Faculty of Resource Science and Technology

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1 Faculty of Resource Science and Technology GENETIC DIVERSITY OF SARAWAK SALTWATER CROCODILE (Crocodylus porosus) USING PCR-RAPD Mohd Khairulazman Bin Sulaiman Bachelor of Science with Honours (Aquatic Resource Science and Management) 2011

2 Genetic Diversity of Sarawak Saltwater Crocodile (Crocodylus porosus) Using PCR- RAPD Mohd Khairulazman Bin Sulaiman This report is submitted in partial fulfillment of the requirement for the degree of Bachelor of Science with Honours (Aquatic Resource Sciences and Management) Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK 2011

3 DECLARATION No portion of the work referred in this dissertation has been submitted in support of an application for another degree qualification of this or any other university or institution of higher learning. Mohd Khairulazman Bin Sulaiman Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resource Science and Technology Universiti Malaysia Sarawak

4 Acknowledgement In the name of Allah The Most Gracious and The Most Merciful. Alhamdulillah, I would like to express my utmost gratitude to Allah SWT, for giving me the strength to complete my Final Year Project although many obstacles experienced prior the completion of this project. Hereby, I wish to take this opportunity to express my gratitude and appreciation to all those that have assisted and guided me throughout the completion of this Final Year Project. First of all, special thanks to Dr Ruhana Hassan as the course coordinator and my supervisor for trusting me do this project and for giving me constant advice, guidance and assistance along the way until the completion of this project. Next, I would like to express my appreciation to all postgraduates of molecular aquatic lab especially Kak Nurhartini Kamalia Yahya for teaching me how to do molecular works, helping me obtaining the NTSYSpc 2.2 software and for her caring and helpful advice. Deepest thanks also to Kak Nursara Shahira Abdullah for providing me references and four extracted DNA samples of C. porosus for this project. I also would like to thanks all my beloved colleagues of molecular aquatic lab whose giving me suggestion and motivation to succeed in this project. I would also like thank all my course mates and friends for their encouragement and support Finally, I would like to take this opportunity to thank my family members especially my parents, Mr. Sulaiman and Mdm. Hatuyah for their endless support and encouragement. i

5 Table of Contents Acknowledgement Table of Contents List of Abbreviations List of Tables List of Figures List of Appendices i ii iv v vi vii Abstract 1 Introduction 2 Literature Review The Crocodilians Saltwater crocodile, Crocodylus porosus Studies of Crocodylus porosus in Sarawak Polymerase Chain Reaction - Random Amplified of 11 Polymorphic DNA (PCR-RAPD) technique molecular approach Materials and Methods Total Genomic DNA Extraction Preparation of Buffer Solution for Modified CTAB 16 Method (Doyle & Doyle, 1987) Preparation of Tissue for Total Genomic DNA 17 Extraction Total Genomic DNA Extraction Using Modified CTAB 18 Method (Doyle & Doyle, 1987) 3.2 Agarose Gel Electrophoresis of DNA Gel Documentation of DNA Bands Optical Density (OD) Reading (Absorbance) Primer Dilution Polymerase Chain Reaction (PCR) RAPD Techniques 23 ii

6 3.7 Gel documentation of RAPD bands Data Analysis 27 Results and Discussions Total Genomic DNA Extraction of Saltwater Crocodile, 28 Crocodylus porosus Agarose Gel Electrophoresis Optical Density Reading PCR - RAPD Oligo 135 Primer OPA-01 Primer OPA-09 Primer Comparisons of RAPD Profiles 41 Conclusion and Recommendation Conclusion Recommendation 43 References 44 Appendix 52 iii

