Genetic Analysis of Stripe Rust Resistance in the Chinese Wheat Cultivar Luomai 163.

Stripe or yellow rust (YR) caused by Puccinia striiformis tritici (Pst) is an important foliar disease affecting wheat production globally. Resistant varieties are the most economically and environmentally effective way to manage this disease. The common winter wheat (Triticum aestivum L.) cultivar Luomai 163 exhibited resistance to Pst races CYR32 and CYR33 at the seedling stage and showed a high level adult plant resistance in the field. To understand the genetic basis of YR resistance in this cultivar, 142 F5 recombinant inbred lines (RILs) derived from cross Apav#1 × LM163 and both parents were genotyped with the 16K SNP array and bulked segregant analysis sequencing (BSA-Seq). The analysis detected a major gene, YrLM163, at the seedling stage associated with the 1BL.1RS translocation. Additionally, three genes for resistance at the adult plant stage were detected on chromosome arms 1BL (Lr46/Yr29/Pm39/Sr58), 6BS and 6BL in Luomai 163, whereas Apav#1 contributed resistance at a QTL on 2BL. These QTL explained YR disease severity variations ranging from 6.9 to 54.8%. KASP markers KASP-2BL, KASP-6BS and KASP-6BL for three novel loci QYr.hzau-2BL, QYr.hzau-6BS and QYr.hzau-6BL were developed and validated. QYr.hzau-1BL, QYr.hzau-2BL and QYr.hzau-6BS showed varying degrees of resistance to YR when present individually or in combination based on genotype and phenotype analysis of a panel of 570 wheat accessions. Six RILs combining resistance alleles of all QTL, showing higher resistance to YR in the field than Luomai 163 with disease severities of 10.7-16.0%, are important germplasm resources for breeding programs to develop YR resistant wheat varieties with good agronomic traits.

Genetic and Biotechnological Approaches to Improve Fruit Bioactive Content: A Focus on Eggplant and Tomato Anthocyanins.

Anthocyanins are a large group of water-soluble flavonoid pigments. These specialized metabolites are ubiquitous in the plant kingdom and play an essential role not only in plant reproduction and dispersal but also in responses to biotic and abiotic stresses. Anthocyanins are recognized as important health-promoting and chronic-disease-preventing components in the human diet. Therefore, interest in developing food crops with improved levels and compositions of these important nutraceuticals is growing. This review focuses on work conducted to elucidate the genetic control of the anthocyanin pathway and modulate anthocyanin content in eggplant (Solanum melongena L.) and tomato (Solanum lycopersicum L.), two solanaceous fruit vegetables of worldwide relevance. While anthocyanin levels in eggplant fruit have always been an important quality trait, anthocyanin-based, purple-fruited tomato cultivars are currently a novelty. As detailed in this review, this difference in the anthocyanin content of the cultivated germplasm has largely influenced genetic studies as well as breeding and transgenic approaches to improve the anthocyanin content/profile of these two important solanaceous crops. The information provided should be of help to researchers and breeders in devising strategies to address the increasing consumer demand for nutraceutical foods.

Nanopore adaptive sampling of a metagenomic sample derived from a human monkeypox case.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Setup of proper genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore (PromethION P24) and Illumina (NextSeq. 2000) Whole Genome Sequencing (WGS) of a monkeypox sample. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. In addition to that, only Nanopore data allowed us to resolve the entire monkeypox virus genome, with respect to two structural variants belonging to the genes OPG015 and OPG208. These SVs in important host range genes seem stable throughout the outbreak and are frequently misassembled and/or misannotated due to the prevalence of short read sequencing or short read first assembly. Ideally, standalone standard Illumina sequencing should not be used for Monkeypox WGS and de novo assembly, since it will obfuscate the structure of the genome, which has an impact on the quality and completeness of the genomes deposited in public databases and thus possibly on the ability to evaluate the complete genetic reason for the host range change of monkeypox in the current pandemic.

Identification of Chromosomal Regions and Candidate Genes for Round leaf Locus in Cucumis melo L.

