WUEMED Drought Course, Bologna, 4-10 July 2006: 5 lectures on Omics and drought by John Bennett, IRRI IRRI. Anthers of field-grown rice cv IR74

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1 Anthers of field-grown rice cv IR74 Apical pore WUEMED Drought Course, Bologna, 4-10 July 2006: 5 lectures on Omics and drought by John Bennett, Basal pore

2 Omics and Drought: Lecture Outline 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 Transcriptomics Proteomics Metabolomics JX Chen GH Salekdeh Cook et al. (2004)

3 WUEMED Drought Course 1. Integration of gene and allele discovery with breeding for drought tolerance John Bennett International Rice Research Institute Los Baños, Philippines

4 1.1: Drought in s breeding program

5 Drought is frequent in rainfed rice eco-systems L.J. Wade

6 s research is organized into 12 projects Breeding - rainfed ecosystems Breeding -irrigated ecosystem Consortium for Unfavorable Rice Environments Natural resource management - for rainfed ecosystems Functional genomics Germplasm conservation and allele mining Activities related to abiotic stresses

7 Abiotic stress is the focus in 5 outputs of Project 7 Outputs: 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 Stop press: s new Strategic Plan features Drought as a Frontier Project

8 Drought tolerance: a complex mix of traits Tolerance retaining water, protecting tissues, maintaining fertility Avoidance deep roots, penetrating roots Escape short duration Early-season drought

9 Reproductive stage drought tolerance Varieties with improved reproductive stage drought tolerance reduce the risk of complete crop failure and increase the floor yield in severe droughts. Raipur, 2005 G. Atlin

10 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) G T PI F M G T PI F M G T PI F M G T PI F M Yield = 10-5 x abcd g m -2 = stress period Boonjung and Fukai (1996) Field Crops Res 48:47-55

11 1.2: International Rice Molecular Breeding Program

12 Biotechnology at : Helping the breeding program Gene bank 1977 Biodiversity of rice DNA technology Genomics Traditional breeding 1993 Tissue culture Gene discovery 1996 Markeraided selection (MAS) 2004 Farmers fields Geneticallyengineered rice 2000 China (Bt, Xa21) 2002 Philippines (Xa21) 1991 Exotic genes

13 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 both parents. Mapping using ~100 markers per genome allows networks of coselected 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: Xu et al. (2005). QTLs for drought escape and tolerance identified in a set of random introgression lines of rice. Theor Appl Genet. 111:

14 Location of institutes participating in International Rice Molecular Breeding Program Z.K. Li

15 1.3: Backcrossing to enhance drought tolerance in popular varieties

16 A six-step molecular breeding strategy 1. Participatory rural appraisal* 2. Mechanism discovery # 3. Gene and allele discovery # 4. Pre-breeding for trait reconstruction 5. Molecular breeding for variety development 6. 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

17 Enhancing breeding efficiency at through molecular approaches Genetic analysis, gene discovery, DNA based selection Four types of traits 1. Simple genetics Simple screen DNA based selection 2. Simple genetics Difficult screen Advanced screens 3. Complex genetics Simple screen 4. Complex genetics Difficult screen Backcross breeding + DNA based selection Pedigree breeding + trait-based selection Package of traits required for adoption by farmers

18 Backcrossing for enhancing drought tolerance Improve drought tolerance of an already popular variety by introgression of major genes or QTLs from different donors using DNA marker-assisted backcrossing (MAB) Donor 1 (with major gene1 or major QTL1) Donor i (with major genei or major QTLi) Recurrent parent (popular variety) RP + 1 RP + i Recurrent parent i There may be a practical upper limit of ~5 genes that can be introgressed simultaneously 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 by mining techniques, and (e) mutant alleles; all must have large effects in diverse backgrounds.

