*2.5. Exon-Intron Organization in CDPK and CRK Groups*

*2.5. Exon-Intron Organization in CDPK and CRK Groups*  The exon-intron organization, as well as the intron numbers, can also provide important evidence to analyze the evolutionary history within gene families [9,11,30]. To obtain further insight into the phylogenetic relationships of two gene families in Cucurbitaceae, gene structures of all 226 CDPKs and CRKs were comparatively depicted, dependent on their gene annotation profiles and genomic sequences (Figure S8). The majority of members in group CDPK I contained six introns with a distinct intron phase pattern 111000, while most CDPKs in group CDPK II had seven introns, sharing a similar intron pattern of 1110020. CDPK III, as a peripheral sister clade of groups CDPK I and II, contained two major intron phases. For instance, 13 out of 17 CDPKs in loci 02 and 25 shared an intron phase 111000, which is identical to that of group CDPK I, while 28 of 37 CDPKs in the other four loci had an intron pattern of 0111000 (Figure S8). CDPK IV, as the smallest group, contained only 10 members with 11 or 12 introns. Among of them, eight CDPKs were constituted of 11 introns with a phase pattern of 02201010000, while the remaining two genes (*CsCDPK6* and *CmoCDPK7*) had 12 introns with an extra intron gain at the 5' or 3' end. In group CRK I, 41 out of 63 members contained 10 introns with a phase pattern 0220110000, showing high similarity with that in group The exon-intron organization, as well as the intron numbers, can also provide important evidence to analyze the evolutionary history within gene families [9,11,30]. To obtain further insight into the phylogenetic relationships of two gene families in Cucurbitaceae, gene structures of all 226 CDPKs and CRKs were comparatively depicted, dependent on their gene annotation profiles and genomic sequences (Figure S8). The majority of members in group CDPK I contained six introns with a distinct intron phase pattern 111000, while most CDPKs in group CDPK II had seven introns, sharing a similar intron pattern of 1110020. CDPK III, as a peripheral sister clade of groups CDPK I and II, contained two major intron phases. For instance, 13 out of 17 CDPKs in loci 02 and 25 shared an intron phase 111000, which is identical to that of group CDPK I, while 28 of 37 CDPKs in the other four loci had an intron pattern of 0111000 (Figure S8). CDPK IV, as the smallest group, contained only 10 members with 11 or 12 introns. Among of them, eight CDPKs were constituted of 11 introns with a phase pattern of 02201010000, while the remaining two genes (*CsCDPK6* and *CmoCDPK7*) had 12 introns with an extra intron gain at the 5' or 3' end. In group CRK I, 41 out of 63 members contained 10 introns with a phase pattern 0220110000, showing high similarity with that in group CDPK IV. Notably, members in the same loci usually had similar exon or intron lengths, such as Locus 25 in group CDPK III.

#### Locus 25 in group CDPK III. *2.6. Duplication and Syntenic Analysis of CDPK and CRK Gene Families*

*2.6. Duplication and Syntenic Analysis of CDPK and CRK Gene Families*  In addition to whole genome duplication (WGD) events, both tandem and segmental In addition to whole genome duplication (WGD) events, both tandem and segmental duplications have also been reported to play vital roles in the expansion and function of a gene family [11,31,32]. To further explore the possible evolutionary relationships of *CDPK* and *CRK* gene families in

duplications have also been reported to play vital roles in the expansion and function of a gene

CDPK IV. Notably, members in the same loci usually had similar exon or intron lengths, such as

Cucurbitaceae, duplication events were investigated in six species (including watermelon, cucumber, bottle gourd, and three *Cucurbita* species). Similar to the melon genome [11], only one or two segmental duplication events were detected in three Benincaceae genomes, with syntenic regions no more than 3.0 Mb (Table S5). However, many more segmental duplication events were observed in *Cucurbita* genus, most of which occurred between sub-genomes. families in Cucurbitaceae, duplication events were investigated in six species (including watermelon, cucumber, bottle gourd, and three *Cucurbita* species). Similar to the melon genome [11], only one or two segmental duplication events were detected in three Benincaceae genomes, with syntenic regions no more than 3.0 Mb (Table S5). However, many more segmental duplication events were observed in *Cucurbita* genus, most of which occurred between sub-genomes. In the Benincaseae tribe, watermelon, melon, and cucumber are important cucurbit crops