7 List of Abbreviations Abbreviation Full Term bp Base pairs CTAB Cethyl-trimethyl Ammonium Bromide Cyt b Cytochrome b DNA Deoxyribonucleic acid ddh 2 O Double distilled water dntp Deoxynucleotide triphosphate EDTA Ethylene diaminetetra-acetic acid EtBr Ethidium bromide EtOH Ethanol g Gram MgCl 2 Magnesium Chloride min Minutes ml Milliliter Mm Millimeter mm Millimole mtdna Mitochondrial DNA NaCl Sodium Chloride PCR Polymerase Chain Reaction RAPD Random Amplification of Polymorphic DNA rpm Revolutions per minute s Seconds SFC Sarawak Forestry Corporation TE Tris-EDTA TBE Tris-borate-EDTA Tris base Tris (hydroxymethyl)-aminomethane µl Microliter UPGMA Unweighted Pair-Group Method with Arithmetic averages V Volt iv

8 List of Tables Title Name Page Table 3.1 2X CTAB buffer recipe for 500 ml stock 16 Table 3.2 Table 3.3 Samples reference of C. porosus for study of genetic diversity in Sarawak Sequences of decamer RAPD primers used in the molecular analysis of C. porosus in this study Table 3.4 PCR master mixture (modified Yau et al., 2002) Table 3.5 Table 3.6 Table 4.1 Amplification process in automated thermocycler for RAPD using Oligo135 and OPA-01 primers Amplification process in automated thermocycler for RAPD using OPA-09 primer Optical density reading for samples involved in this study Table 4.2 RAPD primers showing polymorphism for C. porosus Table 5.1 A Comparison of Relative Densities of Estuarine Crocodile in Sarawak v

9 List of Figures Title Name Page Figure 3.1 Figure 3.2 Figure 3.3 Map showing location of the five populations of C. porosus samples in genetic diversity study. Flow chart of modified CTAB DNA extraction protocol (Doyle and Doyle, 1987). PCR profile for RAPD using Oligo 135 and OPA-01 primers on C. porosus. Figure 3.4 PCR profile for RAPD using OPA-09 primers on C. porosus. 26 Figure 4.1 Figure 4.2 (a) Figure 4.2 (b) Agarose gel electrophoresis photograph showing total genomic DNA extraction product from five tissue samples of C.porosus using modified CTAB extraction method (Doyle and Doyle, 1987) in 1% agarose gel, run in Ix TBE buffer for 60 minutes at 90V. Agarose gel electrophoresis photograph showing RAPD patterns of C. porosus DNA samples, amplified using Oligo 135 in 2% agarose gel, run in Ix TBE buffer for 100 minutes at 80V. Diagrammatical RAPD banding patterns of C.porosus using Oligo 135 primer. Figure 4.3 Phylogenetic dissimilarity distance generated by Oligo 135 using UPGMA procedure according to Nei and Li (1979). Figure 4.4 (a) Figure 4.4 (b) Figure 4.5 Figure 4.6 (a) Figure 4.6 (b) Figure 4.7 Agarose gel electrophoresis photograph showing RAPD patterns of C. porosus DNA samples, amplified using OPA-01 in 2% agarose gel, run in Ix TBE buffer for 100 minutes at 80V.s. Diagrammatical RAPD banding patterns of C.porosus using OPA-01 primer. Phylogenetic dissimilarity distance generated by OPA-01 using UPGMA procedure according to Nei and Li (1979). Agarose gel electrophoresis photograph showing RAPD patterns of C. porosus DNA samples, amplified using OPA-09 in 2% agarose gel, run in Ix TBE buffer for 100 minutes at 80V. Diagrammatical RAPD banding patterns of C.porosus using OPA-09 primer. Phylogenetic dissimilarity distance generated by OPA-09 using UPGMA procedure according to Nei and Li (1979). Figure 5.1 Online DNA Concentration Calculator vi

10 List of Appendices Appendix 1 Title Name Page A Comparison of Relative Densities of Estuarine Crocodile in Sarawak Appendix 2 Online DNA Concentration Calculator vii

