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146 Pages·2015·10.14 MB·Indonesian

MICROSATELLITE MARKER BASED ASSESSMENT OF
GENETIC STRUCTURE OF MACHERLA BROWN SHEEP

By

MOHAN KISHORE MOLABANTI
B.V.Sc. & A.H.
TVM/13-11

Thesis submitted to the
SRI VENKATESWARA VETERINARY UNIVERSITY
In partial fulfilment of the requirements
For the award of the degree of

MASTER OF VETERINARY SCIENCE
In the faculty of veterinary science

DEPARTMENT OF ANIMAL GENETICS AND BREEDING
COLLEGE OF VETERINARY SCIENCE
SRI VENKATESWARA VETERINARY UNIVERSITY
TIRUPATI – 517502. (A.P.) INDIA

December, 2015

i
Certificate

Dr. Mohan Kishore Molabanti, TVM/13-11 has satisfactorily prosecuted the
course of research and that the thesis entitled “MICROSATELLITE MARKER
BASED ASSESSMENT OF GENETIC STRUCTURE OF MACHERLA BROWN
SHEEP” submitted is the result of original research work and is of sufficiently high
standard to warrant its presentation to the examination. I also certify that the thesis or
part thereof has not been previously submitted by him for a degree of any university.

Place: (Dr.B.Punya kumari)
Date: Chairman of the advisory committee

ii
Certificate
This is to certify that the thesis entitled Microsatellite marker based
assessment of genetic structure of MACHERLA BROWN Sheep submitted in
partial fulfilment of the requirements for the degree of “Master of veterinary science”
of Sri Venkateswara Veterinary University, Tirupati is a record of the bonafide research
work carried out by Dr. Mohan Kishore M, TVM/13-11under my guidance and
supervision. The subject of the thesis has been approved by the Student’s advisory
Committee.
No part of the thesis has been submitted for any other degree or diploma. The
published part has been fully acknowledged. All assistance and help received during the
course of Investigations have been duly acknowledged by the author of the thesis.

(Dr. B.Punya Kumari)
Chairman of the advisory committee

Thesis approved by the Student’s advisory committee

Chairman : (Dr. B.Punya Kumari) _______________
Assistant Professor and Head
Department of Animal Genetics and Breeding
College of Veterinary science
Tirupati - 517502

Member : (Dr. P.Jaya laxmi) _______________
Professor
Department of Veterinary Biotechnology
College of Veterinary science
Rajendranagar- 500030

Member : (Dr. J.Suresh ) _______________
Professor and Head
Department of Livestock Production Management
College of Veterinary science
Tirupati – 517502

iii
LIST OF CONTENTS

Chapter
Title Page No
No.
I. INTRODUCTION 1 - 3
II. REVIEW OF LITERATURE 4 – 44
2.1 Isolation of DNA
4
2.2 Molecular Techniques
5
2.2.1 Gel Electrophoresis
5
2.2.2 Polymerase Chain Reaction
7
2.3 Breed Characterization studies using Microsatellites
9
2.3.1 Microsatellite Allele diversity
9
2.3.1.1 Indigenous breeds

