G3: Genes, Genomes, Genetics

Gene model for the ortholog of tango (tgo) in the Sep. 2015 (UC Berkeley ASM127793v1/DbusGB1) Genome Assembly (GenBank Accession: GCA_001277935.1) of Drosophila busckii. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Import of double-stranded RNA (dsRNA) into the germ line can have consequences that last for many generations. However, the role of such transgenerational regulation by extracellular dsRNA is unclear. In the nematode C. elegans, entry of dsRNA into the cytosol requires the transmembrane protein SID-1 and loss of SID-1 for a few generations causes changes in gene expression that can persist for hundreds of generations. Here we report an expanded number of such SID-1-dependent genes (SDGs) and analyze two germline-expressed SDGs: sdg-1 and sdg-2. Deleting sdg-1 reduces brood size in some lineages. An endogenous SDG-1::mCherry fusion protein shows conditional enrichment within nuclei, colocalization with perinuclear germ granules, and colocalization with microtubules. Although animals with SDG-1::mCherry have a normal brood size, they have fewer early progeny with some animals showing defective germline morphology. Deleting the sdg-1 open reading frame eliminates defects in most but not all the animals that express mCherry in a now sdg-1(-) background, suggesting transgenerational consequences of SDG-1::mCherry that persist in some siblings lacking sdg-1. Deleting sdg-2 also reduces brood size in some lineages. An endogenous SDG-2::mCherry fusion protein is constitutively detectable in the cytoplasm and nucleus. The sequence and predicted structure of SDG-2 suggest that it can interact with the Gli-type transcription factor TRA-1, which regulates spermatogenesis. Together, these results suggest that changes in SDG-1 or SDG-2 can impact reproduction. Therefore, the import of extracellular dsRNA or other SID-1 function(s) that regulate SDGs could have evolved to modulate the lingering impacts of ancestral epigenetic changes.

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.

Laboratory cultivation subjects model organisms to selective pressure and genetic drift that can result in the accumulation of many genomic and phenotypic differences over time. The nematode Caenorhabditis elegans has been used for research since the 1970s, and studies comparing the N2 Bristol and CB4856 Hawaiian isolates provided foundational knowledge about metazoan genome evolution. Most comparative genomics studies have used these isolates because their long-term geographical isolation promoted a high degree of genomic divergence within the species. Further, there is growing evidence of phenotypic differences between laboratory lineages of each wild type isolate after repeated independent lab cultivation of these strains. To examine the genomic divergence between different laboratory lineages the Bristol and Hawaiian backgrounds, we first generated de novo genome assemblies of two Bristol and two Hawaiian lineages from Illumina and PacBio sequencing reads. Following genome assembly, we quantified Single Nucleotide Polymorphisms (SNPs), short insertion/deletions (indels), and genomic structural variants (SVs). Between laboratory lineages of the Bristol isolate, we identified 25,432 SNPs, 5,202 indels, and 441 SVs. When aligning laboratory lineages of the Hawaiian isolate, we identified 4,518 SNPs, 1,188 indels, and 387 SVs. For both sets of comparisons, we find that SNPs and indels are broadly enriched in introns and depleted from coding sequences. In contrast to SNPs and indels, we find that genomic SVs are enriched in intergenic sequences. Taken together, our analyses reveal the accumulation of genomic divergence between lineages of Bristol and Hawaiian C. elegans from independent lab cultivation, and how these variants may underpin emergent phenotypic differences observed in the two most popularly used C. elegans wild type isolates. Author SummaryLaboratory model organisms, like natural populations, are subject to evolutionary pressures and genomic changes during prolonged laboratory cultivation. In this study we comprehensively quantify SNPs, indels, and SVs between independent lab cultivations of the C. elegans lineages of the Bristol and Hawaiian isolates.

