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GENETIC DIVERSITY OF FIVE POPULATIONS OF THE NIGERIAN LOCAL BREEDS OF GOAT USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS
ABSTRACT
Genetic diversity and genetic distance of five populations of the Nigerian breeds of goat were investigated using random amplified polymorphic DNA markers. Five breeds of goat were used for the study and each breed constituted a population, hence, the five populations considered in the study. The populations were: Sokoto Red (SR), Sahel (SH), Kano Brown (KB), Bornu White (BW) and West African Dwarf (WAD) goats. The experiment was conducted within four geographical zones of Nigeria: South east, North west, North east and North central. One hundred and twenty (120) blood samples were randomly collected from various locations across the four geographical zones in Nigeria. The samples were collected from the stocks at the central markets, which served as collection point from all the localities and from household goat keepers. Varying numbers of sample sizes (Sokoto Red = 23, Kano Brown = 21, Bornu White = 23, WAD = 26 and Sahel = 27) were collected from each breed. The blood samples were collected from the jugular vein of the animals through a process known as venipuncture. Approximately, 5 ml of blood was collected aseptically from each animal into an EDTA container, using 23 gauge sterile needle and syringe, and was stored at -20oC using (ethylene-di-amine-tetra-acetic acid, EDTA) as anticoagulant containers. The DNA samples were isolated and purified from the 120 blood samples following protocol as recommended by (ZYMO RESEARCH CORPORATION, e-mail: info@zymoresearch.com). The RAPD-PCR reaction followed the procedure described by El Hentati et al., (2012). Seven random primers were used for this study but only three (3) primers produced clearly polymorphic and reproducible bands whereas four (4) failed to produce any band. Allele frequency per population per loci under consideration exceeded the minimum allele frequency (MAF) limit 10%. Level of polymorphism per primer varied from 43 – 80%. Total of 20 scoreable bands were obtained and thirteen (13) out of which were polymorphic to arrive at a total of 65% polymorphism. Genetic diversity of the studied populations was measured with three indices: (Nei’s genetic diversity, Shannon’s information index, Observed and Effective number of alleles). Observed number of allele (Na) was 2.0000 across the populations while Effective number of alleles (Ne) varied from 1.9003 in WAD to 1.9900 in Sahel population. However, SR, KB and BW had Ne values of 1.9897, 1.9287 and 1.9836 respectively. Nei’s heterozygosity varied across the populations with highest values 0.4975 and 0.4974 obtained in Sahel and Kano Brown respectively whereas lowest value 0.4736 was obtained in WAD. Shannon’s Information index followed similar trend across with average of 0.6822. The mean of coefficient of gene differentiation Gst was (0.0139) and mean of gene flow Nm across populations was (35.3710). The highest genetic similarity (0.9995) and lowest genetic distance (0.0005) was recorded between Sokoto Red and Sahel, while the lowest similarity (0.9505) and highest genetic distance (0.0507) was recorded between Bornu White and WAD. Populations with higher similarity indicated that they are of closer descent and closer geographical locations. The unweighted pair group method of arithmetic means (UPGMA) dendrogram based on Nei’s genetic distance clearly separated the five populations into two clusters. The closest relationship was observed between Sokoto Red, Sahel and Bornu White goat populations/breeds and the farthest relationship was observed between WAD and Sokoto Red populations. It was concluded that the high similarity obtained in this study was as a result of loss of heterozygosis in the populatons of Nigerian breeds of goat which may have originated among mates in each population/breed. However, the results of this experiment can offer some crucial scientific data useful for breeding programme of Nigerian breeds of goats.
