Poster Abstracts, Molecular Karyotyping and Gene Mapping

Single-read Sequencing Reveals Synteny Between Ashbya gossypii and Saccharomyces cerevisiae at the Thr4 Locus

Regula Altmann-Johl and Peter Philippsen. Institute for Applied Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, 5056 Basel, Switzerland

Eight clones of a genomic Ashbya gossypii library were identified by Southern analysis to contain inserts derived from the smallest of the seven chromosomes. The termini of these clones were single-read sequenced and compared to data bases. This revealed the following homologies to Saccharomyces cerevisiae genes: THR4 (threonine synthase), PWP2 (periodic tryptophane protein), CYRI (adenylate cyclase), ERGIA (squalene epoxidase), SSK2 (MAPKKK), CPR3 (cyclophilin-3).

For future use as an auxotrophic marker the entire AgTHR4 gene was cloned and sequenced. This represents the first reported sequence for a threonine synthase from a filamentous fungus. The overall identity to the S. cerevisiae protein is 67.4 % on amino acid level. The disruption of the AgTHR4 gene lead to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying the AgTHR4 gene.

The homologies for THR4 and for PWP2 were found at the two termini of one plasmid, In S. cerevisiae these two genes map on chromosome III and are only separated by one other ORF. By sequencing the entire AgTHR4 and the adjacent regions one further ORF and a partial ORF were identified. Surprisingly, these four adjacent ORF's are arranged in A. gossypii and S. cerevisiae in the same order and orientation. We have started sequencing the termini of many more A. gossypii clones to search for homologies and additional cases of synteny between this filamentous fungus and S. cerevisiae.

Vegetative Compatibility Groups and Electrophoretic Karyotype Variation among Isolates of Fusarium oxysporum from Common Bean Fields in Spain

F.M. Alves-Santos and J.M. Diaz-MInguez. Area de Genitica, Departamento de Microbiologia y Genitica, Universidad de Salamanca, 37007 Salamanca, Spain.

We have undertaken a comparative study of the genetic structure and variability of pathogenic and non pathogenic isolates of F. oxysporum from common bean fields in Spain. As a first step here we describe the vegetative compatibility grouping of F. oxysporum isolates and the correlation of these VCGs with electrophoretic karyotype (EK) variation.

F. oxysporum isolates were sampled from rizosphere or colonized tissues of common bean plants (Phaseolus vulgaris L.) from several plots in the zone of Barco de Avila in the province of Avila (Spain). Monoconidial cultures were derived from microconidia from the original isolate and assayed for production of Fusarium wilt in pathogenicity tests carried out on the bean variety "blanca redonda". Isolates which produced no external symptoms of wilt were classified as non pathogenic and used to generate nit mutants on PDC plates, following the procedure described by Correll (1987). nit1, nit3 and nitM mutants from each isolate were tested for vegetative compatibility with complementary nit mutants from other isolates. Up to now we have found 15 VCGs with at least one nitM tester per VCG. No heterokaryon self-incompatible strains have been found among the isolates analyzed so far.

Chromosomal DNAs were prepared from all the isolates belonging to the 15 VCGs and subjected to CHEF electrophoresis. Different conditions were tried in order to resolve the maximum number of chromosomal bands. Most commonly the different EKs were composed of 9 to 11 chromosomes ranging in size from 0.7 Mb to around 10 Mb. EK variation was greater between members of different VCGS. Some VCGs had remarkably uniform EKs. For instance the four members of VCGI showed identical EKs even though these isolates were collected from different plots.

The results obtained show a high number of genetically distinct isolates, as determined by VCGs and EKs, among F. oxysporum colonizing common bean rizosphere. The next step will be to determine whether pathogenic isolates are also highly diverse or they belong to the same clonal lineage.

