A genomic overview of the population structure of Salmonella.

High quality circular genome sequences were generated for the Clostridium chauvoei type strain DSM 7528T (ATCC 10092T) and a field strain 12S0467 isolated in Germany. Comparative genome analysis of the two strains revealed few inversions and translocations in local collinear blocks, indicating a conserved genome with only a small number of accessory genes. Significant homology for C. chauvoei was observed with C. septicum. The species genome shows a large number of genes, the products of which are involved in proteolysis, cleavage of glycosidic linkages (sialidases) and metal ion transportation. Triplicates of fliC were identified in each of the two circular genomes. Sporulation and germination process related genes were homologous to those of the Clostridia cluster I species with highest homology to C. septicum, but novel variations in regulatory genes were also identified. A comparative genomic study was carried out with a total of 64 C. chauvoei strains. The strain collection mostly included strains of European origin whereas few strains were of exotic origin. Pan-genome analysis of the species based on the available C. chauvoei strains shows that the species has an open pan-genome structure. New gene acquisition was observed to be very limited. This probably indicates that the species is undergoing replication only in very isolated areas e.g. inside the host tissue, so that the chances of acquiring foreign genetic material are limited. The predicted prophage identified in the genomes was similar for all strains, indicating that the genomes are immune to any new phage type. A CRISPR type I-B system was identified in all genomes which may contribute to comprehensive phage immunity. There was an obvious correlation of strains from the same region/farm i.e. displaying the same repeat numbers/spacer sequences. The strains originating from outside Europe showed a unique spacer matrix composition. Homologous recombination plays an important role in the evolution of some bacterial pathogens. This study found only a limited number of possible recombination events contributing towards the evolution of C. chauvoei. Maximum Likelihood phylogenetic analysis based on the core genome (Parsnp) identified associated isolates with strong bootstrap support values (100%). These values were highly prominent for strains originating from the same farm/outbreak/animal. Reference genome and alignment free analysis methods (kSNP 3) based on pan-genome SNPs (SNP, Single Nucleotide Polymorphism) were found to be useful for initial clustering of isolates. The strains were clustered based on unique SNPs. The number of allele specific SNPs which are unique for each strain was higher for strains from exotic origin. Read mapping and SNP calling (Snippy) based on a related reference genome (12S0467) was able to provide novel insights to understand the general, outbreak and within-host microevolution of a pathogen. The median pairwise SNP difference value was higher for C. chauvoei strains from Bavaria and Austria as compared to strains from Lower Saxony. The diversity of geographically related strains could therefore be indicative of other environmental factors or herd management influencing strain microevolution. Nonsynonymous SNPs contributed significantly to the observed microevolution of the pathogen. Based on the strain variability observed for strains from the same host and strains from the same outbreak, this study also proved the possibility of genetically different C. chauvoei populations in one host. A very recent study, based on genome sequence data, has pointed out the applicability of strain differentiation based on CRISPR spacer sequences for C. chauvoei. The current study similarly proved the applicability of typing approaches for this pathogen based on CRISPR elements and core genome MLST. The CRISPR spacer diversity was found to be inadequate to differentiate strains of European origin. However core genome MLST was able to differentiate the C. chauvoei strains within Europe and Germany and thus proved to be a valuable tool for strain differentiation. The current study applied the advantage of long read based sequencing technology to generate high quality complete genome reference sequences for a field strain from Germany and the type strain of C. chauvoei. The phylogenetic positioning of the species within the genus was evaluated and the close relatedness to C. septicum was confirmed. Several genome analysis software tools provided novel insights in the genome content and composition of the pathogen. Comparative genome sequence analysis based on 64 strains showed limited horizontal gene transfer and novel gene acquisition. Strain typing based on core genome MLST analysed used the first time for this species could differentiate strains even at farm level.

Cowpea is an important legume crop worldwide. However, the diversity and relationship of current accessions remain unclear due to a lack of robust genetic information. A set of 40,089 single nucleotide polymorphisms (SNPs) adapted to the Kompetitive Allele Specific PCR (KASP) SNP genotyping platform were developed from the existing Illumina Cowpea iSelect Consortium Array for cowpea germplasm genetic diversity assessment and variety identification. Using the genotypic data of 299 cowpea accessions obtained from this SNP assay, a precore pool of 434 SNPs and 50 informative core SNPs was selected and validated for use in future genetic diversity analyses of cowpea germplasm. By further genotyping 75 commercial cultivars using the set of 50 core SNPs, pairwise genotype alignment and dendrogram analysis both showed that each cultivar could be uniquely identified by this core set of markers. The KASP markers developed in this study provide a flexible marker resource for both basic and applied research of cowpea. The precore SNP pool and the core SNP set also provide valuable tools for genetic variation assessment and variety rights protection for cowpea breeders.

