Biology subjects

Johnson, M.

Publications and source records attributed to Johnson, M..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Frequent allopolyploidy with distant progenitors in the moss genera Physcomitrium and Entosthodon (Funariaceae) identified via subgenome phasing of targeted nuclear genes.

Allopolyploids represent a new frontier in species discovery among embryophytes. Within mosses, allopolyploid discovery is challenged by low morphological complexity. The rapid expansion of sub-genome sequencing approaches in addition to computational developments to identifying genome merger and whole-genome duplication using variation among nuclear loci representing homeologs has allowed for increased allopolyploid discovery among mosses. We confirm the intergeneric hybrid nature of Entosthodon hungaricus, and the allopolyploid origin of P. eurystomum and of one population of P. collenchymatum. We also reveal that hybridization gave rise to P. immersum, as well as to yet unrecognized lineages sharing the phenotype of P. pyriforme, and P. sphaericum. Our findings demonstrate the utility of a novel approach to phasing homeologs within loci and phasing loci across subgenomes, or subgenome assignment, called homologizer, when working with polyploid genomes, and its value in identifying progenitor species using target capture data.

evolutionary biology↗

bioRxiv2023

Diverse viral cas genes antagonize CRISPR immunity

Prokaryotic CRISPR-Cas immunity is subverted via anti-CRISPRs (Acrs), small proteins that inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids. CRISPR-Cas defenses are classified into 6 types and 33 subtypes, which employ a diverse suite of Cas effectors and differ in their mechanisms of interference. As Acrs often work via binding to a cognate Cas protein, inhibition is almost always limited to a single CRISPR type. Furthermore, while acr genes are frequently organized together in phage-associated gene clusters, how such inhibitors initially evolve has remained unclear. Here we have investigated the Acr content and inhibition specificity of a collection of Listeria isolates, which naturally harbor four diverse CRISPR-Cas systems (types I-B, II-A, II-C, and VI-A). We observed widespread antagonism of CRISPR, which we traced to 12 novel and 4 known Acr gene families encoded on endogenous mobile genetic elements. Among these were two Acrs that possess sequence homology to type I-B Cas proteins and assemble into a defective interference complex. Surprisingly, an additional type I-B Cas homolog did not affect type I immunity, but instead inhibited the RNA-targeting type VI CRISPR system through sequestration of crRNA. By probing the IMGVR database of viral genomes, we detected abundant orphan cas genes located within putative anti-defense gene clusters. We experimentally verified the Acr activity of one viral cas gene, a particularly broad-spectrum cas3 homolog that inhibits type I-B, II-A, and VI-A CRISPR immunity. Our observations provide direct evidence of Acr evolution via cas gene co-option, and new genes with potential for broad-spectrum control of genome editing technologies.

microbiology↗

bioRxiv2023

Haplotype-resolved, chromosome-level assembly of white clover (Trifolium repens L., Fabaceae)

BackgroundWhite clover (Trifolium repens L.; Fabaceae) is an important forage and cover crop in agricultural pastures around the world, and is increasingly used in evolutionary ecology and genetics to understand the genetic basis of adaptation. Historically, improvements in white clover breeding practices and assessments of genetic variation in nature have been hampered by a lack of high-quality genomic resources for this species, owing in part to its high heterozygosity and allotetraploid hybrid origin. FindingsHere, we use PacBio HiFi and chromosome conformation capture (Omni-C) technologies to generate a chromosome-level, haplotype-resolved genome assembly for white clover totaling 998 Mbp (scaffold N50 = 59.3 Mbp) and 1 Gbp (scaffold N50 = 58.6 Mbp) for haplotypes 1 and 2, respectively, with each haplotype arranged into 16 chromosomes (8 per subgenome). We additionally provide a functionally annotated haploid mapping assembly (968 Mbp, scaffold N50 = 59.9 Mbp), which drastically improves on the existing reference assembly in both contiguity and assembly accuracy. We annotated 78,174 protein-coding genes, resulting in protein BUSCO completeness scores of 99.6% and 99.3% against the embryophyta_odb10 and fabales_odb10 lineage datasets, respectively. ConclusionsWe provide two white clover genome assemblies as part of this project: (1) a haplotype-resolved, chromosome-level assembly, and (2) a functionally annotated haploid mapping assembly. These assemblies place white clover among the best sequenced legumes to date, and one of the best assemblies for a plant of recent polyploid origins. This work promises to facilitate ongoing and future work in agricultural and evolutionary genetics in this agronomically and ecologically important species.

