A Genetic Map of Gibberella zeae (Fusarium graminearum)

Corresponding author: J. F. Leslie, 4002 Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS 66506-5502., jfl{at}plantpath.ksu.edu (E-mail)

Communicating editor: R. H. DAVIS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.

GIBBERELLA zeae (anamorph Fusarium graminearum) is the most important causal agent of Fusarium head blight (scab) of wheat and barley in the United States (MCMULLEN et al. 1997 ) and China (CHEN et al. 2000 ). In the 1990s, scab caused an estimated $3 billion losses to wheat and barley farmers in the United States alone (WINDELS 2000 ). Scab reduces wheat baking quality (SEITZ et al. 1986 ) and harvested grain often is contaminated with mycotoxins such as nivalenol (NIV), deoxynivalenol (DON), and zearalenone (MARASAS et al. 1984 ; TANAKA et al. 1988 ).

G. zeae is homothallic (NELSON et al. 1983 ; YUN et al. 2000 ) and may produce abundant perithecia in the field, but can be outcrossed under laboratory conditions (BOWDEN and LESLIE 1999 ). Despite being homothallic, the amount and distribution of genetic heterogeneity in field populations of this fungus suggest that outcrossing occurs at a significant rate in the field (BOWDEN and LESLIE 1992 ; WALKER et al. 2001 ). Recently, O'DONNELL et al. 2000 used DNA sequences of elongation factor (EF-1), phosphate permease genes (PHO), ß-tubulin (TUB), UTP-ammonia ligase (URA), trichothecene 3-O-acetyltransferase (TRI101), and a putative reductase (RED) to resolve a set of G. zeae strains into at least seven distinct phylogenetic lineages. Differences between strains in distinct lineages include qualitative differences in toxin production (DON, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, NIV, 4-acetylnivalenol, and zearalenone) as well as in DNA sequence-based markers. The degree of genetic isolation and pathogenic specialization among lineages remains unresolved (CARTER et al. 2000 ).

BOWDEN and LESLIE 1999 established that, under laboratory conditions, members of at least three of the phylogenetic lineages described by O'DONNELL et al. 2000 can interbreed and produce viable, recombinant progeny. The fertility of these interlineage crosses can be relatively high and suggests that these strains are members of geographically separated and genetically distinct populations rather than of distinct species. O'DONNELL et al. 2000 described one putative naturally occurring hybrid strain (collected in Nepal by A. E. Desjardins) between lineages 2 and 6. If isolates from these genetically divergent populations interbreed, then there is the potential for the production of new genotypes that carry novel combinations of genes for pathogenicity, host range, or toxin production (BRASIER 2000 ). A cross between isolates from two such distantly related populations also should be rich in polymorphic markers that could be used to generate a detailed genetic map.

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based DNA analysis technique that can detect variations in restriction fragment length polymorphisms (RFLP) on a genome-wide basis (VOS et al. 1995 ). Like restriction fragment length polymorphism analysis, AFLPs can detect size differences in restriction fragments caused by DNA insertions, deletions, or changes in target restriction site sequences. As compared to RFLP analysis, however, the labor required to detect genetic polymorphisms with AFLPs is considerably reduced. AFLP analysis yields dominant band/no-band type markers that can be used to study genetic diversity in fungal populations (e.g., GONZALEZ et al. 1998 ; PURWANTARA et al. 2000 ; ZELLER et al. 2000 ) and to define and distinguish species of Fusarium (MARASAS et al. 2001 ). AFLPs have been used to develop recombination-based genetic maps in mapping populations of higher plants such as barley, soybeans, and maize (VUYLSTEKE et al. 1999 ; YIN et al. 1999 ; HUA et al. 2000 ) and to supplement mapping efforts in fungi, e.g., in Phytophthora (VAN DER LEE et al. 2001 ). When using 2-bp extensions in specific AFLP reactions, as is common in fungi, there are 256 potential primer-pair combinations that can each be used to generate a unique DNA fingerprint pattern for each pair of restriction enzymes used in the initial digestion of the DNA. The genomic distribution of markers generated by AFLPs is limited only by the distribution of the restriction sites used to generate them.