TABLE OF CONTENTS
Page
Title page — — — — — — — — — — i
Certification — — — — — — — — — — ii
Dedication — — — — — — — — — — iii
Acknowledgement — — — — — — — — — iv
Table of Contents — — — — — — — — — v
List of Tables — — — — — — — — — — ix
List of Figures — — — — — — — — — — x
Abstract — — — — — — — — — — xi
CHAPTER ONE
1.0. Introduction — — — — — — — — — 1
1.1. Statement of Problem — — — — — — — — 4
1.2. Objective of the study — — — — — — — — 5
1.3. Justification of the study — — — — — — — — 5
CHAPTER TWO
2.0. LITERATURE REVIEW — — — — — — — 7
2.1. Origin and Distribution of goat — — — — — — — 7
2.1.1. West African Dwarf goat — — — — — — — 7
2.1.2. Sahel goat — — — — — — — — — 8
2.1.3. Red Sokoto goat — — — — — — — — 8
2.1.4. Kano Brown Goat — — — — — — — — 9
2.1.5. Bornu White Goat — — — — — — — — 10
2.2. Taxonomic Classification of Goat — — — — — — 10
2.3. Origin and History of Livestock Diversity — — — — — 11
2.4. State of Genetic Diversity in Nigeria — — — — — — 11
2.5. Molecular Genetic Characterization — — — — — — 15
2.6. Methods for Measuring Genetic Diversity — — — — — 15
2.7. Molecular Genetic Diversity Indices — — — — — — 15
2.7.1. Number and Frequency of Alleles — — — — — — 15
2.7.2. Expected and Observed Heterozygosity — — — — — 16
2.7.3. Polymorphism Information Content (PIC) — — — — — 17
2.7.4. Dendrogram or Phylogenetics or Cluster Diagram — — — — 18
2.7.5. Allele Amplitude — — — — — — — — 18
2.7.6. Genetic distance — — — — — — — — 19
2.7.6.1. Measures of Genetic Distance — — — — — — 19
2.7.7. Shannon’s H and E Indices — — — — — — — 20
2.7.7.1. Shannon’s Index — — — — — — — — 21
2.8. Molecular Markers — — — — — — — — 21
2.8.1. Random Amplification Polymorphism DNA (RAPD) Markers — — 23
2.8.2. Restriction fragment length polymorphism — — — — — 24
2.8.3. Amplified fragment length polymorphism (AFLP) — — — — 25
2.8.4. Definition of SNPs and the generation of single nucleotide polymorphisms– 26
2.8.5. Microsatellite — — — — — — — — — 27
2.8.5.1. Efficiency of Microsatellites — — — — — — — 28
2.9. Uses of Genetic Markers — — — — — — — — 29
2.10. Marker-assisted Breeding for Animal Improvement — — — — 29
2.10.1. Marker-assisted Selection — — — — — — — 30
2.11. Characteristics of a Useful Molecular Marker — — — — — 31
2.12. Non-DNA or Protein markers (Isozyme markers) — — — — 32
2.13. Polymerase Chain Reaction (PCR) — — — — — — 32
2.13.1. Mechanism of Polymerase Chain Reaction — — — — — 33
2.13.2. PCR Based Technique — — — — — — — 34
2.14. Electrophoresis — — — — — — — — — 35
2.15. Agarose Gel Electrophoresis (Component and Function) — — — 36
2.16. Polyacrylamide Gel Electrophoresis — — — — — — 37
2.16.1. Preparing Acrylamide Gels — — — — — — — 38
CHAPTER THREE
3.0. MATERIALS AND METHODS — — — — — — — 39
3.1. Location of the study — — — — — — — — 39
3.2. Experimental animals — — — — — — — — 39
3.3. Sample Collection and Handling — — — — — — — 39
3.3.1. Laboratory Analysis — — — — — — — — 40
3.4. DNA extraction/isolation and purification — — — — — 41
3.5. RAPD-PCR (Polymerase Chain Reaction) conditions — — — — 42
3.6. Gel electrophoresis — — — — — — — — 42
3.6.1. Gel Electrophoreses and scoring of bands — — — — — 43
3.6.2. Preparation of Agarose gel — — — — — — — 43
3.6.3. Pouring the gel — — — — — — — — — 44
3.6.4. Placing the Gel into the gel Tank — — — — — — 44
3.6.5. Loading the samples — — — — — — — — 45
3.6.6. Running the gel — — — — — — — — — 45
3.6.7. Viewing the results — — — — — — — — 45
3.7. Statistical analyses — — — — — — — — 45
3.8. Genetic Diversity Indices estimated — — — — — — 46
CHAPTER FOUR
4.0. RESULT AND DISCUSSION — — — — — — — 47
4.1. Allele frequency per population per locus — — — — — 47
4.2. Summary of Allele frequency across the three loci — — — — 48
4.3. Primer sequences and number of scoreable bands — — — — 48
4.4. Genetic diversity of the five Nigerian local goat populations — — — 50
4.5. Summary of genetic diversity across the loci — — — — — 53
4.6. Analysis of gene diversity (gene differentiation) and gene flow in subdivided
Population — — — — — — — — — 54
4.7. Measure of genetic similarity and genetic distance — — — — 56
