Amplified Fragment Length Polymorphism Research Articles

DNA quality and STR success rate in different formalin-fixed tissues.

Formalin-fixed tissues possess irreplaceable value as a source of DNA for identification, especially when fresh samples are unavailable. Nonetheless, extracting and amplifying DNA from these tissues is challenging, primarily due to formaldehyde-induced cross-linking and nucleic acid fragmentation. In this study, two pre-extraction treatments, gradual dehydration using ethanol and pre-digestion heat treatments, and three DNA extraction methods, the Chelex-100 method, TIANamp FFPE DNA Kit, and ML Ultra-micro DNA extraction kit, were utilized to optimize DNA extraction from different tissues, which were fixed in 4% unbuffered formalin for different durations. The tissues include the heart, liver, spleen, lung, kidney, muscle, and brain. DNA quality was assessed, and quantification was conducted using Spectrophotometer and Quantifiler® Trio DNA Quantification Kits, while the GSTAR™ 25 kit was employed for STR detection. The results indicated that the two pre-extraction treatments exhibited no significant effect on the STR success rate. On day 9, allelic dropout was observed in the heart, liver, spleen, lung, and kidney tissues. Furthermore, allelic dropout was observed in muscle and brain at 12 days and 15 days, respectively. In conclusion, the results underscore the feasibility of effectively extracting DNA from formalin-fixed tissues within 9 days for subsequent STR analysis.

Analyses of the Genetic Diversity and Population Structures of Sporothrix spp. Clinical Isolates from Paraíba, Brazil

Sporotrichosis is a subcutaneous mycosis of global distribution, capable of affecting both humans and animals, and caused by species of the genus Sporothrix spp. This study aimed to evaluate the genetic diversity and mating type distribution of clinical isolates of human sporotrichosis in Paraíba, Brazil, to better understand the population structure, epidemiology, and diversification of this pathogen, as well as to explore possible transmission routes. Methods: A total of 36 clinical isolates were morphologically identified, and clinical and demographic data were collected. Fungal DNA extraction was then performed, followed by species-specific PCR using markers targeting the calmodulin gene. The mating type idiomorph of the species was identified by PCR using primers targeting the MAT1-1 and MAT1-2 loci. Amplified Fragment Length Polymorphism (AFLP) was used to evaluate the genetic variability of Sporothrix spp. Results: The distribution of the disease identified that all cases occurred in João Pessoa and adjacent cities. From the 36 isolates, the majority (75%) being affected females, a prevalent occurrence of the lymphocutaneous form, and 98% zoonotic transmission were confirmed. Micro- and macromorphological structures were similar to each other, confirming Sporothrix spp. All isolates were confirmed as S. brasiliensis and the presence of a single sexual idiomorph, MAT1-2, was detected. The AFLP results indicate the possibility of the circulation of one or two genetic groups in João Pessoa and the metropolitan region. Conclusions: To our knowledge, this is the first time isolates in the Paraíba state are genetically characterised, all identified as Sporothrix brasiliensis. It is likely that this species in Paraiba originated from Rio de Janeiro, as all they possess the MAT1-2 idiomorph, indicating low intergenotypic variation.

Marker Assisted Selection: A Novel Approach for Crop Improvement

The process of marker-assisted selection, or marker-aided selection (MAS), selects a trait of interest indirectly by considering a marker linked to the trait (e.g., quality, productivity, disease resistance, and biotic stress tolerance) rather than the trait itself. This integration of marker data with traditional selection approaches has become a widely studied and recommended method for advancing breeding programs. This technique has been extensively researched and recommended for animal and plant breeding. Here, we combine marker data with conventional selection to choose the best candidate for a future breeding program. Nowadays, the majority of MAS investigations employ DNA-based markers such as microsatellites, single nucleotide polymorphisms (SNPs), random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism (RFLP). In this case, we discussed several kinds, techniques, and other features of marker Assisted Selection.

