Fungal Genomes

15. Molecular karyotypes for Alternaria plant pathogens known to produce host-specific toxins.

H. Akamatsu¹, Y. Itoh¹, M. Kodama¹, H. Otani², K. Kohmoto¹, and M. Taga³. ¹Laboratory of Plant Pathology, Faculty of Agriculture, Tottori University, Tottori 680, Japan. ²The United Graduate School of Agricultural Sciences, Tottori University, Tottori 680, Japan. ³Faculty of Science, Okayama University, Okayama 700, Japan.

There are at least ten plant diseases caused by Alternaria species in which host-specific toxins (HSTS) are responsible for fungal pathogenicity. Of these HST producers, seven are considered distinct pathotypes of the species called A. alternata. Inter- and intraspecific variation among Alternaria taxa, including HST producers, were determined by electrophoretic karyotype using pulsed-field gel electrophoresis. The seven pathotypes of A. alternata and nonpathogenic A. alternata had 9 to 11 chromosomal bands with estimated sizes ranging from 0.6 to 6.5 Mb. In contrast, Altemaria species that are morphologically distinct from A. altemata had 8 to 10 bands with sizes between 0.9 to 6.4 Mb; They could be differentiated from A. alternate on the basis of chromosome size polymorphisms. Comparison of karyotypes following Southern analysis using rDNA as a probe also revealed polymorphisms between A. alternata and other Alternaria species. The results were confirmed by DAPI staining and fluorescence in situ hybridization with the rDNA probe for chromosomes prepared by the germ tube burst method.

16. Replication mechanisms of circular fungal mitochondrial plasmids.

Dipnath Baidyaroy and Helmut Bertrand, Mchigan State University.

Fungal mitochondrial plasmids can be of three major types : (1) linear with a DNA polymerase and/or an RNA polymerase, (2) circular with a gene coding for a reverse transcriptase, and (3) circular with a novel DNA polymerase gene. These plasmids are generally present in high copy numbers, have mitochondrial codon usage and are of unknown physiological significance. The mechanism of their replication remains to be understood. We are trying to characterize the replication mechanisms of the two groups of circular plasmids by analyzing replication intermediates using two-dimensional gel electrophoresis. So far, we have examined the Mauriceville plasmid (Gr. 2) from Neurospora crassa and the pCRYI (Gr. 3) from Cryphonectria parasitica. Contrary to previous studies (Maleszka, 1992, Biochem. Biophy. Res. Comm. 186:1669-1673), we found that the plasmids are present in vivo as multimeric circles instead of linear molecules of heterogenous sizes. In addition, we have obtained data indicating that the Mauriceville plasmid, despite containing a functional reverse transcriptase gene, replicates predominantly by a rolling circle mechanism which effectively generates numerous multimeric forms, some as big as octamers (~28.8 kb), of the unit length plasmid. On the other hand, the pCRYI plasmid, which is assumed to replicate by the action of its own DNA polymerase, seems to have a standard bidirectional replication with the predominant form of the plasmid being the circular monomer. Supported by USDA grant 95-37303-1785,

17. Trichoderma reesei sequences that enhance transformation frequency bind to the nuclear matrix.

Nigel Belshaw^l, Satu Hakola², Helena Nevalainen^2,3, Merj a Penttila⁴, Pirkko Suominen² and David Archer^l. ¹Institute of

Food Research, Norwich, UK, ²Primalco Ltd. Biotec, Rajamaki, Finland, ³Macquarie University, Sydney,

Australia, ⁴VTT Biotechnology and Food Research, Espoo, Finland.

Three DNA fragments (trsl, 2 and 3) were isolated from the T. reesei genome by their ability to promote autonomous replication of plasmids in S.cerevisiae. The presence of the trs elements in transforming plasmids enhanced the frequency of transformation of T. reesei up to 5fold over plasmids without a trs. The trs elements did not promote autonomous replication of plasmids in T. reesei. The trs elements bound specifically to the isolated T. reesei nuclear matrix both in vitro and in vivo indicating that they are matrix attachment regions (MARs). The trs elements were sequenced and shown to contain 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7kb respectively for trs 1, 2 and 3. trs 1 and trs3 were shown to be single copy in the T. reesei genome. A sequence previously isolated from A.nidulans that promotes autonomous replication of plasmids in S.cerevisiae (ansl) was also shown to be a MAR. Each trs contained sequence motifs commonly ascribed to MARs from other eukaryotes although we showed that the motifs were just as common in a series of randomly generated sequences of identical AT richness.

