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WELCOME
Master’s Thesis Presentation
Present By: Komolafe Omotayo BabatundeID No.: 2690
Title: Molecular Characterization using SSR Markers and effects of
Heat stress on Rice (Oryza sativa L.) genotypes
Outline•Introduction•Materials and Methods•Experimental Findings•Conclusions•Recommendations•Future prospects
Brief Introduction
Introduction• Rice (Oryza sativa L.) - most important staple food in Asia and Africa.
• According to Fitzgerald et al. (2009), Rice Provides; • over 21% of the calorific needs of the world’s population • 76% of the calorific intake of the population of South East Asia
• To meet the demand of a growing population, 0.6–0.9% annual increment in rice production is required until 2050 (Carriger and Vallee, 2007)
Fitzgerald M. A., McCouch S. R., and Hall R. D, (2009). Not just a grain of rice: the quest for quality. Trends Plant Sci 14:133–139,Carriger S., and Vallee D. (2007). More crop per drop. Rice Today 6:10–13
Introduction cont’d…•Basmati rice is a class of premium rice
Superfine grainsDistinct aromaExcellent elongation ability• Soft• Flaky• Texture
High market price, Export and Foreign exchange
In summer 2003, heat waves caused loss of 36% corn yield in Italy
Introduction cont’d…• Rice is increasingly being cultivated in more marginal environments that experience warmer temperatures. (Prasad et al. 2006)
• Battisti and Naylor (2009) articulated that “Rapidly warming climate may seriously affect crop yields in the tropics and subtropics by the end of this century”
• Intergovernmental Panel on Climate Change (IPCC, 2014) Predicted that, by the end of the 21st century, the global climate is foreseen to warm by an average of 2.5–5.8 °C
Urgent need to prepare well ahead of time !!!
Overall aims •To identify a basmati variety that is relatively showing heat tolerant trait.
•To attempt understanding the molecular basis of the trait.
Specific Objectives1. To establish diversity relationship within rice
varieties, using molecular characterization through Microsatellite Markers (28 SSR Primers).
2. To Screen and Identify Basmati genotypes showing morphological tolerant traits under control and heat stress conditions at anthesis stage.
3. To decipher the effects of heat treatment on screened varieties from objective 2, for Membrane Stability Index (MSI) and Proline content, after 10 days germinated seedlings were subjected to temperature stress.
4. To evaluate the expression patterns of six Heat stress - inducible genes, subjected to temperature stress at seedling stage by Quantitative Reverse Transcriptase – PCR (qRT-PCR).
Specific Objectives cont’d…
Materials and Methods
Experiment - 1Molecular characterization of 18 rice
genotypes
List of Primers RM- Rice Microsatellites
1. RM 12. RM 103. RM 124. RM 165. RM 206. RM 217. RM 22
8. RM 269. RM 3110.RM 4011.RM 13812.RM 15213.RM 15514.RM 162
15.RM 17116.RM 18217.RM 18918.RM 102419.RM 108920.RM 120921.RM 1367
22.RM 186723.RM 281124.RM 362725.RM 564626.RM 565727.RM 686428.RM 7479
List of Rice genotypes1)Santnang Patna2)Pusa Sugandha –
153)Pusa Basmati –
11214)Pusa Sugandha– 175)Pusa Sugandha – 36)Vallabh Basmati –
247)Pusa Basmati – 18)Basmati – 3869)Vallabh Basmati –
2310)Basmati – 37011)Type – 312)Bas – 564
13)IR – 6414)Super Basmati15)IPB – 116)Shusk Samrat17)Pusa Basmati
150918)Nagina 22
Experiment 1 – Stepwise Procedure•DNA Isolation• Spectrophotometry – 260nm/280nm• Polymerase Chain Reaction (PCR) – Amplification•Gel Electrophoresis• Scoring banding patterns - Zero/One sheet•Ntsys and Exstat – for dendogram• Calculation of Polymorphic Information Content (PIC), Resolving Power, Marker Index etc.
Experiment - 2Effects of heat stress at anthesis
stage
Temperature Data
2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 201426.028.030.032.034.036.038.040.042.0
Meerut District
JuneJulyOctoberNo-vember
Year
Ave.