genomics↗

bioRxiv2023

Transcriptomic Evaluation of Stress Vulnerability Network using Single Cell RNA-Seq in mouse Prefrontal Cortex

Increased vulnerability to stress is a major risk factor for the manifestation of several mood disorders, including major depressive disorder (MDD). Despite the status of MDD as a significant donor to global disability, the complex integration of genetic and environmental factors that contribute to the behavioral display of such disorders has made a thorough understanding of related etiology elusive. Recent developments suggest that a brain-wide network approach is needed, taking into account the complex interplay of cell types spanning multiple brain regions. Single cell RNA-sequencing technologies can provide transcriptomic profiling at the single-cell level across heterogenous samples. Furthermore, we have previously used local field potential oscillations and machine learning to identify an electrical brain network that is indicative of a predisposed vulnerability state. Thus, this study combined single cell RNA-sequencing (scRNA-Seq) with electrical brain network measures of the stress-vulnerable state, providing a unique opportunity to access the relationship between stress network activity and transcriptomic changes within individual cell types. We found especially high numbers of differentially expressed genes between animals with high and low stress vulnerability brain network activity in astrocytes and glutamatergic neurons but we estimated that vulnerability network activity depends most on GABAergic neurons. High vulnerability network activity included upregulation of microglia and mitochondrial and metabolic pathways, while lower vulnerability involved synaptic regulation. Genes that were differentially regulated with vulnerability network activity significantly overlapped with genes identified as having significant SNPs by human GWAS for depression. Taken together, these data provide the gene expression architecture of a previously uncharacterized stress vulnerability brain state, enabling new understanding and intervention of predisposition to stress susceptibility. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/540705v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1ebe036org.highwire.dtl.DTLVardef@cd2131org.highwire.dtl.DTLVardef@13e3519org.highwire.dtl.DTLVardef@1100e62_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Urbanization and a green corridor do not impact genetic divergence in common milkweed (Asclepias syriaca)

Urbanization is altering landscapes globally at an unprecedented rate. While ecological differences between urban and rural environments often promote phenotypic divergence among populations, it is unclear to what degree these trait differences arise from genetic divergence as opposed to phenotypic plasticity. Furthermore, little is known about how specific landscape elements, such as green corridors, impact genetic divergence in urban environments. We tested the hypotheses that: 1) urbanization, and 2) proximity to an urban green corridor influence genetic divergence in Asclepias syriaca (common milkweed) populations for phenotypic traits. Using seeds from 52 populations along three urban-to-rural subtransects in the Greater Toronto Area, one of which followed a green corridor, we grew [~]1000 plants in a common garden and observed >20 ecologically-important traits associated with plant defense/damage, reproduction, and growth over four years. We found significant heritable variation for eight traits within A. syriaca populations and weak phenotypic divergence among populations. However, neither urbanization nor an urban green corridor influenced genetic divergence in individual traits or multivariate phenotype. These findings contrast with the expanding literature demonstrating that urbanization promotes rapid evolutionary change and offer preliminary insights into the eco- evolutionary role of green corridors in urban environments.

evolutionary biology↗

bioRxiv2023

Parallelized screening of virus accessory genes reveals diverse defense and counter-defense mechanisms

Viruses have been evolving host-modifying factors for billions of years. Genomes of bacterial and archaeal viruses are replete with fast-evolving, uncharacterized accessory genes (AGs), most of which likely antagonize host defenses or other viruses1, 2. Systematic investigation of AGs could uncover a multitude of biological mechanisms involved in virus-host competition, but AG identification in genomic databases remains a challenge. We developed an integrated computational and high-throughput discovery platform to identify AGs in virus genomes and assay their functions in complementary phage infection-dependent and -independent contexts. Our approach showcases how phages interact with the principal layers of antiviral immunity, including cell surface modifications, restriction systems, and abortive infection (Abi) mechanisms, which operate simultaneously in the same host. We discovered multiple Enterobacteriophage AGs associated with counter-defense functions that activate rather than inhibit antiviral immunity in cells, including the surprising finding that anti-restriction AGs elicit programmed cell death (PCD) activity of some restriction-modification (R-M) systems. We propose that counter-defense AGs that trigger PCD create a conundrum for phages whereby keeping the AGs causes PCD but losing them exposes the phage to restriction by bacteria. Strategies employed by viruses to avoid this double jeopardy could be an important factor in virus evolution that remains to be explored.

