Copyright  2002 by the Genetics Society of America

A Genetic Map of zeae ( graminearum)

J. E. Jurgenson,* R. L. Bowden,† K. A. Zeller,† J. F. Leslie,†,1 N. J. Alexander‡ and R. D. Plattner‡ *Department of Biology, University of Northern Iowa, Cedar Falls, Iowa 50614, †Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506-5502 and ‡ Research Unit, USDA/ARS National Center for Agricultural Utilization Research, Peoria, Illinois 61604 Manuscript received November 1, 2001 Accepted for publication December 26, 2001

ABSTRACT We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complemen- tary 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 polymor- cM 1300ف phic markers that mapped to 468 unique loci on nine linkage groups. The total map length is 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.

IBBERELLA zeae (anamorph Fusarium graminear- toxin production (DON, 3-acetyldeoxynivalenol, 15-acetyl- G um) is the most important causal agent of Fu- deoxynivalenol, NIV, 4-acetylnivalenol, and ) sarium head blight (scab) of and in the as well as in DNA sequence-based markers. The degree United States (McMullen et al. 1997) and China (Chen of genetic isolation and pathogenic specialization among et al. 2000). In the 1990s, scab caused an estimated $3 lineages remains unresolved (Carter et al. 2000). billion losses to wheat and barley farmers in the United Bowden and Leslie (1999) established that, under States alone (Windels 2000). Scab reduces wheat bak- laboratory conditions, members of at least three of the ing quality (Seitz et al. 1986) and harvested grain often phylogenetic lineages described by O’Donnell et al. is contaminated with such as nivalenol (2000) can interbreed and produce viable, recombinant (NIV), deoxynivalenol (DON), and zearalenone (Mara- progeny. The fertility of these interlineage crosses can sas et al. 1984; Tanaka et al. 1988). be relatively high and suggests that these strains are G. zeae is homothallic (Nelson et al. 1983; Yun et al. members of geographically separated and genetically 2000) and may produce abundant perithecia in the distinct populations rather than of distinct species. field, but can be outcrossed under laboratory conditions O’Donnell et al. (2000) described one putative natu- (Bowden and Leslie 1999). Despite being homothallic, rally occurring hybrid strain (collected in Nepal by A. E. the amount and distribution of genetic heterogeneity in Desjardins) between lineages 2 and 6. If isolates from field populations of this suggest that outcrossing these genetically divergent populations interbreed, then occurs at a significant rate in the field (Bowden and there is the potential for the production of new geno- Leslie 1992; Walker et al. 2001). Recently, O’Donnell types that carry novel combinations of genes for patho- et al. (2000) used DNA sequences of elongation factor genicity, host range, or toxin production (Brasier 2000). ␣ ␤ (EF-1 ), phosphate permease genes (PHO), -tubu- A cross between isolates from two such distantly related lin (TUB), UTP-ammonia ligase (URA), trichothecene populations also should be rich in polymorphic markers 3-O-acetyltransferase (TRI101), and a putative reductase that could be used to generate a detailed genetic map. (RED) to resolve a set of G. zeae strains into at least seven Amplified fragment length polymorphism (AFLP) distinct phylogenetic lineages. Differences between strains analysis is a PCR-based DNA analysis technique that can in distinct lineages include qualitative differences in detect variations in restriction fragment length polymor- phisms (RFLP) on a genome-wide basis (Vos et al. 1995). Like restriction fragment length polymorphism analysis, 1Corresponding author: Department of Plant Pathology, 4002 Throck- morton Plant Sciences Center, Kansas State University, Manhattan, AFLPs can detect size differences in restriction frag- KS 66506-5502. E-mail: jfl@plantpath.ksu.edu ments caused by DNA insertions, deletions, or changes

Genetics 160: 1451–1460 (April 2002) 1452 J. E. Jurgenson et al.

in target restriction site sequences. As compared to 8633] of and the progeny (FGSC 8634–8732) from the map- RFLP analysis, however, the labor required to detect ping cross are available from the Fungal Genetics Stock Center (Department of Microbiology, University of Kansas Medical genetic polymorphisms with AFLPs is considerably re- Center, Kansas City, KS; http://www.fgsc.net). We analyzed 99 duced. AFLP analysis yields dominant band/no-band single -derived progeny from this cross. The fre- type markers that can be used to study genetic diversity quency of nitrate-utilizing was Ͻ1%, because most in fungal populations (e.g., Gonzalez et al. 1998; Pur- of the perithecia were homothallic selfs of Z-11572. wantara et al. 2000; Zeller et al. 2000) and to define We isolated ascospores from mature perithecia by inverting the carrot agar cross plates and collecting ascospores on the and distinguish species of Fusarium (Marasas et al. plate lid. These ascospores were suspended in 5 ml of sterile 2001). AFLPs have been used to develop recombination- water and then dilution plated onto a minimal agar medium based genetic maps in mapping populations of higher (Correll et al. 1987) amended with tergitol and sorbose plants such as barley, soybeans, and maize (Vuylsteke (Bowden and Leslie 1999). Plates were incubated for 5–7 Њ et al. 1999; Yin et al. 1999; Hua et al. 2000) and to days at 24 . Recombinant progeny, identified as nitrate uti- lizers, were collected and transferred to minimal medium supplement mapping efforts in fungi, e.g., in Phytophth- slants. Each of the recombinant progeny was subcultured from ora (van der Lee et al. 2001). When using 