Drought and Irrigated 2013

“IDENTIFICATION OF QTL FOR GRAIN YIELD AND YIELD
RELATED TRAITS UNDER DROUGHT AND IRRIGATED
CONDITIONS IN RICE”
M. Sc. (Ag.) THESIS
By
ASHISH KUMAR TIWARI
DEPARTMENT OF GENETICS AND PLANT BREEDING
COLLEGE OF AGRICULTURE
INDIRA GANDHI KRISHI VISHWAVIDYALAYA,
RAIPUR (C.G.)
2013
“IDENTIFICATION OF QTL FOR GRAIN YIELD AND YIELD
RELATED TRAITS UNDER DROUGHT AND IRRIGATED
CONDITIONS IN RICE”
THESIS
Submitted to the
Indira Gandhi Krishi Vishwavidyalaya, Raipur
By
ASHISH KUMAR TIWARI
IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE
DEGREE OF
Master of Science
In
Agriculture
(GENETICS AND PLANT BREEDING)
Roll No. 15259 ID No. 110307007
JULY 2013
ACKNOWLEDGEMENT
Research is not a single man show. It brings to light patience, vigor and dedication of the
person. It needs the close co-operation of the friends and colleagues and the guidance of experts in
the field to achieve something worthwhile.
Education plays fundamental role in personal and social development and teachers play a
fundamental role in imparting education. Teachers have crucial role in preparing young people not
only to face the further with confidence but also to build up it purpose and responsibility. There in
no substitute for teacher pupil relationship. A teacher is the one who moulds our rough edges, to
give us a smooth mind and you are the world of knowledge, love and care. Once a year, we honor
you all our lives we are grateful to say, you all my teachers.
"A journey is easier when you travel together; interdependence is certainly more valuable
than independence". I bow to Almighty “GOD” who keeps ever burning before my vagrant steps
the kindly light of hopes and always showered blessing on me without whose endless benevolence
and blessing this tedious task could not have been accomplished.
With a sense of high resolve and reverence, I would like to give my sincerest thanks to my
major advisor Professor (Dr.) S.B. Verulkar, Department of Plant Biotechnology, Indira Gandhi
Krishi Vishwavidyalaya, Raipur (C.G.), for his precious instruction, extra efforts, broad and
profound knowledge, unique supervision, his patient instruction, sparing his valuable time has
given me a great inspiration and help at every step during my thesis and pleasure to conduct this
thesis under his supervision.
With a great reverence, I express my sincere thanks to respected members of my advisory
committee Dr. A.K. Sarawgi, Professor, Department of Genetics and Plant Breeding, Dr. (Smt.)
Arti Guhey, Professor, Department of Plant Physiology, Dr. (Smt.) Ritu R. Saxena, Associate
Professor, Department of Genetics and Plant Breeding, and Dr. Ravi R. Saxena, Professor,
Department of Statistics, Mathematics and Computer science, College of Agriculture, Raipur, for
their critical suggestions, keen co-operation and kind help rendered as and when needed.
I wish to record my sincere thanks to Dr. S.K. Patil, Hon’ble Vice Chancellor, Dr.
Director of Research Services Dr. sarnayak and Dr. S. R. Patel, Director of Instructions, IGKV,
Raipur, for providing me the necessary facilities for research work.
Most humbly and respectfully I wish to express my profound sense of gratitude to Dr.
O.P. Kashyap, Dean, College of Agriculture, IGKV, Raipur, for his excellent guidance, valuable
suggestion, memorable advices and encouragement which is the vital source of inspiration in my
life. I pay my sincere thanks to Dr. A.K. Sarawgi, Professor and Head, Department of Genetics
and Plant Breeding for his excellence guidance during course of investigation and providing
necessary facilities.
I would like to specially thanks to Dr. (Smt.) Ritu R. Saxena, Associate Professor,
Department of Genetics and Plant Breeding, Dr. Nandan Mehta Scientist, Dr. Sunil Nair,
Professor, Mrs. Suchita Xalxo, Reaearch Associate, and Smt. Prabha Rani Choudhary, Assistant
Professor, Department of Genetics and Plant Breeding, COA, IGKV, Raipur, for their kind help
and valuable suggestion during the course of investigation.
CONTENTS
CHAPTER PARTICULARS PAGE
I INTRODUCTION 1-3
II REVIEW OF LITERATURE 4-22
2.1 Phenotypic evaluation of segregating population 5-6

for grain yield and related traits under drought
and irrigated condition
2.2 Impact of drought on yield and yield 6-7

contributing characters
2.3 Identification of QTL for grain yield under 7-12

irrigated and drought conditions
2.4 Development of genotypic data based on SSR 12-13

marker
2.4.1 SSRs (Simple Sequence Repeats) 13-14
2.5 Genome mapping 14
2.5.1 Molecular map 14-16
2.6 Development and concept of QTL analysis 16-20
2.7 QTL for drought tolerant 20-22
III MATERIALS AND METHODS 23-36
3.1 Materials 23
3.1.1 Characteristic features of parent 23-24
3.2 Methods 24
3.2.1 Field studies 24
3.2.2 Observations 24-25
3.2.3 Statistical analysis 25-26

3.2.3.1 Mean 25
3.2.3.2 Range 26
3.2.3.3 Standard Deviation 26
3.2.4 Molecular studies 26
3.2.4.1 Genomic DNA isolation 26-27
3.2.4.2 Quantification of DNA 28
3.2.5 PCR amplification using HvSSR primers 28
3.2.5.1 PCR reaction 28
3.2.5.2 Visualization of amplified products in 29

Polyacrylamide gel electrophoresis
3.2.5.3 Assembling and pouring the gel 29
3.2.5.4 Electrophoresis 30
3.2.5.5 Visualization of bands 30
3.2.5.6 Development of genotypic data of the population 31
3.2.5.7 Scoring of data 32
3.2.5.8 Scoring SSR and HvSSR banding pattern in 32

population
3.2.6 QTL analysis 33
3.3 Reagents and solutions 33
3.3.1 Reagents for PCR 33
3.3.2 Stock solutions 33
3.3.3 Solutions for electrophoresis 34
3.3.4 Stocks and solutions for PAGE 35-36
IV RESULTS AND DISCUSSION 37-58

4.1 General observations 37-38
4.2 Development of phenotypic data 38-45
4.3 Development of genotypic data based on SSR 45-37

and HvSSR Markers
4.4 Identification of QTLs 47-58
V SUMMARY, CONCUSIONS AND 59-61

SUGGESTIONS FOR FUTURE RESEARCH
WORK
ABSTRACT 62
REFERENCES 63-79
LIST OF TABLES
Table No. Title Page

No.
3.1 Characteristic features of parents 24
3.2 PCR mix for one reaction (volume 20 l) 28
3.3 Temperature profile used for PCR amplification using 28

microsatellite markers
3.4 SSR and HvSSR primers used for developing genotypic data 31-32
4.1 Phenotypic Data of population 42-43
4.2 Variability under different condition and different characters 44
4.3 Banding pattern of individual markers on segregating 49-50

population
4.4 QTL indentified for different traits 54-58

LIST OF FIGURES
Figure No. Particulars Between pages
4.1 Gel images showing parental polymorphisms 47-48
4.2 Composite marker analysis 50-51
4.3 Single marker analysis 50-51

