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bioRxiv2026

The Dynamics of Long Terminal Repeat Proliferation in the Hesperis matronalis Genome

Genome sizes vary across four orders of magnitude in flowering plants, with consequences for evolution. Much of this variation is due to differences in transposable element content, particularly in long terminal repeat (LTR) retrotransposons. Despite their importance in plant genome evolution, LTRs have long been challenging to characterize because of their repetitive nature, but recent advances in sequencing allow more detailed explorations of their behavior. They are now known to occupy distinct genomic niches, and to evolve and proliferate over time as they escape host controls. In this study, we present a new genome sequence of the largest Brassicaceae genome, Hesperis matronalis, and describe its transposable element complement and how LTRs contributed to its genome expansion. We find evidence for both early proliferation of Ty3 elements and rapid recent expansion of Ty1-copia elements. In addition, we place the H. matronalis LTRs in a broader context of LTR evolution in the Brassicaceae, showing that the dominant copia families are part of an evolutionary radiation endemic to Hesperis. Finally, we describe differences in LTR age, proximity to genes, and apparent removal rate which suggest consistent genomic niches over the lifespan of a TE family. These results shed light on how LTRs evolve dynamically with host genomes, and have contributed to the expansion of the largest genome in the Brassicaceae. Significance StatementOrganisms differ in many ways, including the size of their genomes. Genome size, in turn, can affect many aspects of evolution, such as what kinds of mutations are available to natural selection and how effectively natural selection can act. The "complexity" of an organism does not predict its genome size; rather, much of this variation is explained by the amount of transposable elements, or "jumping genes," that inhabit a genome. This study describes the evolutionary history of the transposable elements in dames rocket (Hesperis matronalis), which has the largest genome of any plant in the mustard family (Brassicaceae). We find that the genome inflation in Hesperis is due to transposable elements that are not found elsewhere in the Brassicaceae, and likely diversified within Hesperis. Characterizing the evolution and behavior of transposable elements in this genome offers a better understanding of the forces that determine genome size and ultimately affect the evolution of life on earth.

genomics↗

bioRxiv2020

Mycobacterium tuberculosis complex lineage 5 exhibits high levels of within-lineage genomic diversity

Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum), from each other. However, genome variability and gene content especially of L5 and L6 strains have not been fully explored and may be potentially important for pathobiology and current approaches for genomic analysis of MTBC isolates, including transmission studies. We compared the genomes of 358 L5 clinical isolates (including 3 completed genomes and 355 Illumina WGS (whole genome sequenced) isolates) to the L5 complete genomes and H37Rv, and identified multiple genes differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found across L5 strains, including a split in the L5.3 sublineage into L5.3.1 and L5.3.2. These gene content differences had a small knock on effect on transmission cluster estimation, with clustering rates influenced by the selection of reference genome, and with potential over-estimation of recent transmission when using H37Rv as the reference genome. Our data show that the use of H37Rv as reference genome results in missing SNPs in genes unique for L5 strains. This potentially leads to an underestimation of the diversity present in the genome of L5 strains and in turn affects the transmission clustering rates. As such, a full capture of the gene diversity, especially for high resolution outbreak analysis, requires a variation of the single H37Rv-centric reference genome mapping approach currently used in most WGS data analysis pipelines. Moreover, the high within-lineage gene content variability suggests that the pan-genome of M. tuberculosis is at least several kilobases larger than previously thought, implying a concatenated or reference-free genome assembly (de novo) approach may be needed for particular questions. Data summarySequence data for the Illumina dataset are available at European Genome-phenome Archive (EGA; https://www.ebi.ac.uk/ega/) under the study accession numbers PRJEB38317 and PRJEB38656. Individual runs accession numbers are indicated in Table S8. PacBio raw reads for the L5 Benin genome are available on the ENA accession SAME3170744. The assembled L5 Benin genome is available on NCBI with accession PRJNA641267. To ensure naming conventions of the genes in the three L5 genomes can be followed, we have uploaded these annotated GFF files to figshare at https://doi.org/10.6084/m9.figshare.12911849.v1. Custom python scripts used in this analysis can be found at https://github.com/conmeehan/pathophy.

genetics↗

bioRxiv2022

Co-transfer of functionally interdependent genes contributes to genome mosaicism in lambdoid phages

