a restriction fragment length polymorphism map and electrophoretic

Copyright 0 1992 by the Genetics Society of America

A Restriction Fragment Length Polymorphism Map and Electrophoretic Karyotype of the Fungal Maize Pathogen Cochliobolus heterostrophus

Tzy-Hwa Tzeng,*.Ty1 Linda K. Lyngholm,* Clark F. Fordtv4 and Charlotte R. Br~nson*~’ Departments of ‘Plant Pathology and tGenetics, Iowa State University, Ames, Iowa 5001 1

Manuscript received April 3, 199 1 Accepted for publication September 12, 199 1

ABSTRACT A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear

genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Toxl , a locus controlling host- specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.

R APID progress in understanding plant disease is dependent on efficient methods for analyzing

plants and their pathogens. In fungi, classical genetic analysis has identified genes controlling pathogenicity to plants (SIDHU 1988) and a few of these genes have been cloned (YODER and TURGEON 1985). The cloning of additional genes should be enhanced by the continuing development of transformation methods for these organisms (LEONG and HOLDEN 1989). Ge- netic maps are also being prepared to permit the cloning of pathogenicity genes by marker-based strategies (MICHELMORE and HULBERT 1987; HULBERT et al . 1988; BUDDE and LEONG 1990; SKINNER, LEUNG and LEONG 1990; B. VALENT, personal communication). This paper describes the development of a genetic map for the maize pathogen, Cochliobolus heterostrophus.

C. heterostrophus (Drechsler) Drechsler is a haploid ascomycete which causes southern corn leaf blight. The anamorphic stage of this fungus is known as Bipolaris maydis (Nisikado and Miyake) Shoemaker [ = Helminthosporium maydis Nisikado and Miyake = Drechslera maydis (Nisikado and Miyake) Subramanian

New York, Buffalo, New York 14260.

Iowa State University, Ames, Iowa 5001 1.

’ Current address: Department of Biological Sciences, State University of

Current address: Department of Food Science and Human Nutrition,

To whom correspondence should be addressed.

Genetics 130: 81-96 (January, 1992)

and Jain] (ALCORN 1983). Interest in causes of pathogenicity in C. heterostrophus was sparked when, in 1970, a previously unknown variant caused a severe epidemic on maize with Texas male-sterile cytoplasm (cms-T) (TATUM 197 1). The variant strains, which are moderately virulent to other maize lines, are now classified as a separate subpopulation, race T. All other known strains are moderately virulent to all maize lines and are classified as race 0.

Biochemical and genetic analyses have shown that the high virulence of race T relative to race 0 is due to a single metabolic difference, race T’s production of a family of linear polyketols collectively known as T-toxin (DALY 1982; TEGTMEIER, DALY and YODER 1982). T-toxin has been shown to specifically inhibit mitochondrial function in cms-T maize (MILLER and KOEPPE 1971; PAYNE et al. 1980; HOLDEN and SZE 1987). To date, genetic analyses of T-toxin synthesis in closely related laboratory strains (LEACH et al. 1982; TEGTMEIER, DALY and YODER 1982) and field isolates collected from various countries (BRONSON, TAGA and YODER 1990) have identified a single naturally variable locus, T o x l , controlling the ability and inability to produce T-toxin. Dominance relationships at Tox l are uncertain (BRONSON 1991); however, based on available evidence, T-toxin production has been proposed to be either a dominant or semidominant trait

82 T.-H. Tzeng et al.

(LEACH, LANG and YODER 1982). Thus, the alleles specifying T-toxin production and the lack of T-toxin production have been named TOXl (dominant) and toxl (recessive), respectively. The Toxl locus has been proposed to be at or near the breakpoint of a reciprocal translocation; it therefore has been suggested that the ability to produce T-toxin may have arisen by a chromosome rearrangement event (BRONSON

Studies of pathogenicity in C. heterostrophus are feasible because of the availability of techniques for its genetic manipulation. Conditions for growing and crossing this fungus in the laboratory have been determined and a set of closely related strains, known as C-strains, have been bred (LEACH, LANC and YODER 1982). A transformation system has been developed (TURCEON, GARBER and YODER 1985, 1987), which has allowed cloning by complementation (YODER et al. 1989b), gene disruption and gene replacement (MUL- LIN, TURCEON and YODER 1990). Methods have also been developed for inducing, selecting and screening mutants and over 50 loci have been defined based on auxotrophy, morphology, pigmentation, mating type, enzyme production and pathogenicity (LEACH, LANC and YODER 1982; TANAKA, KUBO and TSUDA 1988, 1991 ; L. K. LYNGHOLM, P. R. THORSON, C. A. SPIKE and C . R. BRONSON, unpublished results). However, few of these genes have been cloned and attempts to identify linkages have been limited.

The objective of this research was to construct a genetic map of the nuclear genome of C. heterostrophus to aid in studies of pathogenicity. The map was constructed by analyzing segregation patterns of both restriction fragment length polymorphism (RFLP) and phenotypic markers in a single cross. Intact chromosomal DNAs of C. heterostrophus were separated by pulsed field gel electrophoresis and hybridized with the probes used to detect RFLPs. This permitted the confirmation of linkage assignments, the determina- tion of the number of chromosomes, and the associa- tion of linkage groups with chromosomes.

MATERIALS AND METHODS

1988).

Fungal strains: The fungal strains used in this study and their relevant genetic markers are given in Table 1. The field isolates from Iowa were collected in the course of a previous study (KLITTICH and BRONSON 1986). All other field isolates were obtained from 0. C. YODER (Cornell University). The C-strains are a set of fertile, closely related strains bred from field isolates by a program of backcrossing and intercrossing for use in research (LEACH, LANG and YODER 1982). The C-strains used in this study are siblings from cross B30 (BRONSON 1988).

Culture, crossing and storage: Methods for handling cultures of C. heterostrophus were reviewed by YODER (1 988). The fungus was routinely grown on complete medium (CM) (LEACH, LANG and YODER 1982) or CMX [CM with xylose substituted for glucose to improve conidiation (C. R. BRON- SON, unpublished results)] under cool-white fluorescent

TABLE 1

Strains of C. heterostrophus

Strain Genotype" Origin

Field isolates' Bumco6 t o x l MATI-2 North Carolina Dixon toxl MATI-2 Illinois Haw2 13 toxl MATI-2 Hawaii Hm28 toxl MATI-2 North Carolina Hm402 toxl MATI-I Florida Hm404 t o x l MATI-I Costa Rica Hm540 tox l MATI-I North Carolina Hn1648 toxl MATI-2 Florida Hm653 toxl MAT1-2 Florida Hm813 toxl MATI-2 Japan IA 1 t o x l MAT1-1 IA2

Iowa toxl MATI-2 Iowa

IA3 t o x l MATI-2 Iowa 1A4 t o x l MATI-I Iowa Mon2 toxl MATI-I Montana NYSSl toxl MATI-2 New York PRSB toxl MATI-1 Mexico

C-strains B30.A3.R.l TOXl ALBl CyhlS MAT1-I B30.A3.R.22 t o x l albl CyhlR MATI-I B30.A3.R.45 TOXl albl CyhlR MATI-2 B30.A3.R.65 TOXl ALBl CyhlS MATI-2 B30.A3.R.85 toxl AlBI CyhlS MATI-1 B30.A3.R.87 tox l ALBl CyhlS MATI-2 B30.A3.R.88 TOXI albl CyhlR MATI-I

The genetic nomenclature system is as recommended by YODER, VALENT and CHUMLEY ( 1 986). TOXl and toxl are alternate alleles at the Toxl locus for the ability and inability to produce T- toxin, respectively. Alleles at the Albl locus are ALBl for pigmentation and Albl for albinism. CyhtR designates an allele for cycloheximide resistance; Cyh1.S codes for sensitivity to cycloheximide. MATZ-I and MATI-2 are alleles at MAT1 for complementary mating types. ' All field isolates were pigmented and cycloheximide sensitive.

