GENETIC DIVERSITY IN NATURAL POPULATIONS OF DROSOPHILA MELANOGASTER MEIGEN, 1830 FROM SAVANNA ZONE OF NIGERIA USING MICROSATELLITE MARKERS

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ABSTRACT

 

A study was carried out to evaluate the genetic diversity in natural populations of Drosophila melanogaster using seven microsatellite loci. Fermented banana in hand-made bottle traps was used as bait with 10 replicate traps per site of collection. Samples were collected from November, 2015 to March 2016. A total of 42 male flies from Northern Guinea, Sudan and Sahel Savanna were used for the analysis. Genomic DNA was extracted using the phenol chloroform method; PCR products were amplified on 1.5% Agarose gel by Electrophoresis and scored using Molecular Imager® Gel DocTM XR+ system with image LabTM Software of BIO-RAD. GenAlex version 6.501 and MEGA 6 softwares were used to determine the genetic diversity and population structure. The results revealed that the markers were highly polymorphic (PIC˃0.5) in all the sampled populations. The mean observed heterozygosity for all populations (1.000) was greater than the mean expected heterozygosity (0.500), although the populations were in Hardy-Weinberg Equilibrium (P˃0.05). The highest genetic distance was observed in Northern Guinea vs. Sahel (2.639) while no distance was observed in Northern Guinea vs. Sudan (0.000) and Sudan vs Sahel Savanna (0.000). The lowest genetic identity of 0.000 was observed in Sudan vs. Sahel Savanna, followed by identity of 0.071 in Northern Guinea vs. Sahel Savanna and the highest (1.000) was observed in Northern Guinea vs. Sudan Savanna. The values of genetic distance and genetic identity showed that Northern Guinea and Sudan Savanna D. melanogaster are closely related, while Northern Guinea and Sahel D. melanogaster are divergent species. The AMOVA showed an estimated variation of 2163.35 with variation of 9% among vegetation zones, 0.00 among populations within zone and 22320.139 estimated variation with 91% variation within populations. The FIS value was -1.000 which was lower than the FIT (-0.675) indicating random mating and excess heterozygosity and the FST was 0.162. The pairwise FST and gene flow was 0.333and 0.545 respectively for both Northern Guinea vs. Sudan Savanna and Sudan vs. Sahel Savanna, whereas in Northern Guinea vs. Sahel Savanna, a lower FST (0.314) and higher gene flow (0.545) indicating a high population sub-structure and genetic isolation in natural populations of D. melanogaster. Northern Guinea and Sudan Savanna presented a close cluster in the first quadrant while Sahel Savanna presented a distinct quadrant. The Global Spatial Autocorrelation showed that there was no correlation between genetic distance and geographic distance (r = 0.212, P˃0.05). It can be concluded that the used markers are highly polymorphic (PIC˃0.5). The populations of D. melanogaster are highly genetically diversified; outbreeding; excess of heterozygotes, and are highly sub-structured with consistent clusters which quantify the degree of relationships between the zones.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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TABLE OF CONTENTS

 

Content Page Title Page ……………………………………………………………………… i Declaration …………………………………………………………………….. ii

 

Certification  …………………………………………………………………….    iii

 

Dedication       …………………………………………………………………….. iv

 

Acknowledgements    ……………………………………………………………... v

 

Abstract    …………………………………………………………………………. vi

 

Table of Contents ……………………………………………………………….   vii

 

List of Tables                 …………………………………………………………….    x

 

List of Figures               …………………………………………………………….    xi

 

List of Plates …………………………………………………………………….    xii

 

Lists of Abbreviations              …………………………………………………….    xiii

 

CHAPTER ONE

…………………………………………………………….. 1

1.0.

