## 3 Materials and Methods  
### Fruit Material  
Eight peach and nectarine varieties, from early to late harvesting dates, were the objects of analysis: \"Filina\" (peach, mid-June), \"Ufo-4\" (flat peach, white flesh, late June), \"Gergana\" (nectarine, late June), \"Laskava\" (peach, early August), \"July Lady\" (peach, mid-July), \"Flat Queen\" (flat peach, white flesh, early September), \"Evmolipya\" (peach, mid-September), and \"Morsiani 90\" (nectarine, late September). All were grown on the same plantation in the 2020 season. \"Filina\", \"Gergana\", \"Laskava\", and \"Evmolipya\" are Bulgarian varieties created at the Fruit Growing Institute, Plovdiv, through interspecific hybridization. \"Flat Queen\", \"Morsiani 90\", \"July Lady\", and \"Ufo-4\" are introduced varieties. No bactericides were applied to plants during testing. The undamaged peach, nectarine, and flat fruit were harvested at full ripeness, corresponding to their size, skin color, firmness and total soluble solids values. Fruit on the trees was considered rip when the growth of the fruit had stopped and the fruit began to soften, exhibited a yellow or orange ground color (which is also specific to each variety), and was easily detached.  
Figure 7: Clustering result of peach varieties shown as a heatmap. The color scale of the heat map ranges from dark brown (value, \(+2\)) to dark blue (value, \(-2\)). The values were normalized by \(\log_{10}\) transformation.  
Headspace-Solid Phase Micro Extraction (HS-SPME) and Gas Chromatography-Mass Spectrometry Analysis (GC-MS)  
For headspace sampling, a 2 cm SPME fiber assembly Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS, Supelco, Bellefonte, PA, USA) was used.  
The HS-SPME technique was used for the extraction of the peach volatile according the procedure described by [PERSON] et al. [60]. The sampling procedure was automatically performed with a G1888 Network Headspace Sampler that was integrated on-line with the corresponding GC-MS system. An Agilent 7890A GC unit coupled to an Agilent 5975C MSD and a DB-5 ms (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m) column were used to analyze the volatile compounds in all investigated samples. The oven temperature program was as follows: from 40 \({}^{\circ}\)C (hold 1 min) to 250 \({}^{\circ}\)C (hold 5 min) at 2 \({}^{\circ}\)C/min; carrier gas: helium with flow rate: 1.0 mL/min; transfer line temperature: 270 \({}^{\circ}\)C; ion source temperature: 200 \({}^{\circ}\)C, EI energy: 70 eV, mass range: 50 to 550 m/z at 1.0 s/decade.  
AMDIS software, version 2.64 (Automated Mass Spectral Deconvolution and Identification System, NIST, Gaithersburg, MD, USA) aided in the reading of the obtained mass spectra and the identification of the metabolites. For identification, the separated compounds were compared to the GC-MS spectra and Kovats retention index (RI) of reference compounds in the Golm Metabolome Database ([[http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/gmd.html](http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/gmd.html)]([http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/gmd.html](http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/gmd.html)), accessed on 25 August 2021) and NIST'08 database (NIST Mass Spectral Database, PC-Version 5.0, 2008 from National Institute of Standards and Technology, Gaithersburg, MD, USA). The 2.64 AMDIS software recorded the RIs of the compounds with a standard n-hydrocarbon calibration mixture (C8-C36, Restek, Teknokroma, Spain).