2-bp exten- a single macroconidium. Macroconidia were separated with sions in specific AFLP reactions, as is common in fungi, a Cailloux stage-mounted micromanipulator (Stoelting, Chi- there are 256 potential primer-pair combinations that cago). Cultures were maintained on minimal medium and can each be used to generate a unique DNA fingerprint stored as spore/hyphal fragment suspensions in 15% glycerol at Ϫ70Њ at Kansas State University. pattern for each pair of restriction enzymes used in the Analysis of DNA polymorphisms in the mapping population: initial digestion of the DNA. The genomic distribution We inoculated 50 ml of liquid complete medium (Correll -ϫ 105 macroco 5ف of markers generated by AFLPs is limited only by the et al. 1987) in 125-ml Erlenmeyer flasks with ml of a 2.5% aqueous (v/v) solution 1ف distribution of the restriction sites used to generate nidia suspended in them. of Tween 60 (Sigma, St. Louis). Cultures were incubated for 2–3 days at room temperature (22Њ–25Њ) on a rotary shaker Our objective in this study was to establish a recombi- (150 rpm). Tissue from each culture was collected by filtration nation-based genetic linkage map of G. zeae by crossing through a nongauze milk filter (Ken Ag Milk Filter, Ashland, phenotypically and genetically divergent strains from OH), washed with 100 ml sterile water, and blotted dry with different continents. Only one other detailed genetic paper towels. The tissue was frozen at Ϫ20Њ until DNA was map is presently available for any Gibberella species extracted. DNA extraction: DNA was isolated with a cetyltrimethyl am- (Xu and Leslie 1996). Generation of a genetic linkage monium bromide procedure (Kere´nyi et al. 1999) modified map for G. zeae will permit the correlation of physical from that of Murray and Thompson (1980). We estimated sequences with segregating phenotypes, the localization final DNA concentrations (in TE buffer) by comparison of of genes for toxin production, the identification of ge- DNA fluorescence of diluted aliquots of each DNA sample ␭ netically independent markers that can be used in char- against that of HindIII-digested bacteriophage DNA with an acterization of field populations, and the identification IS-1000 version 2.0 digital imaging system (Alpha Innotech, San Leandro, CA). Samples and sample dilutions were run in of genomic sequences that might be of particular impor- 1% agarose gels containing TAE (40 mm Tris-acetate, 1 mm tance in the evolution of this species. EDTA pH 8.0) and 0.5 ␮g/ml ethidium bromide. DNA yields ranged from 100 to 1000 ␮g of DNA per culture. The concen- tration of each DNA sample was adjusted to 20 ␮g/ml for use MATERIALS AND METHODS in AFLP analysis. AFLPs: AFLPs were generated with the protocol of Vos et Mapping cross: One of the parents of this cross was derived al. (1995) as modified by Zeller et al. (2000). AFLP primers from a DON-producing strain, Z-3639, originally isolated from were synthesized by Integrated DNA Technologies (Coralville, wheat in Kansas (Bowden and Leslie 1992) and belonging IA). The EcoRI primers in the final specific amplification reac- to lineage VII as described by O’Donnell et al. (2000). The tions were 5Ј end-labeled with [␥-33P]ATP (NEN Life Sciences, other parent was derived from a NIV-producing strain, R-5470, Boston). Dried gels were exposed to X-ray film (Classic Blue originally isolated from barley in Japan and obtained from Sensitive, Molecular Technologies, St. Louis) for 2–5 days Paul E. Nelson (Department of Plant Pathology, Pennsylvania at room temperature to identify DNA bands. We identified State University, University Park, PA), which belongs to lineage polymorphic bands by eye and scored them manually. We VI as described by O’Donnell et al. (2000). These strains were estimated molecular weights of AFLP fragments by compari- selected because of the difference in toxin production and sons with the Low Mass Ladder (Life Technologies, Bethesda, to give a large number of polymorphic markers, due to the MD) DNA standard that also was 5Ј end-labeled with 33P. Most presumptive isolation of the populations from which they were polymorphisms were characterized as presence/absence of derived. bands although a few occurred in which the polymorphism Crosses were performed essentially as described previously appeared as an apparent difference in molecular weight. Poly- (Bowden and Leslie 1999). Mycelial plugs of a nit1 mutant morphic bands were named using the nomenclature E_M_ (Z-11570) of R-5470 and a nit3 mutant (Z-11572) of Z-3639 0000_, where E_ denotes the EcoRI primer with the two addi- were simultaneously inoculated onto carrot agar medium tional selective nucleotides, M_ denotes the MseI primer with (Klittich and Leslie 1988). nit mutants were generated as the two additional selective nucleotides, the four-digit number previously described (Bowden and Leslie 1992) and charac- is an estimate of the size of the band in base pairs, and the terized by using standard Fusarium protocols (Correll et final blank is either “J” or “K” and denotes the parent that al. 1987; Klittich and Leslie 1988). The parents [Z-11570, was the source of the “band present” allele or was the source Fungal Genetics Stock Center (FGSC) 8632; Z-11572, FGSC of the larger band of a size-difference polymorphism. For G. zeae Genetic Map 1453 example, EAAMGT0234J is an AFLP polymorphism whose used were the following: 5Ј-GGCATGGTTGTATACAGC-3Ј presence allele is a DNA fragment 234 bp in length that origi- and 5Ј-CAGAGTGATCTCATGGCAGG-3Ј. Amplification of nated from the Japanese parent (Z-11570) and was generated the fragment was performed with 30 cycles at 94Њ for 30 sec, by amplification with the primer