Our objective in this study was to establish a recombination-based genetic linkage map of G. zeae by crossing phenotypically and genetically divergent strains from different continents. Only one other detailed genetic map is presently available for any Gibberella species (XU and LESLIE 1996 ). Generation of a genetic linkage map for G. zeae will permit the correlation of physical sequences with segregating phenotypes, the localization of genes for toxin production, the identification of genetically independent markers that can be used in characterization of field populations, and the identification of genomic sequences that might be of particular importance in the evolution of this species.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mapping cross:
One of the parents of this cross was derived from a DON-producing strain, Z-3639, originally isolated from wheat in Kansas (BOWDEN and LESLIE 1992 ) and belonging to lineage VII as described by O'DONNELL et al. 2000 . The other parent was derived from a NIV-producing strain, R-5470, originally isolated from barley in Japan and obtained from Paul E. Nelson (Department of Plant Pathology, Pennsylvania State University, University Park, PA), which belongs to lineage VI as described by O'DONNELL et al. 2000 . These strains were selected because of the difference in toxin production and to give a large number of polymorphic markers, due to the presumptive isolation of the populations from which they were derived.

Crosses were performed essentially as described previously (BOWDEN and LESLIE 1999 ). Mycelial plugs of a nit1 mutant (Z-11570) of R-5470 and a nit3 mutant (Z-11572) of Z-3639 were simultaneously inoculated onto carrot agar medium (KLITTICH and LESLIE 1988 ). nit mutants were generated as previously described (BOWDEN and LESLIE 1992 ) and characterized by using standard Fusarium protocols (CORRELL et al. 1987 ; KLITTICH and LESLIE 1988 ). The parents [Z-11570, Fungal Genetics Stock Center (FGSC) 8632; Z-11572, FGSC 8633] of and the progeny (FGSC 8634–8732) from the mapping cross are available from the Fungal Genetics Stock Center (Department of Microbiology, University of Kansas Medical Center, Kansas City, KS; http://www.fgsc.net). We analyzed 99 single ascospore-derived progeny from this cross. The frequency of nitrate-utilizing ascospores was <1%, because most of the perithecia were homothallic selfs of Z-11572.

We isolated ascospores from mature perithecia by inverting the carrot agar cross plates and collecting ascospores on the plate lid. These ascospores were suspended in 5 ml of sterile water and then dilution plated onto a minimal agar medium (CORRELL et al. 1987 ) amended with tergitol and sorbose (BOWDEN and LESLIE 1999 ). Plates were incubated for 5–7 days at 24°. Recombinant progeny, identified as nitrate utilizers, were collected and transferred to minimal medium slants. Each of the recombinant progeny was subcultured from a single macroconidium. Macroconidia were separated with a Cailloux stage-mounted micromanipulator (Stoelting, Chicago). Cultures were maintained on minimal medium and stored as spore/hyphal fragment suspensions in 15% glycerol at -70° at Kansas State University.

Analysis of DNA polymorphisms in the mapping population:
We inoculated 50 ml of liquid complete medium (CORRELL et al. 1987 ) in 125-ml Erlenmeyer flasks with 5 x 10⁵ macroconidia suspended in 1 ml of a 2.5% aqueous (v/v) solution of Tween 60 (Sigma, St. Louis). Cultures were incubated for 2–3 days at room temperature (22°–25°) on a rotary shaker (150 rpm). Tissue from each culture was collected by filtration through a nongauze milk filter (Ken Ag Milk Filter, Ashland, OH), washed with 100 ml sterile water, and blotted dry with paper towels. The tissue was frozen at -20° until DNA was extracted.

DNA extraction:

DNA was isolated with a cetyltrimethyl ammonium bromide procedure (KERENYI et al. 1999 ) modified from that of MURRAY and THOMPSON 1980 . We estimated final DNA concentrations (in TE buffer) by comparison of DNA fluorescence of diluted aliquots of each DNA sample against that of HindIII-digested bacteriophage DNA with an IS-1000 version 2.0 digital imaging system (Alpha Innotech, San Leandro, CA). Samples and sample dilutions were run in 1% agarose gels containing TAE (40 mM Tris-acetate, 1 mM EDTA pH 8.0) and 0.5 µg/ml ethidium bromide. DNA yields ranged from 100 to 1000 µg of DNA per culture. The concentration of each DNA sample was adjusted to 20 µg/ml for use in AFLP analysis.

