G3: Genes|Genomes|Genetics

Phosphatidylinositol (PI) phosphates (PIPs) are membrane lipids with crucial roles in intracellular signaling, membrane trafficking and cytoskeletal organization. To perturb PIP function in Drosophila melanogaster sperm development, we previously expressed the Salmonella PIP phosphatase SigD (also known as SopB) under control of a spermatocyte- specific promoter. SigD expression results in reduced levels of plasma membrane PI 4,5-bisphosphate [PI(4,5)P2; henceforth PIP2] and male sterility. Transgenic lines expressing high levels of SigD (SigD-high) exhibit defects in spermatocyte cytokinesis, ciliary transition zone maturation and spermatid elongation, whereas lines expressing low levels of SigD (SigD-low) have defects in nuclear shaping and spermatid cell polarization. To further investigate the role of PIPs during Drosophila sperm development, we conducted a sensitized genetic screen to identify suppressors and enhancers of SigD-low phenotypes. From a screen of 760 chromosomal deficiencies spanning 83.6% of chromosomes 2 and 3, we identified 80 interacting regions that were further narrowed down to 40 individual suppressors. Among the strongest interactors were genes involved in microtubule dynamics, nucleocytoplasmic transport and membrane trafficking. The novel genetic interactions identified by our screen pave the way for further investigation into molecular mechanisms underlying the affected aspects of sperm development.

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. Using this sensitized neuroprotective background, we performed a F3 forward genetic screen in C. elegans atfs-1(lf) mutants to identify molecular components associated with the modulation of neurodegeneration 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 (orthologous to human KDM7A, PHF2, and PHF8) and jmjd-3.1 (homologous to yeast CYC8). 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 with DA neurodegeneration, we used neuron-targeted RNA interference, mutants, or both. 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-induced neurotoxicity.

Decades of research has uncovered a wealth of mechanistic information about the development of sensory bristles in Drosophila melanogaster. By studying large-effect, often loss-of-function mutations, many genes have been associated with bristle development, morphology, patterning, and number. Equally, the number of bristles present in certain areas of the fly cuticle is a classic quantitative trait, the genetic basis of which has been studied using a range of tools, from artificial selection to QTL (Quantitative Trait Locus) mapping. Such studies have often implicated well-understood bristle development genes as contributing to natural variation in bristle number. Here we contribute to the study of bristle number genetic variation in flies by executing a GWAS (genomewide association study). We generated whole genome sequences for 897 phenotyped male D. melanogaster individuals derived from a wild-derived, but lab-adapted outbred population, revealing - following quality control and filtering - over 780,000 variants with frequencies greater than 5%. Using these data we estimated the SNP (Single Nucleotide Polymorphism) heritability for ABN (abdominal bristle number) and SBN (sternopleural bristle number) as 0.28 and 0.35, respectively. These values indicate that our set of genotyped variants collectively explain a substantial fraction of the variance in phenotype in the mapping panel. Subsequently, genome scans revealed 1085 (ABN) and 211 (SBN) genomewide significant sites, and - due to extensive LD (Linkage Disequilibrium) in our panel - nearly all these sites are clustered into three locations; We find a GWAS hit for ABN in the middle of chromosome 3L, and hits for SBN at the tip of the X chromosome (where several prior mapping studies have resolved QTL for bristle number), and on 2L. Surveying existing studies that identified genes that control bristle number/development, we highlight several candidates that may segregate for causative, functional variants.

