Plant architecture is a critical agronomic trait in watermelon (Citrullus lanatus), with vine length directly influencing planting density, light interception, and field management efficiency. Short-vine forms have become important agronomic targets in breeding due to their advantages of high-density planting, efficient light utilization, and simplified field management. In this study, a dwarf mutant, designated PKH207, was identified from an ethyl methanesulfonate (EMS)-mutagenized population of the watermelon inbred line G42. The mutant exhibited significantly reduced plant height and shortened internodes due to decreased cell expansion in stem tissues. Genetic analysis indicated that the dwarf phenotype in PKH207 is controlled by a single recessive gene, which was named Cldw2 (Citrullus lanatus dwarf mutant 2). Using a population of 558 F2 plants, bulked segregant analysis sequencing (BSA-seq) and linkage mapping delimited the causal locus to a 540.6 kb region on chromosome 10. Within this interval, a single-nucleotide polymorphism (SNP) mutation was identified in the gene ClG42_10g0100600, encoding an alpha-tubulin, and this gene was determined to be the candidate gene for the dwarf phenotype. Transcriptome analysis revealed that this mutation significantly disrupts key biological processes, including cell wall biosynthesis, microtubule cytoskeleton organization, and auxin signaling pathways, contributing to the dwarfism phenotype. This study identifies a novel dwarfing allele in cucurbits and provides a direct molecular target for breeding compact watermelon cultivars suited to high-density production.

Background Type-B response regulator (ARR-B) genes act as positive regulators of cytokinin signal transduction and exert essential functions in plant growth, development, and abiotic stress tolerance. Nevertheless, systematic genome-wide identification and functional characterization of the ARR-B gene family have not been comprehensively performed in watermelon, an economically important crop vulnerable to diverse abiotic stresses. Results In this study, a total of 10 ARR-B family genes were identified from the watermelon reference genome, and their physicochemical properties, gene structures, conserved cis-elements, tissue expression patterns and abiotic stress response characteristics were systematically analyzed using bioinformatics approaches. Subcellular localization prediction showed that nine ClARR-B proteins were localized in the nucleus, while only ClARR - B4 was distributed in the cytoplasm. Chromosome mapping revealed that ClARR-B genes were unevenly distributed on watermelon chromosomes, with five members located on chromosome 6, accounting for the largest number of family members. Multiple cis-acting elements related to growth and development, hormone response, biotic and abiotic stresses were identified in the promoter regions of ClARR-B genes. The qRT-PCR results indicated that ClARR - B1 and ClARR - B2 were highly expressed in watermelon roots, stems, leaves, flowers and fruits, suggesting their critical roles in the growth and development of major watermelon organs. Compared with the control, ClARR-B4 , ClARR-B7 and ClARR-B9 were markedly upregulated under drought stress, while ClARR-B2 and ClARR-B6 displayed the strongest upregulation under salt stress. ClARR-B4 was the most significantly induced by cold stress. Collectively, ClARR-B4 is involved in the responses of watermelon to drought and cold stresses, laying a solid basis for subsequent studies on abiotic stress tolerance. Conclusion A total of ten ARR-B transcription factor family members were identified in watermelon, which are extensively involved in watermelon growth, development and abiotic stress response. This study provides a theoretical basis for further exploring the molecular functions of the ARR-B gene family in watermelon development and stress resistance.

