NADIAH BINTI HALIM - Universiti Malaysia Sarawak relationship of terubok fishes (24pgs).pdfNADIAH BINTI HALIM This project is submitted in partial fulfilment of the requirements for

Phylogenetic Relationship of Terubok Fishes (Genus Tenualosa: Clupeidae) Inferred

From Sequencing of Cytochrome b Mitochondrial DNA Fragment

NADIAH BINTI HALIM

This project is submitted in partial fulfilment of the requirements for the Degree of Bachelor

of Science with Honours

(Animal Resource Science and Management Programme)

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2012

ACKNOWLEDGEMENTS

First of all, I would like to thank Allah S.W.T for His Blessings for the completion of this

final year project. My sincere appreciation and big thank you to my supervisor, Dr. Yuzine

Esa for his kind guidance, advisory, encouragement, valuable discussion and constructive

criticisms during the course of investigation and preparation of this project.

Besides that, I would like to thank to En. Huzal and other other lab assistants for their

assistances especially in term of technical problems; and master students for their valuable

comments, sharing knowledge and supporting to make this successful project. I am also like

to thank my lab mates that had patient with me, helped and share their knowledge with me. I

would like to dedicate gratefulness to my family for their support and console. Finally, I

would like to thank all people who have helped and inspired me during my final year project.

II

TABLE OF CONTENTS

Acknowledgement..................... .... .............. .............. ........... .................. ....... ........... .... ... II

Declaration..................... .................................. ..... ..... ............... .................................. ..... III

Table of Contents............ ......................... .... ........ .............. .................................. ........ .... IV

List of Abbreviations.............................. .................................. ....................................... VI

List of Figure........................................................... ........................................................ VII

List of Tables................................................................. .................................................. IX

Abstract............................................................................................................................ 1

1.0 Introduction........... ...... .... ................................................................... ......... ........ ... 2

2.0 Literature Review................................................................................................... 4 2.1 Family Clupeidae.......................................................................................... 4 2.2 Tenualosa toli..... ................ .............................. .... ......................................... 5

2.3 Tenualosa macrura......................... ................ ............. .... ...................... .... ... 7

2.4 Tenualosa ilisha....... .......... ................ ............................ ....................... ........ 8

2.5 Polymerase Chain Reaction (PCR).... ..................................................... ...... 9 2.6 DNA Sequencing........................... ............ ...... ................... .... ...... ... ............. 10 2.7 Cytochrome b.......... ............................................ ...... ............................. ....... 10 2.8 Cytochrome b in Aquatic life Study............................................................. 12

3.0 Materials and Methods........................................................................................... 13 3.1 Sample of collection of Fishes.. ....................................................... ............. 13 3.2 Preparation of 2X CT AB buffer.......... ................. ......................................... 14 3.3 DNA extraction............................................................................................. 15 3.4 DNA Amplification by Polymerase Chain Reaction.................................... 16 3.5 Purification................................................................................................... 17 3.6 Sequence Aligment and Phylogenetic Analysis............................................ 18

4.0 Result...................................................................................................................... 20 4.1 DNA extraction and isolation.................... ...... ................ .............................. 20 4.2 PCR Amplification........................................................................................ 21 4.3 Purification..................................... ...................... ......................................... 22 4.4 DNA Sequence Analysis....... .... .......... ....................... ................... ........ ........ 23 4.5 Pairwise Distance.......... ..................... ........................................................... 25 4.6 Phylogenetic Analysis................................................................................... 27

IV

5.0 Discussion................................................................................................................ 32 5.1 Sample Extraction.......................................................................................... 32 5.2 Polymerase Chain Reaction (PCR)................................................................ 33 5.3 Purification..................................................................................................... 35 5.4 DNA Sequence Analysis................................................................................ 35 5.5 Phylogenetic Analysis.................................................................................... 35

6.0 Conclusion and Recommendations.......................................................................... 38

7.0 References................................................................................................................ 39

v

LIST OF ABBREVIATIONS

°C

Bp

CIA

Cyt b

CTAB

DNA

dNTP

MgCb

MtDNA

m

M

NaCI

PCR

RNA

• rpm

Taq

Degree Celcius

Base pair

Chlorofonn-isoamyl alcohol

Cytochrome b

Hexadecyltrimethylammonium bromide

Deoxyribonucleic acid

Deoxynucleoside-5' - triphosphate

Magnesium Chloride

Mitochondrial DNA

Micro- (l0-6)

