The genetic structure of the plant pathogenic fungus Melampsora larici-populina on its wild host is extensively impacted by host domestication

Wild and cultivated plants represent very different habitats for pathogens, especially when cultivated plants bear qualitative resistance genes. Here, we investigated to what extent the population genetic structure of a plant pathogenic fungus collected on its wild host can be impacted by the deployment of resistant cultivars. We studied one of the main poplar diseases, poplar rust, caused by the fungus Melampsora larici‐populina. A thousand and fifty individuals sampled from several locations in France were phenotyped for their virulence profile (ability to infect or not the most deployed resistant cultivar ‘Beaupré’), and a subset of these was genotyped using 25 microsatellite markers. Bayesian assignment tests on genetic data clustered the 476 genotyped individuals into three genetic groups. Group 1 gathered most virulent individuals and displayed evidence for selection and drastic demographic changes resulting from breakdown of the poplar cultivar ‘Beaupré’. Group 2 comprised individuals corresponding to ancestral populations of M. larici‐populina naturally occurring in the native range. Group 3 displayed the hallmarks of strict asexual reproduction, which has never previously been demonstrated in this species. We discuss how poplar cultivation has influenced the spatial and genetic structure of this plant pathogenic fungus, and has led to the spread of virulence alleles (gene swamping) in M. larici‐populina populations evolving on the wild host.     

Characterization of microsatellite loci in White‐chinned Petrel (Procellaria aequinoctialis) and cross‐amplification in six other procellariiform species

Six polymorphic dinucleotide microsatellite loci were isolated and characterized from the White‐chinned Petrel Procellaria aequinoctialis, using a degenerate primer and PCR‐based technique to construct and screen an enriched genomic library. Preliminary data on three populations show heterozygosity levels ranging from 0.22 to 0.67 and allele numbers from three to nine. Preliminary data also suggest genetic distance between these three populations (F_ST 0.088). Cross‐species amplification of these six microsatellite loci and one further locus were tested in six other procellariiform species of the genus Procellaria, Macronectes, Thalassarche and Diomedea.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Genome‐wide association studies of alcohol dependence,DSM‐IV criterion count and individual criteria

Genome‐wide association studies (GWAS) of alcohol dependence (AD) have reliably identified variation within alcohol metabolizing genes (eg, ADH1B) but have inconsistently located other signals, which may be partially attributable to symptom heterogeneity underlying the disorder. We conducted GWAS of DSM‐IV AD (primary analysis), DSM‐IV AD criterion count (secondary analysis), and individual dependence criteria (tertiary analysis) among 7418 (1121 families) European American (EA) individuals from the Collaborative Study on the Genetics of Alcoholism (COGA). Trans‐ancestral meta‐analyses combined these results with data from 3175 (585 families) African‐American (AA) individuals from COGA. In the EA GWAS, three loci were genome‐wide significant: rs1229984 in ADH1B for AD criterion count (P = 4.16E?11) and Desire to cut drinking (P = 1.21E?11); rs188227250 (chromosome 8, Drinking more than intended, P = 6.72E?09); rs1912461 (chromosome 15, Time spent drinking, P = 1.77E?08). In the trans‐ancestral meta‐analysis, rs1229984 was associated with multiple phenotypes and two additional loci were genome‐wide significant: rs61826952 (chromosome 1, DSM‐IV AD, P = 8.42E?11); rs7597960 (chromosome 2, Time spent drinking, P = 1.22E?08). Associations with rs1229984 and rs18822750 were replicated in independent datasets. Polygenic risk scores derived from the EA GWAS of AD predicted AD in two EA datasets (P < .01; 0.61%‐1.82% of variance). Identified novel variants (ie, rs1912461, rs61826952) were associated with differential central evoked theta power (loss ? gain; P = .0037) and reward‐related ventral striatum reactivity (P = .008), respectively. This study suggests that studying individual criteria may unveil new insights into the genetic etiology of AD liability.     

