Characterization Of Genetic Strains In Clariid Species, Clarias Gariepinus And Heterobranchus Bidorsalis Using Microsatellite Markers
ABSTRACT
This study examined the population structure and genetic distance between two Clariid species,
Clarias gariepinus and Heterobranchus bidorsalis using 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 page | i | |
| Certification | ii | |
| Dedication | iii | |
| Acknowledgement | iv | |
| Abstract | v | |
| Table of contents | vi | |
| List of tables | ix | |
| List of figures | x | |
| CHAPTER ONE | ||
| 1.0 | Introduction | 1 |
| 1.1 | Microsatellite markers | 3 |
| 1.2 | Characteristics of microsatellite markers | 5 |
| 1.3.0 | Advantages and Disadvantages of microsatellite markers | 5 |
| 1.3.1 | Advantages of microsatellite markers | 5 |
| 1.3.2 | Disadvantages of microsatellite markers | 6 |
| 1.4 | Uses of microsatellite markers | 6 |
| 1.5.0 | Objectives of the study | 7 |
vi
| 1.5.1 | Specific objectives | 7 |
| 1.6 | Problem statements | 8 |
| 1.7 | Justification of the study | 9 |
| CHAPTER TWO | ||
| 2.0 | Literature review | 10 |
| CHAPTER THREE | ||
| 3.0 | Materials and methods | 15 |
| 3.1 | Selection of samples | 15 |
| 3.2.0 | Collection of blood | 15 |
| 3.2.1 | Apparatus | 15 |
| 3.2.2 | Procedure | 15 |
| 3.3.0 | DNA extraction | 16 |
| 3.3.1 | Apparatus | 16 |
| 3.3.2 | Procedure | 16 |
| 3.4 | Selection of microsatellite markers | 17 |
| 3.5.0 | PCR amplifications | 17 |
| 3.5.1 | Apparatus | 17 |
| 3.5.2 | Procedure | 17 |
| 3.6.0 | Electrophoresis | 18 |
vii
| 3.6.1 | Apparatus | 18 |
| 3.6.2 | Procedure | 19 |
| 3.7 | Precautions | 19 |
| 3.8 | Statistical analysis | 20 |
| CHAPTER FOUR | ||
| 4.0 | Results | 21 |
| CHAPTER FIVE | ||
| 5.0 | Discussion | 30 |
| CHAPTER SIX | ||
| 6.0 | Conclusion and recommendations | 34 |
| 6.1 | Conclusion | 34 |
| 6.2 | Recommendations | 34 |
| References | 36 | |
CHAPTER ONE
1.0 INTRODUCTION
Heterobranchus bidorsalis (Geoffroy Saint-Hilaire, 1809) and Clarias gariepinus (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,
Heterobranchus bidorsalis and Clarias gariepinus are the most common and have received much 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, Clarias gariepinus (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 Clarias anguillaris (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 Heterobranchus longifilis 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 only two 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. Clarias gariepinus culture started almost fifty years ago in Africa and in 1994, Garibaldi (1996) reported a 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, Heterobranchus longifilis has a growth rate which doubles that of Clarias gariepinus. Experiments with Heterobranchus bidorsalis have recently been conducted in Nigeria (Fagbenro et al., 1993), but data on production 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:
- Ability to withstand harsh environmental conditions
- Ability to withstand handling stress
- High fecundity
- Disease resistance
- Fast growth rate
- High yield potential
- 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 Clarias gariepinus and Heterobranchus bidorsalis based on their genetic characteristics which is an important tool in fish breeding and genetics. 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 comprising of 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 Magoulas et 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 (Magoulas et 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
1.5.0 Objectives of the Study
The main objective of this study is to characterize genetic strains in Heterobranchus bidorsalis
and Clarias gariepinus using microsatellite markers.
1.5.1 Specific Objectives
The specific objectives of this study include:
- To acquire basic genetic knowledge on how to improve fish breeding through identification and characterization of desirable traits.
- To obtain preliminary data on genetic variation in Heterobranchus bidorsalis and Clarias gariepinus based on DNA microsatellite loci.
- 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.
- For assessment and comparison of the aquaculture potential of individual species and hybrids of the genus Heterobranchus and Clarias.
- 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; Pante et 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 (Macarañas et al., 1986, Galman et 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 Clarias gariepinus and Heterobranchus bidorsalis 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(Vignal et al.,2002).
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