Promoter variations of ClERF1 gene determines flesh firmness in watermelon.

BACKGROUND: Flesh firmness is a critical factor that influences fruit storability, shelf-life and consumer's preference as well. However, less is known about the key genetic factors that are associated with flesh firmness in fresh fruits like watermelon. RESULTS: In this study, through bulk segregant analysis (BSA-seq), we identified a quantitative trait locus (QTL) that influenced variations in flesh firmness among recombinant inbred lines (RIL) developed from cross between the Citrullus mucosospermus accession ZJU152 with hard-flesh and Citrullus lanatus accession ZJU163 with soft-flesh. Fine mapping and sequence variations analyses revealed that ethylene-responsive factor 1 (ClERF1) was the most likely candidate gene for watermelon flesh firmness. Furthermore, several variations existed in the promoter region between ClERF1 of two parents, and significantly higher expressions of ClERF1 were found in hard-flesh ZJU152 compared with soft-flesh ZJU163 at key developmental stages. DUAL-LUC and GUS assays suggested much stronger promoter activity in ZJU152 over ZJU163. In addition, the kompetitive allele-specific PCR (KASP) genotyping datasets of RIL populations and germplasm accessions further supported ClERF1 as a possible candidate gene for fruit flesh firmness variability and the hard-flesh genotype might only exist in wild species C. mucosospermus. Through yeast one-hybrid (Y1H) and dual luciferase assay, we found that ClERF1 could directly bind to the promoters of auxin-responsive protein (ClAux/IAA) and exostosin family protein (ClEXT) and positively regulated their expressions influencing fruit ripening and cell wall biosynthesis. CONCLUSIONS: Our results indicate that ClERF1 encoding an ethylene-responsive factor 1 is associated with flesh firmness in watermelon and provide mechanistic insight into the regulation of flesh firmness, and the ClERF1 gene is potentially applicable to the molecular improvement of fruit-flesh firmness by design breeding.

Mapping and validation of Fusarium wilt race 2 resistance QTL from Citrullus amarus line USVL246-FR2.

KEY MESSAGE: Fon race 2 resistant QTLs were identified on chromosomes 8 and 9. Families homozygous for resistance alleles at a haplotype of three KASP markers had 42% lower disease severity than those with susceptible alleles in an independent, interspecific validation population confirming their utility for introgression of Fusarium wilt resistance. Fusarium oxysporum f. sp. niveum (Fon) race 2 causes Fusarium wilt in watermelon and threatens watermelon production worldwide. Chemical management options are not effective, and no resistant edible watermelon cultivars have been released. Implementation of marker-assisted selection to develop resistant cultivars requires identifying sources of resistance and the underlying quantitative trait loci (QTL), developing molecular markers associated with the QTL, and validating marker-phenotype associations with an independent population. An intraspecific Citrullus amarus recombinant inbred line population from a cross of resistant USVL246-FR2 and susceptible USVL114 was used for mapping Fon race 2 resistance QTL. KASP markers were developed (N = 51) for the major QTL on chromosome 9 and minor QTL on chromosomes 1, 6, and 8. An interspecific F2:3 population was developed from resistance donor USVL246-FR2 (C. amarus) and a susceptible cultivar 'Sugar Baby' (Citrullus lanatus) to validate the utility of the markers for introgression of resistance from the wild crop relative into cultivated watermelon. Only 16 KASP markers segregated in the interspecific C. amarus/lanatus validation population. Four markers showed significant differences in the separation of genotypes based on family-mean disease severity, but together explained only 16% of the phenotypic variance. Genotypes that inherited homozygous resistant parental alleles at three KASP markers had 42% lower family-mean disease severity than homozygous susceptible genotypes. Thus, haplotype analysis was more effective at predicting the mean disease severity of families than single markers. The haplotype identified in this study will be valuable for developing Fon race 2 resistant watermelon cultivars.

Streptomyces-triggered coordination between rhizosphere microbiomes and plant transcriptome enables watermelon Fusarium wilt resistance.

