fragrance linked-functional dna marker based evaluation of aromatic rice genotypes

99 Pages·2016·11.69 MB·English

FRAGRANCE LINKED-FUNCTIONAL DNA MARKER
BASED EVALUATION OF AROMATIC
RICE GENOTYPES

M. Sc. (Ag) Thesis

by

Sunita Singh

DEPARTMENT OF PLANT MOLECULAR BIOLOGY AND
BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
FACULTY OF AGRICULTURE
INDIRA GANDHI KRISHI VISHWAVIDYALAYA
RAIPUR (Chhattisgarh)
2016
FRAGRANCE LINKED-FUNCTIONAL DNA MARKER
BASED EVALUATION OF AROMATIC
RICE GENOTYPES

Thesis

Submitted to the

Indira Gandhi Krishi Vishwavidyalaya, Raipur

by

Sunita Singh

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF

Master of Science
in
Agriculture
(Plant Molecular Biology and Biotechnology)

UE ID No. 20141520350 ID No. 120114132

JULY, 2016

ACKNOWLEDGEMENT
“A journey is easier when you travel together. Interdependence is
especially more valuable than independence”. I have been accompanied and
supported by many people. It is a pleasant aspect that I got a golden opportunity to
express my gratitude for all of them.

I start in the name of God -who has bestowed upon me all the physical and
mental attributes that I posses and skill to cut through and heal a fellow human.

I take this opportunity to express my humble and deep sense of gratitude to
my Advisor and Chairman of Advisory Committee Smt. Archana S. Prasad,
assistant Professor of the Department Plant Molecular Biology And
Biotechnology, IGKV, Raipur, (C.G.). I have no words to express my thanks to her
for her illuminating guidance, unfailing encouragement, scholarly suggestion,
unique supervision, sympathetic attitude and keen interest during the course of this
investigation.

I express gratefulness thanks to Dr. S.B. Verulkar, Professor and Head,
Department of Plant Molecular Biology and Biotechnology, College of
Agriculture, IGKV, Raipur, whose inspiring suggestions, enthusiastic interest and
encouragement provided me solace during the tenure of investigation and for
providing all necessary facilities during the course of my experiment.

I have immense pleasure in expressing my whole hearted sense of
appreciation for the members of my Advisory Committee, Dr. Zenu Jha, Assistant
Professor, Department of Plant Molecular Biology and Biotechnology, Dr.
Bhawana Sharma, Assistant professor Department of Genetics and Plant
Breeding and Dr. R.R. Saxena, Professor, Department of Mathematics, Statistics
and Social Science (Language) for their co -operation, valuable suggestions and
healthy criticism towards completion of my thesis work.

I am highly indebted to other teaching staffs of my Department Dr. A. S.
Kotasthane, Dr. Girish Chandel, Dr. Shubha Banerjee and Dr. Kanchan Bhan
for their constant co-operation, suggestion, encouragement and help during my
investigation.

I wish to record my sincere thanks to Dr. S.K.Patil, Hon’ble Vice
Chancellor, Dr. S. S. Rao, Dean, College of Agriculture, Raipur, Dr. S. S. Shaw,
Director of Instructions and Dr. J. S. Urkurkar, Director Research Services,
IGKV, Raipur for their administrative and technical help which facilitated my
research work.

A heartfelt thanks to Sonam S. Kale mam for her constant co-operation,
suggestion, encouragement and help during my investigation.

I am deeply privileged to express heartfelt thanks to my classmates Deepak,
Meenakshi, Anjali, Alagrasan, Dayanand, Balwant, Sujata, Geetanjali, Sachin
i

TABLE OF CONTENT
CONTENT PARTICULAR Page No
ACKNOWLEDGEMENT i
TABLE OF CONTENTS i i i
LIST OF TABLES v i
LIST OF FIGURES v i i
LIST OF NOTATIONS v i i i
LIST OF ABBREVIATIONS i x
THESIS ABSTRACT x-xii
I INTRODUCTION 1-3
II REVIEW OF LITERATURE 4 - 1 4
2.1 Aroma 4
2.1.1 Aromatic gene discovery 4
2.1.2 Genetic mapping of grain aroma 5
2.2 Aromatic rice of India 5
2.2.1 Small and medium grained aromatic rice of 6
india
2.3 Economic status of aromatic rice 8
2.3.1 Economic status of aromatic rice in 9
Chhattisgarh
2.4 Conventional method of aroma detection in rice 9
2.4.1 Identification of rice flavour 9
2.4.2 Sensory analysis of aroma and flavour 11
2.5 Molecular markers based identification fragrant gene 11-14
III MATERIALS AND METHODS 15-32
3.1 Location and climate 15
3.2 Study I- Phenotypic characterization 15
3.2.1 Experimental materials 15
3.2.2 Methods 16
3.2.2.1 Observation recorded 16
3.2.2.1.1 Number of tillers / plant 20
3.2.2.1.2 Plant height (cm) 20

