universiti putra malaysia haytham hago abdelwahid bashir fp 2012

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


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


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


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


(Internal Examiner)

Faridah Qamaruz Zaman, PhD

Associate Professor

Faculty of Science


(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


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



(Chairman)

Halimatun Yaakub, PhD

Associate Professor



(Member)

Reuben Sunil Kumar Sharma, PhD Senior Lecturer

Faculty of Veterinary Medicine


(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


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.


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

universiti putra malaysia haytham hago abdelwahid bashir fp 2012

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