AFLPs:

AFLPs were generated with the protocol of VOS et al. 1995 as modified by ZELLER et al. 2000 . AFLP primers were synthesized by Integrated DNA Technologies (Coralville, IA). The EcoRI primers in the final specific amplification reactions were 5' end-labeled with [-³³P]ATP (NEN Life Sciences, Boston). Dried gels were exposed to X-ray film (Classic Blue Sensitive, Molecular Technologies, St. Louis) for 2–5 days at room temperature to identify DNA bands. We identified polymorphic bands by eye and scored them manually. We estimated molecular weights of AFLP fragments by comparisons with the Low Mass Ladder (Life Technologies, Bethesda, MD) DNA standard that also was 5' end-labeled with ³³P. Most polymorphisms were characterized as presence/absence of bands although a few occurred in which the polymorphism appeared as an apparent difference in molecular weight. Polymorphic bands were named using the nomenclature E_M_ 0000_, where E_ denotes the EcoRI primer with the two additional selective nucleotides, M_ denotes the MseI primer with the two additional selective nucleotides, the four-digit number is an estimate of the size of the band in base pairs, and the final blank is either "J" or "K" and denotes the parent that was the source of the "band present" allele or was the source of the larger band of a size-difference polymorphism. For example, EAAMGT0234J is an AFLP polymorphism whose presence allele is a DNA fragment 234 bp in length that originated from the Japanese parent (Z-11570) and was generated by amplification with the primer pair EAA/MGT.

AFLPs were scored based on two DNA preparations that began with independent cultures of each of the progeny. We scored one primer pair from both DNA preparations for all of the progeny. When results could not be scored clearly from the first DNA preparation, the questionable progeny and the parents from both the first and second DNA preparations were run side-by-side on a second gel to resolve discrepancies and to check for reproducibility. Thus the AFLP patterns from the parents were checked with all primer pairs from two DNA preparations, while AFLP patterns from the progeny were all checked with one primer pair and were irregularly tested with the remaining primer pairs. We did not score AFLP polymorphisms based on bands that were <90 bp in length, as they were not always consistent between the two DNA preparations.

Fertility and pigment:
The parental strains differ with respect to pigment production (Kansas strain makes a bright red pigment) and sexual reproduction (Kansas strain produces numerous mature homothallic perithecia on carrot agar, but the Japanese strain does not). These characters (PIG1 and PER1) segregated in the progeny of the cross. PER1 was scored by examination of 4-week-old cultures on carrot agar. PIG1 was scored by examination of 2-week-old cultures on complete medium (CORRELL et al. 1987 ). Progeny were tested at least twice to confirm phenotype designations.

Toxin assay:
For toxin analysis, progeny and parental strains were grown on sterilized cracked corn at 25° for 4 weeks as previously described (LESLIE et al. 1992 ). The standard AOAC method for the extraction of DON was used (SCOTT 1995 ), but the determinative step was modified. To detect the presence of both DON and NIV, gas chromatography/mass spectrometry (GC/MS) of the trimethyl silane (TMS) derivative was used instead of thin layer chromatography or electron capture GC of the heptafluorobutyrate derivative as the detection step. Briefly, the method was the following: Toxins were obtained from cultures by extraction with 4 ml of acetonitrile:water (84:16) per gram of culture material. DON and NIV were detected as TMS derivatives by GC/MS as follows: A 100-µl aliquot of the extract was evaporated to dryness under nitrogen at 60° and derivatized with 100 µl trisil-TBT (trimethylsilylimadzole:bis-trimethylacetamide:trimethylsilylchloride 3:3:2) reagent (Pierce, Rockford, IL) at 60° for 1 hr. A total of 900 µl of iso-octane was added to the solution and 1 µl was injected into the GC/MS at 70° for analysis. Samples were analyzed on a 15-m, 0.25-mm, 0.25-µm Rtx-5MS column (Restec, Belefonte, PA). The column was programmed at 30°/min to 180°, then by 1°/min to 200°, and finally by 30°/min to 270° and held at 270° for 5 min. The TMS derivatives of DON and NIV eluted during the 180°–200° gradient at 14.3 and 20.5 min, respectively.

