Molecular characterization of genotypes for thermotolerance in rice.

Molecular characterization of
genotypes for thermotolerance
in rice
Agricultural College Bapatla
Department Of Genetics And Plant Breeding
Submitted To
Dr . T. SRINIVAS
Professor and Head
Genetics and Plant Breeding
Agricultural College Bapatla.
Presented By:
S. RAVI TEJA
BAM-20-23
M.Sc. 1st year
Genetics and Plant Breeding
1
Course no :- GP-591
Course tittle :- Masters Seminar

Agricultural College Bapatla
Department Of Genetics And Plant Breeding
Rice (Oryza sativa) 2n = 24
Center of Origin = South East Asia
Botanical description
Kingdom Plantae
Division Magnoliophyta
Class Liliopsida
Order Poales
Family Graminae or Poaceae
Tribe Oryzeae
Genus Oryza
Species Sativa
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AGRICULTURAL COLLEGE BAPATLA
DEPARTMENT OF GENETICS AND PLANT BREEDING
Area, Production and Productivity in India (FAOSTAT)
Year Area (m.ha) Production (m.t) Productivity (t/ha)
2016 43.19 163.70 37.90
2017 43.77 168.50 38.49
2018 44.15 174.71 39.56
2019 43.78 177.64 40.57
Area, Production and Productivity in Andhra Pradesh (Kharif + Rabi)
Agricultural Statistics at a Glance - Andhra Pradesh 2018-19
Year Area
(Lakh hectares)
Production
(Lakh tonnes)
Productivity (kg/ha)
2017-2018 22.18 126.91 5722
2018-2019 22.08 123.52 5593
3

Introduction
• Rice (Oryza sativa L.) is a globally important cereal plant, and as a primary source of food it accounts for 35–
75% of the calorie intake of more than 3 billion humans.
• With the likely growth of world’s population towards 10 billion by 2050, the demand for rice will grow faster
than for other crop (Krishnan, et al., 2011), Rice is grown on around one 10th of the arable land and more
than 90% of it is produced and consumed in Asia.
• Global climate change has resulted in an increase in air temperature by 0.5 ºC in the 20th century and
temperature is predicted to be further increased by 1.5 to 4.5 ºC in this century.
• Rice is already growing in areas where temperature has reached optimal for rice growth; therefore, any
additional increase in day or night temperature or exposure to high temperature during sensitive stages
reduces the rice yields.
• It is estimated that the increasing temperature would reduce rice production by 41% at the end of the 21st
century.
4

Why we are searching for thermotolerance ??
5

DEPARTMENT OF GENETICS AND PLANT BREEDING 6

Growth and developmental effects of heat stress
Vegetative stage:-
• Germinability and early seedling growth are major components of seedling vigor.
• Prolonged temperature elevation reduces seed germination potential and leads to poor germination rate and
seedling vigor.
• The optimum growth temperature of rice at the seedling stage is 25–28oC. Heat stress at the seedling stage
results in increased water loss, withered and yellow leaves, impaired seedling and root growth, and even
death of seedlings.
• Rice showed about 35% lower panicle number and 86% lower total yield per plants under HS (40oC day/35oC
night) for 15 days than at 28oC.
• The influence of HS on tiller and panicle number is more severe in japonica than in indica rice.
• Tiller number under HS is often used as a marker for the selection of thermotolerant rice cultivars.
7