18. Genomic organization of genetically defined Pneumocystis populations.

Melanie T. Cushion, Lisa Knapp and Sally Orr. University of Cincinnati College of Medicine, Cincinnati, OH.

Pneumocystis organisms are the cause of a pneumonia in a wide variety of mammals that become immunocompromised. Gene sequence data show they are related to fungi but may represent an independent basal branch in the Ascomycota. Surveys of organisms obtained from the lungs of immunosuppressed rats in commercial colonies using CHEF-PFGE and gene sequencing techniques revealed the rats were infected with either of 2 apparent species of Pneumocystis, "prototype" or "variant" or a combination of both. Eight different forms of prototype (1 -8) and 2 of variant were defined by electrophoretic karyotyping. Prototype forms produced a total of 12-15 chromosome sized bands that separated between 300-700 kb in prototype forms 1-8 while 14-17 bands were observed in the 2 variant forms within the same size range. We are establishing chromosome numbers by hybridization of telomeric probes to Southern blotted restriction enzyme digested chromosome-sized bands excised from low melt gels (Mills et al. In Molec. Methods in Plant Pathol. Lewis Publishers, 1995) and linkage groups by hybridization of gene probes to Southern blotted karyotypes. At least 1 single copy gene has been localized to 8 of 14 bands in the karyotypes of prototype forms and to 4 bands of variant karyotypes. Gene probes hybridized to bands of similar sizes among the prototype karyotypes, but dramatic size differences were observed between prototype and variant populations, supporting the species distinctions detected by gene sequence comparisons.

19. Organization of the nitrate assimilation gene cluster of Stagonospora (Septoria) nodorum.

Simon B. Cutler, R. Neil Cooley and Christopher E. Caten, University of Birmingham, U.K.

The nitrate reductase gene (NIA1) of the phytopathogenic fungus Stagonospora (Septoria) nodorum has been cloned from a cosmid library by homologous hybridization with a PCR-generated probe. A 6.7kb fragment carrying the NIA1 gene was subcloned and partially characterized by restriction mapping. Sequencing of the gene indicated a high degree of homology, both at the nucleotide and amino acid levels, with nitrate reductase genes of other filamentous fungi. In addition, sequencing downstream of the NIA1 gene revealed another open reading frame which proved to be the nitrite reductase gene (NIA1) of the fungus. The nitrate and nitrite reductase-encoding genes of S. nodorum are contiguous and transcribed in the same direction. Furthermore, both genes contain consensus regulatory signals in the 5' flanking regions thought to be involved in the control of nitrogen metabolism.

The cloned NIA1 gene has been used to develop a gene transfer system based on nitrate assimilation. Stable mutants of S. nodorum defective in nitrate reductase activity were isolated by virtue of their resistance to chlorate. These were transformed back to nitrate utilization independently with the Aspergillus niger niaD gene and the S. nodorum NIA1 gene. Southern analyses revealed that transformation occurred as a result of integration of transforming DNA into the fungal genome. Moreover, it was shown that in many cases integration was targeted to the homologous sequence.

20. Preferentially expressed genes from the sexual stage of Neurospora crassa.

Patricia L. Dolan, Donald O. Natvig, Mary Anne Nelson, Department of Biology, University of New Mexico, Albuquerque, NM .

Neurospora crassa, a heterothallic filamentous fungus, undergoes a complex pattern of sexual development to form the perithecium (fruiting body) composed of several kinds of specialized tissue. In the Neurospora Genome Project (NGP) at the University of New Mexico, expressed sequence tags (ESTs) corresponding to three stages of the life cycle of Neurospora crassa (conidial, mycelial and perithecial) are being analyzed. Results of this pilot project identifying genes preferentially expressed during the sexual phase are presented. 557 partial complementary DNA (cDNA) sequences for 425 perithecial clones were determined using single-pass sequencing. For 29.5% of the sequences, highly or moderately significant matches to sequences in the NCBI database were detected. Approximately 59.2% of the ESTs correspond to previously unidentified genes. Genes involved in secondary metabolism were found only in mycelial and perithecial tissues. Genes encoding products required for transport were found primarily in mycelial and perithecial tissues. The majority of the genes from all three tissues were involved in metabolism or protein synthesis. In addition, the serial analysis of gene expression (SAGE) technique is being used to generate more extensive and precise information about mRNA abundance at different stages of the Neurospora life cycle.

