A Combined Amplified Fragment Length Polymorphism and Randomly Amplified Polymorphism DNA Genetic Linkage Map of Mycosphaerella graminicola, the Septoria Tritici Leaf Blotch Pathogen of Wheat

Corresponding author: Gert H. J. Kema, P.O. Box 16, 6700 AA Wageningen, The Netherlands., g.h.j.kema{at}plant.wag-ur.nl (E-mail)

Communicating editor: A. H. D. BROWN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

An F₁ 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.

MAPPING fungal genomes with the aid of molecular markers is a rapidly developing area in biological science. Particularly, the application of PCR-based strategies such as randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs) allows a highly efficient generation of markers. Integration of biological traits and molecular markers into one map allows a quick focus on particular areas of the genome to develop strategies for cloning genes of interest. In plant pathogenic fungi, typical genes of interest are those that control mating, avirulence, and fungicide resistance (WHISSON et al. 1995 ; XU and LESLIE 1996 ; VAN DER LEE et al. 1997 ; TURGEON 1998 ).

More than 1800 species names are in the genus Mycosphaerella, including synonyms. At least 500 are recognized as true species (CORLETT 1991 ), some of which cause huge economic losses in important cash crops. For example, sigatoka disease in banana is caused by M. fijiensis and M. musicola; ringspot disease of Brassica spp. is caused by M. brassicicola; strawberry leaf spot is caused by M. fragariae; and several Mycosphaerella species cause leaf spot diseases on Eucalyptus, Populus, and other tree species (CROUS 1998 ). Septoria tritici leaf blotch is the major disease of wheat in Western Europe and is of considerable importance worldwide in areas with a temperate, high-rainfall environment during the wheat-growing season, such as the Southern Cone of South America (VAN GINKEL and RAJARAM 1993 ; COWGER et al. 2000 ). The causal agent, Septoria tritici, was first described by DESMAZIERES 1842 . One hundred thirty years later Sanderson identified the ascomycete Mycosphaerella graminicola as the sexual stage (teleomorph) of S. tritici (SANDERSON 1972 , SANDERSON 1976 ). The disease is characterized by necrotic blotches on the foliage, which contain asexual (pycnidia) and sexual (pseudothecia) fruiting structures. Recent studies revealed that M. graminicola populations are highly diverse and suggested that sexual reproduction has a major influence on the genetic structure of M. graminicola populations (MCDONALD et al. 1999 ).

Genetic analyses were not possible until we developed a crossing protocol for this haploid ascomycete, which showed that M. graminicola has a heterothallic, bipolar mating system (KEMA et al. 1996B ). Furthermore, AFLP and RAPD markers segregated according to Mendelian expectation so it is now possible to construct a genetic linkage map (KEMA et al. 1996B , KEMA et al. 2000 ). Genetic maps could help to answer important questions about the biology of M. graminicola. For example, the nature of the interaction between M. graminicola and its wheat host has been controversial. Although some researchers have concluded that resistance to this pathogen in wheat and, conversely, avirulence in the pathogen are quantitative traits without specific interactions, others revealed specific interactions between wheat cultivars and particular isolates of the pathogen (KEMA et al. 1999 ). The basis for such specific differences between pathogen isolates could now be addressed in genetic experiments. Recently, we have identified a single avirulence locus in M. graminicola (KEMA et al. 2000 ).

The objective of this study was to generate a high-density genetic linkage map of the M. graminicola genome by integrating AFLP and RAPD data and to map and physically locate loci involved in mating and host-cultivar specificity to assist positional cloning of these and other genes of interest.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fungal isolates and growth conditions:
Two Dutch isolates of M. graminicola with opposite mating types were crossed according to the protocol described by KEMA et al. 1996B . IPO323 was isolated in 1981 from the soft white winter wheat cv. Arminda, has mating type MAT1-1, and is avirulent on the differential cultivars Veranopolis, Kavkaz, and Shafir. IPO94269 was isolated as a single ascospore discharged from wheat stubble in 1994. It has mating type MAT1-2 and is virulent on the aforementioned differential cultivars. The pathogenicity of both isolates has been tested intensively under growth room and field conditions (KEMA and VAN SILFHOUT 1997 ; KEMA et al. 2000 ; BROWN et al. 2001 ). Following co-inoculation of the susceptible cv. Obelisk with both isolates, infected leaves were stimulated to discharge ascospores into sterile water agar plates (KEMA et al. 1996B ). Germinated ascospores were isolated individually with a sterile needle, transferred to 2% V8-juice agar plates, and incubated at 20° under continuous light. Individual colonies were transferred to liquid YG medium (3% yeast and 1% glucose) and incubated in a reciprocal shaker at 18° for 5 days. The resulting spores were collected, concentrated by centrifugation, and maintained at -80° and on silica gel at -20° in freezers. The total progeny population consists of 202 isolates derived from a single cross. Among these, a subset of 68 isolates was chosen for further analyses.

