SummaryNicotiana section Suaveolentes is an almost all‐Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4–5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long‐term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.

Background The allotetraploid tobacco species Nicotiana benthamiana native to Australia has become a popular host for recombinant protein production. Although its usage grows every year, little is known on this plant’s genomic and transcriptomic features. Most N. benthamiana accessions currently used in research lack proper documentation of their breeding history and provenance. One of these, the glycoengineered N. benthamiana line ΔXT/FT is increasingly used for the production of biopharmaceutical proteins. Results Based on an existing draft assembly of the N. benthamiana genome we predict 50,516 protein –encoding genes (62,216 transcripts) supported by expression data derived from 2.35 billion mRNA-seq reads. Using single-copy core genes we show high completeness of the predicted gene set. We functionally annotate more than two thirds of the gene set through sequence homology to genes from other Nicotiana species. We demonstrate that the expression profiles from leaf tissue of ΔXT/FT and its wild type progenitor only show minimal differences. We identify the transgene insertion sites in ΔXT/FT and show that one of the transgenes was inserted inside another predicted gene that most likely lost its function upon insertion. Based on publicly available mRNA-seq data, we confirm that the N. benthamiana accessions used by different research institutions most likely derive from a single source. Conclusions This work provides gene annotation of the N. benthamiana genome, a genomic and transcriptomic characterization of a transgenic N. benthamiana line in comparison to its wild-type progenitor, and sheds light onto the relatedness of N. benthamiana accessions that are used in laboratories around the world. Electronic supplementary material The online version of this article (10.1186/s12864-019-5960-2) contains supplementary material, which is available to authorized users.

Polyploidization is a well-known speciation and adaptation mechanism. Traces of former polyploidization events were discovered within many genomes, and especially in plants. Allopolyploidization by interspecific hybridization between two species is common. Among hybrid plants, many are domesticated species of agricultural interest and many of their genomes and of their presumptive parents have been sequenced. Hybrid genomes remain challenging to analyse because of the presence of multiple subgenomes. The genomes of hybrids often undergo rearrangement and degradation over time. Based on 10 hybrid plant genomes from six different genera, with hybridization dating from 10,000 to 5 million years ago, we assessed subgenome degradation, subgenomic intermixing and biased subgenome fractionation. The restructuring of hybrid genomes does not proceed proportionally with the age of the hybrid. The oldest hybrids in our data set display completely different fates: whereas the subgenomes of the tobacco plant Nicotiana benthamiana are in an advanced stage of degradation, the subgenomes of quinoa (Chenopodium quinoa) are exceptionally well conserved by structure and sequence. We observed statistically significant biased subgenome fractionation in seven out of 10 hybrids, which had different ages and subgenomic intermixing levels. Hence, we conclude that no correlation exists between biased fractionation and subgenome intermixing. Lastly, domestication may encourage or hinder subgenome intermixing, depending on the evolutionary context. In summary, comparative analysis of hybrid genomes and their presumptive parents allowed us to determine commonalities and differences between their evolutionary fates. In order to facilitate the future analysis of further hybrid genomes, we automated the analysis steps within MANTICORE, which is publicly available at https://github.com/MatteoSchiavinato/ manticore.git.

The emergence of resistance against common antibiotics in the gut microbiota is a major issue for both human and livestock health. This highlights the need for understanding the impact of such application on the reservoir of antibiotic resistance genes in poultry gut and devising means to circumvent the potential resistome expansion. Phytogenic feed additives (PFAs) are potential natural alternative to antibiotic to improve animal health and performance, supposedly via positively affecting the gut microbial ecosystem, but there is little systematic information available. In this time-course study, we applied a shotgun meta-transcriptomics approach to investigate the impact of a PFA product as well as the commonly used antibiotic, zinc bacitracin either at AGP concentration or therapeutic concentration on the gut microbiome and resistome of broiler chickens raised for 35 days. Over the course of the trial, PFA treatments increased the abundance of Firmicutes such as Lactobacillus and resulted in a lower abundance of Escherichia, while the latter group increased significantly in the feces of chickens that received either AGP or AB doses of bacitracin. Tetracycline resistance and aminoglycoside resistance were the predominant antibiotic resistance gene (ARG) classes found, regardless of the treatment. PFA application resulted in a decrease in abundance of ARGs compared to those in the control group and other antibiotic treatment groups. In summary, the findings from this study demonstrate the potential of phytogenic feed additives could be an alternative to antibiotics in poultry farming, with the added benefit of counteracting antimicrobial resistance development.

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster‐growing non‐producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long‐term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed‐batch‐like microbioreactor cultivations are conducted under production conditions. The easy‐to‐produce green fluorescent protein (GFP) and a challenging antigen‐binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole‐genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long‐term cultivation under production conditions. Mutations leading to non‐producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase‐based BL21Q expression system remains stable in the production of GFP in long‐term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives have positive effects on long‐term stability. The results indicate that adaptive evolution carried out with genome‐integrated E. coli expression systems in microtiter cultivations under industrial‐relevant production conditions is an efficient strain development tool for production hosts.