Sample collection and DNA extraction
Leaf samples from a total of 280 individual mature trees from 28 natural
jarrah populations across the geographic range of the species (Figure
1), including one outlier population (JIL), were collected during 2019
(Table 1). The sampling, which covered a total area of approximately
80,000 km2, included independent (>50 km
separation) and replicate (across similar climate of origin) populations
over both temperature and precipitation gradients to ensure adequate
partitioning of the adaptive and neutral genetic variation. For each
population, mature leaves were collected from ten trees at least 100m
apart from each other. Leaves were immediately stored in silica gel
until freeze-dried using FreeZone 6 Liter Benchtop Freeze Dryer(Labconco Corporation, USA). Samples were stored in silica gel at room
temperature until DNA extraction could be performed. For each sample,
genomic DNA was extracted from 40mg of freeze-dried leaf material. Each
leaf sample was independently ground into fine powder and a modified
CTAB-DNA extraction protocol was employed (Doyle & Doyle, 1990), with
0.1M sodium sulphite (Byrne et al., 2001) and 2% w/v
polyvinylpyrrolidone (MW 40,000) added to the extraction buffer. Quality
of extracted DNA was estimated using gel electrophoresis and quantified
using the Qubit dsDNA BR assay kit on a Qubit fluorometer
(Invitrogen, Carlsbad, CA).