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STUDIES on MOLECULAR GENETIC DIVERSITY and USEFUL GENOMIC TRAITS of YAM ( DIOSCOREA SPP) GERMPLASM COLLECTIONS from ETHIOPIA

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STUDIES on MOLECULAR GENETIC DIVERSITY and USEFUL GENOMIC TRAITS of YAM ( DIOSCOREA SPP) GERMPLASM COLLECTIONS from ETHIOPIA STUDIES ON MOLECULAR GENETIC DIVERSITY and USEFUL GENOMIC TRAITS of YAM (DIOSCOREA SPP) GERMPLASM COLLECTIONS FROM ETHIOPIA A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES FACULTY OF LIFE SCIENCES ADDIS ABABA UNIVERSITY BY ATNAFUA BEKELE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY (PhD) IN BIOLOGY (APPLIED GENETICS) ADDIS ABABA JUNE 2014 STUDIES ON MOLECULAR GENETIC DIVERSITY and USEFUL GENOMIC TRAITS of YAM (DIOSCOREA SPP) GERMPLASM COLLECTIONS FROM ETHIOPIA A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIESFACULTY OF LIFE SCIENCES ADDIS ABABA UNIVERSITY BY ATNAFUA BEKELE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY (PhD) IN BIOLOGY (APPLIED GENETICS) SUPERVISOR: - PROF. ENDASHAW BEKELE ADDIS ABABA JUNE 2014 ii DECLARATION I declare that the thesis hereby submitted by me for the Degree Doctor of Philosophy (PhD) in Biology (Applied Genetics) to the School of Graduate Studies of Addis Ababa University is my own independent work and has not previously been submitted by me or anybody else at another university. The materials obtained from other sources have been duly acknowledged in the thesis. Signed on June 2014, The School of Graduate Studies, Faculty of Life Sciences, Addis Ababa University, Arat Kilo. PhD Candidate ____________________________ Atnafua Bekele Supervisor ____________________________ Prof. Endashaw Bekele i Acknowledgments I would like to express my sincere gratitude to my advisor Prof. Endashaw Bekele for his scientific guidance, training, support and valuable comments throughout the period of this research. Because of his professional proficiency, this work is in its present shape. I want to also thank Dr. A. Lopez Montes for accepting me to do part of my molecular research at International Institute of Tropical Agriculture (IITA). I want to express my thanks to Dr. R. Bhattacharjee for her training and support during yam molecular diversity study using SSR markers and PAGE system. I want also to express my appreciation to Mrs. Victoria for her assistance and support during my laboratory work. My thanks also go to Dr. Melaku Gedil and Mr. Gezahegn Girma with his wife Tsega Tsergework for their encouragement, support, providing materials and other aspects during my stay in IITA. My sincere thanks also go to all Bioscience Center staff members, Training and Travel office of IITA, who helped me in one way or the other and made my stay at IITA pleasant. I am also grateful to express my gratitude to Ethiopian Health and Nutrition Research Insititute (EHNRI) who allowed me to do Nutritional Analysis in the laboratory. This research was sponsored by Rural Capacity Building Project (RCBP). I express my thanks to this project for the financial support. I am also grateful to the Faculty of Life Sciences of Addis Ababa University for giving me the chance for this study. My sincere thanks go to all Cellular, Microbial and Molecular Biology Department staffs for their assistance during my study period. Specially, I want to express my gratitude to Dr. Kassahun Tesfaye and Ato Abele Teshome for their help and support during my study ii period. I also express my appreciation to Dr. Kifle Dajen who gave me different types of assistance during my request. I am thankful to yam growing farmers of different regions of SNNPRS, Bako and Hawasa Agricultural Research Center for providing me yam tubers. I appreciate Ato Wondmagej Amenu and Ato Tesfaye Abebe for their careful follow up of the experimental field. My special thanks go to Ato Agdew Bekele, Ato Waga Mazengia, Ato Seyum Getaneh, Ato Tamrat Chisa, Ato Tewodros Mulualem, W/ro Tsehay Mengesha, W/rt Amarech Regasa and W/rt Alem Tsegaye for their help in different issues during my study period. I am also grateful to Southern Agricultural Research Institute for granting me the study leave. All my friends who provided me important advice help and encouragement but fail to name all of them, I want to say thank you so much. The encouragement, love and material support from my family has made anything possible. My mother Mulu Ayalew, my brothers and sisters Habte, Getachew, Elsabet, Admas, Meskerem, Hana, Tesfaye and Workneh I say thank you. May God bless you all!!! Above all, I thank the almighty God for every event of my life!!! iii Dedication To my late father Ato Bekele Damtew and my Daughter Ruth iv Table of Contents Page Declaration--------------------------------------------------------------------------------------------i Acknowledgments----------------------------------------------------------------------------------ii Table of