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![Page 1: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/1.jpg)
Ecological and Evolutionary Systems biology:Conceptual and molecular tools for analysis
Justin BorevitzEcology & Evolution
University of Chicagohttp://naturalvariation.org/
Ecological and Evolutionary Systems biology:Conceptual and molecular tools for analysis
Justin BorevitzEcology & Evolution
University of Chicagohttp://naturalvariation.org/
Deer mouse burrow
Finch (large beaked)Sitting in a cotton wood Hemicordate
Sea anemoneStickleback
Aquilegia, Arabidopsis, Mimulus? Indiana Dunes National Lakeshore
![Page 2: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/2.jpg)
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
![Page 3: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/3.jpg)
Developing Model Organisms - Tools
• High throughput Phenotyping– Physiological dissection of 1000s correlated traits– Biological Variation
• Multi species ecological interactions– “extended phenotype”
• Environmental Interaction (GxE)– Local adaptation
• Epistasis (GxG)– Magnify minor QTL in local backgrounds
• Multiple genes under major QTL– High Density markers – Linkage Drag
Functional Genomics•Microarrays •SNPs•Induced deletions
![Page 4: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/4.jpg)
Talk OutlineTalk Outline• Genetic Diversity
– Population structure, Haplotype Mapping set
• Phenotyping in multiple environments– Seasonal Variation in the Lab
• SNP/Tiling microarrays– Splicing
– Methylation
– Very High Density Markers SFPs
– Bulk Mapping
– Deletions
• Genetic Diversity– Population structure, Haplotype Mapping set
• Phenotyping in multiple environments– Seasonal Variation in the Lab
• SNP/Tiling microarrays– Splicing
– Methylation
– Very High Density Markers SFPs
– Bulk Mapping
– Deletions
![Page 5: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/5.jpg)
Global and Local Population Structure
Olivier Loudet
![Page 6: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/6.jpg)
Local adaptation
under strong
selection
![Page 7: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/7.jpg)
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Seasonal Variation
Matt Horton
Megan Dunning
![Page 9: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/9.jpg)
144 Non singleton SNPs >2000 accessions
Global, Midwest, and UK
common haplotypes
Local Population Structure
Megan Dunning, Yan Li
![Page 10: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/10.jpg)
80 Major Haplotypes
Diversity within and between populations
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17 Major Haplotypes
80 Major Haplotypes
Diversity within and between populations
![Page 12: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/12.jpg)
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Variation within a field http://naturalvariation.org/hapmapVariation within a field http://naturalvariation.org/hapmap
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Begin with regions spanning the Native Geographic range
Nordborg et al PLoS Biology 2005Li et al PLoS ONE 2007
Tossa Del MarSpain
LundSweden
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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
GenevaScientific/ 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
1400
sep
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
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Kurt Spokas
Version 2.0a June 2006
USDA-ARS Website Midwest Area (Morris,MN)http://www.ars.usda.gov/mwa/ncscrl
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Flowering time QTL, Kas/Col RILsFlowering time QTL, Kas/Col RILs
![Page 19: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/19.jpg)
Genomic Breeding Path
QTL geneConfirmation
MarkerIdentificationGenotyping
Genomics path
Experimental DesignMapping population PhenotypingQTL AnalysisFine Mapping
Candidate genePolymorphismsgene expressionloss of function
QTL gene
Confirmation
Experimental Design
Mapping population
Phenotyping
QTL Analysis
Fine Mapping
Borevitz and Chory, COPB 2003
![Page 20: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/20.jpg)
Which arrays should be used?
cDNA array
Long oligo array
BAC array
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Which arrays should be used?
Gene array
Exon array
Tiling array35bp tile, 25mers 10bp gaps
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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,
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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
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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
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AT1G07350
AT1G29120
AT1G51350
AT1G53560
AT1G76170
D E
Additive, Dominant, Maternal, Genotype Variation
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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
![Page 27: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/27.jpg)
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 Specific
Expression
Zhang, X., Richards, E., Borevitz, J. Current Opinion in Plant Biology (2007)
65,000 SNPsTranscribedAccession Pairs12,000 genes
>= 1 SNP6,000 >= 2 SNPs
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Potential Deletions
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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
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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
![Page 31: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/31.jpg)
Chip genotyping of a Recombinant Inbred Line
29kb interval
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Map bibb100 bibb mutant plants100 wt mutant plants
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bibb mapping
ChipMapAS1
Bulk segregantMapping usingChip hybridization
bibb maps toChromosome2 near ASYMETRIC LEAVES1
![Page 34: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/34.jpg)
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
![Page 35: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/35.jpg)
Array Mapping
Hazen et al Plant Physiology (2005)
chr1 chr2 chr3 chr4 chr5
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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
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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)
![Page 38: Ecological and Evolutionary Systems biology: Conceptual and molecular tools for analysis Justin Borevitz Ecology & Evolution University of Chicago](https://reader035.fdocuments.net/reader035/viewer/2022062517/56649f265503460f94c3cea2/html5/thumbnails/38.jpg)
eXtreme Array Mapping BurC F2
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XAMLz x Col
F2
QTLLz x Ler
F2
(Werner et al Genetics 2006)
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XRED2 QTL
mark1 mark2
Select recombinants by PCR >200 from >1250 plants
HighLow~2Mb ~8cM
>400 SFPsCol
Kas
Col Col
Col het
Col
~2
Kas
het Col
het het
het
~43
Kas
Kas Col
Kas het
Kas
~268
~43 ~539 ~43
~268 ~43 ~2
Kas
eXtreme Array Fine Mapping
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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
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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)
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Fast Neutron deletions
FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1
Het
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Natural Copy Variation on Tiling Arrays
Segregating self seed from wild ME isolate (Early – Late)
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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
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Aquilegia (Columbines)
Recent adaptive radiation, 350Mb genome
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Genetics of Speciationalong a Hybrid Zone
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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)
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http://www.plosone.org/
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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
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Talking points
• How to clone QTL?
• Why? - ,
• Is it worth it
• Macro evolution vs micro– Large steps vs gradual small steps