UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Multidisciplinary approach to genetic variability, inbreeding and fertility in the endangered Sorraia horse breed Doutoramento em Biologia Biologia da Conservação Helena Josefina Kjöllerström Tese orientada por: Profª Doutora Maria do Mar Jácome Félix Oom Prof. Doutor Bhanu Pratap Chowdhary Documento especialmente elaborado para a obtenção do grau de doutor 2016 UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS Multidisciplinary approach to genetic variability, inbreeding and fertility in the endangered Sorraia horse breed Doutoramento em Biologia Biologia da Conservação Helena Josefina Kjöllerström Tese orientada por: Profª Doutora Maria do Mar Jácome Félix Oom Prof. Doutor Bhanu Pratap Chowdhary Júri: Presidente: ●Doutora Maria Manuela Gomes Coelho de Noronha Trancoso Vogais: ● Doutora Terje Raudsepp ● Doutora Raquel Maria Garcia dos Santos Chaves ● Doutor José António dos Santos Pereira de Matos ● Doutor Luís Lavadinho Telo da Gama ● Doutora Maria Manuela Gomes Coelho de Noronha Trancoso ● Doutora Maria do Mar Jácome Félix Oom Documento especialmente elaborado para a obtenção do grau de doutor SFRH/BD/81502/2011, cE3c UID/BIA/00329/2013, PRODER (57882)(EU)/Action 2.2.3 (2011-2013), LINK Equine Research Endowment, American Quarter Horse Foundation and USDA (#2012-67015-19632) 2016 This work was supported by FCT/MEC (Fundação para a Ciência e Tecnologia) PhD Fellowship (SFRH/BD/81502/2011), cE3c FCT Unit funding (Ref. UID/BIA/00329/2013), PRODER (Contract number 57882)(EU)/Action 2.2.3 (2011-2013) [Conservation and improvement of genetic resources, Sub-action 2.2.3.2. Animal component (Sorraia breed)], LINK Equine Research Endowment, American Quarter Horse Foundation and USDA (grant #2012-67015-19632). This dissertation should be cited as: Kjöllerström, H.J. (2016) Multidisciplinary approach to genetic variability, inbreeding and fertility in the endangered Sorraia horse breed. PhD Thesis, University of Lisbon, Portugal To my Family and Friends The wind of heaven is that which blows between a horse's ears. ~Arabian Proverb Nota prévia Na elaboração da presente dissertação e, nos termos do nº 2, alínea a) do Artigo 25 do regulamento de Estudos de Pós-Graduação da Universidade de Lisboa, publicado em Diário da República nº 57, 2ª Série de 23 de Março de 2015, esclarece-se que apenas foram considerados integralmente artigos científicos originais já publicados (4), submetidos (1) ou em preparação (1), em revistas indexadas de circulação internacional, os quais integram os capítulos da presente tese. A candidata declara que participou integralmente no planeamento, elaboração, análise e discussão dos resultados de todos os trabalhos com primeira autoria. No caso dos trabalhos de colaboração, nomeadamente o intitulado Genetic diversity and demographic structure of the endangered Sorraia horse breed assessed through pedigree analysis (Capítulo 2, Paper 1), a autora participou na análise, interpretação e discussão de resultados, assim como na preparação do manuscrito. A presente dissertação, por ser uma compilação de artigos científicos internacionais, foi redigida em língua Inglesa. No final de cada capítulo é apresentada uma lista de referências em vez de uma única lista no final da tese, razão pela qual poderá ocorrer duplicação das mesmas nos diferentes capítulos. Cada capítulo tem, também, associada a respectiva informação de suporte, quando necessária. Alguns capítulos têm formato diferente devido aos diferentes requisitos das revistas científicas nos quais os mesmos foram publicados, submetidos ou virão a ser submetidos. Preliminary note In the preparation of this dissertation, in accordance with paragraph 2, a) of Article 25 of the Post-Graduate Studies Regulation of the University of Lisbon (Diário da República nº 57, 2ª Série de 23 de Março de 2015), only original scientific articles already published (4), submitted (1) or in preparation (1), in international indexed journals were considered, integrating the chapters of this thesis. The candidate fully participated in the planning, preparation, result analysis, discussion and writing of the manuscripts presented as first author. In case of collaborative work, such as Genetic diversity and demographic structure of the endangered Sorraia horse breed through pedigree analysis (Chapter 2, Paper 1), the author participated in the analysis, interpretation and discussion of results, as well in the manuscript preparation. In addition, because this dissertation is composed of a series of international publications, it has been written in English. A reference list at the end of each chapter, rather than in the end of the dissertation, is presented. This may lead to references duplication between chapters. Each chapter has also their own supplementary information, when necessary. The different format between chapters is a direct