microbiology↗

bioRxiv2023

UBE3A and transsynaptic complex NRXN1-CBLN1-GluD1 in a hypothalamic VMHvl-arcuate feedback circuit regulates aggression

The circuit origins of aggression in autism spectrum disorder remain undefined. Here we report Tac1-expressing glutamatergic neurons in ventrolateral division of ventromedial hypothalamus (VMHvl) drive intermale aggression. Aggression is increased due to increases of Ube3a gene dosage in the VMHvl neurons when modeling autism due to maternal 15q11-13 triplication. Targeted deletion of increased Ube3a copies in VMHvl reverses the elevated aggression adult mice. VMHvl neurons form excitatory synapses onto hypothalamic arcuate nucleus AgRP/NPY neurons through a NRXN1-CBLN1-GluD1 transsynaptic complex and UBE3A impairs this synapse by decreasing Cbln1 gene expression. Exciting AgRP/NPY arcuate neurons leads to feedback inhibition of VMHvl neurons and inhibits aggression. Asymptomatic increases of UBE3A synergize with a heterozygous deficiency of presynaptic Nrxn1 or postsynaptic Grid1 (both ASD genes) to increase aggression. Targeted deletions of Grid1 in arcuate AgRP neurons impairs the VMHvl to AgRP/NPY neuron excitatory synapses while increasing aggression. Chemogenetic/optogenetic activation of arcuate AgRP/NPY neurons inhibits VMHvl neurons and represses aggression. These data reveal that multiple autism genes converge to regulate the VMHvl-arcuate AgRP/NPY glutamatergic synapse. The hypothalamic circuitry implicated by these data suggest impaired excitation of AgRP/NPY feedback inhibitory neurons may explain the increased aggression behavior found in genetic forms of autism. One Sentence SummaryA feedback circuit in the hypothalamus that inhibits aggression is impaired by converging autism genetic defects.

neuroscience↗

bioRxiv2022

A thalamo-parietal cortex circuit is critical for place-action coordination

The anterior and lateral thalamus (ALT) contains head direction cells that signal the directional orientation of an animal within an environment. ALT has direct and indirect connections with the parietal cortex (PC), an area hypothesized to play a role in coordinating viewer-dependent and viewer-independent spatial reference frames. This coordination between reference frames would allow an individual to translate movements toward a desired location from memory. Thus ALT-PC functional connectivity would be critical for moving toward remembered locations. This hypothesis was tested with a place-action task that requires associating an appropriate action (left or right turn) with a spatial location. There are four arms, each offset by 90 degrees, positioned around a central starting point. A trial begins in the central starting point. After exiting a pseudorandomly selected arm, the rat had to displace the correct object covering one of two (left versus right) feeding stations to receive a reward. For a pair of arms facing opposite directions, the reward was located on the left, and for the other pair, the reward was located on the right. Thus, each reward location had a different combination of allocentric location and egocentric action. Removal of an object was scored as correct or incorrect. Trials in which the rat did not displace any objects were scored as no response trials. After an object was removed, the rat returned to the center starting position and the maze was reset for the next trial. To investigate the role of the ALT-PC network, muscimol inactivation infusions targeted bilateral PC, bilateral ALT, or the ALT-PC network. Muscimol sessions were counterbalanced and compared to saline sessions within the same animal. All inactivations resulted in decreased accuracy. Only bilateral PC inactivations resulted in increased no response trials, increased errors, and longer latency responses on the remaining trials. Thus, the ALT-PC network is critical for linking an action with a spatial location for successful navigation.

neuroscience↗

bioRxiv2022

Putting on academic armor: How Black physicians and trainees take stances to make racism visible amidst publishing constraints

