**4. Materials and Methods**

## *4.1. Identification and Biochemical Characterization of CDPKs and CRKs*

For genome-wide identification of *CDPK* and *CRK* genes in Cucurbitaceae species, protein sequences of 18 CmCDPKs, seven CmCRKs, and 19 CsCDPKs were obtained according to recently published studies [9,11]. Then, using BLASTp program with an E-value setting of 1.0 <sup>×</sup> <sup>10</sup>−<sup>5</sup> , these protein sequences were used as queries to search against the predicted protein files of watermelon (*C. lanatus*, v1), cucumber (*C. sativus*, v1), *Cucurbita* genus (*C. moschata,* v1; *C. maxima,* v1.1; *C. pepo*, v4.1), and bottle gourd (*L. siceraria*, v1), which were downloaded from the Cucurbit Genomics Database (http://cucurbitgenomics.org/). Additionally, Hidden Markov Model (HMM) profiles of the core protein kinase domain (PF00069) and EF-hand\_7 domain (PF13499) were downloaded from the Pfam database (http://pfam.xfam.org/), and were also subjected to searches for *CDPKs* and *CRKs* with software HMMER 3.0 (default parameters). The reliability of candidates was verified through searching against the NCBI nr database, and all non-redundant putative genes were tested for the presence of core domains using ScanProsite (http://prosite.expasy.org/scanprosite/). Finally, all candidate genes were further characterized with the following online tools: ProtParam (http://web.expasy.org/protparam/), ScanProsite (http://prosite.expasy.org/scanprosite/), SMART (http: //smart.embl-heidelberg.de/), ExPASy (http://web.expasy.org/myristoylator/), and CSS-plam 4.0 (http://csspalm.biocuckoo.org/). Dot plot analysis was performed using the software Geneious (http://www.geneious.com).

### *4.2. Chromosomal Location of CDPK and CRK Genes*

The genomic distributions of *CDPKs* and *CRKs* on chromosomes were drawn using the software TBtools (http://cj-chen.github.io/tbtools/). To construct an integrated map, all identified CDPKs and CRKs, as well as those reported in recent studies [9,11], were mapped onto watermelon chromosomes based on the syntenic relationships of their flanking genes using BLASTp method, with a stricter E-value setting to 1.0 <sup>×</sup> <sup>10</sup>−10. Then, in-house Perl scripts were used to parse the resulting files and to visualize the chromosomal locations of *CDPK* and *CRK* loci.

#### *4.3. Phylogenetic, Gene Structure, and Syntenic Analyses of CDPKs and CRKs*

Full-length CDPK and CRK protein sequences were aligned using the software Muscle [43], and then were used to construct phylogenetic trees via MEGA 6.0 using the neighbor-joining method with 1000 bootstrap replicates [11]. To perform gene structure analyses, genomic and cDNA sequences of *CDPKs* and *CRKs* were obtained from their corresponding genomes. Then, the exon-intron organization was displayed via the online tool GSDS 2.0 (http://gsds.cbi.pku.edu.cn/). The destination tabular (-m 8) files of the BLASTp program (with an E-value setting of 1.0 <sup>×</sup> <sup>10</sup>−10) and GFF profiles served as input documents for MCScanX to analyze the synteny relationships [44], which were then visualized using software CIRCOS (http://circos.ca/).

#### *4.4. Plant Material and Treatments*

The watermelon inbred line "Y34" provided by the Cucurbits Germplasm Resource Research Group at the Northwest A&F University in China was used in this study. For tissue-specific analysis, germinated seeds of watermelon "Y34" were directly sown in the experimental base, and six organs (roots, stems, leaves, tendrils, and both male and female flowers) were independently sampled during the fruit maturation period (approximately 60–70 days after sowing). For stress experiments, seedlings were cultured in plastic pots (8 cm × 7 cm × 7 cm) filled with commercial peat-based compost (Shaanxi Yufeng Seed Industry Co., Ltd., Yangling, China). All plants were grown under springtime natural light with temperatures of 28–35 ◦C/16–20 ◦C (day/night) in a greenhouse, which were uniformly watered and nourished weekly with half-strength Hoagland's solution before treatments. Four weeks after sowing, seedlings were used for the following treatments.

