Electrophoretic Karyotyping Research Articles

Characterization of a Single‐Spore Isolate Population of Plasmodiophora brassicae Resulting from a Single Club

AbstractThe single‐spore isolates ‘e3’ and ‘e6’ of Plasmodiophora brassicae with different virulence patterns were distinguished by restriction fragment length polymorphisms in fingerprint‐like patterns and by electrophoretic karyotypes using repetitive fragments as hybridization probes. These molecular tools were used to characterize a set of isolates originated from an infected root, which was inoculated with a mixture of the two single‐spore isolates (e3 and e6). Spores harvested from mixed‐infected roots were used to establish 53 new single‐spore isolates. All these single‐spore isolates revealed parental patterns according to their molecular fingerprints and their virulence pattern. No sexual recombination could be detected with repetitive molecular probes. However, one isolate (M36ES49) showed the same fingerprint pattern and virulence pattern but different sizes of small chromosomes than the parental type ‘e6’, which is taken as an indication of chromosome rearrangement during the infection cycle.

Morphotypic and genotypic characterization of sequential Candida parapsilosis isolates from an outbreak in a pediatric intensive care unit

Candidemia outbreaks that due to cross-infection are an emerging problem in hospitals. Typing of microorganisms is an essential tool for understanding the epidemiologic aspects of the infection. Techniques based on phenotypic characteristics are inexpensive and easy to perform but are limited by their lack of reproducibility. This study assessed the value of several phenotypic and genotypic techniques that are used in epidemiologic investigations of Candida parapsilosis in clinical practice and used a combination of these methods to analyze outbreak of C. parapsilosis candidemia. Random amplification of polymorphic DNA polymerase chain reaction with several primers was unsatisfactory because it lacked discriminatory power. By simplifying the reading of the morphotypes, we increased their reproducibility for each malt agar and 2,3,5-triphenyltetrazolium media (97% and 90%) and thus their suitability for its use. The combination of electrophoretic karyotype and the simplified morphotypes was rapid and practical to characterize the different clusters involved in the intensive care unit outbreak.

Chromosome translocation induced by the insertion of the URA blaster into the major repeat sequence (MRS) in Candida albicans.

Electrophoretic karyotype studies have shown that clinical isolates of Candida albicans have extensive chromosome length polymorphisms. Chromosome translocation is one of the causes of karyotypic variation. Chromosome translocation events have been shown to occur very frequently at or near the major repeat sequence (MRS) on chromosomes. The MRS consists of the repeated sequences RB2, RPS and HOK, and the repeated sequences are considered to be the template for recombination. To investigate which element of the MRS is important for chromosome translocation, we constructed three cassettes, each containing a URA blaster and sequences homologous to one of the repeats, for insertion into the MRS region on the chromosomes. The ura3 strain STN22u2, which shows a stable, standard karyotype, was transformed with each construct. Insertion events with each cassette occurred at almost all chromosomes. Insertion into the RB2 repeat, but not into the RPS repeat, was accompanied by chromosome translocation in some transformants: chromosome translocations between chromosomes R and 7 and chromosomes 1 and 7 were found, as well as deletions of 7A and 7C from chromosome 7. We conclude that the insertion at the RB2 region may initiate chromosome translocation in C. albicans.

A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans).

To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers. All but six markers showed no significant (P < 0.05) segregation distortion. At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM. Average marker density is 5.4 cM (range 1-28.7 cM). Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C. neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome. However, only 2 of 12 ungrouped markers hybridized to chromosome 10. The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12. This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance.

