universiti putra malaysia haytham hago abdelwahid bashir fp 2012
TRANSCRIPT
UNIVERSITI PUTRA MALAYSIA
HAYTHAM HAGO ABDELWAHID BASHIR
FP 2012 50
GENETIC CHARACTERIZATION OF KEDAH KELANTAN CATTLE AND ITS CROSSBRED TYPES IN MALAYSIA
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GENETIC CHARACTERIZATION OF KEDAH KELANTAN CATTLE
AND ITS CROSSBRED TYPES IN MALAYSIA
By
HAYTHAM HAGO ABDELWAHID BASHIR
Thesis Submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfilment of the Requirements for the Degree of
Doctor of Philosophy
December 2012
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DEDICATIONS
TO MY FAMILY
WITH LOVE AND RESPECT
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in
fulfilment of the requirement for the degree of Doctor of Philosophy
GENETIC CHARACTERIZATION OF KEDAH KELANTAN CATTLE
AND ITS CROSSBRED TYPES IN MALAYSIA
By
HAYTHAM HAGO ABDELWAHID BASHIR
December 2012
Chairman : Professor Jothi Malar Panandam, PhD
Faculty : Agriculture
The Kedah Kelantan (KK) is the indigenous cattle breed of Malaysia and is
mainly kept by small farmers for meat production because of its small and
compact body, and low maintenance requirement. This breed faces risk of
germplasm dilution due to extensive crossbreeding and breeds replacement
practices in the country. The population size of purebred KK is fast
decreasing and most of the commercial populations are actually crossbreds.
There is a lack of information on the genetic characteristics of KK. The
genetic relationships between the KK, the synthetic breeds developed using
the KK as the maternal line, as well as the nondescriptive KK crossbred types
are also unknown. It is with these in mind that the present study was
conducted. The objective of the study was to evaluate the genetic variability
within and among the indigenous KK cattle and the KK crossbred types in
Malaysia.
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DNA was extracted from the blood of 312 randomly selected animals of six
cattle breed types. These included the Kedah Kelantan (KK), the Brakmas
and Charoke which are synthetic breeds developed through crossbreeding of
the KK, two non descriptive KK crosses (KKX1 & KKX2), and the Brahman
(used as an outgroup). The breed types were assessed for 30 microsatellite
loci recommended by FAO/ISAG for cattle genetic diversity studies. PCR
was accomplished by using a touchdown programme. The PCR products
were separated using capillary electrophoresis. Prior to screening the breed
types, three genotyping methods were evaluated by comparison to the
sequence information for two microsatellite loci, ETH152 and INRA005.
Twelve DNA samples from KK were used for this purpose. The result
indicated that the capillary electrophoresis system using labelled primers
provided higher genotyping accuracy in microsatellite analysis compared to
capillary electrophoresis system requiring no primer labelling primers or
electrophoresis using MetaPhor agarose gel. Therefore, this method was
chosen for fragment analysis in the present study.
The results showed that all 30 microsatellites loci were polymorphic for all
the breed types, with 8 to 18 alleles per locus. Heterozygosity values
observed for all breed types were moderate and ranged from 0.54 (KK) to
0.65 (Charoke), and were lower than the expected heterozygosity values.
Seven loci (BM1818, ETH3, HEL5, HEL9, ILSTS005, INRA063 and TGLA53)
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showed significant (P < 0.05) deviation from Hardy-Weinberg Equilibrium
(HWE) for all the breed types. No breed type was observed to deviate from
HWE for all 30 loci. The mean of inbreeding coefficient (FIS) ranged from
0.149 (Charoke) to 0.232 (KKX2); this indicated inbreeding was present in all
breed types and could lead to loss of genetic diversity if not addressed. No
recent bottleneck effect was detected in any of the breed types.
The genetic distance between KK and the two KK crosses (KKX1 and KKX2)
was lower than those between KK and the two synthetic breeds. Brakmas
and Charoke tended to be the most distantly related. In general, the genetic
differentiation measures were moderate, with a mean FST of 0.054. The
analysis of molecular variance (AMOVA) indicated a greater proportion of
the genetic diversity was within the breed types (96.6%) than between them.
The structure analysis, phylogenetic trees, and principal component analysis
showed that the breed types could be grouped into three clusters: KK and
KKX2 in the first cluster, Brakmas and Brahman in second, and Charoke and
KKX1 in third cluster. Structure analysis also showed that a few of the
Brakmas and Cheroke animals were more KK in their genetic background;
and so were many animals from the two non descriptive KK cross breed
types. Analysis of zebu and taurine diagnostic alleles showed that all breed
types had low proportion of African taurine and European taurine diagnostic
alleles.