11 Genetic Diversity of Sarawak Saltwater Crocodile (Crocodylus porosus) Using PCR- RAPD Mohd Khairulazman Bin Sulaiman Aquatic Resources Sciences and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak ABSTRACT Saltwater crocodile, Crocodylus porosus is abundant in Sarawak rivers and mainly dominating the brackish water ecosystem. Lack of data on C.porosus in Sarawak especially molecular data had limit the knowledge and ability of relevant agencies to manage this potent resource in a sustainable manner. This study is designed to document RAPD profiles of C. porosus sampled from Kuching, Bau, Serian, Sibu and Miri. Methods involved were total genomic DNA extraction using modified CTAB method and Polymerase Chain Reaction (PCR) Random Amplified Polymorphic DNA (RAPD) using three sets of primer namely Oligo 135, OPA-01 and OPA-09. RAPD profiles generated from these three primers had successfully revealed 91.67%, 67.85% and 83.33% polymorphism respectively. Genetic dissimilarities trees generated based on RAPD profiles, indicated RAPD profile did not correlate with the location of the sample obtained and the relationships between nine crocodiles in this study was not fully resolved. Keywords: Crocodylus porosus, DNA extraction, CTAB, RAPD, polymorphism ABSTRAK Buaya Muara, Crocodylus porosus terdapat banyak di sungai Sarawak dan mendominasi ekosistem air payau. Kekurangan data tentang C.porosus terutamanya data molekular telah menghalang pengetahuan dan keupayaan agensi terlibat untuk menguruskan sumber berpotensi tinggi ini secara mapan dan berterusan. kajian ini bertujuan untuk mendokumentasikan profil RAPD daripada sampel C. porosus dari Kuching, Bau, Serian, Sibu dan Miri. Kaedah yang terlibat adalah total pengekstrakan genom DNA dengan menggunakan kaedah CTAB yang telah diubah dan Reaksi Rantai Polimerase (PCR) Pengandaan Rawak Polimorfik DNA (RAPD) menggunakan tiga set primer iaitu Oligo 135, OPA-01 dan OPA-09. Profil RAPD yang dijana daripada ketiga-tiga primer masing-masing telah berjaya mendedahkan 91,67%, 67,85% dan 83,33% polimorfisme. Pokok ketidaksamaan genetik yang dihasilkan berdasarkan profil RAPD, menunjukkan profil RAPD tidak berkorelasi dengan lokasi sampel diperolehi dan hubungan antara sembilan buaya dalam kajian ini tidak sepenuhnya diselesaikan. Kata kunci: Crocodylus porosus, pengekstrakan DNA, CTAB, PCR, RAPD, polimorfisme 1

12 1.0 Introduction Crocodile is a reptile that particularly well adapted for survival. The Saltwater, Estuarine or Indo-Pacific Crocodile, Crocodylus porosus, which can grows to over seven meters, is the largest living reptile on earth (Das, 2002). The saltwater crocodile are found mainly in brackish water or around the river estuaries and mangrove swamps. It also can appear in upstream of rivers or any of the freshwater swamp especially during breeding seasons. According to Das (2002), Saltwater Crocodile, Crocodylus porosus is one of perhaps at least four types of crocodiles that inhabit Borneo along with Freshwater Malayan Gharial, Tomistoma schlegelii, rare endemic Crocodylus raninus, and Siamese Crocodile, Crocodylus siamensis. In Sarawak, Crocodylus porosus and Tomistoma schlegelii are both the protected animals under First Schedule [Section 2(1)] PART II on Protected Animals from the Wild Life Protection Ordinance, 1998 (Forest Department of Sarawak, n.d). Sarawak is located on the Borneo Island and being the largest state of Malaysia. It is located in Southeast Asia between latitudes 4 S and 7 N and longitudes 109 and 119 E, and is also the third largest island in the world (Yasuma & Andau, 1999). One area of critical concern in the management of healthy wild populations is the maintenance of genetic diversity (Thorbjarnarson, 1992; Haig, 1998). Researchers agree that increased genetic variation within local populations may enhance species ability to adapt to changing environmental conditions (Mayr, 1963). From a conservation perspective, detection of recent dramatic changes in population size (population bottlenecks) is another important aspect of any population monitoring program (Amavet et al., 2007). 2

