Diverse mating phenotypes impact the spread of wtf meiotic drivers in Schizosaccharomyces pombe

Meiotic drivers are genetic elements that break Mendel's law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing (mating between individuals from distinct lineages) because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity for cells from distinct clonal lineages to mate varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed levels of preferential mating between cells from the same clonal lineage can slow, but not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs linked to the driver. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

Genetic diversity and potential inoculum sources of Fusarium species causing cankers in bareroot-propagated almond trees in California nurseries

Previous research determined that Fusarium acuminatum and Fusarium avenaceum are important causal agents of a canker disease in bareroot-propagated fruit and nut trees in California that emerges during cold-storage or after transplanting. The disease largely disappeared after 2001, but it reemerged in 2011 in almond trees in at least one nursery. This motivated further study of the etiology and epidemiology of the disease by undertaking studies to determine distribution of the pathogens throughout almond nursery propagation systems and trace possible sources of inoculum. Research initiated in 2013 detected pathogenic Fusarium spp. throughout the almond propagation system, including in healthy trees, in soils, on wheat rotation crops, on equipment, and in the cold storage facility air. In addition to the two Fusarium spp. implicated previously, Fusarium brachygibbosum and a new Fusarium species, Fusarium californicum, were found to be pathogenic on almond trees. Multi-locus sequence typing and somatic compatibility testing confirmed that isolates within a species collected from different materials in the nursery were all highly genetically similar and likely of one clonal lineage. These findings affirm that equipment surfaces, wheat rotation crops, soil, cold storage facility air, and asymptomatic almond tree materials (i.e., rootstock cuttings, budwood, and scions) can potentially contribute inoculum to increase disease prevalence and severity.

Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

Introduction B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction. Methods The B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results Clonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively. Conclusions These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders. For research use only. Disclosures Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

Mefenoxam Sensitivity in US-8 and US-23 Phytophthora infestans from Wisconsin

The contemporary dominant clonal lineage of heterothallic Phytophthora infestans in Wisconsin, US-23, is classified as sensitive to the systemic fungicide mefenoxam and is of the A1 mating type. With the sporadic appearance of clonal lineage US-8, classified as resistant to mefenoxam and of the A2 mating type, there is a need for ongoing monitoring and characterization. Isolates of P. infestans collected from Wisconsin during the 2017 and 2018 growing seasons were tested for sensitivity to mefenoxam with discriminatory dose of 100 ppm. In 2017, both US-23 and US-8 were isolated. On average, isolates of US-23 were significantly more sensitive to mefenoxam than were US-8 isolates (P = 8e-04). There were significant differences in the sensitivity levels among the US-8 isolates (P = 2.02e-06), with a single isolate testing sensitive at 100 ppm of mefenoxam based on the one-way ANOVA. There were significant differences in the sensitivity levels among US-23 isolates (P = 3.75e-09), with two isolates showing resistance. In 2018 only US-23 was found, and isolates were tested for mefenoxam response at 0, 0.1, 1, 10, and 100 ppm. At 0.1 ppm, isolates showed significantly different levels of sensitivity (P = 2.1e-09), and a single isolate showed complete resistance. Isolates from both clonal lineages and years that exhibited moderate levels of resistance had greater variability among replicates. The phenotype of this multigenic trait comes through in the variability seen in isolates that are showing more resistance. Continued screening of P. infestans for mefenoxam sensitivity will help track the development and mechanism of resistance, as well as aid in development of best management approaches.

Genomic insights into methicillin-resistant Staphylococcus pseudintermedius isolates from dogs and humans of the same sequence types reveals diversity in prophages and pathogenicity islands

