UNIVERSITI PUTRA MALAYSIA

DEVELOPMENT OF PCR-BAS ED DNA MARKERS TO IDENTIFY AND CHARACTERISE MALAYSIAN RIVER CATFISH,

MYSTUS NEMURUS (C & V ) : RAPD AND AFLP

CHONG LEE KIM

FSAS 1998 17

DEVE LO PMENT OF PCR-BASED DNA MARKERS TO IDENTIFY AND CHARACTERISE MALAYSIAN RIVER CATFISH,

MYSTUS NEMURUS (C & V) : RAPD AND AFLP

By

CHONG LEE KIM

Thesis submitted in fulfillment of the requirements for the degree of Master of Science in the Faculty of Science and Environmental Studies,

Universiti Putra Malaysia

November 1998

ACKNOWLEDGEMENTS

My sincere gratitude to:

Professor Dr. Tan Soon Guan for his supervision of this thesis as well as his advice

and guidance in this project especially in providing me with all the latest papers and

information.

Dr. Siti Shapor Siraj and Associate Professor Dr. Khatijah Yusoff for access to their

facilities in the laboratory and their support in this work.

Alan Ong Han Kiat for his guidance and technical support since the start of this project

until the thesis writing.

Ms Koh Chew Ling for her company in the dark room and during working at night;

Mr. Vijay for providing me some technical information in the data analysis, Mrs.

Omeima Salleh for her advice, and all the researchers in the Virology Laboratory for

making life in the laboratory more fun and interesting.

Mr. Ng Chong Sing of Bio-diagnostic for regular updates on advances in molecular

techniques.

Finally, and the most important one is to thank the love and support of my family.

This study was funded by Intensification of Research in Priority Areas (IRP A) project

grant no. 0 1-02-04-0074.

II

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF PLATES

LIST OF ABBREVIATIONS

ABSTRACT

ABSTRAK

CHAPTER

I INTRODUCTION

II LITERATURE REVIEW

Mystus Species

Habitat and DistributIon

Taxonomic Classification

Morphology

Importance

Molecular Techniques

Genetic Markers

RAPD Technology

AFLP Technology

Microsatellite Technology

Genetic Marker AnalYSIS

III

Page

II

VI

VII

VIII

IX

X

XIII

5

5

5

5

6

7

8

1 1

1 2

15

19

2 1

III MATERIALS AND METHODS 24

Collection of Samples 24

Sample Collection for Populations Study 24

Sample Collection for Mendelian Inheritance Study 26

Preparation of Genomic DNA 27

Genomic DNA Extraction 27

DNA Purity and Quantity 29

RAPD Procedure 31

AFLP Procedure 32

Statistical Analysis of RAPD and AFLP Markers 34

IV RESULTS 37

RAPD Analysis 37

Optimisation and The Effects of Different Reaction Parameters 37

Reproducibility Testing 47

Selection of RAPD Primers 47

Mendelian Segregation Study of RAPD Markers 50

RAPD Patterns 54

IntrapopulatlOn RAPD Analysis 58

Interpopulation RAPD Analysis 62

AFLP AnalysIs 65 Screening and Selection of AFLP Primers 65

AFLP Banding Patterns 67

Mendelian Inheritance Study of AFLP Markers 70

Intrapopulatlon AFLP AnalysIs 73

Interpopulation AFLP AnalysIs 78

Combination of RAPD and AFLP Data 81

IV

V DISCUSSION

RAPD and AFLP Technology

Mendelian Inheritance Study ofRAPD and AFLP Markels

RAPD and AFLP Population Studies

IntrapopulatlOn Diversity

InterpopulatIon Diversity

ComparIson of The ResolvIng Power between RAPD and AFLP

VI CONCLUSION

REFERENCES

APPENDIX

VITA

Additional Table 14

Additional Table 1 5

V

83

83

86

90

91

94

98

1 00

1 02

1 1 1

1 1 2

1 1 5

1 25

LIST OF TABLES

Tables

Overview of RAPD, AFLP and Microsatell ite Technologies

2 Geographical Region, Sampling Sites and Sample Sizes of M. nemurus

3 LIst of RAPD Primers Used In the Population and Segregation Studies

4 Number of Bands and the S ize Range of the RAPD Fragments in Famil ies A and B of M. nemurus

5 Comparison of Observed and Expected F I Progeny Phenotypic

Page

22

24

49

51

Numbers for Families A and B 5 5

6 Comparison of the Percentage of Similarity within Population Including and Excluding the Unusual Individuals of each Population 59

