Evolutionary gain of oligosaccharide hydrolysis and sugar transport enhanced carbohydrate partitioning in sweet watermelon fruits.

How raffinose (Raf) family oligosaccharides, the major translocated sugars in the vascular bundle in cucurbits, are hydrolyzed and subsequently partitioned has not been fully elucidated. By performing reciprocal grafting of watermelon (Citrullus lanatus) fruits to branch stems, we observed that Raf was hydrolyzed in the fruit of cultivar watermelons but was backlogged in the fruit of wild ancestor species. Through a genome-wide association study, the alkaline alpha-galactosidase ClAGA2 was identified as the key factor controlling stachyose and Raf hydrolysis, and it was determined to be specifically expressed in the vascular bundle. Analysis of transgenic plants confirmed that ClAGA2 controls fruit Raf hydrolysis and reduces sugar content in fruits. Two single-nucleotide polymorphisms (SNPs) within the ClAGA2 promoter affect the recruitment of the transcription factor ClNF-YC2 (nuclear transcription factor Y subunit C) to regulate ClAGA2 expression. Moreover, this study demonstrates that C. lanatus Sugars Will Eventually Be Exported Transporter 3 (ClSWEET3) and Tonoplast Sugar Transporter (ClTST2) participate in plasma membrane sugar transport and sugar storage in fruit cell vacuoles, respectively. Knocking out ClAGA2, ClSWEET3, and ClTST2 affected fruit sugar accumulation. Genomic signatures indicate that the selection of ClAGA2, ClSWEET3, and ClTST2 for carbohydrate partitioning led to the derivation of modern sweet watermelon from non-sweet ancestors during domestication.

Comprehensive Profiling of Alternative Splicing and Alternative Polyadenylation during Fruit Ripening in Watermelon (Citrullus lanatus).

Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening.

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation.

Watermelon (Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid (ABA) signaling pathway gene ClSnRK2.3 might influence watermelon fruit ripening. However, the molecular mechanisms are unclear. Here, we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4 . Furthermore, we determined that the pyrophosphate-dependent phosphofructokinase (ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase (ClGA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4 . Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein (ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9'-cis-epoxycarotenoid dioxygenase 3 (ClNCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4 . Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.

ClZISO mutation leads to photosensitive flesh in watermelon.

KEY MESSAGE: The mutation of ClZISO identified in EMS-induced watermelon leads to photosensitive flesh in watermelon. Watermelon (Citrullus lanatus) has a colorful flesh that attracts consumers and benefits human health. We developed an ethyl-methanesulfonate mutation library in red-fleshed line '302' to create new flesh color lines and found a yellow-fleshed mutant which accumulated ζ-carotene. The initial yellow color of this mutant can be photobleached within 10 min under intense sunlight. A long-term light-emitting diode (LED) light treatment turned flesh color from yellow to pink. We identified this unique variation as photosensitive flesh mutant ('psf'). Using bulked segregant analysis, we fine-mapped an EMS-induced G-A transversion in 'psf' which leads to a premature stop codon in 15-cis-ζ-carotene isomerase (ClZISO) gene. We detected that wild-type ClZISO is expressed in chromoplasts to catalyze the conversion of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene. The truncated ClZISOmu protein in psf lost this catalytic function. Light treatment can partially compensate ClZISOmu isomerase activity via photoisomerization in vitro and in vivo. Transcriptome analysis showed that most carotenoid biosynthesis genes in psf were downregulated. The dramatic increase of ABA content in flesh with fruit development was blocked in psf. This study explores the molecular mechanism of carotenoid biosynthesis in watermelon and provides a theoretical and technical basis for breeding different flesh color lines in watermelon.

Natural variation in the NAC transcription factor NONRIPENING contributes to melon fruit ripening.

The NAC transcription factor NONRIPENING (NOR) is a master regulator of climacteric fruit ripening. Melon (Cucumis melo L.) has climacteric and non-climacteric fruit ripening varieties and is an ideal model to study fruit ripening. Two natural CmNAC-NOR variants, the climacteric haplotype CmNAC-NORS,N and the non-climacteric haplotype CmNAC-NORA,S , have effects on fruit ripening; however, their regulatory mechanisms have not been elucidated. Here, we report that a natural mutation in the transcriptional activation domain of CmNAC-NORS,N contributes to climacteric melon fruit ripening. CmNAC-NOR knockout in the climacteric-type melon cultivar "BYJH" completely inhibited fruit ripening, while ripening was delayed by 5-8 d in heterozygous cmnac-nor mutant fruits. CmNAC-NOR directly activated carotenoid, ethylene, and abscisic acid biosynthetic genes to promote fruit coloration and ripening. Furthermore, CmNAC-NOR mediated the transcription of the "CmNAC-NOR-CmNAC73-CmCWINV2" module to enhance flesh sweetness. The transcriptional activation activity of the climacteric haplotype CmNAC-NORS,N on these target genes was significantly higher than that of the non-climacteric haplotype CmNAC-NORA,S . Moreover, CmNAC-NORS,N complementation fully rescued the non-ripening phenotype of the tomato (Solanum lycopersicum) cr-nor mutant, while CmNAC-NORA,S did not. Our results provide insight into the molecular mechanism of climacteric and non-climacteric fruit ripening in melon.