Reproductive stage
• Rice plants at the reproductive stage, including the processes of panicle initiation, male and female
gametophyte development, to HS, and spikelet fertility is completely lost at this point after HS stress (39oC
day/30oC night) for 7 days.
• After double fertilization, exposure to a short period of HS (39oC for 48 h) can result in abnormal
cellularization of early endosperm development, and impair the subsequent establishment of endosperm.
• The occurrence of HS during the reproductive stage resulted in an up to 80% reduction in spikelet fertility of
rice.
• However, different genetic backgrounds show differing susceptibility to HS.
• For example, high temperature (38oC) reduced the number of pollen grains on the stigma in the Aus type N22
by 55% and the japonica type Moroberekan by 86%, but not in the indica type IR64.
• And the number of grains on the stigmas of superior spikelet's is more sensitive to HS than that on inferior
spikelet's, owing to their different organ temperatures.
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Grain-filling stage
• Grain filling is the completion of growth and development in crop plants and involves the transport and
synthesis of carbohydrates, proteins, and lipids in seeds.
• It has been reported that the grain-filling rate was increased and the total grain-filling duration was reduced by
21.3%–37.1% for different genotypes following exposure to HS at the grain-filling stage.
• Heat stress (35oC for 72 h) at early seed development stage impaired endosperm and embryo development.
• When subjected to HS (33oC day/27 C night) during 3–35 days after flowering, 90.2% of the kernels of
japonica cultivar Koshihikari showed combined chalk, including milky-white and white-back kernels.
• The chalkiness rate was increased while brown rice rate, milled rice rate, and head rice rate were decreased
with an increase of high temperature and prolonged duration at the early grain-filling stage.
• Amylose content is lower under HS (16.1%) than under normal conditions (19.8%), suggesting that lower
activity of amylose synthesis may be involved in chalk formation.
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Physiological effects of heat stress
1. Membrane damage
• Elevated temperature can impair the structure and function of plasma membranes, alter the ratio of saturated to
unsaturated fatty acids, and trigger protein denaturation, leading to increased fluidity and permeability,
compromised membrane integrity, and increased leakage of organic and inorganic ions from cells.
• Cell membrane thermostability, which is a measure of electrolyte leakage, is known to have a direct
association with thermotolerance in many crops and hence is used as screening parameter to differentiate the
tolerant and susceptible genotypes.
2. Reactive oxygen species accumulation
• Intracellular reactive oxygen species (ROS) levels are dramatically increased under HS.
• When rice plants are subjected to HS (38oC day/30oC night) at the meiosis stage, the ROS content in anthers is
over three times higher than that under normal temperature (28oC day/22oC night).
• Accordingly, electrolyte leakage, ROS level, transcript abundance of antioxidative genes, activities of
antioxidant enzymes, and MDA(malondialdehyde) content are frequently used as indicators of membrane and
oxidative damage and also reflect the thermotolerance of plants.
• For example, heat-tolerant rice cultivars such as NERICA-L-44 and Nagina 22 exhibit high membrane stability
and lower ROS and MDA contents because of high antioxidant enzyme activities.
10

3. Photosynthesis damage
• HS results in destroyed permeability of thylakoid membrane or even thylakoid grana disintegration and
triggers a decrease in chlorophyll content, leading to alterations of photochemical reactions with reduction in
the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) and photosynthetic rate.
• High temperature inhibits the activity of ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco), mainly
as a consequence of the inactivation of Rubisco activase.
• Engineering Rubisco activase may be an effective method for thermotolerance breeding.
4. Disturbance of carbohydrate metabolism and partitioning
• Abundances of two key enzymes in the glycolytic pathway, phosphoglucose isomerase and
phosphofructokinase, were very low, and the abundance of phosphoglycerate mutase was also reduced when
rice cells were exposed to high temperature (44oC ), suggesting impaired energy generation in cells under HS.
• A heat tolerant rice cultivar showed higher expression of the sucrose transporter gene OsSUT1 than a
sensitive cultivar, leading to a higher supply of photo assimilates to filling kernels.
• HS at the filling stage led to down-regulation of a series of genes encoding starch synthesis-related proteins
such as granule-bound starch synthase I and branching enzymes, possibly resulting in inhibition of starch
accumulation and increased grain chalkiness.
11

Molecular mechanisms of plant responses to
heat stress
1. Heat stress sensing
• To identify the complex mechanisms underlying HSR, a key question is how plants rapidly sense HS and then
transduce HS signal into intracellular responses.
• The cell wall is the first protective barrier in plants and its structures and properties can be remodeled when
plants are subjected to HS, influencing the release of apoplastic Ca2+ and increasing the content of free
cytosolic Ca2+
2. Heat-induced signal cascades
• A transient cytosolic Ca2+ influx from the extracellular matrix to the cytosol is considered to be an early
event in HSR.
• Ca2+ then acts as a second messenger recognized by calcium sensor proteins, thus rapidly transducing
external HS signals inside the cell.
• HS also impairs the normal function of chloroplasts and mitochondria and disturbs electron transport during
photosynthesis and respiration, thereby promoting the production of ROS and in turn the activation of ROS-
mediated signal transduction in the HSR.
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3.Transcriptional regulatory network of heat stress response
• Rice has 25 HSFs, which can be classified into three classes: HSFA (1a, 2a to 2f, 3, 4b, 4d, 5, 7, 9), HSFB (1,
2a to 2c, 4a to 4d), and HSFC (1a, 1b, 2a, 2b), and the expressions of 22 of these genes are induced by high
temperature.
• Among these HSFs, HSFA1s are considered ‘‘master regulators” in the transcriptional network.
• HSFA2 is a direct target gene of HSFA1s and plays an essential role in HSR.
4.Protein homeostasis under heat stress
• HS disturbs protein homeostasis in the cell, including protein synthesis, folding, quality control, and
subcellular localization, leading to the over-accumulation of unfolded or misfolded proteins, which are toxic
to plant cells.
• The accumulation of such proteins in the endoplasmic reticulum can initiate the unfolded protein response
(UPR).
• rRNA homeostasis is also crucial to translation.
• The DEAD-box RNA helicase TOGR1 is essential for maintaining normal rRNA homeostasis under HS
conditions, and its overexpression can protect rice growth under high temperature.
14