INTRODUCTION

…………………………………………………….. 1

1.1

Background of the study

…………………………………………….. 1

1.2

Statement of the Research Problem

…………………………….. 5

1.3

Justification

…………………………………………………………….. 5

1.4

Aim   …………………………………………………………………….. 6

1.5

Objectives

…………………………………………………………….. 6

1.6

Hypotheses

…………………………………………………………….. 6

CHAPTER TWO

…………………………………………………………….. 7

2.0

LITERATURE REVIEW

…………………………………………….. 7

2.1

The Fruit fly (Drosophila melanogaster)

…………………………….. 7

2.2

DNA Marker and Genotyping…………………………………….. 10

Content

 

 

 

Page

2.3

Genetic Diversity

…………………………………………………….. 10

 

 

 

 

7

 


2.4

Application of Microsatellite Markers

…………………………….. 14

CHAPTER THREE …………………………………………………………….. 17

3.0

MATERIALS AND METHODS   …………………………………….. 17

3.1

The Study Area

…………………………………………………….. 17

3.2

Sampling Locations

…………………………………………………….. 21

3.3

Drosophila melanogaster Collection

……………………………

22

3.4

Loci Sequenced Selection

……………………………………………

25

3.5

Laboratory Analyses

 

……………………………………………

27

3.6

Genomic DeoxyriboNucleic Acids (DNA) Extraction……………

27

3.7

Polymerase Chain Reaction and Simple Sequence Repeats Protocol..

27

3.7.1

Polymerase chain reaction amplification............................................

27

3.8

Electrophoresis

……………………………………………………

28

3.9

Data Analyses

……………………………………………………

28

3.9.1

Standard diversity indices

…………………………………………...

29

CHAPTER FOUR

……………………………………………………………

34

4.0

RESULTS

……………………………………………………………

34

4.1

Amplification of Microsatellite Loci of Drosophila melanogaster from

 

 

Northern Guinea Savanna  ……………………………………………

34

4.2

Amplification of Microsatellite Loci of Drosophila melanogaster from

 

 

Sudan Savanna

……………………………………………………

34

4.3

Amplification of Microsatellite Loci of Drosophila melanogaster from

 

 

Sahel Savanna

 

……………………………………………

34

4.4

Genetic Diversity of Drosophila melanogaster from Northern Guinea

Savanna

 

……………………………………………………………   38

 

4.5

Genetic Diversity of Drosophila melanogaster from Sudan Savanna ..

38

Contents

 

 

 

 

Page

4.6

Genetic Diversity of Drosophila melanogaster from Sahel Savanna…   38

4.7

Analysis of Molecular Variance (AMOVA) and F- Statistics……

38

4.8

Genetic Distance and Genetic Identity

……………………………

43

 

 

 

 

8

 

 


4.9

Genetic Differentiation and Gene Flow

……………………………   43

4.10

Cluster Analysis for Drosophila melanogaster Populations

……   47

CHAPTER FIVE

……………………………………………………………   49

5.0

DISCUSSION

……………………………………………………   49

5.1

Amplicons of Microsatellite Loci of Drosophila melanogaster from

 

Savanna zones of Nigeria   …………………………………………….  49

5.2

Genetic Diversity in Drosophila melanogaster from Savanna zones of

 

Nigeria

…………………………………………………………….  49

5.3

Population Structure of D. melanogaster from Savanna zones of

 

Nigeria

…………………………………………………………….  50

5.3.1

Analysis of Molecular Variance (AMOVA) and F-Statistics

……. 50

5.3.2

Genetic Distance and Genetic Identity

…………………………….  51

5.3.2

Genetic Differentiation and Gene Flow

…………………………….  52

5.3.3

Cluster Analysis for Drosophila melanogaster Populations ……………. 53

CHAPTER SIX

……………………………………………………………. 54

6.0

SUMMARY, CONCLUSIONS, RECOMMENDATIONS AND

 

 

CONTRIBUTIONS TO KNOWLEDGE

……………………………. 54

6.1

Summary

……………………………………………………………. 54

6.2

Conclusions

……………………………………………………………. 55

6.3

Recommendations

……………………………………………………. 55

6.4

Contributions to Knowledge……………………………………. 55

REFERENCES

…………………………………………………………….  57

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF TABLES

Table

 

Page

3.1

Sampling Locations   ……………………………………………………....  21

3.2

Drosophila melanogaster Microsatellite Primers for Genetic Diversity    27

4.1

Genetic Diversity of Drosophila melanogaster from Northern Guinea

 

