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CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR–Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR–Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.

CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.

This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.

Bemisia tabaci, an invasive pest that causes crop damage worldwide, is a highly differentiated species complex, divided into biotypes that have mainly been defined based on mitochondrial DNA sequences. Although endosymbionts can potentially induce population differentiation, specialization and indirect selection on mtDNA, studies have largely ignored these influential passengers in B. tabaci, despite as many as seven bacterial endosymbionts have been identified. Here, we investigate the composition of the whole bacterial community in worldwide populations of B. tabaci, together with host genetic differentiation, focusing on the invasive B and Q biotypes. Among 653 individuals studied, more than 95% of them harbour at least one secondary endosymbiont, and multiple infections are very common. In addition, sequence analyses reveal a very high diversity of facultative endosymbionts in B. tabaci, with some bacterial genus being represented by more than one strain. In the B and Q biotypes, nine different strains of bacteria have been identified. The mtDNA-based phylogeny of B. tabaci also reveals a very high nucleotide diversity that partitions the two ITS clades (B and Q) into six CO1 genetic groups. Each genetic group is in linkage disequilibrium with a specific combination of endosymbionts. All together, our results demonstrate the rapid dynamics of the bacterial endosymbiont-host associations at a small evolutionary scale, questioning the role of endosymbiotic communities in the evolution of the Bemisia tabaci species complex and strengthening the need to develop a metacommunity theory of inherited endosymbionts.

For insects, the prevalence of numerous vertically transmitted viruses can be high in their host populations. These viruses often have few, if any, pathological effects on their hosts, and consequently, many of them can remain unnoticed for long periods, despite their potential role in the evolution of the host phenotype. Some females of Leptopilina boulardi , a solitary parasitoid of Drosophila larvae, are infected by an inherited virus (LbFV) that manipulates the behavior of the wasp by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). This behavioral alteration allows horizontal transmission of the virus within superparasitized Drosophila larvae. Using suppressive subtractive hybridization with infected and uninfected lines, we identified one putative viral sequence. Based on this sequence, we developed a simple PCR test. We tested the correlation between the superparasitism phenotype and PCR amplification of the putative viral marker using several experimental conditions (including horizontal transfers) and several parasitoid genotypes. All of the results revealed that there was a perfect match between the superparasitism phenotype and the amplification profile, which validated use of the molecular marker as a tool to track the presence of the virus and provided the first genomic data for this fascinating virus. The results also show that there was very efficient horizontal and vertical transmission of LbFV, which probably explains its high prevalence in the French populations that we sampled (67 and 70% of infected females). This manipulative virus is likely to play a major role in the ecology and evolution of its parasitoid host.

Summary: Correspondence analysis of codon usage data is a widely used method in sequence analysis, but the variability in amino acid composition between proteins is a confounding factor when one wants to analyse synonymous codon usage variability. A simple and natural way to cope with this problem is to use within-group correspondence analysis. There is, however, no user-friendly implementation of this method available for genomic studies. Our motivation was to provide to the community a Web facility to easily study synonymous codon usage on a subset of data available in public genomic databases. Availability: Availability through the Pôle Bioinformatique Lyonnais (PBIL) Web server at http://pbil.univ-lyon1.fr/datasets/charif04/ with a demo allowing us to reproduce the figure in the present application note. All underlying software is distributed under a GPL licence. Contact: http://pbil.univ-lyon1.fr/members/lobry