7 Genetic Distances of RAPD Markers in Five Populations of M nemurus Based on Lynch' s (1991) Band Sharing Similarity Indices 63

8 AFLP Primer Combinations 66

9 Number of Scorable Bands Produced by Five Sets of AFLP Primer Pairs in the Analysis of the Catfish Genome 69

1 0 Chi-square Test of AFLP Markers 72

11 Percentage of Similanty between IndIviduals of the, Same Populations ?3

12 Genetic Distances of Five Populations of M. nemurus Based on Lynch' s ( 1 99 1 ) Band Sharing Similarity Indices 79

1 3 Genetic Distances of Combmation of RAPD and AFLP Markers in FIve Populations of M nemuru.\ Based on Lynch's ( 1 99 1 ) Band Sharing Simi larity Indices 82

VI

LIST OF FIGURES

Figures Page

The Morphology of M. nemuru.\ 7

2 AFLP Process 1 6

3 Location Map of M nemurus Sampling Sites 25

4 UPGMA Cluster Analysts Based on Nei and Li's Similarity ( 1 979) from 42 Variable RAPD Markers for 78 Individuals of M nemurus 6 1

5 UPGMA Cluster Analysis Based on Lynch's Genetic Distances from 42 Variable RAPD Loci for Five Populations of M nemurus 64

6 UPGMA C luster Analysis Based on Nei and Li's Similarity ( 1 979) from 1 5 8 Variable AFLP Markers for 83 Individuals ofM nemurus 75

7 UPGMA Cluster Analysis B ased on Lynch's Genetic Distances from 1 58 Variable AFLP Loci for Five Populations of M nemurus . 80

8 UPGMA Cluster Analysis Based on Lynch's Genetic Distances from 200 Variable RAPD and AFLP Loci for Five Populations of M nemuru� 82

VJ1

LIST OF PLATES

Plates Page

Quantification of DNA Samples by Comparing with ADNA 30

2 Effects of Genomic DN A from blood and muscle on RAPD patterns 3 8

3 Effects of Different DNA Concentrations and Taq Polymerase on RAPD Patterns 39

4 Effects of Low Anneal ing Temperatures on RAPD Patterns 43

5 Effects of Different MgCh Concentrations on RAPD Patterns 43

6 Effects of Different Thermal Cyclers on RAPD Patterns 45

7 RAPD Patterns on Two Different Types of Gels 46

8 Reproducibi l ity Testing 48

9 RAPD Patterns of M nemurus Genotypes Using Primer OP A08 53

1 0 RAPD Patterns of M nemurus Genotypes from Family B Using Primer OPA1 7 53

1 1 Part of the RAPD Patterns Generated by Primer OPDI8 57

1 2 RAPD B anding Pattern Generated by Primer OPD 1 3 of the Unusual Genotypes Compared to the Majority Genotypes within a Population 60

1 3 AFLP Fingerprints of 1 1 M nemurus Samples Generated by Primer Combination 62 (E-AGG / M-CTC) 68

14 AFLP Banding Pattern Produced by Impure Genomic DNA 7 1

15 Polymorphic AFLP Markers within the UPM Population 76

1 6 Polymorphic AFLP Markers within the Sarawak Population 77

Vlll

Chemicals

EOTA

KCI

MgCh

SOS

TBE

Units

bp

°C

hr

kb

mm

mM

ng

nmole

00 pmole

sec

ug

ul

uM

V

w/v

LIST OF ABBREVIATIONS

Ethylenediamine tetraacetate

Potassium chloride

Magnesium chloride

Sodium dodecyl sulphate

Tris-borate-EOT A buffer

base pair

Celsius

hour

kilobase

minute

mil l imolar

nanogram

nanomole

optical density

picomole

second

mIcrogram

microliter

micromolar

volt

weight / volume

IX

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science.

DEVELOPMENT OF PCR-BASED DNA MARKERS TO [DENTIFY AND CHARACTERISE MALAYSIAN RIVER CATFISH,

MYS'l'l!S NlIMl'JU IS (C & vt RAPD AND AFLP .

By

CHONG LEE KIM

November 1998

Chairman: Professor Tan Soon Guan, Ph.D.

Faculty: Science and Environmental Studies

The main objectives of this study are to evaluate the usefulness of a variety

of recently developed DNA markers to identify and characterise different

populations of Malaysian river catfish, Mystus numerus.

