VNTR Markers Research Articles

Multiple-Locus Variable-Number Tandem Repeat Analysis of Helicobacter pylori Strains Isolated from Biopsy Samples

Background: Multiple-locus variable-number tandem-repeat analysis (MLVA) is a reliable, fast, and useful method for conducting phylogenetic and genotyping analyses. However, its application in Helicobacter pylori typing is limited. Objectives: This study aimed to determine the genetic diversity of H. pylori strains isolated from biopsy samples using six variable loci in MLVA typing. Methods: Over one year, we collected 70 non-duplicative biopsy samples from patients at Rohani Hospital, Babol, in northern Iran. After DNA extraction, H. pylori strains were confirmed through the amplification of the glmM gene in the PCR test. Multiple-locus variable-number tandem-repeat analysis typing utilized six VNTR markers: VNTR-180, VNTR-263, VNTR-614, VNTR-607, VNTR-2181, and VNTR-2457. The PHYLOViZE software was employed to construct the phylogenetic tree. Results: Out of the 70 specimens, 43 tested positive for H. pylori. The phylogenetic tree revealed some strains with completely similar MLVA patterns. Our findings indicated that type 2 is the dominant type in this region, with most patients infected with this type exhibiting gastritis. Conclusions: The high allelic variability observed at VNTR-614, -607, and -2457 indicates high intraspecific variability for these markers in all isolates from our region. Consequently, these VNTRs can be considered ideal sites for typing.

Development of a technique for molecular typing of <i>Bacillus anthracis</i> strains using new VNTR and INDEL markers

Introduction. Bacillus anthracis, the pathogen of a particularly dangerous zoonotic disease known as anthrax, requires strict epidemiological control and is characterized by high genetic homogeneity, which necessitates the development of genotyping methods. The aim of the study were to to find and characterize the VNTR and INDEL loci of B. anthracis and to develop on their basis a genotyping technique by PCR with electrophoretic detection of the results. Materials and methods. Marker search and phylogenetic analysis were performed on a sample of 388 genomes of B. anthracis strains, 322 from the GenBank collection (RefSeq) and 66 from the collection of the Stavropol Anti-Plague Institute of Rospotrebnadzor. Phylogenetic analysis was performed on the basis of SNP crustal alignment using the Parsnp program. The search for markers was carried out using the Mauve program and author's scripts in Python. PCR was performed using a ScreenMix-HS kit (CJSC "Eurogen", Russia). Results. Genomic variations of B. anthracis strains (SNP — 25,664, SNR — 14,387, VNTR — 693, INDEL — 14,667) were found, bioinformatic analysis of which revealed nine new VNTR and six INDEL molecular markers most suitable for genotyping. The genetic (allelic) variants of the markers are described. Primers were selected for the found markers and a PCR protocol with detection by electrophoresis in agarose gel was developed. When typing using VNTR markers was applied, the strains were divided into nine clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.005/006, A.Br.008/009 (Tsiankovskii), A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.008/009 (strain 228/269), B.Br.001/002. When typing using INDEL markers, the strains were divided into six clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.008/009(Tsiankovskii), B.Br.001/002(B.Br.014), as well as a cluster comprising several genetic lineages: A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.005/006 и B.Br.001/002. Conclusion. The use of the developed methodology for the identification of variable VNTR and INDEL loci makes it possible to reliably determine the phylogenetic position of B. anthracis strains and is promising for use in the epidemiological investigation of anthrax outbreaks.

