“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!