Lambdoid (or Lambda-like) phages, are a group of related temperate phages that can infect Escherichia coli and other gut bacteria. A key characteristic of these phages is their mosaic genome structure which served as basis for the "modular genome hypothesis". Accordingly, lambdoid phages evolve by transferring genomic regions, each of which constitutes a functional unit. Nevertheless, it is unknown which genes are preferentially transferred together and what drives such co-transfer events. Here we aim to characterize genome modularity by studying co-transfer of genes among 95 distantly related lambdoid (pro-)phages. Based on gene content, we observed that the genomes cluster into twelve groups, which are characterized by a highly similar gene content within the groups and highly divergent gene content across groups. Highly similar proteins can occur in genomes of different groups, indicating that they have been transferred. About 26% of homologous protein clusters in the four known operons (i.e., the early left, early right, immunity, and late operon) engage in gene transfer, which affects all operons to a similar extent. We identified pairs of genes that are frequently co-transferred and observed that these pairs tend to be in close proximity to one another on the genome. We find that frequently co-transferred genes are involved in related functions and highlight interesting examples involving structural proteins, the CI repressor and Cro regulator, proteins interacting with DNA, and membrane-interacting proteins. We conclude that epistatic effects, where the functioning of one protein depends on the presence of another, plays an important role in the evolution of the modular structure of these genomes. Data summaryThe genomes used in this research are publicly available (Table S1). All supporting data is available in supplementary tables. Source code and documentation to calculate GRR is available under GPLv2 (https://github.com/annecmg/GRRpair). Impact statementTemperate phages, viruses that can integrate their own genetic material into bacterial genomes, are pervasive mobile genetic elements that can influence bacterial fitness in manifold ways. The E. coli phage lambda has been a model phage of molecular biology for decades. Lambdoid phages are highly prevalent in Enterobacteria such as E. coli and Salmonella, have a mosaic-like genome, the same genome architecture as lambda, and can recombine with phage lambda. Nevertheless, these phages can be very distinct, and no lambdoid core genome exits. Although lambdoid phage genomes have been studied for decades, we know relatively little about how they evolve. Early observations led to the modular genome hypothesis, according to which, phages are assemblages of genetic modules. But what determines the structure of these modules and which genes do preferentially occur together in modules? In this study, we provide answers to these questions using a novel computational approach that allows to infer gene transfer events between distantly related phages despite the absence of a core genome. We find that co-transfer of functionally related genes is frequent during the evolution of lambdoid phages. This suggests epistatic interactions among these genes, i.e., the co-transferred genes likely need to function together to ensure a viable phage. A prime example is the co-transfer of structural genes, such as genes encoding for the phage capsid or the phage tail. Additionally, we also find co-transfer of known interacting regulatory genes and co-transfer between functionally related genes that have so far been unknown to interact. Together, our analysis provides novel insights into the evolution of temperate phages. Moreover, our approach, which allows to identify gene transfer in the absence of a core phylogeny might be valuable for studying the evolution of other fast-evolving genomes, including viruses of other hosts.

microbiology↗

bioRxiv2024

Prevalence and Dynamics of Genome Rearrangements in Bacteria and Archaea

The genetic material of bacteria and archaea is organized into various structures and set-ups, attesting that genome architecture is dynamic in these organisms. However, strong selective pressures are also acting to preserve genome organization, and it remains unclear how frequently genomes experience rearrangements and what mechanisms lead to these processes. Here, we assessed the dynamics and the drivers of genomic rearrangements across 121 microbial species. We show that synteny is highly conserved within most species, although several species present exceptionally flexible genomic layouts. Our results show a rather variable pace at which genomic rearrangements occur across bacteria and archaea, pointing to different selective constraints driving the accumulation of genomic changes across species. Importantly, we found that not only inversions but also translocations are highly enriched near the origin of replication (Ori), which suggests that many rearrangements may confer an adaptive advantage to the cell through the relocation of genes that benefit from gene dosage effects. Finally, our results support the view that mobile genetic elements--in particular transposable elements--are the main drivers of genomic translocations and inversions. Overall, our study shows that microbial species present largely stable genomic layouts and identifies key patterns and drivers of genome rearrangements in prokaryotes. Significance statementBacterial and archaeal genomes display stable architectures which ensures the preservation of fundamental cellular processes. However, large genomic rearrangements occasionally occur. Although most of these events are thought to be highly deleterious, they have the potential to lead to adaptive events. Here, we examined the general trends of the dynamic of prokaryotic genomes by exploring the occurrence of genome rearrangements across a broad diversity of bacterial and archaeal species. We find that genomes remain highly syntenic in most species over short evolutionary timescales, although some species appear particularly dynamic. Rearrangements are strongly biased, and most gene blocks are relocated near the origin of replication. We also measured remarkably variables rates at which genome rearrangements occur across species, and transposons and other mobile genetic elements appear to be the main drivers of these variations. Overall, this study provides a comprehensive picture of the dynamic of genome architecture across many microbial species.

