The C4 Urochloa spp (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease amount of land required per unit production and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We therefore set out to look for such alleles in a diverse Urochloa spp and Megathyrsus maximus accessions from the genebank collection held at CIAT.We studied allelic diversity of 20 target genes (11 for digestibility, 9 for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNAseq and then bait-capture DNA-seq to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence.We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified 7 putative LOF alleles with high confidence, including ones in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme.We demonstrated a novel, effective approach to allele discovery in diverse accessions using a draft reference genome from a single species. We used this to find gene variants in a collection of tropical grasses that could help reduce the environmental impact of cattle production.

ChAdOx1 nCoV-19/AZD1222 is an approved adenovirus-based vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) currently being deployed globally. Previous studies in rhesus macaques revealed that intramuscular vaccination with ChAdOx1 nCoV-19/AZD1222 provided protection against pneumonia but did not reduce shedding of SARS-CoV-2 from the upper respiratory tract. Here, we investigated whether intranasally administered ChAdOx1 nCoV-19 reduces detection of virus in nasal swabs after challenging vaccinated macaques and hamsters with SARS-CoV-2 carrying a D614G mutation in the spike protein. Viral loads in swabs obtained from intranasally vaccinated hamsters were decreased compared to control hamsters, and no viral RNA or infectious virus was found in lung tissue after a direct challenge or after direct contact with infected hamsters. Intranasal vaccination of rhesus macaques resulted in reduced virus concentrations in nasal swabs and a reduction in viral loads in bronchoalveolar lavage and lower respiratory tract tissue. Intranasal vaccination with ChAdOx1 nCoV-19/AZD1222 reduced virus concentrations in nasal swabs in two different SARS-CoV-2 animal models, warranting further investigation as a potential vaccination route for COVID-19 vaccines.

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled in the latter half of the twentieth century, new virulent strains of Pgt, such as Ug99, have recently evolved1,2. These strains have caused notable losses worldwide and their continued spread threatens global wheat production. Breeding for disease resistance provides the most cost-effective control of wheat rust diseases3. A number of rust resistance genes have been characterized in wheat and most encode immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class4, which recognize pathogen effector proteins known as avirulence (Avr) proteins5. However, only two Avr genes have been identified in Pgt so far, AvrSr35 and AvrSr50 (refs. 6,7), and none in other cereal rusts8,9. The Sr27 resistance gene was first identified in a wheat line carrying an introgression of the 3R chromosome from Imperial rye10. Although not deployed widely in wheat, Sr27 is widespread in the artificial crop species Triticosecale (triticale), which is a wheat–rye hybrid and is a host for Pgt11,12. Sr27 is effective against Ug99 (ref. 13) and other recent Pgt strains14,15. Here, we identify both the Sr27 gene in wheat and the corresponding AvrSr27 gene in Pgt and show that virulence to Sr27 can arise experimentally and in the field through deletion mutations, copy number variation and expression level polymorphisms at the AvrSr27 locus.

Choosing among types of genomic markers to be used in a phylogenomic study can have a major influence on the cost, design, and results of a study. Yet few attempts have been made to compare categories of next-generation sequence markers limiting our ability to compare the suitability of these different genomic fragment types. Here we explore properties of different genomic markers to find if they vary in the accuracy of component phylogenetic trees and to clarify the causes of conflict obtained from different datasets or inference methods. As a test case, we explore the causes of discordance between phylogenetic hypotheses obtained using a novel dataset of ultraconserved elements (UCEs) and a recently published exon dataset of the cichlid tribe Heroini. Resolving relationships among heroine cichlids has historically been difficult, and the processes of colonization and diversification in Middle America and the Greater Antilles are not yet well understood. Despite differences in informativeness and levels of gene tree discordance between UCEs and exons, the resulting phylogenomic hypotheses generally agree on most relationships. The independent datasets disagreed in areas with low phylogenetic signal that were overwhelmed by incomplete lineage sorting (ILS) and non-phylogenetic signals. For UCEs, high levels of ILS were found to be the major cause of gene tree discordance, whereas, for exons, non-phylogenetic signal is most likely caused by a reduced number of highly informative loci. This paucity of informative loci in exons might be due to heterogeneous substitution rates that are problematic to model (i.e., computationally restrictive) resulting in systematic errors that UCEs (being less informative individually but more uniform) are less prone to. These results generally demonstrate the robustness of phylogenomic methods to accommodate genomic markers with different biological and phylogenetic properties. However, we identify common and unique pitfalls of different categories of genomic fragments when inferring enigmatic phylogenetic relationships.