Enhancing grain carotenoid concentration to increase yellowness and provitamin A content in sorghum (Sorghum bicolor [L.] Moench) is a major breeding objective across sub-Saharan Africa. Carotenoid breeding is constrained by costly post-harvest phenotyping and can be accelerated through genetic markers that capture functional variation in key carotenoid biosynthesis genes. Zeaxanthin epoxidase (ZEP) is a major gene controlling sorghum grain carotenoid accumulation, and SNP-based KASP markers facilitate selection of high-carotenoid grain, although quantitative variation remains among lines carrying the favorable allele. We hypothesized that additional functional variation within ZEP and other carotenoid biosynthesis pathway genes contributes to this variation and can be revealed by a pangenome-informed analysis. Using a 33-member pangenome reference, we characterized sequence and structural variation at ZEP and other key carotenoid biosynthesis genes and applied pangenome-derived genotyping to study marker diversity associated with carotenoid accumulation. Evaluation of ZEP in the pangenome reference revealed previously uncharacterized structural variation that is absent from the BTx623 primary reference genome. A pangenome-based association analysis identified genetic markers associated with the accumulation of carotenoids which were specific to particular pangenome reference members, including in RTx430 and SRN39, both yellow endosperm lines. Machine learning identified markers in ZEP, {beta}-OH, ZDS, and Z-ISO as the most predictive for all carotenoid traits, suggesting a multi-locus genetic architecture underlies the accumulation of carotenoids in sorghum grain. By integrating sorghum pangenomic resources with machine learning, this study establishes a framework for pangenome-accelerated trait discovery and identifies new genetic targets for carotenoid biofortification in sorghum.

Understanding the genetic diversity and population structure of breeding materials is essential for developing stress-resilient cultivars. In tropical maize, where drought and low soil nitrogen (low N) severely limit productivity, continuous development of tolerant varieties remains a priority. This study assessed the genetic diversity and population structure of 250 doubled haploid lines (DHLs) derived from five drought- and low N-tolerant tropical populations. Genotyping was performed using mid-density DArTseq markers, yielding 3,305 high-quality SNPs for analysis. Results revealed a moderate level of diversity among the DHLs, with an average genetic distance of 0.39, a polymorphism information content (PIC) of 0.33, and a minor allele frequency (MAF) of 0.29. These values reflect substantial allelic variation, important for identifying complementary parental combinations in hybrid development. Discriminant analysis of principal components (DAPC) grouped the DHLs into five distinct clusters, largely corresponding to their source populations, although some admixture was observed. This indicates that while the genetic backgrounds of the source populations were mostly retained, recombination introduced useful variation. Overall, the clear population structure and high diversity observed among these DHLs provide a strong genetic foundation for future maize improvement. These lines represent valuable resources for heterotic group formation, hybrid development, and recurrent selection schemes aimed at enhancing drought and low nitrogen tolerance in tropical maize.

In common wheat (Triticum aestivum L.), variation at the Vrn1 and Ppd1 loci changes plant phenology, resulting in differential adaptation useful to breeders. Because the effects of phenology on grain yield are conditional on environmental factors, the incorporation of markers for Vrn1 and Ppd1 alleles into breeding approaches has proven difficult. Historic phenotypic, genotypic, and environmental data can provide insight into the relationship between major phenology alleles and grain yield as mediated by environmental variables. Analyses of eight years of breeding trials (1,038 lines at 219 site-years) determined that weak vrn1 alleles and Ppd1 insensitivity alleles have variable effects. These effects change depending on an environment's winter temperature and latitude, respectively, but the environmentally-driven changes in effect of vrn1 alleles are much more variable than effects of Ppd1 alleles. Mediation analyses showed phenologically-dependent environmental variables are one mechanism through which phenological changes created by vrn1 and Ppd1 variants alter yield across multiple developmental stages. Environmentally-driven relationships between flowering time and yield were modeled to estimate yield allele effects as a function of effects on phenology and environmental variables. Weak winter alleles at vrn1 may play a stabilizing role on yield, due to the correlation of winter conditions that drive larger effects with later-season warm temperatures that penalize later maturity. A better mechanistic understanding of how these loci drive environment-specific yield effects may facilitate utilization of markers for these alleles and improve predictive modeling of yield.

