Download eXtreme Array Mapping - modelspeciesradcliffe

Ecological and Evolutionary Systems biology:
Conceptual and molecular tools for analysis
Justin Borevitz
Ecology & Evolution
University of Chicago
Finch (large beaked)
Sitting in a cotton wood
http://naturalvariation.org/
http://naturalvariation.org/
Hemicordate
Sea anemone
Stickleback
Deer mouse burrow
Aquilegia, Arabidopsis, Mimulus?
Indiana Dunes National Lakeshore
Developing Model Organisms - Community
• Critical mass of labs, hands/eyes/minds
• Coordinated collaboration
– webinar lab meetings
– Open chat/ focus problems/ big picture meetings
• United by questions
– Development: novelty/plasticity
– Adaptation: abiotic/biotic
• United by organism
– Shared tools: genomic/ genetic/ methods
• United by ecoregion – field study site
– Soil/ water testing, weather monitoring,
development/behavioral real time recording
Developing Model Organisms - Tools
• High throughput Phenotyping
– Physiological dissection of 1000s correlated traits
– Biological Variation
• Multi species ecological interactions
– “extended phenotype”
Functional Genomics
•Microarrays
• Environmental Interaction (GxE)
•SNPs
– Local adaptation
•Induced deletions
• Epistasis (GxG)
– Magnify minor QTL in local backgrounds
• Multiple genes under major QTL
– High Density markers
– Linkage Drag
Talk Outline
•• Genetic
Genetic Diversity
Diversity
–– Population
Population structure,
structure, Haplotype
Haplotype Mapping
Mapping set
set
•• Phenotyping
Phenotyping in
in multiple
multiple environments
environments
–– Seasonal
Seasonal Variation
Variation in
in the
the Lab
Lab
•• SNP/Tiling
SNP/Tiling microarrays
microarrays
–– Splicing
Splicing
–– Methylation
Methylation
–– Very
Very High
High Density
Density Markers
Markers SFPs
SFPs
–– Bulk
Bulk Mapping
Mapping
–– Deletions
Deletions
Global and Local Population Structure
Olivier Loudet
Local
adaptation
under
strong
selection
Seasonal Variation
Matt Horton
Megan Dunning
Local Population Structure
common haplotypes
144 Non singleton SNPs >2000 accessions
Megan Dunning, Yan Li
Global, Midwest, and UK
Diversity within and
between populations
80 Major Haplotypes
Diversity within and between populations
17 Major Haplotypes
80 Major Haplotypes
Variation within a field http://naturalvariation.org/hapmap
Begin with regions spanning the
Native Geographic range
Lund
Sweden
Nordborg et al PLoS Biology 2005
Li et al PLoS ONE 2007
Tossa Del Mar
Spain
Seasons in the Growth Chamber
•
•
•
•
Changing Day length
Cycle Light Intensity
Cycle Light Colors
Cycle Temperature
Day Length
Light Intensity
Temperature
1400
Sw eden
Spain
20:00
1200
30
Spain
standard
18:00
25
standard
standard
1000
16:00
600
8:00
Geneva
Scientific/
Percival
15
10
Spain High
5
400
6:00
Spain Low
0
200
0
Spain
standard
month
month
jun
may
apr
mar
feb
jan
dec
nov
oct
aug
jul
jun
may
apr
mar
jan
feb
dec
oct
nov
-10
sep
aug
jul
jun
may
apr
mar
jan
feb
dec
oct
nov
sep
Sweden
month
Sw eden Low
-5
2:00
0:00
Sw eden High
sep
4:00
aug
10:00
800
jul
W/m2
12:00
degrees C
20
14:00
hours
35
Sw eden
22:00
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
Genomic Breeding Path
Borevitz and Chory, COPB 2003
Which arrays should be used?
BAC array
cDNA array
Long oligo array
Which arrays should be used?
Gene array
Exon array
35bp tile, 25mers 10bp gaps
Tiling array
Which 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
Transcriptome Atlas
Improved Genome Annotation
ORFa
ORFb
start
conservation
MMMM M M
AAAAA
SFP
SFP
SFP
SNP
Chromosome (bp)
deletion
MMMM M M
SNP
Universal Whole Genome Array
RNA
Gene/Exon Discovery
Gene model correction
Non-coding/ micro-RNA
DNA
Chromatin
Immunoprecipitation
ChIP chip
Alternative Splicing
Methylation
Antisense transcription
Transcriptome Atlas
Expression levels
Tissues specificity
RNA Immunoprecipitation
RIP chip
Allele Specific Expression
Polymorphism SFPs
Discovery/Genotyping
Comparative Genome
Hybridization (CGH)
Insertion/Deletions
Copy Number Polymorphisms
Control for hybridization/genetic polymorphisms
to understand TRUE expression variation
Additive, Dominant, Maternal, Genotype Variation
AT1G07350
AT1G29120
AT1G51350
D
E
AT1G53560
AT1G76170
Alternative spliced exons
- verification
v
v
v c
RT-PCR
c
FDR for selection
Total exons
tested
Total exons
Tested exons
confirmed
c
v c
gDNA PCR
3%
86,349
69
5
5
Col
Van
Col
Col
No significant
Col
Van
allele specific expression
Van
Van
Van
Col
RNA
Genomic DNA
cis regulatory variation
cis regulatory variation
(Van allele)
(Col allele)
RNA
RNA
Paternal Imprinting
RNA
Global
Allele Specific
Expression
Maternal Imprinting
RNA
65,000 SNPs
Transcribed
Accession Pairs
12,000 genes
>= 1 SNP
6,000 >= 2 SNPs
Zhang, X., Richards,
E., Borevitz, J.