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family [11,31,32]. To further explore the possible evolutionary relationships of *CDPK* and *CRK* gene

In the Benincaseae tribe, watermelon, melon, and cucumber are important cucurbit crops widely cultivated throughout the world. Synteny analyses revealed that similar numbers of syntenic regions were detected among three genomes, with average fragment lengths not exceeding 2.5 Mb (Figure 4 and Table S6). As a close sister lineage of *Citrullus*, approximately 15 collinear regions were found between watermelon and bottle gourd, with the largest one spanning about 16.6 Mb. In the Cucurbiteae tribe, many more collinear regions were detected, with the largest one (21.3 Mb) existing between *C. moschata* and *C. pepo* (Table S6). widely cultivated throughout the world. Synteny analyses revealed that similar numbers of syntenic regions were detected among three genomes, with average fragment lengths not exceeding 2.5 Mb (Figure 4 and Table S6). As a close sister lineage of *Citrullus*, approximately 15 collinear regions were found between watermelon and bottle gourd, with the largest one spanning about 16.6 Mb. In the Cucurbiteae tribe, many more collinear regions were detected, with the largest one (21.3 Mb) existing between *C. moschata* and *C. pepo* (Table S6).

**Figure 4.** Synteny analysis of *CDPK* and *CRK* genes among watermelon, cucumber, and melon. Chromosomes of three species (watermelon, cucumber, and melon) were depicted in different colors (red, green, and yellow) and in circle form. The approximate distributions of each *CDPK* and *CRK* are presented by short black lines on the circle. Colored curves denote the details of syntenic regions **Figure 4.** Synteny analysis of *CDPK* and *CRK* genes among watermelon, cucumber, and melon. Chromosomes of three species (watermelon, cucumber, and melon) were depicted in different colors (red, green, and yellow) and in circle form. The approximate distributions of each *CDPK* and *CRK* are presented by short black lines on the circle. Colored curves denote the details of syntenic regions containing *CDPK* and *CRK* genes among genomes.

#### containing *CDPK* and *CRK* genes among genomes. *2.7. Expression Profiles of ClCDPK and ClCRK Genes in Di*ff*erent Tissues*

accumulations in male flowers [9,11].

*2.7. Expression Profiles of ClCDPK and ClCRK Genes in Different Tissues*  To assess the potential functions of *ClCDPK* and *ClCRK* genes, their expression patterns were investigated in six different tissues, including roots, stems, leaves, tendrils, male flowers, and female flowers. As shown in Figure 5, all identified *ClCDPKs* and *ClCRKs* could be detected in at least one tissue. Some *ClCDPKs* and *ClCRKs*, such as *ClCDPK2*, *ClCDPK3*, *ClCDPK9*, *ClCDPK15*, and *ClCRK1*, showed significantly elevated expression levels in the root, while *ClCDPK6*, *ClCDPK16*, and *ClCDPK17* were strongly expressed in the male flower. Interestingly, *CmCDPK6* in melon, the ortholog of *ClCDPK17* in locus 24, was also reported to have a high transcriptional abundance in the male flower [11]. Moreover, orthologs of *ClCDPK6* and *ClCDPK16* in melon (*CmCDPK9* and *CmCDPK5*) and cucumber (*CsCDPK14* and *CsCDPK9*) were also confirmed to retain high To assess the potential functions of *ClCDPK* and *ClCRK* genes, their expression patterns were investigated in six different tissues, including roots, stems, leaves, tendrils, male flowers, and female flowers. As shown in Figure 5, all identified *ClCDPKs* and *ClCRKs* could be detected in at least one tissue. Some *ClCDPKs* and *ClCRKs*, such as *ClCDPK2*, *ClCDPK3*, *ClCDPK9*, *ClCDPK15*, and *ClCRK1*, showed significantly elevated expression levels in the root, while *ClCDPK6*, *ClCDPK16*, and *ClCDPK17* were strongly expressed in the male flower. Interestingly, *CmCDPK6* in melon, the ortholog of *ClCDPK17* in locus 24, was also reported to have a high transcriptional abundance in the male flower [11]. Moreover, orthologs of *ClCDPK6* and *ClCDPK16* in melon (*CmCDPK9* and *CmCDPK5*) and cucumber (*CsCDPK14* and *CsCDPK9*) were also confirmed to retain high accumulations in male flowers [9,11].

