DOCTOR OF PHILOSOPHY IN ANIMAL GENETICS AND BREEDING By SHAHID AHMAD ...

161 Pages·2016·5.96 MB·English

IDENTIFICATION OF GENETIC POLYMORPHISM IN
IMMUNITY RELATED GENES AND THEIR ASSOCIATION
WITH CLINICAL MASTITIS IN MURRAH BUFFALOES

THESIS SUBMITTED TO THE
ICAR-NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY
IN
ANIMAL GENETICS AND BREEDING
By
SHAHID AHMAD SHERGOJRY
M.V.Sc. (AGB)

DAIRY CATTLE BREEDING DIVISION
ICAR - NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY
KARNAL-132001 (HARYANA), INDIA

2014
Regn. No. : 1071107
IDENTIFICATION OF GENETIC POLYMORPHISM IN IMMUNITY
RELATED GENES AND THEIR ASSOCIATION WITH CLINICAL
MASTITIS IN MURRAH BUFFALOES
By
SHAHID AHMAD SHERGOJRY
Thesis submitted to the
ICAR-National Dairy Research Institute
(Deemed University)
in partial fulfilment of the requirement
for the degree of
DOCTOR OF PHILOSOPHY
IN
ANIMAL GENETICS AND BREEDING
Approved by:
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CHANA~E MA)
Majdt' Advisor & Chairperson
(Guide)
Members of Advisory Committee ~
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1. Dr. I. D. Gupta
Principal Scientist, DCB Division
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2. Dr. A. K. Dang
Principal Scientist, DCP Division
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3. Dr. 5. De
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Senior Scientist, ABTC Division
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4. Dr. M.S. Tantia
Principal Scientist, NBAGR Division ~~~ "
5. Dr. A. Kumaresan
(Director's Nominee) Senior Scientist, LPM Division
DAIRY CATILE BREEDING DIVISION
ICAR - NATIONAL DAIRY RESEARCH INSTITUTE
KARNAL-132001 (HARYANA), INDIA
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Dr. Archana Verma
Principal Scientist
CERTIFICATE
This is to certify that the thesis entitled, "Identification of genetic polymorphism in
immunity related genes and their association with clinical mastitis in Murrah
buffaloes" submitted by Shahid Ahmad Shergojry towards the partial fulfilment of
. the award of the degree of DOCTOR OF PHILOSOPHY in ANIMAL GENETICS
AND BREEDING of the ICAR - NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY), Karnal (Haryana), India, is a bonafide research work
carried out by him under my supervision, and no part of the thesis has been
submitted for any other degree or diploma.
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Dated: ( rchana Verma)
MAJOR DVISOR & & CHAIRPERSON
(GUIDE)