Approaches for improving thermotolerance
1.Agronomic management
• Several agronomic management strategies have been shown to be helpful for alleviating or avoiding heat
damage in rice.
• Proper application of growth regulators such as CTK, SA, BR, and ethylene precursors can alleviate HS-
induced damage to rice plants such as pollen abortion, reduced spikelet's per panicle and kernel weight, and
poor seed-setting rate.
• Glycine betaine or proline can mitigate yield reduction by reducing heat damage to membranes and
maintaining the enzymatic function of Rubisco.
• The moderate increase of nitrogen application and combined application of biochar and phosphorus have also
been shown to alleviate rice yield losses caused by HS at the reproductive stage.
• Mist spray treatment during the flowering period rapidly reduces temperatures in the rice field, delays leaf
senescence, and increases the activities of antioxidant enzymes, thus alleviating HS-caused yield loss.
15

2. Conventional breeding
• Increasing thermotolerance by conventional breeding is a promising approach for reducing the negative
effects of HS on rice yield and quality.
• Conventional breeding is generally based on thermotolerance-related phenotype selection and is applied in a
climatic region similar to that where the crop is to be grown.
• Accurate evaluation of the degree of thermotolerance, selection of elite rice cultivars or breeding lines, and
successful transfer of thermotolerance traits into specific cultivars with good agronomic performance are of
great importance to conventional breeding.
• As described above, the heat-caused influences on seedling growth, tiller number, pollen fertility, seed-setting
rate, grain chalkiness, and grain yield can be used as indices of the thermotolerance of rice germplasm.
• However, the typical index is seed-setting rate, assessed by the naked eye, or the ratio of seed setting rate
under HS to that under normal conditions, a direct, simple, and reliable index for conventional breeding for
HT.
• Using this index, a series of heat-tolerant rice materials have been identified and used as donors to develop
breeding lines, including N22, Giza178, HHT4, 996 , IR2061, and Habataki.
• Another kind of donor, EMF20, is also used to develop cultivars that escape heat at flowering, because of its
early-morning flowering trait.
• Some heat-tolerant hybrid rice, such as Guodao 6, show seed-setting stability under HS conditions owing to
their heat avoidance adaptability based on erratic floral traits, such as shortened flowering phase and
decentralized flowering clock.
16

Identification of heat-tolerant quantitative trait loci and marker
assisted breeding
• Because strategies for agronomic management and conventional breeding for thermotolerance are few, an
urgent task for breeders is to discover heat-resistance genes or quantitative trait loci (QTL) and apply them to
thermotolerance breeding.
• To date, many QTL responsible for thermotolerance at various developmental stages of seedling, booting,
flowering, and grain filling have been identified and validated.
• OsHTAS, a dominant major QTL on chromosome 9, has been cloned and verified, and confers tolerance to
48oC temperatures in rice seedlings.
• Kilasi et al. detected numerous QTL for seedling growth under HS and identified one QTL, RLHT5.1, for root
length under HS with phenotypic contribution up to 20.4%.
• Many QTL associated with heat tolerance at the reproductive stage have been mapped using the phenotypes
of spikelet fertility or seed-setting rate under HS.
• qHTB1-1 has been fine-mapped to a 47.1-kb region near qRRS1 on chromosome 1, and explained 13.1%–
17.8% of the phenotypic variance observed in several generations.
• Zhu et al. identified 12 QTL associated with heat tolerance at the booting stage, with one of the major-effect
QTL (qHTB3-3) located near qTL3.4 and RLPC3.1.
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Different names for a single locus indicate that the locus has been identified in at least two studies, suggesting that the QTL may
have large and stable effects. Red names indicate that these QTL have been cloned and functionally validated by transgenic
approaches.