Exploring the Impact of High-Temperature Stress on Physiological, Anatomical, Molecular, Productive, and Quality Parameters in Diverse Olive Cultivars

Olive (Olea europaea L.) is one of the most important horticultural crops in Saudi Arabia. High temperatures are typical in most regions during the summer season. This study explored the effect of high temperatures on diverse olive cultivars (Manzanillo, Chemlali, and Picual) grown in three different geographical locations in Saudi Arabia (Sakaka, Basita, and Al-Qurayyat). Sakaka region was classified as a hot region, while the Basita and Al-Qurayyat regions were characterized by relatively mild or low temperatures. Genotypic components were assessed by studying Amplified Fragment Length Polymorphism (AFLP) molecular markers and physiological, anatomical, productive, and oil quality parameters. The evaluated genotypes displayed considerable variation across the three studied locations. Picual exhibited more heat tolerance, followed by Chemlali and Manzanillo. The high temperature in Sakaka negatively impacted critical characteristics related to olive growth, production, and quality. Results revealed that linoleic acid, oil acidity, total phenols, upper epidermis, palisade tissue, vascular bundle area, and mesophyll were the most appropriate parameters for predicting the impact of high temperatures. These traits were highly correlated with Sakaka and were highly and significantly associated with all the studied markers. Also, significant interactions between locations and genotypes showed that the means for most of the studied traits were the highest in Basita region.

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis.

Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Microscopic and molecular detection of Trichomonas vaginalis in outpatients seeking medical care in Upper Egypt.

Trichomoniasis remains one of the most significant sexually transmitted disease (STDs) for public health. The disease is caused by parasitic protozoa, Trichomonas vaginalis (T. vaginalis), which is often underestimated in tropical medicine. Despite its public health importance, the epidemiology and molecular characteristics of trichomoniasis in Egypt remains poorly understood, particularly in the southern part of the country (Upper Egypt). This study targeted exploring the genetic variability of T. vaginalis infections in Egyptian women living in Upper Egypt using restriction fragment length polymorphism (RFLP). This cross-sectional study included 150 female patients, who visited the gynaecology and obstetrics outpatient clinics at Sohag General Hospital between 2019 and 2022, exhibiting symptoms of trichomoniasis. Vaginal washout samples were collected from each patient and analyzed using three diagnostic techniques: direct wet mount microscopy, culture on TYM Diamond's medium, and PCR amplification and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) targeting the actin gene, which was applied to all 16 samples that tested positive in culture. The PCR-RFLP results were then visualized through agarose gels electrophoresis to detect DNA fragments. Out of 150 vaginal washout samples, 12 cases (8%) tested positive for T. vaginalis trophozoites via direct wet mount microscopy, while 16 samples (10.6%) were positive in culture. Additionally, PCR-RFLP analysis of the 16 culture-positive samples revealed that 13 samples were confirmed positive using this molecular method. The amplified products were digested with the restriction enzyme Hind II, yielding three DNA fragments of 60, 213, and 827 bp, which were then detected by agarose gel electrophoresis. Digestion with RsaI produced five fragments measuring 87, 103/106, 236, and 568 bp, while MseI digestion resulted in three distinct fragments of 204, 315, and 581 bp. This study provides robust baseline data on the prevalence and microscopic characteristics of T. vaginalis in Upper Egypt, while also presenting, for the first time, molecular detection and genotyping and revealed that genotype E is the only prevalent genotype in the region.

The Emergence of Cat-Transmitted Sporotrichosis Driven by Sporothrix brasiliensis in Piauí, Brazil.

In Brazil, the emergence of feline sporotrichosis, a neglected subcutaneous mycosis primarily transmitted by cats and caused by the fungus Sporothrix brasiliensis, has been monitored via molecular tools. This approach underscores the geographic expansion of this disease and highlights the critical role of molecular surveillance in understanding its epidemiology and guiding public health interventions. We investigated three domestic cats (Felis catus) with multiple skin lesions in Picos, Piauí, Brazil. The cats were examined, and samples were collected for laboratory diagnosis, including cytological evaluation, fungal culture, and molecular characterisation. The molecular analysis involved a one-tube multiplex probe-based qPCR assay for the diagnosis of Sporothrix species, mating-type determination, and amplified fragment length polymorphism (AFLP, EcoRI-GA/MseI-AG) to assess genetic similarity and potential origin. All three cats were diagnosed with sporotrichosis caused by the hypervirulent S. brasiliensis. A probable case of zoonotic transmission has been reported in a 12-year-old girl who developed Parinaud oculoglandular syndrome after contact with one of the cats with sporotrichosis. The molecular analysis revealed that the cat isolates were MAT 1-2 and formed a single cluster according to the AFLP analysis, suggesting direct transmission (cat-cat) and a potential founder effect. The isolates were also closely related to strains from Pernambuco and Southeast Brazil, indicating a possible introduction from these regions. Identifying S. brasiliensis in Piauí emphasises the need for increased awareness and control measures to prevent further spread. The predominance of the MAT1-2 idiomorphs and the genetic similarity among outbreak isolates suggest clonal expansion, which could have significant implications for public health and veterinary practices. Considering its zoonotic potential and environmental adaptability, a One Health approach is crucial for managing and controlling the spread of cat-transmitted sporotrichosis.

