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Genomics of Natural Variation in Arabidopsis thaliana
Justin BorevitzSalk Institutenaturalvariation.org
Talk Outline
• Natural Variation in Light Response– PHYTOCHROME A/QTL mapping– Fine Mapping/ Gene Expression candidates
• Single Feature Polymorphisms– Deletion/ Candidate genes– Bulk Segregant/ eXtreme Mapping
• Haplotype analysis
Light Affects the Entire Plant Life Cycle
de-etiolation
hypocotyl
}
Quantitative Trait Loci
EPI1 EPI2
Epistasis scan
Chr1 Chr2 Chr3 Chr4 Chr5
Chr
1
C
hr2
Chr
3
Chr
4
Chr
5
BQTLhttp://hacuna.ucsd.edu/bqtl43,956 pair-wise tests 163 markers and 133 intervalsPermutation threshold p < 0.05 (5000 permutations)
SNP377
SM184
SM50
SM35
SM106
G2395
SNP65
SM40
SEQ8298
TH1
MSAT7964
MAT7787
CER45
5.50
5.87
6.34
7.01
7.30
7.44
7.60
7.79
7.96
8.13
8.29
8.65
9.32
MbMarker
Near-Isogenic Lines for LIGHT1 Ler / Cvi #3
mm
81N-J 17A-A/J 114 124 189Ler
6 2 4 3 3 3 Plants
Line
RVE7
GI
194
3
5.0 5.8 5.8 5.1 5.9 5.7 5.8 Phenotype
differences may be due to expression or hybridization
Downstream players`
PAG1 down regulated in Cvi
PLALE GREEN1 knock out has long hypocotyl in red light
• Abundant Genetic Variation in Light Response– Quickly map in new crosses (XAM still to come!)
• QTL map to novel loci and candidate genes– New crosses find major loci and new loci
• Gene Expression– NILs, pools of extreme RILs or F2s,– Identify candidate genes at QTL (linked)– or downstream effects of QTL (unlinked)
What is Array Genotyping?
• Affymetrix expression GeneChips contain 202,806 unique 25bp oligo nucleotides.
• 11 features per probset for 21546 genes• New array’s have even more• Genomic DNA is randomly labeled with
biotin, product ~50bp.• 3 independent biological replicates
compared to the reference strain Col
GeneChip
Potential Deletions
Spatial Correction
Spatial Artifacts
Improved reproducibilityNext: Quantile Normalization
physicallocationknown
GeneticMarkers in genes
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
Chip genotyping of a Recombinant Inbred Line
29kb interval
Discovery 6 replicates X $500 12,000 SFPs = $0.25Typing 1 replicate X $500 12,000 SFPs = $0.041
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 MAF1
MAF1
MAF1 natural deletion
Fast Neutron deletions
FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1
Het
Map bibb100 bibb mutant plants100 wt mutant plants
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
• Single Feature Polymorphisms– Improve with replicates (easy)– Improved statistical models
• Genotyping– Precisely define recombination breakpoints– Fine mapping– Gene conversion
• Potential Deletions– Candidate genes/ induced mutations
• Bulk segregant Mapping– eXtreme Array Mapping, F2s etc
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, Ler, Bay, Shah, Cvi, Kas, C24,
Est, Kin, Mt, Nd, Sorbo, Van, Ws2
Array Haplotyping
Inbred lines
Low effectiverecombinationdue to partialselfing
Extensive LDblocks
Col Ler Cvi Kas Bay Shah Lz Nd
Chr
omos
ome1
~50
0kb
(-4,-3.5] (-3,-2.5] (-2,-1.5] (-1,-0.5] (0,0.5] (1,1.5] (2,2.5] (3,3.5]
T statistic
fre
qu
en
cy
0
e+
00
4
e+
04
8
e+
04
Distribution of T-stats
