Magnetic crimp caps

All fruiting progeny were analyzed for volatiles at each harvest except for the RNA-seq combined harvest on December 15, 2011. Five or six fully-ripe fruit (assessed by size and color) per individual were harvested in the morning from the field and stored in a cooler with ice packs until sample processing. Representative ~25 (+/− 3) g samples from each genotype were blended with an equal weight of saturated NaCl (35% NaCl), and the internal volatile standard 3-hexanone was added to a final concentration of 1 ppm. Two 5 ml aliquots from each genotype were aliquoted into 20 ml glass vials and sealed with magnetic crimp caps (Gerstel, Baltimore, MD, USA). Samples were immediately frozen at −80 °C after processing and kept frozen prior to volatile analysis.

All fruiting progeny from the ‘Elyana’ x ‘Mara des Bois’ cross were analyzed for volatiles by GC/MS during the 2010/11 season. Harvest dates were January 20, February 11, February 25, and March 18, 2011. Backcross populations were harvested during the 2012/13 season and were harvested on January 13, January 31, and March 7, 2013. Data from the 2010/11 harvests were used to select genotypes segregating for volatiles of interest. Fruit processing for volatile analysis was conducted as follows. A representative ~25 g sample was collected from five to six fully ripe, clean, and normal-shaped berries from each genotype. The calyx from each berry was removed, and berries were blended with an equal weight of saturated NaCl solution (~35% NaCl in molecular biology grade water). The volatile 3-hexanone was added as an internal standard to a final concentration of 1 ppm prior to blending. Five ml aliquots were dispensed into 20 ml glass vials and sealed with magnetic crimp caps (Gerstel, Baltimore, MD, USA). Two technical replicates were processed for each genotype at each harvest. Samples were frozen at -20°C until analysis by Gas Chromatography/Mass Spectroscopy.

The main metabolite of prilocaine, o-toluidine, was determined in the headspace over arterial blood by means of HS-SPME-GC-MS measurements. Therefore, full blood was withdrawn in Li-Heparin-tubes (S-Monovette^®, 2.7 mL, Lithium-Heparin, Sarstedt AG & Co. KG, Nürnbrecht, Germany), and 1 mL was transferred into 20 mL headspace vials filled with 3 mL phosphate buffer to prevent coagulation. The vials were closed with Teflon-coated rubber septa in combination with magnetic crimp caps (Gerstel GmbH & Co. KG (Mülheim/Ruhr, Germany). Preconcentration was performed by means of Carboxen/polydimethylsiloxane (CAR/PDMS)-SPME-fibres (75 µm, SIGMA, Bellefonte, PA, USA) using a CombiPAL autosampler (CTC analytics AG, Zwingen, Switzerland) with 3 min equilibration time at 42 °C and 7 min adsorption time. For the separation and detection of o-toluidine desorbed from the SPME fibre, a GC-MS system (Agilent 7980A/5975C inert XL MSD, Santa Clara, CA, USA) was used as described before [27 (link)]. Quantification was performed via 5-point calibration in the range of 66.3 to 1060.5 ppbV (0.32–5.07 ug/mL). Two replicates were analysed per concentration level. Relative standard deviation was 4.89% at 530.2 ppbV (n = 4) and calculated value was 99.0% (±4.83%) of spiked value. The limit of detection was determined according to standard practices from the analysis of blank samples (n = 10) (see Table 1).