EVOLUTIONARY DYNAMICS AND BIODIVERSITY IN PAKISTANI

LIVESTOCK SPECIES

FIRST EDITION

Prof. Dr. Masroor Ellahi Babar T.I. Dean, Faculty of Science & Technology

Virtual University of Pakistan

2016

GOVERNMENT OF PAKISTAN HIGHER EDUCATION COMMISSION

ISLAMABAD PAKISTAN

ii

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Very High Cycle Fatigue of Engineering Alloys

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ISBN: 978-969-417-188-3

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Islamabad

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iii

DEDICATION

This monograph is dedicated to

My Father Sheikh Mehboob Ellahi (Late)

Whose habit of meditation to the abyss of the matter

is one of the legacies which I adopted

iv

TABLE OF CONTENTS

Preface……………………………………………………………..... viii Acknowledgements……………………………………………….. ix Foreword……………………………………………………………. x List of Tables……………………………………………………….. xii

List of Figures……………………………………………………… xvi CHAPTER 1 THE CELL 1 Introduction To Evolution Error! Bookmark not defined. 1.1 Basis of Evolution Error! Bookmark not defined. 1.1.1 Biological Overview Error! Bookmark not defined. 1.2 Cells 4 1.2.1 Lysosomes 4 1.2.2 Peroxisome 6 1.2.3 Nucleus 8 1.2.4 Cytoplasm 9 1.2.5 Mitochondria Error! Bookmark not defined. 1.2.6 Plasma membrane Error! Bookmark not defined. 1.2.6.2 Transmembrane channels Error! Bookmark not defined. 1.2.6.3 Endocytosis Error! Bookmark not defined. 1.2.6.4 Exocytosis Error! Bookmark not defined. 1.2.7 The ribosome Error! Bookmark not defined. 1.2.7.1 Treacher Collins Syndrome (TCS) Error! Bookmark not defined. 1.2.8 Golgi Bodies Error! Bookmark not defined. 1.2.9 Endoplasmic reticulum 17 1.3 Types of Cells 19 1.3.1 Stem Cells Error! Bookmark not defined. 1.3.2 Cell development Error! Bookmark not defined. 1.3.3 Cell Senescence Error! Bookmark not defined. 1.3.4 Cell Signalling Error! Bookmark not defined. 1.3.5 Apoptosis Error! Bookmark not defined. 1.3.6 Cell Division Error! Bookmark not defined. 1.3.7 Neuronal and Immune Cells Error! Bookmark not defined. 1.3.8 Transcription and Epigenetics Error! Bookmark not defined. References Error! Bookmark not defined. CHAPTER 2 INHERITANCE 2.1 Mitosis And Meiosis 28 2.1.1 Recombination and Variation 29 2.1.2 DNA/RNA String Manipulation Error! Bookmark not defined. 2.1.2.1 Deletion: amputation of one or more adjacent bases.Error! Bookmark not defined. 2.1.2.2 Insertion Error! Bookmark not defined. 2.1.2.3 Segmental duplication 33 2.1.2.4 Inversion 34 2.1.2.5 Translocation 34 2.1.2.6 Recombination 36 2.1.3 Genes 38 2.1.4 Consequences of Variation: Evolution 39 2.1.5 Corollary of Transposable elements (TE) DynamicsError! Bookmark not defined.

v

2.1.6 Gene Duplication 41 2.2 Mechanisms of duplication 41 2.2.1 Ectopic Recombination 41 2.2.4 Aneuploidy 41 2.2.5 Whole Genome Duplication Error! Bookmark not defined. 2.2.6 Identifying duplications in sequenced genomes 42 2.3 Gene Duplication as an Evolutionary Event 42 2.3.1 Subfunctionalization 42 2.3.2 Neofunctionalization 43 2.3.3 Pseudogene Error! Bookmark not defined. References Error! Bookmark not defined. CHAPTER 3 BIODIVERSITY 3.1 Rare Animal Diversity in Pakistan 49 3.1.1 Species Diversity 49 3.1.1.1 Buffaloes 50 3.1.1.2 Dromedaries 51 3.1.1.3 Sheep 51 3.1.1.4 Yaks 52 3.1.1.5 Goats 52 3.1.2 Ecosystem Diversity 52 3.1.2.1 Genetic Diversity 52 3.3 Valuable Animal Genetic Resources 52 3.4 Genetic characterization at molecular level: 53 3.4.1 Genetic information of livestock 54 3.4.2 Genetic information from sheep and goat: 54 3.4.3 Molecular genetic information from camelids 55 3.4.4 Application of microsatellites in genetic distance measurement within livestock 55 3.4.5 Application of microsatellite for calculating genetic distance in yaks, buffaloes, goats, horses, camelids and rabbits 58 3.5 Assessment of genetic diversity using DNA markers 60 3.5.1 Nuclear DNA markers 60 3.5.2 Microsatellite 60 3.5.3 AFLPs 61 3.5.4 Mitochondrial DNA Markers 61 3.5.5 Quantitative Trait Loci (QTL) Mapping 61 3.5.6 Analysis of gene expression 62 3.5.7 Transcript profiling 62 3.5.8 Protein Profiling 62 3.6 How to Preserve Genetic Resources 63 3.6.1 Genetic Conservation 64 3.6.2 Threats to animal genetic resources 64 3.6.3 Implications for animal genetic resources 65 References 66 CHAPTER 4 LATEST TECHNIQUES IN ANIMAL GENETICS AND GENOMICS 4.1 Genotyping by Sequencing (GBS) 71 4.2 Epigenetics 71 4.3 MicroRNAs Expression Profiling 72 4.4 Genome Wide Association Studies (GWAS) 72 4.5 Whole Genome Sequencing (WGS) 73 4.6 Microarray Technology 73 4.7 Nutrigenetics and Nutrigenomics 73 4.8 Assisted Reproductive Technologies (ART) 74 References 75