The genetic control of T-toxin production is known to be as indicated in Dixon, Hm28, Hm402, Hm540, Hm648, Hm653, Hm813 and NYSSl (BRONSON, TACA and YODER 1990). For the other field isolates, the Toxl allele is assumed to be as indicated because of the phenotype. MAT1 genotypes are also assumed to be as indicated because of the phenotype.

lights (photosynthetically active radiation = 55-65 pmol photon m-2 sec") at 22-24". Crosses were performed according to the methods of LEACH, LANC and YODER (1982) on maize leaves collected from fully senescent plants at 22- 24" on Sachs' medium (LUTTRELL 1958) modified by the inclusion of only 0.85 g of CaCOs and by the omission of FeC13. Ascospores were dissected from asci on day 2 1 to day 24 after crossing. T o facilitate the release of ascospores, asci were treated with 5% P-glucuronidase (v/v of water; type H-2S, Sigma Chemical Co., St. Louis, Missouri) (YODER 1988). Ascospores were placed on sorbose medium (LEACH, LANC and YODER 1982) to regenerate, then on CM to grow. All strains were stored in 15% glycerol at -65' as conidia and mycelial fragments (YODER 1988).

Assays of phenotypic markers: All fungal strains and progeny were tested for cycloheximide resistance and mating type according to the methods of KLITTICH and BRON- SON (1986). Cycloheximide resistance was determined by inoculating 1-2 mm3 blocks of agar with mycelia onto cycloheximide media (CM supplemented with 40 pg/ml cycloheximide) and observing growth after 1 week. Hm540 (CyhlS) and B30.A3.R.45 (CyhlR) were used as sensitive

C. heterostrophus RFLP Map 83

and resistant controls, respectively. Tests of all isolates were performed twice. Mating types were determined by testing the ability of the fungi to form pseudothecia when crossed to two C-strains with opposite mating types (B30.A3.R.45 and B30.A3.R.88). Tests for the mating types of the C- strains, field isolates and the 91 progeny used for making the map were performed twice with identical results.

A plant and a microbiological assay were used to test for T-toxin production. T-toxin production of all strains was tested in a plant assay according to the methods of KLITTICH and BRONSON (1 986) by observing symptom formation on cms-T maize seedlings (W64A-T) inoculated in the whorls with conidia and mycelial fragments. B30.A3.R.45 and B30.A3.R.85 served as TOXl and toxl controls, respectively. Any isolate giving an ambiguous result was retested. T-toxin production of the two parents and the 9 1 progeny used for linkage analysis was confirmed in a microbiological assay utilizing Escherichia coli transformed with T-urf l3 , the mitochondrial gene responsible for T-toxin sensitivity in cms- T maize (DEWEY, TIMOTHY and LEVINGS 1987; WISE et al. 1987). The growth of E. cold transformed with T-ur f l3 is inhibited by T-toxin (DEWEY et a2. 1988). The assay was performed on plates according to the method of YODER et al. (1989a) and BRONSON, TAGA and YODER (1990). PATH 13-T, a plasmid containing T-ur f l3 was generously provided by C. S. LEVINGS 111 (North Carolina State Uni- versity) and Lubrizol Genetics, Inc., Madison, Wisconsin. Inhibition of bacterial growth around mycelial blocks varied from 2 cm in diameter to undetectable with the naked eye; bacterial growth was therefore evaluated using a microscope (30X magnification). Any suppression of bacterial growth indicated the production of T-toxin. Each assay was done twice. B30.A3.R.45 and Hm540 were used as 7'0x1 and toxl controls, respectively. The results from the whorl and microbiological assays were identical.

Sources of probes: Two plasmid libraries, a cosmid library and several cloned genes were used. The plasmid libraries were constructed from total nuclear DNA of C. heterostrophus isolated by the method of YODER (1 988) and GARBER and YODER (1983). The DNA from B30.A3.R.22 and from B30.A3.R.45 was digested to completion with Hind111 (Bethesda Research Laboratories, Gaithersburg, Maryland) and inserted into the HindIII sites of calf intes- tinal alkaline phosphatase (Pharmacia, Piscataway, New Jer- sey) treated plasmid vectors pGEM2 (Promega Biotec, Mad- ison, Wisconsin) and pBSM13+ (Stratagene, La Jolla, Cali- fornia), respectively. The pGEM2 library was transformed into E. coli JM83; the pBSM13+ library was transformed into E. coli JMlOl. Plasmids in the pBSMl3+ library containing repetitive DNA were detected by colony hybridization (GRUNSTEIN and HOGNESS 1975) to 32P-labeled rDNA of C. heterostrophus (in pLR59) and total genomic DNA from B30.A3.R.45. Plasmids which did not have strong hybridization to total genomic DNA and rDNA were used as probes. The clones in the pGEM2 library were not screened for repetitive DNA prior to use.

In the cosmid library, which was kindly provided by B. G. TURGEON and 0. C. YODER (Cornell University), the genomic DNA of strain C2 (TOXl albl Cyh lS MATl -1 ) of C. heterostrophus was cloned into the BamHI site of cosHygl (YODER et al. I989a). Inserts in the library were screened to detect those containing repetitive DNA by colony hybridization to 32P-labeled total genomic DNA (GRUNSTEIN and HOGNESS 1975). Cosmids that did not have strong hybridization were used as probes.

Probes were also prepared from rDNA (in pLR59), a cloned mitochondrial DNA fragment (in pChM A2-7), MATI-1 (in CosMatl), G P D l (in pVW32) and TRPI (in

pChTRP24B), all from C. heterostrophus and all provided by B. G. TURGEON and 0. C. YODER (Cornell University).

Detection of RFLPs: Total genomic DNA of C. heterostrophus was isolated according to the methods of YODER (1988), except that glucose was not omitted in the liquid CM (CM without agar) used for growing the mycelia. The DNA was digested with restriction enzymes using the conditions specified by the supplier (Bethesda Research Labo- ratories). Total genomic DNA of the parents used to make the map was digested with either 18 restriction enzymes (ApaI , BamHI, BglI, BstEII, EcoRI, EcoRV, HindIII, MluI, PstI, PvuII, SalI, ScaI, SmaI, SstI, SstII, StyI, XbaI and XhoI) when plasmids and cloned genes were used as probes or 10 restriction enzymes (ApaI , BamHI, BglI, EcoRI, EcoRV, HindIII, PstI, PvuII, Sty1 and XhoI) when cosmids were used as probes. Progeny DNAs were digested with 13 enzymes (ApaI, BamHI, BglI, EcoRI, EcoRV, HindIII, Mlui , PstI, PuuII, ScaI, SstI, Sty1 and XhoI). Digested DNA fragments were separated on 0.8% agarose gels in 1 X TBE buffer and transferred to nylon membranes (Nytran, Schleicher & Schull Inc., Keene, New Hampshire) (MANIATIS, FRITSCH and SAMBROOK 1982).

Membranes were prehybridized for 2-8 hr at 42 O in 5 X SSPE, 5 X Denhardt's solution, 0.1 % sodium dodecyl sulfate (SDS), 100 pg/ml denatured sheared salmon sperm DNA (Sigma) and 50% (v/v) deionized formamide (MANIATIS, FRITSCH and SAMBROOK 1982). Hybridization was performed at 42" for 14-36 hr with probes labeled with [a- "P]dCT (New England Nuclear, Boston) prepared by the random hexamer labeling method (FEINBERG and VOGEL- STEIN 1983). The membranes were then washed (MANIATIS, FRITSCH and SAMBROOK 1982) and exposed to Kodak XAR- 5 film. The membranes were stripped of label prior to reuse by washing for 30 min in 0.2 M NaOH at 65', and then for 30 min in 0.5 M Tris (pH 8.0), 10 mM Na2EDTA (pH 8.0) with gentle shaking at 22-24'. Readable signals were still obtained from membranes reused more than 20 times. Only RFLPs that appeared reproducibly in parental DNAs run on seven different gels were scored in progeny.

