*2.2. Phylogenetic and Collinearity Relationships, Motif Compositions and Genomic Structure of PGs and PMEs in Grapevine*

The phylogenetic relationships of 36 PGs and 47 PMEs of grapevine and *Arabidopsis* were obtained using MEGA 7.0 with a maximum likelihood approach (ML). The phylogenetic tree revealed that *PG* and *PME* genes can further be divided into six and five major clades (Figure 1a,b). In the phylogenetic tree of both PGs and PMEs, we observed that clade two contained the most number of genes (15 and 14) compared to other clades in grapevine. The phylogenetic analysis suggests that both PGs and PMEs share high similarities and have close genetic relationships with *Arabidopsis*. The observed results of our phylogenetic arrangement were also consistent with previously reported studies [25,27]. In addition, we also constructed phylogenetic trees among *PG* and *PME* genes (Figures 2a and 3a). The results showed consistency among the clades of PGs compared to PMEs, which may be due to variations in tree topologies. We also analyzed the composition of motifs for both PGs and PMEs (Figures 2b and 3b). For PG and PME proteins, we obtained ten conserved motifs using the online server, Multiple Em for Motif Elicitation (MEME). The results revealed that motifs five and two frequently occurred among PG members. Similarly, for PME members, motifs six, five, four, and one were dominantly found in grapevine (Figures 2b and 3b). Hence, these results suggest that most PG and PME protein members carry unique features due to variation in their amino acid sequences. Additionally, we also obtained their LOGOS by the same online server MEME. Ten consensus sequences were acquired for both PG and PME protein members and their distribution patterns are shown in Figures S1 and S2. *Int. J. Mol. Sci.* **2019**, *20*, x; doi: www.mdpi.com/journal/ijms

**Figure 1.** Phylogenetic relationship of *PG* (**A**) and *PME* (**B**) genes between grapevine and *Arabidopsis*. The phylogenetic tree was constructed by MEGA 7.0 using the Maximum Likelihood Method (1000 **Figure 1.** Phylogenetic relationship of *PG* (**A**) and *PME* (**B**) genes between grapevine and *Arabidopsis*. The phylogenetic tree was constructed by MEGA 7.0 using the Maximum Likelihood Method (1000 bootstrap).

bootstrap). Furthermore, based on coding sequence (CDS) and untranslated regions (UTR) of *PG* and *PME* genes in grapevine, gene structures were also resolved using TBtools software (Figures 2c and 3c). The results revealed that both PG and PME members exhibited high divergence and were largely conserved compared to each other. On the other hand, the *PG* and *PME* genes displayed more or fewer similarities among the same clades. This was also observed in a previously reported study focusing on PGs and PMEs in *Brassica rapa* [25].

**Figure 1.** Phylogenetic relationship of *PG* (**A**) and *PME* (**B**) genes between grapevine and *Arabidopsis*.

**Figure 2.** Phylogenetic relationship of *PGs* (**A**). The phylogenetic tree was constructed by MEGA 7.0 using the Maximum Likelihood Method (1000 bootstrap). Motif structure and upstream/downstream regions of *PGs* (**B**). The coding sequences (CDS) and untranslated regions (UTR) for PGs in grapevine (**C**). CDS and UTR are represented by yellow and green boxes. The relative position is proportionally displayed based on the kilobase scale at the bottom of the figures.

**Figure 2.** Phylogenetic relationship of *PGs* (**A**). The phylogenetic tree was constructed by MEGA 7.0 using the Maximum Likelihood Method (1000 bootstrap). Motif structure and upstream/downstream regions of *PGs* (**B**). The coding sequences (CDS) and untranslated regions (UTR) for PGs in grapevine

displayed based on the kilobase scale at the bottom of the figures.

**Figure 3.** Phylogenetic relationship of *PMEs* (**A**). The phylogenetic tree was constructed by MEGA 7.0 using the Maximum Likelihood Method (1000 bootstrap). Motif structure and upstream/downstream regions of *PMEs* (**B**). The coding sequences (CDS) and untranslated regions (UTR) for PMEs in grapevine (**C**). CDS and UTR are represented by yellow and green boxes, respectively. The relative position is proportionally displayed based on the kilobase scale at the bottom of the figures. **Figure 3.** Phylogenetic relationship of *PMEs* (**A**). The phylogenetic tree was constructed by MEGA 7.0 using the Maximum Likelihood Method (1000 bootstrap). Motif structure and upstream/downstream regions of *PMEs*(**B**). The coding sequences (CDS) and untranslated regions (UTR) for PMEs in grapevine (**C**). CDS and UTR are represented by yellow and green boxes, respectively. The relative position is proportionally displayed based on the kilobase scale at the bottom of the figures.

