Random Amplified Microsatellites Research Articles

Use of random amplified microsatellites to type isolates from an outbreak of nosocomial aspergillosis in a general medical ward

Numerous patients were diagnosed with aspergillosis in a nosocomial outbreak caused by Aspergillus fumigatus and Aspergillus flavus. Thirty-three isolates of the former and 28 isolates of the latter were collected from the hospital environment and from the patients and studied for genetic relatedness by random amplified microsatellites (RAMS) analysis, in which two polymorphic regions were tested. Twenty-eight genotypes of A. fumigatus and 23 genotypes of A. flavus were identified. Four patients were infected by two isolates with the same genotype as the environmental isolates. One clinical genotype was shared by three patients and another was shared by two patients. We found that RAMS was useful for fingerprinting Aspergillus spp.

Genetic Diversity Among Alternaria solani Isolates from Potatoes in South Africa.

Genetic diversity among isolates of Alternaria solani, the causal agent of early blight of potato, from various potato-growing regions in South Africa (SA), was determined using virulence assays, vegetative compatibility (VC) tests, and random amplified microsatellite (RAMS) primers. The virulence assays showed low virulence levels for the largest part of the population, but failed to otherwise characterize the population diversity. The VC tests revealed 19 VC groups (VCGs), indicating a relatively high level of diversity among the isolates. There was little correlation between geographic origin of isolates and VCGs. Analysis of RAMS profiles revealed 27% genetic diversity among 46 isolates. This value is relatively high for an asexually reproducing fungus, but is similar to values obtained previously by authors studying A. solani. Distance analysis of the RAMS profiles also provided no evidence for geographical clustering of isolates. VCG and RAMS profiles indicated that isolates are randomly spread across SA. This fact, together with the high diversity of A. solani in SA, indicates that the fungus has a high potential to adapt to resistant cultivars or fungicides. This information can aid in the breeding and deployment of A. solani-resistant potato varieties, and in early blight disease management in SA.

Virulence and Molecular Diversity within Colletotrichum lindemuthianum Isolates from Andean and Mesoamerican bean Varieties and Regions

Virulence on a standard set of 12 common bean differential varieties, DNA sequence of repetitive-elements (Rep-PCR) and random amplified microsatellites (RAMS) were used to assess the genetic variability of 200 Colletotrichum lindemuthianum isolates collected from Andean and Mesoamerican bean varieties and regions. High levels of pathotypic (90 pathotypes) and genetic diversity (0.97) were identified among 200 isolates, revealing that C. lindemuthianum is a highly diverse pathogen. Although a significant number of pathotypes were common to Andean and Mesoamerican regions, many more were only found in the Mesoamerican region. Cluster analysis of virulence and molecular data did not separate isolates into groups that were structured with common bean gene pools. No genetic differentiation (GST=0.03) was apparent between Andean and Mesoamerican isolates of C. lindemuthianum. The diversity exhibited by C. lindemuthianum does not appear to cluster according to common bean gene pools, and the high diversity found in the Mesoamerican region seems to indicate that C. lindemuthianum originated and was disseminated from this region. Due to the high genetic variation exhibited by C. lindemuthianum, stacking major resistance genes appears to be the best option for developing cultivars with durable anthracnose resistance.

A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA

A simple and easy protocol for extracting high-quality DNA from microorganisms and plants is presented. The method involves inactivating proteins by using SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high-molecular-weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous phenol and chloroform, and as many as 100 samples can be processed per day. Absorbency ratios (A260/A280) of 1.6–2.0 indicated a minimal presence of contaminating metabolites. The DNA was completely digested with 5 restriction enzymes:EcoR I,RsaI,TaqI,EcoR V, andHind III. PCR analysis using enterobacterial repetitive intergenic consensus (ERIC) sequence, sequence-characterized amplified region (SCAR), and random amplified microsatellite (RAMS) primers showed the DNA's compatibility with downstream applications. This procedure is applicable to a range of pathogens and plants and thus may find wide application in quarantine services and marker-assisted selection (MAS) breeding.

Identification of Spanish isolates of Rhizoctonia solani from potato by anastomosis grouping, ITS-RFLP and RAMS-fingerprinting

Anastomosis grouping, restriction fragment length polymorphism (RFLP) of the ITS regions including the 5.85 rDNA, and random amplified microsatellites (RAMS) were used to characterize isolates of Rhizoctonia solani collected from Spain and Finland. There was a high similarity between the results obtained with the three techniques. RAMS markers revealed more genetic variation among isolates of R. solani than RFLP. The anastomosis group (AG)–3 isolates were clearly separated from isolates belonging to other AGs by RAMS, RFLPs and anastomosis grouping. Almost all the isolates sampled from potato belonged to AG–3. No differences were observed between Spanish and Finnish AG–3 isolates.

