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Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples. We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S meta-barcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart). The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.

G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2α (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2 poisons at transcribed regions bearing G4 structures.

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host's inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.

Among crop fruit trees, the apricot (Prunus armeniaca) provides an excellent model to study divergence and adaptation processes. Here, we obtain nearly 600 Armeniaca apricot genomes and four high-quality assemblies anchored on genetic maps. Chinese and European apricots form two differentiated gene pools with high genetic diversity, resulting from independent domestication events from distinct wild Central Asian populations, and with subsequent gene flow. A relatively low proportion of the genome is affected by selection. Different genomic regions show footprints of selection in European and Chinese cultivated apricots, despite convergent phenotypic traits, with predicted functions in both groups involved in the perennial life cycle, fruit quality and disease resistance. Selection footprints appear more abundant in European apricots, with a hotspot on chromosome 4, while admixture is more pervasive in Chinese cultivated apricots. Our study provides clues to the biology of selected traits and targets for fruit tree research and breeding.

Arbuscular mycorrhizal (AM) fungi are involved in one of the most widespread plant–fungus interactions. A number of studies on the population dynamics of AM fungi have used mitochondrial (mt) DNA sequences, and yet mt AM fungus genomes are poorly known. To date, four mt genomes of three species of AM fungi are available, among which are two from Rhizophagus irregularis. In order to study intra- and interstrain mt genome variability of R. irregularis, we sequenced and de novo assembled four additional mt genomes of this species. We used 454 pyrosequencing and Illumina technologies to directly sequence mt genomes from total genomic DNA.The mt genomes are unique within each strain. Interstrain divergences in genome size, as a result of highly polymorphic intergenic and intronic sequences, were observed. The polymorphism is brought about by three types of variability generating element (VGE): homing endonucleases, DNA polymerase domain-containing open reading frames and small inverted repeats. Based on VGE positioning, mt sequences and nuclear markers, two subclades of R. irregularis were characterized. The discovery of VGEs highlights the great intraspecific plasticity of the R. irregularis mt genome. VGEs allow the design of powerful mt markers for the typing and monitoring of R. irregularis strains in genetic and population studies.

Microbial interactions occurring on and around seeds are especially important for plant fitness since seed-borne microorganisms are the initial source of inoculum for the plant microbiota. In this study, we analyze structural and functional changes occurring within the plant microbiota at these early stages of the plant cycle, namely germination and emergence. To this purpose, we performed shotgun DNA sequencing of microbial assemblages associated to seeds, germinating seeds and seedlings of two plant species: bean and radish. We observed an enrichment of and during emergence and a set of functional traits linked to copiotrophy that could be responsible for this selection as a result of an increase of nutrient availability after germination. Representative bacterial isolates of taxa that are selected in seedlings showed indeed faster bacterial growth rate in comparison to seed-associated bacteria isolates. Finally, binning of metagenomics contigs results in the reconstruction of population genomes of the major bacterial taxa associated to the samples. Together, our results demonstrate that, although seed microbiota varied across plant species, nutrient availability during germination elicits changes of the composition of microbial communities by potentially selecting microbial groups with functional traits linked to copiotrophy. The data presented here represents the first attempts to empirically assess changes in the microbial community during plant emergence and moves us toward a more holistic understanding of the plant microbiome.

Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct repertoires for IgM, IgD and IgT, respectively, with a few amplified μ and τ junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM(+) and IgT(+) spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences.

Cacao ( Theobroma cacao L.) is an important economic crop, yet studies of its domestication history and early uses are limited. Traditionally, cacao is thought to have been first domesticated in Mesoamerica. However, genomic research shows that T. cacao ’s greatest diversity is in the upper Amazon region of northwest South America, pointing to this region as its centre of origin. Here, we report cacao use identified by three independent lines of archaeological evidence—cacao starch grains, absorbed theobromine residues and ancient DNA—dating from approximately 5,300 years ago recovered from the Santa Ana-La Florida (SALF) site in southeast Ecuador. To our knowledge, these findings constitute the earliest evidence of T. cacao use in the Americas and the first unequivocal archaeological example of its pre-Columbian use in South America. They also reveal the upper Amazon region as the oldest centre of cacao domestication yet identified. Evidence from starch grains, theobromine residues and ancient DNA demonstrate cacao use in the upper Amazon circa 5,300 years ago. This is earlier than previous evidence for cacao domestication in Mesoamerica.

Background Partial endoreplication is a prominent developmental feature and poses a significant challenge for whole genome assembly in orchids. This form of cell cycle results in highly unbalanced cell DNA content, with the highly endoreplicated (P) fraction being overrepresented in sequencing data compared to the non-endoreplicated (F) fraction. Results Here, we report the first genome assembly of Vanilla planifolia into 16 chromosome pairs using axillary buds enriched in non-endoreplicated 2 C-nuclei (55%) as determined by flow cytometry. The assembly was generated using a hybrid approach combining PacBio HiFi sequencing and Omni-C scaffolding generated in this study, together with a GBS-SNP genetic map and Oxford Nanopore Technologies long-read data from the literature. For the first time, we identified P and F regions within reconstructed chromosomes, representing 20.57% and 79.43% of the genome, respectively based on DNA sequencing data from three tissues with varying levels of endoreplicated nuclei. P regions were gene-rich and located at chromosome ends, whereas F regions were SSR-rich and located at central parts of chromosomes. Remarkably, 97.24% of SSRs were found in F regions, predominantly comprising the trinucleotide AAG/CTT motif, which may contribute to the absence of endoreplication in these regions. Protein-encoding genes overrepresented in F regions were associated with negative regulation of flower development, mitotic cycle progression, cell division and histone modification. Conclusions This accurate high-quality chromosome-scale V. planifolia genome assembly provides unprecedented insights into the structural and molecular characteristics of partial endoreplication in Orchids and represents a major step toward the characterization of this complex genome.

Four main evolutionary lineages of A. mellifera have been described including eastern Europe (C) and western and northern Europe (M). Many apiculturists prefer bees from the C lineage due to their docility and high productivity. In France, the routine importation of bees from the C lineage has resulted in the widespread admixture of bees from the M lineage. The haplodiploid nature of the honeybee Apis mellifera, and its small genome size, permits affordable and extensive genomics studies. As a pilot study of a larger project to characterise French honeybee populations, we sequenced 60 drones sampled from two commercial populations managed for the production of honey and royal jelly. Results indicate a C lineage origin, whilst mitochondrial analysis suggests two drones originated from the O lineage. Analysis of heterozygous SNPs identified potential copy number variants near to genes encoding odorant binding proteins and several cytochrome P450 genes. Signatures of selection were detected using the hapFLK haplotype-based method, revealing several regions under putative selection for royal jelly production. The framework developed during this study will be applied to a broader sampling regime, allowing the genetic diversity of French honeybees to be characterised in detail.