Natural Variation in Light Response using Whole Genome Tiling Arrays Justin Borevitz Ecology

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Natural Variation in Light Response using Whole
Genome Tiling ArraysJustin BorevitzEcology
EvolutionUniversity of Chicagohttp//naturalvari
ation.org
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Light Affects the Entire Plant Life Cycle
de-etiolation

hypocotyl
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Light Affects the Entire Plant Life Cycle
Light response variation can be seen under
constant conditions in the lab
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Quantitative Trait Loci
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differences may be due to expression or
hybridization
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PAG1 down regulated in Cvi
PLALE GREEN1 knock out has long hypocotyl in red
light
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Local Population Variation
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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

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Talk Outline

• Single Feature Polymorphisms (SFPs)
• Potential deletions
• Bulk segregant/ eXtreme Mapping
• Haplotype analysis
• Aquilegia

• Single Feature Polymorphisms (SFPs)
• Potential deletions
• Bulk segregant/ eXtreme Mapping
• Haplotype analysis
• Aquilegia

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Which arrays should be used?

• Spotted arrays Arizona 29,000 - 70mers
• ATH1, Affymetrix expression GeneChip
• 202,806 unique 25bp oligo nucleotides features
• AtTILE1, universal whole genome array
• every 35bp, gt 3Million PM features
• Re-sequencing array 120M8bp
• 20 Accessions, Perlegen,
• Max Planck (Weigel), USC (Nordborg)

GeneChip
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Universal Whole Genome Array
RNA
DNA
Chromatin Immunoprecipitation ChIP chip
Gene Discovery Gene model correction Non-coding/
micro-RNA Antisense transcription
Methylation
Transcriptome Atlas Expression levels Tissues
specificity
Polymorphism SFPs Discovery/Genotyping
Comparative Genome Hybridization
(CGH) Insertion/Deletions
Alternative Splicing
35 bp tile, non-repetitive regions, good
binding oligos, evenly spaced
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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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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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False Discovery and Sensitivity














PM only


GeneChip





SAM threshold



SFPs

nonSFPs

Cereon marker accuracy

5 FDR



3806

89118

100




Sequence

817

121

696

Sensitivity



Polymorphic

340

117

223

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Non
-
polymorphic

477

4

473





False Discovery rate

3






Test for independence of all factors






Chisq 177.34, df 1, p
-
value 1.845e
-
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GeneChip





SAM threshold



SFPs

nonSFPs

Cereon marker accuracy

18 FDR



10627

82297

100




Sequence

817

223

594

Sensitivity



Polymorphic

340

195

145

57





Non
-
polymorphic

477

28

449





False Discovery rate

13






Test for indep
endence of all factors






Chisq 265.13, df 1, p
-
value 1.309e
-
59


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100 bibb mutant plants
Map bibb
100 wt mutant plants
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Array Mapping
Hazen et al Plant Physiology 2005
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LUX ARRHYTHMO encodes a Myb domain protein
essential for circadian rhythms Hazen et al
PNAS, 2005 Cloned with Array Mapping
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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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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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Natural Variation on Tiling Arrays
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Array Haplotyping

• What about Diversity/selection across the genome?
• A genome wide estimate of population genetics
  parameters, ?w, p, TajimaD, ?
• LD decay, Haplotype block size
• Deep population structure?
• Col, Lz, Bur, Ler, Bay, Shah, Cvi, Kas, C24, Est,
  Kin, Mt, Nd, Sorbo, Van, Ws2
• Fl-1, Ita-0, Mr-0, St-0, Sah-0

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Array Haplotyping
Inbred lines Low effective recombination due to
partial selfing Extensive LD blocks
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Chromosome Wide Diversity
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Diversity 50kb windows
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Tajimas D like 50kb windows
RPS4
unknown
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R genes vs bHLH
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Review

• Single Feature Polymorphisms (SFPs) can be used
  to
• Identify recombination breakpoints
• eXtreme Array Mapping
• Potential deletions (candidate genes)
• Haplotyping
• Diversity/Selection
• Association Mapping

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Aquilegia (Columbines)
Recent adaptive radiation, 350Mb genome
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Species withgt 20k ESTs 11/14/2003
Animal lineage good coverage
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Aquilegia (Columbines)

• 300 F3 RILs growing (Evadne Smith)
• 85,000 5 3 ESTs -- 51,000 clones, gt3500 SNPs
• TIGR gene index and GenBank
• arrays being designed by Nimblegen

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Genetics of Speciationalong a Hybrid Zone
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NSF Genome Complexity

• 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
• http//www.AQgenome.org

Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus
Nordborg (USC) Justin Borevitz (U Chicago) Jeff
Tompkins (Clemson)
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NaturalVariation.org
NaturalVariation.org
University of Chicago Xu Zhang Evadne Smith Ken
Okamoto Purdue Ivan Baxter UC Davis Julin
Maloof University of Guelph, Canada Dave
Wolyn Sainsbury Laboratory Jonathan Jones
University of Chicago Xu Zhang Evadne Smith Ken
Okamoto Purdue Ivan Baxter UC Davis Julin
Maloof University of Guelph, Canada Dave
Wolyn Sainsbury Laboratory Jonathan Jones
Salk Jon Werner Joanne Chory Joseph Ecker Max
Planck Detlef Weigel UC San Diego Charles
Berry Scripps Sam Hazen Elizabeth Winzeler
Salk Jon Werner Joanne Chory Joseph Ecker Max
Planck Detlef Weigel UC San Diego Charles
Berry Scripps Sam Hazen Elizabeth Winzeler