Abstract
Gibberella moniliformis (Fusarium verticillioides) is primarily a pathogen of maize, but it can also cause disease in other crop species. This pathogenicity, as well as the contamination of food- and feedstuffs with the fumonisin mycotoxins, results in economically significant losses to both farmers and food processors. The dissection of important biological characters in this fungus has been hampered by the lack of a uniformly dense genetic map. The existing restriction fragment length polymorphism-based map contains significant gaps, making it difficult to routinely locate biologically important genes, such as those involved in pathogenicity or mycotoxin production, with precision. We utilized amplified fragment length polymorphisms (AFLPs) to saturate the existing genetic map and added 486 AFLP markers to the ∼150 markers on the existing map. The resulting map has an average marker interval of 3.9 map units and averages ∼21 kb/map unit. The additional markers expanded the map from 1,452 to 2,188 map units distributed across 12 chromosomes. The maximum distance between adjacent markers is 29 map units. We identified AFLP markers less than 1 map unit from the mating type (MAT) locus and 2.5 map units from the spore killer (SK) locus; eight AFLP markers map within 8.5 units of the FUM1 (fumonisin biosynthetic) locus. The increased saturation of this map will facilitate further development of G. moniliformis as a model system for the genetic and population genetic studies of related, but less genetically tractable, plant pathogenic fungi.
Gibberella moniliformis Wineland [anamorph Fusarium verticillioides (Sacc.) Nirenberg] is genetically the most intensively studied species in Fusarium section Liseola. A number of morphological and physiological mutants have been characterized in addition to anonymous restriction fragment length polymorphism (RFLP) markers (3, 23, 25, 37, 38, 47). Members of the genus Fusarium are economically important plant pathogens that cause billions of dollars of damage each year worldwide. G. moniliformis is an economically important pathogen of maize (18, 45) and sorghum (8).
The most prominent toxins produced by G. moniliformis are the fumonisins (10, 29). These toxins have been well studied both in terms of their synthesis (1) and with regard to their effects on animals that consume contaminated grain. Consumption of grain contaminated with fumonisin mycotoxins is correlated with esophageal cancer in humans (40), with several potential carcinogenicity mechanisms proposed (9, 11, 16, 41). Fumonisins also can cause leukoencephalomalacia in horses (21, 30, 42), pulmonary edema in swine (13, 14), kidney and liver damage in nonhuman primates (12), and kidney and liver cancer in rats (9, 15, 16). Thus, G. moniliformis is an economically important pathogen that causes significant losses for both maize and sorghum producers and contaminates food- and feedstuffs for animal and human consumption.
In an earlier version of this map (46, 47), a gene, FUM1, that affects fumonisin biosynthesis was included. Since then three more closely linked loci have been identified genetically, FUM2-4 (5, 6, 34), and one of the loci, FUM1 (also termed FUM5), has been cloned and sequenced (36). These genes appear to be part of a cluster, as is common for the genes encoding other secondary metabolite synthetic pathways in fungi (2, 20, 26), although neither the overall size nor the gene content of the FUM cluster has been determined.
Our map is not the first for G. moniliformis. Puhalla and Spieth (37) had a minimal map of nine markers on four linkage groups, which was consistent with the number of chromosomes that had been identified cytologically (17). They also identified four additional markers that could not be associated with any of these four linkage groups. Xu and Leslie (46, 47) associated 13 genetic linkage groups with 12 chromosome-sized DNA fragments resolved by pulsed-field (CHEF) gel electrophoresis (48); two linkage groups were associated with chromosome 11 by Southern hybridization of RFLP probes to CHEF gel blots (46-48). Linkage group I of Puhalla and Spieth (37) was the same as chromosome 5 of Xu and Leslie (46), but no other correlations could be made between the two maps. The Xu and Leslie (46, 47) map contained 150 biochemical, molecular, and morphological markers that were scored in a 121-member mapping population. Although this map is one of the best for filamentous fungal plant pathogens, it is unsaturated, contains several linkage gaps of more than 25 centimorgans (cM), and has one more linkage group than it does chromosomes. Some of the most interesting genes on the map, e.g., the FUM cluster, SK, and MAT, are relatively distant from at least one if not both of the known flanking markers, and additional markers are needed to facilitate the cloning and identification of these genes. We used the readily available Xu and Leslie (46, 47) mapping population to maintain congruence with previous mapping efforts.