Molecular biology techniques for assessing the loss of HLA heterozygosity after allogeneic hematopoietic stem cell transplantation in children with acute leukemia

According to several observations, up to a third of post-transplant relapses in childhood acute leukemia are associated with the loss of heterozygosity of the major histocompatibility complex (HLA). Furthermore, the inefficacy of the graft-versus-leukemia reaction, as evidenced by the lack of therapeutic effect from the infusion of donor lymphocytes, indicates the need for timely detection of this marker to change the treatment strategy in the post-transplant period. To detect the loss of HLA heterozygosity, the method using the commercial KMR-HLA system and analysis using next-generation sequencing (NGS), as well as the method based on the analysis of highly polymorphic STR and VNTR markers located in the HLA loci region on the short arm of chromosome 6, are widely used. The primary objective of our study was to compare the informativeness of these approaches in diagnosing HLA heterozygosity loss in children during the post-transplant period. The obtained data on the frequency of detecting HLA heterozygosity loss were comparable to the literature data and constituted 23 % of cases of post-transplant relapse of B-cell acute lymphoblastic leukemia, 33 % of cases of T-cell acute lymphoblastic leukemia, and 23% of cases of acute myeloid leukemia. We also demonstrated that the method based on STR marker analysis has sensitivity comparable to allele-specific PCR and NGS sequencing methods. Meanwhile, preliminary sorting of the blast population increases the sensitivity of STR analysis and can be recommended in routine practice.

Genotypic Analysis of the Population Structure in Malassezia globosa and Malassezia restricta

The molecular characterization of Malassezia spp. isolates from animals and humans has not been thoroughly studied. Although a range of molecular methods has been developed for diagnosing Malassezia species, they have several drawbacks, such as inefficiency in differentiating all the species, high cost and questionable reproducibility. The present study aimed to develop VNTR markers for genotyping Malassezia isolated from clinical and animal samples. A total of 44 M. globosa and 24 M. restricta isolates were analyzed. Twelve VNTR markers were selected on seven different chromosomes (I, II, III, IV, V, VII and IX), six for each Malassezia species. The highest discriminatory power for a single locus was obtained with the STR-MG1 marker (0.829) and STR-MR2 marker (0.818) for M. globosa and M. restricta, respectively. After the analysis of multiple loci, 24 genotypes were noted among 44 isolates in M. globosa, with a discrimination index D of 0.943 and 15 genotypes were noted among 24 isolates in M. restricta, with a discrimination index D of 0.967. An endogenous infection was detected in two patients. Different genotypes of M. globosa strains colonized one patient. Interestingly, VNTR markers analysis revealed a carriage between a breeder and his dog in three cases for M. globosa and two for M. restricta. The FST (0.018 to 0.057) values indicate a low differentiation between the three populations of M. globosa. These results suggest a dominant clonal mode of reproduction in M. globosa. The typing of M. restricta showed a genotypic diversity of the strains, which can cause various skin pathologies. However, patient five was colonized with strains having the same genotype collected from different body parts (back, shoulder). VNTR analysis was capable of identifying species with high accuracy and reliability. More importantly, the method would facilitate monitoring Malassezia colonization in domestic animals and humans. It was shown that the patterns are stable and the method is discriminant, making it a powerful tool for epidemiological purposes.

New genetic markers for <i>Burkholderia pseudomallei</i> strains typing

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease in humans and animals that may be asymptomatic long-term and quickly develop to pneumonia and septicemia in immunocompromised state and due to predisposing factors. Melioidosis is endemic in countries with tropical and subtropical climates, where B. pseudomallei is a part of the soil and water microbiota of stagnant water bodies, as well as the plant rhizosphere. The recording of imported melioidosis cases in countries with temperate climate zone, along with the continued threat of using this pathogen as a bioterrorism agent, indicate the relevance of research aimed at developing modern methods for its diagnosing and typing. Current research in the intraspecific differentiation of pathogenic strains of infectious diseases tends to use two or more types of molecular markers. A promising direction for the B. pseudomallei strains genotyping is based on using a combination of VNTR loci, which provides a high discriminating ability of the method of multi-locus variable tandem repeat number analysis (MLVA), and slowly evolving single nucleotide polymorphisms (SNPs). The aim of this work was to identify new VNTR and SNP loci suitable for use as genetic markers in molecular typing of the melioidosis causative agent. 20 strains of B. pseudomallei from the collection of the Volgograd Plague Control Research Institute and whole genome sequences of 85 B. pseudomallei strains from the GenBank NCBI database were analyzed. While typing melioidosis causative agent strains from the GenBank NCBI database for 4 VNTR loci, 74 genotypes were identified, of which 64 were unique (HunterGaston index 0.997). It was found that the high allelic polymorphism of VNTR loci limited a potential to determine the geographical and phylogenetic relationships of B. pseudomallei isolates by using the MLVA-4 scheme, and the identified null alleles increased the risk of homoplasia. In this regard, the MLVA-4 scheme was supplemented with a VNTR marker in the BPSS1974 locus encoding a collagen-like protein as well as with SNP markers in the genes of lipid A biosynthesis lauroyl acyltransferase, RNA polymerase sigma factor RpoH, and glutamine amidotransferase. For amplification of the select loci, primers and probes were designed as part of the developed scheme, which were tested in the typing of B. pseudomallei collection strains. According to the results of a cluster analysis for 105 strains of the melioidosis causative agent, an integrated approach turned out to be optimal, allowing differentiation of strains within SNP groups based on VNTR profiles. The methodological approach developed for B. pseudomallei genetic typing, based on a comprehensive analysis of 4 SNP- and 5 MLVA-markers in our modification, allowed to differentiate strains according to the geographical regions of their origin and establish the clonal origin of isolates upon revealing cases of melioidosis.