Fusarium wilt, caused by the soil-borne Fusarium oxysporum f.sp. niveum (FON), is a devastating disease constraining global watermelon production. While three physiological races (0, 1 and 2) are established, resistance mechanisms to the foundational race remain largely unexplored. This study first evaluated seedling resistance to FON race 0 across 41 diverse watermelon accessions revealing that wild germplasm (Citrullus amarus) possessed superior resistance with 24.4% classified as highly resistant, whereas cultivated accessions were predominantly susceptible. The resistant accession WM216 and susceptible G42 were selected for in-depth analysis. Physiological assessment demonstrated that WM216 mounted a robust and systemic defence response, characterized by a significant post-inoculation induction of ROS-scavenging enzymes like catalase and peroxidase. Integrated transcriptome and BSA-seq analysis identified a major resistance QTL on chromosome 8 (15-22 Mb). Cross-referencing these datasets pinpointed a single key candidate gene, ClG42_08g0072500, encoding a two-component response regulator (ARR family) implicated in cytokinin signalling. Expression profiling suggested this gene may act as a negative regulator of resistance. Our findings underscore the invaluable resistance alleles present in wild watermelon germplasm, provide a foundational genetic map for FON-0 resistance and deliver critical molecular resources for marker-assisted breeding of durable Fusarium wilt-resistant cultivars.

The uniformity and speed of seedling emergence are particularly important in vegetable industrial seedlings. A higher germination rate can shorten the seedling cycle and increase seedling uniformity in melon (Cucumis melo L.). However, the molecular mechanisms underlying the regulation of seed germination in melon remain largely unknown. Golden2-like (CmGLK) has been identified as a key transcription factor involved in chloroplast development, and we found that it also plays a role in regulating seed germination rate in melon. The near-isogenic line of Cmglk (Cmglk-NIL) and CmGLK knockout mutants in melon exhibited slower seed germination rates than those in wild-type lines, while the overexpression of CmGLK in tomato (Solanum lycopersicum L.) showed significantly increased germination rates. Comparative transcriptome analysis revealed that numerous abscisic acid (ABA)-related genes were involved in seed germination. Furthermore, the Cmglk-NIL and CmGLK knockout mutants exhibited increased sensitivity to exogenous ABA during seed germination, while the CmGLK overexpression lines in tomato showed reduced sensitivity to ABA. Bioinformatics analysis and experimental validation indicated that CmGLK could directly bind to the promoter of the H SUBUNIT OF MG-CHELATASE (CmCHLH) gene, thereby activating its expression. Functional validation via CmCHLH overexpression in tomato resulted in a significantly increased seed germination rate, demonstrating its positive regulatory role in germination. These findings identify a CmGLK-CmCHLH regulatory module that regulates seed germination rate through the ABA pathway in melon. They also enhance our understanding of the mechanisms underlying melon seed germination and provide a reference for seed germination regulation in other crops.

Genotyping by Target Sequencing (GBTS) technology, known for its flexibility, high efficiency, high throughput, and low cost, has been increasingly employed in molecular breeding. However, there is still limited study on the design and development of high-throughput genotyping tools in watermelon. In this study, we identified 112 000 high quality SNPs by analyzing the resequencing data of 43 cultivated watermelon accessions. 11 921 and 6094 SNPs were selected for developing two sets of watermelon liquid-phase chips with different marker densities, named Watermelon 10K and 5K, respectively. Furthermore, the SNPs and Indels of most mapped gene/QTLs for many agronomic important traits in watermelon were also integrated into the two chips for foreground selection. These chips have been tested using GBTS technology in various applications in watermelon. The genotyping of 76 accessions by Watermelon 5K liquid-phase chip showed an average detection rate of 99.28 % and 81.78 % for cultivated and wild watermelon accessions, respectively. This provided enough markers information for GWAS and two significant QTLs, ssc1.1 and ssc1.2, associated with soluble sugar content were detected. Furthermore, BSA-seq analysis for non-lobed leaf and dwarf traits were validated by liquid-phase chips, and the candidate region was consistent with our previous studies. Additionally, we precisely introduced the Cldw1 and Clbl genes into an elite inbred line WT2 using Watermelon 5K for assisted selection, resulting in the development of three new germplasm with good plant architecture. As a high-throughput genotyping liquid-phase SNP array, the Watermelon 10K and 5K chips will greatly facilitate functional studies and molecular breeding in watermelon.

