*2.2. Structure Variation Analysis of CpCDPK19 and CpCDPK27*

As mentioned above, CpCDPK19 and CpCDPK27 in *C. pepo* harbored nine and eight EF-hand motifs, respectively. Previous research indicates that *Cucurbita*, containing two sub-genomes A and B, originated from two progenitors that diverged from one another approximately 30.75 million years ago (Mya) [26]. Sequence analysis showed that CpCDPK5 and CpCDPK13, sharing the highest similarity with CpCDPK19 (amino acid identity: 93.74%) and CpCDPK27 (amino acid identity: 95.84%), contained four and three EF-domains, respectively (Table S2). Dot plot analysis indicated that the EF-hand domains were duplicated in CpCDPK19 compared to CpCDPK5, while both STKs\_CAMK protein kinase and EF-hand domains were repeated in CpCDPK27 in contrast to CpCDPK13 (Figure S1). Based on the synteny analysis (Figure 1a), we inferred that a fragment containing four EF-hand motifs in CpCDPK19 was entirely duplicated and inserted into the STKs\_CAMK protein kinase domain, and the fifth EF-hand motif resulted from another duplication event. Compared to ClCDPK13, ClCDPK27 harbored three copies of STKs\_CAMK protein kinase domain. Moreover, the first EF-hand domain located in the second STKs\_CAMK protein kinase, as well as the second and fourth EF-hand domains, were originated from the sixth EF-hand domain, while the third (existed in the third STKs\_CAMK protein kinase) and the fifth EF-hand domains were two copies of the seventh motif (Figure 1b). Using the Cucurbit Genomics Database (CuGenDB) [29], both two genes CpCDPK19 and CpCDPK27 could be detected in the fruits of two different materials at transcriptional level (Figure S2). In summary, self-duplication of conserved domains may contribute to the gene structure variations, and result in sub-functionalization or neo-functionalization.

neo-functionalization.

CpCDPK27

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 4 of 19

domains may contribute to the gene structure variations, and result in sub-functionalization or

**Figure 1.** Gene structure variation analysis of CpCDPK19 and CpCDPK27. Syntenic analysis of CpCDPK5 and CpCDPK19 (**a**), as well as CpCDPK12 and CpCDPK27 (**b**). Blue and green rectangles represent STKs\_CAMK protein kinase and EF-hand domain, respectively. **Figure 1.** Gene structure variation analysis of CpCDPK19 and CpCDPK27. Syntenic analysis of CpCDPK5 and CpCDPK19 (**a**), as well as CpCDPK12 and CpCDPK27 (**b**). Blue and green rectangles represent STKs\_CAMK protein kinase and EF-hand domain, respectively. **Figure 1.** Gene structure variation analysis of CpCDPK19 and CpCDPK27. Syntenic analysis of CpCDPK5 and CpCDPK19 (**a**), as well as CpCDPK12 and CpCDPK27 (**b**). Blue and green rectangles

represent STKs\_CAMK protein kinase and EF-hand domain, respectively.

#### *2.3. Construction of an Integrated Map for CDPK and CRK Genes 2.3. Construction of an Integrated Map for CDPK and CRK Genes*

As the genomic distribution of *CDPKs* and *CRKs* in *Cucumis* [9,11], all identified genes in this study were also unevenly distributed on chromosomes in five species, with a few chromosomes harboring none of them (Figure S3). A recent study has confirmed that there are twenty chromosomes in *Cucurbita* species and nineteen of them could be divided into two sub-genomes A and B, except for chromosome 04, consisting of two segments from sub-genome A and one from sub-genome B [26]. Interestingly, almost equal numbers of *CDPKs* and *CRKs* were found to be retained in two sub-genomes (Table S3), consistent with the similar evolved ratios of gene loss or gain of two sub-genomes after polyploidization [26]. As the genomic distribution of *CDPKs* and *CRKs* in *Cucumis* [9,11], all identified genes in this study were also unevenly distributed on chromosomes in five species, with a few chromosomes harboring none of them (Figure S3). A recent study has confirmed that there are twenty chromosomes in *Cucurbita* species and nineteen of them could be divided into two sub-genomes A and B, except for chromosome 04, consisting of two segments from sub-genome A and one from sub-genome B [26]. Interestingly, almost equal numbers of *CDPKs* and *CRKs* were found to be retained in two sub-genomes (Table S3), consistent with the similar evolved ratios of gene loss or gain of two sub-genomes after polyploidization [26]. *2.3. Construction of an Integrated Map for CDPK and CRK Genes*  As the genomic distribution of *CDPKs* and *CRKs* in *Cucumis* [9,11], all identified genes in this study were also unevenly distributed on chromosomes in five species, with a few chromosomes harboring none of them (Figure S3). A recent study has confirmed that there are twenty chromosomes in *Cucurbita* species and nineteen of them could be divided into two sub-genomes A and B, except for chromosome 04, consisting of two segments from sub-genome A and one from sub-genome B [26]. Interestingly, almost equal numbers of *CDPKs* and *CRKs* were found to be retained in two sub-genomes (Table S3), consistent with the similar evolved ratios of gene loss or gain of two sub-genomes after polyploidization [26].