Existence of viable Mycobacterium leprae in natural environment and its genetic profiling in a leprosy endemic region

IntroductionMolecular epidemiology of leprosy is very important to study leprosy transmission dynamics and to enhance our understanding of leprosy in endemic areas by utilizing the molecular typing method. Nowadays our understanding of leprosy transmission dynamics has been refined by SNP typing and VNTR marker analysis of M. leprae strains.ObjectiveThis study was carried out to find out the presence of viable M. leprae in the soil and water samples from residing areas of leprosy patients staying in different blocks of Purulia district of West Bengal, understanding their genotypes and compared with that of M. leprae present in patients.Material and methodsSlit-skin smear (SSS) samples (n=112) were collected from the active multibacillary leprosy patients from different blocks of leprosy endemic area. Soil samples (n=1060) and water samples (n=620) were collected from residing areas of leprosy patients. SNP subtyping was performed by PCR followed by sequencing. Multiplex PCR was performed using fifteen ML-VNTR loci and results were analysed.ResultsWe observed high PCR positivity in soil samples (344 out of 1060; 32%) and water samples (140 out of 620; 23%). These PCR positive samples when further screened for viability, it was observed that 150 soil samples (44%) and 56 water samples (40%) showed presence of 16S rRNA. SNP typing of M. leprae revealed presence of predominantly type 1. SNP subtype 1D (83%) was most prevalent in all the blocks of Purulia followed by subtype 1C (15%) and subtype 1A (2%). SNP subtype 2F was noted in only one sample. SNP and VNTR combination showed presence of similar strain type in certain pockets of Purulia region which was responsible for transmission.ConclusionPresence of viable M. leprae in the environment, and presence of SNP Type 1 M. leprae in patients and environment suggests both environment and patients play a role in disease transmission.

Mhc-B haplotypes in “Campero-Inta” chicken synthetic line

The major histocompatibility complex-B (MHC-B) in chickens is a cluster of genes located on chromosome 16. The chicken MHC-B is known to be highly associated with resistance to numerous diseases caused by viruses, bacteria, and parasitic pathogens. Since the level of resistance varies with MHC-B haplotypes, identification and classification of different haplotypes within lines is important for sustaining lines. The "Campero-INTA" chicken breed is a meat-type free-range poultry breed that was developed specifically for small producers in Argentina. Campero-INTA was started by selection in populations produced by crosses between a variety of established lines. MHC-B variation was examined in 65 samples obtained in 2002 using the VNTR marker LEI0258, a marker for MHC-B region. These samples plus and an additional 55 samples from 2018 were examined for variation using the MHC-B specific SNP panel that encompasses ∼230,000bp of the MHC-B region. Eleven MHC-B SNP haplotypes with 6 LEI0258 alleles were identified in the 120 samples representing the Campero-INTA AH (male) line. Seven haplotypes originate from the breeds originally used in the development of Campero-INTA AH line. Two appear to be recombinant haplotypes. The origin of the remaining 2 is not known, but may be associated with genes introduced from crosses with the Fayoumi breed conducted more recently to sustain the line.

