Germination and viability assays
Germination rate under standardized conditions was used to quantify seed
dormancy (as in Postma and Ågren 2015). Only lines that had produced at
least 150 seeds were included in germination tests. For seeds produced
in the greenhouse, we monitored changes in seed dormancy over time by
determining the proportion of viable seeds germinating 1, 3, 12, 30 and
54 weeks after harvest. Due to limited seed availability, dormancy of
seeds produced at the two field sites was possible to test only once.
For these seeds, we chose to test germinability 12 weeks after harvest,
because this was the time point at which variation among recombinant
inbred lines derived from a cross between an Italian and a SwedishA. thaliana population was highest in a previous study of
dormancy of seeds produced at the two field sites (Postma and Ågren
2015). In all tests, about 30-100 seeds of each line were sown on 2
filter papers watered with 2 mL of demineralized water in each of three
petri dishes. After sowing, petri dishes were placed in a growth room
and regularly watered for one week (20 °C 16 h light with
photosynthetically active radiation of 150 μmol m-2s-1 and 16 °C 8 h dark). One week after sowing, we
recorded for each line the number of germinated seeds and the total
number of seeds sown.
To quantify germination proportion as number seeds germinating per
viable seed sown, we tested seed viability of lines with a germination
proportion below 95% in a given maternal environment by exposing seeds
to conditions expected to break any seed dormancy. About 50 seeds per
line were sown on agar in petri dishes under sterile conditions. The
petri dishes with sown seeds were kept in darkness at 6 °C for two
weeks, and then moved to randomized positions in a growth room (22 °C 16
h light with photosynthetically active radiation of 150 μmol
m-2 s-1 and 16 °C 8 h dark).
Viability was scored as proportion of seeds that had germinated after
two weeks in the growth room (see Postma & Ågren, 2015). If the
viability of a given line was below 95%, its viability was tested a
second time and the mean viability of the two tests was used in the
analysis. We estimated number of seeds germinating per viable seed using
the expression, number germinated seeds/(number of seeds sown ×
proportion viable seeds), and included only lines that had a mean
viability greater than 95% in the statistical analysis of variation in
seed dormancy. In total, we included in the analysis 214 lines
representing 45 populations grown in the greenhouse, 172 lines
representing 43 populations grown at the Italian site, and 108 lines
representing 28 populations grown at the Swedish site.