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UNIVERSITI PUTRA MALAYSIA
MOLECULAR GENETIC CHARACTERIZATION OF RUSA (CERVUS TIMORENSIS) AND SIKA (CERVUS NIPON) DEER
SPECIES IN MALAYSIA
KOUROSH JOME KHALEDI
FP 2008 21
MOLECULAR GENETIC CHARACTERIZATION OFRUSA (CERVUS TIMORENSIS) AND SIKA (CERVUS NIPON) DEER
SPECIES IN MALAYSIA
KOUROSH JOME KHALEDI
DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA
ii
MOLECULAR GENETIC CHARACTERIZATION OF RUSA (CERVUS TIMORENSIS) AND SIKA (CERVUS NIPON) DEER
SPECIES IN MALAYSIA
By
KOUROSH JOME KHALEDI
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of
Philosophy
April 2008
iii
DEDICATION
TO YOU
MY FATHER AND MOTHER,
MY WIFE AND MY DAUGHTER,
AND ALSO TO
PROFESSOR DR. MOSTAFA CHAMRAN
WHO WAS A REAL SCIENTIST AND RELIGIOUS MAN
AND FINALY WAS MARTYRIZED IN THE WAR
iv
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy
MOLECULAR GENETIC CHARACTERIZATION OF RUSA (CERVUS TIMORENSIS) AND SIKA (CERVUS NIPON) DEER
SPECIES IN MALAYSIA
By
KOUROSH JOME KHALEDI
April 2008
Chairman: Associate Professor Jothi Malar Panandam, PhD
Faculty: Agriculture
The Malaysian livestock industry is an important component of the agricultural
sector providing gainful employment and producing useful animal protein food
to the population. Cattle, buffalo, goat, sheep and swine are the popular
livestock in Malaysia. However, in recent years deer farming for meat, fur and
velvet has become popular as well. Most of the farmed deer are of various
species imported from different sources, such as Australia, New Zealand,
Mauritius, Indonesia, New Caledonia, etc. The genetic background of these
species and populations from different sources are unknown. This study was
conducted to characterize two popular deer species in Malaysia, namely the
rusa deer (Cervus timorensis) and the sika deer (Cervus nippon), using DNA
microsatellite markers.
The use of amelogenin gene primers for sexing of the rusa deer was also
investigated. Random samples of 38 rusa deer from the Deer Breeding Unit of
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the University Research Park, Universiti Putra Malaysia, and 34 sika deer from
Pusat Ternakan Haiwan, Batu Arang, were used in the study to determine and
compare the genetic structures of the two deer species.
One hundred and twenty five sets of microsatellite primer pairs, which had
been reported to have successfully detected variation in deer, cattle or sheep,
were used in the initial screening. Thirty nine primer pairs produced clear and
reproducible amplification products for rusa and 41 primer pairs for sika.
Twenty one primer pairs were polymorphic for the pooled data. However, only
nine microsatellite loci (23.08%) were polymorphic for rusa and 17 loci
(41.46%) were polymorphic for sika. Of these, only five were common to both
deer species (BMS789, BM888, BL4, BM3628 and NVHRT16). Of the
monomorphic loci, 17 were common to both species. Among the 11 reindeer
microsatellite loci screened, nine loci (81%) were amplified for the pooled
data, but only four loci were common to both species. The two white-tailed
deer microsatellite loci (L35582 and L35583) produced amplification in sika
but only L35583 was amplified in rusa. The 17 common monomorphic loci and
the nine polymorphic loci generated in total 53 and 40 microsatellite markers in
the rusa and sika, respectively. Locus BM2113 was amplified exclusively in
rusa (126 bp), and locus NVHRT34 was amplified only in sika (134 bp).
These loci may be used as unique markers to distinguish the two deer species.