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important opportunistic pathogenic bacterium of dogs that also occasionally colonize and infect humans. However, whether MRSP can adapt to human hosts is not clear and whole genome sequences of MRSP from humans are still limited. Genomic comparative analyses of 3 couples of isolates from dogs (n = 3) and humans (n = 3) belonging to ST45, ST112, and ST181, the dominant clones in Thailand were conducted to determine the degree of similarities between human and animal MRSP of a same ST. Among eight prophages, three prophages associated with the leucocidins genes (lukF/S-I), φVB88-Pro1, φVB16-Pro1 and φAP20-Pro1, were distributed in the human MRSPs, while their remnants, φAH18-Pro1, were located in the dog MRSPs. A novel composite pathogenicity island, named SpPI-181, containing two integrase genes was identified in the ST181 isolates. The distribution of the integrase genes of the eight prophages and SpPI-181 was also analysed by PCR in 77 additional MRSP isolates belonging to different STs. The PCR screen revealed diversity in prophage carriage, especially in ST45 isolates. Prophage φAK9-Pro1 was only observed in ST112 isolates from dogs and SpPI-181 was found associated with ST181 clonal lineage. Among the 3 couple of isolates, ST45 strains showed the highest number of single nucleotide polymorphisms (SNP) in their core genomes (3,612 SNPs). The genomic diversity of ST45 isolates suggested a high level of adaptation that may lead to different host colonization of successful clones. This finding provided data on the genomic differences of MRSP associated with colonization and adaption to different hosts.

Resistance to Phytophthora infestans Clonal Lineage US-23 in Potato Cultivars and Its Relationship with Early Blight Resistance and Tuber Yield

Resistance to late blight, caused by Phytophthora infestans clonal lineage US-23, in 217 old and modern potato cultivars was evaluated in field trials in 2016 and 2017 in Pennsylvania. Significant differences in resistance were found among these cultivars (P < 0.0001). Significant interaction between cultivars and environments was found (P < 0.0001). The values of relative area under the disease progress curve ranged from 0 to 0.5841 in 2016 and from 0 to 0.5469 in 2017. Broad-sense heritability of late blight resistance was estimated to be 0.91 with a 95% confidence interval of 0.88 to 0.93. Cluster analysis classified the cultivars into 5 groups: resistant, moderately resistant, intermediate, moderately susceptible, and susceptible. Thirty cultivars showing resistance and 32 cultivars showing moderate resistance were identified. The 217 cultivars were also evaluated for foliar maturity, tuber yield and resistance to early blight, caused by Alternaria solani. A few cultivars with late blight resistance independent of late maturity were found. Late blight resistance and early blight resistance were positively correlated, and 17 cultivars possessed resistance to both diseases. Yield tradeoff associated with late blight resistance was not observed among the cultivars in the absence of disease pressure.

Assessing the Extinction Risk of Heterocypris incongruens (Crustacea: Ostracoda) in Climate Change with Sensitivity and Uncertainty Analysis

Organisms respond to climate change in many different ways and their local extinction risk may vary widely among taxa. Crustaceans from freshwater temporary ponds produce resting eggs to cope with environmental uncertainty and, as a consequence, egg banks have a fundamental role for population persistence. The egg bank dynamics of six clonal lineages of Heterocypris incongruens (Ostracoda) from Northern Italy were simulated. Clonal lineages W1 and W2 are the most common “winter ecotypes”, clonal lineages S1 and S2 are allochthonous “summer ecotypes” and clonal lineages I1 and I2 are relatively rare and generalist in terms of seasonality. Fecundity and proportion of resting eggs vary by clonal lineage, temperature and photoperiod. The clonal extinction risk was estimated in present climate conditions and under climate change. For comparison, and to assess the potential colonization of northern ponds, clonal lineages from Lampedusa Island (Southern Italy), L, were considered. Cohen’s general model was used for simulating egg bank dynamics and the extinction rate of each clonal lineage was estimated with uncertainty analysis. A 30 year simulation in present and climate change conditions was carried out. Extinction rates were lower in climate change conditions than in present conditions. Hydroperiod, hatching rate and egg deterioration rate were the critical factors that affected extinction rates. Extinction rates varied among clonal lineages. This suggests that H. incongruens might be able to have multiple responses to climate change due to its genetic diversity. In climate change conditions, W clonal lineages underwent a niche expansion, while a mismatch between photoperiod and hydroperiod might generate a detrimental effect on the phenology of summer S clonal lineages that might cause their extinction. Southern clonal lineages L, showing an intermediate extinction rate, might colonize northern temporary ponds.