Genetic Evidence for the Occurrence of Dispensable 'B' Chromosomes in the Phytopathogenic Ascomycete Leptosphaeria maculans (Phoma lingam)

Stephane Leclair, Delphine Ansan-Melayah, Thierry Rouxel and Marie-Helene Balesdent. Unite de Pathologie Vegetale, I.N.R.A., 78026 Versailles CEDEX France.

Leptosphaeria maculans electrokaryotypes are easily established using CHEF (Contour-clamped Homogeneous Field) electrophoresis and reveal major chromosome length polymorphism between field isolates or following meiosis. All field isolates displayed a small-sized chromosome clearly separated from the overall electrokaryotype using appropriate electrophoretic conditions. Minichromosome ('MC') size polymorphism ranged from 650 to 950 Kb. Tetrad analyses of crosses between isolates displaying size polymorphism for the MC revealed either parental ditype segregation, monoparental segregation, or generation of MC of intermediate size. MC with higher or lower size than that of the parents were also generated. Genetic analyses thus demonstrated that MC from different field isolates were homologues. Eighteen percent of the tetrads analysed had lost the MC band for 1 or 2 of the 4 genotypes of the tetrads. Crosses between isolates differing in the presence:absence of the MC revealed non-mendelien segregations and demonstrated that some isolates could display at least two copies of the MC. Finally, saprophitic or parasitic fitness was not modified when isolates lacked the MC. All these data strongly suggested that the small-sized chromosome of L. maculans behaves like a dispensable 'B' chromosome.

Chromosomal Rearrangements and Despersed Repetitive Sequences in Fusarium oxysporum

J. M. Daviere, T. Langin, C. Gerlinger, M.J. Daboussi. Institut de Gentetique et Microbiologie, Univeriste Paris-Sud, Bat 400, 91450 Orsay CEDEX France

The discovery of at least seven families of transposable elements in the genome of the phytopathogenic fungus Fusarium oxysporum raises a number of questions on their role in the evolution and adaptation of natural populations. Indeed these elements could represent the origin of multiple chromosomal rearrangements which are responsible for the extraordinary karyotype variation detected among strains of F. oxysporum. These rearrangements (deletion, inversion, translocation) would be the consequence of homologous recombination events among copies of dispersed repetitive sequences. In order to better understand the role of transposable elements on the genome structure of F. oxysporum we developed an experimental system which is based on the introduction, within the same strain, of truncated copies of the niaD gene with a common sequence of about 2 kb, representing the substrate for recombination. The frequent observation of colonies able to utilise nitrate as a consequence of gene runction restoration by mitotic crossing over indicates that this process is highly efficient in F. oxysporum. We therefore analysed the karyotypic variation in order to identify the nature of observed rearrrangements. Pulsed-field gel electrophoresis conditions allowing the separation of chromosomes within 1 and 7-8 Mb range were defined. A polymorphism in the number (11-14) and in size of some chromosomes was observed among subcultures for the same strain and among strains in which a transposition event was selected. The precise nature of events is now under evaluation by probing the electrophoretic karyotypes with different single copy genes or transpoaable elements.

Mucor circinelloides Strains Differing in Mating Type Show Electrophoretic Karyotype Heterogeneity

M. A.Lopez-Matas, A.P. Eslava and J.M. Diaz-Minguez. Area de Genitica, Departamento de Microbiologia y Genitica, Universidad de Salamanca, 37007 Salamanca, Spain.

The electrophoretic karyotypes of strains NRRL 3631 (M. circinelloides f. lusitanicus) and NRRL A-7420 (M. circinelloides f. gryseo-cyanus) have been reported recently. They show a considerable amount of variation, both in chromosomal number and size. It would be desirable to assess whether these polymorphisms extend to other M. circinelloides strains and if they may be correlated with mating type.

