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ABSTRACT

This study examined the population structure and genetic distance between two Clariid species, Clariasgariepinus and Heterobranchusbidorsalisusing microsatellite markers. Genetic strains of 20 domesticated samples of both species were characterized with four microsatellite markers. 95% of the samples amplified upon PCR amplification and 44.3% of the total alleles observed for all the loci were heterozygote. Analysis showed that all the four loci were polymorphic for all the samples, observed and expected heterozygosity had mean values of 0.4438±0.1116 and 0.9025±0.0211 respectively.

Conformity to Hardy-Weinberg Equilibrium using the Chi-Square test showed 81.25% of locus-population relationship conformed to Hardy-Weinberg Equilibrium. The phylogenetic tree obtained gave a bootstrap value of 72 indicating the genetic distance between the two species.

The result obtained in this research will be used to show the genetic differences between the two species, serve as a preliminary data for the improvement of Clariid fishery and characterization of other fish species.

 

 TABLE OF CONTENTS 
Title pagei
Certificationii
Dedicationiii
Acknowledgementiv
Abstract v
Table of contentsvi
List of tablesix
List of figuresx
CHAPTER ONE 
1.0Introduction1
1.1Microsatellite markers3
1.2Characteristics of microsatellite markers5
1.3.0Advantages and Disadvantages of microsatellite markers5
1.3.1Advantages of microsatellite markers5
1.3.2Disadvantages of microsatellite markers6
1.4Uses of microsatellite markers6
1.5.0Objectives of the study7

 

1.5.1Specific objectives7
1.6Problem statements8
1.7Justification of the study9
CHAPTER TWO 
2.0Literature review10
CHAPTER THREE 
3.0Materials and methods15
3.1Selection of samples15
3.2.0Collection of blood15
3.2.1Apparatus15
3.2.2Procedure15
3.3.0DNA extraction16
3.3.1Apparatus16
3.3.2Procedure16
3.4Selection of microsatellite markers17
3.5.0PCR amplifications17
3.5.1Apparatus17
3.5.2Procedure17
3.6.0Electrophoresis18

 

3.6.1Apparatus18
3.6.2Procedure19
3.7Precautions19
3.8Statistical analysis20
CHAPTER FOUR 
4.0Results21
CHAPTER FIVE 
5.0Discussion30
CHAPTER SIX 
6.0Conclusion and recommendations34
6.1Conclusion34
6.2Recommendations34
 References36

LIST OF TABLES

Tables Page
3.1Optimized annealing temperatures of each primer set18
4.1aAlleles size per locus23
4.1bTotal number of alleles per locus23
4.2Basic indicators of allelic variation for the populations sampled24
4.3Genetic diversity indices for the microsatellite markers used in this26
 study 
4.4Global F-statistics estimates for each microsatellite locus27
4.5Chi-Square tests for Hardy-Weinberg Equilibrium28
4.6Cavalli-Sforza and Edwards, (1967) genetic distance for the four29
 populations 

LIST OF FIGURES

Figures Page
1.1Simple microsatellites4
1.2Composite microsatellites5
4.1Agarose gel showing DNA bands for Heterobranchusbidorsalis after21
 electrophoresis 
4.2Agarose gel showing DNA bands for Clariasgariepinus after21
 electrophoresis 
4.3Allele frequencies by population over each loci24
4.4Cavalli-Sforza and Edwards, (1967) phylogenetic dendrogram29

CHAPTER ONE

1.0       INTRODUCTION

Heterobranchusbidorsalis(Geoffroy Saint-Hilaire, 1809) andClariasgariepinus(Burchell,1822) are economically important fresh water fish species of the Clariidae family that contribute immensely to the annual fresh water fish production in Nigeria. They are also readily acceptable among Nigerian fish farmers and consumers, hence command high commercial values. They are commonly referred to as mud fishes or African catfish in various parts of Nigeria and are important source of animal protein. Among the freshwater species for culture in Nigeria,

HeterobranchusbidorsalisandClariasgariepinusare the most common and have receivedmuch attention because of their economic importance and high rate of success in rearing them.

The family Clariidae belongs to the order Siluriformes and contributes significantly to annual freshwater fish production in South and Southeast Asia and Africa (Na Nakorn, 1999). This family is naturally distributed all over Africa, South and South-East Asia with the highest genetic diversity found in Africa. Nearly one fifth of al1 known catfish species occur in Africa and South-East Asia, however, the highest diversity is found in Africa with 14 genera and 92species (Teugels, 1986a), while only 2 genera with some 17 species are presently known from Asia (Teugels, 1996).

Generally, the Clariidae fishes are elongated, have long dorsal and anal fins, and four pairs of barbels. A remarkable character for this family is the presence of a suprabranchial organ, formed by folds of the second and the fourth branchial arches. With this organ, the fishes are able to practice aerial respiration, implicating that they can survive out of the water for a long time. They are also known for walking on land over distances of several hundred meters, breathing atmospheric air and using their pectoral spines as a support (Teugels and Gourène, 1997).

