G3: Genes|Genomes|Genetics

Organismal tolerance of ionizing radiation is a complex trait whose genetic basis has been studied extensively, in large part due to its significance to human health and technological advancement. Conventional mutant screens in model organisms have revealed the paramount role of DNA damage response (DDR) and repair pathways in determining tolerance to ionizing radiation. However, uncovering natural genetic variation in radiotolerance is also of critical importance, as individual differences are associated with the differential susceptibility to cancer as well as differential response to radiation treatment. Genetic variation that underlies phenotype differences in natural populations often occurs in distinct genes and pathways as compared to the genes of major effect revealed by mutant screens, owing to the impact of natural selection on the former. We therefore sought to isolate natural variation in radiotolerance of Drosophila melanogaster by performing extreme QTL mapping. We generated a large genetically diverse multiparental population and exposed 3rd instar larvae to a semi-lethal dose or ionizing radiation. By sequencing surviving adults and comparing their haplotypes to unexposed controls from the same population, we identified a single major effect QTL spanning the 3rd chromosome centromere. The QTL contains 34 genes, none of which are previously implicated in radiotolerance. We interrogated the impact of these genes on radiotolerance through forward genetic analysis and RNA-seq. Our findings implicate diverse processes in radiotolerance including cell-cycle regulation and innate immune function.

Stripe rust and leaf rust, caused by Puccinia striiformis f. sp. tritici and P. triticina, respectively, are the most destructive wheat diseases in the southern Great Plains. Green Hammer is a hard red winter wheat (HRWW) cultivar released by Oklahoma State University in 2018 and has demonstrated a stable adult plant resistance to stripe rust and race-specific seedling resistance to leaf rust. To identify and map rust resistance loci, 109 doubled haploid (DH) lines derived from the cross between Green Hammer and another HRWW cultivar, Lonerider, were developed. Lonerider showed adult plant resistance to stripe rust but was susceptible to multiple P. triticina races. The DH lines were evaluated for stripe rust at the adult plant stage in greenhouse and field environments across Oklahoma, Kansas, and Washington, and for leaf rust at the seedling stage against seven U.S. P. triticina races and at the adult plant stage in Oklahoma and Texas. Genotyping-by-sequencing generated 6,078 polymorphic single-nucleotide polymorphisms used for genetic mapping. Quantitative trait loci (QTL) analysis identified 14 stripe rust and 8 leaf rust resistance QTL. For stripe rust, a major QTL in Green Hammer, QYr.osughln-2AS, was identified in the proximity of the 2NvS translocation. Three other major stripe rust resistance QTL were identified in Lonerider on chromosomes 2AL (two QTL) and 2BS (one QTL). For leaf rust, QLr.osughln-1DS and QLr.osughln-2DS.1 were the two major QTL identified in Green Hammer and most likely correspond to the all-stage resistance genes Lr21 and Lr39, respectively. In this study, we identified previously characterized genes as well as unknown genes that can be utilized in wheat breeding programs to enhance resistance to leaf rust and stripe rust.

In quantitative genetics, candidate SNPs are identified through genotype-phenotype associations inferred with genome-wide association studies (GWAS). In this study, we explore an alternative approach to detect genetic variants with non-neutral effects by tracking temporal trends in allele frequency in a winter wheat (Triticum aestivum L.) breeding population over an eight-year period, from which signals of selection may be inferred. Selection signatures were inferred with a generalized linear model, where we modeled trends in allele frequency as a function of time (crossing year). These signatures of selection were used to prioritize variants. Associations between phenotypic performance and individual load of prioritized variants were then investigated. Furthermore, we assessed whether incorporating selection information into a genomic best linear unbiased prediction (GBLUP) model improves model performance in terms of quality of fit and prediction ability. Our findings indicate that the inferred signals of selection are effective in identifying non-neutral variants. Variants under strong negative selection were associated with a decrease in protein content adjusted for grain yield (p-value < 0.01), while genetic variants that had been under moderate to high levels of positive selection were associated with increased grain yield (p-value < 0.01). However, incorporating selection information did not improve prediction accuracy. In conclusion, temporal trends in allele frequency can be used to detect non-neutral variants. The proposed approach may hence complement traditional quantitative genetic methods for detecting non-neutral genetic variation. This approach may allow breeders to detect non-neutral variants earlier in the breeding cycle, without resorting to phenotypic data.