LIST OF ABBREVIATIONS
% - Per cent
°C - Degree Celsius
µl - Microlitre
Approx. - Approximately
bp - Base pairs
Chr - Chromosome
cm - Centimeter
cM - Centimorgan
DNA - Deoxyribo nucleic acid
dNTPs - Deoxynucleotide triphosphates
dTTP - Deoxy thymidine 5‟ triphosphate
EDTA - Ethylene diamine tetra acetic acid
EtOH - Ethanol
Et br - Ethedium bromide
et al. - And others
g - Gram
H2 O - Water
ha - Hectares
Hrs - Hours
HvSSR - Highly variable single sequence repeat
i.e. - That is
m - Meter
M - Molar
MAS - Marker assisted selection
mb - Megabase pairs (106 bp)
MgCl2 - Magnesium chloride
min - Minutes
ml - Milliliter
NaCl - Sodium chloride
ηg - Nanogram
PCR - Polymerase chain reaction
QTL - Quantitative trait loci
rpm - Rotations per minute
SDS - Sodium dodecyl sulphate
SNP - Single nucleotide polymorphism
SSR - Single sequence repeats
TBE - Tris boric acid EDTA buffer
U - Units
ROS - Rain out shelter
INTRODUCTION
CHAPTER I
INTRODUCTION
Rice (Oryza sativa L.) is one of the most important food crops worldwide
belonging to the family Graminae and subfamily Oryzoidea. Rice is the staple food for
one third of the world‟s population and occupies almost one-fifth of the total land area
covered under cereals. It is grown under diverse cultural conditions and over wide
geographical range. Most of the world‟s rice is cultivated and consumed in Asia, which
constitutes more than half of the global population. Rice is cultivated under diverse
ecologies ranging from irrigated to rainfed upland to rainfed lowland to deep water.
Irrigated rice accounts for 55% of world area and about 75% of total rice production.
Rainfed lowland represents about 25% of total rice area, accounting for 17% of world
rice production. Upland rice covers 13% of the world rice area and accounts for 4% of
global rice production. Deepwater rice, although it has less area, meets the need of
around 100 million people. In India, the total area under irrigated, rainfed lowland and
upland rice is 22.0, 14.4, and 6.3 million ha, respectively (Singh et al., 2009).
Asia is the leader in rice production accounting for about 90% of the world‟s
production. Over 75% of the world supply is consumed by people in Asian countries and
thus rice is of immense importance to food security of Asia. The global production of rice
has been estimated to be at the level of 680 million tonnes and the area under rice
cultivation is estimated at 156 million hectares (FAOSTAT, 2009). India rank 1 st in area
(43.92 million ha) and 2nd in production (91.61 million tonnes) after China (pandey and
veruker, 2010). Chhattisgarh the central eastern state is also called the “Rice bowl of
1
India”. Total area of rice in Chhattisgarh is 3.6 million ha. production is 5.5 million
tonnes and productivity is 1517 kg/ ha. in 2008-09 (Anonymous, 2009).
This productivity is substantially low than the national average. Approximately
70% of rice over in Chhattisgarh is rainfed and frequent drought of different intensity at
different stage often causes substantial loss in productivity. The losses due to drought
have been reported by Verulker et al., (2010).
Breeding for drought tolerance in rice has been difficult mainly because of
complex genetic nature of inheritance, interaction of many secondary trait contributing
toward tolerance. Highly unpredictable nature in terms of stage and intensity which all
male conventional breeding tool less efficient.
In the past half century, rice yield has benefitted from two major genetic
improvements: improved harvest index and plant architecture through use of semidwarf
genes, and production of hybrids that exploit heterosis. Consequently, rice yield has more
than doubled in most parts of the world and even tripled in certain countries and regions
in the last 50 years. The developments in genome mapping, sequencing, and functional
genomic research have provided powerful tools for investigating the genetic and
molecular bases of these quantitative traits. Dissection of the genetic bases of the yield
traits based on molecular marker linkage maps resolved hundreds of quantitative trait loci
(QTLs) for these traits. Molecular characterization of these genes has greatly advanced
the mechanistic understanding of the regulation of these rice yield traits. These findings
have significant implications in crop genetic improvement.
With the increasing world population, rice yield is still a hot topic in rice
breeding despite the increasing grain yield after the green revolution. Particularly under
2
changing climate Most of the improved rice cultivars now grown in the rain fed drought-
prone areas were bred for irrigated conditions and are highly susceptible to drought.
Recently, QTLs with large effects on grain yield under drought stress, namely DTY 1.1,
DTY2.1, DTY2.2, DTY3.1, DTY4.1, DTY9.1 and DTY12.1 have been identified by IRRI
scientists, explaining 31-77% of the phenotypic variance.
This study aims at identification of QTL for yield and yield related traits under
draught as well as irrigated condition using locally adopted doner, which will result in
identification of molecular markers linked to important trait for further use in marker
assisted selection (MAS).
Objectives of investigation:
1. Phenotypic evaluation of segregating population for grain yield and related traits
under drought and irrigated condition.
2. Development of genotypic data based on SSR marker.
3. Identification of QTL for grain yield under different set of conditions.
3
REVIEW OF LITERATURE
CHAPTER-II
REVIEW OF LITERATURE
Drought is one of the major limited factors that affect the growth of rice. The
chromosomal regions associated with drought tolerance in rice using SSR mapping
system were detected in this study. Grain yield in rice is a complex trait multiplicatively
determined by its three component traits: number of panicles, number of grains per
panicle, and grain weight; all of which are typical quantitative traits.
Dissection of the genetic bases of the yield traits based on molecular marker
linkage maps resolved hundreds of quantitative trait loci (QTLs) for these traits. Mutant
analyses and map-based cloning of QTLs have identified a large number of genes
required for the basic processes underlying the initiation and development of tillers and
panicles, as well as genes controlling numbers and sizes of grains and panicles.
Molecular characterization of these genes has greatly advanced the mechanistic
understanding of the regulation of these rice yield traits. These findings have significant
implications in crop genetic improvement.
In this chapter an attempt has been made to review the available literatures on
genetic mechanism and identification of QTLs for yield and various yield contributing
traits in rice. The available literature has been classified into following headings:
2.1 Phenotypic evaluation of segregating population for grain yield and
related traits under drought and irrigated condition.
2.2 Impact of drought on yield and yield contributing characters
2.3 Identification of QTL for grain yield under irrigated and drought
conditions
4
2.4 Development of genotypic data based on SSR marker.
2.4.1 SSRs (Simple Sequence Repeats)
2.4.2 HvSSR (Highly variable Simple Sequence Repeats)
2.5 Genome mapping
2.5.1 Molecular map
2.6 Development and concept of QTL analysis
2.7 QTL for drought tolerant
2.1 Phenotypic evaluation of segregating population for grain yield and related
traits under drought and irrigated condition.
Phenotypic or morphological identification and characterization of segregating
population of rice in different sets of condition (irrigated and drought) for the
development of phenotypic data related to plant height, effective tillers, grain yield, and
other related trait.
Woperesis et al. (1996) conducted an experiment during water stress condition
flower induction and inflorescence development leads to delay in flowering (anthesis)
or even to complete inhibition as apical morphogenesis is sensitive to water deficit.
Hittalmani et al. (2003) conducted an experiment in Asia Genotype-by-
environment (GxE) interaction analysis for 11 growth and grain yield related traits in
nine locations was estimated by AMMI analysis and found that an approximate normal
distribution was observed for phenotypic performance of the traits in all environments
and the mean performance of traits indicates the effect of environment on phenotype.
Deshmukh et al. (2007) observed that in rice, water stress during flowering can
reduce the harvest index by as much as 60%, largely as a result of a reduction in grain set.
5
Panicles in stress plants fail to full exert (emerge) from the flag leaf sheath, flowering is
delayed and the percentage of spikelet that open at anthesis is reduced. The failure of
panicle exertion alone accounts for approximately 25% to 30% of spikelet sterility
because the unexerted spikelet cannot complete anthesis and shed pollen, even when
development is otherwise normal.
2.2 Impact of drought on yield and yield contributing characters
Puckridge et al. (1981) they conducted an experiment in biomass production of
rice is a function of water use. The shortage of water in the soil suppresses the leaf
expansion, tillering and photosynthetic rate along with leaf area due to senescence. All
these factors are responsible for reduction in dry matter accumulation.
Row et al. (1983) they conducted an experiment in yield component of rice
varieties viz, Rasi and IR-20 under moisture condition. The reproductive and ripening
phase were vulnerable and crucial for moisture stress, which resulted in permanent
damage to growth and yield.
Krishnaya and Murty (1991) they conducted an experiment in five upland rice
varieties to soil moisture stress of 25% field capacity at seedling stage. With increase in
soil moisture stress, there was a decrease in stress, relative water content and water
potential of leaf. The cultivar which maintained high relative water content and positive
turgor, in spite of reduced leaf water potential during stress also had optimum
photosynthesis and solute accumulation.
Pantuwan et al. (2002) reported that the drought stress developed prior to
flowering generally delayed the flowering of genotypes and such a delay was associated
with drought susceptibility in rice.
6
Verulkar and Shrivastava (2009) reported that delay in flowering under drought
conditions was related to low water status and was an indicator of drought susceptibility.
Delay in flowering was also associated with higher spikelet sterility.
2.3 Identification of QTL for grain yield under irrigated and drought conditions
Molecular approaches to drought tolerance have been widely applied to rice,
beginning with QTL analysis. QTLs have been identified for many traits that are
associated with drought response such as root characters, membrane stability, osmotic
adjustment and morphological and physiological traits where tolerance is measured as
yield under drought.
Venuprasad et al. (2001) worked on IR64 X Azucena DH mapping population of
rice in three diverse environments and detected QTLs for ten traits at a threshold. The
QTLs were spread across six chromosomes 1, 3, 4, 5, 6 and 7. Three QTLs for grain yield
were detected one each on chromosome 3, 4 and 5.
Hittalmani et al. (2002) detected a total of 34 quantitative trait loci (QTL) for 11
traits across three locations; one QTL was identified for grain yield per plant.
Price et al. (2002) reported 24 QTLs for various root growth traits in population
of a cross between Azucena and Bala in rice for drought tolerance.
Hittalmani et al. (2003) reported a genomic region of 7.9 cM (from 135.8 to 143.7
cM) on chromosome 1 was associated with drought resistance traits such as leaf rolling,
number of spikelet, heading date and harvest index in IR64/ Azucena rice DH lines.
Similar result was found by Kanagraj et al. (2010) by using 23 recombinant inbred (RI)
lines of IR20/ Nootripathu.
7
Babu et al. (2003) reported 47 QTLs for leaf rolling and drying scores, days to
heading (days after sowing), plant height (cm), grain yield, biomass, spikelet fertility,
grains per panicle, harvest index and relative yield by interval mapping via. QTL Mapper
version 1.0 in a doubled-haploid rice population of 154 lines from CT9993 and IR62266
in different trials.
Verulker et al. (2004) reported to identification of 143 QTLs for various traits.
Jonaliza et al. (2004) reported on yield and its stability in rainfed and poorly
irrigated environments. They reported genomic regions influencing the response of yield
and the genetics of drought tolerance and development of more drought tolerant cultivars.
Quantitative trait loci (QTL) for grain yield and its components and other agronomic
traits were identified and found that QTL were identified in different chromosomal
locations for each irrigated condition.
Lafitte et al. (2004) identified 31 QTLs for yield and its components under rice
drought on working with used population of a cross between Bala X Azucena.
Lanceras et al. (2004) used population of a cross between CT9993 and IR62266
and identified 77 QTLs for yield; yield components, panicle sterility etc. in rice.
Xiao et al. (2005) reported identification of 2, 4, 4, 1, 2, 2 and 3 QTLs for grain
yield, 100 kernel weight, kernel number per ear, cob weight per ear, kernel weight per
ear, ear weight and ear number per plant respectively under well-watered regime and 1, 5,
2, 6, 1, 3 and 2 QTLs respectively under water-stressed condition.
Xu et al. (2005) identified 36 QTLs in introgression indica lines of rice for yield
and its components under drought.
8
Yue et al. (2006) reported 39 QTLs for productivity, spikelet fertility, leaf rolling,
biomass, harvest index and grain weight with population of indica lowland X tropical
japonica upland.
Gomez et al. (2006) were identified 24 QTLs for various physio-morphological
and plant production traits under drought stress. The number of QTLs per trait under
stress was: 5 for leaf rolling, 4 for leaf drying, 3 for days to 50% flowering, 5 for plant
height, 2 for number of productive tillers, 1 for panicle length, 3 for grain yield and 1 for
straw yield.
Bernier et al. (2007) using population of 436 random F3 derived line from a cross
between the upland rice cultivar Vandana and Way Rarem reported identification of QTL
(qtl12.1) on chromosome 12 with large effect on grain yield under stress. They also
reported that under stress also increases harvest index, biomass yield and plant height
while reducing the number of days to flowering.
Kumar et al. (2007) reported detection of a QTL on chromosome 1 near sd1 that
explained 32% of the genetic variation for yield under stress, but only 4% under non-
stress. They also found their effect consistence across years.
Kanjivavila et al. (2008) conducted an experiment in India for identifying
genomic regions contributing to drought resistance. Total of 11 QTLs was identified for
various plant phenology and production traits under rainfed and irrigated conditions.
Cui et al. (2008) reported 34 QTLs for plant height etc. by using Zhenshan97/
Minghui63.
Kato et al. (2008) reported identification of 3 QTLs on chromosomes 2, 4 and 7
related to relative growth rate in a backcross inbred population of rice „Akihikari‟
9
(lowland cultivar) × „IRAT109‟. Srinivasan et al. (2008) identified 19 QTLs for plant
height, 50% flowering, grain yield, biomass and harvest index on chromosome 1, 2, 4, 6,
9 and 12 respectively.
Bernier et al. (2009) while studying the effect of qtl12-1 on grain yield and
associated traits in rice over a range of environment in Philippines and eastern India
reported that the relative effect of the QTL on grain yield increased with increasing
intensity of drought stress, from having no effect under well-water conditions to having
additive effect of more than 40% of trail mean in the most severe treatment.
Khowaja et al. (2009) reported QTL for rolling, stomatal conductance,
dimentional traits, drought avoidance, plant height, plant biomass, leaf morphology and
root traits.
Fu et al. (2010) reported 3 regions underling significant QTLs for several yield-
related traits were detected on chromosome 1, 7 and 12 by using an accession of common
wild rice (Oryza rufipogon Griff.) The QTL clusters were found and corresponding
agronomic traits of those QTLs showed highly significant correlation, suggesting the
pleiotropism or tight linkage.
Gomez et al. (2010) reported 22 Consistent QTLs for drought-resistance traits
were detected using locally adapted indica ecotypes, a subset of 250 lines of F8
generation derived from two indica rice lines (IR20 and Nootripathu), which may be
useful for rainfed rice improvement.
Liu et al. (2010) reported 32 quantitative trait loci (QTLs) for panicle traits and
dry grain weight were identified, with contribution rates ranging from 3.33% to 22.66%.
Eleven epistatic QTLs were detected. Cases of collocated QTLs under control and
10
drought were found for panicle length, spikelet density, grain number per plant, primary
branch number and secondary branch number.
Xing et al. (2010) reported QTLs for grain yield on chromosome 1, 5, 6 and 7,
and for grain weight on chromosome 1, 3, 5 and 7 by using Zhenshan97/ Minghui63.
They also identified gene gw2 on short arm of chromosome 2 which controlling grain
width and weight and gene gs3 which responsible for grain length.
Chakraborty et al. (2011) conducted an experiment on QTL Mapping for days to
flowering under drought condition in rice (Oryza sativa L.) and found that rice is very
sensitive to moisture stress during flowering resulting in high floret sterility.
Ghimire et al. (2012) conducted an experiment on identification and introgression
of QTLs for grain yield under drought is a preferred breeding strategy to improve the
drought tolerance of popular elite rice varieties. Crossed with Swarna and IR64 to
develop two recombinant inbred line populations. The two populations were phenotyped
for grain yield under reproductive-stage drought stress and non-stress during DS2010 and
DS2011.
Mishra et al. (2013) conducted an experiment on QTL, qDTY12.1, significantly
associated with grain yield under reproductive-stage drought stress was identified on
chromosome 12 with a consistent effect in two environments: IRRI, Philippines, and
RARS, Nepalgunj, Nepal. This QTL explained phenotypic variance of 23.8% and
contributed an additive effect of 45.3% for grain yield under drought. The positive QTL
allele for qDTY12.1 was contributed by tolerant parent IR74371-46-1-1.
Yadav et al. (2013) conducted an experiment on BC1-derived mapping population
developed from the cross IR77298-5-6-18/2*Sabitri, a large-effect QTL, qDTY3.2, was
11
identified on the short arm of chromosome 3 near RM231. This QTL explained 23.4% of
the phenotypic variance for grain yield under severe lowland drought and had a
consistent effect across varying water stress severities in the Philippines and Nepal in
DS2011, WS2011 and WS2012.
2.4 Molecular markers
A molecular marker can be defined in one of the following ways: (a) a
chromosomal landmark or allele that allows for the tracing of a specific region of DNA;
(b) a specific piece of DNA with a known position on the genome (Semagn et al., 2006)
or (c) a gene whose phenotypic expression is usually easily discerned, used to identify an
individual or a cell that carries it, or as a probe to mark a nucleus, chromosomes, or locus
(Semagn et al., 2006).
Molecular markers are now well established as powerful tools for indirect
selection of difficult traits at the seedling stage during plant breeding, thus speeding-up
the process of conventional plant breeding and facilitating the improvement of difficult
traits that cannot be improved easily by the conventional methods. In this direction, a
large number of genes and QTLs controlling agronomic traits and conferring tolerance to
both abiotic and biotic stresses have been identified and tagged using molecular markers
in several crop species especially cereals (Varshney et al., 2009).
There are many types of DNA markers that are produced by a wide variety of
techniques (Tyagi et al., 2004; Xu et al., 2010) yet only a subset has been widely used for
whole genome mapping projects. RFLPs, RAPDs, AFLP, Simple Sequence Repeats
(SSRs) and more recently SNP are the most popular molecular markers used in genome
mapping. DNA markers are employed to construct the genetic map of organism, to map
12
the genes governing qualitative and quantitative traits, to exercise indirect selection for
various agronomic traits and to isolate the gene based on map position.
2.4.1 SSRs (Simple Sequence Repeats)
McCouch et al., (1997) Simple Sequence Repeats (SSRs), also known as
microsatellites, are present in the genome of all eukaryotes. These are ideal DNA markers
for genetic mapping and population studies because of their abundance. These are