Savanna………………………………………………………….……. 40

4.2

Genetic Diversity of Drosophila melanogaster from Sudan Savanna ……... 41

4.3

Genetic Diversity of Drosophila melanogaster from Sahel  Savanna………. 42

4.4

Analysis of Molecular Variation (AMOVA) and Fixation Indices   .………. 43

4.5

Genetic Distance and Genetic Identity

……………………….………. 45

4.6

Pairwise FST and Gene Flow  ……………………………………….………. 47

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

10


 

 

 

 

 

 

 

 

 

 

 

LIST OF FIGURES

 

Figure

 

Page

3.1

Vegetation map of Nigeria    ……………………………………………... 19

3.2

Sampling Locations   ……………………………………………………... 21

4.1

Correlation between genetic and geographic distances……………... 45

4.2

Dendrogram Representing Phylogenetic Relationship of Drosophila

 

melanogaster from Savanna zones of Nigeria

……………………... 48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF PLATES

 

Plate                                                                                                                                                                    Page

 

I                     Drosophila melanogaster……………………………………………………... 23

 

II            Hand made Bottle Trap             ……………………………………………….. 24

 

III (a)  Amplicons of PCR Analysis of Drosophila melanogaster from

 

Anguwan- Dosa             ……………………………………………………….. 35

 

III (b)  Amplicons of PCR Analysis of Drosophila melanogaster from

 

Danhono            ……………………………………………………………….. 35

 

IV (a)  Amplicons of PCR Analysis of Drosophila melanogaster from

 

Hotoro ……………………………………………………………………….. 36

 

IV (b)  Amplicons of PCR Analysis of Drosophila melanogaster from

 

Gunduwawa    ……………………………………………………………….. 36

 

V (a)    Amplicons of PCR Analysis of Drosophila melanogaster from

 

Kasuwan Azare              ……………………………………………………….. 37

 

V (b)   Amplicons of PCR Analysis of Drosophila melanogaster from

 

Kuzuru                ……………………………………………………………….. 37

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF ABBREVIATIONS


 

AMOVA

 

AZC

 

AZV

 

D

 

DNA

 

FST

 

FIS

 

FIT

 

GSA

 

Ho

 

He

 

HWE

 

I

 

KDC

 

KDV

 

KNC

 

KNV

 

LS

 

NJ

 

Nm

 

Ne

 

PIC

 

PCR

 

SSR

 

Vs


Analysis of Molecular Variance

 

Azare City (Kasuwan Azare)

 

Azare Village (Kuzuru)

 

Nei‟s Genetic Distance

 

Deoxyribonucleic Acids

 

Genetic Differentiation for total population

 

Genetic Differentiation for sub-populations

 

Coefficient of Inbreeding

 

Global Spatial Autocorrelation

 

Observed Heterozygosity

 

Expected Heterozygosity

 

Hardy-Weinberg Equilibrium

 

Nei‟s Genetic Identity

 

Kaduna City (Anguwan-Dosa)

 

Kaduna Village (Danhono)

 

Kano City (Hotoro)

 

Kano Village (Gunduwawa)

 

Level of Significance

 

Neighbor- Joining

 

Gene flow

 

Effective Number of Alleles

 

Polymorphism Information Content

 

Polymerase Chain Reaction

 

Simple Sequence Repeats

 

Versus


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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CHAPTER ONE

 

1.0         INTRODUCTION

 

1.1         Background of the Study

 

Evolution is the dual process of genetic change and diversification of organisms through time resulting in populations diverging from one another in their genetic characteristics thus giving rise to new species. The leading evolutionary forces such as mutation, natural selection, and genetic drift have created a vast diversity of sub populations which led to the formation of many well defined species with different levels of performance (Mahmut, 2012).

 

Drosophila melanogaster (Diptera: Drosophilidae) is generally known as fruit fly or vinegar fly. The fly is said to have probably originated from sub-Sahara Africa (Capy et al., 2004), but is also able to proliferate under temperate climate which could be as a result of the spread of beneficial mutations in non- Africa populations (Kirby and Stephan 1996; Kauer et al., 2003) and selection pressure imposed by man such as the species resistance to insecticides (Daborn et al., 2001). Drosophila melanogaster is probably considered the most differentiated into geographic subpopulations (David et al., 2007).Wild type fruit flies are yellow-brown, with brick red eyes and transverse black rings across the abdomen. They, exhibit sexual dimorphism, females are about 2.5 millimeters (0.098 in) long; males are slightly smaller with a distinct black patch on the abdomen, and a cluster of spiky hairs (claspers) surrounding the reproducing parts used for attachment to the female during mating (Flybase, 2009). The “Drosophila season” as stated by Pavkovic and Kekic (2014) is usually between March to October due to abundance of fruits and vegetables.