The use of microbial inoculant is a promising strategy to improve plant health, but their efficiency often faces challenges due to difficulties in successful microbial colonization in soil environments. To this end, the application of biostimulation products derived from microbes is expected to resolve these barriers via direct interactions with plants or soil pathogens. However, their effectiveness and mechanisms for promoting plant growth and disease resistance remain elusive. In this study, we showed that root irrigation with the extracts of Streptomyces ahygroscopicus strain 769 (S769) solid fermentation products significantly reduced watermelon Fusarium wilt disease incidence by 30% and increased the plant biomass by 150% at a fruiting stage in a continuous cropping field. S769 treatment led to substantial changes in both bacterial and fungal community compositions, and induced a highly interconnected microbial association network in the rhizosphere. The root transcriptome analysis further suggested that S769 treatment significantly improved the expression of the MAPK signalling pathway, plant hormone signal transduction and plant-pathogen interactions, particular those genes related to PR-1 and ethylene, as well as genes associated with auxin production and reception. Together, our study provides mechanistic and empirical evidences for the biostimulation products benefiting plant health through coordinating plant and rhizosphere microbiome interaction.

The SUMOylation pathway regulates the pathogenicity of Fusarium oxysporum f. sp. niveum in watermelon through stabilizing the pH regulator FonPalC via SUMOylation.

SUMOylation is a key post-translational modification, where small ubiquitin-related modifier (SUMO) proteins regulate crucial biological processes, including pathogenesis, in phytopathogenic fungi. Here, we investigated the function and mechanism of the SUMOylation pathway in the pathogenicity of Fusarium oxysporum f. sp. niveum (Fon), the fungal pathogen that causes watermelon Fusarium wilt. Disruption of key SUMOylation pathway genes, FonSMT3, FonAOS1, FonUBC9, and FonMMS21, significantly reduced pathogenicity, impaired penetration ability, and attenuated invasive growth capacity of Fon. Transcription and proteomic analyses identified a diverse set of SUMOylation-regulated differentially expressed genes and putative FonSMT3-targeted proteins, which are predicted to be involved in infection, DNA damage repair, programmed cell death, reproduction, growth, and development. Among 155 putative FonSMT3-targeted proteins, FonPalC, a Pal/Rim-pH signaling regulator, was confirmed to be SUMOylated. The FonPalC protein accumulation was significantly decreased in SUMOylation-deficient mutant ∆Fonsmt3. Deletion of FonPalC resulted in impaired mycelial growth, decreased pathogenicity, enhanced osmosensitivity, and increased intracellular vacuolation in Fon. Importantly, mutations in conserved SUMOylation sites of FonPalC failed to restore the defects in ∆Fonpalc mutant, indicating the critical function of the SUMOylation in FonPalC stability and Fon pathogenicity. Identifying key SUMOylation-regulated pathogenicity-related proteins provides novel insights into the molecular mechanisms underlying Fon pathogenesis regulated by SUMOylation.

A CRISPR/LbCas12a-based method for detection of bacterial fruit blotch pathogens in watermelon.

Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila, Pseudomonas syringae, and Xanthomonas campestris. The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/µL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli, and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli, is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli.

Fine mapping of ClLOX, a QTL for powdery mildew resistance in watermelon (Citrullus lanatus L.).

KEY MESSAGE: ClLOX, is located on chromosome 2 and encodes a lipoxygenase gene, which induced watermelon powdery mildew resistance by inhibiting pathogen spread. Powdery mildew is one of the most severe fungal diseases reducing yield and quality of watermelon (Citrullus lanatus L.) and other cucurbit crops. Genes responsible for powdery mildew resistance in watermelon are highly valuable. In this study, we first identified the QTL pm-lox for powdery mildew resistance in watermelon, located within a 0.93 Mb interval of chromosome 2, via XP-GWAS method using two F2 populations. The F2:3 families from one of the F2 populations were then used for fine-mapping the pm-lox locus into a 9,883 bp physical region between 29,581,906 and 29,591,789, containing only two annotated genes. Of these, only ClG42_02g0161300 showed a significant differential expression between the resistant and susceptible lines after powdery mildew inoculation based on RNA sequencing (RNA-seq) and qRT-PCR analysis, and is designated ClLOX. Derived Cleaved Amplified Polymorphic Sequence (dCAPs) markers were developed and validated. In addition, our tests showed that the resistance was anti-spread rather than anti-infection of the pathogen. This study identified a new resistance gene (ClLOX), provided insights into the mechanism of powdery mildew resistance, and developed a molecular marker for watermelon breeding.