IntroductionWhile there are a growing number of empirical studies of Black physicians and trainees experiences of racism, there are still few accounts from a first-person perspective. These personal commentaries or editorials require taking a delicate stance, balancing the professional, social, and individual. Black authors in the medical publishing space, who already experience microaggressions and racial trauma in their work spaces, must put on their academic armor to further experience them in publishing spaces. Medicine and medical education interpellate each of us, including the authors and readers of this piece, as a particular kind of subject. Beginning with a narrative grounding in the authors personal stances, this study seeks to understand the stances Black physicians and trainees take as they share their personal experiences of racism while protecting the institution of medicine from racisms irreparable harms. MethodsThe authors searched four databases identifying 29 articles authored by Black physicians and trainees describing their experiences. During initial analysis, the authors identified three sets of discursive strategies: identification (introducing and tracking themselves throughout the article); intertextuality (dialoguing and drawing on other "texts" beyond the focus of the articles); and space-time (social constructions of space and time linked to a network of social practices). The authors used a Google form to capture these strategies and direct quotes from the articles. Throughout the study, the authors reflected on their own stances in relation to the experience of conducting the study and its findings. ResultsAuthors engaged in stance taking, which aligned with the concept of donning academic armor, by evaluating and positioning themselves with respect to racism and the norms of academic discourse in response to ongoing conversations both within medicine and in the broader US culture. They did this by (a) positioning themselves as being Black and, therefore, qualified to notice and name personal racist experiences while also aligning themselves with the reader through shared professional experiences and goals, (b) intertextual connections to other related events, people, and institutions that they-and their readers-value and (c) aligning themselves with a hoped-for future rather than a racist present. DiscussionBecause the discourses of medicine and medical publishing interpellate Black authors as Others they must carefully consider the stances they take, particularly when naming racism. The academic armor they put on must not only be able to defend them from attack, but help them slip unseen through institutional bodies replete with mechanisms to eject them. In addition to analyzing their own personal stance, authors leave readers with thought provoking questions regarding this armor as they return to narrative grounding.

scientific communication and education↗

bioRxiv2022

Attenuated humoral responses in HIV infection after SARS-CoV-2 vaccination are linked to global B cell defects and cellular immune profiles

People living with HIV (PLWH) on suppressive antiretroviral therapy (ART) can have residual immune dysfunction and often display poorer responses to vaccination. We assessed in a cohort of PLWH (n=110) and HIV negative controls (n=64) the humoral and spike-specific B-cell responses following 1, 2 or 3 SARS-CoV-2 vaccine doses. PLWH had significantly lower neutralizing antibody (nAb) titers than HIV-negative controls at all studied timepoints. Moreover, their neutralization breadth was reduced with fewer individuals developing a neutralizing response against the Omicron variant (BA.1) relative to controls. We also observed a delayed development of neutralization in PLWH that was underpinned by a reduced frequency of spike-specific memory B cells (MBCs) and pronounced B cell dysfunction. Improved neutralization breadth was seen after the third vaccine dose in PLWH but lower nAb responses persisted and were associated with global, but not spike-specific, MBC dysfunction. In contrast to the inferior antibody responses, SARS-CoV-2 vaccination induced robust T cell responses that cross-recognized variants in PLWH. Strikingly, a subset of PLWH with low or absent neutralization had detectable functional T cell responses. These individuals had reduced numbers of circulating T follicular helper cells and an enriched population of CXCR3+CD127+CD8+ T cells after two doses of SARS-CoV-2 vaccination, which may compensate for sub-optimal serological responses in the event of infection. Therefore, normalisation of B cell homeostasis could improve serological responses to vaccines in PLWH and evaluating T cell immunity could provide a more comprehensive immune status profile in these individuals and others with B cell imbalances.

immunology↗

bioRxiv2022

Cardiac muscle-restricted partial loss of Nos1ap expression has limited impact on electro- and echo-cardiographic features

Genome-wide association studies (GWAS) of QT interval variation have identified common noncoding variants at the NOS1AP gene as the most common genetic regulators of trait variation in the general population. Invoking a cis-regulatory mechanistic hypothesis, we have reported identification of a functional enhancer variant underlying the GWAS signal that influenced human cardiac NOS1AP expression. Functional studies based on in vitro overexpression in murine cardiomyocytes and ex vivo knockdown in zebrafish embryonic hearts, by us and others, have demonstrated that NOS1AP expression levels can alter cellular electrophysiology. Here, to explore the role of NOS1AP in cardiac electrophysiology at an organismal level, we generated and characterized constitutive and heart muscle-restricted Nos1ap knockout mice to assess whether NOS1AP disruption alters the QT interval in vivo. Constitutive loss of Nos1ap led to genetic background-dependent variable lethality at or right before birth. Heart muscle-restricted Nos1ap knockouts generated using cardiac specific alpha-myosin heavy chain promoter-driven tamoxifen-inducible Cre resulted in tissue-level Nos1ap expression reduced by half. This partial loss of expression had no detectable effect on the QT interval, but led to a small yet significant reduction in the QRS interval. Given that challenges associated with defining the end of T wave on murine electrocardiogram can limit identification of subtle effects on QT interval, and that common noncoding NOS1AP variants are also associated with QRS interval, our findings support the role of NOS1AP in regulation of the cardiac electrical cycle.