 

Although there are obvious differences between humans and D. melanogaster, there are many molecular and cellular processes that are common between humans and the fruit fly such as aggression, sleep, learning, memory, circadian rhythm and mating which makes the fruit fly

 

 

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an important model in the investigation on functions of specific genes, diseases and effectiveness of various promising therapeutic drugs (Valente et al., 2004). The short life cycle, large number of offsprings and small genome which was fully sequenced in year 2000 makes genetic manipulation of the fruit flies easy (Adams et al., 2000).

 

D. melanogaster serves as a multiple model organism as its embryo, larva, pupa and adult can be used as models in different toxicological settings. For instance, the embryo and the pupa can be used as models in developmental toxicological studies; the larva can be used as a model for physiological and behavioural studies, while the adult fly posses structures that can mimic the equivalent functions of mammalian reproductive tract, heart, kidney, gut and lung. (Nichols et al., 2002; Wolf and Heberlein, 2003; Andretic et al., 2008). It has been estimated that about 75% of known human disease genes have a recognizable match in the genome of fruit flies (Reiter et al., 2001). Drosophila is nowadays often used as a “test tube” to screen for genetic components of disease-relevant processes or pathways, or to unravel their cellular and molecular mechanisms, covering a wide range of disease mechanisms including neurodegenerative (Parkinson's, Huntington's, spinocerebellar ataxia and Alzheimer's disease), neurotoxicology (Bier, 2005; Rand, 2010; Hu et al., 2011; Jaiswal et al., 2012) and is also being used to study mechanisms underlying aging and oxidative stress, epilepsy, immunity, diabetes, and cancer, as well as drug abuse (Chien et al., 2002). This is to say that the fly has basic biological, biochemical, neurological, and physiological similarities with mammals (Abolaji et al., 2013).

 

The fruit fly may have ten or more generations per year, oviposit in a wide variety of substrates and considered a generalist feeder (Markow and Grady, 2008). D. melanogaster is commonly considered a pest due to its tendency to invade and establish populations where fruit crops are grown. The flies are seen in homes, restaurants, stores, and even in dump sites.

 

 

 

 

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Reduction of an infestation on a fruit farm can be difficult, as larvae may continue to hatch in nearby fruit even as the adult population is eliminated (James, 2009).

 

The use of fruit flies as an invertebrate model organism in the field of classical genetics was introduced more than a century ago due to the fact that it is an omnipresent follower of human culture, easily obtainable and easily maintained in laboratories (Kohler, 1994). Its genetics have been systematically applied to the study of development, physiology and behaviour, generating new understanding of the principal genetic and molecular mechanisms underpinning biology, many being conserved with higher animals and humans (Ashburner, 1993; Keller, 1996; Martinez, 2008; Bellen et al., 2010). Therefore, diversity analysis and identification of genotypes are vital to the D. melanogaster conservation, control and breeding programmes.

 

Genetic diversity which is the total number of genetic characteristics in the genetic makeup of a species is a combination of both variety and variability and a requirement for populations to evolve and cope with environmental changes, new diseases, and pest epidemics (Mahmut, 2012) and which also significantly influences the long-term viability and persistence of local populations (Sushila and Jaya, 2013). Genetic variation is one of the three levels of biodiversity that the World Conservation Union (IUCN) has recommended for conservation, as it is a very important requirement for evolution and a direct linkage to population fitness (Reed and Frankham, 2003). Genetic variation exists within and among members of populations which is brought about by mutation: which is a change in the chemical structure of a gene, random mating, and recombination between homologous chromosomes (Lars et al., 2006).This provides a huge source of information about the biology of an individual species, their history and spatial relationships between populations. The amount and nature of genetic variation in a population allows for the estimation of effective population size,

 

 

 

 

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structure, how selection acts on genes and location of diseases on genes (QTL mapping), (David et al., 2005).

