Publication: Genetic studies of tropical ornamental fish
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Date
1997
Authors
Lim, Chee Whye
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Abstract
The discovery that the use of PCR with an arbitrarily selected primer to amplify a specific set of arbitrarily distributed loci in any genome laid the foundation for high output of genetic markers that can be used for a variety of purposes. In this study, RAPD was utilized as a method in the identification, interrelationship analysis and the construction o f a basic linkage map of medaka (Oryzias latipes). The optimized RAPD-PCR protocol obtained, reveal a moderate degree of genetic diversity among the strains of medaka studied.<br><br>Coefficient of similarity based on RAPDs between individuals from each strain ranged from 93.2% to 99.7% with the two inbred strains, HO5 and HNI, having similarity coefficients of 99.78% and 99.42% respectively. Matching coefficients between strains based on RAPDs ranged from 0.7 to 0.95 suggest a narrow genetic base for the medaka studied. Cluster analysis of RAPDs employing UPGMA grouped the medaka strains examined into 3 main groups. The HNI strain, the local fish obtained from Singapore, and the clustering of HO5 with all the mutant strains examined in one group. Medaka species specific DNA markers were obtained but strain-specific markers were only observed in HNI strain while markers specific for all the mutant strains derived from HO5 strain were also observed. The optimized RAPD-PCR protocol for medaka was also suitable for the analysis of two other organisms.<br><br>Using MAPMAKER, 13 linkage groups were generated with 53 segregating fragments falling into one of the linkage groups that covered 938.1 cM. However, 26 of the total 79 markers still remained unlinked. The two loci investigated, Wy and Red, were successfully included into linkage groups that covered 472.7 cM.<br><br>The cytoplasmic injection into fish egg is performed at the first cell stage, in order to maximize the chance of early integration into all cell lineages. Microinjection technique was employed for transferring the chimeric gene pOBZ-109 into the early stage of zebrafish embryos. Both the linearized and supercoiled forms of the injected DNA were shown to be replicated and expressed in the zebrafish, albeit in a mosaic fashion. Expression of lacz was observed in microinjected fish, with the expression concentrated in the regions surrounding the yolk of the fish suggesting transient expression. The assaying for transgenesis by PCR has made it feasible to screen for large number of sample in this study. Circular and linearized pOBZ109 were detected in 3 day old and 12 week old fish by PCR using the primer pair X-galC and OBA-15 which amplifies a 603 bp DNA fragment. However, variation in the intensity of the band were observed among the fish indicating that the initial concentration of the transgene sequence in the template used in PCR varies among the different fish samples. This result shown that foreign genes which were introduced at the beginning of fish embryogenesis, were not just present but were also replicated through the gastrula stage.