D.J. Penman Institute of Aquaculture, University of Stirling Stirling FK9 4LA, Scotland

OVERVIEW OF AQUACUL ACULTURE GENETICS S RESEARCH IN THE INSTITUTE OF AQUACUL ACULTURE, UNIVERSITY OF STIRLING D.J. Penman Institute of Aquaculture, University of Stirling Stirling FK9 4LA, Scotland PENMAN, D.J. 2001. Overview of aquaculture genetics research in the Institute of Aquaculture, University of Stirling, p. 145-149. In M.V. Gupta and B.O. Acosta (eds.) Fish genetics research in member countries and institutions of the International Network on Genetics in Aquaculture. ICLARM Conf. Proc. 64, 179 p. ABSTRACT The Genetics and Reproduction research group is one of the largest within the Institute of Aquaculture (IOA) at the University of Stirling. The institute works with a broad range of species from diverse geographical areas and habitats and has benefited from collaborations with other institutions in many countries. This overview of activities focuses particularly on areas of relevance to the International Network on Genetics in Aquaculture (INGA) and collaborations with INGA members. Aquaculture Genetics Research Molecular taxonomy, population and conservation genetics Allozyme, mtdna, ribosomal DNA, minisatellite and microsatellite DNA variation have been used in Institute of Aquaculture (IOA) laboratories in studies on the taxonomy and population genetics of wild organisms of interest to aquaculture, and captive aquaculture stocks. Examples of these studies are molecular taxonomy and population genetics of tilapias (Sodsuk et al. 1995; Rognon et al. 1996); population genetics of Penaeus monodon (Klinbunga et al. 1996, 1998a, 1998b, 1999), Macrobrachium nipponense (Wong and McAndrew 1994), Dicentrarchus labrax (Castilho and McAndrew 1998; Martinez Rodriguez et al. 1998), Salmo salar (Taggart et al. 1995a, 1995b; Prodoehl et al. 1997; Stone et al. 1997) and Catla catla. IOA s interest in conservation genetics has focused on the use of cryopreservation and on the potential of androgenesis in gene banking, with tilapia and salmonids as the main experimental subjects (McAndrew et al. 1993; Myers et al. 1995a, 1995b; Rana 1995; Penman et al. 1997). The combination of sperm cryopreservation and diploid androgenesis does allow recovery of viable animals from a sperm gene bank, but successful cryopreservation of eggs is still not possible. Broodstock management and quantitative genetics IOA has been involved in collaboration with the University of Agricultural Sciences (UAS), Bangalore, India, and the University of Wales Swansea on broodstock management and selective breeding of C. catla and Cyprinus carpio in Karnataka, India (Basavaraju et al. 1998). In UK, IOA is involved in Overview of Aquaculture Genetics Research in the Institute of Aquaculture, University of Stirling 145

developing broodstock management and selective breeding programs on molecular markers (principally microsatellites), in Atlantic salmon and other coldwater species. Sex determination and differentiation Research on sex determination mechanisms and their manipulation to produce, e.g., monosex stocks, has been a major interest for IOA for some time. Tilapia, principally Oreochromis niloticus, has been the major model, due to its commercial importance, ease of maintenance under laboratory conditions, amenability to genetic and hormonal manipulations, and the emergence of temperature sex determination (TSD) and autosomal genetic factors affecting sex determination in addition to the major (chromosomal) sex determination. Recently, the chromosomal location of the sex determining region in O. niloticus has been demonstrated (Carrasco et al. 1999a). Fully inbred clonal XX and YY lines in this species have been produced (Sarder et al. in press). Studies now focus on molecular and cytogenetic analysis of the sex determination system and on the role of aromatase in sex differentiation and TSD. Collaborative research with the National Aquaculture Genetics Research Institute, (NAGRI) Thailand and the Bangladesh Fisheries Research Institute has led to the development of genetically monosex female production in the silver barb (Barbodes gonionotus) for aquaculture (Pongthana et al. 1995a, 1999). Genetic manipulations Gynogenesis (Hussain et al. 1993, 1995; Myers et al. 1995; Pongthana et al. 1995b) and androgenesis (Myers et al. 1995a, 1995b) have proven to be useful tools in the study of sex determination and the development of monosex culture, the development of clonal lines (Hussain et al. 1998; Sarder et al. in press) and gene mapping, etc. These tilapia clonal lines have already proved to be extremely useful in studies on nonspecific and specific immune responses, and the institute is planning to use them in research on areas as diverse as nutrition and quantitative genetics. Studies have also been carried out on induced triploidy in tilapia (Hussain et al. 1995, 1996), rainbow trout (Carrasco et al. 1999a, 1999b) and halibut in the last few years. Although triploidy could not currently be used commercially in tilapia due to the difficulties of collecting adequate numbers of unfertilized eggs, studies elsewhere have shown its potential in limiting reproduction in pond culture. Triploidy is, however, used commercially in rainbow trout. A recent Ph.D. study at IOA (Carrasco et al. 1998) revealed differences in meiotic chromosome pairing between genetic male and female triploids which may account for the differences in the success of maturation of male and female triploids (male triploids are generally capable of producing some aneuploid sperm whereas female triploids are generally sterile). IOA carried out a policy study on genetically modified fish for the UK Department of the Environment (Woodwark et al. 1994; Penman et al. 1995), which led to drafting guidelines for the UK government. Genomics Gynogenesis has proven to be a useful tool in gene mapping, in addition to the more conventional crosses. IOA initially studied gene-centromere recombination distances for allozymes, color loci, etc (Hussain et al. 1994). More recently, highly polymorphic DNA markers, such as microsatellites and AFLPs or RFLPs have been developed and, in collaboration with Dr. Tom Kocher at the University of New Hampshire, a linkage map has been developed using haploid gynogenetic embryos (Kocher et al. 1998) and genecentromere linkages are being estimated for a wider variety of loci (Markert et al. 1999). IOA has a particular interest in mapping sex determining loci in tilapias and is pursuing a variety of strategies for this. The genomics capabilities at IOA have been strengthened in the last two years by a new initiative known as Aquagene. Prof. Alan Teale and his group come with a background of mammalian genetics, with a past focus on mapping disease resistance genes in cattle and mice, while Dr. John Taggart, Dr. Margaret Cairney and Dr. Mike Leaver are involved in mapping salmonid genomes and identifying ESTs in these species. IOA is currently developing strategies for the detailed analysis of the genetic basis of a number of traits related to disease resistance, growth performance, nutrition and control of reproduction. 146 Fish Genetics Research in Member Countries and Institutions of INGA

Collaborative Projects with INGA Member Countries Aquaculture Development and Coordination Program The Aquaculture Development and Coordination Program (AADCP) was implemented from 1990 to 1994, with funding from the European Union (EU) and the Association of Southeast Asian Nations (ASEAN). Five components in different ASEAN countries were twinned with partner institutions from EU countries (Penman 1995). In component 5 of AADCP (aquaculture genetics), NAGRI in Thailand was twinned with IOA. The two main areas of research were genetic manipulations and population genetics. DFID Fish Genetics Research Programme The IOA genetics group has been involved in several collaborative projects with INGA member countries through projects funded by the Department of International Development (DFID, formerly Overseas Development Agency) Fish Genetics Research Programme (see Mair and Beardmore, this vol., for more details). The main projects under this program are on population genetics of Penaeus sp. (with University of Putra, Malaysia); monosex culture of B. gonionotus (with NAGRI, Thailand, and the Bangladesh Fisheries Research Institute); and aquaculture genetics of Indian and common carps (with UAS, Bangalore, India, and University of Wales, Swansea, UK). Ph.D. studentships, visiting scientists and others The IOA genetics group has been fortunate to have had several Ph.D. students from INGA countries in the research group over the last decade. The students came from Thailand, Bangladesh and Malaysia and were funded through international programs (e.g., AADCP) and UK or local government, etc. There were also visiting scientists from a number of countries, including India and Thailand, under various schemes. Every year, there are M.S. Aquaculture students carrying out their research projects in IOA. IOA hopes to obtain funding for new projects in the near future, and welcomes new collaborations through INGA. References Basavaraju, Y., B.S. Renuka Devi, G. Mukthayakka, L. Purushotham Reddy, G.C. Mair, E.E. Roderick and D.J. Penman. 