SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Virus-induced gene silencing for in planta validation of gene function in cucurbits.

Virus-induced gene silencing (VIGS) is a powerful tool for high-throughput analysis of gene function. Here, we developed the VIGS vector pCF93, from which expression of the cucumber fruit mottle mosaic virus genome is driven by the cauliflower mosaic virus 35S promoter to produce viral transcripts in inoculated plants. To test the utility of the pCF93 vector, we identified candidate genes related to male sterility (MS) in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. Specifically, we exploited previously reported reference-based and de novo transcriptome data to define 38 differentially expressed genes between a male-sterile line and its fertile near-isogenic line in the watermelon cultivar DAH. We amplified 200- to 300-bp fragments of these genes, cloned them into pCF93, and inoculated DAH with the resulting VIGS clones. The small watermelon cultivar DAH enabled high-throughput screening using a small cultivation area. We simultaneously characterized the phenotypes associated with each of the 38 candidate genes in plants grown in a greenhouse. Silencing of 8 of the 38 candidate genes produced male-sterile flowers with abnormal stamens and no pollen. We confirmed the extent of gene silencing in inoculated flowers using reverse transcription-qPCR. Histological analysis of stamens from male-fertile and male-sterile floral buds and mature flowers revealed developmental defects and shrunken pollen sacs. Based on these findings, we propose that the pCF93 vector and our VIGS system will facilitate high-throughput analysis for the study of gene function in watermelons.

Venn-diaNet : venn diagram based network propagation analysis framework for comparing multiple biological experiments.

BACKGROUND: The main research topic in this paper is how to compare multiple biological experiments using transcriptome data, where each experiment is measured and designed to compare control and treated samples. Comparison of multiple biological experiments is usually performed in terms of the number of DEGs in an arbitrary combination of biological experiments. This process is usually facilitated with Venn diagram but there are several issues when Venn diagram is used to compare and analyze multiple experiments in terms of DEGs. First, current Venn diagram tools do not provide systematic analysis to prioritize genes. Because that current tools generally do not fully focus to prioritize genes, genes that are located in the segments in the Venn diagram (especially, intersection) is usually difficult to rank. Second, elucidating the phenotypic difference only with the lists of DEGs and expression values is challenging when the experimental designs have the combination of treatments. Experiment designs that aim to find the synergistic effect of the combination of treatments are very difficult to find without an informative system. RESULTS: We introduce Venn-diaNet, a Venn diagram based analysis framework that uses network propagation upon protein-protein interaction network to prioritizes genes from experiments that have multiple DEG lists. We suggest that the two issues can be effectively handled by ranking or prioritizing genes with segments of a Venn diagram. The user can easily compare multiple DEG lists with gene rankings, which is easy to understand and also can be coupled with additional analysis for their purposes. Our system provides a web-based interface to select seed genes in any of areas in a Venn diagram and then perform network propagation analysis to measure the influence of the selected seed genes in terms of ranked list of DEGs. CONCLUSIONS: We suggest that our system can logically guide to select seed genes without additional prior knowledge that makes us free from the seed selection of network propagation issues. We showed that Venn-diaNet can reproduce the research findings reported in the original papers that have experiments that compare two, three and eight experiments. Venn-diaNet is freely available at: http://biohealth.snu.ac.kr/software/venndianet.

An evolutionarily conserved non-synonymous SNP in a leucine-rich repeat domain determines anthracnose resistance in watermelon.

KEY MESSAGE: A non-synonymous SNP of CC-NBS-LRR was firstly mapped to confer resistance to anthracnose in watermelon. Newly proposed LRR domain harboring the SNP is evolutionary conserved in the Cucurbitaceae and Fabaceae. Anthracnose disease caused by Colletotrichum devastates many plants. Despite the importance of the disease, the mechanisms of resistance against it are poorly understood. Here, we identified a non-synonymous single-nucleotide polymorphism (SNP) located in a leucine-rich repeat domain as a marker for resistance to anthracnose race 1 in watermelon, using a combination of genetic analyses. We validated this SNP in segregating populations and 59 watermelon accessions using high-resolution melting assays and Sanger sequencing. We demonstrated that the resulting arginine-to-lysine substitution is particularly conserved among the Cucurbitaceae and Fabaceae. We identified a conserved motif, IxxLPxSxxxLYNLQTLxL, found in 1007 orthologues/paralogues from 89 plant species, and discovered that residue 18 of this motif could determine resistance to disease caused by external invaders. This study provides a step forward in understanding anthracnose resistance in watermelon, as well as functional and evolutionary insight into leucine-rich repeat proteins.