vi

CHAPTER 5 EVOLUTION AT COMPUTATIONAL END 79 5.1 Sequential Genetic Algorithms 80 5.1.1 Parallel Genetic Algorithms (PGA) 80 5.1.2 Advantages of using PGA 80 5.1.3 xxGA Description 81 5.1.4 BH87 for Genetic Distance Analyses 81 5.1.5 LOGDET 81 5.1.6 K80 Model 81 5.1.7 K81 Model 82 5.1.8 F81 Model 82 5.1.9 T92 Model 82 5.1.10 TN93 Model 82 5.1.11 GG95 Model 83 5.1.12 Paralin Model Error! Bookmark not defined. 5.1.13 F84 Model 83 5.2 Significance of Evolution 84 5.2.1 Early Diagnosis of Diseases Using Evolution (Distant metastasis) 84 5.2.2 Evidence Based Medicines (EBM) 84 5.2.3 Development of New CNE Regions 85 5.3 Modes of Gene Co-Option 86 5.3.1 Molecular Marker Analyses for Species Identification 88 5.3.2 Origin of 2R Hypothesis 89 References 92 CHAPTER 6 GENOMIC EXPLORATION OF DOMESTIC LIVESTOCK SPECIES 6.1 BUFFALO (Bubalus bubalis) 95 6.1.1 Mitochondrial DNA Diversity Patterns in Pakistani Buffalo 95 6.1.2 DNA Fingerprinting of Pakistani buffalo breeds (Nili-Ravi, Kundi) using microsatellite and cytochrome b gene markers 96 6.1.3 Genetic Characterization of Azakheli Buffalo Breed of Pakistan Using Microsatellites DNA Markers 98 6.1.4 Phylogenetic Analysis of Kundi Buffalo Breed of Pakistan through Mitochondrial D-Loop Region 99 6.1.5 Evaluation of AMPK Genes as Candidates for Production Traits in Buffalo Breeds of Pakistan 99 6.1.6 Genetic Identification of Three Pakistani Buffalo Breeds Through a Homozygosity Pattern in the PRKAG3 GeneError! Bookmark not defined. 6.1.7 Polymorphic Variants of MHC Class II Antigen (BuLA-DRB3) Gene in Pakistani Buffalo Error! Bookmark not defined. 6.2 Cattle Error! Bookmark not defined. 6.2.1 Microsatellite Markers Error! Bookmark not defined. 6.2.3 Genetic Differentiation Error! Bookmark not defined. 6.2.4 Microsatellite Markers Analysis Error! Bookmark not defined. 6.2.5 Phylogenetic Tree of Sahiwal & Cholistani Cattle BreedsError! Bookmark not defined. 6.2.6 Primer for Mitochonrial Cytochrome b Error! Bookmark not defined. 6.2.7 Mitochondrial Cytochrome b Gene Analysis 125 6.2.8 DGAT1 Gene (Bos taurus) Error! Bookmark not defined. 6.2.9 Prolactin Gene Error! Bookmark not defined. 6.2.9.1 Prolactin Gene Polymorphism Error! Bookmark not defined. 6.2.10 POU1FI Primer in Cattle Error! Bookmark not defined. 6.2.11 DGAT I gene Error! Bookmark not defined.

