discovery of qtl for root traits and water use efficiency in rice

201 Pages·2013·18.2 MB·English

DISCOVERY OF QTL FOR ROOT TRAITS AND WATER
USE EFFICIENCY IN RICE (ORYZA SATIVA L.) AND THEIR
VALIDATION- A COMBINED APPROACH OF
ASSOCIATION ANALYSIS AND LINKAGE MAPPING

RAJU, B. R.
PAL 0041

DEPARTMENT OF CROP PHYSIOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
BENGALURU
2013
DISCOVERY OF QTL FOR ROOT TRAITS AND WATER
USE EFFICIENCY IN RICE (ORYZA SATIVA L.) AND THEIR
VALIDATION- A COMBINED APPROACH OF
ASSOCIATION ANALYSIS AND LINKAGE MAPPING

RAJU, B. R.
PAL 0041

Thesis submitted to the
University of Agricultural Sciences, Bengaluru
In partial fulfillment of the requirements
For the award of the Degree of

DOCTOR OF PHILOSOPHY

In

CROP PHYSIOLOGY

BENGALURU JULY, 2013
Dedicated to my beloved parents
and teachers
DEPARTMENT OF CROP PHYSIOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES,
BENGALURU–560 065
C E R T I F I C A T E

This is to certify that the thesis entitled “Discovery of QTL for root traits and water
use efficiency in rice (Oryza sativa L.) and their validation- A combined approach
of association analysis and linkage mapping” submitted in partial fulfilment of the
requirement for the award of Doctor of Philosophy in Crop Physiology to the
University of Agricultural Sciences, Bangalore, is a record of bonafide research work
carried out by RAJU, B. R. bearing ID No. PAL 0041 under my guidance and
supervision and that no part of the thesis has been submitted for the award of any
other degree, diploma, associateship, fellowship or any other similar titles.

Bangalore, July, 2013 (M. S. SHESHSHAYEE)
Department of Crop Physiology
UAS Benguluru-65
Nominated Member: Approved by:

_________________________ Chairman: ____________________________
(S. ROBIN) (M. S. SHESHSHAYEE)
Members:
__________________________
(T. G. PRASAD)

_________________________
(B. MOHAN RAJU)

___________________________

(M. CHENNABYRE GOUDA)

____________________________
(M. P. RAJANNA)
AAAACCCCKKKKNNNNOOOOWWWWLLLLEEEEDDDDGGGGEEEEMMMMEEEENNNNTTTT

Foremost, I thank my family, for their support in every aspect of my life. I would not have
achieved anything without their love.
My sincere feelings, gratitude is expressed to Dr. M. S. Sheshshayee, Associate Professor, Department of
Crop Physiology, UAS, Bangalore and Chairman of my Advisory Committee and my mentor, for his
constant encouragement, enormous patience, and giving of his valuable time.
It is my privilege to express my insightful sense of gratitude to my Advisory Committee members, and
wish to express my whole hearted recognition to Dr.T.G. Prasad , emiratus Professor, Department of
Crop Physiology and Dr. B. Mohanraju, Associate Professor, Department of Crop Physiology, UAS,
GKVK, Bengaluru-65, for their vital suggestion, supports to conduct the experiments and manuscript
valuation. I sincerely owe my deep sense of gratitude to Dr. M.V. Chennabyre Gouda, Project Co-
ordinator, AICRP on small millets, UAS, GKVK, Bengaluru-65, and Dr. M. P. Rajanna, Rice
breeder, V C Farm, Mandya and members of my Advisory Committee for their kind help, constructive
suggestions and encouragement during my investigation.
I record my respectful indebtedness and gratitude to, my lab members for their wishes, ambitious
encouragement, unquantifiable love and affection. It has been a worthy privilege for me to have a
wonderful such lab mates, thanks for being there for me whenever I was in need. Thanks buddies.
On this occasion I whole heartedly thank the team at V. C. Farm, ZARS, Mandya i.e., Dr. P.
Mahadev, Junior Breeder; Mr. Manjunath, Senior Field Assistant and Prathap for the help they
rendered in completing my field work at Mandya. I also express my deep sense of gratitude to Srinivas,
all field boys and Thimme Gowda for the help they rendered for the smooth conduct of the field and Mr.
Lalith kishor vice president of GE health care lab work.
I also have been highly fortunate in having many affectionate class mates friends, seniors and juniors who
always stood with me and shared my anxiety, tensions, happy and sad moments without whom life would
be miserable. A personal note of gratitude to my lovable UG and PG friends, for their encouragement in
each and every step of my life. I gratefully acknowledge Department of Biotechnology for providing me the
financial support (under COE and DBT-HUB) during the due course of my research.
I request that any omission in this brief acknowledgement shall not be taken as lack of gratitude.

