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KIMANI E. N. Msc. Thesis ANALYSIS OF FLAVOUR AND MOLECULAR DIVERSITY OF KENYAN LABLAB BEAN ( Lablab purpureus (L.) Sweet) ACCESSIONS By ESTHER NYAMBURA KIMANI A thesis submitted to the Graduate School in partial fulfilment for the requirements of the Master of Science degree in Biochemistry and Molecular Biology of Egerton University EGERTON UNIVERSITY DECEMBER 2010 ii COPYRIGHT All rights reserved. No part of this document may be reproduced, or distributed in any form or by any means, or stored in a database or retrieval system, without prior permission from the author or by Egerton University. CopyRight © 2010 Kimani Esther iii DEDICATION This work is dedicated to my dear parents Joseph Kimani Wanjama (the late) and Esther Wangare Kimani Wanjama. To God, my ever present help in time of need, to You be the glory. iv ACKNOWLEDGEMENT I would like to thank the Almighty God for seeing me through the challenging times during this study and the life lessons I had to go through. I am greatly indebted to my supervisors, Prof. Francis N. Wachira, Prof Miriam Kinyua, for their encouragement, effort, supervision, insightful criticism and guidance into deep understanding and knowledge of this study and the preparation of this thesis. I wish to express my sincere appreciation to the following institutions for providing support in the course of my study: Kirk House Trust for the financial support, Egerton University and Government Chemist for the laboratory facilities. My gratitude is extended to the Centre Director KARI, Njoro for availing the laboratory facilities, financial support and enabling environment for me to conduct the research. I am grateful to my AWARD mentor- Dr Santie deVillers, for the invaluable advice and effort in scientific writing, and Prof Matasyoh (Chemistry department- Egerton University), who contributed enormously to the analysis of volatile compounds. I also extend my gratitude to the following for their unfailing support during the project; Kimani Paul, Mr. Otieno, Dr. Maling’a, Dr. Karanja, Evans Nyaboga, Loice Kariuki and Kevin Kamanyi for the assistance in the laboratory; Mr Kariuki (Chemistry department, Egerton University) for the support with GC analysis, Dan Orwa for providing the genebank accessions; Dr. Bridigitte Maass for the publications on lablab; Nathan Muasya and Steve Kimno (Moi University) for all the correspondence and Mr. John Kamundia and Bramwel Wanjala for the help with statistical analysis. To all my friends who encouraged me and kept me going to finish this work especially Nelson Ndegwa, John Ndungu, Mwaura Kiarie, Faith Muniale; and Dr. Franklin Nyabuga who made sure I did not lack any scientific paper that I needed, despite his busy schedule. I owe a lot of gratitude to my parents and my siblings for tolerating me during those times I spent long hours in the laboratory, and when the work pressure was too high. You are the best family ever. Last but not least, John Kageche, for the love, spiritual and emotional support and encouragement in the last leg of this work. v ABSTRACT The legume species Lablab purpureus L. Sweet grows in most tropical environments. It is used as a cover crop and green manure and provides a high–protein food for humans and livestock feed. The study was carried out to analyse flavour components and molecular diversity of Kenyan lablab accessions. Twenty four accessions from the National genebank and farmers were evaluated for odour and bitter taste intensities using sensory tests. Analysis of cyanogenic glycosides was carried out using the picrate method and volatile compounds were isolated and separated using gas chromatography. The genetic diversity of 50 accessions was studied using Amplified fragment length polymorphism (AFLP) markers. The sensory evaluations showed significant (p<0.05) differences for the bitter taste but none for odour. Accession 10706 and 13096 exhibited the highest and lowest means respectively for both bitter and odour taste. The levels of cyanogenic glycosides were not different for the 24 accessions, but significant (p<0.05) differences were observed in the volatile compounds isolated from the accessions with upto 89% similarity of the accessions. Two hundred and sixty two volatile compounds were identified using literature databases. The molecular study revealed a total of 180 polymorphic bands. The overall mean expected heterozygosity ( He) for all the populations was 0.189. The Eastern population had the highest He of 0.297. The plot of the first and second principal coordinates for cluster analysis revealed an overlap of the accessions forming a tight cluster, with the exception of four; namely Mwingi-3 and 12000 from Eastern population, 12187R3 and 10706R1 from Coast and Rift Valley populations. The Unweighted pair group using mathematical arithmetic averages (UPGMA) cluster analysis generated from the distance matrix revealed three major groups. Group 1 had accessions 10706R1 and Mwingi-3, group 2 had accessions 12187R3 and 12000, while group 3 had the rest of the accessions. The low diversity revealed from these results may be due to the narrow genetic base for breeding stocks, and exchange of germplasm across the country. Results obtained from this study will be of great help in lablab accession management by ensuring maximization of exploitation of this vital resource as well as in developing breeding strategies for Lablab purpureus. vi TABLE OF CONTENTS DECLARATION AND RECOMMENDATION ........................................................... ii COPYRIGHT...................................................................................................................iii DEDICATION.................................................................................................................. iv ACKNOWLEDGEMENT................................................................................................ v ABSTRACT...................................................................................................................... vi TABLE OF CONTENTS ............................................................................................... vii LIST OF TABLES ............................................................................................................ x LIST OF FIGURES ......................................................................................................... xi LIST OF PLATES .......................................................................................................... xii LIST OF ABBREVIATIONS .......................................................................................xiii CHAPTER ONE ............................................................................................................... 1 INTRODUCTION............................................................................................................. 1 1.1 Background................................................................................................................... 1 1.2 Statement of the Problem.............................................................................................. 2 1.3 Justification................................................................................................................... 3 1.4 Objectives ..................................................................................................................... 4 1.4.1 General Objective .......................................................................................... 4 1.4.2 Specific Objectives ........................................................................................ 4 1.5 Null Hypotheses............................................................................................................ 4 CHAPTER TWO .............................................................................................................. 5 LITERATURE REVIEW ................................................................................................ 5 2.1 Lablab bean crop........................................................................................................... 5 2.2 Importance of Lablab.................................................................................................... 7 2.3 Flavour in lablab ........................................................................................................... 7 2.3.1 Sensory Evaluation of lablab ............................................................................. 8 2.3.2 Cyanogenic Glycosides in lablab....................................................................... 9 2.3.2 Volatile compounds ........................................................................................... 9 2.4 Molecular Markers and their use in crop diversity..................................................... 10 2.4.1 Amplified Fragment Length Polymorphism (AFLP) .................................. 11 vii CHAPTER THREE........................................................................................................ 13 MATERIALS AND METHODS ................................................................................... 13 3.1 Plant material .............................................................................................................. 13 3.2 Sensory Evaluation (Organoleptic Tests) ................................................................... 13 3.3 Biochemical Assays.................................................................................................... 15 3.3.1 Chemical assay of cyanogenic glycosides
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