A unique chromosome translocation disrupting ClWIP1 leads to gynoecy in watermelon.

To understand sex determination in watermelon (Citrullus lanatus), a spontaneous gynoecious watermelon mutant, XHBGM, was selected from the monoecious wild type XHB. Using map-based cloning, resequencing and fluorescence in situ hybridization analysis, a unique chromosome translocation between chromosome 2 and chromosome 3 was found in XHBGM. Based on the breakpoint location in chromosome 2, a putative C2H2 zinc finger transcription factor gene, ClWIP1 (gene ID Cla008537), an orthologue of the melon gynoecy gene CmWIP1, was disrupted. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system 9 to edit ClWIP1, we obtained gynoecious watermelon lines. Functional studies showed that ClWIP1 is expressed specifically in carpel primordia and is related to the abortion of carpel primordia in early floral development. To identify the cellular and metabolic processes associated with ClWIP1, we compared the shoot apex transcriptomes of two gynoecious mutants and their corresponding wild types. Transcriptome analysis showed that differentially expressed genes related to the ethylene and cytokinin pathways were upregulated in the gynoecious mutants. This study explores the molecular mechanism of sex determination in watermelon and provides a theoretical and technical basis for breeding elite gynoecious watermelon lines.

Decreased Protein Abundance of Lycopene ß-Cyclase Contributes to Red Flesh in Domesticated Watermelon.

Red-fleshed watermelons (Citrullus lanatus) that accumulate lycopene in their flesh cells have been selected and domesticated from their pale-fleshed ancestors. However, the molecular basis of this trait remains poorly understood. Using map-based cloning and transgenic analysis, we identified a lycopene ß-cyclase (ClLCYB) gene that controls the flesh color of watermelon. Down-regulation of ClLCYB caused the flesh color to change from pale yellow to red, and ClLCYB overexpression in the red-fleshed line caused the flesh color to change to orange. Analysis of ClLCYB single-nucleotide polymorphisms using 211 watermelon accessions with different flesh colors revealed that two missense mutations between three haplotypes (ClLCYB red , ClLCYB white , and ClLCYB yellow ) were selected and largely fixed in domesticated watermelon. Proteins derived from these three ClLCYB haplotypes were localized in plastids to catalyze the conversion of lycopene to ß-carotene and showed similar catalytic abilities. We revealed that ClLCYB protein abundance, instead of ClLCYB transcript level, was negatively correlated with lycopene accumulation. Different amounts of ClLCYB protein degradation among the ClLCYB haplotypes were found in ClLCYB transgenic Arabidopsis (Arabidopsis thaliana) lines. After treatment with the proteasome inhibitor MG132, the concentration of ClLCYBred increased noticeably compared with other ClLCYB proteins. These results indicate that natural missense mutations within ClLCYB influence ClLCYB protein abundance and have contributed to the development of red flesh color in domesticated watermelon.

Localization shift of a sugar transporter contributes to phloem unloading in sweet watermelons.

Unloading sugar from sink phloem by transporters is complex and much remains to be understood about this phenomenon in the watermelon fruit. Here, we report a novel vacuolar sugar transporter (ClVST1) identified through map-based cloning and association study, whose expression in fruit phloem is associated with accumulation of sucrose (Suc) in watermelon fruit. ClVST197 knockout lines show decreased sugar content and total biomass, whereas overexpression of ClVST197 increases Suc content. Population genomic and subcellular localization analyses strongly suggest a single-base change at the coding region of ClVST197 as a major molecular event during watermelon domestication, which results in the truncation of 45 amino acids and shifts the localization of ClVST197 to plasma membranes in sweet watermelons. Molecular, biochemical and phenotypic analyses indicate that ClVST197 is a novel sugar transporter for Suc and glucose efflux and unloading. Functional characterization of ClVST1 provides a novel strategy to increase sugar sink potency during watermelon domestication.

A Tonoplast Sugar Transporter Underlies a Sugar Accumulation QTL in Watermelon.