PremiseCentaurea melitensis (Asteraceae) is a problematic invader of grasslands globally, but little is known about its genetic makeup. Here we develop a reference genome to facilitate studies of its invasion history, genetic variation, and evolution. MethodsInbred offspring of a single individual of C. melitensis from its invasion of California, USA were used for flow cytometry to estimate genome size, and for genomic DNA extraction. DNA was sequenced with PacBio HiFi technology (yield = 85.7Gb). The genome was assembled with Hifiasm and annotated with BRAKER3. GENESPACE was used to compare gene order (synteny) with three other species within the subfamily Cichorioideae. ResultsWe estimated a mean genome size of 795.0 Mbp for C. melitensis, and our assembly totaled 696.6 Mbp in 48 contigs (N50 = 55.6 Mbp; BUSCO = 98%), with annotation of 25,157 protein-encoding genes. This included four telomere-to-telomere putative chromosomes, nine additional chromosome arms terminated by telomeric repeats, and a complete chloroplast genome. Synteny varied markedly across the genus and subfamily, suggesting a dynamic history of structural variation in the lineage of C. melitensis. DiscussionWe provide a highly complete and contiguous genome assembly to facilitate the further study of genomic variation in C. melitensis.

Gene model for the ortholog of raptor in the D. grimshawi May 2011 (Agencourt dgri_caf1/DgriCAF1) Genome Assembly (GenBank Accession: GCA_000005155.1) of Drosophila grimshawi. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Fungal genomics has expanded rapidly over the past 30 years, and recently the pace and breath has further quickened for many taxa, although many taxonomic gaps persist. With three decades of rapid growth, fungal genomics now merits a re-examination of its history, progress, and unresolved taxonomic gaps. Here, we review the development of fungal genomics from early efforts such as the Fungal Genome Initiative to current progress driven by third-generation long-read sequencing. We have compiled and summarised publicly available fungal genomes to highlight trends in assembly quality, adoption of long-read technologies, and taxonomic representation. Notably, substantial phylogenetic gaps remain, particularly outside Dikarya, and significant challenges persist for unculturable taxa. This review identifies priorities for the fungal community, including: (1) coordinated efforts to close major taxonomic gaps across the fungal tree of life; (2) improved repository metrics to facilitate identification of high-quality assemblies; and (3) improved and standardised genome annotation which is lacking for most assemblies. Together, these steps will support the development of reliable genomic resources that capture the full breadth of diversity across the fungal kingdom, generating foundational data for comparative genomics, evolutionary biology, functional studies, genetic studies and applied research.

At a mere 20 cells, the Caenorhabditis elegans intestine regulates metabolism, energy homeostasis, host defense, yolk production, and genetic aging, all while dynamically responding to its environment. How the intestine develops to carry out these disparate functions is unknown, and how cells differ along the length of the intestine is unclear. To address these questions, we performed single-cell RNA sequencing (scRNA-seq) on FACS-enriched intestinal cells from mixed-stage C. elegans embryos. The resulting single-cell transcriptomes of 974 cells organized into 13 clusters, suggesting a diversity of cell types and states. We used two post hoc approaches to ascribe identities to each cluster. First, genes with known developmental timing in early-, mid-, and late-stages were used to place clusters in time, and smiFISH microscopy was used to fine-tune the assignments. Second, the eight late-stage clusters were assessed for their region of origin. To assign these clusters to anatomical regions, we identified marker genes for each cluster and assessed their expression along the anterior-to-posterior length of the intestine using smiFISH microscopy. Genes associated with growth and cell division were expressed in early stages, whereas genes associated with immune responses and metabolism were expressed later. Genes associated with biotic responses and RNA metabolism were the most likely to vary across the intestines anterior-posterior axis. Finally, perturbation of anterior-localized intestinal transcripts more robustly affected intestinal function compared to central or posterior-localized genes. Overall, this research illustrates the intrinsic heterogeneity across the 20 cells of the embryonic intestine and sets the stage for future works aimed at understanding cell-specific intestinal responses to diet and the environment. ARTICLE SUMMARYWe investigate how the Caenorhabditis elegans intestine develops specialized functions on a spatiotemporal scale. We used single-cell RNA-sequencing to analyze embryonic intestinal cells and identify 13 distinct clusters. Combining gene expression analysis with microscopy, we assigned clusters to developmental stages and anatomical regions. Clusters associated with early intestine development express genes linked to growth and cell division, while later-stage clusters express genes involved in metabolism and immune responses. Genes varied across the intestines anterior-to-posterior axis, and disrupting anterior-specific genes produced stronger functional effects. These findings reveal previously unrecognized intestinal diversity and provide insight into how intestinal cells specialize during development.

