1. “Transcriptional Signatures Of Speciation In NZ Alpine Cress” Palmy, Jan 07 Claudia Voelckel

2. (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

3. expression gene of interest reg 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 X cis-X gene of interest reg X trans-X gene of interest regulator X cis/trans -X Expression Studies – To What End?

4. Cy3=Cy5: equally expressed Cy3>Cy5: up in sample 1 Cy3<Cy5: up in sample 2 cy3-labeled cDNA cy5-labeled cDNA sample 1sample 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

5. 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)

6. 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 across Pachycladon 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: cy3cy5 Status: = all 6 slides hybridized, expecting data anytime (HortResearch)

7. P. fastigiataP. 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)

8. 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)

9. Within-Clade Experiment – Sampling F1 F3 F2 E1 E3 E2

10. 1. Global Expression Patterns Goal: 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 cy3cy5 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

11. 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 leaves polycarpic lateral inflorescences schist serrate leaves monocarpic terminal inflorescences greywacke (after Heenan & Mitchell 2003)

12. 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 Jan07e_se_gn_sn_g200 pl/group Jan08e_se_gn_sn_g0 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

13. 2 Designs: AHow 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.Seedling 2. Rosette Hybridization scheme: 24 arrays/harvest e_se_s e_ge_g n_sn_s n_gn_g n_sn_s e_se_s n_gn_g e_ge_g AB cy3cy5 Status: planning stage Across-Clade Experiment – Array Designs

14. Mustard oil measurements (MPI for Chemical Ecology, Jena) Physiological measurements (Lincoln University?) Microclimate measurements (Landcare?) Scanning electron microscopy of trichomes (Massey?) Other ideas/plans

15. 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!