Internal validation of the Precision ID GlobalFiler NGS STR panel v2 kit with locus-specific analytical threshold, and with special regard to mixtures and low template DNA detection

We performed an internal laboratory validation of the Precision ID GlobalFiler NGS STR panel v2 kit to assist the introduction of the technology into the routine forensic casework practice. The study was designed and evaluated based not only on the key validation standards like sensitivity, stability, reproducibility, repeatability, mixture, and concordance, but we also tested the effect of reduced input DNA, we measured and applied locus-specific analytical threshold values, tested two different PCR cycle conditions, sequence artifacts and stutters were also analysed. During the study we also tested the new method on real casework samples. The sensitivity study confirmed that adding 500 pg template DNA for library preparation can be optimal at base PCR cycle number (that was 24), because the measured average heterozygote balance was not lower than 0.82, and each allele was detected above the analytical threshold. However, contrary to previous communications, increasing the PCR cycle numbers up to 28 has not resulted the significant elevation of the heterozygote imbalance. According to our results, raised PCR cycle condition (i.e. 28) is appropriate at or below 150 pg total input DNA. For most loci, the calculated AT was lower than the manufacturer’s recommended. Applying the newly established ATs with raised PCR cycle conditions the allele detection sensitivity and reliability increased. We observed allele dropouts only at the 15 pg template DNA experiments with 5 % frequency, that is better to previously published studies. This result indicates that this low amount of DNA (i.e. 15 pg) could be a minimum limit of template input for a potentially successful analysis. In the mixture study the minor contributor could be detected up to 1:19 mixture ratio. We detected minor alleles in all measurements and concentrations above the threshold if the template DNA were fixed and only SNP differences were observed between the same alleles of the contributors. To test concordance between the new method and traditional STR genotyping we analysed 58 Hungarian individual samples in parallel. Nearby the detected 248 different length-based alleles on the 31 loci in the sample pool we revealed additional 75 sequence variant alleles, that represent an approximately 23 % increase in the total number of observed alleles. The casework study confirmed that the Precision ID GlobalFiler NGS STR panel v2 kit is effective even in genotyping degraded samples with extremely low levels of DNA, if we apply elevated cycle number for library preparation and use locus-specific analytical thresholds.

Comparison of commercial targeted amplicon sequencing assays for human remains identification casework.

Targeted amplicon sequencing (TAS) facilitates the genotyping of forensically informative single nucleotide polymorphisms (SNPs) using massively parallel sequencing (MPS). For human remains identification, where any extracted DNA is likely to be degraded, TAS may succeed when short tandem repeat (STR) profiling using capillary electrophoresis fails. Further, as well as yielding identity information, SNPs can provide information about ancestry, phenotype, kinship and paternal lineage (Y chromosome haplotypes). Two TAS platforms were compared in this study: Ion AmpliSeq™ panels coupled with Ion Torrent sequencing on an Ion GeneStudio™ S5 Plus System, manufactured by Thermo Fisher Scientific, and the ForenSeq® Kintelligence Kit coupled with Illumina sequencing on the MiSeq FGx® Sequencing System, manufactured by QIAGEN. Four Ion AmpliSeq™ panels (Precision ID Identity, Precision ID Ancestry, DNA Phenotyping and HID Y-SNP) share 177 SNPs with the ForenSeq® Kintelligence Kit and all five were used to profile the DNA extracted from the petrous part of the temporal bone from six skeletonised cadavers. Of the 6 × 177 = 1,062 SNP genotype comparisons, 1,055 (99%) were concordant between the Ion AmpliSeq™ panels and Kintelligence Kit. Of the seven (< 1%) non-concordant SNPs, only three of them (0.3%) would have resulted in erroneous genotypes being reported as a result of allele dropout by either assay, using our optimised relative variant frequency windows for allele calling. We conclude that both the Ion AmpliSeq™ panels and the ForenSeq® Kintelligence Kit were suitable for TAS applied to the human remains in this study.