pair EAA/MGT. 52Њ for 30 sec, and 72Њ for 60 sec in a MJ PTC-100HB Thermocy- AFLPs were scored based on two DNA preparations that cler (MJ Research, Watertown, MA). Hybridization was per- began with independent cultures of each of the progeny. We formed in buffer containing formamide at 42Њ and washes scored one primer pair from both DNA preparations for all were done as recommended by the membrane’s manufac- of the progeny. When results could not be scored clearly from turer. Hybridization was detected by exposing blots to Kodak the first DNA preparation, the questionable progeny and the AR X-ray film at room temperature overnight. parents from both the first and second DNA preparations Marker analysis: Genetic mapping of all characters was were run side-by-side on a second gel to resolve discrepancies performed using Map Manager QTX11 (http://mapmgr. and to check for reproducibility. Thus the AFLP patterns from roswellpark.org/mmQTX) on a Macintosh G4 Power PC com- the parents were checked with all primer pairs from two DNA puter (Manly and Olson 1999). Data from gels were com- preparations, while AFLP patterns from the progeny were all piled as text files and imported into this program. We then checked with one primer pair and were irregularly tested with used Map Manager to distribute the data into linkage groups. the remaining primer pairs. We did not score AFLP polymor- Program settings used for analysis were Kosambi mapping Ͻ function, search and linkage criteria set to a probability of phisms based on bands that were 90 bp in length, as they ϭ were not always consistent between the two DNA preparations. type I error for false linkage of P 0.0001. The authors of Fertility and pigment: The parental strains differ with re- the program suggest that linkage relationships created with spect to pigment production (Kansas strain makes a bright red this setting represent physical chromosomes. The mapping pigment) and sexual reproduction (Kansas strain produces program treated the data as a backcross with codominant numerous mature homothallic perithecia on carrot agar, but markers, with the paternal parent unique, as was necessary the Japanese strain does not). These characters (PIG1 and for analysis of this haploid genome. No user-defined map distances were known, so this function was not used. Following PER1) segregated in the progeny of the cross. PER1 was scored the initial linkage group analysis, we inspected the aligned by examination of 4-week-old cultures on carrot agar. PIG1 phenotype data visually to minimize linkage distance based was scored by examination of 2-week-old cultures on complete on the assumption that single-locus double recombinants were medium (Correll et al. 1987). Progeny were tested at least highly unlikely. These apparent double crossover events may twice to confirm phenotype designations. be due to gene conversion or to errors in data scoring and Toxin assay: For toxin analysis, progeny and parental strains Њ data entry. We also converted unknown data or unscored were grown on sterilized cracked corn at 25 for 4 weeks as markers to their probable phenotypes on the basis of the previously described (Leslie et al. 1992). The standard AOAC character states of scored flanking loci because Map Maker method for the extraction of DON was used (Scott 1995), but V2.0 for the Macintosh program (Lander et al. 1987), which the determinative step was modified. To detect the presence of we used to draw the map figures, treats unscored characters both DON and NIV, gas chromatography/mass spectrometry as a third allele and inserts a crossover on each side of these (GC/MS) of the trimethyl silane (TMS) derivative was used markers, which misrepresents the genetic distances between instead of thin layer chromatography or electron capture GC the markers on the graphical output (Figure 1). 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 RESULTS (84:16) per gram of culture material. DON and NIV were detected as TMS derivatives by GC/MS as follows: A 100-␮l We utilized polymorphic bands generated by PCR aliquot of the extract was evaporated to dryness under nitro- amplification with 34 different AFLP primer pairs. The gen at 60Њ and derivatized with 100 ␮l trisil-TBT (trimethylsilyl- number of polymorphisms detected per primer pair imadzole:bis-trimethylacetamide:trimethylsilylchloride 3:3:2) Ͼ reagent (Pierce, Rockford, IL) at 60Њ for 1 hr. A total of 900 ranged from 23 to 50, with an average of 32 per primer ␮l of iso-octane was added to the solution and 1 ␮l was injected pair. Approximately 0.8% of the scores from the first into the GC/MS at 70Њ for analysis. Samples were analyzed on DNA preparation resulted in unscorable or ambiguous a 15-m, 0.25-mm, 0.25-␮m Rtx-5MS column (Restec, Belefonte, results and were scored on the basis of a second, inde- Њ Њ PA). The column was programmed at 30 /min to 180 , then pendent DNA preparation. We found no duplicate or by 1Њ/min to 200Њ, and finally by 30Њ/min to 270Њ and held at 270Њ for 5 min. The TMS derivatives of DON and NIV completely complementary progeny in the progeny set. eluted during the 180Њ–200Њ gradient at 14.3 and 20.5 min, Map Manager distributed the markers into nine link- respectively. age groups (Figure 1). Chromosome-sized linkage groups TRI5 analysis: The TRI5 gene is part of the trichothecene vary in total genetic length from 281 cM for linkage gene cluster and encodes the enzyme trichodiene synthase group I to 52 cM for linkage group IX (Table 1). We cM 1300ف Hohn and Beremand 1989; Brown et al. 2001). Forty micro- estimate the length of the entire genome as) grams of genomic DNA from each parental and progeny strain was digested with 4 units of MseI (New England BioLabs, with an average distance of 2.8 cM between loci. Beverly, MA) for 4 hr. Fragments were separated on 1% aga- Of the 1070 markers analyzed, 22 were not associated rose gels and blotted onto Nytran SuPer Charge membrane with any of the nine large linkage groups. Of these 22 (Schleicher and Schuell, Keene, NH) as previously described markers, 15 comprise a small linkage group of 10 loci. (Sambrook et al. 1989). To detect polymorphisms in the TRI5 32 Six markers comprise 1 of the loci in this linkage group. region, blots were probed with a P-labeled (Prime-a-Gene, The remaining 7 of these 22 markers show no linkage Promega, Madison, WI) fragment generated by PCR amplifi- bp fragment containing the TRI5 region to each other or to any linkage group. The segregation 590ف cation of an from a larger, sequenced piece of the TRI gene cluster of F. pattern of all 22 markers is distorted, i.e., not 1:1. The graminearum (GenBank accession no. AF359361). The primers Japanese allele is dominant for 18 of these markers and 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. G. zeae Genetic Map 1455

TABLE 1 Distribution of markers, loci, and crossovers across linkage groups

Observed crossover events per linkage group Linkage group None No. No. of No. cM of loci markers Japanese KS 1 2 34567–10 Mean I 281 94 171 7 18 9 15 13 19 8 6 4 2.65 II 263 90 203 33 0 2 24 3 25 1 6 6 2.43 III 219 88 214 19 6 14 16 20 12 7 2 3 2.27 IV 182 69 158 0 24 22 16 23 3 9 1 1 1.95 V 1023171 9 76974210 0 1.09 VI63267157519135000 0 0.61 VII69266928332096102 0 0.73 VIII 55 22 41 20 36 29 10 3100 0 0.63 IX52225327487 95300 0 0.63

Total 1286 468 1048 200 177 151 119 75 66 26 17 14 1.44

the Kansas allele for 4. These markers are probably not group II. Segregation is biased toward the Japanese ge- associated with the mitochondria, since we would expect nome on one end of linkage group III and toward the all of the mitochondrial loci to have originated from Kansas genome at one end of linkage group IV. the Kansas strain that served as the female parent. These Haplotype analysis of linkage groups I, II, and IV bands could originate from multiple sequences that for- (Figure 3) illustrates representative patterns of recombi- tuitously are the same size; i.e., the sequences repre- nation that we observed. On linkage group I, crossovers sented by any given band are not homologous or from appear to be distributed randomly and there is no segre- a multiple-copy DNA sequence, e.g., a transposable ele- gation distortion (Figure 3A). For linkage group II, 33 ment, that is dispersed throughout the genome. of the progeny (Table 1) had no detected crossing over A large number of AFLP markers map to common in this linkage group and contained genetic material genetic loci. On each linkage group multiple loci are from only the Japanese parent. Both terminal portions represented by 2 or more polymorphic markers. We of this linkage group are significantly skewed to the named these loci based on their linkage group number Japanese parent alleles and are fixed at locus 2C for followed by one or more alphabetical characters. This Japanese alleles. Only in the central portion of linkage name is followed in Figure 1 by a number in parentheses group II (Figure 3B) is segregation not distorted. Of the that indicates the number of AFLP markers that map to 66 recombinant progeny, only 7 have an odd number of that location. As many as 22 markers may map to one crossovers. The remaining 59 have an even number location (4K, Figure 1) with 64 loci represented by 4 or of crossovers, with those with two (24) and four (25) more AFLP markers. A total of 485 unique loci are crossovers predominating (Table 1). defined by the 1070 AFLP polymorphisms; 468 of these Linkage group IV’s recombination pattern is similar loci map to unique positions on one of the nine linkage to that for linkage group II, but the direction of skewing groups. is reversed (Figure 3C). In this case the 24 nonrecombi- Haplotype analysis: No loci on linkage groups VII, nant haplotypes are exclusively of the Kansas type. One VIII, and IX have segregation ratios that are statistically end of this linkage group is biased to the Kansas ge- different from 1:1 (Figure 2A). Linkage group I also is nome, and, at locus 4AD, all of the progeny have Kansas generally unbiased, although a few loci have a slight alleles. The central portion of this chromosome also bias toward the Kansas genome. Five of the nine linkage has an apparent excess of even-numbered crossovers. groups (Figure 2B) exhibit a segregation ratio of pater- TRI5 analysis: Hybridization of a PCR probe homolo- nal-to-maternal alleles significantly different from the gous to the TRI5 gene identified a MseI RFLP polymor- 1:1 expected ratio for progeny of a haploid genetic cross phism. The TRI5 probe hybridized to a 2.2-kb MseI frag- (G test, P ϭ 0.05; Weir 1990). Linkage group VI is ment of R-5470 and to a 1.7-kb fragment of Z-3639. in the progeny and 1:1ف severely skewed to the Japanese genome along its entire This polymorphism segregated length. Linkage group V is skewed toward the Japanese maps to linkage group I. genome at one end and toward the Kansas genome Toxin production: Toxin production by the parental at the other. Skewing also occurs toward the Japanese and progeny strains varied greatly under our culture ppm DON 50ف genome for the distal one-third of each end of linkage conditions. The Kansas parent produces 1456 J. E. Jurgenson et al.

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 signifi- cant segregation distortion. Values between the two dotted lines are not statistically different from a 1:1 segregation ratio (G test, P ϭ 0.05).

