Sex Determination and Genetic Management in Nile Tilapia using Genomic Techniques A Thesis Submitted for the Degree of Doctor of Philosophy By Taslima Khanam Institute of Aquaculture, Faculty of Natural Sciences, University of Stirling, Stirling, UK January 2017 Acknowledgements My PhD has been an amazing experience and it would have never been achievable without the generous support and guidance that I received from very important and special persons. The first person I would like to express my heartfelt gratitude to my Principal supervisor Prof. Brendan J. McAndrew for accepting me as a PhD student under his scholastic supervision. I would like to say a very big thank you for all the supports and encouragements during the study period. Thank you very much for being there always and quickly sorted when I needed any support, which made my PhD a wonderful experience. I sincerely appreciate your active participation during thesis/manuscript write up and for valuable suggestions. I would like to express my indebtness to my supervisor Dr. David J. Penman wholeheartedly; you have been a tremendous mentor for me. Your kind support, intellectual stimulation, constant valued guidance, judicious constructive criticism and encouragement made my PhD a pleasing experience and enabled me to grow as a researcher. Thank you very much Dave for all the time invested in me to improve my research thinking and keeping your door always open to talk to you, and active participation while I was doing experiments and writing up articles/thesis. Your willingness to introduce me with the renowned scientists has been priceless. I am very pleased that I did my PhD research under both of your supervisions. I feel short of vocabulary to put across my profound gratitude to Dr. John B. Taggart, who has been truly a dedicated mentor. Thanks very much John for sharing your expertise with me, your guidance during the lab work, fantastic hands-on training on NGS techniques and bioinformatics, and making me understand the technical part of the genetics and genomics field in a very friendly environment. Special thanks to Dr. John Benzie and Dr. Hugues de Verdal (WorldFish Center) for their excellent collaboration during my PhD period. Many thanks to Dr. Stefanie Wehner, it would have never been possible to analyse the NGS data without your expertise. Thanks to Dr. Michaël Bekaert (although you were there at the end of my PhD) to share your expertise with me and for your guidance during the data analysis. I would like to extend my sincere thanks to Dr. Jean- Francois Baroiller and Dr. Helena D'Cotta (CIRAD) for sharing their research experiences and for the discussions we had. My special thanks to Mr. Keith Ranson, Mr. Brian Craig and Miss Kirsten McCallum, Dr. Kerry Bartie and Dr. Sarah-Louise Counter, your support was extremely helpful in the Tropical Aquarium Facilities and in the laboratory. Mr. Charlie Harrower, Miss Jane Lewis and Melanie Cruickshan were very helpful to provide all the research materials and for helping me out to go to different countries for Conference/Training. Taslima Khanam Institute of Aquaculture iii I would like to thank past and present members of the Genetics research group Dr. Milton, Dr. Christos, Dr. Syaifudin, Khalfan, Dr. Munevver, Dr. Joanna, Sienna, Savitree for sharing the ideas and for their friendship. I would like to thank my friends Dr. Aliya, Dr. Olga, Dr. Fani, Aisha, Hayat, Rita, Houra, Munira, Ying, Suleiman and Sumaiya, Ahmed and Shomous, Angela, Yehwa, Margaret, Dr. Greta, Lucas, Michael and the list goes beyond…….., whose company was always there. Many thanks to the Nepali family (Madhab Dada, Saru Didi, Mansi and Prisha) for their support and care. I would like to take the privilege to extend my acknowledgements to the Bengali community in the UK for their constant hospitality and making me feel at home always. I am grateful to Prof. Dr. Md. Abul Khair Chowdhury (BAU) for his endless encouragements, inspirations and supports; and many thanks to Prof. Dr. Mohammad Mahfujul Haque Ripon (BAU) for inspiring me to pursue my PhD at the Institute of Aquaculture. My parents, I am so grateful to you for bringing me up with blessings, love, care, sacrifices, immense patience and inspirations (always with me). My heartfelt thanks to my lovely little sister (Lira), my beloved brothers (Nipu and Nithu), my uncle (Sanu mama) for their love, mental supports, blessings and wanted the best for me always. I would like to thank my mother-in-law and in-laws who supported me with blessings and care during my PhD. My best friend and husband Dr. Atiqur Rahman, thank you very much for your unending and unconditional love and supports, inspiring me always to achieve my dreams, teaching me how to be patient, optimistic and think positive. Thanks for making my every moment enjoyable and being with me always in my success and also when I lost my way. Finally special thanks to the Commonwealth Scholarship Commission (UK) for sponsoring me the PhD Scholarship and University of Stirling for hosting me