## 3 Discussion  
### Genotype-Specific Structural Variation for Isolated VviTPS Paralogues  
Genotypic variations in VviTPS-encoding genes were suggested to have a significant impact on the genetic potential of a given cultivar [20]. By targeting two paralogues in nine genotypes, the extent of the structural differences and sequence variations became evident (Figures 1 and 2). Previous efforts focussed only on the enzyme function, with only two studies reporting on the isolation of new and/or cultivar-specific _VviTPS_ genes [19; 39]. For example, two point mutations in _VviTPS24_ were linked to an altered function in Shiraz [19]. The use of a single reference genome in an \"applies-to-all\" manner is therefore limiting when trying to explore both sequence and structural variations for VviTPS-encoding genes. It was previously suggested that the genotype-specific landscape of VviTPS-encoding genes is greatly underrepresented in the reference genome when compared to next-generation phased diploid genomes [27]. Comparative analyses of the reference [1; 21] and diploid genomes [24; 25; 26] indicated that genotype-specific SV and/or allelic differences likely resulted in a cluster of _VviTPS_ genes on chr. 18 [27]. The results presented here provided functional validation in multiple genotypes for two paralogues in this cluster, namely _VviTPS04_ and _-10_. Once assembled, the diploid genomes should provide new scaffolds for the mapping and mining of SV and SNVs [33; 37; 40; 41; 42; 43; 44] to comprehensively study the allelic differences that impacted the VviTPS functionality. The WGS data provided valuable insight into the events that shaped the genotypic variations [37; 40]. The generation of consensus sequences using gDNA models predicted from phased diploid genomes is advantageous for studying expanded gene families, especially those involved in specialised metabolism. The surveying of WGS data for 11 different genotypes resulted in 147 consensus sequences with >89% identity to the _VviTPS_ gDNA models predicted from the phased diploid [24; 25; 26] and reference genomes [1; 21]. Although the _VviTPS04_ and _VviTPS10_-like sequences formed a distinct clade (Figure 1), extensive conservation at the terminal ends (Table 1 and Figure 1) presented a challenge when designing primers to isolate these genes. The in silico analysis of the primer-binding regions provided important insight into the terminal sequence conservation of the targeted paralogues. The consensus sequences, furthermore, allowed us to identify a phylogenetic clade that is specific to _VviTPS04_ and _-10_. This reduced the number of gDNA models that could likely encode for these two genes. This greatly increased the available resolution in terms of sequence information, where we previously relied largely on the PN40024 reference genome [1; 21]. This approach can be applied to  
Figure 7: Active site alignment of the six functional isolates to VviMATPS10 and VvGwaBer are shown in (A). The position of the DDxXD and NSE/DTE active site motifs are highlighted in green. The active site similarity is shown in the phylo-genetic tree (B). The following cultivar abbreviations were used: Chenin Blanc (CB), Cabernet Sauvignon (CS), Muscat of Alexandria (MA), Pinot noir (PN), and Viognier (VI). identify and cross-reference _VviTPS_ genes of interest in cultivars for which no genome sequence exists. The subtle impact of SNPs, which can render a gene non-functional or alter its catalytic mechanisms, can therefore be explored with a finer focus. The isolated paralogues had extensive homology at the terminal ends; however, when aligned to the aforementioned gDNA sequences, the isolates showed homology to potentially different genomic regions (Figure 5B). The current lack of assembly for the phased diploid genomes does not allow for definitive chromosome positioning. The advantage of PacBio sequencing does, however, allow for the analysis of large contigs.