Detection of carrier state and genetic diversity of Theileria parva in ECF-vaccinated and naturally exposed cattle in Tanzania.

Infection and Treatment Method (ITM) has been practiced in Tanzania for over 20 years as a prevention measure against East Coast Fever disease. It is known that ITM, like natural ECF infection, leads to a carrier state, whereby vaccinated cattle become asymptomatic carriers of the parasite. It is expected that ECF vaccination using ITM also leads to generation of combinations of vaccine specific Theileria parva and local strains that circulate in the field what contributes to an unknown level of parasite diversity. Moreover, the long term impact of ITM on carrier state and parasite diversity in cattle are largely unknown. To address this question blood was collected from ECF-vaccinated (n = 239) and unvaccinated (n = 97) cattle from Loiborsoit, Emboreet, Esilalei, Manyara ranch and Mswakini villages in the Maasai steppe of northern Tanzania, as well as Mruazi and Leila farms in Tanga in eastern Tanzania. Screening for T. parva using nested PCR revealed an overall prevalence of T. parva to be 34.5%, with a significant higher prevalence among ECF-vaccinated cattle. Using three VNTR markers (ms2, ms5 and MS7) higher parasite genetic diversity in terms of higher number of alleles and expected heterozygosity was shown in vaccinated than unvaccinated cattle. These parameters were highest in cattle from Manyara ranch. Nevertheless, the principle component analysis (PCoA) showed no distinct clustering patterns as most T. parva alleles clustered together throughout the four quadrants implying parasite homogeneity among the sampled populations. However, some of the parasite alleles closely clustered with Muguga vaccine alleles in two of the quadrants, consistent with closer genetic relatedness between the vaccine strains and the T. parva populations from the Maasai steppe. Likewise analysis of molecular variance (AMOVA) revealed most of the genetic variation (93%) being contained within populations with only 7% being among populations. This study therefore confirms the role of ECF vaccination in enhancing carrier state and T. parva diversity in vaccinated cattle populations. Higher T. parva diversity may play an important role in carrier cattle by way of restricting breakthrough infections from field parasite strains.

Genotyping of Mycobacterium leprae for better understanding of leprosy transmission in Fortaleza, Northeastern Brazil.

Leprosy is endemic in large part of Brazil with 28,761 new patients in 2015, the second largest number worldwide and reaches 9/10.000 in highly endemic regions and 2.7/10.000 in the city of Fortaleza, Ceará, Northeast Brazil. For better understanding of risk factors for leprosy transmission, we conducted an epidemiologic study supplemented by 17 locus VNTR and SNP 1–4 typing of Mycobacterium leprae in skin biopsy samples from new multibacillary (MB) patients diagnosed at a reference center in 2009 and 2010. Among the 1,519 new patients detected during the study period, 998 (65.7%) were MB and we performed DNA extraction and genotyping on 160 skin biopsy samples, resulting in 159 (16%) good multilocus VNTR types. Thirty-eight of these patients also provided VNTR types from M. leprae in nasal swabs. The SNP-Type was obtained for 157 patients and 87% were of type 4. Upon consideration all VNTR markers, 156 different genotypes and three pairs with identical genotypes were observed; no epidemiologic relation could be observed between individuals in these pairs. Considerable variability in differentiating index (DI) was observed between the different markers and the four with highest DI [(AT)15, (TA)18, (AT)17 and (GAA)21] frequently demonstrated differences in copy number when comparing genotypes from both type of samples. Excluding these markers from analysis resulted in 83 genotypes, 20 of which included 96 of the patients (60.3%). These clusters were composed of two (n = 8), three (n = 6), four (n = 1), five (n = 2), six (n = 1), 19 (n = 1) and 23 (n = 23) individuals and suggests that recent transmission is contributing to the maintenance of leprosy in Fortaleza. When comparing epidemiological and clinical variables among patients within clustered or with unique M. leprae genotypes, a positive bacterial index in skin biopsies and knowledge of working with someone with the disease were significantly associated with clustering. A tendency to belong to a cluster was observed with later notification of disease (mean value of 3.4 months) and having disability grade 2. A tendency for lack of clustering was observed for patients who reported to have lived with another leprosy case but this might be due to lack of inclusion of household contacts in the study. Although clusters were spread over the city, kernel analysis revealed that some of the patients belonging to the two major clusters were spatially related to some neighborhoods that report poverty and high disease incidence in children. Finally, inclusion of genotypes from nasal swabs might be warranted. A major limitation of the study is that sample size of 160 patients from a two year period represents only 15% of the new patients and this could have weakened statistical outcomes. This is the first molecular epidemiology study of leprosy in Brazil and although the high clustering level suggests that recent transmission is the major cause of disease in Fortaleza; the existence of two large clusters needs further investigation.

Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

Intrapatient comparison of Mycobacterium leprae by VNTR analysis in nasal secretions and skin biopsy in a Brazilian leprosy endemic region

This study compares the strains of genotypes of M. leprae from nasal secretions (NS) and skin biopsy (SB) in the same patient, supplementing conventional epidemiology to gain insight into the infection of leprosy in Fortaleza, Brazil. The sample consisted of 38 newly diagnosed leprosy patients attending the National Reference Center of Dermatology Dona Libania (CDERM), in Fortaleza, who tested positive for M. leprae by PCR in DNA extracts of nasal secretions. DNA was also extracted from skin biopsy (SB) scrapings of each patient and used for multiplex PCR amplification of M. leprae VNTR loci. The number of repeats at 15 loci were determined by the fragment length analysis method. Locus VNTR genotypes were achieved in 38 NS, and in 38 SB specimens. M. leprae strains differed in their genotypes in paired specimens in all but two of 38 patients. The genotype similarity in the remainder ranged from 53% to 87%. M. leprae 15 VNTR loci genotypes of paired nasal and biopsy skin samples from five patients were identical, while as many as seven loci differed in the 33 other patients. When the NS and biopsy genotypes were pooled and compared, it was found that there was a great variability among different VNTR markers. It is important to investigate other molecular markers suitable for typing genetic variations of the bacilli.

Successful Linkage Analysis in Classical Phenylketonuria Families Followed by Direct Sequencing and Mutation Detection.

Phenylketonuria (PKU) is the most common disorder of inborn errors of metabolism. Prevalence of PKU is about 1:10000 live births; however, due to high rate of consanguinity in the Middle East and North of Africa the prevalence of PKU is more than in other areas. It is estimated between 1:2600 in Turkey and 1:3672 in Iran. The best way to identify carriers in PKU families is studying causative mutations, but this approach could be costly and time consuming. As a result, linkage analysis can be considered as a reliable way to detect carriers. Ten non-related classical PKU families from Iran-Fars province were enrolled. Linkage analysis was performed through application of highly linked genetic markers to the PAH gene (VNTR, PAHSTR, and XmnI marker) with new designed primers for polymerase chain reaction (PCR). Reliability of approach was assessed by Sanger sequencing, mutation detection, and capillary electrophoresis (CE). Through application of linkage analysis, nine out of ten families were genotyped successfully. Heterozygosity of chromosome 12 was not detected in any of the enrolled PKU patients. Specificity of new designed primers for linkage analysis was confirmed by Sanger dideoxy sequencing. Results obtained from linkage analysis were confirmed by direct sequencing and detecting causative mutations in half of the genotyped families. All the results were the same as the linkage analysis results. Labeled primers were capable and linkage analysis by CE was successful. Linkage analysis is a powerful and reliable approach for detecting carriers in PKU families which have not been previously screened for causative mutations. We suggest studying the feasibility of the approach in preliminary diagnosis of PKU and confirming autozygosity of chromosome 12, prenatal diagnosis, and preimplantation genetic testing. Also, we recommend using labeled primers for constructing faithful local PKU associated haplotype databases to provide fast, cheap, and reliable detection of causative mutations in new cases of hyperphenylalaninemia.