**Figure 5.** Expression profiles of *ClCDPK* and *ClCRK* genes in different tissues. The transcript levels of the respective genes in roots were used as references and set to a value of 1. The data were showed as means value ± SD. All experiments were performed with three independent replicates. R = roots; S = **Figure 5.** Expression profiles of *ClCDPK* and *ClCRK* genes in different tissues. The transcript levels of the respective genes in roots were used as references and set to a value of 1. The data were showed as means value ± SD. All experiments were performed with three independent replicates. R = roots; S = stems; L = leave; FF = female flowers; MF = male flowers; T = tendrils.

#### stems; L = leave; FF = female flowers; MF = male flowers; T = tendrils. *2.8. Expression Patterns of ClCDPK and ClCRK Genes under Abiotic Stresses*

*2.8. Expression Patterns of ClCDPK and ClCRK Genes under Abiotic Stresses*  Accumulation studies showed that *CDPK* and *CRK* genes are widely involved in the adaptations to environmental stimuli, and that their expression levels are affected by drought, salt, and cold [2,9,11]. To investigate the potential roles of *ClCDPKs* and *ClCRKs* in response to abiotic stresses, their dynamic expressions were analyzed under drought, salt, and cold treatments (Figure 6). Compared to cold stimuli, far more *ClCDPKs* and *ClCRKs* could be induced by drought and NaCl treatments, and seven genes could be up-regulated by both drought and NaCl treatments, including *ClCDPK1*, *ClCDPK5*, *ClCDPK6*, *ClCDPK9*, *ClCDPK10*, *ClCDPK12*, and *ClCDPK14*. Following cold treatment, the transcription levels of four genes (*ClCDPK1*, *ClCDPK5*, *ClCDPK16*, and *ClCDPK17*) Accumulation studies showed that *CDPK* and *CRK* genes are widely involved in the adaptations to environmental stimuli, and that their expression levels are affected by drought, salt, and cold [2,9,11]. To investigate the potential roles of *ClCDPKs* and *ClCRKs* in response to abiotic stresses, their dynamic expressions were analyzed under drought, salt, and cold treatments (Figure 6). Compared to cold stimuli, far more *ClCDPKs* and *ClCRKs* could be induced by drought and NaCl treatments, and seven genes could be up-regulated by both drought and NaCl treatments, including *ClCDPK1*, *ClCDPK5*, *ClCDPK6*, *ClCDPK9*, *ClCDPK10*, *ClCDPK12*, and *ClCDPK14*. Following cold treatment, the transcription levels of four genes (*ClCDPK1*, *ClCDPK5*, *ClCDPK16*, and *ClCDPK17*) were down-regulated, while gene *ClCRK2* was continuously up-regulated at all treatment times. Compared to *ClCDPK3* and *ClCDPK18*

against environmental stresses.

down-regulated by drought stress, many more genes were obviously up-regulated, such as *ClCDPK1*, *ClCDPK2*, *ClCDPK8*, *ClCDPK9*, *ClCDPK12*, and *ClCDPK14*. In response to NaCl stress, the majority of *ClCDPK* and *ClCRK* genes were up-regulated, with few exceptions (Figure 6). obviously up-regulated, such as *ClCDPK1*, *ClCDPK2*, *ClCDPK8*, *ClCDPK9*, *ClCDPK12*, and *ClCDPK14*. In response to NaCl stress, the majority of *ClCDPK* and *ClCRK* genes were up-regulated, with few exceptions (Figure 6).