TRI5 analysis:
The TRI5 gene is part of the trichothecene gene cluster and encodes the enzyme trichodiene synthase (HOHN and BEREMAND 1989 ; BROWN et al. 2001 ). Forty micrograms of genomic DNA from each parental and progeny strain was digested with 4 units of MseI (New England BioLabs, Beverly, MA) for 4 hr. Fragments were separated on 1% agarose gels and blotted onto Nytran SuPer Charge membrane (Schleicher and Schuell, Keene, NH) as previously described (SAMBROOK et al. 1989 ). To detect polymorphisms in the TRI5 region, blots were probed with a ³²P-labeled (Prime-a-Gene, Promega, Madison, WI) fragment generated by PCR amplification of an 590 bp fragment containing the TRI5 region from a larger, sequenced piece of the TRI gene cluster of F. graminearum (GenBank accession no. AF359361). The primers used were the following: 5'-GGCATGGTTGTATACAGC-3' and 5'-CAGAGTGATCTCATGGCAGG-3'. Amplification of the fragment was performed with 30 cycles at 94° for 30 sec, 52° for 30 sec, and 72° for 60 sec in a MJ PTC-100HB Thermocycler (MJ Research, Watertown, MA). Hybridization was performed in buffer containing formamide at 42° and washes were done as recommended by the membrane's manufacturer. Hybridization was detected by exposing blots to Kodak AR X-ray film at room temperature overnight.

Marker analysis:
Genetic mapping of all characters was performed using Map Manager QTX11 (http://mapmgr.roswellpark.org/mmQTX) on a Macintosh G4 Power PC computer (MANLY and OLSON 1999 ). Data from gels were compiled as text files and imported into this program. We then used Map Manager to distribute the data into linkage groups. Program settings used for analysis were Kosambi mapping function, search and linkage criteria set to a probability of type I error for false linkage of P = 0.0001. The authors of the program suggest that linkage relationships created with this setting represent physical chromosomes. The mapping program treated the data as a backcross with codominant markers, with the paternal parent unique, as was necessary for analysis of this haploid genome. No user-defined map distances were known, so this function was not used. Following the initial linkage group analysis, we inspected the aligned phenotype data visually to minimize linkage distance based on the assumption that single-locus double recombinants were highly unlikely. These apparent double crossover events may be due to gene conversion or to errors in data scoring and data entry. We also converted unknown data or unscored markers to their probable phenotypes on the basis of the character states of scored flanking loci because Map Maker V2.0 for the Macintosh program (LANDER et al. 1987 ), which we used to draw the map figures, treats unscored characters as a third allele and inserts a crossover on each side of these markers, which misrepresents the genetic distances between the markers on the graphical output (Fig 1).

View larger version (61K): In this window In a new window Download PPT slide   Figure 1. G. zeae linkage map. Loci are named by primer pair as described in the text. Loci that are represented by more than one AFLP marker are indicated in the form xyy, where x is the number of the linkage group and yy is a letter or pair of letters assigned in order along the linkage group. These names are followed in parentheses by the number of polymorphic AFLP markers that map to this location.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We utilized polymorphic bands generated by PCR amplification with 34 different AFLP primer pairs. The number of polymorphisms detected per primer pair ranged from 23 to >50, with an average of 32 per primer pair. Approximately 0.8% of the scores from the first DNA preparation resulted in unscorable or ambiguous results and were scored on the basis of a second, independent DNA preparation. We found no duplicate or completely complementary progeny in the progeny set.

Map Manager distributed the markers into nine linkage groups (Fig 1). Chromosome-sized linkage groups vary in total genetic length from 281 cM for linkage group I to 52 cM for linkage group IX (Table 1). We estimate the length of the entire genome as 1300 cM with an average distance of 2.8 cM between loci.