Endemic and epidemic Acinetobacter baumannii clones: a twelve-year study in a tertiary care hospital.

BackgroundNosocomial outbreaks of multidrug-resistant Acinetobacter baumannii are of worldwide concern. Using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and multiple locus variable number tandem repeat sequence (VNTR) analysis (MLVA), the present work examines the genetic diversity of the endemic and epidemic A. baumannii clones isolated in a single hospital over a twelve-year period.ResultsPFGE analysis of 405 A. baumannii-calcoaceticus complex isolates detected 15 A. baumannii endemic/epidemic PFGE types (EE1 to EE15) that grouped into five clusters: EE1-EE8, EE9, EE10, EE11 and EE12-EE15. The MLST sequence type (ST) distributions were: international clone II (ST-2) 60%, international clone III (ST-3) 26.7%, ST-15 6.7%, and ST-80 6.7%. MLVA-8Orsay returned 17 allelic profiles. The large (L) VNTR marker profiles were fully concordant with the detected STs, and concordant with 14 up to 15 PFGE types. Imipenem resistance was detected in five PFGE types; the prevalence of the blaOXA-58-like and blaOXA-40-like genes was 60% and 40% respectively.ConclusionsPFGE proved to be a vital tool for analysis of the temporal and spatial distribution of the clones. MLST and the VNTR L-markers grouped the isolates into clonal clusters. The wide diversity of MLVA small (S)-markers, however, did not permit clustering. The present results demonstrate the persistence of several endemic PFGE types in the hospital, the involvement of some of them in outbreaks, and the inter hospital transmission of extensively drug-resistant ST-15 and ST-80.

Development of serotype-specific monoclonal antibodies against botulinum neurotoxins A, B and E, based on a trivalent immunization protocol and simultaneous differential robotic screen

Development of serotype-specific monoclonal antibodies against botulinum neurotoxins A, B and E, based on a trivalent immunization protocol and simultaneous differential robotic screen

High-resolution genotyping and mapping of recombination and gene conversion in the protozoan Theileria parva using whole genome sequencing

BackgroundTheileria parva is a tick-borne protozoan parasite, which causes East Coast Fever, a disease of cattle in sub-Saharan Africa. Like Plasmodium falciparum, the parasite undergoes a transient diploid life-cycle stage in the gut of the arthropod vector, which involves an obligate sexual cycle. As assessed using low-resolution VNTR markers, the crossover (CO) rate in T. parva is relatively high and has been reported to vary across different regions of the genome; non-crossovers (NCOs) and CO-associated gene conversions have not yet been characterised due to the lack of informative markers. To examine all recombination events at high marker resolution, we sequenced the haploid genomes of two parental strains, and two recombinant clones derived from ticks fed on cattle that had been simultaneously co-infected with two different parasite isolates.ResultsBy comparing the genome sequences, we were able to genotype over 64 thousand SNP markers with an average spacing of 127 bp in the two progeny clones. Previously unrecognized COs in sub-telomeric regions were detected. About 50% of CO breakpoints were accompanied by gene conversion events. Such a high fraction of COs accompanied by gene conversions demonstrated the contributions of meiotic recombination to the diversity and evolutionary success of T. parva, as the process not only redistributed existing genetic variations, but also altered allelic frequencies. Compared to COs, NCOs were more frequently observed and more uniformly distributed across the genome. In both progeny clones, genomic regions with more SNP markers had a reduced frequency of COs or NCOs, suggesting that the sequence divergence between the parental strains was high enough to adversely affect recombination frequencies. Intra-species polymorphism analysis identified 81 loci as likely to be under selection in the sequenced genomes.ConclusionsUsing whole genome sequencing of two recombinant clones and their parents, we generated maps of COs, NCOs, and CO-associated gene conversion events for T. parva. The data comprises one of the highest-resolution genome-wide analyses of the multiple outcomes of meiotic recombination for this pathogen. The study also demonstrates the usefulness of high throughput sequencing typing for detailed analysis of recombination in organisms in which conventional genetic analysis is technically difficult.