Chromosomal DNAs were prepared from strains CBS 276.49 (incorrectly named CBS 277.49 in previous works), ATCC 1216a (-) and ATCC 1216b (+) and subjected to contour clamped homogeneous electric field gel electrophoresis (CHEF) under different conditions. 276.49 and 1216a, both strains of mating type (-), share the highest chromosomal homology, all the bands resolved being the same size except for the smallest chromosomes. 1216b, mating type (+), has an electrophoretic karyotype that shows greater differences, both in chromosome sizes and number of bands. Disimilarities are more evident among the largest chromosomes. Genome sizes estimated by adding the sizes of individual chromosomes are around 31-33 Mb for the mating type (-) strains and around 46 Mb for the mating type (+) strain.

The degree of homology between chromosomes of similar size was further investigated: several single copy probes were used in Southern hybridizations to detect their chromosomal location. These probes are genes previously described (pyrG and leuA) or recently characterized by our group (chs1 and chs2, genes involved in the chitin biosynthetic pathway). Hybridization data indicate that pyrG, leuA and chs2 are each located in equivalent chromosomes of the three strains analysed, while chs1 shows hybridization against the 5.7 Mb chromosome of (-) strains and the 2.7 Mb chromosome of the (+) strain.

These results suggest that polymorphisms at the chromosomal level may constitute a source of genetic variation among M. circinelloides strains differing in mating type. Whether this variation may be of some importance in hampering the outcome of genetic crosses needs further verification. A survey of M. circinelloides strains showing reasonably homologous chromosomes would be of help to determine the most adequate fertile pairs to perform genetic crosses in this fungus.

Avirulence Genes Cloning in the Rice Blast Fungus Magnaporthe grisea

Waly Dioh 1, Didier Tharreau 2, Rocio Gomez 2, Eddy Roumen 2, Marc Orbach 3, Jean- Loup Notteghem 2, Marc- Henri Lebrun 1. 1Institut de Genetique et de Microbiologie, CNRS UPA 1354, Universite Paris Sud, 91405 Orsay, France. 2Laboratoire de Phytopathologie, CIRADICA, BP 5035, 34032 Montpellier, France. 3Department of plant pathology, University of Arizona, Tucson, 85721 Arizona, USA.

Magnaporthe grisea is an Ascomycete responsible for the major fungal disease of rice. We identified M. grisea isolates pathogenic to rice and fertile in crosses. These isolates were used to identify fungal avirulence genes involved in interactions with race-specific rice resistance genes. Unravelling the mechanisms involved in such an interaction will help us in the identification and deployement of genes that confer durable resistance. Three genetically independent avirulence genes were identified in a cross between two isolates Guy 1 1 and 2/0/3, pathogenic to rice : Avr-MedNoi-1, Avr-Irat7-1, Avr-Ku86-1 (1, 2). These three avirulence genes are likely to interact with so far undescribed resistance genes in rice. In order to clone these avirulence genes by chromosome walking, we constructed a partial genetic map using 77 random progeny from this cross. This map includes 75 RFLP markers corresponding to either repeated, single copy or telomeric sequences and 25 RAPD markers. Two avirulence genes mapped to chromosome tips (Avr-MedNoi-I and AvrKu86-1). Using bulk segregant analysis, we found either one or two RAPD markers linked (I to 4 cM) to each avirulence gene. Most of these RAPD markers were composed of repeated and dispersed sequences alongside with single copy sequences. Single copy sub-clones were obtained from some of these RAPD markers. These single copy probes revealed polymorphisms between the two parents which could be due to chromosomal rearrangements (deletions) around avirulence loci. Linked single copy probes will be used as starting point for chromosome walks toward these genes.

1. Silue al.. 1992. Phytopath., 82:577-580

2. Silue aL. 1992. Phytopath., 82:1402-1407

A Genetic Linkage Map of Phytophthora sojae

S. C. Whisson, A Drenth, D.J. Maclean, and J.A.G. Irwin. Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Queensland, Australia, 4072.

