ABSTRACT
The middle-belt of Nigeria is known for its ethnic diversity and linguistic complexity. Studies on biological variation within the region have been mostly based on anthropometry but with recent advances in anthropological genetics, newer tools now provide greater resolution on human variation. This study examined the efficacy of the mitochondrial DNA (mtDNA) as a genetic marker to characterize the genetic structure of four ethnic groups of Benue-Congo affiliations from Kaduna State. Column and propriety salting based methods were used to extract mtDNA hypervariable segment-I (HVS-I) sequences from samples belonging to four ethnic groups, the Bajju, Chawai, Atyap and Kagoro. Sequences were amplified and amplicons purified using ExoSap. Sequencing for the light strand was done followed by sequence alignment, restriction fragment length polymorphism (RFLP) andsingle nucleotide polymorphism (SNP) analysis. Nucleotide positions 16050-16460 were compared to the revised Cambridge Reference Sequence (rCRS) and 91 haplotypes were observed. A total of 107 polymorphic sites characterized the haplotypes. The African specific HpaIcut site at 3592 defined the L1 and L2 haplotypes which were most frequent but absent for the L0 and L3 haplotypes. Subclade L3e had the highest frequency while other sub clades of the sub Saharan haplogroups werealso present across the study populations in appreciable frequencies, indicative of substantial gene flow between them and other neighbouring populations.A fewsamples, however failed to cluster with the majority as they lacked SNPs belonging to the region and were merely identified as Non-L haplogroups.The Nucleotide diversities (π) were 0.019, 0.026, 0.025 and 0.020 for the Atyap, Bajju, Chawai and Kagoro respectively. The haplotype diversities (HD) were
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high and consistent with the regional and overall African values, with the Atyap having the leastdiverse value (0.960) while the Bajju had the most diverse haplotypes(0.992). Sub-clade analysis based on L0, L1, L2 and L3, for haplotypediversity (HD) and nucleotide diversities, π, exhibited greater diversity for L3 and L2 while L0 had the least diversity. Mismatch distributions for the major haplogroups showed stable demographic patternsfor L0, L1 and L2 but the more recent L3 clade exhibited an expansion pattern as expected. The same expanding demographic was observed for the Atyap, Chawai and Kagoro with the Bajju having a more stable population. Partitioning the genetic variation using the linguistic group model using the analysis of molecular variance (AMOVA) revealed little variation among the populations (3.66%) but showed a high level of variation (94.6 %) within each population.This study has revealed the presence of a shared genetic structure among the Atyap, Bajju, Chawai and Kagorousing molecular markers, which is indicative of close genetic relationship due to common history, substantial gene flowand geographical proximity.
TABLE OF CONTENTS
Cover Page …………………………………………………………………………………………………………… i Title page …………………………………………………………………………………………………………… iii Declaration …………………………………………………………………………………………………………. iv Certification ……………………………………………………………………………………………………….. iii Dedication ……………………………………………………………………………………………………………. v Acknowledgements …………………………………………………………………………………………….. vii Abstract ……………………………………………………………………………………………………………. viii Table of Contents ……………………………………………………………………………………………. ix-xx List of Abbreviations …………………………………………………………………………………………. xxi CHAPTER ONE 1.0 Introduction …………………………………………………………………………………………………… 1 1.1 Background of Study ……………………………………………………………………………………… 1 1.2 Statement of research the Problem …………………………………………………………………. 4 1.3 Justification of the Study ………………………………………………………………………………… 4 1.4 Aims of the Study ………………………………………………………………………………………….. 5 1.5 Objectives of the Study ………………………………………………………………………………….. 5
1.6 Hypotheses of the Study …………………………………………………………………………………. 6
