Genomic resources and gene/QTL discovery in cereals

Roberto TuberosaDept. of Agroenvironmental Sciences & Technology

University of Bologna, Italy

The ABDC Congress1-4 March 2010

Gudalajara, Mexico

OutlineOutline•• GenomicsGenomics approachesapproaches

•• AvailableAvailable geneticgenetic resourcesresources

•• ExamplesExamples ofof target target traitstraits

•• PerspectivesPerspectives

QTL QTL mappingmapping and and cloningcloning strategiesstrategies

LinkageLinkage mappingmapping ((biparentalbiparental))

genome-wide (high LD panel)

AssociationAssociation mappingmapping (set of (set of accessionsaccessions))

candidate gene (low LD panel)

QTL coarse mapping

Near isogenic lines (NILs)

Candidate gene Candidate gene validationvalidation

Positional cloning

Genetic resolution

10-20 cM

1-100 kb

BiparentalRILs

Introgressionlibraries

AB-QTLpopulations

Barley + + + + + + +

Maize + + + + + + no

Millets + no no

Rice + + + + + + + +

Sorghum + + no no

Rye + no no

Triticale + no no

Wheat + + + + + no

Genetic resources for gene/QTL discovery in cereals

BiparentalRILs

Introgressionlibraries

AB-QTLpopulations

Mini-corecollections

Barley + + + + + + + + + +

Maize + + + + + + no + + +

Millets + no no + +

Rice + + + + + + + + + + +

Sorghum + + no no + +

Rye + no no + +

Triticale + no no +

Wheat + + + + + no + + +

Genetic resources for gene/QTL discovery in cereals

BiparentalRILs

Introgressionlibraries

AB-QTLpopulations

Mini-corecollections

Mutagenizedcollections

Barley + + + + + + + + + + + + + +

Maize + + + + + + no + + + + + + +

Millets + no no + + +

Rice + + + + + + + + + + + + + + +

Sorghum + + no no + + + +

Rye + no no + + +

Triticale + no no + +

Wheat + + + + + no + + + + + + +

Genetic resources for gene/QTL discovery in cereals

TTargeting argeting IInduced nduced LLocal ocal LLesions esions ININ GGenomesenomes

+

IDENTIFICATION OF POINT MUTATIONS IDENTIFICATION OF POINT MUTATIONS IN REGIONS OF INTERESTIN REGIONS OF INTEREST

PCRPCR--BASED BASED SCREENINGSCREENING

CHEMICAL CHEMICAL MUTAGENESISMUTAGENESIS

With TILLING, a library of DNA samples from thousands of individWith TILLING, a library of DNA samples from thousands of individuals is uals is screened for screened for artificiallyartificially--inducedinduced or or naturallynaturally--occurringoccurring ((EcoTILLINGEcoTILLING) ) singlesingle--nucleotide polymorphisms (SNPs).nucleotide polymorphisms (SNPs).

TILLINGTILLING

McCallum CM et al. (2000). Nature Biotechnology 1: 455–457.

R --> STOPCGA --> TGA

….AG_GT…

-->….AA_GT…Intron

__ Exon

improper RNA splicing

truncated

nonsense

TILLING recovers a range of mutation types

wild-type protein

Func

tion

alit

y

R --> QCGA --> CAACGA --> CAA

altered

missense

Courtesy of L. Comai, modified

ROOT MUTANTS ROOT MUTANTS –– Reverse Reverse analysisanalysisTILLMoreTILLMore

12/G

5

Mor

exw

ild ty

pe

37/C

7Photos: courtesy of Iwona Szarejko

TILLING with TILLMore in barley

Wild-type 12/G5 37/C7

CloningCloning genesgenes and and QTLsQTLs

…… lookinglooking forfor a a needleneedle in a in a haystackhaystack ……

Salvi & Tuberosa (2005) Trends in Plant Science, 10: 297-302.

QTL cloning as an essential step towards:

• Understanding the functional basis of traits

• Genetic engineering of quantitative traits

To clone or not to clone QTLs?

