Complete genome assembly provides a high-quality skeleton for pan-NLRome construction in melon.

Melon (Cucumis melo L.), being under intensive domestication and selective breeding, displays an abundant phenotypic diversity. Wild germplasm with tolerance to stress represents an untapped genetic resource for discovery of disease-resistance genes. To comprehensively characterize resistance genes in melon, we generate a telomere-to-telomere (T2T) and gap-free genome of wild melon accession PI511890 (C. melo var. chito) with a total length of 375.0 Mb and a contig N50 of 31.24 Mb. The complete genome allows us to dissect genome architecture and identify resistance gene analogs. We construct a pan-NLRome using seven melon genomes, which include 208 variable and 18 core nucleotide-binding leucine-rich repeat receptors (NLRs). Multiple disease-related transcriptome analyses indicate that most up-regulated NLRs induced by pathogens are shell or cloud NLRs. The T2T gap-free assembly and the pan-NLRome not only serve as essential resources for genomic studies and molecular breeding of melon but also provide insights into the genome architecture and NLR diversity.

CmERF1 acts as a positive regulator of fruits and leaves growth in melon (Cucumis melo L.).

Melon (Cucumis melo L.) is an important horticultural and economic crop. ETHYLENE RESPONSE FACTOR1 (ERF1) plays an important role in regulating plant development, and the resistance to multiple biotic and abiotic stresses. In this study, developmental biology, molecular biology and biochemical assays were performed to explore the biological function of CmERF1 in melon. Abundant transcripts of CmERF1 were found in ovary at green-yellow bud (GYB) and rapid enlargement (ORE) stages. In CmERF1 promoter, the cis-regulatory elements for indoleacetic acid (IAA), methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), gibberellic acid (GA), light and low temperature responses were found. CmERF1 could be significantly induced by ethylene, IAA, MeJA, SA, ABA, and respond to continuous light and low temperature stresses in melon. Ectopic expression of CmERF1 increased the length of siliqua and carpopodium, and expanded the size of leaves in Arabidopsis. Knockdown of CmERF1 led to smaller ovary at anthesis, mature fruit and leaves in melon. In CmERF1-RNAi #2 plants, 75 genes were differently expressed compared with control, and the promoter regions of 28 differential expression genes (DEGs) contained the GCC-box (AGCCGCC) or DRE (A/GCCGAC) cis-acting elements of CmERF1. A homolog of cell division cycle protein 48 (CmCDC48) was proved to be the direct target of CmERF1 by the yeast one-hybrid assay and dual-luciferase (LUC) reporter (DLR) system. These results indicated that CmERF1 was able to promote the growth of fruits and leaves, and involved in multiple hormones and environmental signaling pathways in melon.

Characterization and stress-responsive regulation of CmPHT1 genes involved in phosphate uptake and transport in Melon (Cucumis melo L.).

BACKGROUND: Phosphorus (P) deficiency, a major nutrient stress, greatly hinders plant growth. Phosphate (Pi) uptake in plant roots relies on PHT1 family transporters. However, melon (Cucumis melo L.) lacks comprehensive identification and characterization of PHT1 genes, particularly their response patterns under diverse stresses. RESULTS: This study identified and analyzed seven putative CmPHT1 genes on chromosomes 3, 4, 5, 6, and 7 using the melon genome. Phylogenetic analysis revealed shared motifs, domain compositions, and evolutionary relationships among genes with close histories. Exon number varied from 1 to 3. Collinearity analysis suggested segmental and tandem duplications as the primary mechanisms for CmPHT1 gene family expansion. CmPHT1;4 and CmPHT1;5 emerged as a tandemly duplicated pair. Analysis of cis-elements in CmPHT1 promoters identified 14 functional categories, including putative PHR1-binding sites (P1BS) in CmPHT1;4, CmPHT1;6, and CmPHT1;7. We identified that three WRKY transcription factors regulated CmPHT1;5 expression by binding to its W-box element. Notably, CmPHT1 promoters harbored cis-elements responsive to hormones and abiotic factors. Different stresses regulated CmPHT1 expression differently, suggesting that the adjusted expression patterns might contribute to plant adaptation. CONCLUSIONS: This study unveils the characteristics, evolutionary diversity, and stress responsiveness of CmPHT1 genes in melon. These findings lay the foundation for in-depth investigations into their functional mechanisms in Cucurbitaceae crops.

