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Methods: Metabolite extraction for mass specrometry, adapted from (Davey et al. 2008)
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During biomass harvesting a sample of taproot was dissected, wrapped in foil and flash
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frozen in liquid nitrogen. The samples were kept at -80oC. Before metabolite extraction each
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sample was weighed accurately to allow biomass extract concentration correction. 1 ml of
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chilled solvent (MeOH– CHCL3–H2O, 5: 2: 2) was added to the biomass and left for 30
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minutes. The tubes were then vortexed and centrifuged for 3 min, 12000rpm at 0°C. The
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supernatant was removed, added to another tube and put on ice. The pellet was re-extracted
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with 1 ml of solvent (MeOH–CHCL3, 1: 1) and again centrifuged. The second supernatant
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was then added to the first. Addition of 1ml of reverse osmosis H2O separated the organic
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(CHCl3) phase from the aqueous (MeOH–H2O) phase. The aqueous phase was then removed
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from the tube and centrifuged again. The top 1ml of the aqueous phase extraction was
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pipetted into a clean vial and kept at -20oC before mass spectrometry.
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Prior to mass spectrometry analysis the extracts were dilution corrected to a 100 fold
dilution based on 0.1g initial biomass in a solvent (MeOH-H20-HCO2H, 50:49.999:0.001).
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Pheyl a-D
Glucopyranoside
SUCROSE
RAFFINOSE
STACHYOSE
VERBASCOSE
Time
[min]
Name
Standards
C. vulgaris
C. palustre
C. vulgare
V. blattaria
A. minus
S. jacobaea
D. purpurea
C. nutans
V. thapsus
20.74
20.74
20.75
20.74
20.74
20.73
20.73
20.73
20.72
20.74
Area
[µV·s]
84050
91165
110687
88702
105681
65022
51082
93288
94089
96467
Time
[min]
26.31
26.27
26.27
26.26
26.26
26.25
26.26
26.26
26.25
26.25
Area
[µV·s]
3198326
56017
193751
399282
461576
69593
292589
112368
609766
28726
Time
[min]
36.34
36.43
36.44
36.43
36.29
36.42
36.42
36.29
36.41
36.27
Area
[µV·s]
2980188
5826
35612
31480
201802
50007
169002
11272
320274
5769
Time
[min]
43.04
43.03
43.04
0
43.03
43.02
43.02
43.02
43.01
43.01
Area
[µV·s]
1896225
41086
3658
0
841378
38259
5851
6623
3637
13872
Time
[min]
Area
[µV·s]
47.26
0
0
0
47.25
47.24
47.24
47.24
47.22
0
1
2
3
4
Supplementary Table 1
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This table shows the prevalence of the compounds sucrose, raffinose, stachyose and
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verbascose in nine species in a sample root extraction by using GC analysis. The samples
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were chosen at random. The ‘Time’ columns indicate the elution time of compounds off the
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zebron inferno 2B-5HT column in each sample. The area column represents the area of the
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compound peak.
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11
12
13
14
15
16
17
(a)
407457
0
0
0
474107
93277
37483
19940
7508
0
1
2
(b)
3
4
Supplementary Figure 1 (a), (b) A principal component analysis (PC1/PC2) and loading
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scatter plot including plants harvested in H5.
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Principal Component 1: 27%
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Principal Component 2: 15%
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R2X= 61%
2
R2Y=28%
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23
24
1
(a)
2
3
(b)
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1
Supplementary Figure 2 (a), (b). MS: MS profiles of a) A proline standard and b) A C.
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vulgare root extraction.
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Both figures show the targeted ion peak at m/z=116.1 and the resulting product ions after
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fragmentation.
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23
24
1
(a)
2
3
(b)
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Supplementary Figure 3 (a), (b). Score and loadings scatter plots of Fig. 2a,b.
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Scatter plots are often more accessible and easier to interpret than column plots. However,
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please note that caution must be taken when interpreting OPLS scatter plots, as the Y-axis
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shows an orthogonal non-predictive component. The x-axis shows the predictive component.
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Please refer to Figure 2(a) and (b) legends for further details.
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1
1
Supplementary Figure 4: The relationship between species-average growth rate and relative
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sucrose concentration in the taproot.
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The data is made up of mass spectrometer counts of total sugars in the extraction of 0.1g root
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material. Open circles represent raw data and each line represents the model fit through the
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data. Two data points in H4 were an order of magnitude larger than the rest of the (both
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above 23 on the y-axis) data and were removed from the graph; one data point was from the
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fastest growth rate and the one from the cluster of slow growth rates. However, these data
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were included in the ANOVA in Table 2.
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