Leaf morphology plays a crucial role in plant classification and provides a significant model for studying plant diversity while directly impacting photosynthetic efficiency. In the case of melons, leaf shape not only influences production and classification but also represents a key genetic trait that requires further exploration. In this study, we utilized forward genetics to pinpoint a recessive locus, dubbed Cmrl (Round leaf), which is responsible for regulating melon leaf shape. Through bulked segregant analysis sequencing and extensive evaluation of a two-year F2 population, we successfully mapped the Cmrl locus to a 537.07 kb region on chromosome 8 of the melon genome. Subsequent genetic fine-mapping efforts, leveraging a larger F2 population encompassing 1322 plants and incorporating F2:3 phenotypic data, further refined the locus to an 80.27 kb interval housing five candidate genes. Promoter analysis and coding sequence cloning confirmed that one of these candidates, MELO3C019152.2 (Cmppr encoding a pentatricopeptide repeat-containing family protein, Cmppr), stands out as a strong candidate gene for the Cmrl locus. Notably, comparisons of Cmrl expressions across various stages of leaf development and different leaf regions suggest a pivotal role of Cmrl in the morphogenesis of melon leaves.

Advancements in genetic techniques and functional genomics for enhancing crop traits and agricultural sustainability.

Genetic variability is essential for the development of new crop varieties with economically beneficial traits. The traits can be inherited from wild relatives or induced through mutagenesis. Novel genetic elements can then be identified and new gene functions can be predicted. In this study, forward and reverse genetics approaches were described, in addition to their applications in modern crop improvement programs and functional genomics. By using heritable phenotypes and linked genetic markers, forward genetics searches for genes by using traditional genetic mapping and allele frequency estimation. Despite recent advances in sequencing technology, omics and computation, genetic redundancy remains a major challenge in forward genetics. By analyzing close-related genes, we will be able to dissect their functional redundancy and predict possible traits and gene activity patterns. In addition to these predictions, sophisticated reverse gene editing tools can be used to verify them, including TILLING, targeted insertional mutagenesis, gene silencing, gene targeting and genome editing. By using gene knock-down, knock-up and knock-out strategies, these tools are able to detect genetic changes in cells. In addition, epigenome analysis and editing enable the development of novel traits in existing crop cultivars without affecting their genetic makeup by increasing epiallelic variants. Our understanding of gene functions and molecular dynamics of various biological phenomena has been revised by all of these findings. The study also identifies novel genetic targets in crop species to improve yields and stress tolerances through conventional and non-conventional methods. In this article, genetic techniques and functional genomics are specifically discussed and assessed for their potential in crop improvement.

Plant necrotrophic bacterial disease resistance phenotypes, QTL, and metabolites identified through integrated genetic mapping and metabolomics in Solanum species.

Most food crops are susceptible to necrotrophic bacteria that cause rotting and wilting diseases in fleshy organs and foods. All varieties of cultivated potato (Solanum tuberosum L.) are susceptible to diseases caused by Pectobacterium species, but resistance has been demonstrated in wild potato relatives including S. chacoense. Previous studies demonstrated that resistance is in part mediated by antivirulence activity of phytochemicals in stems and tubers. Little is known about the genetic basis of antivirulence traits, and the potential for inheritance and introgression into cultivated potato is unclear. Here, the metabolites and genetic loci associated with antivirulence traits in S. chacoense were elucidated by screening a sequenced S. tuberosum x S. chacoense recombinant inbred line (RIL) population for antivirulence traits of its metabolite extracts. Metabolite extracts from the RILs exhibited a quantitative distribution for two antivirulence traits that were positively correlated: quorum sensing inhibition and exo-protease inhibition, with some evidence of transgressive segregation, supporting the role of multiple loci and metabolites regulating these resistance-associated systems. Metabolomics was performed on the highly resistant and susceptible RILs that revealed 30 metabolites associated with resistance, including several alkaloids and terpenes. Specifically, several prenylated metabolites were more abundant in resistant RILs. We constructed a high-density linkage map with 795 SNPs mapped to 12 linkage groups, spanning a length of 1,507 cM and a density of 1 marker per 1.89 cM. Genetic mapping of the antivirulence and metabolite data identified five quantitative trait loci (QTLs) related to quorum sensing inhibition that explained 8-28% of the phenotypic variation and two QTLs for protease activity inhibition that explained 14-19% of the phenotypic variation. Several candidate genes including alkaloid, and secondary metabolite biosynthesis that are related to disease resistance were identified within these QTLs. Taken together, these data support that quorum sensing inhibition and exo-protease inhibition assays may serve as breeding targets to improve resistance to nectrotrophic bacterial pathogens in potato and other plants. The identified candidate genes and metabolites can be utilized in marker assisted selection and genomic selection to improve soft- rot and blackleg disease resistance.

Experimental evolution of hybrid populations to identify Dobzhansky-Muller incompatibility loci.

