snp/tiling arrays for very high density marker based breeding and qtl candidate gene identification...
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SNP/Tiling arrays for very high density marker based breeding and QTL candidate gene identification
Justin BorevitzEcology & EvolutionUniversity of Chicagohttp://naturalvariation.org/
Major Issues in Breeding Complex Traits
• High throughput Phenotyping– Physiological dissection of 1000s correlated traits– Biological Variation
• Multiple genes under major QTL– High Density markers – High throughput seedling screens– Linkage Drag
• Environmental Interaction (GxE)– Good for optimizing local varieties
• Epistasis (GxG)– Magnify minor QTL in local backgrounds
• Multi species ecological interactions– “extended phenotype”
Genomic Breeding Path
QTL geneConfirmation
MarkerIdentificationGenotyping
Genomics path
Experimental DesignMapping population PhenotypingQTL AnalysisFine Mapping
Candidate genePolymorphismsgene expressionloss of function
QTL gene
Confirmation
Experimental Design
Mapping population
Phenotyping
QTL Analysis
Fine Mapping
Borevitz and Chory, COPB 2003
Talk OutlineTalk Outline• Phenotyping in multiple environments
– Seasonal Variation in the Lab
• Germplasm Diversity– Population structure, Haplotype Mapping set
• SNP/Tiling microarrays– Very High Density Markers
– Mapping Extreme Bulk Segregant
– Expression, splicing, and allelic variation
• Ecological context– Arabidopsis and Aquilegia
• Phenotyping in multiple environments– Seasonal Variation in the Lab
• Germplasm Diversity– Population structure, Haplotype Mapping set
• SNP/Tiling microarrays– Very High Density Markers
– Mapping Extreme Bulk Segregant
– Expression, splicing, and allelic variation
• Ecological context– Arabidopsis and Aquilegia
Begin with regions spanning the Native Geographic range
Nordborg et al PLoS Biology 2005Li et al PLoS ONE 2007
Tossa Del MarSpain
LundSweden
Seasons in the Growth Chamber
• Changing Day length• Cycle Light Intensity• Cycle Light Colors• Cycle Temperature
Sweden Spain
Seasons in the Growth Chamber
• Changing Day length
• Cycle Light Intensity
• Cycle Light Colors
• Cycle Temperature
GenevaScientific/ Percival
Day Length
0:00
2:00
4:00
6:00
8:00
10:00
12:00
14:00
16:00
18:00
20:00
22:00
sep
oct
nov
dec
jan
feb
mar
apr
may jun jul
aug
month
hour
s
Sweden
Spain
standard
standard
Light Intensity
0
200
400
600
800
1000
1200
1400
sep
oct
nov
dec
jan
feb
mar
apr
may jun jul
aug
month
W/m
2
Sweden
Spain
standard
Temperature
-10
-5
0
5
10
15
20
25
30
35
sep
oct
nov
dec
jan
feb
mar
apr
may jun jul
aug
month
degr
ees
C
Spain High
Spain Low
Sweden High
Sweden Low
standard
Kurt Spokas
Version 2.0a June 2006
USDA-ARS Website Midwest Area (Morris,MN)http://www.ars.usda.gov/mwa/ncscrl
Flowering time QTL, Kas/Col RILs
Sweden 1
Col-gl1
Kas1
Sweden 2
Col-gl1
Kas1
Spain 1
Col-gl1
Kas1
Spain 2
Col-gl1
Kas1
Num
ber
of R
ILs
Num
ber
of R
ILs
Flowering time QTL, Kas/Col RILs
FRI
FLM
144 Non singleton SNPs >2000 accessions
Global, Midwest, and UK
common haplotypes
Local Population Structure
Megan Dunning, Yan Li
RNA DNA
Universal Whole Genome Array
Transcriptome AtlasExpression levelsTissues specificity
Transcriptome AtlasExpression levelsTissues specificity
Gene/Exon DiscoveryGene model correctionNon-coding/ micro-RNA
Gene/Exon DiscoveryGene model correctionNon-coding/ micro-RNA
Alternative SplicingAlternative Splicing
Comparative GenomeHybridization (CGH)
Insertion/DeletionsCopy Number Polymorphisms
Comparative GenomeHybridization (CGH)
Insertion/DeletionsCopy Number Polymorphisms
MethylationMethylation
ChromatinImmunoprecipitation
ChIP chip
ChromatinImmunoprecipitation
ChIP chip
Polymorphism SFPsDiscovery/Genotyping
Polymorphism SFPsDiscovery/Genotyping
Control for hybridization/genetic polymorphismsto understand TRUE expression variation
RNA ImmunoprecipitationRIP chip
RNA ImmunoprecipitationRIP chip
Antisense transcription
Allele Specific Expression
SNP SFP MMMMM MSFP
SFP
MMMMM M
Chromosome (bp)
con
serv
atio
n
SNP
ORFa
start AAAAA
Tra
nsc
ripto
me
Atla
s
ORFb
deletion
Improved Genome Annotation
Which arrays should be used?
