Komolafe omotayo's thesis presentation

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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

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


0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

Spikelet fertility (%)

Rice Varieties


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)


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


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


Number of Tillers

Panicle length



Number of Panicle


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.


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.


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

Komolafe omotayo's thesis presentation

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