In the initial methodology development, 1 1 I primers comprising of 40 single

RAPD primers, 64 pairs of AFLP primers and 7 pairs of African catfish

microsatellite primers were screened against M. nemlll'llS samples. RAPD and AFLP

primers gave positive results. Both techniques were also successfully used in

examining the genetic diversity present in five populations of M. nemurus

originating fi'om Kedah. Perak, Johor, Sarawak and one UPM culture population.

However, none of the micro satellite primers developed for the African catfish was

suitable for typing our local M. nemurtls. Therefore, this technique was not used for

the population studies and emphasis was only given to RAPD and AFLP markers in

this thesis.

Nine RAPD primers and four AFLP primers detected a total of 42 and 1 58

polymorphic markers respectively. The modes of inheritance of the bands produced

by four out of the nine RAPD primers and two out of the four AFLP primers were

studied using family samples. The results showed that 9 of the RAPD markers and

24 of the AFLP markers used in population studies segregated as stable Mendelian

loci while 2 RAPD markers and 1 3 AFLP markers showed unusual segregation. The

rest of the markers could not be examined because no segregation was found in the

families.

A correlation coefficient value of 0 .69 between the RAPD and AFLP

distance matrices indicated that the data generated by both methods are not similar

but showed agreement to a certain extent. Both types of markers revealed high

genetic diversities within the UPM and Sarawak populations. B oth types of markers

also showed a low level of genetic variability in the Kedah population. In addition,

RAPD and AFLP showed small genetic distances among the UPM, Kedah and

Perak populations. Based on the position of each popuhtion in the dendrogram, the

Xl

Sarawak population, which is located in East Malaysia, clustered by itself, thus

isolated from the rest of the populations located in Peninsular Malaysia.

In comparison, the AFLP technique showed higher resolving power than

RAPD. Individuals that could be discriminated from one another by AFLP could not

be discriminated by RAPD. Three subgroups each from the Kedah, Perak and

Sarawak populations were revealed by AFLP but not by RAPD .

Overall , AFLP would be advantageous over RAPD in investigating a large

number of loci within a few genotypes in a minimal time while RAPD would be

advantageous in rapid screening of a large number of genotypes since it is cheaper

and less time is required for template DNA preparation.

The results of this study suggest that RAPD and AFLP analysis, if carried

out carefully, give a good indication of the separation between individuals of

different populations and are suitable for identification of closely related genotypes.

XII

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi keperluan lIntuk Ijazah Master Sains.

PERKEMBANGAN PENANDA DNA YANG MENGGUNAKAN PCR UNTUK MENGECAM AND MENGENALPASTI IKAN BAUNG,

/VIysn IS Nt IMI','Rt IS (C & V) DI MALAYSIA: RAPD DAN AFLP

By

CHONG LEE KIM

November 1998

Pengerusi : Profesor Tan Soon Guan, Ph. D.

Fakulti Sains dan Pengajian Alam Sekitar

Tujuan penyelidikan ini ialah untuk menilai kegunaan berbagai jenis penanda

DNA terbaru yang menggunakan tindak balas rantaian polimerase (PCR) untuk

mengecam dan mengenalpasti populasi-populasi ikan baung tempatan, M nemurus.

Pada permulaan perkembangan methodologi, I 1 1 pnmer yang terdiri

daripada 40 primer tunggal RAPD, 64 pasang primer AFLP and 7 pasang primer

mikrosatelit 'African catfish', diuji terhadap beberapa sampel M. nemuf'l(s. Primer

RAPD and AFLP memberi keputusan yang positif. Kedlla-dua teknik ini juga telah

berjaya digllnakan untuk memeriksa kepelbagaian gen dalam lima populasi baung

tempatan yang berasal daripada Kedah, Perak, 10hor, Sarawak dan juga satu

XII I

populasi kultur populasi UPM Walau bagaimanapun, tidak ada satu pun pnmer

mikrosatelit yang diperiksa scsuai digunakan untuk baung tempatan kita. Oleh itu,

teknik ini tidak digunakan dalam kajian populasi dan tumpuan hanya diberikan

kepada penanda RAPD and AFLP dalam tesis ini

Sembilan primer RAPD dan empat primer AFLP masing-masing mengesan

sejumlah 42 and 1 5 8 penanda polimortik Keturunan empat daripada sembilan

primer RAPD dan dua daripada empat primer AFLP dikaji dengan sampel-sampel

sekeluarga. Keputusan menunjukkan 9 daripada penanda RAPD dan 24 daripada

penanda AFLP yang digunakan dalam kajian populasi diasing secara Mendelian

manakala 2 daripada penanda RAPD and 13 daripada penanda AFLP menunjukkan

pengasingan luar biasa Penanda yang lain tidak dapat diperiksa keturunannya

kerana tidak ada pengasingan yang ditunjukkan oleh sampel-sampel sekeluarga.