Comparing quantitative trait differentiation (Q_ST) with neutral genetic differentiation (F_ST) is an established approach to detect locally adaptive trait differentiation, but empirical applications can lose power when Q_ST is deflated by extrinsic trait variance (i.e. from non-genetic sources such as environmental effects and measurement error). We present REDQuanTEA (Replication-Enhanced Detection of Quantitative Traits Evolving Adaptively), a ready-to-use computational workflow that leverages biologically replicated data to disentangle genetic and extrinsic trait variance, while using Approximate Bayesian Computation (ABC) to refine estimates of Q_ST. Instead of comparing all traits with a single F_ST-based cutoff, REDQuanTEA generates trait-specific dynamic outlier Q_ST thresholds from neutral F_ST distributions based on matching experimental properties and the trait-specific level of extrinsic variance. In addition to identification of candidate adaptive traits from empirical data, the package enables simulation-guided assessment of experimental design and statistical analysis options. Using a demographic benchmark based on Drosophila melanogaster, REDQuanTEA outperformed estimators based on analysis of variance (ANOVA), particularly when extrinsic variance was moderate to high, while controlling false positive rates (FPRs). Assuming fixed experimental effort, two replicates with more independent genotypes often outperformed three replicate designs when extrinsic variance was low, and performed similarly as extrinsic variance increased. REDQuanTEA therefore provides a framework for optimizing experimental plans and detecting adaptively differentiated traits with improved power.

Targeted protein turnover of the transcription factor C/EBP is enhanced by the Tribbles (Trib) pseudokinase family of adaptor proteins, which bind to a conserved degron motif in C/EBP substrate and recruit a cognate COP1 E3 ligase to ubiquitinate C/EBP for degradation. We identified a conserved degron in the Drosophila C/EBP ortholog Slbo and fused this motif to a fluorescent protein (GFP or RFP) under the control of a constitutive promoter. Transgenic flies expressing this fluorescent protein (FP)::degron transgene, which we call the Fluorescent Ubiquitination-based Trbl Turnover Indicator (or FUTTI) sensor, showed reduced levels of fluorescence in response to increased levels of Drosophila Tribbles (Trbl) and conversely, increased levels when Trbl activity was reduced. Mutations in the Trbl conserved pseudokinase substrate binding pocket and in a divergent PEST-like motif in the C-terminal tail region disrupted Trbl activity in the FUTTI assay. These data indicate that FUTTI will be a valuable tool to identify sites of Trbl activity in vivo and the critical features of the protein required for function.

Spontaneous mutations are a rare but important source of novel genetic variation, yet their detection and characterization within active breeding programs are seldom documented at gene-level resolution. Grain size and shape are key determinants of rice quality, yield, and market classification. Here, we report the discovery and genetic characterization of a spontaneous short-grain (SG) mutation arising in the long-grain wild-type (WT) advanced breeding line RU2002174 from the LSU AgCenter Rice Breeding Program. The SG phenotype was first observed in 2019 and segregated in subsequent generations as a single recessive gene across both indica and japonica genetic backgrounds. Genetic mapping localized the mutation to a 41.6 kb interval on chromosome 5. Whole-genome sequencing identified a single candidate causal variant: a G[->]T transversion in exon 4 of SRS3 (Os05g06280), introducing a premature stop codon and resulting in a truncated protein. This allele was absent from representative U.S. breeding germplasm and the IRRI 3K SNP database, demonstrating that it represents a novel spontaneous loss-of-function allele of a previously characterized grain-size gene. These findings document the real-time emergence of functional genetic variation in elite rice germplasm and highlight the importance of monitoring off-types during seed increase and purification in breeding programs. They also provide additional insight into the role of kinesin-mediated cell elongation in determining rice grain architecture.

Withdrawal StatementThe authors have withdrawn their manuscript because it was submitted and made public without full consent of all authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Caraway (Carum carvi L., 2n = 2x = 20) is a medicinal and aromatic plant of the Apiaceae family valued for its essential oil with the major terpenoids carvone and limonene. While the essential oil content represents an important quality trait, low seed shattering and high thousand-grain weight (TGW) are key determinants of seed yield. As molecular tools remain scarce within Apiaceae breeding beyond carrot, we conducted a QTL mapping for these seed-related traits using two previously established F2 populations (Effective N(P1) = 236; N(P2) = 266). In P1 with the non-shattering maternal parent Rekord, QTL mapping revealed at least one shattering locus on LG03 (PVE: 9.9%). In addition, loci associated with essential oil (LG08, 10.5% PVE) and TGW (LG09, 11.1% PVE) were detected. In P2, we identified six essential-oil loci (LG02-06, LG10; combined PVE: 49.2%), with five loci affecting both carvone and limonene, and one (LG03) exclusively limonene. Additionally, a major QTL for TGW mapped to LG02 (PVE: 15.6%). For stalk attachment rate one minor QTL was detected (LG07, 8.5% PVE), while no QTL co-localized with the major shattering locus. To pave the path towards fine-mapping, we designed and validated 26 diagnostic PACE markers covering five key QTLs.