Bangalore, July, 2013 RAJU, B.R
Discovery of QTL for root traits and water use efficiency in rice
(Oryza sativa L.) and their validation- A combined approach of
association analysis and linkage mapping
Name: Raju, B. R. ID No.: PAL 0041
Thesis Abstract
Introgression of water mining and water use efficiency (WUE) traits on to a
single elite genetic background is needed while developing aerobic worthy rice
cultivars. Considering the complexity of inheritance of these traits, a focused
molecular breeding is a promising approach.

A set of 173 diverse indica rice accessions were phenotyped for these traits and
significant variability was noticed which was consistent across seasons, indicating a
strong genetic control. Assessment of molecular diversity using 291 SSR markers
detected a total of 2,615 alleles with an average of 8.95 allele per locus and an
average PIC value of 0.6. Population structure analysis identified six main subgroups
which corresponded to the diversity analysis derived from neighbour joining method.
Of the 108 significant marker-trait associations (MTAs) identified using mixed linear
model, 51 were highly significant (P<0.01). There were 24 associations for Root traits
and 1 for ∆13C and 16 for other physiological traits. Many of these markers were
found to be located in QTL regions previously reported for similar traits. A trait
specific mapping population was characterized for root traits and WUE under aerobic
conditions validating the associated markers. Significant phenotypic variations with
transgressive segregants were found for all the traits and 30 QTLs were detected. The
combined approach of association and linkage mapping lead to the identification of
several common markers viz., RM1235, RM8020, RM4477, RM163 and RM307
linked with the traits of interest. These markers can significantly enhance breeding
efficiency in introgressing complex traits. This novel two pronged strategy lead to the
discovery of 13 robust QTLs which on in-silico analysis revealed the presence of
genes that govern variability in cellular metabolism, transport and signal transduction,
transcription and hormonal regulation under abiotic stress adaptation and yield
improvement in rice.