Compared to *ClCDPK3* and *ClCDPK18* down-regulated by drought stress, many more genes were

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 9 of 19

**Figure 6.** Expression patterns of *ClCDPK* and *ClCRK* genes under abiotic stresses. The abiotic stresses are displayed at the low end. The relative transcript level was log2 transformed and visualized as a heat map via Mev4.8.1, using red to indicate increased expression level and green to indicate **Figure 6.** Expression patterns of *ClCDPK* and *ClCRK* genes under abiotic stresses. The abiotic stresses are displayed at the low end. The relative transcript level was log<sup>2</sup> transformed and visualized as a heat map via Mev4.8.1, using red to indicate increased expression level and green to indicate decreased expression level.

#### decreased expression level. *2.9. Expression Patterns of ClCDPK and ClCRK Genes under Hormone Treatments*

*2.9. Expression Patterns of ClCDPK and ClCRK Genes under Hormone Treatments*  Previous studies have indicated that *CDPKs* and *CRKs* are involved in the signaling pathways Previous studies have indicated that *CDPKs* and *CRKs* are involved in the signaling pathways of various plant hormones [19,33]. Here, expression profiles of *ClCDPKs* and *ClCRKs* were investigated in response to four plant hormones ABA, SA, MeJA, and ETH.

of various plant hormones [19,33]. Here, expression profiles of *ClCDPKs* and *ClCRKs* were investigated in response to four plant hormones ABA, SA, MeJA, and ETH. Increasing evidence has shown that *CDPK* and *CRK* genes could participate in ABA-mediated signal transduction in plants [17,34,35]. In the present study, the majority of *ClCDPKs* were found to be induced by ABA treatment, with transcript abundance retaining at higher levels from 6 to 24 hpt (Figure 7a). Interestingly, all *ClCRKs* except for *ClCRK1*, were sharply down-regulated at 6 hpt, which is similar to observations in melon [11]. Following SA treatment, expression levels of most *ClCDPKs* and *ClCRKs* remained either unchanged or slightly changed at 0.5 and 1 hpt (Figure 7b). However, SA application significantly induced or reduced their transcript levels at 6 or 12 hpt. Notably, *ClCRKs* were also found to have decreased at 6 hpt, which is similar to the tendency mentioned above. In contrast to the responses to ABA and SA treatments, almost all *ClCDPKs* and *ClCRKs* showed continuous over-expression in response to ETH and MeJA stimuli, except for genes *ClCDPK16* and *ClCRK3* (Figure 8). Moreover, transcript abundances of most *ClCDPKs* and *ClCRKs* Increasing evidence has shown that *CDPK* and *CRK* genes could participate in ABA-mediated signal transduction in plants [17,34,35]. In the present study, the majority of *ClCDPKs* were found to be induced by ABA treatment, with transcript abundance retaining at higher levels from 6 to 24 hpt (Figure 7a). Interestingly, all *ClCRKs* except for *ClCRK1*, were sharply down-regulated at 6 hpt, which is similar to observations in melon [11]. Following SA treatment, expression levels of most *ClCDPKs* and *ClCRKs* remained either unchanged or slightly changed at 0.5 and 1 hpt (Figure 7b). However, SA application significantly induced or reduced their transcript levels at 6 or 12 hpt. Notably, *ClCRKs* were also found to have decreased at 6 hpt, which is similar to the tendency mentioned above. In contrast to the responses to ABA and SA treatments, almost all *ClCDPKs* and *ClCRKs* showed continuous over-expression in response to ETH and MeJA stimuli, except for genes *ClCDPK16* and *ClCRK3* (Figure 8). Moreover, transcript abundances of most *ClCDPKs* and *ClCRKs* had either sharply increased or decreased at 0.5 hpt, implying their rapid response to ETH and MeJA stimuli. In summary, these expression analyses indicated that *ClCDPKs* and *ClCRKs* could be involved in the regulatory pathways of plant hormones, and thus participate in the plant defense against environmental stresses.