Spines are an important trait of cucumber fruit that affects commercial quality, smoothness, transportation, storage, and pesticide residue retention. Spine Base Size1 (CsSBS1), a C2H2 zinc-finger transcription factor, has been identified as a determinant of cucumber fruit spine size, though its molecular mechanism remains unclear. Here, our study found that exogenous ethylene treatment partially restored the small spine base phenotype in the Cssbs1 near-isogenic line (NIL) S-SB and knockout lines, while the ethylene inhibitor aminoethoxyvinylglycine reduced spine base size in wild-type L-SB plants. Further investigation revealed that CsSBS1 directly bound to the promoter of the ethylene-biosynthesis gene 1-aminocyclopropane-1-carboxylate oxidase 2 (CsACO2) and activated its expression. Silencing CsACO2 through RNA interference inhibited fruit spine base expansion in wild-type plants, whereas enhanced CsACO2 expression resulted in larger spine bases in S-SB plants. Additionally, knockout of CsTTG1 led to smaller fruit spine bases and decreased ethylene release. Genetic and biochemical analyses indicated that CsTTG1 enhances CsSBS1-mediated transcriptional activation of CsACO2. Overall, our results reveal a CsSBS1-CsTTG1 regulatory module that regulates fruit spine base size via CsACO2-mediated ethylene biosynthesis in cucumber.

Photosynthesis serves as the primary source of nutrients synthesized in higher plants, and improving photosynthetic efficiency can significantly increase crop yield and fruit quality. Leaf color mutants represent ideal materials for studying chloroplast development and photosynthesis mechanisms and have been widely characterized in field crops. However, relevant research on watermelon leaf color mutants remains scarce. In this study, we isolated a yellow-green phenotype mutant, PKH352, from an EMS-mutagenized watermelon mutant library. The chlorophyll content and maximal photochemical efficiency in PKH352 were significantly decreased. Genetic analysis showed that the mutated trait was controlled by a single nuclear gene, which was named Clygp (Citrullus lanatus yellow-green plant). Through MutMap and linkage analysis in an F2 population of 440 plants, we identified a single nucleotide polymorphism (SNP) mutation within ClG42_04g0106300, which encoded a signal recognition particle 54 kDa protein, as the causal variant for the yellow-green phenotype. Further validation using a CRISPR/Cas9-mediated system confirmed that knockout of ClG42_04g0106300 results in the yellow-green phenotype in watermelon. In addition, comparative transcriptomic analysis revealed that mutations in ClG42_04g0106300 greatly affected the expression of key genes associated with chloroplast development and photosynthesis, providing strong evidence that this gene plays a critical role in these biological pathways. Taken together, these findings provide insights into the molecular mechanisms underlying chloroplast development and photosynthetic efficiency, offering a theoretical basis for breeding watermelon varieties with high photosynthetic efficiency.

The dwarf structure is an important agronomic trait in watermelon, which can increase plant density and is useful for lab-saving cultivation. Cldw-1 encodes an ABCB transporter protein controlling plant height in watermelon, making it useful in dwarf breeding. However, the regulatory mechanism of Cldw-1 in dwarf formation remains unclear. In this study, a donor parent WM102 carrying the Cldw-1 gene was crossed with a recurrent parent WT4, resulting in BC1F1, BC2F1, and BC2F2 populations. In each generation, the Cldw-1 co-segregating marker dCAPS3 was used for foreground selection and 108 polymorphic SSR markers were used for background selection to develop the near isogenic lines (NIL) of Cldw-1. We ultimately obtained a dw-NIL1 in BC2F2 with a proportion of recurrent parent genome (PRPG) of 98.50 % in WT4 background. Compared to WT4, the plant height, internode number, internode length and tendril length was significantly reduced in the dw-NIL1. Cytological observations indicated that the reduction in cell length lead to shorter stems, while an increase in cell number resulted in thicker stems in the dw-NIL1. Transcriptome analysis of the stems of WT4 and dw-NIL1 identified 518 differentially expressed genes (DEGs) with 392 up-regulated and 126 down-regulated. Among them, a number of DEGs were related to auxin signaling pathway, which are known to regulate plant height. Cell wall biosynthesis and modification, as well as protein modifications, were significantly enriched. These results not only provide important germplasms for dwarf breeding, but also will be helpful for better understanding the molecular mechanisms of plant height development in watermelon.