Genetic diversity and structure of Tunisian and Indian date palm (Phoenix dactylifera and sylvestris) cultivars and genotypes revealed by AFLP markers

The date palm breeding programs need to discover valid genetic fingerprints to characterize cultivars and assess their genetic diversity. This study assessed the genetic diversity among thirty-nine date palm cultivars from Tunisia (Phoenix dactylifera) and India (Phoenix sylvestris) by using six AFLP (Amplified Fragment Length Polymorphism) markers. 360 loci were amplified, with 127 loci polymorphic (34.35 %). The Jaccard's similarity coefficient ranged from 0.161 to 0.931, with the mean genetic distances of 0.568. AFLP's average marker index value was 7.28, with a resolving power of 10.91. The analysis of population structure showed two main clusters with a clear separation between Tunisian and Indian cultivars.Furthermore, the heatmap analysis allowed the identification of 10 bands specific to the Indian accessions, which were not detected in Tunisian genotypes. These loci could be linked to genes involved in adapting the species in Indian lands, which allowed the study of the genetic diversity of date palm resources of different origins, confirming the existence of at least two origins of domestication. Additionally, identifying AFLP loci specific to P. dactylifera and P. sylvestris will significantly contribute to breeding programs by exploiting species-specific polymorphisms.

Molecular epidemiology of seborrheic dermatitis/dandruff associated Malassezia species from northern India.

Malassezia is a commensal that sometimes becomes pathogenic under the influence of diverse factors. Several species of Malassezia are difficult to culture, making traditional methods of identification challenging. The problem with molecular typing of Malassezia in association with seborrheic dermatitis/dandruff (SD/D) arises due to the unavailability of these fastidious yeast cultures. The aim of the study was to investigate the association between fluorescent amplified fragment length polymorphism (FAFLP) genotypes, disease state (SD/D), and the geographic distribution of M. globosa, M. restricta, and M. arunalokei. In total, 154 isolates representing M. globosa (n=85), M. restricta (n=55), and M. arunalokei (n=14) from lesional/non-lesional areas of SD/D patients and healthy controls residing in the rural (n=77) and urban (n=77) areas of northern India were included. A strategy based on the FAFLP methodology was developed using two endonuclease enzymes (EcoRI and HindIII). M. globosa, M. restricta, and M. arunalokei formed 11, 3, and 2 FAFLP clusters, respectively. Disease-specific strains of M. restricta and M. arunalokei preferentially tend to cause SD/D. M. restricta and M. arunalokei showed less genetic variation. M.globosa showed higher genetic diversity. FAFLP clusters revealed the existence of geographically specific strains in M. restricta, M. arunalokei, and M. globosa. Our findings suggest that certain Malassezia strains are not only disease-specific but also geographically distinct.

Changes in global methylation patterns of Mytilus galloprovincialis exposed to microplastics

Microplastics (MPs) disturb the normal activity of aquatic organisms at different levels, causing physiological stress and altering feeding, growth, and reproduction. Alterations of epigenetic patterns due to exposure to MPs have scarcely been studied in invertebrates. In this study, Mytilus galloprovincialis mussels (N = 61) were intermittently exposed to different concentrations of pure polystyrene microbeads for three weeks. The concentrations used in this research were similar to those currently found in certain polluted environments (E1), as well as higher doses to which mussels could be further exposed (E2 and E3). After exposure period, the global methylation patterns were investigated using Amplified Fragment Length Polymorphism (AFLPs). Significantly lower methylation was found in exposed groups compared to the control group. The level of hypomethylation increased with the concentration of microbeads. Similar results were found from field samples inhabiting two sites differentially MPs-polluted. The implications of this discovery were analysed and discussed, noting the already known effects of MPs on metabolism and cell division. Further studies on this and other sentinel organisms are recommended to understand the response of the aquatic species to the currently increasing MPs pollution.