Linkage analysis: Recombination frequencies between adjacent markers were used as measures of map distances. Linkages were calculated using the computer program HAPMAP (BRONSON, CHANC and TZENG 1989); a linkage was considered significant if the upper limit of the 95% binomial confidence interval of recombination frequency (FLEISS 1981) was less than 50 centimorgans (cM). Map distances were not corrected for possible multiple crossovers. The order of markers in the map was chosen such that the number of recombination events was minimized.

Chromosome separation and hybridization: Intact chromosomal DNAs of C. heterostrophus were obtained from protoplasts prepared by growing and digesting mycelia as described by YODER (1 988). The protoplasts were collected by centrifugation at 5000 rpm for 5 min in a SS34 rotor and then washed and imbedded in agarose according to the method of ORBACH et al. (1 988); the final protoplast density was 1-2 X 108/ml. The chromosomes were released from the protoplasts by incubation in NDS buffer (SCHWARTZ and CANTOR 1984). The plugs were washed and stored as described by ORBACH et al. (1 988). The chromosomes did not noticeably degrade when stored under these conditions for 6 months.

Both transverse alternating field electrophoresis (TAFE) (Beckman, Palo Alto, California) (GARDINER and PATTER- SON 1989) and contour-clamped homogeneous electric field (CHEF) (CHU 1989) gel systems were used for the separation of intact chromosomal DNAs. The CHEF apparatus was manufactured by the Engineering Research Institute of

84 T.-H. Tzeng et al .

Iowa State University based on blueprints generously provided by R. w. DAVIS (Stanford University); the distance between opposing electrodes was 28 cm. TAFE gel electrophoresis was performed in 1 X TAFE buffer [20 X TAFE buffer: 24.2 g/liter Tris, 2.9 g/liter EDTA (free acid), 5 ml/ liter glacial acetic acid] at constant current; CHEF gel electrophoresis was performed in 0.5 X TBE (MANIATIS, FRITSCH and SAMBROOK 1982) at constant voltage. The amperage (or voltage), switching interval and total time of electrophoresis varied depending on the size of chromosomes to be resolved. Specific conditions are given in the legends for Figures 3 and 4. DNA in the gels was stained with ethidium bromide (0.5 rg/ml) after electrophoresis.

Sizes of the chromosomes were estimated by running chromosomal DNAs of B30.A3.R.45 and Hm540 on the same gels with chromosomal DNAs of both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Chromosomes X I I , ZV and VII of S. cereutsiue were identified by hybridization with, respectively, rDNA (in pL91), t rp l (in pL35), both from S. cerevisiae and kindly provided by R. L. KEIL (Milton S. Hershey Medical Center), and ade5,7 of S. cereuisiae [in pYeADE5,7(5*2R)] provided by S. HENIKOFF (Hutchinson Cancer Research Center). Chromosomes IIZ and IZ of S. pombe were identified by hybridization with rDNA of C. heterostrophus (in pLR59) and nda3 of S. pombe, respectively. nda3 [in pYIP(NDA3)lI was provided by J.-B. FAN (Univer- sity of California, Berkeley). The size estimates were based on the chromosome separations shown, in part, in Figure 3, a and b.

Methods for transferring and probing the DNA and for stripping the membranes were as described for the detection of RFLPs, except that the labelling reaction mixtures were run through a P-10 (Bio-Rad, Richmond, California) column to separate labeled probes from unincorporated nucleotides. Any ambiguous hybridizations were repeated. Membranes were reused more than 10 times.

Chromosome nomenclature: Chromosomes were named according to their homology to chromosomes in the field isolate Hm540, which were numbered based on their relative migration rates in pulsed field gel electrophoresis (Fig- ure 3). Chromosomes in B30.A3.R.45 that are translocated with respect to their homologous chromosomes in Hm540 are indicated in parentheses by the name of the chromosome homologous to the larger segment (measured as map distance), followed by a semicolon and the name of the chromosome homologous to the smaller segment.

RESULTS

Selection of parental strains: Parental strains were selected to maximize the efficiency of RFLP detection and to insure the usefulness of the map in future research. It was desired that at least one of the parents be a C-strain because a variety of mutants are available and techniques for genetic and molecular manipulation have been developed for this group. Although it would be desirable that both parents be C-strains because of their known high fertility, it was assumed that the close genetic relationships among the C- strains would result in a low number of polymorphisms suitable for mapping. This assumption was tested by hybridizing EcoRI-, HindIII- and XhoI-digested and electrophoretically separated DNAs of six C-strains with two repetitive DNA probes. The C- strains were B30.A3.R.45, B30.A3.R.88, and four

strains prepared by a series of either eight or nine backcrosses of TOXl and CyhlR into C3 (KLITTICH and BRONSON 1986). Although the probes hybridized to over 10 bands in each strain, the C-strains showed few polymorphisms, confirming that they are too closely related for efficient detection of RFLPs.

Field isolates from various states and countries (Table 1) were therefore screened to select one with a high frequency of polymorphism with respect to the C-strains. Because the C-strains had been bred pri- marily from race T (TOXl) strains (LEACH, LANC and YODER 1982), race 0 (toxl) field isolates were chosen for screening to increase the probability of detecting polymorphisms. DNAs from the TOXl C-strain B30.A3.R.88 and 17 toxl field isolates (Table 1) were digested with either 10 (BamHI, EcoRI, EcoRV, HzndIII, PstI, PvuII, Smal, SstII, XbaI and XhoI) or 7 (BamHI, EcoRI, EcoRV, HindIII, PstI, PvuII andXbaI) restriction enzymes and hybridized with 35 probes from the pGEM2 and pBSMl3+ libraries. The frequencies of enzyme-probe combinations revealing RFLPs between B30.A3.R.88 and each field isolate varied from 10/284 to 34/282. Because each probe examined different locations in the genome, the frac- tion of enzymes detecting polymorphisms varied widely between probes (0/10 to 9/10). Therefore, the relative polymorphism of the different field isolates with respect to B30.A3.R.88 was evaluated by analysis of variance, using the probes as blocks. One isolate, Hm648, was significantly less divergent from the C- strain than all the other field isolates; however, no isolate was significantly more divergent than the rest.

Hm540 was chosen as the field isolate parent because it was one of the most polymorphic strains (29 out of 283 enzyme-probe combinations showed RFLPs) and, unlike many other toxl field isolates, did not show segregation distortion for alleles at Toxl when crossed to TOXl C-strains (BRONSON, TACA and YODER 1990). B30.A3.R.88 could not be used as the C-strain parent because it has the incorrect mating type to cross with Hm540. Therefore, B30.A3.R.45, which is a sibling of B30.A3.R.88, was selected as the C-strain parent. It has a complementary mating type to Hm540 and is different from Hm540 at the phenotypic markers Toxl, Albl and Cyhl, permitting the mapping of these loci.