Bioassays and mating type identification:
Bioassays and segregation analysis for avirulence to a number of wheat cultivars in this population were reported elsewhere (KEMA et al. 1996A , KEMA et al. 2000 ). The mating types of 68 progeny were determined by PCR with mating type-specific primers (WAALWIJK et al. 2002 ) and by backcrossing the progeny to both parental strains. Progeny that produced ascospores with the MAT1-2 but not the MAT1-1 parent were scored MAT1-1; those that produced ascospores with the MAT1-1 but not the MAT1-2 parent were scored MAT1-2.

DNA isolation:
For AFLP analysis, isolates were cultured in liquid YG medium for 5 days at 18°. Spores were allowed to sediment overnight and were subsequently collected and lyophilized in cryotubes for 48 hr. DNA from all isolates was extracted using the Puregene DNA isolation kit D6000 (Gentra Systems, Minneapolis) according to the manufacturer's instructions. Tissue for RAPD analysis was grown in 250-ml Erlenmeyer flasks containing 100 ml yeast maltose broth (4 g yeast extract, 4 g malt extract, 4 g sucrose/liter distilled water) at room temperature (22°–24°) on a shaker rotating at 180 rpm. Each flask was inoculated with a lyophilized filter paper disc containing mycelia and spores taken from an actively growing culture. Tissue was harvested after 12 days (occasionally longer for slow-growing isolates) by vacuum filtration, frozen at -80° for at least 30 min, and lyophilized. DNA was extracted essentially as described by BIEL and PARRISH 1986 , except three additional phenol:chloroform extractions were performed instead of the Sephadex-column purification step. Extracted DNA was quantified with a Hoefer DyNAQuant 2000 fluorometer (Hoefer Pharmacia Biotech).

AFLP and RAPD genotyping:
The initial AFLP protocol was as described by VOS et al. 1995 . DNA samples were digested with the restriction enzymes EcoRI and MspI. The latter enzyme was chosen because digests with various restriction enzymes suggest a relatively high CG content of M. graminicola, so MspI should cleave more sites than commonly used alternatives such as MseI, which is specific for AT-rich regions. After screening the parental isolates for optimal polymorphisms with 64 primer combinations, 11 were chosen for the mapping analysis (Table 1). The combinations were screened on the subset of the mapping population following another AFLP protocol in which primers labeled with fluorescent dyes were processed in a thermocycler and analyzed on automated sequencing equipment (BONANTS et al. 2000 ).

RAPD analysis was done essentially as described by WILLIAMS et al. 1990 . PCR reactions (25 µl) contained 7.9 µl deionized H₂O, 2.5 µl 0.01% gelatin, 2.5 µl 10x buffer, 2.5 µl 2.0 mM dNTPs, 3.5 µl 25 mM MgCl₂, 0.1 µl AmpliTaq DNA polymerase, 5 µl 2.0 µM primer, and 1.0 µl template DNA (12.5 ng/µl). PCR amplifications were for 1 min at 94° and then 45 cycles of 94° for 1 min, 37° for 1 min, and 72° for 2 min, followed by 7 min at 72°. In total, 240 primers were screened from Operon primer kits I, J, K, L, M, N, Q, R, S, T, U, and W (Operon Technologies, Alameda, CA).

Amplification products were loaded onto 1.2% agarose gels, run at 150 V for 2 hr and 20 min in 0.5x TBE or TAE buffer, and stained with ethidium bromide. Gel pictures were taken with an electronic photo imaging system (Fotodyne, New Berlin, WI). Fragment sizes were estimated by comparison with a 1-kb DNA ladder (Life Technologies, Grand Island, NY) using Collage image analysis software from Fotodyne. Amplifications were performed in a Perkin-Elmer (Norwalk, CT) GeneAmp PCR system 9600.

