Karyotyping of Fusarium oxysporum by Pulsed-Field Gel Electrophoresis and the Germ Tube Burst Method.

In fungi, karyotyping is fundamental to understanding their genome organization. It is also essential to study various genome- or chromosome-related topics such as karyotype polymorphisms and supernumerary or pathogenicity chromosomes. Here, we describe the protocols of pulsed-field gel electrophoresis and the germ tube burst method for molecular and cytological karyotyping of Fusarium oxysporum. The combined use of the two methods is valuable for determining definitive and comprehensive karyotypes of these fungi.

Karyotype evolution in Fusarium.

The germ tube burst method (GTBM) was employed to examine karyotypes of 33 Fusarium species representative of 11 species complexes that span the phylogenetic breadth of the genus. The karyotypes revealed that the nucleolar organizing region (NOR), which includes the ribosomal rDNA region, was telomeric in the species where it was discernible. Variable karyotypes were detected in eight species due to variation in numbers of putative core and/or supernumerary chromosomes. The putative core chromosome number (CN) was most variable in the F. solani (CN = 9‒12) and F. buharicum (CN = 9+1 and 18-20) species complexes. Quantitative real-time PCR and genome sequence analysis rejected the hypothesis that the latter variation in CN was due to diploidization. The core CN in six other species complexes where two or more karyotypes were obtained was less variable or fixed. Karyotypes of 10 species in the sambucinum species complex, which is the most derived lineage of Fusarium, revealed that members of this complex possess the lowest CN in the genus. When viewed in context of the species phylogeny, karyotype evolution in Fusarium appears to have been dominated by a reduction in core CN in five closely related complexes that share a most recent common ancestor (tricinctum and incarnatum-equiseti CN = 8-9, chlamydosporum CN = 8, heterosporum CN = 7, sambucinum CN = 4-5) but not in the sister to these complexes (nisikadoi CN = 11, oxysporum CN = 11 and fujikuroi CN = 10-12). CN stability is best illustrated by the F. sambucinum subclade, where the only changes observed since it diverged from other fusaria appear to have involved two independent putative telomere to telomere fusions that reduced the core CN from five to four, once each in the sambucinum and graminearum subclades. Results of the present study indicate a core CN of 4 may be fixed in the latter subclade, which is further distinguished by the absence of putative supernumerary chromosomes. Karyotyping of fusaria in the not too distant future will be done by whole-genome sequencing such that each scaffold represents a complete chromosome from telomere to telomere. The CN data presented here should be of value to assist such full genome assembling.

Meiotic inheritance of a fungal supernumerary chromosome and its effect on sexual fertility in Nectria haematococca.

PDA1-conditionally dispensable chromosome (CDC) of Nectria haematococca MP VI has long served as a model of supernumerary chromosomes in plant pathogenic fungi because of pathogenicity-related genes located on it. In our previous study, we showed the dosage effects of PDA1-CDC on pathogenicity and homoserine utilization by exploiting tagged PDA1-CDC with a marker gene. CDC content of mating partners and progenies analyzed by PCR, PFGE combined with Southern analysis and chromosome painting via FISH. In this study, we analyzed mode of meiotic inheritance of PDA1-CDC in several mating patterns with regard to CDC content and found a correlation between CDC content of parental strains with fertility of crosses. The results showed non-Mendelian inheritance of this chromosome followed by duplication or loss of the CDC in haploid genome through meiosis that probably were due to premature centromere division, not by nondisjunction as reported for the supernumerary chromosomes in other species. Correlation of CDC with fertility is the first time to be examined in fungi in this study.

Cytological analyses of the karyotypes and chromosomes of three Colletotrichum species, C. orbiculare, C. graminicola and C. higginsianum.

