Dataset Information

Genome-wide mapping using new AFLP markers to explore intraspecific variation among pathogenic Sporothrix species.

ABSTRACT: Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix species, of which the main aetiological agents are S. brasiliensis, S. schenckii, and S. globosa. Infection occurs after a traumatic inoculation of Sporothrix propagules in mammals' skin and can follow either a classic route through traumatic inoculation by plant debris (e.g., S. schenckii and S. globosa) or an alternative route through zoonotic transmission from animals (e.g., S. brasiliensis). Epizootics followed by a zoonotic route occur in Brazil, with Rio de Janeiro as the epicenter of a recent cat-transmitted epidemic. DNA-based markers are needed to explore the epidemiology of these Sporothrix expansions using molecular methods. This paper reports the use of amplified-fragment-length polymorphisms (AFLP) to assess the degree of intraspecific variability among Sporothrix species. We used whole-genome sequences from Sporothrix species to generate 2,304 virtual AFLP fingerprints. In silico screening highlighted 6 primer pair combinations to be tested in vitro. The protocol was used to genotype 27 medically relevant Sporothrix. Based on the overall scored AFLP markers (97-137 fragments), the values of polymorphism information content (PIC = 0.2552-0.3113), marker index (MI = 0.002-0.0039), effective multiplex ratio (E = 17.8519-35.2222), resolving power (Rp = 33.6296-63.1852), discriminating power (D = 0.9291-0.9662), expected heterozygosity (H = 0.3003-0.3857), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Sporothrix. AFLP markers revealed cryptic diversity in species previously thought to be the most prevalent clonal type, such as S. brasiliensis, responsible for cat-transmitted sporotrichosis, and S. globosa responsible for large sapronosis outbreaks in Asia. Three combinations (#3 EcoRI-FAM-GA/MseI-TT, #5 EcoRI-FAM-GA/MseI-AG, and #6 EcoRI-FAM-TA/MseI-AA) provide the best diversity indices and lowest error rates. These methods make it easier to track routes of disease transmission during epizooties and zoonosis, and our DNA fingerprint assay can be further transferred between laboratories to give insights into the ecology and evolution of pathogenic Sporothrix species and to inform management and mitigation strategies to tackle the advance of sporotrichosis.

SUBMITTER: de Carvalho JA

PROVIDER: S-EPMC7329091 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundSporotrichosis is a chronic (sub)cutaneous infection caused by thermodimorphic fungi in the order, Ophiostomatales. These fungi are characterized by major differences in routes of transmission, host predilections, species virulence, and susceptibilities to antifungals. Sporothrix species emerge in the form of outbreaks. Large zoonoses and sapronoses are ongoing in Brazil and China, respectively. Current diagnostic methods based on morphology and physiology are inaccurate due to closely related phenotypes with overlapping components between pathogenic and non-pathogenic Sporothrix. There is a critical need for new diagnostic tools that are specific, sensitive, and cost-effective.MethodologyWe developed a panel of novel markers, based on calmodulin (CAL) gene sequences, for the large-scale diagnosis and epidemiology of clinically relevant members of the Sporothrix genus, and its relative, Ophiostoma. We identified specific PCR-based markers for S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and O. stenoceras. We employed a murine model of disseminated sporotrichosis to optimize a PCR assay for detecting Sporothrix in clinical specimens.ResultsPrimer-BLAST searches revealed candidate sequences that were conserved within a single species. Species-specific primers showed no significant homology with human, mouse, or microorganisms outside the Sporothrix genus. The detection limit was 10-100 fg of DNA in a single round of PCR for identifying S. brasiliensis, S. schenckii, S. globosa, S. mexicana, and S. pallida. A simple, direct PCR assay, with conidia as a source of DNA, was effective for rapid, low-cost genotyping. Samples from a murine model of disseminated sporotrichosis confirmed the feasibility of detecting S. brasiliensis and S. schenckii DNA in spleen, liver, lungs, heart, brain, kidney, tail, and feces of infected animals.ConclusionsThis PCR-based method could successfully detect and identify a single species in samples from cultures and from clinical specimens. The method proved to be simple, high throughput, sensitive, and accurate for diagnosing sporotrichosis.

