*2.5. Phylogenetic Analysis of PPR Proteins*

A total of 422 PPR protein sequences from watermelon and 44 typical PPR proteins from Arabidopsis, which utilized were in a previous study [3], were used to build a phylogenetic evolutionary tree. The translated sequences of the whole coding regions of PPR proteins were aligned using the MUSCLE method. The tree was constructed using the neighbor-joining (NJ) method with MEGA X software [37] and bootstrap analysis of 1000 replicates.

### *2.6. Expression Pattern of ClaPPR Genes in Watermelon*

To study the expression patterns of *ClaPPR* genes in 97103 watermelon, the transcriptome RNA-seq data (BioProject: SRP012849) from the cucurbit expression atlas (http://cucurbitgenomics.org/rnaseq/ home) were used [38]. These RNA-seq data contained data on fruit flesh (FF) and fruit rind (FR) at four stages (10, 18, 26, and 34 days after pollination (DAP)) during the development of the watermelon cultivar 97103. Furthermore, to examine the contribution and regulatory roles of *ClaPPR* genes, which are involved in fruit ripening, among different flesh-colored watermelons, the comparative watermelon transcript data (BioProject: PRJNA338036), containing five different fruit ripening stages (10, 18, 26, 34, and 42 DAP) of a pale yellow-flesh cultivated watermelon ('COS') and red-flesh cultivated watermelon ('LSW-177'), were used. DEG (differentially expressed gene) values for corresponding *ClaPPR* were obtained using the list of *ClaPPR* accession numbers. The obtained DEG values were log2 base transformed. The visualization of expression in heatmap and hierarchical clustering was investigated using Cluster 3.0 and TreeView software [39].

### *2.7. Identification of Single Nucleotide Polymorphisms (SNPs) for ClaPPR Genes and Match Rate Analysis with Flesh Color*

Single-nucleotide polymorphisms (SNPs) for *ClaPPR* genes in different flesh-colored (red, yellow, and orange) watermelons were identified from the whole genome resequencing (WGRS) data (Bioproject: PRJNA516776) of our recent study [29]. The SNP variant calling procedure was carried out according to our recent study [29] in which the reads of different flesh-colored watermelons from WGRS were mapped on to the watermelon 97103 reference genome. From this SNP variant calling, we searched for the SNPs that were specific to either of the flesh types such as red, yellow, and orange in the SNP subset matrix by identifying protein-coding *ClaPPR* genes bearing SNPs that were 1) monomorphic among a chosen flesh color-type, 2) monomorphic among other unchosen flesh color-types, and 3) polymorphic

between a chosen and an unchosen flesh color-types. Then, the sequences of selected *ClaPPR* SNP variants were converted into CAPS markers using SNP2CAPS software [40] and simultaneously, primers were designed using Primer3Plus software (http://www.bioinformatics.nl/primer3plus). To validate CAPS markers, genomic DNA extraction, PCR assays, restriction digestion of PCR amplicons, and match rate analyses were carried out according to a previous study [29].