vii

6.2.11.1 Restriction Fragment Length Polymorphism Amplification of DGAT Ex.VIII Error! Bookmark not defined. 6.2.11.2 Restriction Digestion Error! Bookmark not defined. 6.2.12 POU1F1 Gene characteristics Error! Bookmark not defined. 6.2.13 Prolactin Gene Orientation Error! Bookmark not defined. 6.2.14 DISCUSSION Error! Bookmark not defined. 6.3 Sheep and Goat Error! Bookmark not defined. 6.3.1 Introduction Error! Bookmark not defined. 6.3.2 Mitochondrial DNA D-Loop and Cytochrome b GeneError! Bookmark not defined. 6.3.2.1 Primer Designing Error! Bookmark not defined. 6.3.2.2 Microsatellites Markers for Genetic Diversity in Sheep and goatError! Bookmark not defined. 6.3.2.3 Microsatellite markers Results in Goat and sheep BreedsError! Bookmark not defined. 6.3.2.4 Discussing Mitochondrial D-Loop and Cytochrome B Analysis 182 6.4 Camel 187 6.4.1 Mitochondrial Cytochrome-b and D-loop Sequence based Genetic Diversity in Mareecha and Breela Camel Breeds of Pakistan 187 6.4.2 Molecular Diversity in ATP6 and ATP8 genes in Dromedaries Camel breeds of Pakistan 190 6.4.3 Discussion on mitochondrial sequences in Pakistani camel 192 6.4.4 Camel DRB3 gene sequence analysis 194 6.4.5 Camel Prion protein Analysis 195 6.4.6 Interferon Alpha Based Genetic Diversity Analysis of Pakistani Camel Breeds 197 6.4.6.1 Alignment of the targeted sequences (Clustal W): 197 6.4.6.2 Identification of SNP’s: 198 6.4.6.3 Phylogenetic analysis 199 6.4.7 Interferon Beta 1 based Genetic Analysis of Pakistani Dromedary Camel Error! Bookmark not defined. 6.4.8 Heat Shock Protein 40/ DNAJA1 Gene Sequence analysis in Mareecha Camel Error! Bookmark not defined. 6.4.8.1 Sample Collection, DNA isolation and QuantificationError! Bookmark not defined. 6.4.8.2 Primer Designing and PCR amplification Error! Bookmark not defined. 6.4.8.2 Sequence Analysis Error! Bookmark not defined. 6.4.9 Results Error! Bookmark not defined. 6.4.9.1 SNP identification Error! Bookmark not defined. 6.4.9.2 The BLAST Analysis Error! Bookmark not defined. 6.4.9.2 Phylogenetic Analysis Error! Bookmark not defined. 6.4.9.3 Pair-Wise Distance Matrix Error! Bookmark not defined. 6.4.9.4 Discussion Error! Bookmark not defined. 6.4.9.5 Conclusion Error! Bookmark not defined. 6.5 Chicken Error! Bookmark not defined. 6.5.1 Genetic Variations Error! Bookmark not defined. 6.5.2 Heterozygosity of Markers Error! Bookmark not defined. 6.5.3 Genetic Distances Error! Bookmark not defined. 6.5.4 Conclusions Error! Bookmark not defined. 6.6 Horse Error! Bookmark not defined. 6.6.1 Horse Parentage Analysis and Breed Characterization by Microsatellite Markers Error! Bookmark not defined. 6.6.2 Conclusion and Suggestions Error! Bookmark not defined. 6.7 Dog Error! Bookmark not defined.

viii

6.7.1 A Panel of Microsatellite Markers for Genetic Diversity and Parentage analysis of German Shepherd and Labrador Retriever Dog Breeds in Pakistan 207 6.8 Wild Punjab Urial Error! Bookmark not defined. 6.8.1 Mitochondrial DNA D-Loop Based Diversity & Evolutionary AnalysisError! Bookmark not defined. 6.8.2 Microsatellites based genetic diversity and relationship of domestic sheep breeds and wild Urial (Ovis orientalis punjabiensis)Error! Bookmark not defined. 6.8.3 Mitochondrial ATP6 and ATP8 Genes Based Molecular Diversity and Phylogeny of Punjab Urial Error! Bookmark not defined. 6.8.4 Interleukin 2 gene sequences based genetic diversity of endangered Punjab Urial Error! Bookmark not defined. 6.8.5 MDS analysis of variation pattern Error! Bookmark not defined. 6.8.6 Interferon alpha of Major Histocompatibility Complex Class I sequences analysis in Punjab Urial (Ovis vignei punjabiensis)Error! Bookmark not defined. References Error! Bookmark not defined.