Gene model for the ortholog of raptor in the May 2011 (Agencourt Dere_CAF1/DereCAF1) Genome Assembly (GenBank Accession: GCA_000005135.1) of Drosophila erecta. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

BackgroundThe black soldier fly (Hermetia illucens) is an emerging model for bioconversion and industrial rearing. Its genome is highly repetitive, yet the contribution of transposable elements (TEs) to population divergence and demographic processes. The sampled populations represent a gradient of demographic histories, including wild and near-wild North American populations, and domesticated European strains with shared industrial origins. Difference in TE composition may influence genome structure, regulatory variation, and evolutionary responses to captive environments. ResultsA comparative analysis of the repetitive landscape was done for four H. illucens genomes, one of which is a wild-caught specimen. Total repeat content was high across all assemblies (67.6% to 70.8%) and dominated by LINE elements. Class-level TE diversity was nearly identical among genomes, but multiple DNA transposon families showed distinct lineage-specific differences. Large families including Maverick and Academ were generally depleted relative to the wild sample. Divergence profiles revealed patterns consistent with recent turnover in several families. Family level turnover, rather than class level change, accounted for the most difference among the genomes. TE-associated structural variants (TESVs) were also not uniformly distributed. Most chromosomes showed mid-chromosome enrichment, and a pronounced TESV peak on chromosome 5 overlapped a histone rich region containing many unclassified repeats. Use of a repeat library derived from multiple genomes increased the number of detected TESVs and improved classification within complex regions, demonstrating that multi-genome libraries enhance annotation accuracy compared to single reference-based models. ConclusionsMultiple DNA transposon families show evidence of recent or lineage-specific amplification in H. illucens, suggesting that TE amplification contributes to genome variation during demography-associated TE turnover. The multi-genome-based library improved TE detection and classification, providing a proof of concept that even a small lineage-inclusive repeat library enhances annotation accuracy and capture TE diversity missed by single-reference approaches. Together, these findings demonstrate that TE family turnover plays a significant role in shaping genome architecture and adaptation in this species.

We present a chromosome-level genome assembly of a female great grey owl (Strix nebulosa lapponica). The assembly comprises two pseudo-haplotypes of 1554 Mb and 1242 Mb, with 83.2% and 91.4% scaffolded into 40 autosomal chromosomes, in addition to the W and Z sex chromosomes both placed in hap1. Assembly completeness is high (BUSCO 99.2% and 94.8%), with 18,493 and 17,279 annotated protein-coding genes for hap1 and hap2, respectively. This genome establishes a reference for investigating genetic variation and chromosome evolution in great grey owls. Compared with the previous S. nebulosa assembly, this assembly includes both sex chromosomes, separates regions that were previously collapsed, and resolves 82 chromosomes total. While larger chromosomes show broadly conserved synteny across owl assemblies, the recovery of additional conserved microchromosome-associated genes suggests that ONT reads improved resolution of the smallest chromosomes relative to HiFi-based assemblies.

In sunflower (Helianthus annuus L.), the composition of fatty acids in the seeds, primarily oleic, linoleic, stearic and palmitic acid, is of utmost importance for oil quality. Despite this, the genetic basis of this trait and its interaction with the environment is poorly understood. Understanding this interaction is critical to improvement of sunflower within the context of climate change. In this work, we incorporated fatty acid composition measurements from the sunflower SAM population and eight environments across an extensive geographic cline into GWAS. The SAM panel consists of 287 varieties representing approximately 90% of sunflower diversity, for which 2.2 million high-quality SNPs with a MAF > 5% are available. For increased power, multivariate GWAS was performed with four different inputs: (i) mean fatty acid composition within each environment, (ii) mean fatty acid composition within each environment omitting high oleic varieties, (iii) trait stability within environments quantified by standard errors among replicate samples ( stability) and (iv) Eberhart and Russells {beta} which quantifies trait stabilities across environments ({beta} stability). All four analyses yielded highly significantly associated SNPs. We found that high oleic varieties exhibited high {beta} trait stability, resulting in substantial overlap in markers between analyses (i) and (iv), with signals being fairly consistent between environments in analysis (i). For analyses (ii) and (iii), significant markers tended to vary between trials. For significant SNPs across all analyses, 147 candidate genes were identified, including promising candidates such as 15 fatty acid metabolism genes, 6 heat shock proteins and 22 transcription factors. Lastly, a large introgression consisting of two flanking inverted sequences on Chromosome 5 was found to coincide with stability in the Georgia trial, suggesting a role in FA composition stability under high heat conditions.

