Within-Clade Experiment – What Expression Differences Evolved Between 2 Greywacke Species?

1. 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)

2. F3 F3 F1 F2 F2 F3 F1 F2 F1 cy3 cy5 E3 E2 E3 E1 E3 E1 E2 E2 E1 Within-Clade Experiment – 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: Results: List of differential genes (x of 25,000?) Fine print: = 36 samples = 18 arrays = 4,500NZ$ = 2 sp. x 3 bioreps x 6 techreps (3 red, 3 green) = less replicates if not enough RNA

3. Within-Clade Experiment – 2. Individual Gene Expression • Goal: • Separate phenotypic plasticity from genetic diversity as a cause for inter-specific variation in gene expression • How: • Common garden experiment • e.g. grow descendants of F1, F2, F3, E1, E2, E3 in the glasshouse and measure expression of genes IDed in array experiment using qPCR • (+ add descendants from F4 which would be the hairy P. fastigiata population) • use 3 plants/pop = 7pop x 3plants = 21 seed sources • grow 10 plants from each seed source = 210 plants = 30 plants/pop • grow them all on greywacke if enough greywacke soil left, otherwise use potting mix • plants could be sampled for qPCR analysis at different time points • if seeds are available from more than the 7 sites they could be included Status: = planning stage