| The present investigation was taken up to assess the resolving power of isozymes, RAPD and SSR fingerprinting techniques. Analysed were 29 materials including 20 parental lines and 9 of the most widely cultivated hybrid rice varieties in China, and to establish a standard procedure for hybrid rice identification. Genetic diversity was estimated and relationships between the 20 parental materials were also analysed.1. The results of the electrophoresis analysis of esterase and peroxidase isozymes extracted from dry seeds were similar to those obtained using isozymes extracted from seedlings. This indicated that seed-isozymes could also be fully used for hybrid rice identification. The best method for DNA extraction was CTAB compared to SDS. The PCR products of DNA extracted with CTAB method were stable. It is economical to use DNA extraction from dry seeds without liquid nitrogen.2. Esterase electrophoresis is convenient at analysing purity and in identifying hybrid rice. Est2 and Est3 revealed co-dominance bands for the parents. Polyacrylamide gel electrophoresis were made using 7.5% separation gel(pH 8.9 ); 2.5% concentration gel (pH 6.7); and Tris bufTer(pH 8.3). The optimum RAPD amplification reaction system was: 12.5 ul of PCR reaction mixture containing 1.25ul of 10 X PCR buffer, 5-50ng of template DNA, 200 ug/L of primers, 2.0 mol/L of Mg2+, 200 umol/L of dNTPs, 0.5U of Taq polymerase. The PCR profile was 40 cycles of 94 "C for 30s, 36 "C for 30s,72"C for lmin,and finally by lOmin at 72'C for the final extension. The amplification products were analysed by 1% agarose gel electrophoresis. The optimum SSR amplification reaction system was: lOul of PCR reaction mixture containing 20ng of template DNA, 1.5mmol/l of Mg2+, 0.36umol/L of primers, 0.2mmol/l of dNTPs, 2.0ul of 5 X SSR buffer and 0.5U of Taq DNA polymerase. The amplification profile was 94XI!for 45s followed by 30 cycles of 94"C for 45s, 56"C for 45s,72"C for lmin,and finally by 8min at 72 for the final extension.3. The level of polymorphism in isozymes was low; it could differentiate only 4 crossing groups out of 9 and their parents. From the 100 RAPD random primers investigated, 28 were polymorphic, producing 48 polymorphic bands, 1-4 polymorphic fragments were amplified per primer, with an average of 1.72 fragment. Out of the 26 pairs of primers, 21 (87.5%) were found to be polymorphic and produced a total of 69 bands. 2-7 polymorphic bands were amplified per pair of primers, with an average of 3.29 bands per pair of primers. The SSRs were found to be the most polymorphic markers and even one pair of primers (eg RM228) could be used to identify all the 9 hybrids and then-parents.4. The cluster analysis of the 29 materials using RAPD and SSR showed similar results. The results revealed that all the parental materials were divided in two main groups. Aparted from parents of two lines hybrids (Liang you pei 9), the sterile lines and restore lines of the other varieties were divided in one group respectively. Varieties II-32A and Gang-46A were found to be closest genetically, while the other sterile lines were grouped together. The 6 sterile lines divided in 3 groups: they are xieqingzao-A; longtepu-A and V20A ^ zhenshan97A> H-32A > Gang-46A. Varieties Gang-46A and 9311 were the most distant genetically.5. The investigations also revealed that, purity analysis based on esterase isozyme, RAPD and SSR could nearly be accorded with field trial results.6. In this study dry seeds were used to extract isozyme and DNA. Compared to using seedlings and leaves, the method is convenient, fast and efficient. The materials eight three-lines-hybrid rice and one two-lines-hybrid rice cover mostly in China. The same materials were analysed using isozymes, RAPD and SSR techniques. The results showed that the selected primers were highly polymorphic, stable and efficient for the differentiation of the main hybrid rice cultivated in China. The techniques (esterase isozymes, RAPD and SSR) used in this study have provided fingerpr...
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