Pathogen-mediated selection favours the maintenance of innate immunity gene polymorphism in a widespread wild ungulate.

Toll-like receptors (TLR) play a central role in recognition and host frontline defence against a wide range of pathogens. A number of recent studies have shown that TLR genes (Tlrs) often exhibit large polymorphism in natural populations. Yet, there is little knowledge on how this polymorphism is maintained and how it influences disease susceptibility in the wild. In previous work, we showed that some Tlrs exhibit similarly high levels of genetic diversity as genes of the Major Histocompatibility Complex (MHC), and signatures of contemporary balancing selection in roe deer (Capreolus capreolus), the most abundant cervid species in Europe. Here, we investigated the evolutionary mechanisms by which pathogen-mediated selection could shape this innate immunity genetic diversity by examining the relationships between Tlr (Tlr2, Tlr4 and Tlr5) genotypes (heterozygosity status and presence of specific alleles) and infections with Toxoplasma and Chlamydia, two widespread intracellular pathogens known to cause reproductive failure in ungulates. We showed that Toxoplasma and Chlamydia exposures vary significantly across years and landscape features with few co-infection events detected and that the two pathogens exert antagonistic selection on Tlr2 polymorphism. By contrast, we found limited support for Tlr heterozygote advantage. Our study confirmed the importance of looking beyond Mhc genes in wildlife immunogenetic studies. It also emphasized the necessity to consider multiple pathogen challenges and their spatiotemporal variation to improve our understanding of vertebrate defence evolution against pathogens.

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing.

BACKGROUND: Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3 V4 hypervariable region of the 16S rRNA gene. RESULTS: We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3 V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3 V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3 V4. We compared the performance of the rpoB and V3 V4 markers in an animal ecosystem model, the infective juveniles of the entomopathogenic nematode Steinernema glaseri carrying the symbiotic bacteria Xenorhabdus poinarii. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium X. poinarii. CONCLUSIONS: Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene. This study will be useful to the growing scientific community describing bacterial communities by metabarcoding in diverse ecosystems.

Both candidate gene and neutral genetic diversity correlate with parasite resistance in female Mediterranean mouflon.

BACKGROUND: Parasite infections can have substantial impacts on population dynamics and are accordingly a key challenge for wild population management. Here we studied genetic mechanisms driving parasite resistance in a large herbivore through a comprehensive approach combining measurements of neutral (16 microsatellites) and adaptive (MHC DRB1 exon 2) genetic diversity and two types of gastrointestinal parasites (nematodes and coccidia). RESULTS: While accounting for other extrinsic and intrinsic predictors known to impact parasite load, we show that both neutral genetic diversity and DRB1 are associated with resistance to gastrointestinal nematodes. Intermediate levels of multi-locus heterozygosity maximized nematodes resistance, suggesting that both in- and outbreeding depression might occur in the population. DRB1 heterozygosity and specific alleles effects were detected, suggesting the occurrence of heterozygote advantage, rare-allele effects and/or fluctuating selection. On the contrary, no association was detected between genetic diversity and resistance to coccidia, indicating that different parasite classes are impacted by different genetic drivers. CONCLUSIONS: This study provides important insights for large herbivores and wild sheep pathogen management, and in particular suggests that factors likely to impact genetic diversity and allelic frequencies, including global changes, are also expected to impact parasite resistance.

Empirical assessment of RAD sequencing for interspecific phylogeny.

Next-generation sequencing opened up new possibilities in phylogenetics; however, choosing an appropriate method of sample preparation remains challenging. Here, we demonstrate that restriction-site-associated DNA sequencing (RAD-seq) generates useful data for phylogenomics. Analysis of our RAD library using current bioinformatic and phylogenetic tools produced 400× more sites than our Sanger approach (2,262,825 nt/species), fully resolving relationships between 18 species of ground beetles (divergences up to 17 My). This suggests that RAD-seq is promising to infer phylogeny of eukaryotic species, though potential biases need to be evaluated and new methodologies developed to take full advantage of such data.

Immunogenetic heterogeneity in a widespread ungulate: the European roe deer (Capreolus capreolus).