4.8. Dendrogram measure of genetic distance — — — — — 58
4.9. Measure of between and within breed genetic distance — — — — 59
CHAPTER FIVE
5.0. CONCLUSION AND RECOMMENDATIONS — — — — 62
5.1. CONCLUSION — — — — — — — — — 62
5.2. RECOMMENDATIONS — — — — — — — — 62
APENDIX
Laboratory practical training on molecular DNA analysis (Fig:a-g) — — — 63
REFERENCES — — — — — — — — — 64
CHAPTER ONE
INTRODUCTION
Nigeria is a country with heavy human population of about 168.8 million (U.S.C.B., 2012), and this population is continuously on the increase. The increase has led to the high demand for the available animal and animal products in all parts of the country, to meet up with the minimum animal protein requirement per individual per day. The protein intake of an average Nigeria is 45.5g per head per day as against the Food and Agricultural Organization’s recommended minimum intake of 70g per head per day, of which 35g should be of animal source (FAOSTAT, 2010). The inadequate supply of animal protein in Nigeria can be attributed to inadequate production potentials of the most common sources of meat which include poultry, goat, cattle, pig, sheep, rabbit etc. Gambo et al. (2004) reported that among the cheapest and mostly affordable protein source for this ever increasing population is mainly the poultry products and chevrons (goat meat).
Goat (Capra hircus) is one of the smallest domesticated ruminants which are managed for the production of milk, meat, wool and leather particularly in arid, semi-tropical or mountainous countries (Morand-Fehr, 2004). Goat is the most prolific ruminant among all domesticated ruminant under tropical and subtropical conditions. It is a resourceful and efficient ruminant producing meat, milk, skin and hair (Morand-Fehr, 2004).
Goats constitute the largest group of small ruminant livestock in Nigeria totalling about 53.8 million and also constituting 6.2 percent of the World’s goat population (FAOSTAT, 2011). Surveys have shown that up to 85 percent of rural households, poor farmers and small-time business people of all age groups and sexes keep goat (FDLPCS, 2007). The ability of goats to tolerate harsh climates, the presence of trypanotolerance in some breeds (Salako, 2004), suitability to traditional systems on account of small size, short generation interval (Abdul-Aziz, 2010) and ability to thrive on poor quality diets provided by scarce grazing on marginal lands (Adedeji et al., 2011) all combine to make small ruminants strategic to increasing livestock productivity in rural agricultural systems (Adedeji et al., 2011). Despite these advantages, little attention has been paid to the genetic characterization and possible improvement of small ruminants in Nigeria.
Detailed knowledge of population structures among and within breeds of livestock is essential for establishing conservation priorities and strategies (Caballero and Toro, 2002). Livestock production is vital to subsistence and economic development (Yakubu and Ibrahim, 2011). The ever increasing demand for livestock production to cater for the nutritional needs of rapidly growing human population has led to indiscriminate crossbreeding in an effort to improve productivity. It has been estimated that up to 1.6 billion people rely on livestock to supply part of their entire daily needs. Consequently, there is the need for conservation and sustainable use of the indigenous livestock genetic resources that are found in the Africa continent (FAO, 2000), precisely Nigeria.
Molecular markers are important tools in tagging desirable loci underlying the expression of traits which have breeding importance. Estimations of genetic variation increasingly are being based upon information at the DNA level by various molecular markers such as, Randomly amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP) (Rincon et al., 2000), Simple Sequence Repeat (SSR) or Microsatellite (Dalvit et al., 2008) etc. Among them, RAPD markers, generated by the polymerase chain reaction (PCR) is widely used since the 1990’s to assess infra-specific genetic variation at nuclear level (Welsh and McClelland, 1990; Williams et al., 1990).