Max
. Tem
pera
ture
(°
c)
Yearly maximum temperature data for Meerut district over the span of 10years
Experiment 2•Anthesis stage – 18 varieties•Temperature - Ambient (control), 34°C and 38°C•Duration - 6hours daily (09:00 – 15:00) for 4days
Observations – At Physiological maturityPlant height (cm)Number of tillersPanicle length (cm)Number of paniclesSpikelet Fertility (%)
PicturesStage1 Stage 2
Stage 3
Stage 4
Experiment - 3, 4 and 5Effects of heat stress at seedling
stage
Experiment 3 & 4•Four (4) varieties•Seedling stage – 10days old•Heat stress – at 30°C and 45°C, for 18hrs respectively.•Observation
Physiological parameterMembrane Stability Index (MSI) – Sairam 1994
Biochemical ParameterProline content – Bates et al. (1973)
Sairam R.K. (1994). Effect of moisture stress on physiological activities of two contrasting wheat genotypes, Indian J Exp Biol 3:584-593.Bates L., Waldren R.P., and Teare I.D., (1973). Rapid determination of free proline for water-stress studies. Plant and Soil, 39, 205-207
Experiment 5 – Gene expression analysis•Seedling stage – 10days old•Heat stress – at 30°C and 45°C•Duration of stress - 0hr and 12hrs•Observation
Expression profile of Heat – inducible genes
Heat – Inducible genes•OsHsfA2a (Os03g0745000) •Dehydrin (Os11g0453900)•DnaJ protein putative (Os06g0195800)•OsHsfB2c (Os09g0526600)•AWPM-19 (Os05g0381400)•Hsp90 (Os04g0107900)•Actin RAc1 – Housekeeping gene
Experiment 5 – Stepwise Procedure•Total RNA Isolation•Spectrophotometry – 260nm/280nm•Gel Electrophoresis of Total RNA•cDNA Synthesis - M-MULV RT-PCR kit•Real-time PCR analysis•Gel Electrophoresis of amplified product•Data analysis - (Livak and Schmittgen, 2001)
Livak K.J., and Schmittgen T.D., (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2^[-delta delta C(T)] Method. Methods.,25(4): 402-408,.
Pictures - Before Stress
Pictures - After Stress
Experimental Findings
Experiment - 1Molecular characterization of 18 rice
genotypes
Gel pictures – Genomic DNA
Gel pictures – PCR Amplified Product
Gel pictures – PCR Amplified Product
Polymorphic Information Content
Bar graph showing SSR –PIC values
RM
1
RM
10
RM
12
RM
16
RM
20
RM
21
RM
22
RM
26
RM
31
RM
40
RM
138
RM
152
RM
155
RM
162
RM
171
RM
182
RM
189
RM
1024
RM
1089
RM
1209
RM
1367
RM
1867
RM
2811
RM
3627
RM
5654
RM
5657
RM
6864
RM
7479
00.10.20.30.40.50.60.70.80.9
Polymorphic Information Content
Primer codes
Pic
Val
ues
Resolving Power of MarkersRM 13% RM 10
3% RM 123% RM 16
3%RM 20
5%RM 21
3%RM 22
3%RM 263%RM 313%
RM 406%
RM 1383%
RM 1523%RM 155
6%RM 1623%
RM 1713%
RM 1826%
RM 1893%
RM 10243%
RM 10894%
RM 12093%
RM 13674%
RM 18674%
RM 28113%
RM 36273%
RM 56543%
RM 56573%
RM 68643%
RM 74793%
Resolving Power
Marker Index of Primers
RM 1
RM 10
RM 12
RM 16
RM 20
RM 21
RM 22
RM 26
RM 31
RM 40
RM 138
RM 152
RM 155
RM 162
RM 171
RM 182
RM 189
RM 1024
RM 1089
RM 1209
RM 1367
RM 1867
RM 2811
RM 3627
RM 5654
RM 5657
RM 6864
RM 7479
0
0.5
1
1.5
2
2.5
3
Marker Index
Primer Codes
Mar
ker
inde
x
Dendrogram
Dendrogram showing clustering of 18 rice varieties constructed using UPGMA based on Jacquard’s dissimilarity coefficient obtained from SSR analysis
Distribution of 18 rice genotypes into different clusters based on Dendrogram result
Groups Sub-group Cluster Sub-cluster No. of Genotypes Genotypes
I A A1 2
Santnang Patna
IR – 64
A2 1 VB – 24
II B 1 PS – 15
III C 2 Pusa Basmati 1509
PS – 3
D
D1
D1(a) 2 VB – 23
PB – 1121
D1(b) 2 PB – 1
PS – 17
D2 D2(a) 4
B – 370
B – 386
Super Basmati
Type – 3
D2(b) 1 IPB – 1
IV E 1 Nagina 22
V F 2 Bas – 564
ShuskSamrat
Distribution of 18 rice genotypes into different clusters
Experiment - 2Effects of heat stress at anthesis stage
Santn
ang P
atna
PS – 1
5
PB – 1
121
PS – 1
7PS
– 3
VB – 24
PB – 1
B – 386
VB – 23
B – 370
Type
– 3
Bas – 5
64IR – 6
4
Supe
r Basm
ati
IPB – 1
Susk
Sumrat
Pusa
Basmati
...