The numbers of observed and effective alleles per polymorphic loci were 2 - 13
and 1.05 - 8.91 for rusa, and 2 - 8 and 1.16 - 5.98 alleles per locus for sika,
respectively. The allele frequencies ranged from 0.01 to 0.97 for rusa, and 0.02
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to 0.92 for sika. The sizes of the alleles at the polymorphic loci ranged from
116 to 389 bp in rusa, and 88 to 364 bp in sika. The mean numbers of effective
alleles were 3.08±2.40 and 2.87±1.65 in rusa and sika, respectively. Six loci in
rusa and three loci in sika exhibited rare alleles. The mean observed
heterozygosity in the rusa and sika populations were 0.48 ± 0.35 and 0.51 ±
0.30, respectively. Seven polymorphic loci in rusa and 14 polymorphic loci in
sika exhibited significant (P<0.01) deviations from Hardy-Weinberg
equilibrium. The Hardy-Weinberg disequilibrium may be due to overlapping of
generations and founder effect, especially in the sika deer population. The
combined discrimination power (cDP) of the nine polymorphic loci in rusa was
0.99 and of the seventeen polymorphic loci in sika was 0.99, thus allowing
individual identification. The inbreeding coefficient (FIS) was very low for the
rusa population (0.06), but for the sika population it was 0.26. The mean value
of FST was 0.67 for rusa and 0.47 for sika. The bottleneck analysis suggested
that the rusa population did not experience any recent bottleneck, whereas the
sika population had encountered a genetic bottleneck in the recent past.
Evaluation of intra-interchromosomal linkage disequilibrium between the
alleles suggested significant (P<0.05) linkage between 10 pairs of alleles in
rusa and 12 pairs of alleles in sika. However, none of the allelic pairs were the
same for the two species. The genetic distance within the rusa population was
lower than that within the sika population (0.088 ± 0.001 vs. 0.184 ± 0.001).
The genetic distance between rusa and sika was 0.35. No distinct clustering
was observed for the rusa population. The sika population displayed two major
clusters of 11 and 23 individuals. The larger cluster in turn had two sub-
clusters.
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The above results show the rusa and sika populations to be genetically different
from each other. High genetic variation exists in both the populations. This
could be due to low inbreeding and no directional selection in these
populations.
Four amelogenin gene primer pairs were used to identify the sexes of the rusa
deer. Three primer pairs, AMEL2, AMGX/Y and AMGX/Y2, exhibited similar
banding patterns for the males and females. The primer pair SE47/48 generated
one band for the females (269 bp) but three bands (223, 269 and 305 bp) for the
males. Therefore, this primer pair is a reliable tool for the identification of the
sexes in the rusa deer.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah
PENCIRIAN GENETIK MOLEKUL SPESIS RUSA (CERVUS TIMORENSIS) DAN SIKA (CERVUS NIPON)
DI MALAYSIA
Oleh
KOUROSH JOME KHALEDI
April 2008
Pengerusi : Profesor Madya Jothi Malar Panandam, PhD
Fakulti : Pertanian
Industri peternakan di Malaysia adalah komponen penting dalam sektor
pertanian dan menyediakan peluang pekerjaan dan sumber makanan protein
haiwan kepada populasi. Lembu, kerbau, kambing, biri-biri dan babi adalah
ternakan yang poular di Malaysia. Walau bagaimana pun, pada beberapa tahun
kini peternakan rusa untuk daging, bulu dan velvet juga menjadi popular.
Kebanyakan rusa yang diternak adalah pelbagai spesis dan diimport dari
sumber berlainan, seperti Australia, New Zealand, Mauritius, Indonesia, New
Caleonia dan sebagainya. Latar belakang genetik spesis dan populasi tersebut
adalah tidak diketahui. Kajian ini telah dijalankan untuk mencirikan dua spesis
rusa yang popular di Malaysia, iaitu rusa (Cervus timorensis) dan sika (Cervus
nippon), dengan menggunakan penanda DNA mikrosatelit.
