually scored as present (1) and absent (0) separately for each individual and each primer. The scores obtained were pooled to create a single data matrix. This was used to estimate polymorphic loci, Nei's (1973), genetic diversity, genetic distance (D) and a UPGMA (Unweighted Pair Group Method with Arithmetic Means) dendrogram using a computer program, POPGENE (Version 1.31) (Yeh et al., 1999). The same programme was also used to perform the test of homogeneity in different loci between population pairs.
Int. Research J. Applied Life Sci. 1(4): 38-65 (2012).
Journal