Tiling/SNP array 2007 250k SNPs, 1.6M tiling probes
SNP array
Ressequencing array
How about multiple species? Microbial communities?
Pst,Psm,Psy,Psx, Agro, Xanthomonas, H parasitica, 15 virus,
Col
Col
Van
Van
Col
Van
Van
Col
Genomic DNA RNA
No significant
allele specific expression
cis regulatory variation
(Van allele)
Paternal Imprinting Maternal Imprinting
cis regulatory variation
(Col allele)
RNA
RNA
RNA
RNA
GlobalAllele Specific
Expression
Zhang, X., Richards, E., Borevitz, J. Current Opinion in Plant Biology (2007)
65,000 SNPsTranscribedAccession Pairs12,000 genes
>= 1 SNP6,000 >= 2 SNPs
Delta p0 FALSE Called FDR
1.00 0.95 18865 160145 11.2%
1.25 0.95 10477 132390 7.5%
1.50 0.95 6545 115042 5.4%
1.75 0.95 4484 102385 4.2%
2.00 0.95 3298 92027 3.4%
SFP detection on tiling arrays
bibb mapping
ChipMapAS1
Bulk segregantMapping usingChip hybridization
bibb maps toChromosome2 near ASYMETRIC LEAVES1
BIBB = ASYMETRIC LEAVES1
Sequenced AS1 coding region from bib-1 …found g -> a change that would introduce a stop codon in the MYB domain
bibb as1-101
MYB
bib-1W49*
as-101Q107*
as1bibb
AS1 (ASYMMETRIC LEAVES1) =MYB closely related toPHANTASTICA located at 64cM
eXtreme Array Mapping
Histogram of Kas/Col RILs Red light
hypocotyl length (mm)
cou
nts
6 8 10 12 14
02
46
81
01
2
15 tallest RILs pooled vs15 shortest RILs pooled
LOD
eXtreme Array Mapping
Allele frequencies determined by SFP genotyping. Thresholds set by simulations
0
4
8
12
16
0 20 40 60 80 100cM
LO
D
Composite Interval Mapping
RED2 QTL
Chromosome 2
RED2 QTL 12cM
Red light QTL RED2 from 100 Kas/ Col RILs (Wolyn et al Genetics 2004)
XRED2 QTL
mark1 mark2
Select recombinants by PCR >200 from >1250 plants
HighLow~2Mb ~8cM
>400 SFPsCol
Kas
Col Col
Col het
Col
~2
Kas
het Col
het het
het
~43
Kas
Kas Col
Kas het
Kas
~268
~43 ~539 ~43
~268 ~43 ~2
Kas
eXtreme Array Fine Mapping
Unite Genetic and Physical Map
• Shotgun genomic or 454 reads• ESTs/ cDNAs/ BAC ends• 1000s of contigs
• Genotype mapping population on arrays– Create very high density genetic map
• Known position of genes/contigs allow QTL candidatet gene identification– Control hybridization variation for gene expression
Potential Deletions
>500 potential deletions45 confirmed by Ler sequence
23 (of 114) transposons
Disease Resistance(R) gene clusters
Single R gene deletions
Genes involved in Secondary metabolism
Unknown genes
Potential Deletions Suggest Candidate Genes
FLOWERING1 QTL
Chr1 (bp)
Flowering Time QTL caused by a natural deletion in FLM
MAF1
FLM natural deletion
(Werner et al PNAS 2005)
Ecological and Evolutionary context
• Abiotic conditions– Light, temperature, humidity– Soil, water
• Biotic conditions– Pathogens and pollinators– Conspecifics, grasses, shrubs, trees
Industrial Agriculture -> Sustainable EcoAgriculture
Green, Super Hybrids!
Aquilegia (Columbine) NSF Genome Complexity
• Microarray floral development – QTL candidates
• Physical Map (BAC tiling path)– Physical assignment of ESTs
• QTL for pollinator preference – ~400 RILs, map abiotic stress
– QTL fine mapping/ LD mapping
• Develop transformation techniques– VIGS
• Whole Genome Sequencing (JGI 2007)
Scott Hodges (UCSB)
Elena Kramer (Harvard)
Magnus Nordborg (USC)
Justin Borevitz (U Chicago)
Jeff Tompkins (Clemson)
NaturalVariation.orgNaturalVariation.orgUSC
Magnus NordborgPaul Marjoram
Max Planck
Detlef Weigel
Scripps
Sam Hazen
University of Michigan
Sebastian Zoellner
USC
Magnus NordborgPaul Marjoram
Max Planck
Detlef Weigel
Scripps
Sam Hazen
University of Michigan
Sebastian Zoellner
University of Chicago
Xu ZhangYan Li
Peter RoycewiczEvadne Smith
Megan DunningJoy Bergelson
Michigan State
Shinhan Shiu
PurdueIvan Baxter
University of Chicago
Xu ZhangYan Li
Peter RoycewiczEvadne Smith
Megan DunningJoy Bergelson
Michigan State
Shinhan Shiu
PurdueIvan Baxter
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