genetics↗

bioRxiv2022

A critical role for touch neurons in a skin-brain pathway for stress resilience

Social touch can act as a stress buffer, reducing behavioral and physiological responses to stressful scenarios. However, skin-brain touch pathways that promote stress resilience remain unknown. Here, we show that mice with an early life genetic ablation of Mrgprb4-lineage touch neurons display stress vulnerability behaviors in adulthood. Chemogenetic activation of these touch neurons reduced corticosterone levels under mild acute stress conditions. In addition, whole-brain c-Fos activity mapping while chemogenetically turning on these neurons uncovered differential neural activity patterns in brain areas relevant to somatosensation, reward, and affect. To gain mechanistic insight into this skin-brain touch pathway for stress susceptibility, we used multi-circuit neurophysiological recordings across seven brain regions at baseline and after stress in mice that had Mrgprb4-lineage touch neurons ablated in early life. Interestingly, the Mrgprb4-lineage neuron-ablated mice have alterations in local field potential phase directionality and power in the theta frequencies in mesolimbic reward regions, which may underlie our observed stress susceptibility phenotype. Together, these studies revealed that sensory neurons in the skin engage networks across the brain to promote stress resilience.

neuroscience↗

bioRxiv2022

The effects of environmental heterogeneity within cities on the evolution of clines

O_LIThere is increasing evidence that environmental change associated with urbanization can drive rapid adaptation. However, most studies of urban adaptation have focused on coarse urban vs. rural comparisons or sampled along a single urban-rural environmental gradient, thereby ignoring the role that within-city environmental heterogeneity might play in adaptation to urban environments. C_LIO_LIIn this study, we examined fine-scale variation in the presence of HCN--a potent anti-herbivore defense--and its two underlying genes (Ac and Li) between park green spaces and surrounding suburban habitats for five city parks in the Greater Toronto Area. C_LIO_LIWe show that fine-scale urbanization has driven the formation of micro-clines in HCN on a scale of < 2 km, though the presence and strength of micro-clines varied across parks. Interestingly, these micro-clines were driven by lower HCN frequencies inside park green spaces, and are therefore in the opposite direction to that predicted based on previously described patterns of HCN frequency change along urban-rural gradients. C_LIO_LISynthesis: These results suggest larger scale, adaptive urban-rural clines occur across a complex matrix of environmental heterogeneity within cities that drives fine-scale adaptive microclines of varying strengths and directions. C_LI

evolutionary biology↗

bioRxiv2022

High neutralizing antibody levels against SARS-CoV-2 Omicron after UB-612 booster vaccination

The highly transmissible Omicron variant has caused high rates of breakthrough infections among vaccinated and convalescent individuals. Here, we demonstrate that a booster dose of UB-612 vaccine candidate delivered 7-9 months after primary vaccination increases neutralizing antibody levels by 131-, 61- and 49-fold against ancestral SARS-CoV-2, Omicron BA.1 and BA.2 variants, respectively. Based on the RBD protein binding antibody responses, we estimated a [~]95% efficacy against symptomatic COVID-19 caused by the ancestral strain after a UB-612 booster. Our results support UB-612 vaccine as a potent booster against current and emerging SARS-CoV-2 variants.

microbiology↗

bioRxiv2022

Urbanization and green corridors influence reproductive success and pollinators of common milkweed

Urbanization exerts many pressures on species, yet little is known about how these pressures impact species interactions. Studies of urban plant-pollinator systems provide mounting evidence that urbanization impairs pollinator movement in fragmented urban landscapes, yet the consequences for pollinator-mediated plant reproduction remains unclear. In non-urban areas, habitat corridors can facilitate the movement of organisms including pollinators, but whether these corridors facilitate plant-pollinator interactions in urban areas remains understudied. To examine how urban environments and green corridors influence plant-pollinator interactions, we measured reproductive success in the native plant common milkweed (Asclepias syriaca), and the community structure of its pollinators, for two years along two urban-rural transects in the Greater Toronto Area, Canada, one of which followed a green corridor. We found that urbanization decreased male fitness (i.e., pollen removal), increased fruit set (i.e., mean no. of follicles per inflorescence), and inconsistently affected female fitness (i.e., no. of follicles) in A. syriaca. Urbanization simultaneously decreased pollinator abundance but increased pollinator richness. Proximity to a green corridor inconsistently affected male fitness but increased reproductive effort (i.e., no. of inflorescences) in A. syriaca, while pollinator diversity and richness was lower in corridors. Notably, there were no consistent relationships between pollinator community structure and reproductive success in A. syriaca in both the presence, and absence, of a green corridor. These results demonstrate the complexity with which urbanization, green corridors, and pollinator communities can shape the reproductive investment and fitness of native plant populations.