We generated the first reference-level nuclear genome assembly of the keystone Caribbean long-spined black sea urchin species, Diadema antillarum (Philippi, 1845). Using whole-genome sequencing data from PacBio HiFi, Oxford Nanopore, and Illumina platforms, we employed multiple assembly strategies to generate a high-quality, near-complete genome. The final assembly spans 1.73 Gbp, consists of 2,964 scaffolds, and has an N50 of 1.56 Mbp. BUSCO analysis (metazoa_odb10) indicates 98.4% completeness. The genome displays a heterozygosity rate of 2.52% and contains 42.85% repetitive elements, of which 29.96% are unclassified. Coverage analysis reveals that while most of the genome was assembled at 11x depth, certain regions exhibit up to 530x coverage. Notably, regions exceeding 33x coverage account for 30.53% of the repetitive content, suggesting localized expansion of repeats. Duplication analysis of the assembled contigs shows that approximately 66% of contigs have duplicated, which supports segmental genome duplication in the past, and is further evidenced by the moderate level of heterozygosity of the assembly. While these characteristics contribute to the complexity of the genome, they do not diminish the quality of our assembly. Despite this complexity, our assembly maintains high completeness and contiguity. Our assembly provides a valuable resource for future genetic studies and serves as a critical framework for conservation, monitoring, and restoration of D. antillarum populations across the Caribbean.

The kynurenine pathway metabolizes tryptophan into 3-hydroxykynurenine (3-HK), a precursor for ommochrome eye pigments synthesized via the cardinal (cd) gene in mosquitoes. While cd disruption was presumed neutral, we observed fitness costs in Anopheles stephensi knock-in but not knock-out cd mutants. Here we investigated this anomaly further by assessing survival, fecundity, and midgut integrity across multiple cd mutant lines. Heterozygous knock-in lines, expressing a fluorescent marker and guide RNA for CRISPR/Cas9, exhibited reduced survival post-blood feeding, larva-to-adult survival deficits, and midgut barrier dysfunction, whereas knock-outs showed no such costs. Oral supplementation with xanthurenic acid partially rescued knock-in mortality, implicating oxidative stress linked to 3-HK metabolism. Expression analyses suggest transgene insertion effects, rather than cd disruption, underlie these fitness costs. These findings highlight the importance of evaluating insertional effects in gene drive target selection and support cd as a viable target for genetic control strategies in An. stephensi.

Gene model for the ortholog of Lst8 (Lst8) in the May 2011 (WUGSC dyak_caf1/DyakCAF1) Genome Assembly (GenBank Accession: GCA_000005975.1) of Drosophila yakuba. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly (GenBank Accession: GCA_000236325.2) of D. eugracilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Gene duplication typically fails to confer a selective advantage to an organism, prompting their removal from a population. In the rare instance that duplication either does not incur a fitness cost or it enhances fitness, gene families can form through repeating the duplication process. While the function of gene duplicates has been studied in detail, little work has explored how repeated duplication impacts paralog redundancy and may restrict the emergence of new paralogs or novel function. Here, we constructed a panel of single deletion mutants for each of the 14 members of the <em>Candida albicans</em> telomere-associated (<em>TLO</em>) gene family to test the redundancy in molecular and biological function among paralogs from a lineage-specific expansion. Tlo proteins function as interchangeable subunits of the Mediator transcriptional regulatory complex and have the potential to alter gene expression and an array of cellular responses. Redundancy was the most common outcome, being observed for approximately 80% of the phenotypic assays in strains lacking single <em>TLO </em>genes. However, mutants for all 14 paralogs displayed non-redundant functions in phenotypes ranging from carbon utilization to <em>in vivo</em> virulence. Analysis of gene expression in single <em>TLO </em>mutants found similar trends in redundancy, and loss of single <em>TLO</em>s disproportionately affected genes involved in filamentation, adhesion, redox reactions, and transporter activity at the cell surface. Importantly, sequence divergence between paralogs positively correlated with the frequency of altered phenotypes in single <em>TLO </em>mutants, indicating the acquisition of non-redundant function with increased evolutionary distance. Double mutants lacking two <em>TLO</em> genes produced both positive and negative synergistic phenotypes, suggesting that crosstalk or coordinated regulation is common among paralogs. Together, this study demonstrates that recently emergent paralogs acquire non-redundant functions despite often retaining redundancy with other gene family members to form a highly interconnected functional network.

The origin recognition complex (ORC) selects origins of replication and directs the loading of the Mcm2-7 replicative helicase at these sites. Five of the six ORC subunits are related to the AAA+ family of ATPases. Although functions for ATP hydrolysis by Cdc6 and the Mcm2-7 complex have been described, the essential role of ORC ATP hydrolysis remains unclear. We performed a genetic screen in Saccharomyces cerevisiae for suppressors of the lethal phenotype of the orc4-R267A allele, which disrupts ORC ATP hydrolysis in vitro. We identified six causative mutations, five of which are distributed across different ORC subunits. The suppressor mutations in Orc1 and Orc4, but not the other ORC subunits, increase the in vitro helicase loading activity of ATPase-defective ORC (ORC4R). Allele specificity studies showed the alleles specifically suppress defects at ATPase interfaces within the ORC-Cdc6 complex. The sixth allele is a mutation in TOA2, a subunit of the TFIIA general transcription factor. Mutations in the general transcription factors TBP and TFIIB, and the large subunit of RNA Polymerase II also suppressed the orc4-R267A lethality, suggesting that reducing transcription is sufficient for suppression. Our study identifies multiple ways to suppress the lethal phenotype of an ATPase defective ORC allele and reveals a connection between ORC ATP hydrolysis and transcription.

Caenorhabditis nematodes are a powerful model clade for evolutionary genetics. Isogenic lines and panels of recombinant inbred lines (RILs) are among the most essential tools for genetic studies in these species. While most Caenorhabditis species are gonochoristic, large RIL panels have only been developed for self-fertilizing species. This gap biases our understanding and limits our ability to address questions related to the genetic architecture of traits in outbred populations, which have radically higher genetic diversity, heterozygosity, and effective recombination than selfers. Having previously identified Caenorhabditis becei as a tractable gonochoristic species due to its moderate inbreeding depression, we generated two panels of advanced-intercross RILs derived from three individual outbred C. becei worms collected from a single locality on Barro Colorado Island, Panama. One panel derives from a pair of worms sampled from a single rotting fig; the other derives from a cross between worms from two different figs. The panels share one founder in common, yielding two half-sib RIL panels. We sequenced and haplotyped the lines, identifying millions of variants and thousands of recombination breakpoints. Using simulations, we demonstrate the suitability of these lines for quantitative genetics studies and QTL mapping. In our single-fig panel, we observe abundant heritable variation in population growth rate, individual body size, and sexual dimorphism for body size. We detected four QTLs associated with population growth rate and show that estimated allelic effects are good predictors of selection that occurred during panel derivation.

Genetically identical cells grown in the same environment show variation in gene expression known as expression noise. Expression noise can be heritable and impact fitness, making it subject to natural selection. Increasing expression noise for the Saccharomyces cerevisiae TDH3 gene was shown to be beneficial in glucose-based media when mean TDH3 expression was far from the fitness optimum but deleterious when it was close to this optimum. Here, we show that growth on different carbon sources alters the effects of new mutations on TDH3 expression noise and examine the fitness effects of changing expression noise. In galactose-based media, we observed the same relationship between expression noise and fitness seen in glucose-based media, but in glycerol- and ethanol-based media, we observed the opposite relationship or no significant relationship, respectively. Using simulations of single-cell organisms, we found that these differences were most likely explained by environment-specific relationships between gene expression and fitness. We also found that, far from the optimum, the fitness effects of noise were greatest when expression was highly heritable between mother and daughter cells. The empirical observations and simulations reported in this study show how environments influence both the production of expression noise and its impacts on fitness.