To analyze genetic features in the Japanese rice population, which is mainly composed of closely related accessions, a core set of single-nucleotide polymorphisms (SNPs) was selected from SNP resources based on Japanese rice cultivars. A total of 25,199 SNPs were newly detected from the comparison of genomic sequences between two cultivars (Eiko and Rikuu132) and Nipponbare as a reference. A total of 81,499 non-redundant SNPs, including 67,051 SNPs of Koshihikari detected in a previous study, were used as candidates to select the core SNPs. Across the entire genome, 3379 SNPs were selected based on the chromosomal position of each SNP and were investigated each allele of 92 Japanese rice accessions and 3 from outside of Japan. As a result, 2551 SNPs were found to be informative for at least 91 cultivars for reducing the potential risks of genotyping error. The Japanese rice accessions were classified into three groups (upland, lowland Hokkaido, and other lowland) using all 2551 SNPs. In addition, a core set of 768 SNPs was selected to provide an even distribution among the chromosomes. Comparison of dendrograms generated by all 2551 SNPs and the core set of 768 SNPs demonstrated that the core SNPs could be used efficiently and reliably in the classification of the Japanese rice population. The core SNPs can be used for diversity analysis and for genetic analysis of the biparental populations of Japanese rice accessions.

BackgroundThe loach (Misgurnus anguillicaudatus) is a significant freshwater economic fish in China, renowned for its tender meat, delicious taste, and high nutritional value. It is widely distributed across the country, except for the western plateau. However, the loach is currently at risk of population decline and degradation of wildlife resources. Research on genetic diversity provides a crucial foundation for the conservation and development of these fish resources. To enhance the protection and utilization of wild loach germplasm resources, we analyzed the genetic structure and diversity of 60 wild loach populations from Xiangtan City (XT), Shaoyang City (SY), and Yueyang City (XY) in Hunan, Guilin City (GL) and Guiping City (GP) in Guangxi, and Wuhan City (WH) in Hubei. Additionally, we mapped the DNA fingerprints of these 60 wild loaches using 12 high-quality SNP sites.ResultsWhole genome resequencing (WGRS) was conducted on 60 loaches from six regions, yielding a total of 1047.17 Gb of raw data and 1046.98 Gb of clean data. From this 2,812,906 high-quality single nucleotide polymorphisms (SNPs) were identified, of which 10,022 core SNPs were selected to analyze the population genetic structure, and 12 core SNPs were used to assess the genetic diversity and DNA fingerprint. The analysis revealed that the 60 loach samples could be grouped into three clusters: Cluster A comprised the XT, SY, and XY groups; Cluster B included the WH group; and Cluster C consisted of the GL and GP groups. The mean nucleic acid diversity (Pi), heterozygosity (Ho), and expected heterozygosity (He) of SNP markers were 0.130, 0.140, and 0.123, respectively. The average inbreeding coefficient was 0.552, indicating high levels of inbreeding.ConclusionsWhole genome resequencing is an effective high-throughput approach for identifying SNP information in loach germplasm and describing genetic relationships within this genus. DNA fingerprinting based on SNP marker technology can accurately assess the genetic structure and variation within natural loach populations, making it a valuable tool for strain and variation identification. Our findings provide a scientific basis for the conservation, development, and optimal breeding of native loach germplasm resources and contribute to expanding the genetic diversity database of wild loach populations.

Development of a core set of SNP markers for the identification of upland cotton cultivars in China

Genetic approaches have proved valuable to the study and conservation of endangered populations, especially for monitoring programs, and there is potential for further developments in this direction by extending analyses to the genomic level. We assembled the genome of the wolverine (Gulo gulo), a mustelid that in Scandinavia has recently recovered from a significant population decline, and obtained a 2.42Gb draft sequence representing >85% of the genome and including >21,000protein-coding genes. We then performed whole-genome resequencing of 10Scandinavian wolverines for population genomic and demographic analyses. Genetic diversity was among the lowest detected in a red-listed population (mean genome-wide nucleotide diversity of 0.05%). Results of the demographic analyses indicated a long-term decline of the effective population size (Ne ) from 10,000well before the last glaciation to <500 after this period. Current Ne appeared even lower. The genome-wide FIS level was 0.089 (possibly signaling inbreeding), but this effect was not observed when analyzing a set of highly variable SNP markers, illustrating that such markers can give a biased picture of the overall character of genetic diversity. We found significant population structure, which has implications for population connectivity and conservation. We used an integrated microfluidic circuit chip technology to develop an SNP-array consisting of 96 highly informative markers that, together with a multiplex pre-amplification step, was successfully applied to low-quality DNA from scat samples. Our findings will inform management, conservation, and genetic monitoring of wolverines and serve as a genomic roadmap that can be applied to other endangered species. The approach used here can be generally utilized in other systems, but we acknowledge the trade-off between investing in genomic resources and direct conservation actions.

