High-Quality Genome Assemblies of 4 Members of the Podospora anserina Species Complex.

The filamentous fungus Podospora anserina is a model organism used extensively in the study of molecular biology, senescence, prion biology, meiotic drive, mating-type chromosome evolution, and plant biomass degradation. It has recently been established that P. anserina is a member of a complex of 7 closely related species. In addition to P. anserina, high-quality genomic resources are available for 2 of these taxa. Here, we provide chromosome-level annotated assemblies of the 4 remaining species of the complex, as well as a comprehensive data set of annotated assemblies from a total of 28 Podospora genomes. We find that all 7 species have genomes of around 35 Mb arranged in 7 chromosomes that are mostly collinear and less than 2% divergent from each other at genic regions. We further attempt to resolve their phylogenetic relationships, finding significant levels of phylogenetic conflict as expected from a rapid and recent diversification.

Evolutionary constraint and innovation across hundreds of placental mammals.

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

Leveraging base-pair mammalian constraint to understand genetic variation and human disease.

Thousands of genomic regions have been associated with heritable human diseases, but attempts to elucidate biological mechanisms are impeded by an inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function, agnostic to cell type or disease mechanism. Single-base phyloP scores from 240 mammals identified 3.3% of the human genome as significantly constrained and likely functional. We compared phyloP scores to genome annotation, association studies, copy-number variation, clinical genetics findings, and cancer data. Constrained positions are enriched for variants that explain common disease heritability more than other functional annotations. Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Leveraging Base Pair Mammalian Constraint to Understand Genetic Variation and Human Disease.

Although thousands of genomic regions have been associated with heritable human diseases, attempts to elucidate biological mechanisms are impeded by a general inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function that is agnostic to cell type or disease mechanism. Here, single base phyloP scores from the whole genome alignment of 240 placental mammals identified 3.5% of the human genome as significantly constrained, and likely functional. We compared these scores to large-scale genome annotation, genome-wide association studies (GWAS), copy number variation, clinical genetics findings, and cancer data sets. Evolutionarily constrained positions are enriched for variants explaining common disease heritability (more than any other functional annotation). Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Bayesian model and selection signature analyses reveal risk factors for canine atopic dermatitis.

Canine atopic dermatitis is an inflammatory skin disease with clinical similarities to human atopic dermatitis. Several dog breeds are at increased risk for developing this disease but previous genetic associations are poorly defined. To identify additional genetic risk factors for canine atopic dermatitis, we here apply a Bayesian mixture model adapted for mapping complex traits and a cross-population extended haplotype test to search for disease-associated loci and selective sweeps in four dog breeds at risk for atopic dermatitis. We define 15 associated loci and eight candidate regions under selection by comparing cases with controls. One associated locus is syntenic to the major genetic risk locus (Filaggrin locus) in human atopic dermatitis. One selection signal in common type Labrador retriever cases positions across the TBC1D1 gene (body weight) and one signal of selection in working type German shepherd controls overlaps the LRP1B gene (brain), near the KYNU gene (psoriasis). In conclusion, we identify candidate genes, including genes belonging to the same biological pathways across multiple loci, with potential relevance to the pathogenesis of canine atopic dermatitis. The results show genetic similarities between dog and human atopic dermatitis, and future across-species genetic comparisons are hereby further motivated.

Rapid adaptive radiation of Darwin's finches depends on ancestral genetic modules.

Recent adaptive radiations are models for investigating mechanisms contributing to the evolution of biodiversity. An unresolved question is the relative importance of new mutations, ancestral variants, and introgressive hybridization for phenotypic evolution and speciation. Here, we address this issue using Darwin's finches and investigate the genomic architecture underlying their phenotypic diversity. Admixture mapping for beak and body size in the small, medium, and large ground finches revealed 28 loci showing strong genetic differentiation. These loci represent ancestral haplotype blocks with origins predating speciation events during the Darwin's finch radiation. Genes expressed in the developing beak are overrepresented in these genomic regions. Ancestral haplotypes constitute genetic modules for selection and act as key determinants of the unusual phenotypic diversity of Darwin's finches. Such ancestral haplotype blocks can be critical for how species adapt to environmental variability and change.

