## 2 Results
### Sequence-Function Relationships of Isolated Paralogues  
Querying the phased diploid _VviTPS_ models [27] for the _VviTPS10_ paralogues revealed that they are part of a cluster consisting of 21 putative proteins that connect to four functionally characterised proteins (Figure 5C). The amino acid sequence similarity of the active sites to that of the characterised enzymes [7; 19; 20] showed that the proteins in this cluster are predicted to use NPP as the initial substrate, with all, except VviMATPS10, predicted to have an initial 1,6-cyclisation, as predicted by reference [27]. A subsequent analysis of the associated sequences (Figure 5A) showed that these proteins are encoded by a set of genotype-specific duplicated genes. These duplications form highly connected networks, illustrating the extent of paralogy, with the most extensive network connectivity (i.e., greatest number of putative duplications) seen in CS (Figure 5A). Complete gene models associated with the gene duplicates _CSTPS083_ and _CSTPS090_ were the closest paralogues of the functional isolates MA-B, CS-1-B, and CB-B (Figure 4B). Genomic regions where the duplication clusters (Figure 5A) are located were analysed for gene synteny with the differences in the _VviTPS_ landscape of the genotypes, as shown in Figure 6. _VviTPS04_ had synteny to two genes located on distinct CS contigs, namely _CSTPS051_ and _CSTPS083_. _VviTPS10_ shared synteny with _CSTPS051_, while it also showed synteny to _CSTPS083_. _CSTPS083_ and _CSTPS086_ are 0.12-Mb apart with three genes in between, two of which are _VviTPS_ genes, in contrast to _VviTPS04_ and _-10_, which are 0.27-Mb apart, with seventeen genes in between them, seven being _VviTPS_ genes. A single CH gene, _CHTP5067_, had synteny to both _VviTPS04_ and _-10_. The gene synteny analysis was therefore in agreement with the protein sequence homology represented in Figure 5A. _CRTP5019_ and _CRTP5199_ were identified as potential gene duplications in CR, with the former not predicted to encode for a functional gene. These two gene models were, furthermore, located on a primary contig and haplotig, respectively, that, unlike the CH and CS paralogues, map to two different chromosomes (chr. 13 and 18). For this reason, we did not include CR for the synteny analysis. The functional genes PN-B and VI-B had the highest _I_' scores with the _CRTP5019_ gene model (97.31 and 97.08, respectively), while VI-A was linked to _CRTP5199_ with a score of 98.99 (Figure 5B). The _I_' score [38] quantifies the extent of paralogy with phylogenetically similar genes (Figure 4B), linking to distinct proteins based on this score (Figure 5B). This allowed for inference of the protein and gene structures most similar to the isolates. Although five of the isolates were connected to CH gene models, none of these isolates were predicted functional (Figure 3B). A closer inspection of the active site amino acids of derived protein sequences revealed that VI-A and MA-B, (_E_)-\(\beta\)-farmesene synthases, had near-identical active sites, with the only difference to MA-B being an amino acid deletion and a single nonsynonymous mutation, shown in Figure 7. Furthermore, pairwise comparisons of the full-length proteins for VI-A, VI-B, and MA-A revealed that these proteins were more similar between cultivars than within a cultivar (Supplementary File S4). The active sites of CB-B and CS-1-B were very similar to that of VvGwaBer, with only two amino acid differences (Figure 7). The heterologous in planta expression (Figure 4A), however, did not result in the synthesis of any compounds similar to those observed in vitro for VvGwaBer [7]. Figure 4: (**A**) Cumulative EIC using the masses 161, 189, and 204 for _N. benthamiana_ transiently expressing genes of interest. Cultivar-specific genes are shown with peaks identified using authentic standards (STD). Wild-type (WT) and mock infiltrations served as controls.