Types of RFLPs detected: The copy number of the DNA hybridized by the probes varied (Figure 1). Because of the presence of multiple restriction sites within the cosmid inserts, copy number could be deduced only for the plasmids. Plasmid probes were considered to detect single copy DNA (Figure la) or multiple copy DNA (data not shown) if they hybridized strongly to a single band or to two to five bands, respectively, in Southern blots of HindIII digested DNA from B30.A3.R.45. Plasmid probes were con-


Progeny

a

b

C

d

B H _". ~

FIGURE 1 .-Different classes of RFLPs and their segregation in progeny. B: BSO.AS.R.45. H: Hm540. (a) RFLP identified by a single copy DNA sequence (G471). (b) RFLPs identified by a repetitive DNA sequence (G238). (c) RFLP identified by a single copy sequence that has no homolog in Hm540 (G241). (d) RFLP identified by a single copy sequence located in different places in the two parental strains (G210).

sidered to detect repetitive DNA if they hybridized strongly to more than five bands in HindIII digested DNA (Figure Ib) or hybridized more strongly to at least one band in HindIII digested DNA and strongly to multiple bands with other enzymes. The majority of the probes hybridized to single copy sequences (Table 2), suggesting that C. heterostrophus has a low frequency of repetitive DNA. Of plasmid probes detecting RFLPs in single copy DNA, 49% did so with only one out of 18 restriction enzymes; most of the variation in single copy DNA thus appears to be due to random base pair substitutions, or small deletions or insertions at the restriction sites.

For some of the probes, the copy number of the DNA hybridized varied from zero to multiple, depending on the strain. These probes detected differences in chromosome arrangement between B30A3.R.45 and Hm540. Ten probes hybridized to DNA from B30A3.R.45, but not Hm540 (Figure I C and Table 3), indicating that they detected large deletions or insertions. Of these, nine detected markers that mapped to chromosome 16, a dispensable (B) chromosome (see below) that is present in B30A3.R.45, but not Hm540. Other probes (four), classified as single copy in the parents, showed variable

TABLE 2

Frequencies of probes that detected RFLPs between strains B30.A3.R.45 and Hm540 of C. heterostrophus

Source of probes enzvrnes tested detecting RFLPs No. of No. of probes Percent of probes

Libraries cosHyg 1 10 74 54 pBSM 1 S+" 18 124 31 pGEM2 18 162 56

Single copy 129 ( 8 0 % ) b 44 Multiple copy 5 (3%) 100 Repetitive 28 (1 7%) 100

Defined sequences' 18 5 60

a These libraries were screened to remove rcpetitive DNA prior to tests for ability to detect RFLPs.

Percent of total detected in pGEM2 library. ' RFLPs were detected by rDNA, M A T I - I , and a mtDNA frag-

ment, but not CPD1 or TRP I .

numbers of copies in the progeny (Figure Id); the segregation of the RFLP bands indicated that the homologous sequences are present at two different locations in the genome. In B30A3.R.45, all of these RFLPs mapped to chromosome 16; in Hm540, one of these mapped to chromosome 4 and three to chromosome 6 .

Frequencies of RFLPs between the parents: Be- cause the preliminary screening of field isolates had indicated a low frequency of RFLPs, the DNAs of B30.A3.R.45 and Hm540 were digested with multiple enzymes to increase the probability that a given probe would detect an RFLP. The DNA was digested with 18 enzymes when plasmids and cloned genes were used as probes and 10 enzymes when cosmids were used as probes. The effectiveness of using large numbers of enzymes for each probe was evaluated to determine whether this strategy for increasing the efficiency of RFLP detection was worthwhile. This was analyzed for the plasmid probes known to hybridize to single copy DNA. The 18 enzymes were grouped into sets based on price at the time of pur- chase, as if to minimize cost, and a count was made of the number of probes that would have detected RFLPs with each set of enzymes. If only the least expensive enzyme (EcoRI) had been used, 10% of the plasmids would have detected RFLPs. If the five least expensive enzymes (listed in order of increasing cost: EcoRI, ApaI, HindIII, XhoI and PstI) had been used, 23% of the plasmids would have detected RFLPs. The percentage with ten enzymes (EcoRI, ApaI, HindIII, XhoI, PstI, BamHI, BstEII, EcoRV, Sty1 and XbaI) would have been 31 %. This is compared to 36% with all 18 enzymes. Thus, although the efficiency of detection increased with increasing numbers of enzymes, there was relatively little added benefit to using more than 5 to 10 restriction enzymes.

The ability of the various restriction enzymes to reveal RFLPs was examined to determine whether

86 T.-H. Tzeng et al. TABLE 3

Segregation and chromosome location of RFLP and phenotypic markers in cross T1 between strains B3O.A3.R.45 and Hm540 of C. heterostrophus

Alleleb Chromosomed Alleleb Chromosomed

Locus" B H xz' B H Locus" B H x2' B H

Albl Cyhl GPD1' MATI' rDNA' TRPI' Ton1 B4' B6 B2 1' B2Y B43' B71' B84' B88' B9 I' B107' B114pl B114p2 B120p B125p B149pl' B 149132' B 149~3 ' B 1 4 9 ~ 4 ' B154 B160' B 195' B246' B257 B264' B272' B277' B28 1 p B283' B285' B288m B30 1' B312-1 B3 12-2 B329p B339' B346m' B380' B403' B405p B41 Ip B416' B421pl' B42 1 p2' B42 1 p3' B42 1 p4' B429p' C3' C9 C28' C50' C53' C60' C64' C67 C70 c72' c74 C76 C84' C113'

39 42

45 42

44 38 44 41 49 40 38 49 43 49 37 43 58 42 60 49 47 35 46 43 46 48 42 60 46 51 45 52 46 42 60 42 60 47 42 40 36 38 37 40 45 41 50 46 51 44 44 45 44 39 51 45 35 44 42 36 47 45 56 38 46

ND

ND

-

52 1.86 49 0.54 ND 46 0.01 49 0.54

47 0.10 52 2.18 46 0.04 50 0.89 42 0.54 51 1.33 53 2.47 42 0.54 48 0.27 42 0.54 54 3.18 46 0.10 31 8.19** 48 0.40 31 9.24** 42 0.54 42 0.28 51 2.98 45 0.01 48 0.27 45 0.01 39 0.93 44 0.05 31 9.24** 45 0.01 39 1.60 46 0.01 39 1.86 45 0.01 49 0.54 31 9.24** 46 0.18 31 9.24** 44 0.10 49 0.54 51 1.33 55 3.97* 47 0.95 54 3.18 51 1.33 45 0.00 50 0.89 40 1.11 44 0.04 38 1.90 45 0.01 46 0.04 42 0.10 43 0.01 50 1.36 39 1.60 44 0.01 56 4.85* 45 0.01 48 0.40 40 0.21 43 0.18 40 0.29 35 4.85* 53 2.47 45 0.01

ND

1 13 14 10 9

10 (6;12) or (12;6)

5 3

14 13

(12;6) 5 3

(6;12) 1

2 or 3 9

16 I

16 2

3 ND

( 1 2 6 ) 7

( 1 2 6 ) 7 4

16 2 1 1 2 I

10 16

7 16

13 8

(6;12) (6;12)

8 3 4 14 I

-

UK ND ND 1

15 I 3 2 8

(6;12) 4 5 1

13 1

I 1 8 8

1 13 14 10 9

10 6 or 12

5 3 14 I3 12 5 3

12 I

2 or 3 9

1 -

"

ND 11

6 7

12 7 4

2 1 1 2 I

10

7

6 13 8 6 6 8 3 4 14

ND

-

"

-

ND ND

1

I 15

1

UK

3 2 8 6 4 5 I 13 1

I 1 8 8

C116' C121 C 155' C173' C188' C192' C 193' C206 C207' C210 C226 C246 C253 C26 1 C270' G19' G29' G32m' G38' G86m G98rl' G98r2' G127' G131' G I 4 4 GI 72' G188 G199' G210-1' G2 10-2' G2 13' G214' G235' G237' 122381-1' G238r2' G238r3' G241 G242' G264rl' G264r2' G300' G308' G309' G3 1 1' G349' G353ml' G353m2' G353m3' G384' G386 G392-1' G392-2' G395' G398' G40 1 G419' G431-1 G431-2 G444 G449m G451' G452m G463' G47 1' G492 G508'