Common scab disease on potato is caused by members of more than 10 pathogenic Streptomyces species. Genome-enabled methods are being increasingly deployed to characterize Streptomyces that cause common scab disease of potato and other tuber and root crops. However, the study of phytopathogenic Streptomyces is constrained by the limited availability of high-quality genome sequences. Here we report improvements to the quality and completeness of genome assemblies for 12 pathogenic type strains of Streptomyces and six closely related non-pathogenic type strains. These assemblies have an average N50 of 7.4 Mbp and with BUSCO scores all greater than 98.5%. Analyses showed that the genomes of phytopathogenic Streptomyces are consistently among the largest Streptomyces genomes sequenced and, relative to those of non-pathogenic strains, are more enriched in genes involved in carbohydrate and amino acid metabolism. Plasmids were not consistently detected across assemblies, suggesting that they are not conserved across species and are not necessary for pathogenicity. Furthermore, comparisons of genome assemblies among both closely and distantly related strains revealed multiple rearrangements within linear chromosomes and reduced synteny near telomeric regions. These improved genome assemblies, many of which correspond to type strains, provide valuable resources for advancing our understanding of the pathogenicity in the genus.

In marine teleosts, gill ionocytes play central roles in the excretion of excess monovalent ions (Na+, Cl-, and K+). Basolateral K+ channels are considered essential for sustaining ion secretion by maintaining K+ homeostasis and membrane potential; however, the molecular identity of basolateral K+ channels in teleost gill ionocytes remains incompletely understood. Kcnj15 is a K+ channel expressed in multiple osmoregulatory tissues of the Japanese pufferfish (Takifugu rubripes), and its localization changes depending on tissue type and osmotic conditions; however, its localization in the gills has remained unresolved. Here, we determined the cellular localization of Kcnj15 in the gills and examined its expression in response to changes in environmental salinity. In seawater-acclimated fish, Kcnj15 was localized to the basolateral membrane of Nka- and Nkcc1-positive ionocytes. Following acclimation to hypoosmotic brackish water (1 ppt), transcript levels of cftr, nkcc1, and kcnj1a decreased, whereas kcnj15 expression remained unchanged. Under this condition, Kcnj15 was localized to the basolateral membrane of Nka-positive ionocytes. These results demonstrate that Kcnj15 is localized to the basolateral membrane of gill ionocytes and suggest that it may function as a basolateral K+ leak channel supporting ionocyte homeostasis under both seawater and low-salinity conditions.

Circadian feeding rhythmicity reflects the distribution of feed intake across the daily 24-hour cycle. Regular feeding-fasting cycles play an important role in regulating metabolism and maintaining health, but endogenous and environmental factors can disrupt the normal circadian pattern of feed intake. Here, we quantified hourly feed intake in 3,470 Swiss Landrace and 15,181 Swiss Large White pigs using 40.5 million records from automated feeding systems. We used the proportion of days exhibiting a significant feeding rhythm after wavelet analysis to investigate variation in circadian feeding organization. Circadian feeding rhythmicity was influenced by sex and age, with higher rhythmicity observed in females and older animals. Variance components analysis revealed a strong additive genetic contribution and a high heritability (0.53-0.57). Pigs with higher circadian feeding rhythmicity had reduced nocturnal intake, improved feed conversion ratio, and lower day-to-day variability in feed intake, suggesting improved feed efficiency and resilience. Genome-wide association testing of 15.7 and 23.1 million imputed sequence variants identified a QTL on chromosome 4 explaining 5.3% and 2.9% of the phenotypic variance of circadian feeding rhythmicity in Swiss Landrace and Swiss Large White pigs, respectively. The QTL overlaps OPRK1 and NPBWR1, two genes implicated in the regulation of feeding behavior, reward and energy homeostasis. Collectively, our findings establish circadian feeding behavior as a heritable trait in pigs and provide a basis to further investigate the genetic regulation of feeding rhythms in mammals and explore its use in animal breeding programs to improve feed efficiency and resilience.

