IN SESAME (Sesamum indicum L.)

190 Pages·2010·2.69 MB·English

MOLECULAR MAPPING AND CHARACTERIZATION
OF YIELD QTL AND TAGGING OF WILT
RESISTANCE GENE(S) IN SESAME
(Sesamum indicum L.)

JYOTHI BADRI
M.Sc. (Ag.)

DEPARTMENT OF GENETICS AND PLANT BREEDING
COLLEGE OF AGRICULTURE
ACHARYA N G RANGA AGRICULTURAL UNIVERSITY
HYDERABAD (A.P.)

DECEMBER, 2009
MOLECULAR MAPPING AND CHARACTERIZATION
OF YIELD QTL AND TAGGING OF WILT
RESISTANCE GENE(S) IN SESAME
(Sesamum indicum L.)

JYOTHI BADRI
M.Sc. (Ag.)

THESIS SUBMITTED TO THE
ACHARYA N G RANGA AGRICULTURAL
UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
(IN THE FACULTY OF AGRICULTURE)

DEPARTMENT OF GENETICS AND PLANT BREEDING
COLLEGE OF AGRICULTURE
ACHARYA N G RANGA AGRICULTURAL UNIVERSITY
HYDERABAD (A.P.)

DECEMBER, 2009
CERTIFICATE

Ms. JYOTHI BADRI has satisfactorily prosecuted the course of
research and that the thesis entitled “MOLECULAR MAPPING AND
CHARACTERIZATION OF YIELD QTL AND TAGGING OF WILT
RESISTANCE GENE (S) IN SESAME (Sesamum indicum L.)” 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 her for a degree of any
university.

Date: (N. A. ANSARI)
Place: Hyderabad Chairman of the Advisory Committee
DECLARATION

I, JYOTHI BADRI, hereby declare that the thesis entitled
“MOLECULAR MAPPING AND CHARACTERIZATION OF YIELD
QTL AND TAGGING OF WILT RESISTANCE GENE (S) IN SESAME
(Sesamum indicum L.)” submitted to Acharya N.G. Ranga Agricultural
University for the degree of DOCTOR OF PHILOSOPHY IN
AGRICULTURE (GENETICS AND PLANT BREEDING) is a result of
original research work done by me. I also declare that the thesis or part
thereof has not been published earlier elsewhere in any manner.

Date: (JYOTHI BADRI)
Place: Hyderbad
CONTENTS

Chapter No. Title Page No.

I INTRODUCTION 1-7

II REVIEW OF LITERATURE 8-43

III MATERIALS AND METHODS 44-72

IV RESULTS 73-109

V DISCUSSION 110-126
VI SUMMARY 127-131
LITERATURE CITED 132-168
APPENDIX 169-175
CERTIFICATE

This is to certify that the thesis entitled “MOLECULAR MAPPING AND
CHARACTERIZATION OF YIELD QTL AND TAGGING OF WILT
RESISTANCE GENE (S) IN SESAME (Sesamum indicum L.)” submitted in partial
fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN
AGRICULTURE (GENETICS AND PLANT BREEDING) of the Acharya N.G.
Ranga Agricultural University, Hyderabad is a record of the bonafide research work
carried out by Ms. JYOTHI BADRI under 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 investigation have been duly acknowledged by the author of the thesis.

(N.A. ANSARI)
Chairman of the Advisory Committee
Thesis approved by the student’s advisory committee
Chairman : Dr. N.A Ansari
Associate Dean, Agricultural college
Aswaraopet. _____________________
Co-chairman : Prof. E. A. Siddiq
Honorary Professor (Biotech),
Institute of Biotechnology,ANGRAU, _____________________
Rajendranagar Hyderabad – 500 030
Member : Dr. G. Anuradha
Principal Scientist (Plant Breeding)
Institute of Biotechnology, ANGRAU _____________________
Rajendranagar, Hyderabad – 500 030