evolutionary biology↗

bioRxiv2025

Improving the benchmark of variant calling in clonal bacteria using more realistic in silico genomes, the case of Mycobacterium tuberculosis

The democratisation of Whole Genome Sequencing data in bacterial genomics requires the benchmarking of associated analytical methodologies such as reference-based variant calling. Current variant calling benchmarks rely either on de novo assembled natural genomes, for which true variants are inferred using a genome aligner, or on genomes evolved in silico by incorporating short variants on reference genomes. We introduce Maketube, a method for evolving realistic genomes of the Mycobacterium tuberculosis complex with the full diversity of variants verified in natural isolates, and describe benchmarking results using Maketube-evolved genomes. We document that Maketube-evolved genomes satisfyingly mimic Mtbc complex genomes. Using Maketube-evolved genomes, we show that genome aligners miss up to 7.5% of the variants, which implies that benchmarkings with natural de novo assembled genomes are biased. Second, we show that recall of popular variant calling pipelines MTBseq, TB-Profiler, and our in-house genomic pipeline genotube, was overestimated by 1 to 10% in benchmarkings relying on simplistic in silico-evolved genomes, and that slight but significant differences in performance exist between pipelines. Finally, we provide evidence that variants are missed in duplicated regions and in regions flanking sequences absent in the reference (displaced insertion sequences or sequences deleted during the evolution of the reference). Altogether, realistic in silico-evolved genomes such as Maketube-derived ones are precious tools for reliable genomic tools benchmarking. We provide new evidence that structural variants interfere with variant-calling, both because of the additional sequences they contain, but also because of misalignments around insertions.

bioinformatics↗

bioRxiv2019

Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods impact on assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or from SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the Enterobacteriaceae family, as these frequently have highly plastic, repetitive genetic structures and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies. Both strategies facilitate high-quality genome reconstruction. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly. IMPACT STATEMENTIllumina short-read sequencing is frequently used for tasks in bacterial genomics, such as assessing which species are present within samples, checking if specific genes of interest are present within individual isolates, and reconstructing the evolutionary relationships between strains. However, while short-read sequencing can reveal significant detail about the genomic content of bacterial isolates, it is often insufficient for assessing genomic structure: how different genes are arranged within genomes, and particularly which genes are on plasmids - potentially highly mobile components of the genome frequently carrying antimicrobial resistance elements. This is because Illumina short reads are typically too short to span repetitive structures in the genome, making it impossible to accurately reconstruct these repetitive regions. One solution is to complement Illumina short reads with long reads generated with SMRT Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) sequencing platforms. Using this approach, called hybrid assembly, we show that we can automatically fully reconstruct complex bacterial genomes of Enterobacteriaceae isolates in the majority of cases (best-performing method: 17/20 isolates). In particular, by comparing different methods we find that using the assembler Unicycler with Illumina and ONT reads represents a low-cost, high-quality approach for reconstructing bacterial genomes using publicly available software. DATA SUMMARYRaw sequencing data and assemblies have been deposited in NCBI under BioProject Accession PRJNA422511 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA422511). We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

bioinformatics↗

bioRxiv2026

Comparative analyses reveal rapid turnover and emergence of transitory 3D genome architectures in the fungal kingdom