Securing sustainable crop production requires the genetic improvement of abiotic stress tolerance. Due to the broad range of environmental factors causing abiotic stress and complex genotype-by-environment interactions, it is crucial to understand the genetic basis of crop yield under suboptimal conditions. Here, we developed a dent maize Multi-parent Advanced Generation Inter-Cross (MAGIC) population comprising 388 doubled haploid (DH) lines. The population was derived from eight founders with varying stress tolerance, selected from a dent diversity panel evaluated for yield performance across a wide range of European environments. The MAGIC DH lines were genotyped via whole-genome sequencing ([~]5X coverage) and evaluated in seven testcross and 14 line per se trials, for grain dry matter yield, leaf senescence, leaf rolling, anthesis-silking interval, and six additional agronomic traits. Genetic dissection identified 22 grain yield QTL, explaining 45% of the genetic variance. Under heat and drought stress, testcross grain yield correlated significantly with leaf senescence and leaf rolling measured in line per se trials. Bivariate multi-trait analysis showed that alleles for delayed senescence and reduced rolling at detected QTL generally exhibited positive effects on grain yield, suggesting that accumulating these favorable alleles could enhance yield performance. Incorporating these proxies into multi-trait genomic prediction models improved yield prediction accuracy, although gains were constrained by modest trait correlations. Given the comprehensive data, we also provide recommendations for optimizing sequencing depth and QTL mapping strategies in experimental maize populations. Key messageThis eight-founder MAGIC population represents a powerful resource for dissecting complex traits in maize, assessing the utility of drought proxy traits, and optimizing low-coverage whole-genome sequencing approaches.

Overexpression of -synuclein (-syn), an inherently disordered protein, triggers chronic activation of the mitochondrial unfolded protein response (UPRmt) pathway in Caenorhabditis elegans with enhanced dopaminergic (DAergic) neurodegeneration. Introduction of a loss-of-function(lf) mutation in atfs-1, the main transcriptional regulator of the UPRmt, into -syn nematodes results in significant neuroprotection from -syn-induced DA neuron loss, indicating that compensatory mechanisms provide neuroprotection. We performed a F3 forward genetic screen in C. elegans atfs-1(lf) mutants to identify molecular components associated with the modulation of neurodegeneration via UPRmt signaling in -syn-expressing DA neurons. Homozygous mutant animals were examined for enhanced neurodegeneration; multiple independent alleles were uncovered. Among these, we identified new nonsense alleles encoding the histone lysine demethylases (H3K27me3), jmjd-1.2 and jmjd-3.1. Another line carried a nonsense allele of twk-14. This gene encodes a conserved protein termed KCNK12 in mammals that facilitates passive background K+ leak currents to set and stabilize resting membrane potential. To further examine the association of these gene products in DA neurodegeneration, mutants and/or RNA interference were employed. DA neurodegeneration was observed in the -syn + atfs-1(lf) background when jmjd-1.2, jmjd-3.1, or twk-14 were individually depleted. These results provide evidence that jmjd-1.2 and jmjd-3.1, which encode previously characterized H3K27me3 demethylases, and the uncharacterized twk-14 gene product, orthologous to human KCNK12, naturally confer protection from -syn neurotoxicity.

Testing for Hardy-Weinberg equilibrium (HWE) is a fundamental component of genetic data analysis, widely used for quality control and model validation. Although HWE testing is well established for autosomal loci, inference on the X chromosome is more complex due to sex-specific genotype structures and potential sex differences in minor allele frequency (sdMAF). Existing tests differ in their assumptions about sdMAF and male sample inclusion, often leading to distinct but poorly characterized null hypotheses. We develop a general statistical framework for HWE inference using the robust allele-based regression model. By formulating HWE testing as an assessment of allele-level dependence, the framework directly parameterizes Hardy-Weinberg disequilibrium, unifies existing Pearson{chi} 2-based tests under explicit modeling assumptions, and clarifies their null hypotheses, degrees of freedom, and sensitivity to sdMAF. The framework also accommodates covariate and population-structure adjustment within a unified regression-based formulation. The proposed framework provides robust, interpretable, and flexible inference, establishing a unified statistical foundation for HWE testing across autosomal and X-chromosomal regions. Simulation studies and analysis of high-coverage 1000 Genomes Project data demonstrate that commonly used X-chromosome tests can exhibit inflated type I error or misleading inference when sdMAF is present.