Amplified fragment length polymorphism (AFLP) analysis (44) allows reliable detection of numerous DNA polymorphisms without prior cloning of restriction fragments. Analysis of total genomes with this method results primarily in polymorphisms scored as dominant presence or absence markers on denaturing polyacrylamide gels. Since the vegetative stage of the G. moniliformis life cycle is haploid, difficulties in distinguishing heterozygotes from homozygotes for the band allele do not occur. We have previously used AFLPs to successfully generate a saturated genetic map of the related fungus Gibberella zeae (19).
Our objective in this study was to further saturate the genetic map of G. moniliformis. We used AFLP markers to increase the number of loci on the map and to increase the general level of map saturation. We also used bulk segregant analysis to identify additional AFLP markers in the regions flanking FUM1. By filling in the unmapped linkage gaps and resolving linkage ambiguities that exist in the Xu and Leslie (46) map, we hope to facilitate other genetic studies of G. moniliformis, the cloning of additional genes from this fungus, to identify markers that can be used for genetic analyses of field strains, and to enable evolutionary comparisons of chromosome synteny and genome organization between various Gibberella species (or their Fusarium anamorphs).
MATERIALS AND METHODS
Analysis of DNA polymorphisms in the mapping population.
We analyzed the progeny from the cross described by Xu and Leslie (46, 47). Both the parents (C = FGSC 7607 = KSU A-0015; N = FGSC 8078 = KSU A-4643) and the progeny of this cross (FGSC 7950-8070 = KSU A-6360-A-6480) are available from the Fungal Genetics Stock Center (Department of Microbiology, University of Kansas Medical Center, Kansas City, Kans. [http://www.fgsc.net]). Approximately 107 microconidia in 1 ml of 2.5% Tween 60 (Sigma, St. Louis, Mo.) were used to inoculate 50 ml of liquid complete media (4) in 125-ml Erlenmeyer flasks. Cultures were grown on a gyratory shaker (150 rpm) at room temperature (23 to 25°C) for 2 days. Tissue from each culture was collected by filtration through a nongauze milk filter (Ken Ag Milk Filter, Ashland, Ohio), washed with 100 ml of sterile water, and blotted dry with paper towels. The tissue was frozen at −20°C until used for DNA extraction.
DNA extraction.
DNA was isolated with a cetyltrimethyl ammonium bromide procedure (22) modified from that of Murray and Thompson (33). Final DNA concentrations (in TE buffer [10 mM TRIS·HCl, 1 mM EDTA, pH 8.0]) were estimated by comparison of DNA fluorescence of diluted aliquots of each DNA sample against that of HindIII-digested bacteriophage λ DNA with an IS 1000 version 2.0 digital imaging system (Alpha Inotech Corp., San Leandro, Calif.). Samples and sample dilutions were run in 1% agarose gels containing 1× TAE (40 mM Tris acetate, 1 mM EDTA [pH 8.0]) and 0.5 μg of ethidium bromide/ml. DNA yields varied between 200 and 400 μg of DNA per culture. The concentration of each DNA sample was adjusted to 20 μg/ml for use in AFLP analysis.
AFLP analysis.
AFLPs were generated with the protocol of Vos et al. (44) as modified by Zeller et al. (49). Two-base-addition, selective AFLP primers were synthesized by Integrated DNA Technologies Corp. (Coralville, Iowa). The EcoRI primers were 5′ end labeled with [γ-33P]ATP (NEN Life Sciences, Boston, Mass.). Dried gels were exposed to X-ray film (Classic Blue Sensitive; Molecular Technologies, St. Louis, Mo.) for 2 to 5 days at room temperature to identify DNA bands. Polymorphic bands were scored by hand. The Low Mass DNA ladder (Life Technologies Inc., Bethesda, Md.) was 5′ end labeled with 33P and used to estimate molecular weights of AFLP fragments. Most polymorphisms were characterized as presence or absence of bands, although a few occurred in which the polymorphism appeared as an apparent difference in molecular weight. From 5 to 24 polymorphisms were observed per primer pair. Polymorphic bands were named following the form EXXMXX0000Y, where EXX and MXX designate the selective bases (X) used with base EcoRI and MseI primers, respectively, followed by a four-digit number giving the size of the band in base pairs, and ending in a letter code, either C or N, that identifies the parental origin of either the amplified polymorphic band (presence or absence) or the larger band of a size difference polymorphism. For example, ECCMGT0361C is a marker of 361 bp generated by amplification with the primer pair ECC and MGT in which the band generated resulted from amplification of a sequence in the California parental DNA.