had either sharply increased or decreased at 0.5 hpt, implying their rapid response to ETH and MeJA stimuli. In summary, these expression analyses indicated that *ClCDPKs* and *ClCRKs* could be involved in the regulatory pathways of plant hormones, and thus participate in the plant defense

**Figure 7.** Expression patterns of *ClCDPK* and *ClCRK* genes under ABA and SA hormone treatments. (**a**) Expression levels of *ClCDPKs* and *ClCRKs* under ABA stress visualized as a heat map (Left). Detailed expression patterns of *ClCRKs* under ABA stress (Right). (**b**) Expressions of *ClCDPKs* and *ClCRKs* under SA stress visualized as a heat map (Left). Detailed expression patterns of *ClCRKs* under SA stress (Right). The relative transcript level was log2 transformed and visualized as a heat map via Mev4.8.1, using red to indicate increased expression level and green to indicate decreased expression level. **Figure 7.** Expression patterns of *ClCDPK* and *ClCRK* genes under ABA and SA hormone treatments. (**a**) Expression levels of *ClCDPKs* and *ClCRKs* under ABA stress visualized as a heat map (Left). Detailed expression patterns of *ClCRKs* under ABA stress (Right). (**b**) Expressions of *ClCDPKs* and *ClCRKs* under SA stress visualized as a heat map (Left). Detailed expression patterns of *ClCRKs* under SA stress (Right). The relative transcript level was log<sup>2</sup> transformed and visualized as a heat map via Mev4.8.1, using red to indicate increased expression level and green to indicate decreased expression level.

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**Figure 8.** Expression patterns of *ClCDPK* and *ClCRK* genes under ETH and MeJA hormone stresses. The abiotic stresses are indicated at the low end. The relative transcript level was log2 transformed and visualized as a heat map by Mev4.8.1, using red to indicate increased expression level and green **Figure 8.** Expression patterns of *ClCDPK* and *ClCRK* genes under ETH and MeJA hormone stresses. The abiotic stresses are indicated at the low end. The relative transcript level was log<sup>2</sup> transformed and visualized as a heat map by Mev4.8.1, using red to indicate increased expression level and green to indicate decreased expression level.