Understanding how immune genetic variation is shaped by selective and neutral processes in wild populations is of prime importance in both evolutionary biology and epidemiology. The European roe deer (Capreolus capreolus) has considerably expanded its distribution range these last decades, notably by colonizing agricultural landscapes. This range shift is likely to have led to bottlenecks and increased roe deer exposure to a new range of pathogens that until recently predominantly infected humans and domestic fauna. We therefore investigated the historical and contemporary forces that have shaped variability in a panel of genes involved in innate and acquired immunity in roe deer, including Mhc-Drb and genes encoding cytokines or toll-like receptors (TLRs). Together, our results suggest that genetic drift is the main contemporary evolutionary force shaping immunogenetic variation within populations. However, in contrast to the classical view, we found that some innate immune genes involved in micropathogen recognition (e.g. Tlrs) continue to evolve dynamically in roe deer in response to pathogen-mediated positive selection. Most studied Tlrs (Tlr2, Tlr4 and Tlr5) had similarly high levels of amino acid diversity in the three studied populations including one recently established in southwestern France that showed a clear signature of genetic bottleneck. Tlr2 implicated in the recognition of Gram-positive bacteria in domestic ungulates, showed strong evidence of balancing selection. The high immunogenetic variation revealed here implies that roe deer are able to cope with a wide spectrum of pathogens and to respond rapidly to emerging infectious diseases.

Reduced microsatellite heterozygosity does not affect natal dispersal in three contrasting roe deer populations.

Although theoretical studies have predicted a link between individual multilocus heterozygosity and dispersal, few empirical studies have investigated the effect of individual heterozygosity on dispersal propensity or distance. We investigated this link using measures of heterozygosity at 12 putatively neutral microsatellite markers and natal dispersal behaviour in three contrasting populations of European roe deer (Capreolus capreolus), a species displaying pre-saturation condition-dependent natal dispersal. We found no effect of individual heterozygosity on either dispersal propensity or dispersal distance. Average heterozygosity was similar across the three studied populations, but dispersal propensity and distance differed markedly among them. In Aurignac, dispersal propensity and distance were positively related to individual body mass, whereas there was no detectable effect of body mass on dispersal behaviour in Chizé and Trois Fontaines. We suggest that we should expect both dispersal propensity and distance to be greater when heterozygosity is lower only in those species where dispersal behaviour is driven by density-dependent competition for resources.

Species or local environment, what determines the infection of rodents by Toxoplasma gondii?

Toxoplasmosis is largely present in rural areas but its spatial distribution in this environment remains poorly known. In particular, it is unclear if areas of high density of cats, the only hosts excreting Toxoplasma gondii, constitute foci of high prevalence. To improve our understanding of the spatial distribution of T. gondii in rural areas, we performed a serological survey in rodents from two villages in France. We trapped 710 rodents including commensal rats and meadow or forest voles and mice. The presence of T. gondii was examined using PCR, mice inoculation and modified agglutination test for antibodies (MAT). We conducted multivariate and discriminant analyses to identify biological, ecological or spatial variables that could explain T. gondii serology in rodents. We then used a logistic regression to assess the relative influence of each explanatory variable. Overall seroprevalence was 4.1%. Commensal-rats were more infected (12.5%) than non-commensal species (3.7%). However, the major determinant of the risk of infection was the distance to the nearest farm (OR = 0.75 for 100 m), which explained the risk in all species or non-commensal species only. We contrast the role of species characteristics and that of the local environment, and discuss the risk of environmental contamination for humans.

Exploring the potential effects of forest urbanization on the interplay between small mammal communities and their gut microbiota.

Urbanization significantly impacts wild populations, favoring urban dweller species over those that are unable to adapt to rapid changes. These differential adaptative abilities could be mediated by the microbiome, which may modulate the host phenotype rapidly through a high degree of flexibility. Conversely, under anthropic perturbations, the microbiota of some species could be disrupted, resulting in dysbiosis and negative impacts on host fitness. The links between the impact of urbanization on host communities and their gut microbiota (GM) have only been scarcely explored. In this study, we tested the hypothesis that the bacterial composition of the GM could play a role in host adaptation to urban environments. We described the GM of several species of small terrestrial mammals sampled in forested areas along a gradient of urbanization, using a 16S metabarcoding approach. We tested whether urbanization led to changes in small mammal communities and in their GM, considering the presence and abundance of bacterial taxa and their putative functions. This enabled to decipher the processes underlying these changes. We found potential impacts of urbanization on small mammal communities and their GM. The urban dweller species had a lower bacterial taxonomic diversity but a higher functional diversity and a different composition compared to urban adapter species. Their GM assembly was mostly governed by stochastic effects, potentially indicating dysbiosis. Selection processes and an overabundance of functions were detected that could be associated with adaptation to urban environments despite dysbiosis. In urban adapter species, the GM functional diversity and composition remained relatively stable along the urbanization gradient. This observation can be explained by functional redundancy, where certain taxa express the same function. This could favor the adaptation of urban adapter species in various environments, including urban settings. We can therefore assume that there are feedbacks between the gut microbiota and host species within communities, enabling rapid adaptation.

Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE).

BACKGROUND: In vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs). RESULTS: We sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini clades. CONCLUSIONS: In consequence of the constraints given by the receptor protein function purifying selection has been a dominant force in evolution of Tlrs. Nevertheless, our results show that episodic diversifying parasite-mediated selection has shaped the present species-specific variability in rodent Tlrs. The intensity of diversifying selection was higher in Tlr4 than in Tlr7, presumably due to structural properties of their ligands.

Invasion genetics of the introduced black rat (Rattus rattus) in Senegal, West Africa.

An understanding of the evolutionary history and dynamics of invasive species is required for the construction of predictive models of future spread and the design of biological management measures. The black rat (Rattus rattus) is a major vertebrate invader with a worldwide distribution. Despite the severe ecological, economic and health impacts of this species, its evolutionary history has been little studied. We carried out extensive specimen sampling in Senegal, West Africa, and used microsatellite markers to describe the pattern and processes of invasion in this large continental area. The genetic data obtained were combined with historical knowledge concerning the presence of this species in Senegal. Data were analysed by a combination of Bayesian clustering and approximate Bayesian computation methods. The invasion pathways closely paralleled the history of human trade routes in Senegal. In several places, we detected the occurrence of multiple introductions from genetically different sources. Long-distance migration between towns and villages was also observed. Our findings suggest that genetic bottlenecks and admixture have played a major role in shaping the genetics of invasive black rats. These two processes may generate genetic novelty and favour rapid evolution along the invasion pathways.

Cytonuclear discordance among Southeast Asian black rats (Rattus rattus complex).

Black rats are major invasive vertebrate pests with severe ecological, economic and health impacts. Remarkably, their evolutionary history has received little attention, and there is no firm agreement on how many species should be recognized within the black rat complex. This species complex is native to India and Southeast Asia. According to current taxonomic classification, there are three taxa living in sympatry in several parts of Thailand, Cambodia and Lao People's Democratic Republic, where this study was conducted: two accepted species (Rattus tanezumi, Rattus sakeratensis) and an additional mitochondrial lineage of unclear taxonomic status referred to here as 'Rattus R3'. We used extensive sampling, morphological data and diverse genetic markers differing in rates of evolution and parental inheritance (two mitochondrial DNA genes, one nuclear gene and eight microsatellite loci) to assess the reproductive isolation of these three taxa. Two close Asian relatives, Rattus argentiventer and Rattus exulans, were also included in the genetic analyses. Genetic analyses revealed discordance between the mitochondrial and nuclear data. Mitochondrial phylogeny studies identified three reciprocally monophyletic clades in the black rat complex. However, studies of the phylogeny of the nuclear exon interphotoreceptor retinoid-binding protein gene and clustering and assignation analyses with eight microsatellites failed to separate R. tanezumi and R3. Morphometric analyses were consistent with nuclear data. The incongruence between mitochondrial and nuclear (and morphological) data rendered R. tanezumi/R3 paraphyletic for mitochondrial lineages with respect to R. sakeratensis. Various evolutionary processes, such as shared ancestral polymorphism and incomplete lineage sorting or hybridization with massive mitochondrial introgression between species, may account for this unusual genetic pattern in mammals.

Estimation of population allele frequencies from next-generation sequencing data: pool-versus individual-based genotyping.