Reliability and repeatability of AFLP polymorphisms.
The reliability of the AFLP polymorphism analysis was tested against the genomes of the parental strains of the cross. Separate cultures of each parent were grown at different times, and DNA was isolated independently. Twenty-four primer pairs were used to amplify AFLP fragments from these cultures. Differences in banding patterns between different cultures of the same strain made up less than 1% of the polymorphisms observed. Those differences that did occur were all for bands of <80 bp in length. Therefore, we used no bands of <80 bp in length in our analysis.
Marker analysis.
Genetic mapping of all characters was performed with Map Manager QTX11 (http://mapmgr.roswellpark.org/mmQTX) on a Macintosh G4 Power PC computer (28). Banding pattern data from gels were compiled as text files and imported into the linkage map analysis software. Map Manager was then used to distribute the data into linkage groups. Program settings used for the analysis presented are the following: 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 “true” linkage groups. The data were treated by the mapping program as a backcross with codominant markers, paternal parent unique, for accurate analysis of this haploid genome.
The data from the map of Xu and Leslie (46, 47) were imported into Map Manager from Map Maker (24) and used to reconstruct the previous linkage map. Marker data from the AFLP analysis was then imported into the program and combined with the previous data. The distribute function was used to assign map positions to the AFLP markers. 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 and probably result from gene conversion or from errors in scoring or bookkeeping. This process also allowed us to identify and correct misscored polymorphisms. We converted unknown data to their probable phenotypes based on their flanking markers because the Map Maker V2.0 for the Macintosh program (24), which we used to draw the maps (see Fig. 1), treats unscored markers as a third allele and places a crossover on each side of these markers.
FIG. 1.
Linkage map of G. moniliformis. Loci that are represented by more than one marker are indicated in the form “XY,” where X is the number of the chromosome and Y is a letter assigned in order along the chromosome. Chromosome 1: 1A, ETCMAT0347N, ETCMCC0393N; 1B, ECCMTA0702C, EAAMAG0714C; FUM1, FUM1, ETCMGA0162C; 1C, EAAMAG0344N, EAAMAT0697N, ETCMGA0138N, EATMCT0188N; 1D, EATMCC0333N; 1F, 6E28, 5E49; 1G, 6E51, 6E81, ECCMTA0369C; 1H, ECCMTA0612C, ECCMTA0561N; 1I, ETTMAG0593N, ETTMAC0649N. Chromosome 2: 2A, ETTMAT0508C, EAAMAT0180C; 2B, EATMAC0225C, ETAMAG0298C. Chromosome 3: 3A, EAAMAA0174N, ETCMGA0562N; 3B, ETCMAG0387C, EACMAC0184C; 3C, 11E, 11P26, 12P38S; 3D, EAAMAC0207C, EAAMAC0519C; 3E, ETTMAT0199C, ETCMGA0203C; 3F, EAAMAC0496C, ECCMGC0311N. Chromosome 4: 4A, ETTMAC0114N, EACMGA0529N; 4B, ETCMAA0121N, EAAMAC0366C; 4C, ATMCC0229C, ECCMTA0193C; 4D, 75E, 5E14; 4E, EACMAG0291N, EATMCC0185N, ETTMAA1007N. Chromosome 5: 5A, 5E23, EATMAA0261N; 5B, ECCMTG0833C, EAAMAA0183N; 5C, P7, 5E34. Chromosome 6: 6A, ETCMCA0113N, ETTMAT0167N, EAAMAA0747N; 6B, ECCMGC0408N, EAAMAG0783N, ETCMCC0547N; 6C, ETCMAC0390N, ECCMTG1200N; 6D, EATMAG0087C, EACMAT0150C; 6E, EATMAG0307N, ETCMAG0150N. Chromosome 7: 7A, ETCMAC0151N, EAAMAT0261N; 7B, 