Perhubungan antara jarak genetik RAPD dan AFLP yang bernilai 0 69

menunjukkan data yang dihasilkan oleh kedua-dua teknik ini tidak seratus-peratus

sama tetapi mematuhi antara satu sarna lain dalam sesuatu tahap Kedua-dua jenis

penanda mempamerkan kepelbagaian gen yang tinggi yang terkandung dalam

populasi dari UPM and Sarawak Kedua-dua jenis penanda juga memberi variasi gen

yang rendah di kalangan populasi dari Kedah. Tambahan pula, RAPD and AFLP

menunjukkan nilai jarak genetik yang kecil antara populasi dari UPM, Kedah dan

XIV

Perak Berdasarkan kedudukan popuiasi-popuiasl dalam dendrogram, popuiasl

dal lpada Sarawak, yang ber a\al dan Malaysia Tlmul, dla�mgkan danpada populasl­

popliiasl lam dl Semenaniling MalaY�la

Danpada perbandmgan kedua-dua tekmk tersebut, tekmk AFLP

menunJukkan daya beza Jelas yang leblh tmggl danpada RAPD Individual Ikan

baung yang dapat dlasmgkan antara satu sam a lam oleh petanda AFLP ttdak dapat

dlaslngkan oleh penanda RAPD Tlga sub-kumpulan yang setlap satunya danpada

populasl Kedah, Perak dan Sarawak Juga dapat dlpamerkan oleh AFLP tetapl tldak

dapat dlpamerkan oleh RAPD

Pada keseluruhannya, AFLP adalah leblh berfaedah danpada RAPD untuk

menYIasat banyak lokus yang terkandung dalam beberapa genotIp dalam masa yang

smgkat SebalIknya, RAPD adalah leblh berfaedah untuk menYlasat sekumpulan

besar genotIp kerana tekmk Inl leblh murah dan kurang masa yang dlperlukan untuk

menyedlakan templat DNA

Keputusan kaJlan 1m menunJukkan bahawa analisls RAPD dan AFLP, Jlka

dlJalankan dengan berhatl-hatl, dapat membenkan keputusan yang balk dalam

mengecam dan mengenalpastl sam pel danpada populasl-populasl yang berbeza la

luga sesual untuk mengecam genotlp yang mempllnyal perhllbungan yang rapat

XV

CHAPTER I

INTRODUCTION

This project is part of the research program entitled' PopulatlOlI GelletIC,\

III MallaKement and ComermtlOlI oj AquatiC Re.wurce.\ alld f)NA {tIlKerpnntlllK

III fishes' headed by Prof Dr Tan Soon Guan of UPM and funded by the

Malaysian Ministry of Science, Technology and Environment under the lRPA

(Intensification of Research in Priority Areas) scheme

Identification and characterisation of population units are imperative for

fisheries management because efficient resource utilisation can best be achieved

when managing at the population level In some countries, genetic markers have

been widely used in fisheries management (Martin et aI, 1992, Ferguson et aI,

1 995) but this approach is still new in Malaysia Not much work has been done on

the genetic characterisation of local fish species through the use of molecular

markers Also, the number of loci used in such studies were small and mainly on

protein electrophoresis (Daud et aI, 1 989, Patimah et aI, 1 989, Siraj et aI, 1 998)

The possible neglect of genetic diversity in fisheries management decisions mIght

have been due to the lack of tools for the determination of genetic variation and

lack of expertise in this field of study Therefore emphasis had been placed on

environmentally influenced charactel s such as morphological characters instead of

molecular genetic markers for evaluating variation among populations As such, it

2

is hoped that the findings of this research wil l provide a better understanding of

our local aquatic resources with respect to genetic diversity so as to improve

fisheries management.

The fish species that was studied here is the local river catfish, ikan baung

with a scientific name of Mystus numerus. It is a freshwater fish that can be found

in the wild. Commercialisation as wel l as farm-raising of this fish are stil l not

widely estab lished yet compared to the other fishes such as tilapia mainly because

of the limited research data on this species. Little is known about the genetic

background of the natural populations and the cultured stocks.