tandemly arranged repeats of mono, di, tri, tetra and penta nucleotides with different
lengths of repeat motifs (A, T, AT, GA, AGG, and AAAC etc.). These repeats are widely
distributed throughout plant and animal genomes that display high levels of genetic
variation based on differences in the number of tandemly repeating units at a locus.
(McCouch et al., (2002) In rice more than 2500 microsatellite markers have been
developed and used to construct genetic map.
Singh et al. (2009) developed 436 HvSSR markers which are evenly distributed in
the rice genome.
SSRs are easier to use than Restriction Fragment Length Polymorphism (RFLP),
owing to the smaller amount of DNA required, higher polymorphism and the ability to
automate assays. SSR markers can easily be exchanged between researchers because each
locus is defined by the primer sequences. SSR assays are more robust than Randomly
Amplified Polymorphic DNA (RAPD) and more transferable than Amplified Fragment
Length Polymorphism (AFLP). SSRs are now replacing RFLPs in genetic mapping of
crop plants. A combination of SSRs with AFLPs is used to produce detailed genetic
maps. The co-dominant nature of SSRs is also an advantage for genetic mapping.
13
The SSR markers have been used widely for rice germplasm evaluation,
interpreting population structure and genetic dissection of quantitative trait loci (QTL) for
important agronomic traits (Akkaya et al. 1992; Yang et al. 1994; McCouch et al. 1997,
2002; Burrow and Blake 1998; Cho et al. 2000; Rahman et al. 2009)
2.5 Genome mapping
The developments in field of molecular biology have opened up avenues for
directed improvement of crop plants, which were unthinkable till recently through
conventional means. Botstein et al. (1980) published the first landmark paper giving a
conceptual framework for construction of saturated genome maps employing DNA based
genetic marker in human. Mapping and sequencing would help to explain gene function,
gene regulation and their expression. High-resolution linkage maps are being developed
for many crops. Construction of detailed genetic map for the crop of interest will make
available a precise but vast amount of information that plant breeders can use to identify,
manipulate and complement traits to their maximum advantage.
Jones et al. (1997) defined mapping as putting markers in order, indicating the
relative genetic distances between them, and assigning them to their linkage groups on
the basis of the recombination values from all their pairwise combinations.
2.5.1 Molecular map
Price et al., (2002) The advent of saturated molecular maps promised rapid
progress towards the improvement of crops for genetic complex traits like drought
resistance via. analysis of QTLs.
Linkage map is a linear or a circular diagram that shows the relative position of
gene on a chromosome as determined by genetic analysis.
14
McCouch et al., (1988) The first restriction fragment length polymorphism
(RFLP) map of rice was constructed in the 1980s at Cornell University.
McCouch et al. (1997) Current genome coverage provided by these simple
sequence length polymorphisms (SSLPs) is sufficient for genotype identification, gene
and quantitative trait locus (QTL) analysis, screening of large insert libraries and marker-
assisted selection in breeding.
Temnykh et al. (2000) developed and evaluated some 188 new microsatellite
markers for allelic diversity. The new simple sequence length polymorphisms (SSLPs)
were incorporated into the existing map previously containing 124 SSR loci. The 312
microsatellite markers reported provide whole-genome coverage with an average density
of one SSLP per 6 cM.
Shanmugasundaram, (2001) Maps are constructed based on the linkage and
crossing over between the genes or markers in a genome. The tendency of two or more
genes to stay together during inheritance is known as linkage. Linkage is the consequence
of the concerned genes being located on the same chromosome.
Shanmugasundaram, (2001) Construction of linkage maps with molecular
markers, parents are chosen that show the maximum of polymorphic loci in order to
ensure the mapping of as many as possible (Nagarajan, 2001). In such studies, it is
desirable, to include only those genes which shows less than 20% preferably, 10% or less
recombination with each other to avoid confusion due to double and triple cross over. If
the crossing over between the genes in test crosses, were more than 20% the linkage
maps would not be very reliable.
15
Mu et al. (2003) was produced a molecular linkage map for drought resistance
with 94 RFLP markers and 71 SSR markers covering 1535.1 cM by using double haploid
(DH) population of IRAT109 x Yuefu.
SNP, RFLPs, RAPDs, simple sequence reports (SSR) and more recently AFLPs
are most popular molecular markers used in genome mapping.
A genetic linkage map was constructed by Gomez et al. (2010) using 101
polymorphic PCR-based markers distributed over the 12 chromosomes covering a total
length of 1529 cM in 17 linkage groups with an average distance of 15.1 cM this study
involve 250 RILs, F8 generation of IR20/ Nootripathu.
2.6 Development and concept of QTL analysis
Quantitative trait (QT) is a term of central importance in the field of biology and
agriculture. As the term indicates, QT refers to characters that can be measured on a
quantitative scale (Arunachalam, 2001). Many important characters are a consequence of
the joint action of several genes. Such characters are often referred to as polygenic or
quantitative. The genetic loci for such characters have been referred to as quantitative
trait loci (QTLs).
Several characters of plant species, including traits of agronomic importance are
inherited quantitatively. Yield, maturity date and drought tolerance are examples of such
characters. Although, the principles of QTL analysis were first outlined and successfully
applied in the early 1920s to map a QTL for seed size in tightly linked to a gene
controlling seed pigmentation (Sax, 1923), a wide scale application of QTL analysis was
not possible at that time due to the paucity of genetic markers available.
16
The systematic identification and characterization of QTLs was finally made
possible more than 50 years later, following the introduction of the first class of
molecular markers (restriction fragment length polymorphism, RFLP) suitable for an
adequately detailed genome wide survey (Bostein et al., 1980). The time consuming
process of map construction was considerably shortened with the introduction of the PCR
based microsatellite (SSR, simple sequence repeat) markers, particularly suited for
mapping purposes due to their high level of polymorphism (Taramino and Tingey, 1996).
From the mid 1990s, the addition of AFLPs (amplified fragment length polymorphism
(Vos et al., 1995) has provided an unprecedented level of map saturation (Vuylsteke et
al., 1999).
In principle, once QTLs have been identified, introgression of the favorable
alleles and their pyramiding into elite germplasm (e.g. parental lines, populations etc.)
becomes possible through marker-assisted selection (MAS) (Ribaut and Hoisington,
1998; Stuber et al., 1999). However, only a few successful applications of MAS for the
improvement of quantitative traits have been described to date (Hu et al., 1997; Ribaut et
al., 2000) due mainly to weak association (in terms of genetic distance) between markers
and target QTLs and/ or the high costs of MAS (Stuber et al., 1999; Salvi et al., 2001).
Certainly, a more rosy picture for MAS emerges considering single-gene traits such as
disease resistance (Witcombe and Hash, 2000).
There are several statistical methods developed to analyze mapping data to search
systematically for major QTLs in experimental organisms. The traditional method known
as single marker analysis or point analysis is used to detect association between a QTL
and a marker. Thoday (1961) and Soller and Brody (1976) described the idea of using
17
single genetic markers one at a time to systematically characterize and map individual
polygene‟s controlling the quantitative trait. For detecting a QTL near a marker,
phenotypic means for the progeny of each marker class are compared (Edwards et al.,
1987). The difference between the means provides an estimate of the phenotypic effect of
substituting a 'B' allele for an 'A' allele at the QTL. A one-way analysis of variance (i.e.,
linear regression) can be performed to test whether the inferred phenotypic effect is
significantly different from zero. A significant value indicates that a QTL is located in the
vicinity of the marker. The additive effect associated with the marker locus can be
estimated by linear regression of marker genotype means on genotypes
Stuber et al. (1992) and Lander and Botstein (1989) proposed a method known as
interval mapping using the likelihood approach (LOD score) which has been in use
widely now-a-days. In this method, instead of using one marker at a time, sets of linked
markers are analyzed simultaneously with regard to their effects on the quantitative traits.
They developed a computer program known as Mapmaker/ QTL (Lincoln et al., 1992)
for implementing interval mapping, which is being used commonly (Redona and Mackill,
1996; Xiao et al., 1996). Typically, a LOD score threshold value of between 2 and 3,
depending on the density of genetic markers, chromosome length and trait distributions,
is required for declaring the significance of a putative QTL (Doerge and Rebai, 1996).
Kruglyak and Lander (1995) recommended that the dense-map threshold always be used,
regardless of the actual density of the map, in order to minimize the false positive rate.
Churchill and Doerge (1994) and Doerge and Churchill (1996) proposed some methods
of estimating empirical threshold values for declaring significant QTL effects in a
18
genome or at any point within a genome by the application of a permutation test. These
methods can also be used for detecting the presence of minor QTLs effects.
Stuber et al. (1992) and Bubeck et al. (1993) compared the one-at-a-time analysis
method with the maximum likelihood-based interval mapping method. Both analytical
methods showed virtually the same results in detecting QTL because maximum
likelihood estimates reduce least squares estimate when data are normally distributed.
Jansen and Stam (1994) proposed a method for multiple linear regression of a
quantitative phenotype (putative QTLs) on genotype (marker).
There are three widely used methods for detecting QTLs, single marker analysis,
simple interval mapping and composite interval mapping (Tanksley, 1993). Single
marker approach, sometime referred to as the single factor analysis of variance (SF-
ANOVA) or simple point analysis, is the simplest method for detecting QTLs associated
with single markers. SF-ANOVA is done for each marker locus independent of
information from other loci. F-test provides evidence whether differences between
marker locus genotype classes are significant or not. The statistical methods used for
single-marker analysis include t-test, analysis of variance (ANOVA) and linear
regression. Linear regression is most commonly used because the coefficient of
determination (R2) from the marker explains the phenotypic variation arising from QTL
linked to the marker. This method does not require a linkage map and can be performed
with basic statistical software programs. However, the major disadvantage with this
method is that the further a QTL is from a marker, the less likely it will be detected. This
is because recombination may occur between the marker and the QTL. This causes the
magnitude of the effect of a QTL to be underestimated (Tanksley, 1993). QGene and
19
MapManager QTX are commonly used computer programmes to perform single-marker
analysis (Nelson, 1997)
Hittalmani et al. (2003) DNA markers have been most useful in dissecting
complex agricultural traits like QTLs controlling drought, disease, salinity, yield traits,
cold tolerance, maturity etc. most of these traits have not only been mapped and QTL
identified, their effect quantified, the alleles detected, some QTLs have been cloned and
characterized. In few cases the most probable genes underlying the QTL have been
identified. This is the most useful area where markers could make an impact on plant
breeding applications with precision. There is now enough information available on
QTLs, which would help us select the superior alleles of different genes controlling the
complex traits via. markers.
Different software was developed for QTL analysis viz., MAPMAKER/ QTL
(Lincoln et al., 1992), QTL cartographer (Basten et al., 1997), Map Manager QT (Manly
and Elliot, 1991), MQTL (Tinker and Mather, 1995).
2.7 QTL for drought tolerant
Vikram P. et al. (2011) reported that major QTL for GY under RS, qDTY1.1, was
identified on rice chromosome 1 flanked by RM11943 and RM431 in all three
populations. In combined analysis over two years, qDTY1.1 showed an additive effect of
29.3%, 24.3%, and 16.1% of mean yield in N22/Swarna, N22/IR64, and N22/MTU1010,
respectively, under RS. qDTY1.1 also showed a positive effect on GY in non-stress (NS)
situations in N22/Swarna, N22/IR64 over both years, and N22/MTU1010 in DS2009.
Mallikarjuna Swamy BP et al. (2011) reported that The integration of 15 maps
resulted in a consensus map with 531 markers and a total map length of 1821 cM. Fifty-
20
three yield QTL reported in 15 studies. Fourteen meta-QTL were obtained on seven
chromosomes. MQTL1.2, MQTL1.3, MQTL1.4, and MQTL12.1 were around 700 kb and
corresponded to a reasonably small genetic distance of 1.8 to 5 cM and they are suitable
for use in marker-assisted selection (MAS). The meta-QTL for grain yield under drought
coincided with at least one of the meta-QTL identified for root and leaf morphology traits
under drought in earlier reports. Validation of majoreffect QTL on a panel of random
drought-tolerant lines revealed the presence of at least one major QTL in each line.
DTY12.1 was present in 85% of the lines, followed by DTY4.1 in 79% and DTY1.1 in
64% of the lines. Comparative genomics of meta-QTL with other cereals revealed that
the homologous regions of MQTL1.4 and MQTL3.2 had QTL for grain yield under
drought in maize, wheat, and barley respectively. The genes in the meta-QTL regions
were analyzed by a comparative genomics approach and candidate genes were deduced
for grain yield under drought. Three groups of genes such as stress-inducible genes,
growth and development-related genes, and sugar transport-related genes were found in
clusters in most of the meta-QTL.
Deming et al. (2004) reported that drought stress during reproductive stages of
rice showed severe drought stress in panicle development stage caused high yield loss
by reducing spikelet per panicle in upland rice.
Jonaliza C. et al. (2004) conducted that in Rice total 77 QTL were identified for
grain yield and its components under varying levels of water stress. Out of the total of 77
QTL, the number of QTL per trait were: 7-grain yield (GY); 8-biological yield (BY); 6-
harvest index (HI); 5-d to flowering after initiation of irrigation gradient (DFAIG); 10-
total spikelet number (TSN); 7-percent spikelet sterility (PSS); 23-panicle number (PN);
21
and 11-plant height (PH). The phenotypic variation explained by individual QTL ranged
from 7.5% to 55.7%. Under wellwatered conditions, and high genetic association for BY,
HI, DFAIG, PSS, TSN, PH, and GY. However, only BY and HI were found to be
significantly associated with GY under drought treatments. QTL flanked by markers
RG104 to RM231, EMP2_2 to RM127, and G2132 to RZ598 on chromosomes 3, 4, and
8 were associated with GY, HI, DFAIG, BY, PSS, and PN under drought treatments. The
aggregate effects of these QTL on chromosomes 3, 4, and 8 resulted in higher grain yield.
Xu JL. et al. (2005) reported that a large set of 254 introgression lines total of 36
quantitative trait loci (QTLs) affecting heading date (HD), plant height (PH), GY and
yield components were identified and most QTLs showed pronounced differential
expression either qualitatively or quantitatively in response to drought. These QTLs could
be grouped into three major types based on their behaviors under control and stress
conditions. Type I included 12 QTLs for stress and non-stress conditions. Type II
comprised 17 QTLs for irrigation but not under stress. Type III included seven QTLs that
were apparently induced by stress.
Kamoshita A. et al. (2008) Reported that identified and listed a number of QTLs
for many droughtresistance traits, such as deep roots and four key genomic regions on
chromosomes 1, 4, 8, and 9 on which are co-located a number of QTLs for traits
considered to be directly or indirectly responsible for grain yield under stress.
Venuprasad et al. (2009) reported DTY3.1 which has been shown to have a strong
influence on grain yield under drought stress in populations derived from the cross Apo/
IR64.
22
MATERIALS AND METHODS
CHAPTER Ш
MATERIALS AND METHODS
The present study entitled “Identification of QTL for grain yield and yield
related traits under drought and irrigated conditions in Rice” was conducted at the
research farm, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya, Raipur
(C.G.), (210 16‟ N and 810 36‟ E at altitude of 289.6 meter above sea level), during Kharif
season 2012, Rice crop was grown in two different conditions: Irrigated and rain out
shelter during the month of June to November to generate the phenotypic data under
managed field conditions.
Genotypic data was generated at Plant Molecular Biology Laboratory
Department of Genetics and Plant Breeding, College of Agriculture, Indira Gandhi Krishi
Vishwavidyalaya, Raipur (C.G.) using microsatellite markers for understanding the
genetics of drought tolerance and identification of QTLs for yield and yield contributing
traits under irrigated and water stress conditions.
3.1 Materials
The planting material was 60 F3 progenies of a cross between MTU1010 and
IR86931-B-6 (derived from Nagina22). Each F3 progeny had 10 plants, under irrigated
condition.
3.1.1 Characteristic features of parent
The characteristic features of parents included in present study are presented in
table 3.1.
23
Table 3.1: Characteristic features of parent
Reaction to water
S. No. Parent Pedigree Salient Features
stress under field
1. MTU-1010 Krishnaveni x Susceptible Semi-dwarf, grains-
IR64 long slender, white,
resistant to blast.
2. IR86931-B-6 Nagina22 Tolerant Semi-tall, grains-
medium slender.
3.2 Methods:
3.2.1 Field Studies
The F3 lines derived from a cross between MTU 1010 X IR86931-B-6 rice
cultivar ware evaluated in the field during wet season 2012 at research cum instructional
farm of College of Agriculture, IGKV, Raipur. The field trials for F3 generation were
conducted under irrigated and drought condition (Rain out shelter). The fields selected
for the study were upland in topology with good drainage with high percolation rate and
had sandy loam soil. The plant material was sown in raised bed nursery on 18 th June 2012
and transplanted after 15 days of sowing under puddled irrigated field condition (60
lines). After 25 days of transplanting two tillers from individual plant was separated and
transplanted under rain out shelters. Under irrigated condition normal package of
practices was followed while under rain out shelter plants was exposed to water stress
after 30 days of transplanting.
3.2.2 Observations
3.2.2.1 Observations under field condition
1. Days of flowering
Number of days for initiation of flowering on individual plants basis.
2. Tillers/plant
Total no. of individual plants tillers were recorded on per plants.
24
3. Plant height (cm)
Plant height was measured in centimeter (cm) from soil surface to tip of the tallest
panicle at maturity of each plant.
4. Panicle length (cm)
Single panicle length of per plants was measured in centimeters from base to tip
of panicle.
5. Effective tiller/plants
Total number of effective tillers bearing panicle was counted on per plants basis.
6. Grain yield (g)
The actual yield of grain in gram per plants was recorded.
7. DII (Drought intensity index)
DII= 1- (Ramírez-Vallejo and Kelly, 1998)
Where s is the mean experiment yield of all genotypes grown under stress, and i is the
mean experiment yield of all genotypes grown under non-stress conditions.
3.2.3 Statistical analysis
3.2.3.1 Mean
Mean is the average value of observation of genotypes of a series. It represents
the standard average value over fluctuation in the environment. Mean was calculated by
the following formula:
Where,
∑Xi = Summation of all the observations
n = Total number of observations
25
3.2.3.2 Range
It is the difference between the highest and the lowest terms of a series of
observation.
Range = XH - XL
Where,
XH = Highest value
XL = Lowest value
3.2.3.3 Standard deviation
A measure of dispersion of variable around the general mean and is a calculated

