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New technologies for plant research and breeding. OECD, Paris 11-12-2009

Technologies of the Future Application in plant research and breeding

Michel Caboche,

OECD, Paris 11-12-2009

A L I M E N T A T I O N

A G R I C U L T U R E

E N V I R O N N E M E N T


Wheat cultivation in France

• 50 BC: Romans introduce wheat in France

• MA-18thc:5 to 9 q/ha (3 seeds from 1)

• 1750-80: Chemistry/fertilisation/restitution principle

• 1880: 11 q/ha

• 1920: 12.5 q/ha

– France imports 1.3M tons of cereals to feed the country.

– Phosphate fertilisers /genetics: high-yielding, disease-resistant varieties / mechanisation

• 1980: 65 q/ha

– France becomes one of the best agricultural power in the world

• 2005: 85 q/ha:

– 50% genetics / 50% agronomy + chemistry


Source : GNISMultilocal testing

Choice of parents, crossing

F1: all plants identical

F2: segregation, choice of plants and selfing

Ear-row

F3: choice of interesting plants within suitable families

F4: choice of families and selection for purity

F5 to F8: continuation of selection and yield trials


Troyer (1995)

The main steps in genetic progress in the States

Net genetic progress is linked to hybrid structure, progeny testing and trial networks. Genetic progress accelerates with F1 hybrids


Issues in classical breeding strategy- Narrow genetic base- Tedious or inaccurate selection methods-> Slow and limited genetic progress

Need for new resources and technologies

Need for responsive crop improvement- World population growth- Climate change- Water scarcity- Land degradation- Continual risks of new disease epidemics

Need for new technologies in plant breeding


Trials and release of variety

Producers

Users

Genetic resources

Traits of interest and

breeding objectives

Introgression into agronomically valuable

variety

Plant breeding synopsis


Broaden the genetic base used in plant breeding1. Genetic diversity mining, population structure analysis2. Generation and management of de novo genetic diversity3. Creation of core collections

Unravel the genetic basis of trait biology1. Gene discovery by sequencing and by expression profiling2. Validation of gene function by functional genomics

Facilitate the transfer of relevant genes to elite crops

1. QTL mapping and marker development2. Advanced backcross between crops and their wild relatives3. Screening by high-throughput genotyping technology4. Gene transfer technologies

Application of new technologies in plant breeding

Trials and release of variety

Producers

Users

Genetic resources

Traits of interest -

breeding objectives

Introgression into agronomically valuable

variety

Plant breeding synopsis


Genetic map and QTL analysis

Genetically simple (conditioned by a single gene) and genetically complex or quantitative traits (conditioned by many genes) can be mapped on chromosomes.

For complex traits:

- only genes with a significant contribution will be detected.

- a genetic interval contributing to the phenotypic difference between two genotypes is called «Quantitative trait locus » or QTL

QTLs generally encompass many genes

( in the order of 10 cMorgans ie 10 x 30,000 genes / 500= 600 genes)


Identification of QTLs for Fusarium resistance by analysis of crosses between tolerant and sensitive accessions

Dedryver et al


Marker-Assisted Selection (MAS)

Resistance

Phoma Erucic Acid

M

1

M

2

M

3M

4

M

1

M

2

M

3M

4

Parent A Parent B


Why identify the genes underlying a QTL?

•Knowing the genes conditioning a trait helps understanding the physiological basis for its function

•In MAS, molecular probes designed in causal genes are perfect markers

•Knowing the genes enables to mine the allelic diversity in genetic resources

•It facilitates the search for QTLs in related species

•Genetic engineering becomes an option


Neighbor-joining trees representing stearoyl-ACP desaturase haplotype relationships.

Major heterotic groups: stiff stalk (blue), non stiff stalk (green) and Lancaster (red).