(M.S. SHESHSHAYEE)
Major advisor
పపపప(cid:2)(cid:2)(cid:2)(cid:2)బబబబంంంంధధధధ (cid:7)(cid:7)(cid:7)(cid:7)ౕౕౕౕ(cid:9)(cid:9)(cid:9)(cid:9)(cid:10)(cid:10)(cid:10)(cid:10)(cid:11)(cid:11)(cid:11)(cid:11)ెెెె::::
భభభభతతతతదద(cid:16)(cid:16)దద(cid:16)(cid:16) (cid:18)(cid:18)(cid:18)(cid:18)ెెెెౕౕౕౕ(cid:19)(cid:19)(cid:19)(cid:19)నననన లలలల(cid:22)(cid:22)(cid:22)(cid:22)ణణణణగగగగ(cid:25)(cid:25)(cid:25)(cid:25)(cid:26)(cid:26)(cid:26)(cid:26)ెెెె మమమమతతతతుుుు (cid:16)(cid:16) (cid:16)(cid:16)జజజజలలలల(cid:30)(cid:30)(cid:30)(cid:30)(cid:31)(cid:31)(cid:31)(cid:31) ౕౕౕౕగగగగ (cid:22)(cid:22)(cid:22)(cid:22)మమమమ!!!!ెెెె(cid:26)(cid:26)(cid:26)(cid:26)ెెెె పపపప(cid:2)(cid:2)(cid:2)(cid:2)""""ాాాా$$$$ాాాాతతతత%%%%కకకక----గగగగుుుుణణణణ----((((ాాాానన))నన)) ----((((QTL)))) గగగగళళళళ ----ెెెె....ౕౕౕౕధధధధ////ెెెె
0000ాాాాగగగగుుుు అఅఅఅవవవవ3333గగగగళళళళ ధధధధృృృృ5555ౕౕౕౕకకకకరరరరణణణణ----ససససహహహహచచచచయయయయ(cid:10)(cid:10)(cid:10)(cid:10)!!!!ెెెెయయయయ ((((association)))) (cid:30)(cid:30)(cid:30)(cid:30)----ెెెెౕౕ;;ౕౕ;; షషషష$$$$ెెెె 0000ాాాాగగగగుుుు కకకకుుుు====(cid:11)(cid:11)(cid:11)(cid:11)ెెెె----
నననన(cid:11)(cid:11)(cid:11)(cid:11)ాాాా----ెెెెయయయయ////ెెెెూూూూ????ళళళళ(cid:26)(cid:26)(cid:26)(cid:26)ెెెెూూూూంంంండడడడ ((((linkage mapping)))) ససససంంంం ౕౕౕౕAAAAతతతత (cid:30)(cid:30)(cid:30)(cid:30)BBBBాాాానననన....
అDె(cid:31)ౕDావ(cid:19) పదEFGH ; బత(cid:16)వను? (cid:18)ెIెయ(cid:18)ెౕ(cid:11)ాదDె భూJయ ఆళLంద (cid:31)ౕరు Mౕరువ మతు(cid:16)
సంNాLOద జల(cid:30)/ెూPగ (cid:22)మ!ెయను? వృLEస(cid:18)ెౕకు. ఈ ఎరడు గుణగళను? ఒంTెౕ బత(cid:16)ద త(cid:25)(cid:26)ె
సం ౕజ/ె "ాడువ3దు అతPవశPక. ఈ ఎరడు V;షWకర గుణగళను? ఒంTెౕ త(cid:25)(cid:26)ె (ెౕ(cid:19)స(cid:18)ెౕ(cid:11)ాదH ;
ఆధు(cid:31)క ఆ=Xక త(cid:25) అYవృL E (cid:30)Bానవను? అనుస(cid:19)స(cid:18)ెౕ(cid:11)ాగుత(cid:16)Tె.
ప(cid:2)సు(cid:16)త అధPయనదH ; సు"ారు ౧౭౪ బత(cid:16)ద త(cid:25)గళ (cid:30)(cid:30)ధ -ా(cid:19)ౕ(cid:19)క గుణగళ "ాపన "ాడ]ా^తు.
