# Color Mutations Alter the Biochemical Composition in the San Marzano Tomato Fruit
## 2 Materials and Methods
### Volatile Detection and Quantification  
For volatile analysis, two biological replicates and two technical replicates were processed and analyzed independently for the two year experiments. Prior to the analysis, frozen fruit powder (0.5 g fresh weight) from each sample was weighed in a 15 mL vial, closed and incubated at 37 \({}^{\circ}\)C for 10 min. Then, 1.1 g of CaCl\({}_{2}\)2H\({}_{2}\)O and 500 \(\mu\)L of EDTA 100 mM (pH 7.5) were added; samples were then gently shaken and sonicated for 5 min. Then,1 mL of the homogenized mixture was transferred into a 10 mL screw cap headspace vial, where volatiles were collected by head space solid-phase microextraction as previously described [27]. A 65 \(\mu\)m PDMS/DVB SPME fiber (Supelco Analytical, Bellefonte, PA, USA) was used for all the analysis. Pre-incubation and extraction were performed at 50 \({}^{\circ}\)C for 10 and 20 min respectively. Desorption was performed for 1 min at 250 \({}^{\circ}\)C in splitless mode. Volatile extraction and injection were performed by means of a CombiPAL autosampler (CTC Analytics AG, Zwingen, Switzerland)). Separation and detection were performed by a 6890N gas chromatograph coupled to a 5975B mass spectrometer (Agilent Technologies Inc., Waldbronn, Germany) with DB-5 ms fused silica capillary column (60 m, 0.25 mm, 1 \(\mu\)m) (J&W Scientific, Agilent Technologies Inc., Santa Clara, CA, USA). Over temperature conditions were 40 \({}^{\circ}\)C for 2 min, 5 \({}^{\circ}\)C/min ramp until 250 \({}^{\circ}\)C and then held isothermally at 250 \({}^{\circ}\)C for 5 min. Helium was used as carrier gas  
\begin{table}
\begin{tabular}{c c c c c} \hline
**Class of Material** & **Class of Variation** & **Name** & **Genetic Symbol** & **Fruit Color** \\ \hline Wild-type & - & San Marzano & SM & Red \\ \hline \multirow{6}{*}{
\begin{tabular}{} \end{tabular} } & Chlorophyll & _green flesh_ & _gf_ & Muddy brown \\  & Carotenoids & _yellow flesh_ & \(r\) & Yellow \\  & Flavonoids & _colorless fruit epidermis_ & \(y\) & Pink \\  & Double mutants & _yellow flesh_ + _green flesh_ & _r\_sf_ & Light yellow \\  & - & _colorless fruit epidermis_ + & _y\_gf_ & Wine-coloured \\  & - & _green flesh_ & _y\_gf_ & Green \\  & - & _colorless fruit epidermis_ + & _y\_r_ & Green \\  & & _yellow flesh_ & _y_r_ & \\ \hline \end{tabular}
\end{table}
Table 1: Extended names, genetic symbols and description of the berry color of the seven studied genotypes, including San Marzano (SM), three single mutant and their respective double mutants.  
at 1.2 mL/min constant flow. Ionization was performed by electron impact (ionization energy, 70 eV; source temperature 230 \({}^{\circ}\)C). Data acquisition was performed in scan mode (mass range _m/z_ 35-250; 6.2 scans per second). Chromatograms and spectra were recorded and processed using the Enhanced ChemStation software (Agilent). Untargeted analysis of all the compounds in the chromatogram was performed by means of the MetAlign 3 software [28]. Compounds were unequivocally identified by comparison of both mass spectra and retention time to those of pure standards (SIGMA-Aldrich, St. Louis, MO, USA). For quantification, peak areas of selected specific ions were integrated for each compound and normalized by comparison with the peak area of the same compound in a reference sample injected regularly, in order to correct for variations in detector sensitivity and fiber aging. This reference sample consisted of a homogeneous mixture of all the samples analyzed. Data for each sample were expressed as the relative content of each metabolite compared to those in the SM reference.