UNIVERSITI PUTRA MALAYSIA

YOW WENG KIT

FP 2012 62

MITOCHONDRIAL DNA POLYMORPHISM IN SEVEN CATTLE BREEDS IN MALAYSIA

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MITOCHONDRIAL DNA POLYMORPHISM IN SEVEN CATTLE BREEDS

IN MALAYSIA

By

YOW WENG KIT

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Master of Science

March 2012

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THIS THESIS IS DEDICATED

TO

MY BELOVED FATHER AND MOTHER,

AND

MY SISTER

THANK YOU

FOR ALL THE SUPPORTS

YOU GAVE TO ME

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia

in fulfillment of the requirement for the degree of Master of Science

MITOCHONDRIAL DNA POLYMORPHISM IN SEVEN CATTLE BREEDS

IN MALAYSIA

By

YOW WENG KIT

March 2012

Chair: Professor Jothi Malar Panandam, PhD

Faculty: Agriculture

Although the Kedah Kelantan (KK) is the indigenous cattle of Malaysia, a number of

cattle breeds have been introduced into the country to enhance the local beef and

dairy cattle industry. The cattle breeds in Malaysia comprise of Bos indicus types

(present in tropical countries), Bos taurus types (indigenous to the European

countries but is also found in Africa and Asia), and the hybrids of these two types of

cattle. There are not many publications available on the origin of Asian cattle. There

is a lack of studies on the molecular variation within and among the cattle breeds in

Malaysia as well as the genetic relationship among these breeds. In addition, the

phylogenetic relationship of cattle breeds in Asia, especially using mitochondrial

DNA (mtDNA), has not been widely conducted. No study has been conducted on

mtDNA polymorphism of Kedah Kelantan (KK), the indigenous cattle breed of

Malaysia.

Mitochondria are cytoplasmic organelle that contain DNA. MtDNA has many special

features which has made it a popular marker to determine evolutionary and

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phylogenetic relationships. It has been used in studies to identify the genetic

relationships between and within species of mammals. Displacement loop (D-loop) is

the highly variable and most rapidly evolving region in the mtDNA. Variation

observed in this region is frequently used for phylogenetic analysis of related breeds

within species. The mtDNA cytochrome b (cyt b) is significantly conserved and,

therefore, suitable for phylogenetic relationship and forensic investigations. It is

suitable for between species comparison. Mitochondrial NADH dehydrogenase 5

(ND5) gene is another highly conserved genes and is commonly used for species

identification.

With the above in mind, this study was conducted to evaluate the mtDNA

polymorphism in seven cattle breeds in Malaysia, namely KK, Brahman, Brakmas,

Brangus, Charoke, Droughtmaster and Jersey at four regions of the mtDNA, which

are the D-loop, cyt b and ND5 regions.

Blood samples were randomly collected from 30 animals of each breed except for

Brangus (n=17). DNA was extracted and quantified using a spectrophotometer. The

D-loop, cyt b and two ND5 regions were amplified using specific primers. These

regions were digested with nine, five and two restriction endonucleases (RE),

respectively, generating 16 polymerase chain reaction-restriction fragment length

polymorphism (PCR-RFLP) loci. DNA cloning was performed on two samples of

KK to obtain the nucleotide sequence for each of the four regions of mtDNA. The

sequence information was compared with that for B. indicus and B. taurus which

were available in the National Center for Biotechnology Information (NCBI)

database.

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The total percentage of polymorphism for the loci was 50%. The ND5 locus recorded

100% polymorphism. Polymorphism at the D-loop locus was 44.44% and for the cyt

b locus it was 40%. The DdeI, EcoNI and MboI RFLP patterns for D-loop, the MspI

and MspR9I RFLP patterns for cyt b, and the HindIII and TasI RFLP patterns for

ND5 loci were clearly able to distinguish between the B. indicus and B. taurus cattle

breeds. Thus, these make them useful markers to detect variation between species.

