Indian Journal of Plant Sciences ISSN: 2319–3824 (Online) An Open Access, Online International Journal Available at http://www.cibtech.org/jps.htm 2014 Vol. 3 (2) April -June, pp. 180-181/Anitalakshmi et al. Research Article © Copyright 2014 | Centre for Info Bio Technology (CIBTech) 180 CHARACTERIZATION OF CULTIVARS BASED ON ELECTROPHORETIC ANALYSIS OF SEED PROTEINS, ISOZYMES AND DNA MARKERS IN RICE (ORYZA SATIVA L.) V. Anitalakshmi, Rame Gowda, *Sathisha, C.S. and Shailaja Hittalmani Department of Seed Science and Technology University of Agricultural Sciences, G.K.V.K, Bangalore-65 *Author for Correspondence ABSTRACT Studies have been carried out for the identification of the genuiness and purity of eighteen rice genotypes using electrophoretic analyses of total soluble seed proteins, isozymes and DNA. Region A, B and D were found useful to identify most of the genotypes studied by protein bands. Malate dehydrogenase isozyme banding pattern showed variation but, Alcohol dehydrogenase banding pattern was similar among the genoypes. RAPD primers showed 73.43 per cent polymorphism between genotypes studied. Variations were observed between these biochemical markers in terms of number of bands, their relative mobility and intensity of bands. Keywords: Electrophoresis, Gel, Genotypes, Identification, Malate Dehydrogenase, Isozymes, Protein, Seed, Rice INTRODUCTION Rice is a major crop in Asia. Genetically divergent rice genotypes are available due to diverse conditions of their cultivation and hence it is very difficult to visually identify cultivars on the basis of phenotypic characters. Uses of biochemical and molecular markers have proved to be of immense help to breeders in improving important agronomic traits in rice. The present investigation was undertaken to characterize the 18 rice genotypes based on the banding pattern of total soluble seed proteins, malate dehydrogenase isozyme and DNA markers as their expression and detection were unaffected by environmental interactions and could be conveniently used as markers in identifying rice genotypes (Cooke, 1987). MATERIALS AND METHODS Seed proteins: SDS-PAGE of total soluble seed proteins was carried out by using 15 per cent gels according to the methods prescribed by Laemmli (1970) with slight modifications. Protein was extracted from single seed by adding 0.2 ml Tris glycine extraction buffer (25 mM, pH 8.5). The suspension was centrifuged at 10000 rpm for 15 minutes. The extract was dissolved in equal amount of sample buffer and kept in boiling water for 2 minutes for denaturation of proteins. Then centrifuged at 10000 rpm for 15 minutes and the supernatant was used for loading on to the gel. A current of 1.5 mA per well with a voltage of 80 volts was applied until the tracking dye crossed the stacking gel. Later the current was increased to 2 mA per well and voltage up to 120 volts. The electrophoresis was stopped when the tracking dye reached the bottom of the resolving gel. Then the gel was stained using coomassie blue solution overnight and destained using a mixture of 227 ml of methanol, 46 ml of acetic acid and 227 ml of distilled water until the bands were clearly visible. Isozymes: Seven day old seedling was ground in 200 μl of extraction buffer (0.1 M Tris-HCl, pH 7.5) and centrifuged at 10000 rpm for 15 minutes and the supernatant was used for loading as prescribed by Glaszman et al., (1988). The gels were stained using Malic acid 15 ml, 15 ml Tris buffer, 50 mg NAD, 50 mg NBT, 25 mg PMS, 75 ml distilled water under dark. DNA analysis: Sixteen days old seedlings were used for DNA extraction. DNA was prepared as per modified CTAB (cetyl trimethyl ammonium bromide) method (Cao and Oard, 1997). The PCR reaction mixture consists of 1.0 μl of template DNA, 22.0 μl primer, 2.0 μl dNTPs, 2.0 μl Tag, 2.0 μl of 1X PCR buffer and 12.6 μl of sterile water in an volume of 20μl and the amplification was carried out as follows: