Summary The present study was conducted on three different colors of Maraz breeds and four different colors of local goat breeds during February, 2015 to January, 2016. A number of 216 does were sampled from the seven groups. Thirty blood samples were taken from each group, except for Maraz brown that were the number was 36 samples. The Whole blood (3 ml) was collected from the jugular vein of each doe into (5 ml) vacutainer tubes containing the anticoagulant, Ethylene Diamine Tetra-Acetic acid (EDTA) and these samples were stored at -20°C until DNA extraction. The
laboratories
works
were
done
at
both
laboratories
of
biotechnology/Animal production- Faculty of Agricultural Sciences- University of Sulaimani and Molecular Genetics in Central Researches/Salahaddin University. Genomic DNA was extracted from the samples using BIONER kit (AccuPrep® Genomic DNA Extraction Unit). After the process of the DNA extraction, quantity and quality of the DNA were checked by Nanodrop spectrophotometer and 1% Agarose gel electrophoresis, respectively. Twenty RAPD primers were applied twelve of them were selected for more analysis depending on band pattern quality, reproducibility, and the presence of bands. Genetic parameters of total fragment numbers, size range of fragments, polymorphic fragment numbers, percentage polymorphic fragment, unique band, Nei's gene diversity, Shannon's information index, gene frequency, genetic distance and phylogenetic tree construction which were calculated using GENEPOP software version 3.3. From the twenty RAPD primers used 12 of them were amplified, for presence of bands. The total fragment number of 12 primers over all the goat breed samples was 485 fragments. The highest number of fragments (121) found in Maraz Brown and the lowest fragment number (39) in local Pnok. The size range of fragments in all goat breeds ranged from 150 to 2000 bp. Out of the I
485 fragments, 90 of them were Polymorphic fragments numbers (PFN). The highest PFN was noticed at 10 in the loci RAP 6 and RAP 8. The lowest PFN was 6 at locus A11. The percentage of polymorphic loci (PPL) for 12 primers in present study was 21.60%. The highest PPL was 31.25% in locus RAP 6 and the lowest was 14.03% at locus D15. From all bands obtained, 20 of them possessed unique bands. The highest unique band was found in locus RAP 6 which have 4 unique bands, three of them in the Maraz Brown and one in the local Koor. Out of the 20 unique bands, half of them (10) found in Maraz goats, seven of the 10 unique bands were found in Maraz Brown goat, and five of them were found in local Koor. Average Nei's gene diversity and Shanon's information index in this study were 0.42 and 0.60, respectively. The gene frequency for all loci ranged from 0.00-0.87 for allele 0 and from 0.13-1.00for allele 1 in all goat breeds. The genetic distance ranged from 0.09 to 0.43%. The highest genetic distance (0.43%) recorded between Maraz goat and other goat breeds and between local Koor and other goat (except Maraz goats) breeds (0.38%). However, the lowest genetic distance recorded between local white and Pnok. The distance between local Black and Pnok at one side and local Black and local white at the other was 0.23%. The distance between local Pnok and local White was 0.09. The distance between all Maraz coat colors was 0.20%.
As the dendrograms
showed, there are four clusters, the 1st cluster brunch including Maraz Brown, Maraz Black, and Maraz White, the 2nd contain White and local Pnok, the 3rd cluster includes local Black and the 4th cluster includes only Local Koor goat. In conclusions, the high distance among these goat breeds, polymorphism and high numbers of unique bands found in the present study indicates that these goat breeds have the required amount of genetic variation to make genetic improvement. This study helps to clarify the image of the genetic diversity of the local goat breeds and breeders can use it for mating system when needed to make the crossing among these goat breeds. II
LIST OF CONTENTS Subject No.
Subject Title
Page No.
Summary ……………………………………………….
I
List of Contents ……………………………….……….
III
List of Tables ………………………………….……….
VI
List of Figures ………………………………………….
VII
List of Appendixes ………………………….………….
VIII
List of Abbreviations ………………………………….
IX
CHAPTER ONE: INTRODUCTION
1
Introduction………………………………………
1
CHAPTER TWO: REVIEW OF LITERATURE
3
Review of Literature……………………………….
3
2.1
Characteristics of Maraz goats………………………....
3
2.2
Characteristics of local goat breeds……………………
4
2.2.1
The Black goat………………………………………….
5
2.2.2
The White goat…………………………………............
5
2.2.3
The Koor goat ………………………………………….
5
2.2.4
The Pnok goat ………………………………………….
5
2.3
Molecular Markers……………………………………...
6
2.3.1
Random amplified polymorphic DNA (RAPD)……….
7
2.3.1.1
principles of the RAPD technique .............................
8
2.3.1.2
The advantages and disadvantages of (RAPD)…………
9
III
2.3.1.3
The total fragment number (TFN)……………………....
10
2.3.1.4
The size range of fragments (SRF)……………………...
11
2.3.1.5
The polymorphic fragment numbers (PFN)……………
12
2.3.1.6
The percentage of polymorphic loci (PPL)…………….
15
2.3.1.7
Nei’s genetic diversity………………………………….
16
2.3.1.8
Shannon's information index……………………………
17
2.3.1.9
Genetic distance estimation…………………………….
17
2.3.1.10
Gene frequency…………………………………………
18
2.3.1.11
Phylogenetic tree ……………………………….
19
.
CHAPTER THREE: MATERIALS AND METHODS
20
Materials and Methods……………………………...
20
3.1
Experimental animal breeds and location………………
20
3.2
Blood collection and DNA extraction………………….
24
3.3
DNA Quantification and Qualification……………….....
25
3.3.1
Loading the gel with DNA samples…………………….
26
3.4
RAPD primers…………………………………………..
27
3.5
PCR amplification for RAPD primers…………………..