46 41 43 42 38 45 45 42 50 49 49 43 47 51 40 45 51 42 41 60 45 51 45 47 43 44 60 50 60 47 37 50 36 35 37 48 47 60 45 44 43 42 60 40 49 48 43 43 46 43 60 60 48 40 49 42 42 60 47 36 29 52 60 51 36 60 45 -

40 49 43 49 53 44 46 40 35 42 41 45 44 39 51 46 40 48 49 31 44 35 46 44 37 47 31 41 31 44 54 41 55 49 53 43 43 31 46 47 44 49 31 51 41 43 48 48 45 48 31 31 41 51 42 49 46 31 44 55 28 35 31 40 55 31 46 -

0.42 0.71 0.00 0.54 2.47 0.01 0.01 0.05 2.65 0.54 0.71 0.05 0.10 1.60 1.33 0.01 1.33 0.40 0.71 9.24** 0.01 2.98 0.01 0.10 0.45 0.10 9.24** 0.89 9.24** 0.10 3.18 0.89 3.97* 2.33 2.84 0.27 0.17 9.24** 0.0 1 0.10 0.01 0.54 9.24** 1.33 0.71 0.27 0.27 0.27 0.01 0.27 9.24** 9.24** 0.55 1.33 0.54 0.54 0.18 9.24** 0.10 3.97* 0.02 3.32 9.24** 1.33 3.97* 9.24** 0.01

5 7 5

2 or 3 2

(12;6) or (6;12) 14

1 4 I3 4 7 5 4 13 14 4

(6;12) 1

16 (12;6) or (6;12)

10 5

7 3

16 11 16

13 5 15 5

13

-

-

ND ND 16 4

(12;6) or (6;12)

13 16 3 1

(6;12) (12;6)

ND

- 1 I

16 16 -

(12;6) 11

( 6 1 2 ) 10 16

15 3

I 1 16

9 7

16 2 or 3

-

5 7 5

2 or 3 2

14 I 4 13 4 7 5 4 13 14 4

1

ND

-

"

ND 10 5 6 7 3

I 1

6 13 5

15 5

7 6 or 12

4

12 13

"

-

ND

"

ND

"

"

I 6 6

12 1 1 "

- 4

12 11 6

I O

6 15 3

I 1

9 7

2 or 3

-

"

"

4

2 4

B421p l * 1

3 6

1 6

2 9

7

1 9

2

1 8

1 2 3

9 10 j B114pl

0 4 6 3 9

rDNA*

C 6 0 . 14

C188’

1 2

0 3 0 9 * B 2 8 4 *

1 1

C. heterostrophus RFLP Map

4 5 2

3 2

2 4

3

2

1 3

3 9

7

1 4

G , 0 2 4 8 .

; 841111 I 6 2 4 2 . , C 8 4 *

2 3

1 2

3 4

2 0

1 0

1 7

871.

0127.

0 2 1 4 -

C166*

C 8 7

C 2 6 3

C118*

8 4 * ’ 0 2 3 7 .

13 14

0213 ‘ 623811.

c 2 7 0 . G P D 1 *

7 8

1 7 2 6

B196*

G 2 3 8 r 2 *

C113. 1 0

87

(12;6) 12

B 1 4 9 p 4 * Q 2 8 : $ r 2 *

G 3 6 3 m l * i i l a [ i - ”_

8 3 1 2 - 2

0 2 1 0 - 2 - 01310 2 2 j

0 4 3 1 - 2 Q349 .

E::::. c3.

A 8114p2 B126p 8 2 6 7 8 2 8 8 m 8 3 1 2 - 1 G88m

0210-10 0 1 8 8

0 3 0 8 * 0 2 4 1

0 3 9 2 - 1 0 0388

0 4 3 1 - 1

0 4 9 2 0 4 6 2 m

~ 3 4 e m -

0 3 2 m -

6 (6;12) 0 4 0 1

FIGURE 2.-Proposed genetic map of C. heterostrophus. T h e hollow lines indicate linkages significant at P = 0.05; the solid lines indicate linkages significant at P = 0.025. The thin lines represent the chromosomes of B30.A3.R.45; the dashed lines represent the chromosomes of Hm540. Gaps between markers on the same chromosome indicate that the markers, although on the same chromosome, were not statistically linked; the order and orientation of the unlinked regions is not known. The stars by the markers indicate that probes detecting these markers were hybridized to separated intact chromosomal DNAs. T w o cloned genes, TRPZ and GPDZ, were assigned to chromosomes based on chromosome hybridization. Linkages for TRPZ and GPDZ were not tested; therefore, their map locations relative to other markers are not known. The map units (cM) are listed on the left of or below the linkage lines. Marker names are generally listed on the right. The dashes itcross the linkage lines indicate the location of markers; half-dashes indicate markers that exist in these locations in only one parent. Only nlarkers whose chromosomal locations were confirmed by hybridization to separated chromosomal DNAs and markers mapping within 20 cM of a confirmed marker are included in the map.

lfootnotes to Table 3)

a RFLP markers identified by probes from the pGEM2 library, the pBSMl3+ library and the cosHygl library begin with G, B and C, respectively. Single copy sequences mapping to different locations in the two parents are denoted by the symbols “-1” and “-2” following the probe name, indicating the allele from B30.A3.R.45 and Hm540, respectively. Markers identified by plasmid probes hybridizing to multiple copy or repetitive sequences are designated by “m” or “r,” respectively. Markers identified by probes that contain more than one insert are indicated by a “p.” If more than one marker identified by a probe were mapped, the “m,” “r” or “p” is followed by a number designation.

Alleles are from B30.A3.R.45 (B) and from Hm540 (H).~“ND” indicates that no RFLP was detected and therefore segregation analysis could not be performed.

* The null hypothesis for the chi-square test is that two alleles segregate in a 1: 1 ratio. Chromosomes in B30.A3.R.45 (B) and Hm540 (H). “-” indicates that the marker does not exist in this strain, although homologous

sequences are present in the genome. “- -” indicates that neither the marker nor homologous sequences are present in this strain. “ND” indicates that the existence of the marker in this strain could not be determined due to the hybridization of the probe to overlapping DNA bands. “UK” indicates that, although the marker is present in this strain, its chromosome location is unknown; it could not be determined by either mapping or hybridization to separated chromosomes.

‘ Markers whose chromosome location was tested by hybridizing to electrophoretically separated chromosomes. * Significantly different from 1 : 1 at P = 0.05. ** Significantly different from 1:l at P = 0.01.

88 T.-H. Tzeng et aE.

some enzymes detected RFLPs more efficiently and therefore whether the trends shown in the preceding analysis would have varied if different sets of enzymes had been chosen. No significant differences were observed in the frequencies of RFLPs revealed by different enzymes using either the plasmid libraries or the cosmid library as probes. When compared as a group, restriction enzymes containing 5’CG3’ in their recognition sequence (MluI , SalI, SmaI, SstII and XhoI) also did not reveal more RFLPs than the other restriction enzymes. This result agrees with observations in lettuce (LANDRY et al. 1987) and rice (MC- COUCH et al. 1988), but contrasts reports for humans, in which a higher frequency of RFLPs was detected with restriction enzymes containing 5‘CG3‘ in their recognition sites (BARKER, SCHAFER and WHITE

The efficiency of RFLP detection varied with the number of restriction fragments to which the probes hybridized (Table 2). Probes that hybridized to larger numbers of fragments, that is, cosmid probes (from the cosHygl library) and multiple copy and repetitive probes, detected RFLPs more efficiently. Under the electrophoretic conditions used, however, the banding patterns produced by the probes hybridizing to repetitive DNA were generally too complicated to score individual bands reliably and, therefore, most of these probes were not useful for mapping.

Cross T1: A cross, T1, between B30.A3.R.45 and Hm540 was made to generate progeny to make the map. A total of 424 ascospores were isolated from 138 asci; all viable progeny in each ascus were usually obtained.