Epistatic interactions between loci that reduce fitness in interspecies hybrids, Dobzhansky-Muller incompatibilities (DMIs), contribute genetically to the inviability and infertility within hybrid populations. It remains a challenge, however, to identify the loci that contribute to DMIs as causes of reproductive isolation between species. Here, we assess through forward simulation the power of evolve-and-resequence (E&R) experimental evolution of hybrid populations to map DMI loci. We document conditions under which such a mapping strategy may be most feasible and demonstrate how mapping power is sensitive to biologically relevant parameters such as one-way versus two-way incompatibility type, selection strength, recombination rate, and dominance interactions. We also assess the influence of parameters under direct control of an experimenter, including duration of experimental evolution and number of replicate populations. We conclude that an E&R strategy for mapping DMI loci, and other cases of epistasis, can be a viable option under some circumstances for study systems with short generation times like Caenorhabditis nematodes.

Linkage mapping of root shape traits in two carrot populations.

This study investigated the genetic basis of carrot root shape traits using composite interval mapping in two biparental populations (n = 119 and n = 128). The roots of carrot F2:3 progenies were grown over 2 years and analyzed using a digital imaging pipeline to extract root phenotypes that compose market class. Broad-sense heritability on an entry-mean basis ranged from 0.46 to 0.80 for root traits. Reproducible quantitative trait loci (QTL) were identified on chromosomes 2 and 6 on both populations. Colocalization of QTLs for phenotypically correlated root traits was also observed and coincided with previously identified QTLs in published association and linkage mapping studies. Individual QTLs explained between 14 and 27% of total phenotypic variance across traits, while four QTLs for length-to-width ratio collectively accounted for up to 73% of variation. Predicted genes associated with the OFP-TRM (OVATE Family Proteins-TONNEAU1 Recruiting Motif) and IQD (IQ67 domain) pathway were identified within QTL support intervals. This observation raises the possibility of extending the current regulon model of fruit shape to include carrot storage roots. Nevertheless, the precise molecular mechanisms through which this pathway operates in roots characterized by secondary growth originating from cambium layers remain unknown.

Unveiling the molecular regulatory mechanisms underlying sucrose accumulation and oil reduction in peanut kernels through genetic mapping and transcriptome analysis.

Sucrose content is a key factor for the flavor of edible peanut, which determines the sweet taste of fresh peanut and also attribute to pleasant flavor of roasted peanut. To explore the genetic mechanism of the sucrose content in peanut, an F2 population was created by crossing the sweet cultivar Zhonghuatian 1 (ZHT1) with Nanyangbaipi (NYBP). A genomic region spanning 28.26 kb on chromosome A06 was identified for the sucrose content through genetic mapping, elucidating 47.5% phenotypic variance explained. As the sucrose content had a significantly negative correlation with the oil content, this region was also found to be related to the oil content explaining 37.2% of phenotype variation. In this region, Arahy.42CAD1 was characterized as the most likely candidate gene through a comprehensive analysis. The nuclear localization of Arahy.42CAD1 suggests its potential involvement in the regulation of gene expression for sucrose and oil contents in peanut. Transcriptome analysis of the developing seeds in both parents revealed that genes involved in glycolysis and triacylglycerol biosynthesis pathways were not significantly down-regulated in ZHT1, indicating that the sucrose accumulation was not attributed to the suppression of triacylglycerol biosynthesis. Based on the WGCNA analysis, Arahy.42CAD1 was co-expressed with the genes involved in vesicle transport and oil body assembly, suggesting that the sucrose accumulation may be caused by disruptions in TAG transportation or storage mechanisms. These findings offer new insights into the molecular mechanisms governing sucrose accumulation in peanut, and also provide a potential gene target for enhancing peanut flavor.

A single locus determines praziquantel response in Schistosoma mansoni.

We previously performed a genome-wide association study (GWAS) to identify the genetic basis of praziquantel (PZQ) response in schistosomes, identifying two quantitative trait loci situated on chromosomes 2 and 3. We reanalyzed this GWAS using the latest (version 10) genome assembly showing that a single locus on chromosome 3, rather than two independent loci, determines drug response. These results reveal that PZQ response is monogenic and demonstrates the importance of high-quality genomic information.

Susceptibility of Toxoplasma gondii to autophagy in human cells relies on multiple interacting parasite loci.