13 In Sarawak, C. porosus are protected under Convention on International Trade in Endangered Species of Wild Flora and Fauna (CITES) Appendix I (official document of CITES) that includes species threatened with extinction. Trade in specimens of these species is permitted only in exceptional circumstances which restricted the accessibility to this potential resource. Ironically, Sarawak Forestry Department reported that in general, C. porosus overpopulate in Sarawak rivers (Tisen & Ahmad, 2010). Thus, it is appropriate for this species to be excluded out from CITES Appendix I so that the accessibility to this potent resource is more open, utilization of this resource can improve the economy of the poor fishermen along the C. porosus habitats. This is because C. porosus are very potential resource because of increasing demand for crocodile leather worldwide (BOSTID, 1983) as well as crocodile meat throughout Asia. In Sarawak, SFC has done the studies to determine the density of crocodiles in Sarawak rivers. There were also some studies related to crocodiles genetic in Sarawak for example Abdullah & Hassan (2008) work on preliminary study of mitochondria Cytochrome b region of Sarawak Saltwater crocodiles and Shoon (2009) Sequencing of Cytochrome b and 12S mtdna Genes of C. porosus from Sarawak. However there is no study on crocodile using RAPD approach yet in Sarawak. In this study, the genetic diversity of C. porosus was determined using the PCR-RAPD method. The objective of this study is to document Random Amplified of Polymorphic DNA (RAPD) profiles of C. porosus from Sarawak using Oligo 135 primer, OPA-01 primer and OPA-09 primer. Respectively, RAPD techniques are recognized for their utility in carrying out initial screenings in many loci due to the minimal amounts of prior 3

14 knowledge about sequences is required and the chance to distinguish several organisms simultaneously (Lynch & Milligan, 1994). The results obtained from this study could be used to provide a genetic framework of conservation units to evaluate, and perhaps to modify, current plans for its sustainable management. 4

15 2.0 Literature Review 2.1 The Crocodilians Crocodilians belong to the great group of archosaurs which includes two extinct clades: the pterosaurs and the dinosaurs (Blake, 1982). Crocodilians are the sole surviving reptilian archosaur, a group of diapsids that include dinosaurs and other ancient reptiles that gave rise to birds (Hedges & Poling, 1999). Crocodylia is a small order within the class Reptilia comprises 23 species belonging to eight genera (King & Burke, 1989). Today, living crocodilians include the 24 species of alligators, caimans, crocodiles and gharials distributed in the warm waters of the worlds (Martin, 2008). The present day crocodilians are grouped in three families; Alligatoridae (Aligators), Crocidilidae (Crocodiles) and Gavialidae (Gharial) the most impoverished one, compared to that Mesozoic (245 to 65 million years before present), when these animals were the dominant life forms (Ritchie et al., 2002). The genus Crocodylus which is the largest, consists of 11 species including the largest living reptile, C. porosus (Meganathan et al., 2010). Despite a long and impressive history, the past century has seen crocodilians face overwhelming threats from human habitation (Miles et al., 2009). Fortunately today, many crocodilians are recovering from the human exploitations that occurred during the first half of the 20 th century (Miles et al., 2009). These exploitations affected crocodilian numbers and inevitably the genetic structure and diversity within these populations (Davis et al., 2002). Crocodilians are large growing, heavily armoured reptiles, associated with wetlands. They are distinguished from all other living reptiles for having an enlarged head with elongated 5