Data analysis and mapping:
AFLP and RAPD markers are designated by the primer combination used for the amplification and the approximate length of the generated fragment. The length of each AFLP marker was estimated by a fluorescently labeled 50-bp ladder (Pharmacia, Piscataway, NJ) and data analysis was performed using ImageMaster 1D Elite (Pharmacia) software. The prefix A or B indicates the origin of the marker; those originating from parent IPO323 have the prefix A while markers from parent IPO94269 are indicated by the prefix B.

Segregation ratios of all markers were analyzed with JoinMap (STAM 1993 ), version 2.0. Markers with segregation ratios significantly different from 1:1 (P < 0.05) were set aside because initial mapping analysis showed that they may cause erroneous grouping of unlinked markers.

Linkage analysis was performed on the remaining markers. Initial assignment to linkage groups was based on the logarithm of the odds (LOD) ratio for each possible marker pair. The LOD value indicates the likelihood of linkage by comparing the probabilities of random association of markers in the progeny to association caused by linkage. We used LOD values in the range of 3–8, whereas the final assembly of linkage groups was completed using a LOD value of 4.5. In the second step, the calculation of a linear order of markers within a linkage group, we used a map LOD value of 0.01 and the Kosambi mapping function. JoinMap 2.0 also offers the possibility to set aside markers, which are difficult to position within a linkage group on the basis of a chi-square test comparing the direct distance of markers with their mapping distance. We used a chi-square threshold of 5, beyond which markers are not positioned on the map. Finally, we tested if the markers with a distorted segregation ratio could contribute to the map. Because most of these markers either were not linked at a LOD threshold of 4.5 or were mapped to previously identified linkage groups only with difficulty, these aberrantly segregating markers were not included in the final map.

Bulked segregant analysis:
Bulked segregant analysis (MICHELMORE et al. 1994 ) was used to identify additional markers closely linked to the avirulence locus. The two bulks were composed of equal amounts of DNA from 34 virulent isolates and 33 avirulent isolates, respectively. The parents and both bulks were screened with the 64 AFLP primer combinations mentioned above. Primer combinations that identified differences between the bulks were then used to analyze the progeny individually and linkage analysis was performed as described above.

Pulsed-field gel electrophoresis:
Contour-clamped homogeneous field electrophoresis (Bio-Rad DRII, Richmond, CA) was used to analyze the electrophoretic karyotypes of both parents. Chromosomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe were used as chromosomal size markers (Bio-Rad). The chromosomes in the small size ranges were separated on 1% SeaKem Gold agarose at 200 V and 14° with pulses increasing from 50 to 90 sec for 24 hr, whereas medium-sized chromosomes were separated at 100 V and 9° with pulses of 3000 sec for 8 hr followed by pulses of 500 sec for 64 hr. Large chromosomes were separated at 45 V and 9° on 0.8% SeaKem Gold agarose using the following consecutive conditions: electrophoresis started with pulses increasing from 600 to 900 sec for 24 hr and continued with pulses increasing from 1500 to 2700 sec for 48 hr, pulses increasing from 2700 to 3600 sec for 96 hr, and finally pulses increasing from 60 to 90 sec for 15 hr at 100 V.

Southern hybridization:
Pulsed-field gels were hybridized to physically map the MAT and AVR loci of M. graminicola. An 850-bp PCR fragment from the MAT1-2 gene of Tapesia yallundae, provided by Dr. P. Dyer (University of Nottingham, United Kingdom), was random-prime labeled (SAMBROOK et al. 1989 ) and used as a probe to map the MAT locus (WAALWIJK et al. 2002 ). A 145-bp AFLP marker identified by the bulked segregant analysis, designated AVIR2, was isolated, cloned in Escherichia coli strain XL-1blue using pGEM-T as cloning vector, radiolabeled as mentioned above, and hybridized to pulsed-field gel blots to physically map the AVR locus on one of the M. graminicola chromosomes.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AFLP and RAPD analysis of the mapping population:
Comparison of the DNA fingerprints identified seven pairs of "twin" genotypes that probably represent genetically identical individuals generated during the mitosis that occurs just before ascospore formation. The presence of these twins enabled us to estimate the reliability of the marker scoring, which was 99.89 and 95.63% for AFLP markers and RAPD markers, respectively. One member of each twin pair was deleted from the final mapping data set, which was thus composed of 61 remaining progeny with different recombination patterns informative for linkage analysis. Application of the 11 selected AFLP primer combinations resulted in a total of 1056 AFLP fragments of which 498 (47%) were polymorphic between the parental isolates (Table 1). All these markers segregated in the progeny; however, only 271 AFLP markers that could be scored clearly on nearly all 61 progeny were used for further analysis. Forty-one of the 240 RAPD primers tested gave scorable polymorphisms between the parents of the mapping population, with an average of 2.5 fragments per primer, resulting in 104 RAPD markers. In the single-marker analyses, 16 AFLP and 20 RAPD markers showed significant deviation from the expected 1:1 segregation ratio. These markers were omitted from further analysis. Hence, eventually 84 RAPD and 255 AFLP markers as well as mating type and avirulence on the wheat cultivars Veranopolis, Kavkaz, and Shafir were used on 61 progeny isolates to construct the genetic linkage map.

