Expression Variation

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Association Mapping of Regulatory Variation in
Arabidopsis thaliana
Andrew Cal Borevitz Group
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Outline

• Genetic Variation
• Maintenance and adaptation to environment
• Cellular molecular phenotype
• Transcription
• Arabidopsis Reference Accessions
• Hap Map
• eQTLs
• Genetic architecture
• Candidate genes, and their regulated pathways

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Geographical/Ecologically Diverse
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Phenotypic Genetic Variation
Flowering time variation controls life history
strategy
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Genetically Diverse

• Short-read sequencing
• (Ossowski et al 2008)
• 0.005 SNPs/non-redundant bp

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Phenotypic Variation by Regulatory Variation
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DNA Microarrays
Quantitative Measure of Transcript
Level Parallel measurement thousands of genes
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Transcriptional Diversity
Vuylsteke et al 2005 Genetics
Zhang et al 2008 Genome Biology
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Genetic Architecture of Transcriptional Variation

• Where are the genetic variants that control
  transcription
• Local vs Distant regulation
• How many loci?
• What is the size of their effects
• Pleiotropy
• Many small mutations, and/or master regulators

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Linkage Mapping
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Expression QTL (eQTL) Mapping

• Map gt1000s Molecular phenotypes
• Where in the genome is transcriptional
  polymorphism??
• West et al. 2007 Genetics

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Association Mapping

• Resolution depends on LD
• Capture common polymorphism

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5707 lines at 139 SNPs
Yan Li
Arabidopsis HAPMAP
Population structure
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Experimental Design
F1 hybrids Gene/exon expression
Association mapping
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Arabidopsis SNP/tiling array
Tiling Array- 1.6M probes,, unbiased
placementSNP probes- 4 probes/SNP, 240,000 SNPs
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Genetic Variation Controlling Transcriptional
Variation

• WHERE are the polymorphisms affecting
  transcription?
• HOW is transcriptional variation inherited?
• Additive
• Dominant
• Over-dominant

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Genetics of Associations
Non-additive dominant
Additive
Over-dominant
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Inheritance Patterns of Gene Expression Variation
Swanson-Wagner et al 2006 PNAS
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Genetic Variation regulating pathways

• WHERE are the polymorphisms affecting
  transcription?
• HOW is transcriptional variation inherited?
• WHAT can we learn about pathway regulation as the
  biological function of master regulatory QTL?

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GPA1
Yvert et al. 2003 Nat Gen
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Data and Results

• 52 F1 lines, 103 parents
• Expression measured on SNP/Tiling array

Association mapping across 217,000 SNPs
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Leaf tissue extracted prior to flowering
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Results

• Single gene- ACD6 (example)
• Characterization of Local regulatory Polymorphism
• cis SNP scan
• Global Transcriptome mapping
• Position, local/distant
• Relative effect size distribution

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Genome-Wide Association Studies
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G -gt C nucleotide R -gt S coding
log(p)
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G/G G/C C/C
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ACD6 Allele Specific Expression
Ratio AB
A
B
Repressive
A B A B A B A B
Promotive
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Local eQTL Positions
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(No Transcript)
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Effect Size
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Results 2

• Inheritance
• Additive
• Dominance (local eQTLs)
• Over-dominance
• mapping
• trans Regulatory HotSpots
• Gene Ontology
• Candidate genes and pathways

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(No Transcript)
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(No Transcript)
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(No Transcript)
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Trans-eQTL Hotspots
Genome Position
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locus
pathway
gene
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Trans SNP 5_4991895_A_G

• GO enrichment Structural constituent of the
  Ribosome, translation
• Associated locally with Amino Acid Permease 6
• High affinity amino acid transporter
• Only AAP transports E
• SOURCE -gt SINK

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SNP 3_19838778_C_T

• GO enrichment defense response, systemic
  acquired resistance, salicylic acid
  biosynthetic process, protein phosph.
• 3 kb from RSZ32- SR Splicing factor
• SNP Associated with altered level of
  RSZ32 3rd exon (p lt 0.004)

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RSZ32

• RSZ32 is has many spliceforms
• Heat induced spliceform change

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Palusa, Ali, and Reddy 2007 Plant J
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Alternate splicing in Defense
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Results 3 (Splicing)

• Splicing index (exon/gene)
• Global patterns
• Local patterns
• Effect size by position
• trans bands
• Hotspot characterization

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Alternative Splicing
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Alternate Splice-form QTL mapping
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Local Splice-form Regulation
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Alternate Splice-form Effect Size
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Alternate Splice-form hotspots
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AS hotspots GO enrichment
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Master Splicing SNP trans (AS-SNP 2_2309060_G_C)

• Gene Set Enrichment (GO terms)
• jasmonic acid biosynthetic process
• jasmonic acid mediated signaling pathway
• response to ethylene stimulus
• induced systemic resistance
• 50 kb from OPR3, essential biosynthesis of JA
• Associated with OPR3 expression (p lt 3x10-5)
• Balancing ODPA and JA response?

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Conclusions

• Regulation acts extremely locally around gene
• Our LD mapping panel need to see this
• Different architectures between expression and
  spliceform regulation
• Our tiling arrays needed to see this
• Effect sizes
• Local vs. trans
• Expression inheritance largely additive
• Over-dominance in trans
• Genomic data identifies candidate genes and
  pathways
• hypothesis generation

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Buffering vs Evolve-ability

• Why dont we confirm more HOT SPOTS?
• Lack of master regulators- Buffered networks?
• Breitling et al. 2008 PLoS Gen
• Shown that pathways related to defense response
• Bergman and Siegal 2003 Nature
• Highly evolvable processes sexual selection and
  pathogen interaction
• GPA1 pheromone hotspot
• Yvert et al. 2003 Nat Gen

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Thank you!!!! Justin Borevitz Committee Borevit
z Lab Bob Haselkorn Xu Zhang
Dave Kovar Jocelyn Malamy Preuss
Lab Plant Group GR training grant greenhouse
staff Kristine Gaston Sue Levison
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• Human study
• gtgt exon/intron enrichment
• -Veyrieras et al. 2008 PLoS Gen
• Intron Size?
• Human 28 kb
• Arabidopsis 0.2 kb
• Unspliced Low

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Jacob and Monod 1961
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NO LACTOSE
Lac Operon
OPERATOR