by summing up the squares of the deviation of each observation from the mean and
dividing by the number of observations and then extracting the square root according to
formula:
Where,
i = an observation or variate value
= Arithmetic mean of the population
n= Number of given observation
3.2.4 Molecular studies
One plant from each progeny row was randomly selected for developing the
genotypic data of the segregating population for subsequent QTL analysis.
3.2.4.1 Genomic DNA isolation
DNA was isolated by modified Miniprip method. Its procedure was as followed:
1. 1-2 g of young, leaves was collected.
2. Leaves were cut into small pieces and transferred to 2ml centrifuge tube
containing 500 µl of extraction buffer along with stainless steel beads.
26
3. These centrifuge tubes containing leaves along with extraction buffer were
fixed in tissue homolyzer (MO. BIO. powerlyzer 24) and it was operated in
two cycles each cycle at 2400 rpm for about 2 minutes having 5 seconds pause
between each cycle.
4. Then, the centrifuge tubes were removed from homolyzer, after that the
stainless steel beads were taken out with help of magnetic rods.
5. These tubes were centrifuged at 14000 rpm for about 5 minute using mini
centrifuge.
6. After that 400 µl of 24:1 choloroform: Iso amyl alchohol was added and
mixed well using spinix.
7. Later tubes were centrifuged at 14000 rpm for about 5 minutes to settle down
the debris.
8. Supernatant was taken out into fresh new 1.5ml centrifuge tube carefully with
micro pipettes.
9. Steps 6, 7 and 8 were repeated once again.
10. Double the amount of supernatant, 100% absolute ethanol was added and
kept it at -20O C for about 30 minutes to precipitate the DNA. After that it was
centrifuged at 14000 rpm for about 5minutes to settle and form the pellet of
DNA and Pellet was washed with 70% ethanol.
11. Pellet was kept for air dry until ethanol smell in tubes was gone.
12. After drying 100 µl of TE buffer was added and tap it gently to dissolve the
DNA pellet.
13. DNA was stored at -200C until use.
27
3.2.4.2 Quantification of DNA
The DNA samples isolated from each line were quantified using Nano Drop
Spectroscopy (NANODROP 2000c). After quantification, the DNA was diluted with TE
buffer such that the final concentration of DNA was 40ηg/μl for PCR analysis.
3.2.5 PCR amplification using SSR and HvSSR markers

