Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an economically important vegetable crop grown extensively worldwide. To facilitate the identification of agronomically important traits and provide new information for genetic and genomic research on this species, a high-density genetic linkage map of watermelon was constructed using an F2 population derived from a cross between elite watermelon cultivar K3 and wild watermelon germplasm PI 189225. Based on a sliding window approach, a total of 1,161 bin markers representing 3,465 SNP markers were mapped onto 11 linkage groups corresponding to the chromosome pair number of watermelon. The total length of the genetic map is 1,099.2 cM, with an average distance between bins of 1.0 cM. The number of markers in each chromosome varies from 62 in chromosome 07 to 160 in chromosome 05. The length of individual chromosomes ranged between 61.8 cM for chromosome 07 and 140.2 cM for chromosome 05. A total of 616 SNP bin markers showed significant (P < 0.05) segregation distortion across all 11 chromosomes, and 513 (83.3 %) of these distorted loci showed distortion in favor of the elite watermelon cultivar K3 allele and 103 were skewed toward PI 189225. The number of SNPs and InDels per Mb varied considerably across the segregation distorted regions (SDRs) on each chromosome, and a mixture of dense and sparse SNPs and InDel SDRs coexisted on some chromosomes suggesting that SDRs were randomly distributed throughout the genome. Recombination rates varied greatly among each chromosome, from 2.0 to 4.2 centimorgans per megabase (cM/Mb). An inconsistency was found between the genetic and physical positions on the map for a segment on chromosome 11. The high-density genetic map described in the present study will facilitate fine mapping of quantitative trait loci, the identification of candidate genes, map-based cloning, as well as marker-assisted selection (MAS) in watermelon breeding programs.

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus ] is an economically important vegetable belonging to the Cucurbitaceae family. Genotypes that exhibit agronomically important traits are selected for the development of elite cultivars. Understanding the genetic diversity and the genotype population structure based on molecular markers at the genome level can speed up the utilization of diverse genetic resources for varietal improvement. In the present study, we carried out an analysis of genetic diversity based on 3882 SNP markers across 37 core watermelon genotypes, including the most widely used watermelon varieties and wild watermelon. Based on the SNP genotyping data of the 37 watermelon genotypes screened, gene diversity and polymorphism information content values across chromosomes varied between 0.03 -0.5 and 0.02 -0.38, with averages of 0.14 and 0.13, respectively. The two wild watermelon genotypes were distinct from cultivated varieties and the remaining 35 cultivated genotypes were differentiated into three major clusters: 20 genotypes were grouped in cluster I; 11 genotypes were grouped in cluster II; three advanced breeding lines of yellow fruit flesh and genotype SW043 were grouped in cluster III. The results from neighbour-joining dendrogram, principal coordinate analysis and STRUCTURE analysis approaches were consistent, and the grouping of genotypes was generally in agreement with their origins. Here we reveal the genetic relationships among the core watermelon genotypes maintained at the Jiangsu Academy of Agricultural Sciences, China. The molecular and phenotypic characterization of the existing core watermelon genotypes, together with specific agronomic characteristics, can be utilized by researchers and breeders for future watermelon improvement.

RNA-binding proteins (RBPs) form ribonucleoprotein (RNP)complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

Brassinosteroids (BRs), a group of naturally occurring plant steroidal compounds, are essential for plant growth, development and stress tolerance. Recent studies showed that BRs could induce systemic tolerance to biotic and abiotic stresses; however, the molecular mechanisms by which BRs signals lead to responses in the whole plant are largely unknown. In this study, 24-epibrassinosteroid (EBR)-induced systemic tolerance in Cucumis sativus L. cv. Jinyan No. 4 was analyzed through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation. Expression of defense/stress related genes were induced in both treated local leaves and untreated systemic leaves by local EBR application. With the suppressive subtractive hybridization (SSH) library using cDNA from the phloem sap of EBR-treated plants as the tester and distilled water (DW)-treated plants as the driver, 14 transcripts out of 260 clones were identified. Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) validated the specific up-regulation of these transcripts. Of the differentially expressed transcripts with known functions, transcripts for the selected four cDNAs, which encode an auxin-responsive protein (IAA14), a putative ankyrin-repeat protein, an F-box protein (PP2), and a major latex, pathogenesis-related (MLP)-like protein, were induced in local leaves, systemic leaves and roots after foliar application of EBR onto mature leaves. Our results demonstrated that EBR-induced systemic tolerance is accompanied with increased transcript of genes in the defense response in other organs. The potential role of phloem mRNAs as signaling components in mediating BR-regulated systemic resistance is discussed.