Nagina
220.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
Plant Height (cm)
Panicle length (cm)
Rice Varieties
(cm
)
Effect of genotypes on Morphology
Santn
ang P
atna
PS – 1
5
PB – 1
121
PS – 1
7PS
– 3
VB – 24
PB – 1
B – 386
VB – 23
B – 370
Type
– 3
Bas – 5
64IR – 6
4
Supe
r Basm
ati
IPB – 1
Susk
Sumrat
Pusa
Basmati
...
Nagina
220.001.002.003.004.005.006.007.008.009.00
Number of Pan-iclesNumber of Tillers
Rice Varieties
Effect of genotypes on Morphology
0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00
Spikelet fertility (%)
Rice Varieties
Effect of genotypes on Morphology
Effects of heat stress on Morphology
Control (Ambient) 34°C 38°C0.00
10.0020.0030.0040.0050.0060.0070.0080.0090.00
100.00110.00
Plant Height (cm)Panicle length (cm)
Temperature
Effect of varying temperatures on plant height (cm) & panicle length (cm)
Effects of heat stress on Morphology
Control (Ambient) 34°C 38°C0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Number of Pani-cles
Number of Tillers
Temperature
Effect of varying temperatures on Number of Panicles & Number of Tillers
Effects of heat stress on Morphology
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Control (Ambient)
34°C
38°C
Temperature
Spik
elet
Fer
tilit
y (%
)
Effect of varying temperatures on spikelet fertility
Interacting effects of heat stress on genotypes
Plant Height
Interacting effects of heat stress on genotypes
Number of Tillers
Panicle length
Interacting effects of heat stress on genotypes
Interacting effects of heat stress on genotypes
Number of Panicle
Interacting effects of heat stress on genotypes
Spikelet Fertility
Four Selected varieties•Pusa Basmati - 1•Nagina 22•Basmati - 370• IR – 64
Experiment – 3 & 4Effects of heat stress at seedling stage
Membrane stability index (MSI) of selected genotypes
0.000
5.000
10.000
15.000
20.000
25.000
30.000
PB1N22B370IR64
Varieties
Mem
bran
e St
abili
ty In
dex
(%)
Effect of temperature on Membrane Stability Index in Rice
seedlings
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
18.000
30°c45°c
Temperature
Mem
bran
e St
abili
ty In
dex
(%)
Interacting effect of Heat stress and selected varieties on MSI
PB1 N22 B370 IR640.000
5.000
10.000
15.000
20.000
25.000
30.000
35.000
30°c
45°c
Rice Varieties
Mem
bran
e St
abili
ty
Inde
x (%
)
Proline content of selected genotypes
.000
.050
.100
.150
.200
.250
.300
.350
.400
.450
.500
PB1N22B370IR64
Varieties
Prol
ine
(µ m
ol g
-1)
.000
.100
.200
.300
.400
.500
.600
30°c45°c
Temperature
Prol
ine
(µ m
ol g
-1)
Effect of temperature on Proline content in Rice seedlings
Proline content at seedling stage
PB1 N22 B370 IR64.000
.100
.200
.300
.400
.500
.600
.700
30°c
45°c
Rice Varieties
Prol
ine
(µ
mol
g-1
)
Experiment - 5Expression of heat – inducible genes
Gel picture – Total RNA
Gel picture – Total RNA cont’d…
qRT – PCR Amplification curves
Gel pictures – Amplified product
Varieties
PB - 1 N22 B - 370 IR - 64
Treatments
Temperature/duration
Temperature/duration Temperature/durationTemperature/
duration
Genes 30°C/0hr
30°C/12hrs
45°C/12hrs30°C/0hr
30°C/12hrs 45°C/12hrs30°C/0hr
30°C/12hrs 45°C/12hrs30°C/0hr
30°C/12hrs
45°C/12hrs
OsHsfA2a
01.00 0.09 7.41 01.00 0.12 0.37 01.00 4.49 333.00 01.00 7.34 62.67
Dehydrin
01.00 0.17 3.21 01.00 0.31 0.07 01.00 4.05 42.32 01.00 2.97 10.32
OSHsfB2c
01.00 0.28 0.35 01.00 0.61 0.03 01.00 3.27 4.23 01.00 1.93 0.11
AWPM - 19
01.00 0.18 0.83 01.00 0.09 0.01 01.00 72.42 325.90 01.00 74.78 14.59
HSP 90 01.00 9.18 640.15 01.00 14.90 75.44 01.00 554.66656704.