Phytophthora sojae belongs to the oomycetes which are characterised by gametangial meiosis, thus having a diploid life cycle. Until recently the genetics of virulence/avirulence in P. sojae was considered intractable due to its homothallic nature. Two crosses between genetically different isolates of P. sojae were established by coculturing parental isolates followed by screening the predominantly selfed progeny to identify hybrids using RAPD markers. Hybrids were recovered at a rate of approximately 2 % for both crosses. One isolate was used as a common parent in both crosses. F2 populations comprising over 200 individuals were generated for each cross. A subset of 53 F2 individuals from each cross was selected at random for analysis of segregations of avirulence genes and molecular markers, and finally the construction of a genetic linkage map. The genetic linkage map developed for P. sojae is based on 35 RFLP, 229 RAPD, and 7 dominant avirulence markers. The linkage map comprises ten major linkage groups and fourteen small linkage pieces covering a total of 968.4 cM. Close linkage (0.2 cM) of a RAPD marker to two avirulence genes (Avr4 and Avr6) may form the starting point for chromosome walking strategies directed towards cloning avirulence genes from P. sojae.

Chromosomal Mapping of an Endochitinase Gene from Trichoderma hamatum

Fekete, C. and Hornok, L. Agricultural Biotechnology Center, 2101 Godollo and Department of Microbiology, University of Agricultural Sciences, 2100 Godollo, Hungary

A PCR-amplified DNA fragment (1,450 bp) from the antagonistic fungus, Trichoderma hamatum was subjected to sequence analysis. This sequence proved to be highly homologous (93.1 %) to that of a chitinase gene from Trichoderma harzianum, but much less homology (66 %) was found with the chitinase gene from Aphanocladium album. The chitinase gene of T hamatum contains three introns, 57, 65 and 66 bp in sizes, as well as two highly conserved regions located in the catalytic domain and probably code for the active site of the enzyme. When chromosome-sizd DNA-fractions of four other Trichoderma species (T. atroviride, T. harzianum, T. koningli, T. viride) were probed with the chitinase gene from T. hamatum, hybridisation signals developed in all cases on a chromosome aggregate of -5.5 Mb in size.

A Repetitive DNA Sequence from Fusarium poae

Hornok L., Papp, I., and Fekete, C. Department of Microbiology, University of Agricultural Sciences, 2100 Godollo and Agricultural Biotechnology Center, 21 01 Godollo, Hungary

Fusarium poae is a trichothecene producing asexual fungus and lacks a complete parasexual cycle, as well; all strains of this species harbour virus-like particles with double-stranded RNA genome. A 1209 bp clone, named ZITI isolated from F. poae strain 72.187 was found to be a moderately repetitive DNA sequence, which selectively hybridised to the polymorphic chromosomal regions of various F. poae strains. Sequence analysis of ZITI revealed retrotransposon-like structures, among them a characteristic zinc-finger motif was the most prominent. ZITI may play a role in the genome plasticity of F. poae.

Differentiating Interspecies Hybrids of Aspergilli by RAPD Analysis and Electrophopetic Karyotyping.

J. Hothersall and J. F. Peberdy. Department of Life Science, University of Nottingham, Nottingham NG7 2RD, United Kingdom.

Interspecific hybridisation between auxotrophic mutants of the filamentous fungi Aspergillus nidulans, A. rugulosus and A. ochraceus was studied using polyethylene glycol induced protoplast fusion. Viable prototrophic fusion products from crosses between A. nidulans and A. rugulosus were exposed to benomyl containing medium which promotes haploidisation and resulted in the formation of haploid segregants. The aim of this investigation was to assess the suitability of Randomly Amplified Polymorphic DNA (RAPD) and electrophoretic karyotyping in distinguishing interspecific hybrid haploid progeny from their parental species.

Several 10-mer oligonucleotide primers which gave unique RAPD profiles for the parental species were used to analyse over twenty segregants. RAPD profiles different to those of either parent were generated demonstrating that recombinant hybrids had been isolated through an induced parasexual cycle.