The three-dimensional architecture of genomes plays major roles in biological processes such as gene expression and DNA replication. The architecture of genomes has evolved substantially with distinct 3D genome shapes being identified in different lineages. The factors driving the evolution of genome architectures have primarily been assessed in animals and plants, yet large parts of the tree of life remain poorly explored. Fungi offer excellent models to assess the evolution of 3D genome architecture in a phylogenetic context given rapid genome size changes and chromosomal sequence turnover. Here, we analyzed chromosome conformation data (Hi-C) of 55 fungal species with completely assembled genomes. We identified ten species with Rabl, one species with chromosome territories and ten with a novel, intermediate chromosomal architecture, where centromeres and telomeres are at opposites in the nucleus (Rabl-like) but with a distinct 3D organization. This "bean" shape likely evolved several times independently. The discovery of a genome with a chromosome territories conformation was unexpected, as this was thought to be associated with condensin II subunits in the animal kingdom. We investigated whether 3D conformations correlated with genome size and repeat content using phylogenetic independent contrasts, however we found no genomic feature to be significantly associated with changes in genome architecture. Overall, we report the first large-scale comparison of 3D genome architecture in the fungal kingdom and identify a novel "bean" configuration. SignificanceThree-dimensional genome architecture strongly influences gene regulation, yet little is known about 3D genome architecture in an organismal group that has adapted to nearly all ecosystems on our planet, Fungi. We reconstructed 3D genome architectures from 55 fungal species covering three different phyla and demonstrate that most species do not conform to the existing definitions of 3D architectures. We identified the first case of Chromosome Territories in the Fungal Kingdom and a previously undescribed organization that we label "bean-shaped", and show that some fungal species do not conform to the canonical 3D-architecture categories of the animal and plant kingdoms. The diversity of genome architectures observed in the study could reflect the diverse gene regulatory mechanisms known from Fungi and marks the beginning of mapping out 3D genome organizations in this diverse clade. Further research in this area will uncover the diverse strategies employed by Fungi in light of their rapid adaptation.

evolutionary biology↗

bioRxiv2026

Strain handling exposes IS-mediated recurrent genome reduction at a dynamic accessory locus in Komagataeibacter rhaeticus

Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement.

microbiology↗

bioRxiv2016

Bacterial genome reduction as a result of short read sequence assembly

High-throughput comparative genomics has changed our view of bacterial evolution and relatedness. Many genomic comparisons, especially those regarding the accessory genome that is variably conserved across strains in a species, are performed using assembled genomes. For completed genomes, an assumption is made that the entire genome was incorporated into the genome assembly, while for draft assemblies, often constructed from short sequence reads, an assumption is made that genome assembly is an approximation of the entire genome. To understand the potential effects of short read assemblies on the estimation of the complete genome, we downloaded all completed bacterial genomes from GenBank, simulated short reads, assembled the simulated short reads and compared the resulting assembly to the completed assembly. Although most simulated assemblies demonstrated little reduction, others were reduced by as much as 25%, which was correlated with the repeat structure of the genome. A comparative analysis of lost coding region sequences demonstrated that up to 48 CDSs or up to ~112,000 bases of coding region sequence, were missing from some draft assemblies compared to their finished counterparts. Although this effect was observed to some extent in 32% of genomes, only minimal effects were observed on pan-genome statistics when using simulated draft genome assemblies. The benefits and limitations of using draft genome assemblies should be fully realized before interpreting data from assembly-based comparative analyses.