The rails (Family Rallidae) are the most diverse and widespread group in the Gruiformes. Their extensive fossil history, global geographic distribution, and tendency to rapidly evolve flightless species on islands make them an attractive subject of evolutionary studies, but the rarity of modern museum specimens of so many rail species has, until recently, limited the scope of molecular phylogenetics studies. As a result, the classification of rails remains one of the most unsettled among major bird radiations. We extracted DNA from museum specimens of 82 species, including 27 from study skins collected as long ago as 1875, and generated nucleotide sequences from thousands of homologous ultra-conserved elements (UCEs). Our phylogenetic analyses, using both concatenation and multispecies coalescent approaches, resulted in well-supported and highly congruent phylogenies that resolve the major lineages of rails and reveal several currently recognized genera to be polyphyletic. A fossil-calibrated time tree is well-resolved and supports the hypothesis that rails split into 2 major lineages (subfamilies Himantornithinae and Rallinae) ~34 mya, but clade age estimates have wide confidence intervals. Our results, combined with results of other recently published phylogenomics studies of rails and other Gruiformes, form the basis for a proposed classification of the Rallidae that recognizes 40 genera in 9 tribes.• Rails are a diverse, globally distributed group of birds that are of great interest to ornithologists that study fossil birds and the evolution of flightless species on islands. But because so many rail species are rare or extinct, studying rail evolution using genomic data requires that DNA be obtained from very old museum specimens.• We extracted DNA from museum specimens, including study skins of rare and extinct species collected as long ago as 1875, and generated DNA sequences from thousands of genes.• The DNA-based phylogeny indicates that rails underwent an initial split into 2 groups ~34 mya, and that 9 major extant rail lineages have evolved since then.• Our genetic analyses clarify the relationships of some enigmatic species that have not been studied with DNA before and are the basis for a revised taxonomic classification of rails.

The sustainability of many crops is hindered by the lack of genomic resources and a poor understanding of natural genetic diversity. Particularly, application of modern breeding requires high-density linkage maps that are integrated into a highly contiguous reference genome. Here, we present a rapid method for deriving haplotypes and developing linkage maps, and its application to Mentha suaveolens, one of the diploid progenitors of cultivated mints. Using sequence-capture via DNA hybridization to target single nucleotide polymorphisms (SNPs), we successfully genotyped ∼5,000 SNPs within the genome of > 400 individuals derived from a self cross. After stringent quality control, and identification of non-redundant SNPs, 1,919 informative SNPs were retained for linkage map construction. The resulting linkage map defined a total genetic space of 942.17 cM divided among 12 linkage groups, ranging from 56.32 to 122.61 cM in length. The linkage map is in good agreement with pseudomolecules from our preliminary genome assembly, proving this resource effective for the correction and validation of the reference genome. We discuss the advantages of this method for the rapid creation of linkage maps.

Reports of P. vivax infections among Duffy-negative hosts have accumulated throughout sub-Saharan Africa. Despite this growing body of evidence, no nationally representative epidemiological surveys of P. vivax in sub-Saharan Africa have been performed. To overcome this gap in knowledge, we screened over 17,000 adults in the Democratic Republic of the Congo (DRC) for P. vivax using samples from the 2013-2014 Demographic Health Survey. Overall, we found a 2.97% (95% CI: 2.28%, 3.65%) prevalence of P. vivax infections across the DRC. Infections were associated with few risk-factors and demonstrated a relatively flat distribution of prevalence across space with focal regions of relatively higher prevalence in the north and northeast. Mitochondrial genomes suggested that DRC P. vivax were distinct from circulating non-human ape strains and an ancestral European P. vivax strain, and instead may be part of a separate contemporary clade. Our findings suggest P. vivax is diffusely spread across the DRC at a low prevalence, which may be associated with long-term carriage of low parasitemia, frequent relapses, or a general pool of infections with limited forward propagation.

Technological advances in genome sequencing have improved our ability to catalog genomic variation and led to an expansion of the scope and scale of genetic studies. Yet, for agronomically important plant pathogens such as the downy-mildews the scale of genetic studies remains limited. This is, in part, due to the difficulties associated with maintaining obligate pathogens, and the logistical constraints involved in the genotyping of these species. To study the genetic variation of two Pseudoperonospora species (P. cubensis and P. humuli), we describe a targeted enrichment (TE) protocol able to genotype isolates using less than 50 ng of mixed pathogen and plant DNA for library preparation. We enriched 830 target genes across 128 samples and identified 2,514 high-quality SNP variants. We detected significant genetic differentiation (p=0.01) between P. cubensis subpopulations from Cucurbita moschata (clade I) and Cucumis sativus (clade II) in Michigan. No evidence of location-based differentiation was detected within the P. cubensis (clade II) subpopulation. A significant effect of location on the genetic variation of the P. humuli subpopulation was detected in the state (p=0.01). Mantel tests found evidence that the genetic distance among P. humuli samples was associated with the physical distance of the hop yards from which the samples were collected (p=0.005). The differences in the distribution of genetic variation of the P. humuli and P. cubensis subpopulations of Michigan suggest differences in the dispersal of these two species. Our TE protocol provides an additional tool for genotyping obligate pathogens and the execution of new genetic studies.

It is established that for CRISPR-Cas9 applications guide RNAs with 17–20 bp long spacer sequences are optimal for accurate target binding and cleavage. In this work we perform cell-free CRISPRa (CRISPR activation) and CRISPRi (CRISPR inhibition) experiments to demonstrate the existence of a complex dependence of CRISPR-Cas9 binding as a function of the spacer length and complementarity. Our results show that significantly truncated or mismatched spacer sequences can form stronger guide–target bonds than the conventional 17–20 bp long spacers. To explain this phenomenon, we take into consideration previous structural and single-molecule CRISPR-Cas9 experiments and develop a novel thermodynamic model of CRISPR-Cas9 target recognition.