Isolation of New Delhi metallo‐β‐lactamase 1‐producing Vibrio cholerae non‐O1, non‐O139 strain carrying ctxA,st and hly genes in southern Vietnam

Vibrio cholerae non‐O1, non‐O139 (VC_NAG) organisms are universally present in the aquatic environment and regarded as non‐pathogenic bacteria. However, considering that they do occasionally induce gastroenteritis, a study of their virulence and antibiotic resistance genes is important. The presence of enteropathogenic genes, including ctxA, VC_NAG‐specific heat‐stable toxin gene (st), hemolysin (hly), and zona occludens toxin (zot) was determined by PCR in 100 VC_NAG strains isolated in southern Vietnam in 2010–2013 from 94 environmental and six human origins. These 100 VC_NAG strains were also tested phenotypically and genotypically for the presence of the New Delhi metallo‐β‐lactamase (NDM‐1). Of the 100 VC_NAG strains tested, six were positive for ctxA; five from the environment and one of human origin. The st gene was detected in 17 isolates, 15 and two of which were of environmental and human origins, respectively. Gene hly was detected in 19 VC_NAG strains examined, two of which were isolated from humans and 17 from environments. The zot gene was not detected in any of the strains tested. Three VC_NAG strains of environmental origin were confirmed to produce NDM‐1 and the bla_NDM‐1 gene was detected in those strains by PCR. Of note, one of the three NDM‐1‐producing VC_NAG strains was confirmed to carry ctxA, st and hly genes concurrently. This is the first report of isolation of NDM‐1‐producing VC_NAG strains in Vietnam.     

A multiplex panel of microsatellite markers for widespread sub‐Saharan rodents of the genus Mastomys

We isolated and characterized 12 polymorphic microsatellite loci in the sub‐Saharan rodent Mastomys huberti. We tested cross‐species amplification of all these loci in three closely related Mastomys species: M. coucha, M. erythroleucus and M. natalensis. Multiplex panels comprising 11 loci were developed and their application to a set of individuals in each species allowed clear and easy characterization of allele sizes. Statistics from 31 M. huberti coming from one locality in Mali showed no deviation from Hardy–Weinberg equilibrium except for one locus, and no significant linkage disequilibria between loci.     

Time scale matters: genetic analysis does not support adaptation‐by‐time as the mechanism for adaptive seasonal declines in kokanee reproductive life span

Seasonal declines of fitness‐related traits are often attributed to environmental effects or individual‐level decisions about reproductive timing and effort, but genetic variation may also play a role. In populations of Pacific salmon (Oncorhynchus spp.), seasonal declines in reproductive life span have been attributed to adaptation‐by‐time, in which divergent selection for different traits occurs among reproductively isolated temporal components of a population. We evaluated this hypothesis in kokanee (freshwater obligate Oncorhynchus nerka) by testing for temporal genetic structure in neutral and circadian‐linked loci. We detected no genetic differences in presumably neutral loci among kokanee with different arrival and maturation dates within a spawning season. Similarly, we detected no temporal genetic structure in OtsClock1b, Omy1009uw, or OmyFbxw11, candidate loci associated with circadian function. The genetic evidence from this study and others indicates a lack of support for adaptation‐by‐time as an important evolutionary mechanism underlying seasonal declines in reproductive life span and a need for greater consideration of other mechanisms such as time‐dependent, adaptive adjustment of reproductive effort.     

Inferring the history of speciation in house mice from autosomal,X‐linked,Y‐linked and mitochondrial genes

Patterns of genetic differentiation among taxa at early stages of divergence provide an opportunity to make inferences about the history of speciation. Here, we conduct a survey of DNA‐sequence polymorphism and divergence at loci on the autosomes, X chromosome, Y chromosome and mitochondrial DNA in samples of Mus domesticus, M. musculus and M. castaneus. We analyzed our data under a divergence with gene flow model and estimate that the effective population size of M. castaneus is 200 000–400 000, of M. domesticus is 100 000–200 000 and of M. musculus is 60 000–120 000. These data also suggest that these species started to diverge approximately 500 000 years ago. Consistent with this recent divergence, we observed considerable variation in the genealogical patterns among loci. For some loci, all alleles within each species formed a monophyletic group, while at other loci, species were intermingled on the phylogeny of alleles. This intermingling probably reflects both incomplete lineage sorting and gene flow after divergence. Likelihood ratio tests rejected a strict allopatric model with no gene flow in comparisons between each pair of species. Gene flow was asymmetric: no gene flow was detected into M. domesticus, while significant gene flow was detected into both M. castaneus and M. musculus. Finally, most of the gene flow occurred at autosomal loci, resulting in a significantly higher ratio of fixed differences to polymorphisms at the X and Y chromosomes relative to autosomes in some comparisons, or just the X chromosome in others, emphasizing the important role of the sex chromosomes in general and the X chromosome in particular in speciation.     