6E7, 7E26, 7E37; 7C, ETTMAG1200N, ECCMTT0486N; 7D, ETCMAG0368N, EATMCC0319N, EATMCC0099N, ETCMAA0121N; 7F, 7E77, 7E51. Chromosome 8: 8A, P37, EAAMAA0597N; 8B, ETCMCC0297C, ECCMTA0188C, ETCMAA0336C; 8C, 22E, 6E10; 8D, 6E46, 11P45. Chromosome 9: 9A, EAAMAA0562N, ETTMAT0287N; 9B, ETCMAC0853C, ETTMAT0287N; 9C, 37E, 6E62, 14E; 9D, P39, ETCMCG0278C. Chromosome 10: 10A, ETGMCG0362N, ETAMCT0780N; 10B, ETCMAA0590N, ETCMCA0598C; 10C, P13, P15; 10D, ETTMAA0398C, ETTMAA0439N, EATMAA0356N; 10F, ETAMCT0556C, ECCMTT0094C; 10G, P25, 10P25S; 10H, 12P18, 6E79; 10I, 12P28, 6E37. Chromosome 11: 11A, ECCMTG0653N, EAAMAC0434C; 11B, ECCMTA0731N, ECCMTT0158C; 11C, ETCMCC0682C, ETTMAT0429C; 11D, EATMAG0164C, EATMAA0153C. Chromosome 12: 12A, EAAMACO941C, EATMCC0189C, ETCMAT0453C; 12B, ETGMCG0554N, ETCMGA0174N, EAAMCG0291N; 12C, EACMAT0515C, EATMAG0815C; 12D, ETCMAA1087C, ECCMGC0648C; 12F, 11P43, 5E32.
Bulk segregant analysis.
One of our objectives in refining the genetic map of G. moniliformis was to more precisely locate the FUM1 locus on chromosome 1. After addition of ∼420 AFLP markers to the G. moniliformis genetic map, large linkage gaps still flanked FUM1. We identified AFLP markers closer to FUM1 with a bulk segregant analysis protocol (32).
For bulk segregant analysis we made two mixed AFLP templates. Each template consisted of mixtures of equal volumes (5 μl) of the preamplification templates of 10 progeny. In one bulk template, the progeny were predicted to contain only the California genome in a 10-cM region on either side of FUM1. The C template pool contained preamplification templates of progeny: FGSC 7951, 7966, 7971, 7976, 7977, 7980, 7991, 8011, 8047, and 8056. The N template pool, in which the progeny were predicted to contain sequences from the Nepalese parent in the FUM1 region, contained preamplification templates of progeny: FGSC 7959, 7962, 7981, 7983, 7999, 8010, 8012, 8036, 8045, and 8053. In both bulk templates, the flanking regions, based on mapping data, of the pooled DNA samples were heterogeneous for the remainder of the parental genome. These two pools and preamplification templates from each parent were used for AFLP reactions. If an AFLP band was polymorphic and located near FUM1, then the band would appear in one pool amplification (either C or N) and the corresponding parental amplification. Polymorphic bands not near FUM1 would appear in both pool amplifications and one of the two parental amplifications. We tested 18 additional primer pair combinations on these four AFLP templates and identified polymorphic bands in five: ECCMTT, ECCMGA, ECCMGC, ECCMTA, and ECCMTG. All detectable polymorphisms resulting from amplification with any of these primer pairs were scored and added to the map.
RESULTS
We identified and scored 486 AFLP polymorphisms from 37 primer pairs (Table 1). The number of polymorphic markers per primer pair varied from a low of 3 for EAAMGA to a high of 23 for primer pair ETTMAT with an average of 13 per primer pair. Of the AFLP bands scored, we found 25 to 30% were polymorphic in this mapping population, which is consistent with observed genetic differences between strain pairs from other species associated with the Gibberella fujikuroi complex (31). The distribution of markers from individual primer pairs appears to be random with respect to chromosome.
TABLE 1.