Project description:BACKGROUND AND AIMS: Aglaonema is an important ornamental foliage plant genus, but genetic relationships among its species and cultivars have not been reported. This study analysed genetic relatedness of 54 cultivars derived from nine species using amplified fragment length polymorphism (AFLP) markers. METHODS: Initially, 48 EcoRI + 2/MseI + 3 primer set combinations were screened, from which six primer sets that showed clear scoreable and highly polymorphic fragments were selected and used for AFLP reactions. AFLP fragments were scored and entered into a binary data matrix as discrete variables. Jaccard's coefficient of similarity was calculated for all pair-wise comparisons among the 54 cultivars, and a dendrogram was constructed by the unweighted pair-group method using the arithmetic average (UPGMA). KEY RESULTS: The number of AFLP fragments generated per primer set ranged from 59 to 112 with fragment sizes varying from 50 to 565 bp. A total of 449 AFLP fragments was detected, of which 314 were polymorphic (70 %). All cultivars were clearly differentiated by their AFLP fingerprints. The 54 cultivars were divided into seven clusters; cultivars within each cluster generally share similar morphological characteristics. Cluster I contains 35 cultivars, most of them are interspecific hybrids developed mainly from A. commutatum, A. crispum or A. nitidum. However, Jaccard's similarity coefficients among these hybrids are 0.84 or higher, suggesting that these popular hybrid cultivars are genetically much closer than previously thought. This genetic similarity may imply that A. nitidum and A. crispum are likely progenitors of A. commutatum. CONCLUSIONS: Results of this study demonstrate the efficiency and ease of using AFLP markers for investigating genetic relationships of ornamental foliage plants, a group usually propagated vegetatively. The AFLP markers developed will help future Aglaonema cultivar identification, germplasm conservation and new cultivar development.

Project description:BackgroundAmplified fragment length polymorphism (AFLP) markers are frequently used for a wide range of studies, such as genome-wide mapping, population genetic diversity estimation, hybridization and introgression studies, phylogenetic analyses, and detection of signatures of selection. An important issue to be addressed for some of these fields is the distribution of the markers across the genome, particularly in relation to gene sequences.ResultsUsing in-silico restriction fragment analysis of the genomes of nine eukaryotic species we characterise the distribution of AFLP fragments across the genome and, particularly, in relation to gene locations. First, we identify the physical position of markers across the chromosomes of all species. An observed accumulation of fragments around (peri) centromeric regions in some species is produced by repeated sequences, and this accumulation disappears when AFLP bands rather than fragments are considered. Second, we calculate the percentage of AFLP markers positioned within gene sequences. For the typical EcoRI/MseI enzyme pair, this ranges between 28 and 87% and is usually larger than that expected by chance because of the higher GC content of gene sequences relative to intergenic ones. In agreement with this, the use of enzyme pairs with GC-rich restriction sites substantially increases the above percentages. For example, using the enzyme system SacI/HpaII, 86% of AFLP markers are located within gene sequences in A. thaliana, and 100% of markers in Plasmodium falciparun. We further find that for a typical trait controlled by 50 genes of average size, if 1000 AFLPs are used in a study, the number of those within 1 kb distance from any of the genes would be only about 1-2, and only about 50% of the genes would have markers within that distance.ConclusionsThe high coverage of AFLP markers across the genomes and the high proportion of markers within or close to gene sequences make them suitable for genome scans and detecting large islands of differentiation in the genome. However, for specific traits, the percentage of AFLP markers close to genes can be rather small. Therefore, genome scans directed towards the search of markers closely linked to selected loci can be a difficult task in many instances.

Project description:BackgroundThe genus Sporothrix belongs to the order Ophiostomatales and contains mainly saprobic soil and plant fungi, although pathogenic species capable of causing human infections are also present. The whole-genomes of disease-causing species have already been sequenced and annotated but no comprehensive genomic resources for environmental Sporothrix species are available, thus limiting our understanding of the evolutionary origin of virulence-related genes and pathogenicity.ResultThe genome assembly of four environmental Sporothrix species resulted in genome size of ~ 30.9 Mbp in Sporothrix phasma, ~ 35 Mbp in S. curviconia, ~ 38.7 Mbp in S. protearum, and ~ 39 Mbp in S. variecibatus, with a variable gene content, ranging from 8142 (S. phasma) to 9502 (S. variecibatus). The analysis of mobile genetic elements showed significant differences in the content of transposable elements within the sequenced genomes, with the genome of S. phasma lacking several class I and class II transposons, compared to the other Sporothrix genomes investigated. Moreover, the comparative analysis of orthologous genes shared by clinical and environmental Sporothrix genomes revealed the presence of 3622 orthogroups shared by all species, whereas over 4200 genes were species-specific single-copy gene products. Carbohydrate-active enzyme analysis revealed a total of 2608 protein-coding genes containing single and/or multiple CAZy domains, resulting in no statistically significant differences among pathogenic and environmental species. Nevertheless, some families were not found in clinical species. Furthermore, for each sequenced Sporothrix species, the mitochondrial genomes was assembled in a single circular DNA molecule, ranging from 25,765 bp (S. variecibatus) to 58,395 bp (S. phasma).ConclusionIn this study, we present four annotated genome assemblies generated using PacBio SMRT sequencing data from four environmental species: S. curviconia, S. phasma, S. protearum and S. variecibatus with the aim to provide a starting point for future comparative genome evolution studies addressing species diversification, ecological/host adaptation and origin of pathogenic lineages within the genus Sporothrix.

Dataset Information

Genome-wide mapping using new AFLP markers to explore intraspecific variation among pathogenic Sporothrix species.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets