The mitochondrial genome of the ethanol-metabolizing, wine cellar mold Zasmidium cellare is the smallest for a filamentous ascomycete.

Fungi in the class Dothideomycetes often live in extreme environments or have unusual physiology. One of these, the wine cellar mold Zasmidium cellare, produces thick curtains of mycelia in cellars with high humidity, and its ability to metabolize volatile organic compounds is thought to improve air quality. Whether these abilities have affected its mitochondrial genome is not known. To fill this gap, the circular-mapping mitochondrial genome of Z. cellare was sequenced and, at only 23 743 bp, is the smallest reported for a filamentous fungus. Genes were encoded on both strands with a single change of direction, different from most other fungi but consistent with the Dothideomycetes. Other than its small size, the only unusual feature of the Z. cellare mitochondrial genome was two copies of a 110-bp sequence that were duplicated, inverted and separated by approximately 1 kb. This inverted-repeat sequence confused the assembly program but appears to have no functional significance. The small size of the Z. cellare mitochondrial genome was due to slightly smaller genes, lack of introns and non-essential genes, reduced intergenic spacers and very few ORFs relative to other fungi rather than a loss of essential genes. Whether this reduction facilitates its unusual biology remains unknown.

A New Map Location of Gene Stb3 for Resistance to Septoria Tritici Blotch in Wheat.

Septoria tritici blotch (STB), caused by Mycosphaerella graminicola (synonym: Zymoseptoria tritici; asexual stage: Septoria tritici), is an important disease of wheat worldwide. Management of the disease usually is by host resistance or fungicides. However, M. graminicola has developed insensitivity to most commonly applied fungicides so there is a continuing need for well-characterized sources of host resistance to accelerate the development of improved wheat cultivars. Gene Stb3 has been a useful source of major resistance, but its mapping location has not been well characterized. Based on linkage to a single marker, a previous study assigned Stb3 to a location on the short arm of chromosome 6D. However, the results from the present study show that this reported location is incorrect. Instead, linkage analysis revealed that Stb3 is located on the short arm of wheat chromosome 7A, completely linked to microsatellite (SSR) locus Xwmc83 and flanked by loci Xcfa2028 (12.4 cM distal) and Xbarc222 (2.1 cM proximal). Linkage between Stb3 and Xwmc83 was validated in BC1F3 progeny of other crosses, and analyses of the flanking markers with deletion stocks showed that the gene is located on 7AS between fraction lengths 0.73 and 0.83. This revised location of Stb3 is different from those for other STB resistance genes previously mapped in hexaploid wheat but is approximately 20 cM proximal to an STB resistance gene mapped on the short arm of chromosome 7Am in Triticum monococcum. The markers described in this study are useful for accelerating the deployment of Stb3 in wheat breeding programs.

Accidental amplification and inactivation of a methyltransferase gene eliminates cytosine methylation in Mycosphaerella graminicola.

A de novo search for repetitive elements in the genome sequence of the wheat pathogen Mycosphaerella graminicola identified a family of repeats containing a DNA cytosine methyltransferase sequence (MgDNMT). All 23 MgDNMT sequences identified carried signatures of repeat induced point mutation (RIP). All copies were subtelomeric in location except for one on chromosome 6. Synteny with M. fijiensis implied that the nontelomeric copy on chromosome 6 served as a template for subsequent amplifications. Southern analysis revealed that the MgDNMT sequence also was amplified in 15 additional M. graminicola isolates from various geographical regions. However, this amplification event was specific to M. graminicola; a search for MgDNMT homologs identified only a single, unmutated copy in the genomes of 11 other ascomycetes. A genome-wide methylation assay revealed that M. graminicola lacks cytosine methylation, as expected if its MgDNMT gene is inactivated. Methylation was present in several other species tested, including the closest known relatives of M. graminicola, species S1 and S2. Therefore, the observed changes most likely occurred within the past 10,500 years since the divergence between M. graminicola and S1. Our data indicate that the recent amplification of a single-copy MgDNMT gene made it susceptible to RIP, resulting in complete loss of cytosine methylation in M. graminicola.

Discovery of a functional Mycosphaerella teleomorph in the presumed asexual barley pathogen Septoria passerinii.