The fertility of the cross, measured as numbers of viable ascospores per ascus, was low. Cochliobolus undergoes a postmeiotic mitosis to produce eight ascospores. However, in cross T 1, the average number of viable ascospores in asci containing at least one viable spore was 3.1. Asci containing no viable spores could not be distinguished ‘from immature asci and were not counted. The majority of the asci contained two or four viable ascospores; 38% of asci were of each type. Only 6.5% of the asci contained more than four viable ascospores. Among asci with four viable spores, 84% segregated 2:2 for alternate alleles at Toxl.

Segregation of markers: Segregation of RFLP and phenotypic markers were examined among 91 progeny. The randomness of the progeny was assured by selecting one random ascospore from each of 91 random asci.

Asci were taken from both albino (B30.A3.R.45 maternal parent) and pigmented (Hm540 maternal parent) ascocarps to permit tests of maternal inheritance. Marker B14 and pChM A2-7, a mitochondrial DNA clone known to detect mitochondrial RFLPs

1984).

between Hm540 and the C-strains (GARBER and YODER 1984), showed absolute maternal inheritance. The detection of a clone, B14, showing maternal inheritance in the pBSMl3+ library suggests mitochondrial contamination of the nuclear DNA preparations. The mitochondrial origin of B14 was confirmed by its hybridization to a band resolved by TAFE (Figure 3a) corresponding to the mitochondrial chromosome, but not to bands corresponding to nuclear chromosomes.

The remaining 132 markers (128 RFLP and 4 phenotypic) were assumed to be nuclear and were tested for Mendelian segregation by chi-square analysis with one degree of freedom (Table 3). All but 21 of the markers segregated normally (1: 1). Fifteen markers deviated from 1: 1 segregation at a significance level of P = 0.01 with an excess of the allele from B30.A3.R.45. These markers mapped to chromosome 16, the apparent B chromosome (see below) and may be expected to segregate abnormally. The other six markers (B346m, C60, C76, G235, G444 and G471) deviated from 1:l segregation at P = 0.05. The apparent distorted segregation by these six markers may not reflect true anomalous behavior; at a significance level of P = 0.05, 7 normally segregating markers (5% of 132) are expected to test statistically as though not segregating 1: 1. Another possible ex- planation for the deviation is that the 6 markers might be linked to loci that cause segregation distortion. A locus causing segregation distortion was detected linked to Toxl in several field isolates, although not in Hm540 (BRONSON, TACA and YODER 1990).

Linkage analysis: Linkage analysis was performed on the 132 nuclear markers in order to construct the map (Figure 2). The maximum detectable map distance at P = 0.05 (two-sided test) was 39 cM. However, because of the large number of marker comparisons and therefore the risk that numerous unlinked markers might test as statistically linked, only linkages that were less than 20 cM and/or confirmed by hybridization of the probes to electrophoretically separated, intact chromosomal DNAs were included in the map and used to calculate the total map distance. Linkages among 126 markers met at least one of these criteria. Of these, 120 markers, distributed among 26 linkage groups, were included in the map and comprised 941 cM.

The remaining six markers showed tight linkage, but were not included in the map and not used to calculate the total map length. These were RFLPs whose chromosome location could not be determined because the probes revealing them hybridized to many chromosome bands. Of these RFLPs, B421p2 was linked to B421p4 by 3 cM but neither was linked to other markers. C192, G98r1, G238r3 and G353m2 were linked to Toxl at 1, 1, 3 and 3 cM, respectively,


1.2.3 (12:6)

5 (6: 12). 7

4.8. Q

10,11,13

14 15,16

mt

- 1,2,3 2\ 3- 1-

/ 4

- 8 5- -9.10 (6: 12) -

7’

1.6M b - 14,15

1.3M b

b C

B H P B H B H S

-3.6 Mb P -1 ‘2,3

8 - -4

-5

-6 10,13- 4’9 -1 8 9

10

1 1 , 1 2

13

FIGURE 3.--lntact chromos1~n1;11 DNAs of S. cerevisiae (S). S . pomhr (1’) and C. heterostrophvs strains 1330.A3.R.4.5 (R) and Hm540 (H) separated by TAFE and CHEF electrophoresis. Chromosome bands of B3O.M.R.45 and Hm540 are indicated on the left and right, respectively. Mitochondrial DNA is indicated by “mt.” (a) Separation of smaller chromosomal DNAs by TAFE. The separation conditions were I % agarose. I 1 ”, 60 mA, 4-sec switching interval for 30 min and then IO-min switching interval for 110 hr. (b) Separation o f larger chromosonlal DNAs by TAFE. The separation conditions were 0.9% agarose, 9”, 45 mA, 4-sec switching interval for 30 min, then 15-min switching interval for 72 hr and 20-min switching interval for 7 2 hr. (c) Separation of chromosomal DNAs o f intermediate size by CHEF electrophoresis. T h e separation conditions were 0.9% agarose. 1 1 ’, 2.9 V/cm, and 1 I-min switching interval for 141 hr.

but their order relative to the other markers in that region was not clear.

The markers identified by a repetitive or multiple copy DNA clone usually mapped to more than one site in the genome, indicating dispersion of homologous sequences. I t could not always be determined whether these homologous sequences were present in both parents at any particular site because of the overlapping of restriction bands.

Electrophoretic karyotypes: The chromosomes of B30.A3.R.45, Hm540 (Figure 3) and five of their progeny (Figure 4) were separated by pulsed field gel electrophoresis and distinguished by hybridization with probes used to recognize RFLPs.

The banding and hybridization patterns were highly reproducible for each strain, but differed among the strains. Consistent banding and hybridization patterns were obtained with four different preparations of B30.A3.R.45 chromosomal DNAs, and two different preparations of Hm540 chromosomal DNAs. The banding patterns of chromosomal DNAs of the progeny (Figure 4) were different from those of B30.A3.R.45 and Hm540 (Figure 3). Several new

chromosome bands were identified in the progeny, presumably due to recombination between parental chromosomes of different size.

The number of identified chromosome bands in the parental strains was maximized by comparison of the banding and hybridization patterns obtained after separation under a variety of electrophoretic conditions. No electrophoretic condition tested, for either TAFE or CHEF electrophoresis, resolved all the identified bands in a single run. However, four different combinations of switching time and amperage or voltage for TAFE and CHEF were identified which resolved chromosomes in different size ranges (Figures 3 and 4). The identities of the various bands in the different runs were determined by hybridization with the probes used to make the RFLP map. Examples of the hybridizations are given in Figure 5. In instances in which DNAs representing different linkage groups migrated with similar speed as a broad band, comparisons were made on the same membranes of the hybridization positions of probes representing the linkage groups. When probes representing one linkage group always hybridized to the upper part of the band,

T.-H. Tzeng et al. 90

a b H B 1 2 3 4 5

FIGURE 4."Electrophoretic karyotypes of f ive progeny from cross T1 between BSO.AS.R.45 (B) and Hm540 (H). ( 1 ) T1.AS.S.l [tox-] (2) TI.P2.1.1 [tox-] (3) TI.P5.2.1 [tox-] (4) Tl.A20.9.1 [ tox+] and ( 5 ) T1 .P8.2.1 [tox-1. (a) Separation of smaller chromosome bands by TAFE. The separation conditions were 0.9% agarose, 8". 60 mA, 4-sec switching interval for 30 min, then 10-min switching interval for 72 hr and 9-min switching interval for 68 hr. (b) Separation of larger chromosome bands by TAFE. The separation conditions are given in the legend for Figure 3b.

whereas probes representing the other linkage group always hybridized to the lower part of the band, it was concluded that the band contained at least two chromosomes. Using these techniques, a total of fourteen bands were resolved for B30.A3.R.45 and twelve bands for Hm540 (Figures 3 and 4).

