Scientific highlights

Supergenes govern multi-trait-balanced polymorphisms in a wide range of systems; however, our understanding of their origins and evolution remains incomplete. The reciprocal placement of stigmas and anthers in pin and thrum floral morphs of distylous species constitutes an iconic example of a balanced polymorphism governed by a supergene, the distyly S-locus. Recent studies have shown that the Primula and Turnera distyly supergenes are both hemizygous in thrums, but it remains unknown whether hemizygosity is pervasive among distyly S-loci. As hemizygosity has major consequences for supergene evolution and loss, clarifying whether this genetic architecture is shared among distylous species is critical. Here, we have characterized the genetic architecture and evolution of the distyly supergene in Linum by generating a chromosome-level genome assembly of Linum tenue, followed by the identification of the S-locus using population genomic data. We show that hemizygosity and thrum-specific expression of S-linked genes, including a pistil-expressed candidate gene for style length, are major features of the Linum S-locus. Structural variation is likely instrumental for recombination suppression, and although the non-recombining dominant haplotype has accumulated transposable elements, S-linked genes are not under relaxed purifying selection. Our findings reveal remarkable convergence in the genetic architecture and evolution of independently derived distyly supergenes, provide a counterexample to classic inversion-based supergenes, and shed new light on the origin and maintenance of an iconic floral polymorphism.

Personalised medicine (PM) presents a great opportunity to improve the future of individualised healthcare. Recent advances in -omics technologies have led to unprecedented efforts characterising the biology and molecular mechanisms that underlie the development and progression of a wide array of complex human diseases, supporting further development of PM. This article reflects the outcome of the 2021 EATRIS-Plus Multi-omics Stakeholder Group workshop organised to 1) outline a global overview of common promises and challenges that key European stakeholders are facing in the field of multi-omics research, 2) assess the potential of new technologies, such as artificial intelligence (AI), and 3) establish an initial dialogue between key initiatives in this space. Our focus is on the alignment of agendas of European initiatives in multi-omics research and the centrality of patients in designing solutions that have the potential to advance PM in long-term healthcare strategies.

Although physical activity and sedentary behavior are moderately heritable, little is known about the mechanisms that influence these traits. Combining data for up to 703,901 individuals from 51 studies in a multi-ancestry meta-analysis of genome-wide association studies yields 99 loci that associate with self-reported moderate-to-vigorous intensity physical activity during leisure time (MVPA), leisure screen time (LST) and/or sedentary behavior at work. Loci associated with LST are enriched for genes whose expression in skeletal muscle is altered by resistance training. A missense variant in ACTN3 makes the alpha-actinin-3 filaments more flexible, resulting in lower maximal force in isolated type IIA muscle fibers, and possibly protection from exercise-induced muscle damage. Finally, Mendelian randomization analyses show that beneficial effects of lower LST and higher MVPA on several risk factors and diseases are mediated or confounded by body mass index (BMI). Our results provide insights into physical activity mechanisms and its role in disease prevention.

Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20-25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20-25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

DNA methylations are one of the most well-known epigenetic modifications along with histone modifications and noncoding RNAs. They are found at specific sites along the DNA in all domains of life, with 5-mC and 6-mA/4-mC being well-characterized in eukaryotes and bacteria respectively, and they have not only been described as contributing to the structure of the double helix itself but also as regulators of DNA-based processes such as replication, transcription, and recombination. Different methods have been developed to accurately identify and/or map methylated motifs to decipher the involvement of DNA methylations in regulatory networks that affect the cellular state.Although DNA methylations have been detected along archaeal genomes, their involvement as regulators of DNA-based processes remains the least known. To highlight the importance of DNA methylations in the control of key cellular mechanisms and their dynamics in archaea cells, we have used single-molecule real-time (SMRT) sequencing. This sequencing technology allows the identification and direct mapping of the methylated motifs along the genome of an organism. In this chapter, we present a step-by-step protocol for detecting DNA methylations in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius using SMRT sequencing. This protocol can easily be adapted to other prokaryotes.