ix

PREFACE The livestock industry plays a pivotal role in the economy of Pakistan by contributing 56.3

percent of the agriculture value added and 11.8 percent to national GDP during 2014-15 (Economic survey of Pakistan, 2014-15). Currently, Pakistan has a herd size of around 76.8 million animals (41.2 million cattle, 35.6 million buffaloes) with an estimated production of around 52 billion liter of milk per annum (Economic survey of Pakistan, 2014-15). About 36 million people are involved in dairy farming, deriving more than 40% of their total income from livestock. This sector is central to the livelihood of the rural people in the country and can play an important role in poverty alleviation. It can uplift the socioeconomic condition of Pakistan’s rural masses. The major problem for small holding dairy farmers is the dreary milk productivity of Pakistani cattle and buffalos which is less than 4 liters to 5 liters per day for the whole duration of the lactation cycle of around 305 days. On average a dairy animal in Pakistan yields 6-8 times less milk than a dairy animal of the developed world; approximately 8 Pakistani milk producing animals are equal to 1 animal of the developed world. One of the reasons of this low production potential of Pakistani animals is the involvement of infectious agents including bacteria, viruses and protozoa.

Genomics of livestock species had advanced significantly in recent years. The completion of whole genome sequencing projects has brought a wealth of information about the structure of animal genome. High throughput technologies made feasible by sequencing and re-sequencing of some economically significant livestock species. All biotechnological tools combine with bioinformatics tools would be of massive assist in understanding of livestock genetic architecture for efficient production through genetic analyses and Markers Assisted Selection. The applications of these technologies have their significant impact for rapid improvement of livestock production. In future, the proficient livestock production would rely on existing and promising biotechnological advances for better improvement in productivity. Here we have included the genomic work done, reported from Pakistan by different researchers as well on indigenous livestock and wild species for better understanding of readers about the current status of animal genomics in Pakistan.

This monograph reflects our humble endeavor to spotlight the potential of economically valuable animal species, but at the same time reader’s suggestions, novel ideas and constructive criticism will be welcome to enhance the quality of the later version of this manuscript.

Prof.Dr. Masroor Ellahi Babar (TI)

x

ACKNOWLEGMENTS

Gratitude to the most gracious almighty ALLAH, who bequeathed me health, ease and opportunities to accomplish this piece of work. I pray for holy prophet MUHAMMAD (salalla hu alahe wa'aalahi wasalam) contemplation on doomsday, which is ever an ember of guidance and knowledge for me and for whole of the humanity. I also implore for all success in the life hereafter, like the way he has granted me with all the blessings here.

I would also like to express my appreciation to the many people who came to know me through this book; to all those who provided me support, talked things over, read, wrote, offered comments, allowed me to quote their research findings and assisted in the editing, proofreading and designing of this book.

I am also very thankful to the worthy Rector, Virtual University, Dr. Naveed Malik, for trusting in me profoundly and encouraged all my ambitions to uplift VU to the horizons of prosperity. I am also grateful to my patron Prof. Dr. Muhammad Nawaz, whose personality is permanent source of inspiration for me.

Above all I want to thank my family, who supported and encouraged me in spite of all the time it took me away from them.

I appreciate Dr. Rashid Saif, who has major contribution of his efforts in editing and composing the material of this book. I also acknowledge Dr. Tanveer Hussain and Mr. Muhammad Aqeel for helping me in the process of gathering the material. All the researchers whose reports on animal genetics and genomics have been included in this monograph are also acknowledged. I also want to pay my regards to Higher Education Commission HEC-Pakistan as publisher and providing the platform and encouraged me to complete this monograph.

Last but not least, I hope that you will find this book informative and contributing towards ever increasing knowledge pool.

Thank you

Prof.Dr. Masroor Ellahi Babar (TI)

xi

FOREWORD

The monograph "Evolutionary Dynamics and Biodiversity in Pakistani Livestock Species” by Professor Dr. Masroor Ellahi Babar stands poised to significantly influence the understanding of this very important area, both for students and teachers alike.

Professor Babar has been in the forefront of the evolution of the Virtual University of Pakistan from a teaching-only institution to a teaching and research oriented university that is undertaking major projects in various fields, even outside the virtual classrooms. Despite his many duties as Registrar of the University, he has been consistently involved in research efforts and has also inspired other departments at the University to follow his lead. The young and dynamic team that he has brought together in his own area of expertise has the potential to place the Virtual University in a leadership position and several collaborative efforts with sister institutions are already underway or in the pipeline. Given its unique pedagogical strengths, the University is also offering services through its free to air broadcast television channels to support a wider understanding of important social and technical issues. These include the livestock and dairy domains through the efforts of Professor Babar.

I would like to take this opportunity to congratulate Dr. Babar for authoring this important treatise and I look forward to seeing many more contributions from him in the future.