2.3.1.2 Exotic breeds

2.3.2 Heterozygosity
20
2.3.2.1 Indigenous breeds

2.3.2.2 Exotic breeds

2.3.3 Polymorphism Information Content
29
2.3.31 Indigenous breeds

2.3.3.2 Exotic breeds

2.3.4 Within Population Inbreeding Estimates
34
2.3.4.1 Indigenous breeds

2.3.4.2 Exotic breeds

2.3.5 Hardy-Weinberg Equilibrium
39
2.3.5.1 Indigenous breeds

2.3.5.2 Exotic breeds

III. MATERIALS AND METHODS 45 – 59
3.1 Experimental material
45
3.2 Sample collection
45
3.3 DNA Extraction 47
3.3.1 Isolation of DNA by Phenol-chloroform method 47
3.4 Quality check and quantification of DNA
50
3.5 Agarose gel electrophoresis
50
3.6 Spectrophotometric measurement 51
iv
3.7 Preparation of template DNA for PCR 51
3.8 Selection of Microsatellite markers 51
3.9 PCR amplification of microsatellite loci
52
3.10 Components of PCR reaction mixture
52
3.10.1 Primers
52
3.10.2 PCR assay Buffer
52
3.10.3 dNTPs
52
3.10.4 Taq polymerase
52
3.11 PCR Protocol
56
Checking PCR products and genotyping using
3.12 56
agarose gel electrophoresis
3.13 Statistical analysis for microsatellite data
58
3.13.1 Estimation of heterozygosity
58
3.13.2 Estimation of Polymorphism information content 59
3.13.3 Testing for Hardy-Weinberg equilibrium 59
IV. RESULTS 60 – 95
4.1 Isolation of DNA
60
4.2 Polymerase Chain Reaction
60
4.3 Genotyping of animals
60
4.3.1 Microsatellite allele diversity
65
4.3.2 Number of effective alleles
88
4.3.3 Genetic diversity
88
4.3.4 Heterozygosity
88
4.3.5 Within Population Inbreeding Estimates(F )
IS 88
4.3.6 Percentage of polymorphic loci
95
4.3.7 Polymorphism information content (PIC)
95
4.3.8 Hardy-Weinberg equilibrium
95
V. DISCUSSION 96 – 111
5.1 Quality and Quantity of Genomic DNA
96
5.2 PCR amplification
96
5.3 Genotyping of animals
96
5.3.1 Allele diversity
97
5.3.2 Effective number of alleles
106
5.3.3 Heterozygosity
107
v
5.3.4 Within population inbreeding estimate
108
5.3.5 Polymorphism Information Content
109
5.3.6 Hardy–Weinberg Equilibrium
110
VI. SUMMARY 112 – 115
BIBLIOGRAPHY 116 – 127
ANNEXURE – I 128 – 129
ANNEXURE – II 130

vi
LIST OF TABLES

Table No. Title Page No.
1 The primer sequences, chromosomal localization, gene 53 - 55
accession number and annealing temperature of the used
microsatellites
2 Components of PCR mix 57
3 Cyclical conditions used for amplification of the 57
Microsatellite markers by PCR
4 Genomic DNA concentration of Macherla Brown sheep 61
5 Standardisation of PCR protocol for twenty four 63 – 64
microsatellite markers
6 Genotypes (bp) of Macherla Brown sheep at various 66 – 71
microsatellite loci
7 Allele size, allele number and allele frequencies of the 74 – 77
microsatellite loci
8 Observed heterozygosity, expected heterozygosity and 90
within population inbreeding estimate in Macherla Brown
Sheep
9 Polymorphism information content and test for Hardy- 93
Weinberg Equilibrium of the microsatellite loci

vii
LIST OF PLATES AND FIGURES

S.No. Title Page No.
Plate 1 Macherla Brown Ram 46
Plate 2 Macherla Brown Ewe 46
Plate 3 Macherla Brown grazing flock 48
Plate 4 Macherla Brown flock in pen 48
Plate 5 Map showing the distribution of Macherla Brown sheep 49
population in Andhra pradesh and Telangana
Plate 6 Agarose gel image showing Genomic DNA 62
Plate 7a Agarose gel image showing Allele sizes of twelve 72
microsatellites
Plate 7b Agarose gel image showing Allele sizes of twelve 73
microsatellites
Figure 1 Allele frequencies at twenty four microsatellite loci in Macherla 78,80,82,
Brown Sheep
84,85,87
Figure 2 Observed (N ) and effective (N ) number of alleles for twenty 89
a e
four microsatellite loci in Macherla Brown sheep
Figure 3 Observed and expected heterozygosity for twenty four 91
microsatellite loci in Macherla brown sheep
Figure 4 Inbreeding estimates (F ) for twenty four microsatellite loci in 92
IS
Macherla Brown sheep
Figure 5 PIC values for twenty four microsatellite loci in Macherla 94
Brown sheep

viii
DECLARATION

I Dr. Mohan Kishore Molabanti, TVM/13-11 hereby declare that the thesis
entitled “MICROSATELLITE MARKER BASED ASSESSMENT OF GENETIC
STRUCTURE OF MACHERLA BROWN SHEEP” submitted to Sri Venkateswara
Veterinary University, Tirupati for the degree of Master of Veterinary Science is the
result of original research work done by me. I also declare that the materials contained
in this thesis have not been published earlier.

Date: (Mohan Kishore Molabanti)

ix
LIST OF ABBREVATIONS

Symbol Full form
% Per cent
@ At the rate of
°C Degree Celsius
µg Microgram
µl Microlitre
µM Micromoles
bp Base pair
Cm Centimeter
Df degrees of freedom
DNA Deoxyribo Nucleic acid
dNTPs Deoxy Nitrogen Triphosphate
EDTA Ethylene Diamine Tetra Acetic acid
et al. and others
FAO Food and Agricultural Organization
F Inbreeding coefficient
IS
F Overall fixation index
IT
F Fixation index
ST
G Gram
H Expected Heterozygosity
e
H Observed Heterozygosity
o
HWE Hardy-Weinberg Equilibrium
i.e that is
ISAG International Society for Animal Genetics
Kb Kilo base pair
Kg Kilogram
M Molarity
mg Milligram
MgCl Magnesium chloride
2
min Minute
ml Millilitre
mM Millimoles
N Normality
x