Scientific highlights

The European green toad (Bufotes viridis) is geographically widely distributed. While the species global conservation status is labeled as of least concern by the IUCN, it is declining in many parts of its range where populations are fragmented and isolated. A high-quality reference genome is an important resource for conservation genomic researchers who are trying to understand and interpret the genomic signals of population decline, inbreeding, and the accumulation of deleterious mutations. Here, we assembled and annotated a chromosome-level reference genome for B. viridis as part of the European Reference Genome Atlas pilot project. The genome assembly, with a size of ∼3.89 Gb consists of 11 chromosomes and an additional 2,096 unplaced scaffolds. The final assembly had a scaffold N50 value of 478.39 Mb and covered 90.4% single copy tetrapod orthologs, and 46.7% repetitive elements. Finally, a total of 23,830 protein-coding genes matching a known gene, together with 56,974 mRNAs were predicted. This high-quality reference genome will benefit amphibian evolutionary genomics research and enable conservation genetic studies to inform practical conservation work on this species.

The early evolution of sex chromosomes has remained obscure for more than a century. The Vandiemenella viatica species group of morabine grasshoppers is highly suited for studying the early stages of sex chromosome divergence and degeneration of the Y chromosome. This stems from the fact that neo-XY sex chromosomes have independently evolved multiple times by X-autosome fusions with different autosomes. Here, we generated new chromosome-level assemblies for two chromosomal races representing karyotypes with and without neo-sex chromosomes (P24XY and P24X0), and sequence data of a third chromosomal race with a different neo-XY chromosome system (P25XY). Interestingly, these two neo-XY chromosomal races are formed by different X-autosome fusions (involving chr1 and chrB, respectively), and we found that both neo-Y chromosomes have partly ceased to recombine with their neo-X counterpart. We show that the neo-XY chromosomes have diverged through accumulation of SNPs and structural mutations, and that many neo-Y-linked genes have degenerated since recombination ceased. However, the non-recombining regions of neo-Y chromosomes host non-degenerated genes crucial for sex determination, such as sex-lethal and transformer, alongside genes associated with spermatogenesis, fertility, and reproduction, illustrating their integrative role as a masculinizing supergene. Contrary to expectations, the neo-Y chromosomes showed (slightly) lower density of transposable elements (TEs) compared to other genomic regions. The study reveals the unique dynamics of young sex chromosomes, with evolution of recombination suppression and pronounced decay of (some) neo-sex chromosome genes, and provides a compelling case illustrating how chromosomal fusions and post-fusion mutational processes contribute to the evolution of supergenes.

Morabine grasshoppers in the Vandiemenella viatica species group, which show karyotype diversity, have been studied for their ecological distribution and speciation in relation to their genetic and chromosomal diversity. They are good models for studying sex chromosome evolution as "old" and newly emerged sex chromosomes co-exist within the group. Here we present a reference genome for the viatica19 chromosomal race, that possesses the ancestral karyotype within the group. Using PacBio HiFi and Hi-C sequencing, we generated a chromosome-level assembly of 4.09 Gb in span, scaffold N50 of 429 Mb, and complete BUSCO score of 98.1%, containing 10 pseudo-chromosomes. We provide Illumina datasets of males and females, used to identify the X chromosome. The assembly contains 19,034 predicted protein-coding genes, and a total of 75.21% of repetitive DNA sequences. By leveraging HiFi reads, we mapped the genome-wide distribution of methylated bases (5mC and 6 mA). This comprehensive assembly offers a robust reference for morabine grasshoppers and supports further research into speciation and sex chromosome diversification within the group and its related species.

Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.

Spiders produce nature's toughest fiber using renewable components at ambient temperatures and with water as solvent, making it highly interesting to replicate for the materials industry. Despite this, much remains to be understood about the bioprocessing and composition of spider silk fibers. Here, we identify 18 proteins that make up the spiders' strongest silk type, the major ampullate fiber. Single-cell RNA sequencing and spatial transcriptomics revealed that the secretory epithelium of the gland harbors six cell types. These cell types are confined to three distinct glandular zones that produce specific combinations of silk proteins. Image analysis of histological sections showed that the secretions from the three zones do not mix, and proteomics analysis revealed that these secretions form layers in the final fiber. Using a multi-omics approach, we provide substantial advancements in the understanding of the structure and function of the major ampullate silk gland as well as of the architecture and composition of the fiber it produces.