Penggunaan primer gen amelogenin untuk pengelasan jantina rusa turut
disiasat. Sampel rawak 38 ekor rusa dari Unit Pembiakbakaan Rusa, Taman
Penyelidikan Universiti, Universiti Putra Malaysia, dan 34 ekor sika dari Pusat
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Ternakan Haiwan, Batu Arang, telah digunakan dalam kajian ini untuk
menentukan dan membandingkan struktur genetik kedua-dua spesis rusa.
Sejumlah satu ratus dua puluh lima set pasangan mikrosatelit, yang telah
dilaporkan sebagai berjaya mengesan variasi dalam rusa, lembu, kambing atau
biri-biri, telah digunakan dalam pengujian awalan. Tiga puluh sembilan
pasangan primer menghasilkan produk amplifikasi yang jelas dan boleh
dihasilkan semula untuk rusa dan 41 pasangan primer untuk sika. Dua puluh
pasangan primer adalah polimorfik bagi data terkumpul. Walaubagaimanapun,
hanya sembilan (23.08%) lokus mikrosatelit adalah polimorfik untuk rusa dan
17 lokus ( 41.46%) adalah polimorfik untuk sika. Antara ini, hanya lima adalah
sama untuk kedua-dua spesis rusa (BM5789, BM888, BL4, BM3628 dan
NVHKT16). Antara lokus monomorfik, 17 adalah sama bagi kedua-dua spesis.
Antara 11 lokus mikosatelit reindeer yang dikaji, sembilan lokus (81%) adalah
diamplifikasi bagi data terkumpul, tetapi hanya empat lokus adalah sama bagi
kedua-dua spesis. Dua lokus mikosatelit rusa white-tail (L35582 dan L35583)
hasilkan amplifikasi pada sika tetapi hanya L35583 diamplifikasi dalam rusa.
Tujuh belas lokus monomorfik yang sepunya dan sembilan lokus polimorfik
telah menghasilkan sejumlah 53 dan 40 penanda mikrosatelit dalam rusa dan
sika, masing-masing. Lokus BM2113 diamplifikasi khusus dalam rusa (126
bp), dan lokus NVHR734 diamplifikass hanya dalam sika (134bp). Lokus ini
boleh digunakan sebagai penanda unik bagi membezakan kedua-dua spesis
rusa tersebut.
Bilangan alel yang dicerap dan efektif per lokus polimorfik adalah 2 - 13 dan
1.05 - 8.91 untuk rusa, dan 2 - 8 dan 1.16 - 5.98 alel per lokus untuk sika,
x
masing-masing. Frekuensi alel berjulat antara 0.01 ke 0.97 bagi rusa, dan 0.02
ke 0.92 bagi sika. Saiz alel pada lokus polimorfik berjulat antara 16 ke 389 bp
dalam rusa, dan 88 ke 364 bp dalam sika. Purata bilangan alel yang efektif
adalah 3.08± 2.90 dan 2.87±1.65 dalam rusa dan sika, masing-masing. Enam
lokus dalam rusa dan tiga dalam sika menunjukkan alel yang jarang. Purata
heterozigositi yang dicerap dalam populasi rusa dan sika adalah 0.48 ± 0.35
dan 0.51 ± 0.30, masing-masing. Tujuh lokus polimorfik dalam rusa dan 14
lokus polimorfik dalam sika menunjukkan sisihan signifikan (p<0.01) daripada
keseimbangan Hardy-Weinberg. Ketidakseimbangan Hardy-Weinberg
mungkin adalah kerana pertindihan generasi dan kesan penubuh, terutamanya
bagi populasi sika. Kuasa diskriminasi bersatu (cDp) sembilan lokus
polimorfik dalam rusa adalah 0.99 dan bagi 17 lokus polimorfik dalam rusa
adalah 0.99, maka membenarkan pengenalpastian individu. Koefisyen
pembiakbakaan dalam (FIS) adalah sangat rendah bagi populasi rusa (0.06),
tetapi bagi populasi sika ia adalah 0.26. Nilai purata Fst adalah 0.67 bagi rusa
dan 0.47 bagi sika. Analisis ‘bottleneck’ mencadangkan populasi rusa tidak
mengalami masalah ‘bottleneck’ yang baru-baru ini, manakala populasi sika
telah mengalami ‘bottleneck’ genetik pada masa baru-baru ini. Penilaian
ketidakseimbangan ‘intra-interchromosomal linkage’ di antara alel
mencadangkan rangkaian signifikan (p<0.05) di antara 10 pasangan alel dalam
rusa dan 12 pasangan alel dalam sika. Walau bagaimanapun, tiada sebarang
pasangan alel yang sama untuk kedua-dua spesis. Jarak genetik dalam populasi
rusa adalah lebih rendah berbanding dengan yang dalam populasi sika
(0.088±0.001 melawan 0.184±0.001). Jarak genetik di antara rusa dan sika
adalah 0.35. Tiada kluster yang yang diperhatikan untuk populasi rusa.
