Diversity Arrays Technology

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New Plant Breeding Strategies Using an Affordable and Effective Whole-Genome

Profiling MethodEric Huttner

Diversity Arrays Technology, Canberra, Australia

Diversity Arrays Technology

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Molecular technologies for crop improvementMolecular technologies for crop improvement

Transgenic (GM) plantsTransgenic (GM) plantsSuccessful deployment for simple traitsSmall number of widely used useful genes

Molecular genetic markersMolecular genetic markersIdentification of polymorphism associated with specific traitsSmall number of markers used in routine breeding (substitute to phenotyping)

Mostly used in “first world” cropsMostly used in “first world” crops

Can we do better ?Can we do better ?For complex traitsFor orphan crops

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Multigenic: many genes involvedMultigenic: many genes involved

Genes are interactingGenes are interacting

Many possible combinations of alleles for the Many possible combinations of alleles for the genes involvedgenes involved

Allele-specific quantitative contribution, Allele-specific quantitative contribution, modulated by the other genesmodulated by the other genes

Epigenetic variation (e.g. methylation)Epigenetic variation (e.g. methylation)plays a role not fully establishedplays a role not fully established

Whole-genome molecular marker profiles: a Whole-genome molecular marker profiles: a useful tool to deal with this complexityuseful tool to deal with this complexity

Complex traitsComplex traits

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The need for whole-genome analysisThe need for whole-genome analysis

Complex traitsComplex traitsNeed to recombine several regions simultaneously

New sources of genesNew sources of genesUsing more exotic germplasmAccelerate the transfer of small genomic regions

Speed to marketSpeed to market

Capture the results of genomics programsCapture the results of genomics programs

Diversity Arrays Technology

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Molecular markers and their limitationsMolecular markers and their limitations

HistoricHistoricBiochemical, morphologicalGenetic (DNA based): RFLP, RAPD

CurrentCurrentSSR, AFLP®, coming … soon ? SNPAnd DArT !

LimitationsLimitationsDiscovery: cost, choice of genetic diversityAssay: cost, throughput, data integrityFreedom To Operate

Diversity Arrays Technology

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Diversity Arrays TechnologyDiversity Arrays Technology

Andrzej Kilian, the inventor of Diversity Arrays Technology, with Norman Borlaug

Diversity Arrays Technology

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Diversity Arrays Technology (DArT)Diversity Arrays Technology (DArT)DArT genome scansDArT genome scans

Parallel analysis of several hundred markers on microarrays Sequence independent, affordable for orphan cropsDiscovery and Typing on the same platform: no assay development requiredAmenable to full automation → data integrityApproximately 10-fold cost reduction

Technology described at the last BioVision conferenceTechnology described at the last BioVision conference

Description and references: www.DiversityArrays.comDescription and references: www.DiversityArrays.com

Demonstrated on 18 cropsDemonstrated on 18 crops

This talk: Applying the technology to wheat and bananaThis talk: Applying the technology to wheat and banana

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DArTFWCentromer

1H 2H 3H 4H 5H 6H 7H

Workflowlibrary and target preparation

slide spotting

hybridization with targets

slide scanning

DArTsoft:dataanalysis

results

DArTdb: LIMS

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Current statusCurrent statusSpecies Marker collections Note

Rice 14 Service available Barley (H. vulgare and H. spontaneum) 10 Triticarte service available

Wheat (T. aestivum) 5 Triticarte service available Triticum durum and other Triticum sp 4 Triticarte service available

Sorghum 3 Service available. Lupin 1 Service available

Chickpea 1 POC done. Routine typing under development Sugarcane 10 POC done. Routine typing under development Cassava 4 Generation Challenge Program Coconut 1 Generation Challenge Program

Musa sp (Banana) 2 Generation Challenge Program Pigeon pea (Cajanus cajan) 4 POC done.

Apple 3 Used for varietal ID service Tomato* 2 POC done One breeding application.

Arabidopsis*** 2 Wittenberg et al, Mol. Genet. Genom. 274, 30-39 Perennial rye grass 5 POC done. Routine typing under development

Quinoa** 2 Asplenium sp (fern) and Garovaglia sp (moss) 2 Biodiversity.