Current Opinion in
Plant Biology (2007)
Potential Deletions
SFPs and CC*GG Methylome
SFP
A)
Extract genomic 100ng
*
*
mSFP
*
*
HpaII digestion
DNA (single leaf)
*
*
*
Digest with either
Random labeling
msp1 or hpa2 CC*GG
Label with biotin
B)
*
*
*
*
Random primers
Hybridize to array
MspI digestion
Hpa msp
Hpa msp
Hpa msp
Van
Van
Col
Col
Van
Van
Col
Col
Intensity
Random labeling
Hpa msp
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
0.95
3298
92027
3.4%
Chip genotyping of a Recombinant Inbred Line
29kb interval
Map bibb
100 bibb mutant plants
100 wt mutant plants
bibb mapping
Bulk segregant
Mapping using
Chip hybridization
bibb maps to
Chromosome2 near
ASYMETRIC LEAVES1
AS1
ChipMap
BIBB = ASYMETRIC LEAVES1
AS1 (ASYMMETRIC LEAVES1) =
MYB closely related to
PHANTASTICA located at 64cM
bibb
as1
Sequenced AS1 coding region from
bib-1 …found g -> a change that
would introduce a stop codon in the
MYB domain
bib-1
W49*
MYB
as-101
Q107*
bibb
as1-101
Array Mapping
chr1
chr2
chr3
chr4
Hazen et al Plant Physiology (2005)
chr5
eXtreme Array Mapping
Histogram of Kas/Col RILs Red light
6
4
2
0
counts
8
10
12
15 tallest RILs pooled vs
15 shortest RILs pooled
6
8
10
hypocotyl length (mm)
12
14
eXtreme Array Mapping
Allele frequencies
determined by SFP
genotyping. Thresholds
set by simulations
RED2 QTL 12cM
LOD
Chromosome 2
16
12
RED2 QTL
LOD
8
4
0
0
20
40
cM
60
80
100
Composite Interval Mapping
Red light QTL RED2 from 100 Kas/ Col RILs (Wolyn et al Genetics 2004)
eXtreme Array Mapping BurC F2
QTL
Lz x Ler
F2
XAM
Lz x Col
F2
(Werner et al Genetics 2006)
eXtreme Array Fine Mapping
~2Mb ~8cM
Col
Kas
mark1
~268
Col
het
~43
het
~2
Col
het
Kas
het
het
Col
~43
Kas
het
~268
~43
Kas
RED2 QTL
mark2
~539
~2
Col
X
~43
Col
Col
Low
High
>400 SFPs
Kas
Kas
Kas
Select recombinants by PCR >200 from >1250 plants
Potential Deletions
>500 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
Potential Deletions Suggest Candidate Genes
FLM natural deletion
FLOWERING1 QTL
Chr1 (bp)
MAF1
Flowering Time QTL caused by a natural deletion in FLM
(Werner et al PNAS 2005)
Fast Neutron deletions
FKF1 80kb deletion CHR1
Het
cry2 10kb deletion CHR1
Natural Copy Variation on Tiling Arrays
Segregating self seed from wild ME isolate (Early – Late)
Unite Genetic and Physical Map
• Shotgun genomic or 454 reads
• ESTs/ cDNAs/ BAC ends
• 1000s of contigs
• Genotype mapping population on arrays
– Create very high density genetic map
• Known position of genes/contigs allow QTL
candidatet gene identification
– Control hybridization variation for gene expression
Aquilegia (Columbines)
Recent adaptive radiation, 350Mb genome
Genetics of Speciation
along a Hybrid Zone
Aquilegia (Columbine) NSF Genome Complexity
• Microarray floral 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 2007)
Scott Hodges (UCSB)
Elena Kramer (Harvard)
Magnus Nordborg (USC)
Justin Borevitz (U Chicago)
Jeff Tompkins (Clemson)
http://www.plosone.org/
NaturalVariation.org
USC
Magnus Nordborg
Paul Marjoram
Max Planck
Detlef Weigel
Scripps
Sam Hazen
University of Michigan
Sebastian Zoellner
University
University of
of Chicago
Chicago
Xu Zhang
Yan Li
Peter Roycewicz
Evadne Smith
Megan Dunning
Joy Bergelson
Michigan
Michigan State
State
Shinhan Shiu
Purdue
Ivan Baxter
Talking points
• How to clone QTL?
• Why? - ,
• Is it worth it
• Macro evolution vs micro
– Large steps vs gradual small steps