Metabolite profiles of the flour of 40 wheat varieties grown at three location.

A total of 18 old (year of release 1962-1999) and 22 modern (2005-2014) winter wheat
varieties were used for this study (Suppl. Fig. 1). All varieties, which will be designated in the
following as genotypes, have been cultivated in the season 2015/16 in 5m2 plots in Germany
at o=3 locations. Two locations were located in Baden-Wuerttemberg, namely Hohenheim
(HOH) and Ihinger Hof (IHO), as well as at Gatersleben (GAL) in Saxony-Anhalt. The
experimental design was an incomplete block design with one replication per location. The metabolite analyses of our study were based on the wheat flour samples collected for
the 40 wheat varieties from all three locations. Each of the 120 samples was analyzed one
time via gas chromatography-mass spectrometry (GC-MS) using an adapted protocol from
Lisec et al. (2006). Metabolites were extracted from 45-55 mg dry flour samples with 750 μl
of a 1:2.5:1 H2 O:methanol:chloroform (v:v:v) mixture pre-cooled to -20 ◦ C, then mixed on a
rotator for 10 min and centrifuged at 20,000 g for 2 min (both at 4 ◦C). A total of 50 μl of
the supernatant were dried completely in a vacuum concentrator and derivatized in two steps
via an MPS-Dual-head autosampler (Gerstel): (1) with 10 μl methoxyamine hydrochloride
(Acros organics; freshly prepared at 20 mg/ml in pure pyridine (Sigma-Aldrich)) and shaking
◦at 37 C for 90 min, (2) adding 90 μl N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA;
◦Macherey-Nagel) and shaking at 37 C for 30 min. After incubation for 2 hours at room
temperature, 1 μl of derivatized compounds was injected at a flow of 1 ml/min with an
automatic liner exchange system in conjunction with a cold injection system (Gerstel) in
◦ ◦ ◦splitless mode (ramping from 50 C to 250 C at 12 C/s) into the GC. Chromatography
was performed using a 7890B GC system (Agilent Technologies) with a 30 m long, 0.25
mm internal diameter, HP-5MS column with 5% phenyl methyl siloxane film (Agilent
19091S-433). The oven temperature was held constant at 70◦ C for 2 min and then ramped at
12.5◦C/min to 320◦ C at which it was held constant for 5 min; resulting in a total run time of
27 minutes.