The dominant white color trait of the melon fruit rind is associated with epicuticular wax accumulation.

MAIN CONCLUSION: Microscopic analyses and chemical profiling demonstrate that the white rind phenotype in melon fruit is associated with the accumulation of n-alkanes, fatty alcohols, aldehydes and wax esters. Serving as an indicator of quality, the rind (or external) color of fruit directly affects consumer choice. A fruit's color is influenced by factors such as the levels of pigments and deposited epicuticular waxes. The latter produces a white-grayish coating often referred to as "wax bloom". Previous reports have suggested that some melon (Cucumis melo L.) accessions may produce wax blooms, where a dominant white rind color trait was genetically mapped to a major locus on chromosome 7 and suggested to be inherited as a single gene named Wi. We here provide the first direct evidence of the contribution of epicuticular waxes to the dominant white rind trait in melon fruit. Our light and electron microscopy and gas chromatography-mass spectrometry (GC-MS) comparative analysis of melon accessions with white or green rinds reveals that the rind of melon fruit is rich in epicuticular waxes. These waxes are composed of various biochemical classes, including fatty acids, fatty alcohols, aldehydes, fatty amides, n-alkanes, tocopherols, triterpenoids, and wax esters. We show that the dominant white rind phenotype in melon fruit is associated with increased accumulation of n-alkanes, fatty alcohols, aldehydes and wax esters, which are linked with the deposition of crystal-like wax platelets on their surfaces. Together, this study broadens the understanding of natural variation in an important quality trait of melon fruit and promotes the future identification of the causative gene for the dominant white rind trait.

Pan-genome analysis sheds light on structural variation-based dissection of agronomic traits in melon crops.

Sweetness and appearance of fresh fruits are key palatable and preference attributes for consumers and are often controlled by multiple genes. However, fine-mapping the key loci or genes of interest by single genome-based genetic analysis is challenging. Herein, we present the chromosome-level genome assembly of 1 landrace melon accession (Cucumis melo ssp. agrestis) with wild morphologic features and thus construct a melon pan-genome atlas via integrating sequenced melon genome datasets. Our comparative genomic analysis reveals a total of 3.4 million genetic variations, of which the presence/absence variations (PAVs) are mainly involved in regulating the function of genes for sucrose metabolism during melon domestication and improvement. We further resolved several loci that are accountable for sucrose contents, flesh color, rind stripe, and suture using a structural variation (SV)-based genome-wide association study. Furthermore, via bulked segregation analysis (BSA)-seq and map-based cloning, we uncovered that a single gene, (CmPIRL6), determines the edible or inedible characteristics of melon fruit exocarp. These findings provide important melon pan-genome information and provide a powerful toolkit for future pan-genome-informed cultivar breeding of melon.

Nontarget site-based resistance to nicosulfuron and identification of candidate genes in Cucumis melo L. var. agrestis Naud. via RNA-Seq transcriptome analysis.

Herbicide resistance is a worldwide concern for weed control. Cucumis melo L. var. agrestis Naud. (C. melo) is an annual trailing vine weed that is commonly controlled by nicosulfuron, acetolactate synthase (ALS)-inhibiting herbicides. However, long-term use of this herbicide has led to the emergence of resistance and several nicosulfuron resistant populations of C. melo have been found. Here we identified a resistant (R) C. melo population exhibiting 7.31-fold resistance to nicosulfuron compared with a reference sensitive (S) population. ALS gene sequencing of the target site revealed no amino acid substitution in R plants, and no difference in enzyme activity, as shown by ALS activity assays in vitro. ALS gene expression was not significantly different before and after the application of nicosulfuron. Pretreatment with the cytochrome P450 monooxygenase (P450) inhibitor malathion reduced nicosulfuron resistance in the R population. RNA-Seq transcriptome analysis was used to identify candidate genes that may confer metabolic resistance to nicosulfuron. We selected genes with annotations related to detoxification functions. A total of 20 candidate genes (7 P450 genes, 1 glutathione S-transferase (GST) gene, 2 ATP-binding cassette (ABC) transporters, and 10 glycosyltransferase (GT)) were identified; 12 of them (7 P450s, 1 GST, 2 ABC transporters, and 2 GTs) were demonstrated significantly differential expression between R and S by quantitative real-time RT-PCR (qRT-PCR). Our findings revealed that the resistance mechanism in C. melo was nontarget-site based. Our results also provide a valuable resource for studying the molecular mechanisms of weed resistance.