Watermelon is an economically important horticultural crop cultivated worldwide. The ERF family is one of the largest transcription factor families in plants and plays an extensive role in regulating growth, development, hormone signaling, and stress responses. Although the ERF family has been characterized in various plant species, it remains unexplored in watermelon. In this study, we identified 118 ClERF family members in Citrullus lanatus and classified them into ten subgroups. We conducted comprehensive analyses of their physicochemical properties, evolutionary relationships, chromosomal locations, collinearity, gene structures, conserved motifs, and promoter cis-regulatory elements. Furthermore, we comprehensively analyzed the expression patterns of subgroup VIII genes and transcriptional responses to abiotic stresses (cold, salt), a biotic stress (Fusarium wilt), and hormone treatments (ABA, MeJA, SA, BR and ETH). Crucially, functional analysis demonstrated that overexpression of ClERF054 significantly enhanced plant susceptibility to cold and salt stresses. This research establishes a theoretical foundation for understanding the ClERF gene family in watermelon and provides important insights into the functional divergence of subgroup VIII members in hormone signaling and biotic/abiotic stress responses, while also facilitating investigations into the regulatory mechanisms of ClERF054 under cold and salt stresses.

Senescence is a complex biological process coordinately regulated by multiple genes at the molecular level. Deciphering its regulatory mechanisms holds significant potential for enhancing crop yield and stress resistance. However, the study on identification of senescence-related genes in watermelon has been limited by low genetic diversity. In this study, we identified an early-senescence watermelon inbred line, WM103, which displayed a pale green phenotype at the seedling stage that transitions to yellow at maturity. Genetic analysis indicated the early-senescence phenotype was controlled by a single recessive gene. Combined by BSA-seq and linkage analysis in a large F2 population, we identified Cla97C10G186360 as the candidate gene, which encoded a BALANCE OF CHLOROPHYLL METABOLISM (ClBCM) protein. Further functional validation through virus-induced gene silencing and CRISPR/Cas9-mediated knockout confirmed that the down-regulation and loss of function of ClBCM can accelerate senescence. RNA-seq analysis revealed that the ClBCM was involved in the chlorophyll metabolism pathway, and these chlorophyll degradation-related genes were significantly up-regulated in WM103. Molecular interaction assays revealed a direct physical interaction between ClBCM and ClSGR. Furthermore, we found WRKY family transcription factors were significantly enriched in differentially expressed genes. In vivo and in vitro experiments showed ClWRKY53 directly bound to the ClBCM promoter and suppressed its transcription, thereby promoting chlorophyll degradation and senescence. These findings provide novel insights into the molecular regulation of senescence in watermelon and establish a theoretical framework for genetic improvement of fruit yield and stress tolerance in cucurbit crops.

The early flowering and less lateral branches in watermelon hold significant agricultural value. The synergistic effects of these traits provide an ideal template for watermelon plant architecture improvement. However, the molecular regulatory networks underlying the development of lateral organs (including branches and flowers) in watermelon remain unclear. In this study, we found ClTFL1 knockout lines significantly promote flowering time and inhibit lateral branching and tendril formation, while also leading to a mild apical flower phenotype. These findings indicate that the function of ClTFL1 in watermelon is more extensive than that of its homologous genes in Arabidopsis, rice, and tomato. Through yeast two-hybrid screening, we identified the interacting proteins of ClTFL1, including members of the 14-3-3 family ClGRF8, ClGRF9, and ClGRF12. Bimolecular fluorescence complementation (BiFC) assays further demonstrated ClTFL1 could directly interact with the ClGRF8, ClGRF9, and ClGRF12 protein. The knockout of ClGRF8 and ClGRF12 leads to reduced lateral branches and early flowering. These phenotypes are highly consistent with those of ClTFL1 knockout mutants. Our findings demonstrate the important role of the ClTFL1-ClGRFs module in regulating lateral organ development and flowering time in watermelon, offering important targets for watermelon plant architectural modification and molecular breeding.