_ౕల`ండ ఎరడు గుణగ(cid:25)(cid:26)ె ఉత(cid:16)మbాద bై(cid:30)ధP!ె ఇదు,e ఈ గుణగళg ధృడbాద అనువం(cid:7)ౕయ
(cid:31)యంత(cid:2)ణదH;bె ఎందు కండుబంLతు. ౨౯౧ ఎj.ఎj.ఆk గురుతు(cid:11)ారకగళను? బళO ౨౬౧౫
వంశbాM రూపగళను? ప!ె(cid:16)"ాడ]ా^తు.(ప(cid:2)F (ా)న(cid:11)ె` ౮.౯౫ వంశbాM రూపగళg, o.ఐ.O=౦.౬).
/ెDె0ెూంTా=(cid:11)ె (neighbor joining method) (cid:30)Bానవను? బళO సంతF-రచ/ెయను? (cid:30)ంగ5OTాగ ౬
గుంప3గ(cid:25)రువ3దు కండుబంతు. ౧౦౮ ఆ=Xక గురుతుగళg మతు(cid:16) అవ3గ(cid:25)(cid:26)ె 0ెూంL(cid:11)ెూండ గుణగళను?
Jశ(cid:2)-Dెౕsాత%క-సూత(cid:2) (mixed linear model) వను? బళO ప(cid:19)ౕtOTాగ ౫౧ 0ెూంTా=(cid:11)ెగళg
అథ(cid:10)గY(cid:10)తbాదువ3 ఎందు కండుబంLతు. ఈ ౫౧ రH,; ౨౪ గురుతుగళg ఉత(cid:16)మbాద (cid:18)ెౕ(cid:19)రువ
గుణ(cid:11)ె` 0ెూంL(cid:11)ెూం5దeDె, ౧ గురుతు ౧౩O(cid:26)ె, మతు(cid:16) ౧౬ గురుతుగళg ఇ(cid:31)?తర -ా(cid:19)ౕ(cid:19)క గుణగ(cid:25)(cid:26)ె
0ెూంL(cid:11)ెూం5దeవ3. ఈ MంTె కండుబంద 0ెూౕంTా=(cid:11)ెగళను? మరు (cid:31)రూoస]ా^తు. ఒందు
గు$ాBా(cid:19)త సంతFయను? ఆళbాద (cid:18)ెౕరుగ(cid:25)(cid:26)ె మతు(cid:16) జల(cid:30)(cid:31) ౕగ-(cid:22)మ!ె(cid:26)ె అDె(cid:31)ౕDావ(cid:19)
పదEFయH ; ప(cid:19)ౕtOTాగ, ఈ అధPయనLంద ౩౦ ప(cid:2)"ా$ాత%క-గుణ-(ా)న-(QTL)గ(cid:25)రువ3దు
కండుబంతు. ఈ సం ౕAత అధPయనLంద ముఖPbాద గుణగ(cid:25)(cid:26)ె O)రbాద గురుతు(cid:11)ారకగ(cid:25)రువ3దు
(RM1235, RM8020, RM4477, RM163 & RM307) (ాxౕ!ా^తు. ఈ గురుతు(cid:11)ారకగళను? భత(cid:16)ద
త(cid:25) అYవృL(cid:26)E ె బళసబహుదు. ఈ న(cid:30)ౕకృత అధPయనLంద ౧౩ అథ(cid:10)గY(cid:10)తbాద QTLగళను?
యశOXyాz ప!ె(cid:16)yాzbె. ఈ గుణ-(ా)నగళ ఇ{-OH(cid:11)ెూ (in silico) (cid:30)-ె;ౕష$ె(cid:26)ె ఒళప5OTాగ
అAౕ(cid:30)క ఒత(cid:16)డగళH; ఇళgవ(cid:19) సుBార$ె "ాడువ, (cid:11)ెూౕషగళH;న చyాపచయ V(cid:2)Gగళను?
(cid:11)ాయుe(cid:11)ెూళg|వ, సం(cid:11)ెౕత-చల/ె, (ా(cid:19)(cid:26)ె మతు(cid:16) ప(cid:2)}ెూౕదకగ(cid:25)(cid:26)ె సంభంLOద వంశbాMగ(cid:25)రువ3దు
కండుబంLతు.

