## 3 Results and Discussion
### Principal Component Analysis and Hierarchical Cluster Analysis  
A Principal Component Analysis was performed using the mean GC-peak area values of individual VOCs, total levels, and the content in sinigrin for each population (Figure 2). The first two components explained together 61.1% of the total variance, with the first component contributing to 43.1% of variation. The first component was positively correlated to the ITCs compounds, where values for component loadings were \(>\)0.2 (Figure 2a). Allyl ITC (I1) grouped together with the total level of ITCs and the total level of VOCs. This result is in agreement with the previous identification of allyl ITC as the main volatile in wall rocket and therefore the greatest contributor to the total added area of VOCs. The first component also had a positive correlation with the content in sinigrin. On the contrary, esters were not grouped by neither the first nor the second component. As exception, _cis_-3-hexenyl isovalerate (E4) and _cis_-3-hexenyl valerate (E5) clustered together; indeed, the Spearman ordinal correlation between both compounds was highly significant and positive (\(\rho\) = 0.945; \(p\) < 0.001). On the other hand, the second component accounted for 18.0% of total variance (Figure 2a). It was also positively correlated to the content in sinigrin. In addition, this component clustered three of the four compounds included in the miscellaneous group. _Cis_-3-hexen-1-ol (O1) was closely grouped with the total sum of this group as it was the greatest contributor. The relationship between individual compounds was also evaluated by means of the Hierarchical Cluster Analysis (Figure 3). Two main clusters were established, C1 and C2, and were powerful in grouping together the esters (cluster C1) and, separately, the ITCs (cluster C2). The compounds of the miscellaneous group were included in the previous groups as described: decanal (O2) and tetradecane (O3) within the cluster C1, and _cis_-3-hexen-1-ol (O1) and \(\beta\)-ionone (O4) as part of the cluster C2. Within the cluster C1, the compounds _cis_-3-hexenyl isovalerate (E4) and _cis_-3-hexenyl valerate (E5) clustered together, as it was observed in the PCA loading plot (Figure 2a, Figure 3). Regarding the cluster C2, here it was included the GSL sinigrin, which mainly grouped with allyl ITC (I1), hexyl ITC (I5), benzyl ITC (I6) and phenylethyl ITC (I7) (Figure 3). Figure 1: Chromatogram of the GLSs profile in wall rocket obtained with UHPLC by the external service. 1: glucoiberin; 2: sinigrin; 3: glucotropaeolin; 4: glucobrassicin; 5: gluconasturtin/methoxyglucobrassicin; 6: neoglucobrassicin. On the other hand, the score plot of the PCA revealed the relationship between the materials (Figure 2b). Most of the populations grouped close in the analysis, although some populations were separated along either the first or second component. Thus, DER045 clearly separated in the first component, placed on the left of the graphic. As previously described, this population had the lowest level in ITCs combined with high levels of esters (Table 2). Its different profile was also observed in  
Figure 3: Hierarchical Cluster Analysis for the identified volatile compounds and content in sinigrin in the 24 populations of wall rocket. Data represent the average value for each population (\(n=3\)). For compound identities refer to Table 3.  
Figure 2: Principal Component Analysis of the 24 populations of wall rocket evaluated in the study, in the first (PC1) and second (PC2) components. (**a**) PCA loading plot for the volatile organic compounds and content in sinigrin. (**b**) PCA score plot for the populations. Data were averaged for each accession. For compound identities refer to Table 3.  
the Hierarchical Cluster Analysis (Figure 3). Similarly, DER068 was also separated within this first component (Figure 2b). This population showed the highest level of esters, but also an increase in the level of ITCs compared to DER045 (Table 2).