In previous isozyme studies of local Mystus numerus, only 4 polymorphic

loci out of 1 7 loci and 16 polymorphic loci out of 24 loci were found by Tay

( 1 997) and Siraj et at. ( 1 998) respectively. Therefore, more effective techniques

with respect to the ease of detecting genetic polymorphisms are required to

support the existing isozyme results as well as to provide new information which

is not found in the isozyme work. Hence, in our current studies, attempts were

made to apply different types of PCR-based DNA markers to identify and

characterise five populations of local Mystus numerus. These populations

originated from Kedah, Perak, lohor and Sarawak state representing the wild

groups, and one UPM cultured population whose parents were purchased from a

fish farm in Selangor state.

The scopes of this study are divided into three parts:

(i) methodology development,

(ii) Mendelian inheritance study, and

(iii) population study.

3

In the first part of the study, I I I primers representing three types of PCR­

based methods were tested . They are random amplified polymorphic DNA

(RAPD, 40 single primers), amplified fragment length polymorphism (AFLP, 64

pairs of primer combinations), and microsatellites (7 pairs of African catfish,

Clarias gariepinus primers,) . Unfortunately, only RAPD and AFLP primers gave

positive results while none of the microsatell ite primers produced any true

microsatel lite fragments. Because of this, the microsatellite technique could not be

applied in the subsequent segregation and population studies. Therefore, emphasis

is only given for RAPD and AFLP analyses in this thesis.

The primers that gave positive results in the first part of the study wil l

then be used to screen against the families of M nemurus in order to evaluate the

inheritance of the ON A markers from the parents to the offspring. This study will

determine the usefulness of the primers in the breeding program.

In the third part of the study, five populations of M. nemurus provided by

Unit Hatchery of UPM were used to evaluate the utility of the methods developed

above in identifying and characterising ditTerent populations of M. nemurus.

4

Based on the scope of the study, the objectives of this project could be

summarised as follows

(i) to develop DNA marker typing methods for M. nemul'llS,

(ii) to evaluate the inheritance of the DNA markers from the parents to the

offspring,

(iii) to investigate the population structures of M flemlll'llS inhabiting five

different geographical regions in Malaysia using the markers developed

above

(iv) to identify specific gene markers (diagnostic markers) that would characterise

the different M nemurus populations, and

(v) to compare the utility as well as the data generated by different DNA markers

It is hoped that this thesis will provide some valuable information for

future studies In addition, the population genetic data presented here together

with the existing isozyme data could provide some sort of guidelines in the choice

of stocks for breeding purposes

Habitat and Distribution

CHAPTER II

LITERA TURE REVIEW

Mystus Species

Mystus is widely distributed in the East Indies (now Indonesia), and also in

the Asian mainland such as Peninsular Malaysia, Indo-China, and Thailand (Smith,

1 945) . This species is also found in North Borneo (now Sabah) (Inger and Chin,

1 962). It is normally found in rivers, swamps, lakes and other forms of water bodies.

Inger ( 1 955) reported that some specimens were found in the tidal zones at river

mouths where it may actually live [rum the brackish water to the head waters of

most water basins.

In Malaysia, it can be found in most of the states in Peninsular Malaysia such

as Pahang, Selangor, Perak, Terengganu (Mohsin and Ambak, 1 983) as well as in

Sabah and Sarawak. It is locally known as 'ikan baung'.

Taxonomic Classification

The catfish is classified under the following (Smith, 1 945; Inger and Chin,

1 962; Mohsin and Ambak, 1 983) :

5

Phylum ('hordata

Superclass

Class

Pisces

O.\lelchthyes

Subclass Teleos/oml

Order C'yprm�formes

Family Hagrldae

Genus

Species

Mystus

Mystus baramensls

Mystus mgrlceps

Mystus nemurus

Mystus wycku

Mystus Vlttatus

6

The catfishes have borne with many generic names The earliest available

name is Mystus, which was first used by Gronow in 1 763 and was validated by

Scopoli in 1 977 Dumerill in 1 856 replaced Mystus with Macrones which is still in

use in most of the books The name Macrone5 is preoccupied in entomology

Several other names i e Aorta, Hem I bagrus, Hypselobagrus and Aspldobagrus are

also used as synonyms for this fish (Mohsin and Ambak, 1 983)