The aim of this project was to implement long-read sequencing for BCR-ABL1 TKI resistance mutation screening in a clinical setting for patients undergoing treatment for chronic myeloid leukemia. Processes were established for registering and transferring samples from the clinic to an academic sequencing facility for long-read sequencing. An automated analysis pipeline for detecting mutations was established, and an information system was implemented comprising features for data management, analysis and visualization. Clinical validation was performed by identifying BCR-ABL1 TKI resistance mutations by Sanger and long-read sequencing in parallel. The developed software is available as open source via GitHub at https://github.com/pharmbio/clamp. The information system enabled traceable transfer of samples from the clinic to the sequencing facility, robust and automated analysis of the long-read sequence data, and communication of results from sequence analysis in a reporting format that could be easily interpreted and acted upon by clinical experts. In a validation study, all 17 resistance mutations found by Sanger sequencing were also detected by long-read sequencing. An additional 16 mutations were found only by long-read sequencing, all of them with frequencies below the limit of detection for Sanger sequencing. The clonal distributions of co-existing mutations were automatically resolved through the long-read data analysis. After the implementation and validation, the clinical laboratory switched their routine protocol from using Sanger to long-read sequencing for this application. Long-read sequencing delivers results with higher sensitivity compared to Sanger sequencing and enables earlier detection of emerging TKI resistance mutations. The developed processes, analysis workflow, and software components lower barriers for adoption and could be extended to other applications.

Malignant gliomas, the most common malignant brain tumors in adults, represent a heterogeneous group of diseases with poor prognosis. Retroviruses can cause permanent genetic alterations that modify genes close to the viral integration site. Here we describe the use of a high-throughput pipeline coupled to the commonly used tissue-specific retroviral RCAS-TVA mouse tumor model system. Utilizing next generation sequencing, we show that retroviral integration sites can be reproducibly detected in malignant stem cell lines generated from RCAS-PDGFB-driven glioma biopsies. A large fraction of common integration sites contained genes that have been dysregulated or misexpressed in glioma. Others overlapped with loci identified in previous glioma-related forward genetic screens, but several novel putative cancer-causing genes were also found. Integrating retroviral tagging and clinical data, Ppfibp1 was highlighted as a frequently tagged novel glioma-causing gene. Retroviral integrations into the locus resulted in Ppfibp1 upregulation, and Ppfibp1-tagged cells generated tumors with shorter latency upon orthotopic transplantation. In human gliomas, increased PPFIBP1 expression was significantly linked to poor prognosis and PDGF treatment resistance. Altogether, the current study has demonstrated a novel approach to tagging glioma genes via forward genetics, validating previous results, and identifying PPFIBP1 as a putative oncogene in gliomagenesis.

The Nexilin F-Actin Binding Protein (Nexilin) encoded by NEXN is a cardiac Z-disc protein important for cardiac function and development in humans, zebrafish, and mice. Heterozygote variants in the human NEXN gene have been reported to cause dilated and hypertrophic cardiomyopathy. Homozygous variants in NEXN cause a lethal form of human fetal cardiomyopathy, only described in two patients before. In a Swedish, four-generation, non-consanguineous family comprising 42 individuals, one female had three consecutive pregnancies with intrauterine fetal deaths caused by a lethal form of dilated cardiomyopathy. Whole-exome sequencing and variant analysis revealed that the affected fetuses were homozygous for a NEXN variant (NM_144573:c.1302del;p.(Ile435Serfs*3)). Moreover, autopsy and histology staining declared that they presented with cardiomegaly and endocardial fibroelastosis. Immunohistochemistry staining for Nexilin in the affected fetuses revealed reduced antibody staining and loss of striation in the heart, supporting loss of Nexilin function. Clinical examination of seven heterozygote carriers confirmed dilated cardiomyopathy (two individuals), other cardiac findings (three individuals), or no cardiac deviations (two individuals), indicating incomplete penetrance or age-dependent expression of dilated cardiomyopathy. RNA sequencing spanning the variant in cDNA blood of heterozygote individuals revealed nonsense-mediated mRNA decay of the mutated transcripts. In the current study, we present the first natural course of the recessively inherited lethal form of human fetal cardiomyopathy caused by loss of Nexilin function. The affected family had uneventful pregnancies until week 23-24, followed by fetal death at week 24-30, characterized by cardiomegaly and endocardial fibroelastosis.

Transgenerational inheritance of environmentally induced epigenetic marks can have significant impacts on eco-evolutionary dynamics, but the phenomenon remains controversial in ecological model systems. We used whole-genome bisulfite sequencing of individual water fleas (Daphnia magna) to assess whether environmentally induced DNA methylation is transgenerationally inherited. Genetically identical females were exposed to one of three natural stressors, or a de-methylating drug, and their offspring were propagated clonally for four generations under control conditions. We identified between 70 and 225 differentially methylated CpG positions (DMPs) in F1 individuals whose mothers were exposed to a natural stressor. Roughly half of these environmentally induced DMPs persisted until generation F4. In contrast, treatment with the drug demonstrated that pervasive hypomethylation upon exposure is reset almost completely after one generation. These results suggest that environmentally induced DNA methylation is non-random and stably inherited across generations in Daphnia, making epigenetic inheritance a putative factor in the eco-evolutionary dynamics of freshwater communities.