In contrast to the recent accomplishments of genome projects, cytological information on chromosomes and genomes of the genus Colletotrichum is very scarce. In this study, we performed mitotic cytological karyotyping for the three species, C. orbiculare, C. graminicola, and C. higginsianum by fluorescence microscopy and compared the results with those from genome projects. Chromosome number (CN) of C. orbiculare was determined for the first time to be n=10 with no minichromosomes (MCs) in the genome, while CNs of C. graminicola and C. higginsianum were consistent with those from their genome project including the number of MCs. Regarding chromosome features, C. orbiculare was peculiar in that each chromosome was distinctly partitioned into a highly AT-rich pericentromeric region and the remaining highly GC-rich regions, and the pericentromeric region was judged to be constitutive heterochromatin. Integrating all the discernible morphological characteristics such as chromosome length, nucleolar organizing region, and DAPI-stained regions, idiograms were constructed for the three species. The overall cytological features of the chromosomes and genomes fit well with the data from the genome projects in terms of genome size, GC-content, and the occurrence of AT-rich regions. This study represents the most comprehensive and detailed mitotic cytological karyotyping of fungi ever reported.

Cytological karyotyping and characterization of a 410 kb minichromosome in Nectria haematococca MPI.

Karyotypes of the cucurbit pathogen Nectria haematococca MPI (anamorph Fusarium solani f. sp. cucurbitae race 1) was studied using the two standard strains ATCC18098 and ATCC18099. Complete separation of all chromosomes was difficult with pulsed field gel electrophoresis due to both the large size and co-migration of chromosomes. In contrast, cytological karyotyping was done successfully with fluorescence microscopy combined with the germ tube burst method for sample preparation to visualize mitotic metaphase chromosomes. For each strain the basic chromosome number (CN) was nine, which revises previous chromosome estimates of n = 4. Chromosomes were morphologically characterized by their sizes, intensely fluorescing segments, and protrusion of rDNA. In addition to the basic chromosome complement, ATCC18098 had a mini-chromosome of ~410 kb present as a single copy in somatic nuclei. Chromosome fluorescence in situ hybridization indicated that this mini-chromosome is not a derivative from the other chromosomes in the genome. In addition, crossing experiments suggested that it was transmitted in a Mendelian manner to the ascospore progeny.

Karyotyping methods for fungi.

Pulsed field gel electrophoresis enables separation of fungal chromosomes up to several megabases and is a worthwhile tool for fungal karyotyping. The germ tube burst method is a technique to separate fungal chromosomes of any size for chromosome number determination as well as in situ hybridization. Here we provide detailed protocols for both complementary methods that have many applications in fungal biology including chromosome size and chromosome number polymorphisms, and in situ localization of genes on chromosomes.

Fluorescence in situ hybridization for molecular cytogenetic analysis in filamentous fungi.

Fluorescence in situ hybridization (FISH) is a powerful technology for studying eukaryotic chromosomes and genomes from the combined view of cytogenetics and molecular biology, but its use in filamentous fungi has been limited. In this chapter, we describe protocols to perform three basic FISH techniques in filamentous fungi: (a) FISH mapping of unique sequences on the somatic chromosomes and interphase nuclei, (b) chromosome painting to detect a specific chromosome in the genome by fluorescent painting of the whole chromosome, and (c) fiber FISH on the stretched DNA fibers for physical mapping. The ways of preparing target specimens unique to filamentous fungi are included in the protocols.

The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion.

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the "Fusarium solani species complex". Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.

Cytological and electrophoretic karyotyping of the chestnut blight fungus Cryphonectria parasitica.

The karyotypes of nine strains including three transformants of the chestnut blight fungus Cryphonectria parasitica were analyzed by pulsed-field gel electrophoresis (PFGE) and cytology using a fluorescence microscope. Cytology of the mitotic metaphase showed n=9 for both standard strain EP155 and field strain GH2 infected by Cryphonectria hypovirus 3. Chromosomes were morphologically characterized by size, heterochromatic segment, and constriction. PFGE resolved 5 or 6 chromosomal DNA bands ranging from 3.3Mbp to 9.7Mbp, but accurate determination of the chromosome number was hampered by clumping of some bands. Banding profiles in PFGE were similar among the strains except for GH2, in which a chromosome translocation was detected by Southern blot analysis. By integrating the data from cytology and PFGE, the genome size of C. parasitica was estimated to be ca. 50Mbp. This is the first report of a cytological karyotype in the order Diaporthales.