Acknowledgement
I condescend my head with great adoration to Almighty Allah, the one Who is omnipotent
and omniscient and Who is the cause behind every effect (Alhamdullilah-i-rabbil aalamin).
This thesis is a result of a year of research that was done since I completed my academics
in July 2012. Since then, I have worked with a number of people whose contribution in assorted
ways to the research and the making of the thesis deserves special mention. It is a pleasure to
convey my gratitude to them all in my humble acknowledgment.
In the first place I would like to record with immense pleasure and a feeling of privilege
my esteemed gratitude to my respected guide and Chairperson of the Advisory committee Dr.
Archana Verma, Principal Scientist , Dairy Cattle Breeding Division, NDRI, Karnal for her
wholehearted cooperation, inspiration, blessings, encouragement, valuable guidance, everlasting
affection and pertinent suggestions throughout my research work and help in conducting my
thesis work and preparation of every part of this manuscript. Her patience and support helped me
to overcome many crisis situations and finish this dissertation. Her everlasting smiling face is
like an evergreen refreshing magic which always boosted my body with tonnes of energy to work
efficiently. Her calm nature, politeness and down to Earth personality make her a special person.
She always treats all the students like her children. Without her deep involvement, care and
support at every stage, this PhD work would not have been accomplished. I feel honored to be
researcher under her judicious guidance.
I extend my grateful acknowledgement to the members of the advisory committee, Dr. I.
D. Gupta, PS, DCB Division, Dr. A. K. Dang, PS, DCP Division, Dr. S. De, PS, ABTC
Division, Dr. A. Kumaresan, SS, LPM Division and Dr. M. S. Tantia Principal Scientist
NBAGR Karnal for their useful advice and suggestions during research work.
I extend an elite memorandum of thanks to Dr. A. K. Srivastava, Director & Vice
Chancellor, National Dairy Research Institute, Karnal for providing me the opportunity to
pursue higher studies from such a great institute and granting approval for my research topic.
I express my warmest regards to all the faculty members of the DCB Division, NDRI, Karnal;
Dr. A. K. Chakravarty, Head, Dairy Cattle Breeding Division, Dr. Avtar Singh, Principal
scientist and Dr. A.K.Gupta, Principal scientist for their affection, expert guidance and needful
suggestions that helped me in improving the eminence of my work. I am fortunate to learn from
the knowledge and experience of my master’s degree advisor Dr .K. P. Ramesha, PS, (AGB),
Southern campus of NDRI Bangalore who has been always the source of inspiration.
People whose contributions have helped me and have left an everlasting impression of being
the wonderful people who acquainted me with their scholastic ideas, include, Dr. Ovais Aarif
and Dr. Nazir Ahmad Mir for their advice and their willingness to share their bright thoughts
with me, which were very fruitful for shaping up my ideas and research. I express my sincere
love and affection to my friends cum lab mates Dr. Ankit Magotra, Krishnender, , Tavsief
Ahmad, Mahesh, Nishant, Rakesh, Dhaman, Sailo, Ramendra Alka Chopra, Shaktivel,
Bharti, Arun, Richa, Santosh,Vineeth, and special thanks to jnyanashree for providing black
tea as refreshment in lab during exhaustive ours of thesis preparation. I extend warm personal
appreciation to Dr.Neeraj Kashyap and Dr. Ashwani Arya for their collective effort for
preparation of Hindi abstract. I am also very much thankful to all the office staff members of the
division for their timely help and unconditional support during my research work. I would like to
pay my heartiest regards to my beloved friends at home Dr. Bashir Ahmad, Dr. Muzaffer, Dr.
Gulzar Dr. Fayaz Ahmad sheikh and Dr. Iqbal Dar for their unconditional support when I
needed the most. A word of special thanks goes to my NDRIan friends Dr. Aejaz sir, Nazaim
sir, Rayees sir Muzamil, Syedmohd, Shahid iqbaL, Sajad Bilal, Mohsin, Iqbal and
Hujaz, for their persistent and permanent love& affection, understanding, compassion and
positive reciprocity in everything.
Besides thanking, I also have a feeling of compunction and so would like to extend my
sincere apologies for all the troubles and difficulties I might have, intentionally or
unintentionally, caused to anyone of you.
Where would I be without my family? My parents deserve special mention for their
inseparable support, encouragement and prayers throughout the period of my life. My Mother,
Gasha Bano, in the first place is the person who put the foundation of my learning character,
showing me the joy of intellectual pursuit ever since I was a child. My Father, Mohd Ahsan, is
the one who sincerely raised me with his caring and gentle love. Words fail me to express my
appreciation to my beloved wife Mussavir-ul-nissa whose dedication, love and persistent
confidence in me, during critical hours when I needed the most, has taken the load off my
shoulders. This work could not have been done without her understanding and support.
Finally, I would like to thank everybody who was important to the successful realization
of thesis, as well as expressing my apology that I could not mention personally one by one. May
God bless u all. Wasalam.