21. Structural analysis of an unstable chromosome in Nectria haematococca.

J. Enkerli¹, G. Bhatt² and S. F. Covert², ¹ Dept. of Botany, ²Warnell School of Forest Resources, University of Georgia, Athens, GA 30602, USA

Certain isolates of Nectria haematococca mating population VI contain a dispensable 1.6 Mb chromosome that is unstable during sexual reproduction. Previous karyotype analysis of genetic crosses suggested that specific regions of this chromosome are particularly susceptible to chromosome breakage (Miao et al., 1991 Science 254: 1773 - 1776). We are testing the hypothesis that repeated sequences contribute to the chromosome's instability. Using a chromosomespecific cosmid library, we created a contiguous cosmid map of the 1.6 Mb chromosome. The map consists of 8 contigs covering 1.3 Mb of the chromosome. We are using PCR and restriction enzyme mapping to order the contigs. Three repeated sequences that are each present in 5 to 7 copies on the chromosome were cloned during the mapping process. One of these repeats is homologous to Fot1, a transposable element in Fusarium oxysporum. To identify breakpoints and missing fragments, a variety of chromosomal derivatives are being used to probe the chromosome-specific cosmid library. Identified breakpoints will be mapped on a fine scale to determine if they are associated with repeated sequences.

22. Identifying sites of duplication-dependent DNA methylation in Coprinus cinereus.

Sylvia A. Frazier and Patricia J. Pukkila, University of North Carolina at Chapel Hill.

CpG methylation is known to be involved in gene inactivation and to be responsible for genomic imprinting for most eukaryotes. We wish to understand the consequences of cytosine methylation in the fungal system Coprinus cinereus. It was shown previously that cytosine methylation can occur as a result of a duplication of the trp1 gene in the C. cinereus genome. Using a genomic sequencing protocol, we have analyzed the trp1 gene sequence of several clones representing one tetrad known to contain methylated cytosines. This tetrad which has both Trp⁺ and Trp^- progeny was used to make a direct comparison of differences in methylation between silenced and unsilenced genes. The objective is to determine if specific methylated cytosines are necessary and sufficient to render the trp1 gene inactive. Sequence analysis has confirmed that there is site specificity for cytosine methylation, and that this methylation occurs almost exclusively at CpG dinucleotides. Information on the location and extent of methylation in C. cinereus may give us clues about how gene silencing occurs in this species and in eukaryotes as a whole.

23. Generation and characterization of selenate-resistant mutants of Magnaporthe grisea.

Tyler L. Harp and James C. Correll, University of Arkansas, Fayetteville.

Numerous mutants have previously been recovered and genetically characterized in the fungus Magnaporthe grisea. In this study, selenate-resistant (sel) mutants of M. grisea were generated and characterized. Isolates representing several different MGR586 DNA fingerprint groups of M. grisea were grown on either rice bran agar (RBAS), potato dextrose agar, or a defined minimal medium, all amended with 0. 1% sodium selenate. Spontaneous fast-growing sectors were recovered from the restricted colonies after 7 - 28 days. All sectors were then transferred back to RPAS to verify that they were selenate-resistant (sel) mutants. All sel mutants were further characterized for their ability to utilize sulfur by growing them on NM amended with K₂S0₄ or a reduced sulfur (L-methionine) source. Mutants unable to utilize sulfate-sulfur were designated as sulfate non utilizing (sul) mutants. The majority of the sectors recovered were sul mutants. Broth growth-rate studies confirmed the inability of sul mutants to utilize sulfate sulfur; furthermore, growth of sul mutants were comparable to wild-type when given a reduced sulfur source. Generation frequency varied from 0.1 to 1.3 sectors per colony after 28 days on RBAS and mean sector frequency corresponded with MGR586 DNA fingerprint group. Sul mutants were stable and comparable in virulence to the corresponding wild type isolates on rice in greenhouse pathogenicity tests.

24. Analysis of expressed sequence tags (ESTS) corresponding to conidial, mycelial, and sexual stages of Neurospora crassa.

Seogchan Kang, Mary Anne Nelson, and Donald O. Natvig, Department of Biology, University of New Mexico, Albuquerque, NM.