genomics↗

bioRxiv2020

A comprehensive and high-quality collection of E. coli genomes and their genes

Escherichia coli is a highly diverse organism which includes a range of commensal and pathogenic variants found across a range of niches and worldwide. In addition to causing severe intestinal and extraintestinal disease, E. coli is considered a priority pathogen due to high levels of observed drug resistance. The diversity in the E. coli population is driven by high genome plasticity and a very large gene pool. All these have made E. coli one of the most well-studied organisms, as well as a commonly used laboratory strain. Today, there are thousands of sequenced E. coli genomes stored in public databases. While data is widely available, accessing the information in order to perform analyses can still be a challenge. Collecting relevant available data requires accessing different sources, where data may be stored in a range of formats, and often requires further manipulation, and processing to apply various analyses and extract useful information. In this study, we collated and intensely curated a collection of over 10,000 E. coli and Shigella genomes to provide a single, uniform, high-quality dataset. Shigella were included as they are considered specialised pathovars of E. coli. We provide these data in a number of easily accessible formats which can be used as the foundation for future studies addressing the biological differences between E. coli lineages and the distribution and flow of genes in the E. coli population at a high resolution. The analysis we present emphasises our lack of understanding of the true diversity of the E. coli species, and the biased nature of our current understanding of the genetic diversity of such a key pathogen. Author NotesAll supporting data have been provided within the article or through supplementary data files. All supporting code is provided in the git repository https://github.com/ghoresh11/ecoli_genome_collection. Significance as a BioResource to the communityAs of today, there are more than 140,000 E. coli genomes available on public databases. While data is widely available, collating the data and extracting meaningful information from it often requires multiple steps, computational resources and expert knowledge. Here, we collate a high quality and comprehensive set of over 10,000 E. coli genomes, isolated from human hosts, into a set of manageable files that offer an accessible and usable snapshot of the currently available genome data, linked to a minimal data quality standard. The data provided includes a detailed synopsis of the main lineages present, including their antimicrobial and virulence profiles, their complete gene content, and all the associated metadata for each genome. This includes a database which enables the user to compare newly sequenced isolates against the assembled genomes. Additionally, we provide a searchable index which allows the user to query any DNA sequence against the assemblies of the collection. This collection paves the path for many future studies, including those investigating the differences between E. coli lineages, following the evolution of different genes in the E. coli pan-genome and exploring the dynamics of horizontal gene transfer in this important organism. Data SummaryO_LIThe complete aggregated metadata of 10,146 high quality genomes isolated from human hosts (doi.org/10.6084/m9.figshare.12514883, File F1). C_LIO_LIA PopPUNK database which can be used to query any genome and examine its context relative to this collection (Deposited to doi.org/10.6084/m9.figshare.12650834). C_LIO_LIA BIGSI index of all the genomes which can be used to easily and quickly query the genomes for any DNA sequence of 61 bp or longer (Deposited to doi.org/10.6084/m9.figshare.12666497). C_LIO_LIDescription and complete profiling the 50 largest lineages which represent the majority of publicly available human-isolated E. coli genomes (doi.org/10.6084/m9.figshare.12514883, File F2). Phylogenetic trees of representative genomes of these lineages, presented in this manuscript, are also provided (doi.org/10.6084/m9.figshare.12514883, Files tree_500.nwk and tree_50.nwk). C_LIO_LIThe complete pan-genome of the 50 largest lineages which includes: O_LIA FASTA file containing a single representative sequence of each gene of the gene pool (doi.org/10.6084/m9.figshare.12514883, File F3). C_LIO_LIComplete gene presence-absence across all isolates (doi.org/10.6084/m9.figshare.12514883, File F4). C_LIO_LIThe frequency of each gene within each of the lineages (doi.org/10.6084/m9.figshare.12514883, File F5). C_LIO_LIThe representative sequences from each lineage for all the genes (doi.org/10.6084/m9.figshare.12514883, File F6). C_LI C_LI

genomics↗

bioRxiv2024

The linear correlation between genome size and the size of the non-transcribing region

BackgroundThe genome sizes of organisms vary widely (C-value paradox). There are non-transcribing regions in the genome that neither encode proteins nor RNA entities. There are several hypotheses about the function of these regions: one suggests that they are unannotated functional areas, while another views them as genomic isolation zones that reduce mutations in coding regions. MethodStatistical analysis was conducted on the transcribing regions (including areas annotated as genes and transcribed pseudogenes) and non-transcribing regions, protein-coding regions (Coding sequence, CDS), and genome sizes using annotation files from 63,866 species genomes in the NCBI RefSeq database. ResultsThere is a significant linear relationship between the size of non-transcribing genomic regions and overall genome size across species, with varying proportional coefficients among different phyla (realms for viruses). As genome size increases, the proportion of non-transcribing regions gradually rises, eventually approaching a linear proportional limit, resembling one arm of hyperbolic functions. Eukaryotes show high linear correlation, with the highest in Streptophyta and the lowest in Apicomplexa. In eukaryotes, the size of the coding region increases with genome size, but the increasing trend diminishes (proportionally decreases). In non-eukaryotes, the size of the coding region maintains a linear relationship with genome size. ConclusionThe size of non-transcribing region in species may be subject to some strict quantitative control mechanism, showing that genome and non-transcribing genome sizes increase proportionally with the expansion of the transcribing genome, indicating a strict balance between expansion and energy conservation. The proportion of non-transcribed genomes in eukaryotes is conservative (although the sequences are not), and the presence of non-transcribing genomes has significant implications for the evolution or survival of species. Thus, I propose a new hypothesis about the non-transcribing genome, that it is a space for generating new genes from scratch, and the different proportional coefficients among phyla are due to their different positions in energy transfer. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/613789v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@dc3e88org.highwire.dtl.DTLVardef@18d70e8org.highwire.dtl.DTLVardef@efb92corg.highwire.dtl.DTLVardef@66068b_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Genome-Guided Characterization of Weissella Species Highlights Strain-Specific Functional Traits Relevant to Food Fermentation and Probiotics

