## 3 Results
### Multivariate Analysis
Blue color: Japanese chestnut (“Ginyose”, \"Ishizuki”, \"Ishizuki Preocce\", \"Ishizuki Tardiva”, \"Tsukuba”), green color: sweet chestnuts (\"Contessa\", \"Gabiana\", \"Garrone Rosso\", \"Ruiana\", \"Savoye\"); fuchsia color. “marrone-type” chestnuts (“Marron Comballe”, \"Marron de Lyon\", \"Marron de Redon\", \"Marron de Chiusa Pesio\", \"Marrone della Val di Susa”); grey color: Euro-Japanese hybrids (“Bouche de Betizac”, \"Colossal”, \"Maridone”, \"Marigoule\", \"Marissard”). showed a correlation with the content of cinnamic acids, vitamin C, tannins, and catechins, whereas PC2 was related to flavonols, monoterenes, and benzoic acids. In the loading plot (Figure 11), the phytochemical classes are displayed as vectors in the PCA plane. Based on the obtained score plot (Figure 10), the different _Castanae_ species were distributed along the horizontal axis (PC1): C. _crenata_ cultivars were on the left of the plot, the European chestnuts (\"chestnut-type\" and \"marrone-type\") on the right, and Euro-Japanese hybrids in the middle. This distribution is associated with the content of vitamin C (increasing to the right) and the composition of cinnamic acids and tannins. The cultivars belonging to the same species were vertically distributed (PC2); therefore, variables related to PC2 were more discriminant for intraspecific characterization, especially benzoic acids and flavonoids for the group of \"marrone-type\" cultivars. The spatial placement suggested that Euro-Japanese hybrids were more similar to the European chestnuts than to the Japanese chestnuts in terms of chemical profile. Since the aim of the breeding work was to obtain genotypes with improved disease tolerance, maintaining nut quality comparable to that of the European chestnuts [58], this result seems to be a validation of this purpose. Furthermore, the PCA plots provided information about specific biomarkers for each group: \"marrone-type\" cultivars and Euro-Japanese hybrids presented higher sugar content, Japanese cultivars were mainly characterized by catechins content, and European sweet chestnuts were mainly characterized by monoterenes content. Some unknown or unsafe cultivars, always collected in the germplasm repository of the Chestnut R&D Center, were considered and then added to the model to test its effectiveness; the results of morphological and genetic analysis confirmed the genotype reported by the placement in the score plot. For example, the sample O14, defined as \"Ishizuki Precoce\" cv, was placed on the top right in the PCA score plot, not in the _C. crentata_ group, even if amounts of some bioactive compounds were consistent with the chemical profile of the other _C. crentata_ cultivars. This result confirmed that the evaluation of single markers is not enough to distinguish cultivars with an unknown origin, but it is necessary for a multivariate evaluation. Further genetic analysis proved that this sample did not match with the \"Ishizuki Precoce\" cv, supporting the efficacy of the statistical  
Figure 11: PCA loading plot of considered variables. multivariate model. Moreover, unsafe samples B4 and T11, incorrectly reported as _C. sativa_ \"chestnut-type\" cultivars in the list of Chestnut R&D Center, were included in the Euro-Japanese group by the PCA model. This result was confirmed by the morphological and genetic analysis. Finally, sample O2 (described as _C. sativa_ \"chestnut-type\" in the list) and samples V7, W6, and X3 (reported as _C. sativa_ \"marrone-type\" in the list) were confirmed in their original description both by the PCA model and morpho-genetic analysis. In any case, further unknown or unsafe cultivars will be tested in the future to confirm these preliminary results.