Dr. Naveed A. Malik Rector, Virtual University of Pakistan,

Lahore.

xii

FOREWORD

Prof. Dr. Masroor Ellahi Babar the author of this monograph is an active researcher in the field of animal genetics and genomics and is regarded as an authority in animal biotechnology. His current book entitled “Evolutionary Dynamics and Biodiversity in Pakistani Livestock Species” is an excellent resource for not only young researchers and students at all levels, but for anyone interested in livestock in general and animal breeding in particular. This book integrates insights regarding the Cell, Inheritance, Biodiversity, Techniques in Animal Genetics and Genomics, Molecular Evolution and Genetic and Genomic exploration of valuable Pakistani Livestock species. Dr. Babar has the gift of presenting complex concepts in a simplified manner so that it becomes useful to both students and teachers. The book is replete with wonderful amalgamation of basics and applied trends within the field of genetics/genomics and bingers of bioinformatics/computational biology.

Based on the latest molecular approaches of genetics/genomics, biotechnology and bioinformatics contents have been distributed into various chapters. Chapter:1 describes the introduction to basics unit of life and cell organelles in the context of evolution; Chapter:2 defines inheritance from classical techniques to physical manipulations and its molecular approaches; Chapter:3 shed light on the different forms of the life and their peculiar characteristics encrypted in their DNA, hidden potentials of particular species and breeds; Chapter:4 introduces computational biology and bioinformatics tools and latest trends to analyze genotyping and sequencing data for meaningful interpretation; Chapter:5 explains the computational background of evolution, its prediction and reliability, latest trends in this significant field of knowledge; Chapter: 6 spot lights on the genomic exploration to Pakistani domestic livestock species what he and his colleagues has done for the last 10 years. The discussions of mentors and active researchers, biographical study, journaling make Chapter 6 particularly useful for teachers and early career researchers to take guidance for project write-ups which will help them to familiarize the lab techniques beyond the boundaries of classroom.

I am sure, all readers will benefit from this book. This monograph will definitely provide a road map to the readers that how to explore the mysteries encrypted in animal life

Dr. Kauser Adbulla Malik HI, SI,TI

HEC Distinguished National Professor Forman Christian College

(A Chartered University), Lahore

xiii

LIST OF TABLES

S. No. CAPTION OF TABLES Page No.

1. 1.1 Model organisms used in research laboratories ranges from bacteria to higher organisms

4

2. 1.2 Some important functions governed by peroxisomes in approximately all forms of eukaryotic life

8

3. 3.1 Investigation of various genetic markers in cattle 54

4. 3.2 Investigation of genetic markers in sheep 54

5. 3.3 Investigation of genetic markers in sheep

55

6. 3.4 Investigation of genetic markers in camelids 55

7. 3.5 FAO-recommended projects used these microsatellite loci 56

8. 3.6 FAO-recommended projects used these microsatellite loci in sheep 57

9. 3.7 Genetic distance studies involving these microsatellites in buffalo 58

10. 3.8 Genetic distance studies used involving these microsatellites in goats 58

11. 3.9 Genetic distance studies involving microsatellites in camelids 59

12. 6.1 List of 21 microsatellite markers selected from ISAG (International Society of Animal Genetics) and Food and Agriculture Organization (FAO) recommended markers for cattle

103

13. 6.2 Summary of genetic diversity statstics of 21 microsatellite markers in selected Pakistani cattle breeds

105

14. 6.3 Total number of alleles abserved on all 21 microsatellite loci in selected cattle breeds of Pakistan

106

15. 6.4 Descriptive statistics of the 21 microsatellite markers in each of selected cattle breeds

107

16. 6.5 Summery of parentage confirmation parameters of all 21 microsatellite markers applied in cattle breeds of Pakistan using Power Stat 2.1 software

113

17. 6.6 Distance method: No. of different alleles (Fst) values in selected cattle breeds of Pakistan

114

18. 6.7 AMOVA design and results for genetic variations in Pakistani cattle breeds

114

xiv

19. 6.8 Mitochondrial D-Loop primers for Sahiwal & Cholistani Breeds based on NC006853

117

20. 6.9 Mitochondrial cytochrome b gene primers for Pakistani cattle breeds 119

21.

6.10 The nucleotide changes in Cytochrome b sequences of Pakistani cattle breeds and the amino acid changes. Shaded changes are found typically in taurine cattle

120

22. 6.11 Estimates of evolutionary divergence of Pakistani cattle breeds over sequence pairs between groups.