Milli- (l0-3)

Molar

Sodium Chloride

Polymerase Chain Reaction

Ribonucleic acid

Rotation per time

Thermus aquaticus

VI

LIST OF FIGURES

Figure I: Picture of Tenualosa toli

Figure 2: Picture of Tenualosa macrura

Figure 3: Picture of Tenualosa Share

ilisha adapted from Bangladesh Fisheries Information

Figure 4: Picture of Structure of Cytochrome b

Figure 5: Map showing the location of study areas in Sarawak

Figure 6: DNA extraction of 11 samples of Tenualosa toli which lane I to 5 are from Bintulu region, lane 6 to 7 are from Mukah and from lane 8 to II are from Batang Lassa. M is GeneRuler™ lKb ladder. There were some samples that do not show bands at all. Also show DNA band with bright single band on gel.

Figure 7: PCR products of 8 samples of selected extracted DNA. M represented I Kb ladder. Lane 8 represent negative control. Lane 1 to 2 represent samples of selected extracted DNA from Mukah and lane 3 to 7 represent samples of selected extracted DNA from Bintulu. All samples showed presence of bright single band.

Figure 8: Gel electrophoresis of purification products. The products run in 1 % gel with I x TAE buffer. Lane M represents Fermentas™ IKb DNA ladder. Lane 1 to lane 4 were samples of T. toli from Batang Lassa. Lane 5 to lane 7 were samples of T. toli from Bintulu.

Figure 9: Neighbour - joining (NJ) Tree showing the Cyt b relationship among T. toli, T. macrura, T. ilisha and two out-group analysed. Values at major nodes refer to bootstrap support with 1000 interations. Numbers at nodes indicate the bootstrap values in percentage. There are two major group, (group 1 and group 2). Group I are samples from Sarawak. Group 2 for T. macrura are from Sarawak and T. ilisha are sequence retrieved from Genbank from Bangladesh. Only bootstrap values >50% are shown.

Figure 10: Phylogenetic tree of among T. toli, T. macrura and T. ilisha based on nucleotide sequences of partofthe mitochondrial Cyt b gene using maximum-parsimony (MP) clustering method. There are two major group, (group 1 and group 2). Tree constructed using Kimura two- parameter model of evolution. Values at major nodes refer to the bootstrap support. Only bootstrap values >50% are shown.

VII

Figure 11: Phylogenetic tree of among T toli, T macrura and T ilisha based on nucleotide sequences of part of the mitochondrial Cyt b gene using maximum-parsimony (MP) clustering method. There are two major group, (group 1 and group 2). Tree constructed using Kimura two- parameter model of evolution. Values at major nodes refer to the bootstrap support. Only bootstrap values >50% are shown.

Figure 12: Bayesian inference of 50 % rule-majority consensus tree of nucleotide sequence of part of the mitochondrial cyt b gene was constructed using Bayesian Inferences analyses (Mr. Bayes) with Sardinella maderensis as outgroup. The Bayesian Posterior Probability value (pp) values are indicated by the number at nodes.

VIII

LIST OF TABLES

Table I : specific classification of Tenualosa toli.

Table 2 : The variety of Tenualosa sp. in tropical Asian Region

Table 3 : Description of fish study sites, number of fish, and abbreviations in this study

Table 4: The Mastermix for PCR Reaction Mixture

Table 5 : Description of Tenualosa toli and Tenualosa macrura samples sent for sequencing

Table 6 : Samples that were obtained from gene bank and their abbreviation

Table 7 : Nucleotide compositions based on full length Cytochrome b for the three species of Tenualosa toli, Tenualosa macrura and Tenualosa ilisha.

Table 8 : Pairwise distances (or genetic distance) ca1culated using Kimura 2 - parameter in Percentage

..