Development and characterization of microsatellite markers for the poplar rust fungi Melampsora medusae and Melampsora larici‐populina

Melampsora species and their hybrids are obligate parasites of Populus and Salix species worldwide. The increasing interest in Populus and Salix for biomass and fibre production necessitates genetic markers for population studies of Melampsora spp. Libraries enriched for simple sequence repeats were used to develop five microsatellite markers for Melampsora medusae and Melampsora larici‐populina. The variation detected by these markers will be valuable for phylogenetic and population genetic studies, substantiate putative hybrids, and deployment of resistant poplar clones.     

Dissection of the genetic architecture of three seed‐quality traits and consequences for breeding in Brassica napus

Genome‐wide association studies (GWASs) combining high‐throughput genome resequencing and phenotyping can accelerate the dissection of genetic architecture and identification of genes for plant complex traits. In this study, we developed a rapeseed genomic variation map consisting of 4 542 011 SNPs and 628 666 INDELs. GWAS was performed for three seed‐quality traits, including erucic acid content (EAC), glucosinolate content (GSC) and seed oil content (SOC) using 3.82 million polymorphisms in an association panel. Six, 49 and 17 loci were detected to be associated with EAC, GSC and SOC in multiple environments, respectively. The mean total contribution of these loci in each environment was 94.1% for EAC and 87.9% for GSC, notably higher than that for SOC (40.1%). A high correlation was observed between phenotypic variance and number of favourable alleles for associated loci, which will contribute to breeding improvement by pyramiding these loci. Furthermore, candidate genes were detected underlying associated loci, based on functional polymorphisms in gene regions where sequence variation was found to correlate with phenotypic variation. Our approach was validated by detection of well‐characterized FAE1 genes at each of two major loci for EAC on chromosomes A8 and C3, along with MYB28 genes at each of three major loci for GSC on chromosomes A9, C2 and C9. Four novel candidate genes were detected by correlation between GSC and SOC and observed sequence variation, respectively. This study provides insights into the genetic architecture of three seed‐quality traits, which would be useful for genetic improvement of B. napus.     

Selection of broad‐spectrum cephalosporin‐resistant Escherichia coli in the feces of healthy dogs after administration of first‐generation cephalosporins

Although antimicrobial products are essential for treating diseases caused by bacteria, antimicrobial treatment selects for antimicrobial‐resistant (AMR) bacteria. The aim of this study was to determine the effects of administration of first‐generation cephalosporins on development of resistant Escherichia coli in dog feces. The proportions of cephalexin (LEX)‐resistant E. coli in fecal samples of three healthy dogs treated i.v. with cefazolin before castration and then orally with LEX for 3 days post‐operation (PO) were examined using DHL agar with or without LEX (50 µg/mL). LEX‐resistant E. coli were found within 3 days PO, accounted for 100% of all identified E. coli 3–5 days PO in all dogs, and were predominantly found until 12 days PO. LEX‐resistant E. coli isolates on DHL agar containing LEX were subjected to antimicrobial susceptibility testing, pulsed‐field gel electrophoresis (PFGE) genotyping, β‐lactamase typing and plasmid profiling. All isolates tested exhibited cefotaxime (CTX) resistance (CTX minimal inhibitory concentration ≥4 µg/mL). Seven PFGE profiles were classified into five groups and three β‐lactamase combinations (bla_CMY‐4‐bla_TEM‐1, bla_TEM‐1‐bla_CTX‐M‐15 and bla_TEM‐1‐bla_CTX‐M‐15‐bla_CMY‐4). All isolates exhibited identical PFGE profiles in all dogs on four days PO and subsequently showed divergent PFGE profiles. Our results indicate there are two selection periods for AMR bacteria resulting from the use of antimicrobials. Thus, continuing hygiene practices are necessary to prevent AMR bacteria transfer via dog feces after antimicrobial administration.     

Binding of spermine and ifenprodil to a purified,soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D ‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The K_d values for binding of [¹⁴C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [³H]Ifenprodil bound to NR1‐R (K_d, 0.18 μM) and NR2B‐R (K_d, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These K_d values were confirmed by circular dichroism measurements. The K_d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.     

Novel polymorphic microsatellites for the red‐legged partridge (Alectoris rufa) and cross‐species amplification in Alectoris graeca

The red‐legged partridge, Alectoris rufa, is an endemic species of the southwestern Mediterranean, and the most popular game bird in the Iberian Peninsula. A total of 27 microsatellite loci was isolated from an enriched genomic library of A. rufa. Six perfect GT microsatellites were characterized and optimized in 45 individuals of A. rufa. All loci revealed high levels of polymorphism with a number of alleles that ranged from three to 13. Observed heterozygosity ranged from 0.2 to 0.6. Cross‐species amplification showed that all loci were also polymorphic in rock partridge, Alectoris graeca. The new markers will be useful in determining hybridization between both species of Alectoris.     