The two most rapidly migrating bands in B30.A3.R.45 were resolved in only a single gel (Fig- ure 4a); the presence of two bands, representing chromosomes 15 and 16, was verified by their resolution in the progeny. This was possible because chromosome 15 from the two strains migrates at different rates and chromosome 16 is absent in Hm540. A membrane prepared from a gel similar to the one shown in Figure 4a was hybridized with probes G235 and G210, which identify chromosomes 15 and 16, respectively. Two of five progeny tested contained both chromosomes; in both progeny, chromosome 15 migrated similarly to the homologous chromosome in Hm540, whereas chromosome 16 migrated more slowly, similarly to the one in B30.A3.R.45. The resolution of these bands in the progeny confirmed the presence of two bands in B30.A3.R.45.

In a given strain, certain bands were deduced to contain more than one chromosome even though these chromosomes were not resolved under any of the conditions tried. These were bands containing chromosomes 4 and 9 and chromosomes 10 and I3 in

a b C

B H B H B H B H

FIGURE 5.--1 lybridization of separated intact chrornosornal DNAs of C. hettrostrophus strains B30.A3.R.45 (B) and Hrn540 (H) with probes used for making the RFLP map. (a) Ethidium bromide stained TAFE gel. The lanes are from the same gel as shown in Figure 3a. (b) Hybridbation of chromosomes in gel in (a) with probe G451. (c) Hybridization of chromosomes in gel in (a) with probe G392. The marker detected by this probe mapped to chromosome 16 in B30.A3.R.45 and chromosome 4 in Hm540.

B30.A3.R.45 and bands containing chromosomes 2 and 3, chromosomes I I and 12, and chromosomes 14 and 15 in Hm540. These chromosomes were in resolved bands in the opposite parent. If each of the unresolved bands had contained a single chromosome, the chromosome in one parent would have had to pair at meiosis with two chromosomes in the other parent and, therefore, one quarter of the progeny should have had duplicate copies of the markers for the chromosome whose centromere did not pair. Any such duplication progeny would likely survive. In cross T1, 50% of progeny had duplications of markers associated with the presence of B3 12, G2 10 and G43 1 on chromosome 6 in Hm540 and on chromosome 16 in B30.A3.R.45, and the presence of G392 on chromosome 4 in Hm540 and on chromosome 16 in B30.A3.R.45. These duplications caused no deleteri- ous effect as measured by growth rate. In addition, duplication progeny are viable in the related ascomycete Neurospora crassa (PERKINS 1974). In cross T1, none of the 91 progeny analyzed showed the duplication of any single copy markers except B3 12, G2 10, G392 and G431. We therefore reasoned that the unresolved bands contained at least two chromo-


TABLE 4

Estimated sizes of the chromosomes of strains B30.A3.R.45 and Hm540 of C. heterostrophus

B30.A3.R.45 Hm540

Chromosome Size (Mb) Chromosome Size (Mb)

1 3.1 1 3.3 2 3.7 2 3.2 3 3.6 3 3.2 4 2.0 4 2.7 5 2.5 5 2.4

(6;12) 2.3 6 2.2 7 2.2 7 2.1 8 2.0 8 2.0 9 2.0 9 1.9

10 1.9 10 1.9 11 1.9 11 1.8

( 1 2 6 ) 2.6 12 1.8 13 1.9 13 1.7 14 1.5 14 1.4 15 1.3 15 1.4 16 1.3

The sizes of the chromosomes were estimated from their migration rates in the gels shown in Figure 3, a and b, using the chromosomes of S . cerevisiae and S . pombe as size standards.

somes, bringing the total number of identified chromosomes to 16 in B30.A3.R.45 and 15 in Hm540. This deduction has been confirmed for chromosomes 4 and 9 in B30.A3.R.45 and related strains by their resolution in other experiments (H.-R. CHANG, L. K. LYNGHOLM and C. R. BRONSON, unpublished results).

The sizes of the chromosomes of C. heterostrophus were estimated to range from approximately 1.3 megabases (Mb) to approximately 3.7 Mb (Table 4). The sizes of the chromosomes in each band were estimated by comparison to chromosomes ZV and VZZ of S. cerevisiae (Beckman) and chromosomes ZZ and ZZZ of S. pombe (Beckman). Chromosomes Wand VZZ have been estimated to be 1.6 and 1.3 Mb, respectively (MORTI- MER and SCHILD 1985). Chromosome XZZ was not used to estimate the sizes of C. heterostrophus chromosomes because it did not form a well defined band under our separation conditions. When hybridized with rDNA it formed a broad smear running most of the length of the gel; similar behavior by chromosome XZZ has been reported by CARLE and OLSON (1985). Chro- mosomes ZZ and ZZZ of S. pombe have been estimated to be 4.6-4.7 and 3.5 Mb, respectively (FAN et al. 1988). The chromosomes used as size standards bounded those of C. heterostrophus and were resolved in the same gel, suggesting that the chromosomes of C. heterostrophus were in a region of high resolution.

The mitochondrial chromosome, identified by its hybridization to a mtDNA specific probe (pChM A2- 7) and not to probes which hybridized to the various nuclear chromosomes, migrated faster and separated from the nuclear chromosomes as a diffuse band (Figure 3a). Since the mobility of circular DNA is

independent of switching interval (HIGHTOWER, METGE and SANTI 1987), it is impossible to estimate the size of the mitochondrial chromosome based on its mobility in the gel. The size of the mitochondrial genome has been estimated to be 1 15 kb by restriction mapping (GARBER and YODER 1984).

Confirmation of linkage assignments: The probes used for making the RFLP map were hybridized to the separated chromosomal DNAs in order to confirm the linkage assignments and to associate linkage groups with chromosomes (Table 3). All of the linkages between adjacent markers that were greater than 20 cM were tested. Chromosome rearrangements detected by linkage analysis were confirmed (Figure 2). Twelve of the chromosomes were shown to hybridize with probes for more than one linkage group and/or unlinked marker, revealing a total of 14 linkage gaps. The order and orientation of unlinked regions on the same chromosome is not known.

Three cloned genes of known function, i e . , G P D l , MATl-1 and T R P l from C. heterostrophus, were also associated with chromosomes by hybridization (Table 3 and Figure 2). No RFLPs were identified using GPDl and T R P l as probes; therefore, their positions relative to other markers in the map could not be determined. The MATl-1 clone was not hybridized to restricted progeny DNAs because this clone was al- ready known to cosegregate with MAT1 (YODER et al. 1989b).

A reciprocal translocation associated with Toxl: A branched linkage, which suggests a reciprocal translocation, was detected associated with Toxl (Figure 2). This translocation was confirmed by hybridization of ,)robes on the four arms of the translocation to different chromosomes in the two parents.

No crossing over was observed between Toxl and the translocation breakpoint; all progeny that produced T-toxin had the chromosome arrangement of the TOXl parent and all progeny that did not produce T-toxin had the chromosome arrangement of the toxl parent. The chromosome arrangements of the progeny were determined by utilizing the fact that, in crosses between strains that differ by a reciprocal translocation, half of all ascospores are expected to abort (PERKINS 1974). The progeny were crossed to two strains of known chromosome arrangement: a TOXl C-strain (B30.A3.R. 1 or B30.A3.R.65, depending on mating type) and a toxl C-strain (B30.A3.R.85 or B30.A3.R.87). All the progeny with the allele toxl were more fertile when crossed to the toxl C-strains; all the progeny with the allele TOXl were more fertile when crossed to the TOXl C-strains. The majority of asci in crosses homozygous for Toxl (TOXl X TOXl or toxl X t ox l ) had six to eight viable ascospores; in heterozygous crosses (TOXl X toxl or toxl X T O X l ) , the majority of asci had four or fewer viable asco-


spores. Similar abortion levels were reported for homozygous and heterozygous crosses among C- strains (BRONSON 1988). This result suggests that no crossing over occurred between Toxl and the breakpoint of the translocation.