Single Nucleotide Polymorphisms (SNPs) are now popular for a myriad of applications in animal and plant species including, ancestry assignment, conservation genetics, breeding, and traceability of animal products. The objective of this study was to develop a customized cost-effective SNP panel for genetic characterisation of Macrobrachium species in Cameroon. The SNPs identified in a previous characterization study were screened as viable candidates for the reduced panel. Starting from a full set of 1,814 SNPs, a total of 72 core SNPs were chosen using conventional approaches: allele frequency differentials, minor allele frequency profiles, and Wright’s Fst statistics. The discriminatory power of reduced set of informative SNPs were then tested using the admixture analysis, principal component analysis, and discriminant analysis of principal components. The panel of prioritised SNP markers (i.e., N = 72 SNPs) distinguished Macrobrachium species with 100% accuracy. However, large sample size is needed to identify more informative SNPs for discriminating genetically closely related species, including M. macrobrachion versus M. vollenhovenii and M. sollaudii versus M. dux. Overall, the findings in this study show that we can accurately characterise Macrobrachium using a small set of core SNPs which could be useful for this economically important species in Cameroon. Given the results obtained in this study, a larger independent validation sample set will be needed to confirm the discriminative capacity of this SNP panel for wider commercial and research applications.

Identification of genetic variants of Brucella spp. through genome-wide association studies

Effective use of rapid and inexpensive whole genome sequencing for microbes requires fast, memory efficient bioinformatics tools for sequence comparison. The kSNP v2 software finds single nucleotide polymorphisms (SNPs) in whole genome data. kSNP v2 has numerous improvements over kSNP v1 including SNP gene annotation; better scaling for draft genomes available as assembled contigs or raw, unassembled reads; a tool to identify the optimal value of k; distribution of packages of executables for Linux and Mac OS X for ease of installation and user-friendly use; and a detailed User Guide. SNP discovery is based on k-mer analysis, and requires no multiple sequence alignment or the selection of a single reference genome. Most target sets with hundreds of genomes complete in minutes to hours. SNP phylogenies are built by maximum likelihood, parsimony, and distance, based on all SNPs, only core SNPs, or SNPs present in some intermediate user-specified fraction of targets. The SNP-based trees that result are consistent with known taxonomy. kSNP v2 can handle many gigabases of sequence in a single run, and if one or more annotated genomes are included in the target set, SNPs are annotated with protein coding and other information (UTRs, etc.) from Genbank file(s). We demonstrate application of kSNP v2 on sets of viral and bacterial genomes, and discuss in detail analysis of a set of 68 finished E. coli and Shigella genomes and a set of the same genomes to which have been added 47 assemblies and four “raw read” genomes of H104:H4 strains from the recent European E. coli outbreak that resulted in both bloody diarrhea and hemolytic uremic syndrome (HUS), and caused at least 50 deaths.

