Watermelon is a diploid (2n = 2x = 22) vegetative fruit crop belonging to the family Cucurbitaceae and genus Citrullus. The global watermelon cultivation area and production are 4,459,239 ha and 161,837,308 tons, respectively, which account for approximately 10% of the major vegetable production (Food and Agriculture Organization, 2020). Watermelon is native to Africa and has been found in temperate and Mediterranean regions outside Africa (Jeffrey, 1975; Paris, 2015). After more than 4,000 years of domestication, wild-type watermelon species with small fruit size, firm flesh, and bitter taste were improved to modern cultivars such as Citrullus lanatus (Thunb.) Matsum. & Nakai, which have consumable traits, including the large size, thin rind, and sweet red flesh (Paris, 2015). The whole-genome sequencing (WGS) was completed for ‘97103,’ a Chinese cultivar (Ren et al., 2012; Guo et al., 2013), and ‘Charleston Gray,’ a US heirloom (Wu et al., 2019), which revealed an approximate genome size of 410 Mb and 22,500 genes (Cucurbit Genome Database, CuGenDB, http://cucurbitgenomics.org).

The fruit flesh color of watermelon is categorized as red (scarlet or coral red), yellow (canary, salmon, or pale yellow), orange, and white (UPOV). Different flesh colors result from the accumulation of specific carotenoid components in flesh (Grassi et al., 2013). The accumulation of all-trans-lycopene (lycopene) and xanthophylls (zeaxanthin, neoxanthin, and violaxanthin) in the chromoplasts results in red and canary yellow flesh colors, whereas the accumulation of ξ-carotene and prolycopene or ß-carotene results in the salmon yellow and orange flesh colors, respectively (Tadmor et al., 2005; Perkins-Veazie et al., 2006; Bang et al., 2010; Jin et al., 2019). White flesh is resulted from the accumulation of phytofluene, a colorless carotenoid, and light-yellow-colored ξ-carotene (Zhao et al., 2013).

Watermelon flesh color is genetically ascertained by several loci (genes) involved in the carotenoid biosynthesis pathways. According to Henderson (1989), three alleles (Y, y ^o , and y) at the Y locus are associated with coral red, orange with a high prolycopene content, and salmon yellow flesh color. Coral red, regulated by locus Y, is dominant over the other two colors. Lou (2009) reported that scarlet red and coral red are regulated by the Y ^SCR and Y alleles, respectively, and Y ^SCR was dominant over all other alleles of the Y locus (Y ^SCR > Y > y ^o > y). Contrarily, a major QTL on Chr. 1 is responsible for orange flesh with a high ß-carotene content, which is dominant to scarlet red flesh (Branham et al., 2017).

The canary yellow flesh (CY) color in watermelon is conferred by the C locus, which is dominant over the red (scarlet or coral red) flesh color (Henderson et al., 1996). The lycopene ß-cyclase (LCYB) gene of the C locus catalyzes the conversion of lycopene to ß-carotene, but its mutant allele suppresses LCYB gene function, leading to the accumulation of lycopene and the appearance of red flesh (Bang et al., 2007; Bang et al., 2014). However, Henderson et al. (1998) described that the homozygous recessive i-C locus results in red flesh irrespective of the presence of the C allele, but the gene responsible for i-C is unknown. In addition, the homozygous recessive py locus prevents the formation of canary yellow flesh but develops pale yellow flesh (Bang et al., 2010). In certain genetic backgrounds, red and yellow colors appear as a mixed pattern in the same flesh. Although Bang et al. (2007) reported a complete dominant control for canary yellow flesh, incomplete yellow (mixed with red) flesh appears frequently in F₁ and segregates in the progeny of crosses between red and canary yellow flesh cultivars, which implies that unknown genetic factors other than the C locus may be involved in the determination of canary yellow. Based on our knowledge, there are no research reports on the genetic control of the mixed color pattern in incomplete yellow flesh.

For the marker-assisted selection of fruit flesh color, LCYB gene-based markers have been developed to distinguish red from canary yellow (Bang et al., 2007). Jin et al. (2019) also suggested the development of a CRTISO gene-based marker for orange flesh with high prolycopene content. Recently, Li et al. (2020) and Lee et al. (2021) described the markers distinguishing scarlet and coral red. However, no markers have been reported for selecting incomplete yellow, which is essential for breeding canary yellow- or red-fleshed watermelons.

Whole-genome resequencing (WGRS) based on next-generation sequencing (NGS) provides comprehensive genome sequence variations that are ideal for identifying DNA markers and genes associated with phenotypic variations (Meuwissen and Goddard, 2010; Saidi and Hajibarat, 2020). The WGRS of a large number of watermelon accessions and genome-wide association study (GWAS) revealed sequence polymorphisms and genes for fruit-related traits, such as fruit shape, flesh color, rind patterns (Park et al., 2018; Subburaj et al., 2019), and disease resistances, including gummy stem blight (Lee et al., 2018). The WGRS of near-isogenic lines successfully revealed a locus related to the high lycopene content in a scarlet red-fleshed watermelon cultivar (Lee et al., 2021).