In recent years, a range of innovations in molecular genetics have been developed for the study of genetic variation and evolution of populations using DNA marker genotype information. The study of genetic variation plays an important role in developing rational breeding strategies for economical animal species (Maudet et al., 2002). Genetic analysis of livestock species have been performed using of polymorphic markers such as restriction fragment length polymorphisms (RFLPs) and microsatellites (Rincon et al., 2000; Dalvit et al., 2008). However, their use is limited since designation of these genetic markers is expensive, technically demanding and is time consuming (Beuzen et al., 2000). However, Random amplified polymorphic DNA (RAPD) assay which uses short oligonucleotide primers of arbitrary sequence to amplify genomic DNA by Polymerase Chain Reaction (PCR) enables an approach for identifying polymorphic and genetic markers faster (Cushwa and Medrano, 1996). A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze the process, DNA polymerase, can only add new nucleotides to an existing strand of DNA. These markers have been used for genotype identification (Tinker et al., 1993), construction of genetic maps (Maddox and Cockett, 2007), etc. The RAPD technique has also been used in analysis of genetic variations between different breeds of animals such as fish (Ambak et al., 2006), chicken (Okomus and Kaya, 2005), cattle (Hassen et al., 2007), buffaloes (Abdel-Rahman and Elsayed, 2007), goat (Yadav and Yadav, 2007) and sheep (Kunene et al., 2009).
Random amplified polymorphic DNA (RAPD) molecular markers, has proved to be an efficient tool in the quantification of genetic diversity and genetic characterization of various species and populations (Rahman et al., 2006; Barker et al., 2001). Random amplified polymorphic DNA (RAPD) was developed by Williams et al. (1990) and was proved to be a powerful tool in different genetic analyses. This approach detects DNA polymorphisms based on amplification using single DNA fragments. They are specific and quick and do not require previous DNA sequence information (Williams et al., 1990; Ragot and Hoisington, 1993).
Genetic diversity may be measured through genetic markers. These have been used to determine evolutionary relationship within and between species, genera or higher taxonomic categories (Paterson et al., 1991). However, breeders tend to concentrate on specific genotypes for determination of genetic diversity which combine traits of interest and may be used as progenitors in several breeding programmes in order to introduce important genetic traits. In an attempt to solve the problem of maintaining pure breeds using the observed morphological characteristics that require a lot of time and effort, the use of molecular markers in maintaining goat breeds is more suitable and less time consuming.
Gene diversity is an appropriate measure of genetic variability within a population. Genetic variation between and within breeds is described as diversity. Low genetic variability may be caused by selection practices in farms. Populations showing higher intra-breed similarity and lower proportion of polymorphic loci are likely to have less heterozygosity, i.e. possess lower level of genetic variation compared to those showing less intra-breed similarity and high proportion of polymorphic loci. In other words, genotypes having higher similarity have more homozygous groups (Yasmin et al., 2006).
Genetic diversity is shaped by past populations and the future. The maintenance of genetic diversity is a key to the long-term survival of most species (Hall and Bradley, 1995). Farm animal genetic diversity is required to meet current production needs in various environments, to allow sustained genetic improvement and to facilitate rapid adaptation to changing breeding objective (Kumar et al., 2006). It is essential to characterize a breed for its conservation. If genetic diversity is very low, none of the individuals in a population may have the characteristics needed to cope with the new environmental conditions or challenges. Such a population could be suddenly wiped out. Low amount of genetic diversity increases the vulnerability of populations to catastrophic events such as disease outbreaks. Also, low genetic diversity may indicate high levels of inbreeding with its associated problems of expression of deleterious alleles or loss of over-dominance. Change in the distribution of the pattern of genetic diversity can destroy local adaptation and break up co-adapted gene complexes. These problems combined, lead to an increasing poorer ‘match’ of the population to its habitat, and eventually lead to the probability of population or species extinction (Bizhan et al., 2010).
The genetic diversity of goat population in Nigeria needs a thorough investigation so as to identify the undisclosed genetic potentials associated with these animals, conserve the genetic resource and develop better breeding strategies. Molecular markers (RAPD markers) are the useful tools for the divulgence of these genetic attributes and can also be employed in formulating long term inference or plans for genetic improvement programmes.