Intraspecific Variation in Phytophthora citrophthora from Citrus Trees in Eastern Corsica

Isolates of Phytophthora pathogenic to citrus crops on Eastern Corsica and associated with gummosis were identified by PCR-RFLP of internal transcribed spacers (ITS) sequences and characterized by the random amplified microsatellites (RAMS) technique. A sample of 114 isolates collected from diseased trunks and fruits, and from soil, were overwhelmingly Phytophthora citrophthora. Further analysis indicated that the P. citrophthora population was not homogeneous in citrus groves. There were two groups, with a few (4%) atypical isolates in two marginal groups. The major groups have been re-examined in the light of mating behaviour, RFLPs of mitochondrial DNA and sequence comparisons of ITS regions of rDNA. They were found distinct with all these criteria and perhaps constitute distinct taxa. The results indicate that important modifications occurred in the population structure of P. citrophthora over time in Corsican groves. These changes may have impact on the recent outbreaks of gummosis.

Sexuality and intersterility within the Heterobasidion insulare complex

The sexual system of Heterobasidion insulare was elucidated and 40 specimens originating from the mainland of China, Taiwan, and Japan were studied with the aid of mating tests. Most of the material was also investigated with DNA fingerprinting using random amplified microsatellite (RAMS) and M13 minisatellite primers. Denaturing gradient gel electrophoresis (DGGE) and sequencing were applied for detecting variation in the internally transcribed spacer (ITS) region of the ribosomal DNA cluster. H. insulare proved to be a species complex with bipolar mating system. Clamp connections occurred in both heterokaryotic and homokaryotic mycelia. Three intersterility groups were found: the 'T group' included specimens from Taiwan and Guizhou Province (southern China), the 'N group' specimens from northern China and Japan, and the 'Y group' specimens from Yunnan (south-west China). The T group seems to be totally intersterile with the two other groups, and was grouped separately in neighbour joining analysis based on DNA fingerprints. As concluded from mating experiments, groups N and Y are partially intersterile, but they are able to produce hybrid heterokaryons, no group specific DNA markers were found, and they were grouped together in neighbour joining analysis. The identity of H. insulare sensu stricto was studied from the type collections. It seems that none of the above-presented taxa can be referred to it, and we propose the term H. insulare sensu lato to be used in future studies.

Microsatellite and RAPD polymorphisms in Ontario corn hybrids are related to the commercial sources and maturity ratings

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were used to estimate the genetic relationships among 37 Ontario corn hybrids. Almost all (95%) of the 160 RAPD fragments and all of the 79 microsatellite alleles were polymorphic across the 37 hybrids. Similarity values among the hybrids ranged from 31% to 86% when based on the RAPD data. The similarities based on microsatellite markers ranged from 12% to 77%. The genetic diversity revealed by microsatellite marker analysis was higher than that obtained from RAPD analysis. The similarity matrices for the microsatellite data and the RAPD data were moderately correlated (0.43). Cluster analyses based on either type of marker showed that most of the hybrids from the same company were closely related to each other. Both dendrograms clustered similar pairs or groups of hybrids. A principal component analysis, based on the combined RAPD and microsatellite data, yielded a good separation of the hybrids with Ontario Corn Heat Unit (OCHU) values 2800. Seventeen RAPD markers and 5 microsatellite markers were significantly associated with the OCHU ratings of the hybrids.

Assessment of Diversity in Claviceps africana and Other Claviceps Species by RAM and AFLP Analyses

ABSTRACT Genetic diversity among isolates of Claviceps africana, the sorghum ergot pathogen, and isolates of other Claviceps spp. causing ergot on sorghum or other hosts, was analyzed by random amplified microsatellite (RAM) and amplified fragment length polymorphism (AFLP) analyses. Of the RAM primer sets tested, one revealed polymorphism in C. africana isolates, with Australian and Indian isolates possessing a unique fragment. AFLP analysis, in addition to clearly distinguishing Claviceps spp., revealed polymorphisms in C. africana. A group of isolates from the United States, Puerto Rico, and South Africa exhibited 95 to 100% similarity with one another. Several isolates from Isabela, Puerto Rico were 100% similar to an isolate from Texas, and another isolate from Puerto Rico was identical with one from Nebraska. Australian and Indian isolates showed greater than 90% similarity with isolates from the United States., Puerto Rico, and South Africa. A number of polymorphisms existed in the United States group, indicating that the recently introduced population contains multiple genotypes. Isolates of C. sorghicola, a newly described sorghum pathogen from Japan, were very distinct from other species via RAM and AFLP analyses, as were isolates from outgroups C. purpurea and C. fusiformis. Both RAM and AFLP analysis will be useful in determining future patterns of intercontinental migration of the sorghum ergot pathogen, with the AFLP method showing greater ability to characterize levels of intraspecific variation.