Duplication of a conditionally dispensable chromosome carrying pea pathogenicity (PEP) gene clusters in Nectria haematococca.

A supernumerary chromosome called a conditionally dispensable chromosome (CDC) is essential for pathogenicity of Nectria haematococca on pea. Among several CDCs discovered in N. haematococca, the PDA1 CDC that harbors the pisatin demethylation gene PDA1 is one of the best-studied CDCs and serves as a model for plant-pathogenic fungi. Although the presence of multiple copies is usual for supernumerary chromosomes in other eukaryotes, this possibility has not been examined well for any CDCs in N. haematococca. In this study, we produced strains with multiple copies of the PDA1 CDC by protoplast fusion and analyzed dosage effects of this chromosome. Using multiple methods, including cytological chromosome counting and fluorescence in situ hybridization, the fusion products between two transformants derived from the same strain that bears a single PDA1 CDC were shown to contain two PDA1 CDCs from both transformants and estimated to be haploid resulting from the deletion of an extra set or sets of A chromosomes in the fused nuclei. In phenotype assays, dosage effects of PDA1 CDC in the fusion products were evident as increased virulence and homoserine-utilizing ability compared with the parents. In a separate fusion experiment, PDA1 CDC accumulated up to four copies in a haploid genome.

Electrophoretic and cytological karyotyping of the foliar wheat pathogen Mycosphaerella graminicola reveals many chromosomes with a large size range.

The karyotypes of three isolates of Mycosphaerella graminicola, the septoria tritici blotch pathogen of wheat, were analyzed with both pulsed field gel electrophoresis (PFGE) and the cytological technique called germ tube burst method (GTBM). These analyses revealed a chromosome length polymorphism among these isolates. The estimated genome size was 31-40 Mb depending on the isolates, indicating 17-22% redundancy in the genome of the standard strain IP0323 because such differences do not affect development, pathogenicity and sexual reproduction of the other isolates. The chromosome numbers in the three isolates were 18-20 and the chromosome size was 0.3-6 Mb. These data show that M. graminicola has the highest chromosome number and the smallest autosomes (A chromosomes) in filamentous ascomycetes. Our data also confirmed a large (> or =6 Mb) chromosome that was assembled recently in the IPO323 genome sequence. GTBM analyses revealed the mitotic metaphase chromosomes, enabling chromosome quantification, which was fully congruent with the PFGE analyses. These data will be instrumental in the final assembly of the M. graminicola genome.

Scanning electron microscopy of mitotic nuclei and chromosomes in filamentous fungi.

A new method for scanning electron microscopy (SEM) of fungal mitotic nuclei and chromosomes was established for two ascomycetes, Cochliobolus heterostrophus and Neurospora crassa. Nuclei and chromosomes discharged from germling cells by the germ-tube burst method were spread on a surface of a glass slide. The spreads were impregnated with osmium-thiocarbohydrazide for conductive staining, followed by coating with platinum, and observed by field-emission SEM. Ultrastructure of fungal chromosomes and nuclei was visualized by SEM for the first time.

Dynamic changes of rDNA condensation state during mitosis in filamentous fungi revealed by fluorescence in situ hybridisation.

We visualised rDNA of two ascomycetes, Cochliobolus heterostrophus and Haematonectria haematococca, by fluorescence in situ hybridisation (FISH) and analysed the condensation state of rDNA during mitosis. Both fungi showed a similar course of change in rDNA condensation corresponding to different mitotic stages. rDNA was decondensed in its entire length at interphase, and became increasingly condensed during prophase. The condensation reached a maximum at metaphase, remaining in that state through anaphase. Metaphase observation revealed the single distal location on a chromosome of rDNA in each fungus. This study provides the first visual evidence of the cyclic change of the condensation/decondensation state of rDNA during mitosis in filamentous fungi by FISH.