1998. Evaluation of marking and tagging methods for genetic studies in carp. J. Biosci. (India) 23: 585-593. Carrasco, L.A.P., D.J. Penman and N. Bromage. 1999a. Evidence for the presence of sex chromosomes in the Nile tilapia (Oreochromis niloticus) from synaptonemal complex analysis of XX, XY and YY genotypes. Aquaculture 173: 207-218. Carrasco, L.A.P., D.J. Penman and N. Bromage. 1999b. Morphometric parameters of commercial interest in triploid rainbow trout, Oncorhynchus mykiss. Aquatic 6: http://aquatic.unizar.es/ (In Spanish). Carrasco, L.A.P., D.J. Penman, S. Doroshov and N. Bromage. 1998. Long-term, quantitative analysis of gametogenesis in autotriploid rainbow trout, Oncorhynchus mykiss. J. Reprod. Fertility 113: 197-210. Castilho, R. and B. McAndrew. 1998. Two polymorphic microsatellite markers in the European seabass, Dicentrarchus labrax (L.). Anim. Genet. 29: 151. Hussain, M.G., B.J. McAndrew and D.J. Penman. 1995b. Phenotypic variation in meiotic and mitotic gynogenetic diploids of the Nile tilapia (Oreochromis niloticus L.). Aquacult. Res. 26: 205-212. Hussain, M.G., D.J. Penman and B.J. McAndrew. 1996. Effects of triploidy on sexual maturation and reproduction in Nile tilapia, Oreochromis niloticus L., p. 320-325. In R.S.V. Pullin, J. Lazard, M. Legendre, J.B. Amon Kothias and D. Pauly (eds.) Proceedings of the Third International Symposium on Tilapia in Aquaculture. ICLARM, Manila, Philippines. 575 p. Hussain, M.G., D.J. Penman and B.J. McAndrew. 1998. Production of heterozygous and homozygous clones in the Nile tilapia. Aquacult. Int. 6: 197-205. Hussain, M.G., B.J. McAndrew, D.J. Penman and P. Sodsuk. 1994. Estimating gene-centromere recombination frequencies in gynogenetic diploids of Oreochromis niloticus L., using allozymes, skin colour and a putative sex-determination locus (SDL-2), P. 502-509. In A. Beaumont (ed.) Genetics and evolution of aquatic organisms. Chapman and Hall. Hussain, M.G., D.J. Penman, B.J. McAndrew and R. Johnstone. 1993. Suppression of first cleavage in the Nile tilapia, Oreochromis niloticus L. - a comparison of pressure and heat shocks. Aquaculture 111: 263-270. Hussain, M.G., G.P.S. Rao, N.M. Humayan, C.F. Randall, D.J. Penman, D. Kime, N.R. Bromage, J.M. Myers and B.J. McAndrew. 1995. Comparative performance of growth, biochemical composition and endocrine profiles in diploid and triploid Oreochromis niloticus L. Aquaculture 138: 87-98. Klinbunga, S., D.J. Penman and B.J. McAndrew. 1998b. A preliminary study of ribosomal DNA polymorphism in Overview of Aquaculture Genetics Research in the Institute of Aquaculture, University of Stirling 147

the tiger shrimp, Penaeus monodon. J. Mar. Biotechnol. 6: 186-188. Klinbunga, S., D.J. Penman, B.J. McAndrew and A. Tassanakajon. 1999. Mitochondrial DNA diversity in three populations of the giant tiger shrimp Penaeus monodon. Mar. Biotechnol. 1: 113-121. Klinbunga, S., S. Sodsuk, D.J. Penman and B.J. McAndrew. 1996. An improved protocol for total DNA isolation and visualisation of mtdna RFLP(s) in tiger prawn, Penaeus monodon. Thai J. Aquat. Sci. 3: 36-41. Klinbunga, S., D.J. Penman, B.J. McAndrew, A. Tassanakajon and P. Jarayabhand. 1998a. Genetic variation, population differentiation and gene flow of the giant tiger shrimp (Penaeus monodon) inferred from mtdna- RFLP data, p. 51-59. In T.W. Flegel (ed.) Advances in shrimp biotechnology. National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand. Kocher, T.D., W.-J. Lee, H. Sobolewska, D. Penman and B. McAndrew. 1998. A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus). Genetics 148: 1225-1232. Markert, J.A., T.D. Kocher, D. Penman and I. Karayucel. 1999. Gene-centromere distances in tilapia, p.33. In Plant and animal genome. Vol. 7: Abstracts. San Diego, California, USA. Martinez Rodriguez, G., M.C. Alvarez and B.J. McAndrew. 1998. Genetic variability of European sea bass, Dicentrarchus labrax L.: data from a hatchery stock. Aquacult. Res. 29: 851-853. McAndrew, B.J., K.J. Rana and D.J. Penman. 1993. Conservation and preservation of genetic variation in aquatic organisms, p. 295-336. In J.F. Muir and R.J. Roberts (eds.) Recent advances in aquaculture. Vol. 4. Croom Helm. Myers, J.M., S.F. Powell and B.J. McAndrew. 