Krill are vital as food for many marine animals but also impacted by global warming. To learn how they and other zooplankton may adapt to a warmer world we studied local adaptation in the widespread Northern krill (Meganyctiphanes norvegica). We assemble and characterize its large genome and compare genome-scale variation among 74 specimens from the colder Atlantic Ocean and warmer Mediterranean Sea. The 19 Gb genome likely evolved through proliferation of retrotransposons, now targeted for inactivation by extensive DNA methylation, and contains many duplicated genes associated with molting and vision. Analysis of 760 million SNPs indicates extensive homogenizing gene-flow among populations. Nevertheless, we detect signatures of adaptive divergence across hundreds of genes, implicated in photoreception, circadian regulation, reproduction and thermal tolerance, indicating polygenic adaptation to light and temperature. The top gene candidate for ecological adaptation was nrf-6, a lipid transporter with a Mediterranean variant that may contribute to early spring reproduction. Such variation could become increasingly important for fitness in Atlantic stocks. Our study underscores the widespread but uneven distribution of adaptive variation, necessitating characterization of genetic variation among natural zooplankton populations to understand their adaptive potential, predict risks and support ocean conservation in the face of climate change.

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6::RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-yr-old at relapse, the cell line offers a practical model for the study of pediatric B-ALL. In recent years, short- and long-read DNA and RNA sequencing have emerged as a complement to karyotyping techniques in the resolution of structural variants in an oncological context. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole-genome sequencing, IsoSeq RNA sequencing, and short-read Illumina sequencing to create a detailed genomic and transcriptomic characterization of the REH cell line. Whole-genome sequencing clarified the molecular traits of disrupted ALL-associated genes including CDKN2A, PAX5, BTG1, VPREB1, and TBL1XR1, as well as the glucocorticoid receptor NR3C1 Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.

The European sprat is a small plankton-feeding clupeid present in the northeastern Atlantic Ocean, in the Mediterranean Sea, and in the brackish Baltic Sea and Black Sea. This species is the target of a major fishery and, therefore, an accurate characterization of its genetic population structure is crucial to delineate proper stock assessments that aid ensuring the fishery's sustainability. Here, we present (i) a draft genome assembly, (ii) pooled whole genome sequencing of 19 population samples covering most of the species' distribution range, and (iii) the design and test of a single nucleotide polymorphism (SNP)-chip resource and use this to validate the population structure inferred from pooled sequencing. These approaches revealed, using the populations sampled here, three major groups of European sprat: Oceanic, Coastal, and Brackish with limited differentiation within groups even over wide geographical stretches. Genetic structure is largely driven by six large putative inversions that differentiate Oceanic and Brackish sprats, while Coastal populations display intermediate frequencies of haplotypes at each locus. Interestingly, populations from the Baltic and the Black Seas share similar frequencies of haplotypes at these putative inversions despite their distant geographic location. The closely related clupeids European sprat and Atlantic herring both show genetic adaptation to the brackish Baltic Sea, providing an opportunity to explore the extent of genetic parallelism. This analysis revealed limited parallelism because out of 125 independent loci detected in the Atlantic herring, three showed sharp signals of selection that overlapped between the two species and contained single genes such as PRLRA, which encodes the receptor for prolactin, a freshwater-adapting hormone in euryhaline species, and THRB, a receptor for thyroid hormones, important both for metabolic regulation and the development of red cone photoreceptors.

Distyly is an iconic floral polymorphism governed by a supergene, which promotes efficient pollen transfer and outcrossing through reciprocal differences in the position of sexual organs in flowers, often coupled with heteromorphic self-incompatibility. Distyly has evolved convergently in multiple flowering plant lineages, but has also broken down repeatedly, often resulting in homostylous, self-compatible populations with elevated rates of self-fertilization. Here, we aimed to study the genetic causes and genomic consequences of the shift to homostyly in Linum trigynum, which is closely related to distylous Linum tenue. Building on a high-quality genome assembly, we show that L. trigynum harbors a genomic region homologous to the dominant haplotype of the distyly supergene conferring long stamens and short styles in L. tenue, suggesting that loss of distyly first occurred in a short-styled individual. In contrast to homostylous Primula and Fagopyrum, L. trigynum harbors no fixed loss-of-function mutations in coding sequences of S-linked distyly candidate genes. Instead, floral gene expression analyses and controlled crosses suggest that mutations downregulating the S-linked LtWDR-44 candidate gene for male self-incompatibility and/or anther height could underlie homostyly and self-compatibility in L. trigynum. Population genomic analyses of 224 whole-genome sequences further demonstrate that L. trigynum is highly self-fertilizing, exhibits significantly lower genetic diversity genome-wide, and is experiencing relaxed purifying selection and less frequent positive selection on nonsynonymous mutations relative to L. tenue. Our analyses shed light on the loss of distyly in L. trigynum, and advance our understanding of a common evolutionary transition in flowering plants.