1.1. Statement of Problem
Production performances of Nigerian breeds are limited compared to those of their counterparts reared in temperate countries. The limited performances may not only be explained by the breeds’ genetic potentials but also traditional management practices. On the other hand, local breeds are well adapted to the rather difficult conditions. These unique characteristics are results of the evolutionary forces and their interactions over long periods of time. However, resistance and adaptation capabilities might have declined due to indiscriminate mating, crossbreeding, sub-structuring and consequent genetic drift in these goat populations over time.
For more than four decades now, the efforts of breeders in Nigeria to improve the performance of our native breeds of goats have virtually proven abortive due to limited knowledge of the genetic diversity of these animals, hence, the inevitability role of this experiment. Furthermore, as the demand for certified products such as meat and milk is increasing, the focus is now on local indigenous breeds. Consequently, indigenous populations will be at a higher risk of genetic erosion due to selection pressure, indiscriminate mating and lack of characterization, unless adequate steps are taken to characterize and conserve their genetic diversity. A detail genetic study of local goat populations is therefore imperative in quantifying their structure of genetic diversity and optimizing conservation and utilization strategies.
Majority of countries in the developed world have the genetic information/status of their local breeds stored in the genebank to be accessed by everybody throughout the world, reason being that there have been substantial research works carried out on the subject matter, but such information is lacking with regard to Nigerian local breeds of goats due the fact that there are limited research work. Unfortunately, this is due to non-involvement in biotechnologically oriented researches as a result of their high cost, lack of governmental incentives on research and lack of interest.
Establishment of genebank in Nigeria is of paramount importance as this could serve as benchmark or reference point for accessibility of phenotypic and genotypic information on Nigerian native breed of goats. However, to feed the rapidly increasing population in Nigeria as well as to counter the challenges imposed by global climate change and fragile natural resource base, animal production need to be doubled in the next three decades, through exploration of their genetic potentials and marker assisted selection (MAS).
Therefore, the investigation of genetic diversity and similarity between and within breeds is necessary to provide useful genetic information essential for developing effective management plans for the conservation and improvement of the genetic resources. Furthermore, there is a worldwide recognition of the need for the conservation of livestock diversity (FAO, 1995) and for the genetic characterization of breeds and populations including their genetic differentiation and relationships.
1.2. Objective of the study
General Objective of the study
The general objective of this study was to evaluate the genetic diversity of five populations of Nigerian breeds of goat (Sahel, West African Dwarf, Red Sokoto, Kano Brown and Bornu white), using random amplified polymorphic DNA markers.
Specific Objectives of the study
To evaluate the allele frequencies of the populations and primer loci used
To evaluate the genetic diversity of Nigerian local breeds of goat across populations and marker loci.
To determine the gene differentiation and gene flow in the five goat populations.
To determine the between and within breed genetic similarity and genetic distance among goat populations, and classify them using dendrogram into their most likely populations of origin.
1.3. Justifications of the study
Goats, particularly Nigerian breeds, are genetically diverse, so it is important to analyze their genetic diversity in order to identify populations and individuals of particular merit. Genetic diversity of indigenous goat breeds in Nigeria has not been sufficiently studied or understood clearly. Considering the fact that much research works have not been carried out on the genetic diversity of the Nigerian breeds of goat, coupled with the fact that this nature of research is a novel and still at its infancy stage in the country, it becomes imperative that this study be carried out to create an insight on the genetic integrity of the Nigerian breeds of goat, which can also serve as a benchmark to conservation, selection and improvement strategies. For instance, low genetic distance among the populations is an indication of incessant inbreeding which on the contrary reduces heterosis, whereas, high genetic distance is as a result of long term divergence and lack of genetic mixing and inter-migration between the breeds, groups and populations. Uncontrolled breeding and indiscriminate mating of these animals to other breeds (exotic or local) of goat has led to the dilution of these enormous genetic potentials. Therefore, the genetic study of the goat populations in Nigeria is crucial, so as to integrate the result into the livestock sector data base and as well conserve the superior genotypes, and also, to provide information at the molecular level about genetic structure and diversity of Nigerian goat breeds reared across the six geographical zones in Nigeria, employing RAPD-PCR molecular markers.
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