#### to indicate decreased expression level. **3. Discussion**

#### **3. Discussion**  *3.1. Characteristic Features of CDPK and CRK Genes in Cucurbitaceae*

*3.1. Characteristic Features of CDPK and CRK Genes in Cucurbitaceae*  Functioning as both Ca2+ sensors and effectors, the *CDPK* gene family has been identified throughout the plant kingdom, as well as in several protozoa, but are absent in animals [4,28]. For instance, 34 *CDPKs* and eight *CRKs* were identified genome-wide in *Arabidopsis* [13], 29 *CDPKs* and six *CRKs* in tomato [10], 31 *CDPKs* and five *CRKs* in pepper [15], 30 *CDPKs* and nine *CRKs* in poplar [36], and 31 *CDPKs* and five *CRKs* in rice [12,14]. Moreover, approximately 19, 41, and 40 *CDKPs* have been detected in the genomes of grape [19], cotton [30], and maize [37], respectively. In Cucurbitaceae, 18 *CDPKs* and seven *CRKs* have been found in the melon genome [11], while 19 *CDPKs* were identified in cucumber by Xu et al. [9]. In this study, a total of 128 *CDPK* and 56 *CRK* genes were identified in six Cucurbitaceae species, including *C. lanatus*, *C. sativus*, *C. moschata*, *C. maxima*, *C. pepo*, and *L. siceraria* (Table 1). The numbers of *CDPKs* and *CRKs* are much higher in three *Cucurbita* species than in four Benincaseae tribe species (*C. lanatus*, *C. melon*, *C. sativus*, and *L. siceraria*), which may be due to a WGD event during the origin of this genus [25,26]. Moreover, the numbers of *CRKs* in three *Cucurbita* genomes are much higher than in other species, such as tomato, Functioning as both Ca2<sup>+</sup> sensors and effectors, the *CDPK* gene family has been identified throughout the plant kingdom, as well as in several protozoa, but are absent in animals [4,28]. For instance, 34 *CDPKs* and eight *CRKs* were identified genome-wide in *Arabidopsis* [13], 29 *CDPKs* and six *CRKs* in tomato [10], 31 *CDPKs* and five *CRKs* in pepper [15], 30 *CDPKs* and nine *CRKs* in poplar [36], and 31 *CDPKs* and five *CRKs* in rice [12,14]. Moreover, approximately 19, 41, and 40 *CDKPs* have been detected in the genomes of grape [19], cotton [30], and maize [37], respectively. In Cucurbitaceae, 18 *CDPKs* and seven *CRKs* have been found in the melon genome [11], while 19 *CDPKs* were identified in cucumber by Xu et al. [9]. In this study, a total of 128 *CDPK* and 56 *CRK* genes were identified in six Cucurbitaceae species, including *C. lanatus*, *C. sativus*, *C. moschata*, *C. maxima*, *C. pepo*, and *L. siceraria* (Table 1). The numbers of *CDPKs* and *CRKs* are much higher in three *Cucurbita* species than in four Benincaseae tribe species (*C. lanatus*, *C. melon*, *C. sativus*, and *L. siceraria*), which may be due to a WGD event during the origin of this genus [25,26]. Moreover, the numbers of *CRKs* in three *Cucurbita* genomes are much higher than in other species, such as tomato, pepper, and rice, although these species contain similar copies of *CDPKs*.

pepper, and rice, although these species contain similar copies of *CDPKs*. Tandem, segmental, and whole genome duplication events are confirmed to play important roles in the expansion of gene families. Approximately 12, 13, and seven segmental duplications were reported to exist in poplar, cotton, and rice genomes [14,30,36], while many more events were found in three *Cucurbita* species that mainly occurred between two sub-genomes (Table S5). However, only a few segmental duplication events (one or two) were detected in genomes of Tandem, segmental, and whole genome duplication events are confirmed to play important roles in the expansion of gene families. Approximately 12, 13, and seven segmental duplications were reported to exist in poplar, cotton, and rice genomes [14,30,36], while many more events were found in three *Cucurbita* species that mainly occurred between two sub-genomes (Table S5). However, only a few segmental duplication events (one or two) were detected in genomes of watermelon, cucumber, bottle gourd, and melon [11], which may cause the low copy numbers of *CDPK* and *CRK*

watermelon, cucumber, bottle gourd, and melon [11], which may cause the low copy numbers of *CDPK* and *CRK* genes. In the present study, the majority of CDPKs contained four EF-hand motifs genes. In the present study, the majority of CDPKs contained four EF-hand motifs (Table S2), which is consistent with observations found in other species [7,11,19]. However, two CDPKs (CpCDPK19 and CpCDPK27) in *C. pepo* with detectable transcriptional levels had been confirmed to contain nine and eight EF-hands, respectively. Dot plot analysis showed that self-duplications of the STKs\_CAMK protein kinase or EF-hand domains resulted in the gene structure variations (Figure 1), which may affect their functional specificity.

#### *3.2. Conserved Evolution of CDPK and CRK Genes in Cucurbitaceae*

Generally, *CDPK* and *CRK* genes are randomly distributed in genomes [10,11,15,36], which has also been validated in Cucurbitaceae species (Figure S3). Using watermelon chromosomes as reference, an integrated map was obtained and contained 16 *CDPK* and nine *CRK* loci, harboring almost all *CDPK* and *CRK* genes identified in Cucurbitaceae (Figure 2 and Table S4). Of these, nine loci (four *CDPK* and five *CRK*) exhibited Presence/Absence polymorphisms, while the remaining 16 loci (12 *CDPK* and four *CRK*) were shared by all seven species, implying that the flanking regions of most *CDPK* and *CRK* genes were conserved in these species during the evolutionary process.