while the Japanese parent produces much lower levels tion (PER1) and red pigment production (PIG1) both ppm). Fifty-four of the progeny produced mapped near the locus controlling high levels of toxin 1ف) of NIV levels of toxin that were high enough to characterize the production on linkage group IV (Figure 1). toxin by GC/MS. Of these high producers, 28 produced DON and 26 produced NIV. No progeny produced both DISCUSSION toxin types at high levels. All high-level NIV producers produce trace amounts of DON, but high-level DON This map of G. zeae is the second genetic map for a producers make no detectable NIV. Both toxin type Gibberella species and for any species with a Fusarium (DON/NIV) and toxin level (TOX1) segregated in the anamorphic state. The G. zeae map includes many loci cross as single Mendelian characters. TOX1 maps to one that are represented by more than one AFLP polymor- end of linkage group IV and the locus controlling toxin phism. Some explanations for this clustering include type cosegregated with the MseI polymorphism associ- map saturation (the average distribution of loci is 2.8 ated with the TRI5 gene on linkage group I (Figure 1). cM/locus); nonrandom distribution of AT-rich nucleo- Fertility and pigment: The loci for perithecia produc- tide regions in the genome, which would contain a G. zeae Genetic Map 1457

eny. 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 avail- able to obtain sufficient ascospores from the rare hybrid perithecia for the analysis. Because the selection we applied selected for only one class of recombinant prog- eny, 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 cross- overs 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 com- pared to an expected Poisson distribution (␹2 test, P ϭ 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 sup- pressed in our cross. Recombination suppression has been reported on chromosome 1 in the pseudohomo- thallic species Neurospora tetrasperma (Gallegos et al. 2000), but no mechanism to explain these observations has been proposed. Grell (1962) proposed that recom- bination might be essential for a chromosome to find its homolog and pass properly through meiosis. This Figure 3.—Haplotype plots of linkage groups I (A), II (B), phenomenon has been studied in Drosophila (Hawley and IV (C). Each vertical column represents an individual et al. 1993) and more recently in yeast (Haber 1998). progeny haplotype; some haplotypes represent more than one Chromosomes without partners go through a distribu- of the progeny. Haplotypes are sorted by the number of cross- tive disjunction process where pairing apparently occurs overs and the relative position of the first crossover. Solid, Kansas parental genome; open, Japanese parental genome. on the basis of size. In filamentous fungi, “B” chromo- somes are thought to segregate in this manner (Miao et al. 1991; Xu and Leslie 1996). In our cross, the number higher number of EcoRI and MseI restriction sites; re- of progeny carrying an intact parental linkage group is combination suppression, perhaps caused by heterozy- high (Table 1), and we presume that distributive dis- gous inversions, deletions, or insertions that interfere junction must be functioning for the corresponding with recombination; or nonrandom distribution of re- chromosomes to pass through meiosis. We do not know striction sites due to methylation, as has been observed the physical sizes for any of the chromosomes in G. in soybeans (Young et al. 1999). zeae and thus cannot determine whether the size of the Segregation distortion in the progeny: As G. zeae is chromosome is playing a role in the segregation patterns homothallic we crossed strains carrying complementary we observed or if aberrant segregation is reducing the auxotrophic nit mutants in the parents and selected number of viable progeny produced. We also cannot nitrate-utilizing recombinant random ascospore prog- determine if the relatively high number of intact paren- 1458 J. E. Jurgenson et al. tal linkage groups observed in the progeny is the result because the responsible gene(s) in the trichothecene of a lack of pairing and/or synapsis that could lead to gene cluster might already be named (Brown et al. crossing over or if nonviable progeny result if crossing 2001; Lee et al. 2001). over occurs other than in a few specific patterns. We A locus controlling toxin amount was located on link- are currently testing some of these alternative explana- age group IV. This gene was not previously described tions for our results. and was designated TOX1. Since this gene has a large Linkage groups with unusual properties: Linkage effect on toxin biosynthesis, it deserves further study. group II has several unusual characteristics. The only We mapped several AFLP markers within 15 cM of TOX1 nonrecombinant haplotype for linkage group II is from that could be useful for cloning the gene. Loci control- the Japanese parent, which is found in 33 of the 99 ling red pigment production and perithecium forma- progeny (Table 1). The remaining 66 progeny also con- tion also map near TOX1, but these distances and the tain a small region near each end of this linkage group gene order might be affected by the putative inversion that is predominantly Japanese genome in origin. on linkage group IV. Among the progeny in which detectable recombination Map utilization: In future studies our map can be has occurred on linkage group II, the central part of utilized to locate genes of interest in several ways. If the linkage group has a near 1:1 segregation ratio (Fig- mutations arise or are induced in one of the strains, ure 2). Of the 66 recombinant progeny, 59 have an even these mutations can be mapped by performing a new