How sugar transporters regulate sugar accumulation in fruits is poorly understood and particularly so for species storing high-concentration Suc. Accumulation of soluble sugars in watermelon (Citrullus lanatus) fruit, a major quality trait, had been selected during domestication. Still, the molecular mechanisms controlling this quantitative trait are unknown. We resequenced 96 recombinant inbred lines, derived from crossing sweet and unsweet accessions, to narrow down the size of a previously described sugar content quantitative trait locus, which contains a putative Tonoplast Sugar Transporter gene (ClTST2). Molecular and biochemical analyses indicated that ClTST2 encodes a vacuolar membrane protein, whose expression is associated with tonoplast uptake and accumulation of sugars in watermelon fruit flesh cells. We measured fruit sugar content and resequenced the genomic region surrounding ClTST2 in 400 watermelon accessions and associated the most sugar-related significant single-nucleotide polymorphisms (SNPs) to the ClTST2 promoter. Large-scale population analyses strongly suggest increased expression of ClTST2 as a major molecular event in watermelon domestication associated with a selection sweep around the ClTST2 promoter. Further molecular analyses explored the binding of a sugar-induced transcription factor (SUSIWM1) to a sugar-responsive cis-element within the ClTST2 promoter, which contains the quantitative trait locus (QTL) causal SNP. The functional characterization of ClTST2 and its expression regulation by SUSIWM1 provide novel tools to increase sugar sink potency in watermelon and possibly in other vegetable and fruit crops.

High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon.

Chromoplast development plays a crucial role in controlling carotenoid content in watermelon flesh. Modern cultivated watermelons with colorful flesh are believed to originate from pale-colored and no-sweet progenitors. But the molecular basis of flesh color formation and regulation is poorly understood. More chromoplasts and released carotenoid globules were observed in the red-fleshed fruit of the 97103 cultivar than in the pale-colored fruits of the PI296341-FR line. Transcriptome profiles of these two materials identified Cla017962, predicted as ClPHT4;2, was dramatically up-regulated during flesh color formation. High ClPHT4;2 expression levels were closely correlated with increased flesh carotenoid contents among 198 representative watermelon accessions. Down-regulation of ClPHT4;2 expression in transgenic watermelons reduced the fruit carotenoid accumulation. ClPHT4;2 as a function of chromoplast-localized phosophate transporter was tested by heterologous expression into a yeast phosphate-uptake-defective mutant, western blotting, subcellular localization, and immunogold electron microscopy analysis. Two transcription factors, ClbZIP1 and ClbZIP2, were identified, which responded to ABA and sugar signaling to regulate ClPHT4;2 transcription only in cultivated watermelon species. Our findings suggest that elevated ClPHT4;2 gene expression is necessary for carotenoid accumulation, and may help to characterize the co-development of flesh color and sweetness during watermelon development and domestication.

Efficient CRISPR/Cas9-based gene knockout in watermelon.

KEY MESSAGE: CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Mutation in the gene encoding 1-aminocyclopropane-1-carboxylate synthase 4 (CitACS4) led to andromonoecy in watermelon.

Although it has been reported previously that ethylene plays a critical role in sex determination in cucurbit species, how the andromonoecy that carries both the male and hermaphroditic flowers is determined in watermelon is still unknown. Here we showed that the watermelon gene 1-aminocyclopropane-1-carboxylate synthase 4 (CitACS4), expressed specifically in carpel primordia, determines the andromonoecy in watermelon. Among four single nucleotide polymorphism (SNPs) and one InDel identified in the coding region of CitACS4, the C364W mutation located in the conserved box 6 was co-segregated with andromonoecy. Enzymatic analyses showed that the C364W mutation caused a reduced activity in CitACS4. We believe that the reduced CitACS4 activity may hamper the programmed cell death in stamen primordia, leading to the formation of hermaphroditic flowers.

An integrated genetic map based on four mapping populations and quantitative trait loci associated with economically important traits in watermelon (Citrullus lanatus).

BACKGROUND: Modern watermelon (Citrullus lanatus L.) cultivars share a narrow genetic base due to many years of selection for desirable horticultural qualities. Wild subspecies within C. lanatus are important potential sources of novel alleles for watermelon breeding, but successful trait introgression into elite cultivars has had limited success. The application of marker assisted selection (MAS) in watermelon is yet to be realized, mainly due to the past lack of high quality genetic maps. Recently, a number of useful maps have become available, however these maps have few common markers, and were constructed using different marker sets, thus, making integration and comparative analysis among maps difficult. The objective of this research was to use single-nucleotide polymorphism (SNP) anchor markers to construct an integrated genetic map for C. lanatus. RESULTS: Under the framework of the high density genetic map, an integrated genetic map was constructed by merging data from four independent mapping experiments using a genetically diverse array of parental lines, which included three subspecies of watermelon. The 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel), 36 structure variation (SV) and 386 SNP markers from the four maps were used to construct an integrated map. This integrated map contained 1339 markers, spanning 798 cM with an average marker interval of 0.6 cM. Fifty-eight previously reported quantitative trait loci (QTL) for 12 traits in these populations were also integrated into the map. In addition, new QTL identified for brix, fructose, glucose and sucrose were added. Some QTL associated with economically important traits detected in different genetic backgrounds mapped to similar genomic regions of the integrated map, suggesting that such QTL are responsible for the phenotypic variability observed in a broad array of watermelon germplasm. CONCLUSIONS: The integrated map described herein enhances the utility of genomic tools over previous watermelon genetic maps. A large proportion of the markers in the integrated map are SSRs, InDels and SNPs, which are easily transferable across laboratories. Moreover, the populations used to construct the integrated map include all three watermelon subspecies, making this integrated map useful for the selection of breeding traits, identification of QTL, MAS, analysis of germplasm and commercial hybrid seed detection.