Linkage and mapping analysis:
Using a LOD threshold value of 4.5, 302 of the 341 markers analyzed (nearly 90%) showed linkage to at least 3 other markers, allowing reliable mapping by three-point analysis. As expected, all linkages were in coupling phase if markers originated from the same parent and in repulsion phase if markers were from different parental origin. This indicates that a LOD value of 4.5 is sufficiently selective to discriminate between true linkage and experimental noise. After the markers were assembled into linkage groups they were ordered within the linkage groups using a chi-square jump restriction of five. Nine AFLP and 11 RAPD markers caused larger chi-square jumps and were, therefore, not positioned on the map.

Hence, eventually 282 markers were positioned on the map in 23 linkage groups (Fig 1). The average spacing between markers is 4.3 cM, but there is a significant clustering of markers, which is particularly evident for the AFLP markers (Fig 1).

View larger version (60K): In this window In a new window Download PPT slide   Figure 1. Genetic linkage map of the M. graminicola genome. Markers beginning with A originated from the IPO323 parent; those beginning with B were from the IPO94269 parent. AFLP markers are indicated by their origin followed by the EcoRI (E) and MspI (Mp) primer combination and the size of the marker, e.g., (white "1" in black circle) AFLP marker (150 bp) on linkage group 21 originates from IPO323 and is therefore designated as AEGAMpCA, 150. RAPD markers are indicated by the primer set and number followed by a dash and the approximate size of the fragment in base pairs, e.g., BM14-918 is a 918-bp fragment originating from IPO94269 using Operon primer M14. The (black star) MAT and (white star) AVR locus mapped to linkage groups 16 and 22, respectively.

Avirulence and mating types:
The genetics of avirulence in M. graminicola isolate IPO323 to the wheat cvs. Shafir, Kavkaz, and Veranopolis was recently discussed (KEMA et al. 2000 ) and appeared to be controlled by a single locus (; Table 2). This avirulence locus mapped to linkage group 22 along with four AFLP markers (Fig 1).

Backcrossing to both parents successfully identified the mating type of progeny isolates (Table 2). In all cases, ascospores were produced only from crosses with either one or the other parent. MAT genotypes were later successfully verified using MAT-specific PCR primers (WAALWIJK et al. 2002 ). Both mating type idiomorphs appeared in nearly equal frequencies (; Table 2), confirming the heterothallic, bipolar nature of the mating system in M. graminicola (KEMA et al. 1996B ). The MAT locus mapped to linkage group 16, which also contained four AFLP and two RAPD markers (Fig 1).

Bulked segregant analysis:
Due to the size of the bulks only two additional AFLP markers were generated by bulked segregant analysis, one with primer combination EcoRI + GA/MspI + CA (AVIR1, 122 bp) and one with primer combination EcoRI + AT/MspI + CA (AVIR2, 145 bp). Both markers cosegregated perfectly with the avirulence locus (Fig 1).

Electrophoretic karyotypes:
Pulsed-field gel electrophoresis (PFGE) under different conditions revealed chromosomal polymorphisms between the parental strains (Fig 2). The maximal count of chromosomal bands in IPO323 was 15, whereas in IPO94269 13 bands were visible. The largest chromosomal band of the PFGE gels coincides with the compression zone that, as a result of the applied PFGE technique, usually contains a variety of chromosomal fragments and thus most likely is not an individual chromosome. Southern analysis revealed a discrepancy between physical and genetic linkage. Linkage groups 16 and 22 appear to be among the smallest of the entire genome (Fig 1). However, Southern analyses with the MAT1-2 probe of T. yallundae as well as with a probe derived from AVIR2 showed that both loci are indeed on different, large chromosomes of the M. graminicola genome (Fig 3).

