molecular characterisation of honeybees, apis mellifera races in east africa

276 Pages·2017·2.81 MB·English

MOLECULAR CHARACTERISATION OF HONEY
BEES, APIS MELLIFERA SUBSPECIES IN EAST
AFRICA

HARRISON GATHENGA KIBOGO

DOCTOR OF PHILOSOPHY
(Biotechnology)

JOMO KENYATTA UNIVERSITY OF
AGRICULTURE AND TECHNOLOGY

2017
Molecular characterisation of honey bees, Apis mellifera
subspecies in East Africa

Harrison Gathenga Kibogo

A thesis submitted in fulfillment for the Degree of Doctor of
Philosophy in Biotechnology in Jomo Kenyatta University of
Agriculture and Technology

2017

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DECLARATION
I, Harrison Gathenga Kibogo declare that this thesis is my original work and has not
been presented to any other university for the award of a degree
Sign…………………………. Date…………………………………
This Thesis has been presented for examination with our approval as supervisors

Prof Anne Muigai
JKUAT, Kenya

Sign…………………………. Date…………………………………

Dr Joseph Ng’ang’a
JKUAT, Kenya

Sign…………………………. Date…………………………………

Dr Daniel Masiga
ICIPE, Kenya

Sign…………………………. Date…………………………………

Prof Suresh Raina
ICIPE, Kenya

Sign…………………………. Date…………………………………

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DEDICATION
This work is dedicated to my parents, late Mr Kibogo Mwangi and Mrs Hannah
Wanjiru Kibogo who supported me in light and difficult moments
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ACKNOWLEDGEMENTS
My gratitude is to Almighty God for enabling me to start and finish these studies in
good health and with greater strength. My utmost appreciation goes to my
supervisors; Prof Suresh Raina (ICIPE), Prof Anne Muigai (JKUAT), Dr Daniel
Masiga (ICIPE), and Dr Joseph Ng’ang’a (JKUAT) for their guidance and invaluable
support in these studies. My heartfelt thanks are to Prof Anne Muigai for her
assistance in the analysis, organization and interpretation of molecular statistical
data. Thanks for helping me understand the underlying concepts.
My candid acknowledgements are to the ICIPE’s African Regional Post-graduate
Programme in Insect Science (ARPPIS) under the German Academic Exchange
Service (DAAD) in-country fellowship for sponsoring my studies. This work
wouldn’t have been possible without the IFAD project fund under the Commercial
Insect Programme and Molecular Biology and Biotechnology Department for their
direct support financially, fieldwork, miscellaneous and the general welfare of my
studentship.
My honest appreciations are to my colleagues Nelly Mucugu, Paul Mireji, Fathiya
Mbarak, Catherine Muturi, David Omondi, Simon Ngao and Joel Bargul for their
encouragement, advice and support in the most difficult time of my studies. I’m
greatly indebted to you. It is tough to wait for something that you know might not
happen but it’s even harder to give up when you know it’s everything you ever
wanted. You made me understand that science is organized knowledge but wisdom is
organized life. Collectively, may God richly bless all those who assisted me in one
way or another.