Analysis of ESTs has been widely applied in many model organisms as a rapid method of gene discovery. We have undertaken an analysis of ESTs corresponding to three stages of the life cycle of Neurospora crassa. Three unidirectional cDNA libraries were constructed, using mRNA isolated from germinating conidia, mycelia, and perithecia. Single-pass, partial sequencing of cDNA clones was used to rapidly identify duplicate clones present in the three libraries and to determine the nature of the encoded gene products. 1879 partial cDNA sequences for 1423 clones were determined. Contig analysis allowed the identification of 838 unique ESTs and 156 ESTs present in multiple cDNA clones. For about 33% of the sequences, highly or moderately significant matches to sequences in the NCBI database were detected. Approximately 57% of the ESTs show no similarity to previously identified genes, which was unexpected given the presence of the complete genome sequence of Saccharomyces cerevisiae in the database. Our result demonstrates the wealth of genes yet to be discovered, and supports the assertion that sequencing of genes from phylogenctically diverse organisms will yield novel insights into gene structure and function.

25. In vitro reconstruction of the Neurospora crassa genome.

H. Kelkar, M.E. Case, S. Covert, J. Griffith, C. Keith, M. Orbach, M. Sachs, and J. Arnold.

Physical maps of fungal genomes are opening up new research areas in genetics. We are building a physical map of the Neurospora crassa genome to study its distribution of repeated DNA and to contrast its distribution of repeats with that of the Aspergillus nidulans genome. We have expanded the Orbach-Sachs Library of 4800 clones in the Fungal Genetics Stock Center by 10,000 cosmid clones in the cosmid vector, pMOcosX. Average insert size is estimated to be 34 kb +/- 1 kb from EcoRI digestions. A second new cosmid vector has been created called LoristX6h. The cosmid vector Lorist6 was improved by inserting an adaptor (Orbach, 1994, Figure 1, Gene 150: 159-162) at the BamHI cloning site to allow half site fill ins (automatic insert size selection) as well as a hygromycin cassette (Carrol et al., 1994; Fungal Genetics Newsletter 41: 22) from plasmid pCB1004 for fungal transformation. A second cosmid library of 10,000 clones is being made in Lorist6Xh. The seven N. crassa chromosomes have been separated into 5 bands on CHEF gels, and the libraries are being probed with intact whole chromosomes corresponding to linkage groups I, V, IV, and III initially. A robot serving as an Intelligient System for Automated Assembly of Chromosomes (I.S.A.A.C.), for use by the fungal genetics community, is being constructed for ordering these libraries. (supported by NSF MCB-9630910 and BIR-9512887)

26. Proposal to sequence the genome of Aspergillus nidulans.

Doris M. Kupfer*, Sandra W. Clifton*, Rolf A. Prade, Bruce A. Roe*. *University of Oklahoma, Oklahoma State University.

A.nidulans is an ideal model organism for molecular and genetic studies of filamentous fungi. With the development of methods for rapid and accurate large scale DNA sequencing, the Aspergillus community has proposed undertaking sequencing the A. nidulans genome. We have begun three pilot projects as a prelude to the sequencing of the entire genome.

First, the sequence of the 38.8 Kbp cosmid, SW06EO8, from Chromosome VIIII was completed as a feasibility study. Thirteen ORFs were observed. Seven had homology with fungal and other higher eucaryotic genes. Six had no homologs in the public databases. Second, we are creating an Expressed Sequence Tag (EST) database by sequencing the vegetative and asexual A. nidulans cDNA library constructed by Rudolfo Aramayo. To date we have isolated over 5000 clones and sequenced 600. This data will be useful for analysis of the future genomic sequences and will be available on our www site, http://www.genome.ou.edu.

Third, sequencing of the 2.9Mbp Chromosome IV will commence upon completion of the EST database. We will shotgun shear and clone the entire chromosome via an approach similar to that used with great success in our laboratory to sequence the genomes of both Neisseria gonorrhoeae and Streptococcus pyogenes. Members from an ordered set of A. nidulans cosmids will be end sequenced to facilitate ordering of contiguous sequences generated from the random sequencing. At the culmination of the Chromosome IV project, a consortium of labs from the Aspergillus community will sequence the remaining seven chromosomes.

27. Optional introns in mitochondrial DNA of Podospora anserina are the primary source of observed polymorphisms.

Jill L. Salvo, Birgit Rodeghier, Arnon Rubin and Taylor Troischt; Dept. of Biology, Union College, Schenectady, New York 12308.