Development of a multilocus variable number of tandem repeat typing method for Oenococcus oeni

Oenococcus oeni is responsible for the malolactic fermentation of wine. Genomic diversity has already been established in this species. In addition, winemakers usually report varying starter culture efficiency. The monitoring of indigenous and selected strains is essential for understanding strain survival and implantation during the winemaking process. In this study, we report the development of the first typing scheme for O. oeni using multiple-locus variable number of tandem repeat analysis (VNTR). The discriminatory power of 14 out of 44 tandem repeat loci in the genome of the PSU-1 strain was initially evaluated with a test collection of 18 genotypically distinct starter strains. Then five VNTR loci, which can be easily scored with the technology used here, were identified and used to genotype a collection of 236 strains, previously classified by restriction endonuclease analysis-pulsed-field gel electrophoresis (REA-PFGE) and multilocus sequence typing (MLST) into 136 REA-PFGE types or 110 MLST types. The discriminatory power of VNTR (as determined by Simpson's index of discrimination) was higher than that of the other two methods, with 201 VNTR types. The targeted VNTR markers were found to be stable and did not change for the clones of the same strain deposited in a collection at intervals of several years. Strains isolated from the different wine producing areas or the products were assigned to phylogenetic groups and were statistically linked with the VNTR profiles. Another interesting observation was that the loci were found in sequences homologous to regions encoding for membrane-anchored proteins.

Emergence and Pathogenicity of Highly Virulent Cryptococcus gattii Genotypes in the Northwest United States

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Geographic substructure of Fusarium asiaticum isolates collected from barley in China

Fusarium head blight (FHB) can affect wheat and barley and is a devastating disease caused by a complex of Fusarium species. Here we report on a large-scale survey on the genetic diversity of isolates collected from barley in China. Ten VNTR markers were tested on a representative set of 40 isolates covering 14 sampling areas along the Yangtze River. VNTR4 and VNTR7, with 13 and 6 alleles, each were applied to a total of 1106 single-spore isolates to reveal the population structure of F. asiaticum. The F. asiaticum population showed high genetic diversity and a clear genotypic substructure within China. Pair-wise comparisons of allele frequencies between the mountainous provinces of Sichuan and Chongqing in Western China, Hubei Province in the centre or the eastern provinces of Zhejiang, Jiangsu and Shanghai showed significant differences. Even between counties of the same province, significant differences between allele frequencies were found (P < 0.001). Our results indicate serious constraints for migration of this pathogen in the major cereal-growing areas of China.

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp)

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.

Estimation Haplotype Frequency of BglII/EcoRI/VNTR Markers at the PAH Gene Region in Iranian Population

Deficiency in phenylalanine hydroxylase (PAH) is the main molecular characteristic of phenylketonuria (PKU). So far over 500 different mutations in the PAH gene have been identified as associated with the disease. Mutation analysis of the PAH gene is a time-consuming and cost-effective procedure. Therefore, molecular markers which are highly linked to the PAH gene, have been used in carrier detection and prenatal diagnosis in PKU families. These markers show a population dependent based haplotype frequency. In the present study, the haplotype frequency of three markers including BglII, EcoRI and VNTR, at the PAH gene region were investigated in Iranian population. Nine different alleles for VNTR marker with 3, 5, 6, 7, 8, 9, 10, 11 and 13 core repeats were detected. Alleles 4 and 13 were found specific to the Iranian population. The haplotype frequency was calculated using FBAT, PHASE and Arlequin (haplotype frequency estimation computer programs). Among the 36 possible estimated haplotypes identified for 2 alleles (+ and -) for BglII and EcoRI; and nine alleles for VNTR, ten haplotypes showed relatively high frequency (e 5%), based on the above programs. Therefore, a combination of BglII-EcoRI-VNTR could be suggested as an informative haplotype in performing carrier and prenatal diagnosis of the PAH gene mutations in Iranian population.