Maximum utilization of existing genetic variability in a breeding program depends on the efficient classification of the inbred lines into heterotic groups, particularly under stress conditions. This study applied practical breeding approaches to determine the mode of genetic inheritance for Striga resistance and proposes a weighted heterotic grouping method based on the general combining ability of multiple traits (WHGCAMT) and compares its effectiveness with other existing methods in classifying the inbred lines into heterotic groups in Striga-infested and optimum environments. Using Diallel design IV, 300 crosses were generated from 21 inbred lines and 4 standard testers. The crosses, along with six checks, were evaluated in an 18 x 17 alpha lattice design with two replications at two locations, in both artificial Striga-infested and Striga-free environments. The inbred lines were genotyped using DArTtag SNP markers. Phenotypic and genotypic data were analyzed using R. Analysis of variance revealed significant mean squares for hybrid, general combining ability (GCA), specific combining ability (SCA) and their interactions with environment. Significant positive and negative GCA and SCA effects were detected for grain yield and other measured traits. However, a larger proportion of additive gene action than non-additive gene action was observed for grain yield and most measured traits. The analysis of molecular variance also showed substantial genetic differences within and between clusters. Except for HSCA, the mean grain yield between the inter-group and intra-group hybrids was significant for each method. Pairwise comparison of the inter- and intra-group hybrids of all the methods showed significant differences between the WHGCAMT and all other methods in most cases. WHGCAMT consistently produced higher-yielding inter-group hybrids and lower-yielding intra-group hybrids, achieving breeding efficiency improvements of 55.8%, 4.3%, 15.7%, and 11.4% over the HSCA, HSGCA, HGCAMT and molecular marker methods, respectively, under Striga infestation. Thus, WHGCAMT offers more precise, reliable and biologically meaningful heterotic groups among early-maturing maize inbred lines.

The fungal pathogen Zymoseptoria tritici poses a major global threat to wheat production, causing severe yield losses and necessitating intensive and costly fungicide applications. The increasing demand for durable genetic resistance has intensified interest in quantitative resistance loci, particularly those exhibiting multi-stage resistance (MSR), which suppress pathogen development continuously throughout the wheat life cycle. Many previously effective resistance genes are now showing declining efficacy, underscoring the urgent need for novel and long-lasting sources of resistance. In this study, we report the identification and genetic mapping of two quantitative resistance loci that address this need. The first locus, designated Stb23, is a major QTL on chromosome 1DS, with LOD scores exceeding 9 and explaining 6-36% of phenotypic variation at the seedling stage and 2-16% at the adult-plant stage. The second locus, designated Stb24, is a major QTL on chromosome 3DL, with LOD scores of approximately 10 and accounting for 11-30% of seedling-stage variation and 9-23% of adult-plant variation. Furthermore, two tightly linked KASP markers-snp_1D1217527 for Stb23 and snp_3D1077880 for Stb24-were developed and validated across three popular Australian bread wheat cultivars, providing practical tools for deploying these loci in breeding programs targeting improved resistance to Z. tritici. Key messageTwo significant major-effect resistance loci on chromosomes 1DS (proposed as Stb23) and 3DL (proposed as Stb24) were identified and characterized. Two tightly linked KASP markers with these loci were also discovered and validated for molecular-assisted breeding programs.