Increasing evidence indicates that topological structures of *CDPK* and *CRK* gene families are conserved, with four *CDPK* and one *CRK* groups in phylogenetic trees [10,11,28,36]. In the present study, group CDPK IV was found to be close to CRK I rather than the other three CDPK groups in distance trees (Figure 3 and Figures S4, S5 and S7), confirming that CDPK IV and CRK I may originate from a common ancestor [10,11,30]. Moreover, the five groups can be further divided into 25 loci, according to the integrated map. The evolutionary scenario of seven modern Cucurbitaceae species revealed that watermelon diverged from bottle gourd around 10.4–14.6 Mya and from *Cucumis* 17.3–24.3 Mya; however, the progenitor B of *Cucurbita* diverged from Benincaseae around 25.5–27.0 Mya, and progenitor A diverged from the common ancestor of progenitor B and Benincaseae around 29.9–31.6 Mya [26,27]. In agreement with this evolutionary scenario, orthologs from watermelon and bottle gourd usually gathered together in phylogenetic trees, and genes from sub-genome B were preferentially clustered with those from Benincaseae tribe genomes (Figure 3 and Figure S7).

Both exon-intron structures and intron numbers can reflect the evolution, expansion, and functional relationships within a gene family, which were caused by three main types of mechanisms, including exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion [11,19,38]. Exon-intron organization analyses revealed that each group contained one or two major intron phase patterns (Figure S8). For instance, the majority of homologs in group CDPK I contained six introns with a distinct intron phase pattern of 111000, while most members in CDPK II had seven introns sharing a similar intron phase 1110020. As a peripheral sister clade of CDPK I and II, CDPK III contained two major intron phases and one of them was identical to that of group CDPK I. Moreover, the major intron pattern (02201010000) of eight members in CDPK IV is similar to that (0220110000) of most *CRK* homologs in CRK I (Figure S8). Combined with the topological structures of CDPK IV and CRK I, we infer that group CDPK IV is the ancient lineage of CDPK gene family, which may have diverged from the last common ancestor with CRK I before the divergence of monocots and dicots [28].

#### *3.3. Functional Comparison of CDPK and CRK Genes*

*CDPKs* and *CRKs* have been confirmed to play crucial roles in the signal pathways in response to various environmental stresses [4,5,28]. The systemic expression profiles of *CDPK* and *CRK* genes in *Cucumis* species under different stimuli have been reported recently [9,11]. Consequently, dynamic expression levels of *CDPKs* and *CRKs* under different stimuli were investigated in detail in watermelon. Compared to cold stimuli, far more *ClCDPKs* and *ClCRKs* were induced under drought and NaCl treatments (Figure 6), which differed from the expression trends of most homologs that were up-regulated by cold stress in cucumber and melon [9,11]. In watermelon, expression levels of *ClCDPK14* were up-regulated by all three stresses (cold, drought, and salt); its ortholog *CmCDPK1* in melon, could be also induced by cold and salinity treatments [11]. Completely different expression