901.00 778.50
15755.09
Relative expression of all genes
Relative expression of OsHsfA2a gene
B370 IR640.000
50.000
100.000
150.000
200.000
250.000
300.000
350.000
0hr12hrs/3012hrs/45
Rice varieties
Rela
tive
exp
ress
ion
PB - 1 N220.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
0hr12hrs/3012hrs/45
Rice varieties
Rel
ativ
e ex
pres
sion
Relative expression of Dehydrin gene
B370 IR640.000
5.000
10.000
15.000
20.000
25.000
30.000
35.000
40.000
45.000
0hr12hrs/3012hrs/45
Rice varieties
Rel
ativ
e ex
pres
sion
PB - 1 N220.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0hr12hrs/3012hrs/45
Rice varieties R
elat
ive
expr
essi
on
Relative expression of OsHsfB2c gene
B370 IR640.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
4.500
0hr12hrs/3012hrs/45
Rice varieties
Rel
ativ
e ex
pres
sion
PB - 1 N220.000
0.200
0.400
0.600
0.800
1.000
1.200
0hr12hrs/3012hrs/45
Rice varieties
Rel
ativ
e ex
pres
sion
Relative expression of AWPM-19 gene
B370 IR640.000
50.000
100.000
150.000
200.000
250.000
300.000
350.000
0hr12hrs/3012hrs/45
Rice varieties
Rel
ativ
e ex
pres
sion
PB - 1 N220.000
0.200
0.400
0.600
0.800
1.000
1.200
0hr12hrs/3012hrs/45
Rice varieties
Rel
ativ
e ex
pres
sion
Relative expression of Hsp90 gene
B370 IR640.000
100000.000
200000.000
300000.000
400000.000
500000.000
600000.000
700000.000
0hr12hrs/3012hrs/45
Rice varieties
Rela
tive
exp
ress
ion
PB - 1 N220.000
100.000
200.000
300.000
400.000
500.000
600.000
700.000
0hr12hrs/3012hrs/45
Rice varietiesR
elat
ive
expr
essi
on
1. Molecular characterization – Traditional basmati varieties have very high degree of similarity between them
Cross – bred varieties and other non – aromatic varieties showed varying level of similarity with the traditional Basmati rice group.
Conclusion
2. Effect of High temperature at anthesis stage
Increases Plant height Number of tillers, and
Panicle length
Reduces Spikelet fertility (%)
No significant effect
Number of panicles
Conclusion Cont’d…
•Membrane Stability Index of all the •Selected varieties differ significantly•B – 370 possess the most stable plasma membrane under heat stress.
•Proline content accumulates distinctively under heat stress condition•B – 370 accumulated more proline.
Conclusion Cont’d…
4. All the six heat – inducible genes were expressed in all the selected varieties (except DnaJ protein putative gene) under heat stress at seedling stage.
•B – 370 expressed all genes much more than all other selected varieties.
Conclusion Cont’d…
Contribution to knowledge•Widely known heat tolerant variety N22 has been identified from the findings of this investigation to be susceptible to heat stress at seedling stage.•Membrane Stability Index that has been used over time to screen for heat tolerant varieties has been noted not significant (p<0.05) for rice at seedling stage.
•For future breeding program, a basmati variety (B – 370) has been identified to show tolerance based on biochemical and molecular responses to heat stress at seedling stage.•For local farmers, ahead of global warming effect a variety of basmati can be proposed as tolerant to heat stress at seedling stage.
Future prospects
Addition achievements•Computer Applications for laboratory usage•Name – Molecular Calculator
Dos basedUser-friendly
Recommendations•Protein profiling of the seedling of B – 370 under heat stress at seedling stage, to be able to ascertain if, the relatively expressed genes would correspond to the translated protein.•Expression profile analysis of the six heat – inducible genes on selected varieties under heat stress at anthesis, to compare their relative expression with that of seedling stage.
Issue of Concern – N22
Thank You for Listening