Distribution of AFLP markers on chromosomes
| Primer paira | No. of markers on chromosome:
|
Total no. of markers | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
| EAAMAA | 2 | 0 | 1 | 0 | 3 | 2 | 0 | 2 | 1 | 0 | 0 | 0 | 11 |
| EAAMAC | 2 | 1 | 4 | 3 | 0 | 2 | 0 | 2 | 0 | 1 | 1 | 1 | 17 |
| EAAMAG | 5 | 0 | 3 | 1 | 0 | 1 | 0 | 3 | 1 | 2 | 0 | 0 | 16 |
| EAAMAT | 2 | 1 | 1 | 2 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 8 |
| EAAMCG | 2 | 0 | 1 | 0 | 1 | 2 | 0 | 0 | 0 | 0 | 1 | 1 | 8 |
| EAAMGA | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 3 |
| EACMAC | 0 | 0 | 3 | 2 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 8 |
| EACMAG | 2 | 0 | 1 | 4 | 2 | 3 | 0 | 1 | 1 | 2 | 1 | 1 | 18 |
| EACMAT | 0 | 0 | 1 | 1 | 0 | 4 | 2 | 0 | 1 | 1 | 0 | 1 | 11 |
| EACMCT | 0 | 0 | 0 | 0 | 0 | 2 | 1 | 1 | 1 | 2 | 0 | 0 | 7 |
| EACMGA | 1 | 2 | 1 | 1 | 0 | 0 | 2 | 0 | 0 | 1 | 1 | 0 | 9 |
| EATMAA | 2 | 1 | 0 | 1 | 1 | 0 | 1 | 4 | 2 | 1 | 4 | 0 | 17 |
| EATMAC | 1 | 1 | 2 | 1 | 0 | 3 | 0 | 1 | 1 | 3 | 0 | 0 | 13 |
| EATMAG | 0 | 0 | 1 | 2 | 0 | 4 | 0 | 0 | 0 | 0 | 1 | 3 | 11 |
| EATMCC | 3 | 1 | 2 | 2 | 0 | 0 | 3 | 0 | 1 | 0 | 2 | 1 | 15 |
| EATMCT | 3 | 2 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 7 |
| ECCMGA | 3 | 2 | 0 | 0 | 1 | 0 | 4 | 0 | 1 | 0 | 0 | 0 | 11 |
| ECCMGC | 1 | 1 | 1 | 0 | 3 | 5 | 0 | 0 | 1 | 2 | 0 | 1 | 15 |
| ECCMTA | 4 | 3 | 0 | 1 | 1 | 0 | 0 | 5 | 0 | 0 | 1 | 0 | 15 |
| ECCMTG | 0 | 0 | 2 | 1 | 1 | 2 | 0 | 0 | 1 | 1 | 2 | 0 | 10 |
| ECCMTT | 2 | 0 | 1 | 0 | 1 | 1 | 3 | 1 | 2 | 5 | 1 | 0 | 17 |
| ETAMAG | 1 | 2 | 0 | 2 | 0 | 0 | 1 | 2 | 1 | 0 | 1 | 0 | 10 |
| ETAMCT | 2 | 2 | 0 | 0 | 1 | 0 | 2 | 2 | 0 | 4 | 0 | 0 | 13 |
| ETCMAA | 0 | 1 | 1 | 2 | 2 | 1 | 3 | 2 | 3 | 1 | 3 | 1 | 20 |
| ETCMAC | 2 | 0 | 1 | 1 | 2 | 2 | 2 | 0 | 3 | 0 | 1 | 0 | 14 |
| ETCMAG | 0 | 3 | 3 | 4 | 2 | 4 | 2 | 0 | 1 | 0 | 0 | 0 | 19 |
| ETCMAT | 1 | 1 | 0 | 2 | 1 | 1 | 0 | 0 | 1 | 0 | 2 | 1 | 10 |
| ETCMCA | 2 | 3 | 2 | 2 | 3 | 2 | 3 | 3 | 0 | 1 | 0 | 1 | 22 |
| ETCMCC | 1 | 2 | 1 | 0 | 0 | 2 | 0 | 1 | 1 | 2 | 1 | 0 | 11 |
| ETCMCG | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 6 |
| ETCMGA | 2 | 1 | 3 | 2 | 0 | 0 | 1 | 0 | 3 | 1 | 0 | 1 | 14 |
| ETGMCA | 1 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 6 |
| ETGMCG | 2 | 1 | 1 | 0 | 0 | 2 | 1 | 2 | 1 | 2 | 2 | 1 | 15 |
| ETTMAA | 1 | 1 | 0 | 5 | 1 | 0 | 0 | 1 | 2 | 5 | 4 | 0 | 20 |
| ETTMAC | 3 | 0 | 3 | 1 | 1 | 2 | 1 | 1 | 1 | 0 | 0 | 1 | 14 |
| ETTMAG | 3 | 3 | 0 | 1 | 6 | 2 | 1 | 1 | 1 | 2 | 1 | 1 | 22 |
| ETTMAT | 2 | 3 | 1 | 3 | 0 | 3 | 2 | 0 | 4 | 3 | 2 | 0 | 23 |
| Total | 60 | 38 | 42 | 48 | 36 | 55 | 38 | 35 | 39 | 45 | 34 | 16 | 486 |
AFLP primer pairs use the foundation sequence of Vos et al (44) (E-EcoRI or M-MseI). Two bases were added to each foundation sequence to make the AFLP selective primers.