We have demonstrated that the significant differences in mitochondrial genome size among six races (M, T, U, B, W and Y) of Podospora anserina are primarily due to the presence and/or absence of introns, including four introns not previously known to be optional. Information from physical mapping of races M and T was used to identify regions likely to contain insertions or deletions, which were then characterized using PCR and sequence analysis. Newly confirmed optional introns are: the single intron in ATPase subunit 6 (ATPase6), the fifth intron of NADH dehydrogenase subunit 3 (ND3i5), the first intron of the large ribosomal RNA (r1i1), and the fifth intron of cytochrome oxidase subunit I (COIi5). In addition, an isolate resulting from a cross between races A and s (provided by L. Belcour), was shown to contain an novel intron pattern, resulting from a probable mobility event during mating (L. Belcour). We have also discovered that race M apparently exists in two forms.

These results bring to eight (including races A and s) the number of races characterized by mitochondrial intron content with a total of six known optional introns. Seven of the eight races contain a distinct set of introns, thus providing a reliable means for identification and comparison.

28. Multiple introns in the mitochondrial small subunit rDNA Gene of Cryphonectria parasitica.

Denise B. Searles, Claudia B. Monteiro-Vitorell and Helmut Bertrand, Michigan State University.

Ubiquity and conservation of the small subunit ribosomal DNA (SrDNA) have led to its extensive use in phylogeneties. However, this gene shows considerable variability in length and sequence composition, due in part to insertions. We are sequencing the mitochondrial SrDNA of C. parasitica, an ascomycetous fungus which causes chestnut blight in the American chestnut (Castanea dentata). Two introns have been found to date. The first is a 2.2 kb Group I intron, containing a 285 amino acid open reading frame (ORF). 1.8kb region of a second intron has also been sequenced and contains an ORF of 501 amino acids. Both ORFs contain a pair of LAGLI-DADG sequences, indicating that they encode maturases,

Insertions have been found in at least 17 positions in SrDNA sequences, and many fungi have multiple inserts in the nuclear SrDNA. However, ther have been few reported introns in mitochondrial SrDNA and this is the fir report of multiple introns in mitochondrial SrDNA. Further information regarding sequence analysis and secondary structure will be provided.

29. The Candida albicans genome project- First steps.

EJA Tait, MC Simon, S. King, AJP Brown, NAR Gow, DJ Shaw. University of Aberdeen.

A cosmid library of approximately 10-fold coverage has been constructed from Candida albicans strain 1161 and will serve as a starting point for physically mapping the entire C. albicans genome. The breadth and depth of genome coverage has been confirmed and investigations into the stability of the clones are described. DNA has been prepared from each clone and fingerprints generated busing a combination of restriction digestion and fluorescent labelling techniques. By alignment of these fingerprints sets of contibuous clones can be built up and then assigned to particular chromosomes. This will be folllowed by assignment of genes to individual clones thereby positioning these markers on the C. albicans genome. The computer software and data processing required to build the contigs are discussed.

30. Genome plasticity in Mycosphaerella graminicola (anamorph Septoria tritici).

Cees Waalwijk, Jos G.P. Koeken and Gert H.J. Kema, DLO-Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands.

Mycosphaerella graminicola is a bipolar heterothallic plant pathogenic ascomycete. A recently described crossing procedure enables genome analysis of this fungus showing specificity for Triticum species and cultivars. A molecular tetrad analysis enabled the identification of the pairs of ascospores within a single ascus. All isolates within this ascus contain a 800 bp RAPD marker produced with primer OPB-6. However, analysis of a progeny derived from a cross between isolates IP094265 and IP094266 revealed that the 800 hp RAPD markers inherited independently. Sequence analysis of both 800 hp fragments showed 91% identity. We hypothesize that both fragments inherited independently as a result of a translocation event, particularly since no segregation for the 800 bp fragment was observed in a cross between isolate IP094265 and isolate EP094268, also from the same ascus.

Pulsed field gel electrophoresis (PFGE) of the ascospore isolates derived from a single ascus, and RFLP analyses of these isolates and progenies of crosses between them were used to test this hypothesis and to study the inheritance of the supposed translocation. PFGE showed polymorphism among the monoascospore isolates, supporting the translocation hypothesis. The 800 hp fragment hybridized to multiple sequences on genomic blots. On PFGE blots these hybridization signals appeared to be evenly distributed among the chromosomes. Southern analysis with truncated sequences of the 800 bp fragment revealed less complex but still polymorphic multilocus haplotypes. The inheritance of these haplotypes and their relationship with the hypothesized translocation will be discussed.

