Electrophoretic Karyotypes of Fusarium spp.

Migheli, Q., Berio, T., and Gullino, M. L. 1993. Electrophoretic karyotypes of Fusarium spp. Experimental Mycology 17, 329-337. The electrophoretic karyotype of 17 antagonistic and pathogenic strains of Fusarium spp. has been established by using contour-clamped homogeneous electric field gel electrophoresis. Intact chromosomal DNA was prepared from fungal protoplasts with standard procedures. Up to 11 distinct chromosomal bands were resolved after 184 h of migration at 50 V. Polymorphic karyotypes were observed in different species of Fusarium, formae speciales of F. oxysporum , and races of F. oxysporum f.sp. dianthi. Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as size standards, the size of the Fusarium genome was estimated to range from approximately 18.1 to 51.5 Mb. The suitability of electrophoretic karyotyping as a tool for strain characterization, as well as some applications in hybridization analysis of Fusarium spp., is discussed.     

Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans

By using pulsed-field gel electrophoresis, we have separated the entire chromosome bands and examined the electrophoretic karyotypes of 27 strains of Candida albicans. The electrophoretic karyotype varied widely among these strains. Their chromosomal DNAs were resolved into 7-12 bands ranging in size from 0.42 to 3.0 Mb. Most of the separated chromosomal bands were assigned by eight cloned C. albicans DNA probes. These results suggest that the haploid number of C. albicans chromosomes is eight. Each of the probes hybridized specifically to one or two bands of similar size in most strains. With the exception of the MGL1 probe, when two bands were detected by one probe, the size of one of them was very conserved whilst the other was of fairly variable size. The sizes of the chromosome bands assigned by the MGL1 probe were much more variable. As C. albicans is considered to be a diploid organism, it is inferred that the karyotype polymorphism between strains is mainly derived from wide size heterogeneity in one of the homologous chromosomes. Furthermore, we have confirmed species-specific and strain-specific variation in medically important Candida species (C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata). Electrophoretic karyotype analysis is thus useful for species assignation. The TUB2 probe, encoding C. albicans beta-tubulin, hybridized to the chromosomal DNA of all the Candida species examined, but four C. albicans probes exhibited cross-species hybridization with C. stellatoidea only. The karyotype of C. stellatoidea seems to be within the range of the intraspecies variation observed in C. albicans.     

Pulsed field gel electrophoresis reveals chromosome length differences between strains of Cladosporium fulvum (syn. Fulvia fulva)

Summary Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.     

Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma

In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two -carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutant strains affecting carotenogenesis obtained in our laboratory.     

Chromosomal polymorphism in the yeast species Debaryomyces hansenii

Pulse field gel electrophoresis karyotypes of 41 strains of the genus Debaryomyces, including 35 strains confirmed as D. hansenii species by D1/D2 ribosomal DNA sequence analysis, were performed. Electrophoretic karyotypes of the 41 strains exhibited 4 to 10 chromosomal bands ranging between 0.7 Mb and 4.2 Mb. Among D. hansenii species, the patterns of strains obtained from the CBS collection and cheese isolates differed strongly from D. hansenii var. hansenii CBS767^T. Both D. hansenii var. hansenii and D. hansenii var. fabryii showed chromosome length polymorphism. Electrophoretic karyotypes of the D. hansenii strains were analyzed by Southern hybridization with various species-specific probes isolated from D. hansenii var. hansenii CBS767^T. Repeated sequences including the F01pro, M18pro, the Ty1-copia retrotransposon Tdh5 and hypothetical telomeric sequence hybridized to several chromosomal bands, while a D1/D2 probe derived from the large ribosomal sub-unit hybridized only to the largest chromosome. Unique probes such as those hybridizing to actin ACT1, glycerol-3-phosphate dehydrogenase GPD1 and β-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767^T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosomal polymorphism in the yeast species Debaryomyces hansenii