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Populasi sika menunjukkan dua kluster major dengan 11 dan 23 individu.
Kluster yang lebih besar mempunyai dua sub-kluster
Keputusan di atas menunjukkan populasi rusa dan sika adalah berbeza dari segi
genetik. Variasi genetik yang besar wujud dalam kedua-dua populasi. Ini
mungkin disebabkan oleh pembiakbakaan dalam yang rendah dan tiada
pemilihan berhala dalam populasi tersebut.
Empat pasangan primer gen amelogenin telah digunakan untuk mengenalpasti
jantina rusa. Tiga pasangan primer, AMEL2, AMGX/Y dan AMGX/Y2,
menunjukkan kesamaan corak ’banding’ bagi jantan dan betina. Pasangan
primer SE47/48 menghasilkan satu Jalurn untuk betina (269 bp) tetapi tiga jalur
(223,269 dan 305 bp) untuk jantan. Maka, pasangan primer ini adalah alat yang
sesuai untuk mengenalpasti jantina rusa.
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ACKNOWLEDGEMENTS
Glory and praise to Allah (SWT), the Omnipotent, Omniscient and
Omnipresent, for opening doors of opportunity to me throughout my life and
for giving me the strength and health to achieve what I have so far.
First and foremost, I would like to express my utmost gratitude to my highly
respected supervisor, Assoc. Prof. Dr. Jothi Malar Panandam, chairman of my
supervisory committee, for her advice, invaluable guidance, hospitality,
support and encouragement throughout the period of the study.
I would like to express my deepest thanks and gratitude to Assoc. Prof. Dr.
Maheran Abdul Aziz and Assoc. Prof. Dr. Siti Shapor Siraj for their
suggestions and guidance towards the completion of this study.
I would like to acknowledge Mr. Kamal Basha and the staff of the deer farm at
Pusat Ternakan Haiwan (Batu Arang), the Deer Breeding Unit of University
Research Park, Universiti Putra Malaysia (UPM) for their kind cooperation and
help in the collection of the samples for this study.
I would also like to extend my thanks to the Department head and all staff
members of the Department of Animal Science, Faculty of Agriculture, and the
staff of the School of Graduate Studies of Universiti Putra Malaysia for helping
me in one way or another during the course of my study at UPM.
xiii
My heart felt thanks and appreciation goes to my understanding wife, Maryam
and her family, to whom I am indebted for their sacrifice, patience and
understanding, throughout the course of my study.
My deepest gratitude to my mother and father (God bless them) who advised
and supported me in my pursuit for higher education and academic excellence
and expressed understanding and consideration towards me. Words cannot
express my gratitude for their love, support, and patience that have sustained
me during my life and study. What can I say, except thank you.
My deepest appreciation to my sisters and their families for their kindness.