Pathogenicity, morphology and genetic variation of Phytophthora cactorum from strawberry, apple, rhododendron, and silver birch

The analysis of random amplified microsatellite (RAMS) markers of Phytophthora cactorum from strawberry showed that leather rot of strawberry fruits and crown rot are not caused by genetically different strains of this species. UPGMA-analysis showed that the populations of P. cactorum on strawberry differ between North America and Europe and that no clear genetic separation between isolates from different plants can be made among these populations. Slight morphological variation exists in the diameters of oogonia and oospores among the isolates but is insufficient for the identification of genetic groups or host specificity of P. cactorum isolates. Pathogenicity experiments proved that strains show a tendency towards host specialization.

Genetic Evidence for Somatic Haploidization in Developing Fruit Bodies of Armillaria tabescens

Armillaria spp. have vegetative hyphae with diploid uninucleate cells, but the fruit bodies of many species contain clamped dikaryotic hyphae. Earlier observations suggest that somatic haploidization takes place in developing fruit bodies. To verify this, a uninucleate diploid cell was isolated from each of the 49 mating combinations between single-spore isolates of Armillaria tabescens and they were fruited. Twenty-four isolates produced fruit bodies with at least a partially dikaryotic subhymenium. Dikaryotic hyphae were isolated from fruit-body primordia and homokaryons were obtained by micromanipulation or by protoplasting. Approximately half of the isolates proved to represent recombinant mating types in respect to parent homokaryons, and most of them contained recombinant haploid DNA, based on random-amplified microsatellite markers. The results show that the nuclei in dikaryotic hyphae found in fruit bodies result from somatic haploidization. The mechanism of haploidization remains unclear.

Genetic differentiation between European and North American populations of Phlebiopsis gigantea

We investigated the degree of genetic differentiation between geographically isolated populations of the saprophytic fungus Phlebiopsis gigantea from Europe and North America using two different molecular approaches: (i) denaturing gradient gel electrophoresis (DGGE) and sequence analyses of the ITS region, and (ii) genetic fingerprinting using random amplified microsatellite (RAMS) markers. The analysis included 84 homokaryotic single spore isolates of 28 European strains, 13 heterokaryotic strains from North America, and the Rotstop strain used as a biocontrol agent against Heterobasidion annosum. Three different ITS-alleles (designated as ITS/A, ITS/B and ITS/C) were separable by the DGGE, two of them occurring both in Europe and North America. However, sequence determination of these allele types showed that while the ITS/A alleles were identical between Europe and North America, the ITS/C alleles contained differences. Analysis of molecular variation (AMOVA) and neighbor joining (NJ) clustering analysis using altogether 28 RAMS markers revealed a considerable degree of differentiation between the European and North American populations. The results show that recent human-mediated P. gigantea migration between the continents is unlikely and that strains should not be transferred between continents for biocontrol purposes in order to avoid the introduction of novel genotypes.

Genetic Differentiation between European and North American Populations of Phlebiopsis gigantea

We investigated the degree of genetic differentiation between geographically isolated populations of the saprophytic fungus Phlebiopsis gigantea from Europe and North America using two different molecular approaches: (i) denaturing gradient gel electrophoresis (DGGE) and sequence analyses of the ITS region, and (ii) genetic fingerprinting using random amplified microsatellite (RAMS) markers. The analysis included 84 homokaryotic single spore isolates of 28 European strains, 13 heterokaryotic strains from North America, and the Rotstop strain used as a biocontrol agent against Heterobasidion annosum. Three different ITS-alleles (designated as ITS/A, ITS/B and ITS/C) were separable by the DGGE, two of them occurring both in Europe and North America. However, sequence determination of these allele types showed that while the ITS/A alleles were identical between Europe and North America, the ITS/C alleles contained differences. Analysis of molecular variation (AMOVA) and neighbor joining (NJ) clustering analysis using altogether 28 RAMS markers revealed a considerable degree of differentiation between the European and North American populations. The results show that recent human-mediated P. gigantea migration between the continents is unlikely and that strains should not be transferred between continents for biocontrol purposes in order to avoid the introduction of novel genotypes.

Genetic variation of the resin top fungus in Finland as determined by random amplified microsatellites (RAMS)

SummaryThe analysis of random amplified microsatellite (RAMS) markers among aecia of the causal agent of the resin top disease (Peridermium pini) on Scots pine (Pinus sylvestris) suggested that the genetic variation between the populations of this rust fungus is low in Finland. The method used allowed the identification of heterozygotic aecia in two loci, where the degree of heterozygosity was, however, low. The RAMS patterns of the Finnish aeciospores, other aeciospores from Thetford, UK and Cronartium flaccidum from Italy were highly similar suggesting that the autoecious and heteroecious forms of these rust fungi would be genetically closely related.