The dendogram generated using the PCR-RFLP mtDNA data showed two clusters

for the seven cattle breeds. The KK, Brakmas, Charoke and Droughtmaster breeds

were grouped together while the other cluster comprised of Brahman, Brangus and

Jersey breeds. Alignment of the KK nucleotide sequences with those of B. indicus

and B. taurus for the D-loop, cyt b and ND5/HindIII regions showed KK to be more

similar to B. indicus than to B. taurus. However, comparison of the ND5/TasI region,

showed KK to be similar to B. taurus instead of B. indicus.

More regions of the mtDNA should be investigated and more RE should be used in

order to determine the reliability of the result. Sequencing of more samples and from

all breeds for the various regions would provide more information on the genetic

structures and relationships among the cattle breeds.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master Sains

POLIMORFISMA DNA MITOKONDRIA DALAM TUJUH BAKA LEMBU

DI MALAYSIA

Oleh

YOW WENG KIT

Mac 2012

Pengerusi: Profesor Jothi Malar Panandam, PhD

Fakulti: Pertanian

Walaupun Kedah Kelantan (KK) adalah lembu asli Malaysia, beberapa baka lembu

telah diperkenalkan ke negara ini untuk meningkatkan industri lembu pedaging dan

tenusu tempatan. Baka lembu di Malaysia terdiri daripada jenis Bos indicus (sedia

ada di negara-negara tropika), jenis Bos taurus (asli kepada negara-negara Eropah

tetapi juga didapati di Afrika dan Asia), dan hibrid daripada kedua-dua jenis lembu.

Tidak banyak penerbitan yang boleh diperolehi pada asal usul lembu Asia. Terdapat

kekurangan kajian tentang variasi molekul di dalam dan di antara baka lembu di

Malaysia serta hubungan genetik antara baka. Di samping itu, hubungan filogenetik

ternakan lembu di Asia, terutamanya menggunakan DNA mitokondria (mtDNA),

tidak dijalankan secara meluas. Tiada kajian yang dijalankan ke atas polimorfisma

mtDNA Kedah Kelantan (KK), baka lembu asli Malaysia.

Mitokondria merupakan organel sitoplasmik yang mengandungi DNA. MtDNA

mempunyai banyak ciri-ciri istimewa yang menjadikan ia penanda yang popular

untuk menentukan hubungan evolusi dan filogenetik. Ia telah digunakan dalam

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kajian untuk mengenal pasti hubungan genetik antara dan dalam spesies mamalia.

Displacement loop (D-loop) merupakan bahagian yang mempunyai perubahan tinggi

dan yang paling pesat berkembang dalam mtDNA. Perubahan yang diperhatikan di

bahagian ini sering digunakan untuk analisis filogenetik baka yang berkaitan dalam

spesies. MtDNA cytochrome b (cyt b) adalah dipelihara dengan ketara dan, oleh itu,

sesuai untuk hubungan filogenetik dan siasatan forensik. Ia adalah sesuai untuk

perbandingan antara spesies. Gen mitochondrial dehydrogenase NADH 5 (ND5)

merupakan satu lagi gen yang sangat dipelihara dan biasanya digunakan untuk

mengenal pasti spesies.

Dengan di atas dalam fikiran, kajian ini telah dijalankan untuk menilai polimorfisma

mtDNA dalam tujuh baka lembu di Malaysia, iaitu KK, Brahman, Brakmas,

Brangus, Charoke, Droughtmaster dan Jersey di empat bahagian mtDNA, yang ada

di bahagian D-loop , cyt b dan ND5.

Darah diambil secara rawak daripada 30 haiwan setiap baka kecuali Brangus (n =

17). Sampel DNA diekstrak dan kuantitinya diperiksa mengguna spektrofotometer.

D-loop, cyt b dan dua bahagian ND5 diamplifikasi mengguna primer khusus.