IMPORTANCE: Autophagy is a process used by cells to recycle organelles and macromolecules and to eliminate intracellular pathogens. Previous studies have shown that some stains of Toxoplasma gondii are resistant to autophagy-dependent growth restriction, while others are highly susceptible. Although it is known that autophagy-mediated control requires activation by interferon gamma, the basis for why parasite strains differ in their susceptibility is unknown. Our findings indicate that susceptibility involves at least five unlinked parasite genes on different chromosomes, including several secretory proteins targeted to the parasite-containing vacuole and exposed to the host cell cytosol. Our findings reveal that susceptibility to autophagy-mediated growth restriction relies on differential recognition of parasite proteins exposed at the host-pathogen interface, thus identifying a new mechanism for cell-autonomous control of intracellular pathogens.

Genetic basis of ear length in sheep breeds sampled across the region from the Middle East to the Alps.

Ear length in sheep (Ovis aries) shows a wide range of natural variation, from the absence of an outer ear structure (anotia), to small outer ears (microtia), to regular ear length. Up until now, the underlying genetics of this phenotype has been studied in four sheep breeds from China, Jordan and Italy. These studies revealed a broad range of genes significantly associated with ear length, potentially indicating genetic heterogeneity across breeds or geographic regions. In the current study, we performed genome-wide SNP genotyping and haplotype-based mapping, in a population of 340 individuals, to identify loci influencing ear length variation in additional sheep breeds from Slovenia, Croatia, Cyprus and Greece. Additionally, two previously described candidate variants were also genotyped in our mapping population. The mapping model without candidate variant genotypes revealed only one genome-wide significant signal, which was located next to HMX1 on OAR6. This region was previously described as being associated with ear length variation in the Altay and Awassi sheep breeds. The mapping model including the candidate duplication genotype near HMX1 as a fixed effect explained the phenotypic variance on OAR6 and revealed an additional genome-wide significant locus on OAR13 associated with ear length. Our results, combined with published evidence, suggest that a duplication in the evolutionarily conserved region near HMX1 is the major regulator of ear length in sheep breeds descended from a larger region from Central Asia, to the Middle East, Cyprus, Greece and to the Alps. This distribution suggests an ancient origin of the derived allele.

Opioid trail: Tracking contributions to opioid use disorder from host genetics to the gut microbiome.

Opioid use disorder (OUD) is a worldwide public health crisis with few effective treatment options. Traditional genetics and neuroscience approaches have provided knowledge about biological mechanisms that contribute to OUD-related phenotypes, but the complexity and magnitude of effects in the brain and body remain poorly understood. The gut-brain axis has emerged as a promising target for future therapeutics for several psychiatric conditions, so characterizing the relationship between host genetics and the gut microbiome in the context of OUD will be essential for development of novel treatments. In this review, we describe evidence that interactions between host genetics, the gut microbiome, and immune signaling likely play a key role in mediating opioid-related phenotypes. Studies in humans and model organisms consistently demonstrated that genetic background is a major determinant of gut microbiome composition. Furthermore, the gut microbiome is susceptible to environmental influences such as opioid exposure. Additional work focused on gene by microbiome interactions will be necessary to gain improved understanding of their effects on OUD-related behaviors.

A key mutation in magnesium chelatase I subunit leads to a chlorophyll-deficient mutant of tea (Camellia sinensis).

Tea (Camellia sinensis) is a highly important beverage crop renowned for its unique flavour and health benefits. Chlorotic mutants of tea, known worldwide for their umami taste and economic value, have gained global popularity. However, the genetic basis of this chlorosis trait remains unclear. In this study, we identified a major-effect quantitative trait locus (QTL), qChl-3, responsible for the chlorosis trait in tea leaves, linked to a non-synonymous polymorphism (G1199A) in the magnesium chelatase I subunit (CsCHLI). Homozygous CsCHLIA plants exhibited an albino phenotype due to defects in magnesium protoporphyrin IX and chlorophylls in the leaves. Biochemical assays revealed that CsCHLI mutations did not affect subcellular localization or interactions with CsCHLIG and CsCHLD. However, combining CsCHLIA with CsCHLIG significantly reduced ATPase activity. RNA-seq analysis tentatively indicated that CsCHLI inhibited photosynthesis and enhanced photoinhibition, which in turn promoted protein degradation and increased the amino acid levels in chlorotic leaves. RT-qPCR and enzyme activity assays confirmed the crucial role of asparagine synthetase and arginase in asparagine and arginine accumulation, with levels increasing over 90-fold in chlorotic leaves. Therefore, this study provides insights into the genetic mechanism underlying tea chlorosis and the relationship between chlorophyll biosynthesis and amino acid metabolism.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

The gain-of-function mutation blf13 in the barley orthologue of the rice growth regulator NARROW LEAF1 is associated with increased leaf width.

