**5. Conclusions**

In this study, a total of 422 PPR protein genes were identified in the watermelon genome. Based on the PPR motif type, *ClaPPR* genes were divided into five subgroups. Most of the genes were intronless and distributed widely across all watermelon chromosomes, and encoding proteins were targeted to organelles of chloroplast or mitochondria which gives valuable information for future studies on characterization of *ClaPPR* genes. Duplication analyses suggested that 11 segmentally duplicated *ClaPPR* pairs exist in the genome. We conducted an in-silico expression pattern analysis in watermelon fruit rind and flesh tissues. In addition, a comparative expression study was performed in the fruit ripening stages of red- and pale yellow-fleshed watermelons, which provides preliminary understanding about *ClaPPR* participation in fruit development. Based on sequence variation analyses of *ClaPPR* genes, four CAPS markers were developed and found to have co-segregation with distinct flesh types, which could be used to distinguish different flesh colors, including red, yellow, and orange. Taken together, the findings of this study provide comprehensive understanding of the *ClaPPR* gene family and clarify candidate *ClaPPR* genes for functional validation in the future.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/10/1125/s1, Figure S1. Conserved motifs in *ClaPP*R proteins using MEME Suite, Figure S2. Conserved motifs of *ClaPPR* proteins. The consensus sequence of motif logos shown in the left panel. The number of motifs in the different subfamilies and/or subgroups of the *ClaPPR* proteins are shown in the right panel, Figure S3. Subcellular localizations of *ClaPPR* genes, using on-line tools of TargetP 2.0 and Predotarv.1.04. The locations of each *ClaPPR* subfamily and its subgroups in the chloroplast (blue), mitochondria (orange), endoplasmic reticulum (ER) (grey), and other locations (yellow) were determined, Figure S4. GO enrichment analysis of *ClaPPR* genes. (A) Graphical results of biological process. (B) Graphical results of molecular function, Figure S5. A comparative expression profiles of *ClaPPR* genes during watermelon fruit development between red-fleshed (LSW177) and pale-yellow-fleshed cultivated watermelons (BioProject: PRJNA338036). The heat map with clustering (labeled as roman numerals) was generated based on the log2- transformed FPKM value of *ClaPPR* genes at developmental stages of 10, 18, 26, 34, and 42 days after pollination (DAP). (A) Expression profiles of *ClaPPR* genes in the P subfamily, (B–F) Expression profiles of *ClaPPR* genes in the PLS subfamily of subgroups (B) DYW, (C) E2, (D) PLS, (E) E1, and (F) E+. Differences in transcript abundances such as high (red) and low (blue) levels are shown in color as the scale, Table S1. List of watermelon commercial cultivars or inbred lines used in this study for CAPS marker validation and their representative fruit characteristics, Table S2. Detailed information of PPR genes in watermelon genome, Table S3. Segment duplication of PPR genes in watermelon genome, Table S4. Subcellular localization of predicted PPR genes in watermelon, Table S5. Details of GO annotation of *ClaPPR* genes, Table S6. Log2 FPKM values for *ClaPPR* genes in different fruit ripening stages of two cultivated watermelon varieties with red-fleshed (LSW-177) and pale-yellow-fleshed (COS), Table S7. Identification of putative sequence variation (SNPs) between lines with flesh-colored (red, yellow, and orange) watermelons from the WGRS data, Table S8. Summary of result analysis of match rate between validated *ClaPPR* CAPS markers and SNPs in 70 watermelons.

**Author Contributions:** Conceptualization, S.S. and G.-J.L.; data curation, S.S.; formal analysis, S.S., and G.-J.L.; funding acquisition, Y.-P.L. and G.-J.L.; investigation, L.T., K.L., G.-S.P., H.L., and J.-P.C.; methodology, S.S., C.M., and G.-J.L.; project administration, G.-J.L.; resources, M.-W.P.; software, S.S.; visualization, S.S.; writing—original draft, S.S.; writing—review and editing, C.M. and G.-J.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Golden Seed Project (Center for Vegetable Seed Development, No. 213003-05-1-SBW30), Ministry of Agriculture, Food and Rural Affairs (MAFRA) and Basic Science Research Program (NRF-2020R1A2C1015119) through the National Research Foundation (NRF) of Korea funded by the Ministry of Science and ICT.

**Acknowledgments:** We thank Ian Small, The University of Western Australia, for the technical assistance in classification of PPR genes in this study.

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