“convergence, constraint and the role of gene expression during adaptive radiation:
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“Convergence, constraint and the role of gene expression during adaptive radiation: Floral anthocyanins in Aquilegia ”. JUSTEN B. WHITTALL, CLAUDIA VOELCKEL DAN J. KLIEBENSTEIN, SCOTT A. HODGES Ecology, Evolution & Marine Biology University of California Santa Barbara. A. formosa. - PowerPoint PPT PresentationTRANSCRIPT
“Convergence, constraint and the role of gene expression during adaptive radiation:
Floral anthocyanins in Aquilegia ”
Chicago, July 2006
JUSTEN B. WHITTALL, CLAUDIA VOELCKEL DAN J. KLIEBENSTEIN, SCOTT A. HODGES
Ecology, Evolution & Marine BiologyUniversity of California Santa Barbara
A. formosa A. pubescens
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Bob SkowronBob Skowron
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Aquilegia As An Evolutionary Model System
Why Aquilegia?
Floral & ecological diversity Recent radiation (interfertility) Small genome (350 Mbp, n=7) Basal lineage in the eudicots
Resource Development
EST database (TIGR Gene Index: 17,800 unique sequences)
Microarrays (NimbleGen) for both expression & genotyping studies
SNPs (ca 3500 assays by Sequenom) Physical map (CUGI) Transformation system (Kramer Lab)
to manipulate candidate gene expression ! Genome Project (JGI 2007) !
Introduction Methods & Results Discussion Outlook
(Whittall & Hodges, in prep)
Do similar phenotypes evolve by similar molecular mechanisms?convergent/parallel phenotypes as a consequences of similar selection pressures (e.g. succulence, albinisms)
Rapid phenotypic evolution mediated by changes in regulatory rather than enzyme-coding regions?
Questions Of General Interest…
Loss-of-phenotype mutations more diverse than gain-of-phenotype mutations?
(e.g. insecticide resistance)
floral anthocyanins – phenotypically and moleculary tractable
Aquilegia – multiple losses of floral anthocyanins
Introduction Methods & Results Discussion Outlook
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Floral Anthocyanins (A) : How Does Evolution Repeat Itself?
Which genes are mutated in A-?
Structural versus regulatory mutations?
Degree of convergence across lineages?
Any constraints to the evolution of A-?
Phylogenetic Character Mapping:
6 independent losses of floral anthocyanins (A-)
A+ A- A+/A-
Introduction Methods & Results Discussion Outlook
Coumaroyl CoA + Malonyl CoA
Chalcones
Flavanones
3-OH Flavonols
Leucanthocyanidins
Anthocyanins
Anthocyanidins
The Anthocyanin Biosynthetic Pathway (ABP)
CHS
CHI
F3H
DFR
ANS
UF3GT
6 ABP loci
Experiment:
Monitor expression of these 6 loci in multiple A- species via RT-PCR
Feeding repellents,
UV protectants
…
Hypothesis:
Pleiotropy constrains A-mutations to later stages of the ABP
Floral pigments
Introduction Methods & Results Discussion Outlook
A. canadensis (A+)
12
1 2 3 4 5
Preliminary Study Or Timing Is Everything!
Peak expression mostly in stages 3 & 4 and tissue 2
CHS
ACTIN Control
CHI
F3H
UF3GT
DFR
ANS
degenerate primers for
6 loci
Introduction Methods & Results Discussion Outlook
All loci expressed
Little variation between stages, tissues and individuals
5 stages 2 tissue types3 individuals
highly significant correlation between DFR and ANS (p=0.0001)
ABP Gene Expression In 13 Aquilegia Species
2 – expression like in A+ species
2 main patterns in A- species:
1 – reduced expression in one or more loci
LA CA LO PI CH MI BA CO PU FL FO FP OW
CHS
Actin control
CHI
F3H
UF3GT
DFR
ANS
Species
Pattern 2 A+ 1 1 1 2 2 A+ 1 1 A+ 2 1
Introduction Methods & Results Discussion Outlook
LA CA LO PI CH MI BA CO PU FL FO FP OW
Significant Patterns In A-
Introduction Methods & Results Discussion Outlook
X – expressed
– reduced
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CHS/ACT
CHI/ACT
F3H/ACT
DFR/ACT
ANS/ACT
UGT/ACT
More down-regulated genes in the late part of the ABP (F3H early: p=0.0172, F3H late: p=0.0096)
Pleiotropy
Mutated trans-regulator
Strongly correlated expression of DFR & ANS (p=4.76x10-5)
All genes expressed in A. micrantha lineage Non-functional enzyme(s)
expression
ABP GeneRegA+
2 Models To Explain The A-Phenotype
– e.g. A. formosa
Introduction Methods & Results Discussion Outlook
Ancestral
ABP Gene
ABP Gene
Reg
Reg
X
X
A-
A-
ABP GeneA-
no expression
Common – e.g. A. pubescens
Derived 1
- ABP GeneRegXA
impaired function Rare – e.g. A. micrantha
Derived 2
What Is Known In Other Systems?