Pulse field gel electrophoresis karyotypes of 41 strains of the genus Debaryomyces, including 35 strains confirmed as D. hansenii species by D1/D2 ribosomal DNA sequence analysis, were performed. Electrophoretic karyotypes of the 41 strains exhibited 4 to 10 chromosomal bands ranging between 0.7 Mb and 4.2 Mb. Among D. hansenii species, the patterns of strains obtained from the CBS collection and cheese isolates differed strongly from D. hansenii var. hansenii CBS767^T. Both D. hansenii var. hansenii and D. hansenii var. fabryii showed chromosome length polymorphism. Electrophoretic karyotypes of the D. hansenii strains were analyzed by Southern hybridization with various species-specific probes isolated from D. hansenii var. hansenii CBS767^T. Repeated sequences including the F01pro, M18pro, the Ty1-copia retrotransposon Tdh5 and hypothetical telomeric sequence hybridized to several chromosomal bands, while a D1/D2 probe derived from the large ribosomal sub-unit hybridized only to the largest chromosome. Unique probes such as those hybridizing to actin ACT1, glycerol-3-phosphate dehydrogenase GPD1 and β-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767^T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electrophoretic karyotypes of some species of the genus Leptomonas (kinetoplastida, trypanosomatidae), homoxenous trypanosomatids parasites of insects

Electrophoretic karyotypes of homoxenous trypanosomatids Leptomonas peterhoffi, L. mycophilus, L. nabiculae and Leptomonas sp. have been studied by transverse alternating-field electrophoresis under varying electrophoretic conditions. From 12 to 17 chromosomal DNA bands, ranging from 370 to more than 1500 kb were detected in the karyograms of the species compared. In each pattern, some intensely stained bands could represent more than one chromosome. Taking into account the number of intensely stained bands, the karyotype of L. peterhoffi was estimated to contain at least 18 chromosomes, the karyotypes of L. mycophilus and L. nabiculae, at least 21 chromosome each, and the karyotype of Leptomonas sp. up to 20 chromosomes. Interclonal variations of electrophoretic karyotypes of 10 clones of Leptomonas sp. (cfmI-cfmX) were studied. Seven of ten clones had identical electrophoretic patterns. In the karyograms of three clones (cfmI, cfmVI, cfmVII), additional chromosomal DNA bands were observed. The obtained results suggest, that electrophoretic karyotypes cannot be used as reliable markers of species of homoxenous trypanosomatids, since intraspecies variability does occur in these parasites.     

Electrophoretic karyotype of Saprolegnia monoica

Abstract The electrophoretic karyotype of Saprolegnia monoica was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Eight chromosomal bands were separated. The size of these bands, based on migration relative to those of chromosomal DNA of Saccharomyces cerevisiae , Schizosaccharomyces pombe and Hansenula wingei , is estimated to be between 0.9 and 5.8 Mb. The genome size is estimated to be 51 Mb.     

Insertion of transformation vector DNA into different chromosomal sites of Dictyostelium discoideum as determined by pulse field electrophoresis

Chromosomes of the cellular slime mold Dictyostelium discoideum were fractionated on three pulse field gel electrophoresis systems (pulse field, orthogonal field and C.H.E.F. (Contour-clamped Homogeneous Electric Fields] into a series of 13 bands ranging from 0.1 Mb to over 2 Mb in size. Since this organism has only seven chromosomes (estimated to be 1-10 Mb), and -90 copies of an 88-kilobase linear ribosomal DNA molecule (14% of genome), it was apparent that not all of these bands were whole chromosomes. However these bands were reproducibly obtained with the cell preparation used. They fell into three categories: i) four large poorly resolved DNA molecules (-2 Mb in size) which represent very large fragments or intact chromosomes, ii) eight faint bands ranging from 0.1 Mb to 2 Mb, iii) a prominent band in the apparent size range of about 0.15 Mb. Cloned Fragment V of an EcoR1 digest of the ribosomal DNA, hybridized to the 0.15 Mb band indicating it contained the linear ribosomal DNA. This chromosomal banding pattern was used to examine the stability and location of vector DNA in 16 transformed strains of D. discoideum. Each transformed strain was initially selected on the basis of G418 resistance with an integrating vector containing pBR322 sequences. Eleven transformants still carried pBR322 sequences after more than 60 generations of growth without selection on G418. All four strains transformed with constructs containing regions of the D. discoideum plasmid Ddp1 had lost their pBR322 insert, indicating that integration of Dictyostelium plasmid DNA into chromosomes leads to instability. Orthogonal field electrophoresis of the eleven strains still carrying pBR322 sequences revealed at least seven different integrating sites for the transforming DNA. We conclude that these vectors have many possible sites of integration in the D. discoideum genome.     