Genetic Dissection of ToLCNDV Resistance in Resistant Sources of Cucumis melo.

Tomato leaf curl New Delhi virus (ToLCNDV) is a begomovirus causing significant melon (Cucumis melo) crop losses globally. This study aims to map the ToLCNDV resistance in the PI 414723 melon accession, previously identified and characterized through phenotypic studies, thereby exploring shared genomic regions with the established resistant source WM-7. In the present study, WM-7 and PI 414723 were crossed with the susceptible accessions 'Rochet' and 'Blanco' respectively, to generate F1 hybrids. These hybrids were self-pollinated to generate the populations for mapping the ToLCNDV resistance region and designing markers for marker-assisted selection. Disease evaluation included visual symptom scoring, viral-load quantification and tissue printing. Genotyping-by-sequencing and SNP markers were used for quantitative trait loci (QTL) mapping. For genetic analysis, qPCR and bulked segregant RNA-seq (BSR-seq) were performed. Gene expression was assessed using RNA-seq, and qRT-PCR was used for confirmation. The research narrows the candidate region for resistance in WM-7 and identifies overlapping QTLs on chromosome 11 in PI 414723, found in the region of the DNA primase large subunit. BSR-seq and expression analyses highlight potential regulatory roles of chromosome 2 in conferring resistance. Differential expression was confirmed for six genes in the candidate region on chromosome 2. This study confirms the existence of common resistance genes in PI 414723 and WM-7.

Molecular Markers for Marker-Assisted Breeding for Biotic and Abiotic Stress in Melon (Cucumis melo L.): A Review.

Melon (Cucumis melo L.) is a globally grown crop renowned for its juice and flavor. Despite growth in production, the melon industry faces several challenges owing to a wide range of biotic and abiotic stresses throughout the growth and development of melon. The aim of the review article is to consolidate current knowledge on the genetic mechanism of both biotic and abiotic stress in melon, facilitating the development of robust, disease-resistant melon varieties. A comprehensive literature review was performed, focusing on recent genetic and molecular advancements related to biotic and abiotic stress responses in melons. The review emphasizes the identification and analysis of quantitative trait loci (QTLs), functional genes, and molecular markers in two sections. The initial section provides a comprehensive summary of the QTLs and major and minor functional genes, and the establishment of molecular markers associated with biotic (viral, bacterial, and fungal pathogens, and nematodes) and abiotic stress (cold/chilling, drought, salt, and toxic compounds). The latter section briefly outlines the molecular markers employed to facilitate marker-assisted backcrossing (MABC) and identify cultivars resistant to biotic and abiotic stressors, emphasizing their relevance in strategic marker-assisted melon breeding. These insights could guide the incorporation of specific traits, culminating in developing novel varieties, equipped to withstand diseases and environmental stresses by targeted breeding, that meet both consumer preferences and the needs of melon breeders.

Novel Allelic Gene Variations in CmCLAVATA3 (CmCLV3) Were Identified in a Genetic Population of Melon (Cucumis melo L.).

Carpel number (CN) is an important trait affecting the fruit size and shape of melon, which plays a crucial role in determining the overall appearance and market value. A unique non-synonymous single nucleotide polymorphism (SNP) in CmCLAVATA3 (CmCLV3) is responsible for the variation of CN in C. melo ssp. agrestis (hereafter agrestis), but it has been unclear in C. melo ssp. melo (hereafter melo). In this study, one major locus controlling the polymorphism of 5-CN (multi-CN) and 3-CN (normal-CN) in melo was identified using bulked segregant analysis (BSA-seq). This locus was then fine-mapped to an interval of 1.8 Mb on chromosome 12 using a segregating population containing 1451 progeny. CmCLV3 is still present in the candidate region. A new allele of CmCLV3, which contains five other nucleotide polymorphisms, including a non-synonymous SNP in coding sequence (CDS), except the SNP reported in agrestis, was identified in melo. A cis-trans test confirmed that the candidate gene, CmCLV3, contributes to the variation of CNs in melo. The qRT-PCR results indicate that there is no significant difference in the expression level of CmCLV3 in the apical stem between the multi-CN plants and the normal-CN plants. Overall, this study provides a genetic resource for melon fruit development research and molecular breeding. Additionally, it suggests that melo has undergone similar genetic selection but evolved into an independent allele.