Taxonomic identification of Nigerian Terminalia superba and Terminalia ivorensis Combretaceae) using AFLP molecular markers

Terminalia superba and T. ivorensis are difficult to identify with morphological markers. Molecular characterization is very effective to correct the delimitation of timber species, especially when the discriminatory power of morphological markers is weak. In this study, four amplified fragment length polymorphism (AFLP) marker combinations were used to determine the taxonomic relationship among a total of 33 accessions of the genus Terminalia, consisting of two species, Terminalia ivorensis (12 accessions) and T. superba (21). The primer combinations produced a total of 740 fragments out of which 619 (83.6%) were polymorphic. Among all the primer combinations used, E-ACT/M-CTC was the most informative, which generated 11 unique markers for clear identification of the two species. The average polymorphic information content (PIC) ranged from 0.24 (E-ACT/M-CAT) to 0.28 (E-ACT/M-CTC) while the resolving power (RP) varied from 40.73 (E-AAC/M-CTA) to 63.76 (E-ACT/M-CTC). The genetic difference between the two species was significant (PhiPT = 0.610, p&lt;0.001). All 33 accessions were delimited by both the UPGMA cluster and principal component analysis. Based on the results, it was concluded that E-ACT/M-CTC is the best among the marker combinations used for molecular study of the two tree species and used significantly to distinguish them. STRUCTURE analysis identified two accessions of Terminalia superba with alleles derived from T. ivorensis as well as revealed five putative hybrid accessions, which requires further genetic investigation to substantiate the finding.

A wide range of abiotic habitat factors and genetic diversity facilitate expansion of Trapa natans within its native range

Climate change and intense human activity are exacerbating changes in species' ranges. While the rapid spread of invasive alien species is well documented worldwide, the phenomenon of the spread of native species is poorly understood. To explain the problem of rapidly spreading species in the changing world, it is necessary to understand their ecology, genetic diversity and habitat limitation. The aim of our study was to analyze the ecological requirements and genetic diversity in the population of the macrophyte Trapa natans s. l., an invasive alien species in North America but native in Europe and Asia. We investigated the populations in its native range (Central and Northeastern Europe), where the species is defined as rare or extinct. We found the occurrence of T. natans in Northeastern Europe aquatic habitats where, up to now, it was described as an extinct species. The results of our environmental studies showed that the species has a wide range of tolerance to habitat conditions and lives in medium to highly nutrient-rich water with low and high salinity. Using Amplified Fragment Length Polymorphism (AFLP) analysis, we revealed high genetic variability within populations with relatively limited differentiation between populations. We showed that some populations are highly diverse (possibly refugia; Central Europe) and others are homogeneous (new sites, commercial reintroduction; Northeastern Europe). Conservation status of T. natans in its native range should be reconsidered, as the species has spread rapidly in recent decades and could be detrimental to aquatic habitats. The conclusion is that expansion/invasion can start from small populations, but under favorable conditions these populations spread rapidly. The introduction of species (even native) should be done carefully, if at all, as uncontrolled introduction to new locations, e.g. private ponds, could be the start of dispersal (native habitats) or invasion (non-native area).

Troubleshooting apparent allelic dropout using two methods: of next generation sequencing

Troubleshooting apparent allelic dropout using two methods: of next generation sequencing

Genomic Association and Prediction Study for Yield Traits in a Sugarcane (Saccharum spp. Hybrids) Mapping Population ‘LCP 85‐384’