Chromosomal Polymorphism in Botrytis Cinerea Strains

The electrophoretic karyotypes of five strains of Botrytis cinerea from different locations were determined. A unique karyotype was observed for each strain: although general features of the banding pattern of chromosome sized DNA were conserved between strains not two strains had identical karyotypes, indicating that considerable length polymorphism exists in this species. The size of the hands ranged from 1.88 Mb to 3.72 Mb and the genome sizes from 11.22 to 22.49 Mb using chromosomal standards from Hansenula wingeii. Three of the strains were quite similar both in number and size of bands and genome total size and would be the diploid form of the species. The other two presented lower band number and, according to the total genome size, one of them could be haploid and the other an anenploid form. The mitochondrial DNA hand was also shown on gels and its origin confirmed by the cytochrome c oxidase specific reaction. Infection assays were carried out both in Nicotiana tabacum and Vifis uinifera and differences in pathogenicity were shown by the strains, although parasitic degree could not be related to any ploidy state or specific band.

Mating Within the Meiotic Tetrad and the Maintenance of Genomic Heterozygosity

Abstract Mating among the products of a single meiosis (automixis or meiotic parthenogenesis) is found in diverse groups of plant, animal, and fungal taxa. Restoration of the diploid stage is often strictly controlled and brings together products separated at the first meiotic division. Despite apparent similarities to diploid selfing, the theoretical prediction is that heterozygosity should be maintained on all chromosomes when it is linked to the centromeres and thus also segregates at the first meiotic division. Using the fungus Microbotryum, we directly test this prediction by linear tetrad analysis. The patterns of meiotic segregation for chromosome size variation (electrophoretic karyotypes) and PCR products (AFLP procedures) were determined for Microbotryum lineages native to North America and Europe. Our data reveal a surprisingly dynamic genome that is rich in heterozygosity and where size-dimorphic autosomes are common. The genetic variation agrees with the prediction of centromere-linked heterozygosity. This was observed to the greatest extent in the lineage of Microbotryum native to North America where there was consistent first-division segregation and independent assortment of multiple linkage groups. The data also show properties that distinguish the fungal sex chromosomes from the autosomes in both lineages of Microbotryum. We describe a scenario where the mating system of automixis with first-division restitution is the result of feedback mechanisms to control exposure of genetic load.

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America.

The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.

Speciation in the large-spored Metschnikowia clade and establishment of a new species, Metschnikowia borealis comb. nov.

The reproductive boundaries among species in the large-spored Metschnikowia clade were studied by prototrophic recombinant selection, electrophoretic karyotyping, mitochondrial DNA restriction analysis, and DNA sequence analysis. Inviable ascospores arose from crosses between the two varieties of Metschnikowia continentalis, indicating that they should be recognized as separate species. Prototrophic recombinants were recovered from crosses between auxotrophic mutants of Metschnikowia borealis, M. continentalis, Metschnikowia lochheadii, Metschnikowia sp. UWO(PS)00-154.1, and Candida ipomoeae, showing that some genetic exchange is possible in spite of the sterility of the asci formed in interspecific crosses. Metschnikowia hawaiiensis, although capable of ascus formation when its h(-) mating type is crossed with the h(+) mating type of the other species, did not give rise to recombinants. In the other species, some recombinants acquired the ability to form asci directly from single cells. These often contained the chromosomes of both parents, suggesting formation of allodiploid hybrids. Other recombinants behaved as haploids and were similar to one parent except for having inherited the selectable wild-type allele from the other parent. In most, but not all cases, inheritance of the mitochondrial genome was uniparental and correlated with the inheritance of the nuclear chromosome complement. In some cases, what appeared to be a recombinant mitochondrial genome was observed. Phylogenies derived from the sequences of various DNA regions were not congruent, indicating that hybridization may have taken place in nature as the large-spored species diverged from their common ancestor. Further evidence that C. ipomoeae arose from a natural recombination event was obtained, but a pair of Metschnikowia species that might represent derived forms of the parents could not be identified conclusively. C. ipomoeae and most of its closely related Metschnikowia species contained a group-II intron in the mitochondrial small-subunit ribosomal gene. The intron was absent in M. borealis, M. hawaiiensis, and other species in the genus Metschnikowia.