null (permutation)actual
Not Col ColNA NA duplications
Accession FDR Sensitivity SNP Totalbay 0.0% 43% 51 563c24 0.2% 39% 64 580cvi 0.0% 38% 91 543est 0.0% 59% 39 548kas 1.9% 44% 66 577kendl 3.1% 33% 57 545ler 0.0% 49% 43 562lz 0.0% 53% 51 573mt 0.2% 61% 49 570nd 0.0% 47% 49 568shah 0.0% 24% 80 548sorbo 0.0% 45% 55 526van 0.2% 29% 92 571ws2 0.0% 49% 57 514
Sequence confirmation of SFPs
SFPs for reverse genetics
http://naturalvariation.org/sfp
14 Accessions 30,950 SFPs`
-20
24
Chromosome 1
-20
24
Chromosome 2
-20
24
Chromosome 3
-20
24
Chromosome 4
-20
24
Chromosome 5
Chromosome Wide Diversity
Diversity 50kb windows
18100000 18200000 18300000 18400000 18500000 18600000
-10
12
34
5
Chromosome 5 (Mb)
Div
ers
ity
perm95%
sfp.countav.pair-wise
18100000 18200000 18300000 18400000 18500000 18600000
-10
12
34
5
Chromosome 5 (Mb)
Div
ers
ity
perm95%
sfp.countav.pair-wise
18100000 18200000 18300000 18400000 18500000 18600000
-10
12
34
5
Chromosome 5 (Mb)
Div
ers
ity
perm95%
sfp.countav.pair-wise
Tajima’s D like 50kb windows
18100000 18200000 18300000 18400000 18500000 18600000
-3-2
-10
12
Chromosome 5 (Mb)
Ta
jima
's D
like
perm95%
• Remember to think about hybridization polymorphism in RNA analysis (affy or cDNA)
• Keep in mind that DNA can be used on many arrays
• Example for mapping ESTs
• Haplotyping
• Diversity/Selection
• Association Mapping– Population Genomics (hybrid zones)
20 40 60 80 100 120 140
20
40
60
80
10
01
20
14
0
features
fea
ture
s
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
ChipViewer: Mapping of transcriptional units of ORFeome
From 2000v At1g09750 (MIPS) to the latest AGI At1g09750
2000 v Annotation (MIPS)
The latest AGI Annotation
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
Haplotype Map –Linkage Disequilibrium, Gene Family (R genes)
Association Studies – Whole Genome Arrays
192 Accessions, > 200,000 SFPs (~600bp resolution)
Confirm Associations in specific crosses with
eXtreme Array Mapping
Future Projects DNA
True natural variation in gene expression polymorphism accounted for, alternative splicing
Cis regulatory variation/ Imprintingreciprocal F1s 3 replicates
Transcriptome QTL Map –VanC Advanced Intercross Recombinant Inbred Lines
How many loci control the variation in gene transcription? Candidate TF and binding sites?
Future Projects RNA
Future work with Natural Variation
• VanC advanced intercross RIL population• Backcross collections
Salk
Jon WernerTodd MocklerSarah LiljegrenOlivier LoudetHuaming ChenJoanne ChoryDetlef WeigelJoseph Ecker
UC San Diego
Charles Berry
Scripps
Sam HazenElizabeth Winzeler
UC Davis
Julin Maloof
University of Guelph, Canada
Dave WolynSainsbury Laboratory
Jonathan Jones
USC
Magnus NordborgTina Hu
Syngenta
Hur-Song ChangTong Zhu
NaturalVariation.orgSalk
Jon WernerTodd MocklerSarah LiljegrenOlivier LoudetHuaming ChenJoanne ChoryDetlef WeigelJoseph Ecker
UC San Diego
Charles Berry
Scripps
Sam HazenElizabeth Winzeler
UC Davis
Julin Maloof
University of Guelph, Canada
Dave WolynSainsbury Laboratory
Jonathan Jones
USC
Magnus NordborgTina Hu
Syngenta
Hur-Song ChangTong Zhu
Helen Hay Whitney FoundationHelen Hay Whitney Foundation