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It may be concluded that there is still some genetic variation present in the
KK and KK crossbred types. However, this genetic diversity is at risk of
being lost if no appropriate breeding practices are implemented. The
Charoke and Brakmas may be considered as genetically distinct breeds.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan untuk ijazah Doktor Falsafah
PENCIRIAN GENETIK LEMBU KEDAH KELANTAN DAN JENIS
KACUKAN NYA DI MALAYSIA
Oleh
HAYTHAM HAGO ABDELWAHID BASHIR
Disember 2012
Pengerusi : Profesor Jothi Malar Panandam, PhD
Fakulti : Pertanian
Kedah Kelantan (KK) adalah baka lembu asli Malaysia dan kerap kali
diperlihara oleh peternak kecil untuk pengeluaran daging kerana badannya
yang kecil dan padat serta keperluan penyelenggaraan yang rendah. Baka ini
menghadapi risiko pencairan germplasma disebabkan pengkacukan meluas
dan amalan penggantian baka di negara ini. Saiz populasi KK adalah
berkurangan dengan kadar cepat, dan kebanyakan populasi komersial
sebenarnya adalah kacukan. Terdapat kekurangan maklumat tentang ciri
genetik KK. Persaudaraan genetik antara KK, baka sintetik yang
dikembangkan dengan menggunakan KK sebagai titis maternal, serta jenis
kacukan KK yang non descriptive adalah juga tidak diketahui. Ia adalah
dengan pertimbangkan perkara ini, yang kajian ini dijalani. Objektif kajian
ini adalah untuk menilai kepelbagaian genetik dalam dan di antara lembu
asli KK dan jenis kacukan KK di Malaysia.
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DNA diekstrak daripada darah 312 ekor lembu yang dipilih secara rawak
dari enam jenis baka lembu. Ini terdiri daripada baka Kedah Kelantan (KK),
Brakmas dan Charoke yang adalah baka sintetik yang terhasil daripada
kacukan KK, dua kacukan KK yang nondescriptive (KKX1 & KKX2), dan
Brahman (digunakan sebagai kumpulan luar). Semua jenis baka dinilai
untuk 30 lokus mikrosatelit yang syorkan oleh FAO/ISAG untuk kajian
kepelbagaian genetic lembu. PCR telah dicapai dengan menggunakan satu
program touchdown. Produk PCR telah dipisahkan menggunakan
elektroforesis kapilari. Sebelum pengskrinan jenis baka, tiga kaedah
penggenotipan telah dinilai dengan membandingkan kepada maklumat
sekuens dua lokus mikrosatelit, ETH152 dan INRA005. Duabelas sampel
DNA daripada KK telah digunakan untuk tujuan ini. Keputusan telah
menunjukkan sistem elektroforesis kapilari dengan primer berlabel memberi
lebih ketepatan penggenotipan dalam analisis mikrosatelit berbanding
dengan sistem elektroforesis kapilari yang tidak memerlukan pelabelan
primer dan elektroforesis menggunakan gel MetaPhor agarose. Oleh itu,
kaedah ini telah dipilih untuk analisis fragmen dalam kajian ini.
Keputusan telah menunjukkan bahawa semua 30 lokus mikrosatelit adalah
polimorfik untuk semua jenis baka, dengan 8 - 18 alel per lokus. Nilai
heterozigositi yang telah diperhatikan untuk semua jenis baka adalah
sederhana dan berada dalam lingkungan 0.54 (KK) hingga 0.65 (Charoke),
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dan adalah lebih rendah daripada nilai heterozigositi jangkaan. Tujuh lokus
(BM1818, ETH3, HEL5, HEL9, ILSTS005, INRA063 and TGLA53)
menunjukkan sisihan ketara (P<0.05) daripada keseimbangan Hardy-
Weinberg (HWE) untuk semua jenis baka. Tiada jenis baka yang didapati
menunjukkan sisihan daripada HWE untuk kesemua 30 lokus. Min koefisien
pembiakbakaan (FIS) berjulat antara 0.149 (Charoke) hingga 0.232 (KKX2); ini
menunjukkan pembiakbakaan dalam terdapat dalam semua jenis baka dan
boleh membawa kepada kehilangan kepelbagaian genetic jika tidak
ditangani.
Jarak genetik antara KK dan kedua-dua kacukan KK (KKX1 dan KKX2)
adalah lebih rendah daripada yang di antara KK dan kedua-dua baka
sintetik. Brakmas dan Charoke didapati bersaudara berpaling jauh. Secara
umumnya, pengukuran pembezaan genetik adalah sederhana, dengan min
FST sebagai 0.054. Analisis varians molekul (AMOVA) menunjukkan
kebanyakan kepelbagaian genetik adalah dalam jenis baka (96.6%)
berbanding dengan di antara mereka. Analisis struktur, pokok filogenetik,
dan analisis komponen utama menunjukkan bahawa jenis baka tersebut
boleh dikumpulkan dalam tiga kluster; KK dan KKX2 dalam kluster
pertama, Brakmas dan Brahman dalam yang kedua, dan Charoke dan KKX1
dalam kluster ketiga. Analisis struktur juga menunjukkan bahawa
sebilangan kecik ternakan Brakmas dan Cheroke adalah lebih KK dalam
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latarbelakang genetik mereka; dan begitu juga banyak ternakan daripada
dua jenis baka kacukan KK nondescriptive. Analisis alel diagnostik zebu dan
taurine menunjukkan kesemua jenis baka mempunyai proporsi rendah alel
diagnostik African taurine dan European taurine.