• Although many heat-resistance QTL have been detected in rice, identification of the causal genes underlying
these QTL remains challenging, and only a few causal genes have been successfully cloned and functionally
validated to date.
• TT1 is a major QTL for thermotolerance at the seedling stage identified in African rice, and the more
thermotolerant TT1CG14 allele, as revealed by nearisogenic lines (NILs) and transgenic plants, appears to have
great potential for thermotolerance breeding.
• The natural variations of SLG1 confer high-temperature tolerance in indica rice.
• Compared with SLG1Tej-carrying plants, SLG1Ind-carrying plants show strong thermotolerance at both
seedling and reproductive stages with similar yield traits, suggesting the high potential of the SLG1Ind allele
for improving thermotolerance in japonica cultivars.
• QTL OsHTAS and Sus3, involved in thermotolerance at the seedling and grain filling stages, respectively,
have also been cloned and functionally verified.
• Identified QTL can be introduced into recipient cultivars using DNA markers linked to the QTL, even if the
underlying gene is unknown.
• Markers RM11633 and RM11642, linked to the qHTB1- 1 locus, were used to improve rice thermotolerance
at the booting stage by marker-assisted selection.
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• qMW4.1-introgressed NILs genotyped with the linked marker RM16424 showed higher grain quality than
recipient cultivars when exposed to HS.
• Identification and validation of thermotolerance QTL with stable effects across different genetic background
and environments, and pyramiding of these nonallelic QTL, are targets of thermotolerance breeding.
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Transgenic approach and genome editing
technologies
• Genetic engineering is an efficient and time-saving approach to generating heat-tolerant rice.
• The receptor-like kinase ERECTA is the best-known gene used for producing thermotolerant rice.
• Most leaves and tillers of ERECTA-overexpressing plants remained green and survived after exposure to HS
(42oC day/35oC night) for 10 days at the reproductive stage, whereas tillers in control lines became dried and
withered, and the seed-setting rate of ERECTA-overexpressing plants was 55%– 70% higher than that of the
control line (~35%) after HS.
• Field trials in multiple locations during a summer heat wave confirmed that ERECTA-overexpressing rice
plants showed higher seed setting rate and yield potential than control plants.
• Despite the promise of transgenic rice for improving thermotolerance, its use and commercialization are still
strongly affected by public concerns about unsubstantiated health and environmental safety questions,
hindering the application of transgenic technology in practical breeding.
• The emergence of genome editing technology provides a new opportunity for the application of plant
molecular breeding, as it can produce plants harboring only mutations in target genes without expression
cassettes.
• Genome editing has been successfully used to engineer plant thermotolerance and identify the molecular
mechanism of heat resistance.
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CASE STUDIES

Case study-1
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1. Department of Genetics and Plant Breeding, S.V. Agricultural College, Acharya NG Ranga Agricultural
University (ANGRAU), Tirupati, AP 517502, India.
2. Department of Plant Breeding, Institute of Frontier Technology, RARS, Acharya NG Ranga Agricultural
University (ANGRAU), Tirupati, AP 517502, India.
3. Department of Crop Physiology, Institute of Frontier Technology, RARS, ANGRAU, Tirupati, AP 517502, India.
JOURNAL :-MOLECULAR BIOLOGY REPORTS NAAS SCORE:- 8.11

