genomic resources and gene/qtl discovery in cereals · genomic resources and gene/qtl discovery in...
TRANSCRIPT
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.