Salinity is a major crop production constraint in dry pea (Pisum sativum L.), making the development of salt-tolerant varieties essential to improve crop productivity and land-use efficiency. The genetic mechanisms of salt tolerance in dry pea is largely unknown, and research on salt-tolerant genes is limited. In this study, we established comprehensive genomic and transcriptomic resources, along with a robust screening protocol, to dissect the genetic basis of salinity tolerance using two germplasm sets: the USDA pea diversity panel, consisting of approximately 200 globally sourced accessions, and a set of 300 modern elite lines from the NDSU Pulse Crops Breeding Program. Genetic variation for the salinity response was assessed based on ten phenotypic traits, with root dry weight, shoot dry weight, and specific root length identified as key indicators based on their heritability. Genome-wide association mapping uncovered significant genomic regions and several candidate genes linked to salt stress, with the strongest association found on chromosome 6. Overlapping QTL signals across traits suggest a shared genetic architecture underlying salinity tolerance. Field-based transcriptomic analysis further identified five putative genes involved in salinity response conserved across multiple crop species. Notably, Psat5g000800, encoding a glycosyl hydrolase gene, was markedly upregulated under salinity stress. These findings highlight the complex, multi-gene regulatory nature of salinity tolerance in dry pea and underscore the importance of functional validation of candidate genes. This study provides key insights and practical tools to support breeding efforts aimed at improving salt tolerance in dry pea.

Animals sleep more when they are sick. In C. elegans, stress-induced sleep (SIS) follows cellular injury such as exposure to ultraviolet (UV) light. The genetic regulators of SIS remain incompletely defined. Using a worm-picking robot, multi-well WorMotel imaging, and association analysis we performed a semi-automated screen of 941 whole-genome-sequenced Million Mutation Project (MMP) strains. We quantified behavioral activity and quiescence before and after ultraviolet (UV) radiation. We applied the Sequence Kernel Association Test (SKAT) to this behavioral data to prioritize 6,663 genes and observed significant enrichment of known SIS genetic regulators. Based on these results, we conducted a candidate validation screen for additional genes regulating SIS. We identified three genes (strd-1, egl-8, cla-1), mutations in which reproducibly influence SIS. Further exploration of these genes holds potential for enhancing our understanding of the molecular basis of SIS. These findings establish a pipeline for automated behavioral phenotyping coupled with gene-based association to accelerate studies of C. elegans neurogenetics.

Viral haemorrhagic septicaemia (VHS) is a severe disease affecting rainbow trout (Oncorhynchus mykiss) and a wide range of wild freshwater and marine fish species. VHSV threatens rainbow trout aquaculture, as it may cause 100% mortality in fry. Previous studies identified a quantitative trait locus (QTL) on chromosome 3 associated with resistance to VHSV waterborne challenge and reduced viral replication in fin explants, although these findings were obtained using limited genetic diversity. The objective of this study was to validate and extend the identification of genomic regions associated with resistance to VHSV in the genetically diverse rainbow trout line designated "synthetic." A genome-wide association study (GWAS) was conducted using whole-genome sequences from parents of progeny classified as resistant or susceptible to a VHSV waterborne challenge. While the QTL on chromosome 3 was not validated in the synthetic line, four novel suggestive SNPs associated with survival following VHSV waterborne challenge were identified on chromosomes 6, 8, 17, and 32. Notably, one SNP on chromosome 17 was located within a gene potentially involved in antiviral defence, a paralog of lrp1 (low-density lipoprotein receptor-related protein 1). To further investigate its role, lrp1 function was analysed in vitro using CRISPR-Cas9 genome editing. Three independent lrp1-/- CHSE-EC cell lines were generated and challenged with VHSV. The results showed that lrp1 is not essential for viral entry but may modulate the inflammatory response during VHSV infection in epithelial cell lines.

Organismal survival depends on coordinated responses to oxidative stress and DNA damage. Using Caenorhabditis elegans, we investigate mul-1, a robust transcriptional target of ionizing radiation and reactive oxygen species. Although annotated as a mucin, MUL-1 is a small ShKT domain-containing protein belonging to an invertebrate expanded family of cysteine-rich proteins. mul-1 is selectively induced by oxidative stress, including IR, hydrogen peroxide (H2O2), Pseudomonas aeruginosa infection, or loss of the peroxiredoxin PRDX-2, via the p38 MAPK-ATF-7 pathway in intestinal cells. Loss of mul-1 and its paralogs increases ROS accumulation, oxidative stress sensitivity, and CEP-1/p53 dependent germ cell apoptosis. Combined deletion of mul-1 paralogs causes constitutive apoptosis, reduced fecundity, and compensatory activation of DAF-16/Foxo and SKN-1/Nrf2 stress response pathways. Together with genetic analysis of SYSM-1, these findings suggest MUL-1-like ShKT proteins buffer oxidative stress.