Kesimpulan boleh dibuat bahawa masih terdapat sedikit variasi genetik
pada KK dan jenis kacukan KK. Walau bagaimanapun, kepelbagaian genetik
tersebut adalah dalam risiko mungkin dihilang sekiranya tiada amalan
pembiakan yang sesuai dilaksanakan. Brakmas dan Charoke boleh dianggap
sebagai baka yang berbeza secara genetik.
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ACKNOWLEDGEMENTS
The Prophet Muhammad, peace be upon him, said: He has not thanked Allah
who has not thanked people. My sincere thanks to the Almighty Allah for
giving me the health and patience to complete this work.
I am grateful to my father and mother for their sacrifices and supporting me
all my life by their supplication and money. I’m grateful to my wife Amira
Abdallah, for her patience, encouragement and support, and for caring for
me and my daughters. I would also like to extend my sincere thanks to my
brother Khalid and my sister Sarah for their encouragement and support.
I’m indebted to my wonderful supervisor, Prof. Dr. Jothi Malar Panandam. It
is difficult to find suitable words to show my deepest thanks and gratitude,
because she gave me a chance to work with her, taught me many things that I
know today about Animal Genetics, supporting me with her knowledge,
guidance, time and money.
I would like to express my sincere thanks to Dr. Rueben Sharma for allowing
the use of his laboratory, for his suggestions and guidance during DNA
cloning and throughout the study. I also extend my appreciation to his
laboratory staff and his student Col Lin Lee for teaching me DNA cloning.
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I am also thankful to the members of my supervisory committee, Assoc. Prof.
Dr. Mohamad Hilmi Hj. Abdullah, Assoc. Prof. Dr. Halimatun Yaakub and
Assoc. Prof. Dr. Johari Jiken Abdullah, for their suggestions and support
during my study.
My sincere thanks and appreciation to my friends and my lab mates,
Sulieman Elhori, Abdelrahim Abubakr, Kamal Abu Elfatah, Zeiad Amjad,
Mohamed Idris Ali, Arash Javanmard, Alireza Majidi, Saeid Nikbin,
Hamidah Ali Kamarulzaman, Reza Tohidi, Yow Weng Kit and Kamariah
Jamhari, for their friendly behaviour, help during blood collection and
encouragement.
I would like to extend my sincere thanks to the Sudanese community in
UPM, especially Siddig Ibrahim, Elshafie Ibrahim and Majdi Abdellatif for
their help and support during my illness and throughout the study. I am also
thankful to Dr. Salah Awadallah and Dr. Kamal Hassan Ali for their support
and encouragement.
I would like to acknowledge the contributions made by the following
organizations: The Ministry of Science, Technology and Innovation Malaysia
(MOSTI) for funding this project; Malaysian Agriculture Research and
Development Institute (MARDI) and their field staff at MARDI stations in
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Muadzam Shah, Pahang, and Kluang, Johor; Department of Veterinary
Services Malaysia (DVS) and their field staff at the livestock centres (Pusat
Ternakan Haiwan) in Tanah Merah, Kelantan, and Kuala Berang,
Terengganu; and Kulim Livestock Sdn Bhd. I would like to place on record
the cooperation and support of these organisations for giving permission to
use their herds for the study and their help in blood sample collection.
I would like to express my thanks and appreciation to the academic and
supporting staff of Department of Animal Science, Faculty of Agriculture,
Universiti Putra Malaysia for their hospitality and kindness.
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I certify that a thesis Examination Committee has met on 21th December 2012
to conduct the final examination of HAYTHAM HAGO ABDELWAHID
BASHIR on his thesis entitled " Genetic Characterization of Kedah Kelantan
Cattle and its Crossbred Breed Types in Malaysia" in accordance with
Universities and university Colleges 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 degree of Doctor of Philosophy
(PhD).
Members of the Examination Committee were as follows:
Ismail Idris, PhD
Associate Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Chairman)
Tan Soon Guan, PhD
Professor Dr.
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Faridah Qamaruz Zaman, PhD
Associate Professor
Faculty of Science
Universiti Putra Malaysia
(Internal Examiner)
Ronny Rachman Noor, PhD
Professor Dr.
