S2: Additional information on date of sowing/day length, climate conditions, plot size
and statistical design of individual experiment is provided
Flowering time measurements
(i) Natural variation for flowering time under field conditions
Flowering times were evaluated under birdcage conditions over two years (2010 and 2011) at
the WWAI research farm located at Wagga Wagga, NSW (59°N, 90 m above sea level). In
three different experiments, we used a range of genotypes across growing environments
depending upon the availability of seed while keeping the majority of the core set of 188
genotypes constant.
In the first experiment (DTF-10(P), 188 diverse genotypes were arranged in 4 rows by
94 columns on a bench in replicates of 2 rows by 94 columns in 1 row by 8 column trays.
Each column of a tray held seven seeds from each genotype. The experiment design software
DiGGer (Coombes, 2002) was used to randomise genotypes to tray blocks and spatially
rearrange genotypes within trays. Seedlings were raised in plastic trays (7 × 8 well) under
glasshouse conditions maintained at 18±1°C as described previously (Raman et al., 2013).
Seeds were sown on 21 June 2010. After 30 days from sowing, 5 seedlings per genotype were
transplanted (evenly spaced) into plastic pots and moved to a ‘birdcage’ under the natural
conditions maintaining their spatial arrangement. Plants were watered daily fertilised weekly
using in-line liquid fertilizers and sprayed with fungicides as necessary (Raman et al., 2013).
Days to flower were recorded when 40% of plants showed their first flower from the day of
sowing.
We have previously described two trials (namely SHT195 field experiment and GD
200 experiment) to determine the extent of genetic variation in pod shatter resistance in a set
of 200 genotypes (188 test genotypes plus 12 controls) (Raman et al., 2014). Both trials were
scored for genetic variation in flowering time in order to estimate G × E interactions across
years. Flowering time was evaluated as described above. SHT195 (2010) and GD200 (2011)
experiments are labelled as DTF-10 (P) and DTF-11 (F), respectively in this study. For DTF10 (P), accessions were sown at the WWAI research farm located at Wagga Wagga on 20
May 2010. DTF-11(F) trial was sown on 8 April 2011 in single rows (1 meter length) spaced
0.5 m apart at the rate of 30 seeds/row.
(ii) Natural variation for flowering time and vernalisation response under glasshouse
conditions
Seeds of 188 lines were sown in plastic trays (7 × 8 well) as described above. The tworeplicate design for 188 genotypes and vernalisation was set-out in plastic trays on benches in
a glasshouse. Each tray held a row of seven genotypes, with each genotype a column of 8
plants. Trays were paired (placed side by side), with and without vernalisation treatment, with
genotype main plot columns across two trays in a strip-strip plot arrangement. Vernalisation
treatments were randomised to trays, and genotypes randomised within pairs of trays. For
vernalisation treatment, seeds were planted in a controlled environment cabinet (CE)
maintained at 18±1°C for two weeks. Seedlings were then moved to the cool room at 4±1°C
and subsequently grown under fluorescent bulbs (4000 K, Osram) with light intensity of
approximately 150µM/m2/s, with a 16-h photoperiod for 9 weeks. Two sowings for the nonvernalisation treatments (control) were done in two growth cabinets as described above. The
plants which synchronised to the same developmental stage as vernalised plants after 9 wks
of vernalisation treatment were selected. Both vernalised and non-vernalised plants were
grown in the CEs for 2 wks before transferring them in pots under glasshouse conditions.
Flowering for 188 lines was recorded for up to 180 days. Plants without any flower bud at the
end of the experiments were assigned as value to 180 days. The response to vernalisation was
the calculated as the difference between 50% flowering in vernalised and non-vernalised
plants as described previously (Raman et al., 2013).
(iii) Natural variation for flowering time under controlled environment conditions
All the 188 diverse lines were raised in plastic trays without transplanting to pots in CE
maintained at 18±1°C, and illuminated with cool white fluorescent irradiance of 150µM/m2/s
with 16-h photoperiod. Two partially replicated experiments were conducted with 64
genotypes duplicated and 124 genotypes unreplicated. In the first experiment, plants were not
vernalised (control), while in the second experiment; plants were vernalised for 9 weeks at 4
± 1°C. Plants were illuminated with cool white fluorescent tubes with an irradiance of
150µmol/s-2, with a 16-h photoperiod. Each experiment was set out on two benches in a 4
row by 63 column arrangement, with 1 row by 7 column trays of genotypes. Each genotype
experimental unit was a column of 8 plants. Days to first flower were calculated for each
genotype across treatments and measured as the number of days from the end of vernalisation
to the appearance of first flower on 50% of plants. The response to vernalisation was
calculated as described previously (Raman et al., 2013). Genotypes were treated as random in
the analysis.
Rainfall and temperatures during the growing season (Wagga Wagga, NSW, Australia)
in 2010 and 2011. Between both years, there was no difference in daylength.
Rainfall and Temperature 2010 and 2011
180
160
140
120
Date of sowings
mm, oC
100
80
60
40
20
0
Rainfall
2010
Rainfall
2011
Max Temp
2010
Max temp
2011
Average Daylength 2010 and 2011
16:48:00
14:24:00
12:00:00
09:36:00
07:12:00
Average Daylength 2010
Average Daylength 2011
04:48:00
02:24:00
00:00:00