We added the AFLP markers to the linkage map of Xu and Leslie (46, 47) and modified several features of their map (Fig. 1; Table 2). In general, the order and relative genetic distances of the linkage map of Xu and Leslie (46, 47) are unchanged. As before, segregation differs significantly from 1:1 only in the region near SK on chromosome 5, in a small region near 73E and 12P27 on chromosome 6, and in another small region on chromosome 7 that includes 6E7, 7E26, 7E37, and 6E18. Several large recombination gaps in the map have been filled, and all of the previously described ambiguities have been resolved.
TABLE 2.
Comparison of the linkage map of Xu and Leslie (46) with the updated map from this study
| Chromosome | Physical chromosome size (Mb)a | Xu and Leslie (46) map
|
This study
|
No. of chromosomes with frequency of crossovers/chromosome of:
|
Mean crossover frequency | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. of markers | cM | No. of markers | No. of loci | cM | 0Cb | 0Nc | 1 | 2 | 3 | 4 | ≥5 | |||
| 1 | 10 | 15 | 173 | 77 | 65 | 241 | 2 | 5 | 32 | 32 | 27 | 10 | 13 | 2.5 |
| 2 | 6.5 | 15 | 196 | 53 | 51 | 189 | 3 | 7 | 39 | 38 | 14 | 14 | 6 | 2.1 |
| 3 | 4.9 | 15 | 90 | 58 | 51 | 190 | 10 | 10 | 44 | 22 | 13 | 10 | 12 | 2.0 |
| 4 | 4.1 | 14 | 120 | 62 | 56 | 214 | 10 | 5 | 27 | 43 | 18 | 8 | 10 | 2.2 |
| 5 | 4.0 | 11 | 110 | 48 | 45 | 189 | 17 | 0 | 20 | 35 | 28 | 11 | 10 | 2.1 |
| 6 | 3.6 | 13 | 146 | 68 | 61 | 195 | 8 | 12 | 40 | 30 | 11 | 9 | 11 | 1.9 |
| 7 | 3.0 | 12 | 113 | 50 | 42 | 169 | 12 | 8 | 38 | 31 | 20 | 7 | 5 | 1.8 |
| 8 | 2.6 | 14 | 134 | 49 | 44 | 184 | 7 | 8 | 35 | 38 | 21 | 7 | 5 | 1.7 |
| 9 | 2.5 | 10 | 132 | 49 | 44 | 173 | 12 | 9 | 41 | 28 | 18 | 3 | 10 | 1.8 |
| 10 | 2.2 | 14 | 137 | 60 | 51 | 216 | 9 | 0 | 39 | 31 | 15 | 17 | 10 | 2.0 |
| 11 | 2.0 | 6 | 86 | 42 | 38 | 150 | 10 | 9 | 50 | 33 | 11 | 4 | 4 | 1.6 |
| 12 | 0.7 | 4 | 15 | 20 | 13 | 78 | 36 | 33 | 27 | 5 | 12 | 3 | 1 | 0.8 |
| Total | 46.1 | 143 | 1,452 | 636 | 568 | 2,188 | 139 | 109 | 441 | 357 | 208 | 101 | 96 | 1.9 |
From Xu et al. (48).
No observed crossovers. Identical to California parent.
No observed crossovers. Identical to Nepalese parent.
On chromosome 1, the previously large recombination distances flanking FUM1 have been reduced to less than 20 cM on both sides. Eight AFLP markers map within 7 cM of FUM1 in a 43-cM region that formerly contained no genetic marker other than FUM1. RFLP markers 11P25 and 15P44, previously known to be genetically linked to each other and to hybridize to chromosome 1, are distal to the RFLP marker 6E51, as predicted (46).
