*4.5. Analysis of Cis-Acting Elements and Gene Ontology*

To examine the possible regulatory mechanisms of B3 genes, a 1.5-kb promoter region upstream of the start codon of each gene was retrieved from the Grape Genome Database (http://www.genoscope. cns.fr) and analyzed through the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/) online server. Gene ontology analysis of B3 protein sequences was acquired from Blast2GO (http://www.blast2go.com).

## *4.6. Plant Materials*

This study used one seeded cultivar, Red Globe, and one seedless cultivar, Thompson Seedless (both *V. vinifera*). Plants were maintained in the grape germplasm resource orchard of Northwest A&F University, Yangling, China (34◦200 N 108◦240 E). The obtained plant structures were young roots, stems, leaves, tendrils, and fruits (42 days after full bloom, DAF). All samples were collected from plants under natural conditions. Developing seeds were dissected from fruits at 28, 31, 34, 37, 40, and 43 DAF. All samples were immediately frozen in liquid nitrogen and stored at −80 ◦C for RNA extraction and expression analysis.

## *4.7. RNA Extraction and Expression Analysis by PCR*

Total RNA was extracted from samples using an EZNA Plant RNA Kit (R6827-01, OMEGA Biotek, Norcross, GA, USA), according to the manufacturer's guidelines. First-strand cDNA was synthesized by reverse transcription of 500 ng total RNA using Prime Script RTase (Trans Gen Biotech, Beijing, China). After this, cDNA was diluted six-fold and preserved at −40 ◦C for further analysis. The grapevine *ACTIN1* gene (Genbank Accession NC\_012010) and *EF1-*α gene (Genbank Accession NC\_012012) were used as internal controls; *ACTIN1* was amplified with the oligonucleotide primers (50 -GAT TCT GGT GAT GGT GTG AGT-30 ) and (50 -GAC AAT TTC CCG TTC AGC AGT-30 ), and *EF1-*α was amplified using (50 -AGG AGG CAG CCA ACT TCA CC-30 ) and (50 -CAA ACC CTG CAT CAC CAT TC-30 ). Gene-specific primers were designed for selected B3 genes using Primer Premier 6.0 (Table S5). The specificity of primers was checked in the NCBI (https://www.ncbi.nlm.nih.gov/) database, using the Primer-BLAST program. Semiquantitative RT-PCR assays were carried out in a volume of 20 µL per reaction containing 1 µL of cDNA template, 2 µL of gene-specific primers (1.0 µM), 7 µL sterile distilled water, and 10 µL PCR Master Mix (BIOSCI BIOTECH CO. LTD, Hangzhou, China). PCR conditions were 94 ◦C for 2 min, 35–38 cycles of 94 ◦C for 30 s, 51–63 ◦C for 30 s (depending on the specific gene), and 72 ◦C for 30 s, with a final extension of 72 ◦C for 7 min. In each case, 10 µL of the resulting product was resolved on a 1.5% (*w*/*v*) agarose gel and visualized using ethidium bromide and then imaged under ultraviolet light using GeneSnap software. Each assay was performed with three biological replicates. Semiquantitative RT-PCR expression data were visualized using GeneTools software. The mean expression value for each gene was determined in all tissues or seed developmental stages in all cultivars, and was then log2-transformed to generate heat maps using Multi Experiment Viewer software (Mev 4.8.1). Quantitative real-time PCR was performed for selected genes using SYBR Green (Trans Gen Biotech, Beijing, China) on an IQ5 real-time PCR machine (Bio-Rad, Hercules, CA, USA). *ACTIN1* was used as the internal reference gene and each reaction was performed with triplicate technical and biological repeats. The reaction mixture was 1 µL of cDNA template, 0.8 µL each primer (1.0 µM), 0.4 µL Rox reference dye, 7 µL sterile distilled water, and 10 µL of SYBR green. PCR was performed following the parameters 95 ◦C for 30 s, followed by 42 cycles of 95 ◦C for 5 s and 60 ◦C for 30 s. Relative expression levels were determined by the comparative CT method also referred to as 2−∆∆*C*<sup>T</sup> method, where ∆∆*C*T = [(C<sup>T</sup> target gene–C<sup>T</sup> control gene) Sample A–(C<sup>T</sup> target gene–C<sup>T</sup> control gene) Sample B] [69]. After this, the RT-PCR values were used to create graphs using Sigma Plot 10.0 [33].
