genetic diversity and molecular characterization of jatropha curcas and its allied species using pcr

279 Pages·2013·7.47 MB·English

“MOLECULAR ANALYSIS FOR DROUGHT
TOLERANCE IN UPLAND RICE (Oryza sativa L)”

A
THESIS
SUBMITTED TO
ANAND AGRICULTURAL UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF DEGREE
OF
Doctorate of Philosophy
IN

PLANT BIOTECHNOLOGY

BY
AMIT BALLANI
M. Sc. (BIOTECHNOLOGY)

DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY
ANAND AGRICULTURAL UNIVERSITY
ANAND-388 110

2013

Registration No. 04–0692-2008
“Molecular analysis for drought tolerance
in upland rice (Oryza sativa L)”

Name of Student Major Advisor
Ballani Amit C. Dr. G. C. Jadeja

Department of Agricultural Botany and Biotechnology
B. A. College of Agriculture
Anand Agricultural University
Anand-388110

ABSTRACT

Rice is one of the three major food crops of the world and contributes
significantly to India’s food grain production (42%). Globally, drought represents
a major constraint on rice production. Molecular and phenotypic studies (PVC
pipes and Field) were conducted in the present investigation for drought tolerance
in forty rice genotypes, ten NILs and six hybrids along with their parents.
PVC experimentation for two seasons revealed significant differences among
genotypes for several traits wherein GR-9, AAU DR-1, DDR-105 (Kharif -2009);
DDR-43 and SK-20 (Kharif -2011), exhibited better performance.
Field evaluation of forty genotypes revealed significant differences due to
genotypes and moisture regimes (stress and non-stress) for plant height, panicle
number and tiller number. Among genotypes, Anjali, N-22 and IR-64 showed
better performance. Grain yield showed positive and significant association of
high magnitude with maximum root length, root number and root volume
indicating their important role for increased yield. The path coefficient analysis
revealed importance of direct effects of panicle number and spikelet fertility and
indirect contributions of tiller number which resulted in positive association of
high order with grain yield.
During field evaluation of NILs & IR-64, NIL-4 was found superior for various
drought tolerance traits and can be used as potential parent for combining drought
tolerance with better grain quality and blast resistance. The cluster analysis based
on phenotypic and molecular data confirmed the improvement of NIL-4 over the
recurrent parent, IR-64.
The SSR analysis of forty genotypes with 27 microsatellite markers showed
polymorphism for all markers producing 195 alleles. The clustering pattern was
in line with geographical origin and parental gene pool of the genotypes. SSR
analysis of genotypes validated the use of few SSR markers for marker assisted
selection viz., RM205 for panicle length, RM206 for panicle number, RM212 for
maximum root length and RM263 for grain yield. The marker RM3825 was
found efficient in distinguishing the genotypes for grain yield under stress.
Significant and positive correlation (r = 0.069; P < 0.05) was observed between
matrix of molecular and morphological data as revealed by Mantel’s test.
SSR analysis of NILs with 17 polymorphic markers generated 194 alleles. The
molecular size of the amplified PCR products ranged from 90 (RM168) to 245bp
(RM242). Eighteen polymorphic microsatellite markers were used to analyze six
hybrids and their parents. The cross, GR-9 x Azucena was identified as the
potential hybrid for improving maximum root length, an important root trait
under stress condition.
Root length specific SCAR marker was validated for its discriminatory
efficiency, among the genotypes for the root length. Genotypes GR-9, Azucena
and UPLRI-7 were identified as potential parents for increasing root length. This
SCAR marker can be used in Marker Assisted Selection (MAS).
For the analysis of genetic diversity at nucleotide level, ten genotypes were
evaluated with six Single Nucleotide Polymorphism (SNP) markers and resulted
in the identification of 27 synonymous and 139 non synonymous SNPs.
The PVC pipes experimentation is a useful strategy to study root morphological
traits in rice and their role in drought tolerance. The association of these traits
with novel SSR and SNP markers can be explored for discrimination of drought
tolerance and marker assisted breeding. The need for efficient and functional
markers for discrimination of genotypes for drought tolerance is emphasized.
Dr. G. C. Jadeja
Professor and Head
Department of Agricultural Botany
Anand Agricultural University
Anand- 388 110
Gujarat, India.