From A Rafalski, Du Pont


- Individual allelic values not necessarily detectable on the phenotype

- Mutagenesis can help to design new alleles (TILLING)

-->

Broaden the genetic base of plant breeding

From Tanksley and McCouch, 1997

a ---> b ---> c ---> d ---> LycopeneX1 2 3 4

select genetic resources based on genotype evaluation


Samson

Brunaud et al

Sequencing

And

annotating

Genomes

A Thaliana

Rice

Poplar

Grapevine

Corn

Finishing….

Tomato

Medicago


INRA foresight

Technologies for the Future

Context:• « (…) INRA must anticipate and facilitate major scientific and technological

breakthroughs likely to emerge in the next decades for a significant impacton agriculture and food. »


Objectives

• Identify and analyse emerging technologies– Relevant for agriculture research

• Animal and plant production• Green chemistry and biomass production

• Food and microbiology– Likely to meet the needs of tomorrow’s agriculture

• Deliverables:– Establish a list of emerging technologies– Brainstorming seminar / validation / broadening– Collection of technological worksheets– Final report summarising the conclusions of the work


2. Consultation of 53 research scientists and technology experts (1h30 to 3-hour meetings with each expert)

– INRA or not INRA– Contacts (colleagues, wider network, conferences)– Publication authors– Recommendations (from INRA scientific directors/department directors)

-> Enrich, correct, validate our choices

Strategy

1. Reviewing and monitoring science and technology- Scientific literature

- Research projects

- Conferences, workshops and seminars

- Various web sites

-> - Explore science and technologies

- Identify, analyse, assess emerging technologies relevant to agriculture, food and environment research

- Draft of technological worksheets


1 High-throughput genotyping

2 High-throughput "next-generation" sequencing

3 ex aequo Metagenomics

3 ex aequo Targeted genetic engineering

3 ex aequo Mass spectrometry-based proteomics and metabolomics

4 Imaging and single-molecule tracking

5 Molecular engineering for novel enzymatic activities

6 Synthetic biology and metabolic engineering

7 Nutrigenetics and nutrigenomics

8 Induced pluripotent stem cells

New technologies sorted by order of interest and priority by our guest experts


High-throughput "next-generation" sequencing

High-throughput genotyping

Metagenomics

Targeted genetic engineering

Molecular engineering for novel enzymatic activities

Synthetic biology and metabolic engineering

New technologiesfor plant breeding


- GENOME LEVELHTP de novo sequencingFacilitated sequencing of related genomes Resequencing - genetic diversity and evolutionary studies: - genome-wide discovery of genetic polymorphisms- Low-cost alternatives to whole-genome resequencing:

- TRANSCRIPTOME LEVELTranscript discovery and gene expression profilingAccurate gene annotation ( splicing sites)Deep sequencing of small RNAs (miRNA, si RNAs, etc)ChIP sequencing: Epigenome analysis

LimitationsCost. Danger of simplistic views on research. Needs some thinking

High-throughput sequencing technologies


Metagenomics

Metagenomics exploits HTP sequencing to study an ecological niche and create an inventory of the microrganisms there

** Microbes are ubiquitous and essential to life. Metagenomics gives access ….

- … to the uncultured (>99% bacteria)- … to whole microbe communities in a variety of natural environments

• Environment: Biosensors, bioremediation of contaminated soils, industrial treatment of wastewater

• Agriculture: rapid identification of pathogens responsible of emerging diseases (Ex; honey bee)

• Human nutrition and health: Search for new antibiotics, role of human gut microbes (microbiome) in nutrition and obesity

Consulted experts on Metagenomics:•Dusko Ehrlich, Génétique microbienne, INRA Jouy-en-Josas, France•Denis Le Paslier, Génoscope, Evry, France•Jean Weissenbach, Génoscope, Evry, France


High-Throughput genotyping

Genotyping and phenotyping are at the root of genetic analysis.

•All genotyping technologies exploit the variations occurring in genomic sequences.

•Among DNA polymorphisms, SNP variations are found in all genomes and can be identified by high-throughput genome re-sequencing.