Electrophoretic karyotypes were established by pulsed field gel electrophoresis using the contour-clamped homogenous electric field (CHEF) system. Unique karyotypes were obtained for the parental species. A. nidulans and A. rugitlosits were each separated into five chromosomal mobility groups. In both cases some groups contain more than one chromosome of similar size, which may be further identified by Southern analysis using chromosome specific probes. Nine segregants karyotyped to date indicate chromosomal reassortment has occurred.

Following protoplast fusion the established procedures for confirming and determining the extent of hybridisation between two species are based on genetic marker recombination studies. However in some circumstances it may be undesirable to introduce several genetic markers into the parental species through mutagenesis. The presented results clearly demonstrate both RAPD analysis and electrophoretic karyotyping to be valuable in the differentiation of interspecies hybrids and would provide a rigorous non-invasive analysis of hybridisation.

Characterisation of a Gene of the Blackleg Fungus, Leptosphaeria maculans Conferring Virulence on Indian Mustard

Howlett, B. J., Chen C.Y. and Cozijnsen, T. Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, VIC, 3052, Australia

Leptosphaeria maculans causes blackleg disease of oilseed Brassicas worldwide. The blackleg fungus is a model system for studying the genetic basis of host-pathogen interactions as it has a haploid vegetative state, is outcrossing, can be cultured on defined media, has a relatively small genome size and a high efficiency DNA transformation system. We are examining the interaction of blackleg isolates on Brassica juncea (Indian mustard). In a cross between a virulent blackleg isolate (attacks varieties of Indian mustard) and an aviruient isolate (cannot attack), the Fl and backcross progeny segregate in a 1:1 ratio, suggesting the presence of a single locus for virulence (Chen et al. 1995). We are determining the molecular basis of this trait.

One way that virulent L. maculans isolates may invade Indian mustard is by breaking down preformed host defence compounds. Seeds and leaves of this plant contain high levels of glucosinolates, sulphur-containing compounds that give mustard a pungent taste. Upon wounding of the plant, these compounds are cleaved into glucose and volatile compounds such as isothiocyanates, which are toxic to some insects and fungi. We have shown that volatiles from Indian mustard seed meal (a rich source of glucosinolates) inhibit the growth of avirulent blackleg isolates (cannot attack Indian mustard) much more strongly than they inhibit the growth of virulent isolates. We are currently testing the progeny of a cross between avirulent and virulent blackleg isolates to see if the ability to grow in the presence of the volatiles segregates with the virulence trait. We are also looking at the ability of virulent blackleg isolates to metabolise isothiocyanates.


Chen CY, Plummer KM and Howlett BJ (1995) Ability of an Leptosphaeria maculans isolate to form stem cankers on Indian mustard segregates as a single locus. European Journal of Plant Pathology, in press.

Mapping the Genome of Claviceps purpurea by Means of RFLP Analysis and Electrophoretic Karyotyping

Frank Luerweg, Sabine Giesbert and Paul Tudzynski. Westfalische Wilhelms-Universitat, Institut fur Botanik, SchloBgarten 3, 48149 Munster

Claviceps purpurea (Fries) Tulasne, belonging to the pyrcnomycetous ascomycetes, is a phytopathogenic fungus with a wide host range parasitizing more than 200 species of Poaceac. It is not known whether this is due to the existence of subspecies or physiological races.

Different strains of Claviceps purpurea vary frequently in their chromosomal equipment. It is possible to perform sexual crossings between strains with a different karyotype resulting in a fertile progeny.

In this work, the progeny of a crossing between two Claviceps-strains differing largely in their karyotypes were examined by means of Pulsed Field Gel Electrophoresis (PFGE), the analysis of Restriction Fragment Length Polymorphisms (RFLP) and Random Amplified Polymorphic DNA (RAPD). The majority of the molecular markers showed a 1: 1 -segregation. Pulsed Field Gel Electrophorcsis revealed six chromosomes for each parental strain, whereas nine linkage groups could be found by the analysis of the segregation patterns of the molecular markers (65 RFLP-markers, 32 RAPD-markers).