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CHAPTER TWO 2.0 Literature Review ………………………………………………………………………………………….. 7 2.1 Origins of Man and Anthropological Genetics ………………………………………………… 7 2.2 Biological Sources of Human Variation…………………………………………………………. 11 2.2.1 Base Substitutions ……………………………………………………………………………………… 12 2.2.2 Insertions and Deletions …………………………………………………………………………….. 13 2.3 Linguistic Affiliations …………………………………………………………………………………… 13 2.3.1 The Niger-Kordofanian family …………………………………………………………………… 16 2.3.2 Benue-Congo …………………………………………………………………………………………….. 19 2.4 Historical Background of the Bajju, Atyap, Chawai and Kagoro …………………… 21 2.4.1The Chawai (Atsam) …………………………………………………………………………………… 23 2.4.2 The Bajju ………………………………………………………………………………………………….. 25 2.4.3 The Atyap (Kataf) ……………………………………………………………………………………… 25 2.4.4 Kagoro (Oegworok) …………………………………………………………………………………… 27 2.5 Genetic Markers ………………………………………………………………………………………….. 29 2.5.1ClassicalMarkers ……………………………………………………………………………………….. 29 2.5.2 Molecular Markers ……………………………………………………………………………………. 31 2.5.3 Autosomal Markers …………………………………………………………………………………… 33
2.6 Y-Chromosome ……………………………………………………………………………………………. 33
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2.7 Mitochondrial DNA ……………………………………………………………………………………… 34 2.8 History of Mitochondria ……………………………………………………………………………….. 39 2.9 Biology of the Mitochondria …………………………………………………………………………. 41 2.9.1 Replication ………………………………………………………………………………………………… 41 2.9.2 Energy Generation …………………………………………………………………………………….. 43 2.10 mtDNA Variation in Humans ……………………………………………………………………… 44 2.11 DNA sequencing …………………………………………………………………………………………. 47 2.12 Polymerase Chain Reaction (PCR) …………………………………………………………….. 49 2.13 Gel Electrophoresis (Agarose) …………………………………………………………………….. 50 2.14 Restriction Fragment Length Polymorphisms (RFLPS) ………………………………. 51 2.15 Phylogenetics ……………………………………………………………………………………………… 53 2.16 Networks ……………………………………………………………………………………………………. 57 CHAPTERTHREE 3.0 Materials and Method ………………………………………………………………………………….. 60 3.1 Subject Sampling …………………………………………………………………………………………. 60 3.1.1 Comparative Populations …………………………………………………………………………… 61 3.1.2 Ethical Approval ……………………………………………………………………………………….. 61 3.1.3 Comparative Populations …………………………………………………………………………… 61
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3.2 Buccal Cell Collection …………………………………………………………………………………… 63 3.3 DNA Extraction and Polymerization …………………………………………………………….. 63 3.4 Amplification (Polymerase Chain Reaction) ………………………………………………….. 67 3.5 Electrophoresis Procedure ……………………………………………………………………………. 70 3.5.1 Preparing the Gel ………………………………………………………………………………………. 70 3.5.2 Running the Gel ………………………………………………………………………………………… 71 3.6 Purification of PCR Product and Sequencing ………………………………………………… 71 3.7 HVS-I Sequencing ………………………………………………………………………………………… 73 3.8 Restriction Fragment Length Polymorphism (RFLP) ……………………………………. 73 3.9 Precautionary Measures ……………………………………………………………………………….. 76 3.10 Haplogroup Assignment ……………………………………………………………………………… 77 3.11 Data Analysis ……………………………………………………………………………………………… 77 3.11.1 Genetic Diversity ……………………………………………………………………………………… 77 3.11.2 Fst Distance Matrix …………………………………………………………………………………. 78 3.11.3 Intra-population Diversity Measures ………………………………………………………… 78 3.11.4 Neutrality Test …………………………………………………………………………………………. 78 3.11.5 Interpopulation Diversity Measures (Networks) ……………………………………….. 78 3.11.6 Mismatch Distribution …………………………………………………………………………….. 79
3.11.7 Analysis of Molecular Variance(AMOVA) ……………………………………………….. 79
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3.11.8 Phylogenetic Analysis ………………………………………………………………………………. 80 3.11.9 Principal Component Analysis (PCA) ……………………………………………………… 80 CHAPTER FOUR 4.0 Result …………………………………………………………………………………………………………… 82 4.1 DNA Extraction ……………………………………………………………………………………………. 82 4.2 HVS-I Sequencing and Gel Electrophoresis …………………………………………………… 84 4.3 HVS-I Sequences and Haplogroup Characterization ……………………………………… 87 4.4 Diversity Indices …………………………………………………………………………………………… 97 4.5 Analysis of Molecular Variance (AMOVA) …………………………………………………. 102 4.6 Neighbour-Joining (NJ) Tree ………………………………………………………………………. 107 4.7 Mismatch Analysis ……………………………………………………………………………………… 108 4.8 Principal Component Analysis ……………………………………………………………………. 110 4.9 Median-Joining network …………………………………………………………………………….. 120 CHAPTER FIVE 5.0 Discussions …………………………………………………………………………………………………. 122 CHAPTER SIX 6.0 Summary, Conclusion and Recommendation ………………………………………………. 134