For the salinity treatment, seedlings irrigated with 300 mM NaCl solution (80 mL per plant) were sampled at 6, 24, 48, 72, 96, and 120 h post-treatment (hpt), while plants irrigated with distilled water were used as control. Compared to seedlings planted in a growth chamber at 27 ± 1 ◦C and 80% humidity under a light intensity of 300 mmol·m−<sup>2</sup> ·s <sup>−</sup><sup>1</sup> PPFD, leaves of plants kept at 4 ◦C were sampled at 1, 3, 6, 12, 24, and 48 hpt for cold treatment. To simulate a national drought treatment [45], all seedlings were uniformly well-watered to 70 ± 5% field capacity based on their weight. Then, leaves of drought-treated (unwatered) and control plants were sampled at 24, 48, 96, and 192 hpt. Leaves sprayed with 100 µM abscisic acid (ABA) [46], 1 mM salicylic acid (SA) [47], 100 µM methyljasmonate (MeJA), and 10 mM ethephon (ETH) [48] were collected at 0.5, 1, 6, 12, 24, and 48 hpt for hormone treatments, while control seedlings were only sprayed with equal volumes of the corresponding solution without hormones. In this study, leaves of four plants were pooled at each time point for each treatment with three biological replicates, which were immediately frozen in liquid nitrogen and stored at −80 ◦C until further analysis.

## *4.5. RNA Isolation and qRT-PCR*

The total RNA of samples was extracted using the RNASimple Total RNA Kit (TIANGEN, China) following the manufacturer's instructions. Then, approximately 1 µg of total RNA was used to synthesize the first strand of cDNA using the FastKing RT Kit with gDNase (TIANGEN, China). Gene-specific primers for *ClCDPKs* and *ClCRKs* are listed in Table S1. Amplification was conducted in a 20 µL reaction volume, containing 10.0 µLSYBR Green Premix, 0.8 µL of each primer (10 µM), and 1.0 µL cDNA template (80 ng/µL), which was diluted with ddH2O to 20 µL. The PCR conditions consisted of pre-denaturing at 95 ◦C for 5 min, followed by 40 cycles of 95 ◦C for 10 s and 60 ◦C for 30 s. The watermelon β*-actin* gene (*Cla007792*) was used as the internal control gene [49]. Each treatment was repeated thrice, and all data were calculated for relative expressions following the 2−∆∆Ct method, as described by Livak and Schmittgen [50]. The relative expressions were then log2 transformed and visualized in a heat map using Mev 4.8.1 (http://www.mybiosoftware.com/). All data were analyzed via IBM SPSS Statistics 21 and values were presented as the means ± SD of three biological and three technical replicates (Table S7). The significance of expression between treatments and controls was evaluated by one-way ANOVA and Duncan's multiple range tests.

## **5. Conclusions**

In the present study, a total of 128 *CDPK* and 56 *CRK* genes were identified in six Cucurbitaceae species. Using the watermelon genome as reference, an integrated map containing 25 loci (16 *CDPK* and nine *CRK* loci) was obtained, 16 of which (12 *CDPK* and four *CRK*) were shared by all seven Cucurbitaceae species. Combined with exon-intron organizations, topological analyses indicated an ancient origination of groups CDPK IV and CRK, which will contribute to elucidating the evolutionary history of these two gene families in Cucurbitaceae. Moreover, expression patterns of *ClCDPKs* and *ClCRKs* under different abiotic stresses were also performed in this study. Importantly, comparative analyses indicated a subset of valuable orthologous genes for future functional characterizations. For instance, both*ClCDPK6* and its ortholog*CsCDPK14* in cucumber could be induced by salinity, while *ClCDPK6* and *ClCDPK16*, as well as their orthologs in *Cucumis*, maintained high expression levels in male flowers, which may play important roles in plant organ development and response to environmental stresses.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/10/ 2527/s1.

**Author Contributions:** C.W. and X.Z. designed the study. C.W., R.Z., X.Y., and C.Z. contributed to the experiments and data analysis. J.M. provide the seed for the experiment. Y.Z. and J.Y. provided guidance throughout the study. C.W. wrote, and H.L. revised the manuscript. All authors reviewed and approved the final manuscript.

**Funding:** This work was supported by National Key R&D Program of China (2018YFD0100704), Scientific Startup Foundation for Doctors of Northwest A&F University (Z109021604), National Natural Science Foundation of China Grant No. 31701939, and the Modern Agro-industry Technology Research System of China (No. CARS-26-18).

**Acknowledgments:** We wish to thank all the reviewers and editors for their careful reading and helpful comments on this manuscript.

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