3.2.5.1 PCR reaction
About 2 l of diluted template DNA of each genotype was dispensed in the
bottom of PCR plates. Separately cocktail was prepared in an eppendorf tube as described
in table 3.2. 18 l of cocktail was added to each tube to make final volume 20 l.
Table 3.2: PCR mix for one reaction (Volume 20 l)
Reagent Stock concentration Volume ( l)

1) Nanopure H2O - 13.5
2) PCR buffer 10 X 2.0
3) dNTPs (Mix) 10 Mm 1.0
4) Primer (forward) 5 pmol. 0.5
5) Primer (reverse) 5 pmol. 0.5
6) Taq polymerase 1 unit/ l 0.5
7) DNA template 40 ηg/ l 2.0
Total 20
Table 3.3: Temperature profile used for PCR amplification using microsatellite
markers
Steps Temperature ( C) Duration (min.) Cycles Activity
1 95 5 1 Denaturation
2 94 1 Denaturation
3 55 1 34 Annealing
4 72 2 Extension
5 72 10 1 Final Extension
6 4 24 hrs 1 Storage
28
3.2.5.2 Visualization of amplified products in Polyacrylamide gel electrophoresis
Five percent polyacrylamide gels (vertical) were used for better separation and
visualization of PCR amplified microsatellite products, since polyacrylamide gels have
better resolution for amplified products. Gels were casted in CBS-SCIENTIFIC
electrophoresis unit. Glass plates were prepared before making the gel solution. Both
glass plates (outer and inner notched glass plates) were cleaned thoroughly with warm
water,detergent and then with deionised water.
3.2.5.3 Assembling and pouring the gel
Gasket was fixed to the three sides of the outer plate (without notches).
Spacers of 1.5mm thickness were placed along the sides by just attaching
the gasket of outer plate.
Later, notch plate was kept on the outer plate so that spacers were between
the two plates. Clamps were put on the three sides of plates leaving notch
side of unit. It was checked with water to found any leakages.
For casting each gel, 65 ml of acrylamide gel (5%) solution was prepared
just prior to pouring. For each 65 ml of solution, 60 μl of TEMED (N-N-
N-N-Tetramethylethylene diamine) and 600 μl of (freshly prepared)
ammonium per sulphate (10 %) (APS) were added to initiate the
polymerization process.
The contents were mixed gently by swirling, but bubbles were avoided.
Before pouring, assembly was kept on the bench top so that it made 45
degree angle with bench top.
29
Then gel solution was poured from notch side with maximum care to
avoid air bubbles. Comb of 1.5 mm thickness (60 and 63 wells) was
inserted with tooth side in the gel.
Later assembly was kept for polymerization for 20-30 min.
3.2.5.4 Electrophoresis
After polymerization process, gasket was removed and assembly was kept
in the electrophoresis unit with electrophoresis unit clamps so that notch
side facing inner side of the unit and facing other plate without notch to
outer side
TBE (1x) was poured in upper tank in the unit and the rest was poured in
bottom chamber.
Comb was removed with care so that it does not disturb the wells formed.
At last, 4 l loading dye (10x) was added to PCR products.
Finally, 6 l of each sample were loaded into the wells for facilitating the
sizing of the various alleles. Ladder (50bp) was loaded in the first well.
Gel was run at 180 volts till the dye reached bottom of the gel.
3.2.5.5 Visualization of bands
After electrophoresis, clamps were removed and glass plates were separated
without damaging the gel.
a) Gel was taken out from plate into staining box with care by flipping the
gel with help of spatula and by pouring little amount of water for easy
removal.
30
b) Ethidium bromide solution (prepared by adding 10 l to 200 ml double
distilled water was poured into the staining box to stain the gel.
c) It was agitated for about five minutes to stain the gel.
d) Gel stained with Ethdium Bromide was washed two times with double
distilled water to have clear images.
e) The gels were scanned with the help of BIO-RAD gel doc XR+..
f) Care was taken while using TEMED and staining with Ehtidium bromide
solution as they are carcinogenic and mutagenic agents, respectively.
3.2.5.6 Development of genotypic data of the population
The primer exhibiting polymorphism on parents were selected and use in
segregating population for PCR amplification on all 60 lines of population along with
parents. Genotypic data was generated with a set of 48 polymorphic primers i.e. SSR and
HvSSR both presented in table 3.4.
Table 3.4: SSR and HvSSR primers used for developing genotypic data
S.N. NAME CHROMOSOME POSITION(cM)

1 RM1 1 29.7
2 RM11943 1 37.85
3 RM12146 1 40.71
4 RM490 1 51
5 RM9 1 92.4
6 RM486 1 153.5
7 RM109 2 0
8 HvSSR2-12 2 4.5
9 HvSSR2-27 2 7.72
10 HvSSR2-44 2 17
11 RM279 2 17.3
12 RM174 2 33.8
13 RM530 2 158
14 HvSSR3-9 3 3.8
15 HvSSR3-41 3 15.51
16 RM231 3 15.7
17 RM232 3 76.7
31
S.N. NAME CHROMOSOME POSITION(cM)
18 RM55 3 168.2
19 RM168 3 171.2
20 RM468 3 202.3
21 RM3471 4 16.7
22 RM335 4 21.5
23 RM153 5 0.5
24 HvSSR5-13 5 3.11
25 HvSSR5-48 5 19.82
26 HvSSR5-65 5 27.22
27 RM169 5 34.7
28 RM164 5 91.4
29 RM190 6 7.4
30 RM225 6 26.2
31 RM111 6 35.3
32 RM340 6 133.5
33 RM481 7 3.2
34 RM152 8 9.4
35 RM25 8 52.2
36 RM483 8 60.9
37 RM444 9 3.3
38 RM242 9 73.3
39 RM553 9 76.7
40 RM278 9 77.5
41 RM228 10 130.3
42 RM21 11 85.7
43 RM206 11 104.2
44 RM1233 11 112.9
45 RM309 12 74.5
3.2.5.7 Scoring of data
The banding pattern of each line was developed by each HvSSR and SSR
markers were scored separately.
3.2.5.8 Scoring SSR and HvSSR banding pattern in population
The female parent band was scored as „A‟ while male parent band was
scored as „B‟ the bands of individual scored either as A or B depending on its position
32
like A and B parent, respectively. The bands other than A and B were termed as E and
both type bands present in single locus they called h type band.
S. No. Code Type of band

1. A MTU1010 like allele
2. B IR86931-B-6 like allele
3. E Other type
4. H Heterozygous
3.2.6 QTL analysis
(a) Single marker analysis using QTL cartographer
(b) Interval mapping using QTL cartographer
3.3 Reagents and solutions
3.3.1 Reagents for PCR
a. Primers: Highly variable microsatellite markers from ILS, USA.
b. dNTPs: (dATP/dCTP/dGTP/dTTP)
1 mM stock of dNTP (Genei) was used.
c. PCR buffer (10X)
10X Genaxy buffer was used
d. Taq polymeras
1 unit/µl, Taq polymerase (Genaxy) was used for PCR.
3.3.2 Stock solutions
a. DNA extraction buffer
Tris HCl (1M; pH-8) 5 ml
EDTA (0.5M; pH-8) 10 ml
NaCl (4M) 7.5 ml
33
SDS (20% W/V) 5 ml
Final volume was adjusted to 100ml with distilled water.
b. TE buffer
1M Tris-Hcl (pH-8) 1 ml
0.25 EDTA 0.4 ml
Final volume was adjusted to 100 ml and autoclaved.
c. EDTA (0.5M; pH-8)
186.12 g of EDTA was dissolved in 700 ml of distilled water. The pH was set
to 8 using NaOH. Final volume was adjusted to 1000 ml with distilled water and
sterilized by autoclaving.
d. 4M NaCl
23.36 g of NaCl was dissolved in 80 ml of distilled water. Final volume was
adjusted to 100 ml and sterilized by autoclaving.
e. 1M Tris HCl (pH 8.3 at 25°C)
30.28 g of Trizma base was dissolved in 200 ml of distilled water. The pH
was set to 8.3 using concentrated HCl. The final volume was adjusted to 250 ml with
distilled water and sterilized by autoclaving.
3.3.3 Solutions for electrophoresis
a. 10X TBE buffer
Tris base 104 g
EDTA (0.5M) 40 ml
Boric Acid 55 g
Distilled water 500 ml
Final volume was adjusted to 1 liter with distilled water.
34
b. 1X TBE buffer
100 ml of 10 X TBE + 900 ml of distilled water was taken to make 1 lt of 1X
TBE.
c. 10X loading dye
Sucrose 667 mg
Bromophenol Blue 4.2 mg
Water 1.0 ml
3.3.4 Stocks and solutions for PAGE
A) 5% Acrylamide gel solution (1000ml)
S.No. Stock For 1000ml