The present study aimed to identify the genetic control responsible for incomplete yellow flesh in watermelon. Using the WGRS and linkage mapping approach, we identified a novel locus named C ₂ that controls incomplete yellow flesh, which is required for the development of canary yellow flesh in addition to the previously reported locus C. The molecular markers for C ₂ established in this study will be beneficial for MAS of canary yellow or red-fleshed watermelons.

Differences in the flesh color of watermelons are mostly due to the composition and amount of carotenoids. The selection of specific flesh color is an important breeding target in terms of the consumer’s preferences and nutritional aspects. Previous studies have suggested that those genes involved in the metabolism of carotenoids are associated with various colors of watermelon flesh. The canary yellow flesh (CY) is regulated by the dominant C locus, and a homozygous recessive mutation of C results in red (scarlet and coral red) flesh (Bang et al., 2010). Molecular studies have characterized the C locus as the gene Cllcyb (Cla97C04G070940) encoding LCYB (Wang P. et al., 2019; Wang C. et al., 2019; Zhang et al., 2020), and gene-based markers have been used for the MAS of canary yellow and red flesh. However, some watermelon inbred lines with the Cllcyb marker genotype of dominant homozygosity (CC) have been found to show a mixture of CY and red flesh (incomplete canary yellow, ICY). Although it has been suggested that environmental variables might contribute to ICY (Bang et al., 2007), no genetic factors that are involved in the expression of ICY are currently known.

In this study, to identify possible genetic factors determining ICY and CY, we compared genome sequences of six ICY and CY inbred lines to identify genomic regions specific to this trait (ICY-specific genomic region) and mapped this region using an F₂ population. A whole-genome sequence comparison of the ICY and CY inbred groups identified a genomic region containing 66 genes on Chr. 2 that was polymorphic between these groups. Genetic inheritance analysis using an F₂ population indicated that ICY is controlled by the homozygous recessive alleles at a single locus C ₂ , named in this study. Genetic linkage mapping using a set of CAPS markers derived from the SNPs in the ICY-specific genomic region showed that these markers are tightly linked to C ₂ . In this map, a CAPS (M7) derived from a non-synonymous SNP in the gene encoding PPR cosegregated with the flesh color of 135 F₂ plants, possibly implying that ClPPR (Cla97C02G039880) may be a putative candidate gene for C ₂ . Furthermore, genotyping of 28 watermelon accessions using the two Cllcyb gene-based markers and ClPPR-based CAPS markers (M7) indicated that all CY inbreds require dominant alleles for both C and C ₂ and that c ₂ is dominantly epistatic to C.

Bang et al. (2010) identified that the pale yellow flesh color in watermelon is due to a locus distinct from i-C, indicating the involvement of at least two loci in the variation of canary yellow, pale yellow, and red flesh. This resonates with our findings on the influence of gene interactions in the expression of ICY. Bang et al. (2010) also found that the expression of pale yellow flesh required a dominant allele at the C locus, analogous to the necessity for that allele in the expression of ICY, with the presence of a necessary allele at the C ₂ locus. We observed that all CY plants demonstrated dominant homozygosity or heterozygosity for both the LCYB and the C ₂ locus (PPR gene). However, the presence of a recessive homozygosity at the C ₂ locus (c ₂ c ₂ ) indicates an epistatic relationship over the C locus gene, partially masking the effect of the LCYB gene for canary yellow flesh. In essence, our study is consistent with the multi-gene influences and epistatic relationships found by Bang et al. (2010), highlighting the complex gene interactions influencing watermelon flesh color.

PPRs are a diverse group of proteins predominantly present in plants (Lurin et al., 2004), play crucial roles in organelle formation, plant growth and development (Sun et al., 2018), and expression of cytoplasmic male sterility (CMS) (Jo et al., 2016), and affect fruit development and flesh color formation (Eriksson et al., 2004). Moreover, PPRs are linked to the early chloroplast formation and plastid gene expression (Pyo et al., 2013). Protein deficiency of PPR generally caused embryo development delay in maize (Sun et al., 2019; Yuan et al., 2019), abnormal chloroplast development, and aberrant pigmentation in seeds in Arabidopsis (Bryant et al., 2011). The significance of PPR proteins lies in their role as essential proteins that bind to RNA and can regulate gene expression in multiple post-transcriptional processes, including shearing, RNA editing, splicing, degradation, and translation in the chloroplasts (Wang et al., 2021a), plastids (Wang et al., 2021b), and mitochondria (Binder et al., 2013). The PPR protein family has been categorized into two subfamilies, namely, P type and PPR-like long and short (PLS) type (Lurin et al., 2004). Several studies have demonstrated that PPR proteins belonging to these subfamilies, such as the P-type PPR protein ACM1 (Wang et al., 2021b) and the PLS-type PPR protein MORF9 (Yan et al., 2017), have an impact on plastid development. Some PPR members in the P-subfamily of watermelon (ClPPR) had high expression levels in the fruit flesh, fruit rind, or both rind and flesh tissues, indicating that they might be crucial in coordinating the development of flesh and rind in watermelons (Subburaj et al., 2020). In contrast, the majority of subgroups in the PLS-subfamily showed preferential expression in the rind; high expression levels were observed at all fruit development stages for rind, but only at 10-day post anthesis (DPA) for flesh. Subburaj et al. (2020) suggested that these PLS-subfamily genes may play significant roles in the fruit rind and early fruit flesh development of watermelon. Taken together, these outcomes suggest that the ClPPR genes are likely involved in fruit development of watermelon including chloroplast-to-chromoplast transition in fruit flesh (Subburaj et al., 2020).