as a PhD researcher and to have all the experiences mentioned above and beyond. Many thanks to the Genetic Society (GS), the Fisheries Society of the British Isles (FSBI) for providing grants to attend the international conferences, and the European Aquaculture Society (EAS) for their support in the Aquaculture Europe Conference 2016. I humbly express my gratefulness to the Almighty and I sincerely believe that without His blessings, I would not have come all this way. Taslima Khanam January 2017 Taslima Khanam Institute of Aquaculture iv Dedicated To The departed soul of my father Taslima Khanam Institute of Aquaculture v Published Articles, Conferences and Trainings List of Published Papers in Peer Reviewed Journals Taslima K., Davie A., McAndrew B.J., Penman D.J. 2016. DNA sampling from mucus in the Nile tilapia, Oreochromis niloticus: minimally invasive sampling for aquaculture- related genetics research. Aquaculture Research, 47:4032-4037. Taslima K., Taggart J.B., Wehner S., McAndrew B.J., Penman D.J. 2016. Suitability of DNA sampled from Nile tilapia skin mucus swabs as a template for ddRAD-based studies. Conservation Genetics Resources, doi: 10.1007/s12686-016-0614-z. Conference Presentations Taslima K., Khan M.G.Q., Taggart J.B., Wehner S., de Verdal H., Benzie J., McAndrew B.J., Penman D.J. 2016. Sex determination in Nile tilapia (Oreochromis niloticus) varies among populations. Aquaculture Europe, Edinburgh, Scotland, 20-23 September. Taslima K., Taggart J.B., Wehner S., Andrew D., McAndrew B.J., Penman D.J. 2016. Minimally invasive DNA sampling from fish mucus for standard genotyping and next generation sequencing. Annual Symposium of the Fisheries Society of the British Isles- Fish, Genes & Genomes: Contributions to Ecology, Evolution & Management in Bangor University, North Wales UK, 18-22 July. Taslima K., Taggart J.B., Wehner S., de Verdal H., Benzie J., McAndrew B.J., Penman D.J. 2016. Identifying the location of the major sex determining locus in Genetically Improved Farmed Tilapia using BSA-ddRAD sequencing. 4th International Symposium on Genomics in Aquaculture (GIA), Greece, 20-22 April. Taslima K., Taggart J.B., Bekaert M., Penman D.J., McAndrew B.J. 2015. The use of RADseq for marker-assisted selection for controlling sex-ratio in Nile tilapia. 4th PhD Research Conference, Institute of Aquaculture, University of Stirling UK, 18th February. Training Courses Attended An introduction to solving biological problems with Perl, University of Cambridge, UK, September 2016. An introductory R course, Institute of Aquaculture, University of Stirling, UK, November 2015. Introduction to RNAseq and ChIPseq data analysis, University of Cambridge, UK, August 2015. From genome-wide association studies (GWAS) to function, Italy, June 2015. Introduction to RADseq data analysis workshop, Edinburgh Genomics, University of Edinburgh, UK, May 2015. A general introduction to Minitab, Institute of Aquaculture, University of Stirling, UK, April 2015. Home Office animal handling modules 1-3 (Fish) under the Animals (Scientific Procedures) Act 1986, University of Glasgow, UK, October 2013. Taslima Khanam Institute of Aquaculture vi Abstract The PhD research studied two aspects in tilapia, firstly the analysis of sex determination in Nile tilapia (evidence of complex sex-determining systems) and secondly the genetic management of the tilapia species, using different genomic analysis approaches. This research started with the development of two techniques: minimally invasive DNA sampling from fish mucus, which was found to be suitable for standard genotyping and double-digest restriction-site associated DNA sequencing – ddRADseq; and pre-extraction pooling of tissue samples for ddRADseq (BSA-ddRADseq), which was found to be suitable for identifying a locus linked to a trait of interest (sex in this case). The first molecular evidence concerning the sex determination in genetically improved farmed tilapia (GIFT) was described using BSA-ddRADseq. Given the multiple stock origin of GIFT, surprisingly only a single locus (in linkage group 23) was found to be associated with the phenotypic sex across the population. The first evidence of LG23 influence on phenotypic sex in the Stirling population of Nile tilapia was also found. Different combinations of estrogen hormones and high temperature were tested for feminising Nile tilapia: a combined treatment of estrogen hormone and high temperature was found to be more efficient in feminising Nile tilapia than the estrogen alone. A set of species- diagnostic SNP markers were tested which were found to be suitable to distinguish pure species (O. niloticus, O. mossambicus and O. aureus), and these were used to analyse species contribution to GIFT and a selected tilapia hybrid strain. The results of the current research added novel information to our understanding of sex determination in Nile tilapia, which will be helpful in the development of marker-assisted selection in GIFT and other Nile tilapia strains towards the production of all male offspring.