During the pre-anthesis stage, high chlorophyll levels in petals result in a green hue in many plants. Upon anthesis, chlorophyll degradation uncovers other pigments, thereby influencing pollinator attraction and reproductive success in insect-pollinated crops. In the watermelon accession WM109, a novel petal phenotype characterized by a yellow-green hue was observed, significantly different from the canonical yellow petal phenotype typically associated with this species. Genetic analysis using F2 populations revealed that this yellow-green petal trait is controlled by a single recessive gene. By screening SSR primer pairs with the constructed yellow and yellow-green DNA pools and genotyping F2 individuals, the responsible gene was mapped to a 139.7 kb interval on watermelon chromosome 11, containing two candidate genes. Through sequence analysis, expression profiling, and functional verification of these candidates, a gene encoding a MYB transcription factor with a base insertion was identified as the key determinant of this unusual phenotype, which is characterized by elevated chlorophyll levels and increased chloroplast density. Given the limited current knowledge regarding the relationship between MYB transcription factors and chlorophyll biosynthesis, these findings enhance our understanding of the molecular mechanisms underlying chlorophyll production.

The color and pattern of watermelon rind are crucial external traits that directly affect consumer preferences. Watermelons with stripes having a stronger color than the background rind are ideal for studying stripe patterns in plants, while there is still limited knowledge about the genetic mechanisms underlying stripe coloration due to the lack of germplasm resources. In this study, we focused on a watermelon germplasm with colorless stripes, and genetic analysis revealed that the trait is controlled by a single recessive gene. The gene Clsc (Citrullus lanatus stripe coloration), which is responsible for the colorless stripe, was localized into a 147.6 kb region on Chr9 by linkage analysis in a large F2 mapping population. Further analysis revealed that the Cla97C09G175170 gene encodes the APRR2 transcription factor, plays a crucial role in determining the watermelon colorless stripe phenotype and was deduced to be related to chlorophyll synthesis and chloroplast development. Physiological experiments indicated that Cla97C09G175170 may significantly influence chloroplast development and chlorophyll synthesis in watermelon. The results of this study provide a better understanding of the molecular mechanism of stripe coloration in watermelon and can be useful in the development of marker-assisted selection (MAS) for new watermelon cultivars.

Plant trichome development is influenced by diverse developmental and environmental signals, but the molecular mechanisms involved are not well understood in most plant species. Fruit spines (trichomes) are an important trait in cucumber (Cucumis sativus L.), as they affect both fruit smoothness and commercial quality. Spine Base Size1 (CsSBS1) has been identified as essential for regulating fruit spine size in cucumber. Here, we discovered that CsSBS1 controls a season-dependent phenotype of spine base size in wild-type plants. Decreased light intensity led to reduced expression of CsSBS1 and smaller spine base size in wild-type plants, but not in the mutants with CsSBS1 deletion. Additionally, knockout of CsSBS1 resulted in smaller fruit spine base size and eliminated the light-induced expansion of spines. Overexpression of CsSBS1 increased spine base size and rescued the decrease in spine base size under low light conditions. Further analysis revealed that ELONGATED HYPOTCOTYL5 (HY5), a major transcription factor involved in light signaling pathways, directly binds to the promoter of CsSBS1 and activates its expression. Knockout of CsHY5 led to smaller fruit spine base size and abolished the light-induced expansion of spines. Taken together, our study findings have clarified a CsHY5-CsSBS1 regulatory module that mediates light-regulated spine expansion in cucumber. This finding offers a strategy for cucumber breeders to develop fruit with stable appearance quality under changing light conditions.

The SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box genes encode a plant-specific transcription factor family that plays pivotal roles in various growth and developmental processes, including phase transition, flowering time, and plant architecture. Although the SPL gene family has been identified and characterized in several species, the studies of SPL genes and their function in watermelon are still unknown. The watermelon genome comprises a total of 15 ClSPL genes. Through comprehensive analysis of the physicochemical properties, gene structures, chromosome locations, conserved motifs, collinearity, and expression patterns of these ClSPLs, we observed diverse and specific characteristics among them. Notably, the genes exhibited diverse spatial-temporal expression patterns. Target prediction analysis confirmed that 6 out of the 15 ClSPLs are targeted by miRNA156b in watermelon. Quantitative PCR analysis further confirmed inhibitory effect of ClmiR156b on ClSPL9. Knocking out ClSPL9 in watermelon resulted in growth retardation, characterized by delayed flowering, reduced internode count, decreased plant height, and fewer lateral branches at the stem base, indicating the essential role of ClSPL9 in watermelon growth. Overall, this research provides a comprehensive understanding of the SBP-box gene family in watermelon and establishes a crucial foundation for future investigations into the functionality and evolution of SPL genes in watermelon.

Watermelon (Citrullus lanatus) is a widely cultivated cucurbitaceae crop appreciated by consumers worldwide. However, the long vine and abundant lateral branches of currently cultivated watermelon varieties hinder light simplification and mechanized cultivation, affecting plant spacing and row spacing requirements. To address this, the development of watermelon with dwarf and branchless traits has become a crucial direction for the industry. In previous studies, the genes controlling dwarf (Cldw-1) and branchless (Clbl) traits were mapped and cloned. Marker-assisted selection markers, dCAPS3 and dCAPS10, were developed for these traits, respectively. In this study, the dwarf germplasm WM102 and the branchless germplasm WCZ were crossed to obtain F1 .Further self-crossing of the F1 individuals resulted in the F2 population. Through multiple generations of self-pollination, a new watermelon germplasm DM with double mutation (dwarf and branchless) was obtained. DM exhibited stable inheritance without segregation. Moreover, DM was used as a donor parent for crossing with commercial watermelon materials, and near-isogenic lines (NILs) with the dwarf and branchless traits were developed. These NILs carry additional desirable agronomic traits and provide valuable genetic resources for future watermelon breeding programs, particularly in improving plant architecture and overall quality. The development and application of DM and NILs hold great potential for advancing the watermelon industry toward industrialization, large-scale cultivation, and enhanced plant architecture.

Leaf is a vital organ of plants that plays an essential role in photosynthesis and respiration. As an important agro-nomic trait in leaf development, leaf shape is classified into lobed, entire (no-lobed), and serrated in most crops. In this study, two-lobed leaf watermelon inbred lines WT2 and WCZ, and a no-lobed leaf watermelon inbred line WT20 were used to create two F2 populations. Segregation analysis suggested that lobed leaves were dominant over the no-lobed leaves, and it was controlled by a signal gene. A locus on watermelon chromosome 4 controlling watermelon lobed/no-lobed leaves was identified through BSA-seq strategy combined with linkage analysis. The candidate gene was fine-mapped to a 61.5 kb region between 21,224,481 and 21,285,957 bp on watermelon chro-mosome 4 using two F2 populations. Four functional genes were annotated in the candidate region, while sequences blast showed that there was a single-base deletion (A/-) only in the exon of Cla018360, which resulted in premature termination of translation in the no-lobed leaf lines. Function prediction showed that Cla018360 encodes an HD-Zip protein that has been reported to regulate the development of leaf shape. The single-base deletion also occurred in the HD-Zip domain. We inferred that the Cla018360 gene is the candidate gene for reg-ulating the development of lobed/no-lobed leaves in watermelon. Gene expression analysis showed that Cla018360 was highly expressed in young leaves. Phylogenetic analysis showed that Cla018360 had a close genetic relationship with AtHB51, which had been reported to regulate the formation of leaf shape in Arabidopsis. Furthermore, transcriptome analysis showed that a total of 333 differentially expressed genes were identified between WT2 and WT20, of which 115 and 218 genes were upregulated and downregulated in no-lobed leaved watermelon WT20. This study not only provides a good entry point for studying leaf development but also pro-vides foundational insights into breeding for special plant architecture in watermelon.