Genetic analysis of apomictic wine yeasts

The Saccharomyces cerevisiae wine yeast IFI256 was selected because of its high fermentative capacity and tolerance to ethanol. Sporulation of the IFI256 strain produced two-spore asci unable to conjugate, but able to sporulate again and the spores produced two-spore asci in all cases. That process was studied for at least five generations. The electrophoretic karyotype showed a pattern of 21 chromosomal bands, which was identical both in the parental and in all the descendants analyzed, from the first to the fifth generation. The DNA content of the parental and the descendants was of 1.7 n, which indicates that the capacity for sporulation shown by all descendants was due to apomixis rather than homothallism of the strain. Different concentrations of glucose and acetate and the addition of zinc salts to the presporulation and sporulation media increased the frequency of four-spore asci by up to 9%. However, the tetrads formed were in fact two dyads that resulted from induced endomitosis. Crosses of IFI256 with laboratory strains produced hybrids giving four-spore asci after sporulation, thus indicating the mutation to be recessive. Transformation of IFI256 with plasmids carrying either SPO12 or SPO13 functional genes and crosses with strains carrying functional or mutated SPO12 and/or SPO13 genes indicated that IFI256 carries several mutations, one of which was located to the SPO12 gene. Parasexual cycles and chromosome loss induced after crossing IFI256 with cir0 strains indicated that apomictic mutations were exclusively located at chromosome VIII. The high frequency of wine strains which are apomictic suggests apomixis to be an advantageous phenotype which allows the formation of stress-resistant asci but prevents the loss of favored chromosomal rearrangements.

Electrophoretic karyotype of two Micromucor species.

Electrophoretic karyotype analysis was applied to obtain information on the organisation and intrageneric variability of the nuclear genome in three Micromucor isolates of two different species (M. isabellina and M. ramanniana). A protoplast formation protocol, conditions for the preparation of highly-intact chromosome-size DNA molecules and for the separation of DNA molecules were established. The chromosomal banding patterns revealed substantial variability among the isolates: 11 to 14 chromosomal mobility groups were resolved. The DNA in the Micromucor chromosomes were rather small; their estimated sizes were calculated to be between 2.60 and 0.4 Mb. Using Saccharomyces cerevisiae and Schizosaccharomyces pombe as size standard, the minimum total genome sizes were estimated to be between 24.19 and 24.9 Mb.

Electrophoretic molecular karyotype of the dermatophyte Trichophyton rubrum

The electrophoretic karyotype of the dermatophyte Trichophyton rubrum was established using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Five chromosomal bands of approximately 3.0 to 5.8 megabase pairs (Mbp) each were observed and together indicated that 22.05 Mbp of the total genome are organized as chromosomal macromolecules. In addition to establishing the number and size of T. rubrum chromosomes, these results open perspectives for the construction of chromosome-specific libraries and for the physical mapping of genes of interest, thus permitting future gene linkage studies in this pathogen. A detailed understanding of the karyotype and genomic organization of T. rubrum should contribute to further genetic, taxonomic and epidemiological studies of this dermatophyte.

Genetic transformation, electrophoretic karyotyping and isolation of insertional mutants in the tree pathogenic fungus Neonectria galligena

SummaryThe ascomycete tree pathogen Neonectria galligena was transformed using several plasmid vectors containing the bacterial hygromycin phosphotransferase gene (hph) conferring resistance to hygromycin B. Protoplasts were produced by digesting germinating conidia in a Novozym 234 solution osmotically stabilized with NaCl. The protoplasts regenerated at ca. 70% on complete medium supplemented with either sucrose or NaCl. Up to 115 hygromycin B‐resistant transformants per microgram of transforming DNA per 108 protoplasts were obtained. Southern analysis of genomic DNA from the transformants, separated by either standard agarose gel electrophoresis or by pulsed field gel electrophoresis, confirmed that hygromycin B resistance resulted from integration of the vector. Transformants expressing the green fluorescent protein were also obtained. A total of 209 transformants were then screened for aggressiveness and mycelial radial growth. Four mutants were identified including one mutant with reduced aggressiveness but normal growth in vitro, two mutants with reduced growth in vitro but normal aggressiveness, and one mutant in which both aggressiveness and in vitro growth were reduced.