31. Analysis of nuclear and mitochondrial DNA RFLPs of Colletotrichum orbiculare and allied species.

Lusike A. Wasilwa, James C. Correll, Doug D. Rhoads, and Teddy E. Morelock. Depts. of Plant Pathology, Biological Sciences and Horticulture. University of Arkansas, Fayetteville.

We have been using genetic and molecular markers to examine population diversity in several economically important Colletotrichum species. In this study, mtDNA and nuDNA RFLPs were analyzed from a worldwide collection of cucurbit isolates of C. orbiculate and allied Colletotrichum species. All isolates of C. orbiculare that were pathogenic on cucurbits belong to a single mtDNA haplotype. Nonpathogenic cocklebur isolates from Australia had a similar mtDNA haplotype, but could be distinguished by one additional restriction site. Several isolates of C. lindemuthianum and C trifolii had a similar mtDNA haplotype to pathogenic isolates of C. orbiculate. C. acutatum, C. capsici, C. cingulata, C. gloesporiodes, C. graininicola, and C. truncatum, each had distinct mtDNA haplotypes. Multiple mtDNA haplotypes were identified among isolates of C. magna, Glomerella cingulata, and Colletotrichum spp. recovered from cucurbit fruit, all of which were nonpathogenic on cucurbits. There was a correspondence between vegetative compatibility group and nuDNA RFLP haplotype among pathogenic races of C. orbiculare.

32. A novel approach to identify centromeric DNA of the filamentous fungal pathogen Ashbya gossypii.

Jurgen Wendland, Christine Mohr, and Peter Philippsen, Institute of Applied Microbiology, Biozentrum, University of Basel, CH-Basel, Switzerland.

Ashbya gossypii has been isolated as a pathogen of cotton and citrus fruits. Additionally it has been shown to be a potent overproducer of riboflavin. Recent developments on its molecular genetics include a transformation system based on a dominant as well as on auxotrophic marker genes (1,2,3,4) and the demonstration of efficient homologous recombination in this fungus (5). In addition S. cerevisiae ARS-elements serving as origins of replication in yeast allowed the introduction of freely replicating plasmids in A. gossypii (1). These plasmids could be stably maintained within a mycelium only upon selective pressure. S. cerevisiae centromeric (CEN) DNA positioned closely to the ARS-element rather decreased the stabilty of freely replicating plasmids as could be visualized under non-selective conditions. The identification of A. gossypii CEN DNA was fostered by the concurrent effort to gain insight into protein coding sequences of this fungus. This approach has been based on sequence analyses of a plasmid library containing genomic inserts of a size range from 3.5 kb to 5.5 kb. Up to now 650 plasmids were investigated by single-read sequencing 400-700 bases from both ends. Subsequent bioinformatics com-pared these single-read sequences (SRS) to the entries of available databases. At least 25 % of the open reading frames deduced from pairs of SRS derived from single plasmids displayed the same gene order as the homologous genes found in S. cerevisiae (synteny). Out of this data two pairs of SRS could be recognized that share significant homology and colinearity to genes located on both sides of cen-tromeric regions in S. cerevisiae. We have initiated a functional as well as structural analysis of these presumptive centromeric regions of A. gossypii .

(1) Wright and Philippsen, 1991 Gene 109: 99-105 (2) Steiner and Philippsen, 1994 MGG 242: 263-271

(3) Altmann-Jhl and Philippsen, 1996 MGG 250: 69-80 (4) Mohr, 1995 PhD-thesis, University of Basel, Switzerland

(5) Steiner et al., 1995 Genetics 140: 973-987

33. Physical mapping of a developmental mutant deficient in appressorium formation in Magnaporthe grisea using BAC clones.

Heng Zhu and Ralph A. Dean, Clemson University, SC. 29634-0377.

Magnaporthe grisea (Hebert) Barr. causes rice blast, one of the most devastating diseases on rice (Oryza sativa) world wide. Previously, it was shown that an appressorium deficient mutant 243-7 was under single gene control, namely APPI. The gene was mapped to a central region of chromosome 2 flanked by several RFLP markers within 0.5 cM of the gene. To physically map this region, a BAC library of M. grisea was constructed containing 9216 clones with an average insert size of 120 kb, which represents greater than 25 genome equivalents. Four single-copy DNA probes were used to screen 4608 clones in the library and at least 6 overlapping BAC clones were found with each probe. These data indicate that the BAC library should be suitable for map-based cloning of M. grisea genes and physical mapping of the genome. A BAC contig covering the APP1 region has been constructed.