Discovery of dual PKS involved in sclerotiorin biosynthesis in Penicillium meliponae using genome mining and gene knockout.

Fungi of the genus Penicillium section Sclerotiora have as their main characteristic the presence of orange-pigmented mycelium, which is associated with sclerotiorin, a chlorinated secondary metabolite of the azaphilone subclass of polyketides. Sclerotiorin presents anti-diabetes, antioxidant, anti-inflammatory, anti-Alzheimer, antiviral, and antimicrobial activities, which has always attracted the attention of researchers worldwide. During our ongoing search for azaphilone-producing Amazonian fungi, the strain of Penicillium MMSRG-058 was isolated as an endophyte from the roots of Duguetia stelechantha and showed great capacity for producing sclerotiorin-like metabolites. Using multilocus phylogeny, this strain was identified as Penicillium meliponae. Moreover, based on the genome mining of this strain through the reverse approach, a cluster of putative biosynthetic genes (BGC) responsible for the biosynthesis of sclerotiorin-like metabolites (scl cluster) was identified. The knockout of the sclA (highly reducing PKS) and sclI (non-reducing PKS) genes resulted in mutants with loss of mycelial pigmentation and terminated the biosynthesis of sclerotiorin-like metabolites: geumsanol B, chlorogeumsanol B, 7-deacetylisochromophilone VI, isochromophilone VI, ochrephilone, isorotiorin, and sclerotiorin. Based on these results, a biosynthetic pathway was proposed considering the homology of BGC scl genes with the azaphilone BGCs that have already been functionally characterized.

The plant organs and rhizosphere determine the common bean mycobiome.

The plant microbiota diversity is often underestimated when approaches developed mainly for the identification of cultivable microorganisms are used. High-throughput sequencing allows a deeper understanding of the microbial diversity associated with plants. The amplification of ITS1 was used to analyze fungal diversity in several plant organs and rhizosphere of three common bean (Phaseolus vulgaris) varieties grown in a greenhouse. The fungal diversity diverged between those plant organs and the rhizosphere, with the highest found in the rhizosphere and the lowest in the stem. In each organ different numbers of genus, OTUs were identified, in a total of 283 OTUs evenly distributed among the varieties. In the co-occurrence network, a larger number of positive interactions were found in the organs of the aerial part in all varieties. We observed that the diversity of the endophytic microbiota differed more between plant organs than between common bean varieties. Our results show that the diversity of endophytic fungi can be efficiently accessed with the sequencing of ITS amplicons and that this diversity may vary among distinct plant organs and the rhizosphere of a single plant variety.

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. CONCLUSION: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

Distribution and Genetic Diversity of Bacteriocin Gene Clusters in Rumen Microbial Genomes.

Some species of ruminal bacteria are known to produce antimicrobial peptides, but the screening procedures have mostly been based on in vitro assays using standardized methods. Recent sequencing efforts have made available the genome sequences of hundreds of ruminal microorganisms. In this work, we performed genome mining of the complete and partial genome sequences of 224 ruminal bacteria and 5 ruminal archaea to determine the distribution and diversity of bacteriocin gene clusters. A total of 46 bacteriocin gene clusters were identified in 33 strains of ruminal bacteria. Twenty gene clusters were related to lanthipeptide biosynthesis, while 11 gene clusters were associated with sactipeptide production, 7 gene clusters were associated with class II bacteriocin production, and 8 gene clusters were associated with class III bacteriocin production. The frequency of strains whose genomes encode putative antimicrobial peptide precursors was 14.4%. Clusters related to the production of sactipeptides were identified for the first time among ruminal bacteria. BLAST analysis indicated that the majority of the gene clusters (88%) encoding putative lanthipeptides contained all the essential genes required for lanthipeptide biosynthesis. Most strains of Streptococcus (66.6%) harbored complete lanthipeptide gene clusters, in addition to an open reading frame encoding a putative class II bacteriocin. Albusin B-like proteins were found in 100% of the Ruminococcus albus strains screened in this study. The in silico analysis provided evidence of novel biosynthetic gene clusters in bacterial species not previously related to bacteriocin production, suggesting that the rumen microbiota represents an underexplored source of antimicrobial peptides.

Draft Genome Sequence of Streptococcus equinus (Streptococcus bovis) HC5, a Lantibiotic Producer from the Bovine Rumen.

Streptococcus equinus (Streptococcus bovis) HC5 is a bacteriocinogenic lactic acid bacterium with simple growth requirements. The draft genome sequence of S. equinus HC5 consists of 1,846,241 bp, with a G+C content of 37.04%. In silico analysis indicated that S. equinus HC5 might be useful to control bacteria that are detrimental to livestock animals.