Gummy stem blight (GSB) is one of the most destructive foliar diseases of cucurbits, worldwide. To identify and characterize the pathogen which causes GSB on watermelon and muskmelon in East China, morphological characteristics, pathogenicity assays as well as sequence characterization of the rDNA internal transcribed spacer (ITS) were performed on 41 isolates collected from Jiangsu, Zhejiang, Anhui and Jiangxi provinces. The mycelia of all the isolates were white on top and olivaceous green to black with concentric circles at the bottom on potato-dextrose agar medium. The isolates differed significantly on aggressiveness based on pathogenicity assays. rDNA-ITS sequences and phylogenetic analysis confirmed the isolates as Didymella bryoniae. The isolates were found to be highly identical with the exception of 13 isolates, which had a guanine substitution instead of adenine at position 131 of the ITS.

Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

Gummy stem blight caused by Didymella bryoniae (anamorph Phoma cucurbitacearum) is a major fungal disease of watermelon (Citrullus lanatus) and other cucurbits. Thirty-five isolates of Didymella and Phoma spp. associated with symptoms of gummy stem blight on watermelon, Canary melon (Cucumis melo), muskmelon (C. melo), and winter squash (Cucurbita maxima) from Florida and Georgia were characterized based on morphology on agar media, pathogenicity on ‘Melody’ watermelon, the internal transcribed spacer (ITS) sequence of ribosomal DNA (rDNA), random amplified polymorphic DNA (RAPD) analysis, and polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis. All of the isolates were pathogenic on watermelon but differed in virulence. RAPD and ITS sequence analysis indicated genetic variability among the isolates but PCR-RFLP analysis did not show any variability. ITS sequence phylogenetic analysis identified two isolates, DB-05 and DB-33, which had a greater identity to that of D. bryoniae isolates from China (98 to 100% sequence homology) than other isolates from Florida and Georgia (95 to 98%). These two isolates possessed a single nucleotide substitution of A to G at position 131 of the ITS1 region. The study characterized the genetic profile of a collection of D. bryoniae isolates from Florida and Georgia in relation to isolates from other U.S. states and countries.

Precocious leaf senescence can reduce crop yield and quality by limiting the growth stage. Melatonin has been shown to delay leaf senescence; however, the underlying mechanism remains obscure. Here, we show that melatonin offsets abscisic acid (ABA) to protect photosystem II and delay the senescence of attached old leaves under the light. Melatonin induced H2O2 accumulation accompanied by an upregulation of melon respiratory burst oxidase homolog D (CmRBOHD) under ABA‐induced stress. Both melatonin and H2O2 induced the accumulation of cytoplasmic‐free Ca2+ ([Ca2+]cyt) in response to ABA, while blocking of Ca2+ influx channels attenuated melatonin‐ and H2O2‐induced ABA tolerance. CmRBOHD overexpression induced [Ca2+]cyt accumulation and delayed leaf senescence, whereas deletion of Arabidopsis AtRBOHD, a homologous gene of CmRBOHD, compromised the melatonin‐induced [Ca2+]cyt accumulation and delay of leaf senescence in Arabidopsis under ABA stress. Furthermore, melatonin, H2O2 and Ca2+ attenuated ABA‐induced K+ efflux and subsequent cell death. CmRBOHD overexpression and AtRBOHD deletion alleviated and aggravated the ABA‐induced K+ efflux, respectively. Taken together, our study unveils a new mechanism by which melatonin offsets ABA action to delay leaf senescence via RBOHD‐dependent H2O2 production that triggers [Ca2+]cyt accumulation and subsequently inhibits K+ efflux and delays cell death/leaf senescence in response to ABA.