We studied the possibility of a teleomorph associated with the genotypically diverse septoria speckled leaf blotch (SSLB) pathogen of barley, Septoria passerinii. A teleomorph in the genus Mycosphaerella had been predicted previously based on phylogenetic analyses. This prediction was tested with experiments in the Netherlands and the United States by co-inoculating isolates with opposite mating types onto susceptible barley cultivars and monitoring leaves for sexual structures and for the discharge of ascospores. Characterization of putative hybrid progeny by both molecular (AFLP, RAPD, mating type, and ITS sequencing) and phenotypic analyses confirmed that a Mycosphaerella teleomorph of S. passerinii has been discovered approximately 125 years after the description of the anamorph. Progeny had recombinant genotypes of the molecular alleles present in the parents, and the identities of representative progeny isolates as S. passerinii were confirmed by ITS sequencing. A previously unknown sexual cycle explains the high degree of genetic variation among isolates found in nature. The experimental identification of a predicted teleomorph for S. passerinii indicates that cryptic sexual cycles may be common for many other "asexual" fungi with high levels of genotypic diversity.

Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the septoria tritici blotch pathogen Mycosphaerella graminicola.

A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned with a custom software pipeline for di- and trinucleotide units repeated tandemly six or more times. The bioinformatics analysis identified 109 putative SSR loci, and for 99 of them, flanking primers were developed successfully and tested for amplification and polymorphism by PCR on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 23 were polymorphic between the parents of the mapping population. Twenty-one of these as well as two previously published microsatellite loci were positioned on the existing genetic linkage map of M. graminicola on 13 of the 24 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also revealed polymorphisms when tested with DNA from the related banana black leaf streak (Black Sigatoka) pathogen, M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of M. graminicola and related species.

Molecular Mapping of the Stb4 Gene for Resistance to Septoria tritici Blotch in Wheat.

ABSTRACT Breeding wheat for resistance is the most effective means to control Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici). At least eight genes that confer resistance to STB in wheat have been identified. Among them, the Stb4 locus from the wheat cv. Tadinia showed resistance to M. graminicola at both seedling and adult-plant stages. However, no attempt has been made to map the Stb4 locus in the wheat genome. A mapping population of 77 F10 recombinant-inbred lines (RILs) derived from a three-way cross between the resistant cv. Tadinia and the susceptible parent (Yecora Rojo x UC554) was evaluated for disease resistance and molecular mapping. The RILs were tested with Argentina isolate I 89 of M. graminicola for one greenhouse season in Brazil during 1999, with an isolate from Brazil (IPBr1) for one field season in Piracicaba (Brazil) during 2000, and with Indiana tester isolate IN95-Lafayette-1196-WW-1-4 in the greenhouse during 2000 and 2001. The ratio of resistant:susceptible RILs was 1:1 in all three tests, confirming the single-gene model for control of resistance to STB in Tadinia. However, the patterns of resistance and susceptibility were different between the Indiana isolate and those from South America. For example, the ratio of RILs resistant to both the Indiana and Argentina isolates, resistant to one but susceptible to the other, and susceptible to both isolates was approximately 1:1:1:1, indicating that Tadinia may contain at least two genes for resistance to STB. A similar pattern was observed between the Indiana and Brazil isolates. The gene identified with the Indiana tester isolate was assumed to be the same as Stb4, whereas that revealed by the South American isolates may be new. Bulked-segregant analysis was used to identify amplified fragment length polymorphism (AFLP) and microsatellite markers linked to the presumed Stb4 gene. The AFLP marker EcoRI-ACTG/MseI-CAAA5 and microsatellite Xgwm111 were closely linked to the Stb4 locus in coupling at distances of 2.1 and 0.7 centimorgans (cM), respectively. A flanking marker, AFLP EAGG/ M-CAT10, was 4 cM from Stb4. The Stb4 gene was in a potential supercluster of resistance genes near the centromere on the short arm of wheat chromosome 7D that also contained Stb5 plus five previously identified genes for resistance to Russian wheat aphid. The microsatellite marker Xgwm111 identified in this study may be useful for facilitating the transfer of Stb4 into improved cultivars of wheat.

A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic kinkage map of Mycosphaerella graminicola, the septoria tritici leaf blotch pathogen of wheat.

An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.