Genome Structure and Chromosomal Polymorphisms in Field Isolates of Colletotrichum lindemuthianum

Donal O’Sullivan 1,2, F. Creusot2, B.M. Cooke1, and M. Dron2. 1 Dept of Env. Resource Management, Faculty of Agriculture, University College Dublin, Dublin 4. 2 Laboratoire de Phytopathologie Moleculaire, Institut de Biotechnologie des Plantes, Batiment 630, Universite de Paris-Sud, 91450 ORSAY Cedex

The filamentous ascomycete Collelotrichrim lindemuthiuanum causes anthracnose of the common bean Phaseolus vulgaris, and is responsible for significant yield losses in tropical regions. The fungus lacks a sexual stage but exhibits nevertheless a large degree of diversity in both pathotype, as determined by pathogenicity testing on a set of differential bean cultivars, and genotype, as measured by RAPD and RFLP analyses (Fabre el al, 1995). The genome structure was investigated at different levels. Flow cytometry was used to determine the haploid DNA content of the nucleus, as well as ploidy levels in the growing mycelium. Strains representing genotype groups I and 11 (as defined by ITS and RFLP analyses of the ribosomal DNA unit) had similar DNA contents, suggesting that the evolutionary divergence between these groups has not resulted in large-scale reductions or expansions of the genome. At a finer level, physical techniques (notably pulsed-field separation of chromosomes, separation of rare cutter digests, and hybridisation analysis) have been used to identify variable regions of the genome, with the aim of finding evidence of structural plasticity which might explain in part the high level of variability found in the fungal population.

The results of pulsed-field separations of nuclear DNA of twenty strains (representative of a worldwide collection offield isolates) reveal molecules of two remarkably disparate size classes, the first consisting of chromosomes which are larger than 10 Mb in size and not resoluble by pulsed-field electrophoresis, while the other comprises between one and four chromosomes in the range of 0,5 - 2,0 Mb. Within this latter class, there is great variation in both number and size of chromosomes indicating that at least a portion of the genome is subject to frequent rearrangements. Chromosome-specific sub-libraries are currently being prepared to obtain probes for the investigation of homology among chromosomes from different isolates.

Reference :

Fabre, J. V., Julien. J., Parisot D., & Dron. M. (I 995) Myc. Res. 99 (4), 429-43 5

AFLPc Mapping of the Phytophthora infestans Genome

Theo van der Lee, Ijfke de Witte and Francine Govers. Department of Phytopathology, Graduate School Experimental Plant Sciences, Wageningen Agricultural University, Binnenhaven 9, 6709 PD, Wageningen, The Netherlands.

A new, powerful DNA fingerprinting technique called AFLP (Vos et al. NAR 1995 in press) has been used to generate molecular markers of the Oomycetous plant pathogen Phytophthora infestans. The AFLP technique is based on PCR amplification of genomic DNA restriction fragments to which linkers with known sequence are ligated. The PCR primers consist of the linker sequence but are extended with 1, 2 or 3 bases to select a subpopulation of the genomic fragments for amplification. In this way up to a hundred different amplified fragments can be obtained in a single PCR reaction. Electrophoresis of the radioactive labelled fragments on polyacrylarnide and autoradiography results in DNA fingerprinting patterns which are reproducible and easy to score. With the AFLP technique it is possible to analyse many markers in a short time on a population large enough to establish a linkage map saturated with molecular markers. Currently, we are analysing the segregation of AFLP markers in the Fl progeny of a cross between two P. infestans isolates (80029x88133). In this progeny there is also segregation for several avirulence genes, among which avrl. If there is a Mendelian segregation of AFLP markers in this cross then DNA from AVR+and AVRI- progeny will be pooled and used for Bulked Segregant Analysis (BSA). In this way AFLP markers linked to the avri gene can be obtained and these will be used as starting point for map based cloning of the avrl gene. A preliminary AFLP linkage map of P. infestans will be presented.