122

23. 6.12 DGAT1 Gene Primers for Pakistani cattle breeds

126

24. 6.13 Primers of Prolactin Gene study in Pakistani cattle breeds 128

25. 6.14 POU1F1 Gene Primers used on Pakistani cattle breeds 128

26. 6.15 DGAT Ex. VIII Genotype observed in the Sahiwal cattle breed 129

27. 6.16 DGAT EX.VIII Genotype observed in Dhanni cattle breed 129

28. 6.17 POU1F1 SNPs in Holstein Fresian, Sahiwal and Dhanni 131

29. 6.18 Prolactin gene SNPs found in Sahiwal cattle breed 134

30. 6.19 Prolactin gene SNPs found in Holstein Friesian cattle breed 135

31. 6.20 Prolactin gene SNPs found in Dhani cattle breed 136

32. 6.21 Prolactin gene SNPs found in Red-Sindhi cattle breed 137

33. 6.22 Mitochondrial Cytochrome b and D-loop Primers for Goat 140

34. 6.23 Mitochondrial Cytochrome b and D-loop Primers for Sheep 141

35. 6.24 Haplotypes detected in Cytochrome b and the D-loop region of Pakistani Goat breeds

142

36. 6.25 Haplotypes detected in Cytochrome b and the D-loop region of Pakistani Sheep breeds

142

37. 6.26 Mitochondrial Conserved regions in Pakistani goat and sheep 143

38. 6.27 Comparison of haplotypes based on Cytochrome b region 1140 bp of Pakistani Goat with Capra family

148

39. 6.28 Comparison of haplotypes based on D-loop region 720 bp of Pakistani Sheep with Ovine family

150

40. 6.29 Worldwide comparisons of Goat D-loop haplotypes with newly found goat haplotypes (Mega 4.1) based on 481 bp of D-Loop HVR1

152

41. 6.30 Worldwide comparisons of Sheep D-loop haplotypes with newly found sheep haplotypes (Mega 4.1) based on 523 bp of D-Loop HVR2

155

xv

42. 6.31 Microsatellite Markers Description used on goat and sheep characterization

157

43. 6.32 Overall Allele Frequency of microsatellite markers in selected Pakistani Goat Breeds

158

44. 6.33 Summary of Genic Variation Statistics for all Loci in selected Pakistani Goat Breeds

159

45. 6.34 Summary of Heterozygosity Statistics for all Loci in selected Pakistani Goat Breeds

160

46. 6.35 Summary of F-Statistics and gene flow for all loci in selected Pakistani goat breeds

161

47. 6.36 Nei's Original Measures of Genetic Identity and Genetic distance in selected Pakistani Goat Breeds

162

48. 6.37 Forensic application values of Microsatellites markers in pakistani Goat breeds (Power Stat)

163

49. 6.38 Overall allele frequency of microsatellite markers in twelve selected sheep breeds of Pakistan

170

50. 6.39 Summary of genetic variation statistics for all loci in 12 Pakistani sheep breeds [See Nei (1987) Molecular Evolutionary Genetics (p. 176-187)]

171

51. 6.40 Summary of heterozygosity statistics for all microsatellite loci 12 Pakistani sheep breeds

172

52. 6.41 Summary of F-Statistics and Gene Flow for All Loci in 12 Pakistani Sheep Breeds

173

53. 6.42 Nei's Original Measures of Genetic Identity and Genetic distance in 12 Pakistani Sheep Breeds

174

54. 6.43 Forensic application values of microsatellites markers in Pakistani sheep breeds (Power Stat)

175

55. 6.44 Polymorphic sites detected in mt cytochrome b gene of Pakistani camel breeds

189

56. 6.45 Polymorphic sites detected in the IFN-α region of Pakistani Camel 198

57. 6.46 Population Genetic Distance and Genetic Identity among Percheron, Pak Arab and Thorough bred horse breeds in Pakistan.

206

58.

6.47 The average values of Ne = Number of effective alleles, PIC= Polymorphism Information Content, PE = Power of Exclusion, Fst = Wright’s fixation index, Nm = Gene Flow, based on 13 microsatellite markers in four horse breeds in Pakistan.

207

59. 6.48 Number of effective alleles, polymorphism information content, power of exclusion, Differentiation in subpopulations in dog

208

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60. 6.49 Mean basic diversity indices, number of loci deviating from Hardy-Weinberg equilibrium and average within breed inter-individual allele sharing distance in different sheep populations

217

61. 6.50 Analysis of molecular variance among different sheep populations 218

62. 6.51 Polymorphic sites detected in the IFNα-A region of Punjab Urial Sequences

224

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LIST OF FIGURES

S. No. CAPTION OF FIGURES

Page No.