Of the 1070 markers analyzed, 22 were not associated with any of the nine large linkage groups. Of these 22 markers, 15 comprise a small linkage group of 10 loci. Six markers comprise 1 of the loci in this linkage group. The remaining 7 of these 22 markers show no linkage to each other or to any linkage group. The segregation pattern of all 22 markers is distorted, i.e., not 1:1. The Japanese allele is dominant for 18 of these markers and the Kansas allele for 4. These markers are probably not associated with the mitochondria, since we would expect all of the mitochondrial loci to have originated from the Kansas strain that served as the female parent. These bands could originate from multiple sequences that fortuitously are the same size; i.e., the sequences represented by any given band are not homologous or from a multiple-copy DNA sequence, e.g., a transposable element, that is dispersed throughout the genome.

A large number of AFLP markers map to common genetic loci. On each linkage group multiple loci are represented by 2 or more polymorphic markers. We named these loci based on their linkage group number followed by one or more alphabetical characters. This name is followed in Fig 1 by a number in parentheses that indicates the number of AFLP markers that map to that location. As many as 22 markers may map to one location (4K, Fig 1) with 64 loci represented by 4 or more AFLP markers. A total of 485 unique loci are defined by the 1070 AFLP polymorphisms; 468 of these loci map to unique positions on one of the nine linkage groups.

Haplotype analysis:
No loci on linkage groups VII, VIII, and IX have segregation ratios that are statistically different from 1:1 (Fig 2A). Linkage group I also is generally unbiased, although a few loci have a slight bias toward the Kansas genome. Five of the nine linkage groups (Fig 2B) exhibit a segregation ratio of paternal-to-maternal alleles significantly different from the 1:1 expected ratio for progeny of a haploid genetic cross (G test, P = 0.05; WEIR 1990 ). Linkage group VI is severely skewed to the Japanese genome along its entire length. Linkage group V is skewed toward the Japanese genome at one end and toward the Kansas genome at the other. Skewing also occurs toward the Japanese genome for the distal one-third of each end of linkage group II. Segregation is biased toward the Japanese genome on one end of linkage group III and toward the Kansas genome at one end of linkage group IV.

View larger version (15K): In this window In a new window Download PPT slide   Figure 2. Marker segregation by linkage group. (A) Linkage groups (I, VII, VIII, and IX) with no significant segregation distortion. (B) Linkage groups (II, III, IV, V, and VI) with significant segregation distortion. Values between the two dotted lines are not statistically different from a 1:1 segregation ratio (G test, P = 0.05).

Haplotype analysis of linkage groups I, II, and IV (Fig 3) illustrates representative patterns of recombination that we observed. On linkage group I, crossovers appear to be distributed randomly and there is no segregation distortion (Fig 3A). For linkage group II, 33 of the progeny (Table 1) had no detected crossing over in this linkage group and contained genetic material from only the Japanese parent. Both terminal portions of this linkage group are significantly skewed to the Japanese parent alleles and are fixed at locus 2C for Japanese alleles. Only in the central portion of linkage group II (Fig 3B) is segregation not distorted. Of the 66 recombinant progeny, only 7 have an odd number of crossovers. The remaining 59 have an even number of crossovers, with those with two (24) and four (25) crossovers predominating (Table 1).

View larger version (36K): In this window In a new window Download PPT slide   Figure 3. Haplotype plots of linkage groups I (A), II (B), and IV (C). Each vertical column represents an individual progeny haplotype; some haplotypes represent more than one of the progeny. Haplotypes are sorted by the number of crossovers and the relative position of the first crossover. Solid, Kansas parental genome; open, Japanese parental genome.

Linkage group IV's recombination pattern is similar to that for linkage group II, but the direction of skewing is reversed (Fig 3C). In this case the 24 nonrecombinant haplotypes are exclusively of the Kansas type. One end of this linkage group is biased to the Kansas genome, and, at locus 4AD, all of the progeny have Kansas alleles. The central portion of this chromosome also has an apparent excess of even-numbered crossovers.

TRI5 analysis:
Hybridization of a PCR probe homologous to the TRI5 gene identified a MseI RFLP polymorphism. The TRI5 probe hybridized to a 2.2-kb MseI fragment of R-5470 and to a 1.7-kb fragment of Z-3639. This polymorphism segregated 1:1 in the progeny and maps to linkage group I.