trends were also observed among species. For example, both genes*ClCDPK6* and*ClCDPK12*, with a close relationship in group CDPK II, could be induced by salt stress (Figure S4 and Figure 6), while expression levels of their orthologs in melon (*CmCDPK9* and *CmCDPK11*) and cucumber (*CsCDPK14* and *CsCDPK5*) exhibited contrary responses to salinity stimuli [9,11]. Similarly, transcriptional levels of *ClCRK2* and its paralog *ClCRK3*, as well as their orthologs *CmCRK3* and *CmCRK6* in melon, were increased under low temperature; however, these two groups of genes showed different expression trends under salt stress between watermelon and melon. Taken together, we inferred that the functional fates of some orthologs may be diversified during species evolution. The phytohormone ABA has been reported to be widely involved in the response of plants to biotic and abiotic stresses [17,34,35]. For instance, AtCPK4 and AtCPK11 in *Arabidopsis* can positively mediate the CDPK/calcium-mediated ABA signaling pathways via phosphorylation of two ABA-responsive transcription factors, ABF1 and ABF4, and loss-of-function mutations decrease the tolerance of seedlings to salt stress [17]. As their closest homolog in the phylogenetic tree (Figure S7), transcript abundance of *ClCDPK14* was also up-regulated under ABA and salt stimuli (Figures 6 and 7). The biosynthesis of ABA could be induced by drought, leading to stomatal closure [17,39]. In *Arabidopsis*, the AtCPK4 and AtCPK11 are partially involved in ABA-induced stomatal closure, and the double mutant lost more water from leaves compared to single mutants [17]; gene *ClCDPK14*, as their closed homolog in watermelon, was also activated by drought (Figure 6). Additionally, gene *AtCPK6* has been proven to be involve in the response to drought and salt stress as a positive regulator [40]. Intriguingly, *ClCDPK5* in watermelon, sharing the highest sequence similarity with *AtCPK6*, was also up-regulated by drought (or salt) and ABA stresses, indicating that they might function in similar pathways. In this study, the transcription levels of most *ClCDPKs* were increased by exogenous ABA (Figure 7), similar to the expression trend in grape but different to that in *Cucumis* species [9,11,19].

In watermelon, transcription levels of four genes (*ClCDPK1*, *ClCDPK5*, *ClCDPK16*, and *ClCDPK17*) were down-regulated by cold treatment, while only one gene (*ClCRK2*) was continuously up-regulated at all treatment times (Figure 6). Moreover, complex expression patterns were also observed under cold stimuli, such as *ClCRK5* and *ClCRK6*. Three up-regulated genes (*ClCDPK1*, *ClCDPK2*, and *ClCDPK5*) and one down-regulated gene (*ClCDPK18*) under continuous drought treatment in this study have also been detected and regarded as different expression genes in our previous study [41]. Following salt treatment, gene *ClCDPK6* was significantly up-regulated at all treatment times, which is similar to its ortholog *CsCDPK14* in cucumber but in contrast with its ortholog *CmCDPK9* in melon [9,11]. Additionally, two pairs of segmental duplications were detected in watermelon: *ClCDPK7*/*ClCDPK8* and *ClCDPK6*/*ClCDPK16* (Table S5). Interestingly, *ClCDPK7* and *ClCDPK8* had similar expression patterns under most treatments, while expression tendency of *ClCDPK6* was usually opposite to *ClCDPK16* (Figures 6–8), inferring that genes *ClCDPK6* and *ClCDPK16* may have undergone sub-functionalization after duplication.

Generally, *CDPK* genes are ubiquitously expressed in plant organs, with some showing organ- or tissue-specific expression [19,21]. In the present study, most identified *ClCDPKs* and *ClCRKs* could be detected in at least one tissue, with at least five genes showing extremely high expression levels in specific organs (Figure 5). For instance, *ClCDPK17* in locus 24 showed a high expression level in the male flower, similar to its ortholog *CmCDPK6* in melon, which preferentially accumulated in male flowers [11]. In *Arabidopsis*, both genes *AtCPK17* and *AtCPK20* are reported to be preferentially expressed in mature pollen to regulate the growth of pollen tubes [21,42]. Interestingly, their phylogenetically-close homologs *ClCDPK6* and *ClCDPK16* in watermelon, as well as that in melon (*CmCDPK9* and *CmCDPK5*) and cucumber (*CsCDPK14* and *CsCDPK9*) (Figure S7), were also detected with high transcriptional abundances in male organs [9,11], indicating their conserved and important roles in the development of male flowers.