Heterochiasmy and the establishment of gsdf as a novel sex determining gene in Atlantic halibut.

Atlantic Halibut (Hippoglossus hippoglossus) has a X/Y genetic sex determination system, but the sex determining factor is not known. We produced a high-quality genome assembly from a male and identified parts of chromosome 13 as the Y chromosome due to sequence divergence between sexes and segregation of sex genotypes in pedigrees. Linkage analysis revealed that all chromosomes exhibit heterochiasmy, i.e. male-only and female-only meiotic recombination regions (MRR/FRR). We show that FRR/MRR intervals differ in nucleotide diversity and repeat class content and that this is true also for other Pleuronectidae species. We further show that remnants of a Gypsy-like transposable element insertion on chr13 promotes early male specific expression of gonadal somatic cell derived factor (gsdf). Less than 4.5 MYA, this male-determining element evolved on an autosomal FRR segment featuring pre-existing male meiotic recombination barriers, thereby creating a Y chromosome. Our findings indicate that heterochiasmy may facilitate the evolution of genetic sex determination systems relying on linkage of sexually antagonistic loci to a sex-determining factor.

A genomic and morphometric analysis of alpine bumblebees: Ongoing reductions in tongue length but no clear genetic component.

Over the last six decades, populations of the bumblebees Bombus sylvicola and Bombus balteatus in Colorado have experienced decreases in tongue length, a trait important for plant-pollinator mutualisms. It has been hypothesized that this observation reflects selection resulting from shifts in floral composition under climate change. Here we used morphometrics and population genomics to determine whether morphological change is ongoing, investigate the genetic basis of morphological variation, and analyse population structure in these populations. We generated a genome assembly of B. balteatus. We then analysed whole-genome sequencing data and morphometric measurements of 580 samples of both species from seven high-altitude localities. Out of 281 samples originally identified as B. sylvicola, 67 formed a separate genetic cluster comprising a newly-discovered cryptic species ("incognitus"). However, an absence of genetic structure within species suggests that gene flow is common between mountains. We found a significant decrease in tongue length between bees collected between 2012-2014 and in 2017, indicating that morphological shifts are ongoing. We did not discover any genetic associations with tongue length, but a SNP related to production of a proteolytic digestive enzyme was implicated in body size variation. We identified evidence of covariance between kinship and both tongue length and body size, which is suggestive of a genetic component of these traits, although it is possible that shared environmental effects between colonies are responsible. Our results provide evidence for ongoing modification of a morphological trait important for pollination and indicate that this trait probably has a complex genetic and environmental basis.

Functional differences between TSHR alleles associate with variation in spawning season in Atlantic herring.

The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of TSHR alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate TSHR mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2 kb retrotransposon insertion upstream of the TSHR transcription start site, near an open chromatin region, which is likely to affect TSHR expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the TSHR locus may both contribute to the regulation of seasonal reproduction in herring.

The Enterprise, a massive transposon carrying Spok meiotic drive genes.

The genomes of eukaryotes are full of parasitic sequences known as transposable elements (TEs). Here, we report the discovery of a putative giant tyrosine-recombinase-mobilized DNA transposon, Enterprise, from the model fungus Podospora anserina Previously, we described a large genomic feature called the Spok block which is notable due to the presence of meiotic drive genes of the Spok gene family. The Spok block ranges from 110 kb to 247 kb and can be present in at least four different genomic locations within P. anserina, despite what is an otherwise highly conserved genome structure. We propose that the reason for its varying positions is that the Spok block is not only capable of meiotic drive but is also capable of transposition. More precisely, the Spok block represents a unique case where the Enterprise has captured the Spoks, thereby parasitizing a resident genomic parasite to become a genomic hyperparasite. Furthermore, we demonstrate that Enterprise (without the Spoks) is found in other fungal lineages, where it can be as large as 70 kb. Lastly, we provide experimental evidence that the Spok block is deleterious, with detrimental effects on spore production in strains which carry it. This union of meiotic drivers and a transposon has created a selfish element of impressive size in Podospora, challenging our perception of how TEs influence genome evolution and broadening the horizons in terms of what the upper limit of transposition may be.