number of crossovers on linkage group II, with the two cross between Z-3639 and R-5470 or their mutagenized and four crossover classes being approximately equally derivatives. Linkage to the AFLP markers on the map frequent (Table 1). This pattern could result if there is should be readily determined by analysis with as few as a large heterozygous inversion that includes most of the seven primer pairs. For example, the seven primer pairs central portion of the linkage group. Chromosomes that use the EAA primer generate a skeleton map that with odd numbers of crossovers within the inverted re- defines 70% of the total linkage map. Economically gion would be duplicated for one region distal to the important traits could include virulence, toxin produc- inversion and deficient for the other. If the deficient tion, competitive ability, and fungicide sensitivity. The region carries essential genes, then the duplication/ density of the map is high enough that it should be deficiency progeny will be dead. The 7 progeny with an possible to analyze these traits as quantitative trait loci odd number of crossovers on linkage group II all appear (QTL). If the gene of interest has been cloned or if it to have an even number of crossovers within the putative is an expressed sequence tag (EST) sequence, either inverted region (a span of 220 cM from locus 2F to 2AJ) hybridization with a PCR-amplified probe or the AFLP and an odd number, usually a single, in one of the two mapping technique of Cato et al. (2001), which uses a distal regions (Figure 3). The Japanese parent carries single 4-bp recognition-site restriction enzyme digest of the nit3 wild-type allele that was selected for in all of the ESTs, can be used to localize the gene within the the progeny. We expect nit3 to be in the small region present mapping population. If the map is used to order (locus 2C) for which all of the progeny have the Japanese genomic libraries, then the process of sequencing the genome. G. zeae genome could be greatly simplified. Linkage group IV recombination patterns also are Our map provides markers with known linkage rela- somewhat unusual. The 24 nonrecombinant progeny tionships that can be used for population studies. Such for this linkage group are all of the Kansas type. It is studies are required to understand the current structure possible that the crossover type pattern between 4C and and future changes in the pathogen’s populations and 4P (56 cM) could be due to a heterozygous inversion. their correlation with disease epidemics. AFLPs provide This linkage group also is fixed near one end at locus a well-defined, relatively large set of markers that can 4AD for the Kansas genome. This region is the only one be used to monitor populations on a genome-wide basis. in the map that is 100% Kansas genome, and, therefore, These studies should more accurately reflect the popula- we predict that the nit1 gene maps on this linkage group tion being studied since the bias created when linked in or very near locus 4AD. markers are treated as unlinked can be removed (Bra¨n- Trichothecene gene analyses: We mapped TRI5, and dle et al. 1997). presumably the rest of the trichothecene gene cluster, The seven lineages described by O’Donnell et al. near the middle of linkage group I in G. zeae. TRI5 (2000) appear genetically divergent based on the se- encodes trichodiene synthase, the first step in the tricho- quences of six genes. The fertility between various lin- thecene toxin biosynthetic pathway (Hohn and van eages needs to be quantified to help estimate the risk Middlesworth 1986; Hohn and Beremand 1989; of generating novel parasitic phenotypes when lineages Brown et al. 2001). The locus controlling toxin type are commingled and to determine their degree of ge- (DON or NIV) cosegregated with TRI5. This is the first netic isolation. We estimate that Ͻ1% of the perithecia genetic proof that toxin type is controlled by a single were heterozygous outcrosses when Z-3639 and R5470 locus that is linked to the trichothecene gene cluster. were mated. Up to 35% heterozygous perithecia were We did not give a new name to the toxin-type locus produced in crosses of Z-3639 with two other Kansas G. zeae Genetic Map 1459 strains that belong to lineage VII (Bowden and Leslie torting our perception of the genomic organization of 1999), so there may be lower fertility in crosses between G. zeae. For example: lineages. The map could be used to identify naturally occurring hybrids between lineages and to estimate the i. Are the putative chromosome rearrangements we amount of genetic material within a hybrid strain that observed peculiar to one of the strains in the present originated from the different lineages. cross? On the basis of our AFLP study, the two parental ii. Is the suppression of crossing over a general property ?of of G. zeae outcrosses? Peculiar to interlineage crosses %50ف strains of the mapping population differ at the observed bands. In the G. fujikuroi species complex Specific for one (or both) of the strains used in the (Leslie et al. 2001; Marasas et al. 2001), we would present cross? conclude that these strains were in the same species, We thank Amy Beyer, Ann Clouse, and Amy Hanson for technical but perhaps not the same subspecies. [In the G. fujikuroi assistance. J. E. Jurgenson was supported by a Professional Develop- species complex, strains in the same subspecies always ment Leave grant from the University of Northern Iowa. This work share 65% or more AFLP identity and those in different was supported in part by U.S. Wheat and Barley Scab Initiative project species share Ͻ40% AFLP identity. The only case in 59-0790-9-029 and by the Kansas Agricultural Experiment Station. This is contribution no. 02-67-J from the Kansas Agricultural Experiment which an intermediate value (55%) was observed (K. Station, Manhattan, KS. 