SLE is a systemic autoimmune disease with a large number of common risk gene variants, but several rare gene variants can cause monogenic SLE. The relationship between common and rare variants in SLE is unclear. We therefore investigated the occurrence of rare deleterious variants in patients with childhood-onset SLE (cSLE) and adult-onset SLE (aSLE) and compared the frequency of these variants with their individual SLE polygenic risk score (PRS). Targeted sequencing of 1832 gene regions, including coding regions of 31 genes associated with monogenic SLE, was performed in 958 patients with SLE and 1026 healthy individuals. A total of 116 patients with SLE had disease onset before the age of 18 (cSLE). An SLE common variant PRS was created from 37 SLE genome-wide association study single nucleotide variants (SNVs). Rare coding deleterious SNVs (RD SNVs) were observed in 23 of the monogenic SLE-associated genes. Six per cent of patients with cSLE, compared with 3.2% of controls and 4.6% of patients with aSLE, carried rare deleterious alleles. In cSLE, RD SNVs were observed in the C1S, DDX58, IFIH1, IKZF1, RNASEH2A and C8A genes. A PRS analysis showed that patients with cSLE with any of these gene variants had a similar average PRS as control individuals. RD SNVs were observed in a small proportion of cSLE and carriers of these RD SNVs had a PRS similar to healthy individuals, suggesting the importance of rare coding heterozygous variants in driving disease risk in a subset of children with SLE.

The genetic contributions to early onset heart failure (HF) are incompletely understood. Genetic testing in advanced HF patients undergoing heart transplantation (HTx) may yield clinical benefits, but data is limited. We performed deep-coverage whole genome sequencing (WGS) in 102 Swedish HTx recipients. Gene lists were compiled through a systematic literature review. Variants were prioritized for pathogenicity and classified manually. We also compared polygenic HF risk scores to a population-based cohort. We found a pathogenic (LP/P) variant in 34 individuals (34%). Testing yield was highest in hypertrophic (63% LP/P carriers), dilated (40%) and arrhythmogenic right ventricular (33%) cardiomyopathy and lower in ischemic cardiomyopathy (10%). A family history was more common in LP/P variant carriers than in non-carriers but was present in less than half of carriers (44% vs 13%, P < 0.001), whereas age was similar. Polygenic risk scores were similar in HTx recipients and the population cohort. In conclusion, we observed a high prevalence of pathogenic cardiomyopathy gene variants in individuals with early-onset advanced HF, which could not accurately be ruled out by family history and age. In contrast, we did not observe higher polygenic risk scores in early onset advanced HF cases than in the general population.

How gene expression evolves to enable divergent ecological adaptation and how changes in gene expression relate to genomic architecture are pressing questions for understanding the mechanisms enabling adaptation and ecological speciation. Furthermore, how plasticity in gene expression can both contribute to and be affected by the process of ecological adaptation is crucial to understanding gene expression evolution, colonisation of novel niches and response to rapid environmental change. Here, we investigate the role of constitutive and plastic gene expression differences between host races, or host-specific ecotypes, of the peacock fly Tephritis conura, a thistle bud specialist. By cross-fostering larvae to new buds of their natal host plant or the alternative, novel host plant, we uncover extensive constitutive differences in gene expression between the host races, especially genes associated with processing of host plant chemicals. However, evidence for expression plasticity was minimal and limited to the ancestral host race. Genes with host race-specific expression are found more often than expected within a large inversion in the T. conura genome, adding to evidence that inversions are important for enabling diversification in the face of gene flow and underscores that altered gene expression may be key to understanding the evolutionary consequences of inversions.