Contents-----------------------------------------------------------------------------------v List of Tables---------------------------------------------------------------------------------------ix List of Figures-------------------------------------------------------------------------------------xiii Acronyms and abbreviations------------------------------------------------------------------xvii Abstract---------------------------------------------------------------------------------------------xix 1. General Introduction --------------------------------------------------------------------------1 2. Literature Review--------------------------------------------------------------------------------7 2.1 Taxonomy and geographical distribution of yam (Dioscorea spp)-----------------------9 2.2 Some yam (Dioscorea species) and their origin-------------------------------------------11 2.3 Cultivation & socio economic importance of yam ( Dioscorea spp)-------------------17 2.4 Yam species in Ethiopia-----------------------------------------------------------------------18 2.5 Previous researches on Ethiopian yam (Dioscorea spp) diversity ---------------------20 2.6 Statement of the problem and objective of the study--------------------------------------24 2.7 Objectives --------------------------------------------------------------------------------------25 2.7.1 General objective of the study--------------------------------------------------------------25 2.7.2 Specific objectives --------------------------------------------------------------------------25 3. Methodology-------------------------------------------------------------------------------------26 3.1. Morphological characterization and yam landraces--------------------------------------26 3.2. Characters evaluated by farmers ------------------------------------------------------------34 3.3. Statistical analysis for morphological characterization ---------------------------------35 3.4. Samples preparation for proximate and minerals content analysis ---------------------35 3.4.1.Moisture determination----------------------------------------------------------------------38 v 3.4.2. Protein determination-----------------------------------------------------------------------38 3.4.3.Fiber determination--------------------------------------------------------------------------39 3.4.4. Fat and minerals determinations ---------------------------------------------------------40 3.5.. Statistical analysis for proximate compositions and mineral contents-----------------------------------------------------------------------------------------41 3.6. Microsatellites (SSR)-------------------------------------------------------------------------42 3.6.1. DNA Extraction -----------------------------------------------------------------------------42 3.6.2. PCR amplification---------------------------------------------------------------------------43 3.6.3. Microsatellite markers and detection of PCR products--------------------------------44 3.6.4. Details of germplasm studied and micro Satellite Primers----------------------------45 3.6.5 Data scoring and statistical data analysis-------------------------------------------------47 3.7. Combined data analysis-----------------------------------------------------------------------49 3.7.1 Combined data sets and statistical data analysis ----------------------------------------49 4. Results--------------------------------------------------------------------------------------------54 4.1 Morphological variability based species identification-----------------------------------55 4.2 Morphological diversity data of yam determined based on cluster analysis-------------------------------------------------------------------60 4.2.1. Principal component analysis-----------------------------------------------------------65 4.3. Proximate and mineral composition of Ethiopian yam (Dioscorea spp)-----------------------------------------------------------------------68 4.3.1. Nutritional value variations among yam ( Dioscorea Species) of Ethiopia-------------------------------------------------------------------------------------68 4.3.1.1. Protein Content (%)-----------------------------------------------------------------------68 vi 4.3.1.2. Fat content (%)----------------------------------------------------------------------------69 4.3.1.3. Fiber (%) content -------------------------------------------------------------------------69 4.3.1.4. Moisture (%) Content--------------------------------------------------------------------70 4.3.1.5. Iron Content (Mg/100g)------------------------------------------------------------------71 4.3.1.6. Ash (%) Content --------------------------------------------------------------------------72 4.3.1.7. Zink content (g/100mg)------------------------------------------------------------------72 4.3.1.8. Calcium content (g/100mg)--------------------------------------------------------------73
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