genomic systems underlying the genetics of adaptation in arabidopsis thaliana justin borevitz...
Post on 22-Dec-2015
216 views
TRANSCRIPT
Genomic Systems underlying the genetics of adaptation in Arabidopsis thaliana
Justin BorevitzEcology & EvolutionUniversity of Chicagohttp://naturalvariation.org/
Light Affects the Entire Plant Life Cycle
de-etiolation
hypocotyl
}
Talk OutlineTalk 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
Begin with regions spanning the native geographic range
Nordborg et al PLoS Biology 2005
Regional/Seasonal Variation
• What is Local Adaptation?
• Predictable Seasonal changes unique to each location.
Tossa Del MarSpain
LundSweden
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
Geneva Scientific/ Percival
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
month
degr
ees
C
Spain High
Spain Low
Sweden High
Sweden Low
standard
Developmental Plasticity == BehaviorDevelopmental Plasticity == Behavior
Kurt Spokas
Version 2.0a June 2006
USDA-ARS Website Midwest Area (Morris,MN)http://www.ars.usda.gov/mwa/ncscrl
Flowering time QTL, Kas/Col RILs
Sweden 1
Col-gl1
Kas1
Sweden 2
Col-gl1
Kas1
Spain 1
Col-gl1
Kas1
Spain 2
Col-gl1
Kas1
Num
ber
of R
ILs
Num
ber
of R
ILs
Flowering time QTL, Kas/Col RILs
FRI
FLM
Kas/Col flowering time QTL GxE
Chr4 FRIChr1 FLM Chr4 FRI
Environment and Epistasis
Globally Distributed
http://www.inra.fr/qtlat/NaturalVar/NewCollection.htm
Olivier Loudet
Current collections• 807 Lines from 25 Midwest Populations
– (Diane Byers IL state) – pics published!
• 1101 Lines from UK, 51 populations– (Eric Holub Warwick, UK) – growing!
• > 500 lines N and S Sweden (Nordborg)• > 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
134 Non singleton SNPs of 1234 accessions
Global, Midwest, and UK
common haplotypes
Local Population Structure
Megan Dunning, Yan Li
17 Major Haplotypes
80 Major Haplotypes
Diversity within and between populations
Variation within a field http://naturalvariation.org/hapmapVariation within a field http://naturalvariation.org/hapmap
RNA DNA
Universal Whole Genome Array
Transcriptome AtlasExpression levelsTissues specificity
Transcriptome AtlasExpression levelsTissues specificity
Gene/Exon DiscoveryGene model correctionNon-coding/ micro-RNA
Gene/Exon DiscoveryGene model correctionNon-coding/ micro-RNA
Alternative SplicingAlternative Splicing
Comparative GenomeHybridization (CGH)
Insertion/DeletionsCopy Number Polymorphisms
Comparative GenomeHybridization (CGH)
Insertion/DeletionsCopy Number Polymorphisms
MethylationMethylation
ChromatinImmunoprecipitation
ChIP chip
ChromatinImmunoprecipitation
ChIP chip
Polymorphism SFPsDiscovery/Genotyping
Polymorphism SFPsDiscovery/Genotyping
Control for hybridization/genetic polymorphismsto understand TRUE expression variation
RNA ImmunoprecipitationRIP chip
RNA ImmunoprecipitationRIP chip
Antisense transcription
Allele Specific Expression
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
Which arrays should be used?
cDNA array
Long oligo array
BAC array
Which arrays should be used?
Gene array
Exon array
Tiling array35bp tile, 25mers 10bp gaps
Which arrays should be used?
Tiling/SNP array 2007 250k SNPs, 1.6M tiling probes
SNP array
Ressequencing array
How about multiple species? Microbial communities?
Pst,Psm,Psy,Psx, Agro, Xanthomonas, H parasitica, 15 virus,
Col
Col
Van
Van
Col
Van
Van
Col
Genomic DNA RNA
No significant
allele specific expression
cis regulatory variation
(Van allele)
Paternal Imprinting Maternal Imprinting
cis regulatory variation
(Col allele)
RNA
RNA
RNA
RNA
GlobalAllele SpecificExpression
65,000 SNPsTranscribedAccession Pairs12,000 genes
>= 1 SNP6,000 >= 2 SNPs
Potential Deletions
SFPs and CC*GG Methylome
Extract genomic 100ng DNA (single leaf)
Digest with either msp1 or hpa2 CC*GG
Label with biotinRandom primers
Hybridize to array
HpaII digestion
Random labeling
Random labeling
MspI digestion
* * *
* * *
* * *
A)
B)
Hpa msp
Inte
nsit
y
*
*
Col
Col
Hpa msp
Col
Col
Hpa mspV
an
Van
mSFP
Hpa msp
Van
Van
SFP
Col♀ x Col♂
Van ♀ x Van ♂
Col ♀ x Van ♂
Van ♀ x Col ♂
Experimental design
Four genotypes, each with four biological replicates
4 day old seedlings, white light
Fit modelIntensity ~ additive + dominant + maternal +
additive:enzyme + dominant:enzyme + maternal:enzyme
Methylation Polymorphisms, mSFPs
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
methylated features and mSFPs
>10,000 of 100,000 at 5% FDR
Enzyme effect, on CCGG features GxE
276 at 15% FDR
mQTL?
