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 Arrays
Justin BorevitzEcology & EvolutionUniversity of Chicagohttp://naturalvariation.org
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
Quantitative Trait Loci
differences may be due to expression or hybridization
PAG1 down regulated in Cvi
PLALE GREEN1 knock out has long hypocotyl in red light
Local Population Variation
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
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
1400se
p
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
monthde
gree
s C
Spain High
Spain Low
Sweden High
Sweden Low
standard
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
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, > 3Million PM features• Re-sequencing array 120M*8bp
– 20 Accessions, Perlegen,
– Max Planck (Weigel), USC (Nordborg)
GeneChip
RNA DNA
Universal Whole Genome Array
Transcriptome AtlasExpression levelsTissues specificity
Transcriptome AtlasExpression levelsTissues specificity
Gene DiscoveryGene model correctionNon-coding/ micro-RNAAntisense transcription
Gene DiscoveryGene model correctionNon-coding/ micro-RNAAntisense transcription
Alternative SplicingAlternative Splicing Comparative GenomeHybridization (CGH)
Insertion/Deletions
Comparative GenomeHybridization (CGH)
Insertion/Deletions
MethylationMethylation
ChromatinImmunoprecipitation
ChIP chip
ChromatinImmunoprecipitation
ChIP chip
Polymorphism SFPsDiscovery/Genotyping
Polymorphism SFPsDiscovery/Genotyping
~35 bp tile, non-repetitive regions, “good” binding oligos, evenly spaced
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
Potential Deletions
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
False Discovery and Sensitivity
PM only
SAM threshold
5% FDR
GeneChip SFPs nonSFPs Cereon marker accuracy 3806 89118 100% Sequence 817 121 696 Sensitivity
Polymorphic 340 117 223 34% Non-polymorphic 477 4 473
False Discovery rate: 3% Test for independence of all factors: Chisq = 177.34, df = 1, p-value = 1.845e-40 SAM threshold 18% FDR
GeneChip SFPs nonSFPs Cereon marker accuracy 10627 82297 100% Sequence 817 223 594 Sensitivity
Polymorphic 340 195 145 57% Non-polymorphic 477 28 449
False Discovery rate: 13% Test for independence of all factors: Chisq = 265.13, df = 1, p-value = 1.309e-59
3/4 Cvi markers were also confirmed in PHYB
90% 80% 70%
41% 53% 85%
90% 80% 70%
67% 85% 100%
Cereonmay be asequencingError
TIGRmatch isa match
Map bibb100 bibb mutant plants100 wt mutant plants
Array Mapping
Hazen et al Plant Physiology 2005
LUX ARRHYTHMO encodes a Myb domain
protein essential for circadian rhythms
Hazen et al PNAS, 2005
Cloned with Array Mapping
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)
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)
Fast Neutron deletions
FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1
Het
Natural Variation on Tiling Arrays
Array Haplotyping
• What about Diversity/selection across the genome?
• A genome wide estimate of population genetics parameters, θw, π, Tajima’D, ρ
• LD decay, Haplotype block size• Deep population structure?• Col, Lz, Bur, Ler, Bay, Shah, Cvi, Kas,
C24, Est, Kin, Mt, Nd, Sorbo, Van, Ws2Fl-1, Ita-0, Mr-0, St-0, Sah-0
Array Haplotyping
Inbred lines
Low effectiverecombinationdue to partialselfing
Extensive LDblocks
Col Ler Cvi Kas Bay Shah Lz Nd
Chr
omos
ome1
~50
0kb
Chromosome Wide Diversity
Diversity 50kb windows
Tajima’s D like 50kb windows
RPS4 unknown
R genes vs bHLH
(-1,-0.8] (-0.6,-0.4] (-0.2,0] (0.2,0.4] (0.6,0.8]
Selection
Tajima's D like statistic
freq
uen
cy
01
02
03
04
05
06
07
0
RgenesbHLH
Review
• Single Feature Polymorphisms (SFPs) can be used to
• Identify recombination breakpoints• eXtreme Array Mapping• Potential deletions (candidate genes)
• Haplotyping• Diversity/Selection
• Association Mapping
Aquilegia (Columbines)
Recent adaptive radiation, 350Mb genome
Species with> 20k ESTs 11/14/2003
Animal lineage: good coverage
Plant lineage: crop plant coverage
• 300 F3 RILs growing (Evadne Smith)• 85,000 5’ 3’ ESTs -- 51,000 clones, >3500 SNPs• TIGR gene index and GenBank• arrays being designed by Nimblegen
Aquilegia (Columbines)
Genetics of Speciationalong a Hybrid Zone
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)
NaturalVariation.orgNaturalVariation.orgSalk
Jon WernerJoanne ChoryJoseph Ecker
Max Planck
Detlef Weigel
UC San Diego
Charles Berry
Scripps
Sam HazenElizabeth Winzeler
Salk
Jon WernerJoanne ChoryJoseph Ecker
Max Planck
Detlef Weigel
UC San Diego
Charles Berry
Scripps
Sam HazenElizabeth Winzeler
University of Chicago
Xu ZhangEvadne SmithKen Okamoto
PurdueIvan Baxter
UC Davis
Julin Maloof
University of Guelph, Canada
Dave Wolyn
Sainsbury Laboratory
Jonathan Jones
University of Chicago
Xu ZhangEvadne SmithKen Okamoto
PurdueIvan Baxter
UC Davis
Julin Maloof
University of Guelph, Canada
Dave Wolyn
Sainsbury Laboratory
Jonathan Jones