Evidence Supporting a Role of Alternative Splicing Participates in Melon (Cucumis melo L.) Fruit Ripening.

One key post-transcriptional modification mechanism that dynamically controls a number of physiological processes in plants is alternative splicing (AS). However, the functional impacts of AS on fruit ripening remain unclear. In this research, we used RNA-seq data from climacteric (VED, Harukei 3) and non-climacteric (PI, PS) melon cultivars to explore alternative splicing (AS) in immature and mature fruit. The results revealed dramatic changes in differential AS genes (DAG) between the young and mature fruit stages, particularly in genes involved in fruit development/ripening, carotenoid and capsaicinoid biosynthesis, and starch and sucrose metabolism. Serine/arginine-rich (SR) family proteins are known as important splicing factors in AS events. From the melon genome, a total of 17 SR members were discovered in this study. These genes could be classified into eight distinct subfamilies based on gene structure and conserved motifs. Promoter analysis detected various cis-acting regulatory elements involved in hormone pathways and fruit development. Interestingly, these SR genes exhibited specific expression patterns in reproductive organs such as flowers and ovaries. Additionally, concurrent with the increase in AS levels in ripening fruit, the transcripts of these SR genes were activated during fruit maturation in both climacteric and non-climacteric melon varieties. We also found that most SR genes were under selection during domestication. These results represent a novel finding of increased AS levels and SR gene expression during fruit ripening, indicating that alternative splicing may play a role in fruit maturation.

Functional Analysis of Cucumis melo CmXTH11 in Regulating Drought Stress Tolerance in Arabidopsis thaliana.

The CmXTH11 gene, a member of the XTH (xyloglucan endotransglycosylase/hydrolase) family, plays a crucial role in plant responses to environmental stress. In this study, we heterologously expressed the melon gene CmXTH11 in Arabidopsis to generate overexpressing transgenic lines, thereby elucidating the regulatory role of CmXTH11 in water stress tolerance. Using these lines of CmXTH11 (OE1 and OE2) and wild-type (WT) Arabidopsis as experimental materials, we applied water stress treatments (including osmotic stress and soil drought) and rewatering treatments to investigate the response mechanisms of melon CmXTH11 in Arabidopsis under drought stress from a physiological and biochemical perspective. Overexpression of CmXTH11 significantly improved root growth under water stress conditions. The OE lines exhibited longer roots and a higher number of lateral roots compared to WT plants. The enhanced root system contributed to better water uptake and retention. Under osmotic and drought stress, the OE lines showed improved survival rates and less wilting compared to WT plants. Biochemical analyses revealed that CmXTH11 overexpression led to lower levels of malondialdehyde (MDA) and reduced electrolyte leakage, indicating decreased oxidative damage. The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were significantly higher in OE lines, suggesting enhanced oxidative stress tolerance. The CmXTH11 gene positively regulates water stress tolerance in Arabidopsis by enhancing root growth, improving water uptake, and reducing oxidative damage. Overexpression of CmXTH11 increases the activities of antioxidant enzymes, thereby mitigating oxidative stress and maintaining cellular integrity under water deficit conditions. These findings suggest that CmXTH11 is a potential candidate for genetic improvement of drought resistance in crops.

Effect of Green Light Replacing Some Red and Blue Light on Cucumis melo under Drought Stress.