Canopy architecture in cereals plays an important role in determining yield. Leaf width represents one key aspect of this canopy architecture. However, our understanding of leaf width control in cereals remains incomplete. Classical mutagenesis studies in barely identified multiple morphological mutants, including those with differing leaf widths. Of these, we characterized the broad leaf13 (blf13) mutant in detail. Mutant plants form wider leaves due to increased post-initiation growth and cell proliferation. The mutant phenotype perfectly co-segregated with a missense mutation in the HvHNT1 gene which affected a highly conserved region of the encoded protein, orthologous to the rice NARROW LEAF1 (NAL1) protein. Causality of this mutation for the blf13 phenotype is further supported by correlative transcriptomic analyses and protein-protein interaction studies showing that the mutant HvNHT1 protein interacts more strongly with a known interactor than wild-type HvHNT1. The mutant HvHNT1 protein also showed stronger homodimerization compared with wild-type HvHNT1, and homology modelling suggested an additional interaction site between HvHNT1 monomers due to the blf13 mutation. Thus, the blf13 mutation parallels known gain-of-function NAL1 alleles in rice that increase leaf width and grain yield, suggesting that the blf13 mutation may have a similar agronomic potential in barley.

Soybean Variety Saedanbaek Confers a New Resistance Allele to Phytophthora sojae.

Phytophthora root and stem rot (PRSR) disease results in substantial losses in soybean production worldwide. The occurrence of PRSR caused by Phytophthora sojae Kaufmann & Gerdemann has become increasingly important for soybean production in the Republic of Korea, but domestic soybean-P. sojae interaction has been less studied. The disease has been managed by developing varieties harboring resistance to the Phytophthora sojae (Rps) gene. The present study aimed to identify a major gene locus conferring resistance to new P. sojae isolate 2858 in the recombinant inbred line population derived from a cross between parental lines 'Daepung' (susceptible) and 'Saedanbaek' (resistant). Seventy-three recombination inbred lines (RILs) were evaluated for resistance to P. sojae isolate 2858. A resistance locus was identified in the approximate 3.3-4.3 megabase pair region on chromosome 3 using both single-marker and linkage analyses. The Rps of Saedanbaek (RpsSDB) was located on the well-known Rps gene/allele cluster region, which also partially overlapped with a locus previously identified in the Korean soybean variety, 'Daewon', resistant to another P. sojae isolate 2457 (RpsDW). Approximately 402 kilobase pairs of the interval region overlapped, including six nucleotide-binding site-leucine-rich repeat (NBS-LRR)-coding genes. Additional phenotypic assays revealed that Saedanbaek was susceptible to isolate 2457 and that Daewon was susceptible to isolate 2858, indicating that RpsSDB and RpsDW are different genes or alleles that confer race-specific resistance to the two P. sojae isolates. These results provide information that will be helpful for breeders developing P. sojae-resistant cultivars.

Mapping and Functional Dissection of the Rumpless Trait in Piao Chicken Identifies a Causal Loss of Function Mutation in the Novel Gene Rum.

Rumpless chickens exhibit an abnormality in their tail development. The genetics and biology of this trait has been studied for decades to illustrate a broad variation in both the types of inheritance and the severity in the developmental defects of the tail. In this study, we created a backcross pedigree by intercrossing Piao (rumpless) with Xianju (normal) to investigate the genetic mechanisms and molecular basis of the rumpless trait in Piao chicken. Through genome-wide association and linkage analyses, the candidate region was fine-mapped to 798.5 kb (chromosome 2: 86.9 to 87.7 Mb). Whole-genome sequencing analyses identified a single variant, a 4.2 kb deletion, which was completely associated with the rumpless phenotype. Explorations of the expression data identified a novel causative gene, Rum, that produced a long, intronless transcript across the deletion. The expression of Rum is embryo-specific, and it regulates the expression of MSGN1, a key factor in regulating T-box transcription factors required for mesoderm formation and differentiation. These results provide genetic and molecular experimental evidence for a novel mechanism regulating tail development in chicken and report the likely causal mutation for the tail abnormity in the Piao chicken. The novel regulatory gene, Rum, will, due to its role in fundamental embryo development, be of interest for further explorations of a potential role in tail and skeletal development also in other vertebrates.

Cell morphology QTL reveal gene by environment interactions in a genetically diverse cell population.

Genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening as they offer power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.

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.