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 LITERATURE CITED species need to be examined before concluding that Anderson, W., J. Arnold, D. Baldwin, A. Beckenbach, C. Brown et the criteria applicable to G. fujikuroi also are applicable al., 1991 Four decades of inversion polymorphism in Drosophila pseudoobscura. Proc. Natl. Acad. Sci. USA 88: 10367–10371. to G. zeae. Bowden, R. L., and J. F. Leslie, 1992 Nitrate-nonutilizing mutants The finding of at least two putative chromosome re- of Gibberella zeae (Fusarium graminearum) and their use in de- arrangements in our cross also is suggestive of significant termining vegetative compatibility. Exp. Mycol. 16: 308–315. Bowden, R. L., and J. F. Leslie, 1999 Sexual recombination in genetic differentiation. Heterozygous inversions are post- Gibberella zeae. Phytopathology 89: 182–188. zygotic fertility blocks that reduce fertility and progeny Bra¨ndle, U. E., U. A. Hammerli, J. M. McDermott and M. S. Wolfe, variability. These differences are certainly important in 1997 Interpreting population genetic data with the help of ge- netic linkage maps, pp. 157–171 in The Gene-for-Gene Relationship Drosophila speciation (Anderson et al. 1991), but prob- in Plant-Parasite Interactions, edited by I. R. Crute,E.B.Holub ably not in Neurospora speciation (Perkins 1997). and J. J. Burdon. CAB International Press, Wallingford, UK. .of the Brasier, C., 2000 The rise of hybrid fungi. Nature 405: 134–135 %90ف ,Within the G. fujikuroi species complex Brown, D. W., S. P. McCormick, N. J. Alexander, R. H. Proctor and RFLP markers tested remained on the same chromo- A. E. Desjardins, 2001 A genetic and biochemical approach some across six biological species (Xu et al. 1995). These to study trichothecene diversity in Fusarium sporotrichioides and results indicate that translocations and transpositions Fusarium graminearum. Fungal Genet. Biol. 32: 121–133. Carter, J. P., H. N. Rezanoor, A. E. Desjardins and P. Nicholson, are not widespread, but provide no information on in- 2000 Variation in Fusarium graminearum isolates from Nepal as- versions, as an inversion would not alter the chromo- sociated with their host of origin. Plant Pathol. 49: 452–460. some to which a marker hybridizes. To the degree that Cato, S. A., R. C. Gardner, J. Kent and T. E. Richardson, 2001 A rapid PCR-based method for genetically mapping ESTs. Theor. chromosome rearrangements alter gene position, they Appl. Genet. 102: 296–306. might alter expression due to specific position effects. Chen, L.-F., G.-H. Bai and A. E. Desjardins, 2000 Recent advances We have no evidence for this type of effect in Fusarium, in wheat head scab research in China. National Agricultural Li- brary (http://www.nal.usda.gov/pgdic/WHS/whsindex.html). although rearrangements that affected toxin gene clus- Correll, J. C., C. J. R. Klittich and J. F. Leslie, 1987 Nitrate ters or pathogenicity gene clusters might show such nonutilizing mutants of Fusarium oxysporum and their use in vege- effects. Limitations to recombination also could be an tative compatibility tests. Phytopathology 77: 1640–1646. Gallegos, A., D. J. Jacobson, N. B. Raju, M. P. Skupski and D. O. effective means to lock particular allele combinations Natvig, 2000 Suppressed recombination and a pairing anom- into gene complexes that might have selective value and aly on the mating-type chromosome of Neurospora tetrasperma. could not easily be broken apart, e.g., the spore killer Genetics 154: 623–633. Gonzalez, M., M. E. Z. Rodriguez, J. L. Jacabo, F. Hernandez, complex in Neurospora (Raju 1994). J. Acosta et al., 1998 Characterization of Mexican isolates of To test some of these hypotheses, follow-up studies Colletotrichum lindemuthianum by using differential cultivars and will be needed. Cytological studies have identified no molecular markers. Phytopathology 88: 292–299. Grell, R. F., 1962 A new model for secondary nondisjunction: the more than four chromosomes (Howson et al. 1963), role of distributive pairing. Genetics 47: 1737–1754. but studies with pulse-field chromosome separation are Haber, J. E., 1998 Searching for a partner. Science 279: 823–824. likely to be more successful, given the relatively small Hawley, R. S., K. S. McKim and T. Arbel, 1993 Meiotic segregation in Drosophila melanogaster females: molecules, mechanisms, and size of Fusarium chromosomes. Additional maps are myths. Annu. Rev. Genet. 27: 281–317. needed to confirm linkage group configurations and to Hohn, T. M., and M. N. Beremand, 1989 Isolation and nucleotide map other economically important traits, e.g., pathoge- sequence of a sequiterpene cyclase gene from the trichothecene- producing fungus Fusarium sporotrichioides. Gene 79: 131–138. nicity, host range, or toxin production, and to deter- Hohn, T. M., and F. van Middlesworth, 1986 Purification and mine if the segregation distortion we observed is dis- characterization of the sesquiterpene cyclase trichodience syn- 1460 J. E. Jurgenson et al.