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Transcriptome profiling of differentially expressed genes in floral buds and flowers of male sterile and fertile lines in watermelon.

BACKGROUND: Male sterility is an important mechanism for the production of hybrid seeds in watermelon. Although fruit development has been studied extensively in watermelon, there are no reports on gene expression in floral organs. In this study, RNA-sequencing (RNA-seq) was performed in two near-isogenic watermelon lines (genic male sterile [GMS] line, DAH3615-MS and male fertile line, DAH3615) to identify the differentially expressed genes (DEGs) related to male sterility. RESULTS: DEG analysis showed that 1259 genes were significantly associated with male sterility at a FDR P-value of < 0.01. Most of these genes were only expressed in the male fertile line. In addition, 11 functional clusters were identified using DAVID functional classification analysis. Of detected genes in RNA-seq analysis, 19 were successfully validated by qRT-PCR. CONCLUSIONS: In this study, we carried out a comprehensive floral transcriptome sequence comparison of a male fertile line and its near-isogenic male sterile line in watermelon. This analysis revealed essential genes responsible for stamen development, including pollen development and pollen tube elongation, and allowed their functional classification. These results provided new information on global mechanisms related to male sterility in watermelon.

Infectivity and complete nucleotide sequence of cucumber fruit mottle mosaic virus isolate Cm cDNA.

Three isolates of cucumber fruit mottle mosaic virus (CFMMV) were collected from melon, cucumber, and pumpkin plants in Korea. A full-length cDNA clone of CFMMV-Cm (melon isolate) was produced and evaluated for infectivity after T7 transcription in vitro (pT7CF-Cmflc). The complete CFMMV genome sequence of the infectious clone pT7CF-Cmflc was determined. The genome of CFMMV-Cm consisted of 6,571 nucleotides and shared high nucleotide sequence identity (98.8 %) with the Israel isolate of CFMMV. Based on the infectious clone pT7CF-Cmflc, a CaMV 35S-promoter driven cDNA clone (p35SCF-Cmflc) was subsequently constructed and sequenced. Mechanical inoculation with RNA transcripts of pT7CF-Cmflc and agro-inoculation with p35SCF-Cmflc resulted in systemic infection of cucumber and melon, producing symptoms similar to those produced by CFMMV-Cm. Progeny virus in infected plants was detected by RT-PCR, western blot assay, and transmission electron microscopy.

Physiological and pathological roles of lipogenesis.

Lipids are essential metabolites, which function as energy sources, structural components and signalling mediators. Most cells are able to convert carbohydrates into fatty acids, which are often converted into neutral lipids for storage in the form of lipid droplets. Accumulating evidence suggests that lipogenesis plays a crucial role not only in metabolic tissues for systemic energy homoeostasis but also in immune and nervous systems for their proliferation, differentiation and even pathophysiological roles. Thus, excessive or insufficient lipogenesis is closely associated with aberrations in lipid homoeostasis, potentially leading to pathological consequences, such as dyslipidaemia, diabetes, fatty liver, autoimmune diseases, neurodegenerative diseases and cancers. For systemic energy homoeostasis, multiple enzymes involved in lipogenesis are tightly controlled by transcriptional and post-translational modifications. In this Review, we discuss recent findings regarding the regulatory mechanisms, physiological roles and pathological importance of lipogenesis in multiple tissues such as adipose tissue and the liver, as well as the immune and nervous systems. Furthermore, we briefly introduce the therapeutic implications of lipogenesis modulation.

Brief report: genome sequence and construction of an infectious cDNA clone of Ribgrass mosaic virus from Chinese cabbage in Korea.

Ribgrass mosaic virus (RMV) has severely decreased the production and lowered quality of Chinese cabbage co-infected with Turnip mosaic virus (63.4%) in Korea. The complete genome sequence of RMV isolated from Brassica rapa ssp. pekinensis was determined. The full genome consisted of 6,304 nucleotides and showed sequence identities of 91.5-94.2% with the corresponding genome of other RMV strains. Full-length cDNA of RMV-Br was amplified by RT-PCR with a 5'-end primer harboring a T7 promoter sequence and a 3'-end RMV specific primer. Subsequently, the full-length cDNA was cloned into plasmid vectors. Capped transcripts synthesized from the cDNA clone were highly infectious and caused characteristic symptoms in B. rapa ssp. pekinensis and several indicator plants, similar to wild type RMV. Since there has not been found RMV resistant Chinese cabbage yet and the virus has been prevalent already throughout the natural fields of Korea, the identification of full sequence and development of infectious clone would help developing breeding program for RMV resistant crops.