IX

Phylogenetic Relationship of Terubok Fishes (Genus Tenualosa: Clupeidae) Inferred

From Sequencing of Cytochrome b Mitochondrial DNA Fragment

N adiah Binti Halim

Animal Resource Science and Management Program Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

This study examines the phylogenetic relationship among three different species of Tenualosa toli, Tenualosa macrura and Tenualosa ilisha. A total of ten T toli, two T macrura and three T ilisha nucleotide sequences were analysed. The sequences of Tenualosa ilisha obtained from Genbank was used in this study. Phylogenetic results supports the monophyletic status between the three terubok with high genetic distances (13.943-16.033%) between them. The nucleotide sequences were subjected to phylogenetic analyses by using the neighbor-joining, maximum parsimony, and Bayesian analyses produced similarity tree topologies with only the maximum likelihood tree was slightly different on the position of T ilisha. Overall, this study has managed to provide useful informations on the phylogenetic status of terubok in Sarawak.

Keywords Tenualosa toli. Tenualosa ilisha. Tenualosa macrura, phylogenetic relationships, nucleotide sequences

ABSTRAK

Kajian ini telah dijalankan untuk mengkaji perhubungan filogenetik diantara tiga species yang berbeza ialtu Tenualosa toli. Tenualosa macrura and Tenualosa ilisha. Sejumlah sepuluh dua T macrura dan liga T ilisha sekuens telah dianalisis. Penjujukan DNA telah diambil daripada Genbank digunakan di dalam kajian ini. Keputusan analysisfilogenetik telah menyokong hubungan monofiletik status diantara tiga terubok dengan nilai genetik yang linggi (13.943-16.033%). Analysis filogenetik menggunakan kaedah neighbor-joining, maximum parsimony dan analysis Bayesian lelah menghasilkan topology pokok yang hampir sama dengan hanya kaedah analysis maximum likelihood telah menghasilkan sedikit kelainan terhadap kedudukan T i/isha di dalam topology pokok. Secara keseluruhanya. kajtan ini telah berjaya menghasilkan info yang berguna di dalam status hubunganfilogenetik terubok di Sarawak,

Kata kunci : Tenualosa lOli. Tenualosa ilisha. Tenualosa mao'ura. hubunganfilogenetik •

..

1

I

1.0 INTRODUCTION

Tenualosa is a genus of Clupeidae family that can be divided into few species. One of the

most studied species is the Tenualosa ilisha that was widely found from north Sumatra to

Kuwait. In addition to that, it is also one of the economically important fish in the coastal

areas of countries such as Bangladesh, India, Burma, and Pakistan (Blaber et al., 2003). In

Sarawak, two of the Tenualosa species was recorded; i) Tenualosa toli normally found in

estuaries and also adjacent coastal areas of Sarawak (north coast of Borneo) and ii)

Tenualosa macrura, lives in the coastal area of Sumatra and Borneo.

Furthermore, Tenualosa species exhibit several type of life history strategies; for example,

T ilisha and T reeevesii are gonochoristic, whereas T. toli and T macrura are protandrous

hermaphordites. Habitat preferences of Tenualosa species movement patterns and

maximum age also vary between species. Two species of Tenualosa ( terubok ) are found

in Sarawak are T. toli (big-mouthed shad) and T macrura (long-tailed shad) (Raj ali,

1992).

Besides that, all Tenualosa are subjected to heavy fishing pressure and have suffered major

declines. Although T toli and T macrura have great cultural significance in Sarawak and

Sumatra respectively, both are still commercially fished for their eggs, which command

very high prices.

Fish systematic has historically depended on morphological techniques for identifying

groups of evolutionary related species (Stepien and Kocher, 1997). However, the

development of molecular techniques has helped to invigorate the study of fish systematics

(

2

III

"

(Stepien and Kocher, 1997). Very little molecular techniques had been done to

systematically sort out Tenualosa species for proper reference. One characteristics that

interest to this genus is that it shows few geographical variation in morphological traits.

Thus, confusion remains in the recognization and classification of this genus, especially in

dealing with specimen from other regions.