Bahagian-bahagian ini telah dicerna dengan sembilan, lima dan dua restriction

endonuclease (RE), masing-masing menjana 16 lokus polymerase chain reaction-

restriction fragment length polymorphism (PCR-RFLP). DNA pengklonan telah

dilakukan ke atas dua sample KK untuk mendapatkan urutan nukleotida bagi setiap

empat bahagian mtDNA. Maklumat urutan dibandingkan dengan B. indicus dan B.

taurus yang terdapat dalam pangkalan data National Center for Biotechnology

Information (NCBI).

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Jumlah peratusan polimorfisma untuk lokus adalah 50%. Lokus ND5 mencatat 100%

polimorfisma. Polimorfisma pada lokus D-loop adalah 44.44% dan bagi lokus cyt b

ia adalah 40%. Corak RFLP DdeI, EcoNI dan MboI untuk D-loop, corak RFLP MspI

dan MspR9I untuk cyt b, dan corak RFLP HindIII dan TasI untuk lokus ND5 dengan

jelasnya dapat membezakan antara baka lembu B. indicus dan B. taurus. Oleh itu, ini

menjadikan mereka petanda berguna untuk mengesan variasi di antara spesies.

Dendogram yang dijana mengguna data mtDNA PCR-RFLP menunjukkan dua

kelompok tujuh baka lembu tersebut. Baka KK, Brakmas, Charoke dan

Droughtmaster dikumpulkan bersama manakala kelompok yang lain terdiri daripada

baka Brahman, Brangus dan Jersey. Penjajaran jujukan nukleotida KK dengan B.

indicus dan B. taurus untuk bahagian D-loop, cyt b dan ND5/HindIII menunjukkan

KK akan lebih serupa dengan B. indicus berbanding dengan B. taurus. Walau

bagaimanapun, perbandingan di bahagian ND5/TasI, menunjukkan KK sama dengan

B. taurus dan bukannya B. indicus.

Lebih banyak bahagian-bahagian mtDNA harus disiasat dan lebih RE harus

digunakan untuk menentukan kebolehpercayaan keputusan. Sequencing sampel yang

lebih banyak dan dari semua baka bagi pelbagai bahagian akan memberi maklumat

yang lebih mengenai struktur genetik dan hubungan antara baka lembu.

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ACKNOWLEDGEMENTS

First and foremost, I would like to express my thank to GOD for His blessings and

help in making sure the completion of this thesis. Without His consent, I would not

have able to finish the thesis.

I would like to express my sincere gratefulness and gratitude to my supervisor, Prof.

Dr. Jothi Malar Panandam, and co-supervisors, Assoc. Prof. Dr. Ismail Idris and

Assoc. Prof. Dr. Norihan Mohd Saleh who gave invaluable supervision and

encouragement throughout the completion of this research. I would like to thank

Arash Javanmard, Alireza Majidi, Haytham Hago Abdelwahid, Saeid Nibkin, Reza

Tohidi, Hamidah Ali Kamarulzaman and Kamariah Jamhari who also helped and

advised me. I would also like to thank Assoc. Prof. Dr. Parameswari Namasivayam

for her permission to use the lab and her student, Conie Toh who helped me with the

cloning of samples.

Very special thank goes to my beloved family for their love, never-ending support

and encouragement in making sure that I did my best in my research at Universiti

Putra Malaysia (UPM) and completed the thesis. I would like to acknowledge the

Ministry of Science, Technology and Innovation (MOSTI) Malaysia, who granted

me with the scholarship of National Science Fellowship (NSF) throughout my

studies.

Lastly, to all who were involved directly or indirectly in making this research a

possible success. Million thanks and may GOD bless you all.

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I certify that a Thesis Examination Committee has met on 1st March 2012 to conduct

the final examination of Yow Weng Kit on his thesis entitled “Mitochondrial DNA

Polymorphism in Seven Cattle Breeds in Malaysia” in accordance with the

Universities and University College Act 1971 and the Constitution of the Universiti

Putra Malaysia [P.U.(A) 106] 15 March 1998. The committee recommends that the

student be awarded the Master of Science.