Sequence-tagged sites (STS) for studies of molecular epidemiology of scleroderris canker of conifers

Scleroderris canker is a very damaging disease of conifers caused by a fungal pathogen, Gremmeniella abietina var ‘abietina’. This fungal pathogen is now known to comprise a number of distinct races and biotypes. In North America, two races, an indigenous North American race and an introduced European race, are present. In Europe, three distinct biotypes have been reported within the European race: one in the Alps, another in Fennoscandia, and a third that overlaps with the first two. We used random amplified microsatellites (RAMS) and DNA sequencing with arbitrary primer pairs (SWAPP) to design five PCR primer pairs flanking polymorphic regions of the genome of the European race of G. abietina. Length polymorphisms produced by repeats of basic units in microsatellites were distinguished by electrophoresis of PCR products in agarose gels, and point mutations were identified by low-ionic-strength single-strand conformation polymorphisms (LIS-SSCP). Some primers generated private alleles in the European biotype and the psychrophilic Alpine and Fennoscandian biotypes, i.e., alleles that were fixed within the two groups but polymorphic between them. Conversely, one pair of primers amplified at least 3, 4, and 7 alleles in the Fennoscandian, Alpine, and European biotypes, respectively. The Alpine and Fennoscandian biotypes, although geographically separated, were genetically more closely related to one another than to European biotype, which has an overlapping distribution. However, both Alpine and Fennoscandian biotypes have similar ecotypic adaptation. The evolution of these biotypes could be explained by their geographic separation following the end of the last glaciation.

Genetic variation in Phlebiopsis gigantea as detected with random amplified microsatellite (RAMS) markers

Random Amplified Microsatellite (RAMS) markers were used to detect variation among Phlebiopsis gigantea isolates from seven European countries. The scoring of 26 bands revealed five markers common to all isolates and 21 variable markers, of which six occurred in only one strain. A considerable amount of variation was observed among the progeny isolates of single strains. The analysis of variable markers resulted in 75 different banding patterns among the 86 isolates studied. The high degree of polymorphism and heterozygosity observed in RAMS markers suggests that the amount of genetic variation within this fungus is considerable. The equal distribution of markers in strains from different locations and the lack of country-specific markers suggests that genetic differentiation among the populations is low. The lack of distinct groups of similar banding patterns supports the idea of P. gigantea being a true biological species consisting of a single intersterility group.

Genetic variation and host specificity of Phytophthora cactorum isolated in Europe

Analysis of Phytophthora cactorum using Random Amplified Microsatellites (RAMS) revealed considerable variation among isolates, most of which correlated with the original host plants. The lack of variation among isolates originating from strawberry suggests that crown rot of strawberry is caused by a single clone within the geographical area studied. It was also shown that P. cactorum isolates form a unique group different from other Phytophthora spp. from Group I. P. cactorum isolates from necrotic stem lesions on Betula pendula seedlings or Fragaria x ananassa plants suffering from crown rot were highly pathogenic to their original host plants. P. cactorum isolates from strawberry inoculated via wounds also caused necrotic lesions on B. pendula. On the other hand isolates from B. pendula did not cause disease symptoms on strawberry plants. On Alnus glutinosa the percentage of successful inoculations with a birch isolate was 40.

Variation within Gremmeniella abietina in Finland and other countries as determined by Random Amplified Microsatellites (RAMS)

Genetic variation within Gremmeniella abietina var. abietina was studied using the Random Amplified Microsatellite-technique (RAMS). Ninety-three isolates were investigated, originating mostly from Finland, but also from Canada, U.S.A., Japan, Norway, Italy, Iceland and Sweden. Four banding pattern types were observed corresponding to the present division of this species in Asian, North American and two types of European races. Furthermore, an additional banding pattern was also observed. Intraracial variation was observed within all races, the North American one being the most polymorphic. Isolates of the large tree type (LTT) Gremmeniella from North America, Italy and Iceland contained RAMS alleles not observed in Finland, Sweden or Norway. Therefore, the isolates of LTT Gremmeniella should not be transported even within the area of its natural occurrence.

Random amplified microsatellites (RAMS) — a novel method for characterizing genetic variation within fungi

SummaryA novel method, Random Amplified Microsatellites (RAMS, due to the nature of amplified markers as two randomly amplified microsatellites with the intervening sequence), was applied to generate DNA markers in a variety of fungi (Armillaria cepistipes, Gremmeniella abietina, Heterobasidion annosum, Pbytophthora cactorum, Phlebiopsis gigantea, and Stereum sanguinolentum). It is based on the polymerase chain reaction (PCR), and uses primers containing microsatellite sequences and degenerate anchors at the 5' end. The method is highly reproducible, applicable to all tested fungal species including members of the Phycomycetes, Ascomycetes and Basidiomycetes, and allows detection of interspecific and intraspecific DNA‐polymorphisms.