Dietary factors may regulate the epigenome. We aimed to explore whether a diet intervention, including excess sugar, affects the methylome in human sperm, and to describe the sperm methylome. We used Whole Genome Bisulfite Sequencing (WGBS) to analyze DNA methylation in sperm taken at three time points from 15 males during a diet intervention; i) at baseline, ii) after one week on a standardized diet, and iii) after an additional week on a high-sugar diet providing 150% of their estimated total energy expenditure. We identified seven nominal diet-associated differentially methylated regions in sperm (p < 0.05). The diet was nominally associated with methylation of 143 sites linked to fertility (e.g. AHRR, GNAS, and HDAC4), 313 sites in imprinted genes (e.g. GLIS3, PEG10, PEG3, and SNURF), and 42 sites in top 1%-expressed genes (e.g. CHD2) (p < 0.05). In sperm, 3'UTRs and introns had the highest levels of methylation, while 5'UTRs and CpG islands had the lowest levels. Non-expressed genes in human sperm were hypomethylated in exons compared with transcribed genes. In human sperm, DNA methylation levels were linked to gene expression, and excess sugar had modest effects on methylation on imprinted and highly expressed genes, and genes affecting fertility.

Copy number variation (CNV) of the amyloid-β precursor protein gene (APP) is a known cause of autosomal dominant Alzheimer disease (ADAD), but de novo genetic variants causing ADAD are rare. We report a mother and daughter with neuropathologically confirmed definite Alzheimer disease (AD) and extensive cerebral amyloid angiopathy (CAA). Copy number analysis identified an increased number of APP copies and genome sequencing (GS) revealed the underlying complex genomic rearrangement (CGR) including a triplication of APP with two unique breakpoint junctions (BPJs). The mosaic state in the mother had likely occurred de novo. Digital droplet PCR (ddPCR) on 42 different tissues, including 17 different brain regions, showed the derivative chromosome at varying mosaic levels (20-96%) in the mother who had symptom onset at age 58 years. In contrast, the derivative chromosome was present in all analyzed cells in the daughter whose symptom onset was at 34 years. This study reveals the architecture of a de novo CGR causing APP triplication and ADAD with a striking difference in age at onset between the fully heterozygous daughter compared to the mosaic mother. The GS analysis identified the complexity of the CGR illustrating its usefulness in identifying structural variants (SVs) in neurodegenerative disorders.

Newts have large genomes harboring many repeat elements. How these elements shape the genome and relate to newts' unique regeneration ability remains unknown. We present here the chromosome-scale assembly of the 20.3 Gb genome of the Iberian ribbed newt, Pleurodeles waltl, with a hitherto unprecedented contiguity and completeness among giant genomes. Utilizing this assembly, we demonstrate conserved synteny as well as genetic rearrangements, such as in the major histocompatibility complex locus. We provide evidence suggesting that intronic repeat elements drive newt-specific circular RNA (circRNA) biogenesis and show their regeneration-specific expression. We also present a comprehensive in-depth annotation and chromosomal mapping of microRNAs, highlighting genomic expansion profiles as well as a distinct regulatory pattern in the regenerating limb. These data reveal links between repeat elements, non-coding RNAs, and adult regeneration and provide key resources for addressing developmental, regenerative, and evolutionary principles.

Cancer is the leading cause of death from disease in children. Survival depends not only on surgery, cytostatic drugs, and radiation but also on systemic immune responses. Factors influencing these immune responses in children of different ages and tumor types are unknown. Novel immunotherapies can enhance anti-tumor immune responses, but few children have benefited, and markers of effective responses are lacking. Here, we present a systems-level analysis of immune responses in 191 children within a population-based cohort with diverse tumors and reveal that age and tumor type shape immune responses differently. Systemic inflammation and cytotoxic T cell responses correlate with tumor mutation rates and immune cell infiltration. Clonally expanded T cell responses are rarely detected in blood or tumors at diagnosis but are sometimes elicited during treatment. Expanded T cells are similarly regulated in children and adults with more immunogenic cancers. This research aims to facilitate the development of precision immunotherapies for children with cancer.

