Basal pore

Apical pore

Anthers of field-grown rice cv IR74

WUEMED Drought Course,Bologna, 4-10 July 2006:5 lectures on “Omics and drought”by John Bennett, IRRI IRRI

1. Integration of gene and allele discovery with breeding for drought tolerance

2. Microarrays and proteomics as tools to generate new hypotheses

3. Microarrays: experimental design, statistical analysis and gene clustering

4. Reversible and irreversible responses of gene networks to drought stress

5. Potential and limitations of proteomics and metabolomics in drought research

Omics and Drought: Lecture Outline

Transcriptomics Proteomics Metabolomics

JX Chen GH Salekdeh Cook et al. (2004)

IRRI

John BennettInternational Rice Research Institute

Los Baños, Philippines

1. Integration of gene and allele discoverywith breeding for drought tolerance

WUEMED – Drought Course

IRRI

1.1: Drought in IRRI’s breeding program

IRRI

L.J. Wade

Drought is frequent in rainfed rice eco-systems

IRRI

IRRI

IRRI’s research is organized into 12 projects

1 2 3 7 8 9

Functional genomics

Germplasm conservation and allele mining

Breeding - irrigated ecosystem

Breeding - rainfed ecosystems

Consortium for UnfavorableRice Environments

Activities relatedto abiotic stresses

IRRI

Natural resource management- for rainfed ecosystems

Abiotic stress is the focus in 5 outputs of Project 7

1

2

3

4

5

6

IRRI

Lowlands – drought and submergence

Lowlands – salinity, deep-water, poor soils

Uplands – drought, poor soils

Aerobic rice – water deficit, water saving

Highly nutritious rice

Novel breeding methods for national programs

Outputs:

Stop press: IRRI’s new Strategic Plan features Drought as a Frontier Project

Early-seasondrought

Drought tolerance: a complex mix of traits• Tolerance – retaining water, protecting tissues, maintaining fertility • Avoidance – deep roots, penetrating roots• Escape – short duration

IRRI

Reproductive stage drought tolerance

Varieties with improved reproductive stage drought tolerance reduce the risk of complete crop failure and increasethe “floor” yield in severe droughts.

Raipur, 2005

G. Atlin IRRI

Focus on understanding how drought stress affects yield components differently

a = panicles m-2

b = spikelets panicle-1

c = filled grain (%)

d = single grain weight (mg)

Yield = 10-5 x abcd g m-2

G T PI F M

G T PI F M

G T PI F M

G T PI F M

= stress period

Boonjung and Fukai (1996) Field Crops Res 48:47-55 IRRI

1.2: International Rice Molecular Breeding Program

IRRI

Biodiversityof rice

Farmers’fields

Traditional breeding

Genebank Tissue

culture

DNAtechnology

Marker-aided

selection(MAS)

GenomicsGenetically-engineered

rice Gene

discovery Exoticgenes

1977 1985

1988

1998

1991

Biotechnology at IRRI:Helping the breeding program

1960

1994

1999

2000

2000 China (Bt, Xa21)2002 Philippines (Xa21)

19961993

2004

IRRI

International Rice Molecular Breeding Program

• A method of identifying genes and alleles important for drought tolerance (applicable to other traits also).

• It combines gene/allele discovery and gene mapping with backcross breeding and gene network discovery

• Many institutions may be involved, backcrossing a common set of ~300 donors to ~3 local elite varieties

• The reproductive-stage drought stress is strong enough to kill bothparents.

• Mapping using ~100 markers per genome allows networks of co-selected unlinked genes to be discovered and novel alleles effective in many genetic backgrounds identified.