Cellular Senescence: The Villain of Metabolic Disease?: Discovery of a distinct senescent cell population in obesity-induced metabolic dysfunction.

Senescent p21high cells in epididymal white adipose tissue (eWAT) aggravate metabolic dysfunction in obese animals. In obesity, p21high cells are specifically accumulated in stromal vascular fraction of eWAT and they have increased expression of inflammatory genes and NFκB signaling pathway. Transplantation of p21high cells provokes glucose intolerance whereas clearance of p21high cells by senolytic agents relieves insulin resistance in obese animals.

Genome-wide core sets of SNP markers and Fluidigm assays for rapid and effective genotypic identification of Korean cultivars of lettuce (Lactuca sativa L.).

Lettuce is one of the economically important leaf vegetables and is cultivated mainly in temperate climate areas. Cultivar identification based on the distinctness, uniformity, and stability (DUS) test is a prerequisite for new cultivar registration. However, DUS testing based on morphological features is time-consuming, labor-intensive, and costly, and can also be influenced by environmental factors. Thus, molecular markers have also been used for the identification of genetic diversity as an effective, accurate, and stable method. Currently, genome-wide single nucleotide polymorphisms (SNPs) using next-generation sequencing technology are commonly applied in genetic research on diverse plant species. This study aimed to establish an effective and high-throughput cultivar identification system for lettuce using core sets of SNP markers developed by genotyping by sequencing (GBS). GBS identified 17 877 high-quality SNPs for 90 commercial lettuce cultivars. Genetic differentiation analyses based on the selected SNPs classified the lettuce cultivars into three main groups. Core sets of 192, 96, 48, and 24 markers were further selected and validated using the Fluidigm platform. Phylogenetic analyses based on all core sets of SNPs successfully discriminated individual cultivars that have been currently recognized. These core sets of SNP markers will support the construction of a DNA database of lettuce that can be useful for cultivar identification and purity testing, as well as DUS testing in the plant variety protection system. Additionally, this work will facilitate genetic research to improve breeding in lettuce.

SREBP1c-PARP1 axis tunes anti-senescence activity of adipocytes and ameliorates metabolic imbalance in obesity.

Emerging evidence indicates that the accretion of senescent cells is linked to metabolic disorders. However, the underlying mechanisms and metabolic consequences of cellular senescence in obesity remain obscure. In this study, we found that obese adipocytes are senescence-susceptible cells accompanied with genome instability. Additionally, we discovered that SREBP1c may play a key role in genome stability and senescence in adipocytes by modulating DNA-damage responses. Unexpectedly, SREBP1c interacted with PARP1 and potentiated PARP1 activity during DNA repair, independent of its canonical lipogenic function. The genetic depletion of SREBP1c accelerated adipocyte senescence, leading to immune cell recruitment into obese adipose tissue. These deleterious effects provoked unhealthy adipose tissue remodeling and insulin resistance in obesity. In contrast, the elimination of senescent adipocytes alleviated adipose tissue inflammation and improved insulin resistance. These findings revealed distinctive roles of SREBP1c-PARP1 axis in the regulation of adipocyte senescence and will help decipher the metabolic significance of senescence in obesity.

Adipocyte HIF2α functions as a thermostat via PKA Cα regulation in beige adipocytes.

Thermogenic adipocytes generate heat to maintain body temperature against hypothermia in response to cold. Although tight regulation of thermogenesis is required to prevent energy sources depletion, the molecular details that tune thermogenesis are not thoroughly understood. Here, we demonstrate that adipocyte hypoxia-inducible factor α (HIFα) plays a key role in calibrating thermogenic function upon cold and re-warming. In beige adipocytes, HIFα attenuates protein kinase A (PKA) activity, leading to suppression of thermogenic activity. Mechanistically, HIF2α suppresses PKA activity by inducing miR-3085-3p expression to downregulate PKA catalytic subunit α (PKA Cα). Ablation of adipocyte HIF2α stimulates retention of beige adipocytes, accompanied by increased PKA Cα during re-warming after cold stimuli. Moreover, administration of miR-3085-3p promotes beige-to-white transition via downregulation of PKA Cα and mitochondrial abundance in adipocyte HIF2α deficient mice. Collectively, these findings suggest that HIF2α-dependent PKA regulation plays an important role as a thermostat through dynamic remodeling of beige adipocytes.