3.2.2.1.3 Panicle length (cm) 20
iii
3.2.2.1.4 Flag leaf length (cm) 20
3.2.2.1.5 Flag leaf width (cm) 20

3.2.2.1.6 Number of grains per 20
panicle
3.2.2.1.7 Number of sterile 20
spikelet’s
3.2.2.1.8 Total number of 20
spikelet’s per panicle
3.2.2.1.9 Seed length (mm) 21
3.2.2.1.10 Seed breadth (mm) 21
3.2.2.1.11 Seed L: B ratio 21
3.2.2.1.12 1000 seeds weight (g) 21
3.2.2.2 Statistical analysis 21
3.2.2.2.1 Mean 21
3.2.2.2.2 Frequency graphs 21
(Histograms)
3.3 Study II- Phenotyping of aroma using KOH method 22
3.3.1 Protocol followed for aroma detection using 22
KOH method
3.4 Study III- genotypic characterization using 22
functional/gene tagged markers
3.4.1 Genomic DNA isolation 22
3.4.1.1 Protocol for isolation of genomic 24
DNA (Miniprep method)
3.4.2 Quantification of genomic DNA 25
3.4.3 Polymerase chain reaction (PCR) 25
amplification
3.4.4 Agarose gel electrophoresis 29
3.4.4.1 Visualization of bands 30
3.4.4.2 Data scoring 30
3.4.4.3 Data analysis 30
3.5 Stock solution 30
3.5.1 Genomic DNA extraction buffer: 30
3.5.2 Solutions for electrophoresis 31
iv
3.5.3 solution for aroma detection 32
IV RESULTS AND DISCUSIONS 33-66
4.1 Phenotypic characterization 34
4.1.1 Charaterization of selected aromatic rice 34
genotypes and mapping population for yield
related traits
4.1.2 mapping population 39
4.1.3 Frequency distribution of aromatic rice 47
genotypes and mapping population
4.1.4 Mapping population 49
4.1.5 Aroma assay in aromatic rice genotypes and 53
mapping population
4.1.5.1 Aromatic rice genotypes 53
4.1.5.2 Mapping population 55
4.2 Genotypic characterization 55
4.2.1 Genotypic characterization of aromatic rice 57
genotypes
4.2.2 Genetic diversity 61
4.2.3 Genotyping of F individuals of mapping 61
2
populations
4.3 Study III- Survey of promoter of aroma 66
4.3.1 Identification of MITE elements in promoter 66
V SUMMARY AND CONCLUSIONS 67-69
REFERENCES 70-78
RESUME 79

v
LIST OF TABLE
Table Particular Page
3.1 List of selected 140 aromatic rice genotypes 17-18
3.2 Mapping populations used in the study 18
3.3 General information about field condition of mapping population 20
3.4 List of 29 markers used for genotypic characterization 23
3.5 List of DNA primers and their sequences 26-28
3.6 Reaction mixture used for polymerase chain reaction (PCR) 29
3.7 Temperature profiles used for PCR 29
Mean performance of aromatic rice genotypes for 13 yield and its
4.1 components traits in Kharif 2015 35
Mean performance of mapping population for 13 yield related
4.2 traits 40
Classification of aromatic rice genotypes based on their fragrance
4.3 score 54
Mean diversity analysis of 140 aromatic rice genotypes using
4.4 functional/gene tagged marker for aroma locus 57
Details of functional/gene tagged aroma locus specific markers
used for genotyping in the 140 aromatic rice genotypes and their
genetic diversity parameter using POWERMARKER v 3.25
4.5 program 59
4.6 MITE element present in 1 kb upstream of aroma gene 66

vi