Introduction
• An experiment has been conducted to identify differentially expressed genes in rice under heat stress
conditions by employing a diverse set of 32 rice genotypes that includes reported heat tolerant genotypes
Nagina 22 (N22) and Dular.
• Screening of the genotypes at field conditions during Summer-2018 for reproductive stage heat tolerance
(wherein the mean minimum (29.8°C) and maximum (38.4°C) temperatures surpassed optimum temperatures
(25°C night/30°C day) required for rice flowering and grain filling stages) and lab conditions employing
thermal induction response (TIR) technique to know the genotype’s acquired thermal tolerance revealed that
the genotype FR13A (indica landrace) showed highest overall performance for multitude of traits viz.,
95.29% of spikelet fertility (SF-%) at field level and 100% seedling survival percentage (SSP) at sub-lethal
temperatures under laboratory conditions.
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Materials and methods
• The plant material comprised of 32 genetically diverse genotypes of rice (Oryza sativa L.) which includes two
proven varieties for heat tolerance viz., Nagina22 (N22, aus ssp.), Dular (aus ssp.); landraces, cultivated
varieties etc., were used for the screening of genotype tolerance towards heat stress both at field and
laboratory conditions.
• Phenotyping for reproductive stage heat tolerance at field
conditions
• The chosen set of genotypes were screened for their reproductive stage stress tolerance under field conditions,
during Summer, 2018.
• such that to expose the temperature sensitive flowering and grain filling stages of the genotypes with the peak
summer temperatures i.e., from April to May, 2018.
• The data was recorded for the following parameters on five plants per genotype per replication. (i) Panicle
length (PL, cm)—length of the panicle from panicle node to the tip of the panicle, (ii) panicle number/plant
(PNP, no.), (iii) filled grain/panicle (FGP, no.), (iv) chafy grain number/ panicle (CGP, no.), (v) spikelet
fertility (SF, %)—the ratio of filled grains to ill filled grains expressed in percentage, (vi) 1000 seed weight
(TSW, g) and (vii) yield per plant (YP, g).
• The genotype tolerance towards high temperature was analyzed by considering the ratio of fully matured
grain number in contrast to chafy/ill flled grain number per panicle.
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• Phenotyping for seedling stage heat tolerance
at laboratory conditions
• Phenotyping for heat tolerance at lab level was carried out using ‘Thermal Induction
Response (TIR) technique.
• The experiment was conducted by arranging ten germinated seeds per genotype that consists
of 0.5 to 1.0 mm of coleoptile length, in the TIR plate.
• The ‘TIR’ technique involves three treatments, where in one acts as control maintained at
room temperature, second treatment directly exposed to lethal temperature of 55 °C for 2 h
and the third treatment which is sub-lethal, that involves exposing to gradually increased
temperatures from 38 to 55 °C i.e., with an increase of 4 °C /45 min up to 50 °C, then the
samples were kept at room temperature for 30 min followed by a direct exposure to the
challenging lethal temperature of 55 °C for 2 h in a growth chamber (Nano lab, India).
• Later the two treatments along with the control were maintained at room temperature for
7 days to revive shoot and root growth, by providing adequate moisture.
• On the eighth day after treatment, data was recorded for the traits, survival percentage (SP,
%), maximum root length (MRL, cm), shoot length (SHTL, cm), seedling dry weight (SDW,
mg) under both control and sub-lethal treatments.
• The relative root length (RRL), relative shoot length (RSHTL) and relative seedling dry
weight (RSDW) over control conditions were calculated and expressed in percentage, to
reveal the thermotolerance ability of a genotype.

• Genotyping of the germplasm employing reported candidate gene
based markers.
 Selection of genotypes for molecular analysis.
 Isolation of total RNA and cDNA synthesis.
 Selection of primers.
 Amplification of cDNA using semi-quantitative RT-PCR.
 Identification of differentially expressed genes (DEGs) upon heat stress in resistant and susceptible
genotypes.
27

Results and discussion
Screening of genotypes under field conditions
• Out of the 32 genotypes screened, four genotypes viz., Jagannadh, RPBio226, RNR15048 and MTU3626
showed no flowering and hence, it was not possible to screen their reproductive stage tolerance.
• Further, it can be assumed that these genotypes are photo-sensitive in nature.
• The genotypes FR13A (26.93 cm) and MTU 1010 (21.47 cm) have showed highest and lowest performance
respectively for Panicle length (cm).
• Genotypes BPT5204 (17.80) and the Konark (5.07) have showed highest and lowest performance respectively
for Panicle number/plant.
• Filled grain per panicle (No) was found highest in FR13A (247.87) and the lowest in Pusa1121 (37.87).
• Chafy grain number/panicle was highest in Pusa1121 (56.33) and the lowest in Sona Mashuri (4.73) followed
by FR13A (12.46).
• Spikelet fertility was highest in Sona Mashuri (96.95%) followed by FR13A (95.29%) and the lowest in
Pusa1121 (43.59%).
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• 1000 Seed weight (g) was highest in the reported heat tolerant genotype N22 (26.00 g) and the lowest in
BPT1235 (10.40 g).
• Yield per plant (g) was highest in Sona Mashuri (33.82 g) and the lowest in Pusa1121 (4.15 g).
• The frequency distribution of the traits filled grain/panicle and 1000 seed weight (g) showed nearly normal
distribution with skewness and kurtosis values of −0.19, 0.43 and 0.18, −0.77; respectively, whereas the other
traits did not fit the normal distribution (Supplementary Fig. 2).
• In the current study also N22 and Dular showed around 86% spikelet fertility under stress conditions,
as reported by Jagadish et al.
• The genotypes Sona Mashuri and FR13A showed very high spikelet fertility (around 96%) when
compared to the reported genotypes.
• Hence, these can be used as donor parents in the heat tolerance breeding. Further, the molecular
mechanism behind the rice spikelet fertility/pollen activity can be studied by employing such
genotypes.
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Conclusions
• Until recently, N22 and Dular that belongs to aus ssp., only have been widely used as donors to improve heat
tolerance in rice.
• However, in the current study the landrace, FR13A that belongs to indica ssp., has out-performed over the
reported checks N22 and Dular for vast list of traits under heat stress.
• Thus, this genotype can be used as potent donor source in heat tolerance breeding programmes from indica
sub-group.
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Agricultural college Bapatla