Hepatic GSK3ß-Dependent CRY1 Degradation Contributes to Diabetic Hyperglycemia.

Excessive hepatic glucose production (HGP) is a key factor promoting hyperglycemia in diabetes. Hepatic cryptochrome 1 (CRY1) plays an important role in maintaining glucose homeostasis by suppressing forkhead box O1 (FOXO1)-mediated HGP. Although downregulation of hepatic CRY1 appears to be associated with increased HGP, the mechanism(s) by which hepatic CRY1 dysregulation confers hyperglycemia in subjects with diabetes is largely unknown. In this study, we demonstrate that a reduction in hepatic CRY1 protein is stimulated by elevated E3 ligase F-box and leucine-rich repeat protein 3 (FBXL3)-dependent proteasomal degradation in diabetic mice. In addition, we found that GSK3ß-induced CRY1 phosphorylation potentiates FBXL3-dependent CRY1 degradation in the liver. Accordingly, in diabetic mice, GSK3ß inhibitors effectively decreased HGP by facilitating the effect of CRY1-mediated FOXO1 degradation on glucose metabolism. Collectively, these data suggest that tight regulation of hepatic CRY1 protein stability is crucial for maintaining systemic glucose homeostasis.

Targeted erasure of DNA methylation by TET3 drives adipogenic reprogramming and differentiation.

DNA methylation is a crucial epigenetic modification in the establishment of cell-type-specific characteristics. However, how DNA methylation is selectively reprogrammed at adipocyte-specific loci during adipogenesis remains unclear. Here, we show that the transcription factor, C/EBPδ, and the DNA methylation eraser, TET3, cooperatively control adipocyte differentiation. We perform whole-genome bisulfite sequencing to explore the dynamics and regulatory mechanisms of DNA methylation in adipocyte differentiation. During adipogenesis, DNA methylation selectively decreases at adipocyte-specific loci carrying the C/EBP binding motif, which correlates with the activity of adipogenic promoters and enhancers. Mechanistically, we find that C/EBPδ recruits a DNA methylation eraser, TET3, to catalyse DNA demethylation at the C/EBP binding motif and stimulate the expression of key adipogenic genes. Ectopic expression of TET3 potentiates in vitro and in vivo adipocyte differentiation and recovers downregulated adipogenic potential, which is observed in aged mice and humans. Taken together, our study highlights how targeted reprogramming of DNA methylation through cooperative action of the transcription factor C/EBPδ, and the DNA methylation eraser TET3, controls adipocyte differentiation.

Alteration of floral organ identity by over-expression of IbMADS3-1 in tobacco.

The MADS-box genes have been studied mainly in flower development by researching flower homeotic mutants. Most of the MADS-box genes isolated from plants are expressed exclusively in floral tissues, and some of their transcripts have been found in various vegetative tissues. The genes in the STMADS subfamily are important in the development of whole plants including roots, stems, leaves, and the plant vascular system. IbMADS3-1, which is in the STMADS subfamily, and which has been cloned in Ipomoea batatas (L.) Lam., is expressed in all vegetative tissues of the plant, particularly in white fibrous roots. Sequence similarity, besides the spatial and temporal expression patterns, enabled the definition of a novel MADS-box subfamily comprising STMADS16 and the other MADS-box genes in STMADS subfamily expressed specifically in vegetative tissues. Expression of IbMADS3-1 was manifest by the appearance of chlorophyll-containing petals and production of characteristic changes in organ identity carpel structure alterations and sepaloidy of the petals. In reverse transcription-polymerase chain reaction analysis with a number of genes known to be key regulators of floral organ development, the flowering promoter NFL1 was clearly reduced at the RNA level compared with wild type in transgenic line backgrounds. Moreover, NtMADS5 showed slight down-regulation compared with wild-type plants in transgenic lines. These results suggest that IbMADS3-1 could be a repressor of NFL1 located upstream of NtMADS5. IbMADS3-1 ectopic expression is suggested as a possible means during vegetative development by which the IbMADS3-1 gene may interfere with the floral developmental pathway.

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea.

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.

Genetic Diversity Assessment and Cultivar Identification of Cucumber (Cucumis sativus L.) Using the Fluidigm Single Nucleotide Polymorphism Assay.