A Near-Isogenic Fusarium oxysporum f. sp. lycopersici Strain with a Novel Combination of Avirulence Characteristics

ABSTRACT A novel Fusarium oxysporum f. sp. lycopersici strain (F1-27) was obtained from protoplast fusions between race 1 Fol004 (putative avirulence genotype A1a2a3) and race 2 Fol007 (a1A2A3). Bioassays using different tomato cultivars revealed new virulence characteristics for F1-27 that were mitotically stable. The corresponding avirulence genotype for F1-27 was assigned a1A2a3. Despite their distinction in avirulence genotype, molecular analysis revealed that parent Fol007 and F1-27 were near-isogenic strains. The electrophoretic karyotype of F1-27 was identical to that observed for Fol007. Foxy-amplified fragment length polymorphism (AFLP) marker analysis showed that all Fol007-specific bands were present in F1-27. In addition, 11 new F1-27-specific Foxy insertions were identified. Segregation of both virulence and these new Foxy-AFLP markers was observed in a backcross between F1-27 and its parent Fol007. One marker was found to cosegregate with the a3 allele. The nature of the genetic change in this strain is discussed.

The Italian Green Certification market between uncertainty and opportunities : Lorenzoni, A. Energy Policy, 2003, 31, (1), 33–42

To breed or select banana cultivars with durable resistance to Fusarium wilt it is necessary to determine the genetic variation that exists within the pathogen, Fusarium oxysporum f. sp. cubense, as potentially resistant cultivars ideally should have resistance to all variants of the pathogen. We have analysed genetic variation within a world-wide collection of isolates of F. oxysporum f. sp. cubense which represented races 1, 2 and 4 and 11 different vegetative compatibility groups (VCGs) using RAPD-PCR. Comparison of the RAPD-PCR banding patterns both visually and by phenetic analysis sub-divided isolates of F. oxysporum f. sp. cubense into two major groups. Group 1 contained isolates belonging in VCGs 0120, 0121, 0122, 0126, 01210, 01211 and 01212 and group 2 contained isolates belonging in VCGs 0123, 0124, 0124/5 and 0125. The RAPD-PCR banding patterns were generally VCG specific and it was possible to determine the genetic relatedness between the different VCGs and of isolates within each VCG. The two groups differentiated by RAPD-PCR analysis correspond with previous classifications based on volatile production and electrophoretic karyotyping. In contrast, there was no correlation between RAPD-PCR banding pattern and race. The results support the hypothesis that F. oxysporum f. sp. cubense co-evolved with edible bananas and their wild diploid progenitors in South East Asia.

Chromosomal polymorphism, syntenic relationships, and ploidy in the pathogenic fungus Paracoccidioides brasiliensis

Pulsed field gel electrophoresis (PFGE) and DNA hybridization were used to establish and compare the electrophoretic karyotypes of 12 clinical and environmental Paracoccidioides brasiliensis isolates from different geographic areas. Gene mapping allowed the identification of synteny groups and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome. This represents the first comparative mapping study among different isolates. The results are indicative of the existence of genetic differences among natural isolates. DNA content of DAPI-stained nuclei of each isolate was estimated by confocal microscopy. Comparison of the genome sizes estimated by PFGE with those calculated by microfluorometry indicated the possible existence of haploid and diploid (or aneuploid) isolates of the fungus.

Electrophoretic karyotype analysis of Crinipellis perniciosa, the causal agent of witches' broom disease of Theobroma cacao

Pulse-field gel electrophoresis (PFGE) was used to determine the genome size and characterize karyotypic differences in isolates of the cacao biotype of Crinipellis perniciosa (C-biotype). The karyotype analysis of four isolates from Brazil revealed that this biotype could be divided into two genotypes: one presenting six chromosomal bands and the other presenting eight. The size of the chromosomes ranged from 2.7 to 5.3 Mb. The different genotypes correlate with telomere-based PCR analysis. The isolates with six chromosomal bands had two that appeared to be doublets, as shown by densitometric analysis, indicating that the haploid chromosome number for this biotype is eight. The size of the haploid genomes was estimated at approximately 30 Mb by both PFGE and Feulgen-image analysis. DNA hybridization revealed that the rDNA sequences are clustered on a single chromosome and these sequences were located on different chromosomes in an isolate dependent manner. This is the first report of genome size and chromosomal polymorphism for the C-biotype of C. perniciosa.

