Thinopyrum intermedium possesses many desirable agronomic traits that make it a valuable genetic source for wheat improvement. The precise identification of individual chromosomes of allohexaploid Th. intermedium is a challenge due to its three sub-genomic constitutions with complex evolutionary ancestries. The non-denaturing fluorescent in situ hybridization (ND-FISH) using tandem-repeat oligos, including Oligo-B11 and Oligo-pDb12H, effectively distinguished the St, J and JS genomes, while Oligo-FISH painting, based on seven oligonucleotide pools derived from collinear regions between barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), was able to identify each linkage group of the Th. intermedium chromosomes. We subsequently established the first karyotype of Th. intermedium with individual chromosome recognition using sequential ND-FISH and Oligo-FISH painting. The chromosome constitutions of 14 wheat–Th. intermedium partial amphiploids and addition lines were characterized. Distinct intergenomic chromosome rearrangements were revealed among Th. intermedium chromosomes in these amphiploids and addition lines. The precisely defined karyotypes of these wheat–Th. intermedium derived lines may be helpful for further study on chromosome evolution, chromatin introgression and wheat breeding programs.

Abstract Cell-free expression systems provide a suite of tools that are used in applications from sensing to biomanufacturing. One of these applications is genetic circuit prototyping, where the lack of cloning is required and a high degree of control over reaction components and conditions enables rapid testing of design candidates. Many studies have shown utility in the approach for characterizing genetic regulation elements, simple genetic circuit motifs, protein variants or metabolic pathways. However, variability in cell-free expression systems is a known challenge, whether between individuals, laboratories, instruments, or batches of materials. While the issue of variability has begun to be quantified and explored, little effort has been put into understanding the implications of this variability. For genetic circuit prototyping, it is unclear when and how significantly variability in reaction activity will impact qualitative assessments of genetic components, e.g. relative activity between promoters. Here, we explore this question by assessing DNA titrations of seven genetic circuits of increasing complexity using reaction conditions that ostensibly follow the same protocol but vary by person, instrument and material batch. Although the raw activities vary widely between the conditions, by normalizing within each circuit across conditions, reasonably consistent qualitative performance emerges for the simpler circuits. For the most complex case involving expression of three proteins, we observe a departure from this qualitative consistency, offering a provisional cautionary line where normal variability may disrupt reliable reuse of prototyping results. Our results also suggest that a previously described closed loop controller circuit may help to mitigate such variability, encouraging further work to design systems that are robust to variability. Graphical Abstract

Abstract Bonytongues (Osteoglossomorpha) constitute an ancient clade of teleost fishes distributed in freshwater habitats throughout the world. The group includes well-known species such as arowanas, featherbacks, pirarucus, and the weakly electric fishes in the family Mormyridae. Their disjunct distribution, extreme morphologies, and electrolocating capabilities (Gymnarchidae and Mormyridae) have attracted much scientific interest, but a comprehensive phylogenetic framework for comparative analysis is missing, especially for the species-rich family Mormyridae. Of particular interest are disparate craniofacial morphologies among mormyrids which might constitute an exceptional model system to study convergent evolution. We present a phylogenomic analysis based on 546 exons of 179 species (out of 260), 28 out of 29 genera, and all six families of extant bonytongues. Based on a recent reassessment of the fossil record of osteoglossomorphs, we inferred dates of divergence among transcontinental clades and the major groups. The estimated ages of divergence among extant taxa (e.g., Osteoglossomorpha, Osteoglossiformes, and Mormyroidea) are older than previous reports, but most of the divergence dates obtained for clades on separate continents are too young to be explained by simple vicariance hypotheses. Biogeographic analysis of mormyrids indicates that their high species diversity in the Congo Basin is a consequence of range reductions of previously widespread ancestors and that the highest diversity of craniofacial morphologies among mormyrids originated in this basin. Special emphasis on a taxon-rich representation for mormyrids revealed pervasive misalignment between our phylogenomic results and mormyrid taxonomy due to repeated instances of convergence for extreme craniofacial morphologies. Estimation of ancestral phenotypes revealed contingent evolution of snout elongation and unique projections from the lower jaw to form the distinctive Schnauzenorgan. Synthesis of comparative analyses suggests that the remarkable craniofacial morphologies of mormyrids evolved convergently due to niche partitioning, likely enabled by interactions between their exclusive morphological and electrosensory adaptations. [Africa; ancestral state estimation; diversity; exon capture; freshwater fishes; Phylogenomics.]