Genetic diversity analysis and cultivar identification were performed using a core set of single nucleotide polymorphisms (SNPs) in cucumber (Cucumis sativus L.). For the genetic diversity study, 280 cucumber accessions collected from four continents (Asia, Europe, America, and Africa) by the National Agrobiodiversity Center of the Rural Development Administration in South Korea and 20 Korean commercial F1 hybrids were genotyped using 151 Fluidigm SNP assay sets. The heterozygosity of the SNP loci per accession ranged from 4.76 to 82.76%, with an average of 32.1%. Population genetics analysis was performed using population structure analysis and hierarchical clustering (HC), which indicated that these accessions were classified mainly into four subpopulations or clusters according to their geographical origins. The subpopulations for Asian and European accessions were clearly distinguished from each other (FST value = 0.47), while the subpopulations for Korean F1 hybrids and Asian accessions were closely related (FST = 0.34). The highest differentiation was observed between American and European accessions (FST = 0.41). Nei's genetic distance among the 280 accessions was 0.414 on average. In addition, 95 commercial F1 hybrids of three cultivar groups (Baekdadagi-, Gasi-, and Nakhap-types) were genotyped using 82 Fluidigm SNP assay sets for cultivar identification. These 82 SNPs differentiated all cultivars, except seven. The heterozygosity of the SNP loci per cultivar ranged from 12.20 to 69.14%, with an average of 34.2%. Principal component analysis and HC demonstrated that most cultivars were clustered based on their cultivar groups. The Baekdadagi- and Gasi-types were clearly distinguished, while the Nakhap-type was closely related to the Baekdadagi-type. Our results obtained using core Fluidigm SNP assay sets provide useful information for germplasm assessment and cultivar identification, which are essential for breeding and intellectual right protection in cucumber.

Characterization of QTLs and Candidate Genes for Days to Heading in Rice Recombinant Inbred Lines.

Understanding the gene mechanisms controlling days to heading (DH) is important in rice breeding for adaption in the target environment. Using a recombinant inbred line population derived from the cross between two japonica rice cultivars, Koshihikari and Baegilmi, we identified three consistent quantitative trait loci (QTLs) for DH for two years, qDH3, qDH6, and qDH7 on chromosomes 3, 6, and 7, respectively. While Baegilmi contributed the allele for early heading at qDH6 and qDH7 with the additive effect of five days each, Koshihikari contributed the allele for early heading at qDH3 with the additive effect of three days. Notably, pyramiding two or more alleles for early heading at these QTLs accelerated heading effectively. Sequencing of Hd16, Hd1, and Ghd7, the previously known heading date genes underlying qDH3, qDH6, and qDH7, respectively, revealed that Baegilmi and Koshihikari carry different alleles at the three genes. Molecular markers were developed to screen the allelic compositions of the three genes among 295 Korean commercial rice cultivars. The results showed that few cultivars carry alleles for early heading at the three genes, highlighting that DH can be further accelerated and fine-tuned in breeding programs by combining the desirable alleles of Hd16, Hd1, and Ghd7.

Genome-wide SNP discovery and core marker sets for assessment of genetic variations in cultivated pumpkin (Cucurbita spp.).

Three pumpkin species Cucurbita maxima, C. moschata, and C. pepo are commonly cultivated worldwide. To identify genome-wide SNPs in these cultivated pumpkin species, we collected 48 F1 cultivars consisting of 40 intraspecific hybrids (15 C. maxima, 18 C. moschata, and 7 C. pepo) and 8 interspecific hybrids (C. maxima x C. moschata). Genotyping by sequencing identified a total of 37,869 confident SNPs in this collection. These SNPs were filtered to generate a subset of 400 SNPs based on polymorphism and genome distribution. Of the 400 SNPs, 288 were used to genotype an additional 188 accessions (94 F1 cultivars, 50 breeding lines, and 44 landraces) with a SNP array-based platform. Reliable polymorphisms were observed in 224 SNPs (78.0%) and were used to assess genetic variations between and within the four predefined populations in 223 cultivated pumpkin accessions. Both principal component analysis and UPGMA clustering found four major clusters representing three pumpkin species and interspecific hybrids. This genetic differentiation was supported by pairwise Fst and Nei's genetic distance. The interspecific hybrids showed a higher level of genetic diversity relative to the other three populations. Of the 224 SNPs, five subsets of 192, 96, 48, 24, and 12 markers were evaluated for variety identification. The 192, 96, and 48 marker sets identified 204 (91.5%), 190 (85.2%), and 141 (63.2%) of the 223 accessions, respectively, while other subsets showed <25% of variety identification rates. These SNP markers provide a molecular tool with many applications for genetics and breeding in cultivated pumpkin.