TAPBP is a key chaperone of the peptide-loading complex that facilitates peptide loading onto major histocompatibility complex class I (MHC I) molecules. This study characterized TAPBP alleles in Korean Native Chickens (KNCs), identified novel variants, and evaluated haplotypic associations with BF2. Thirty-six samples representing six KNC lines were genotyped using LEI0258 and the MHC-B SNP panel, and individuals homozygous at both markers were classified into 16 groups. The same samples were subjected to Sanger sequencing of TAPBP exons 3-8. Sequences were assembled and aligned against MHC-B reference haplotypes and the Red Junglefowl reference. Additional comparisons with "tapasin allele" datasets enabled the identification of novel variants. Six novel nucleotide variants were detected across exons 3-6, including one nonsynonymous substitution in exon 4 (D251H). This residue corresponds to position Q265 in human TAPBP and lies adjacent to residues involved in MHC I interaction, suggesting potential functional relevance. Furthermore, TAPBP exhibited high haplotype diversity (Hd = 0.93) and moderate nucleotide diversity ({pi} = 0.00892), with exon 5 showing the highest diversity ({pi} = 0.01). B9 was the most frequent haplotype at the nucleotide level, whereas B6/B24 predominated at the amino acid level. Comparison with BF2 data revealed haplotype-dependent pairing patterns: BF2-B9 consistently matched TAPBP-B9, whereas BF2-B6 was associated with distinct TAPBP nucleotide variants, indicating allelic diversification within a shared haplotypic background. Homozygosity at LEI0258 and the SNP panel corresponded with TAPBP homozygosity, supporting marker-based prediction. These findings highlight potential BF2-TAPBP associations and provide a foundation for understanding variation in MHC I peptide loading.

Genetic-background studies require defined perturbations that can be crossed reproducibly into many recipient backgrounds. We generated a Drosophila dilp2GS-rpr donor line for adult-inducible ablation of insulin-producing cells (IPCs), which secrete insulin-like peptides and provide a tractable model of insulin-deficient metabolic physiology. This line carries dilp2-GeneSwitch-GAL4 and UAS-reaper in cis on the same second chromosome homolog over a balancer. PCR genotyping and sequencing confirmed both transgenic elements in the candidate recombinant line. RU486 induction reduced dilp2 mRNA expression, supporting partial IPC ablation. Treatment-duration testing identified 8 days of RU486 as sufficient to increase whole-body glucose in the dilp2GS-rpr line but not in the background-matched control; food intake did not differ between RU486- and vehicle-treated flies. Across metabolic assays, whole-body glucose showed the clearest RU486- and line-dependent phenotype. This validated dilp2GS-rpr line enables testing how recipient genetic backgrounds modify inducible IPC/DILP metabolic phenotypes and provides a framework for similar linked donor-line resources.

Nuclear receptors, transcription factors essential for organism growth, development, and reproduction, are expressed in a variety of tissues, with some exhibiting differential expression between males and females. The Estrogen-related receptor (ERR) is a conserved metabolic nuclear receptor required for energy metabolism and lipid accumulation. While previous studies in Drosophila have identified potential ERR targets from mixed sex larval populations and adult males, it is unclear whether transcriptional targets and biological pathways downstream of ERR are altered in a sex-specific manner. Here, we took an RNA sequencing approach to identify candidate ERR targets specifically in adult females and compared differentially expressed genes to a published male-specific dataset. Whole body conditional knockout of ERR significantly downregulated transcription of enzymes associated with glycolysis and the pentose phosphate pathway. In contrast, components of the DNA replication machinery were selectively downregulated in adult females, whereas ribosome biogenesis transcription was increased. Our results have further defined the metabolic targets of ERR between males and females, as well as suggest that ERR regulates DNA replication and global translation in females. SUMMARYIn this manuscript, we used RNA sequencing to identify differential expression of transcripts dependent on the nuclear receptor ERR in Drosophila adult females. We find that ERR is required for activating transcription of glycolytic and pentose phosphate pathway enzymes, as observed in larvae and adult males. Furthermore, compared to males, loss of ERR in females specifically decreased DNA replication enzyme components while upregulating ribosomal components. Our results suggest that nuclear receptors have common and sex-specific targets, which will be of interest for those in the nuclear receptor and sexual dimorphism fields.