Chagas disease remains the most important parasitic infection in Latin America. The aetiological agent, Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), is a complex vector-borne zoonosis transmitted in the faeces of hematophagous triatomine bugs (Hemiptera: Reduviidae: Triatominae), and maintained by mammalian reservoir hosts ranging from the southern United States to Argentinean Patagonia. In the absence of chemotherapy, infection is life-long and can lead to a spectrum of pathological sequelae ranging from subclinical to lethal cardiac and/or gastrointestinal complications in up to 30% of patients. T. cruzi displays remarkable genetic diversity, which has long been suspected to contribute to the considerable variation in clinical symptoms observed between endemic regions. Currently, isolates of T. cruzi can be assigned to a minimum of six stable genetic lineages or discrete typing units (DTUs) (TcI-TcVI), which are broadly associated with disparate ecologies, transmission cycles and geographical distributions. The principal mode of reproduction among T. cruzi strains is the subject of an intense, decades-old debate. Despite the existence of two recent natural hybrid lineages (TcV and TcVI), which resemble meiotic F1 progeny, a pervasive view is that recombination has been restrained at an evolutionary scale and is of little epidemiological relevance to contemporary parasite populations. The aim of this PhD project was to investigate T. cruzi genetic diversity through significant development of phylogenetic markers and their application to the characterization of natural parasite population structures and genetic exchange mechanisms. Multiple, single-copy, chromosomally-independent, nuclear housekeeping genes were assessed initially for their ability to allocate isolates to DTU-level, to facilitate higher resolution intra-lineage analyses and finally for their inclusion alongside additional targets in a standardized T. cruzi multilocus sequence typing (nMLST) scheme. For the immediate future, nuclear MLST, using a panel of four to seven nuclear loci, is a robust, reproducible and highly discriminatory method that has potential to become the new gold standard for T. cruzi DTU assignment. To investigate natural parasite population structures and uncover evidence of genetic exchange, a high resolution mitochondrial MLST (mtMLST) scheme, based on ten gene fragments, was developed and evaluated against current nuclear markers (multilocus microsatellite typing; MLMT) using isolates belonging to the oldest and most widely distributed lineage (TcI). Observations of gross nuclear-mitochondrial phylogenetic incongruence indicate that recombination is ongoing, geographically widespread and continues to influence natural populations, challenging the traditional paradigm of clonality in T. cruzi. Application of this combined nuclear-mitochondrial methodology to intensively sampled, minimally-subdivided TcI populations revealed extensive mitochondrial introgression within a disease focus in North-East Colombia as well as among arboreal transmission cycles in Bolivia. Failure to detect any reciprocal nuclear hybridization among recombinant strains ! 4 may be indicative of alternate, cryptic mating strategies in T. cruzi, which are challenging to reconcile with both in vitro parasexual mechanisms of genetic exchange described, and patterns of Mendelian allele inheritance among natural hybrid DTUs. High resolution genotyping of TcI populations was also undertaken to explore the interaction between parasite genetic heterogeneity and ecological biodiversity, exposing the significant impact human activity has had on T. cruzi evolution. Reduced genetic diversity, accelerated parasite dissemination between densely populated areas and mitochondrial gene flow between domestic and sylvatic populations, suggests humans may have played a crucial role in T. cruzi dispersal across the Bolivian highlands. Parallel reductions in genetic diversity were observed among isolates from the Brazilian Atlantic Forest, attributable to ongoing anthropogenic habitat fragmentation. By comparison domestic TcI isolates (TcIDOM) are divergent from their sylvatic counterparts, but also genetically homogeneous, and likely to have originated in North/Central America before distribution southwards. Molecular dating of Colombian TcIDOM clones confirmed that this clade emerged 23,000 ± 12,000 years, coinciding with the earliest human migration into South America. Lastly, Illumina amplicon deep sequencing markers were developed to explore the interaction between parasite multiclonality and clinical status of chronic Chagas disease. An unprecedented level of intra-host genetic diversity was detected, highlighting putative diversifying selection affecting antigenic surface proteases, which may facilitate survival in the mammalian host. In lieu of comparative genomics of representative T. cruzi field isolates, not yet a reality, as is the case with other more experimentally-tractable trypanosomatids, presented herein are some of the highest resolution genotyping techniques developed in T. cruzi to date, which have the potential to expand our current understanding of parasite genetic diversity and its relevance to clinical outcome of Chagas disease.

Clostridioides difficile is an important nosocomial pathogen increasingly observed in the community and in different non-human reservoirs. The epidemiology and transmissibility of C. difficile has been studied using a variety of typing methods, including more recently developed whole-genome sequence (WGS) analysis that is becoming used routinely for bacterial typing worldwide. Here we review the schemes for WGS-based typing methods available for C. difficile and their applications in the field of human C. difficile infection (CDI). The two main approaches to discover genomic variations are single nucleotide variant (SNV) analysis and methods based on gene-by-gene comparisons (frequently called core genome or whole genome MLST, cgMLST, or wgMLST). SNV analysis currently provides the ultimate resolution, however, typing nomenclature and standardized methodology are missing. On the other hand, gene-by-gene approaches allow portability and standardized nomenclature, and are therefore becoming increasingly popular in bacterial epidemiology and outbreak investigation. Two commercial software packages (BioNumerics and Ridom SeqSphere+) and an open source database (EnteroBase) for allele and sequence type determination for C. difficile are currently available. Proof-of-concept WGS studies have already enabled advances in the investigation of the population structure of C. difficile species, microevolution within the epidemic strains, intercontinental transmission over time and in tracking of transmission events. WGS of clinical C. difficile isolates demonstrated a considerable genetic diversity suggesting diverse reservoirs for CDI. WGS was also shown to aid in resolving relapses and reinfections in recurrent CDI and has potential for use as a tool for assessing hospital infection prevention and control performance.

