Cytogenetic Study of Diploid and Induced Tetraploid in Korean Rose Bitterling, Rhodeus Uyekii
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Gil et al. SpringerPlus (2016) 5:186 DOI 10.1186/s40064-016-1830-4 RESEARCH Open Access Cytogenetic study of diploid and induced tetraploid in Korean rose bitterling, Rhodeus uyekii Hyun Woo Gil1*, Hee Jeong Kong2, Cheul Min An2, Bong‑Seok Kim3, Sang‑Gu Lim3 and In‑Seok Park1 Abstract In this study, we induced tetraploidy in Korean rose bitterling, Rhodeus uyekii, by applying various hydrostatic pres‑ sure shock conditions. Tetraploidy was not induced under 4500 psi pressure treatment in any experimental group. Instead, the induction rate of tetraploidy was highest under 7500 psi hydrostatic pressure treatment. As a result, when the processing method was similar and as the process time increased, the induction rate of each experimental group increased; however, there was no significant difference (P > 0.05). The production rate was 3.1 %, which was highest in all experimental groups exposed to 6000 psi for 10 min after being fertilized for 100 min. The production rate was highest in the experimental groups treated with hydrostatic pressure alone, whereas the production rate was lowest in groups treated under hydrostatic pressure with chemical treatment. The abnormal rate of all experimental groups treated with 7500 psi for 20 min was very high, at about 5 %. Based on these studies, only hydrostatic pressure shock was considered effective at inducing tetraploidy based on the calculated hatching, abnormal, and induction rates. The most effective condition for inducing tetraploidy was 6000 psi of hydrostatic pressure shock for 10 min after being fer‑ tilized for 100 min. The chromosome number of the induced tetraploid Korean rose bitterling was 4n 96, while that of the diploid was 2n 48. In the diploid, there were 1 or 2 nucleoli in the cells, whereas the induced =tetraploids con‑ tained 1, 2, 3, or 4. The= DNA content of tetraploids and diploids were 3.68 0.009 pg/nucleus and 1.84 0.019 pg/ nucleus, respectively, according to flow cytometric analysis. The DNA content± and chromosome number± of the tetra‑ ploids were twice that of the diploids. Keywords: Chromosome, Diploid, DNA content, Induced tetraploid, NORs, Rhodeus uyekii Background considered an important method for reproductive con- Tetraploids can artificially produce triploid progeny after tainment of genetically modified fish through the gen- fertilization through normal diploids (Refstie 1981). eration of sterile triploid transgenics using tetraploids Hence, tetraploid breeding lines are beneficial to aqua- and diploids (Nam et al. 2001a). Moreover, it has been culture because they provide a convenient approach to important to develop methods to produce infertile fish produce large numbers of sterile triploid fish through (triploid) to overcome the detrimental effects of sexual simple interploidy crosses between tetraploids and dip- maturation on the growth of many fish species (Malison loids (Chourrout et al. 1986). Successful production of et al. 1986). triploids by crossing diploids and tetraploids has only Several attempts have been made to induce tetraploidy been observed once in fish (Chourrout et al.1986 ). in selected salmonids, cyprinids, and cichlids; however, Since the 1980s, the interploidy technique has also been almost all attempts have failed in cichlids (Pandian and Koteeswaran 1998). Viable induced tetraploids have *Correspondence: [email protected] been generated in a few cyprinids, in which the natu- 1 Division of Marine Bioscience, College of Ocean Science ral occurrence of tetraploids has been reported (e.g., and Technology, Korea Maritime and Ocean University, Busan 606‑791, Korea cyprinid loach, Misgurnus anguillicaudatus, Arai et al. Full list of author information is available at the end of the article 1991; spinous loach, Cobitis biwae, Kusunoki et al. 1994; © 2016 Gil et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Gil et al. SpringerPlus (2016) 5:186 Page 2 of 10 ginbuna, Carassius auratus langdorfii, Kobayashi 1971; Induction of tetraploidy crucian carp, C. auratus gibelio, and common carp, We regulated the photoperiod (13 h light and 11 h dark; Cyprinus carpio, Cherfas et al. 1994). Inhibition of the lights on: 06:00, lights out: 22:00), and fed Artemia an first cleavage, as a technique for chromosome duplica- artificial assorted feed twice a day in the morning and tion, can theoretically induce diploidy in gynogenetically evening. We selected adult Korean rose bitterling (20 activated eggs, as well as in triploid and tetraploid normal females and males), and obtained about 