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics.

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (dXY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.

A novel canine reference genome resolves genomic architecture and uncovers transcript complexity.

We present GSD_1.0, a high-quality domestic dog reference genome with chromosome length scaffolds and contiguity increased 55-fold over CanFam3.1. Annotation with generated and existing long and short read RNA-seq, miRNA-seq and ATAC-seq, revealed that 32.1% of lifted over CanFam3.1 gaps harboured previously hidden functional elements, including promoters, genes and miRNAs in GSD_1.0. A catalogue of canine "dark" regions was made to facilitate mapping rescue. Alignment in these regions is difficult, but we demonstrate that they harbour trait-associated variation. Key genomic regions were completed, including the Dog Leucocyte Antigen (DLA), T Cell Receptor (TCR) and 366 COSMIC cancer genes. 10x linked-read sequencing of 27 dogs (19 breeds) uncovered 22.1 million SNPs, indels and larger structural variants. Subsequent intersection with protein coding genes showed that 1.4% of these could directly influence gene products, and so provide a source of normal or aberrant phenotypic modifications.

The Human Adenovirus Type 2 Transcriptome: An Amazing Complexity of Alternatively Spliced mRNAs.

We have used the Nanopore long-read sequencing platform to demonstrate how amazingly complex the human adenovirus type 2 (Ad2) transcriptome is with a flexible splicing machinery producing a range of novel mRNAs both from the early and late transcription units. In total we report more than 900 alternatively spliced mRNAs produced from the Ad2 transcriptome whereof more than 850 are novel mRNAs. A surprising finding was that more than 50% of all E1A transcripts extended upstream of the previously defined transcriptional start site. The novel start sites mapped close to the inverted terminal repeat (ITR) and within the E1A enhancer region. We speculate that novel promoters or enhancer driven transcription, so-called eRNA transcription, is responsible for producing these novel mRNAs. Their existence was verified by a peptide in the Ad2 proteome that was unique for the E1A ITR mRNA. Although we show a high complexity of alternative splicing from most early and late regions, the E3 region was by far the most complex when expressed at late times of infection. More than 400 alternatively spliced mRNAs were observed in this region alone. These mRNAs included extended L4 mRNAs containing E3 and L5 sequences and readthrough mRNAs combining E3 and L5 sequences. Our findings demonstrate that the virus has a remarkable capacity to produce novel exon combinations, which will offer the virus an evolutionary advantage to change the gene expression repertoire and protein production in an evolving environment.IMPORTANCE Work in the adenovirus system led to the groundbreaking discovery of RNA splicing and alternative RNA splicing in 1977. These mechanisms are essential in mammalian evolution by increasing the coding capacity of a genome. Here, we have used a long-read sequencing technology to characterize the complexity of human adenovirus pre-mRNA splicing in detail. It is mindboggling that the viral genome, which only houses around 36,000 bp, not being much larger than a single cellular gene, generates more than 900 alternatively spliced mRNAs. Recently, adenoviruses have been used as the backbone in several promising SARS-CoV-2 vaccines. Further improvement of adenovirus-based vaccines demands that the virus can be tamed into an innocent carrier of foreign genes. This requires a full understanding of the components that govern adenovirus replication and gene expression.

Reconstruction of the birth of a male sex chromosome present in Atlantic herring.

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an ∼500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination.

A chromosome-level assembly of the Atlantic herring genome-detection of a supergene and other signals of selection.