Light quality not only directly affects the photosynthesis of green plants but also plays an important role in regulating the development and movement of leaf stomata, which is one of the key links for plants to be able to carry out normal growth and photosynthesis. By sensing changes in the light environment, plants actively regulate the expansion pressure of defense cells to change stomatal morphology and regulate the rate of CO2 and water vapor exchange inside and outside the leaf. In this study, Cucumis melo was used as a test material to investigate the mitigation effect of different red, blue, and green light treatments on short-term drought and to analyze its drought-resistant mechanism through transcriptome and metabolome analysis, so as to provide theoretical references for the regulation of stomata in the light environment to improve the water use efficiency. The results of the experiment showed that after 9 days of drought treatment, increasing the percentage of green light in the light quality significantly increased the plant height and fresh weight of the treatment compared to the control (no green light added). The addition of green light resulted in a decrease in leaf stomatal conductance and a decrease in reactive oxygen species (ROS) content, malondialdehyde MDA content, and electrolyte osmolality in the leaves of melon seedlings. It indicated that the addition of green light promoted drought tolerance in melon seedlings. Transcriptome and metabolome measurements of the control group (CK) and the addition of green light treatment (T3) showed that the addition of green light treatment not only effectively regulated the synthesis of abscisic acid (ABA) but also significantly regulated the hormonal pathway in the hormones such as jasmonic acid (JA) and salicylic acid (SA). This study provides a new idea to improve plant drought resistance through light quality regulation.

Identification and characterization analysis of the HDM gene family in melon (Cucumis melo L.).

Histone demethylase (HDM) play crucial roles in regulating plant growth and environmental adaptation. In this study, the HDM gene family in melon was identified by bioinformatics methods and the expression patterns of the CmHDM family members in different melon tissues were analyzed using transcriptome data. The results showed that 20 CmHDM genes were identified in the melon genome, which were unevenly distributed across each chromosome. These members fall into two major categories: LSD1 and JmjC. The JmjC group could be further divided into five subgroups with different numbers. The results of collinearity analysis of intraspecific and interspecific relationships showed that there were only one pair of segmental duplication in melon HDM genes, and more collinearity in genetic relationship of HDM genes between melon and tomato. The numbers of conserved domains, exons and introns in each member vary and various cis-acting elements responding to hormones and environmental signals existed in the respective promoter regions. Expression analysis showed that the respective gene members were expressed at different levels in male flowers, female flowers, roots, stems, leaves, ovary, and mature fruits of melon. These results will contribute to the understanding on the potential functions of the HDM genes and their potential functions in regulating melon growth and environmental adaptation.

Pan-genome and multi-parental framework for high-resolution trait dissection in melon (Cucumis melo).

Linking genotype with phenotype is a fundamental goal in biology and requires robust data for both. Recent advances in plant-genome sequencing have expedited comparisons among multiple-related individuals. The abundance of structural genomic within-species variation that has been discovered indicates that a single reference genome cannot represent the complete sequence diversity of a species, leading to the expansion of the pan-genome concept. For high-resolution forward genetics, this unprecedented access to genomic variation should be paralleled and integrated with phenotypic characterization of genetic diversity. We developed a multi-parental framework for trait dissection in melon (Cucumis melo), leveraging a novel pan-genome constructed for this highly variable cucurbit crop. A core subset of 25 diverse founders (MelonCore25), consisting of 24 accessions from the two widely cultivated subspecies of C. melo, encompassing 12 horticultural groups, and 1 feral accession was sequenced using a combination of short- and long-read technologies, and their genomes were assembled de novo. The construction of this melon pan-genome exposed substantial variation in genome size and structure, including detection of ~300 000 structural variants and ~9 million SNPs. A half-diallel derived set of 300 F2 populations, representing all possible MelonCore25 parental combinations, was constructed as a framework for trait dissection through integration with the pan-genome. We demonstrate the potential of this unified framework for genetic analysis of various melon traits, including rind color intensity and pattern, fruit sugar content, and resistance to fungal diseases. We anticipate that utilization of this integrated resource will enhance genetic dissection of important traits and accelerate melon breeding.

CmLHP1 proteins play a key role in plant development and sex determination in melon (Cucumis melo).