A dense array of SNPs (Ex: an average of 10 SNPs per gene and a total of 1M SNPs) can be used to genotype all gene alleles present in one genome in a single step

Consulted experts: A. Eggen, D. Brunel, A. Charcosset, I. Gut


Genome-wide Association Genetics

Linkage studiesFamilies and pedigrees with known ancestry

Few opportunities for recombinationLower mapping resolution

Association (LD) studiesNatural genetic diversityHistorical recombination

Higher mapping resolution

Linkage Disequilibrium (LD): Non-random co-segregation of alleles at two loci

Phenotype (ex diabete) correlated with a specific allele of one of the 30000 genes in the genome

Comparison of strategies to detect non-random association between a genotype and a phentoype:


| Association statistics from one of the five type 2 diabetes genome-wideassociation studies. The y axis represents the –log10 p value and the x axisrepresents each of the ~400,000 SNPs used in this scan. The point of each arrowindicates the location of the most strongly associated SNP in each of nine known type2 diabetes gene regions. From Frayling, Nat. Rev. Genet., 2007.

Same approach to identify an allele of a gene involved in a disease and an allele of a gene improving an agronomic trait!

Limitations: only one or two individuals analysed on one array. Large numbers

(1000 genotypes) required for statistical significance. Very expensive…


Rationale for genetic engineering

DNA is the support of genetic informationin all living organisms

DNA can be transferred from an organism to anotherspontaneously (horizontal transfer)

or by molecular techniques

Genes can be expressed in heterologous hosts provided different rules are respected

Genetic engineering is used to make genes functional in heterologous hosts


Drought toleranceAharoni, A., Dixit, S., Jetter, R., Thoenes, E., van Arkel, G. and Pereira, A. (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell, 16, 2463-2480.Hu, H., Dai, M., Yao, J., Xiao, B., Li, X., Zhang, Q. and Xiong, L. (2006) Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci U S A, 103, 12987-12992.Jeanneau, M., Gerentes, D., Foueillassar, X., Zivy, M., Vidal, J., Toppan, A. and Perez, P. (2002) Improvement of drought tolerance in maize: towards the functional validation of the Zm-Asr1 gene and increase of water use efficiency by over-expressing C4-PEPC. Biochimie, 84, 1127-1135.Nelson, D.E., Repetti, P.P., Adams, T.R., Creelman, R.A., Wu, J., Warner, D.C., Anstrom, D.C., Bensen, R.J., Castiglioni, P.P., Donnarummo, M.G., Hinchey, B.S., Kumimoto, R.W., Maszle, D.R., Canales, R.D., Krolikowski, K.A., Dotson, S.B., Gutterson, N., Ratcliffe, O.J. and Heard, J.E. (2007) Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. Proc Natl Acad Sci U S A, 104, 16450-16455.Qin, F., Sakuma, Y., Tran, L.S., Maruyama, K., Kidokoro, S., Fujita, Y., Fujita, M., Umezawa, T., Sawano, Y., Miyazono, K., Tanokura, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2008) Arabidopsis DREB2A-interacting proteins function as RING E3 ligases and negatively regulate plant drought stress-responsive gene expression. Plant Cell, 20, 1693-1707.Umezawa, T., Fujita, M., Fujita, Y., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future. Curr Opin Biotechnol, 17, 113-122.Wang, C.R., Yang, A.F., Yue, G.D., Gao, Q., Yin, H.Y. and Zhang, J.R. (2008) Enhanced expression of phospholipase C 1 (ZmPLC1) improves drought tolerance in transgenic maize. Planta, 227, 1127-1140.


Targeted gene modification

•Reverse genetics and targeted gene inactivation •Induction of allelic diversity to optimise a trait of agronomical value.

Applications of RNAi:•The technique relies on the production of transgenic plants. •No expected toxicity.•The production of an Ami-RNA by the host can lead to the inactivation of an essential gene in a pest that feeds on this host, resulting in the protection of the plant against the pest.