The Atlantic herring is a model species for exploring the genetic basis for ecological adaptation, due to its huge population size and extremely low genetic differentiation at selectively neutral loci. However, such studies have so far been hampered because of a highly fragmented genome assembly. Here, we deliver a chromosome-level genome assembly based on a hybrid approach combining a de novo Pacific Biosciences (PacBio) assembly with Hi-C-supported scaffolding. The assembly comprises 26 autosomes with sizes ranging from 12.4 to 33.1 Mb and a total size, in chromosomes, of 726 Mb, which has been corroborated by a high-resolution linkage map. A comparison between the herring genome assembly with other high-quality assemblies from bony fishes revealed few inter-chromosomal but frequent intra-chromosomal rearrangements. The improved assembly facilitates analysis of previously intractable large-scale structural variation, allowing, for example, the detection of a 7.8-Mb inversion on Chromosome 12 underlying ecological adaptation. This supergene shows strong genetic differentiation between populations. The chromosome-based assembly also markedly improves the interpretation of previously detected signals of selection, allowing us to reveal hundreds of independent loci associated with ecological adaptation.

Maintenance of High Genome Integrity over Vegetative Growth in the Fairy-Ring Mushroom Marasmius oreades.

Most mutations in coding regions of the genome are deleterious, causing selection to favor mechanisms that minimize the mutational load over time [1-5]. DNA replication during cell division is a major source of new mutations. It is therefore important to limit the number of cell divisions between generations, particularly for large and long-lived organisms [6-9]. The germline cells of animals and the slowly dividing cells in plant meristems are adaptations to control the number of mutations that accumulate over generations [9-11]. Fungi lack a separated germline while harboring species with very large and long-lived individuals that appear to maintain highly stable genomes within their mycelia [8, 12, 13]. Here, we studied genomic mutation accumulation in the fairy-ring mushroom Marasmius oreades. We generated a chromosome-level genome assembly using a combination of cutting-edge DNA sequencing technologies and re-sequenced 40 samples originating from six individuals of this fungus. The low number of mutations recovered in the sequencing data suggests the presence of an unknown mechanism that works to maintain extraordinary genome integrity over vegetative growth in M. oreades. The highly structured growth pattern of M. oreades allowed us to estimate the number of cell divisions leading up to each sample [14, 15], and from this data, we infer an incredibly low per mitosis mutation rate (3.8 × 10-12 mutations per site and cell division) as one of several possible explanations for the low number of identified mutations.

Regulatory changes in pterin and carotenoid genes underlie balanced color polymorphisms in the wall lizard.

Reptiles use pterin and carotenoid pigments to produce yellow, orange, and red colors. These conspicuous colors serve a diversity of signaling functions, but their molecular basis remains unresolved. Here, we show that the genomes of sympatric color morphs of the European common wall lizard (Podarcis muralis), which differ in orange and yellow pigmentation and in their ecology and behavior, are virtually undifferentiated. Genetic differences are restricted to two small regulatory regions near genes associated with pterin [sepiapterin reductase (SPR)] and carotenoid [beta-carotene oxygenase 2 (BCO2)] metabolism, demonstrating that a core gene in the housekeeping pathway of pterin biosynthesis has been coopted for bright coloration in reptiles and indicating that these loci exert pleiotropic effects on other aspects of physiology. Pigmentation differences are explained by extremely divergent alleles, and haplotype analysis revealed abundant transspecific allele sharing with other lacertids exhibiting color polymorphisms. The evolution of these conspicuous color ornaments is the result of ancient genetic variation and cross-species hybridization.

Allele specific chromatin signals, 3D interactions, and motif predictions for immune and B cell related diseases.