In monoecious melon (Cucumis melo), sex is determined by the differential expression of sex determination genes (SDGs) and adoption of sex-specific transcriptional programs. Histone modifications such as H3K27me3 have been previously shown to be a hallmark associated to unisexual flower development in melon; yet, no genetic approaches have been conducted for elucidating the roles of H3K27me3 writers, readers, and erasers in this process. Here we show that melon homologs to Arabidopsis LHP1, CmLHP1A and B, redundantly control several aspects of plant development, including sex expression. Cmlhp1ab double mutants displayed an overall loss and redistribution of H3K27me3, leading to a deregulation of genes involved in hormone responses, plant architecture, and flower development. Consequently, double mutants display pleiotropic phenotypes and, interestingly, a general increase of the male:female ratio. We associated this phenomenon with a general deregulation of some hormonal response genes and a local activation of male-promoting SDGs and MADS-box transcription factors. Altogether, these results reveal a novel function for CmLHP1 proteins in maintenance of monoecy and provide novel insights into the polycomb-mediated epigenomic regulation of sex lability in plants.

Genome-wide identification and expression analysis of the JMJ-C gene family in melon (Cucumis melo L.) reveals their potential role in fruit development.

BACKGROUND: Proteins with the jumonji (JMJ)-C domain belong to the histone demethylase family and contribute to reverse histone methylation. Although JMJ-C family genes have an essential role in regulating plant growth and development, the characterization of the JMJ-C family genes in melon has not been uncovered. RESULTS: In this study, a total of 17 JMJ-C proteins were identified in melon (Cucumis melo L.). CmJMJs were categorized into five subfamilies based on the specific conserved domain: KDM4/JHDM3, KDM5/JARID1, JMJD6, KDM3/JHDM2, and JMJ-C domain-only. The chromosome localization analyses showed that 17 CmJMJs were distributed on nine chromosomes. Cis-acting element analyses of the 17 CmJMJ genes showed numerous hormone, light, and stress response elements distributed in the promoter region. Covariance analysis revealed one pair of replicated fragments (CmJMJ3a and CmJMJ3b) in 17 CmJMJ genes. We investigated the expression profile of 17 CmJMJ genes in different lateral organs and four developmental stages of fruit by RNA-seq transcriptome analysis and RT-qPCR. The results revealed that most CmJMJ genes were prominently expressed in female flowers, ovaries, and developing fruits, suggesting their active role in melon fruit development. Subcellular localization showed that the fruit-related CmJMJ5a protein is specifically localized in the cell nucleus. CONCLUSIONS: This study provides a comprehensive understanding of the gene structure, classification, and evolution of JMJ-C in melon and supports the clarification of the JMJ-C functions in further research.

QTL mapping of resistance to Pseudoperonospora cubensis clade 2, mating type A1, in Cucumis melo and dual-clade marker development.

KEY MESSAGE: This is the first identification of QTLs underlying resistance in Cucumis melo to an isolate of Pseudoperonospora cubensis identified as Clade 2/mating type A1. Pseudoperonospora cubensis, causal organism of cucurbit downy mildew (CDM), causes severe necrosis and defoliation on Cucumis melo (melon). A recombinant inbred line population (N = 169) was screened against an isolate of P. cubensis (Clade 2/mating type A1) in replicated greenhouse and growth chamber experiments. SNPs (n = 5633 bins) identified in the RIL population were used for quantitative trait loci (QTL) mapping. A single major QTL on chromosome 10 (qPcub-10.3-10.4) was consistently associated with resistance across all experiments, while a second major QTL on chromosome 8 (qPcub-8.3) was identified only in greenhouse experiments. These two major QTLs were identified on the same chromosomes (8 and 10) but in different locations as two major QTLs (qPcub-8.2 and qPcub-10.1) previously identified for resistance to P. cubensis Clade 1/mating type A2. Kompetitive allele-specific PCR (KASP) markers were developed for these four major QTLs and validated in the RIL population through QTL mapping. These markers will provide melon breeders a high-throughput genotyping toolkit for development of melon cultivars with broad tolerance to CDM.

Natural allelic variation in the EamA-like transporter, CmSN, is associated with fruit skin netting in melon.