Identification of allelic relationship and translocation region among chromosomal translocation lines that leads to less-seed watermelon.

Less-seed and seedless traits are desirable characteristics in watermelon (Citrullus lanatus). Hybridization between watermelon chromosomal translocated lines and wild lines significantly reduced seed counts in the hybrid fruits, approaching even seedless. However, the allelic relationships and the chromosomal translocation breakpoints from different sources are unclear, which limits their utility in breeding practices. This study focused on three groups of chromosomal translocation materials from different sources and conducted inheritance and allelic relationship analysis of translocation points. The results from third-generation genome sequencing and fluorescence in situ hybridization (FISH) revealed that the specific translocations in the naturally mutated material MT-a involved reciprocal translocations between Chr6 and Chr10. The Co60γ radiation-induced mutant material MT-b involved reciprocal translocations between Chr1 and Chr5, Chr4 and Chr8. The Co60γ radiation-induced mutant material MT-c involved complex translocations among Chr1, Chr5, and Chr11. Cytological observation showed that heterozygous translocation hybrids showed chromosomal synapsis abnormalities during meiotic diakinesis. Further, dominant and codominant molecular markers were developed on both sides of the translocation breakpoints, which could facilitate rapid and efficient identification of chromosome translocation lines. This study provides technical guidance for utilizing chromosomal translocation materials in the development of less-seed watermelon varieties.

Quantitative Transcriptomic and Proteomic Analysis of Fruit Development and Ripening in Watermelon (Citrullus lanatus).

Fruit ripening is a highly complicated process, which is modulated by phytohormones, signal regulators and environmental factors playing in an intricate network that regulates ripening-related genes expression. Although transcriptomics is an effective tool to predict protein levels, protein abundances are also extensively affected by post-transcriptional and post-translational regulations. Here, we used RNA sequencing (RNA-seq) and tandem mass tag (TMT)-based quantitative proteomics to study the comprehensive mRNA and protein expression changes during fruit development and ripening in watermelon, a non-climacteric fruit. A total of 6,226 proteins were quantified, and the large number of quantitative proteins is comparable to proteomic studies in model organisms such as Oryza sativa L. and Arabidopsis. Base on our proteome methodology, integrative analysis of the transcriptome and proteome showed that the mRNA and protein levels were poorly correlated, and the correlation coefficients decreased during fruit ripening. Proteomic results showed that proteins involved in alternative splicing and the ubiquitin proteasome pathway were dynamically expressed during ripening. Furthermore, the spliceosome and proteasome were significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, suggesting that post-transcriptional and post-translational mechanisms might play important roles in regulation of fruit ripening-associated genes expression, which might account for the poor correlation between mRNAs and proteins during fruit ripening. Our comprehensive transcriptomic and proteomic data offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of fruit ripening.

Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles.

BACKGROUND: Cultivated watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues. RESULTS: We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. De novo assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression profiles helped elucidating molecular mechanisms governing these important quality-related traits during watermelon fruit development. CONCLUSION: We have generated a large collection of watermelon ESTs, which represents a significant expansion of the current transcript catalog of watermelon and a valuable resource for future studies on the genomics of watermelon and other closely-related species. Digital expression analysis of this EST collection allowed us to identify a large set of genes that were differentially expressed during watermelon fruit development and ripening, which provide a rich source of candidates for future functional analysis and represent a valuable increase in our knowledge base of watermelon fruit biology.

CRISPR/Cas9-mediated mutagenesis of ClBG1 decreased seed size and promoted seed germination in watermelon.

Abscisic acid (ABA) is a critical regulator of seed development and germination. ß-glucosidases (BGs) have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA. However, whether BGs are involved in seed development is unclear. In this study, a candidate gene, ClBG1, in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system. Seed size and weight were significantly reduced in the Clbg1-mutant watermelon lines, which was mainly attributed to decreased cell number resulting from decreased ABA levels. A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination (DAP), respectively. Cytoskeleton- and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1-mutant lines during seed development. Moreover, the expression of genes in the major signaling pathways of seed size control was also changed. In addition, seed germination was promoted in the Clbg1-mutant lines due to decreased ABA content. These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-, cell cycle- and signaling-related genes. Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size, a key agricultural trait of significant economic importance.