Title: Genomic Systems underlying the genetics of adaptation in Arabidopsis thaliana Justin Borevitz Ecology
1Genomic Systems underlying the genetics of
adaptation in Arabidopsis thalianaJustin
BorevitzEcology EvolutionUniversity of
Chicagohttp//naturalvariation.org/
2Light Affects the Entire Plant Life Cycle
de-etiolation
hypocotyl
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4Talk Outline
Talk Outline
- Predictable to Seasonal Variation
- Local Adaptation in the Lab
- Population Genetics
- Population structure
- Extant diversity and new mutation
- Arrays
- Genetic, epigenetic, expression,
- splicing, and allelic variation
- Ecological context
- Arabidopsis and Aquilegia
- Predictable to Seasonal Variation
- Local Adaptation in the Lab
- Population Genetics
- Population structure
- Extant diversity and new mutation
- Arrays
- Genetic, epigenetic, expression,
- splicing, and allelic variation
- Ecological context
- Arabidopsis and Aquilegia
5Begin with regions spanning the native geographic
range
Nordborg et al PLoS Biology 2005
6Regional/Seasonal Variation
- What is Local Adaptation?
- Predictable Seasonal changes unique to each
location.
Tossa Del Mar Spain
Lund Sweden
7Sweden 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
8Solar Calc II
Kurt Spokas Version 2.0a June 2006
USDA-ARS Website Midwest Area (Morris,MN) http//
www.ars.usda.gov/mwa/ncscrl
9Flowering time QTL, Kas/Col RILs
Flowering time QTL, Kas/Col RILs
10Kas/Col flowering time QTL GxE
Chr4 FRI
Chr1 FLM Chr4 FRI
11Environment and Epistasis
12Globally Distributed
Olivier Loudet
http//www.inra.fr/qtlat/NaturalVar/NewCollection.
htm
13Current collections
- 807 Lines from 25 Midwest Populations
- (Diane Byers IL state) pics published!
- 1101 Lines from UK, 51 populations
- (Eric Holub Warwick, UK) growing!
- gt 500 lines N and S Sweden (Nordborg)
- gt 400 Lines France and Midwest (Bergelson)
- 400 lines Midwest (Borevitz)
- 857 Accessions stock center (Randy Scholl)
- pics published
- Others welcome
- Genotyped with Sequenom 149 SNPs 0.03 per
14Local Population Structure
common haplotypes
134 Non singleton SNPs of 1234
accessions Global, Midwest, and UK
Megan Dunning, Yan Li
15Diversity within and between populations
17 Major Haplotypes
80 Major Haplotypes
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19Variation within a field http//naturalvariation.o
rg/hapmap
Variation within a field http//naturalvariation.o
rg/hapmap
20Universal 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
21Improved 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)
22Which arrays should be used?
BAC array
cDNA array
Long oligo array
23Which arrays should be used?
Gene array
Exon array
Tiling array
35bp tile, 25mers 10bp gaps
24Which 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
25Global Allele Specific Expression 65,000
SNPs Transcribed Accession Pairs 12,000 genes
gt 1 SNP 6,000 gt 2 SNPs
26Potential Deletions
27SFPs and CCGG Methylome
A)
- Extract genomic 100ng
- DNA (single leaf)
- Digest with either
- msp1 or hpa2 CCGG
- Label with biotin
- Random primers
- Hybridize to array
HpaII digestion
Random labeling
B)
MspI digestion
Random labeling
Intensity
Col
Col
Hpa msp
28Experimental design
Van ? x Col ?
Col? x Col?
Col ? x Van ?
Van ? x Van ?
Four genotypes, each with four biological
replicates 4 day old seedlings, white light
29Methylation Polymorphisms, mSFPs
Fit model Intensity additive dominant
maternal additiveenzyme dominantenzyme
maternalenzyme
30SFP 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
31methylated features and mSFPs
Enzyme effect, on CCGG features
GxE
mQTL?
gt10,000 of 100,000 at 5 FDR
276 at 15 FDR
32Genomic Distribution of nonPolymorphic
methylation sites
q-value enzyme addenz domenz matenz
0.001 2671 31 0 0
0.005 4000 141 0 481
0.01 4387 251 0 504
0.05 5950 725 0 507
0.1 6733 1263 43 507
33Verification of additive x enzyme by genomic PCR
34Verification of additive x enzyme by genomic PCR
35Verification of additive x enzyme by epiTyper
Col Col Col Van Van Van Col? x Van? Col? x Van ?
Van? x Col? Van ? x Col ? Van? x Col ?
CCGG
CCGG
chromomethylase 2 (CMT2) exon19
36Next Questions
- What is the genetic architecture of methylation
variation? - How does it change with the environment and
through development? - Regional patterns, eg chromatin remodeling
- When does methylation effect expression?
37Transcription subUnits (TUs)Intensity(gene/tu)
add dommat error
Expression Analysis with Annotation
Intron1
Exon1
Exon2
?
cDNA1
cDNA2
cDNA3
X
Tu1
Tu2
Tu3
38Additive, Dominant, Maternal, Genotype Variation
D
E
39Alternative spliced introns
v v v c c c v c
RT-PCR gDNA PCR
FDR for selection 3.5 5
Total introns tested 62,051 62,051
Total introns 43 228
Tested introns 32 72
Confirmed 20 36
percentage 71 56
40Alternative spliced exons - verification
v v v c c c v c
RT-PCR gDNA PCR
FDR for selection 3
Total exons tested 86,349
Total exons 69
Tested exons 5
confirmed 5
41Ecological and Evolutionary context
- Abiotic conditions
- Light, temperature, humidity
- Soil, water
- Biotic conditions
- Pathogens and polinators
- Conspecifics, grasses trees
42Local Population Variation
43Local adaptation under strong selection
44Seasonal Variation
Matt Horton
Megan Dunning
45Aquilegia (Columbines)
Recent adaptive radiation, 350Mb genome
46Genetics of Speciationalong a Hybrid Zone
47NSF Genome Complexity
- Microarray 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?)
Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus
Nordborg (USC) Justin Borevitz (U Chicago) Jeff
Tompkins (Clemson)
48http//www.plosone.org/
49NaturalVariation.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