#### *2.3. Chromosomal Localization and Gene Duplication Analysis of PG and PME Genes 2.3. Chromosomal Localization and Gene Duplication Analysis of PG and PME Genes*

A total of 36 *PG* genes were distributed unevenly across different chromosomal locations of grapevine genomes (i.e., Chr01–Chr19). The majority of the chromosomes of PGs showed inconsistency in terms of genes. Both Chr05 and Chr08 exhibited the highest number (8) of genes, followed by Chr1 and Chr07 with five genes. The others varied in number (Figure 4a and Figure S3). A total of 36 *PG* genes were distributed unevenly across different chromosomal locations of grapevine genomes (i.e., Chr01–Chr19). The majority of the chromosomes of PGs showed inconsistency in terms of genes. Both Chr05 and Chr08 exhibited the highest number (8) of genes, followed by Chr1 and Chr07 with five genes. The others varied in number (Figure 4a and Figure S3). Moreover, the chromosomal localization for PME members also displayed high variation in the number of genes. The highest number of genes (seven) was found on both Chr11 and Chr16 each, followed by Chr5 containing five genes. The others largely varied from 1–4 per chromosome (Figure 4b and Figure S3). In majority, we observed non-random distribution patterns of *PG* and *PME* genes in the grapevine genome. The 36 *PG* and 47 *PME* genes were also clustered for collinearity between

among PME members compared to PGs.

grapevine and *Arabidopsis* using Circos (Figure 4a,b). The results illustrated high conservation among PME members compared to PGs. However, we also found four pairs of PGs (i.e., *VvPG29-VvPG30*, *VvPG5-VvPG7*, *VvPG14-VvPG15*, and *VvPG18-VvPG21*) and five pairs of PMEs (*VvPME5-VvPME16*, *VvPME22-VvPME23*, *VvPME35-*

*VvPME36*, *VvPME6-VvPME7*, and *VvPME14-VvPME26*) that indicate positive selection.

were less than one, implicating a purifying selection and a reduction in divergence after duplications.

observed may play a major role in the expansion of PG and PME members.

*Int. J. Mol. Sci.* **2019**, *20*, x; doi: www.mdpi.com/journal/ijms Moreover, the chromosomal localization for PME members also displayed high variation in the number of genes. The highest number of genes (seven) was found on both Chr11 and Chr16 each, followed by Chr5 containing five genes. The others largely varied from 1–4 per chromosome (Figure 4b and Figure S3). In majority, we observed non-random distribution patterns of *PG* and *PME* genes in the grapevine genome. The 36 *PG* and 47 *PME* genes were also clustered for collinearity between grapevine and *Arabidopsis* using Circos (Figures 4a and 4b). The results illustrated high conservation

To study evolutionary rates and types of duplications among *PG* and *PME* genes in grapevines, we used MEGA7.0 and MCScanX. Among the 36 PGs, we identified 10 dispersed, 1 proximal, 12 tandem, and 13 segmental genes. Furthermore, we determined 23 dispersed, 1 proximal, 16 tandem, and 7 segmental genes in the 47 PMEs (Table 1). As gene duplications are vital for discovering novel biological functions, evolutions, and gene expansion [30], the segmental and dispersed duplications

To estimate the selection pressure among various types of duplications for both PGs and PMEs, we also intended their synonymous (*Ks*) and non-synonymous substitution rate (*Ka*) values. During evolutionary implications, genes are typically exposed to different types of selection processes (i.e.,

**Figure 4.** The collinear correlation for all genes of PGs (**A**) and PMEs (**B**) is displayed between grapevines and *Arabidopsis*. The localization of chromosomes was shown for grapevine and *Arabidopsis* in different random colors. **Figure 4.** The collinear correlation for all genes of PGs (**A**) and PMEs (**B**) is displayed between grapevines and *Arabidopsis*. The localization of chromosomes was shown for grapevine and *Arabidopsis* in different random colors.

**Table 1.** Duplications of the *PG* and *PME* genes in grapevine. **Gene 1 Gene 2** *Ks Ka Ka/Ks* **Selection Pressure Gene Duplications**  Between *PG* genes *VvPG1 VvPG2* 0.029 0.022 0.77 Purifying Selection Tandem *VvPG3 VvPG4* 0.034 0.016 0.47 Purifying Selection Tandem To study evolutionary rates and types of duplications among *PG* and *PME* genes in grapevines, we used MEGA7.0 and MCScanX. Among the 36 PGs, we identified 10 dispersed, 1 proximal, 12 tandem, and 13 segmental genes. Furthermore, we determined 23 dispersed, 1 proximal, 16 tandem, and 7 segmental genes in the 47 PMEs (Table 1). As gene duplications are vital for discovering novel biological functions, evolutions, and gene expansion [30], the segmental and dispersed duplications observed may play a major role in the expansion of PG and PME members.