Toxin production:
Toxin production by the parental and progeny strains varied greatly under our culture conditions. The Kansas parent produces 50 ppm DON while the Japanese parent produces much lower levels of NIV (1 ppm). Fifty-four of the progeny produced levels of toxin that were high enough to characterize the toxin by GC/MS. Of these high producers, 28 produced DON and 26 produced NIV. No progeny produced both toxin types at high levels. All high-level NIV producers produce trace amounts of DON, but high-level DON producers make no detectable NIV. Both toxin type (DON/NIV) and toxin level (TOX1) segregated in the cross as single Mendelian characters. TOX1 maps to one end of linkage group IV and the locus controlling toxin type cosegregated with the MseI polymorphism associated with the TRI5 gene on linkage group I (Fig 1).

Fertility and pigment:
The loci for perithecia production (PER1) and red pigment production (PIG1) both mapped near the locus controlling high levels of toxin production on linkage group IV (Fig 1).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This map of G. zeae is the second genetic map for a Gibberella species and for any species with a Fusarium anamorphic state. The G. zeae map includes many loci that are represented by more than one AFLP polymorphism. Some explanations for this clustering include map saturation (the average distribution of loci is 2.8 cM/locus); nonrandom distribution of AT-rich nucleotide regions in the genome, which would contain a higher number of EcoRI and MseI restriction sites; recombination suppression, perhaps caused by heterozygous inversions, deletions, or insertions that interfere with recombination; or nonrandom distribution of restriction sites due to methylation, as has been observed in soybeans (YOUNG et al. 1999 ).

Segregation distortion in the progeny:
As G. zeae is homothallic we crossed strains carrying complementary auxotrophic nit mutants in the parents and selected nitrate-utilizing recombinant random ascospore progeny. This procedure was used by BOWDEN and LESLIE 1999 to demonstrate the potential for outcrossing and hybridization between G. zeae strains. Selection of wild-type recombinants was the most practical method available to obtain sufficient ascospores from the rare hybrid perithecia for the analysis. Because the selection we applied selected for only one class of recombinant progeny, we expected and observed segregation distortion in our cross due to the selection for the wild-type nit1 and nit3 alleles. All of the progeny in the 99-member mapping population have at least two recombinant chromosomes (98 have 3 or more) and no more than 80% of the markers in any one of the progeny are from a single parent.

Distribution of crossovers:
The distribution of crossovers across the linkage groups of the progeny is not random (Table 1). In particular, there is an excess of progeny with no crossovers within a linkage group and a reduction in the number of progeny with linkage groups in which recombination has occurred as compared to an expected Poisson distribution (² test, P = 0.05). For each of linkage groups VI, VII, VIII, and IX, at least half of the progeny have no detectable crossover, which could make estimates of linkage distances less accurate than expected, given the number of progeny analyzed.

There also is no evidence for chromosome loss as no progeny have a linkage group on which all of the alleles are of the no-band type. Thus, unlike F. moniliforme (XU and LESLIE 1996 ) and F. solani (MIAO et al. 1991 ), we found no evidence for dispensable "B" chromosomes in G. zeae.

We think that recombination might be generally suppressed in our cross. Recombination suppression has been reported on chromosome 1 in the pseudohomothallic species Neurospora tetrasperma (GALLEGOS et al. 2000 ), but no mechanism to explain these observations has been proposed. GRELL 1962 proposed that recombination might be essential for a chromosome to find its homolog and pass properly through meiosis. This phenomenon has been studied in Drosophila (HAWLEY et al. 1993 ) and more recently in yeast (HABER 1998 ). Chromosomes without partners go through a distributive disjunction process where pairing apparently occurs on the basis of size. In filamentous fungi, "B" chromosomes are thought to segregate in this manner (MIAO et al. 1991 ; XU and LESLIE 1996 ). In our cross, the number of progeny carrying an intact parental linkage group is high (Table 1), and we presume that distributive disjunction must be functioning for the corresponding chromosomes to pass through meiosis. We do not know the physical sizes for any of the chromosomes in G. zeae and thus cannot determine whether the size of the chromosome is playing a role in the segregation patterns we observed or if aberrant segregation is reducing the number of viable progeny produced. We also cannot determine if the relatively high number of intact parental linkage groups observed in the progeny is the result of a lack of pairing and/or synapsis that could lead to crossing over or if nonviable progeny result if crossing over occurs other than in a few specific patterns. We are currently testing some of these alternative explanations for our results.