6.1 Summary ……………………………………………………………………………………………………. 134
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6.2 Conclusions ………………………………………………………………………………………………… 135 6.3 Recommendations ………………………………………………………………………………………. 135 References ………………………………………………………………………………………………………. 137
CHAPTER ONE
1.0 INTRODUCTION 1.1 BACKGROUND OF STUDY
Linguistic inclinations, anthropometric records and geographical background of human populations are known to provide a historical basis for human evolution and variation, as well as the reasons underlying such changes (Tishkoff et al., 2007; Adebisi, 2008). The diversity exhibited by Homo sapiens arose during their processes of dispersal into their present regions, thus, the subsets of variation tend to be associated with particular geographic areas and populations (Rosa et al., 2004). Human variation has been measured using simple visual characters like size, form and skin colour leading to the conclusion that sharing one or more features is an indication of common descent (Molnar, 1998), but advances in the science of genetics have revealed greater distinctions (Relethford, 1990). Thus, anthropological genetics, a comparatively new discipline makes attempt at answering questions that concern human origin and variation using methods and theories of genetics (Crawford, 2007).
The mapping of the human genome and the emergence of technologies has made it possible to identify variation at the level of the individual (Underhill et al., 2000) and more recently, the haploid characteristics and of some genetic markers allow the successful application of phylogenetic and phylogeographic approaches to population genetics (Ennafaa et al., 2009). This has moved the focus to more detailed methods like the genome wide characterization of natural variation (Thangaraj et al., 2005). These advances in
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human genetics have supplied researchers with tools for the investigation of the variation between language groups and haploid markers such as the mitochondrial DNA (mtDNA) and non-recombining region of the Y-chromosome (NRY) (Hurles et al., 2002; Wood et al., 2005). The focus of these studies has ranged from explorations of disease factors to historically focused research on the genetic relations between African populations (MacEachern, 2006). A striking factor in many of these researches is the search for relationship between populations within Africa with that elsewhere. Some of these studies are germane to the considerations of ancient relations and migrations which indicate strong correlations between genetic and linguistic relationships among globally distributed human populations (Chen, 1995). From an evolutionary view point, such relationships spread across the world‟s genetic map have led to efforts on illuminating the origin and dispersal of anatomically modern man across the world (Saccone et al., 1992). Postulations based on developed models have shown different origins for man, out of which the Recent African origin (RAO) also known as the “out of Africa” model asserts a common descent for all populations from an anatomically modern Homo sapiens ancestor (Ramachandran et al., 2005; Relethford 2008). This makes the African continent, the ancestral home of all humans today.
Reconstructing the history of the West African population is considered complex (Rosa et al., 2004), this is due to short and long migration events within the region (Tishkoff and Williams, 2002). One of the earliest indications of West Atlantic occupation by modern humans goes back to about 40 KYA (Wood et al., 2005), but subsequent changes in the climatic conditions resulted in the significant movement of these occupants (Aumassip et al., 1994).