*VvPG6 VvPG25* 0.852 0.579 0.68 Purifying Selection Tandem *VvPG28 VvPG31* 0.738 0.693 0.94 Purifying Selection Tandem *VvPG33 VvPG34* 0.663 0.197 0.30 Purifying Selection Tandem *VvPG8 VvPG9* 0.5 0.458 0.92 Purifying Selection Dispersed To estimate the selection pressure among various types of duplications for both PGs and PMEs, we also intended their synonymous (*Ks*) and non-synonymous substitution rate (*Ka*) values. During evolutionary implications, genes are typically exposed to different types of selection processes (i.e., positive, neutral, and purifying selection). For understanding these selection pressures, we selected 15 duplicated pairs of genes among PGs and PMEs (Table 1). The *Ka*/*Ks* ratios for most PGs and PMEs were less than one, implicating a purifying selection and a reduction in divergence after duplications. However, we also found four pairs of PGs (i.e., *VvPG29-VvPG30*, *VvPG5-VvPG7*, *VvPG14-VvPG15*, and *VvPG18-VvPG21*) and five pairs of PMEs (*VvPME5-VvPME16*, *VvPME22-VvPME23*, *VvPME35-VvPME36*, *VvPME6-VvPME7*, and *VvPME14-VvPME26*) that indicate positive selection.

#### *2.4. Gene Ontology Enrichment (GO) and Cis-Regulatory Elements in Grapevine*

To study the regulatory functions of the 36 PGs and 47 PMEs, we performed GO annotation and GO enrichment analyses. The GO terms were largely based on three groupings, including molecular functions (MF), cellular component (CC), and biological process (BP). In brief, GO enrichments validate that PGs are enriched in various MF terms, such as "polygalacturonase activity" (GO:0004650), "hydrolase activity" (GO:0016787), and "hydrolyzing O-glycosyl compounds" (GO:0016798). The term CC was enriched in the plant-type cell wall, such as "integral component of membrane" (GO:0016021), and "extracellular region" (GO:0005576). The BP term was mainly responsive, such as "carbohydrate metabolic processes" (GO:0005975) and various other "metabolic processes" (GO:0008152), which are briefly listed in Table S3. Moreover, the PME results reveal that

MF is enriched in "enzyme inhibitor activity" (GO:0004857) "pectinesterase activity" (GO:0030599), "aspartyl esterase" (GO:0045330). The CC and BP were also found to be enriched in "membrane" (GO:0016020), "proteolysis" (GO:0006508), and "cell wall modification" (GO:0042545). Our results from the GO enrichment analysis further hinted the role of PG and PME members in grapevine.

The 36 *PG* and 47 *PME* genes were also tested for pathway enrichment analysis using the KEGG database, where the results showed enrichment in three major pathways (Table S4). These pathways include "carbohydrate metabolism" followed by "metabolism" and "pentose and glucuronate interconversions" in grapevines.

In addition, we also observed cis-acting elements by utilizing the promoter regions of both PG and PME members using the PlantCARE database. The various types of cis-regulatory elements were analyzed and are described in Figure 5 and Table S5. In brief, the majority of the genes participated in numerous signaling pathways, such as phytohormones, biotic-abiotic and other regulatory stress factors. For instance, 27.61% of the genes of PGs and PMEs were responsive to light regulations (e.g., GTI-motif, G-Box, GATA-motif, and others), followed by phytohormones (25.65%) (CGTCA, TGACG, ABRE). Other observed key regulatory elements include TC-Rich repeats, and HD-ZIP 3, which were reactive to defense stress and protein binding, respectively. These results inferred that the *PG* and *PME* genes have diverse gene functions and are indirectly involved in various biotic-abiotic/hormone signaling. *Int. J. Mol. Sci.* **2019**, *20*, x; doi: www.mdpi.com/journal/ijms the *PG* and *PME* genes have diverse gene functions and are indirectly involved in various bioticabiotic/hormone signaling.