Electrophoretic karyotype of Flammulina velutipes and its variation among monokaryotic progenies

The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0Mb or more.

Karyotypic similarity identifies multiple host-shifts of a pathogenic fungus in natural populations

The detection of incipient host-shifts is important to the study of emergent diseases because it allows the examination of ecological and genetic conditions that favor novel inter-species transmission. Mixed populations of Silene latifolia and Silene vulgaris were investigated for the putative occurrence of host-shifts by the fungal plant pathogen Microbotryum violaceum (the cause of anther-smut disease) between S. latifolia (a common host for the pathogen) and S. vulgaris (a rare host). Samples of the fungus from mixed and pure host populations were studied for variation in their electrophoretic karyotypes. A karyotype distance matrix showed that fungal samples clustered by locality, but not by host species. Fungal samples from S. vulgaris were indistinguishable from sympatric samples from S. latifolia in multiple cases. The results indicated at least two independent host-shifts, one in the US and perhaps two in Italy. The karyotype and ecological data indicate that the direction of the host-shifts is from S. latifolia to S. vulgaris.

Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone.

Chytridiomycosis is a recently identified fungal disease associated with global population declines of frogs. Although the fungus, Batrachochytrium dendrobatidis, is considered an emerging pathogen, little is known about its population genetics, including the origin of the current epidemic and how this relates to the dispersal ability of the fungus. In this study, we use multilocus sequence typing to examine genetic diversity and relationships among 35 fungal strains from North America, Africa and Australia. Only five variable nucleotide positions were detected among 10 loci (5918 bp). This low level of genetic variation is consistent with the description of B. dendrobatidis as a recently emerged disease agent. Fixed (i.e. 100%) or nearly fixed frequencies of heterozygous genotypes at two loci suggested that B. dendrobatidis is diploid and primarily reproduces clonally. In contrast to the lack of nucleotide polymorphism, electrophoretic karyotyping of multiple strains demonstrated a number of chromosome length polymorphisms.

Electrophoretic Karyotype Analysis in Fungi

The resolution of chromosomal-sized DNAs by PFGE has many applications that include karyotyping, strain identification of similar species, characterization of transformed strains, building of linkage maps, and preparation of DNA for genomic analysis. Successful electrophoretic separation of chromosomes is an empiric process in which the initial concentration of intact chromosome-sized DNA and the optimization of electrophoretic parameters are the most important experimental variables. Nonetheless, inherent attributes of the genome architecture of certain species may thwart success. When a karyotype contains numerous chromosomes of the same size and/or many large (greater than 8 Mb) chromosomes, no amount of manipulation of the electrophoretic parameters will resolve individual chromosome bands using present technology. Further, fungi display a surprising amount of intraspecific variation in both chromosome number and size, making it difficult to establish a standard "reference" karyotype for many species. Although PFGE is not a panacea for bringing genetics to species that lack classical genetic systems, it often does provide a way for developing a molecular linkage map in the absence of a formal genetic system. It is far faster than parasexual analysis in the discovery of linkage relationships. For genomics projects, DNA can be recovered from pulsed field gels and used to prepare chromosome-specific libraries. Where whole genome sequencing strategies are used, chromosomes separated by PFGE provide an anchor for sequencing data. Electrophoretic karyotypes can be probed with anonymous pieces of DNA from bacterial artificial chromosome (BAC) contigs, thereby facilitating the building of physical maps. In conclusion, despite its shortcomings, the PFGE technique underlies much of our current understanding of the physical nature of the fungal genome.