Linkage groups with unusual properties:
Linkage group II has several unusual characteristics. The only nonrecombinant haplotype for linkage group II is from the Japanese parent, which is found in 33 of the 99 progeny (Table 1). The remaining 66 progeny also contain a small region near each end of this linkage group that is predominantly Japanese genome in origin. Among the progeny in which detectable recombination has occurred on linkage group II, the central part of the linkage group has a near 1:1 segregation ratio (Fig 2). Of the 66 recombinant progeny, 59 have an even number of crossovers on linkage group II, with the two and four crossover classes being approximately equally frequent (Table 1). This pattern could result if there is a large heterozygous inversion that includes most of the central portion of the linkage group. Chromosomes with odd numbers of crossovers within the inverted region would be duplicated for one region distal to the inversion and deficient for the other. If the deficient region carries essential genes, then the duplication/deficiency progeny will be dead. The 7 progeny with an odd number of crossovers on linkage group II all appear to have an even number of crossovers within the putative inverted region (a span of 220 cM from locus 2F to 2AJ) and an odd number, usually a single, in one of the two distal regions (Fig 3). The Japanese parent carries the nit3 wild-type allele that was selected for in all of the progeny. We expect nit3 to be in the small region (locus 2C) for which all of the progeny have the Japanese genome.

Linkage group IV recombination patterns also are somewhat unusual. The 24 nonrecombinant progeny for this linkage group are all of the Kansas type. It is possible that the crossover type pattern between 4C and 4P (56 cM) could be due to a heterozygous inversion. This linkage group also is fixed near one end at locus 4AD for the Kansas genome. This region is the only one in the map that is 100% Kansas genome, and, therefore, we predict that the nit1 gene maps on this linkage group in or very near locus 4AD.

Trichothecene gene analyses:
We mapped TRI5, and presumably the rest of the trichothecene gene cluster, near the middle of linkage group I in G. zeae. TRI5 encodes trichodiene synthase, the first step in the trichothecene toxin biosynthetic pathway (HOHN and VAN MIDDLESWORTH 1986 ; HOHN and BEREMAND 1989 ; BROWN et al. 2001 ). The locus controlling toxin type (DON or NIV) cosegregated with TRI5. This is the first genetic proof that toxin type is controlled by a single locus that is linked to the trichothecene gene cluster. We did not give a new name to the toxin-type locus because the responsible gene(s) in the trichothecene gene cluster might already be named (BROWN et al. 2001 ; LEE et al. 2001 ).

A locus controlling toxin amount was located on linkage group IV. This gene was not previously described and was designated TOX1. Since this gene has a large effect on toxin biosynthesis, it deserves further study. We mapped several AFLP markers within 15 cM of TOX1 that could be useful for cloning the gene. Loci controlling red pigment production and perithecium formation also map near TOX1, but these distances and the gene order might be affected by the putative inversion on linkage group IV.

Map utilization:
In future studies our map can be utilized to locate genes of interest in several ways. If mutations arise or are induced in one of the strains, these mutations can be mapped by performing a new cross between Z-3639 and R-5470 or their mutagenized derivatives. Linkage to the AFLP markers on the map should be readily determined by analysis with as few as seven primer pairs. For example, the seven primer pairs that use the EAA primer generate a skeleton map that defines 70% of the total linkage map. Economically important traits could include virulence, toxin production, competitive ability, and fungicide sensitivity. The density of the map is high enough that it should be possible to analyze these traits as quantitative trait loci (QTL). If the gene of interest has been cloned or if it is an expressed sequence tag (EST) sequence, either hybridization with a PCR-amplified probe or the AFLP mapping technique of CATO et al. 2001 , which uses a single 4-bp recognition-site restriction enzyme digest of the ESTs, can be used to localize the gene within the present mapping population. If the map is used to order genomic libraries, then the process of sequencing the G. zeae genome could be greatly simplified.