View larger version (85K): In this window In a new window Download PPT slide   Figure 2. Estimation of the number of chromosomes of the M. graminicola genome by pulsed-field gel electrophoresis. (A) Small, (B) medium, and (C) large chromosomes were separated as described in MATERIALS AND METHODS.

View larger version (47K): In this window In a new window Download PPT slide   Figure 3. Physical mapping of AVR and MAT loci to some of the largest M. graminicola chromosomes by Southern analysis of PFGE filters. (A) Hybridization with probe AVIR2, which is derived from a perfectly cosegregating AFLP marker, is shown. The probe hybridizes to a chromosome of 3.0 Mb in M. graminicola isolate IPO323 but not to any chromosome in isolate IPO94269. (B) Hybridization with a heterologous probe from the fungus T. yallundae specific for the HMG box of the MAT1-2 allele is shown. The probe hybridizes to a chromosome of 1.8 Mb in size.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Despite the economic importance of plant pathogenic fungi, the number of genetic maps available remains limited. Among the four major groups of true fungi, genetic linkage maps are available for only 6 species of basidiomycetes, 1 zygomycete, 1 chytridiomycete, and 13 ascomycetes (O'BRIEN 1993 ; XU and LESLIE 1996 ; CHIBANA et al. 1998 ; COZIJNSEN et al. 2000 ; FORCHE et al. 2000 ). Among these 21, only 8 are plant pathogens. Current taxonomy divides the Ascomycetes into 46 orders (HAWKSWORTH et al. 1995 ), among which Saccharomycetales is represented by the complete genome sequence of the yeast S. cerevisiae (GOFFEAU et al. 1996 ). However, genetic linkage maps are available only for representatives from 6 of the remaining 45 orders. Within the Dothideales, the only map available is from Cochliobolus heterostrophus (TZENG et al. 1992 ), but that species is in a different family from Mycosphaerella (HAWKSWORTH et al. 1995 ). The present results provide the first genetic map for any species within Mycosphaerella and also within the family Mycosphaerellaceae. The genus Mycosphaerella contains over 500 species (CORLETT 1991 ), most of which are pathogenic on one or more plant hosts, including both monocots and dicots, gymnosperms, ferns, horsetails, and lycopods (FARR et al. 1995 ). As mentioned before, M. graminicola is a major wheat disease with a large annual economic impact in Western Europe. A linkage map of this organism facilitates the elucidation of crucial biological aspects of the pathosystem, such as avirulence, pathogenicity, and mating. This map, together with other indispensable tools, such as a crossing and transformation protocol as well as high throughput genomic analyses, make M. graminicola a useful model for other host-pathogen relationships in the Dothideales.

AFLP analysis was a fast way to generate a large number of polymorphic markers that provided the overall framework for the map. However, these markers were often clustered, which reduced the overall length of the final map. Similar clustering of AFLP markers has been reported in many other species, including animals (YOUNG et al. 1998 ), plants (SPADA et al. 1998 ; BOIVIN et al. 1999 ; REMINGTON et al. 1999 ), and oomycetes (VAN DER LEE et al. 1997 ). Clustering of AFLP markers in plants is often localized at centromeric regions (QI et al. 1998 ; HAANSTRA et al. 1999 ). If the same phenomenon occurs in M. graminicola, then the probable positions of the centromeres can be inferred for about half of the 23 linkage groups.

The large number of AFLP markers permitted easy identification of identical progeny that subsequently could be removed from the analysis. In M. graminicola twin progeny isolates may arise from the mitotic division that follows meiosis II in the ascus, giving four pairs of genetically identical ascospores. Genetically identical isolates according to the AFLP analyses in this study usually had consecutive or near-consecutive numbers, indicating that they were isolated from the same part of the water-agar plate. This is consistent with the suggested simultaneous ejection of ascospores from the same ascus (KEMA et al. 1996B ).

In our study, AFLP markers appeared to be more reliable than RAPD markers. Therefore, RAPD markers are less likely to cluster and JoinMap tends to position them at the end of linkage groups to cope with the friction created by the scoring errors. Indeed, we observed that RAPD markers were at the ends of 13 of the 18 linkage groups in which they occurred. This suggests that RAPDs inflated the total mapped region of the genome by 22%, from 995 cM with the AFLP markers alone to 1216 cM for the combined map. Hence, differences in the reliability of different marker types may lead to an overestimation of the mapped genomic region. Nevertheless, inclusion of multiple marker types may be critical for thorough genome coverage. Although almost four times as many AFLP as RAPD markers were on the map, RAPD markers were present on the 17 largest linkage groups and on 18 of the 23 groups in total.