Hence, the applications of molecular techniques such as DNA sequencing offer better

solution to the problem on taxonomy and population size of Tenualosa species. Molecular

marker provides reliable and consistent results for rapid species identification (Ryan and

Esa, 2006). In this study, the cytochrome b (cyt b) of the mitochondrial gene was used to

analyze the phylogenetic relationship of several selected species of fish from family

Clupeidae. Similarly report has shown that cytochrome b was one of the best-studied

mtDNA genes in fishes (Zhu et al., 1998; Jansson and Ost, 1997). The aim of this study

was conducted to provide scientific basis and clarification of the phylogenetic relationship

from three different species of (Genus Tenualosa). In addition, study of molecular data can

help to strengthen the taxanomy of Tenualosa species based on morphology or can assist in

solving the question of species origin. It has been acknowledge that a molecular

phylogenetic study is essential for better understanding towards the evolution of a

particular species (Mindel et ai., 1997)

The objectives of this study were:

I) To amplify cytochrome b mtDNA gene in three species of Tenualosa from family Clupeidae.

2) To determine phylogenetic relationship among the three species of Tenuaiosa, using cyt b mtDNA gene.

3

I

2.0 LITERATURE REVIEW

2.1 Family Clupeidae

d

The Family Clupeidae (subfamily Alosinae) comprehends some of the wolrd's most

important commercial fish species. Alosinae is composed of about 19 species among seven

genera : Alosa. Brevoortia, Ethmalosa, Ethmidium, Gudusia, Hi/sa and Tenualosa (John

Waldman). The majority of the Clupeidae are marine pelagic species, but some of them

occasionally venture into rivers (sometimes for spawning), and a few species are

exclusively found in freshwater. Besides that, clupeidaes special characterization is their

head is not cover by scales, a terminal or inferior mouth without or very weakly

development of small teeth.

Family Clupeidae are fishes which are body form are usually fusiform, round to strongly

compressed. A single dorsal fin, small and near midpoint of body; pelvic fins more or less

below dorsal fin base; dorsal and pelvic fins absent in some species; soft rays only. Lateral

line spanning a few scales behind the head in some species, missing in others; scales

cycloid (smooth to touch); abdominal scutes usually present (a single pelvic scute in the

Dussumieriinae). Branchiostegal rays usually 5-10. Most feed on small planktonic animals.

Size range (adult): from 2 to 75 cm. One of the most important family of commercial

fishes, processed for food, oil or fish meal. All this information based on the (Whitehead,

1985). The distribution are global (mostly tropical) from 70° N to about 600 S. chieflt

marine coastal and schooling fishes; some freshwater and anadromous.

Colour; back usually bluel green, sides silvery, sometimes with a distinct silver band.

Darker markings include a black spot behind gil opening, sometimes continued as a series

4

of spots along sides, a dark spot at dorsal fin origin ( Sardinella ), dark dots or spots along

back and dark pigmentation on parts of fins ( especially outer margins). Most clupeids are

marine, but some can tolerate low salinities and some shads (Alosinae) and gizzard shads

(Dorosomatinae) live temporarily or permanently in freshwater.

2.2 Tellualosa IoU

Figure 1 : Picture of Tenualosa tali

Tenualosa toli (T toli) or locally known as Tembok (Figure 3.2) is one of the important

fish in Malaysia. Tenualosa toli is unique among the Clupeiformes because it is a

protandrous hermaphrodite. It is anadromous fishes (mature at the sea and return to

ascending rivers to reproduce) and spent most of their juvenile stage at freshwaters. It is an

endemic species that currently can only be found in estuaries and adjacent coastal area of

Sarawak (northern coast of Borneo) (Blaber et aI., 2003). T toli are classified as pelagic

fishes and can be found in schools in the coastal waters, brackish and also in marine areas

(Anon., 2004 b and Anon., 2004 d).

T toli can be easily distinguished from other Tenualosa sp as it lacks the series of black

spots on the side which characterize that species. Ttoli can grow to about 400mm SL

5

(Standard length) and live from 2 to 3 years (Milton et ai., 1997). Table 1 showed the

taxanomy of T toli.

Table 1 : Specific classification of Tenualasa tali.

Kingdom Animalia

phylum Chordata

Class Actinopterygii

Order Clupeiformes

Family Clupeidae

Genus Tenualosa

Species Tenualosa toli

T toli become one of the important consumption tropical shad in Malaysia because this

fish is rich in lipid and provide valuable fatty acids which playa significant role in human

health (Rahman and Salimon, 2006). Variety of Tenualosa sp. with different scientific

name has been found in tropical Asian Region as shown in Table 2 (Blaber et al., 1996).