#### **Figure 5.** Various cis-elements identified in grapevine by using PlantCARE. *2.5. Tanscriptional Profiling of PGs and PMEs in Di*ff*erent Organs and Developmental Stages in Grapevine*

*2.5. Tanscriptional Profiling of PGs and PMEs in Different Organs and Developmental Stages in Grapevine*  To understand the spatiotemporal expression levels of *PG* and *PME* genes in grapevines, the global transcriptomic data of developmental phases of 19 different tissues and organs were retrieved from NCBI (GSE36128) [31]. Figures 6a and 6b represent the heat maps, indicating expression patterns of PGs and PMEs in grapevines. Among PGs, V*vPG8, VvPG10, VvPG13, VvPG17, VvPG18, VvPG19, VvPG20, VvPG22,* and *VvPG23* showed tissue- or organ-specific expression across many tissues during development. In contrast, the remaining PGs demonstrated weak tissue-specific response in any of the selected grapevine organs. Likewise, in PMEs, *VvPME3, VvPME4, VvPME5, VvPME6, VvPME19, VvPME21, VvPME23, VvPME29, VvPME31*, and *VvPME32* suggested higher tissue-specific response in all the tissues. In contrast, the others showed either moderate to weak expression or no expression in any of the grapevine tissues (Figure 6b). Overall the *PG* and *PME* genes showed enriched expression in flower (*VvPG6-7*, *VvPG31-34*, *VvPME-28*) and fruit ripening PMEs. **Figure 5.** Various cis-elements identified in grapevine by using PlantCARE. To understand the spatiotemporal expression levels of *PG* and *PME* genes in grapevines, the global transcriptomic data of developmental phases of 19 different tissues and organs were retrieved from NCBI (GSE36128) [31]. Figure 6a,b represent the heat maps, indicating expression patterns of PGs and PMEs in grapevines. Among PGs, *VvPG8, VvPG10, VvPG13, VvPG17, VvPG18, VvPG19, VvPG20, VvPG22,* and *VvPG23* showed tissue- or organ-specific expression across many tissues during development. In contrast, the remaining PGs demonstrated weak tissue-specific response in any of the selected grapevine organs. Likewise, in PMEs, *VvPME3, VvPME4, VvPME5, VvPME6, VvPME19, VvPME21, VvPME23, VvPME29, VvPME31*, and *VvPME32* suggested higher tissue-specific response in all the tissues. In contrast, the others showed either moderate to weak expression or no expression in any of the grapevine tissues (Figure 6b). Overall the *PG* and *PME* genes showed enriched expression in flower (*VvPG6-7*, *VvPG31-34*, *VvPME-28*) and fruit ripening (*VvPG4-5*, *VvPME7-8*, and *VvPME44-45*), where the PGs suggested a more profound response than PMEs.

(*VvPG4-5*, *VvPME7-8*, and *VvPME44-45*), where the PGs suggested a more profound response than

*Int. J. Mol. Sci.* **2019**, *20*, x; doi: www.mdpi.com/journal/ijms

**Figure 6.** Expression profiles of the *PG* (**A**) and *PME* (**B**) genes in different grapevine organs, tissues, and, developmental stages. Data were normalized based on the mean expression values of each gene in all analyzed tissues. BerryPericarp-FS: berry pericarp fruit set; BerryPericarp-PFS: berry pericarp post-fruit set; BerryPericarp-V: Bud-S: bud swell; Bud-B: bud burst (green tip); Bud-AB: bud afterburst (rosette of leaf tips visible); Bud-L: latent bud; Bud-W: winter bud; Flower-FB: flowering begins (10% caps off); Flower-F: flowering (50% caps off); Leaf-Y: young leaf (pool of leaves from shoot of 5 leaves); Leaf-FS: mature leaf (pool of leaves from shoot at fruit set); Rachis-FS: rachis fruit set; Rachis-PFS: rachis post fruit set; Stem-G: green stem; Stem-W: woody stem; Tendril-Y: young tendril (pool of tendrils from shoot of 7 leaves); Tendril-WD: well developed tendril (pool of tendrils from shoot of 12 leaves); and Tendril-FS: mature tendril (pool of tendrils at fruit set). **Figure 6.** Expression profiles of the *PG* (**A**) and *PME* (**B**) genes in different grapevine organs, tissues, and, developmental stages. Data were normalized based on the mean expression values of each gene in all analyzed tissues. BerryPericarp-FS: berry pericarp fruit set; BerryPericarp-PFS: berry pericarp post-fruit set; BerryPericarp-V: Bud-S: bud swell; Bud-B: bud burst (green tip); Bud-AB: bud after-burst (rosette of leaf tips visible); Bud-L: latent bud; Bud-W: winter bud; Flower-FB: flowering begins (10% caps off); Flower-F: flowering (50% caps off); Leaf-Y: young leaf (pool of leaves from shoot of 5 leaves); Leaf-FS: mature leaf (pool of leaves from shoot at fruit set); Rachis-FS: rachis fruit set; Rachis-PFS: rachis post fruit set; Stem-G: green stem; Stem-W: woody stem; Tendril-Y: young tendril (pool of tendrils from shoot of 7 leaves); Tendril-WD: well developed tendril (pool of tendrils from shoot of 12 leaves); and Tendril-FS: mature tendril (pool of tendrils at fruit set).