Chromosome Polymorphism of the Obligate Biotrophic Parasite Plasmodiophora brassicae

Electrophoretic karyotypes of different isolates of Plasmodiophora brassicae, the causative agent of clubroot of cabbage, using contour‐clamped homogeneous electric field gel electrophoresis (CHEF) revealed chromosome polymorphism in this obligate parasite. Ribosomal RNA (rRNA) genes have been localized on three chromosomes (I, IV and V) of P. brassicae of the 16 chromosomal bands, which can be distinguished for the single‐spore isolate ‘e₃’. In comparison with this isolate three other single‐spore isolates showed chromosome polymorphism by size of chromosomal bands and by hybridization pattern with rRNA gene fragments and other Plasmodiophora‐specific DNA fragments.     

Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato)

Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less agressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESSI6K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes.     

Separation of megabased-sized DNA molecules of Aspergillus niger using pulsed field gel electrophoresis

In this study, the chromosomal DNAs were extracted from Aspergillus niger Z10 wild type strain and these DNAs were separated using the contour clamped homogeneous electric field gel electrophoresis (CHEF) system. This system is laboratory-made and is operated by a computer program. Total DNAs resolved into five distinct chromosomal bands. The size of the chromosomes was estimated as being between 3.3 Mb to 6.4 Mb.     

Molecular karyotype analysis and mapping of housekeeping genes to chromosomes of selected species complexes of Leishmania.

The molecular karyotypes for 20 reference strains of species complexes of Leishmania were determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Determination of number/position of chromosome-sized bands and chromosomal DNA locations of housekeeping genes were the two criteria used for differentiating and classifying the Leishmania species. We have established two gel running conditions for optimal separation of chromosomes, which resolved DNA molecules as large as 2,500 kilobase pairs (kb). Chromosomes were polymorphic in number (22-30) and size (200-2,500 kb) of bands among members of five complexes of Leishmania. Although each stock had a distinct karyotype, in general the differences found between strains and/or species within each complex were not clear enough for parasite identification. However, each group showed a specific number of size-concordant DNA molecules, which allowed distinction among the Leishmania complex parasites. Clear differences between the Old and New world groups of parasites or among some New World Leishmania species were also apparent in relation to the chromosome locations of beta-tubulin genes. Based on these results as well as data from other published studies the potential of using DNA karyotype for identifying and classifying leishmanial field isolates is discussed.     

Meiotic Instability of Pythium Sylvaticum as Demonstrated by Inheritance of Nuclear Markers and Karyotype Analysis