 

The assessment of genetic diversity may be done at molecular level by using different markers based techniques such as allozymes (Biochemical marker), Random Amplified Polymorphic DNA- Polymerase Chain Reaction (RADP-PCR), Restriction Fragment Length Polymorphism (RFLP), Microsatellite (Molecular markers) (Penzes et al., 2002). The molecular markers are more accepted because they overcome many of the limitations morphological and biochemical techniques poise since they are not affected by the environment or developmental stages and can detect a variation at the DNA level.

 

Microsatellite marker is among the most recently developed molecular marker which gives a much higher estimate resolution even at small spatial scales when compared with other markers such as allozymes, RAPD (Turlure et al., 2014). It is currently the marker of choice for molecular genetic studies such as reconstruction of phylogenetics and relationships among populations (MacHugh et al., 1997), determination of paternity and kinship analyses, forensic studies, linkage analysis and population structures (Arora and Bhatia, 2004; Schlotterer, 2004) because they are highly polymorphic, highly abundant, co-dominantly inherited, easy to analyze and score. However, null alleles, or size homoplasy could be seen in using the marker (Schlotterer, 2004).

 

Microsatellite also known as Simple Sequence Repeats (SSR) is a class of repetitive DNA elements, which according to Kahl (2001) is any one of a series of very short (2-10 base pairs), middle repetitive, tandemly arranged, highly variable DNA sequences which are dispersed throughout living organisms genomes. They are generally found in nuclear genome, usually in the introns of genome. Microsatellites are "junk" DNA, and are selectively neutral (Li et al., 2002). Microsatellite alleles when amplified are of variable lengths which can be separated by gel electrophoresis and visualised by silver-staining,

 

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autoradiography (if primers are radioactively labelled) or via automation (if primers are fluorescently labelled) (FAO/IAEA, 2002).

 

1.2     Statement of the Research Problem

 

Every organism in its natural habitat is faced with constantly changing pressure from natural forces, such as temperature, light, competition, predation, or from human impacts such as pollution, habitat destructions which result in a highly variable environment (Sofija and Vladimir, 2014). In order for a species to survive, part of the population of that species must exhibit sufficient genetic variability to adapt to the changing environment; this forms the basis of natural selection (Bader, 1998).

 

Genetic variations among D. melanogaster population have been analyzed using different genetic markers in different parts of the world (Kaurer et al., 2003; Scholotter et al., 2005). No reported studies have been conducted in the Savanna zone of Nigeria thereby leaving the genetic structure and genetic relationship of this species unexplored in this geographic area.

 

1.3    Justification

 

The level of genetic variation among populations has received considerable attention, because it is indicative of overall species fitness and potential for evolutionary responses to environmental changes (Mateus and Sene, 2003).

 

The recent ethical issues on the use of Mice, Bacteria, Nematodes and Zebra fish have led scientists to seek for a cost effective research organisms that can be studied for many, if not all perspective with little ethical concerns (Koushik and Krishna, 2013). Drosophila especially D. melanogaster may be such an organism whose genetics have revealed it to be a powerhouse for unraveling concepts and fundamental understanding of basic biology.

 

Knowledge on the genetic diversity in natural populations of D. melanogaster would provide relevant information for developing strategies to conserve its genetic resources, for genetic

 

 

 

 

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control, developing new strains for neuroscience, for breeding programmes, and would also provide insights regarding the natural history and evolutionary relationship of this insect.

 

1.4     Aim of the Study

 

The aim of this study is to investigate the genetic diversity in natural populations of D.

 

melanogaster from Savanna zones of Nigeria using Microsatellite markers.

 

1.5     Objectives of the Study

 

i.            To determine the degree of polymorphism in markers from D. melanogaster populations.

 

ii.             To determine the genetic diversity within and among natural populations of D. melanogaster from Savanna zone of Nigeria.

 

iii.             To determine the genetic relationships within and among D. melanogaster populations from Savanna zone of Nigeria.

 

1.6    Hypotheses

 

i.            There is no polymorphism in markers from D. melanogaster populations.

 

ii.             There is no genetic diversity within and among natural populations of D. melanogaster from Savanna zone of Nigeria.

 

iii.             D. melanogaster populations from Savanna zone of Nigeria are not genetically related.

 

 

 

 

 

 

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