The genus Clarias is the most common and popular of the family Clariidae containing 32 species in Africa (Teugels, 1986b). One of them, Clariasgariepinus (Burchell, 1822) is of great economic importance as it is the most cultured catfish in Africa and the third most cultured

catfish species in the World (Garibaldi, 1996). Another economic important species of this genus is Clariasanguillaris (Linnaeus, 1758), which is also cultured in Nigeria.

Genus Heterobranchus is mainly recognized and differentiated from Clarias by the presence of an arrayed dorsal fin. Four species of this genus are known, these are Heterobranchuslongifilis

(Valenciennes, 1840), H. bidorsalis (Geoffroy. 1809), H. boulengen (Pallegrin, 1922) and H.isopterus(Bleeker, 1863) which is the smallest member of the genus (Reed et. al, 1967), but onlytwo species are available in Nigeria, H. bidorsalis and H.longifilis

Aluko and Shaba (1999) stated that African catfish, Clarias and Heterobranchus, are widely cultured in Africa and Europe and recently, African catfish is being cultured in Asia. Clariasgariepinusculture started almost fifty years ago in Africa and in 1994, Garibaldi (1996) reporteda production of 3,978 metric tons in Africa, whereas Heterobranchus was recently introduced in aquaculture and has been reported to show promising results (Teugels and Gourène, 1997). Legendre et al., (1992) demonstrated that under identical conditions, Heterobranchuslongifilis has a growth rate which doubles that of Clariasgariepinus. Experiments with Heterobranchusbidorsalishave recently been conducted in Nigeria (Fagbenro et al., 1993), but data onproduction of Heterobranchus species are not yet available. They are intensively and extensively cultured in Africa where they have exhibited high rate of success as a result of the following attributes:

  1. Ability to withstand harsh environmental conditions
  2. Ability to withstand handling stress
  3. High fecundity
  4. Disease resistance
  5. Fast growth rate
  6. High yield potential
  7. High palatability

Despite the popularity of these two species, and the great market potentials, the production is still low, basically at subsistence level due majorly to inadequate availability of seed for stocking, feed and marketing problems. The fingerlings supplied by hatcheries in Nigeria are not sufficient to meet farmers fingerlings needs, hence, there is need for improvement of fingerlings production majorly through genetically modified fish breeding which ensures a high success rate of quality fingerlings production.

The aim of this study is therefore to differentiate Clariasgariepinus and Heterobranchusbidorsalisbased on their genetic characteristics which is an important tool in fish breeding andgenetics. The precise description and characterization of strains in these species is sine qua non to the sustainable management of their cultivated and natural stocks and to guide conservation efforts of these economically important resources.

1.1       Microsatellite Markers

Microsatellite markers are DNA sequences or simple sequence repeats (SSR) genetic markers with a known location on the chromosome that can be used to identify associated strains in organisms. They are used to identify loci on the chromosome where short sequences of DNA nucleotides (Adenine, Guanine, Cytosine, Thymine) are repeated one after the other (in tandem arrays).

They are also called simple sequences (Tautz 1989) and short tandem repeats (STRs) (Edwards et al., 1991). They are Highly polymorphic DNA marker comprisingof  mononucleotides, di-nucleotides, tri-nucleotides or tetra-nucleotides that are repeated in  tandem arrays and distributed throughout the genome. Microsatellite markers are sometimes referred to as variable number of tandem repeats (VNTRs) upon which certain analyses may be based. They are used to identify segments of the DNA that have repeated sequence such as ACACACAC or GTGTGTGT.

According to Magoulaset al., (1997), microsatellites are much more numerous in the genome (particularly of vertebrates) and have a mutation rate between 10-3 and 10-4. They are ideal for mapping “causal” genes responsible for single factor conditions (e.g. muscular dystrophy in humans) or for multifactorial traits (e.g. quantitative trait loci, QTL). They are also the best

markers for determining parenthood in mass-crosses (Magoulaset al., 1997). The basic drawback of microsatellite markers remains the high cost and labour intensiveness of the first phase of the technique, i.e. the development of primers.

The majority of microsatellite loci in fish genomes are composed of the GT motifs, similar to that of higher vertebrates, e.g. rat and human. In addition, microsatellite markers are often conserved among closely related species, e.g. Salmonids and Cyprinids.

Microsatellites can either be classified as simple or composite

  • Simple microsatellite contain only one kind of repeat sequence while composite contains more than one.

Figure 1.1   Simple Microsatellites

Figure 1.2   Composite Microsatellites

  • Characteristics of Microsatellite Markers
  1. They are associated with a specific locus
  2. They are highly polymorphic
  3. They are short sequences, with a length of 1-6 bp
  4. They are locus specific
  5. They are codominat markers i.e. they amplify at only one particular locus.
  • Advantages and Disadvantages of Microsatellite Markers
  • Advantages of Microsatellite Markers
  1. They are abundant; studies have shown that microsatellite exists in 30-40kb DNA in a well studied mammal.
  2. They are evenly distributed on all segments of the chromosome.
  3. They have small locus sizes which allows them to be easily assayed by polymerase chain reaction, hence only a small amount of tissue or blood is required. This also allows it to work on degraded DNA.
  4. The use of markers derived from microsatellite loci leads to a deeper knowledge about the domestication and improvement of desirable traits in breeds.