Case study- 2
31
JOURNAL :-ORYZA NAAS SCORE:- 4.4

INTRODUCTION
• Yield based indices are required for the evaluation of abiotic stress tolerant genotype and in the present
experiment, the response of 60 diverse rice germplasm lines to high temperature stress was carried out.
• Indices which measures the yield loss under high temperature conditions compared to normal can be used for
screening genotypes as done in case of drought stress by Mitra (2001).
• So, based on this yield-based indices were calculated from our experiment for identification of high
temperature stress tolerant genotype and also to identify suitable selection indices that could be used as
screening criteria for selecting tolerant genotypes.
32

MATERIALS AND METHODS
• A diverse set of sixty rice genotypes comprising of germplasm lines (2), green super rice (11), wild
introgression (2), landraces (8), tropical japonica (2), released varieties (35) were grown in the field.
• The genotypes were sown at different sowing dates (normal and late sowing) to enable crop to be exposed to
different temperature regimes naturally during reproductive stage (anthesis to physiological maturity).
• The first sowing was considered as control and second sowing as high temperature stress.
• At physiological maturity, panicles from each genotype in both normal and high temperature condition from
demarcated area of one-meter square were collected, dried, threshed, cleaned and the weight of grains was
recorded and expressed in kg ha-1.
• Spikelet fertility was worked out as number of filled spikelet's/ total number of spikelet's x 100 and expressed
in percentage.
• Based on the grain yield under control (Yp) and grain yield under stress (Ys) heat-tolerant indices were
computed namely, Stress Susceptibility Index (SSI), Relative Heat Index (RHI), Stress tolerance index (STI),
Geometric Mean Production (GMP), Mean Production (MP), Yield Index (YI), Heat resistance index (HI),
Yield Stability Index (YSI), Stress Susceptibility Percentage Index (SSPI) and Modified stress tolerance
(K1STI and K2STI) (Moosavi et al., 2008; Farshadfar and Sutka, 2002).
• Correlation between grain yield under both the conditions and computed yield-based indices were performed
in MS-Excel
33

RESULTS AND DISCUSSION
Grain yield (kg ha-1) and spikelet fertility (%)
• There was a substantial reduction of 26.7 % in grain yield during the 2nd sowing.
• Grain yield was higher in Rasi followed by Akshayadhan, HKR47, IR64 and Assanchidiya, whereas lower in
ADT43, Vandana, NDR359, MTU1001 and IR36 under high temperature stress.
• Lesser reduction in grain yield was evident in N22 (9.7 %), IR64 (12.0 %), Rasi (14.5 %) and Khudaridhan
(14.9 %).
• On the contrary, higher reduction was in Jaya (36.2%), MTU1001 (36.3 %), Lalat (36.5 %), Varadhan (36.5
%), IR36 (37.7 %), Vandana (45.4 %) and ADT43 (54.0 %).
• Elevated temperature led to a reduction of spikelet fertility and the differences among the treatments were
highly significant.
• Spikelet fertility under high temperature was higher in Rasi, N22, Akshayadhan and IR64. Lower spikelet
fertility was noted in Vandana, IR36 and ADT43.
34

S.NO CHARACTER HIGHEST/MAXIMUM LOWEST/MINIMUM
1 Stress Susceptibility Index (SSI) ADT43 (1.98) N22 (0.34)
2 Relative Heat Index (RHI) N22 (1.24) ADT43 (0.64)
3 Stress Tolerance Index (STI) GSR310 (1.18) ADT43 (0.28)
4 Geometric Mean Production (GMP) GSR310 ADT43
5 Mean Production (MP) GSR310 ADT43
6 Yield Index (YI) Rasi (1.25) ADT43 (0.49)
7 Heat Resistance Index (HI) Rasi (1.09) ADT43 (0.23)
8 Yield Stability Index (YSI) N22 (0.91) ADT43 (0.47)
9 Modified stress tolerance (K1STI and K2STI) GSR310,Rasi Vandana, ADT43
10 Stress Susceptibility Percentage Index (SSPI Pantdhan4 (23.45) N22 (4.43)

CONCLUSION
• In this study, out of the computed 11 indices STI, GMP, MP, YI, K1STI, K2STI were selected as most
effective and suitable stress indices for identifying tolerant rice genotypes with high yield potential under high
temperature conditions as correlation analysis revealed that these indices were significantly correlated under
both Ys and Yp.
• When a large germplasm is available computing these yield based indices would help in preliminary
screening for high temperature tolerant genotypes.
36