Pyruvate dehydrogenase complex (PDHc) deficiency (PDCD) is a primary mitochondrial disorder characterized by neurodevelopmental disability, altered intermediary metabolism and early mortality. Dichloroacetate (DCA), a pyruvate analogue, is a well-described PDHc activator that remains under clinical investigation for treatment of PDCD. Here, we studied the in vivo efficacy of a 5-point log concentration range of DCA on animal health and metabolism in C. elegans with feeding RNA interference (RNAi) expression knockdown of either PDHA-1 or DLD-1 homologues at graded degrees to model variable disease severity. These worm models recapitulate phenotypic features of PDCD observed in human patients, including reduced survival, delayed growth, locomotor impairment, and elevated lactate and/or pyruvate tissue levels. DCA treatment appeared well-tolerated, with no gross morphologic toxicity seen at doses up to 25 mM. Significantly improved health, survival, tissue lactate levels, and mitochondrial physiology were observed at 25 mM in pdha-1(RNAi) knockdown animals. DCA treatment in dld-1(RNAi) C. elegans models (undiluted, 1:20 dilution, and 1:100 dilution) showed significant therapeutic benefits on survival, neuromuscular function and metabolic phenotypes primarily in the moderate (1:20) and/or mild (1:100) dld-1(RNAi) deficiency strains, but not in full-dose dld-1(RNAi). Importantly, linear growth, neuromuscular activity, and mitochondrial physiology were significantly improved with DCA treatment even in the most severe dld-1(RNAi) undiluted model. Overall, preclinical modeling provides objective evidence of DCA therapeutic efficacy in C. elegans expression knockdown strains for two well-conserved homologues of PDHA1 and DLD that represent distinct genetic etiologies of PDHc deficiency, with demonstrated beneficial effects on survival, healthspan, tissue lactate, and mitochondrial physiology. These data further confirm that DCA's therapeutic effect correlates with PDHc disease phenotype severity in dld-1(RNAi) animals.

An expectation of a hypothesis that proposes cell-to-cell signalling pathways are redundant due to the redundancy of pathway terminal transcription factors (TFs) was tested by screening 35 signalling ligands (SLs) for rescue of a decapentaplegic (dpp) hypomorphic wing growth phenotype. The screen identified three examples of partial rescue: Hedgehog (HH), Semphorin 1a (SEMA1A) and Wnt ortholog 2 (WNT2). HH overexpression with dppGAL4 may increase the expression of DPP activity from the hypomorphic dpp alleles. However, SEMA1A and WNT2 did not phenocopy ectopic expression of HH or DPP and neither SEMA1A nor WNT2 were required for wing growth suggesting substitution of DPP for partial restoration of wing growth. The WNT2 rescue was dependent on the Frizzled 4 (FZ4) WNT receptor excluding the possibility that WNT2 weakly binds the DPP receptor. Although examples of phenotypic nonspecificity of SL function were identified, this is an expectation, and not direct proof, of the hypothesis of TF redundancy. Screen Report SummaryAn expectation of a hypothesis proposing that cell-to-cell signalling pathways are redundant due to the redundancy of the pathway terminal transcription factors was tested by screening for replacement of one signalling ligand (DPP; SLa) with another SLb for wing growth. Three non-DPP SLs were identified in the screen of 35SLs: HH, SEMA1A and WNT2. Genetic analysis of Sema1a and Wnt2 suggests functional complementation of dpp for wing growth suggesting that SEMA1A and WNT2 partially replace DPP for wing growth. Therefore, an expectation of the hypothesis is met.