Our map provides markers with known linkage relationships that can be used for population studies. Such studies are required to understand the current structure and future changes in the pathogen's populations and their correlation with disease epidemics. AFLPs provide a well-defined, relatively large set of markers that can be used to monitor populations on a genome-wide basis. These studies should more accurately reflect the population being studied since the bias created when linked markers are treated as unlinked can be removed (BRANDLE et al. 1997 ).

The seven lineages described by O'DONNELL et al. 2000 appear genetically divergent based on the sequences of six genes. The fertility between various lineages needs to be quantified to help estimate the risk of generating novel parasitic phenotypes when lineages are commingled and to determine their degree of genetic isolation. We estimate that <1% of the perithecia were heterozygous outcrosses when Z-3639 and R5470 were mated. Up to 35% heterozygous perithecia were produced in crosses of Z-3639 with two other Kansas strains that belong to lineage VII (BOWDEN and LESLIE 1999 ), so there may be lower fertility in crosses between lineages. The map could be used to identify naturally occurring hybrids between lineages and to estimate the amount of genetic material within a hybrid strain that originated from the different lineages.

On the basis of our AFLP study, the two parental strains of the mapping population differ at 50% of the observed bands. In the G. fujikuroi species complex (LESLIE et al. 2001 ; MARASAS et al. 2001 ), we would conclude that these strains were in the same species, but perhaps not the same subspecies. [In the G. fujikuroi species complex, strains in the same subspecies always share 65% or more AFLP identity and those in different species share <40% AFLP identity. The only case in which an intermediate value (55%) was observed (K. ZELLER and J. LESLIE, unpublished data) is also a case where some members of the two groups can occasionally cross and produce perithecia with viable ascospores.] However, many more G. zeae populations and related species need to be examined before concluding that the criteria applicable to G. fujikuroi also are applicable to G. zeae.

The finding of at least two putative chromosome rearrangements in our cross also is suggestive of significant genetic differentiation. Heterozygous inversions are postzygotic fertility blocks that reduce fertility and progeny variability. These differences are certainly important in Drosophila speciation (ANDERSON et al. 1991 ), but probably not in Neurospora speciation (PERKINS 1997 ). Within the G. fujikuroi species complex, 90% of the RFLP markers tested remained on the same chromosome across six biological species (XU et al. 1995 ). These results indicate that translocations and transpositions are not widespread, but provide no information on inversions, as an inversion would not alter the chromosome to which a marker hybridizes. To the degree that chromosome rearrangements alter gene position, they might alter expression due to specific position effects. We have no evidence for this type of effect in Fusarium, although rearrangements that affected toxin gene clusters or pathogenicity gene clusters might show such effects. Limitations to recombination also could be an effective means to lock particular allele combinations into gene complexes that might have selective value and could not easily be broken apart, e.g., the spore killer complex in Neurospora (RAJU 1994 ).

To test some of these hypotheses, follow-up studies will be needed. Cytological studies have identified no more than four chromosomes (HOWSON et al. 1963 ), but studies with pulse-field chromosome separation are likely to be more successful, given the relatively small size of Fusarium chromosomes. Additional maps are needed to confirm linkage group configurations and to map other economically important traits, e.g., pathogenicity, host range, or toxin production, and to determine if the segregation distortion we observed is distorting our perception of the genomic organization of G. zeae. For example:

1. Are the putative chromosome rearrangements we observed peculiar to one of the strains in the present cross?

2. Is the suppression of crossing over a general property of G. zeae outcrosses? Peculiar to interlineage crosses? Specific for one (or both) of the strains used in the present cross?

	ACKNOWLEDGMENTS

We thank Amy Beyer, Ann Clouse, and Amy Hanson for technical assistance. J. E. Jurgenson was supported by a Professional Development Leave grant from the University of Northern Iowa. This work was supported in part by U.S. Wheat and Barley Scab Initiative project 59-0790-9-029 and by the Kansas Agricultural Experiment Station. This is contribution no. 02-67-J from the Kansas Agricultural Experiment Station, Manhattan, KS.

Manuscript received November 1, 2001; Accepted for publication December 26, 2001.

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