16 snout, muscular, laterally compressed tail, the codonth teeth set into sockets, and osteoderms, the heavy plates of bone that underlie the dorsal scales. Crocodilian are poikilotherms, but they like to maintain body temperature within a narrow range of 28 o C 33 o C by using the thermogradient in their natural environment consisting of sunshine and shade, warm surface and cold deep water, as well as burrows (Huchzermeyer, 2002). According to Ritchie et al., (2002) all crocodiles are adapted to aquatic life by having webbed feet, nostrils on top of their snout that are equipped with air-tight valves, a fleshy valve at the base of the tongue that permit the mouth to be opened underwater. They also possess eyes with a nictitating membrane, which called third eyelid that is drawn across the eye when submerged. Other feature which is unique among reptiles include a muscular partition separating the pectoral and abdominal cavities as the like diaphragm in mammals, alveoli in the lungs and deoxygenated blood. Crocodilians are more closely related to birds than to any living reptiles (Ritchie et al., 2002). According to Meganathan et al., (2010), in the past, the genus Crocodylus was known to consist of 12 species including Crocodylus cataphractus but recent studies (Brochu, 2000; McAliley et al., 2006) have provided consistent evidence for this species as a non- Crocodylus member and thus the name, Mecistops cataphractus was resurrected. Although recovery programs have bolstered crocodilian numbers, 17 of the 23 species are still listed as CITES Appendix I in various regions, and the pressures of illegal hunting, habitat fragmentation and human encroachment continue to loom for a range of vulnerable crocodilians (Miles et al., 2009). In addition to previous threats, the elimination of spatial and temporal boundaries through modern anthropogenic pressures has facilitated hybridization in crocodiles by bringing together crocodilian species that would otherwise 6

17 not breed due to a lack of opportunity (Fitzsimmons et al., 2002). This hybridization has been known in several Crocodylus species such as C. rhombifer, C. moreletti, C. siamensis and C. porosus (Ramos et al., 1994; Fitzsimmons et al., 2002; Ray et al., 2004). Problems such as these demonstrate the need for further polymorphic markers to assist in population studies to assess the vulnerability status of some species (Miles et al., 2009). The genus Crocodylus has been included in many phylogenetic analyses, which have established the basic structure of the crocodilian phylogeny which mostly aimed to resolve the interfamilial and intergeneric problems with few of them focusing on the intrageneric relationships of Crocodylus (Gatesy et al., 2003, 2004; Harshman et al., 2003; Janke et al., 2005; McAliley et al., 2006; Roos et al., 2007). However, the relationships between species within Crocodylus remained poorly understood (Meganathan et al., 2010). All crocodilian species may be considered as totally water dependent since they can only mate in water (Martin, 2008). Crocodilians appear to be very important for freshwater ecosystems as they maintain, during the dry season waterholes that are used as reservoir for many arthropods, crustacean, fishes and amphibians (Kushlan, 1974; Gans, 1989; Martin, 2008). From an economic point of view, crocodilians play an essential role in modern agriculture, as well as forming a basis for tourism, with management programs in more than 40 nations worldwide (Thorbjarnarson, 1999). 7

18 2.2 Saltwater Crocodile, Crocodylus porosus The saltwater crocodile (C. porosus), which is distributed throughout much of South East Asia, is relatively uncharacterized genetically (Miles et al., 2008). C. porosus is the largest and most broadly distributed crocodilian species; occurring in coastal and estuarine habitats across the Indo-Pacific region, from Northern Australia, throughout Southeast Asia, to India and Sri Lanka, and in the western Pacific Ocean the distribution ranges from the Solomon Islands to Vanuatu, and in the Republic of Palau (Russello et al., 2007). C. porosus possess special glands on tongue surface, that are modified salivary glands, aid in the excretion of high concentration of salt, allowing them to invade marine and estuarine environments (Ritchie et al., 2002). They are dangerous to man and livestock and are more active than freshwater crocodiles. Below is the taxanomic classification of the Saltwater Crocodile adapted from World Register of Marine Species (Uetz, 2010); Kingdom: Animalia Phylum: Chordata Subphylum: Vertebrata Class: Reptilia Order: Crocodylia Family: Crocodylidae Subfamily: Crocodylinae Genus: Crocodylus Species: Crocodylus porosus (Schneider, 1801) 8