First Detection of Human ST131-CTX-M-15-O25-B2 Clone and High-Risk Clonal Lineages of ESBL/pAmpC-Producing E. coli Isolates from Diarrheic Poultry in Tunisia

Circulation of a multi-resistance clone of bacteria associated with genetic elements in diseased animals constitutes a global public health problem. Our study focused on the characterization of the support of ESBL in cefotaxime resistant E. coli (CTXR) isolates recovered from poultry with diarrhea, analysis of their clonal lineage, and virulence-associated genes. The study was carried out on 130 samples of chickens with diarrhea, collected in 2015 from poultry farms in Tunisia. Isolates of 20 CTXR E. coli strains were identified as ESBL and AmpC β- lactamase producers. The following β-lactamase genes (number of isolates) were detected: blaCTX-M-15+ blaOXA1 (4), blaCTX-M-15 + blaOXA1 + blaTEM-1b (2), blaCTX-M-1 + blaTEM-1b (9), blaCTX-M-1 (2), blaCMY2 + blaTEM-1b (3). Six E. coli harboring blaCTXM-15 were allocated to ST131-B2-O25b-; six and three blaCTX-M-1 were grouped in ST155, ST10, and ST58, respectively, related to the phylogroup D and A. The qnrB gene, the variant aac(6′)-Ib-cr, and the class 1 integrons with different gene cassettes, were detected amongst our 20 isolated strains, which were classified as ExPEC and aEPEC. Our findings highlighted the emergence of the human pandemic ST131-CTX-M-15-O25-B2 clone and the high risk of such clonal lineage strains in diarrheic poultry, in Tunisia, which could constitute a risk of their transfer to healthy animals and humans.

Examining Phenotypic Traits Contributing to the Spread in Northern European Potato Crops of EU_41_A2, a New Clonal Lineage of Phytophthora infestans

Until recently, genotypes of Phytophthora infestans were regionally distributed in Europe, with populations in western Europe being dominated by clonal lineages and those in northern Europe being genetically diverse due to frequent sexual reproduction. However, since 2013, a new clonal lineage (EU_41_A2) has successfully established itself and expanded in the sexually recombining P. infestans populations of northern Europe. The objective of this study was to study phenotypic traits of the new clonal lineage of P. infestans, which may explain its successful establishment and expansion within sexually recombining populations. Fungicide sensitivity, aggressiveness and virulence profiles of isolates of EU_41_A2 were analyzed and compared to those of the local sexual populations from Denmark, Norway, and Estonia. None of the phenotypic data obtained from the isolates collected from Denmark, Estonia and Norway independently explained the invasive success of EU_41_A2 within sexual Nordic populations. Therefore, we hypothesize that the expansion of this new genotype could result from a combination of fitness traits and more favorable environmental conditions that have emerged due to climate change.

Transmission, adaptation and geographical spread of the Pseudomonas aeruginosa Liverpool epidemic strain

The Liverpool epidemic strain (LES) is an important transmissible clonal lineage of Pseudomonas aeruginosa that chronically infects the lungs of people with cystic fibrosis (CF). Previous studies have focused on the genomics of the LES in a limited number of isolates, mostly from one CF centre in the UK, and from studies highlighting identification of the LES in Canada. Here we significantly extend the current LES genome database by genome sequencing 91 isolates from multiple CF centres across the UK, and we describe the comparative genomics of this large collection of LES isolates from the UK and Canada. Phylogenetic analysis revealed that the 145 LES genomes analysed formed a distinct clonal lineage when compared with the wider P. aeruginosa population. Notably, the isolates formed two clades: one associated with isolates from Canada, and the other associated with UK isolates. Further analysis of the UK LES isolates revealed clustering by clinic geography. Where isolates clustered closely together, the association was often supported by clinical data linking isolates or patients. When compared with the earliest known isolate, LESB58 (from 1988), many UK LES isolates shared common loss-of-function mutations, such as in genes gltR and fleR. Other loss-of-function mutations identified in previous studies as common adaptations during CF chronic lung infections were also identified in multiple LES isolates. Analysis of the LES accessory genome (including genomic islands and prophages) revealed variations in the carriage of large genomic regions, with some evidence for shared genomic island/prophage complement according to clinic location. Our study reveals divergence and adaptation during the spread of the LES, within the UK and between continents.