Herbivory meets fungivory: insect herbivores feed on plant pathogenic fungi for their own benefit

Plants are regularly colonised by fungi and bacteria, but plant‐inhabiting microbes are rarely considered in studies on plant–herbivore interactions. Here we show that young gypsy moth (Lymantria dispar) caterpillars prefer to feed on black poplar (Populus nigra) foliage infected by the rust fungus Melampsora larici‐populina instead of uninfected control foliage, and selectively consume fungal spores. This consumption, also observed in a related lepidopteran species, is stimulated by the sugar alcohol mannitol, found in much higher concentration in fungal tissue and infected leaves than uninfected plant foliage. Gypsy moth larvae developed more rapidly on rust‐infected leaves, which cannot be attributed to mannitol but rather to greater levels of total nitrogen, essential amino acids and B vitamins in fungal tissue and fungus‐infected leaves. Herbivore consumption of fungi and other microbes may be much more widespread than commonly believed with important consequences for the ecology and evolution of plant–herbivore interactions.     

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Development of EST‐SSRs from the Alpine Lady‐fern,Athyrium distentifolium

The utility of EST‐simple sequence repeats (EST‐SSRs) was evaluated in the fern Athyrium distentifolium. From 1152 frond cDNA clones, 165 microsatellites, including di‐, tri‐, tetra and penta‐nucleotide repeat motifs, were identified. Primer design was possible for 74 of the SSRs; subsequent screening of 10 loci on 186 individuals from six natural populations revealed between two and seven alleles per locus and expected heterozygosity (H_E) estimates ranging from 0.027 to 0.809. Eight of these loci were further examined for cross‐species and cross‐generic amplification in other Woodsiaceae species, and polymorphic products were detected. EST‐derived SSRs provide robust, informative and potentially transferable polymorphic markers suitable for biodiversity research.     

Time‐kill studies with a ceftazidime‐treated mixed culture consisting of Pseudomonas aeruginosa,Burkholderia cepacia and Staphylococcus aureus

So far, time‐kill analyses were mostly done with isolates of bacteria. Here, we used a mixed culture consisting of Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus to investigate the impact of ceftazidime treatment. Following an earlier study with the same strains, the influence of different ceftazidime concentrations as well as repeated ceftazidime treatment was tested. In order to assess the influence of substrate competition, which might be relevant to interpret mixed‐culture time‐kill studies, the major metabolites of the chemically defined cultivation medium were measured additionally. Time‐kill experiments were conducted in shake flasks with the chemically defined and modified medium M199. The cell concentration in the mixed culture was analyzed on the single‐species level using a quantitative terminal restriction fragment (qT‐RFLP) method. The amount of gluconate produced in mixed culture positively correlated with increased ceftazidime concentrations (5, 15, 30, 60 mg/L). Burkholderia cepacia developed resistance after repeated ceftazidime addition and reached the highest cell concentration of the three cultivated strains. Pseudomonas aeruginosa showed a pronounced regrowth phase after removal of ceftazidime, while growth of S. aureus was not influenced by medium exchange. In conclusion, growth of B. cepacia was dominant in the ceftazidime‐treated mixed culture over the observed time range, due to low susceptibility against ceftazidime as well as advantages in substrate usage.     

Characterization of expressed sequence tag‐derived microsatellites from the kuruma prawn,Marsupenaeus japonicus

The kuruma prawn, Marsupenaeus japonicus, is a commercially important benthic marine crustacean in East Asia. Understanding the species’ population structures will be very important for its proper stock assessment and management strategy. Herein, 13 polymorphic microsatellite loci originating from expressed sequence tag libraries of M. japonicus were isolated and characterized. Number of alleles per locus ranged from two to 15, observed and expected heterozygosities ranged from 0.250 to 0.906 and from 0.310 to 0.932, respectively. The adequate level of variability within the population renders these microsatellite loci ideal markers for population genetic analysis of M. japonicus.     

Genomic characterization of sex‐identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes

Fish have evolved a variety of sex‐determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex‐specific genotypic markers, double digest restriction site – associated DNA sequencing (ddRAD‐seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male‐specific loci met the criteria in at least one species and 17 in both. Conversely, no female‐specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex‐specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex‐specific and autosomal loci. A PCR‐RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex‐determining gene in other teleosts gonadal soma‐derived factor (gsdf) was present in the model fish chromosomes that spanned our sex‐specific markers.