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
Genomic Distribution of nonPolymorphic methylation sites
AT4G19020 tu19
AT1G27320 tu9
AT2G37080 tu1
AT4G27910 tu9
Col Van F1v F1c Col Van F1v F1c Col Van F1v F1c
Control HpaII MspI
AT1G51790 intron9
AT1G53240 intron4
AT2G01220 tu5
AT5G14600 intron3
AT1G49730 tu10
AT1G79990 intron9
AT1G20780 tu4
AT2G35350 tu2
AT2G45270 intron9
AT3G11460 tu1
AT4G04340 tu15
AT5G56370 tu3
AT2G18100 tu9
AT3G07740 tu2
AT3G48730 tu3
AT5G63190 tu6
AT5G13960 intron8
AT1G27900 intron7
AT2G45620 intron5
AT4G31140 tu2
Verification of additive x enzyme by genomic PCR
AT5G67130 tu7
AT1G19450 tu10
Col Van F1v F1c Col Van F1v F1c Col Van F1v F1c
Control HpaII MspI
AT2G32730 tu3
AT2G45670 tu11
AT3G04820 tu1
AT3G04610 intron5
AT3G53100 tu2
AT3G23590 intron4
AT1G10910 intron9
AT3G28880 tu8
AT4G23570 tu9
AT4G02500 tu1
AT4G17140 tu8
AT5G14950 tu1
AT5G04710 tu1
AT1G73730 tu2
AT1G19715 intron2
Verification of additive x enzyme by genomic PCR
ColColColVanVanVan
Col♂ x Van♀Col♂ x Van ♀
Van♂ x Col♀Van ♂ x Col ♀Van♂ x Col ♀
CC*GG
chromomethylase 2 (CMT2) exon19
0
1
2
3
4
5
6
hpaII mspI
log
inte
nsity
col van col♂xvan♀ van♂xcol♀
CC*GG
Verification of additive x enzyme by epiTyper
Next 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?
Transcription subUnits (TUs)Intensity(gene/tu) ~ add + dom+mat + error
Exon1 Exon2Intron1
Tu1 Tu2 Tu3
?
cDNA1
cDNA2
cDNA3
X
Expression Analysis with Annotation
9.37%141150801508Maternal
1.2%22184901849Allele Additive
1.2%91741742cis Regulatory
1.1%40323323Imprinting
3.50%24561129690Dominant
3.50%5541411881602Additive
FDRExpected from perm
-Sig. Features
+ Sig. Features
Total Sig. Features
9.37%141150801508Maternal
1.2%22184901849Allele Additive
1.2%91741742cis Regulatory
1.1%40323323Imprinting
3.50%24561129690Dominant
3.50%5541411881602Additive
FDRExpected from perm
-Sig. Features
+ Sig. Features
Total Sig. Features
AT1G07350
AT1G29120
AT1G51350
AT1G53560
AT1G76170
D E
Additive, Dominant, Maternal, Genotype Variation
v v v c c c v c RT-PCR gDNA PCR
Alternative spliced introns
ColVan
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%
FDR for selection 3%
Total exons tested
86,349
Total exons 69
Tested exons 5
confirmed 5
Alternative spliced exons
- verification
ColVan
v v v c c c v c RT-PCR gDNA PCR
Ecological and Evolutionary context
• Abiotic conditions– Light, temperature, humidity– Soil, water
• Biotic conditions– Pathogens and polinators– Conspecifics, grasses trees
Local Population Variation
Local adaptation
under strong
selection
Seasonal Variation
Matt Horton
Megan Dunning
Aquilegia (Columbines)
Recent adaptive radiation, 350Mb genome
Genetics of Speciationalong a Hybrid Zone
NSF 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)
http://www.plosone.org/
NaturalVariation.orgNaturalVariation.orgUSC
Magnus NordborgPaul Marjoram
Max Planck
Detlef Weigel
Scripps
Sam Hazen
University of Michigan
Sebastian Zoellner
USC
Magnus NordborgPaul Marjoram
Max Planck
Detlef Weigel
Scripps
Sam Hazen
University of Michigan
Sebastian Zoellner
University of Chicago
Xu ZhangYan Li
Peter RoycewiczEvadne Smith
Megan DunningJoy Bergelson
Michigan State
Shinhan Shiu
PurdueIvan Baxter
University of Chicago
Xu ZhangYan Li
Peter RoycewiczEvadne Smith
Megan DunningJoy Bergelson
Michigan State
Shinhan Shiu
PurdueIvan Baxter