CCEERRTTIIFFIICCAATTEE

This is to certify that the thesis entitled “Molecular analysis

for drought tolerance in upland rice (Oryza sativa L)” submitted by
AMIT BALLANI., Reg. No. 04-0692-2008 in partial fulfillment of the
requirements for the award of the degree of DOCTORATE IN
PHILOSOPHY in PLANT BIOTECHNOLOGY of the Anand
Agricultural University is a record of bonafide research work carried out
by him under my personal guidance and supervision. The thesis has not
previously formed the basis for the award of any degree, diploma or other
similar title.

Place: Anand (G. C. Jadeja)
Date: / / Major Advisor
DDEECCLLAARRAATTIIOONN

This is to declare that the whole of the research work reported here in
the thesis for the partial fulfillment of the requirements for the degree of
DOCTORATE OF PHILOSOPHY in PLANT BIOTECHNOLOGY,
by the undersigned is the results of investigation done by him under the
direct guidance and supervision of Dr. G. C. JADEJA, Professor and
Head, Department of Agricultural Botany, Anand Agricultural
University, Anand and no part of the work has been submitted for any
other degree so far.

Place: Anand
Date: / 06 / 2013 (Amit Ballani.)

Counter signed by

((DDrr.. GG.. CC.. JJaaddeejjaa))
Professor & Head
Department of Agricultural Botany
Anand Agricultural University
Anand-388 110
ACKNOWLEDGEMENT