1. Acrylamide 47.5g
2. Bis-Acrylamide 2.5g
3. 10X TBE 100ml
Autoclaved milliQ water (to make up volume to 1000ml) Acrylamide and
bis-acrylamide were weighed and dissolved in 500ml distilled water and then
added to the beaker containing 100 ml of 10X TBE and the volume was made upto
1000ml by adding autoclaved double distilled water. The solution was sterilized
by passing through 0.22 micron and stored in amber colour bottle at 4 0 C.
B) 10% Ammonium persulphate (APS) solution was prepared by mixing following
components
S.No. Component Final concentration
1. Ammonium persulphate 1.0g
2. Distilled water 10ml
35
3.4 Instruments used in the laboratory
o Veriti 96 well thermal cycler (Applied Biosystems)
o Refrigerated centrifuge
o Microwave oven
o C.B.S. PAGE unit with power pack
o Transilluminator and Gel documentation system
o Micropipettes
o Eppendorf tubes
o Electronic balance.
36
RESULTS AND DISCUSSION
CHAPTER-IV
RESULTS AND DISCUSSION
The present investigation entitled “Identification of QTL for grain yield and
yield related traits under drought and irrigated conditions in Rice” was carried out
with the main objectives of genetic analysis and identification of the QTLs for grain yield
and yield contributing characters for drought tolerance. The segregating population was
phenotyped under two set of environmental conditions, one under irrigated condition and
another under rain out shelter condition during wet season 2012. The mean data of
irrigated and rain out shelter condition were used for genetic and QTL analysis. The
DNA from selected plants per lines of segregating population along with parents were
extracted, quantified and amplified in PCR using SSR and HvSSR primers to generate the
genotypic data. The phenotypic and genotypic data were further used for QTL analysis.
The results thus obtained are presented under following headings:
4.1 General observations
4.2 Development of phenotypic data
4.3 Development of genotypic data based on SSR and HvSSR Markers
4.4 Identification of QTLs
4.1 General observations
Overall, during the crop season total rainfall of 1716.7 mm was received, which is
approximately 22% more than normal. With good rainfall along with normal agronomic
practices under irrigated condition, the mean productivity of all the genotype was about
48 quintal per hectare indicating normal production potential as there genotype at least
under normal condition.
37
Since the drought stress is highly unpredictable in nature therefore genotype
specifically designed for rainfed condition should have normal productivity under normal
condition (Verulker et al. 2010).
On the other hand under both the rainout shelters water stress was imposed at the
time of reproductive phase which resulted in substantial reduction in yield. The mean
yield per plant was 15.03gm in ROS1 and 10.5gm in ROS2 which resulted in drought
intensity index of about 0.6 to 0.7 respectively. At reproductive phase the mean
tensiometer reading was ~ -32 K Pascal and gravimetric soil moister content of about 20-
30% at soil depth of 30cm.
There was only sporadic incidence of disease and pest. Under irrigated condition
5 plants from each F3 progeny row ware selected and observation recorded on number of
agronomic trait while under ROS1 and 2 phenotypic observation were received on both
the plants of each F3 progeny.
4.2 Development of phenotypic data
Observations were recorded on various morphological traits under both the
condition i.e. under irrigated and ROS#1 and #2. Under irrigated condition 5 plants were
randomly selected and mean of these 5 plants was further used for analysis. Phenotypic
data represented in table 4.1 and population variability measurement presented in table
4.2.
38
4.2.1 Condition - Irrigated
(a) Total tillers at 45 DAS
Total tiller of F3 population ranged from 21 maximum (in line no. 121) to 3
minimum (in line no. 26, 27, 29, 30, 31, 32, 34, 35, 36, 39, 40, 44, 45, 48, 49, 50) with
mean of 5.2 and there standard deviation was 3.4 indicating wide range of variability.
(b) Seedling Plant height (cm)
Within total population, mean plant height was recorded as 69.79cm with
maximum plant height in line no. 18 which had height of single plant as 89cm and
minimum plant height in line no. 20 and 25 and calculated standard deviation was 9.15. A
wide variability for seedling height was also reported by Bernier et al. (2007),
Kanjivavila et al. (2008) and Bai et al. (2011).
(c) Days of flowering
Mean days to flowering was recorded as 98 days in total population which rang
from early flowering of 89 days in line no.16 and 48 and highest value of 105 days in
line no. 22 and 37 with standard deviation of 4.66. This variation was expected as both
parents involved vary greatly for flowering.
(d) Effective tiller
Calculated mean of effective tiller was 5.6 in the total population which ranged
from 15 (highest) counted in line no. 21 and 1 (lowest) in line 37, with calculated
standard variation as 3.4. Similar result has been reported by Hittalmani et al. (2003) Gomez
et al. (2006) and Bernier et al. (2007).
39
(e) Panicle length (cm)
The mean of panicle length was counted 25.7cm and in total population it ranged
from 30cm. (highest in line no. 41) and lowest 19.7cm (in line no. 32) with the 2.18 of
standard deviation.
(f) Plant height at maturity (cm)
The mean value of plant height was recorded to be 103.9cm and it ranged from
highest value of 148 cm in line no. 21 and lowest value of 72 cm in line no. 18 and 35;
with standard deviation of 13.86.
(g) Grain yield (g)
Mean of grain yield was recorded to be 41.21g in total population raging between
84g highest in single plant line 45 and lowest 10g in single plant line no. 16 with standard
deviation of 79.96. The wide variation for grain yield recorded in this study indicates that
this population could be an ideal population for selection. Wide variability for grain yield
has been reported by Bernier et al. (2007), Kanjivavila et al. (2008), Then et al. (2011)
Bai et al. (2011) and Mishra et al. (2013).
4.2.2 Condition - Rain out shelter1st
(a) Days to flowering
The mean of days to flowering in rain out shelter 1 st was observed to be 98 days
and in total population it ranged from 108 days highest observed in single plant line no.
22, and lowest 93 days observed in single plant line no. 16, with the 22.0 of standard
deviation.
40
(b) Effective tiller
Mean number of effective tillers in total population was 3.6 which ranged from 7
in line no. 48 and lowest value of 2 in line no. 22, 25, 32, 33, 41, 44, 49. The overall
mean number of effective tillers was lower compared to irrigated condition as rainout
shelter has light soil, upland condition and numbers of tillers are highly influenced by this
condition.
(c) Panicle length (cm)
In total population, mean of panicle length was calculated as 22.7cm ranging from
26.5cm highest value in single plant line no. 21and lowest value in single plant line no 25
along with 5.3 standard deviation.
(d) Grain Yield (g)
The mean of rain out shelter 1st yield was observed 15.03g and in total population
it ranged from 37.3g highest observed in single plant line no. 33, and lowest 2g was
observed in single plant line no. 18, with the 8.61 of standard deviation. Again under
ROS condition with water stress, good range of variability was observed for this trait.
4.2.3 Condition - Rain out shelter 2nd
(a) Days to flowering
In rainout shelter 2nd mean value for days to flowering was calculated as100 days
in the total population, it ranged from highest of 108 days in single plant line no. 22 and
lowest in single plant line no. 16 with standard deviation of 22.01.
41
Table 4.1: Phenotypic Data of population
ROS1
Total Tiller Grain ROS1 ROS1 ROS1 ROS2 ROS2 ROS2 ROS2
S.N. Seedling PH (cm) DF ET PL (cm) PH at maturity Yield
45 Das Yield(g) DF ET PL(cm) DF ET PL(cm) Yield(g)
(g)
1 5 66 104 14 25 97 120 107 4 24 22.8 106 4 22 12.4
2 5 62 95 3 27.5 102 140 96 3 25 7.1 96 3 26 10.3
3 5 59 102 8 26 112 140 101 4 22.5 26 101 7 24 19
4 4 68 103 6 26 116 200 0 0 0 - 101 6 23 -
5 8 66 98 8 26 116 180 102 3 22.5 18.4 101 4 27 11.2
6 5 72 100 4 27 119 250 96 3 23 19.3 97 5 24 9.3
7 8 87 89 6 27.5 108 50 93 4 23 23 93 6 22.8 18.3
8 7 76 102 6 28 123 270 103 3 24 14.4 104 5 26.5 11.3
9 6 89 92 2 23 72 60 97 3 21 2 98 3 16 9.2
10 5 63 104 3 25 83 250 98 4 22.5 9.6 98 4 23.5 13.2
11 5 55 95 3 21 98 150 99 3 24.5 21 99 8 21.5 9.1
12 21 86 102 15 28 148 240 107 4 26.5 27.7 107 8 22 -
42
13 4 61 105 6 28 117 290 108 2 19.5 17.5 108 5 22.5 4.8
14 7 66 95 7 28 107 160 98 5 23 5.4 98 5 22 7.5
15 11 59 96 12 23.5 94 200 95 4 21.5 10.1 95 4 23 14.2
16 5 55 95 5 26.5 100 180 106 2 18 12.7 107 5 21 4
17 3 72 102 3 26.5 121 300 102 3 22.5 9.8 102 3 23 8.2
18 3 68 100 6 24 119 160 95 3 24 14.9 96 4 23 14.5
19 8 66 91 11 25 105 230 97 6 24.5 8.8 97 4 28 9.1
20 3 58 104 3 23.5 98 170 0 0 0 0 0 4 21 -
21 3 58 104 3 25 97 290 100 6 26 9.4 100 0 0 18.6
22 3 70 96 4 27 104 150 98 3 18.5 4.6 98 4 21.5 7.2
23 3 60 91 6 19.7 100 210 99 2 23.5 18 99 4 25 6.6
24 13 60 99 11 26 115 380 98 2 24.5 37.3 98 10 26 8.6
25 3 71 97 5 27.5 92 110 99 3 23.5 14.8 98 4 23.5 12.2
26 3 77 96 5 28 72 200 96 3 22.5 18.4 96 4 27 10.5
27 3 86 101 2 28 92 230 98 4 25.2 15.1 98 4 25.5 12.1
ROS1
Total Tiller Grain ROS1 ROS1 ROS1 ROS2 ROS2 ROS2 ROS2
S.N. Seedling PH (cm) DF ET PL (cm) PH at maturity Yield
45 Das Yield(g) DF ET PL(cm) DF ET PL(cm) Yield(g)
(g)
28 4 58 105 1 26 96 250 102 2 25 12.2 102 4 24.2 4.2
29 5 63 104 6 27.5 99 330 102 4 19 9.7 103 4 22 12.1
30 3 61 96 3 27 113 100 103 4 22.5 11.5 102 4 25 7.7
31 3 66 94 4 25 101 230 97 4 21.8 23.2 97 5 25 8.5
32 7 74 102 4 30 108 220 107 2 22.5 18.1 106 8 22.5 3.2
33 7 60 95 7 25.5 103 160 96 4 22.5 20.6 98 6 22 10.1
34 4 70 91 13 23.5 103 130 94 4 23 - 94 0 0 0
35 3 70 99 4 28.5 101 180 100 2 23 17.9 99 5 22 7.4
36 3 70 93 5 24 108 420 96 5 22 9.3 96 3 23 8.8
37 4 60 95 3 23 97 140 95 5 21 5.5 95 2 24 10.7
38 5 85 99 3 23 116 340 97 6 26 14 97 4 23.5 24.7
39 3 64 89 4 22.5 89 240 97 7 23 5 96 2 22 15.2
40 3 74 99 2 26 90 180 102 2 19 18.3 102 5 20.5 5.5
41 3 76 95 4 26.2 112 220 100 4 23.5 - 101 7 23 11.9
43
TIL. – Tiller
PH – Plant height
DF- Days to flowering
EF- Effective tillers
PL- Panicle length
ROS- Rain out shelter
Table 4.2: Statistical Data of Population
Irrigated
S.N. Trait Mean Minimum Maximum S.D.
1 Seedling stage tiller 5.2 3 21 3.4
2 Plant height 67.9 55 89 9.15
3 Days to flowering 98 89 105 4.66

4 Effective tillers 5.6 1 15 3.4
5 Panicle length 25.7 19.7 30 2.18
6 Ph with panicle 103.9 72 148 13.86
7 Direct sowing yield 41.21 10 84 15.99
Rain out shelter 1st