Applications of ZFN and Meganucleases:•More efficient than TILLING and RNAi concerning the diversity of target modifications. •New therapies against DNA virus infection.•Introduction of genes by using ZFN recently proven in maize

Limitations: ZNF and Meganuclease technologies are work intensive and expensive .

Consulted experts: A. Choulika, Cellectis; B. Dujon, Pasteur


An AmiRNA complementary to a transcript of the insect is produced by the host plant. When the insect eats plant tissues his gut is made non functional by silencing of a n ATPase of the insect

Ami RNA

Transgenic plant Expressing Ami RNA in root tissues

ATPase involved in gut functioning

Ami RNA

AAA

AAA


Pathogen: Diabrotica vigifera vigifera (Western corn rootwormBaum, WCR)

Target: V ATPase A from the insect

Ref: JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plastics G, Munyikwa T, Pleau M, Vaughn T, Roberts J. (2007) Control of coleopteran insect pests through RNA interference. Nature Biotechnology. 25:1322-6.


Recombination events

Target

Donor DNA

Sequence substitution

Applications: specific change in a gene of interest


How to induce homologous recombination in the nuclear genome of a plant

1° Provide donor DNA sharing sequence identity with a specific genomic sequence

2° Induce double strand breaks in a genome sequence sharing homologies with the donor DNA by action of a nuclease (mega nuclease, zinc finger nuclease specific for a 14nt sequence in the plant genome)

3° Select recombination events


Nature. 2009 May 21;459:437-41 Dow AgroSciences, and Sangamo biosciences,USA


Creation of novel enzymatic activities

-Combination rational design / directed evolution-Computational design based on crystal structure or homology modeling, and phylogenetic analysis

10,000 fold improvement in catalytic efficiency

Example: DNA shuffling of GAT

-> Novel/improved biocatalysts for chemical, food and pharmaceutical industries

Ite

rative

rou

nd

s

(After Johannes and Zhao, 2006)

Random/targeted mutagenesis or Gene shuffling

Selection or HTP screening

Novel substrate specificity or

Improved catalytic activity

Target gene(s)

Goal achieved

Library of mutant genes

Library of mutant enzymes

Functionally improved enzymes

The diversity of X-ray-establishedenzyme 3D structures seem to reach a plateau. How to create diversity?

Goal:Creation of new enzyme specificities by active site random mutagenesis and htp testing of these activities on new substrates is a novel avenue


Long chain Omega-3 PUFA (Ex EPA, eicosa pentaenoic acid ) are essential for health They derive from phytoplankton

Synthetic biology

Source: DuPont


Synthetic Biology…

Source: DuPont


Source: DuPont


Thank you for your attention

A L I M E N T A T I O N

A G R I C U L T U R E

E N V I R O N N E M E N T

Thanks to Alain Charcosset, Catherine Golstein


New technologies and their refinement lead to new discoveries and innovations

Their emergence is a rare phenomenon

Nothing is telling us that this process is slowing down


Other emerging technologies identified by our guest participants in order of priority

1. High-throughput phenotyping, from the lab to the field

2. Mathematics, computing and modelling of biological systems

3. Imaging

4. Use of synchrotron

5. Chips and biosensors

6. Nanotechnologies (Microfluidics, Lab-on-chip, Nanocatalysts)

7. Metabolomics (Serach for metabolic markers and structure determination)

8. CLICK chemistry

9. Optogenetics


Selection of relevant technologies for TF

New technology?

Scientific publication?

no yes

Application potential reached?

no yes

Application field relevant for agriculture?

no yes

no yes

Technological worksheet

Technological worksheet

Technology

Monitoring

(Synchrotron)

(PCR)(Artificial

photosynthesis)

(Anti-cancer treatment)

Extended or renewed interest

(Metabolic engineering)

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