1. 1.1 Phylogenetic relationship established between three major domains of life. Out of these three divisions, Archaebacteria and Eukaryotes are evolved from Eubacteria

3

2. 1.2 Structure of a typical animal cell showing the origin and working mechanism of a Lysosome

5

3. 1.3 Lysosomes are very important for human health concerning their metabolic activities whether it is heterocytosis or autocytosis

6

4. 1.4 Peroxisome is acting as a transporter where small molecules are transported with the zero consumption of ATPs

6

5. 1.5 Peroxisomes evolving from rough endoplasmic reticulum 7

6. 1.6 Peroxisome catalyzing long chain fatty acid involved a number of steps 8

7. 1.7 Anatomy of the nucleus 9

8. 1.8 Structure of cell in which major biological processes are shown 10

9. 1.9 Mitochondria provide plenty of energy in the form of adenosine triphosphate (ATP) molecules.

11

10. 2.1 Fertilization of n+n cells resulted in 2n zygote 28

11. 5.1 Approaches of gene co-option. There are two processes non-duplicative and duplicative

87

12. 6.1 Neighbour-joining phylogeny of 52 Pakistani, nine swamp (Carabao), 10 Mediterranean and 17 Indian buffalo haplotypes

97

13. 6.2 Reduced median network of 52 Pakistani buffalo haplotypes 98

14. 6.3 Phylogenetic tree using UPGMA method of Pakistani buffaloDRB3 gene haplotypes constructed with Murrah breed reported in GenBank

101

15. 6.4 Phylogenetic tree using UPGMA method of Pakistani buffalo DRB3 gene haplotypes constructed with Murrah buffalo and other reported sequences in different mammals in GenBank

102

16. 6.5 Dendrogram Based Nei's (1978) Genetic distance. UPGMA Modified from NEIGHBOR procedure of PHYLIP Version 3.5

115

17. 6.6 Neighbour Joining Tree of Pakistani cattle breeds based on mt D-loop sequences

118

xviii

18. 6.7 Phylogenetic Tree (UPGMA Method) by MEGA 5.1 (Circular view) of Pakistani cattle breeds based on mt cytochrome b gene sequences

123

19. 6.8 POU1F1 Gene Primers used on Pakistani cattle breeds 129

20. 6.9 Gel Electrophoresis of PCR product. Lanes 2, 3, 4, 5, 6 showing amplification of required product, Lane 1- Negative control for DNA, Lane 8- Negative control for primer

130

21. 6.10 Gel Electrophoresis after Restriction digestion (Lanes 1-7 showing samples of Sahiwal breed, Lanes 9-14 showing samples of Dhanni breed, Lane 8 showing 50bp DNA marker

131

22. 6.11 Sequence results of prolactin gene in Pakistani cattle 133

23. 6.12 Neighbour Joining phylogenetic tree constructed by MEGA 4.1 from 50 haplotypes of Pakistani Goat (Cytochrome b and mtD-loop sequences)

145

24. 6.13 Neighbour Joining phylogenetic tree constructed by MEGA 4.1 from 50 haplotypes of Pakistani Sheep (Cytochrome b and mtD-loop sequences)

147

25. 6.14 Neighbour Phylogenetic Tree based on Cytochrome b and mtD-loop sequences of Goat and Sheep in comparison with Bos indicus, Bos Taurus

148

26. 6.15 Phylogenetic Comparison of haplotypes based on Cytochrome b region 1140 bp of Pakistani goat with Capra family (UPGMA MEGA 4.1)

149

27. 6.16 Phylogenetic Comparison of haplotypes based on D-loop region 720 bp of Pakistani Sheep with Ovine family Relationship between different sheep of Ovine Family. (UPGMA MEGA 4.1)

151

28. 6.17 Worldwide comparison of goat d-loop haplotypes with Pakistani goats (Mega 4.1)

154

29. 6.18 Worldwide comparison of sheep d-loop haplotypes with Pakistani sheep (Mega 4.1)

156

30. 6.19 Allele frequencies of all microsatellite markers in Lehri goat 163

31. 6.20 Allele frequencies of all microsatellite markers in Local Hairy goat 164

32. 6.21 Allele frequencies of all microsatellite markers in Barbari goat 164

33. 6.22 Allele frequencies of all microsatellite markers in Beetal goat 165

34. 6.23 Allele frequencies of all microsatellite markers in Damani goat 165

35. 6.24 Allele frequencies of all microsatellite markers in Pahari goat 166

36. 6.25 Allele frequencies of all microsatellite markers in Teddy goat 166

37. 6.26 Allele frequencies of all microsatellite markers in Kamori goat 167

xix

38. 6.27 Allele frequencies of all microsatellite markers in Khurasani goat 167

39. 6.28 Allele frequencies of all microsatellite markers in Nachi goat

168

40. 6.29 Allele frequencies of all microsatellite markers in Jattal goat 168

41. 6.30 Allele frequencies of all microsatellite markers in Dera Din Panah goat 169