10 Panicle length 22.7 18 26.5 5.3
11 Yield 15.03 2 37.3 8.61
Rain out shelter 2nd
14 Panicle length 23.3 16 28 5.51
15 Yield 10.5 3.2 24.7 5.13
44
(b) Effective tiller
The mean of effective tiller was 4.7 and ranged from 10 highest (in plant line
no.33), and lowest 2 (in single plant line no. 46, 48), with the 1.96 standard deviation.
(c) Panicle length (cm)
Mean of panicle length was calculated to be 23.3cm, in total population it ranged
from 28cm highest counted in single plant line no. 28, and lowest 16cm was counted in
single plant line no. 18, with the standard deviation of 5.51.
(d) Grain Yield (g)
The mean of rain out shelter 2nd yield was observed to be 14.79g and in total
population it ranged from 37.3g highest observed in single plant line no. 33, and lowest
2g was observed in single plant line no. 18, with the 5.13 of standard deviation.
A comparison of grain yield under irrigated condition with grain yield under ROS
#1 and #2 clearly indicated substantial reduction in yield which indicated quit high
drought intensity index. This level of stress is required for effective selection (Kumar et
al. 2009)
4.3 Development of genotypic data based on SSR and HvSSR markers
4.3.1 Extraction of genomic DNA and quantification
Total genomic DNA was extracted from 62 plants (one plant each from individual
F3 progeny row) of rice along with both the parent using Miniprip method. Fresh and
healthy leaves were used for extraction of DNA. The DNA samples quantification was
done by using Nano Drop Spectroscopy. The quantity of the samples was found in the
47
range from 200-1000 ηg/µl. DNA samples were then diluted with sigma (sterilized) water
such that the final concentration of DNA becomes 40ηg/µl.
4.3.2 SSR
The standardized PCR protocol for SSR was used for all subsequent studies. The
DNA of selected lines along with the parents was subjected to PCR based simple
sequence repeat (SSR) amplification technique to generate genotypic data.
4.3.3 HvSSR
The DNA of selected lines along with the parents was subjected to PCR based
HvSSR amplification technique to generate genotypic data.
4.3.4 Parental polymorphism analysis using SSR and HvSSR primers
SSR and HvSSR primers were used in this study for amplification of genomic
DNA of segrigating population through PCR. The PCR products were loaded on 5%
PAGE gel and electrophoresis was done at 180 volts for 1 hours. The DNA was stained
with Ethidiam bromide. Gels were visualized and photographed by using Gel Doc Unit.
Out of 150 primers 45 primer exhibited parental polymorphism and were subsequently
used to generat genotyping data. The overall percent polymorphism for SSR and HvSSR
comes to around 30%. Similar level of percentage polymorphism was reported by other
workers Fu et al. (2010) and Bai et al. (2011).
4.3.5. SSR and Hv SSR based population analysis (Genotyping)
Primer showing polymorphisms were further used for PCR amplification with all
of the 62 lines along with parents using standardized PCR protocol. PCR products were
loaded on 5 % PAGE gel. Electrophoresis was carried for 1 hour at 180 volts to allow
separation of amplified product. The bands observed were designated as A, B, H and E,
where A represents female like allele, B represent male like allele, H represents
45
heterozygous and E represents other type allele. The gel pictures for different primer SSR
and HvSSR with population are presented in Fig 4.1.
The segregation pattern of most of the marker deviated from the normal
mendelian 1:1 ratio, and exhibited distorted segregation pattern. Distorted and skewed
segregation of molecular markers was also reported in many mapping populations
(Causse et al., 1994; Xiao et al., 1996). The genetic basis of segregation distortion may
be the abortion of male or female gametes, or the selective fertilization of particular
gametic genotypes (Xu et al., 1997) or because of less number of population size used for
generating genotypic data. The marker HvSSR5-48, RM-340, RM-483 produces more
female type alleles (100%) with 0% male 0% heterozygous and 0% other type alleles. On
the other hand RM444, RM25, HvSSR5-65, RM231, RM174, HvSSR2-44 produces 99%
male type allele respectively and high percentage of heterozygosity was observed in
primer no. RM1, RM490 with 2% and high percentage of extra or other type band
presented (10%) in primer no. RM279. However, if we consider all the primers the
overall allelic segregation pattern for male and female alleles come close to 1:1 presented
on Table 4.2.
4.4 Identification of QTLs
QTLs linked to drought resistance have been mapped in at least 15 different
populations (Komashita et al., 2008). However it has been emphasized to use mapping
population derived from same ecotype as well as adapted to target environments.
Considering these facts in the present investigation mapping population derived from
cross between MTU1010 and IR86931-B-6 parents both indica types were used for QTL
analysis.
46
Fig.4.1: Gel images showing parental polymorphisms
The genotypic data of the whole mapping population was developed however for
the purpose of QTL identification, genotypes were selected from extreme classes and
thus selective genotyping method was used to detect the association of QTLs with traits.
Bernier et al, (2008) also report to used selective genotyping for QTL detection. Test for
QTL association with traits was performed by single marker approach.
The map developed by Singh et al. (2009), Causse et al. (1994), McCouch et al.
(2002) and the International Rice Genomic Sequencing project (2005) helped to identify
relative position of markers on chromosome.
The genotypic data thus generated along with phenotypic date recorded in the
field was used for identification of QTLs for different trait. The result of this analysis is
presented in Table 4.3 and graphical presentation has been done in fig 4.2 and 4.3.
A perused of table no. 3.4 revealed that putative QTLs for plant height were
identified on chromosome no. 1, 2, 3, 5, 6, 7, 8 and 11 under irrigated condition and the
marker showing significant association with plant height were RM11943, RM109,
RM279, RM530, RM174, RM242, RM169, HvSSR5-65, HvSSR5-13, RM111, RM481,
RM25, RM206, RM21. In agreement for over findings Gomez et al. (2010) also
identified QTL for plant height on chromosome no. 1, 4, and 5 by using recombinant
inbred (RI) lines of Bala × Azucena. Babu et al. (2003) has also identified QTLs on
chromosome no 1, 2, 4, 7, 8, and 9 under control and stress condition using doubled-
haploid (DH) population of 154 rice lines from the cross CT9993-5-10-1-M/ IR62266-
42-6-2.
QTLs for grain yield were found on chromosome 6 under irrigated and 2, 7, 8,
and 11 chromosomce under drought (rain out shelter 1st) condition. The markers which
48
Table 4.3: Banding pattern of individual markers on segregating population
Marker A (female) B (male) H (heterozygous) E (extra) Total

RM1 2 36 2 1 41
RM11943 40 1 0 0 41
RM12146 27 14 0 0 41
RM490 37 2 2 0 41
RM9 40 0 0 1 41
RM486 26 15 0 0 41
RM109 32 9 0 0 41
HvSSR2-12 8 32 0 1 41
HvSSR2-27 22 16 1 2 41
HvSSR2-44 1 40 0 0 41
RM279 29 2 0 10 41
RM174 0 40 0 1 41
RM530 39 2 0 0 41
HvSSR3-9 37 4 0 0 41
HvSSR3-41 39 1 1 0 41
RM231 0 40 0 1 41
RM232 1 39 0 1 41
RM55 40 0 0 1 41
RM168 2 39 0 0 41
RM468 40 1 0 0 41
RM3471 5 36 0 0 41
RM335 40 0 0 1 41
RM153 38 2 0 1 41
HvSSR5-13 39 1 0 1 41
HvSSR5-48 41 0 0 0 41
HvSSR5-65 1 40 0 0 41
RM169 40 1 0 0 41
RM164 40 1 0 0 41
RM190 40 1 0 0 41
RM225 13 20 0 8 41
RM111 2 38 0 1 41
49
Marker A (female) B (male) H (heterozygous) E (extra) Total
RM340 41 0 0 0 41
RM481 20 21 0 0 41
RM152 2 38 1 0 41
RM25 1 40 0 0 41
RM483 41 0 0 0 41
RM444 0 40 0 1 41
RM242 38 1 0 2 41
RM553 29 11 1 0 41
RM278 38 3 0 0 41
RM228 13 20 0 8 41
RM21 0 39 0 2 41
RM206 1 31 0 9 41
RM1233 36 2 0 3 41
RM309 5 36 0 0 41
50
Fig. 4.2: Composite marker analysis
Seedling stage tillers Effective tillers Days of flowering (rain out shelter 1 st)
Panicle length (rain out shelter 1st) Days of flowering (rain out shelter 2nd)
Plant height Yield (rain out shelter 1st) Panicle length (rain out shelter 2nd)
Days to flowering Panicle length Plant height with panicle Direct sowing yield
Effective tillers (rain out shelter 1st) Effective tillers (rain out shelter 2nd)
Yield (rain out shelter 2nd)