The genus Weissella comprises heterofermentative lactic acid bacteria widely distributed across fermented foods, environmental niches, and host-associated habitats, yet the genomic basis underlying their functional diversity and evolutionary plasticity remains incompletely resolved. Here, we performed a large-scale comparative genomic analysis of 347 high-quality genomes representing 16 Weissella species to characterize pan-genome architecture, phylogenomic structure, and the distribution of functional traits relevant to fermentation performance, safety, and probiotic potential. Pan-genome modeling revealed a strongly open pan-genome (P = 3419.89*N0.455), dominated by shell and cloud genes, indicating extensive genomic plasticity and ongoing gene acquisition. Average amino acid identity analysis delineated clear species boundaries while highlighting close evolutionary relationships among several taxa. Functional profiling demonstrated conserved carbohydrate-active enzyme repertoires dominated by glycoside hydrolases and glycosyltransferases, accompanied by species-specific variation in carbohydrate-binding modules. Comparative synteny analysis resolved six major architectures of exopolysaccharide biosynthesis loci, with W. cibaria enriched in regulator-rich and potentially active operons. In silico safety screening detected antimicrobial resistance genes in only three strains and no virulence-associated genes across the dataset, supporting a generally low-risk genomic profile. Probiotic-associated traits, including vitamin biosynthesis and stress resistance, were broadly conserved, whereas adhesion, {gamma}-aminobutyric acid biosynthesis, and secondary metabolite gene clusters were rare and strain-specific. Collectively, these results provide a systems-level view of genome diversification within the Weissella genus and establish a genome-guided framework for rational strain selection in food, health, and biotechnological applications. ImportanceMicrobial traits relevant to food fermentation, probiotic performance, and safety often emerge from complex interactions among core metabolism, accessory genes, and genome plasticity, yet these relationships remain poorly resolved at the genus scale. By analyzing 347 high-quality genomes spanning 16 Weissella species, this study reveals how an open pan-genome, extensive accessory gene diversity, and lineage-specific gene architectures collectively shape functional potential across this important lactic acid bacterial genus. We demonstrate that key traits, including exopolysaccharide biosynthesis, carbohydrate utilization capacity, stress resilience, and biosynthetic gene cluster distribution, are unevenly structured across species and strains, highlighting the necessity of genome-guided strain selection rather than taxonomic assumptions. The integration of phylogenomic relationships, gene content variation, and functional profiling provides a systems-level framework for linking genome evolution with applied phenotypes. These findings advance our understanding of how microbial genomic diversity underpins ecological adaptation and biotechnological performance and provide a scalable blueprint for rational selection of safe and functionally optimized strains in food and health applications.

genomics↗

bioRxiv2026

Whole-genome sequencing data of a diverse grapevine germplasm collection maintained in Bordeaux, France