Several Genome Wide Association Studies (GWAS) have reported variants associated to immune diseases. However, the identified variants are rarely the drivers of the associations and the molecular mechanisms behind the genetic contributions remain poorly understood. ChIP-seq data for TFs and histone modifications provide snapshots of protein-DNA interactions allowing the identification of heterozygous SNPs showing significant allele specific signals (AS-SNPs). AS-SNPs can change a TF binding site resulting in altered gene regulation and are primary candidates to explain associations observed in GWAS and expression studies. We identified 17,293 unique AS-SNPs across 7 lymphoblastoid cell lines. In this set of cell lines we interrogated 85% of common genetic variants in the population for potential regulatory effect and we identified 237 AS-SNPs associated to immune GWAS traits and 714 to gene expression in B cells. To elucidate possible regulatory mechanisms we integrated long-range 3D interactions data to identify putative target genes and motif predictions to identify TFs whose binding may be affected by AS-SNPs yielding a collection of 173 AS-SNPs associated to gene expression and 60 to B cell related traits. We present a systems strategy to find functional gene regulatory variants, the TFs that bind differentially between alleles and novel strategies to detect the regulated genes.

ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells.

Zinc finger BED domain containing protein 6 ( Zbed6) has evolved from a domesticated DNA transposon and encodes a transcription factor unique to placental mammals. The aim of the present study was to investigate further the role of ZBED6 in insulin-producing cells, using mouse MIN6 cells, and to evaluate the effects of Zbed6 knockdown on basal ß-cell functions, such as morphology, transcriptional regulation, insulin content, and release. Zbed6-silenced cells and controls were characterized with a range of methods, including RNA sequencing, chromatin immunoprecipitation sequencing, insulin content and release, subplasma membrane Ca2+ measurements, cAMP determination, and morphologic studies. More than 700 genes showed differential expression in response to Zbed6 knockdown, which was paralleled by increased capacity to generate cAMP, as well as by augmented subplasmalemmal calcium concentration and insulin secretion in response to glucose stimulation. We identified >4000 putative ZBED6-binding sites in the MIN6 genome, with an enrichment of ZBED6 sites at upregulated genes, such as the ß-cell transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene homolog A and Nk6 homeobox 1. We also observed altered morphology/growth patterns, as indicated by increased cell clustering, and in the appearance of axon-like Neurofilament, medium polypeptide and tubulin ß 3, class III-positive protrusions. We conclude that ZBED6 acts as a transcriptional regulator in MIN6 cells and that its activity suppresses insulin production, cell aggregation, and neuronal-like differentiation.-Wang, X., Jiang, L., Wallerman, O., Younis, S., Yu, Q., Klaesson, A., Tengholm, A., Welsh, N., Andersson, L. ZBED6 negatively regulates insulin production, neuronal differentiation, and cell aggregation in MIN6 cells.

Chromosomal inversions associated with environmental adaptation in honeybees.

Chromosomal inversions can facilitate local adaptation in the presence of gene flow by suppressing recombination between well-adapted native haplotypes and poorly adapted migrant haplotypes. East African mountain populations of the honeybee Apis mellifera are highly divergent from neighbouring lowland populations at two extended regions in the genome, despite high similarity in the rest of the genome, suggesting that these genomic regions harbour inversions governing local adaptation. Here, we utilize a new highly contiguous assembly of the honeybee genome to characterize these regions. Using whole-genome sequencing data from 55 highland and lowland bees, we find that the highland haplotypes at both regions are present at high frequencies in three independent highland populations but extremely rare elsewhere. The boundaries of both divergent regions are characterized by regions of high homology with each other positioned in opposite orientations and contain highly repetitive, long inverted repeats with homology to transposable elements. These regions are likely to represent inversion breakpoints that participate in nonallelic homologous recombination. Using long-read data, we confirm that the lowland samples are contiguous across breakpoint regions. We do not find evidence for disruption of functional sequence by these breakpoints, which suggests that the inversions are likely maintained due to their allelic content conferring local adaptation in highland environments. Finally, we identify a third divergent genomic region, which contains highly divergent segregating haplotypes that also may contain inversion variants under selection. The results add to a growing body of evidence indicating the importance of chromosomal inversions in local adaptation.