The complete mitochondrial genome of the Carniolan honeybee (Apis mellifera carnica) from Slovenia, a homeland of this subspecies, was acquired in two contigs from WGS data and annotated. The newly obtained mitochondrial genome is a circular closed loop of 16,447 bp. It comprises 37 genes (13 protein coding genes, 22 tRNA genes, and 2 rRNA genes) and an AT-rich control region. The order of the tRNA genes resembles the order characteristic of A. mellifera. The mitogenomic sequence of A. m. carnica from Slovenia contains 44 uniquely coded sites in comparison to the closely related subspecies A. m. ligustica and to A. m. carnica from Austria. Furthermore, 24 differences were recognised in comparison between A. m. carnica and A. m. ligustica subspecies. Among them, there are three SNPs that affect translation in the nd2, nd4, and cox2 genes, respectively. The phylogenetic placement of A. m. carnica from Slovenia within C lineage deviates from the expected position and changes the perspective on relationship between C and O lineages. The results of this study represent a valuable addition to the information available in the phylogenomic studies of A. mellifera-a pollinator species of worldwide importance. Such genomic information is essential for this local subspecies' conservation and preservation as well as its breeding and selection.

Peatlands are generally important sources of methylmercury (MeHg) to adjacent aquatic ecosystems, increasing the risk of human and wildlife exposure to this highly toxic compound. While microorganisms play important roles in mercury (Hg) geochemical cycles where they directly and indirectly affect MeHg formation in peatlands, potential linkages between net MeHg formation and microbial communities involving these microorganisms remain unclear. To address this gap, microbial community composition and specific marker gene transcripts were investigated along a trophic gradient in a geographically constrained peatland chronosequence. Our results showed a clear spatial pattern in microbial community composition along the gradient that was highly driven by peat soil properties and significantly associated with net MeHg formation as approximated by MeHg concentration and %MeHg of total Hg concentration. Known fermentative, syntrophic, methanogenic and iron-reducing metabolic guilds had the strong positive correlations to net MeHg formation, while methanotrophic and methylotrophic microorganisms were negatively correlated. Our results indicated that sulfate reducers did not have a key role in net MeHg formation. Microbial activity as interpreted from 16S rRNA sequences was significantly correlated with MeHg and %MeHg. Our findings shed new light on the role of microbial community in net MeHg formation of peatlands that undergo ontogenetic change.

Fibroblast growth factor 21 (FGF21) is a hepatokine that produces metabolic benefits, such as improvements of lipid profile. We performed a genome-wide association study (GWAS) to identify genetic variants associated with circulating FGF21 and investigated the causal effects of FGF21 on pertinent outcomes using Mendelian randomization (MR). We conducted a GWAS testing ∼7.8 million DNA sequence variants with circulating FGF21 in a discovery cohort of 6259 Swedish adults with replication in 4483 Swedish women. We then performed two-sample MR analyses of genetically predicted circulating FGF21 in relation to alcohol and nutrient intake, cardiovascular and metabolic biomarkers and diseases, and liver function biomarkers using publicly available GWAS summary statistics data. Our GWAS identified multiple single-nucleotide polymorphisms with genome-wide significant associations (P < 5 × 10-8) with circulating FGF21 on chromosomes 2 and 19 in or near the GCKR and FGF21 genes, respectively. The strongest signal at the FGF21 locus (rs2548957, β = 0.181, P < 2.18 × 10-42) displayed in two-sample MR analyses robust associations with lower alcohol intake, lower circulating low-density lipoprotein cholesterol, apolipoprotein B, C-reactive protein, gamma-glutamyl transferase, and galectin-3 concentrations, and higher circulating insulin-like growth factor-I and alkaline phosphatase concentrations after correcting for multiple testing (P < 0.0018) whereas associations with fat mass, type 2 diabetes, and cardiovascular disease were largely null. We identified robust associations of certain genetic variants in or near the GCKR and FGF21 genes with circulating FGF21 concentrations. Furthermore, our results support a strong causal effect of FGF21 on improved lipid profile, reduced alcohol consumption and C-reactive protein concentrations, and liver function biomarkers including fibrosis. We found largely null or weak positive associations with fat mass, diabetes, and cardiovascular disease as well as higher insulin-like growth factor-I concentrations, which could indicate a compensatory increase to regulate the above FGF21 resistant states in humans.

Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.

Pulmonary manifestations in RA are common comorbidities, but the underlying mechanisms are largely unknown. The added value of a multiplex of ACPA and genetic risk markers was evaluated for the development of pulmonary fibrosis (PF) in an inception cohort. A total of 1184 patients with early RA were consecutively included and followed prospectively from the index date until death or 31 December 2016. The presence of 21 ACPA fine specificities was analysed using a custom-made microarray chip (Thermo Fisher Scientific, Uppsala, Sweden). Three SNPs, previously found related to PF were evaluated, rs2609255 (FAM13A), rs111521887 (TOLLIP) and rs35705950 (MUC5B). ACPA and genetic data were available for 841 RA patients, of whom 50 developed radiologically defined PF. In unadjusted analyses, 11 ACPA specificities were associated with PF development. In multiple variable analyses, six ACPA specificities were associated with increased risk of PF: vimentin (Vim)60-75, fibrinogen (Fib)β62-78 (72), Fibα621-635, Bla26, collagen (C)II359-369 and F4-CIT-R (P < 0.01 to P < 0.05). The number of ACPA specificities was also related to PF development (P < 0.05 crude and adjusted models). In multiple variable models respectively adjusted for each of the SNPs, the number of ACPA specificities (P < 0.05 in all models), anti-Vim60-75 (P < 0.05, in all models), anti-Fibβ62-78 (72) (P < 0.001 to P < 0.05), anti-CII359-369 (P < 0.05 in all models) and anti-F4-CIT-R AQ4 (P < 0.01 to P < 0.05), anti-Fibα621-635 (P < 0.05 in one) and anti-Bla26 (P < 0.05 in two) were significantly associated with PF development. The development of PF in an inception cohort of RA patients was associated with both presence of certain ACPA and the number of ACPA specificities and risk genes.

The endosperm is a nutritive tissue supporting embryo growth in flowering plants. Most commonly, the endosperm initially develops as a coenocyte (multinucleate cell) and then cellularizes. This process of cellularization is frequently disrupted in hybrid seeds generated by crosses between different flowering plant species or plants that differ in ploidy, resulting in embryo arrest and seed lethality. The reason for embryo arrest upon cellularization failure remains unclear. In this study, we show that triploid Arabidopsis thaliana embryos surrounded by uncellularized endosperm mount an osmotic stress response that is connected to increased levels of abscisic acid (ABA) and enhanced ABA responses. Impairing ABA biosynthesis and signalling aggravated triploid seed abortion, while increasing endogenous ABA levels as well as the exogenous application of ABA induced endosperm cellularization and suppressed embryo growth arrest. Taking these results together, we propose that endosperm cellularization is required to establish dehydration tolerance in the developing embryo, ensuring its survival during seed maturation.

The genomes of immortalized cell lines (and cancer cells) are characterized by multiple types of aberrations, ranging from single nucleotide polymorphisms (SNPs) to structural rearrangements that have accumulated over time. Consequently, it is difficult to estimate the relative impact of different aberrations, the order of events, and which gene functions were under selective pressure at the early stage towards cellular immortalization. Here, we have established novel cell cultures derived from Drosophila melanogaster embryos that were sampled at multiple time points over a one-year period. Using short-read DNA sequencing, we show that copy-number gain in preferentially stress-related genes were acquired in a dominant fraction of cells in 300-days old cultures. Furthermore, transposable elements were active in cells of all cultures. Only a few (<1%) SNPs could be followed over time, and these showed no trend to increase or decrease. We conclude that the early cellular responses of a novel culture comprise sequence duplication and transposable element activity. During immortalization, positive selection first occurs on genes that are related to stress response before shifting to genes that are related to growth.