Transforming Growth Factor beta (TGF-{beta}) signaling helps orchestrate multiple organismal traits in animals by regulating target gene expression. However, how this pathway's transcriptional regulators, called Smads, control target gene expression to generate different traits is not well understood. Using the C. elegans system, we identified the master chromatin remodeler B-lymphocyte maturation protein-1 (BLMP-1, a Kruppel-like zinc finger protein homologous to human PRDM1/BLIMP1) as a partner in Smad-mediated gene expression regulation. Previous research showed BLMP-1/PRDM1 controls organismal traits that the C. elegans TGF-{beta} superfamily member DBL-1 also affects. We identified genetic interactions between the DBL-1 pathway and blmp-1 for these traits. While BLMP-1/PRDM1 and DBL-1 pathway signaling had an additive effect on body size, blmp-1 mutants were epistatic to DBL-1 pathway mutants for male tail development, hermaphrodite gonad development, brood size, lipid levels, movement, and survival traits. We showed DBL-1 signaling transcriptionally regulates blmp-1 expression. We also identified a physical interaction between the DBL-1 pathway Smads and BLMP-1/PRDM1 using yeast-two hybrid analyses. Using bioinformatics and qPCR analysis, we showed that Smads and BLMP-1/PRDM1 regulate expression of common downstream target genes. Our work identifies novel interactions between the DBL-1 signaling pathway and BLMP-1, two conserved major transcriptional regulators, that ultimately influence a spectrum of organismal traits. We propose a model whereby BLMP-1/PRDM1 regulates DBL-1 signaling by acting as a gatekeeper, remodeling the chromatin architecture to control the access of the Smad complex to target genes. Together, our results reveal a direct link between a TGF-{beta} signaling pathway and a PRDM1-family master transcription regulator and offer clues to how they interact to control larval and adult traits. Our work suggests a conserved mechanism of transcriptional control of developmental traits by the Smad-PRDM axis.

Amongst measures of linkage disequilibrium, the correlation measure r2, due originally to Sewall Wright, has several advantages. Although basically a haploid statistic, it can readily be estimated using covariance and correlation from diploid data. Its population expectation can also be found from probability methods. We show that the probability of linked identity by descent, LIBD, directly estimates the expectation for r2 for Wright-Fisher models. The simplest such model for two loci is the haploid model, where each haplotype is chosen independently from haplotypes of the previous generation. This gives an equilibrium between recombination (recombination frequency c) and drift (population size N) where the expected value of r2 is approximately 1/(1+4Nc). For diploid models, the expectation is similar for low values of c, but contains a term in c2 and rises to double the haploid value for the limiting value of c = 0.5. An increase in r2 is also expected for the model of monogamous mating, as opposed to random mating for each offspring. The expectation for r2 is unaffected by the use of diploid rather than haploid data, an exception being the case of monogamous mating. Computer simulation shows close agreement of r2 with probability expectations, with minor discrepancies observed for unlinked loci. Using the probabilistic definition, r2 can be intuitively understood as a parameter shaping the effective sample size in genome-wide association studies.

Allelic heterogeneity is a term from genetics which means that alleles that differ in primary sequence can have a similar phenotypic outcome. In other words, they are functional equivalents, and they naturally appear through convergent evolution under selection. Current GWAS has trouble detecting these instances, as allelic heterogeneity leads to signal dilution in these analyses, often leading to a LOD score that stays below detection thresholds. In this paper, we show a method that can overcome this problem by bundling haplotypes into Artificial Combined Markers. The created marker matrix can then easily be used in existing GWAS software.

