Genomic DNA isolation
Seeds of four sesame varieties were obtained from Bangladesh Agricultural Research Institute, Gazipur, Bangladesh and grown in the experimental field of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh. Genomic DNA was isolated following protocol described by Saghai-Maroof et al. (1984) with some modifications. Juvenile leaves (unfolded) of 30 days old plants were used in genomic DNA isolation. Leaf tissues were cut into small pieces, homogenized and digested with extraction buffer (pH= 8.0): 50 mM Tris-HCl, 25 mM EDTA (Ethylenediaminetetraacetic acid), 300 mM NaCl and TEN buffer + 5% SDS (Sodium Dodecyl Sulfate) +10% PVP (Poly Vinyl Pyrolideone) +20% CTAB (Cetyl Trimethyl Ammonium Bromide). After incubation for 20 minutes at 65°C with intermittent swirling, the mixture was emulsified with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, v/v/v). DNA was precipitated using two volume of absolute alcohol in presence of 0.3 M 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) and finally treated with 2 μl of RNAse. Quality and quantity of DNA were controlled via gel electrophoresis and spectrophotometer, repectively.
Primers identification
A set of five Random primers were chosen to estimate the potential of these marker for variety identification. Finally three primers, OPA-09, OPC-05 and OPL-07 were selected based on their performance for RAPD data analysis.
Polymerase Chain Reaction
Polymerase Chain Reactions were done in a volume of 10 μl containing 10 x PCR Buffer, 0.25 mM each of the dNTPs, 25 pmol of primer, 1 unit ampli Taq DNA polymerase, 100 ng template DNA and a suitable amount of sterile deionized water. Amplification were carried out in a oil free thermal cycler (Thermal cycler gradient, Eppendorf) with the following thermal profile: initial denaturation step at 94oC for 4 min followed by 45 cycles at 94 oC for I min, 34-36oC for 1 min, and 72 oC for 2 min and a final cycle at 72 oC for 7 min. PCR was the confirmed by electrophoresis on 2% agarose gel.
RAPD data analysis
Following electrophoresis, the sizes of the PCR products were estimated by comparisons of distance travelled by each fragment with distance travelled by known size fragments of the DNA molecular weight markers (100 bp DNA ladder, Genei, India). All distinct bands or fragments (RAPD markers) were thereby given identification numbers according to size and scored visually on the basis of their presence (1) or absence (0), separately for each variety for each primer. The scores obtained using all primers in the RAPD analysis were then pooled to create a single data matrix. This was used to compare the frequencies of all polymorphic RAPD markers among populations with 1000 simulated samples using POPGENE (version 1.31) (Yeh et al., 1999) computer program. The size of the RAPD markers were estimated by using the software DNAfrag, version 3.03 (Nash, 1991). Nei (1972) genetic distance values were computed from frequencies of polymorphic markers using the POPGENE (Version 1.31) computer package (Yeh et al., 1999). The dendrogram was constructed using POPGENE (Version 1.31) (Yeh et al., 1999).