KEY MESSAGE: A SNP mutation in CmSN, encoding an EamA-like transporter, is responsible for fruit skin netting in melon. In maturing melon (Cucumis melo L.), the rind becomes reticulated or netted, a unique characteristic that dramatically changes the appearance of the fruit. However, little is known about the molecular basis of fruit skin netting formation in this important cucurbit crop. Here, we conducted map-based cloning of a skin netting (CmSN) locus using segregating populations derived from the cross between the smooth-fruit line H906 and the netted-fruit line H581. The results showed that CmSN was controlled by a single dominant gene and was primarily positioned on melon chromosome 2, within a physical interval of ~ 351 kb. Further fine mapping in a large F2 population narrowed this region to a 71-kb region harboring 5 genes. MELO3C010288, which encodes a protein in the EamA-like transporter family, is the best possible candidate gene for the netted phenotype. Two nonsynonymous single nucleotide polymorphisms (SNPs) were identified in the third and sixth exons of the CmSN gene and co-segregated with the skin netting (SN) phenotype among the genetic population. A genome-wide association study (GWAS) determined that CmSN is probably a domestication gene under selective pressure during the subspecies C. melo subsp. melo differentiation. The SNP in the third exon of CmSN (the leading SNP in GWAS) revealed a bi-allelic diversity in natural accessions with SN traits. Our results lay a foundation for deciphering the molecular mechanism underlying the formation of fruit skin netting in melon, as well as provide a strategy for genetic improvement of netted fruit using a marker-assisted selection approach.

A recessive gene Cmpmr2F confers powdery mildew resistance in melon (Cucumis melo L.).

KEY MESSAGE: Identified a recessive gene (Cmpmr2F) associated with resistance to infection by the powdery mildew causing agent Podosphaera xanthii race 2F. Powdery mildew (PM) is one of the most destructive fungal diseases of melon, which significantly reduces the crop yield and quality. Multiple studies are being performed for in-depth genetic understandings of PM-susceptibility or -resistance mechanisms in melon plants, but the holistic knowledge of the precise genetic basis of PM-resistance is unexplored. In this study, we characterized the recessive gene "Cmpmr2F" and found its association with resistance against the PM causative agent "Podosphaera xanthii race 2F." Fine genetic mapping revealed the major-effect region of a 26.25-kb interval on chromosome 12, which harbored the Cmpmr2F gene corresponding to the MELO3C002403, encoding allantoate amidohydrolase. The functional gene annotation, expression pattern, and sequence alignment analyses were carried out using two contrast parent lines of melon "X055" PM-susceptible and "PI 124112" PM-resistant. Further, gene silencing of Cmpmr2F using virus-induced gene silencing (VIGS) significantly increased PM-resistance in the susceptible plant. In contrast to the previously reported studies, we identified that Cmpmr2F-silenced plants showed no impairment in growth due to less apparent negative effects in silenced melon plants. So, it is believed that the Cmpmr2F gene has great potential for further breeding studies to increase the P. xanthii race 2F resistance in melon. In short, our study provides new genetic resources and a solid foundation for further functional analysis of PM-resistance genes in melon, as well as powerful molecular markers for marker-assisted breeding aimed at developing new melon varieties resistant to PM infection.

CRISPR/Cas9-Mediated Cytosine Base Editing Using an Improved Transformation Procedure in Melon (Cucumis melo L.).

Melon is a recalcitrant plant for stable genetic transformation. Various protocols have been tried to improve melon transformation efficiency; however, it remains significantly low compared to other plants such as tomato. In this study, the primary focus was on the optimization of key parameters during the inoculation and co-culture steps of the genetic transformation protocol. Our results showed that immersing the explants in the inoculation medium for 20 min significantly enhanced transformation efficiency. During the co-culture step, the use of filer paper, 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), and a temperature of 24 °C significantly enhanced the melon transformation efficiency. Furthermore, the impact of different ethylene inhibitors and absorbers on the transformation efficiency of various melon varieties was explored. Our findings revealed that the use of these compounds led to a significant improvement in the transformation efficiency of the tested melon varieties. Subsequently, using our improved protocol and reporter-gene construct, diploid transgenic melons successfully generated. The efficiency of plant genetic transformation ranged from 3.73 to 4.83%. Expanding the scope of our investigation, the optimized protocol was applied to generate stable gene-edited melon lines using the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated cytosine base editor and obtained melon lines with editions (C-to-T and C-to-G) in the eukaryotic translation initiation factor 4E, CmeIF4E gene. In conclusion, the optimized melon transformation protocol, along with the utilization of the CRISPR/Cas9-mediated cytosine base editor, provides a reliable framework for functional gene engineering in melon. These advancements hold significant promise for furthering genetic research and facilitating crop improvement in this economically important plant species.