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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

Faculty of Resource Science and Technology

Genetic Diversity of Sarawak Saltwater Crocodile (Crocodylus porosus) Using PCR-

RAPD


This report is submitted in partial fulfillment of the requirement for the degree of

Bachelor of Science with Honours

(Aquatic Resource Sciences and Management)


UNIVERSITI MALAYSIA SARAWAK

2011

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.

______________________________


Aquatic Resource Science and Management

Department of Aquatic Science


Universiti Malaysia Sarawak

i

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.

ii

Table of Contents

Acknowledgement i

Table of Contents ii

List of Abbreviations iv

List of Tables v

List of Figures vi

List of Appendices vii

Abstract 1

Introduction 2

Literature Review 5

2.1 The Crocodilians 5

2.2 Saltwater crocodile, Crocodylus porosus 8

2.3 Studies of Crocodylus porosus in Sarawak 10

2.4 Polymerase Chain Reaction - Random Amplified of

Polymorphic DNA (PCR-RAPD) technique molecular approach

11

Materials and Methods 15

3.1 Total Genomic DNA Extraction 15

3.1.1 Preparation of Buffer Solution for Modified CTAB

Method (Doyle & Doyle, 1987)

16

3.1.2 Preparation of Tissue for Total Genomic DNA

Extraction

17

3.1.3 Total Genomic DNA Extraction Using Modified CTAB

Method (Doyle & Doyle, 1987)

18

3.2 Agarose Gel Electrophoresis of DNA 20

3.3 Gel Documentation of DNA Bands 21

3.4 Optical Density (OD) Reading (Absorbance) 21

3.5 Primer Dilution 22

3.6 Polymerase Chain Reaction (PCR) – RAPD Techniques 23

iii

3.7 Gel documentation of RAPD bands 26

3.8 Data Analysis 27

Results and Discussions 28

4.1 Total Genomic DNA Extraction of Saltwater Crocodile,

Crocodylus porosus

28

4.1.1 Agarose Gel Electrophoresis 28

4.1.2 Optical Density Reading 30

4.2 PCR - RAPD 31

4.2.1 Oligo 135 Primer 34

4.2.2 OPA-01 Primer 37

4.2.3 OPA-09 Primer 39

4.3 Comparisons of RAPD Profiles 41

Conclusion and Recommendation 43

5.1 Conclusion 43

5.2 Recommendation 43

References 44

Appendix 52

iv

List of Abbreviations

Abbreviation Full Term

bp Base pairs

CTAB Cethyl-trimethyl Ammonium Bromide

Cyt b Cytochrome b

DNA Deoxyribonucleic acid

ddH2O Double distilled water

dNTP Deoxynucleotide triphosphate

EDTA Ethylene diaminetetra-acetic acid

EtBr Ethidium bromide

EtOH Ethanol

g Gram

MgCl2 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

v

List of Tables

Title Name Page

Table 3.1 2X CTAB buffer recipe for 500 ml stock 16

Table 3.2 Samples reference of C. porosus for study of

genetic diversity in Sarawak

17

Table 3.3 Sequences of decamer RAPD primers used in the

molecular analysis of C. porosus in this study

23

Table 3.4 PCR master mixture (modified Yau et al., 2002) 24

Table 3.5 Amplification process in automated thermocycler

for RAPD using Oligo135 and OPA-01 primers

24

Table 3.6 Amplification process in automated thermocycler

for RAPD using OPA-09 primer

25

Table 4.1 Optical density reading for samples involved in this

study

30

Table 4.2 RAPD primers showing polymorphism for C.

porosus

33

Table 5.1 A Comparison of Relative Densities of Estuarine

Crocodile in Sarawak

52

vi

List of Figures

Title Name Page

Figure 3.1 Map showing location of the five populations of C. porosus

samples in genetic diversity study.

15

Figure 3.2 Flow chart of modified CTAB DNA extraction protocol

(Doyle and Doyle, 1987).

19

Figure 3.3 PCR profile for RAPD using Oligo 135 and OPA-01 primers

on C. porosus.

25

Figure 3.4 PCR profile for RAPD using OPA-09 primers on C. porosus. 26

Figure 4.1 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.

34

Figure 4.2 (a) 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.

34

Figure 4.2 (b) Diagrammatical RAPD banding patterns of C.porosus using

Oligo 135 primer.

34

Figure 4.3 Phylogenetic dissimilarity distance generated by Oligo 135

using UPGMA procedure according to Nei and Li (1979).

35


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.

37


OPA-01 primer.

37

Figure 4.5 Phylogenetic dissimilarity distance generated by OPA-01


38


patterns of C. porosus DNA samples, amplified using OPA-09

in 2% agarose gel, run in Ix TBE buffer for 100 minutes at

80V.

39


OPA-09 primer.

39

Figure 4.7 Phylogenetic dissimilarity distance generated by OPA-09


40

Figure 5.1 Online DNA Concentration Calculator 53

vii

List of Appendices

Title Name Page

Appendix 1 A Comparison of Relative Densities of Estuarine

Crocodile in Sarawak

52

Appendix 2 Online DNA Concentration Calculator 53

1

Genetic Diversity of Sarawak Saltwater Crocodile (Crocodylus porosus) Using PCR-

RAPD


Aquatic Resources Sciences and Management


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

2

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).

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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

4

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.

5

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 20th

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

6

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 oC –

33 oC 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

7

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).

8

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)

9

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).

10

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

11

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;

12

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

13

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

14

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).

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