ABSTRACTSugarcane (Saccharum spp. hybrids) are complex polyploid and aneuploid interspecific hybrids with 110–130 chromosomes. A traditional sugarcane breeding cycle takes 13–15 years and involves multiple years and locations testing of yield. To identify molecular markers associated with yield‐related traits, the LCP 85‐384 cultivar and its mapping population of 263 self‐progenies were planted in two randomly replicated field plots. The mapping population was genotyped with amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR) and target region amplification polymorphism (TRAP) markers. Data on plant height, stalk number, stalk diameter and stalk weight were collected. A large variation was observed for each trait. A genome‐wide association study (GWAS) was conducted using mixed linear model (MLM), generalized linear model (GLM) and single marker regression (SMR) programmes of TASSEL 5 and FarmCPU of GAPIT 3. A total of 64 yield trait‐associated alleles were identified, including 11 for stalk number, 36 for stalk weight, 21 for stalk diameter and 5 for plant height. Of the 64 alleles, seven were linked to two traits and one to three traits. Genomic prediction (GP) was also performed by cross‐prediction with five models, namely, ridge regression best linear unbiased prediction (rrBLUP), Bayesian ridge regression (BRR), Bayesian A (BA), Bayesian B (BB), and Bayesian least absolute shrinkage and selection operator (BL). Prediction accuracy (r value) reached 0.40 for plant height, 0.36 for stalk number, 0.44 for stalk diameter and 0.54 for stalk weight with the standard errors from 0.009 to 0.012. Once verified, these markers will be a valuable tool to aid in the selection of yield‐related traits in sugarcane improvement programmes.

Genetic Diversity and Population Genetic Structure of Jatropha curcas L. Accessions from Different Provenances Revealed by Amplified Fragment-Length Polymorphism and Inter-Simple Sequence Repeat Markers

The genetic diversity and structure of 17 populations of J. curcas, including 92 accessions from different provenances (tropical and subtropical), were investigated and effectively evaluated using twelve inter-simple sequence repeats (ISSRs) and seven pairs of florescence-amplified fragment-length polymorphism (AFLP) primers. Genetic diversity, at the overall level among populations of J. curcas based on the ISSR markers, showed that the observed number of alleles (Na) was 1.593, the effective number of alleles (Ne) was 1.330, Nei’s gene diversity (H) was 0.200, Shannon’s information index (I) was 0.303, and the percentage of polymorphic loci was 59.29%, indicating moderate genetic diversity between and within the different populations of J. curcas. Based on the genetic diversity analysis of AFLP markers, there were 1.464 (Na) and 1.216 (Ne) alleles, Nei’s gene diversity (H) was 0.132, Shannon’s information index (I) was 0.204, and the percentage of polymorphic loci was 46.40%. The AMOVA analysis showed that this large variance was due to differences within the populations, with genetic distinctions and limited gene flow among those from varied regions. The 17 populations were clustered into five main groups via UPGMA clustering analysis based on Nei’s genetic distance, and the genetic relationships among the populations exhibited no significant correlations with geographical provenances. The genetic variation among Chinese populations of J. curcas distributed in dry-hot valley areas was remarkable, and the American germplasm presented with distinct genetic differentiation.

Performance comparison of a previously validated microhaplotype panel and a forensic STR panel for DNA mixture analysis

Short Tandem Repeats (STRs) are the most widespread markers in forensic genetics. However, STR stutter peaks can mask alleles from a minor contributor when analysing mixtures, hindering the interpretation of complex profiles. In this study we compared the performance of a previously described panel of microhaplotypes (MHs), an alternative type of forensic marker, against a standard STR kit. The parameters evaluated included: capability of determining the minimum number of contributors in the mixture; percentages of allele drop-outs and drop-ins; retrieval of alleles belonging to the minor contributor, and estimation of likelihood ratio (LR) values. In addition, the capacity of EuroForMix software to estimate each donor’s percentage of contribution was tested, as well as the impact on results when using manually, or automatically prepared libraries. The MH panel showed better performance than STRs for the detection of 2-contributor mixtures, but the lower degree of polymorphism per MH marker hindered the task of deconvolution with multiple contributors. MHs presented higher drop-in rates and lower drop-out rates, a higher capability to recover the minor contributor’s alleles and provided higher LR values than STRs, likely due to the much higher number of loci combined in the panel. Estimations of contributor ratios using EuroForMix showed promising results and marginal differences were found in these values between manually and automatically prepared libraries. Overall, results showed that the mixture detection performance of the MH panel was better or equal to the standard forensic autosomal STR panel, indicating microhaplotypes are informative markers for this purpose.

The Emergence of New Sporothrix brasiliensis Genotypes in Current Epidemic of Sporotrichosis in Southeastern Brazil.