42. 6.31a Dendrogram Based Nei's (1972) Genetic distance: Method = UPGMA Modified from NEIGHBOR procedure of PHYLIP Version 3.5 of Pakistani goat breeds

169

43. 6.31b Allele frequencies of all microsatellite markers in Karakul sheep 176

44. 6.32 Allele frequencies of all microsatellite markers in Bulkhi sheep 176

45. 6.33 Allele frequencies of all microsatellite markers in Salt Range sheep 177

46. 6.34 Allele frequencies of all microsatellite markers in Awasi sheep 177

47. 6.35 Allele frequencies of all microsatellite markers in Shenwari sheep 178

48. 6.36 Allele frequencies of all microsatellite markers in Kuchi sheep 178

49. 6.37 Allele frequencies of all microsatellite markers in Kaghani sheep 179

50. 6.38 Allele frequencies of all microsatellite markers in Lohi sheep 179

51. 6.39 Allele frequencies of all microsatellite markers in Dumri sheep 180

52. 6.40 Allele frequencies of all microsatellite markers in Thalli sheep 180

53. 6.41 Allele frequencies of all microsatellite Markers in Buchi Sheep 181

54. 6.42 Allele Frequencies of All Microsatellite Markers in Hushtnagri Sheep 181

55. 6.43 Dendrogram Based Nei's (1972) Genetic distance: Method = UPGMA Modified from NEIGHBOR procedure of PHYLIP Version 3.5 for 12 pakistani sheep breeds

182

56.

6.44 Neighbor Joining Phylogenetic tree of Pakistani Mareecha and Breela breeds based on cytochrome b sequences with already reported Dromedary Bactrian camel, Ovis aries, Capra hircus, Bos taurus, Bos indicus, Bubalus bubalis (river and swamp type) using bootstrap value 1000.

189

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57.

6.45 Neighbor Joining Phylogenetic tree of Pakistani Mareecha and Breela breeds based om mt D-loop sequences with already reported Dromedary, wild Bactrian and Bactrian camel, Ovis aries, Capra hircus, Bos taurus, Bos indicus, Bubalus bubalis (river and swamp type) using the bootstrap value 1000.

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58. 6.46 Neighbor Joining tree of selected camel breeds of Pakistan with other reported sequences from different mammalian species, constructed using MEGA 6.1.

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59. 6.47 Phylogenetic Analysis of prion protein gene in Paksitani camel in comparison to other mammals

196

60. 6.48 UPGMA method based Phylogenetic tree based on IFN-α nucleotide sequences from Pakistani Camel breeds using MEGA6.

199

61. 6.49 UPGMA method based Phylogenetic tree based on IFN-α nucleotide sequences of camel from Pakistan and worldwide reported camels along with other species using MEGA6.

200

62. 6.50 NJ method based Phylogenetic tree for dromedary camel breeds of Pakistan based on INFβ1 nucleotide sequences from different species using MEGA6.

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63. 6.51 Bayesian phylogeny of mitochondrial haplotypes (212bp) of wild and domestic sheep

210

64. 6.52 Three dimensional scatterplot of average pairwise differences derived from mitochondrial D-Loop haplotypes (212bp) of wild and domestic sheep

211

65.

6.53 Distribution of allele sharing distance (IBS) between pairs of individuals. Distance was plotted separately where pairs are drawn from within the same population (red bars) and from across the populations (blue bars)

212

66. 6.54 a-b UPGMA dendrogram (top) and radial tree (below) based on pairwise Nei’s genetic distance among different sheep populations

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67. 6.55 Multidimensional scaling (MDS) plot of pairwise FST among different sheep populations (S stress = 0.03854)

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68. 6.56 Three dimensional scattergram of principal components derived from pairwise allele sharing distance among individuals of different sheep populations

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69. 6.57 Bayesian clustering of 471 sheep under assumption of 2 to 9 clusters without a priori population information

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70. 6.58 Phylogenetic Analysis of Punjab Urial in comparison with mammalian species using MEGA6.1 based on Mitochondrial ATP6 and ATP8 Genes

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71. 6.59 Neighbor Joining Treeconstructed with MEGA6 using IL-2 sequences from Punjab Urialand other different mammalian species.

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72. 6.60 Evolutionary distance was calculated between selected species by making pair-wise comparisons among them

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73. 6. 61 Multidimensional scaling for IL2 gene was plotted to see within-species pattern variations

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74. 6.62 Interferon alpha-A based phylogenetic tree (Neighbor joining method) constructed by MEGA 6.1 for Punjab Urial in comparison with other mammalian species sequences available on GenBank NCBI.

223