**1. Introduction**

As a ubiquitous second messenger, Calcium (Ca2+) plays an important role in sophisticated signal transduction pathways to survive frequently occurring environmental stresses during plant growth and development [1,2]. Transient changes of the Ca2<sup>+</sup> concentration in the cytoplasm can be sensed by four types of calcium sensors: calmodulins (CaM), calmodulin-like proteins (CaML), calcineurin B-like proteins (CBL), and the calcium-dependent protein kinase (CDPK) [3,4]. Among these sensors, CDPKs not only sense but also directly translate Ca2<sup>+</sup> signals into a downstream phosphorylation pathway, thus functioning both as Ca2<sup>+</sup> sensors and effectors [2,5,6].

The CDPKs (also referred to as CPKs) are ser/thr protein kinases, which consist of four typical domains: a variable N-terminal domain (containing the myristoylation and palmitoylation sites), a catalytic ser/thr protein kinase domain, an auto-inhibitory domain (acting as a pseudosubstrate combined with the kinase domain to inhibit activity), and a C-terminal regulatory calmodulin-like domain that contains one to four EF-hand motifs for Ca2<sup>+</sup> binding [7–9]. CDPK-related kinases (CRKs) have similar domain structures than CDPKs (such as the ser/thr kinase domain); however, they do not have EF-hand domains [10,11]. To date, genome-wide identification of *CDPKs* has been widely performed in a large number of plants, e.g., 34 *CDPKs* have been identified in *Arabidopsis* [7], 31 in rice [12], 29 in tomato [10], 18 in melon [11], and 19 in cucumber [9]. Similarly, *CRKs* have also been identified in genomes, e.g., eight *CRKs* in *Arabidopsis* [13], five in rice [14], six in tomato [10], five in pepper [15], and seven in melon [11].

Accumulating evidence indicates that *CDPK* and *CRK* genes are not only involved in plant growth and development, but also in the plant response to abiotic and hormone stresses. For example, AtCPK32 in *Arabidopsis* has been reported to interact with the calcium channel protein CNGC18, thus controlling the polar growth of pollen tubes [16]. In addition, *AtCPK4* and its homolog *AtCPK11* have been reported to function in seed germination and growth, stomatal movement, and response to salt stress [17]. The gene *AtCPK23* acts as a negative regulator and plays important roles in response to drought and salt stresses via controlling K<sup>+</sup> channels; its overexpression can increase stomatal apertures [18]. In grape plants, a large number of *CDPKs* have been reported to be induced in response to various abiotic and biotic stresses, as well as to hormone treatments [19]. A previous study showed that the *CDPK* genes in cucumber are extensively regulated by various stimuli, including salt, cold, heat, waterlogging, and abscisic acid stresses, possibly following different mechanisms [9]. Furthermore, *CmCDPKs* and *CmCRKs* in melon were also shown to be differentially expressed in response to exogenous stresses, such as biotic stress (*Podosphaera xanthii* inoculation), abiotic stresses (salt and cold), and hormone (abscisic acid) treatment [11]. In general, *CDPK* genes are ubiquitously expressed in most of the different plant organs. However, several genes show organ- or tissue-specific expression patterns. For instance, *VvCDPK5* in grape plants can only be detected in pollen [20]. Similarly, *AtCPK17* and *AtCPK34* are both preferentially expressed in mature pollen, regulating the growth of pollen tubes [21].

The Cucurbitaceae family contains several economically important species with already published genomes, including watermelon (*Citrullus lanatus*), melon (*Cucumis melon*), cucumber (*Cucumis sativus*), and bottle gourd (*Lagenaria siceraria*), which belong to the Benincaseae tribe and three *Cucurbita* species (*Cucurbita maxima*, *Cucurbita moschata*, and *Cucurbita pepo*) of the Cucurbiteae tribe [22–27]. Although genome-wide identifications of *CDPKs* and *CRKs* have been performed in the genus *Cucumis* [9,11], comparative evolutionary analysis of both gene families in Cucurbitaceae is lacking. In this study, we identified a total of 128 *CDPK* and 56 *CRK* genes in six Cucurbitaceae species (*C. lanatus*, *C. sativus*, *C. moschata*, *C. maxima*, *C. pepo*, and *L. siceraria*). After mapping these identified genes onto chromosomes of watermelon, we obtained an integrated map including 25 loci (16 *CDPK* and nine *CRK* loci). Evolutionary analyses revealed that four CDPK groups and one CRK group in phylogentic trees could be further divided into loci, consistent with the integrated map. In addition, expression patterns of *ClCDPKs* and *ClCRKs* under different abiotic stresses were also analyzed. Our results provide insights into the evolutionary history of both gene families in Cucurbitaceae, and indicate a subset of candidate genes for future functional analysis.

#### **2. Results**

#### *2.1. Genome-Wide Identification of CDPK and CRK Genes*

Our previous study verified 18 *CmCDPK* and seven *CmCRK* genes in melon [11], while only 19 *CDPK* homologs were identified genome-wide in cucumber [9]. Hence, to comparatively analyze the *CRK* gene family in Cucurbitaceae, identification of *CRK* genes in cucumber genome was also performed in this study. As a result, a total of 128 *CDPK* (typically containing both STKs\_CAMK protein kinase and EF-hand domains) and 56 *CRK* (solely harboring STKs\_CAMK protein kinase domain) genes were identified in six Cucurbitaceae species (*C. lanatus*, *C. sativus*, *C. moschata*, *C. maxima*, *C. pepo*, and *L. siceraria*). All the identified genes were designated based on their chromosomal locations (Table S2). It is worth noting that two *CDPKs*, referred to as *CsCDPK18* (*Csa018149*) and *CsCDPK15* (*Csa008536*) in the previous study [9], were renamed to *CsCRK6* and *CsCRK7* due to their lack of EF-hand domains. Compared to the similar copy numbers of *CDPK* and *CRK* genes in four species (*C. lanatus*, *C. melon*, *C. sativus*, and *L. siceraria*) of the Benincaseae tribe (Table 1), many more homologs were identified in three Cucurbiteae tribe genomes (*C. moschata*, *C. maxima*, and *C. pepo*). This may have been caused by the whole-genome duplication (WGD) that only occurred in the progenitor of the *Cucurbita* genus [25,26].


**Table 1.** Numbers and characteristic properties of *CDPKs* and *CRKs* in Cucurbitaceae species.

Physico-chemical properties of CDPKs and CRKs, including predicted amino acids, molecular weight (MW), and isoelectric point (pI), showed similar ranges in watermelon, melon, and cucumber; however, these characteristics exhibited broader intervals in the remaining four species (Table 1). In this study, six CDPKs were predicted to have long (>1000) amino acid sequences, possibly due to their long CT domains [28]. Compared to CRKs with no EF-hand domain, the majority of CDPKs were predicted to contain four EF-hands, with few exceptions harboring two or three EF-hand motifs (Table S2). Strikingly, two CDPKs (CpCDPK19 and CpCDPK27) in *C. pepo* were confirmed to have more than five EF-hand domains via both online tools ScanProsite and SMART. More detailed information, for example for myristoylation and palmitoylation sites, is also provided in Table S2.
