*4.5. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomics (KEGG) and Cis-Elements Predictions of PGs and PMEs*

The GO enrichment was carried using an online panther server (http://pantherdb.org/) and TBtools software [52]. KEGG enrichment analysis was carried out by the online server (https: //www.genome.jp/kegg/pathway.html) and their enriched pathways were further analyzed by TBtools software [52]. The promoter sequences of PGs and PMEs (i.e., selected as 1500 bp) were imported in Generic File Format (GFF) file from the grapevine genome. Subsequently, the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [54] was utilized for identifying various cis-regulatory elements for each promoter sequence of PGs and PMEs.

## *4.6. Chromosomal Location and Collinearity Analysis*

The chromosomal locations of PGs and PMEs were mapped based on information available at the Grape Genome Database (CRIBI. Available online: http://genomes.cribi.unipd.it/grape/, V2.1). and were illustrated using TBtools software [52]. For collinearity analysis, the relationships between grapevine and *Arabidopsis* homologs were verified and visualized by the Circos tool in TBtools software.

## *4.7. Principal Component Analysis (PCA)*

The principal component analysis was implemented using Rstudio (R program) for qRT-PCR at a significance level of 0.05 (*p*-value) [37,55].

### *4.8. Plant Material and Methods*

Six-year-old *V. vinifera* cv. Summer Black plants grown under standard field conditions were selected from Jiangsu Academy of Agricultural Sciences (JAAS), Nanjing-China. In brief, the 4th unfolded leaf was selected for tissue extraction. Various grapevine tissues/organs, such as the root, stem, tendril, inflorescence, flesh, and skin, were collected at different developmental stages. Tissue samples were immediately frozen in liquid nitrogen and stored at −80 ◦C for further use and RNA extraction.

#### *4.9. RNA Isolation and Expression Profiling of PGs and PMEs in Grapevine*

Total RNA was extracted from various organs using Trizol (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. RNA was reverse-transcribed into cDNA using the Primer Script RT reagent kit (TAKARA, Dalian, China) according to the manufacturer's instructions. Specific primers were designed using Becan Designer 7.9, and are presented in Table S7. In order to check the specificity of the primers, the BLAST tool was used against the grapevine genome for confirmation. RT-PCR was performed according to the guidelines of previous studies [56,57]. The relative fold expression was calculated with the comparative Ct-method. The expression patterns of all *PG* and *PME* genes were analyzed based on a previous study [58,59]. The housekeeping and grapevine actin gene (AB073011 and XM\_010659103) was used as the reference gene for qRT-PCR.

In brief, the RT-PCR amplification reactions were performed on an ABI 7500 RT-PCR System (Applied Biosystems, CA, USA) using SYBR Green (Applied Biosystems, Carlsbad, CA, USA) with three replicates. PCR was conducted as follows: denaturation at 95 ◦C for 2 min, 40 cycles of denaturation at 95 ◦C for 10 s, annealing at 60 ◦C for 40 s, and extension at 72 ◦C for 15 s, followed by melting curve analysis (61 cycles at 65 ◦C for 10 s).

Transcriptomic data were utilized for various organs and developmental stages from NCBI GEO server (https://www.ncbi.nlm.nih.gov/geo/) under the series entry GSE36128. Additionally, gene expression levels were quantified by FPKM (fragments per kilobase of transcript per million fragments mapped), and heat maps were visualized by using Rstudio (R program, Boston, MA, USA).

#### **5. Conclusions**

In conclusion, we systematically carried out a genome-wide exploration of grapevines through various bioinformatic analyses, which include elucidating the physicochemical properties of PGs and PMEs, phylogenetic characterization, collinearity of PGs and PMEs, gene structure and motif composition, evolutionary rates, and gene duplications. The GO and KEGG enrichment, and cis-elements prediction analysis extended our repositories on the putative functions of PGs and PMEs in plant developments during pectin and carbohydrate metabolism, and various stress-related activities in grapevine. Additionally, expression profiling of various organs during developmental stages and their correlation by principal component analysis highlights the essential role of PGs and PMEs for plant improvements in grapevines.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/20/13/3180/s1.

**Author Contributions:** Conceptualization, methodology, performed the experiments, N.K.; managed the research work, writing original draft, figures, review and editing, N.K., F.F., and M.S.H.; software, H.S.; validation, Z.L. and T.Z.; and supervision, funding acquisition, project administration, J.F.

**Funding:** This work was supported by the funding of National Key R&D Projects 2018YFD1000200, Jiangsu Agricultural Science and Technology Independent Innovation Project CX (18)2008, National Natural Science Foundation of China (31672131, 31801809).

**Conflicts of Interest:** The authors declare no conflict of interest.