Molecular markers produced by next-generation sequencing (NGS) technologies are revolutionizing genetic research. However, the costs of analysing large numbers of individual genomes remain prohibitive for most population genetics studies. Here, we present results based on mathematical derivations showing that, under many realistic experimental designs, NGS of DNA pools from diploid individuals allows to estimate the allele frequencies at single nucleotide polymorphisms (SNPs) with at least the same accuracy as individual-based analyses, for considerably lower library construction and sequencing efforts. These findings remain true when taking into account the possibility of substantially unequal contributions of each individual to the final pool of sequence reads. We propose the intuitive notion of effective pool size to account for unequal pooling and derive a Bayesian hierarchical model to estimate this parameter directly from the data. We provide a user-friendly application assessing the accuracy of allele frequency estimation from both pool- and individual-based NGS population data under various sampling, sequencing depth and experimental error designs. We illustrate our findings with theoretical examples and real data sets corresponding to SNP loci obtained using restriction site-associated DNA (RAD) sequencing in pool- and individual-based experiments carried out on the same population of the pine processionary moth (Thaumetopoea pityocampa). NGS of DNA pools might not be optimal for all types of studies but provides a cost-effective approach for estimating allele frequencies for very large numbers of SNPs. It thus allows comparison of genome-wide patterns of genetic variation for large numbers of individuals in multiple populations.

Evaluation of 96-well high-throughput DNA extraction methods for 16S rRNA gene metabarcoding.

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.

Candidatus Neoehrlichia mikurensis in bank voles, France.

To further assess the geographic occurrence, possible vectors, and prevalence of Candidatus Neoehrlichia mikurensis, we analyzed spleen tissues from 276 voles trapped close to human settlements in France; 5 were infected with the organism. Sequencing showed the isolates carried the same genotype as the bacteria that caused disease in humans and animals elsewhere in Europe.

SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing.

SUMMARY: Characterizing genetic diversity through genotyping short amplicons is central to evolutionary biology. Next-generation sequencing (NGS) technologies changed the scale at which these type of data are acquired. SESAME is a web application package that assists genotyping of multiplexed individuals for several markers based on NGS amplicon sequencing. It automatically assigns reads to loci and individuals, corrects reads if standard samples are available and provides an intuitive graphical user interface (GUI) for allele validation based on the sequences and associated decision-making tools. The aim of SESAME is to help allele identification among a large number of sequences. AVAILABILITY: SESAME and its documentation are freely available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported Licence for Windows and Linux from http://www1.montpellier.inra.fr/CBGP/NGS/ or http://tinyurl.com/ngs-sesame.

Increasing helminth infection burden depauperates the diversity of the gut microbiota and alters its composition in mice.

The gut microbiota constitutes a diverse community of organisms with pervasive effects on host homeostasis. The diversity and composition of the gut microbiota depend on both intrinsic (host genetics) and extrinsic (environmental) factors. Here, we investigated the reaction norms of fecal microbiota diversity and composition in three strains of mice infected with increasing doses of the gastrointestinal nematode Heligmosomoides polygyrus. We found that α-diversity (bacterial taxonomic unit richness) declined along the gradient of infective doses, and ß-diversity (dissimilarity between the composition of the microbiota of uninfected and infected mice) increased as the infective dose increased. We did not find evidence for genotype by environment (host strain by infective dose) interactions, except when focusing on the relative abundance of the commonest bacterial families. A simulation approach also showed that significant genotype by environment interactions would have been hardly found even with much larger sample size. These results show that increasing parasite burden progressively depauperates microbiota diversity and contributes to rapidly change its composition, independently from the host genetic background.

Small terrestrial mammals (Rodentia and Soricomorpha) along a gradient of forest anthropisation (reserves, managed forests, urban parks) in France.

