In silico and quantitative reverse transcription–PCR analysis

Two putative biosynthetic genes for SL, namely the Carotenoid Cleavage Dioxygenases (CCD) 7 and 8, were identified by BLAST searching the grapevine ‘PN40024’ 12X genome draft, V1 annotation, at the Grape Genome Database (http://genomes.cribi.unipd.it/grape/) with the Arabidopsis sequences.

Concentration changes of target transcripts were quantified on powdered berry skin samples (1.5 g) by quantitative reverse transcription–PCR (RT–qPCR). Total RNA was extracted following a cetyltrimethylammonium bromide (CTAB)-based protocol (Carra et al., 2007). RNA integrity and quantity were checked using a 2100 Bioanalyzer (Agilent Technologies). RNA samples were treated with RNase-free DNase I (Fermentas: 50 U µl⁻¹ UAB, Vilnius, Lithuania) to avoid any risk of genomic DNA contamination, and first-strand cDNA was synthesized starting from 5 µg of total RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. cDNA integrity and primer specificity were then checked by gradient PCR and agarose gel electrophoresis. RT–qPCR was conducted in triplicate using a StepOnePlus™ System (Applied Biosystems), and the SYBR Green method (Power SYBR^® Green PCR Master Mix, Applied Biosystems) was used for quantifying amplification results (Giordano et al., 2016; Pagliarani et al., 2017). Each reaction contained 1 μl of 5 μM primer mix, 100 ng of template cDNA, 5 μl of 2× SYBR Green mix, and 3 μl of diethylpyrocarbonate (DEPC)-treated water for a total reaction volume of 10 μl. Thermal cycling conditions were as follows: 95 °C for 10 min before the beginning of the amplification (holding stage), followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Specific annealing of primers was further checked on dissociation kinetics at the end of each RT–qPCR run. Expression of target transcripts was quantified after normalization to the geometric mean of the Ubiquitin (VvUBI) and Actin (VvACT1) transcripts used as endogenous controls. Expression changes were analysed for VvMybA1 (encoding a Myb transcription factor controlling anthocyanin biosynthesis in grapevine: Walker et al., 2007), VvUFGT (terminal gene of anthocyanin biosynthesis in grapevine, encoding UDP-glucose:flavonoid 3-O-glucosyltransferase: Ford et al., 1998), VvNCED1 (rate-limiting gene of ABA biosynthesis, encoding 9-cis-epoxycarotenoid dioxygenase: Wheeler et al., 2009), two genes encoding ABA 8'-hydroxylases (VvHYD1 and VvHYD2; Speirs et al., 2013), an ABA-UDPG glycosyl transferase (VvGT1; Sun et al., 2015), a β-glucosidase that hydrolyses ABA-glucose ester (VvBG1; Sun et al., 2015), and the grapevine orthologues of the Arabidopsis ABC Transporter G Family Protein (ABCG) ABA membrane transporters VvABCG25 (Kuromori et al., 2010) and VvABCG40 (Kang et al., 2010). Transcript quantification of the putative grapevine CCD7 and CCD8 was performed on non-treated berry samples only. Gene-specific primer pairs used in RT–qPCR experiments are listed in Supplementary Table S1 at JXB online.