The goal of this study was to investigate the prevalence and genetic relatedness of Salmonella enterica in meat and contact surfaces from two processing lines at a pork processing plant over a commercial production schedule. Across 192 samples, there was no significant difference in Salmonella prevalence between Bootjack Trim (BJ) and Boston Butt Trim (BBT) meat (11.5% vs. 11.5%, P=1.0), though prevalence was higher in meat than on contact surfaces for both the BJ (11.5% vs. 0%, P=0.01) and BBT (11.5% vs. 3.1%, P=0.08) processing lines. Both Salmonella prevalence and identified serotypes clustered within four distinct processing windows that spanned multiple dates and processing lines. Phylogenetic analysis using core single nucleotide polymorphisms (SNPs) identified a highly related Salmonella I4,[5],12:i:- strain (N=33, 0-2 SNPs difference across all isolates) in both the BJ and BBT lines, persisting over consecutive days within one processing window. Similarly, a highly related Salmonella London strain (N=18, 0-1 SNPs) was found across both processing lines on three processing dates that spanned 28days. Additional highly related strains of Salmonella Typhimurium (N=8, 0-1 SNPs) and Salmonella Agona (N=7, 0-3 SNPs) were also detected across multiple dates. Strains of S. I4,[5],12:i:- and S. London were genetically distinct (>30 SNPs) from publicly available genomes from isolates obtained from other pork processing plants located in the Upper Midwest. Overall, findings suggested that Salmonella prevalence varies across processing lines and production schedules. However, the high phylogenetic relatedness among the Salmonella serotypes suggests a common source may have been present prior to each primal cut being processed into subprimal cuts.

Rapid, economical, and reliable genotyping is an important requirement for germplasm analysis and cultivar identification in crop species. Chinese cabbage (Brassica rapa L. subsp. pekinensis (Lour.) Hanelt) originated in China and is now an economically important vegetable crop worldwide, especially in East Asia. In this study, we evaluated 1167 single nucleotide polymorphisms (SNPs) among 166 representative Chinese cabbage inbred lines using a KASP genotyping assay. On the basis of polymorphisms and principal component analysis, we selected 60 core SNPs distributed on all Brassica rapa chromosomes with allele frequencies sufficiently balanced so as to provide adequate information for genetic identification. The core set of SNPs was used for construction of a neighbor-joining dendrogram, in which the 166 inbred lines were clustered into spring, summer, and autumn ecotype groups. Clustering of the ecotype groups was better resolved than that achieved with 1167 and 360 polymorphic SNP datasets. Stability and resolution of the core SNP markers were tested using 178 commercial hybrid Chinese cabbage cultivars to confirm their utility in genetic identification. The set of 60 informative and stable SNP markers showed high discriminatory power and relatively uniform genomic distribution (4–9 markers per chromosome). The SNPs represent a cost-efficient and accurate marker set for germplasm analysis and cultivar identification and are suitable for molecular marker-assisted breeding in Chinese cabbage.

Genome-wide networks reveal emergence of epidemic strains of Salmonella Enteritidis

We previously mapped the 41rst gene locus (DFNA41) for autosomal dominant hearing loss on chromosome 12q24-qter in a large multi-generational Chinese family. We determined that DFNA41 is located in a 15 cM region, proximal to the marker D12S1609. A maximum two point LOD score of 6.56 at theta = 0.0 was obtained with marker D12S343. In the current study, screening of eight candidate genes within the DFNA41 interval did not reveal the mutation causing deafness in this family. Eight highly informative single nucleotide polymorphisms (SNPs) in the region of D12S343 were selected for linkage and association study. Because the pedigree studied here is a large family with many founders, we applied the transmission/disequilibrium (TDT) test. To account for the dependence of small families and the relatively small sample size, simulations were performed to obtain P-values. For three nearby SNPs spanning a 7 kb interval, we found significant evidence of linkage and association. The highest Z score of linkage and association of 3.6 (P < or = 0.0001) was obtained for SNP rs1566667. Haplotype analysis revealed that affected individuals were heterozygous for one core SNP (rs1027560-rs1027557-rs1566667-rs1463865-rs2078105) CAGTC haplotype, confirming location and autosomal dominant inheritance of the DFNA41 locus. Examination of pairwise LD calculation identified a major haplotype block defined by the four most centromeric SNPs. This study represents a significant refinement of the DFNA41 locus and should facilitate positional cloning of the disease gene.