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TABLE OF CONTENTS
DECLARATION ....................................................................................................... II
DEDICATION .......................................................................................................... III
ACKNOWLEDGEMENTS ..................................................................................... IV
TABLE OF CONTENTS .......................................................................................... V
LIST OF TABLES .................................................................................................... X
LIST OF FIGURES ............................................................................................... XII
LIST OF APPENDICES ...................................................................................... XIII
ABBREVIATIONS AND ACRONYMS ............................................................. XIV
ABSTRACT ........................................................................................................... XVI
CHAPTER ONE ........................................................................................................ 1
INTRODUCTION ...................................................................................................... 1
1.1 Background information .................................................................................... 1
1.2 Statement of the problem ................................................................................... 6
1.3 Justification ........................................................................................................ 8
1.4 Hypotheses ....................................................................................................... 10
1.4.1 Main hypothesis .................................................................................... 10
1.4.2 Specific hypotheses ............................................................................... 10
1.5. Objectives of the study .................................................................................... 10
1.5.1 General Objective.................................................................................. 10
1.5.2 Specific Objectives................................................................................ 10
1.6 Research Questions .......................................................................................... 11
CHAPTER TWO ..................................................................................................... 12
LITERATURE REVIEW ........................................................................................ 12
2.1 Honey bees’ classification ................................................................................ 12
2.2 Life history of the honey bee ........................................................................... 12
2.3 Haplodiploid sex determination in honey bees ................................................ 14
2.4 Economic Importance of Honey bees .............................................................. 14
2.4.1 Honey bee products ............................................................................... 14
2.4.2 Honey bees as pollinators of crop plants............................................... 18
2.4.3 Role of beekeeping in biodiversity conservation .................................. 19
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2.4.4 Honey bee as a model of behavioral genetic studies............................. 20
2.5 The African Honey bee .................................................................................... 21
2.6 Apiculture in Africa ......................................................................................... 24
2.7 Declining bee health ......................................................................................... 25
2.8 Molecular markers ........................................................................................... 27
2.9 DNA Barcoding ............................................................................................... 29
2.10 Genetic Diversity Studies ............................................................................... 30
2.11 Quantitative trait loci Analysis ....................................................................... 32
CHAPTER THREE ................................................................................................. 34
MATERIALS AND METHODS ............................................................................ 34
3.1 Basic methodology ........................................................................................... 34
3.1.1 Honey bee sampling sites ...................................................................... 34
3.1.2 Collection of honey bee specimens ....................................................... 35
3.1.3 Total DNA extraction ............................................................................ 38
3.1.4 Selection of mitochondrial and microsatellite markers ......................... 38
3.1.5 Amplification of mitochondrial DNA genes ......................................... 41
3.1.6 Amplification of microsatellite markers ............................................... 41
3.1.7 Microsatellite genotyping ..................................................................... 42
3.2 Phylogenetic relationship analysis of honey bee subspecies ........................... 43
3.2.1 Purification of PCR products for sequencing ....................................... 43
3.2.2 Sequencing of purified PCR products ................................................... 43
3.2.3 Delineation of honey bees using COI-COII intergenic sequences ........ 45
3.3 Allelic proportions and genetic diversity of populations ................................. 46
3.3.1 Hardy Weinberg Equilibrium and linkage disequilibrium .................... 46
3.3.2 Genetic diversity analysis ..................................................................... 47
3.3.3 Genetic bottleneck ................................................................................. 48
3.4 Population differentiation, admixture and gene flow studies ........................... 49
3.4.1 Population differentiation by F-statistics .............................................. 49
3.4.2 Genetic distance and phylogenetic relationship .................................... 51
3.4.3 Analysis of Molecular Variance (AMOVA) ......................................... 52
3.4.4 Population and racial admixture ........................................................... 53
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3.4.5 Population Assignments and Exclusion ................................................ 54
3.5 Dissection of foraging and stinging behaviors in honey bees .......................... 55
3.5.1 Selection for pollen and nectar foraging behavior ................................ 55
3.5.2 Selection for stinging behavior ............................................................. 55
3.5.3 Association between variables .............................................................. 56