Subburaj et al. (2020) identified and characterized 422 ClPPR genes in the watermelon genome, and out of them, four ClPPR genes are associated with orange, two genes with yellow, and one gene with red flesh color among marker genotypes and phenotypes of orange, yellow, and red flesh color in all surveyed lines. Various studies suggested that ClaPPR11 co-localized with ß-carortene-related QTL on Chr.1 (Branham et al., 2017), whereas ClaPPR140 co-localized with lycopene-related QTL on Chr.4 (Liu et al., 2015). However, ClaPPR25 and ClaPPR95 were not co-localized with any QTL for flesh color. Similarly, another PPR member ClPPR (Cla97C06G122120) on Chr. 6 has been also reported as a candidate gene for the locus Cyf that regulates the carotenoid accumulation level in watermelon (Liu et al., 2023). The mutant allele of Cyf was responsible for pale yellow flesh (Liu et al., 2023). However, the molecular mechanism for genetic control of the PPR on fruit flesh color formation is not yet fully elucidated. PPR proteins have so far been discovered to be cis-element-recognizing site-specific RNA factors in the plastid (Okuda et al., 2006). PPR (CRR4) specifically those nucleotide sequences nearby cis-acting element of the ndhD-1 site, allowing the scaffold for RNA editing (conversion of C to U) and likely making it easier for the putative RNA editing enzyme to access these sequences (Okuda et al., 2006). The deficiency of a single PPR protein can have an impact on RNA editing at multiple locations in the gene, consequently influencing gene expression within the plastid (Okuda et al., 2006). In a recent study by Galpaz et al. (2018), it was found that CmPPR1 participates in RNA editing processes within plastids, which are responsible for the accumulation of carotenoids and chlorophyll, consequently giving rise to white, orange, and green flesh formation in melon. It has also been suggested that the CmPPR1 gene might be involved in retrograde signaling from plastids to the nucleus, influencing the expression of plastid-targeted genes, exhibiting that PPR proteins are involved in the variation of fruit flesh color (Galpaz et al., 2018).

Plastids can be categorized into three groups based on their pigments: chloroplasts, chromoplasts, and leucoplasts. The development of chloroplasts through the involvement of PPR proteins has been reported by numerous studies (Bryant et al., 2011; Pyo et al., 2013). The ClPPR gene might be involved in chromoplast development, carotenoid accumulation, and flesh color regulation as well (Liu et al., 2023). However, there is limited research available on the role of PPR proteins in chromoplast development and the formation of flesh color in watermelon. Notably, there are noticeable differences between globular and crystalloid chromoplasts in terms of their structure and color. The presence of red flesh in watermelon is primarily associated with the presence of crystalloid plastid structures, whereas white, yellow, or orange flesh indicates the presence of globular chromoplasts (Fang et al., 2020). These structural differences are likely attributed to variations in pigment components. In a study by Fang et al. (2020), a direct relationship was observed between the formation of crystalloid chromoplasts and lycopene accumulation. Conversely, excessive accumulation of violaxanthin, lutein, γ-carotene, and ß-carotene led to the development of globular chromoplasts, and they have a potential role in the regulation of carotenoid accumulation and flesh color. The chromoplast’s plastoglobulus serves as a specialized structure responsible for the mechanism of producing and storing carotenoids (Brehelin and Kessler, 2008). The PPR gene may specifically influence the number of plastoglobuli, thereby contributing to differences in flesh color. Fang et al. (2020) observed a progressive increase in the accumulation of carotenoids during the fruit flesh development of pale yellow-fleshed ‘COS’ and canary yellow-fleshed ‘PI 635597’watermelon varieties. This increase in carotenoid accumulation was accompanied by an increase in both the quantity and size of chromoplasts and plastoglobuli. Importantly, they reported that the size and number of plastoglobuli were significantly greater in PI 635597 than those in COS, suggesting that the enlarged size and number of plastoglobuli may contribute to the formation of canary yellow flesh color.

In conclusion, we identified a novel locus, C ₂ , which is required for CY flesh color in watermelon in addition to the previously known C locus cloned as the Cllcyb. Our genetic mapping results indicated that C ₂ is located on Chr. 2, and ClPPR (Cla97C02G039880) encoding a member of PPR protein is a putative candidate gene for the locus. Using a ClPPR-based CAPS developed in this study and Cllcyb-based markers, watermelon cultivars with CY, ICY, and red flesh could be successfully discerned, implying that the combined use of these markers will be efficient for MAS of flesh color in watermelon breeding. Nevertheless, the molecular mechanism for the involvement of ClPPR in carotenoid synthesis and its interaction with the Cllcyb remains to be elucidated.