Special thanks to my friends, Iranians, Malaysians, and those from other
places, in particularly to Zawawi Ismail, Dr Habiba Ali El-jaafari, Reza
Solimani, Arash Javanmard, Alireza Majidi, Mohammad Reza Taheri,
Mohammad Reza Momayezi, Hamideh, Mamat Hamidi Kamalludin, Behnam
Kamali, Dwi Yulistiani, Nguyen Tinh Than, Abdullah Ali Abdullah, Aziz, Cai
Pui Wah, Dr Hossein Sasan, Mohammad Golizadeh, Dr Mahdi Mohamadi,
Habib Naderi, Yousef Rostami, Dr Zamani, Fallah, Moradi, Zakipour, Dr
Safayei, Reza Khakvar, Reza Motaleb, Morteza Karami and others for their
help when needed.
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I certify that an Examination Committee has met on 11th April 2008 to conduct the final examination of Kourosh. J. Khaledi on his Doctor of Philosophy thesis entitled ‘’Molecular Genetic Characterization of Rusa (Cervus timorensis) and Sika (Cervus nipon) Deer Species in Malaysia’’ in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the degree of Doctor of Philosophy.
Members of the Examination Committee are as follows:
Abdul Razak Alimon, PhDProfessorFaculty of AgricultureUniversiti Putra Malaysia(Chairman)
Tan Soon Guan, PhDProfessorFaculty of Biotechnology and Bimolecular ScienceUniversiti Putra Malaysia(Internal Examiner)
Ismail Idris, PhDAssociate ProfessorFaculty of AgricultureUniversiti Putra Malaysia(Internal Examiner)
Christopher Moran, PhDProfessorFaculty of Veterinary ScienceUniversity of SydneyAustralia(External Examiner)
HASANAH MOHD. GHAZALI, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date: 26 May 2008
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This thesis was submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows:
Jothi Malar Panandam, PhDAssociate ProfessorFaculty of AgricultureUniversiti Putra Malaysia(Chairman)
Siti Shapor Siraj, PhDAssociate Professor Faculty of Science Universiti Putra Malaysia(Member)
Maheran Abdul Aziz, PhDAssociate ProfessorFaculty of AgricultureUniversiti Putra Malaysia(Member)
AINI IDERIS, PhD Professor and Dean
School of Graduate Studies Universiti Putra Malaysia
Date: 12 June 2008
xvi
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 other institution.
KOUROSH JOME KHALEDI
Date: 26 June 2008
xvii
TABLE OF CONTENTS
Page
DEDICATION iiiABSTRACT ivABSTRAK viiiACKNOWLEDGEMENTS xiiAPPROVAL xivDECLARATION xviLIST OF TABLES xixLIST OF FIGURES xxLIST OF ABBREVIATIONS xxi
1 INTRODUCTION 11.1 Research Problem 51.2 General Objective 61.3 Specific Objective 61.4 Significance of the Study 7
2 LITERATURE REVIEW 82.1 Deer 82.2 Deer in Malaysia 9