19 The C. porosus is considered an endangered or threatened species throughout the majority of its range, and is of special conservation and economic interest (Ross, 1998; Russello et al., 2007). The remaining species of the Indo-pacific Crocodylus are found in freshwater and marsh environment and rarely in brackish water (Martin, 2008). According to Meganathan et al. (2010), C. porosus is one of the species having ambiguous phylogenetic position within genus Crocodylus. C. porosus has not been included in many phylogenetic analyses and those analyses that included this species could not provide a consistent placement for C. porosus (Meganathan et al., 2010). Based on analysis by Meganathan et al. (2010), previous molecular studies on C. porosus described its close association with C. palustris (Densmore and Owen, 1989; Poe, 1996; Gatesy and Amato, 2008; Willis, 2009), whereas some studies have combined C. porosus within the monophyletic clade of other Indo-pacific crocodilians excluding C. siamensis and C. palustris (Densmore, 1983; Densmore and Owen, 1989; Brochu, 2000). While many of the phylogenetic relationships within the genus Crocodylus are unknown, recent study by Meganathan et al., (2010) had proposed that the saltwater crocodile, C. porosus is the sister taxon to C. siamensis. Due to this, further molecular studies are needed to provide a better understanding of the relationships among Crocodylus species and to establish the phylogenetic position of C. porosus (Meganathan et al., 2010). 9

20 2.3 Studies on Crocodylus porosus in Sarawak According to Cox & Gombek, (1985) C. porosus in Sarawak were distributed along the sluggish rivers and brackish swamps of Batang Lupar, the mangrove areas and Batang Samarahan. They could also be found around major rivers in a totally unrelated habitat, with sighting of the crocodile hatchlings in Loagan Bunut, a natural lake, Upper Belaga and Kelauh, the freshwater tributary of Batang Lupar. In contrast to the information obtained by Cox & Gombek (1985) who claimed that population of crocodiles in Sarawak was decreasing, Tisen & Ahmad, (2010) reported that the saltwater crocodile population has shown general trend of increased density in its natural habitat (refer Appendix 1). Equivalently, Gani et al. (2011) stated that in Batang Samarahan, the population was strongly biased towards immature animals (hatchlings and yearlings) which indicative of a recovering population, while the presence of hatchlings indicating that there is some successful nesting occurring along Batang Samarahan. There was also study on human-crocodile conflict in Sarawak by Landong & Zaini (2010) which seek to find the problems and potential solutions on issues of crocodile attacks. In their study, Landong & Zaini (2010) also proposed in depth studies of certain issues like (1) crocodile population and distribution study and (2) socio-economic study that will enable the Sarawak state government to prepare comprehensive crocodile management plan. Based on Cyt b gene analysis, Abdullah et al. (2009) had suggested that the population of C.porosus from Sarawak and Australia were sharing the same genetic pool due to very low 10

21 genetic divergence between C. porosus Australia and C. porosus Sarawak. This finding is consistent with Russello et al. (2007) as they reported that based on Cyt b gene analyses, Palau C. porosus and Australia C. porosus possibly share the same population. However, Shoon (2009), had suggested that Northern Sarawak (Miri) crocodiles and Western Sarawak (Kuching and Sibu) crocodiles from were from different genetic pool as both showed high genetic divergence values. 2.4 Polymerase Chain Reaction - Random Amplified of Polymorphic DNA (PCR- RAPD) technique molecular approach According to Karim et al. (2010), molecular markers have been proved to be valuable tools in the characterization and evaluation of genetic diversity inter- and intraspecies and within or between populations. Different markers might reveal different classes of dissimilarity (Powell et al., 1996; Russell et al., 1997). Characterization and evaluation of genetic diversity is correlated with the genome fraction surveyed by each kind of marker, their distribution all over the genome and the size of the DNA target which is analysed by each specific test (Dávila et al., 1999). The advent of the polymerase chain reaction (PCR) preferred the development of different molecular techniques such as Random Amplified of Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR or microsatellite), Restriction Fragment Length Polymorphism (RFLP), Sequence Tagged Sites (STS), Random Amplified Microsatellite Polymorphism (RAMP), Amplified fragment length polymorphism (AFLP) and Inter-simple Sequence Repeat Polymorphic DNA (ISSR) (Saiki et al., 1988; Welsh and McCleland 1990; 11