Rind pattern is one of the most important appearance qualities of watermelon, and the mining of different genes controlling rind pattern can enrich the variety of consumer choices. In this study, a unique intermittent rind stripe was identified in the inbred watermelon line WT20. The WT20 was crossed with a green stripe inbred line, WCZ, to construct F2 and BC1 segregating populations and to analyze the genetic characterization of watermelon stripe. Genetic analysis showed that the intermittent stripe was a qualitative trait and controlled by a single dominant gene, ClIS. Fine mapping based on linkage analysis showed that the ClIS gene was located on the 160 Kb regions between 25.92 Mb and 26.08 Mb on watermelon chromosome 6. Furthermore, another inbred watermelon line with intermittent stripe, FG, was re-sequenced and aligned on the region of 160 Kb. Interestingly, only two SNP variants (T/C, A/T) were present in both WT20 and FG inbred lines at the same time. The two SNPs are located in 25,961,768 bp (T/C) and 25,961,773 bp (A/T) of watermelon chromosome 6, which is located in the promoter region of Cla019202. We speculate that Cla019202 is the candidate gene of ClIS which controls the intermittent stripe in watermelon. In a previous study, the candidate gene ClGS was proved to control dark green stripe in watermelon. According to the verification of the two genes ClIS and ClGS in 75 watermelon germplasm resources, we further speculate that the ClGS gene may regulate the color of watermelon stripe, while the ClIS gene regulates the continuity of watermelon stripe. The study provides a good entry point for studying the formation of watermelon rind patterns, as well as providing foundation insights into the breeding of special appearance quality in watermelon.

The stripe pattern is an important agronomic trait in watermelon, which determines the fruit rind pattern and consumer choice. However, the genes controlling these traits are still largely unknown. In the present study, a dark-green stripe inbred line WT2 and a netted stripe inbred line WM204 were used for genetic analysis, which revealed that the dark-green stripe is controlled by a single dominant gene ClGS. By bulked-segregant analysis (BSA), the ClGS was primarily mapped on watermelon chromosome 6 by using F2 plants developed from a cross between WT2 and WM204. Next-generation sequencing-aided marker discovery and a large mapping population consisting of 1206 F2 plants was used for fine mapping of the ClGS gene, and it was further mapped into a 107 kb candidate region. There were 11 genes predicated in this candidate region and 10 of them were differentially expressed in the 1-DAP fruit rind between two parental lines. Furthermore, 64 SNPs and 3 Indels were detected in the CDS region of these candidate genes. To further confirm the candidate gene of ClGS, we investigated the sequence variations among 74 re-sequenced natural watermelon accessions by in silico bulk segregant analysis. A 3-bp insertion was identified in all the non-dark green stripe watermelon accession group, which was located on the 8th exon of Cla019205. A Indel marker developed harboring the insertion showed co-segregation with the phenotype in the F2 mapping population, and it was also in completely agreement with another 25 watermelon accessions by electrophoretic analysis. These evidences suggested Cla019205 is probably the candidate gene controlling dark-green stripe in watermelon. The results of this study will be helpful for better understanding of the stripe formation and marker-assisted selection in watermelon.