Place: Karnal
Date: Shahid Ahmad
LIST OF CONTENTS
Chapter Page
Title
No. No.
1 INTRODUCTION 1-4
2 REVIEW OF LITERATURE 5-20
2.1 Mastitis 5
2.2 Mammary Defense Mechanism 5
2.3 Candidate Genes Associated With Mastitis Resistance 6
2.4 Single Nucleotide Polymorphism (SNP) 7
2.5 Marker-Assisted Selection for disease resistance 8
2.6 Genetic Basis of Mastitis 9
2.7 Mannose-Binding Lectin (MBL) 11
2.8 Overview of the Complement System 11
2.9 The Function of MBL in the Lectin Pathway 12
2.9.1 Mechanism of MBL 13
2.9.2 Role of MBL in Activation of Complement System 13
2.9.3 Molecular Structure of Mannose Binding Protein (MBP) 13
Organization and Localization of Bovine Mannose-Binding
2.9.4 14
Lectin (MBL1 and MBL2) Genes
Organization and Localization of Bovine Complement
2.10 14
Components 4A (C4A) Gene
2.11 MBL Gene Polymorphisms and Disease Association 14
2.12 MBL Gene Polymorphisms and Association With Mastitis 15
2.13 C4A Gene Polymorphisms and Disease Association 19
2.14 C4A Gene Polymorphisms and Association With Mastitis 19
3 MATERIALS AND METHODS 21-34
3.1 Experimental Animals 21
3.1.1 Source of Data 21
3.2 Isolation of Genomic DNA 21
3.2.1 Blood Collection 21
3.2.2 DNA Extraction 21
3.2.3 Phenol-chloroform Extraction Method 21
3.3 Quality and Quantity Checking of DNA 23
3.4 Amplification of MBL1 Gene in Murrah Buffalo 23
3.4.1 Primer Designing 23
3.5 Amplification of MBL2 Gene in Murrah Buffalo 24
3.5.1 Primer Designing 24
Amplification of Targeted region of C4A Gene in Murrah
3.6 24
Buffalo
3.6.1 Primers Designing 24
3.7 Reaction Mixture 24
3.8 PCR programme of MBL1 Gene in Murrah Buffalo 29
3.9 PCR programme of MBL2 Gene in Murrah Buffalo 29
3.10 PCR programme of C4A Gene in Murrah Buffalo 30
3.11 Checking of PCR Product 31
3.11.1 Gel Assembly 31
3.11.2 Agarose Gel Electrophoresis 31
3.11.3 Visualization of Gel 32
3.12 Sequencing of PCR Product 32
3.13 Sequence Data Analysis and SNP Detection 32
3.14 Genotyping of the Animals 33
3.15 Genotypic and Allelic Frequencies 33
3.16 Molecular Data Analysis 33
3.17 Association Studies 34
4.0 RESULT AND DISCUSSIONS 35-78
4.1 Genomic DNA Extraction 35
4.2 PCR Standardization 35
4.3 DNA Amplification of MBL1 Gene in Murrah Buffalo 35
Sequence Data Analysis and SNPS Detection Of Mbl1 Gene
4.4 36
In Murrah Buffalo
Genotype and allele frequencies in promoter region of MBL1
4.5 39
gene
4.5.1 Population genetic indices 42
Haplotype distribution of MBL1 promoter region gene in
4.6 43
Murrah buffalo
Linkage disequilibrium analysis of MBL1 gene in Murrah
4.7 45
buffalo
4.8 Association analysis 45
Association between SNP, haplotype of MBL1 gene and
4.8.1 45
clinical mastitis in Murrah buffalo
4.8.2 Allelic association analysis 45
4.8.3 Genotypic association analysis 46
4.8.4 Haplotype association analysis 46
Genotype and allele frequencies in MBL1 gene other than
4.9 47
promoter region of MBL1 gene
Linkage disequilibrium analysis other than promoter region of
4.10 51
MBL1
Haplotype distribution other than promoter resgion of MBL1
4.11 51
gene in Murrah buffalo
4.12 Association analysis 53
4.12.1 Allelic association analysis 53
4.12.2 Genotypic association analysis 53
4.12.3 Haplotype association analysis 54
4.13 Basic local alignment search tool (BLAST) analysis 54
4.14 Construction of phylogenetic tree 54
4.15 DNA amplification of MBL2 gene in Murrah buffalo 55
Sequence data analysis and SNPs detection of MBL2 gene in
4.16 56
Murrah buffalo
4.17 Genotype and allele frequencies distribution of MBL2 gene 59
4.17.1 Population genetic indices 60
4.18 Haplotype distribution of MBL2 gene in Murrah buffalo 61
Linkage disequilibrium analysis of MBL2 gene in Murrah
4.19 62
buffalo
4.20 Association analysis 62
Associations between SNP, haplotype of MBL2 gene and
4.20.1 62
clinical mastitis in Murrah buffalo
4.20.2 Allelic association analysis 62
4.20.3 Genotypic association analysis 63
4.20.4 Haplotype association analysis 64
4.21 Basic local alignment search tool (BLAST) analysis 66
4.2.1 Construction of phylogenetic tree 66
4.22 DNA amplification of C4A gene in Murrah buffalo 68
Sequence data analysis and SNP detection of C4A gene in
4.23 68
Murrah buffalo
4.24 Genotype and allele frequencies distribution of C4A gene 70
4.24.1 Population genetic indices 71
4.25 Haplotype distribution of C4A gene in Murrah buffalo 72
4.26 Linkage disequilibrium analysis of C4A gene in Murrah buffalo 73
4.27 Association analysis 73
Association between SNP, haplotype of C4A gene and
4.27.1 73
clinical mastitis in Murrah buffalo
4.27.2 Allelic association analysis 73
4.27.3 Genotypic association analysis 74
4.27.4 Haplotype association analysis 76
4.28 Basic local alignment search tool (BLAST) analysis 76
4.29 Construction of phylogenetic tree 77
5 SUMMARY AND CONCLUSIONS 79-83
BIBLIOGRAPHY i-ix
APPENDIX i-vi