On chromosome 3, the addition of 43 AFLP markers enabled the mapping of Xu and Leslie's RFLP marker 11P28, which was reported to hybridize to this chromosome but which had not been localized on the map. In addition, the cluster of markers from 5E60 to STE1 is now inverted relative to its previous order to minimize the total length of this linkage group.
Chromosome 5, as presented, has 37 additional markers plus the 11 previously mapped. One RFLP marker, 5E56, known to hybridize to this chromosome was placed on the map in addition to 36 AFLP markers.
On linkage group 10, we mapped 45 AFLP markers and one additional RFLP marker, 86E, that had not previously been localized.
The map of chromosome 11 as described by Xu and Leslie (46, 47) contained two linkage groups. Both linkage groups were assigned to chromosome 11 because the RFLP probes all hybridized to the same band on CHEF gels. We added 34 AFLP markers to this chromosome. In so doing we fused these two linkage groups and mapped RFLP markers 7E58 and 10P16, which hybridize to this chromosome. The length of chromosome 11 is now estimated to be at least 150 cM (Table 2).
The number of markers on chromosome 12 has increased from 4 to 20, and they now collectively define a linkage group of 78 cM.
We also added additional markers [number of markers added] to chromosomes 2 [38], 4 [48], 6 [55], 7 [38], 8 [35], and 9 [39], without altering the fundamental nature of these chromosomes or the gene order on them (Fig. 1; Table 2).
With the addition of the AFLP markers, the genetic map is now defined by 636 polymorphic markers; however, 56 loci are represented by more than one polymorphic marker (Fig. 1). We named these 56 loci based on their chromosome number followed by an alphabetical character. This name is followed in Fig. 1 by a number in parentheses that indicates the number of markers that map to that location. As many as four markers may map to a single location, e.g., locus 1C. Thus, 568 unique loci are defined by the 636 polymorphisms.
DISCUSSION
General map properties.
We used AFLP analysis to add 486 anonymous genetic markers to the linkage map of G. moniliformis (formerly termed F. moniliforme or G. fujikuroi mating population A; see reference 43 for a discussion of nomenclature for this group of fungi) and localized all of the RFLP polymorphisms previously detected by Xu and Leslie (46). The addition of these markers expanded the map by 736 cM, an increase of approximately 50%, while correspondingly reducing the estimated average ratio of 32 kb/cM to 21 kb/cM. The linkage maps of chromosomes 11 and 12, in particular, are much denser. The average distance between identified markers has been reduced from 10 cM/interval to 3.4 cM/interval. The number of gaps greater than 20 cM in length has been reduced from 24 to 5, with no interval larger than 29 cM. The remaining gaps tend to be on the distal portions of chromosomes. Addition of markers for telomeres, e.g., those of Powell and those of Kistler (35), is needed to determine if the ends of the chromosomes have been adequately saturated with markers. The present map also lacks centromeres, although they too could be added through the analysis of the unordered tetrads produced by this fungus (7, 39).
The average crossover frequency for each chromosome varies from a low of 0.77 for chromosome 12 to a high of 2.5 for chromosome 1 (Table 2) and is similar to that seen in G. zeae (19). The distribution of the number of crossovers per chromosome (Table 2) appears to be random (χ2 test; P = 0.05). The physical size of the chromosome is not strongly correlated with the number of markers (r = 0.72), the number of loci (r = 0.71), or the length of a chromosome in map units (r = 0.68). The recombinational length of the chromosomes is strongly correlated with the number of markers (r = 0.94) and with the number of loci (r = 0.95). The numbers of markers and loci per chromosome also are strongly correlated (r = 0.98).
Progeny with limited recombination.
All of the nonrecombinant chromosomes 5 and 10 are of the California parental type. This result is not surprising for chromosome 5, which carries SK. Selection for the SKK allele during meiosis would also select for the California version of this chromosome, which carries the SKK allele. We do not know why all nine of the nonrecombinant chromosome 10s (Table 2) that were recovered in the progeny were the California version. A few of the loci at one end of this chromosome had segregation ratios that were significantly different from 1:1 (ECCMTT0678N-ECCMTT0141N) and skewed towards the California parental type. Two adjacent loci on chromosome 7 (ETAMAG0875C and ETCMAC0168C) also are significantly distorted (84:37 and 85:36) in favor of the California allele.