Bemisia tabaci, an invasive pest that causes crop damage worldwide, is a highly differentiated species complex, divided into biotypes that have mainly been defined based on mitochondrial DNA sequences. Although endosymbionts can potentially induce population differentiation, specialization and indirect selection on mtDNA, studies have largely ignored these influential passengers in B. tabaci, despite as many as seven bacterial endosymbionts have been identified. Here, we investigate the composition of the whole bacterial community in worldwide populations of B. tabaci, together with host genetic differentiation, focusing on the invasive B and Q biotypes. Among 653 individuals studied, more than 95% of them harbour at least one secondary endosymbiont, and multiple infections are very common. In addition, sequence analyses reveal a very high diversity of facultative endosymbionts in B. tabaci, with some bacterial genus being represented by more than one strain. In the B and Q biotypes, nine different strains of bacteria have been identified. The mtDNA-based phylogeny of B. tabaci also reveals a very high nucleotide diversity that partitions the two ITS clades (B and Q) into six CO1 genetic groups. Each genetic group is in linkage disequilibrium with a specific combination of endosymbionts. All together, our results demonstrate the rapid dynamics of the bacterial endosymbiont-host associations at a small evolutionary scale, questioning the role of endosymbiotic communities in the evolution of the Bemisia tabaci species complex and strengthening the need to develop a metacommunity theory of inherited endosymbionts.

Wolbachia are symbiotic intracellular bacteria, which are classified as reproductive parasites. Although generally facultative, Wolbachia is necessary for Asobara tabida (Hymenoptera), because aposymbiotic females do not produce any offspring. Interestingly, the ovarian phenotype of aposymbiotic females is variable: some females do not produce any eggs, whereas others do produce some eggs, but these are aborted. Here, we show that the ovarian phenotype of aposymbiotic females is highly polymorphic within populations, although dependence remains complete in both cases. We also identified some lines in which aposymbiotic females were able to produce a very few viable offspring, further extending the range of variation observed. These results suggest that various factors actively maintain polymorphism. We demonstrated that Wolbachia is necessary to trigger oogenetic processes, but that the ovarian phenotype was determined by the host only. Phenotypic variation was also correlated with the differential expression of genes controlling iron homeostasis and oxidative stress, which are potentially involved in the evolution of dependence. This suggests that variation in the ovarian phenotype could reflect selection for different levels of compensatory mechanisms in response to Wolbachia infection, and that polymorphism is maintained through selection on different antagonist traits influenced by oxidative stress.

Background Persistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome). Results To study bacteriome immune specificity, we first identified immune-relevant genes of the weevil Sitophilus zeamais by using suppressive subtractive hybridization (SSH) and then analyzed their full-length coding sequences obtained by RACE-PCR experiments. We then measured immune gene expression in the bacteriome, and in the aposymbiotic larvae following S. zeamais primary endosymbiont (SZPE) injection into the hemolymph, in order to consider the questions of bacteriome immune specificity and the insect humoral response to symbionts. We show that larval challenge with the endosymbiont results in a significant induction of antibacterial peptide genes, providing evidence that, outside the bacteriome, SZPE are recognized as microbial intruders by the host. In the bacteriome, gene expression analysis shows the overexpression of one antibacterial peptide from the coleoptericin family and, intriguingly, homologs to genes described as immune modulators (that is, PGRP-LB, Tollip) were also shown to be highly expressed in the bacteriome. Conclusion The current data provide the first description of immune gene expression in the insect bacteriome. Compared with the insect humoral response to SZPE, the bacteriome expresses few genes among those investigated in this work. This local immune gene expression may help to maintain the endosymbiont in the bacteriome and prevent its invasion into insect tissues. Further investigations of the coleoptericin, the PGRP and the Tollip genes should elucidate the role of the host immune system in the maintenance and regulation of endosymbiosis.

Summary: Comparing genetic and physical maps (the so-called Marey map approach) is still the most widely used approach to estimate genome-wide recombination rates. Remarkably, there is no available bioinformatics tool specifically devoted to Marey map approach. Here, we developed such a tool called MareyMap based on GNU R and Tcl/Tk. MareyMap offers a user-friendly graphical interface and includes useful features, such as data cleaning process, sophisticated interpolation methods to estimate local rates, possibility of complex queries, various range of import–export files. Moreover, MareyMap comes with ready-to-use maps for human, Drosophila, Caenorhabditis elegans and Arabidopsis. MareyMap has been made so that it can be easily upgraded with new data and interpolation methods.Availability: http://pbil.univ-lyon1.fr/software/mareymap/Contact: marais@biomserv.univ-lyon1.fr