MpSaci is a widespread gypsy-Ty3 retrotransposon highly represented by non-autonomous copies in the Moniliophthora perniciosa genome.

Transposons are an important source of genetic variation. The phytopathogen Moniliophthora perniciosa shows high level of variability but little is known about the role of class I elements in shaping its genome. In this work, we aimed the characterization of a new gypsy/Ty3 retrotransposon species, named MpSaci, in the M. perniciosa genome. These elements are largely variable in size, ranging from 4 to 15 kb, and harbor direct long terminal repeats (LTRs) with varying degrees of similarity. Approximately, all of the copies are non-autonomous as shifts in the reading frame and stop codons were detected. Only two elements (MpSaci6 and MpSaci9) code for GAG and POL proteins that possess functional domains. Conserved domains that are typically not found in retrotransposons were detected and could potentially impact the expression of neighbor genes. Solo LTRs and several LARDs (large retrotransposon derivative) were detected. Unusual elements containing small sequences with or without interruptions that are similar to gag or different pol domains and presenting LTRs with different levels of similarities were identified. Methylation was observed in MpSaci reverse transcriptase sequences. Distribution analysis indicates that MpSaci elements are present in high copy number in the genomes of C-, S- and L-biotypes of M. perniciosa. In addition, C-biotype isolates originating from the state of Bahia have fragments in common with isolates from the Amazon region and two hybridization profiles related to two chromosomal groups. RT-PCR analysis reveals that the gag gene is constitutively expressed and that the expression is increased at least three-fold with nutrient depravation even though no new insertion were observed. These findings point out that MpSaci collaborated and, even though is primarily represented by non-autonomous elements, still might contribute to the generation of genetic variability in the most important cacao pathogen in Brazil.

Analysis of Tc1-Mariner elements in Sclerotinia sclerotiorum suggests recent activity and flexible transposases.

BACKGROUND: Sclerotinia sclerotiorum is a necrotrophic fungus that is pathogenic to many plants. Genomic analysis of its revealed transposable element expansion that has strongly influenced the evolutionary trajectory of several species. Transposons from the Tc1-Mariner superfamily are thought to be ubiquitous components of fungal genomes and are generally found in low copy numbers with large numbers of deleterious mutations in their transposase coding sequence. RESULTS: This study shows that the genome of S. sclerotiorum has a large number of copies of Tc1-Mariner transposons, and in silico analysis shows evidence that they were recently active. This finding was confirmed by expressed sequence tag (EST) analysis. Fourteen new Tc1-Mariner transposon families that were distributed throughout the genome were identified, and in some cases, due to the excision/retention of introns, different transcripts were observed for the same family, which might be the result of an efficient strategy to circumvent mutations that generate premature stop codons in the RNA sequence. In addition, the presence of these introns shows that the transposase protein has a flexible coding sequence and, consequently, conformation. No evidence for RIP-like gene silencing mechanisms, which are commonly found in fungi, was found in the identified Tc1-Mariner elements, and analysis of the genomic insertion sites of these elements showed that they were widely distributed throughout the genome with some copies located near the 3' regions of genes. In particular, EST analysis demonstrated that one of these copies was co-expressed with a gene, which showed the potential for these elements to undergo exaptation. CONCLUSIONS: Fourteen novel Tc1-Mariner families were characterized. Some families had evidence of introns, which might or might not be excised depending on the family or element in question, and this finding demonstrates a possible strategy for overcoming possible mutations that generate premature stop codons in a RNA sequence. Tc1-Mariner elements likely play an important role in the structure and evolution of the S. sclerotiorum genome.

Characterization and potential evolutionary impact of transposable elements in the genome of Cochliobolus heterostrophus.

BACKGROUND: Cochliobolus heterostrophus is a dothideomycete that causes Southern Corn Leaf Blight disease. There are two races, race O and race T that differ by the absence (race O) and presence (race T) of ~ 1.2-Mb of DNA encoding genes responsible for the production of T-toxin, which makes race T much more virulent than race O. The presence of repetitive elements in fungal genomes is considered to be an important source of genetic variability between different species. RESULTS: A detailed analysis of class I and II TEs identified in the near complete genome sequence of race O was performed. In total in race O, 12 new families of transposons were identified. In silico evidence of recent activity was found for many of the transposons and analyses of expressed sequence tags (ESTs) demonstrated that these elements were actively transcribed. Various potentially active TEs were found near coding regions and may modify the expression and structure of these genes by acting as ectopic recombination sites. Transposons were found on scaffolds carrying polyketide synthase encoding genes, responsible for production of T-toxin in race T. Strong evidence of ectopic recombination was found, demonstrating that TEs can play an important role in the modulation of genome architecture of this species. The Repeat Induced Point mutation (RIP) silencing mechanism was shown to have high specificity in C. heterostrophus, acting only on transposons near coding regions. CONCLUSIONS: New families of transposons were identified. In C. heterostrophus, the RIP silencing mechanism is efficient and selective. The co-localization of effector genes and TEs, therefore, exposes those genes to high rates of point mutations. This may accelerate the rate of evolution of these genes, providing a potential advantage for the host. Additionally, it was shown that ectopic recombination promoted by TEs appears to be the major event in the genome reorganization of this species and that a large number of elements are still potentially active. So, this study provides information about the potential impact of TEs on the evolution of C. heterostrophus.