Directing metabolic flux into tannin synthesis turns pink tobacco flowers white
Nicotiana tabacum(Xie et al. 2003)
Anthocyanin polymorphisms caused by different alleles in regulatory loci
(Epperson and Clegg 1988, Quattrocchio et al. 1999, Chang et al. 2005)
Petunia
Ipomoea
Gradual degeneration of the ABPStructural mutation followed by loss of expressionIpomoea(Zufall and Rausher 2004)
Introduction Methods & Results Discussion Outlook
The role of regulatory loci in the evolution of the A-phenotype Identify ABP regulators, monitor their expression in A+ & A- species,
search for interspecific polymorphisms
Molecular mechanism for A-phenotype in A. pubescens? Do any of the ABP loci map to a QTL for spur color?
Independent origins, repeated fixing of an ancestral polymorphism or introgression via hybridization?
Compare alleles of ABP loci from A+ & A- species
Establish causal links between genotype and A-phenotype Replace A- alleles with A+ alleles to rescue A+ phenotype via genetic
engineering
Next Steps
Introduction Methods & Results Discussion Outlook
ESTs 85,039
TC sequences 11,985Singleton sequences 5,816Total unique 17,801
Output Sequences
Input Sequences
TIGR Aquilegia Gene Index (Sept 20th, 2005)
A. formosa x pubescens
mixed tissue normalized cDNA
library
High-density oligonucleotide arrays (NimbleGen) for genotyping and expression analysis
Large Scale Array Research in Aquilegia
First array: 20 probes/unigene (17,800x20 = 356000 spots)
Expression in 5 floral whorls of 6 A. formosa individuals (30 samples)
1. sepals 2. petals (spurs) 3. anthers (♂) 4. carpels (♀), 5. staminodia (???)
Within population genetic variation?
2 Goals for my Chicago visit...
Matrix of colored dots
What’s the big picture? ??
Matrix of sample-dependent expression values (x)
1. Make sense of data! 2. See the Art Institute!
• Whorl-specific genes?
• Which floral organs are most similar?
• How does Aquilegia compare to Gerbera or Poppy?
• Intra-specific SFPs?
Georges Seurat 1884
samples
probes 1 2 3 4 5 ... 301 X X X X X ... ...2 X X X X X ... ...3 X X X X X ... ...4 X X X X X ... ...5 X X X X X ... ...
... ... ... ... ... ... ... ...
350,000 ... ... ... ... ... ... ...
Acknowledgements
Justen B. Whittall
Daniel A. Kliebenstein
Scott HodgesJustin BorevitzElena Kramer
Magnus NordborgJeff Tomkins
NSF (EF-0412727)
Thank you for your attention!
LA CA LO PI CH MI BA CO PU FL FO FP OW
Significant Patterns In A-
X – expressed – reduced
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CHS/ACT
CHI/ACT
F3H/ACT
DFR/ACT
ANS/ACT
UGT/ACT
More down-regulated genes in the late part of the ABP (F3H early: p=0.0172, F3H late: p=0.0096)
Pleiotropy
Mutated trans-regulator
Strongly correlated expression of DFR & ANS (p=4.76x10-5)
All genes expressed in A. micrantha lineage
Non-functional enzyme(s)
expression
ABP GeneRegA+
2 Models To Explain The A-Phenotype
– e.g. A. formosa
Introduction Methods & Results Discussion Outlook
ANCESTRAL
ABP Gene
ABP Gene
Reg
Reg
X
X
A-
A-
ABP GeneA-
no expression
Common – e.g. A. pubescens
DERIVED 1
- ABP GeneRegXA
impaired function Rare – e.g. A. micrantha
mutationX
DERIVED 2
The role of regulatory loci in the evolution of the A-phenotype
Identify ABP regulators, monitor their expression in A+ & A- species, search for interspecific polymorphisms
Molecular mechanism for A-phenotype in A. pubescens?
Do any of the ABP loci map to a QTL for spur color?
Independent origins, repeated fixing of an ancestral polymorphism or introgression via hybridization?
Compare alleles of ABP loci from A+ & A- species
Establish causal links between genotype and A-phenotype
Replace A- alleles with A+ alleles to rescue A+ phenotype via genetic engineering
Next Steps
Introduction Methods & Results Discussion Outlook
ABP Gene Expression In 13 Aquilegia Species
2 – expression like in A+ species
2 main patterns in A- species:
1 – reduced expression in one or more loci
Actin control
Pattern 2 A+ 1 1 1 2 2 A+ 1 1 A+ 2 1
LA CA LO PI CH MI BA CO PU FL FO FP OW
CHS
CHI
F3H
UF3GT
DFR
ANS
Species