The injectable contraceptive, depot medroxyprogesterone acetate (DMPA), is associated with compromised cervical mucosal barriers. High-resolution spatial transcriptomics is applied here to reveal the spatial localization of these altered molecular markers. Ectocervical tissue samples from Kenyan sex workers using DMPA, or non-hormonal contraceptives, underwent spatial transcriptomics and gene set enrichment analyses. Integrated systemic estradiol levels and bulk tissue gene expression data from a larger cohort enhanced the study's scope. Unsupervised clustering unveiled four epithelial and seven submucosal layers, showcasing spatially restricted and diverse functional epithelial responses, and a less structured submucosal spatial ordering. DMPA associated with mucosal-wide immunoglobulin gene upregulation, verified by CD20+ B-cell immunostaining, and upregulated immune markers adjacent to the basal membrane. Downregulated genes represented spatially restricted disrupted epithelial barrier integrity and submucosal extracellular matrix dysfunction. The transcriptional profile was associated with markers of estrogen regulation. Collectively, our findings reveal estrogen-modulated distinct ectocervical transcriptional profiles associated with DMPA usage. While upregulation of immunoglobulin genes occurs throughout the mucosa, activation of innate immune responses and dysregulation of barrier integrity markers are spatially restricted. These results extend previous analyses using bulk transcriptomics and provide insights into the molecular landscape influenced by DMPA, shedding light on contraceptive effects and health implications.

Hydroxyurea (HU) is frequently used in the early phase of chronic myeloid leukemia (CML) to achieve cytoreduction prior to tyrosine kinase inhibitor therapy. However, its impact on CML stem and progenitor cells (SPC) remains largely unknown. This study utilized targeted proteo-transcriptomic expression data on 596 genes and 51 surface proteins in 60,000 CD14-CD34+ cells from chronic phase CML patients to determine effects of short-term HU treatment (4-19 days) on CML SPC. Peripheral blood and bone marrow samples were obtained from 17 CML patients eligible for short-term HU treatment (3 patients before and after HU, 7 patients before HU and 7 patients after HU) and subjected to single-cell CITE-sequencing and/or flow cytometry analysis. The analysis revealed enhanced frequencies of hemoglobin-expressing (HBA1, HBA2, HBB) erythroid progenitor cells in blood and bone marrow following HU treatment. In addition, there was an accumulation of cell subsets with S/G2/M phase-related gene and protein expression, likely representing cells arrested in, or progressing slowly through, the cell cycle. The increased frequency of cells in S/G2/M phase after HU was observed already among the most immature leukemic stem cells (LSC), and patients with a large fraction of LSC in the S/G2/M phase showed poor responsiveness to tyrosine kinase inhibitor treatment. We conclude that short-term HU treatment entails differentiation of erythroid progenitor cells and alters the characteristics of LSC in CML. The results imply that studies of LSC and progenitor populations in CML should take effects of initial HU therapy into account.

CO2 fixation (i.e. primary production) is a key function of all ecosystems, providing the carbon and energy that fuel the entire food web. It also plays an important role in mitigating climate change as CO2 is the most important greenhouse gas. While photosynthesis is regarded as the most important carbon fixation pathway, prokaryotes able to fix carbon in the absence of light (chemolithoautotrophs) can also be a significant source of energy in a light-limited ecosystem. Boreal lakes, notoriously colored and stratified with respect to oxygen and nutrients, present ideal conditions for this so-called dark carbon fixation by the chemolithoautotrophs. However, the prevalence of dark carbon fixation in boreal lakes remains unknown. Here, we measured dark carbon fixation in Swedish lakes from the boreal and boreo-nemoral zones, during summer stratification. We detected dark carbon fixation in 16 out of the 17 lakes studied, and concluded that dark fixation is a widespread phenomenon in boreal lakes. Moreover, the average dark primary production ranged from 18.5 % in the epilimnion to 81.4 % in the hypolimnion of all tested lakes. Our data further suggests that chemolithoautotrophic activity is mostly driven by iron-oxidizing bacteria. The chemolithoautotrophic guild is diverse and seems to be composed of both ubiquitous bacteria, like Gallionellaceae or Chromatiaceae, and endemic taxa, such as Ferrovaceae, which appears to be favored by a low pH. These results are particularly exciting as they suggest that dark carbon fixation could partly compensate for the low photosynthetic capacity in lakes with dark-colored water.