Reproductive stage drought disrupts flag leaf metabolism in rice (Oryza sativa), limiting photoassimilate translocation to developing grains and reducing grain yield (GY). How natural variation in this response relates to GY remains poorly understood. This study combines untargeted metabolomics of flag leaves from 271 indica rice accessions grown under well-watered (WW) and reproductive-stage water-limited (WL) field conditions with genome-wide association studies of 89 annotated polar metabolites. A total of 180 metabolite quantitative trait loci (mQTLs) were identified: 93 detected only under WL, 68 only under WW, and 19 under both conditions, indicating a predominant stress-dependent genetic control; mapping of GY itself detected only four marginal QTLs, none overlapping the mQTLs. Four high-density regions (HDRs) of mQTLs were identified: HDR1-1, detected only under WW conditions and associated mainly with raffinose family oligosaccharides; HDR1-2, HDR4, and HDR11, detected only under WL conditions and associated mainly with tricarboxylic acid cycle intermediates and amino acids. After correcting for genetic relatedness, accessions carrying the favourable haplotype at both HDR1-2 and HDR4 yielded 21.5% more under WL conditions than accessions carrying the unfavourable haplotype at both loci and lost 11.4 percentage points less GY to stress. Accessions carrying the favourable haplotype at only one locus showed a comparable reduction in yield loss. Haplotype-guided variant detection against the 3,000 Rice Genomes Project panel identified major-impact variants in candidate genes, including a polyphenol oxidase at HDR1-2 and tandem cytochrome P450s at HDR4. These hubs are promising, metabolically defined targets for breeding drought-resilient rice.

Copy number variants (CNVs) are a major source of genetic diversity and could contain some of the missing heritability for mouse models of human disease. However, mouse CNVs have not been comprehensively characterized because they are difficult to resolve in repeat-rich, segmentally duplicated or reference sequence-absent regions of the genome. Here we analyzed long range sequence (LRS) data for 40 inbred mouse strains and characterized CNVs using pangenome graph-based (and other) methods and a C57BL/6J telomere to telomere (T2T) genome reference sequence. We resolved 1,594 high-confidence CNVs that often overlap tandem repeats (60.3%), segmental duplications (44.8%) or pericentromeric regions (11.5%); and 131 CNVs were T2T sequence-specific. CNVs affected 384 protein-coding genes, which spanned a range of important functional classes. The 40-strain pangenome map expanded the genome sequence from 2.29 to 3.32 Gb, with the wild-derived strains accounting for the largest sequence increments. Two different AIs were sequentially used to analyze this database and identify a 29-kb deletion CNV within the Nlrp1b locus of KK mice that contributed to the metabolic syndrome they develop. Human NLRP1 alleles also were associated with metabolic syndrome features in human populations. Hence, AI analyses of this comprehensive T2T pangenome-based resource could uncover some of the missing heritability for mouse models of human diseases and biomedical traits.

Genome visualization has existed for as long as assembled genomes, with an array of tools and approaches to meet researchers' needs. The genome browser is one of these tools, and it has become a critical analytical resource for geneticists, genome biologists, and breeders. The evolution of genome browser capabilities is ongoing, and the USDA-ARS Maize Genetics and Genomics Database (MaizeGDB) has implemented JBrowse2, the most recent iteration of JBrowse. It includes multiple genome browser and alignment views, demonstrating pangenome visualization capability and permitting sophisticated functional characterization of maize loci. Described here also is MaizeGDB's new Pangenome Viewer, which integrates pangenome graph views with linear browser visualization to obtain an on-the-fly, interactive visual snapshot of structural variation across a pangenome at user-selected loci, with zoom capabilities and statistical and variant information for each subgraph. Finally, we demonstrate how different MaizeGDB pangenome pipelines complement one another to help guide accurate analyses. This functionality can lead to more precise characterization of loci that confer important agronomic traits, resulting in better outcomes for farmers and the public.