result of the different requirements from the scientific journals in which they were published, or will be submitted. TABLE OF CONTENTS ACKNOWLEDGMENTS III RESUMO / PALAVRAS-CHAVE VI ABSTRACT / KEYWORDS XI CHAPTER 1: INTRODUCTION 1 1.1 - The Sorraia horse breed 2 1.2 - Genetic variability 9 1.2.1 - Pedigree analysis 9 1.2.2 - Molecular analysis 11 1.3- Reproductive fitness and inbreeding 16 1.4- Specific aims 20 1.5- Thesis Structure 21 REFERENCES 24 CHAPTER 2: BREED CHARACTERIZATION 33 PAPER 1 - Pinheiro M., Kjöllerström H.J. & Oom M.M. (2013) Genetic diversity and demographic structure of the endangered Sorraia horse breed assessed through 34 pedigree analysis. Livestock Science 152, 1-10 PAPER 2 - Kjöllerström H.J. & Oom M.M. Morphological characterization and inbreeding effects in the endangered Sorraia Horse breed (submitted to the Journal of 45 Animal Science) PAPER 3 - Kjöllerström, H.J., Raudsepp, T., Khanshour, A., Gosh, S., Oom, M.M., Chowdhary, B.P. Genome-wide variability in the Sorraia horse (In preparation for PLoS 70 ONE) CHAPTER 3: REPRODUCTIVE FITNESS 115 PAPER 4 - Kjöllerström H.J., Gama L.T. & Oom M.M. (2015) Impact of inbreeding on fitness-related traits in the highly threatened Sorraia horse breed. Livestock Science 116 180, 84-9 I PAPER 5 - Kjöllerström H.J., Collares-Pereira M.J. & Oom M.M. (2011) First evidence of sex chromosome mosaicism in the endangered Sorraia Horse breed. Livestock Science 123 136, 273-6 PAPER 6 - Kjöllerström H.J., Oom M.M., Chowdhary, B.P & Raudsepp, T. (2016) Fertility assessment in Sorraia stallions by sperm-FISH and FKBP6 genotyping. Reproduction in 128 Domestic Animals 51, 351-9 CHAPTER 4: DISCUSSION 143 4.1 - Breed Characterization 144 4.2 - Reproductive fitness 152 CHAPTER 5: FINAL REMARKS 157 REFERENCES 160 GLOSSARY 166 II Acknowledgements III Acknowledgements First and foremost, I wish to thank my supervisor, Prof. Maria do Mar Oom: without your help, encouragement, support and faith in me, none of this would have been possible. Thank you for pushing me through all the hardship and chaos that happened a little bit throughout these years. A special thank you to my co-supervisor, Dr. Bhanu P. Chowdhary: you gave my PhD a new route and I learned so much because of that. Thank you for the opportunity you gave me to join your lab at Texas A&M. My work and knowledge greatly improved because of that. To Dr. Terje Raudsepp, a big thank you for all you taught me while I was at your lab. Thank you for all the discussions we had and how much you helped me when I was lost in what sense to make of my results. Thank you for making feel part of your family and for always welcoming me in your house and lab. To Prof. Maria João Collares-Pereira, for all the help and wisdom shared regarding cytogenetics. To my dad, a special thank you - your “paitrocínio” made my PhD happen! It made field work possible, travelling to Texas and also supported me for half of my PhD. Thank you for always supporting me and “forcing” me to do this because “the family business was not my future”, as you like to put it, except when it comes to meetings in Spain, ahahah. To my mom for putting up with my meltdowns when push came to shove, and for always having wise words of encouragement to share with me. To my sisters, publish or perish forever!! I love you, thank you for your support and faith in me, even in times when I didn’t believe in myself. To my nephews and niece, you always find a way to brighten up my days. Thank you for all the laughter, smiles and unconditional love. To Raquel because putting up with me for 6 years, through all the hard times I’ve been through, can’t have been easy. Thank you for never giving up on me and always being there for me, for whatever I needed at whatever time. I couldn’t have done it without you (literally!). Thank you for all the help: you’ve taught me to be a better person. To Carla, for the companionship and scientific discussions. Thank you for all you’ve taught me and for being there for me when I needed. To Ana Catarina, because Texas (and clearly the Universe) decided we needed to meet and help each other. You know you have a very special place in my life and heart; you’ve helped me through this journey. I’ve grown to be a different and better person because of you and your teachings. Thank you! To my Portuguese friends, because I know I’m a pain and have made your lives harder at points because of my meltdowns and doubts. To my Texas family, I cannot imagine my life without you. You have made my life so much better. At the same time I’ve discovered so many new things about myself. I’ve become a better person from meeting you. To Kathy, thank you for making me a part of your family, for all the experiences shared and for all you’ve done for me throughout the years. Thank you! To Doug, Lori and Clementine, thank you for everything and for making me part of your family.