L. Das
Professor
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202.
M. M. Islam
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh-2202
K. M. Nasiruddin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202.
S. N. Begum
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh-2202
M. A. Haque
Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202
RAPD, Characterization Brassica.
Department of Fisheries Management, BAU, Mymensingh-2202
Variety and Species
The experiment was conducted at the Biotechnology Laboratory of the Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. The present investigation was carried out during the period from August, 2007 to May, 2008. Eight different varieties of Brassica spp. namely, Binasharisa-3, Binasharisa-4, Binasharisa-5, Binasharisa-6, Agrani, Tori-7, Safal and BARI sharisa-14 were used to analyze the molecular characteristics and relationships among them. Immature, vigorously growing fresh leaf samples from 15 days old seedlings were used as the source material of genomic DNA. The collected leaf samples were stored in –80oC refrigerator into polythene bags. DNA extraction was done by using the mini preparation Modified CTAB method (IRRI, 1997). 670 μl extraction buffer, 50 μl 20% SDS, 100 μl 5M NaCl, 100 μl CTAB were used and incubated at 65oC in different stages of DNA extraction. For purification equal volume (900 μl) of chloroform : isoamylalcohol (24:1) was added and centrifuged for 15 min. at 12000 rpm then transferred the supernatant into a new eppendorf tube. 600 μl ice cold isopropanol was added and DNA became visible as white strands by flicking the tube several times with fingers. DNA was washed with 200 μl 70% ethanol and stored in 30μl 1x TE buffer and placed in -200C. DNA samples were evaluated both quantitatively and qualitatively (was it higher molecular weight or was there substantial shearing or degradation) using spectrophotometer and agarose gel, respectively. A target DNA sequence is exponentially amplified with the help of arbitrary primers, a thermo stable DNA polymerase, deoxy nucleotide tri-phosphates, magnesium chloride and reaction buffer. The reaction involves repeated cycles, each consisting of a denaturation (94oC for 3 minutes), a primer annealing (34oC for 1 minute) and an elongation (72oC for 2 minutes) step. Amplification products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Twelve primers of random sequence were screened on a sub sample of two randomly chosen individuals from eight different cultivars to evaluate their suitability for amplification of DNA sequences, which could be scored accurately. Primers were evaluated based on resolution intensity of bands consistency within individual, presence of smearing, and potential for population discrimination. A final subset of three primers exhibiting good quality banding patterns and sufficient variability were selected for further analysis. 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 mixture containing the following reagents: Genomic DNA (25 ng/μl) = 1μl, Dilute primer = 2.5μl, Taq buffer = 1μl, dNTPs (250μM) = 1μl, Taq DNA polymerase = 0.2μl and Deionized water = 4.33 μl. The PCR buffer, dNTPs, primer and DNA samples solutions were thawed from frozen stocks, mixed by vortexing and kept on ice. DNA template were pipetted first into PCR tubes compatible with the thermocycler used (0.2 ml). A pre-mixture was then prepared in the course of the following order: reaction buffer, dNTPs, DNA template and sterile distilled water. Agarose gel (1.5%) was prepared and 0.5X TBE buffer into the tank to submerge the gel. The PCR products were mixed with 3 ml of 2X gel loading dye. The molecular weight marker (20 bp DNA ladder) was loaded and electrophoresis machine run for 1.5-2.0 hr at 120 volts. The separation process was monitored by the migration of the dyes in the loading buffer. When the bromophenol blue dye had reached about three-fourth of the gel length, the electrophoresis was switched off. After completion of electrophoresis the gel was soaked in ethidium bromide (10 mg/ml) solution for 20-25 minutes. The gel was then taken out carefully from the staining tray and placed on high performance ultraviolet light box (UV transilluminator) of gel doc for checking the DNA bands. The DNA was observed as band and saved the records. All distinct bands or fragments (RAPD markers) were thereby given identification numbers according to their position on the gel and scored visually on the basis of their presence (1) or absence (0), separately for each individual and each primer. The scores obtained using all primers in the RAPD analysis were then combined to create a single data matrix. This was used for estimating polymorphic loci, Nei’s (1973) gene diversity, genetic distance (GD) and constructing a UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram among the populations using computer program POPGENE (Version 1.31). Gene frequency estimation for RAPD loci was based on the assumption of a two-allele system. Under the assumption of Hardy-Weinberg equilibrium, the null allele frequency (q) may be (N/n) 1/2 where N and n are the number of band negative individuals observed and the sample size, respectively. The frequency of the other allele (P) is 1-q. The assumption of the two allele system enables us to calculate the Nei’s genetic distance (Nei, 1972) from the RAPD pattern. Genetic-similarity was calculated manually from RAPD markers of the same molecular weight on the data matrix according to Similarity index (SI) = (2 Nxy/Nx + Ny) X 100. Where, Nxy is the number of RAPD bands shared by individuals x and y respectively, and Nx and Ny are the number of bands in individual x and y, respectively (Chapco et al., 1992). Gene flow (Nm) was estimated according to the Nm = 0.5 (1- Gst)/ Gst formula. Where, Gst is the proportion of total genetic diversity attributable to subpopulation. It is also known as coefficient of gene differentiation. The Gst values were calculated by using Gst = 1- Hs/Ht. Where, Ht is the mean average heterozygosity of the total population and Ht is the mean of Hardy-Weinberg exception of heterozygosity obtained with population average allele frequencies. Nei’s genetic distance and identity values were computed from frequencies of polymorphic markers to estimate genetic relationship between the studied eight Brassica species using the unweighted pair-group method of arithmetic means (UPGMA). The dendrogram was constructed using the POPGENE (Version 1.31) computer package.
Bangladesh J. Seed Sci. & Tech. 15 (1 &2): 195-202 (2011), ISSN 1029 - 8800
Journal