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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
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