universiti putra malaysia molecular genetic ...psasir.upm.edu.my/id/eprint/4725/1/fp_2008_21.pdf ·...

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


DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

ii


SPECIES 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

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


SPECIES IN MALAYSIA

By


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

v

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

vi

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.

vii

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.

viii

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


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

ix

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.

xi

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.

xii

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.

xiv

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

xv

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.


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


4.5 Electrophoretic banding pattern for locus BMS789 in rusa and sika 68

4.6 Electrophoretic banding pattern for locus NVHRT22 in rusa and sika 69




4.10 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

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