Li et al. (2005). Genome-wide introgression lines and their use in genetic and molecular dissection of complex phenotypes in rice (Oryza sativa L.). Plant Mol Biol. 59:33-52. Xu et al. (2005). QTLs for drought escape and tolerance identified in a set of randomintrogression lines of rice. Theor Appl Genet. 111:1642-50. IRRI

Location of institutes participating in International Rice Molecular Breeding Program

Z.K. Li IRRI

1.3: Backcrossing to enhance drought tolerance in popular varieties

IRRI

A six-step molecular breeding strategy

1. Participatory rural appraisal*2. Mechanism discovery#

3. Gene and allele discovery#

4. Pre-breeding for trait reconstruction5. Molecular breeding for variety development6. Participatory variety selection

*Varieties popular with farmers often contain useful genes- salt-tolerance in Bangladesh- drought tolerance in Bangladesh

#Mechanisms, genes or alleles can be discovered any order

IRRI

1.Simple geneticsSimple screen

2.Simple geneticsDifficult screen

4.Complex geneticsDifficult screen

3.Complex geneticsSimple screen

Enhancing breeding efficiency at IRRI throughmolecular approaches

DNA based selection

Genetic analysis, gene discovery, DNA based selection

Advancedscreens

Package of traitsrequired for

adoption by farmers

Pedigree breeding +trait-based selectionBackcross breeding +

DNA based selection

Four types of traits

IRRI

Backcrossing for enhancing drought tolerance

• Improve drought tolerance of an already popular variety by introgression of major genes or QTLs from different donorsusing DNA marker-assisted backcrossing (MAB)

Donor 1(with major gene1or major QTL1)

Donor i(with major geneior major QTLi)

Recurrent parent(popular variety)

RP + 1

RP + i

Recurrent parent+ 1 + … + i

• There may be a practical upper limit of ~5 genes that can be introgressedsimultaneously into a recurrent parent using MAB.

• The best genes to use may be (a) well-characterized transgenes, (b) major genes, (c) QTLs of large effect, (d) novel alleles identified bymining techniques, and (e) mutant alleles; all must have large effects in diverse backgrounds. IRRI

1.4: QTLs of large effect

IRRI

Biomass production is greatly reduced in dry fields

G. Atlin

Enhancing biomass in dry fields at flowering may help increase yield

IRRI

Screening in paddies drained after transplanting can identify cultivars with improved tolerance to

long periods of dry soil conditions

Raipur, India, 2005G. Atlin

IRRI

410116370.40PSBRC 82421013080.31IR 36485416710.34PSBRC 14519715550.30PSBRC 80733618740.26IR 79670-125-1-1-3757621080.28IR 70213-10-CPA 4-2-2-2

Biomass (kg/ha)

Grain yield (kg/ha)

Harvest indexDESIGNATION

Cultivar differences in biomass production in intermittently-dried field: IRRI WS 2005

G. Atlin

IRRI

• Some varieties have much greater tolerance to flowering–stage stress than IR 36, IR 64, and MTU 1010.

• Tolerance to extreme stress at flowering may be affected by major QTLs.

Tolerance to reproductive-stage stress

R. Venuprasad, J. Bernier, A. Kumar, G. Atlin IRRI

Genes for drought tolerance at IRRI

IRRI*IRMBP = International Rice Molecular Breeding Program

Rice germplasm Genes fromother sources

Gene discovery

Allele pyramids

Allele mining

New drought-tolerant varieties

Promoterdiscovery

Phenotyping, mutants,expression analysis, map-based cloning

TILLING, DArT

Marker-aided selection

Marker-aided backcrossing

Novel alleles

Promoterswitching

Transformation

IRMBP*

Phenotypingof advancedbackcrosslines Rice orthologues

1.5: Allele mining

IRRI

Finding alleles through whole-genome sequencing

McNally et al. (2006) Plant Physiol. IRRI

Finding mutants and alleles throughhigh-throughput TILLING and eco-TILLING

Colbert et al. (2001). High-throughput screening for induced point mutations. Plant Physiol. 126: 480-484.

TILLING: Targeting Induced Local Lesions IN Genomes

IRRI

Colbert et al. (2001). High-throughput screening for induced point mutations. Plant Physiol. 126: 480-484.