Background: Understanding the relationships between wildlife biodiversity and zoonotic infectious diseases in a changing climate is a challenging issue that scientists must address to support further policy actions. We aim at tackling this challenge by focusing on small mammal-borne diseases in temperate forests and large urban green spaces. Small mammals are important reservoirs of zoonotic agents, with a high transmission potential for humans and domestic animals. Forests and large urban green spaces are ecosystems where efforts are undertaken to preserve biodiversity. They are put forward for their contribution to human well-being in addition to other ecosystem services (e.g. provisioning and regulating services). Moreover, forests and large urban green spaces are environments where small mammals are abundant and human/domestic-wildlife interactions are plausible to occur. These environments are, therefore, focal points for conservation management and public health issues. New information: The European Biodiversa BioRodDis project (https://www6.inrae.fr/biodiversa-bioroddis) aims at better understanding the relationships between small terrestrial mammal biodiversity and health in the context of global change and, in particular, of forest anthropisation and urbanisation. Here, we present the data gathered in France. The dataset will enable us to describe the diversity of small terrestrial mammal communities in forested areas corresponding to different levels of anthropisation and to evaluate the variability of this diversity over time, between seasons and years.The dataset contains occurrences of small terrestrial mammals (Rodentia and Soricomorpha) trapped in forested areas in eastern France (administrative Departments: Rhône, Ain, Jura). The sampling sites correspond to different degrees of anthropisation. Forests included in biological reserves are the least anthropised sites. Then, public forests and urban parks experience increasing levels of anthropisation. Data were collected during spring and autumn 2020 (three to four sampling sites), 2021 (six sampling sites) and 2022 (four sampling sites). These variations in the number of sites between years were due to lockdown restrictions in 2020 or to the legal authorisation to trap around biological reserves granted in 2021 only. The capture of animals was carried out in various types of forests (pine, deciduous, mixed) and in different habitats within urban parks (wooded areas, buildings, hay storage yards, riverside vegetation, restaurants, playground for kids, botanical garden, landfills). Animals were captured using live traps that were set on the ground for one to 11 nights. During this study period, 1593 small mammals were trapped and identified. They belong to 15 species, amongst which were nine species of rodents (Muridae, Cricetidae, Gliridae) and six species of shrews (Soricidae). They were weighted (gram) and measured (cm): head-body length, tail length and hind-foot length. Sexual characteristics were also recorded.

Isolation and Genetic Characterization of Puumala Orthohantavirus Strains from France.

Puumala orthohantavirus (PUUV) causes a mild form of haemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica (NE), regularly diagnosed in Europe. France represents the western frontier of the expansion of NE in Europe with two distinct areas: an endemic area (north-eastern France) where PUUV circulates in rodent populations, with the detection of many human NE cases, and a non-endemic area (south-western France) where the virus is not detected, with only a few human cases being reported. In this study, we describe the different stages of the isolation of two PUUV strains from two distinct French geographical areas: Ardennes (endemic area) and Loiret (non-endemic area). To isolate PUUV efficiently, we selected wild bank voles (Myodes glareolus, the specific reservoir of PUUV) captured in these areas and that were seronegative for anti-PUUV IgG (ELISA) but showed a non-negligible viral RNA load in their lung tissue (qRT-PCR). With this study design, we were able to cultivate and maintain these two strains in Vero E6 cells and also propagate both strains in immunologically neutral bank voles efficiently and rapidly. High-throughput and Sanger sequencing results provided a better assessment of the impact of isolation methods on viral diversity.

A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies.

BACKGROUND: High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. RESULTS: DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. CONCLUSIONS: This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied. It opens up new perspectives for the study of evolutionary and functional genetics of highly polymorphic genes like major histocompatibility complex genes in vertebrates or loci regulating self-compatibility in plants. Important applications in biomedical research will include the detection of individual variation in disease susceptibility. Similarly, agronomy will benefit from this approach, through the study of genes implicated in productivity or disease susceptibility traits.

Associations between MHC genes and Puumala virus infection in Myodes glareolus are detected in wild populations, but not from experimental infection data.

We analysed the influence of MHC class II Dqa and Drb genes on Puumala virus (PUUV) infection in bank voles (Myodes glareolus). We considered voles sampled in five European localities or derived from a previous experiment that showed variable infection success of PUUV. The genetic variation observed in the Dqa and Drb genes was assessed by using single-strand conformation polymorphism and pyrosequencing methods, respectively. Patterns were compared with those obtained from 13 microsatellites. We revealed significant genetic differentiation between PUUV-seronegative and -seropositive bank voles sampled in wild populations, at the Drb gene only. The absence of genetic differentiation observed at neutral microsatellites confirmed the important role of selective pressures in shaping these Drb patterns. Also, we found no significant associations between infection success and MHC alleles among laboratory-colonized bank voles, which is explained by a loss of genetic variability that occurred during the captivity of these voles.