Table 2 : The variety of Tenualasa sp. in tropical Asian Region (Blaber et aI., 1996)

Common name Scientific name Origin

Terubolrl Tervbok sullgai Tenualosa toli Malaysia

Terubukl Terubok laut Tenualosa macrura Indonesia

Hilsa Tenualosa ilisha India

Pha MakPang Tenualosa thibaudeani Mekong

nil Tenualosa reeves;i Southern China

6

2.3 Tenualosa macrura.

Tenualosa macrura was previously found throughout the estuaries and coastal waters of

Sumatra and Borneo where it formed the basis of flourishing fisheries. T macrura feeds on

zooplankton (Vidthayanon, 2005). Schooling in coastal waters and ascending rivers to

breed. Presumably its biology is similar to that of Tenualosa ilisha, but the fewer gilirakers

suggest that it takes larger food organisms (Whitehead., 1985). T macrura is a protandrous

hermaphrodite (Allsop, and West, 2003). It changes from male to female mainly between

14 and 20 cm SL (standard length) (six month to one year in age), after the male has

spawned.

Figure 2: Picture of T macrura

T macrura is an anadromous fish (Riede, 2004). The distribution of the T macrura is at

western Central Pacific; Malaysia, Indonesia (Java Sea and Sarawak, also affluent rivers)

and Thailand (southern tip). T macrura caudal fin long, the lobes long and pointed. No

series of dark spots along flank. Resembles T.toli, which has longer head but shorter tail.

7

2.4 Tenualosa ilisha

Tenualosa ilisha (Hilsa shad), one of the most important tropical fish of the Clupeidae

family, is the national fish of Bangladesh. It is primarily an anadromous species and occurs

in the coastal shelf, brackish, estuarine, and fresh water rivers (Rei de, 2004) of the western

division of the Indo-Pacific faunistic region . Its geographical distribution extends from

Northern part of Indian Ocean (from the 'Gulf' eastward to Myanmar, including western

and eastern coast of India, also rivers) (Talwar and Jhingran, 1991).

Figure 3 : Picture of Tenualosa ilisha adapted from Bangladesh Fisheries Information Share Home

http://www.searchfish.orglfish/33628Itenualo 'a-iJishaJ

During the commencement of the south-west monsoon, they start the spawning migration

towards the estuaries and rivers (from July till October, also from January to February),

(Rahman, 1989 and 2005). Feeds on zooplankton in the early hours of the day (Bhuiyan,

1964). Attain sexual maturity at 1-1.5 years of age (Shati and Quddus, 2001).

8

Ti

2.5 Polymerase Chain Reaction (PCR) Amplification.

Polymerase Chain Reaction (PCR) is a techniques used to make a huge number of copies

of gene or the target DNA. The concept ofPCR was developed by Kary B. Mullis in 1980s

(Rabinow, 1998). These include DNA cloning and sequencing, DNA-based phylogeny, or

functional analysis of gene. PCR is a technique which amplifies a specific region of DNA

gene for genetics studies. From a small amount of tissues or blood sample, millions copies

of a specific genes can be amplified by using a pair of specific primers (forward and

reverse) (Hillis et al., 1996).

Other than that, it is a cell- free method of DNA replication, and requires no cleanup of I

1,unwanted cellular debris or vector DNA. PCR can be end labeled with a variety of ~ ~ ~, I

modifications and specific process that targets only the desired DNA to be copied. PCR ,.'

technique is suitable for population genetic studies (Silva and Russo, 2000). Beside that, ,I'

PCR requires a repetitive series cycle of heating and cooling which, involved three ",

.' ,/

I' I l~

fundamental steps that defines as one PCR cycle. With each cycle of denaturing, annealing,

and synthesis the specific DNA fragment is amplified exponentially. PCR procedure

usually involved various components such as template DNA, primers, Taq polymerase,

deoxynucleoside tryphosphate (dNTPs), Tris- HCL (ph 8.3) and MgCb, However, PCR is a

very sensitive procedure which may show false result if a slight error occurs such as

contaminants (Rudin and Inman, 2001).

9

2.6 DNA sequencing

DNA sequencing refers to sequencing methods for determining the order of the nucleotide

bases. Sequencing refers to the technique used to determine the order of the constituent

bases (i.e., adenine, thymine, guanine, and cytosine) of deoxyribonucleic acid (DNA) or

protein. Protein sequencing determines the order of the constituent amino acids (Hoyle,

2004). DNA sequencing of PCR products is important in constructing phylogenetics trees

to understand the genetic relationship between species in taxa. In general, phylogenetic

trees evaluate the number of shared and differing characters between groups of organisms

(Scott and Graham, 2001).