1995a. Induction of tetraploidy in brown trout, Salmo trutta L., using hydrostatic pressure. Aquacult. Res. 26: 229-232. Myers, J.M., D.J. Penman, Y. Basavaraju, S.F. Powell, P. Baoprasertkul, K.J. Rana, N. Bromage and B.J. McAndrew. 1995b. Induction of diploid androgenetic and mitotic gynogenetic Nile tilapia (Oreochromis niloticus). Theor. Appl. Genet. 90: 205-210. Penman, D.J. 1995. The ASEAN-EC Aquaculture Development and Coordination Programme (AADCP). EC Fish. Coop. Bull. 8(2): 13-17. Penman, D.J., J.M. Myers and B.J. McAndrew. 1997. Restoration of diploid genotypes by androgenesis, p. 469-473. Sciences et Techniques du Froids. Actes du Colloques Refrigeration and Aquaculture, Bordeaux Aquaculture 96, 20-22 March 1996. Penman, D.J., M. Woodwark and B.J. McAndrew. 1995. Genetically modified fish populations, p. 22-27. In Environmental impacts of aquatic biotechnology. OECD, Paris. Pongthana, N., P. Baoprasertkul and D.J. Penman. 1995b. Hormonal feminisation in the catfish Clarias macrocephalus. Aquaculture 137: 158-159. Pongthana, N., D.J. Penman, J. Karnasuta and B.J. McAndrew. 1995a. Induced gynogenesis in the silver barb (Puntius gonionotus Bleeker) and evidence for female homogamety. Aquaculture 135: 267-276. Pongthana, N., D.J. Penman, P. Baoprasertkul and B. Tongmee. 1995b. Sex identification in tilapia. Ext. Pap. No. 3, 20 p. National Aquaculture Genetics Research Institute, Department of Fisheries, Thailand. (In Thai/ English). Pongthana, N., D.J. Penman, P. Baoprasertkul, M.G. Hussain, M.S. Islam, S.F. Powell and B.J. McAndrew. 1999. Monosex female production in the silver barb (Puntius gonionotus Bleeker). Aquaculture 173: 247-256. Prodoehl, P.A., A.F. Walker, R. Hynes, J.B. Taggart and A. Ferguson. 1997. Genetically monomorphic brown trout (Salmo trutta L.) populations, as revealed by mitochondrial DNA, multilocus and single-locus minisatellite (VNTR) analyses. Heredity 79: 208-213. Rana, K. 1995. Cryopreservation of aquatic gametes and embryos: recent advances and applications, p. 85-89. In F.W. Goetz and P. Thomas (eds.) Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish, 2-8 July 1995, University of Texas at Austin, USA. Rana, K.J., B.J. McAndrew, G. Wohlfarth and I. Macgowan. 1996. Observations on intergeneric hybrids in tilapias, p. 391-397. In R.S.V. Pullin, J. Lazard, M. Legendre, J.B. Amon Kothias and D. Pauly (eds.) Proceedings of the Third International Symposium on Tilapia in Aquaculture. ICLARM, Manila, Philippines. 575 p. Rognon, X., M. Andriamanga, B. McAndrew and R. Guyomard. 1996. Allozyme variation in natural and cultured populations in two tilapia species: Oreochromis niloticus and Tilapia zillii. Heredity 76: 640-650. Sarder, M.R.I., D.J. Penman, J.M. Myers and B.J. McAndrew. Production and propagation of fully inbred clonal lines in the Nile tilapia (Oreochromis niloticus L.). J. Exp. Zool. (In press). Sodsuk, P.K., B.J. McAndrew and G.F. Turner. 1995. Evolutionary relationships of the Lake Malawi Oreochromis species: evidence from allozymes. J. Fish Biol. 47: 321-333. Stone, C.E., J.B. Taggart and A. Ferguson. 1997. Single locus minisatellite DNA variation in European populations of Atlantic salmon (Salmo salar L.). Heredity 126: 269-275. Taggart, J.B., P.A. Prodoehl and A. Ferguson. 1995a. Genetic markers for Atlantic salmon (Salmo salar L.): single locus inheritance and joint segregation analyses of minisatellite (VNTR) DNA loci. Anim. Genet. 26: 13-20. Taggart, J.B., E. Verspoor, P.T. Galvin, P. Moran and A. Ferguson. 1995b. A minisatellite DNA marker for discriminating between European and North American Atlantic salmon (Salmo salar). Can. J. Fish. Aquat. Sci. 148 Fish Genetics Research in Member Countries and Institutions of INGA

52: 2305-2311. Trillo, E.M., Z.Z. Rutczynska, B. McAndrew, V. Vincek, F. Figueroa, H.S. Ultmann and J. Klein. 1998. Linkage relationships and haplotype polymorphism among cichlid mhc class ii b loci. Genetics 149:1527-1537. Wong, J.T.Y. and B.J. McAndrew. 1994. Allozyme variation in riverine and lacustrine populations of Macrobrachium nipponense (de Haan). Aquacult. Fish. Manage. 25: 393-400. Woodwark, M., D.J. Penman and B.J. McAndrew. 1994. Genetic modification of fish: a UK perspective. Genetically Modified Organisms Res. Rep. No. 2, UK Department of the Environment. 84 p. Overview of Aquaculture Genetics Research in the Institute of Aquaculture, University of Stirling 149

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