Fig. 4.3: Single marker analysis
exhibited significant association with grain yield were RM111 under irrigated condition,
while under drought condition HvSSR 2-12, HvSSR2-27, RM109, RM481, RM152,
RM206, RM1233 were significantly associated. In agreement to our finding Venuprasad
et al. (2009b), Yano et al. (2001), Courtois et al. (2000), Yadav et al. (1997) and Yue et
al. (2006b) reported DTY 2.1 for grain yield by using RIL population of Apo/ Swarna.
The QTL linked to RM324 (DTY2.1) had a highly significant effect on grain yield in
lowland drought stress. Srinivasan et al. (2008) identified QTLs gy1.1 for grain yield on
chromosome 1. Fu et al. (2010) reported QTLs for grain yield on chromosome 1, 2, 8 and
12. Similarly Venuprasad et al. (2009) identified three QTLs for grain yield one each on
chromosome 3, 4 and 5 by using IR64 X Azucena DH mapping population. (Bernier et
al. (2007) has also identified QTL on chromosome 12 with large effect on grain yield
under stress by using Vandana/ Way Rarem). (Venuprasad et al. (2009b) has also
reported DTY3.1 which has been shown to have a strong influence on grain yield under
drought). Fu et al. (2010) reported 3 regions underling significant QTLs for several yield-
related traits were detected on chromosome 1, 7 and 12 by using an accession of common
wild rice (Oryza rufipogon Griff.). Xing et al. (2010) reported QTLs for grain yield on
chromosome 1, 5, 6 and 7 by using Zhenshan97/ Minghui63. Similarly Lafitte et al.
(2004b), in a population derived from the cross Azucena/ Bala, identify 31 QTLs
responsible for grain yield.
Several experiments have reported to identify QTL for yield and yield
contributing traits under type of drought stress in rice. Babu et al. (2003) reported to
detect five QTLs related to grain yield under drought stress in the population CT9993/
IR62266. In eastern India using same population a single large effect of QTL affecting
51
grain yield under stress condition, was reported to detect at 206 cM on chromosome 1,
between marker EM-11-11 and RG 109. Bernier et al. (2007) reported a very large QTL
for grain yield under stress. Centromeric region of chromosome 12 (qtl12.10), using CIM
analysis over 2 year resulted in the localization of this QTL in interval between RM28048
(45.2 cM) and RM511 (55.5cM). For better understanding the effects of this (qtl 12.1)
they performed single-marker analysis for all traits related to grain yield under both stress
and non stress condition for RM511.
QTL for effective tiller were found in chromosome no. 1, 2, 3, 4, 5, 6, 8, 9 and 11
under irrigated condition associated with RM1, RM11943, RM490, RM12146, RM9,
RM174, RM55, HvSSR2-44, HvSSR3-41, RM232, RM168, RM169, RM164, RM3471,
HvSSR5-13, HvSSR5-65, RM225, RM25, RM111, RM340, RM152, RM444, RM242,
RM206, RM278, RM21 markers. Under water stress condition QTLfound in
chromosome no. 3, 6, 7, 9 and 11 associated with RM232, RM481, RM225, RM553, and
RM206 markers. Similarly Gomez at al. (2006) identification of QTL for effective tiller
were detected one each on chromosome 1, 3 by using recombinant inbred (RI) lines of
Bala × Azucena. Kanjivavila et al. (2008) identified QTL for days to flowering were
detected on chromosome no. 3 by using single-seed descent progenies from a cross
between IR58821-23-B-1-2-1 (abbreviated as IR58821, a lowland indica accession which
has high root penetration index and thicker roots) and IR52561-UBN-1-1-2 (abbreviated
as IR52561, a lowland indica line with low root penetration index and thinner roots).
QTL for days to flowering was found on chromosome no. 3, 6, 9 and 10 under
irrigated condition associated with RM168, RM468, RM225, RM242, and RM228
markers. Under water stress condition QTL found on chromosome no. 1, 2, 3, 4, 5, 6, 8, 9
52
and 11 were associated with marker no. RM9, HvSSR2-27, HvSSR2-44, RM530,
HvSSR3-41, RM232, RM55, RM168, RM153, HvSSR5-13, HvSSR5-48, HvSSR5-65,
RM164, RM190, RM225, RM111, RM340, RM25, RM444 and RM242. Similarly
Chakraborty at al. (2011) identification of QTL for days to flowering on chromosome no.
1, 2, 3, 5, 7, 8, 9, 11 and 12 by using 154 double haploid (DH) lines derived from a cross
between a deep-rooted upland adapted japonica rice genotype, „CT9993-5-10-1-M‟ and a
lowland indica rice genotype with shallow roots having moderate drought tolerance,
„IR62266-42-6-2. Kanjivavila et al. (2008) identified QTL for days to flowering on
chromosome no. 5 by using single-seed descent progenies from a crossbetween IR58821-
23-B-1-2-1 (abbreviated as IR58821, a lowland indica accession which has high root
penetration index and thicker roots) and IR52561-UBN-1-1-2 (abbreviated as IR52561, a
lowland indica line with low root penetration index and thinner roots).
Overall number of QTL was identified for grain yield and other related
agronomic traits, both under irrigated as well as water stress condition. These QTL with
major effective can be used for improvement of yield under these defined conditions
using closely linked molecular marker.
53
Table 4.4: QTL indentified for different traits
Single
marker
Chro 1% 0.1% 0.01%
SN Trait Condition Composite Marker analysis LOD Value Analysis
no. signific. signifi. Signi.
5%
significant
Seedling
1. stage Irrigated 1 RM1 7
tiller
1 RM11943
2 RM174 7.8 RM174
HvSSR2-12,
2
2-27,2-44, RM530
Within HvSSR3-41,
3 RM232,RM55 RM168, 8
RM468
3 HvSSR3-9 RM242
4 6 RM3471
HvSSR5-
HvSSR5-13,
5 7.8 13,5-65,
5-65 RM169
RM169
5 RM153, HvSSR5-48 7.8
6 RM111 5.9 RM 111
6 RM340 5.9
7 RM 481
8 RM 25 6 RM 25
9 Within RM444, RM242 9.4
9 RM 278
11 RM 1233 RM 21
Plant
2. Irrigated 1 RM11943 RM12146
height
2 RM530 3.6 RM530
2 RM174
HvSSR5-13,
5 HvSSR5-65,
RM169
54
Single
marker
Chro 1% 0.1% 0.01%
5%
significant
8 RM 25
9 RM 242 3.6
Days to
3. Irrigated 3 Within RM168,RM468 3.2
flowering
6 RM 225
9 RM 242
10 RM 228
9 RM 242 3
Effective RM1,
4. Irrigated 1 RM1,RM11943 RM490, 6.6 RM11943
tillers RM490
RM12146,
1 6.6
RM 9
2 RM174 7 RM 174
2 HvSSR2-44 7
HvSSR3-41,
3 HvSSR3-41, RM232,RM168 7.8 RM 232
RM168
3 RM55 7.8
4 RM 3471
5 RM169,RM164 6.8 RM 164 RM169
HvSSR5-
5
13,5-65
6 RM225,RM111 RM340 6
8 RM25 6.4 RM 25
8 RM152 6.4
9 Within RM444,RM242 6.6
9 RM 278
11 6 RM 206 RM 21
Panicle
5. Irrigated 7 RM 481
length
11 0 3.1
6. Ph with
Irrigated 1 RM 11943
panicle
55
Single
marker
Chro 1% 0.1% 0.01%
5%
significant
RM109,
2 RM279, RM174
RM530
3 RM 242
RM169,
5 HvSSR5-
65,5-13
6 RM 111
7 RM 481
8 RM 25
11 RM 206 RM 21
Direct
7. sowing Irrigated 6 RM 111 3.2 RM 111
yield
Days of Rain out
8. 1 RM 9 14.8
flowering shelter 1st
HvSSR2-27,
2 19
2-44 RM530
HvSSR3-41, RM232,
3 14.5
RM55,RM168
4 0 15
5 RM153 17 RM 153
HvSSR5-13,
5 17
5-48,5-65, RM168,RM164
RM190,RM225
6 16
RM111,RM340
8 RM 25 16
9 RM444,RM242 15
11 0 15
Effective Rain out
9. 6 RM 225
tillers shelter 1st
10 RM 228
56
Single
marker
Chro 1% 0.1% 0.01%
5%
significant
Panicle Rain out
10. 1 RM490,RM9, RM486 9.2
length shelter 1st
HvSSR2-12,
2 10
2-27,2-44, RM174,RM530
HvSSR3-9,
3 3-41, RM232,RM55, 9
RM168,RM468
4 0 10
5 RM153 15 RM 153
HvSSR5-13,
5 15
5-48,5-65, RM168,RM164
6 RM111,RM340 14
8 RM25 13.8
9 RM444,RM242 8.6
11 0 8
Rain out HvSSR2-12,
11. Yield 2 3.8
shelter 1st 2-27, RM109
7 RM 481
8 RM 152
RM206,
11
RM1233
Days of Rain out RM490,RM9,
12. 1 15
flowering shelter 2nd RM486
HvSSR2-12,
2 2-27,2-44 19
RM174,RM530 and within
HvSSR3-41, RM232,RM55
3 14
RM168,RM468
5 RM153 14 RM 153
HvSSR5-13,
5 14
5-48,5-65, RM169,RM164
6 RM190,RM225RM111,RM340 15
57
Single
marker
Chro 1% 0.1% 0.01%
5%
significant
8 RM25 15
9 RM444,RM242 and Within 15.5
Effective Rain out
13. 3 No found QTL RM 232
tillers shelter 2nd
7 RM 481
9 RM 553
11 RM 206
Panicle Rain out
14. 1 0 11
length shelter 2nd
2 HvSSR2-44, RM174,RM530 14
HvSSR3-9, RM232,
3 11
RM55,RM168
5 RM153 13 RM 153
HvSSR5-13,
5 5-48,5-65, within 13
RM169,RM164
6 RM225,RM111 10
8 RM 25 12
9 Within RM 444 RM242 11
Rain out
15. Yield QTL not found
shelter 2nd
58
SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR
FUTURE RESEARCH WORK
CHAPTER-V
SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR FUTURE
RESEARCH WORK
Among the abiotic stresses drought stress is a major problem in production and
yield under rainfed rice ecosystem. It acts as a serious limiting factor in agriculture
production by preventing a crop from reaching the genetically determined theoretical
maximum yield. The development of cultivars with improved drought tolerance is thus an
important element in increasing productivity and alleviating poverty in communities
depended on rainfed ecosystem. The complex nature of drought tolerance, genotype x
environment interaction, lack of understanding of inheritance of drought tolerance, poor
understanding of physiological basis of yield under water limited condition and difficulty
of effective drought tolerance screening complicate the development of drought tolerance
varieties. Therefore, the present study was undertaken to carry out the genetic analysis for
understanding the genetics basis of drought tolerance and identification of QTLs for yield
and yield contributing traits under water stress condition.
Sixty lines of F3 segregating population were evaluated under irrigated and rain
out shelter condition during wet season 2012. Derived from a cross between MTU1010
and IR86931-B-6 for genetic analysis and generating phenotypic data for QTLs analysis.
The data was statistically analyzed to calculate genetic parameters, mean, coefficient of
variance and identification of QTLs. Identification of QTLs was performed using “Single
Marker Analysis” and “Composite Marker Analysis” method for developing genotypic
data. DNA from each line two plants along with parents was extracted using miniprep
method and each of which were subjected to DNA quantification. Finally DNA was
59
diluted to approximate concentration of 40 g/ l. The diluted DNA was then subjected to
PCR base amplification using SSR and HvSSR primers. The PCR products were then
analyzed on 5% page gel. A set of 150 SSR and HvSSR primers were screen to detect
parental polymorphism, out of which 45 primers exhibited polymorphism and 45 primers
were selected to generate genotypic data. The genotypic data thus obtained was used for
single marker analysis and composite marker analysis for detecting the association
between marker and grain yield and yield contributing traits.
Conclusions:
The mean grain yield of population was 41.21g/plant and 15.03g/plant,
10.5g/plant under direct sowing and rain out shelter 1st and 2nd condition
respectively, indicating high level of stress.
The mean of irrigated condition (seedling stage total tiller was 5.2/line, plant
height was 67.97cm days to flowering was 98 days, effective tiller was counted
5.6, panicle length was 25.7cm and plant height was 103.9cm), rain out shelter 1st
condition the mean of days to flowering was 98 days, effective tillers was 3.6 and
panicle length was 22.7cm, the mean days to flowering in rain out shelter 2nd was
100 days, effective tiller were 4.7 and panicle length was 23.3cm.
Screening of parental polymorphism using SSR and HvSSR primer reveal low
level of polymorphism between two parents of the population.
Number of the markers showed significant association with grain yield and yield
contributing characters under irrigated and rain out shelter condition.
60
RM 111(direct sowing condition) and RM481, RM152, RM109, HvSSR2-12,
HvSSR2-27(rain out shelter 1st condition) markers were found to be associated
with grain yield.
Suggestions for future work:
Use these markers for subsequent marker assisted selection.
Candidate gene approach to identify gene controlling drought related traits.
61
ABSTRACT
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“IDENTIFICATION OF QTL FOR GRAIN YIELD AND YIELD

RELATED TRAITS UNDER DROUGHT AND IRRIGATED

M. Sc. (Ag.) THESIS

ASHISH KUMAR TIWARI

DEPARTMENT OF GENETICS AND PLANT BREEDING

Indira Gandhi Krishi Vishwavidyalaya, Raipur

ASHISH KUMAR TIWARI

IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE

(GENETICS AND PLANT BREEDING)

Roll No. 15259 ID No. 110307007

CHAPTER PARTICULARS PAGE

II REVIEW OF LITERATURE 4-22

2.1 Phenotypic evaluation of segregating population 5-6

2.2 Impact of drought on yield and yield 6-7

2.3 Identification of QTL for grain yield under 7-12

2.4 Development of genotypic data based on SSR 12-13

2.4.1 SSRs (Simple Sequence Repeats) 13-14

2.5 Genome mapping 14

2.5.1 Molecular map 14-16

2.6 Development and concept of QTL analysis 16-20

2.7 QTL for drought tolerant 20-22

III MATERIALS AND METHODS 23-36

3.1.1 Characteristic features of parent 23-24

3.2.1 Field studies 24

3.2.2 Observations 24-25

3.2.3 Statistical analysis 25-26

3.2.3.3 Standard Deviation 26

3.2.4 Molecular studies 26

3.2.4.1 Genomic DNA isolation 26-27

3.2.4.2 Quantification of DNA 28

3.2.5 PCR amplification using HvSSR primers 28

3.2.5.1 PCR reaction 28

3.2.5.2 Visualization of amplified products in 29

3.2.5.3 Assembling and pouring the gel 29

3.2.5.5 Visualization of bands 30

3.2.5.6 Development of genotypic data of the population 31

3.2.5.7 Scoring of data 32

3.2.5.8 Scoring SSR and HvSSR banding pattern in 32

3.2.6 QTL analysis 33

3.3 Reagents and solutions 33

3.3.1 Reagents for PCR 33

3.3.2 Stock solutions 33

3.3.3 Solutions for electrophoresis 34

3.3.4 Stocks and solutions for PAGE 35-36

IV RESULTS AND DISCUSSION 37-58

4.1 General observations 37-38

4.2 Development of phenotypic data 38-45

4.3 Development of genotypic data based on SSR 45-37

4.4 Identification of QTLs 47-58

V SUMMARY, CONCUSIONS AND 59-61

Table No. Title Page

3.1 Characteristic features of parents 24

3.2 PCR mix for one reaction (volume 20 l) 28

3.3 Temperature profile used for PCR amplification using 28

4.1 Phenotypic Data of population 42-43

4.2 Variability under different condition and different characters 44

4.3 Banding pattern of individual markers on segregating 49-50

4.4 QTL indentified for different traits 54-58

Figure No. Particulars Between pages

4.1 Gel images showing parental polymorphisms 47-48

4.2 Composite marker analysis 50-51

4.3 Single marker analysis 50-51