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The dearth of archeological evidence (attributable to differences in sea level which may have buried such artifacts) for reconstructing the past has also contributed to this situation, as such language groups were used to genetically classify populations into groups of common descent (Brown and Ogilvie, 2009). This is evident in the fact that cultures may spread without attendant spread of genes, but languages are not easily acquired in later life than other cultural transformations (Bellwood, 2001). The recent development of genetic tools has proven that linguistic groups within Africa share common gene pools (Excoffier etal., 1992)which have become useful in probing phylogenies. The genetic variation in modern man occurred during the events of early migration into new territories, with concomitant localization of these variations to particular regions (Ingman et al., 2000; Atkinson et al., 2009). Studies are ongoing to understand the past events involving population expansion, contraction, genetic drift and substructure. Some of these studies employ genetic methods to probe the human genome to investigate and analyse single nucleotide polymorphisms (SNPs) in conjunction with restriction fragment length polymorphism (RFLP) techniques obtained from the hypervariable region of the d-loop (Chen et al., 1995, 2000; Salas et al., 2002, 2004). These studies have demonstrated that human mtDNA is geographically structured and may be classified into groups of related haplotypes (Chen et al., 1995; Wallace et al., 2007).
All these approaches attempt to provide the historical perspectives of genetic lineages by using various human population groups as the focus of their investigations. It is estimated that more than 2,000 distinct ethnic groups and languages are spoken in Africa (www.ethnologue.com, 2013); however, they belong to comparatively few language families. Studies have shown extensive genetic diversity among geographically close
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African populations. Some 50 African languages have more than half a million speakers each, but many others are spoken by relatively few people. Yet, many studies rely on very few populations within Africa as a representation of the diversity within it (Tishkoff and Williams, 2007). 1.2 STATEMENT OF THE PROBLEM Human variation based on features under selection, often lead to spurious inferences. The push is now towards understanding variability founded on available non-recombining genetic systems, which represent a fundamental part of a population‟s evolutionary history. Thus the focus on the Atyap, Bajju, Chawai and Kagoro located within the ethnically diverse but little studied Middle-belt region. These populations are described as a homogenous population due to shared oral traditions and proximity in linguistics, culture and geography. But this nondescript lumping together has been rejected by these groups as they see themselves as ethnically distinct. This study, however, is undertaken to apply mtDNA markers in testing the nature of relationship within and among these groups and to also establish if there is substantial gene flow between them. 1.3 JUSTIFICATION OF THE STUDY
Africa has played a principal role in the origin of diverse human populations. Therefore, understanding the patterns of genetic variation and the demographic history of populations within Africa is important for understanding the demographic history of global human populations. Out of a vast array of population genetic studies, only a handful of studies
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have been carried out within Africa. Relatively, Nigeria has recorded only a handful of studies on genetic variation at the level of its populations. Thus, there is the need to fill up the gap by carrying out more studies on the available subsets of ethnic populations. The present study aims at providing such needed information to add to existing data on the diverse genetic landscape in Nigeria, and also the African region as a whole. 1.4 AIMS OF STUDY The study is aimed at the identification of the composition and distribution of inherited mtDNA haplogroups of four ethnic groups from Southern Kaduna area. It also seeks to corroborate their origins and to determine the basis for the shared ethnocultural similarities between them as well as neighbouring populations within the Niger-Kordofanian construct. 1.5 OBJECTIVES OF STUDY The objectives of the study are to
i. Isolate mitochondrial hypervariable segment I (HVS-I) sequences from sampled populations of the Bajju, Atyap, Chawai and Gworok ethnic groups.
ii. Sequence the hypervariable segment I (HVS-I) of each individual with respect to their ethnic group.
iii. Identify and compare polymorphisms in these ethnic groups using the revised Cambridge reference sequence (rCRS).
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iv. Assign each individual to a specific haplogroup based on confirmation from characteristic restriction fragment length polymorphisms (RFLP) and single nucleotide polymorphism (SNP) markers
v. Characterize the genetic structure within each study ethnic population using the haplogroup assignments.
vi. Investigate the genetic variability of the study populations which may explain their evolution into different ethnic groups.
vii. Compare the obtained sequences from this study to those in previously published works on Africans.
1.6 HYPOTHESES OF STUDY The following hypothesis would be tested using statistiical variability measures.
i. mtDNA haplogroups markers will indicate genetic structure within each ethnic populations.
ii. There is shared genetic structure between Bajju, Atyap, Chawai and Gworok due to geographical proximity
iii. There is a statistically significant relationship between language family, territory and genetics across the study populations.
iv. There is gene flow between the study populations and other comparative African populations.
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