Construction of a Kluyveromyces lactis ku80⁻ host strain for recombinant protein production: extracellular secretion of pectin lyase and a streptavidin-pectin lyase chimera.

In several organisms used for recombinant protein production, integration of the expression cassette into the genome depends on site-specific recombination. In general, the yeast Kluyveromyces lactis shows low gene-targeting efficiency. In this work, two K. lactis ku80⁻ strains defective in the non-homologous end-joining pathway (NHEJ) were constructed using a split-marker strategy and tested as hosts for heterologous gene expression. The NHEJ pathway mediates random integration of exogenous DNA into the genome, and its function depends on the KU80 gene. KU80-defective mutants were constructed using a split-marker strategy. The vectors pKLAC1/Plg1 and pKLAC1/cStpPlg1 were used to evaluate the recovered mutants as hosts for expression of pectin lyase (PNL) and the fusion protein streptavidin-PNL, respectively. The transformation efficiency of the ku80⁻ mutants was higher than the respective parental strains (HP108 and JA6). In addition, PNL secretion was detected by PNL assay in both of the K. lactis ku80⁻ strains. In HP108ku80⁻/cStpPlg1 and JA6ku80⁻/Plg1 cultures, the PNL extracellular specific activity was 551.48 (±38.66) and 369.04 (±66.33) U/mg protein. This study shows that disruption of the KU80 gene is an effective strategy to increase the efficiency of homologous recombination with pKLAC1 vectors and the production and secretion of recombinant proteins in K. lactis transformants.

Abundance, distribution and potential impact of transposable elements in the genome of Mycosphaerella fijiensis.

BACKGROUND: Mycosphaerella fijiensis is a ascomycete that causes Black Sigatoka in bananas. Recently, the M. fijiensis genome was sequenced. Repetitive sequences are ubiquitous components of fungal genomes. In most genomic analyses, repetitive sequences are associated with transposable elements (TEs). TEs are dispersed repetitive DNA sequences found in a host genome. These elements have the ability to move from one location to another within the genome, and their insertion can cause a wide spectrum of mutations in their hosts. Some of the deleterious effects of TEs may be due to ectopic recombination among TEs of the same family. In addition, some transposons are physically linked to genes and can control their expression. To prevent possible damage caused by the presence of TEs in the genome, some fungi possess TE-silencing mechanisms, such as RIP (Repeat Induced Point mutation). In this study, the abundance, distribution and potential impact of TEs in the genome of M. fijiensis were investigated. RESULTS: A total of 613 LTR-Gypsy and 27 LTR-Copia complete elements of the class I were detected. Among the class II elements, a total of 28 Mariner, five Mutator and one Harbinger complete elements were identified. The results of this study indicate that transposons were and are important ectopic recombination sites. A distribution analysis of a transposable element from each class of the M. fijiensis isolates revealed variable hybridization profiles, indicating the activity of these elements. Several genes encoding proteins involved in important metabolic pathways and with potential correlation to pathogenicity systems were identified upstream and downstream of transposable elements. A comparison of the sequences from different transposon groups suggested the action of the RIP silencing mechanism in the genome of this microorganism. CONCLUSIONS: The analysis of TEs in M. fijiensis suggests that TEs play an important role in the evolution of this organism because the activity of these elements, as well as the rearrangements caused by ectopic recombination, can result in deletion, duplication, inversion and translocation. Some of these changes can potentially modify gene structure or expression and, thus, facilitate the emergence of new strains of this pathogen.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene.

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao.

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthora perniciosa, the causal agent of witches' broom disease of Theobroma cacao

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.

Genetic variability and chromosome-length polymorphisms of the witches' broom pathogen Crinipellis perniciosa from various plant hosts in South America.

