## 5 Materials and Methods
### Isolation and Cloning of VviTPS04 and -10 Paralogues  
Primers were designed to target putative paralogues: forward primer A (_VviTPS10_ paralogues): \(\mathrm{5^{\prime}}\)-ATGGCCTTAAATTCTCCTACCAGCAACGGG-\(\mathrm{3^{\prime}}\), forward primer B (_VviTPS04_ paralogues): \(\mathrm{5^{\prime}}\)-ATGTTGAGGTCTCGGAGTGAGTTTACACATGGCC-\(\mathrm{3^{\prime}}\)), and a single conserved reverse primer: \(\mathrm{5^{\prime}}\)-TCATATTGGCACAGGGCTATAGCAATATAT-\(\mathrm{3^{\prime}}\).  
RNA extractions from 100-mg flower tissue of nine _V. vinifera_ genotypes were performed using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) with an on-column DNase I (Thermo Fisher Scientific, Waltham, MA, USA) treatment. RNA was checked for genomic DNA (gDNA) contamination through PCR, followed by reverse strand synthesis (cDNA) using the SuperScript VILO Master Mix (Thermo Fisher Scientific, USA). Genomic DNA (gDNA) was isolated from the same genotypes according to the method described by reference [68]. RNA extractions from 100-mg flower tissue of nine _V. vinifera_ genotypes were performed using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) with an on-column DNase I (Thermo Fisher Scientific, Waltham, MA, USA) treatment. RNA was checked for genomic DNA (gDNA) contamination through PCR, followed by reverse strand synthesis (cDNA) using the SuperScript VILO Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA (gDNA) was isolated from the same genotypes according to the method described by reference [68]. Flower genotypes were collected at stage 17/18 according to the modified Eichhorn and Lorenz (EL) growth classification system [69] for the following cultivars: Chardonnay (CH), Chenin Blanc (CB), Muscat of Alexandria (MA), Pinot nor (PN), Pinotage (PI), Sauvignon Blanc (SB), Shiraz (SH), Viognier (VG), and Weisser Riesling (WR). Samples were a composite of six to eight flowers clusters that were frozen in liquid nitrogen, with the rachis structures separated before homogenisation. All genotypes were planted in close geographical proximity at a mother block in the Stellenbosch area (\(\mathrm{33^{\circ}5^{\prime}73.50^{\circ}}\) S, \(\mathrm{18^{\circ}51^{\prime}38.09^{\circ}}\) E), South Africa.  
PCR reactions using cDNA or gDNA as the template was performed using TaKaRa ExTaq proofreading polymerase, as per the product specifications (Separations, Roode-poor, South Africa). PCR reaction conditions were as follows: 2 min initial denaturation, followed by 35 cycles of 30 sec denaturation, 30 s annealing, 2 min of 30-s extension, and a final extension of 7 min. cDNA amplicons were purified after electrophoretic separation using the Zymoclean Gel DNA Recovery Kit (Inquad Biotech, Pretoria, South Africa), TA-cloned, screened for positives through colony PCR, and the plasmids isolated from overnight cultures. Plasmid DNA was diluted to 1/50 and used as a template for two-step Gateway PCR with the primers modified accordingly to build in _attB_ sites, as described in the Gateway Technology Manual (Thermo Fisher Scientific, Waltham, MA, USA). Gateway cloning was performed according to the aforementioned manual using the appropriate clonases to generate entry clones with pDONR-Zeocin (Thermo Fisher Scientific, Waltham, MA, USA) and expression clones with pEAQ-HT-DEST1 [70,71]. The respective clones were transformed into chemically competent DH5\(\alpha\)_Escherichia coli_ using appropriate antibiotics and screened for positives through colony PCR. Entry clones were bidirectionally sequenced according to the standard methods of the Central Analytical Facility, Stellenbosch University, South Africa using M13 sequencing primers and a walking primer (\(\mathrm{5^{\prime}}\)-CTCTTCTTGTGTGTATGATTTTC-\(\mathrm{3^{\prime}}\)).