Grapevine (Vitis vinifera) is one of the worlds most economically important fruit crops and a model species for perennial fruit tree genetics and genomics. The extensive genetic diversity found in cultivated and wild Vitis species provides a valuable resource for studies of domestication, adaptation, trait evolution, and breeding. This article presents a standardized whole-genome variant dataset comprising 547 grapevine accessions maintained in the INRAE Bordeaux grapevine germplasm collection, including 397 domesticated V. vinifera cultivars and 150 wild Vitis accessions. Whole-genome sequencing data were generated at a target sequencing depth of approximately 20x, and sequence variants were identified using a standardized Genome Analysis Toolkit (GATK) workflow against the reference genome PN40024v4 (40X). Variant discovery was carried out simultaneously across the complete sample set to ensure consistent genotype calling. All accessions were sequenced using the same technology and processed using the same reference genome, sequence alignment, variant-calling, and filtering workflow to produce a standardized variant dataset comprising ca. 9.1M SNPs and 0.77M INDELs. The resulting VCF files provide a harmonized genomic resource that can be readily reused for studies of grapevine genetics, germplasm characterization, population genomics, comparative genomics, genome-wide association studies, and the development and benchmarking of bioinformatic methods. SPECIFICATIONS TABLE O_TBL View this table: org.highwire.dtl.DTLVardef@14f2b12org.highwire.dtl.DTLVardef@94b5acorg.highwire.dtl.DTLVardef@1232f80org.highwire.dtl.DTLVardef@1038e85org.highwire.dtl.DTLVardef@81ec71_HPS_FORMAT_FIGEXP M_TBL C_TBL VALUE OF THE DATAO_LIThis dataset provides whole-genome raw sequencing for 118 wild Vitis accessions originating from North America and Asia and a sequencing-derived variant data for these accessions and 429 Vitis vinifera cultivars and wild accessions from the INRAE Bordeaux germplasm collection, previously published by Dong et al. 2023[1]. The dataset captures genetic variation across a total of 547 grapevine accessions, including domesticated and wild grapevine germplasm using a common variant-calling pipeline, facilitating direct comparisons among accessions. C_LIO_LIThe inclusion of wild Vitis species together with cultivated grapevine accessions provides a resource for studies of grapevine diversity, domestication, phylogenetic relationships, and comparative genomics. The dataset enables the investigation of genetic variation across multiple Vitis species using a standardized set of genomic variants. C_LIO_LIThe variant call format (VCF) file can be reused for population genetics, phylogenetic analyses, genetic diversity assessments, introgression analyses, and the identification of genomic regions of interest. The dataset is compatible with widely used bioinformatics software and can be integrated with other publicly available grapevine genomic resources. C_LIO_LIThis dataset constitutes a genomic resource for grapevine breeding and conservation research. Researchers can use these data to identify genetic diversity in wild relatives, compare allelic variation between cultivated and wild germplasm, investigate candidate loci associated with traits of interest, and support the management and characterization of grapevine germplasm collections. C_LI

genomics↗

bioRxiv2026

Rapid whole genome- and annotation-based multilocus sequence typing of the fungal pathogen Histoplasma capsulatum

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

genomics↗

bioRxiv2017

A near complete haplotype-phased genome of the dikaryotic wheat stripe rust fungus Puccinia striiformis f. sp. tritici reveals high inter-haplome diversity

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here we present a diploidaware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long-reads using the FALCON-Unzip assembler. RNA-seq datasets were used to infer high quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high inter-haplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When investigating genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors, and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales. In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.\n\nImportanceCurrent representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organisms evolutionary potential and ability to adapt to stress conditions. Yet it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which leads to allelic variation and the presence of genes lacking allelic counterparts. Non-allelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.

microbiology↗

bioRxiv2022

Ecological effects of genome size in yellow starthistle (Centaurea solstitialis) vary between invaded and native ranges

Invasive species have become a pervasive threat on every continent and across a broad array of environments. Several traits predicted to promote invasion success, such as small seed size, rapid vegetative growth and short time to reproduction, are correlated with smaller genome sizes in a number of systems. To understand the influence of genome size on plant invasion dynamics, we compared genome sizes and traits in Centaurea solstitialis (YST) genotypes from the Californian invasion to those from their native source region in Spain. We conducted a common garden experiment and genome size survey to ask: (1) Is the invasion associated with genome size reduction? (2) To what extent can differences in genome size explain previously observed increases in plant size and reproduction in YST invasions? (3) Finally, we tested for expected evolutionary patterns in genome size across populations, including evidence of selection favouring reduced genome sizes at higher elevations, and evidence of stochastic processes leading to increases in genome sizes where effective population sizes are smaller. We found a reduction in corrected genome size in the invaded range, as well as significant interaction effects of range x elevation on genome size, and range x genome size on flowering time variation. Specifically, larger genomes tended to flower later and genome size decreased with increasing elevation in the invasion only. These emergent relationships in invading YST suggest potential selection for smaller genomes following introduction of YST to its invaded range. We also found a significant effect of measurement date on genome size estimation by flow cytometry, and this effect was more pronounced among native range genotypes.

evolutionary biology↗

bioRxiv2019

Insights from the revised complete genome sequences of Acinetobacter baumannii strains AB307-0294 and ACICU belonging to global clone 1 and 2