3.6 Identification of candidate genes ..................................................................... 57
3.6.1 Marker polymorphism and population admixture................................. 57
3.6.2 Linkage disequilibrium ......................................................................... 57
3.6.3 Association between phenotypic variables ........................................... 59
3.6.4 Marker-Trait Association ...................................................................... 59
3.6.5 Candidate genes search ......................................................................... 60
CHAPTER FOUR .................................................................................................... 61
RESULTS ................................................................................................................. 61
4.1 Phylogenetic relationships of honey bee subspecies ........................................ 61
4.1.1 Amplification of COI gene.................................................................... 61
4.1.2 COI sequence analysis .......................................................................... 61
4.1.3 Sequence alignment of COI .................................................................. 63
4.1.4 Phylogenetic relationships of COI gene ................................................ 63
4.1.5 Amplification of Cytb-tRNAser intergenic region ................................. 65
4.1.6 Sequence analysis of Cytb-tRNAser intergenic region .......................... 65
4.1.7 Phylogenetic relationships of Cytb-tRNAser sequences ........................ 66
4.1.8 Amplification and sequence analysis of COI-COII intergenic region .. 68
4.1.9 Multiple sequence alignment of COI-COII intergenic region .............. 68
4.1.10 COI-COII intergenic region haplotype and lineage determination ..... 68
4.1.11 COI-COII phylogenetic relationships and cluster analysis ................. 70
4.2 Allelic proportions and genetic diversity of honey bee subspecies ................. 72
4.2.1 Hardy-Weinberg Equilibrium (HWE) using Fisher’s exact test ........... 72
4.2.2 Loci testing for linkage disequilibrium (LD) ........................................ 74
4.2.3 Allele counts per locus and population ................................................. 76
4.2.4 Mean Number of Alleles (MNA) and Heterozygosities ....................... 78
4.2.5 Genetic bottleneck ................................................................................. 80
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4.2.6 Genetic diversity per locus and population ........................................... 83
4.2.7 Allelic richness for all loci and populations .......................................... 85
4.2.8 Locus by population estimate null alleles frequencies .......................... 87
4.2.9 Private allele frequencies ...................................................................... 89
4.3 Population differentiation, admixture and gene flow ....................................... 91
4.3.1 F-statistics ............................................................................................. 91
4.3.2 Within-population inbreeding estimates, F ........................................ 93
IS
4.3.3 Pairwise F differentiation ................................................................... 95
ST
4.3.4 Nei’s standard genetic distances ........................................................... 98
4.3.5 Nei’s chord distances .......................................................................... 101
4.3.6 Population differentiation by AMOVA .............................................. 104
4.3.7 PCA analysis among populations ....................................................... 107
4.3.8 PCA analysis between altitude levels ................................................. 110
4.3.9 PCA analysis between honey bee subspecies ..................................... 112
4.3.10 Population differentiation by admixture analysis ............................. 114
4.3.10.1 K Clusters determination ............................................................... 114
4.3.10.2 Cluster analysis from structure results ........................................... 118
4.3.10.3 Subcluster analysis ......................................................................... 121
4.3.11 Genetic assignment of individual to populations .............................. 123
4.4 Characterization of foraging and stinging behaviors ..................................... 125
4.4. 1 Mean, range and standard deviation of phenotypic variables ............ 125
4.4.2 Spatial autocorrelation analysis........................................................... 126
4.4.3 Association between variables ............................................................ 128
4.5 Candidate genes associated with foraging and stinging behaviors ................ 130
4.5.1 Marker polymorphism ......................................................................... 130
4.5.2 Linkage disequilibrium ....................................................................... 131
4.5.3 Determining the optimum population structure .................................. 133
4.5.4. Identification of marker-phenotype association ................................. 136
4.5.5 Candidate gene identification .............................................................. 138
CHAPTER FIVE .................................................................................................... 143
DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS .................... 143
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5.1 Discussion ...................................................................................................... 143
5.1.1 Phylogenetic relationships of honey bee subspecies ........................... 143
5.1.2 Allelic proportions and Genetic diversity of honey bees .................... 145
5.1.3 Population differentiation, admixture and gene flow .......................... 153
5.1.4 Characterization of honey bee foraging and stinging behaviors ......... 162
5.1.5 Candidate genes identification based on foraging and stinging QTLs 163
5.2 Conclusions .................................................................................................... 166
5.2.1 Phylogenetic relationships of honey bee subspecies ........................... 166
5.2.2 Genetic diversity of honey bees .......................................................... 167
5.2.3 Population genetic differentiation of honey bee subspecies ............... 168
5.2.4 Honey bee foraging and stinging behavioral traits ............................. 168
5.2.5 Candidate genes Identification ............................................................ 169
5.3 Recommendations .......................................................................................... 169
REFERENCES ....................................................................................................... 172
APPENDICES ........................................................................................................ 219

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