CASE STUDY-3
37
Department of genetics and plant breeding
• Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou
310021, China
• Rice Research Institute, Fujian High Quality Rice Research & Development Center, Fujian Academy of Agricultural
Sciences, Fuzhou 350019, China
• Collage of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
JOURNAL :- PLANT MOLECULAR BIOLOGY REPORTER NAAS SCORE:- 7.60

Introduction
• Breeding heat-tolerant rice using genes affording thermotolerance is a fundamental way to address heat stress
challenge.
• In this study, a major QTL, TT1-2, was found to regulate heat tolerance in rice; this QTL was controlled by a
single dominant gene.
• Using F2:3 populations, we narrowed TT1-2 to a 26.0-kb region containing three putative genes, one of which
encodes an α2 subunit of the 26S proteasome.
• This gene was considered the TT1-2 candidate, and the TT1 gene involved in rice heat tolerance was present
at this locus.
• Further analysis showed that the amino acid sequence of the TT1-2 gene had one amino acid difference:
arginine (R)-99 was changed to histidine (H), which leads to the formation of a normal alpha-helix.
• Moreover, through marker-assisted selection and conserved breeding selection, we developed a new male-
sterile line, Zhehang 10A, which had a high outcrossing rate, good quality, and strong heat tolerance.
• They used Zhehang 10A as the female parent and the restorer line Fuhui 1586 as the male parent and bred a
new hybrid, Zhehangyou 1586, which showed high yield, good quality, and strong heat tolerance.
38

Materials and Methods
Plant Materials and Construction of the Mapping Population
• In the summer of 2014, the rice cultivar Nipponbare was used as a pollen donor in crosses with Y1502.
• Twenty true F1 hybrid seeds were sown at the Sanya Experimental Station in Hainan Province in the spring,
and the resulting F2 seeds were harvested.
• A total of 3464 individual plants of the F2 population were planted at the Hangzhou Experimental Station in
Zhejiang Province in the summer of 2015.
• A total of 3438 F2:3 seeds were ultimately harvested, and a total of 804 non-heat-tolerant plants in the F2:3
population were selected for fine mapping.
Plant Cultivation and Thermotolerance Identification
• The rice seedlings used in this study were cultivated by hydroponic culture in Yoshida solution (pH 5.8).
• For the survival rate analysis of Y1502, Nipponbare, Zhehangyou 1586, and Tianxie 1, 12- day-old seedlings
were treated with 45 °C for 45 h followed by recovery at 28 °C for 2 weeks, and the number of plants in each
line and their corresponding survival were recorded and used to calculate survival rates.
39

Molecular Mapping of the TT1-2 Gene
• The physical map of the target gene was constructed via bioinformatic analysis using BAC and P1-derived
artificial chromosome (PAC) clones of the cultivar Nipponbare released by the International Rice Genome
Sequencing project.
• The clones were anchored with the target gene-linked markers, and then, alignment of the sequences was
carried out using the pairwise Basic Local Alignment Search Tool (BLAST).
40

Results
Evaluation of the Heat Tolerance of Y1502 Under Laboratory
Conditions.
• To evaluate the heat tolerance of Y1502 in the laboratory, a heat tolerance experiment was carried out
involving Y1502 and Nipponbare.
• We found that Y1502 (Oryza sativa ssp. indica) was much more heat tolerant than Nipponbare (Oryza sativa
ssp. japonica); the survival rates of Y1502 and Nipponbare were 100 and 0, respectively.
Genetic Analysis of the Heat Tolerance Gene
• To map the heat tolerance gene in Y1502, the resistant donor Y1502 was crossed with Nipponbare.
• All the F1 individuals exhibited a heat-tolerant phenotype.
• The F2 individuals were used to analyze the inheritance of the heat tolerance gene in Y1502.
• The segregation of the heat-tolerant and non-heattolerant progeny in the F2 population fit a 3:1 ratio.
• The segregation ratio suggested that Y1502 harbors a major dominant heat tolerance gene, which was
tentatively designated TT1-2.
41

