## 4 Discussion
### Metabolite Profiles Successfully Discriminate Jalapeno and Serrano Type Chile Peppers  
Though crop metabolite profiling is an important area of nutritional and health research, this information can also be used for the classification of peppers. Principal components and partial least discriminant analyses are relevant for the classification of metabolomic datasets based on different factors such as variety, location, and their interactions [32]. In the current study, the jalapeno and serrano type chile peppers were  
Figure 6: Rich factor (RF) values for the differentially expressed metabolites with annotated functions on the KEGG database. RF is the ratio of the number of differentially expressed metabolites in a pathway to the total number of metabolites annotated in the same pathway (RF for a pathway = no. of differentially expressed metabolites/total number of metabolites). Terpenoid backbone biosynthesis had significant enrichments, with an RF value of 1.  
differentiated based on their metabolite profiles. Both PCA and OPLS-DA showed consistent clustering based on cultivars, with total variation explained by the first two PCs reaching a total of 63%, indicating that the cultivars were significantly different from each other. The values for \(Q^{2}\), \(R^{2}X\), and \(R^{2}Y\) derived from performing OPLS-DA further support the good predictive ability and reproducibility of the model in classifying the cultivars based on metabolomic information. Correlation analyses within sample treatments were significant (\(p<0.0001\)), demonstrating clustering based on the cultivars. Genomic information using 66,650 single nucleotide polymorphism markers, nonetheless, previously classified 'NuMex LotaLutein' and 'NuMex Pumpkin Spice' in the same cluster [33], indicating high genetic relatedness; yet, implementing a metabolomic approach can resolve differences between the cultivars.  
Metabolite profiles have been previously used for species differentiation and cultivar identification in Chile peppers. Capsaicinoid and flavonoid contents, for example, were recently used as biochemical markers to distinguish Jize from Korean chile peppers [34]. In another study, an untargeted metabolomics approach identified biomarkers, including flavonoids, steroids, terpenoids, anthocyanin, and carboxylic acids, and characterized three _Capsicum_ species (_C. bucactum_, _C. chinense_, and _C. frutscens_) from a collection of native South American chile peppers [35]. Eleven Mexican _C. annuum_ cultivars were differentiated based on their metabolomic fingerprints, which consisted of sugars, amino acids, organic acids, polyphenic acids, and alcohols [36]. Serrano peppers from Mexico were grouped in two different clusters based on their metabolite profiles, which included organic acids, sugars, amino acids, and polyphenols [37]. In contrast, using a single metabolite (i.e., capsaicin) alone did not successfully discriminate 'Superhot' chile peppers cultivated across multi-year trials in New Mexico [38]. Taken together, our results and those of previous studies indicate that a'macroscale' metabolomic profiling would be more advantageous than using only a single metabolite in cultivar identification and species differentiation in _Capsicum_.