2.The Acinetobacter baumannii global clone 1 (GC1) isolate AB307-0294, recovered in the USA in 1994, and the global clone 2 (GC2) isolate ACICU, isolated in 2005 in Italy, were among the first A. baumannii isolates to be completely sequenced. AB307-0294 is susceptible to most antibiotics and has been used in many genetic studies and ACICU belongs to a rare GC2 lineage. The complete genome sequences, originally determined using 454 pyrosequencing technology which is known to generate sequencing errors, were re-determined using Illumina MiSeq and MinION (ONT) technologies and a hybrid assembly generated using Unicycler. Comparison of the resulting new high-quality genomes to the earlier 454-sequenced version identified a large number of nucleotide differences affecting protein coding features, and allowed the sequence of the long and highly-repetitive bap and blp1 genes to be properly resolved for the first time in ACICU. Comparisons of the annotations of the original and revised genomes revealed a large number of differences in the protein coding features (CDSs), underlining the impact of sequence errors on protein sequence predictions and core gene determination. On average, 400 predicted CDSs were longer or shorter in the revised genomes and about 200 CDS features were no longer present.\n\n3. Impact statementThe genomes of the first 10 A. baumannii strains to be completely sequenced underpin a large amount of published genetic and genomic analysis. However, most of their genome sequences contain substantial numbers of errors as they were sequenced using 454 pyrosequencing, which is known to generate errors particularly in homopolymer regions; and employed manual PCR and capillary sequencing steps to bridge contig gaps and repetitive regions in order to finish the genomes. Assembly of the very large and internally repetitive gene for the biofilm-associated proteins Bap and BLP1 was a recurring problem. As these strains continue to be used for genetic studies and their genomes continue to be used as references in phylogenomics studies including core gene determination, there is value in improving the quality of their genome sequences. To this end, we re-sequenced two such strains that belong to the two major globally distributed clones of A. baumannii, using a combination of highly-accurate short-read and gap-spanning long-read technologies. Annotation of the revised genome sequences eliminated hundreds of incorrect CDS feature annotations and corrected hundreds more. Given that these revisions affected hundreds of non-existent or incorrect CDS features currently cluttering GenBank protein databases, it can be envisaged that similar revision of other early bacterial genomes that were sequenced using error-prone technologies will affect thousands of CDS currently listed in GenBank and other databases. These corrections will impact the quality of predicted protein sequence data stored in public databases. The revised genomes will also improve the accuracy of future genetic and comparative genomic analyses incorporating these clinically important strains.\n\n4. Data summaryO_LIThe corrected complete genome sequence of A. baumannii AB307-0294 has been deposited in GenBank; GenBank accession number CP001172.2 (chromosome url - https://www.ncbi.nlm.nih.gov/nuccore/CP001172.2).\nC_LIO_LIThe corrected complete genome sequence of ACICU has been deposited in GenBank under the GenBank accession numbers CP031380 (chromosome; url - https://www.ncbi.nlm.nih.gov/nuccore/CP031380), CP031381 (pACICU1; url - https://www.ncbi.nlm.nih.gov/nuccore/CP031381) and CP031382 (pACICU2; url - https://www.ncbi.nlm.nih.gov/nuccore/CP031382).\nC_LI\n\nThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2019

The Collaborative Cross Graphical Genome

The mouse reference is one of the most widely used and accurately assembled mammalian genomes, and is the foundation for a wide range of bioinformatics and genetics tools. However, it represents the genomic organization of a single inbred mouse strain. Recently, inexpensive and fast genome sequencing has enabled the assembly of other common mouse strains at a quality approaching that of the reference. However, using these alternative assemblies in standard genomics analysis pipelines presents significant challenges. It has been suggested that a pangenome reference assembly, which incorporates multiple genomes into a single representation, are the path forward, but there are few standards for, or instances of practical pangenome representations suitable for large eukaryotic genomes. We present a pragmatic graph-based pangenome representation as a genomic resource for the widely-used recombinant-inbred mouse genetic reference population known as the Collaborative Cross (CC) and its eight founder genomes. Our pangenome representation leverages existing standards for genomic sequence representations with backward-compatible extensions to describe graph topology and genome-specific annotations along paths. It packs 83 mouse genomes (8 founders + 75 CC strains) into a single graph representation that captures important notions relating genomes such as identity-by-descent and highly variable genomic regions. The introduction of special anchor nodes with sequence content provides a valid coordinate framework that divides large eukaryotic genomes into homologous segments and addresses most of the graph-based position reference issues. Parallel edges between anchors place variants within a context that facilitates orthogonal genome comparison and visualization. Furthermore, our graph structure allows annotations to be placed in multiple genomic contexts and simplifies their maintenance as the assembly improves. The CC reference pangenome provides an open framework for new tool chain development and analysis.

bioinformatics↗