“transcriptional signatures of speciation in nz alpine cress” palmy, jan 07 claudia voelckel
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“Transcriptional Signatures Of Speciation In NZ Alpine Cress”
Palmy, Jan 07
Claudia Voelckel
(after Heenan & Mitchell 2003)
Why Pachycladon?
• recent radiation, species interfertile• morphologically and ecologically diverse• South Island endemic related to Arabidopsis
Genetic basis of speciation
• finding the genes underlying adaptative differences between species• QTL studies (Vaughan), association studies, expression studies, transformation studies, fitness studies, etc.
Introduction
Beringia bursifolia
P. novae-zealandiae
P. wallii
P. radicata
P. sp.
P. fastigiata
P. latisiliqua
P. enysii
P. stellata
P. exilis
P. cheesemanii
greywacke
schist
generalist
expression
gene of interestreg
promoter
Gene expression =• proxy for cis- and trans mutations (genetic differences)• allows speculation about adaptations (phenotypic differences)• can be measured for thousands of genes simultaneously using arrays
gene of interest
reg
Xcis-X
gene of interest
regX
trans-X
gene of interest
regulatorX
cis/trans-X
Expression Studies – To What End?
Cy3=Cy5: equally expressed Cy3>Cy5: up in sample 1Cy3<Cy5: up in sample 2
cy3-labeled cDNA
cy5-labeled cDNA
sample 1 sample 2
(After Quantifoil Tutorial)
gene probes
Array Procedure And Source
Arrays from
25,000 Arabidopsis genes
70mer oligos
2-channel platform
Cy signal proportional to:
A. Amount of mRNA in sample
B. Sequence similarity between gene probe and sample mRNA
2. Within-clade experiment –
What expression differences evolved between two greywacke species?
3. Across-clade experiment –
Is soil adaptation driving schist/greywacke split?
Expression Studies In Pachycladon
Beringia bursifolia
P. novae-zeal. (1,3)
P. wallii
P. radicata
P. sp.
P. fastigiata (1,2)
P. latisiliqua
P. enysii (1,2,3)
P. stellata
P. exilis
P. cheesemanii
greywacke
schist
generalist
1. Do Arabidopsis arrays work with Pachycladon?
(after Heenan & Mitchell 2003)
Do Arabidopsis Arrays Work With Pachycladon?
P1
E1
P2
N1
E2
N2
Goal: Find the spots that a) give decent signals and b) give similar signals acrossPachycladon species
How: Hybridize DNA from 2 populations of P. enysii (P1, P2), P. fastigiata (F1, F2) and P.novae-zealandiae (N1, N2) to slides with ~ 25,000 Arabidopsis probes
Hybridization scheme:
cy3 cy5
Status:= all 6 slides hybridized, expecting data anytime (HortResearch)
P. fastigiata P. enysii
monocarpic perennials with serrate leaves and terminal inflorescences on greywacke
Alpine (1485m) High alpine (1885m) Glabrous Hairy
Within-Clade Experiment – What Expression Differences Evolved Between 2 Greywacke Species?
Beringia bursifolia
P. novae-zeal.
P. wallii
P. radicata
P. sp.
P. fastigiata
P. latisiliqua
P. enysii
P. stellata
P. exilis
P. cheesemanii
greywacke
schist
generalist
(after Heenan & Mitchell 2003)
Within-Clade Experiment – What Expression Differences Evolved Between 2 Greywacke Species?
Goal: Infer from differential gene expression to genetic differences between P. enysii and P. fastigiata
How:
1. Global expression patterns (arrays)Measure inter-specific expression differences in the field using 3 populations of each species (differences may be due to genetic diversity or phenotypic plasticity)
2. Individual gene expression (qPCR)Raise descendants of each field population under similar conditions and test if differential genes from 1 are still different (and thus due to genetic differences) or not (and thus due to phenotypic plasticity)
Within-Clade Experiment – Sampling
F1F2F3
E1E2E3
Pop Location LENZ
F1 Ohau Skifield P1.2cF2 Twin Stream P1.2dF3 Mt. Hodgkinson P1.2dE1 Mt. Potts P1.2dE2 Mt. Hutt P1.2cE3 Broken River P1.2c
F1
F3
F2
E1
E3
E2
1. Global Expression PatternsGoal: Measure inter-specific differences
How:Field experiment – 3 pop/species (F1, F2, F3, E1, E2, E3; 15 plants/pop)
Hybridization scheme: 18 arrays
Within-Clade Experiment – 2 Step Design
F1
E1
F2
E2
F3
E3
F1
E2
F1
E3
F2
E1
F2
E3
F3
E1
F3
E2
cy3 cy5
Status:= samples await RNA extraction
2. Individual Gene Expression Goal: Separate phenotypic plasticity from genetic diversity as a cause for inter-specific differences
How:Common garden experiment e.g. grow 30 descendants of F1, F2, F3, E1, E2, E3 in the glasshouse and measure expression of genes identified in step 1 using qPCR
• Time course?• Different tissues?• Different glasshouse conditions?• Add candidate genes from Arabidopsis?• Fail safe array experiment?
Status:= planning stage
P. novae-zealandiae
P. enysii
Across-Clade Experiment – Is Soil Adaptation Driving Schist/Greywacke Split?
Beringia bursifolia
P. novae-zeal.
P. wallii
P. radicata
P. sp.
P. fastigiata
P. latisiliqua
P. enysii
P. stellata
P. exilis
P. cheesemanii
greywacke
schist
generalist
lobed leavespolycarpic
lateral inflorescences
schist
serrate leavesmonocarpic
terminal inflorescences
greywacke
(after Heenan & Mitchell 2003)
Status:• seeds have germinated in Landcare glasshouse in Lincoln• first harvests (dry mass, expression) and transition from flats to pots planned for early March
Goal: Monitor fitness & expression patterns of a greywacke species (P. enysii) & a schist species (P. novae-zealandiae) on both soils
How:
Across-Clade Experiment – Overview
Jan07 e_s e_g n_s n_g 200 pl/group
Jan08 e_s e_g n_s n_g 0 pl/group
• Measure growth, physiology, secondary metabolism, gene expression, dry mass, male and female fitness over time
• Harvest subsets of each group at different time points for above analyzes
2 Designs: A How do 2 different soils (s,g) influence expression in the same species?B How do 2 different species (e,n) respond to the same soil?
2 Harvests: 1. Seedling2. Rosette
Hybridization scheme: 24 arrays/harvest
e_s
e_g
n_s
n_g
n_s
e_s
n_g
e_g
A B
cy3 cy5
Status:• planning stage
Across-Clade Experiment – Array Designs
• Mustard oil measurements
(MPI for Chemical Ecology, Jena)
• Physiological measurements
(Lincoln University?)
• Microclimate measurements
(Landcare?)
• Scanning electron microscopy of trichomes
(Massey?)
Other ideas/plans
MPI
Ch
em
isc
he
Ökologie
Acknowledgements
Pachycladon folks - Pete, Simon, Vaughan, Trish, Richard…
Landcare botanists – Peter Heenan & Kerry Ford
Array specialist from HortResearch – Bart Janssen
Sponsors – Marsden & Humboldt Foundation
You!
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