Members of the Examination Committee were as follows:

Zainal Aznam Bin Mohd Zelan, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Reuben Sunil Kumar Sharma, PhD

Senior Lecturer

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Internal Examiner)

Tan Soon Guan, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal Examiner)

Subha Bhassu, PhD

Senior Lecturer

Faculty of Science

Universiti Malaya

(External Examiner)

_______________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 21 May 2012

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Jothi Malar Panandam, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Ismail Idris, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Member)

Norihan Mohd Saleh, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

____________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.

________________

YOW WENG KIT

Date: 1 March 2012

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TABLE OF CONTENTS

Page

DEDICATIONS ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL x

DECLARATION xii

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF ABBREVIATIONS xix

CHAPTER

1 INTRODUCTION 1

1.1 Problem statement 3

1.2 Objectives 4

2 LITERATURE REVIEW 5

2.1 Cattle 5

2.1.1 Bos indicus 6

2.1.2 Bos taurus 7

2.2 Cattle breeds in Malaysia 8

2.2.1 Kedah Kelantan (KK) 8

2.2.2 Brahman 9

2.2.3 Brakmas 10

2.2.4 Brangus 11

2.2.5 Charoke 12

2.2.6 Droughtmaster 13

2.2.7 Jersey 14

2.3 Mitochondrial DNA (mtDNA) 15

2.3.1 Displacement loop (D-loop) 17

2.3.2 Cytochrome b (Cyt b) 20

2.3.3 NADH dehydrogenase subunit 5 (ND5) 21

2.4 Studies using mitochondrial DNA 22

2.5 Genetic variation studies in cattle using other marker

systems

24

3 MATERIALS AND METHODS 27

3.1

3.2

Experimental materials

Flow chart of methods

27

28

3.3 Blood sampling 29

3.3.1 Genomic DNA extraction 29

3.3.2 Testing and quantification of the DNA 30

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3.3.3 Loci and primer sequences 31

3.3.4 Polymerase chain reaction (PCR) 32

3.3.5 Digestion of PCR product 34

3.3.6 DNA purification and cloning 35

3.4 RFLP fragment size estimation using restriction mapper 43

3.5 Statistical analysis 44

3.5.1 Allele frequency 44

3.5.2 Effective number of alleles (Ne) 45

3.5.3 Nei’s gene diversity (h) 45

3.5.4 Shannon Information Index (I) 46

4 RESULTS 47

4.1 Percent polymorphism 47

4.2 Displacement loop (D-loop) PCR amplification 48

4.2.1 Digestion with ApaI 49

4.2.2 Digestion with BamHI 50

4.2.3 Digestion with BstXI 51

4.2.4 Digestion with DdeI 52

4.2.5 Digestion with EcoNI 55

4.2.6 Digestion with HinfI 56

4.2.7 Digestion with MboI 56

4.2.8 Digestion with MspI 58

4.2.9 Digestion with TaqI 58

4.3 Cytochrome b (cyt b) PCR amplification 59

4.3.1 Digestion with AluI 60

4.3.2 Digestion with ApaI 60

4.3.3 Digestion with MboI 61

4.3.4 Digestion with MspI 62

4.3.5 Digestion with MspR9I 63

4.4 NADH dehydrogenase subunit 5 (ND5) PCR amplification 65

4.4.1 Digestion with HindIII 65

4.4.2 Digestion with TasI 67

4.5 Genetic variation and relation between breeds 69

4.6 Sequence alignment of Kedah Kelantan, Bos indicus and

Bos taurus

71

4.7 D-loop sequence variation within and between Kedah

Kelantan, Bos indicus and Bos taurus

74

4.8 Cytochrome b sequence variation within and between

Kedah Kelantan, Bos indicus and Bos taurus

74

4.9 ND5/HindIII sequence variation within and between

Kedah Kelantan, Bos indicus and Bos taurus

75

4.10 ND5/TasI sequence variation within and between

Kedah Kelantan, Bos indicus and Bos taurus

76