Genetic variability can be generated by different mechanisms, and across the life cycle. Many basidiomycete fungi have an extended somatic stage, during which each cell carries two genetically distinct haploid nuclei (dikaryosis), resulting from fusion of two compatible monokaryotic individuals. Recent findings have revealed remarkable genome stability at the nucleotide level during dikaryotic growth in these organisms, but whether this pattern extends to mutations affecting large genomic regions remains unknown. Furthermore, despite high genome integrity during dikaryosis, basidiomycete populations are not devoid of genetic diversity, begging the question of when this diversity is introduced. Here, we used a Marasmius oreades fairy ring to investigate the rise of large-scale variants during mono- and dikaryosis. By separating the two nuclear genotypes from four fruiting bodies and generating complete genome assemblies, we gained access to investigate genomic changes of any size. We found that during dikaryotic growth in nature the genome stayed intact, but after separating the nucleotypes into monokaryons, a considerable amount of structural variation started to accumulate, driven to large extent by transposons. Transposon insertions were also found in monokaryotic single-meiospore isolates. Hence, we show that genome integrity in basidiomycetes can be interrupted during monokaryosis, leading to genomic rearrangements and increased activity of transposable elements. We suggest that genetic diversification is disproportionate between life cycle stages in mushroom-forming fungi, so that the short-lived monokaryotic growth stage is more prone to genetic changes than the dikaryotic stage.

Tandem repeat expansions (TREs) are associated with over 60 monogenic disorders and have recently been implicated in complex disorders such as cancer and autism spectrum disorder. The role of TREs in schizophrenia is now emerging. In this study, we have performed a genome-wide investigation of TREs in schizophrenia. Using genome sequence data from 1154 Swedish schizophrenia cases and 934 ancestry-matched population controls, we have detected genome-wide rare (<0.1% population frequency) TREs that have motifs with a length of 2-20 base pairs. We find that the proportion of individuals carrying rare TREs is significantly higher in the schizophrenia group. There is a significantly higher burden of rare TREs in schizophrenia cases than in controls in genic regions, particularly in postsynaptic genes, in genes overlapping brain expression quantitative trait loci, and in brain-expressed genes that are differentially expressed between schizophrenia cases and controls. We demonstrate that TRE-associated genes are more constrained and primarily impact synaptic and neuronal signaling functions. These results have been replicated in an independent Canadian sample that consisted of 252 schizophrenia cases of European ancestry and 222 ancestry-matched controls. Our results support the involvement of rare TREs in schizophrenia etiology.

The aim of this study was to investigate fungal communities associated with leaves and roots of healthy-looking and declining U. glabra trees. The study was expected to demonstrate whether and how the diversity and composition of fungal communities change in these functional tissues following the infection by Dutch elm disease-causing fungi. The study sites included six U. glabra sites in Lithuania, where leaves and roots were sampled. DNA was isolated from individual samples, amplified using ITS2 rRNA as a marker, and subjected to high-throughput sequencing. The sequence analysis showed the presence of 32,699 high-quality reads, which following clustering, were found to represent 520 non-singleton fungal taxa. In leaves, the fungal species richness was significantly higher in healthy-looking trees than in diseased ones (p < 0.05). In roots, a similar comparison showed that the difference was insignificant (p > 0.05). The most common fungi in all samples of roots were Trichocladium griseum (32.9%), Penicillium restrictum (21.2%), and Unidentified sp. 5238_7 (12.6%). The most common fungi in all samples of leaves were Trichomerium sp. 5238_8 (12.30%), Aureobasidium pullulans (12.03%), Cladosporium sp. 5238_5 (11.73%), and Vishniacozyma carnescens (9.86%). The results showed that the detected richness of fungal taxa was higher in samples collected from healthy-looking trees than from diseased ones, thereby highlighting the negative impact of the Dutch elm disease on the overall fungal diversity.

The honeybee gut microbiome is thought to be important for bee health, but the role of the individual members is poorly understood. Here, we present closed genomes and associated mobilomes of 102 Apilactobacillus kunkeei isolates obtained from the honey crop (foregut) of honeybees sampled from beehives in Helsingborg in the south of Sweden and from the islands Gotland and Åland in the Baltic Sea. Each beehive contained a unique composition of isolates and repeated sampling of similar isolates from two beehives in Helsingborg suggests that the bacterial community is stably maintained across bee generations during the summer months. The sampled bacterial population contained an open pan-genome structure with a high genomic density of transposons. A subset of strains affiliated with phylogroup A inhibited growth of the bee pathogen Melissococcus plutonius, all of which contained a 19.5 kb plasmid for the synthesis of the antimicrobial compound kunkecin A, while a subset of phylogroups B and C strains contained a 32.9 kb plasmid for the synthesis of a putative polyketide antibiotic. This study suggests that the mobile gene pool of A. kunkeei plays a key role in pathogen defense in honeybees, providing new insights into the evolutionary dynamics of defensive symbiont populations.