Morphology

River catfish or baung as it is locally known has cat-like small eyes with

whisker-like barbels around the mouth somewhat resembling the cat, hence it has

been given the name river catfish (Kamarudin el al., 1 987) Its head is broader thaI!

the high, and the upper jaw is slightly longer It has four pairs of barbels, nasal

7

il.,,-ilcls re'lching Ihe eycs, l1la:-;illary b<Jrbcl.s reachlng III Ihe nlr end or Ihe anal lin,

l1l'lndiblllary barbels reaching Ihe the base Ill" peclorals and Incntal harbels ,"-e

�h()rlcr II has a long or moderare adipose jill The dUl"sai lill has a pUtlg�111 spine

\Vhieh is scrrated in its hind bonkr and the pectoral lins have pungent spines which

IS serrated behind. The caudal tin is deeply forked with the upper lobe more or less

produced and pointed. The body is scalelcss und brown or black in colour (Mohsin

:lnd Ambak. 1983).

r

Figure I : The morphology of II/f I/ClI1l1l'11S

lmpo.-tance

Thc river cattish is an economically important food that has gained

popularity among consumers due to ils high nutritional value and good taste. Three

COl1lmon Malaysian li-eshwarcr catfish namely AI. IWII/III'11.\'. C. I)(I/rucilll.\' and ('.

,\/ucro('('IJ/wills have higher average percentage 01' the lean portion than beef and

pork (Karnaruclin cl (//., 1987). Besides Ihat . they wntain the highest crude prolcin

8

and the lowest crude fat compared to beef, pork and chicken (Kamarudin et aI.,

1 987).

The river catfish are unable to reproduce in captivity and their seasonal seed

availability cause them to achieve high prices in the market (Siraj et aI., 1 995). The

price of one kilogram of catfish is between RM 1 0 to RM 1 5 three years ago (Saberi

Mawi from Pusat Penyelidikan Perikanan air Tawar, 1 995) . Recently, the price had

been increased to RMI8/kg. Due to its high potential in the market, the river catfish

had gained popularity among fish aquaculturist and research had been carried out

pertaining to reproductive biology (Khan et al., 1 990), diet (Khan et al., 1 993 ,

1994), nutrition (Kamarudin et aI., 1987) and recently genetics (Daud et al., 1989;

Dodson, 1 995; Siraj et aI. , 1 995; Tay, 1997).

Molecular Techniques

Molecular techniques allowing differentiation between strains, genotypes, or

individuals have become indispensable tools in population genetic survey. Several

molecular techniques are available, but these differ in their ease of use and their

power to resolve genetic differences and may sometimes produce contradictory

results (Martivez et aI., 1 997). For example, enzyme electrophoresis indicated two

different breeding populations of minke whales from West Greenland and Iceland

(Danielsdottir et aI., 1992), while restriction fragment analysis of mitochondrial and

ribosomal DNAs did not reveal any significant differences (Palsboll, 1 989).

9

Before the introduction of the polymerase chain reaction (peR) technology,

protein electrophoresis and classical hybridization-based technique using RFLP had

been widely used in detecting polymorphic genetic markers (Ladizinsky et aI., 1984;

Apuya et aI., 1988 ; Theilmann et aI., 1 989). However, since the introduction of

peR (Saiki, 1988), a series of increasingly effective peR-based techniques with

respect to ease of detecting genetic polymorphisms have been developed. In general,

these techniques used one or two small primers to amplity a region of DNA flanked

by them. The amplification takes place in a thermocycler and is mediated by a

thermostable DNA polymerase. In principle, if primers which flank a polymorphic

region of the genome are available, then this region can be amplified and

polymorphism can be scored by the presence versus absence of the amplified

products as observed from gel electrophoresis. These polymorphisms are the result

of point mutations or rearrangements in the DNA such as insertions or deletions. As

the variations in the banding patterns would be a direct reflection of the genetic

relationship between the organisms examined, these banding patterns can be

considered as 'genomic fingerprints' allowing numerical analysis for characterization

(typing) and identification purposes (Janssen et ai., 1996).

In general, there are two types of PCR-based molecular techniques:

(i) peR with arbitrary primers such as RAPD (Random Amplificd

Polymorphic DNA Williams et ai, 1 990), AP-PCR (Arbitary Primed PCR: Welsh

and McClelland, 1 990), DAF (DNA Amplification Fingerprinting; Caetano-Anolls et