Ashbya gossypii - a Filamentous Fungus with a Genome of less than 10 Mb

Sabine Steiner, Karin Gaudenz, Jurgen Wendland, Christine Mohr and Peter Philippsen. Institut fur Angewandte Mikrobiologie, Biozentrum Basel, Klingelbergstrafle 70, CH-4056 Basel, Switzerland

Ashbya gossypii is a phytopathogenic ascomycete which was first isolated from cotton but which also can infect citrus fruits and tomato. Furthermore, about 30 % of the word industrial riboflavin output is produced with this organism. A. gossypii is an unusual filamentous fungus. According to sequence homologies and behaviour of transforming DNA it resembles more the yeast Saccharonzyces cerevisiae than other filamentous ascomycetes. In addition, its genome is extremely small. Electrophoretic separation of chromosome sized DNA revealed seven chromosomal bands which range between 690 kb and 2020 kb and add up to a total genome size of 9.7 Mb. This value is very low compared with the genomes of other filamentous fungi (generally between 25 Mb to 40 Mb) and even compared with the genomes of unicellular fungi like S. cerevisiae (13.5 Mb) and Schizosaccharonyces ponibe (13.8 Mb). We therefore investigated if A. gossypii has larger chromosomes which did not enter the gel. Furthermore, we analysed if the seven bands really represent only seven chromosomes or if they consist of equally sized chromosomes comigrating within the gels. With hybridisation analyses using 80 randomly chosen clones as probes the seven bands were shown to represent the whole nuclear genome. To investigate the existence of double bands, the 18 bp recognition site for the restriction enzyme I-SceI was integrated by homologous recombination into chromosomes corresponding to each of the seven bands. Cleavages at these I-SceI-sites revealed that each band represents a single chromosome. Hence, the A. gossypii genome really consists of only 9.7 Mb and is therefore one of the smallest eukaryotic genomes described so far.

Molecular Analysis of Chromosome Polymorphism in Beauveria bassiana by Using Electrophoretic Karyotype and a Telomeric Probe

M. Viaudl, S. Masloff2, C. Levis3, Y. Couteaudierl and G. Ribal l . l Station de Lutte Biologique, INRA La Miniere, 78285 Guyancourt, France 2 Lehrstuhifar Allgemeine Botanik, Ruhr-Universitdi, D-44780 Bochum, Germany. 3 Station de Pathologie Vegetale, INRA, Route de Saint-Cyr, 78026 Versailles, France

The imperfect fungus Beauveria bassiatia is an entomopathogenic species widely used as biological control agent against several insects. The karyotypes of isolates from different host insects have been studied using two complementary approaches :

- Pulsed-Field Gel Electrophoresis was used to separate chromosomes. The electrophoretic karyotypes present bands between 1,2 and 7,5 megabase pairs and a high chromosome length polymorphism between isolates from different host insects. On the other hand, isolates from the European Corn Borer (Ostrinia nubilalis) could not be distinguished.

By Southern Hybridization with homologous probes, the chromosomal location of known genes (Nitrate reductase, -tubuline, Histone 4, rDNA) and gene of the Proteinase I (which is implicated in penetration of the host cuticule) have been mapped. Results show that similar sized chromosomes do not always bear the same information.

- The second approach involved a telomeric probe from Botrytis cinerea. By Southern hybridization on total DNA digested by restriction enzymes, the minimum number of chromosomes of each isolate has been estimated. This probe was also used onto Southern blots of restriction digests of individual chromosomes in order to detect doublets.

Molecular knowledge of the genome organisation in B. bassiatia is a milestone in the genetic improvement by protoplast fusion. These studies would facilitate the identification of strains with pathogenicity-related genes -on different chromosomes which would be good candidates for somatic hybridization in order to increase gene copy number and the diversity of pathogenicity genes.

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