Genome-Wide Identification and Expression Analysis of Chitinase Genes in Watermelon under Abiotic Stimuli and Fusarium oxysporum Infection.

Chitinases, which catalyze the hydrolysis of chitin, the primary components of fungal cell walls, play key roles in defense responses, symbiotic associations, plant growth, and stress tolerance. In this study, 23 chitinase genes were identified in watermelon (Citrullus lanatus [Thunb.]) and classified into five classes through homology search and phylogenetic analysis. The genes with similar exon-intron structures and conserved domains were clustered into the same class. The putative cis-elements involved in the responses to phytohormone, stress, and plant development were identified in their promoter regions. A tissue-specific expression analysis showed that the ClChi genes were primarily expressed in the roots (52.17%), leaves (26.09%), and flowers (34.78%). Moreover, qRT-PCR results indicate that ClChis play multifaceted roles in the interaction between plant/environment. More ClChi members were induced by Race 2 of Fusarium oxysporum f. sp. niveum, and eight genes were expressed at higher levels on the seventh day after inoculation with Races 1 and 2, suggesting that these genes play a key role in the resistance of watermelon to Fusarium wilt. Collectively, these results improve knowledge of the chitinase gene family in watermelon species and help to elucidate the roles played by chitinases in the responses of watermelon to various stresses.

Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation.

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.

Transcriptome and Metabolome Analyses Reveal That Jasmonic Acids May Facilitate the Infection of Cucumber Green Mottle Mosaic Virus in Bottle Gourd.

Cucumber green mottle mosaic virus (CGMMV) is a typical seed-borne tobamovirus that mainly infects cucurbit crops. Due to the rapid growth of international trade, CGMMV has spread worldwide and become a significant threat to cucurbit industry. Despite various studies focusing on the interaction between CGMMV and host plants, the molecular mechanism of CGMMV infection is still unclear. In this study, we utilized transcriptome and metabolome analyses to investigate the antiviral response of bottle gourd (Lagenaria siceraria) under CGMMV stress. The transcriptome analysis revealed that in comparison to mock-inoculated bottle gourd, 1929 differently expressed genes (DEGs) were identified in CGMMV-inoculated bottle gourd. Among them, 1397 genes were upregulated while 532 genes were downregulated. KEGG pathway enrichment indicated that the DEGs were mainly involved in pathways including the metabolic pathway, the biosynthesis of secondary metabolites, plant hormone signal transduction, plant-pathogen interaction, and starch and sucrose metabolism. The metabolome result showed that there were 76 differentially accumulated metabolites (DAMs), of which 69 metabolites were up-accumulated, and 7 metabolites were down-accumulated. These DAMs were clustered into several pathways, including biosynthesis of secondary metabolites, tyrosine metabolism, flavonoid biosynthesis, carbon metabolism, and plant hormone signal transduction. Combining the transcriptome and metabolome results, the genes and metabolites involved in the jasmonic acid and its derivatives (JAs) synthesis pathway were significantly induced upon CGMMV infection. The silencing of the allene oxide synthase (AOS) gene, which is the key gene involved in JAs synthesis, reduced CGMMV accumulation. These findings suggest that JAs may facilitate CGMMV infection in bottle gourd.

From Fruit Waste to Medical Insight: The Comprehensive Role of Watermelon Rind Extract on Renal Adenocarcinoma Cellular and Transcriptomic Dynamics.