• More effective marker-assisted selection

• Unlocking the allelic richness at target loci via direct sequencing of germplasm

Stress Barley Maize Rice Sorghum WheatDrought in progress in progress in progress in progress in progress

Flooding - in progress yes - -

Salinity in progress - yes - yes

Low N in progress yes in progress - in progress

Low P - in progress in progress - -

Aluminum - yes in progress yes in progress

Boron yes - - - yes

Low temps yes - - - yes

High temps - - - - in progress

Summary of QTL cloning for abiotic stress tolerance in cereals

PerspectivesPerspectivesin gene/QTL in gene/QTL mappingmapping

and and cloningcloning

Maize Diversity Project TeamThe Nested Association Mapping (NAM) project

Courtesy of Mike McMullenCourtesy of Mike McMullen

Nested Association Analysis

Yu et al. (2008) Genetics 178: 539

25 DL

× B73

F1 s

SSD

NAM

1

2

200

B97

CM

L103

CM

L228

CM

L247

CM

L277

CM

L322

CM

L333

CM

L52

CM

L69

Hp3

01

Il14H

Ki1

1

Ki3

Ky2

1

M16

2W

M37

W

Mo1

8W

MS

71

NC

350

NC

358

Oh4

3

Oh7

B

P39

Tx30

3

Tzi8

Courtesy of Mike McMullen

Linkage mappingLinkage mapping Association mapping Association mapping Recent recombinationRecent recombinationHigh powerHigh powerLow resolutionLow resolutionAnalysis of two allelesAnalysis of two allelesModerate marker densityModerate marker density

Historic recombinationHistoric recombinationLow powerLow power

High resolutionHigh resolutionAnalysis of many allelesAnalysis of many alleles

High marker densityHigh marker density

Nested Association Mapping

Recent and ancient recombinationRecent and ancient recombinationHigh powerHigh power

High resolutionHigh resolutionAnalysis of many allelesAnalysis of many alleles

Moderate genetic marker densityModerate genetic marker densityHigh projected marker densityHigh projected marker density

COS markers have been derived from orthologous Expressed Sequence Tags (EST) that have been conserved across evolutionary related species.

Advantages• Transferable among species• Mostly codominant• Gene-based markers

Disadvantages• Need expertised inference of synteny• Low level of polymorphism

Conserved Orthologous Set (COS) markers(J. Salse & coworkers, INRA, France)

• Set of ca. 170,000 gene sequences available for cereals• Rice, maize, sorghum, Brachypodium genomes have been sequenced

Bolot et al. (2009) COPB, 12: 11-15

Salse & co-workers: Paleogenomics in cereals for trait improvement

Salse & co-workers (INRA, France)9,138 COS markers for the monocots

NUE

Tiller

Size

Starch

Viscosity

Hardiness

Bolot et al. (2009) COPB, 12: 11-15

•• HighHigh--throughputthroughput genotypinggenotyping ((SNPsSNPs) & ) & phenotypingphenotyping

•• ResequencingResequencing and and managingmanaging the data the data

•• In In silicosilico analysisanalysis, , annotationannotation and comparative and comparative genomicsgenomics

•• TissueTissue--specificspecific expressionexpression profilesprofiles

•• FunctionalFunctional mapsmaps ((ESTsESTs, , FLcDNAsFLcDNAs, , eQTLeQTL) )

•• QTL QTL cloningcloning

Future challenges/opportunities forfor unlockingunlockingvaluablevaluable naturalnatural variationvariation forfor target target traitstraits

Evolution of QTL mapping and cloning strategies

Little or no Little or no constraintsconstraints in in mappingmapping information information

and and resolutionresolutionIntegrationIntegration of of linkagelinkage mappingmapping and and associationassociation (LD) (LD) mappingmapping

biparental crosses

multiparental crosses

genome-wide association

LinkageLinkage mappingmapping

AssociationAssociation mappingmapping

association at candidate genes

Candidate genes (-omics, EST, etc.)Genome-wide genotyping (SNP, DArT haplotypes) Genome sequencing (Solexa, Solid, etc.)

Genomics tool box

Past Future

Genotyping

Phenotyping Genotyping

Phenotyping

ConclusionsConclusions• Plenty of genetic resources are available in cereals

for gene/QTL discovery.• Devising the right phenotyping screening and choosing the

right materials is key to the discovery of loci relevant for the desired trait in the target environment.

• The cost of molecular profiling and sequencing will continue to decrease, making genomics approaches more affordable.

• Association mapping will play an increasingly important role for relatively simple traits; it is more questionable to what extent it will work with physiologically complex traits.

• The contribution of wild germplasm to gene discovery will increase.