MORPHOLOGICAL AND MOLECULAR DIVERSITY IN ETHIOPIAN LENTIL (LENS CULINARIS MEDIKUS) LANDRACE ACCESSIONS AND THEIR COMPARISON WITH SOME EXOTIC GENOTYPES A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES, ADDIS ABABA UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGY (APPLIED GENETICS) BY EDOSSA FIKIRU JULY 2006 ADDIS ABABA UNIVERSITY SCHOOL OF GRADATE STUDIES MORPHOLOGICAL AND MOLECULAR DIVERSITY IN ETHIOPIAN LENTIL (LENS CULINARIS MEDIKUS) LANDRACE ACCESSIONS AND THEIR COMPARISON WITH SOME EXOTIC GENOTYPES BY EDOSSA FIKIRU Approved by Board of Examiners: Examiner Signature Date Examiner Signature Date Advisor Signature Date Chairperson Signature Date DEDICATION This thesis work is dedicated to my mother. ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my advisor Professor Endashaw Bekele for his kind and continuous guidance and supervision starting from designing the setup of the research to its completion. His patience in traveling to Sinana Agricultural Research Center to supervise my research field is highly acknowledged. He also closely supervised my activities in the laboratory. His comments during the entire research activities and write up of the manuscript were the key to my success. Dr. Amsalu Ayana, too, is acknowledged for his guidance during the thesis research and detailed comments on the manuscript. My especial thanks go to Mr. Kassahun Tesfaye for shaping my proposal, for his technical support during laboratory work, facilitating the purchase of laboratory supplies and above all for working almost all the laboratory activities with me. I am also thankful to Dr. Tadesse W/Mariam, manager of the CoCE project, for facilitating the purchase of laboratory supplies for me. Mr. Tamiru Oljira, too, deserves my sincere thanks for his technical guidance and continuous and unreserved assistance during the laboratory work. I am thankful to Mr. Kedir Nefo and the stuff of the Finance Section of Sinana Agricultural Research Center (SARC) for appropriately handling my research budget. Mr. Tadele Tadesse and Mr. Tamiru Tadesse, too, are acknowledged for their continuous follow up of my research field during my absence. I also would like to extend my thanks to all people in the Pulse and Oil Crop’s Section of the Field Crops’ Breeding and Genetics Research Division of SARC for their assistance from planting to harvesting. Mr. Zerihun Jaleta and Mr. Mahamed Abinassa deserve my heart felt thank for all their support during my study. Mr. Genene Gezu is acknowledged for lending me a laptop computer at critical time. I am also grateful to all individuals and institutions who/that have directly or indirectly contributed to the thesis work. I would like to thank the Agricultural Research and Training Project of the Ethiopian Agricultural Research Institute, the former Ethiopian Agricultural Research Organization, for sponsoring my M. Sc. Thesis Research. Addis Ababa University is also acknowledged for covering some of the research costs and provision of computer and internet services. My special thanks go to Oromiya Agricultural Research Institute for allowing me for the study. I I am greatly indebted to Institute of Biodiversity Conservation for kindly providing me the seeds of all selected lentil landrace accessions. I am also thankful to Debre-Zeit Agricultural Research Center for provision of seeds of nine improved varieties of lentil. SARC is also acknowledged for providing me seeds of one improved and one candidate varieties. Above all, I thank the almighty God for every thing. II TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................................. I LIST OF TABLES ......................................................................................................................... III LIST OF FIGURES ........................................................................................................................ V LIST OF APPENDICES ................................................................................................................ VI LIST OF ACRONYMS ................................................................................................................ VII ABSTRACT ................................................................................................................................... IX 1. INTRODUCTION ....................................................................................................................... 1 2. LITERATURE REVIEW ............................................................................................................ 4 2.1. DIVERSITY: ITS IMPORTANCE AND THREAT ........................................................................... 4 2.2. METHODS OF DIVERSITY ASSESSMENT .................................................................................. 4 2.2.1. Morphological Method .................................................................................................. 4 2.2.2. Molecular Markers ......................................................................................................... 4 2.2.3. Sampling Strategy for DNA Extraction ......................................................................... 6 2.3. DIVERSITY STUDIES IN LENTIL ............................................................................................... 8 2.3.1. Morphological Diversity ................................................................................................ 8 2.3.2. Molecular Diversity ....................................................................................................... 8 2.3.3. Comparison of Morphological and Molecular Markers .............................................. 10 3. OBJECTIVES ............................................................................................................................ 11 4. MATERIALS AND METHODS ............................................................................................... 12 4.1. DESCRIPTION OF THE STUDY AREA ...................................................................................... 12 4.2. PLANT MATERIALS .............................................................................................................. 12 4.3. EXPERIMENTAL SETUP AND SAMPLING PROCEDURE ............................................................ 16 4.4. MORPHOLOGICAL DATA ...................................................................................................... 16 4.5. MOLECULAR DATA .............................................................................................................. 17 4.5.1. Leaf Sampling and DNA Extraction ............................................................................ 17 4.5.2. Test Gel Electrophoresis and Staining ......................................................................... 17 4.5.3. Selection and Dilution of DNA Extract ....................................................................... 18 4.5.4. PCR Amplification and Electrophoresis ...................................................................... 19 4.5.4.1. PCR Reaction Components and Profiles .............................................................. 19 4.5.4.2. Primer Screening ................................................................................................... 19 4.5.4.3. ISSR-PCR Product Gel Electrophoresis ............................................................... 25 4.6. DATA ANALYSIS .................................................................................................................. 25 4.6.1. Morphological Data Analysis ...................................................................................... 25 4.6.1.1. Analysis of Variance ............................................................................................. 25 4.6.1.2. Estimation of Phenotypic and Genotypic Variances ............................................ 25 4.6.1.3. Euclidian Distance and Clustering Analysis ......................................................... 26 4.6.2. Molecular Data Analysis ............................................................................................. 26 4.6.2.1. Scoring Bands ....................................................................................................... 26 4.6.2.2. Genetic Diversity Analysis ................................................................................... 27 4.6.2.3. Genetic Structure .................................................................................................. 28 4.6.2.3.1. Analysis of Molecular Variance .................................................................... 28 4.6.2.3.2. Clustering Analysis ........................................................................................ 28 4.6.3. Correlation of Morphological and Molecular Distances ............................................. 28 5. RESULTS .................................................................................................................................. 29 5.1. MORPHOLOGICAL VARIABILITY ........................................................................................... 29 5.1.1. Analysis of Variance (ANOVA) .................................................................................. 29 5.1.2. Phenotypic and Genotypic Coefficients of Variation .................................................. 29 5.1.3. Euclidian Distances .....................................................................................................