Research and Community Services Institute
Bogor Agricultural University, Indonesia
(External Examiner)
SEOW HENG FONG, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has
been accepted as fulfilment of the requirements for the degree of Doctor of
Philosophy. The members of the Supervisory Committee were as follows:
Jothi Malar Panandam, PhD
Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Chairman)
Halimatun Yaakub, PhD
Associate Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Member)
Reuben Sunil Kumar Sharma, PhD Senior Lecturer
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
Johari Jiken Abdullah, PhD
Senior Research Officer and Deputy Director
Strategic Livestock Research Centre
MARDI
Malaysia
(Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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 institutions.
HAYTHAM HAGO ABDELWAHID BASHIR
Date: 21 December 2012
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TABLE OF CONTENTS
Page
ABSTRACT iii
ABSTRAK vii
ACKNOWLEDGEMENTS xi
APPROVAL xiv
DECLARATION xvi
TABLE OF CONTENT xvii
LIST OF TABLES xx
LIST OF FIGURES xxiii
LIST OF ABBREVIATIONS xxv
CHAPTER
1 INTRODUCTION 1
1.1 Problem Statement 3
1.2 Objectives of the Study 3
2 LITERATURE REVIEW 5
2.1 Indigenous Breeds of Livestock 5
2.2 Cattle 8
2.3 Beef Industry in Malaysia 12
2.4 Beef Cattle Breeds in Malaysia 12
2.4.1 Indigenous beef cattle 12
2.4.2 Imported exotic beef cattle 16
2.4.3 Crossbred beef cattle 17
2.5 Genetic Diversity 21
2.6 Genetic Characterization 22
2.7 Microsatellites 26
2.7.1 Distribution and sequences 28
2.7.2 Advantages, disadvantages and applications 29
2.7.3 Mutation Mechanisms 30
2.7.4 Genotyping of Microsatellite Loci 32
2.8 Animal Genetics Conservation 34
2.9 Genetic Characterisation of Cattle Breeds in Malaysia 37
2.10 Studies of Cattle Diversity Using Microsatellite Markers 41
3 MATERIALS AND METHODS 49
3.1 Experimental materials 49
3.2 Sample Collection 52
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3.3 DNA Extraction 53
3.4 DNA Quality and Quantification 53
3.5 Microsatellite Primers 53
3.6 Polymerase Chain Reaction (PCR) Optimization 53
3.7 DNA Pooling 54
3.8 PCR Amplification of Microsatellites 54
3.9 Comparison between Microsatellites Genotyping
Techniques
55
3.9.1 Microsatellite genotyping using MetaPhor agarose
gel electrophoresis
56
3.9.2 Microsatellite genotyping using CEQ8000 57
3.9.3 Microsatellite genotyping using QIAxcel System 58
3.9.4 Microsatellite cloning and sequencing 60
3.10 Microsatellite Analysis of Individual Samples 63
3.10.1 Poolplexing 63
3.11 Statistical Analysis 66
3.11.1 Genetic diversity within KK cattle and its crossbred
breed types
66
3.11.2 Genetic variation and relationship between KK
cattle and its crossbred populations
69
3.11.3 Comparison between KK and other cattle breeds 73
4 RESULTS 75
4.1 DNA Extraction and PCR Optimization 75
4.2 DNA Pooling 76
4.3 Poolplexing Technique 80
4.4 Comparisons between Microsatellite Genotyping
Techniques
82
4.5 Allele Frequency Distributions 94
4.6 Genotype Frequency 97
4.7 Microsatellite Loci Analysis 101
4.8 Genetic Diversity within KK Cattle and KK Breed Types 104
4.8.1 Observed and effective number of alleles 104
4.8.2 Heterozygosity 109
4.8.3 Hardy-Weinberg Equilibrium (HWE) 112
4.8.4 Estimation of inbreeding 114
4.8.5 Bottleneck analysis 116
4.9 Genetic Variation and Relationship Between KK Cattle and
KK Crossbred BreedTypes
119
4.9.1 F-statistics and gene flow 119
4.9.2 Analysis of molecular variance 122
4.9.3 Estimation of genetic admixture in the KK and KK
crossbred types
123
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4.9.4 Genetic distance and phylogenetic analysis 137
4.9.5 Principal component analysis 142
4.10 Comparison between KK and other Cattle Breeds 143
5 DISCUSSION 146
5.1 Comparisons of Microsatellites Genotyping Techniques 146
5.2 DNA Pooling and Poolplexing 153
5.3 Genetic Diversity at the Microsatellite Loci 156
5.4 Genetic Diversity within KK Cattle and KK Crossbred Breed
Types
157
5.5 Genetic Variation and Relationship Between KK and Its
Crossbred Types
169
5.6 Genetic Relationship between KK and other Cattle Breeds 179
6 CONCLUSION 181
REFERENCES 184
APPENDICES 201
BIODATA OF STUDENT 225
LIST OF PUBLICATIONS 226