2.7 Cytochrome b (cyt b)

Cytochrome b is one of the cytochromes involved in the electron transport in the

respiratory chain of mitochondria. It contains eight transmembrane helices connected by

intramembrane or extramembrane domains (Esposti et al., 1993). It is the only cytochrome

coded by mitochondrial DNA

10

INTRAMEMBRANE SPACEN-1enninuJ

A B c E F

~----" EXTRAMEMBRANE SPACE

Figure 4. Structure of the cytochrome b protein. The gene region used in this study corresponds the shaded

parts of the protein. Adapted from journal of Phylogeny and phylogeography of European Parids.

The cytochrome b gene is the most widely used gene for phylogenetics work for several

reasons. Although it evolves slowly in tenns of non-synonymous substitutions, the rate of

evolution in silent positions is relatively fast (Irwin et al. 1991). The wide use of

cytochrome b has created a status as a universal metric, in the sense that studies can be

easily compared. Cytochrome b is thought to be variable enough for population level

questions, and conserved enough for clarifying deeper phylogenetic relationships.

However, the cytochrome b gene is under strong evolutionary constraints because some

parts of the gene are more conserved than other due to functional restrictions (Meyer

1994). Most of the variable positions seem to be located within the coding regions for

transmembrane domains or for the amino- and carboxyl-tenninal ends (Irwin et al.1991).

11

2.8 Cytochrome b (Cyt b) in Aquatic life Study

There are many literature review that had been done usmg Cytochrome h in the

phylogenetic study. The mitochondrial cytochrome h (cyt h) gene is widely used in

systematic studies to resolve divergence at many taxanomic. The previous phylogenetic

study by Lydeard and Roe (1997) based on the studied of phylogenetic utility of the cyt h

gene for inferring high level relationships among the Actinopterygian fishes. Although

strong support was shown for the monophyl of certain groups within the Perciformes, no

robust pattern ofrelationship among groups was found.

Based on study which have done by Song et al., (1998) on Phylogenetic Relations among

Percid Fishes, the cytochrome h dataset for 21 species of Percidae strongly supports the

monophyly of the three major groups, Etheostomatinae, Luciopercinae, and Percinae.

Another study using cytochrome h as genetic marker was done by Jerome et al., (2003) on

the Molecular Phylogeny and Species Identification of Sardines. This work was to study

the genetic divergence between Clupeiformes species labeled as Sardines type products

and to define relevant tools to discriminate these pelagic fishes. In this study, 8 species of

Sardines were examined by 1141 bp sequences with 430 evenly distributed variable sites

were recorded, 353 of which were phylogenetically informative. The result show good

distinction between the main genera on different scales of cytochrome h.

Cytochrome h has been considered one of the most useful genes for phylogenetic work,

and is probably the best-known mitochondrial gene with respect to structure and function

of its protein product (Esposti et al., 1993). Therefore, this gene has been used for a

diversity of systematic questions from "deep" phylogeny (e.g., Meyer and Wilson, 1990;

Irwin et aI., 1991; L ydeard and Roe, 1 997)

12

3.0 MATERJALS AND METHODS

3.1 Sample collection

The terubok fishes samples have been taken from available collections in, the Fish

Museum, Department of Aquatic Science, Faculty of Resource Science and Technology of

UNIMAS. Samples of T tali and T macrura have been collected from various localities in

Sarawak such as Muara Tuang, Batang Lassa, Sebangan, Mukah, Bintulu and Sebuyau.

The sequences of T ilisha were taken from (GenBank). All the samples have kept in eitl1er

-20oe and preserve in 70% ethanol. Whole fish were identified with the keys based on

(Kottelat et ai. , 1993).

Table 3 : Description of fish study sites and number of fish in this study.

Study sites Number of fish for available for the study

Sebangan 4

Muaratuang 9

Batang Lassa 5

Sebuyau 2

Mukah 2

Bintulu 6

Total 28

The samples from Mukah and Bintulu were fresh samples and the other samples were from

voucher specimen collection in the Fish Museum, Department of Aquatic Science, Faculty

of Resource Science and Technology ofUNIMAS.