This memorable occasion provides me a unique privilege to express my deep
sense of gratitude and indebtedness to my Honorable Major Advisor
Dr. G. C. Jadeja, Professor and Head, Department of Agricultural Botany and
Biotechnology, B. A. College of Agriculture, Anand Agricultural University, Anand,
for his valuable guidance, constant encouragement, sustained interest, expert guidance,
constructive criticism and judicious supervision throughout the course of this
investigation and scrutinizing the manuscript right from the selection of my research
problem up to the final shaping of the thesis in the present form.
I am very grateful to my Minor Advisor Dr. N. Subhash, Professor & Head,
“ANKUR” Department of Agril. Biotechnology, AAU, Anand for his valuable
suggestions and support during the entire investigation.
I express my gratitude to Dr. C. L. Patel, Chairman, CVM for granting us study
leave for the Ph.D. and providing us an opportunity to upgrade our academic
qualifications. I am also very much thankful to Dr. Basudeb Bakshi, Principal,
NVPAS, & Dr. Madhumati Bora, Head, Dept. of BT, GT & BNF, NVPAS,
VV Nagar.
I also thank to Dr. R. S. Fougat, Professor & Unit officer, Department of Agril.
Biotechnology, Dr. Atul Mehta, Research Scientist (Rice), Main Rice Research
Station, A. A. U., Nawagam, Dr. S.K.Dixit, Professor, Department of Agricultural
Statistics, for rendering his kind help and valuable suggestions for statistical analysis.
I am equally thankful to Dr. J.G Talati, Professor & Head, Dept of Biochemistry,
Dr.Y.M.Shukla, Associate Professor, Department of Biochemistry, BACA, AAU,
Anand for their kind help and guidance during my research work.
I express my heartfelt thanks to Dr. M.G. Mackwana, Main Rice Research
Station, A. A. U., Nawagam & Dr. S.H.Patel & Dr. Vishal Sharma, Derol research
sub-station, AAU, for providing seed material and concern during primary screening
for my research purpose without which my task could not have been accomplished.
My special thanks are due to Dr. M. M. Bhatt, Dr. D. B. Patel and Dr. N. Sasidharan,
Department of Ag. Botany and Biotechnology.
I owe my special thank to Dr. J.S.Patel, Professor & Head, Dr. V.B.Darji and
Dr.D.J.Parmar, Department of Agricultural Statistics and all the non teaching staff of
the Department of Ag. Botany and Biotechnology for their active co-operation during
the course of this study.
The limited world of words hampers to express the feeling of my indebtedness towards
my friends Dr. Kundan Mishra, Dr.Urvish Chhaya and Mr. Niraj kumar for their
affection, concern and unconditional support.
I would also like to express my gratitude to Mr. Amar Sakure & Mr. Adhir Ahir,
Dr. Yachana Jha, Asst. Professor and labmates particularly Sandeep Raj, R. Ravikiran,
Praveen and Vivek for helping me at numerous occasions during the course of my
research. I extend my thanks to Dilraj, Kewal, Awadh, Ruchi, George, Vijay, Sharad,
Indrapratap, Arpan, Kalyani, Ankita, Amrita, Bhooshan, Arun, Nilam, Maria, Mayur
and Gayatriben for their support.
I owe my reverence and gratefulness to my Parents without whose moral support and
sacrifice, my dream would not have come true. Particularly My Grandparents Smt.
Taradevi & Shri L.R.Ballani and parents Smt. Madhuri & Shri Chandraprakash
Ballani who first dreamed of this achievement and encouraged me to fulfill it with
vigour and enthusiasm. The encouragement and support provided by my wife sheetal,
daughter Niharika, brothers Sumit and Punit (my brothers), and other members of my
family might not be explained by words.
Place: Anand
Date : / / ( Amit C Ballani )
CONTENTS
PAGE
CHAPTER SECTION CHAPTER TITLE
NO.
I INTRODUCTION 1-6
II REVIEW OF LITERATURE 7-38
2.1 Rice statistics 7
2.2 Drought statistics 8
Various traits conferring drought tolerance
2.3 11
in rice
2.4 Importance of phenotyping 20
Statistical tools for morphological data and
2.5 23
SSR analysis
2.6 SSR markers studies 25
2.7 SNP primers 36
III MATERIALS AND METHODS 39-65
3.1 Experimental materials 39
3.2 Phenotypic studies 43
Study of root morphological traits in PVC
3.3 44
pipes:
3.4 Laboratory wares and reagents 50
3.5 Microsatellites (SSR) analysis 52
3.6 SNP analysis 61
IV RESULTS AND DISCUSSION 66-159
Screening of 40 rice genotypes in PVC
4.1 66
pipes (Season-2011)
Main evaluation of 40 rice genotypes in
4.2 78
PVC pipes.
Field evaluation of 40 rice genotypes
4.3 94
(Season-2011).
Field evaluation of Near Isogenic Lines of
4.4 102
rice (Season 2011)
Simple Sequence Repeat (SSR) marker
4.5 112
analysis
4.6 SCAR marker analysis 147
4.7 Single Nucleotide Polymorphism Studies 149
V SUMMARY AND CONCLUSION 160-172
VI REFERENCES 173-201
APPENDIX I-VIII
Appendix-1 I
Appendix-2 II
Appendix-3 IV
Appendix-4 IV
Appendix-5 V
Appendix-6 VI
LIST OF TABLES
Table Page
Title
No. No.
2.1 Putative traits for drought tolerance 14
Root traits and their functional characteristics in root QTL
2.2 17
mapping studies
2.3 List of SSR markers with associated QTL & traits 27
3.1 List of genotypes, their source and origin 40
3.2 List of NILs used in the present study 42
3.3 List of hybrids along with their parents 42
3.4 Scheme of field experimentation 43
3.5 Scheme of PVC pipe experimentation 45
3.6 Preparation of stock solutions for DNA isolation 53
3.7 Methods of preparation of buffers 54
3.8 List of SSR markers and their sequences 55
List of components of PCR master mix (25 µl) used for
3.9 57
amplification reaction of SSR primers
3.10 List of SNPs and gene specific primers 62
3.11 List of genotypes for SNP studies 62
3.12 Component of cycle sequencing reaction 64