29
3.5.1
PCR protocols…………………………………………...
30
3.6
Agarose gel check for PCR amplification……………....
32
3.7
Scoring of gel …………………………………………..
32
CHAPTER FOUR: RESULTS AND DISCUSSION
34
Results and Discussion…………………………
34
4.1
Quality checks and quantified of DNA samples……….
34
4.2
Primer amplification……………………………………
34
4.3
The total Fragment Number (TFN)…………………….
41
4.4
The size range of fragments (bp)………………………..
41
IV
4.5
Polymorphic Fragment Numbers (PFN)………………..
42
4.6
Percentage Polymorphic Loci (%) (PPL)……………..
42
4.7
Unique Bands ………………………………………….
45
4.8
Nei’s Gene Diversity (h) ……………………………….
48
4.9
Shannon's information index (I)……………………….
48
4.10
Gene frequency…………………………………………
49
4.11
Genetic Distance………………………………………...
50
4.12
Phylogenetic tree construction…………………………..
52
Conclusions and Recommendations………………….....
54
Conclusions……………………………………………...
54
Recommendations ……………………………………....
55
References……………………………………………….
56
Appendixes………………………………………………
63
Summary in Arabic…………………………………..
i,ii
Summary in Kurdish………………………………...
A,B
V
LIST OF TABLES Table No.
Table Title
Page No.
1
Features of RAPD nuclear DNA marker………….
9
2
The TFN in different goat and sheep breeds……...
11
3
The SRF in different goat and sheep breeds………
13
4
The PFN in different goat and sheep breeds ……... 14
5
The PPL in different goat and sheep breeds………
6
Number of animal/ goat breeds from different
16
locations…………………………………………..
20
7
Primers name, sequence and GC % for all primers.
28
8
Band numbers and fragments size ranges (bp) in goat breeds………………………………………… 44
9
Overall polymorphic fragment numbers and percentage polymorphic fragments for all primers used……………………………………………….
10
45
Unique band numbers and fragments size in goat breeds……………………………………………… 47
11
Estimation of Nei's gene diversity and Shannon's information index for all goat breeds……………... 49
12
Gene frequency for all loci in the goat breeds.......... 50
13
Nei's genetic identity (above diagonal) and genetic distance (below diagonal) ………………………… 51
VI
LIST OF FIGURES Figure
Figure Title
No.
Page No.
1
Local black goat ……………………………………
21
2
Local white goat……………………………………
21
3
Local Koor goat…………………………………….
22
4
Local Pnok goat………………………………….…
22
5
The black Maraz goat………………………………
23
6
The brown Maraz goat ……………………………...
23
7
The white Maraz goat………………………………
24
8
Genomic DNA image for 24 samples, performed on (1%) agarose gel electrophoresis……........................
9
Agarose Gel electrophoresis (1.7%) for primer (A11) in all goat samples…………………………………...
10
35
Agarose Gel electrophoresis (1.7%) for primer (C1) in all goat samples…………………………………...
12
35
Agarose Gel electrophoresis (1.7%) for primer (A18) in all goat samples…………………………………...
11
34
36
Agarose Gel electrophoresis (1.7%) for primer (C20) in all goat samples…………………………………… 36
13
Agarose Gel electrophoresis (1.7%)for primer (B5) in all goat samples……………………………….......
14
Agarose Gel electrophoresis (1.7%) for primer (B8) in all goat samples…………………………………..
15
37 37
Agarose Gel electrophoresis (1.7%) for primer (D15) in all goat samples…………………………………... VII
38
16
Agarose Gel electrophoresis (1.7%) for primer (RAP3) in all goat samples………………………….. 38
17
Agarose Gel electrophoresis (1.7%) for primer (RAP6) in all goat samples………………………….. 39
18
Agarose Gel electrophoresis (1.7%) for primer (RAP8) in all goat samples………………………….. 39
19
Agarose Gel electrophoresis (1.7%) for RAPD (4) primers in all goat samples………………………….. 40
20
Agarose Gel electrophoresis (1.7%) for RAPD (21) primers in all goat samples………………………….. 40
21
UPGMA
dendogram
showing
differentiation
between the goat breeds based on Nei's (1972) genetic distance……………………………………...
53
LIST OF APPENDEX Appendixes No.
A
Title
Page No.
Images for all goat breeds with (different colors) from different locations……………
B
63
Concentration and purity of DNA samples, measured by Nanodrop spectrophotometer...
VIII
64
LIST OF ABBREVIATIONS Abbreviation
Detail
bp
Base pair
oC
Celis degree.
DNA
Deoxyribonucleic acid
dNTP
Deoxy nucleotide triphosphate
EDTA
Ethylene diamine tetra acetic acid
FAO
Food and Agriculture Organization of the United Nations
G+C
Guanine and Cytosine
I
Shanons information index
LB
Local Black
LE
Elution buffer
LK
Local Koor
LP
Local Pnok
LW
Local White
MB
Maraz Black
MR
Maraz Brown
MW
Maraz White
M
Mol
MgCL2
Magnesium chloride
μl
Microlitre
ml
Milliliter
ng
Nanogram
No
Number
PCR
Polymerase Chain Reaction
PFN
Polymorphic fragment number
PH
Potential of Hydrogen IX
PPL
Percentage Polymorphic Loci
rpm
Rotation per minute
RAPD
Random Amplified polymorphic DNA
SRF
Size Range Fragment
Taq
Thermus aquaticus
TBE
Tris-Boric Acid-EDTA Buffer
TFN
Total fragment number
UPGMA
Unweighted Pair Group Method with Arithmetic mean
UV
Ultra Violet
V
Volts
W1
Washer buffer one
W2
Washer buffer two
X