13

Figure 5 : Map showing the location of study areas in Sarawak.

3.2 Preparation of 2x CT AB buffer

Before DNA extraction can be done, hexadecyltrimethyolammnium bromide (CT AB)

solution had to be prepared . 40.9g NaCI was placed in a lOOOml beaker. Tris Base was put

into the beaker then followed by CT AB and EDTA into the beaker. 400ml of ddH20 was

then poured into the beaker to dissolve the substances. The substances then stirred until it

become clear on a hotplate. After it become clear, let it cool down. Then the clear solution

was poured into 500ml bottle. After the solution had been poured, the bottle was wrapped

with aluminium foil. The bottle was later added with 1OOO~1 2-mercaptoethanol-B­

mercapto. This was because the volume in the bottle was less than 50OmI, ddH20 was

added till it exceeds 500ml.

14

3.3 DNA Extraction

DNA extraction method was done using modified CT AB method (Grewe et at., 1993)

with Proteinase K. About 2 cubic millimeters of the sample tissue was macerated into

small bits and transferred into I.S eppendorf tube. Then, 700lli CT AB buffer and 20111

Proteinase K was added into the eppendorf tube and mixed welLAfter that, the tube have

been incubated in the water bath at 6S0C for 1-2 hours until tissues are completely

dissolved. After the tissues had dissolved, 600 III of chloroform-isomyl alcohol (CIA)

(24: 1) was added and shaken for 2 to 3 monutes. It then centrifuged at 13 000 rpm for 20

minutes.

After centrifugation had be done, the SOO III upper layer of the supernatant were taken and

transfer to a newly labeled tube. Then add SOO III of cold absolute ethanol (99%), mixed

and the tube sit let on the bench for a few minutes (preferably), then the tube centrifuged

for 20 minutes at 13 000 rpm. The ethanol discarded by pouring it or by using pipettes. The

pellets was washed and precipitated with SOOIlI of cold 70% ethanol, and 2S III of 3M

NaOAc / NaCI and spinned at 13 OOOrpm for 20 minutes. The ethanol then discarded

carefully making sure that the pellet is not not disloged during the process. Small droplets

of liquid that were still present are left for evaporation or sucked using tissue after taking

consideration about the position of the pellet in the tube. The remaining pellet is suspended

in SO to IOOIlI of distilled water (ddH20). Next, DNA extraction had been kept in the

freezer (-20°C). After DNA had been extracted, it was electrophoresis on 1 % agarose gel

containing 0.4 micro-litres of ethidium bromide. Then it was photographed under UV

illumination. This was done to determine the quality of the DNA extracted and to ensure

whether there are contaminants exist

15

3.4 DNA Amplification by Polymerase Chain Reaction

The mitochondrial DNA genome from the complete length cytochrome b gene have been

amplify using standard PCR protocol (Sambrook et al., 1989). The 2 primers used for

amplifying complete length cytochrome b in this study were; H 15915- (AAC TGC AGT

CAT CTC GGG TTT ACA AGA C - reverse) (Irwin et al., 1991) and L 14724- ( CGA

AGC TTG A TA TGA AAA ACC A TC GTT G - forward) (PAAbo, 1990).

A master mix in this study made in 1.5 ml eppendorf tube, as below:

Compenant 1 x reaction (pi)

5 X PCR buffer 5.00

MgCh 1.5

dNTPs mix (10mM) 0.5

ddH20 13.3

primer Cyt b f 1.25

primer Cyt b e' 1.25

Template DNA 2.0

Taq polymerase 0.2

Total Volume 25.0

Table 4 : The Mastermix for peR Reaction Mixture.

Then, the tube was spined down by pulsing in a centrifuge. The amplification was carried

out in standard thermocycler (BIORAD) machine for 3 main cycles. Before the main

cycles start, the samples were pre- heated at 94°C for 30 seconds. The first cycles consist 1·

of 30 cycles of denaturation of double stranded 95°C for 30 seconds per cycles. The second

cycles involved 35 cycles of annealing at 47.3°C for 30 seconds, which the extension

primers had been annealed to the specific site. The third cycle or the extension will be

16