Fungal wheat*** and barley pathogen**** various Map built for wheat pathogen

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WheatWheatAustralian company Triticarte delivering the service Australian company Triticarte delivering the service since Dec 2004 (since Dec 2004 (www.triticarte.com.auwww.triticarte.com.au))

DNA-to-data serviceDNA-to-data service

6545 samples analysed over past 12 months6545 samples analysed over past 12 months

About 2.7 millions data pointAbout 2.7 millions data point

Average cost to customers: 8 US c. / data pointAverage cost to customers: 8 US c. / data point

35 customers, 9 countries35 customers, 9 countries

32 mapping populations, 2000 markers located to 21 32 mapping populations, 2000 markers located to 21 chromosomeschromosomes

Initial marker-trait associations reportedInitial marker-trait associations reported

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000011100832wPt0028

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0X001183772wPt0026

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F1E1D1C1B1A1Call RateQualityChromosomeMarker

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Banana and plantainsBanana and plantains4th most important crop in the world4th most important crop in the world

food for millions of people

a herb not a tree a herb not a tree belong to the Musaceae (monocot)Musa acuminata (A) – dessert bananaMusa balbisiana (B) – cooking banana

Genome size 540-600 MbGenome size 540-600 Mb(1.5 x rice genome size)(1.5 x rice genome size)

Complex genomeComplex genomestructure and evolutionstructure and evolution

Image © INIBAP

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Establishing DArT for BananaEstablishing DArT for BananaA project of Generation Challenge ProgramA project of Generation Challenge Program

One visitor from CIRAD (France), 3 monthsOne visitor from CIRAD (France), 3 monthsIdentified about 1000 polymorphic markersIdentified about 1000 polymorphic markersTyped 187 samples of diverse banana clonesTyped 187 samples of diverse banana clones

Triploid AAA (dessert), AAB (plantain) and ABB (cooking) genomeDiploid AA and BB genomes and others

Markers now availableMarkers now available

Management of collectionsManagement of collections

Understand the phenotypic variation in the plantain Understand the phenotypic variation in the plantain groupgroup

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Diversity analysis of 187 cultivars based on 373 markersDiversity analysis of 187 cultivars based on 373 markers

BBBB ABB AAB AA AAAASOthers

Predominance of the A vs B differentiationWide diversity within A: two domestication routes ?

NB Possible bias due to excessive weight of triploid clones

Axis 1 42.1 %

Axis 2 9.5 %

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A close up on the plantain groupA close up on the plantain groupA clone

Several origins

• Fragment of Neighbor Joining dendrogram based on Dice Index• Source of variation among the clonal material: epigenetics and/or genome rearrangements

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ApplicationsApplications of DArT of DArT““Whole-genome profile” applicationsWhole-genome profile” applications

Comprehensive single-step characterisation of germplasmGenetic background screening in Back CrossesAccelerated introgression from exotic germplasmIdentification of multiple regions responsible for complex traits (QTL and association mapping)Mapping of genome rearrangementsBulk segregant analysis in a single stepMarker-assisted selection for many traits simultaneously

Will this enable new breeding methodologies ?Will this enable new breeding methodologies ?“Bolder” use of genetic diversity, increased use of exotic germplasmData mining approaches to marker-trait association

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AcknowledgementsAcknowledgements

The Grains R&D Corporation of AustraliaThe Grains R&D Corporation of Australia

Dr Neil Howes, Plant Breeding Institute of the Dr Neil Howes, Plant Breeding Institute of the University of SydneyUniversity of Sydney

The Generation Challenge Program of the CGIARThe Generation Challenge Program of the CGIAR

Dr Ange-Marie Risterucci and colleagues, CIRAD Dr Ange-Marie Risterucci and colleagues, CIRAD (Montpellier, France)(Montpellier, France)

BioVision AlexandriaBioVision Alexandria

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Triticarte / DArT teamTriticarte / DArT team

Andrzej

Eric

Ling

Grzegorz

Vanessa

Jason

Shi Ying

Cyril

www.Triticarte.com.au / www.DiversityArrays.com

Brent

Peter

Mona

Margaret

KasiaDamian

Sujin