19 1.4: QTLs of large effect

20 Biomass production is greatly reduced in dry fields Enhancing biomass in dry fields at flowering may help increase yield G. Atlin

21 Screening in paddies drained after transplanting can identify cultivars with improved tolerance to long periods of dry soil conditions G. Atlin Raipur, India, 2005

22 Cultivar differences in biomass production in intermittently-dried field: WS 2005 DESIGNATION Harvest index Grain yield (kg/ha) Biomass (kg/ha) IR CPA IR PSBRC PSBRC IR PSBRC G. Atlin

23 Tolerance to reproductive-stage stress Some varieties have much greater tolerance to flowering stage stress than IR 36, IR 64, and MTU Tolerance to extreme stress at flowering may be affected by major QTLs. R. Venuprasad, J. Bernier, A. Kumar, G. Atlin

24 Genes for drought tolerance at Phenotyping of advanced backcross lines IRMBP* Rice germplasm Phenotyping, mutants, expression analysis, map-based cloning Gene discovery TILLING, DArT Allele mining Marker-aided selection Rice orthologues Promoter discovery Promoter switching Genes from other sources Transformation Allele pyramids Novel alleles Marker-aided backcrossing New drought-tolerant varieties *IRMBP = International Rice Molecular Breeding Program

25 1.5: Allele mining

26 Finding alleles through whole-genome sequencing McNally et al. (2006) Plant Physiol.

27 Finding mutants and alleles through high-throughput TILLING and eco-tilling TILLING: Targeting Induced Local Lesions IN Genomes Colbert et al. (2001). High-throughput screening for induced point mutations. Plant Physiol. 126:

28 TILLING using acrylamide gels Colbert et al. (2001). High-throughput screening for induced point mutations. Plant Physiol. 126:

29 TILLING of WAXY gene of wheat Slade et al. (2004). A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nature Biotechnology 23:

30 Screen for mutations in the wheat waxy genes 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 properties and 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 crop improvement by TILLING. Nature Biotechnology 23:

31 TILLING of WAXY gene of wheat Slade et al. (2004). A reverse genetic, non-transgenic approach to wheat crop improvement by TILLING. Nature Biotechnology 23:

32 1.6: Transgenes with promoter switching

33 Model for induction of rd29a Gene expression under abiotic stress conditions Liu et al. (1998) Plant Cell 10:

34 RNA gel blot analysis of DREB1A and DREB2A transcripts Liu et al. (1998) Plant Cell 10:

35 Freezing and drought tolerance of 35S:DREB1Ab and 35S:DREB1Ac transgenic plants Liu et al. (1998) Plant Cell 10:

36 Phenotypes of 35S:DREB1A and rd29a:dreb1a tobacco plants in relation to control plants Kasuga et al. (2004) Plant Cell Physiol. 45:

37 RB DREB Constructs for Rice Transformation Lip9::AtDREB1A LB pag7 HPT P-Nos OsLip9 Promoter AtDREB1A T-Nos RB Lip9::OsDREB1B B H H : Hind III E: Eco R I B: Bam H I E LB pag7 HPT P-Nos OsLip9 Promoter OsDREB1B T-Nos Rice Genotypes used for this work H B E Variety Genotype Drought tolerance Palmar Indica, Lowland + Cica 8 Indica, Lowland ++ CT6241 Japonica, Upland +++ Z. Lentini, M. Ishitani, CIAT

38 Transgenic rice with OsLIP9::AtDREB1A construct 10 days 30 days Z. Lentini, M. Ishitani, CIAT 80 days Maturity

39 Applying advanced ideas in drought physiology to transgenic rd29a::atdreb1a groundnut NTR NTR: Normalized transpiration rate FTSW: fraction of transpirable soil water JL 24: Non-transgenic control plants RD 2: rd29a::dreb1a transgenic line JL 24 RD FTSW K. Sharma, V. Vadez, ICRISAT

40 Asian Rice Biotechnology Network: Working together on biosafety 1. Adopt science-based biosafety regulations 2. Harmonize regulations to facilitate bidirectional trade 3. Encourage post-graduate training of regulators 4. Optimize costs of regulation 5. Focus of transparency and public information s greenhouse for GM rice has operated since 1994

41 Genes for drought tolerance at Phenotyping of advanced backcross lines IRMBP* Rice germplasm Phenotyping, mutants, expression analysis, map-based cloning Gene discovery TILLING, DArT Allele mining Marker-aided selection Rice orthologues Promoter discovery Promoter switching Genes from other sources Transformation Allele pyramids Novel alleles Marker-aided backcrossing New drought-tolerant varieties *IRMBP = International Rice Molecular Breeding Program

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