thase from Fusarium sporotrichioides. Arch. Biochem. Biophys. 251: Raju, N. B., 1994 Ascomycete spore killers: chromosomal elements 756–761. that distort genetic ratios among the products of meiosis. Myco- Howson, W. T., R. C. McGinnis and W. L. Gordon, 1963 Cytologi- logia 86: 461–473. cal studies on the perfect stages of some species of Fusarium. Can. Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Clon- J. Genet. Cytol. 5: 60–64. ing: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Hua, J., L. L. Domier, S. Xuejen and F. L. Kolb, 2000 Combined Press, Cold Spring Harbor, NY. AFLP and RFLP mapping in two hexaploid oat recombinant Scott, P. M. (Editor), 1995 AOAC official method 986.18. Deoxyni- inbred populations. Genome 43: 94–101. valenol in wheat. Gas chromatographic method, pp. 49–36–49.37 Kere´nyi, Z., K. Zeller, L. Hornok and J. F. Leslie, 1999 Molecular in Official Methods of the Association of Analytical Chemists, Vol. II, standardization of mating type terminology in the Gibberella fuji- Ed. 16. AOAC International, Gaithersburg, MD. kuroi species complex. Appl. Environ. Microbiol. 65: 4071–4076. Seitz, L. M., W. D. Eustace, H. E. Mohr, M. D. Shogren and W. T. Klittich, C. J. R., and J. F. Leslie, 1988 Nitrate reduction mutants of Yamazaki, 1986 Cleaning, milling, and baking tests with hard Fusarium moniliforme (Gibberella fujikuroi). Genetics 118: 417–423. red winter wheat containing deoxynivalenol. Cereal Chem. 63: Lander, E. S., P. Green, J. Abrahamson, A. Barlow, M. J. Daly 146–150. et al., 1987 Mapmaker: an interactive computer package for Tanaka, T., A. Hasegawa, S. Yamamota, U.-S. Lee, Y. Sugiura et constructing primary genetic linkage maps of experimental and al., 1988 World-wide contamination of cereals by Fusarium my- natural populations. Genomics 1: 174–181. cotoxins nivalenol, deoxynivalenol, and zearalenone. 1. Survey Lee, T., D. W. Oh, H.-S. Kim, J. Lee, Y.-H. Kim et al., 2001 Identifica- of 19 countries. J. Agric. Food Chem. 36: 979–983. tion of deoxynivalenol- and nivalenol-producing chemotypes of van der Lee,T.,A.Robold,A.Testa,J.W.van ‘t Klooster and Gibberella zeae by using PCR. Appl. Environ. Microbiol. 67: 2966– F. Govers, 2001 Mapping of avirulence genes in Phytophthora 2972. infestans with amplified fragment length polymorphisms selected Leslie, J. F., F. J. Doe, R. D. Plattner, D. D. Shackelford and J. Jonz, by bulk segregant analysis. Genetics 157: 949–956. 1992 Fumonisin B1 production and vegetative compatibility of Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee et al., strains from Gibberella fujikuroi mating population “A” (Fusarium 1995 AFLP: a new technique for DNA fingerprinting. Nucleic moniliforme). Mycopathologia 117: 37–45. Acids Res. 23: 4407–4414. Leslie, J. F., K. A. Zeller and B. A. Summerell, 2001 Icebergs and Vuylsteke, M., R. Mank, R. Antonise, E. Bastiaans, M. L. Senior speciation in species of Fusarium. Physiol. Mol. Plant Pathol. 59: et al., 1999 Two high-density AFLP linkage maps of mays L.: 107–117. analysis of distribution of AFLP markers. Theor. Appl. Genet. Manly, K. F., and J. M. Olson, 1999 Overview of QTL mapping software and introduction to Map Manager QT. Mamm. Genome 99: 921–935. 10: 327–334. Walker, S. L., S. Leath, W. M. Hagler, Jr. and J. P. Murphy, 2001 Marasas, W. F. O., P. E. Nelson and T. A. Toussoun, 1984 Toxigenic Variation among isolates of Fusarium graminearum associated with Fusarium Species: Identity and Mycotoxicology. The Pennsylvania State Fusarium head blight in North Carolina. Plant Dis. 85: 404–410. University Press, University Park, PA. Weir, B. S., 1990 Genetic Data Analysis. Sinauer Associates, Sunder- Marasas, W. F. O., J. P. Rheeder, S. C. Lamprecht, K. A. Zeller land, MA. and J. F. Leslie, 2001 Fusarium andiyazi sp.nov., a new species Windels, C. E., 2000 Economic and social impacts of Fusarium head from sorghum. Mycologia 93: 1203–1210. blight: changing farms and rural communities in the Northern McMullen, M. P., R. Jones and D. Gallenberg, 1997 Scab of wheat Great Plains. Phytopathology 90: 17–21. and barley: a re-emerging disease of devastating impact. Plant Xu, J.-R., and J. F. Leslie, 1996 A genetic map of Gibberella fujikuroi Dis. 81: 1340–1348. mating population A (Fusarium moniliforme). Genetics 143: 175– Miao, V. P., S. F. Covert and H. D. Van Etten, 1991 A fungal gene 189. for antibiotic resistance on a dispensable (“B”) chromosome. Xu, J.-R., K. Yan, M. B. Dickman and J. F. Leslie, 1995 Electropho- Science 254: 1773–1776. retic karyotypes distinguish the biological species of Gibberella Murray, M. G., and W. F. Thompson, 1980 Rapid isolation of high fujikuroi (Fusarium section Liseola). Mol. Plant-Microbe Interact. molecular weight plant DNA. Nucleic Acids Res. 8: 4321–4325. 8: 74–84. Nelson, P. E., T. A. Tousson and W. F. O. Marasas, 1983 Fusarium Yin, X., P. Stam, C. J. Dourleijn and M. J. Kropff, 1999 AFLP Species: An Illustrated Manual for Identification. The Pennsylvania mapping of quantitative trait loci for yield-determining physiolog- State University Press, University Park, PA. ical characters in spring barley. Theor. Appl. Genet. 99: 244–253. O’Donnell, K., H. C. Kistler, B. K. Tacke and H. H. Casper, 2000 Young, W. P., J. M. Schupp and P. Keim, 1999 DNA methylation Gene genealogies reveal global phylogeographic structure and and AFLP marker distribution in the soybean genome. Theor. reproductive isolation among lineages of Fusarium graminearum, Appl. Genet. 99: 785–790. the fungus causing wheat scab. Proc. Natl. Acad. Sci. USA 97: Yun, S. H., T. Arie, I. Kaneko, O. C. Yoder and B. G. Turgeon, 7905–7910. 2000 Molecular organization of mating type loci in heterothal- Perkins, D. D., 1997 Chromosomal rearrangements in Neurospora lic, homothallic, and asexual Gibberella/Fusarium species. Fungal and other filamentous fungi. Adv. Genet. 36: 239–398. Genet. Biol. 31: 7–20. Purwantara, A., J. M. Barrins, A. J. Cozijnsen, P. K. Ades and B. J. Zeller, K. A., J. E. Jurgenson, E. M. El-Assiuty and J. F. Leslie, Howlett, 2000 Genetic diversity of the Leptosphaeria maculans 2000 Isozyme and amplified fragment length polymorphisms species complex from Australia, Europe and North America using from Cephalosporium maydis in Egypt. Phytoparasitica 28: 121–130. amplified fragment length polymorphism analysis. Mycol. Res. 104: 772–781. Communicating editor: R. H. Davis