ecology↗

bioRxiv2022

Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOC) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOC. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses against VOC, as compared to non-human primates or humans, suggesting caution should be exercised when interpreting data for this animal model.

immunology↗

bioRxiv2021

Mutational robustness changes during long-term adaptation in laboratory budding yeast populations

As an adapting population traverses the fitness landscape, its local neighborhood (i.e., the collection of fitness effects of single-step mutations) can change shape because of interactions with mutations acquired during evolution. These changes to the distribution of fitness effects can affect both the rate of adaptation and the accumulation of deleterious mutations. However, while numerous models of fitness landscapes have been proposed in the literature, empirical data on how this distribution changes during evolution remains limited. In this study, we directly measure how the fitness landscape neighborhood changes during laboratory adaptation. Using a barcode-based mutagenesis system, we measure the fitness effects of 91 specific gene disruption mutations in genetic backgrounds spanning 8,000-10,000 generations of evolution in two constant environments. We find that the mean of the distribution of fitness effects decreases in one environment, indicating a reduction in mutational robustness, but does not change in the other. We show that these distribution-level patterns result from biases in variable patterns of epistasis at the level of individual mutations, including fitness-correlated and idiosyncratic epistasis.

evolutionary biology↗

bioRxiv2021

Meta Analysis of the Ralstonia solanacearum species complex (RSSC) based on comparative evolutionary genomics and reverse ecology

Ralstonia solanacearum species complex (RSSC) strains are bacteria that colonize plant xylem and cause vascular wilt diseases. However, individual strains vary in host range, optimal disease temperatures, and physiological traits. To increase our understanding of the evolution, diversity, and biology of the RSSC, we performed a meta-analysis of 100 representative RSSC genomes. These 100 RSSC genomes contain 4,940 genes on average, and a pangenome analysis found that there are 3,262 genes in the core genome ([~]60% of the mean RSSC genome) with 13,128 genes in the extensive flexible genome. Although a core genome phylogenetic tree and a genome similarity matrix aligned with the previously named species (R. solanacearum, R. pseudosolanacearum, R. syzygii) and phylotypes (I-IV), these analyses also highlighted an unrecognized sub-clade of phylotype II. Additionally, we identified differences between phylotypes with respect to gene content and recombination rate, and we delineated population clusters based on the extent of horizontal gene transfer. Multiple analyses indicate that phylotype II is the most diverse phylotype, and it may thus represent the ancestral group of the RSSC. Additionally, we also used our genome-based framework to test whether the RSSC sequence variant (sequevar) taxonomy is a robust method to define within-species relationships of strains. The sequevar taxonomy is based on alignments of a single conserved gene (egl). Although sequevars in phylotype II describe monophyletic groups, the sequevar system breaks down in the highly recombinogenic phylotype I, which highlights the need for an improved cost-effective method for genotyping strains in phylotype I. Finally, we enabled quick and precise genome-based identification of newly sequenced Ralstonia strains by assigning Life Identification Numbers (LINs) to the 100 strains and by circumscribing the RSSC and its sub-groups in the LINbase Web service. IMPACT STATEMENTThe Ralstonia solanacearum species complex (RSSC) includes dozens of economically important pathogens of many cultivated and wild plants. The extensive genetic and phenotypic diversity that exists within the RSSC has made it challenging to subdivide this group into meaningful subgroups with relevance to plant disease control and plant biosecurity. This study provides a solid genome-based framework for improved classification and identification of the RSSC by analyzing one hundred representative RSSC genome sequences with a suite of comparative evolutionary genomic tools. The results also lay the foundation for additional in-depth studies to gain further insights into evolution and biology of this heterogeneous complex of destructive plant pathogens. DATA SUMMARYThe authors confirm that all raw data and code and protocols have been provided within the manuscript. All publicly available sequencing data used for analysis have been supplemented with accession numbers to access the data. The assembled genome of strain 19-3PR_UW348 was submitted to NCBI under Bioproject PRJNA775652 Biosample SAMN22612291. This Whole Genome Shotgun project has been deposited at GenBank under the accession JAJMMU000000000. The version described in this paper is version JAJMMU010000000.

genomics↗