Physical mapping of plasmid and cosmid clones in filamentous fungi by fiber-FISH.

Fluorescence in situ hybridization to extended DNA fibers (fiber-FISH) serves as a powerful tool for direct physical mapping in plants and animals. Here, we show that fiber-FISH is useful for contig mapping as well as for estimating the physical distance between genetic markers in fungi. A five-cosmid contig from a chromosome of Nectria haematococca and four cloned genetic markers from a linkage map of Cochliobolus heterostrophus were chosen as models for the application of this technology. In N. haematococca, overlapping and non-overlapping clones were visually mapped on individual DNA fibers, confirming the results from conventional physical mapping perfectly. Fiber-FISH concomitantly indicated the gap size or the extent of overlap between two clones. In C. heterostrophus, the physical distance between the two pairs of genetic markers could be estimated from the microscopic measurements of the intervals. Chromosomal DNA isolated from a pulsed field gel was suitable for preparing the DNA fibers.

Electrophoretic karyotyping and gene mapping of seven formae speciales in Fusarium solani.

Chromosomal DNAs of 22 strains in 7 formae speciales (f. spp.) of Fusarium solani were compared by pulsed field gel electrophoresis (PFGE) and gene mapping on the chromosomes. Using PFGE, complete separation of the full components of the genome was not attained, due to the limited resolution of large chromosomes, but 5-12 chromosomes with sizes of 0.6-5.7 Mbp were resolvable for every strain. Although each strain had a unique banding profile, similarity in the banding profile was noticed among strains of the same (f. sp.). In gene mapping, the ribosomal RNA gene (rDNA) and putative pathogenesis-related genes encoding kievitone hydratase (khs), pisatin demethylase (pda) and pectate-degrading enzyme (pelA) were located on the chromosomes separated by PFGE. rDNA was always detected on the stacked large bands of 5.2-5.7 Mbp. The khs gene was detected on a chromosome of 2.8-5.4 Mbp in all f. sp. phaseoli strains and one strain of f. sp. pisi. The pda gene was detected on a chromosome of 1.4-5.6 Mbp in f. sp. pisi and pelA was localized on a chromosome of 2.3-2.9 Mbp in f. spp. pisi, xanthoxyli, batatas and mori. The results of PFGE and Southern blot hybridization supported the idea that each f. sp. of F. solani (or mating population of the teleomorph Nectria haematococca) has a distinctive genomic organization, as previously inferred from molecular phylogenetic analyses.

Application of fibre-FISH (fluorescence in situ hybridization) to filamentous fungi: visualization of the rRNA gene cluster of the ascomycete Cochliobolus heterostrophus.

Fibre-FISH (fluorescence in situ hybridization) has not been used in filamentous fungi before to the authors' knowledge. In this study, this technique was applied to a filamentous ascomycete, Cochliobolus heterostrophus, to visualize the organization of the rRNA gene clusters (rDNA). Using protoplasts embedded in agarose, DNA fibres were released from interphase nuclei and extended on a glass slide. Four kinds of probes (0.5-9.0 kb in size) that correspond to specific regions in the repeat unit of rDNA were hybridized singly or in combination to the DNA fibres, and the hybridization was detected with fluorescein- and/or rhodamine-conjugated antibodies after one round of signal amplification. The alternating arrangement of 18S and 28S rRNA genes as well as the tandem repetitive nature of the repeat units were clearly visualized by this single- or two-colour fibre-FISH. With a probe targeting the 5.8S or 18S rRNA gene, a region spanning over 800 kb could be visualized in a single fibre, allowing estimation of both the copy number of the repeat unit in rDNA and the stretching degree of the DNA fibre. It was shown that C. heterostrophus has more than 90 copies of the repeat unit in its rDNA and the stretching degree was similar to the value based on the Watson-Crick model. Visualization of individual genes on an extended DNA fibre was accomplished in filamentous fungi by this study.