22 Williams et al., 1990; Akkaya et al,. 1992; Tragoonrung et al., 1992; Zietkiewicz et al., 1994; Wu et al., 1994; Vos et al., 1995; Nagaoka & Ogihara 1997). In this study, PCR- RAPD technique was chosen because it has several advantages such as the simplicity and low cost of the RAPD technique (Bardacki, 2000), in addition to the low quantities and medium quality of DNA needed, and their simple method of acquiring data on variation in genomic DNA (Amavet et al., 2007). The development of RAPD markers, generated by the polymerase chain reaction (PCR), allows the examination of genomic variation without prior knowledge of DNA sequences (Williams et al., 1990; Welsh and McClelland, 1990; Hadrys et al., 1992). The RAPD technique is PCR based, permitting scores of markers to be assayed on DNA extracted from a single organism (Wilkerson et al., 1993). However, instead of using primer pairs as in traditional PCR, RAPD reactions use a single short primer usually ten bases in length of randomly chosen sequence. Williams et al. (1990), clarified that the standard RAPD technology utilises short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. For a RAPD band to be generated, the primer needs to match a binding site that is within approximately 2 to 3 kilobase pairs of another, oppositely oriented binding site, so that the single oligonucleotide can prime replication in both the forward and reverse direction (Wilkerson et al., 1993). The number and the size of amplified fragments depend on length and sequence of short, single and arbitrary primers (Bardacki & Skibinski, 1994). RAPD analysis is a multilocus technique detecting polymorphism based on an amplification of random DNA segments (Amavet et al., 2007). Arnold et al. (1991), reported that RAPD band may display a high degree of polymorphism. In addition, they 12

23 reported that screening multiple primers against taxa of interest has proven to be a means of quickly identifying species-specific markers. The presence or absence of the polymorphism is either caused by nucleotide sequence divergence in primer sites or by insertions or deletions in the amplified segment of template DNA, hence, RAPD primers can provide information of point mutations (Amavet et al., 2007). Williams et al. (1990) stated that RAPD bands typically represent dominant genetic markers, which are inherited in a Mendelian fashion and can be used as molecular diagnostic characters at different taxonomic levels. They have been successfully applied for taxonomic identification and in population genetic surveys (Hadrys et al., 1992). RAPD markers have been successfully used to detect genetic variability in mata-raton plants, Gliricidia (Chalmers et al., 1992), mosquito species and populations (Kambhampati et al.,1992), closely related species of black Aspergilli (Megnegneau, 1993), cocoa (Russel et al., 1993), medfly (Baruffi et al., 1995) and parasitic protozoa (Tibayrenc et al., 1993). The technique has also been used to study genetic variation in several fish species. Bardakci & Skibinski (1994) and Naish et al. (1995) used RAPD markers to discriminate between commercially important tilapia species, subspecies and strains of tilapia. RAPD markers were also generated for several tropical fish species representing 7 families (Dinesh et al., 1993). Furthermore, RAPD analysis revealed high levels of genetic variations among individuals from the same broodstock of European sea bass, Dicentrarchus labrax (Allegrucci et al., 1995). Finally, 721 strain-specific RAPD markers were identified in 2 laboratory strains of zebrafish (Johnson et al., 1994). However, RAPD markers may not have been as effective at detecting variation as microsatellite amplification in populations of crocodilians (Dessauer et al., 2002), which is 13

24 probably because their polymorphism levels are lower because of the type of variation analyzed. RAPD detects point mutations in homozygosis, whereas microsatellite amplification detects variation in the length of alleles generated by slippage or unequal crossing over (Glenn et al., 1996). 14