The large number of noncrossover chromosomes for chromosome 12 suggests that this chromosome may have difficulty in successfully completing meiosis if recombination is required to ensure that a chromosome has found its proper mate before the reductional division occurs. As noted by Xu and Leslie (46, 47), progeny 34 (FGSC 7983), 118 (FGSC 8067), and 121 (FGSC 8070) appear to have lost this chromosome, and progeny 64 (FGSC 8013) has a duplication/deletion for portions of this chromosome. Analysis of the 20 markers on this chromosome confirmed that all of the alleles present in progeny 34, 118, and 121 were “absence of band,” confirming the deletion of chromosome 12 in these strains. The only portion of chromosome 12 present in progeny 64 is between 12A and 7E19, since all eight loci in this region were represented by the band allele and no other loci appeared as bands for the rest of chromosome 12 in this strain.
Loci with known functions.
Several of the markers we examined have a known function. A 20-kb region (∼1 cM) containing the putative coding sequence of the polyketide synthase in the fumonisin biosynthetic pathway has been cloned and sequenced from G. moniliformis (36). This sequence contains three EcoRI sites, none of which corresponds to any of the markers we mapped in the FUM1 region. The FUM1 locus is somewhat difficult to map, as the toxin production phenotype of some of the progeny is not easy to score unambiguously. However, our map has reduced the proximal linkage gap near FUM1 to 10 cM, between EACMAG0248C and FUM1, and placed seven markers within 7 cM distal to FUM1. With respect to MAT on chromosome 6, the RFLP marker 6E75 is still the closest marker on one side (1.7 cM), but the nearest marker on the other side is now the AFLP marker EATMAG0087C (0.8 cM), with the nearest RFLP marker on that side (73E) more than 20 cM away. We also amplified the intergenic spacer region of the ribosomal DNA coding region but did not detect useful polymorphisms between the parental strains and thus could not map this gene cluster. We also did not place any AFLP markers closer to SK than the existing RFLP markers, although we have expanded the map of chromosome 5.
Gaps in the map.
Some important gaps remain even in this relatively saturated map. One end of chromosome 5 now has eight markers that are in a cluster separated by 26 cM from the next-nearest locus. Other large recombination gaps exist on chromosome 1 (21 cM) and at the ends of chromosomes 7, 8, and 10 (Fig. 1). We estimate that the 10 terminal markers of chromosomes 2, 4, 7, 8, and 10 add ∼120 cM to the genetic map. Elimination of these markers would reduce the average map distance to 3.2 cM/marker. Even with these large gaps it appears that the map is relatively saturated, as 56 loci (10%) are represented by more than one marker. We think that primarily the ends of the chromosomes would be more clearly defined with the addition of more markers.
Map utilization.
AFLP markers are defined by the sequences at the ends of the amplified fragments, and the DNA of these bands can be cloned and sequenced (27). Use of probes derived from these markers could facilitate efforts to clone and identify genes for traits such as toxin production, spore killer, and vegetative incompatibility, which cannot be selected for directly. AFLP markers also can be used to order cosmid or BAC clones into contigs (44). AFLP markers also allow the identification of genetic exchange on a genomic scale following meiosis. This information can be of use in studies of general or localized changes in recombination frequency or in the selection of progeny that are near isogenic with either each other or one of the parents. A dense genetic map will allow readier detection of genome rearrangements, e.g., inversions, and should allow us to compare the genome organization of the numerous species in the G. fujikuroi species complex, to which G. moniliformis belongs.
In conclusion, we expect that this genetic map of G. moniliformis is close to becoming saturated, as 10% of loci are represented more than once. This map will be useful for localizing and cloning genes of interest in this economically important fungus. Of particular interest to us is the isolation of genes involved in the vegetative incompatibility reaction. This multifactorial character has so far eluded isolation without benefit of a dense genetic map.
Acknowledgments
We thank Amy Beyer, Ann Clouse, and Amy Hanson for technical assistance and Robert L. Bowden and Scot H. Hulbert for critically reading the manuscript.
J.E.J. was supported by a Professional Development Leave grant from the University of Northern Iowa. This work was supported in part by the Sorghum and Millet Collaborative Research Support Program (INTSORMIL) AID/DAN-1254-G-00-0021-00 from the U.S. Agency for International Development, USDA-NRI 97-35201-4680, and by the Kansas Agricultural Experiment Station.
Footnotes
Contribution no. 02-128-J from the Kansas Agricultural Experiment Station, Manhattan, Kans.
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