F. Easmin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
M. S. Rahman
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
M. S. Islam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
M. A. Samad
Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh
M. S. Alam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh
Genetic variation, RAPD markers, Rice
BAU, Mymensingh
Variety and Species
Study Materials Six high yielding rice varieties viz. Binadhan 4, Binadhan 5, Binadhan 6, Binasail, BRRI dhan28 and BRRI dhan29 were used in the present study. The seeds of the varieties were collected from Genetic Resource Centre (GRS), Bangladesh Rice Research Institute (BRRI) and Bangladesh Institute of Nuclear Agriculture (BINA). Varieties were different in breeding strategies, production potentials, cultivation season etc. Plants were regenerated from the seeds in the laboratory of BINA. Leaf tissues of the plants were used as source of DNA in the experiment. Extraction of Genomic DNA Total genomic DNA was isolated from young actively growing fresh leaf tissues following SDS extraction, phenol: chloroform: isoamyl alcohol purification and ethanol precipitation method. Approximately 25 mg leaf tissues was cut into small pieces, homogenized and digested with extraction buffer [50 mM Tris-HCl, 25 mM ethylenediaminetetraacetic acid (EDTA), 300 mM NaCl and 1% sodium dodecyl sulphate (SDS)] at 65ºC for 15 min. DNA was purified by extraction with phenol: chloroform: isoamyl alcohol (25 : 24 : 1, v:v:v). DNA was precipitated using absolute alcohol in the presence of 0.3M sodium acetate and pelleted by centrifugation. The pellets were then washed with 70% ethanol, air dried and resuspended in an appropriate volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH=8.0). DNA quality was checked by electrophoresis in a minigel. DNA samples were then quantified using a UV-spectrophotometer. Primer selection Initially, 20 decamer random primers from three kits (5 from kit A, 10 from kit B and 5 from kit C) of random sequence (Operon Technologies, USA) were screened on a sub sample of two randomly chosen individuals to test their suitability for amplification of the DNA sequences, that could be scored accurately. Primers were evaluated based on intensity of bands, consistency within individual, presence of smearing, and potential for population discrimination. A final subset of four primers exhibiting good quality banding patterns and sufficient variability were selected for further analysis. PCR amplification The amplification conditions were based on Williams et al. (1990) with some modifications. PCR reactions were performed on each DNA sample in a 10 μl reaction mix containing 1 μl of 10X Ampli Taq polymerase buffer, 2 μl of 10 μM primer, 1 μl of 10 mM dNTPs (Bangalore Genei, India), 1 unit of Ampli Taq DNA polymerase (Bangalore Genei, India), 100 ng genomic DNA and a suitable amount of sterile deionized water. DNA amplification was performed in an oil-free thermal cycler (Master Cycler Gradient, Eppendorf). The reaction mix was preheated at 94oC for 3 minutes followed by 40 cycles of 1 min denaturation at 94oC, 1 min annealing at 34oC and elongation or extension at 72oC for 2 minutes. After the last cycle, a final step of 7 minutes at 72oC was added to allow complete extension of all amplified fragments. After completion of cycling program, reactions were held at 4oC. Agarose gel electrophoresis The amplified product from each sample was separated electrophoretically on 1.4% agarose gel (Nacalai tesque, Inc., Kyoto, Japan) containing ethidium bromide in 1X TBE buffer at 120 V for 1 hour and 15 min. Two molecular weight marker DNA (Phi X 174 DNA / HaeIII digest and / or 100 bp DNA ladder) was electrophoresed alongside the RAPD reactions. DNA bands were observed on UV-transilluminator and photographed with a Gel Cam Polaroid camera. Data analysis The RAPD markers were scored visually on the basis of their presence (1) or absence (0), separately for each individual and each primer. For more accuracy, band scoring was performed by two independent persons. Bands not identified by the two readers were considered as non-scorable. The scores obtained using all primers in the RAPD analysis were then pooled for constructing a single data matrix. This was used for estimating polymorphic loci, Nei’s (1973) gene diversity, co-efficient of gene differentiation (Gst), genetic distance (D) and constructing a UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram using computer program POPGENE (Version 1.31) (Yeh et al., 1999). The same program was also used to perform pair wise homogeneity test across different loci.
Bangladesh J. Genet Pl. Breed., 21(1): 07-14, 2008
Journal