2.2.1 Cervus timorensis (Rusa) 102.2.2 Cervus nippon (Sika) 11
2.3 Genetic variation 132.4 Molecular marker system2.5 Microsatellite
1819
2.5.1 Identification of microsatellite2.5.2 Microsatellite evolution2.5.3 Potential problems associated with
microsatellite marker2.5.4 Application of microsatellite marker
202224
27 2.6 Molecular marker in deer 28
2.7 Genetic and molecular evidence for sex determination in mammals
31
2.8 Amelogenin gene and sex determination 34 2.9 Sex identification in deer species 36
3 MATERIALS AND METHODS 383.1 Study I 38
3.1.1 Experimental material 383.1.2 Sample collection 383.1.3 DNA extraction 393.1.4 DNA quantification 413.1.5 Microsatellite primers 433.1.6 PCR procedures and optimization 473.1.7 Electrophoresis 49
xviii
3.1.8 Interpretation of microsatellite loci 503.1.9 Statistical analysis 50
3.2 Study II 583.2.1 Experimental material 583.2.2 Sample collection 583.2.3 DNA extraction 583.2.4 Amelogenin gene primers 583.2.5 Amelogenin gene amplification 59
4 RESULTS 61 4.1 Microsatellite study in rusa and sika deer 61
4.1.1 Amplified of microsatellite primers4.1.2 Number of alleles and allele frequencies4.1.3 Genotypes and genotypic frequencies4.1.4 Heterozygosity4.1.5 Hardy-Weinberg equilibrium4.1.6 Analysis of molecular variance4.1.7 Genetic bottleneck 4.1.8 Genetic distance within and between rusa and
sika deer population4.1.9 Cluster analysis4.1.10 Linkage disequilibrium analysis
618890
96 99 101 104
106
106109
4.2 Sex determination in rusa deer using amelogenin gene Primers
111
5 DISCUSSION 1145.1 Microsatellite polymorphism in rusa and sika deer5.2 Unique specific microsatellite markers between rusa and
sika deer5.3 Number of alleles and allele frequencies5.4 Heterozygosity in rusa and sika deer5.5 Hardy-Weinberg equilibrium5.6 Analysis of molecular variance5.7 Genetic bottleneck5.8 Linkage disequilibrium5.9 Sex determination in rusa
114121
123126128129130131133
6 CONCLUSION 135REFERENCES 138APPENDICES 158BIODATA OF STUDENT 190
xix
LIST OF TABLES
Table Page
2.1 Recognized model of gene flow between populations. 16
3.1 Microsatellite marker primers which amplify in rusa and sika deer. 44
3.2 Concentrations of PCR reaction components. 47
3.3 Amelogenin gene primer sequences. 59
4.1 Polymorphism at the microsatellite loci amplified for rusa and sika deer. 63
4.2 Allele size of monomorphic loci in the rusa and sika deer. 80
4.3 Observed and effective number of alleles, and range of allele sizes and frequencies for the polymorphic microsatellite loci in rusa and sika deer.