Understanding patterns of diversification, genetic exchange, and pesticide resistance in arthropod disease vectors is necessary for effective population management. With the availability of next-generation sequencing technologies, one of the best approaches for surveying such patterns involves the simultaneous genotyping of many samples for a large number of genetic markers. To this end, the targeting of gene sequences of known function can be a cost-effective strategy. One insect group of substantial health concern are the mosquito taxa that make up the Culex pipiens complex. Members of this complex transmit damaging arboviruses and filariae worms to humans, as well as other pathogens such as avian malaria parasites that are detrimental to birds. Here we describe the development of a targeted, gene-based assay for surveying genetic diversity and population structure in this mosquito complex. To test the utility of this assay, we sequenced samples from several members of the complex, as well as from distinct populations of the relatively under-studied Culex quinquefasciatus . The data generated was then used to examine taxonomic divergence and population clustering between and within these mosquitoes. We also used this data to investigate genetic variants present in our samples that had previously been shown to correlate with insecticide-resistance. Broadly, our gene capture approach successfully enriched the genomic regions of interest, and proved effective for facilitating examinations of taxonomic divergence and geographic clustering within the Cx . pipiens complex. It also allowed us to successfully survey genetic variation associated with insecticide resistance in Culex mosquitoes. This enrichment protocol will be useful for future studies that aim to understand the genetic mechanisms underlying the evolution of these ubiquitous and increasingly damaging disease vectors.

Across herbivorous insect clades, species richness and host-use diversity tend to positively covary. This could be because host-use divergence drives speciation, or because it raises the ecological limits on species richness. To evaluate these hypotheses, we performed phylogenetic path model analyses of the species diversity of Nearctic aphids. Here, we show that variation in the species richness of aphid clades is caused mainly by host-use divergence, whereas variation in speciation rates is caused more by divergence in non-host-related niche variables. Aphid speciation is affected by both the evolution of host and non-host-related niche components, but the former is largely caused by the latter. Thus, our analyses suggest that host-use divergence can both raise the ecological limits on species richness and drive speciation, although in the latter case, host-use divergence tends to be a step along the causal path leading from non-host-related niche evolution to speciation.