Cancer researchers are fascinated by the chemistry of diverse natural products that show exciting potential as anticancer agents. In this study, we aimed to investigate the anticancer properties of watermelon rind extract (WRE) by examining its effects on cell proliferation, apoptosis, senescence, and global gene expression in human renal cell adenocarcinoma cells (HRAC-769-P) in vitro. Our metabolome data analysis of WRE exhibited untargeted phyto-constituents and targeted citrulline (22.29 µg/mg). HRAC-769-P cells were cultured in RPMI-1640 media and treated with 22.4, 44.8, 67.2, 88.6, 112, 134.4, and 156.8 mg·mL-1 for 24, 48, and 72 h. At 24 h after treatment, (88.6 mg·mL-1 of WRE) cell proliferation significantly reduced, more than 34% compared with the control. Cell viability decreased 48 and 72 h after treatment to 45% and 37%, respectively. We also examined poly caspase, SA-beta-galactosidase (SA-beta-gal), and wound healing activities using WRE. All treatments induced an early poly caspase response and a significant reduction in cell migration. Further, we analyzed the transcript profile of the cells grown at 44.8 mg·mL-1 of WRE after 6 h using RNA sequencing (RNAseq) analysis. We identified 186 differentially expressed genes (DEGs), including 149 upregulated genes and 37 downregulated genes, in cells treated with WRE compared with the control. The differentially expressed genes were associated with NF-Kappa B signaling and TNF pathways. Crucial apoptosis-related genes such as BMF, NPTX1, NFKBIA, NFKBIE, and NFKBID might induce intrinsic and extrinsic apoptosis. Another possible mechanism is a high quantity of citrulline may lead to induction of apoptosis by the production of increased nitric oxide. Hence, our study suggests the potential anticancer properties of WRE and provides insights into its effects on cellular processes and gene expression in HRAC-769-P cells.

Molecular Mapping of Putative Genomic Regions Controlling Fruit and Seed Morphology of Watermelon.

The genetic regulatory basis of qualitative and quantitative phenotypes of watermelon is being investigated in different types of molecular and genetic breeding studies around the world. In this study, biparental F2 mapping populations were developed over two experimental years, and the collected datasets of fruit and seed traits exhibited highly significant correlations. Whole-genome resequencing of comparative parental lines was performed and detected single nucleotide polymorphism (SNP) loci were converted into cleaved amplified polymorphic sequence (CAPS) markers. The screened polymorphic markers were genotyped in segregating populations and two genetic linkage maps were constructed, which covered a total of 2834.28 and 2721.45 centimorgan (cM) genetic lengths, respectively. A total of 22 quantitative trait loci (QTLs) for seven phenotypic traits were mapped; among them, five stable and major-effect QTLs (PC-8-1, SL-9-1, SWi-9-1, SSi-9-1, and SW-6-1) and four minor-effect QTLs (PC-2-1 and PC-2-2; PT-2-1 and PT-2-2; SL-6-1 and SSi-6-2; and SWi-6-1 and SWi-6-2) were observed with 3.77-38.98% PVE. The adjacent QTL markers showed a good fit marker-trait association, and a significant allele-specific contribution was also noticed for genetic inheritance of traits. Further, a total of four candidate genes (Cla97C09G179150, Cla97C09G179350, Cla97C09G180040, and Cla97C09G180100) were spotted in the stable colocalized QTLs of seed size linked traits (SL-9-1 and SWi-9-1) that showed non-synonymous type mutations. The gene expression trends indicated that the seed morphology had been formed in the early developmental stage and showed the genetic regulation of seed shape formation. Hence, we think that our identified QTLs and genes would provide powerful genetic insights for marker-assisted breeding aimed at improving the quality traits of watermelon.

A Genome-Wide Identification and Comparative Analysis of the Heavy-Metal-Associated Gene Family in Cucurbitaceae Species and Their Role in Cucurbita pepo under Arsenic Stress.