 

  • Disadvantages of Microsatellite Markers
  1. Previous genetic information is needed before use
  2. Huge upfront work is required for development of the markers
  3. Due to high rate of mutation, it is rarely useful for higher level systematic
  4. In some situation, these markers give bias information since there are relatively few loci to work with.

 

  • Uses of Microsatellite Markers
  1. Microsatellites markers are used to assess level of inbreeding in a population.
  2. They are used for genetic studies such as population genetics, quantitative genetics, diversity and phylogeny studies
  3. They give information about allelic variations at similar loci within individuals of a population and between species of different populations.
  4. They are the prerequisite for the identification of functional and positional genes responsible for quantitative traits in organisms.
  5. They are used to determine genetic distances between various species of a family or organisms.
  6. They play a role in genetic engineering for the production of genetically modified organisms.
  7. They are used for gene mapping because of their high heterozygousity and ease of typing via PCR.
  8. Through genetic linkage, microsatellites alleles are associated to certain mutations or diseases in regions of the DNA, hence can be used to detect genetically induced diseases.
  9. They are the primary marker in use in forensics, both human and wildlife cases(Evett and Weir, 1998).
  10. Microsatellites are also used to trace and verify cytogenetic treatments, such as induction of polyploidy or gynogenesis. It has been used for this purpose in Japanese oyster (Magoulaset al., 1997).

 

Other genetic markers used are:

  • Restriction fragment length polymorphism(RFLP)
  • Amplified sequence length polymorphism(AFLP)
  • Simple sequence length polymorphism(SSLP)
  • Allozymes
  • Randomly amplified polymorphic DNA(RAPD)
  • Minisatellites
  • Mitochondrial DNA(mtDNA) variation
  • Single nucleotide polymorphism(SNP)
  • Single feature polymorphism(SFP)
  • Diversity arrays technology(DArT)

 

1.5.0    Objectives of the Study

The main objective of this study is to characterize genetic strains in Heterobranchusbidorsalis and Clariasgariepinus using microsatellite markers.

 

1.5.1    Specific Objectives

The specific objectives of this study include:

  1. To acquire basic genetic knowledge on how to improve fish breeding through identification and characterization of desirable traits.
  2. To obtain preliminary data on genetic variation in Heterobranchusbidorsalis and Clariasgariepinusbased on DNA microsatellite loci.
  3. To obtain basic knowledge on how to enhance the utilization of catfish and other fish species through correct identification and characterization of cultured and captured fish species.
  4. For assessment and comparison of the aquaculture potential of individual species and hybrids of the genus Heterobranchus and Clarias.
  5. To serve as a model for studies in other fish species in Nigeria.

 

  • Problem Statements

Durnham et al (2001) made an exhaustive review on the status of genetics in aquaculture for the new millennium and its would-be positive impact to aquaculture sustainability. In his review he mentioned the advances in fish breeding programmes in several countries using knowledge of breeding and inheritance (Mendelian principles) and the emerging science of molecular genetics as applied to enhancement programmes in fish and invertebrate species such as salmon, trout, carp, catfish, sea-bass, tilapia, oysters, prawns etc (Omitogun, 2005). In Nigeria, research on fish stock development and management is limited; also, enhancement programs available for fish breeding have not been fully maximized by local farmers.

Morphological description and morphometric analyses were the first tools used to define Tilapiine species (Galman and Avtalion, 1983; Panteet al., 1988). But these techniques are rather arbitrary, and biochemical means (i.e. electrophoresis of expressed isozymes) soon found a more reliable use in such studies (Macarañaset al., 1986, Galmanet al., 1988). Biochemical investigations, however, are still limited in that most of the isozymes are affected by environmental and/or developmental conditions (Galman and Cariño, 1979). DNA-level investigations were thus developed for fish genetic studies. Such approach provides direct investigations of the genetic make-up of several fish species, thus eliminating the effects of extraneous factors. Furthermore, polymorphisms (variant forms) in the DNA are highly numerous as compared to that of isozymes. (Omitogun, 2005).

 

Hence, the following questions were raised in this study.

  • What are the advantages of microsatellite markers and why are they preferred to morphometric or biochemical analysis?
  • Is the application of microsatellite analysis fully maximized in fish’s stock management?
  • Can microsatellite DNA analysis be applied to both cultured and captured fish species?
  • Can molecular genetics help to improve the poor state of Nigeria’s aquaculture?
  • Can genetically improved breeding be practiced by farmers in Nigeria?
  • Justification of the study

The African catfish Clariasgariepinus and Heterobranchusbidorsalis are economically important species, but little is known about the genetic background of the natural populations and cultured stocks of these species. Also, genetic study is needed for proper identification of the two species and determination of the genetic connection between them. Although, morphometric parameters have been used in the past to identify these too species but more specific tool is needed for a more concise differentiation.

Microsatellite DNA marker has been the most widely used for genetic studies, due to its easy use by simple PCR, followed by a denaturing gel electrophoresis for allele size determination, and to the high degree of information provided by its large number of alleles per locus(Vignaletal.,2002).

 

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