Progeny from a sexual outcross between opposite mating types of Pythium sylvaticum were analyzed for inheritance of RFLP and random amplified polymorphic DNA (RAPD) markers. Although most were inherited in expected Mendelian frequencies, several were not. Pulsed field gel electrophoresis was employed to examine these unexpected patterns of marker inheritance at a karyotypic level. Parental oogonial and antheridial isolates had different electrophoretic karyotypes and minimum number of chromosome-sized DNAs (13 and 12, respectively), however, summation of the sizes of all chromosomal bands for each isolate was similar at ~37 Mb. Progeny karyotypes differed significantly from each other and the parental isolates, ranging in estimated minimum number of chromosome-sized DNAs from 9 to 13 and the summation of band sizes within each isolate from 28.1 to 39.0 Mb. For the eight isolates most extensively analyzed, 80% of the progeny chromosome-sized DNAs were nonparental in size or hybridization grouping of cDNA clones and isolated RAPD markers. Based on the results of Southern analysis it appears that length mutations and perhaps aneuploidy and translocations have contributed to generation of karyotypic polymorphisms. Nineteen field isolates of P. sylvaticum collected from the same location also exhibited significantly different karyotypes, suggesting that the meiotic instability observed in the laboratory also is occurring in field populations.     

Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Electrophoretic karyotyping of the two most widely studied strains of Phanerochaete chrysosporium, BKMF-1767 and ME-446, has been determined using transverse alternating field etectrophoresis. The genomic DNA of BKMF-1767 was resolved into 10 chromosomes ranging in size from 1.8–5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME-446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF-1767 and to four chromosomes in strain ME-446.     

一种新的脉冲电泳分子量标记*

在脉冲电泳（ｐｕｌｓｅｄ－ｆｉｅｌｄ　ｇｅｌ　ｅｌｃｔｒｏｐｈｏｒｅｓｉｓ,ＰＦＧＥ）研究中,经常使用的分子量标记有啤酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｅｓｌａｅ）和粟酒裂殖酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）的染色体完整ＤＮＡ。其中,啤酒酵母（如菌株ＹＮＮ２９５）有１６条染色体,分子量变化范围为０．２５Ｍｂ～２．２Ｍｂ（ＭｃＣｌｕｓｋｅｙｅｌａｌ,１９９０）,适于作为小于２．２Ｍｂ的染色体ＤＮＡ的分子量标记;粟酒裂殖酵母（如菌株９７２ｈ－）有３条染色体,分子量…     

Assignment of RAPD marker probes designed from 12 linkage groups of Flammulina velutipes to CHEF-separated chromosomal DNAs

Electrophoretic karyotype analyses of Flammulina velutipes FSB and its monokaryotic progeny, omFSB1 and omFSB2, obtained from oidia were performed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. At least 11 chromosome-sized DNA bands (CB 1 through CB 11) for FSB, 6 bands for omFSB1, and 7 bands for omFSB2, respectively, were resolved on a CHEF gel. Southern hybridization analysis on CHEF-separated chromosomal DNA of FSB was carried out using RAPD marker probes prepared from each of the 12 linkage groups. The bands CB 1, 2, and 4 each hybridized to two or three probes for different linkage groups. The bands CB 5 and 6 both hybridized to a common probe. The bands CB 3, 7, 8, and 9 each hybridized to a single specific probe for different linkage groups. The two smallest bands (CB 10 and 11) did not hybridize with any probes.     

Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato)

Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less agressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESSI6K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes.     

An electrophoretic karyotype of Aspergillus niger

Summary An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomesized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage groupspecific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5–38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.     

An electrophoretic karyotype forPhytophthora megasperma

Chromosomal DNA has been prepared from mycelial spheroplasts ofPhytophthora megasperma (isolate 63, chromosome numbern = 13–14)(E. M. Hansen, C. M. Brasier, D. S. Shaw, and P. B. Hamm, 1986.Trans Brit. Mycol. Soc.87, 557–573) and resolved by a pulsed field gel electrophoresis system which uses contour-clamped homogeneous electric fields. Nine chromosomal DNA bands were separated, the smallest being about 1.4 Mb in size: several larger DNAs were unresolved under all conditions tested. The estimated size was based on migration rates relative to those of chromosomal DNA ofSaccharomyces cerevisiae, Schizosaccharomyces pombe, and aNeurospora crassa translocation strain which has a minichromosome.