Member : Dr. R. Sudhakar
Senior Scientist (Plant Pathology)
Seed Research and Technology Centre _____________________
ANGRAU, Rajendranagar,
Hyderabad – 500 030
LIST OF TABLES
S. No Title Page
No
1 2.1 Genetic Diversity based on D2 statistics 13-14
2 2.2 Simple measures of variability (Genotypic and Phenotypic Coefficient of 15-16
Variation)
3 2.3 Correlation analysis 27-28
4 2.4 Genetic architecture of yield contributing traits 29
5 2.5 Mode of inheritance of some yield related traits in sesame 30
6 3.1 Accessions used for studying the properties of newly developed SSRs 60
7 3.2 Characteristic features of parents chosen for the development of mapping 62
population for yield QTL
8 3.3 Description of yield and its components and their codes 65
9 3.4 Parental polymorphism using SSRs from various sources 66
10 3.5 Genotypes screened for tolerance to wilt 68
11 3.6 Scale of disease ratings 69
12 4.1 DNA quantification using spectrophotometer readings 73
13 4.2 Summary of the SSRs identified in the study 80
14 4.3 Properties of the isolated SSRs (Selective Hybridization) 82-83
15 4.4 Properties of the isolated SSRs (EST derived SSRs) 84-89
16 4.5 Tests of significance for assessing variability between parents 94
17 4.6 Transgressive segregants, Heterosis and Heterobeltiosis for 12 95
traits
18 4.7 Phenotypic correlations of 12 traits of 176 BC F plants 97
1 2
19 4.8 Salient features of six linkage groups 99
20 4.9 QTL detected in BC F population of the cross Swetha/BB-3-8 103
1 2
21 4.10 Reaction of the Genotypes screened for tolerance to wilt 105
22 4.11 Mode of inheritance of wilt resistance in the cross between TKG-22 X 108
TMV-3
LIST OF FIGURES
S. No Title Page
No
1 3.1 Schematic representation of microsatellite isolation using selective 45
hybridization method.
2 3.2 Pictorial representation of microsatellite isolation 48
3 3.3 Confirmation of heterozygosity of the F hybrid of Swetha/BB-3-8. 63
1
4 . 3.4 Plant types of Swetha, F and BB-3-8 63
1
5 . 3.5 Capsule shape of Swetha, F and BB-3-8 63
1
6 3.6 Schematic representation of development of the mapping population and QTL 64
mapping
7 3.7 Development of the mapping population for tagging of wilt resistance gene (s) 70
8 4.1 Genomic DNA on 1% Agarose gel 73
9 4.2 Restriction digestion smear on 1 % Agarose gel 74
10 4.3 Double stranded linker with GTTT “pig-tail” 74
11 4.4 PCR check for successful ligation of linkers to the restriction fragments 75
12 4.5 PCR recovery of enriched DNA 76
13 4.6 Blue-white screening of the colonies after transformation and overnight 77
incubation at 370C
14 4.7 Colony PCR for selection of colonies with insert in the size range of 300-1000 77
bp
15 4.8 Raw sequence of clone no- M82 containing (TC)17(AC)17 repeat 78
16 4.9 Primer designing using Primer 3.0 software in clone no.M93 79
17 4.10 Characterization of repeat motifs recovered from the process 81
18 4.11 Distribution of total SSRs in different motifs 81
19 4.12 Evaluation of SSR primer pairs for polymorphism in 16 sesame accessions 91
20 4.13 Dendrogram showing genetic diversity among 16 sesame accessions using the 92
newly isolated SSRs
21 4.14 Phenotypic distributions of yield and its components of BC F Population 96
1 2
S. No Title Page
No
22 4.15 Polymorphism between Swetha and BB-3-8. 98
23 4.16 Segregation pattern of BC F plants at SM15 in 3% agarose gel 99
1 2
24 4.17 Framework linkage map of sesame of cross Swetha X BB-3-8 104
25 4.18 QTL cartographer LOD peak for capsule length and seed yield 105
26 4.19 Evaluation of BC F population of TKG-22 X TMV-3 under green house 107
1 2
conditions
27 4.20 Bulked segregant analysis with SEM 248 108
28 4.21 Segregation pattern of BC F plants at SEM 248 in 3% agarose gel 109
1 2
29 4.22 Tagging of wilt resistance gene on linkage group 4 109

APPENDIX
S. No Title Page No
1 Vectors and enzymes 169
2 Oligo primers and other molecular biology chemicals 170
3 Buffers and solutions 171-174
4 Software used 175
Acronyms and Abbreviations

% : Per cent
AFLP : Amplified Fragment Length Polymorphism
APS : Ammonium per Sulphate
bp : Base pairs
BSA : Bulked Segregant Analysis
CIM : Composite interval mapping
CL : Capsule length (cm)
cM : Centi morgan
CTAB : Cetyl-Trimethyl-Ammonium Bromide
DAS : Days after sowing
DF : Days to flowering
DH : Doubled Haploid
DM : Days to maturity
DNA : Deoxy-Ribonucleic Acid
dNTP : Deoxy Nucleotide Tri Phosphate
EDTA : Ethylene Diamine Tetra Acetic Acid
EMS : Ethyl Methane Sulphonate
EST : Expressed Sequence Tag
FAOSTAT : Food and Agriculture Organisation Statistics
g : Gram
ha : Hectare
HCl : Hydro Chloric Acid
HI : Harvest index
HPTLC : High Pressure Thin Layer Chromatography
hr : Hour
i.e., : Which is to say in other words
IPTG : Isopropyl-Beta-D-Thiogalactopyranoside
ISSR : Inter-Simple Sequence Repeat
kb : Kilo Base Pairs
kDa : Kilo Dalton
L X T : Line X Tester
LB : Luria Broth
LG : Linkage Group
LOD : Logarithm of Odds
LOD : Likelihood of Odds Ratio/ Limit of detection
M : Molar
MAS : Marker Assisted Selection
MCS : Multiple Cloning Site
MDS : Multi Dimensional Scaling
mg : Milli Gram
MICAS : Microsatellite Analysis Server
min : Minute
ml : Milli Litre
mM : Milli Molar