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Molecular and Morphologicalgenetic Diversity of Potato (Solanum Tuberosum) Clones Conserved in Ethiopia Using Simple Sequence Repeat (Ssr) Markers

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Molecular and Morphologicalgenetic Diversity of Potato (Solanum Tuberosum) Clones Conserved in Ethiopia Using Simple Sequence Repeat (Ssr) Markers MOLECULAR AND MORPHOLOGICALGENETIC DIVERSITY OF POTATO (SOLANUM TUBEROSUM) CLONES CONSERVED IN ETHIOPIA USING SIMPLE SEQUENCE REPEAT (SSR) MARKERS. HULUBANCHE TADELE KASSA MB 300-0003/2015 A Thesis submitted to Pan African University, Institute for Basic Sciences, Technology and Innovation in partial fulfillment of the requirements for the Degree of Master of Science in Molecular Biology and Biotechnology 2017 i DECLARATION This thesis is my original work and has not been submitted to any other University for examination. Signature:……………………………………....... Date:………......................... Hulubanche Tadele Kassa This thesis report has been submitted for examination with our approval as University supervisors. Signature:.................................................... Date:................................. Dr. Remmy W. Kasili Institute for Biotechnology Research (IBR), Jomo Kenyatta University of Agriculture and Technology, Kenya Signature:.................................................... Date:................................. Dr. Kassahun Tesfaye Institute of Biotechnology (IOB), Addis Ababa University, Ethiopia ii ACKNOWLEDGEMENTS First I want to thank God for giving strength and wisdom from t during my research work. I would like to say thank you to my supervisors for their support. I would like to acknowledge African Union (AU) and Pan African University, Institute for Basic Sciences, Technology and Innovation (PAUSTI) for giving me this scholarship. I also want to acknowledge Africa-ai-JAPAN JKUAT office for their financial support. I would like to say thank you to all Amhara Agricultural Research Institute plant tissue culture laboratory Bahirdar Ethiopia staffs for their support during my sample preparation and collection and the Institute for giving me a study leave and allowing me to access the potato clones and the laboratory and greenhouse facility. I want to say thank you to all my friends and colleges who was always available and supportive, Biniam Mesfine, Amanuel, Yosef and Temitope. I am very grateful to have you as my Friends. My deepest heartfelt gratitude to my Fiancé Mr. Gashaw Belay for his support and inspiration starting from applying for the scholarship and guiding me during my course work, research and also not letting me to give up during my hard times and also for his unconditional love. I am deeply indebted to my family my mother, my brothers and my sister for their unconditional love and support throughout my life. iii DEDICATION This thesis is dedicated to my mother and memories of my beloved Father. iv ABSTRACT Potato (Solanum tuberosum L.) is the fourth most important food crop in the world after rice, wheat, and maize. Potato plays a significant role in human nutrition worldwide, where more than 320 million tons of potato is produced annually on 20 million hectares of land. In this study, a total of 53 potato genotypes which are conserved in vitro in Amhara Regional Agricultural Research Institute, Ethiopia, for further research and development purpose were used. These in vitro conserved Potatoes had not been investigated for their genetic diversity or relatedness. So, the aim of this study was to assess the molecular and morphological genetic diversity of in vitro conserved potato genotypes. Molecular genetic diversity was done using twelve fluorescently labeled simple sequence repeat (SSR) markers while Morphological diversity was done using morphological descriptors from CIP Potato catalog. The dendrogram analysis using the morphological data clustered the potato genotypes in to four main cluster and five sub clusters. The first cluster (CI) was the smallest cluster with 3 genotypes all from Exotic. The second cluster (CII) had 5 genotypes four Ethiopian local varieties and one from CIP. The third cluster (CIII) was the largest cluster with five sub-clusters (SC1 to SC5) It had 27 genotypes. Cluster four (CIV) contained 7 genotypes all from Ethiopian nationally released varieties. Using SSR markers a total of 79 alleles were observed with an average of 6.58 per marker. The polymorphism level of the markers varied from 3 to 10 for markers STM1053 and STM1052, respectively. The polymorphic information content of the markers ranged from 0.93 for markers STI0030 and STI0004 to 0.57 for marker STM1053 with an average value of 0.85. The highest percentage of polymorphic loci was 83.33% observed in CIP populations and the lowest was 58.33% recorded in Ethiopian local Varieties. The number of different (Private) Alleles varied from 1.792 in Ethiopian local genotypes to 3.292 in CIP genotypes. The highest genetic distance was observed between Ethiopian local varieties and the Exotic varieties which was 0.8236; the lowest genetic distance was observed between Exotic varieties and CIP Populations which was 0.6256. Principal coordinate analysis (PCoA) explained 27.63% of the total variation. Samples from CIP and the Exotics were scattered in two coordinates. The Neighbor-joining tree generated from SSR markers divided the 53 studied potato genotypes into three main clusters and 5 sub-clusters. The first cluster (C1) contained 19 genotypes with 2 v sub-clusters and was dominated by samples from CIP. The second cluster (C2) contained 29 genotypes with 2 sub-clusters; all Ethiopian local varieties are clustered in this group. The third cluster (C3) constituted 5 genotypes from CIP only. The analysis of molecular variation showed that 4 % total variation was among populations and 96% of the variation was within individuals. This study revealed that there is genetic diversity among conserved potato genotypes. The information generated from this study can serve as a basis for the Ethiopian potato breeding program and in vitro potato conservation management strategies. Keywords: Potato, simple sequence repeat (SSR), genetic diversity, in vitro conservation vi TABLE OF CONTENTS DECLARATION........................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iii DEDICATION.............................................................................................................................. iv ABSTRACT ................................................................................................................................... v TABLE OF CONTENTS ........................................................................................................... vii LIST OF TABLES ....................................................................................................................... xi LIST OF FIGURES .................................................................................................................... xii LIST OF PLATES ..................................................................................................................... xiii LIST OF APPENDIX ................................................................................................................ xiv ACRONYMS ............................................................................................................................... xv CHAPTER 1: INTRODUCTION ................................................................................................ 1 1.1 Background of the study .................................................................................................. 1 1.1.1 Potato Production Constraints in Ethiopia...................................................................... 1 1.1.2 Agronomic practices of potato in Ethiopia ..................................................................... 2 1.1.3 Potato breeding strategies in Ethiopia ............................................................................ 3 1.2. Statement of the problem ..................................................................................................... 7 1.3 Justification ........................................................................................................................... 7 1.4. General objective of the study:............................................................................................. 8 1.4.1. Specific objectives ......................................................................................................... 8 1.5. Significance of the study ...................................................................................................... 8 1.6 Hypothesis ............................................................................................................................. 8 CHAPTER 2: LITERATURE REVIEW ................................................................................... 9 2.1. Origin and Distribution of Potato ......................................................................................... 9 2.2. Taxonomy of Potato (Solanum tuberosum L.) ..................................................................... 9 2.2.1. Taxonomic hierarchy of Potato ..................................................................................... 9 vii 2.3. Botany of potato ................................................................................................................. 10 2.3.1 Reproduction ................................................................................................................ 11 2.4 Ecological requirements of potato......................................................................................
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