(cid:30)BాP~(cid:10)య సM ముఖP సల0ె(cid:26)ారర సM
(Dాజు, x. అk) ((cid:127)ా.ఎం.ఎj. -ెౕష-ా^)
CONTENTS

SL.
TITLE PAGE NO.
NO
1 INTRODUCTION 1-4
2 REVIEW OF LITERATURE 5-28
3 MATERIAL AND METHODS 29-52
4 RESULTS 53-99
5 DISCUSSION 100-112
6 SUMMARY 113-115
7 REFERENCES 116-144
8 APPENDIXS
LIST OF TABLES

Table Page
Title
No. No.
List of important mechanism/ traits associated with drought
2.1 8
adaptation in rice for which variability was reported in literature
List of putative drought adaptive traits in rice for which variability
2.2 9
and QTLs has been reported
List of root traits in rice for which variability and QTLs were
2.3 12
reported
List of phenotyping methods for root traits reported in the
2.4 13
literature for rice
Several common definitions of water-use efficiency (WUE) for
2.5 17
which phenotyping methods are reported in the literature
Comparison between Bi-parental mapping and Association
2.6 22
mapping
Factors that control LD in a population are reported in the
2.7 22
literature
Progress in association mapping studies reported in rice using co-
2.8 24
dominant marker systems
List of rice databases reported in literature for crop improvement
2.9 26
studies
3.10 Climatic variable between the two locations 34
3.11 Details of traits recorded in root structure under aerobic condition 36
Details of traits recorded under both aerobic and puddled
3.12 37
conditions.
4.13 Summary statistics of molecular diversity in germplasm accessions 53
Analysis of molecular variance (AMOVA) for the six clusters of
4.14 55
rice accessions inferred populations
Genetic distance between subpopulation. Allele–frequencies
4.15 divergence among population (K=6 net nucleotide distance),

computed using point estimates on Structure 2.2.3.
Descriptive and correlation statistics for root and WUE traits in
4.16 57
germplasm accessions
Descriptive and correlation statistics for yield and components
4.17 57
traits
Descriptive and correlation statistics for gas exchange and other
4.18 59
morpho-physiological traits
4.19 Principal components analysis of germplasm accessions 61
Maker trait associations for root traits with MLM approach using
4.20 63
TASSEL 3.0
Maker trait associations for yield and component traits with MLM
4.21 63
approach using TASSEL 3.0
Table Page
Title
No. No.
Maker trait associations for gas exchange and other morpho-
4.22 63
physiological with MLM approach using TASSEL 3.0
4.23 Markers associated with different traits by LD mapping 66
4.24 Common markers associated for root, yield and other traits in rice 66
In-silico validation of markers associated with root traits using
4.25 68
gramene database
In-silico validation of markers associated with yield and
4.26 69
components traits using gramene database
In-silico validation of markers associated with WUE, leaf and
4.27 70
biometric traits using gramene database
Mean phenotypic variation of trait donor lines identified for trait
4.28 71
introgression.
Performance of parents (IET 15963 x Thanu) and RILs for root
4.29 traits and also for various morpho-physiological traits under 74
aerobic condition at Bangalore
Estimation of genetic variability for root traits and other morpho-
4.30 76
physiological traits of RILs under aerobic condition at Bangalore
Performance of parents (IET 15963 and Thanu) and RILs for yield
4.31 and other morpho-physiological traits under aerobic condition 79
(AC) at ZARS, Mandya.
Performance of parents (IET 15963 and Thanu) and RILs for yield
4.32 and other morpho-physiological traits under puddled condition 80
(PC) at ZARS, Mandya.
Estimation of genetic variability in RILs for yield and other
4.33 morpho-physiological traits under aerobic condition (AC) at 81
ZARS, Mandya
Estimation of genetic variability in RILs for yield and other
4.34 morpho-physiological traits under puddled condition (PC) at 82
ZARS, Mandya.
4.35 Classification of RIL population based on ∆13C and root biomass 90
Differences in various yield traits of RILs classified based on
4.36 relative performance of harvest index between aerobic and puddled 90
conditions
Summary of significant QTLs identified by composite interval
4.37 93
mapping in the RIL population.
Summary of common markers between association mapping and
4.38 95
bi-parental mapping
Genomic information of a few QTL selected for map based gene
4.39 97
mining
Summary of genes having non-synonymous SNP and their
4.40 98
description.