Zoonotic sporotrichosis caused by Sporothrix brasiliensis has become the main subcutaneous mycosis in Brazil. Minas Gerais (MG) is located in southeast Brazil and since 2015 has experienced an epidemic of zoonotic sporotrichosis. This study aimed to reconstruct the epidemiological scenario of sporotrichosis from S. brasiliensis in recent epizooty in the Metropolitan Region of Belo Horizonte (MRBH), MG. A total of 95 Sporothrix spp. isolates (Sporothirx brasiliensis n = 74, S. schenckii n = 11 and S. globosa n = 10) were subjected to Amplified Fragment Length Polymorphism (AFLP) genotyping and mating-type analysis to determine genetic diversity and population structure. Of these, 46 S. brasiliensis isolates were recovered from animals (cats n = 41 and dogs n = 5) from MRBH. Our study describes the high interspecific differentiation power of AFLP-based genotyping between the main phylogenetic Sporothrix groups. S. brasiliensis presents high genetic variability and pronounced population structure with geographically focused outbreaks in Brazil. The genetic groups include older genotypes from the prolonged epidemic in Southeast (Rio de Janeiro and São Paulo), South (Rio Grande do Sul), Northeast (Pernambuco) and new genotypes from the MRBH. Furthermore, we provide evidence of heterothallism mating strategy in pathogenic Sporothrix species. Genotypes originating in Rio de Janeiro and Pernambuco carry the predominant MAT1-2 idiomorph as opposed to genotypes from Rio Grande do Sul, which have the MAT1-1 idiomorph. We observed an overwhelming occurrence of MAT1-1 among MRBH isolates. Our study provides clear evidence of the predominance of a genetic group profile circulating in animals in Minas Gerais, independent of that disseminated from Rio de Janeiro. Our data can help us understand the genetic population processes that drive the evolution of this fungus in Minas Gerais and contribute to future mitigation actions for this ongoing epidemic.

Pythium and Globisporangium species associated with cucumber rhizosphere causing damping-off and their effects on cucumber seed decay in Oman.

Pythium sensu lato (s.l.) is a pathogenic oomycete. The present study was conducted to isolate and identify Pythium s.l. species associated with the rhizosphere and roots of greenhouse-growing cucumbers showing damping-off symptoms in 10 Omani governorates (provinces). A total of 166 isolates were recovered from 276 rhizosphere soil and root samples and were identified based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit I (COX I) gene region. Pythium aphanidermatum, P. myriotylum, Globisporangium spinosum, Globisporangium sp.1 (isolates Kb003/PySyCu-1 and Kb004/PySyCu-2), and Globisporangium sp.2 (isolate Ib002R) were identified. Among these species, P. aphanidermatum was the most abundant species, represented by 143 isolates (86.1%), followed by G. spinosum with 18 isolates (10.8%), Globisporangium sp.1 and P. myriotylum each with 2 isolates (2.4%), and Globisporangium sp.2 with 1 isolate (0.6%). Pathogenicity tests were also conducted for 38 isolates, including P. aphanidermatum (25), P. myriotylum (2), Globisporangium sp.2 (1), G. spinosum (8), and Globisporangium sp.1 (2). Among the tested isolates, only Globisporangium sp.2 isolate was avirulent, and none of the seeds were rotted at the end of the treatment. However, the other species induced the symptoms of seed decay with the incidence ranged from 86.7 to 100%. Phylogenetic analyses were conducted based on 222 ITS and 53 COX I sequences, and confirmed morphological identification. In addition, the genetic diversity of 93 P. aphanidermatum isolates was assessed via the amplified fragment length polymorphism (AFLP) method. The analysis produced 93 genotypes and 449 polymorphic loci. Pythium aphanidermatum populations were found to have moderate levels of genetic diversity (H = 0.2) and a moderate Shannon information index (I = 0.3793). Analysis of molecular variance (FST = 0.1, P = 0.0) revealed a moderate level of genetic differentiation among P. aphanidermatum isolates between Oman governorates. The sensitivity of 15 P. aphanidermatum isolates was evaluated against hymexazol at different concentrations (10, 100, and 1000ppm). The results revealed that P. aphanidermatum could grow well at concentrations of up to 100ppm hymexazol. However, hymexazol at 1000ppm retarded the growth of P. aphanidermatum. This study showed that P. aphanidermatum is the most prevalent species in greenhouses in Oman and exhibited a moderate level of genetic diversity. Most of the isolates exhibited differences in tolerance to hymexazol but showed no resistance.