The mTOR pathway is a central regulator of cellular metabolism and growth whose downregulation extends life span across taxa. In C. elegans, mTOR acts cell non-autonomously to influence organismal longevity, yet underlying mechanisms remain elusive. Here, we show that deletion of the TORC1 regulator, raga-1/RRAGA, enhances production of the bile acid-like hormone, dafachronic acid (DA), and extends life span dependent on DA-hormone biosynthetic genes and DA-cognate nuclear hormone receptor DAF-12, a homolog of mammalian farnesoid X receptor (FXR). Through functional genomic screens, we identify the evolutionarily conserved short chain dehydrogenase DHS-26/DHRS1 as a previously uncharacterized downstream regulatory target and effector of the mTOR-steroid axis essential for organismal longevity. Worm DHS-26 is expressed prominently in the canal associated neurons, cells which are essential to growth and development, suggesting a neuroendocrine mechanism. Murine DHRS1 also exhibits regulation by mTOR signaling and nuclear receptor FXR suggesting that the mTOR-DHS-26/DHRS1 axis is evolutionarily conserved. These findings suggest that mTOR signaling systemically impacts metazoan longevity through the regulation of bile acid-like hormone availability and nuclear receptor signal transduction.

Scyphozoan jellyfish have a complex life cycle that includes a characteristic transition known as strobilation. Retinoid signaling has been suggested to be involved in jellyfish metamorphosis and development. However, the genomic basis of signaling pathways associated with metamorphosis has not been sufficiently compared at the class level. Experimental studies have reported that indole compounds can induce metamorphosis in some jellyfish species. Indole- and tryptophan-derived metabolites are known to function as ligands for the aryl hydrocarbon receptor (AhR) in other organisms. However, the potential role of AhR signaling in jellyfish metamorphosis has not been previously explored. We compared the distribution of retinoid- and AhR-associated gene families across multiple scyphozoan genomes. This analysis aimed to characterize their distribution patterns in relation to signaling pathways associated with development and environmental responses. A standard gene prediction and annotation pipeline was applied to 20 species from 21 publicly available scyphozoan reference genome assemblies retrieved from the NCBI database. The distribution and copy number of these gene families were compared across species. Retinoid-associated gene families were detected across almost all Scyphozoa genomes, and core components of AhR signaling (AhR, ARNT) were identified in most species. These results suggest that scyphozoan genomes contain genetic components of retinoid- and AhR-related signals. This study presents the distribution of gene families related to developmental signaling across Scyphozoa using a comparative genomic approach. It does not imply direct functional involvement of retinoid or AhR signaling, but instead focuses on potential signaling pathways at the genome level. It also provides an overview of currently available scyphozoan genomic data. These findings provide a basis for future hypothesis generation and functional validation in jellyfish metamorphosis research.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most destructive diseases of wheat worldwide. Although the Z. tritici genome encodes hundreds of predicted effector proteins, functional characterization through the use of genome-editing techniques has been limited due to low homologous recombination efficiency and extensive effector redundancy. In this study, we established and evaluated a CRISPR/Cas9-based genome editing procedure for targeted effector gene disruption in Z. tritici using in vitro-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes combined with short (60 bp) homologous donor DNA flanks. Using this approach, we successfully generated knockout mutants for a selected candidate effector gene, the Hce2 domain-containing effector Mycgr3107904. Virulence assays on the susceptible wheat cultivar Taichung 29 revealed that two independent{Delta} Mycgr3107904 mutants exhibited a pronounced delay in symptom development compared to the wild-type strain IPO323, with disease onset and progression delayed by approximately 4-5 days. While mutant strains ultimately followed a similar disease trajectory, wild-type-infected leaves displayed extensive necrosis and pycnidia formation at earlier time points, indicating a significant reduction in virulence upon loss of Mycgr3107904. Together, our results demonstrate the feasibility of CRISPR/Cas9-mediated effector gene knockout in Z. tritici and provide functional evidence that Mycgr3107904 contributes to timely disease progression. This work advances genome editing tools for Z. tritici and facilitates systematic dissection of effector functions underlying fungal virulence.