The heavy-metal-associated (HMA) proteins are a class of PB1-type ATPases related to the intracellular transport and detoxification of metals. However, due to a lack of information regarding the HMA gene family in the Cucurbitaceae family, a comprehensive genome-wide analysis of the HMA family was performed in ten Cucurbitaceae species: Citrullus amarus, Citrullus colocynthis, Citrullus lanatus, Citrullus mucosospermus, Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, and Legenaria siceraria. We identified 103 Cucurbit HMA proteins with various members, ranging from 8 (Legenaria siceraria) to 14 (Cucurbita pepo) across species. The phylogenetic and structural analysis confirmed that the Cucurbitaceae HMA protein family could be further classified into two major clades: Zn/Co/Cd/Pb and Cu/Ag. The GO-annotation-based subcellular localization analysis predicted that all HMA gene family members were localized on membranes. Moreover, the analysis of conserved motifs and gene structure (intron/exon) revealed the functional divergence between clades. The interspecies microsynteny analysis demonstrated that maximum orthologous genes were found between species of the Citrullus genera. Finally, nine candidate HMA genes were selected, and their expression analysis was carried out via qRT-PCR in root, leaf, flower, and fruit tissues of C. pepo under arsenic stress. The expression pattern of the CpeHMA genes showed a distinct pattern of expression in root and shoot tissues, with a remarkable expression of CpeHMA6 and CpeHMA3 genes from the Cu/Ag clade. Overall, this study provides insights into the functional analysis of the HMA gene family in Cucurbitaceae species and lays down the basic knowledge to explore the role and mechanism of the HMA gene family to cope with arsenic stress conditions.

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.

Clorf Encodes Carotenoid Isomerase and Regulates Orange Flesh Color in Watermelon (Citrullus lanatus L.).

Flesh color is a significant characteristic of watermelon. Although various flesh-color genes have been identified, the inheritance and molecular basis of the orange flesh trait remain relatively unexplored. In the present study, the genetic analysis of six generations derived from W1-1 (red flesh) and W1-61 (orange flesh) revealed that the orange flesh color trait was regulated by a single recessive gene, Clorf (orange flesh). Bulk segregant analysis (BSA) locked the range to ∼4.66 Mb, and initial mapping situated the Clorf locus within a 688.35-kb region of watermelon chromosome 10. Another 1,026 F2 plants narrowed the Clorf locus to a 304.62-kb region containing 32 candidate genes. Subsequently, genome sequence variations in this 304.62-kb region were extracted for in silico BSA strategy among 11 resequenced lines (one orange flesh and ten nonorange flesh) and finally narrowed the Clorf locus into an 82.51-kb region containing nine candidate genes. Sequence variation analysis of coding regions and gene expression levels supports Cla97C10G200950 as the most possible candidate for Clorf, which encodes carotenoid isomerase (Crtiso). This study provides a genetic resource for investigating the orange flesh color of watermelon, with Clorf malfunction resulting in low lycopene accumulation and, thus, orange flesh.

Genome-wide association study of soluble solids content, flesh color, and fruit shape in citron watermelon.

Fruit quality traits are crucial determinants of consumers' willingness to purchase watermelon produce, making them major goals for breeding programs. There is limited information on the genetic underpinnings of fruit quality traits in watermelon. A total of 125 citron watermelon (Citrullus amarus) accessions were genotyped using single nucleotide polymorphisms (SNPs) molecular markers generated via whole-genome resequencing. A total of 2,126,759 genome-wide SNP markers were used to uncover marker-trait associations using single and multi-locus GWAS models. High broad-sense heritability for fruit quality traits was detected. Correlation analysis among traits revealed positive relationships, with the exception of fruit diameter and fruit shape index (ratio of fruit length to fruit diameter), which was negative. A total of 37 significant SNP markers associated with soluble solids content, flesh color, fruit length, fruit diameter, and fruit shape index traits were uncovered. These peak SNPs accounted for 2.1%-23.4% of the phenotypic variation explained showing the quantitative inheritance nature of the evaluated traits. Candidate genes relevant to fruit quality traits were uncovered on chromosomes Ca01, Ca03, Ca06, and Ca07. These significant molecular markers and candidate genes will be useful in marker-assisted breeding of fruit quality traits in watermelon.

Genome-Wide Identification and Expression Patterns of Cucumber Invertases and Their Inhibitor Genes.