Fine Mapping of the TT1-2 Gene
• To map the TT1-2 gene to a smaller region, 804 individuals with no heat tolerance were identified from the
F2:3 population derived from Y1502×Nipponbare.
• Additional maps were constructed using published markers of the region between Indel-3-18 and Indel-3-22.
• All recombinants were genotyped using five polymorphic markers.
• The results showed that the TT1-2 gene was located between the molecular markers RM6594 and RM6931 on
chromosome 3, and the genetic distance between the two markers was 5.9 cM.
Phylogenetic Tree of the TT1-2 Gene
• To gain insight into the function of TT1-2, a phylogenetic tree was generated using the 26S
proteasome protein sequences from rice and other plant species.
• The phylogenetic tree showed highly homologous genes of TT1-2 in more than 20 different
species, and all these genes encoded the same 26S proteasome protein.
• These results indicated that the TT1-2 gene was highly homologous and conserved across
different plant species.
42

Conclusion
TT1-2 Regulates the Heat Tolerance of Rice
Utilization of the TT1-2 Gene in Rice Breeding
• Plant breeding, which aims to improve the genetic basis of new varieties of crops with increased productivity
and quality, combines art and science (Xi et al. 2006).
• In general, traditional breeding is predominantly based on phenotypic assays (Xu et al. 2010), but this
approach targets only traits with an easily observable phenotype for genetic improvement.
• Moreover, heat tolerance is an unobservable trait that requires further phenotypic identification. However, we
can improve the heat resistance of rice by molecular marker-assisted selection.
43

JOURNAL :- RICE(SPRINGER) NAAS SCORE:- 9.51
44

• Analysis of genetic mutations is one of the most effective techniques for investigating gene function.
• Genes controlling developmental and metabolic processes have been discovered in plants by mutational
analysis (Miroslaw and Iwona 2003).
• Mapping a novel mutation to a well defined chromosomal region is an important step in genetic analysis.
• The International Rice Functional Genomics Consortium announced the public availability of more than
200,000 rice mutant lines, which represent mutations in about half of the known functional genes mapped for
rice to date (Krishnan et al. 2009).
• Though Nagina 22 (N22) is deep rooted, drought and heat tolerant aus rice variety (Jagadish et al. 2010b),
there are very few genetic studies using N22 mutants.
• Characterization of Ethyl Methane Sulphonate (EMS) induced mutants of N22 for water stress and heat
tolerance was reported by Panigrahy et al. (2011).
• A dark green leaf mutant, N22-H-dgl219 (NH219) was isolated under prolonged drought.
• During dark-induced senescence, NH219 maintained higher chlorophyll and carotenoid content and
photochemical efficiency of photosystem II in comparison with N22.
BACKGROUND

• Detached leaves of NH219 accumulated less reactive oxygen species (H2O2 and superoxide radicals) and
maintained higher chlorophyll content than N22 after 40°C heat treatment for 3 days.
• The present study reports further characterization of NH219 for heat tolerance under field conditions in
comparison with its wild type N22.
• The mutant NH219 was crossed with another moderately heat tolerant variety IR64 (Khush and Virk 2005,
Jagadish et al. 2010b) to map the mutation causing the drought and heat tolerant phenotype in the mutant and
heat tolerance associate traits.
• The results of initial mapping of mutations in NH219 using F2 segregants with extreme phenotype for eight
traits derived from both IR64 × NH219 and its reciprocal cross are also reported.
46

Results
Characterization of N22, NH219 and IR64 for heat tolerance
• Morphological and physiological traits (plant height, tiller number, number of panicles, panicle length,
yield/plant, pollen viability, spikelet fertility, chlorophyll a/b ratio (chl a/b), relative water content (RWC),
electron transport rate (ETR) and Fv/Fm were studied in N22 and NH219 in field in 2 sets, one in ambient
conditions and the other in heat stress conditions.
• All the observed trait values in ambient conditions were higher in mutant compared to N22 and IR64 except
marginal difference in Chl a/b ratio and ETR.
47

Two-dimensional gel electrophoresis
48

Conclusion
• They conclude that the EMS induced mutant NH219 can tolerate heat stress more when compared with its
wild type N22.
• NH219 showed lesser reduction in yield/plant and related traits compared to N22.
• Ribulose bisphosphate carboxylase large chain precursor (EC 4.1.1.39) was present in NH219 leaves and
absent in N22 under ambient growth conditions.
• Both pollen viability and spikelet fertility were significantly reduced in IR64 but not in N22 and NH219.
• Marker RM1089 was associated with number of tillers and yield per plant, RM423 with leaf senescence,
RM584 with leaf width and RM229 with yield per plant, based on single marker analysis of F2 mapping
population from the cross between IR64 and NH219.
• Dense genotyping of mapping population can help to map traits related to heat tolerance.
49

Thank you
50

Molecular characterization of genotypes for thermotolerance in rice.

Molecular characterization of genotypes for thermotolerance in rice.

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