The evolutionary scenario of Cucurbitaceae paleohistory hypothesizes that the modern chromosomal structures in cucurbits were derived from an ancestral Cucurbitaceae karyotype (ACK) that consisted of 12 protochromosomes and experienced different times of chromosomal fission and fusion events [27]. Hence, to investigate the conserved loci of *CDPK* and *CRK* in Cucurbitaceae, an integrated map was constructed using watermelon chromosomes as reference, including 16 *CDPK* and nine *CRK* loci (Figure 2). Of these, nine loci (four *CDPK* and five *CRK*) exhibited Presence/Absence polymorphism among genomes, while the remaining 16 loci (12 *CDPK* and four *CRK*) were conserved and shared by all seven species (Figure 2 and Table S4). For example, the first locus on chromosome 01 was lost in *L. siceraria*, while the second locus on chromosome 02 was only absent in watermelon genome. The evolutionary scenario of Cucurbitaceae paleohistory hypothesizes that the modern chromosomal structures in cucurbits were derived from an ancestral Cucurbitaceae karyotype (ACK) that consisted of 12 protochromosomes and experienced different times of chromosomal fission and fusion events [27]. Hence, to investigate the conserved loci of *CDPK* and *CRK* in Cucurbitaceae, an integrated map was constructed using watermelon chromosomes as reference, including 16 *CDPK* and nine *CRK* loci (Figure 2). Of these, nine loci (four *CDPK* and five *CRK*) exhibited Presence/Absence polymorphism among genomes, while the remaining 16 loci (12 *CDPK* and four *CRK*) were conserved and shared by all seven species (Figure 2 and Table S4). For example, the first locus on chromosome 01 was lost in *L. siceraria*, while the second locus on chromosome 02 was only absent in watermelon genome. The evolutionary scenario of Cucurbitaceae paleohistory hypothesizes that the modern chromosomal structures in cucurbits were derived from an ancestral Cucurbitaceae karyotype (ACK) that consisted of 12 protochromosomes and experienced different times of chromosomal fission and fusion events [27]. Hence, to investigate the conserved loci of *CDPK* and *CRK* in Cucurbitaceae, an integrated map was constructed using watermelon chromosomes as reference, including 16 *CDPK* and nine *CRK* loci (Figure 2). Of these, nine loci (four *CDPK* and five *CRK*) exhibited Presence/Absence polymorphism among genomes, while the remaining 16 loci (12 *CDPK* and four *CRK*) were conserved and shared by all seven species (Figure 2 and Table S4). For example, the first locus on chromosome 01 was lost in *L. siceraria*, while the second locus on chromosome 02 was only absent in watermelon genome.

**Figure 2.** An integrated map of *CDPK* and *CRK* loci in Cucurbitaceae. All *CDPK* and *CRK* genes in Cucurbitaceae were mapped onto 11 chromosomes of watermelon 97103. Loci from *C. lannatus* (red), **Figure 2.** An integrated map of *CDPK* and *CRK* loci in Cucurbitaceae. All *CDPK* and *CRK* genes in Cucurbitaceae were mapped onto 11 chromosomes of watermelon 97103. Loci from *C. lannatus* (red), **Figure 2.** An integrated map of *CDPK* and *CRK* loci in Cucurbitaceae. All *CDPK* and *CRK* genes in Cucurbitaceae were mapped onto 11 chromosomes of watermelon 97103. Loci from *C. lannatus* (red), *C. melon* (light green), *C. sativus* (dark green), *C. maxima* (pink), *C. moschata* (yellow), *C. pepo* (dark yellow), and *L. siceraria* (blue) have been marked with different colors, as indicated in brackets. Black dots and triangles represent *CDPK* and *CRK* loci, respectively.