Invertases and their inhibitors play important roles in sucrose metabolism, growth and development, signal transduction, and biotic and abiotic stress tolerance in many plant species. However, in cucumber, both the gene members and functions of invertase and its inhibitor families remain largely unclear. In this study, in comparison with the orthologues of Citrullus lanatus (watermelon), Cucumis melo (melon), and Arabidopsis thaliana (Arabidopsis), 12 invertase genes and 12 invertase inhibitor genes were identified from the genome of Cucumis sativus (cucumber). Among them, the 12 invertase genes were classified as 4 cell wall invertases, 6 cytoplasmic invertases, and 2 vacuolar invertases. Most invertase genes were conserved in cucumber, melon, and watermelon, with several duplicate genes in melon and watermelon. Transcriptome analysis distinguished these genes into various expression patterns, which included genes CsaV3_2G025540 and CsaV3_2G007220, which were significantly expressed in different tissues, organs, and development stages, and genes CsaV3_7G034730 and CsaV3_5G005910, which might be involved in biotic and abiotic stress. Six genes were further validated in cucumber based on quantitative real-time PCR (qRT-PCR), and three of them showed consistent expression patterns as revealed in the transcriptome. These results provide important information for further studies on the physiological functions of cucumber invertases (CSINVs) and their inhibitors (CSINHs).

Construction and application of a new watermelon germplasm with the phenotype of dwarf and branchless.

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.

GRAS family member LATERAL SUPPRESSOR regulates the initiation and morphogenesis of watermelon lateral organs.

The lateral organs of watermelon (Citrullus lanatus), including lobed leaves, branches, flowers, and tendrils, together determine plant architecture and yield. However, the genetic controls underlying lateral organ initiation and morphogenesis remain unclear. Here, we found that knocking out the homologous gene of shoot branching regulator LATERAL SUPPRESSOR in watermelon (ClLs) repressed the initiation of branches, flowers, and tendrils and led to developing round leaves, indicating that ClLs undergoes functional expansion compared with its homologs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum). Using ClLs as the bait to screen against the cDNA library of watermelon, we identified several ClLs-interacting candidate proteins, including TENDRIL (ClTEN), PINOID (ClPID), and APETALA1 (ClAP1). Protein-protein interaction assays further demonstrated that ClLs could directly interact with ClTEN, ClPID, and ClAP1. The mRNA in situ hybridization assay revealed that the transcriptional patterns of ClLs overlapped with those of ClTEN, ClPID, and ClAP1 in the axillary meristems and leaf primordia. Mutants of ClTEN, ClPID, and ClAP1 generated by the CRISPR/Cas9 gene editing system lacked tendrils, developed round leaves, and displayed floral diapause, respectively, and all these phenotypes could be observed in ClLs knockout lines. Our findings indicate that ClLs acts as lateral organ identity protein by forming complexes with ClTEN, ClPID, and ClAP1, providing several gene targets for transforming the architecture of watermelon.

Identification of watermelon H3K4 and H3K27 genes and their expression profiles during watermelon fruit development.

BACKGROUND: The ClaH3K4s and ClaH3K27s gene families are subfamilies of the SET family, each with a highly conserved SET structure domain and a PHD structural domain. Both participate in histone protein methylation, which affects the chromosome structure and gene expression, and is essential for fruit growth and development. METHODS AND RESULTS: In order to demonstrate the structure and expression characteristics of ClaH3K4s and ClaH3K27s in watermelon, members of the watermelon H3K4 and H3K27 gene families were identified, and their chromosomal localization, gene structure, and protein structural domains were analyzed. The phylogeny and covariance of the gene families with other species were subsequently determined, and the expression profiles were obtained by performing RNA-Seq and qRT-PCR. The watermelon genome had five H3K4 genes with 3207-8043 bp nucleotide sequence lengths and four H3K27 genes with a 1107-5499 bp nucleotide sequence. Synteny analysis revealed the close relationship between watermelon and cucumber, with the majority of members displaying a one-to-one covariance. Approximately half of the 'Hua-Jing 13 watermelon' ClaH3K4s and ClaH3K27s genes were expressed more in the late fruit development stages, while the changes were minimal for the remaining half. H3K4-2 expression was observed to be slightly greater on day 21 compared to other periods. Moreover, ClaH3K27-1 and ClaH3K27-2 were hardly expressed throughout the developing period, and ClaH3K27-4 exhibited the highest expression. CONCLUSION: These results serve as a basis for further functional characterization of the H3K4 and H3K27 genes in the fruit development of watermelon.