Crinipellis perniciosa has been classified into at least four known biotypes associated with members of unrelated plant families. In this study, genetic variability is shown for 27 C (Cacao), 4 S (Solanum), and 7 L biotype (Liana) isolates of C. perniciosa collected from different regions of Brazil and South America. The objective was to investigate the genetic variability of the pathogen in the cacao-producing region of Bahia, Brazil, and elsewhere, through microsatellite analysis, and attempt to identify possible correlations between host specificity and electrophoretic karyotypes. The PCR-banding patterns were found to vary both within and between the different biotypes, and a correlation was established between the PCR-banding patterns and the chromosomal-banding patterns of each isolate. Microsatellite and chromosomal patterns among all of the L and S biotype isolates were distinctly different from the C biotypes analysed. A higher degree of genetic and chromosomal variability was found among C biotype isolates from the Amazon in comparison with C biotype isolates from Bahia, which seems to be comprised of only two main genotypes. This finding has important implications to the current cacao-breeding programme in Brazil.

Molecular characterization and expression profile of pectin-lyase-encoding genes from Penicillium griseoroseum.

Penicillium griseoroseum has been studied by our group because of its good pectinase production. Attempts have been done to clone pectinolytic genes, aiming to obtain pectinase-overproducing strains for industrial purposes. Here, two genes coding for pectin lyase were isolated from the P. griseoroseum genome. The plg1 gene has an open reading frame of 1341 bp coding for a putative protein of 374 amino acids with a calculated molecular mass of 40.1 kDa. The plg2 gene is characterized by an open reading frame of 1400 nucleotides and codes for a polypeptide of 383 amino acids. The plg1 gene 5'-flanking region contains putative binding sites for the transcription factors involved in regulation by ambient pH and catabolite repression. The primary structure of Plg1 and Plg2 proteins showed a relatively high homology (varying between 32.4% and 74.8%) to fungal pectin lyases characterized to date. Southern blotting analysis revealed that both genes are present as single copies in the fungus genome. Expression studies revealed a differing pattern of gene expression of plg1 and plg2 when mycelium was cultivated on medium containing different pectic components. Citric pectin followed by apple pectin were the carbon sources that best induced plg1 expression, and transcripts were detected from 24 to 76 h. The expression of the plg2 gene was monitored by reverse transcriptase - polymerase chain reaction, since Northern analysis failed to detect hybridization signals. The differential expression of these genes may provide means for the fungus to adapt to various growth conditions.

Easy detection of green fluorescent protein multicopy transformants in Penicillium griseoroseum.

Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/mug of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.

Easy detection of green fluorescent protein multicopy transformants in Penicillium griseoroseum

Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/µg of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.

Structural organization of polygalacturonase-encoding genes from Penicillium griseoroseum

The pectinolytic system of Penicillium griseoroseum has been studied as a model to investigate aspects of gene organization in filamentous fungi. Here we show that the endopolygalacturonase-coding genes previously isolated exist as single copies in the fungus genome. DNA blot analysis revealed the presence of corresponding genes in other Penicillium species, although only one or two genes were found in opposition to the endoPG gene family reported for other filamentous fungi. The nucleotide and amino acid sequences of Penicillium PG genes of retrieved from data banks were compared for intron length and number, codon usage, and consensus sequences for translation initiation sites. The introns are conserved in the same position, although there was no conservation of their nucleotide sequences. Other sequence features resemble those seen in Aspergillus and Neurospora genes

Electrophoretic characterization of Aspergillus nidulans strains with chromosomal duplications

Linhagens de Aspergillus nidulans que apresentam duplicaçäo cromossômica Dp(I-II) foram caracterizadas por eletroforese em campo pulsado. Foram analisados variantes morfologicamente deteriorados e melhorados. O cariótipo eletroforético demonstrou que em ambas as linhagens duplicadas (A e B) a banda de 4,2 Mb, que corresponde ao cromossomo II, näo estava presente e foi encontrada uma nova banda. Foram feitas hibridizaçöes usando os genes uapA (cromossomo I) e wA (cromossomo II), que demonstraram que a nova banda corresponde ao cromossomo II mais o segmento duplicado do cromossomo I. O tamanho da duplicaçäo foi determinado como aproximadamente 1,0 Mb. A análise das bandas cromossômicas da linhagem morfologicamente melhorada mostrou que o segmento duplicado do cromossomo I foi completamente perdido. Os variantes morfologicamente deteriorados V9 e V17 mostraram o mesmo cariótipo eletroforético apresentado pelas linhagens duplicadas. Contudo, o variante deteriorado V5 perdeu parte do cromossomo I e apresentou um rearranjo envolvendo o cromossomo V. Esse rearranjo pode ter resultado do tratamento mutagênico usado para a obtençäo dos marcadores genéticos. Os resultados obtidos nesse trabalho demonstram que a técnica de eletroforese em campo pulsado é uma ferramenta excelente para a localizaçäo de rearranjos cromossômicos.