SNP/Tiling arrays for very high density marker based breeding and QTL candidate gene identification Justin Borevitz Ecology

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SNP/Tiling arrays for very high density marker
based breeding and QTL candidate gene
identificationJustin BorevitzEcology
EvolutionUniversity of Chicagohttp//naturalvari
ation.org/
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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

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Genomic Breeding Path
Experimental Design Mapping population
Marker Identification Genotyping
Phenotyping QTL Analysis Fine Mapping
Candidate gene Polymorphisms gene expression loss
of function
QTL gene Confirmation
QTL gene Confirmation
Phenotyping QTL Analysis Fine Mapping
Genomics path
Experimental Design Mapping population
Borevitz and Chory, COPB 2003
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Talk Outline
Talk 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

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Begin with regions spanning the Native Geographic
range
Lund Sweden
Nordborg et al PLoS Biology 2005 Li et al PLoS
ONE 2007
Tossa Del Mar Spain
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Sweden Spain
Seasons in the Growth Chamber
Seasons in the Growth Chamber

• Changing Day length
• Cycle Light Intensity
• Cycle Light Colors
• Cycle Temperature

• Changing Day length
• Cycle Light Intensity
• Cycle Light Colors
• Cycle Temperature

Geneva Scientific/ Percival
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Solar Calc II
Kurt Spokas Version 2.0a June 2006
USDA-ARS Website Midwest Area (Morris,MN) http//
www.ars.usda.gov/mwa/ncscrl
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Flowering time QTL, Kas/Col RILs
Flowering time QTL, Kas/Col RILs
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Kas/Col flowering time QTL GxE
Chr4 FRI
Chr1 FLM Chr4 FRI
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Global and Local Population Structure
Olivier Loudet
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Local Population Structure
common haplotypes
144 Non singleton SNPs gt2000 accessions Global,
Midwest, and UK
Megan Dunning, Yan Li
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Diversity within and between populations
80 Major Haplotypes
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Universal Whole Genome Array
DNA
RNA
Gene/Exon Discovery Gene model correction Non-codi
ng/ micro-RNA
Chromatin Immunoprecipitation ChIP chip
Alternative Splicing
Methylation
Antisense transcription
Polymorphism SFPs Discovery/Genotyping
Transcriptome Atlas Expression levels Tissues
specificity
Comparative Genome Hybridization
(CGH) Insertion/Deletions Copy Number
Polymorphisms
RNA Immunoprecipitation RIP chip
Allele Specific Expression
Control for hybridization/genetic
polymorphisms to understand TRUE expression
variation
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Improved Genome Annotation
ORFa
Transcriptome Atlas
ORFb
start
AAAAA
deletion
M
M
M
M
M
M
SFP
M
M
M
M
M
M
SNP
SNP
SFP
SFP
conservation
Chromosome (bp)
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Which arrays should be used?
BAC array
cDNA array
Long oligo array
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Which arrays should be used?
Gene array
Exon array
Tiling array
35bp tile, 25mers 10bp gaps
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Which arrays should be used?
SNP array
How about multiple species? Microbial
communities?
Pst,Psm,Psy,Psx, Agro, Xanthomonas, H parasitica,
15 virus,
Ressequencing array
Tiling/SNP array 2007 250k SNPs, 1.6M
tiling probes
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Global Allele Specific Expression
65,000 SNPs Transcribed Accession Pairs 12,000
genes gt 1 SNP 6,000 gt 2 SNPs
Zhang, X., Richards, E., Borevitz, J. Current
Opinion in Plant Biology (2007)
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Potential Deletions
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SFP detection on tiling arrays
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 0.9
5 3298 92027 3.4
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Chip genotyping of a Recombinant Inbred Line
29kb interval
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100 bibb mutant plants
Map bibb
100 wt mutant plants
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bibb mapping
Bulk segregant Mapping using Chip hybridization
bibb maps to Chromosome2 near ASYMETRIC LEAVES1
AS1
ChipMap
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BIBB ASYMETRIC LEAVES1
AS1 (ASYMMETRIC LEAVES1) MYB closely related
to PHANTASTICA located at 64cM
as1
bibb
Sequenced AS1 coding region from bib-1 found g
-gt a change that would introduce a stop codon in
the MYB domain
bib-1 W49
as-101 Q107
bibb
as1-101
MYB
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Array Mapping
chr1 chr2 chr3 chr4
chr5
Hazen et al Plant Physiology (2005)
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eXtreme Array Mapping
15 tallest RILs pooled vs 15 shortest RILs pooled
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eXtreme Array Mapping
LOD
Allele frequencies determined by SFP genotyping.
Thresholds set by simulations
Red light QTL RED2 from 100 Kas/ Col RILs
(Wolyn et al Genetics 2004)
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eXtreme Array Mapping BurC F2
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XAMLz x Col F2
QTLLz x Ler F2
(Werner et al Genetics 2006)
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eXtreme Array Fine Mapping
2Mb 8cM
Low
Col
RED2 QTL
gt400 SFPs
High
Kas
X
mark2
mark1
2
268
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Col
Col
het
Col
Kas
Col
43
43
539
539
Kas
het
Col
het
het
het
het
het
2
43
268
Kas
Kas
Kas
Col
het
Kas
Select recombinants by PCR gt200 from gt1250 plants
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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

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Potential Deletions
gt500 potential deletions 45 confirmed by Ler
sequence 23 (of 114) transposons Disease
Resistance (R) gene clusters Single R gene
deletions Genes involved in Secondary
metabolism Unknown genes
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Potential Deletions Suggest Candidate Genes
FLOWERING1 QTL
Chr1 (bp)
MAF1
Flowering Time QTL caused by a natural deletion
in FLM
(Werner et al PNAS 2005)
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Fast Neutron deletions
FKF1 80kb deletion CHR1
cry2 10kb deletion CHR1
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Ecological and Evolutionary context

• Abiotic conditions
• Light, temperature, humidity
• Soil, water
• Biotic conditions
• Pathogens and pollinators
• Conspecifics, grasses, shrubs, trees
• Industrial Agriculture -gt
• Sustainable EcoAgriculture
• Green, Super Hybrids!

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Local adaptation under strong selection
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Seasonal Variation
Matt Horton
Megan Dunning
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Aquilegia (Columbines)
Recent adaptive radiation, 350Mb genome
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Genetics of Speciationalong a Hybrid Zone
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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)
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http//www.plosone.org/
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NaturalVariation.org
NaturalVariation.org
USC Magnus Nordborg Paul Marjoram Max
Planck Detlef Weigel Scripps Sam
Hazen University of Michigan Sebastian Zoellner
USC Magnus Nordborg Paul Marjoram Max
Planck Detlef Weigel Scripps Sam
Hazen University of Michigan Sebastian Zoellner
University of Chicago Xu Zhang Yan Li Peter
Roycewicz Evadne Smith Megan Dunning Joy
Bergelson Michigan State Shinhan
Shiu Purdue Ivan Baxter
University of Chicago Xu Zhang Yan Li Peter
Roycewicz Evadne Smith Megan Dunning Joy
Bergelson Michigan State Shinhan
Shiu Purdue Ivan Baxter