300 fertilized fertilized eggs. Methods used previously include expos- eggs through artificial fertilization (wet method) by artifi- ing fertilized eggs to temperature shock (hot or cold; cial spawning and feeding. Cassanni and Caton 1985; Seol et al. 2008), hydrostatic First, 30 fertilized eggs at 90, 100, 110, and 120 min pressure shock (Cassanni and Caton 1986), or chemicals, after fertilization were treated under each condition such as colchicine (Smith and Leoine 1979), cytocha- (4500, 6000, and 7500 psi) for 10, 20, and 30 min to lasin B (CB; Refstie 1981), and nitrous oxide (Shelton investigate the optimal first treatment time, duration of et al. 1986). Physical treatments include hydrostatic pres- treatment, and pressure treatment. Pressure treatment sure and temperature shock. Compared with tempera- was administered using a pressure treatment instrument ture shock, hydrostatic pressure can produce a higher (Otake 5502-R; Otake, Japan). We used cold water at 4 °C triploidy rate and is easier to administer; therefore, it is to process pressure + cold shock, and used a concentra- commonly used for triploid induction in fish (Lou and tion of 10 ppm solution of CB (Sigma, USA) to process Purdom 1984; Goudie et al. 1995). pressure + chemical treatment. Nam et al. (2001b) reported the long-term viabil- ity and growth performance of induced tetraploid mud Hatching, survival, induction, abnormal, and production loach, Misgurnus mizolepis. Erythrocyte cellular and rates of tetraploids nuclear dimensions of tetraploid mud loach were sig- We determined the survival, abnormal, and induction nificantly higher than those of diploids, as expected. rates in each experiment. The hatching and abnormal Chromosomal and flow cytometric analyses revealed rates were also investigated 24 h after hatching of the the genomic structure of tetraploids as originating from first larvae. The induction and production rates were the doubling of 2n chromosomes, as evidenced by their investigated 2 days after hatching of the last larvae. modal chromosome number and average cellular DNA After investigation of the abnormal rate, the shape of content, which were double the diploid values (Nam the abnormal individual was filmed using a microscope et al. 2001b). In addition, Nam et al. (2001b) confirmed camera (Axioskop; Zeiss, Germany). The production rate that tetraploids showed a significantly depressed growth of tetraploids was calculated based on the hatching and performance based on body weight loss between tetra- induction rates. The survival rates of each group were ploids and diploids. investigated at 10 days after hatching. We determined Korean rose bitterling, Rhodeus uyekii, is an endemic the optimal first treatment time, duration of treatment, Korean fish belonging to the Acheilognathinae subfam- and pressure treatment based on the survival, hatching, ily of the Cyprinidae family. It is distributed in rivers that abnormal, and induction rates. For a synergistic effect empty into the Yellow and Southern Seas in Korea (Kang of the induction method, we investigated various induc- et al. 2005). Recently, this species has been considered a tion methods by treating 30 fertilized eggs with pressure candidate for developing ornamental fish in Korea (Kang treatment, pressure + cold shock, and pressure + chemi- et al. 2005). Therefore, in this study, the cytogenetic char- cal treatment. acteristics, including DNA content, chromosome, and nucleolar organization regions (NORs) between the dip- Ploidy level determination and cytogenetic analysis loid and induced tetraploid were investigated using vari- of diploids and tetraploids ous pressure treatments; then we determined the optimal Flow cytometry cytogenetic analysis was performed tetraploid induction method (4 °C cold shock and CB). based on measurement of the DNA content and deter- mination of triploid induction. Fish were sampled peri- Methods odically and triploid induced samples were determined Experimental fish by flow cytometry (Ploid Analyzer II, Partec, Germany) Korean rose bitterling, Rhodeus uyekii, was supplied of the nuclear DNA content in erythrocytes or fin cells. by the Jinhae Inland National Institute of Fisheries Sci- Chromosome metaphases of each group were observed ence, Korea. A total of 100 females and males were reared for certain determination of triploid induction. We des- at 20 °C in two 100 L square water tanks in the Fishery ignated the experimental groups with the highest induc- Genetic and Breeding Science laboratory of Korea Mari- tion rate as tetraploids. Two days before hatching, Korean time and Ocean University, Korea. rose bitterling larvae were managed by