TILLING using acrylamide gels

IRRI

Slade et al. (2004). A reverse genetic, nontransgenic approach to wheat cropimprovement by TILLING. Nature Biotechnology 23: 75-81.

TILLING of WAXY gene of wheat

IRRI

1. Waxy genes encode granule-bound starch synthase1 (GBSS1) 2. Wheat with only one or two functional GBSSI genes produces starch with

intermediate levels of amylose and is referred to as partial waxy wheat.3. Waxy starches, including those of maize and rice, are composed almost entirely of

amylopectin with little or no amylose; they have unique physiochemical propertiesand economically valuable functional qualities.

4. Commercial uses of full waxy wheat exist in the food, chemical and paper industries.

Slade et al. (2004). A reverse genetic, nontransgenic approach to wheat cropimprovement by TILLING. Nature Biotechnology 23: 75-81.

Screen for mutations in the wheat waxy genes

IRRI

TILLING of WAXY gene of wheat

Slade et al. (2004). A reverse genetic, non-transgenic approach to wheat cropimprovement by TILLING. Nature Biotechnology 23: 75-81. IRRI

1.6: Transgenes with promoter switching

IRRI

Model for induction of rd29A Gene expression under abiotic stress conditions

Liu et al. (1998) Plant Cell 10:1391-1406 IRRI

RNA gel blot analysis of DREB1A and DREB2Atranscripts

Liu et al. (1998) Plant Cell 10:1391-1406

IRRI

Freezing and drought tolerance of 35S:DREB1Aband 35S:DREB1Ac transgenic plants

Liu et al. (1998) Plant Cell10:1391-1406 IRRI

Phenotypes of 35S:DREB1A and rd29A:DREB1Atobacco plants in relation to control plants

Kasuga et al. (2004) Plant Cell Physiol. 45:346-350 IRRI

OsLip9 Promoter T-NosHPT

LBRB

pAg7

H H : H : Hind IIIHind IIIE: E: Eco R IEco R IB: B: Bam H IBam H I

AtDREB1AP-Nos

EB

Lip9::AtDREB1A

Lip9::OsDREB1B

DREB Constructs for Rice Transformation

OsLip9 Promoter T-NosHPT

LBRB

pAg7

H

OsDREB1BP-Nos

EB

Variety Genotype Drought tolerance

Palmar Indica, Lowland +Cica 8 Indica, Lowland ++CT6241 Japonica, Upland +++

Rice Genotypes used for this work

Z. Lentini, M. Ishitani, CIAT IRRI

10 days 30 days

80 days Maturity

Z. Lentini, M. Ishitani, CIAT

Transgenic rice with OsLIP9::AtDREB1A construct

IRRI

0.00.20.40.60.81.00.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

NTR

JL 24RD 2

FTSWK. Sharma, V. Vadez, ICRISAT

Applying advanced ideas in drought physiology to transgenic rd29A::AtDREB1A groundnut

NTR:Normalized transpiration rate

FTSW: fraction of transpirable soil waterJL 24:Non-transgenic control plantsRD 2:rd29A::DREB1A transgenic line

IRRI

Asian Rice Biotechnology Network: Working together on biosafety

1. Adopt science-based biosafety regulations2. Harmonize regulations to facilitate bidirectional trade3. Encourage post-graduate training of regulators4. Optimize costs of regulation5. Focus of transparency and public information

IRRI’s greenhouse for GM rice has operated since 1994

IRRI

Genes for drought tolerance at IRRI

IRRI*IRMBP = International Rice Molecular Breeding Program

Rice germplasm Genes fromother sources

Gene discovery

Allele pyramids

Allele mining

New drought-tolerant varieties

Promoterdiscovery

Phenotyping, mutants,expression analysis, map-based cloning

TILLING, DArT

Marker-aided selection

Marker-aided backcrossing

Novel alleles

Promoterswitching

Transformation

IRMBP*

Phenotypingof advancedbackcrosslines Rice orthologues

Can we discover other suitable genes, alleles and transgenes using “omics” approaches?

IRRI