We observed a significant discrepancy between the number of linkage groups and the estimated number of chromosomes. The total number of chromosomes in M. graminicola is not known for certain. In our study 13–15 chromosomes could be discerned, whereas 17 or 18 chromosomes were observed by MCDONALD and MARTINEZ 1991 . Although comigration of similar-sized chromosomes is a legitimate explanation of the observed discrepancy, we expect that some of the smallest linkage groups probably will be joined with others once additional markers are added to the map. This was supported by Southern analyses of the PFGE gels using probes for the mating type and avirulence loci that hybridized to two of the larger chromosome bands, despite these loci being mapped on two of the smallest linkage groups. Differences may exist among electrophoretic karyotypes of individual isolates within species (COOLEY and CATEN 1991 ; KEMA et al. 1999 ). However, in M. graminicola the difference in numbers and sizes of the chromosomes apparently does not preclude sexual recombination and viability of the offspring. So far, generated segregating populations only erratically contained few nongerminating ascospores (this study and KEMA et al. 2000 ).

The current map density in the relatively small M. graminicola genome is sufficiently high to identify genetic and physical linkages with the biological traits analyzed in this study. On average, the current map contains one marker every 4.3 cM. However, this overestimates the functional density of the map. If cosegregating markers are reduced to 1 per cluster, the number of independent markers on the map is 229, or 1 every 5.3 cM. Also, 39 of the 302 markers (12%) were unlinked to the linkage groups, indicating that parts of the genome contain low marker densities. Due to the strong clustering of markers it is difficult to calculate the total genome size on the basis of these mapping data.

The mapping process effectively identified molecular markers linked to the mating type and avirulence loci. Subsequent bulked segregant analysis identified several additional markers. Inclusion of >30 progeny in each bulk resulted in markers cosegregating with avirulence. The recent cloning of the mapped mating type gene (WAALWIJK et al. 2002 ) and the progress in cloning the avirulence locus demonstrate the potential utility of the map for map-based cloning of selected genes. This approach now can be used to identify markers linked to any gene that segregates in this cross.

Little is known about syntenic relationships among fungal genetic maps. This is mainly due to the small number of species that have been compared and the lack of common marker sets mapped in multiple species. A common set of markers is greatly needed to test for synteny among fungal genomes. Recent sequence analyses have shown a high degree of synteny between the genetic maps of S. cerevisiae and Ashbya gossypii in the Saccharomycetales (DIETRICH et al. 1999 ). The Neurospora genome project resulted in the first entire DNA sequence of a filamentous fungus, which is of huge interest for synteny studies among this group of organisms (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/). As synteny among fungal species in the same order may be high, the current map of M. graminicola can be used to develop anchor points for comparative mapping in other species within the Dothideales. Knowledge of syntenic relationships provides information about gene clusters in general, but synteny near the avirulence locus of M. graminicola might particularly provide information about the evolution of host-pathogen relationships in closely related species that are destructive pathogens of important crop hosts.

The results of this study confirmed that avirulence to the cultivars Kavkaz, Shafir, and Veranopolis is under single-gene control and that a single locus may control pathogenicity to all three (KEMA et al. 2000 ). The ability to perform genetic analyses in the pathogen will complement analyses of resistance in wheat and for the first time will permit thorough analyses of host-pathogen interactions in this economically important pathosystem (BRADING et al. 2002 ).

	FOOTNOTES

² Present address: Institut National Agronomique de Tunisie (INAT), 43 Av. Charles Nicolle, 1082 Tunis El Mahrajène, Tunisia.
³ Present address: Plant Research International B.V., P.O. Box 16, 6700 AA Wageningen, The Netherlands.

	ACKNOWLEDGMENTS

We thank Paul Dyer for supplying the T. yallundae mating type probe. This work was supported in part by EU-BIOTECH grant BIO4-CT96-0352, the Dutch Ministry of Agriculture, Nature Management and Fisheries, and the U.S. Department of Agriculture CRIS project 3602-22000-009-00D.

Manuscript received August 16, 2001; Accepted for publication May 16, 2002.

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