89
4.4 Allele and genotype frequencies of polymorphic loci common to the rusa and sika deer.
91
4.5 Allele and genotype frequencies of polymorphic loci not shared by the rusa and sika deer.
94
4.6 Summary of heterozygosity for the polymorphic loci in rusa and sika. 97
4.7 Deviation from Hardy-Weinberg equilibrium of the polymorphic loci in the rusa and sika deer populations.
100
4.8 Smouse’s multilocus analysis for Hardy-Weinberg Equilibrium 100
4.9 AMOVA design and results of rusa and sika deer. 102
4.10 F-Statistic analysis based on polymorphic loci. 102
4.11 Genetic variation for polymorphic loci in rusa and sika based on Shannon’s information index (Nei, 1987).
103
4.12 Wilcoxon test for bottleneck detection in the rusa and sika population. 105
4.13 Evaluation of the detected intra- and interchromosomal linkage disequilibrium (LD).
110
xx
LIST OF FIGURES
Figure Page
2.1 Photographs of the rusa and sika deer species. 12
4.1 Graphical display of number of common and unique microsatellite loci between rusa and sika deer.
61
4.2 Electrophoretic banding pattern for locus BL4 in rusa and sika. 65
4.3 Electrophoretic banding pattern for locus BM888 in rusa and sika 66
4.4 Electrophoretic banding pattern for locus BM3628 in rusa and sika 67
4.5 Electrophoretic banding pattern for locus BMS789 in rusa and sika 68
4.6 Electrophoretic banding pattern for locus NVHRT22 in rusa and sika 69
4.7 Electrophoretic banding pattern for locus BM1260 in rusa and sika 70
4.8 Electrophoretic banding pattern for locus BM4208 in rusa and sika 71
4.9 Electrophoretic banding pattern for locus BM4513 in rusa and sika 72
4.10 Electrophoretic banding pattern for locus IDVGA-37 in rusa and sika 73
4.11 Electrophoretic banding pattern for locus NVHRT21 in rusa and sika 74
4.12 Electrophoretic banding pattern for uncommon polymorphic loci 75
4.13 Electrophoretic banding pattern for uncommon polymorphic loci in sika. 77
4.14 Monomorphic loci amplified in both rusa and sika deer. 86
4.15 Banding pattern produced by primer pair of BM2113 locus in rusa. 87
4.16 Banding pattern produced by primer pair of NVHRT34 locus in sika. 87
xxi
4.17 L-shaped graph of allele frequencies in rusa and sika deer populations. 105
4.18 Estimation membership coefficient for individuals in each cluster. 106
4.19 Dendogram of the rusa deer population generated by cluster analysis of genetic distance according to Nei (1972).
107
4.20 Dendogram of the sika deer population generated by cluster analysis of genetic distance according to Nei (1972).
108
4.21 Amelogenin bands generated by AMEL2 primer. 112
4.22 Amelogenin bands generated by SE47/48 primer. 112
4.22 Amelogenin bands generated by AMGXY primer. 1113
4.23 Amelogenin bands generated by AMGXY2 primer. 113
xxii
LIST OF ABBREVIATIONS
A Adenine
AE Elution buffer
AFLP Amplified Fragment Length Polymorphism
AL Lysis buffer
AMELX amelogenin gene in chromosome X
AMELY amelogenin gene in chromosome Y
AMG amelogenin gene
AMOVA analyses of molecular variance
AW1 Wash buffer 1
AW2 Wash buffer 2
bp Base pair
C Cytosine
cDP combined discrimination power
CSB clone sequence based
dNTP dinucleotide triphosphate
DP Discrimination Power
DVS Department of Veterinary Services
EDTA ethylene diamine tetra acetic Acid
EPA Extreme Preferential Amplification
EtBr ethidium bromide
F Fixation Index
G Guanine
GATA4 gata binding protein 4
Ho Observed heterozygosity
He Expected heterozygosity
HMG high mobility group
HWE Hardy-Weinberg equilibrium
IAM Infinite Allele Model
LD linkage disequilibrium
MAS marker assisted selection
MgCl2 magnesium chloride
ml Milliliter
mM Millimolar
mRNA messenger ribonucleic acid
xxiii
MSA Microsatellite Analyser
mtDNA mitochondrial DNA
Na observed number of alleles
Ne effective number of alleles
ng nanogram
P Panmictic Index
PAGE Polyacriramid Gele Electrophoresis
PCR Polymerase Chain Reaction
PIC Polymorphism Information Content
QTL Quantitative Trait Loci
RAHM Random Amplified Hybridization Microsatellite
RAMPO Random Amplified Microsatellite Polymorphism
RAPD Random Amplification of Polymorphic DNA,
RFLP Restriction Fragment Length Polymorphism
rpm rotation per minute
SF1 Steroidogenic Factor 1
SMM Stepwise Mutation Model
SNP Single Nucleotide Polymorphism
SOX9 Sry-related HMG box gene 9
SRX Sex Determination Region in chromosome X
SRY Sex Determination Region in chromosome Y
SSR Simple Sequence Repeats
STR Short Tandem Repeats
T Thymine
TBE Tris borate ethylene diamine tetra acetic acid
TDF Testis Determination Factor
Tm Melting temperature
TPM Two-Phase Model
TSPY Testis- Specific Protein Y-encoded
U/µL unit per microlitter
UPGMAUnweighted Pair Group Method with Arithmetic mean
UPM Universiti Putra Malaysia
UV ultraviolet
VNTRs variable number of tandem repeats
WT1 Wilm’s Tumor gene 1
ZFX Zinc Finger protein in chromosome X
xxiv
ZFY Zinc Finger protein in chromosome Y
µl microliter
µM micromolar0C Centigrade Celsius
1X one time