To combat future severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles that present randomly arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against epitopes that are conserved and relatively occluded rather than variable, immunodominant, and exposed. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD nanoparticles in mice and macaques and observed stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains, including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants, including Omicrons, and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest that mosaic-8 RBD nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers. , A mosaic approach to protection The COVID-19 pandemic has been ongoing for more than 2 years now, and new variants such as Omicron are less susceptible to the vaccines developed against earlier lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition, there is continued risk of spillovers of other animal sarbecoviruses into humans. There is thus a need for vaccines that will give broader protection. Cohen et al . developed mosaic nanoparticles that display the receptor-binding domains (RBDs) from SARS-CoV-2 and seven other animal sarbecoviruses. Mosaic nanoparticles protected against both SARS-CoV-2 and SARS-CoV challenges in animal models even though the SARS-CoV RBD was not present on the mosaic-8 RBD nanoparticles. By contrast, a homotypic SARS-CoV-2 RBD nanoparticle (presenting only SARS-CoV-2 RBDs) only protected against a SARS-CoV-2 challenge. —VV , A mosaic sarbecovirus nanoparticle protects against SARS-2 and SARS-1, whereas a SARS-2 nanoparticle only protects against SARS-2. , INTRODUCTION Two animal coronaviruses from the severe acute respiratory syndrome (SARS)–like betacoronavirus (sarbecovirus) lineage, SARS coronavirus (SARS-CoV) and SARS-CoV-2, have caused epidemics or pandemics in humans in the past 20 years. SARS-CoV-2 triggered the COVID-19 pandemic that has been ongoing for more than 2 years despite rapid development of effective vaccines. Unfortunately, new SARS-CoV-2 variants, including multiple heavily mutated Omicron variants, have prolonged the COVID-19 pandemic. In addition, the discovery of diverse sarbecoviruses in bats raises the possibility of another coronavirus pandemic. Hence, there is an urgent need to develop vaccines and therapeutics to protect against both SARS-CoV-2 variants and zoonotic sarbecoviruses with the potential to infect humans. RATIONALE To combat future SARS-CoV-2 variants and spillovers of sarbecoviruses threatening global health, we designed nanoparticles that present 60 randomly arranged spike receptor-binding domains (RBDs) derived from the spike trimers of eight different sarbecoviruses (mosaic-8 RBD nanoparticles) to elicit antibodies against conserved and relatively occluded—rather than variable, immunodominant, and exposed—epitopes. The probability of two adjacent RBDs being the same is low for mosaic-8 RBD nanoparticles, a feature chosen to favor interactions with B cells whose bivalent receptors can cross-link between adjacent RBDs to use avidity effects to favor recognition of conserved, but sterically occluded, RBD epitopes. By contrast, nanoparticles that present 60 copies of SARS-CoV-2 RBDs (homotypic RBD nanoparticles) are theoretically more likely to engage B cells with receptors that recognize immunodominant and sterically accessible, but less conserved, RBD epitopes. RESULTS We compared immune responses elicited by mosaic-8 (SARS-CoV-2 RBD plus seven animal sarbecoviruses RBDs) and homotypic (only SARS-CoV-2 RBDs) nanoparticles in mice and macaques and observed stronger responses elicited by mosaic-8 to mismatched (not represented with an RBD on nanoparticles) strains, including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization produced antisera that showed equivalent neutralization of SARS-CoV-2 variants, including Omicron variants, and protected from both SARS-CoV-2 and SARS-CoV challenges in mice and nonhuman primates (NHPs), whereas homotypic SARS-CoV-2 immunization protected from SARS-CoV-2 challenge but not from SARS-CoV challenge in mice. Epitope mapping of polyclonal antisera by using deep mutational scanning of RBDs demonstrated targeting of conserved epitopes after immunization with mosaic-8 RBD nanoparticles, in contrast with targeting of variable epitopes after homotypic SARS-CoV-2 RBD nanoparticle immunization, which supports the hypothesized mechanism by which mosaic RBD nanoparticle immunization can overcome immunodominance effects to direct production of antibodies against conserved RBD epitopes. Given the recent plethora of SARS-CoV-2 variants that may be arising at least in part because of antibody pressure, a relevant concern is whether more conserved RBD epitopes might be subject to substitutions that would render vaccines and/or monoclonal antibodies targeting these regions ineffective. This scenario seems unlikely because RBD regions conserved between sarbecoviruses and SARS-CoV-2 variants are generally involved in contacts with other regions of spike trimer and therefore less likely to tolerate selection-induced substitutions. CONCLUSION Together, these results suggest that mosaic-8 RBD nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers—in particular, highlighting the potential for a mosaic nanoparticle approach to elicit more broadly protective antibody responses than those with homotypic nanoparticle approaches. Mosaic RBD nanoparticle vaccination protects and elicits antibodies against conserved epitopes. Mosaic-8 elicited broader cross-reactive responses than those of homotypic nanoparticles. In a stringent infection model [K18-human angiotensin-converting enzyme 2 (K18-hACE2)], both protected against matched challenge (SARS-CoV-2 Beta), but only mosaic-8 also protected against a mismatch (SARS-CoV). Mosaic-8–immunized NHPs were protected against mismatched SARS-CoV-2 (Delta) and SARS-CoV. Mosaic-8–elicited antibodies predominantly bound conserved epitopes, whereas homotypic-elicited antibodies predominantly bound variable epitopes.

Abstract Animal species differ considerably in their ability to fight off infections. Finding the genetic basis of these differences is not easy, as the immune response is comprised of a complex network of proteins that interact with one another to defend the body against infection. Here, we used population- and comparative genomics to study the evolutionary forces acting on the innate immune system in natural hosts of avian influenza virus (AIV). For this purpose, we used a combination of hybrid capture, next generation sequencing and published genomes to examine genetic diversity, divergence and signatures of selection in 127 innate immune genes at a micro- and macroevolutionary time scale in 26 species of waterfowl. We show across multiple immune pathways (AIV-, toll-like-, and RIG-I like receptors signalling pathways) that genes involved in pathogen detection (i.e. toll-like receptors) and direct pathogen inhibition (i.e. antimicrobial peptides and interferon-stimulated genes), as well as host proteins targeted by viral antagonist proteins (i.e. MAVS) are more likely to be polymorphic, genetically divergent and under positive selection than other innate immune genes. Our results demonstrate that selective forces vary across innate immune signalling pathways in waterfowl, and we present candidate genes that may contribute to differences in susceptibility and resistance to infectious diseases in wild birds, and that may be manipulated by viruses. Our findings improve our understanding of the interplay between host genetics and pathogens, and offer the opportunity for new insights into pathogenesis and potential drug targets.