The location of the translocation breakpoint was further verified by testing the chromosome arrangements of a sample of progeny by hybridization of probes to electrophoretically separated chromosomal DNAs. Chromosomal DNAs of five progeny were separated by TAFE (Figure 4b) and hybridized with probes identifying the four arms of the translocation, i .e. , B88, G210, G349 and G395. Three progeny with known crossovers between Toxl and its flanking markers were included to increase the chance of detecting a crossover between the translocation and Toxl. Any progeny with a crossover between Toxl and the breakpoint should have the chromosome hybridization patterns of one parent and the Toxl allele of the other. For all progeny tested, the chromosome hybridization patterns were the same as for the parent with the corresponding Toxl allele. Therefore, no crossovers were detected between Toxl and the breakpoint, suggesting tight linkage.

Chromosome 26 is a dispensable (B) chromosome: Several differences in chromosome arrangement between the parents were detected that were associated with chromosome 16. This chromosome, to which 15 probes hybridized, is present in B30.A3.R.45, but not Hm540 (Figure 2). Two-thirds of the progeny inherited this chromosome (Table 3). Ten of these probes did not hybridize or hybridized very faintly to Hm540. One of the probes (B114) contained multiple inserts and detected sequences in both parents. The remaining four probes detected RFLPs that mapped and hybridized to other chromosomes in Hm540, indicating insertions in Hm540 or deletions in B30.A3.R.45. In Hm540, G392 mapped to chromosome 4 (Figure 5) and B3 12, G2 10 and G43 1 mapped 14 cM from Toxl on chromosome 6 . No sequences homologous to these probes mapped to B30.A3.R.45 at either of these points. Chromosomes 4 and 6 as- sorted randomly with respect to chromosome 16 at meiosis. Therefore, some of the progeny (33% for G392 and 31% for B312, G210 and G431) inherited both copies of the sequences hybridized by these four probes, and some (21% for G392 and 16% for B312, G210 and G431) inherited neither. Since no crossovers were found between any of the markers on chromosome 16, this chromosome probably did not pair with any of the homologous regions during meiosis. No obvious differences in growth rate, morphology or pathogenicity were detected associated with the presence or absence of any of these sequences; thus chromosome 16 and its homologous sequences are dispensable. The dispensability and

aberrant segregation of chromosome 16 suggest that it is a B chromosome.

DISCUSSION

A genetic map has been constructed for the maize pathogen C. heterostrophus (Figure 2). The total map length exceeds 1500 cM. Linkages totalling 941 cM among 120 markers were confirmed by hybridization of probes to electrophoretically separated chromosomal DNAs. Fourteen gaps between linkage groups and between linkage groups and unlinked markers on individual chromosomes were identified. Because the maximum distance which can be detected at the 5% significance level using 91 progeny is 39 cM, the minimum size of each gap should be 40 cM. There- fore, the 14 gaps should cover at least 560 cM. The total map length of C. heterostrophus can therefore be estimated to be at least 1501 cM, suggesting that the current map covers about 63% of the total map distance.

Fifteen chromosomes were identified in one strain of C. heterostrophus and 16 chromosomes were identified in the other; the chromosomes were estimated to range in size from 1.3 to 3.7 Mb. The numbers of identified chromosomes were maximized by combin- ing information about marker segregation with information about the locations of the markers on electrophoretically separated chromosomal DNAs. These estimates of the numbers of chromosomes should, however, be considered minima. If, for example, two chromosomes had migrated as a single band in both parents, unlinked markers hybridizing to such a band would have been misinterpreted as being on the same chromosome. The sizes of the chromosomes were estimated by comparisons of their migration rates in TAFE to those of the chromosomes of S. cerevisiae and S. pornbe. These estimates of chromosome sizes may be imprecise because most of the chromosomes of C. heterostrophus migrated at a rate intermediate to that of the chromosomes used as size standards.

The number of chromosomes in C. heterostrophus determined in this study is different from that previously reported. A haploid chromosome number of eight had been reported based, in one instance, on observations of an unspecified nature (NELSON and KLINE 1969) and, in a second instance, on counts of iron-hematoxylin-stained pachytene chromosomes under a light microscope (GUZMAN, GARBER and YODER 1982). This difference in reported chromosome numbers might be due to the fact that some of the chromosomes are too small to count reliably by light microscopy. Most of the chromosomes we identified in C. heterostrophus were smaller than those in fungi whose chromosome number has been successfully determined by light microscopy. For example, the chromosomes of N. crassa, which can be counted by light


microscopy (PERKINS and BARRY 1977), have been estimated to be 4.0 to 10.3 Mb (M. J. ORBACH, personal communication).

The genome of Hm540 was chosen as a reference for numbering homologous chromosomes and desig- nating differences in chromosome arrangement. The establishment of a reference strain was necessary because of numerous differences in chromosome arrangement and size between the strains and progeny used to prepare the map. Hm540 was chosen as a reference because, of the two strains mapped, its karyotype is most likely to be representative of the general C . heterostrophus population. Unlike B30.A3.R.45, Hm540 is a naturally occurring isolate, unmodified by genetic manipulation. It is also race 0, and race 0 is the only race currently prevalent in nature. The genome of Hm540 is similar to that of all of seven additional race 0 field isolates whose karyotypes have been studied. All show identical chromosome hybridization patterns for probes specific for chromosome arms associated with Toxl; six lack chromosome 16, as does Hm540 (H.-R. CHANG, L. K. LYNGHOLM and C. R. BRONSON, unpublished results). In addition, future efforts to map genes identified in C-strains will require crosses to this strain. The genome of Hm540 was therefore chosen as a standard for naming chromosomes.

Chromosome 16 in B30.A3.R.45 appears to be a B chromosome, that is, an extra chromosome without any essential function UONES and REES 1982). This chromosome, marked by 15 RFLPs, was present in B30.A3.R.45, but not Hm540. Although four single copy sequences homologous to 16 were detected at other chromosomal locations in Hm540, the sequences were not essential; 9 of the 91 progeny contained no copies of any of the single copy or moderately repetitive sequences on this chromosome. Thus, chromosome 16 appears to be completely dispensable. The detection of two insertions or deletions associated with chromosome 16 suggest that it may be unstable. The chromosome was also preferentially transmitted among the progeny, as has been reported for B chromosomes in many plants and animals UONES and REES 1982). Chromosome 16 thus appears to be a B chromosome. To our knowledge, this constitutes the most complete evidence to date for a B chromosome in a fungus. The occurrence of B chromosomes has been suggested for other plant pathogenic fungi (MILLS and MCCLUSKEY 1990). A meiotically unstable chromosome, at least part of which has been shown to be dispensable, has been recently reported in the pea pathogen Nectria haematococca. Dispensability was shown for pda, a gene controlling virulence on pea (V. P. W. MIAO and H. D. VANETTEN, personal communication). Small, presumably nonessential chromosomes have also been observed in Magnaporthe

grisea (M. J. ORBACH, personal communication). The strains used to prepare the map were shown to

differ by a reciprocal translocation, the breakpoint of which mapped to the same site as the virulence locus Toxl. These results support a previous proposal that TOXl strains differ from toxl strains by a reciprocal translocation tightly linked to Toxl (BRONSON 1988). This proposal was based on studies of ascospore abortion in crosses homozygous and heterozygous for Toxl among both C-strains and field isolates (BRONSON 1988). Demonstration of the translocation in these strains was achieved by hybridization of probes to electrophoretically separated chromosomal DNAs (H.-R. CHANG, L. K. LYNGHOLM and C. R. BRONSON, unpublished results).

The reciprocal translocation could account for some of the low fertility of the cross used to make the map and some of the similar low fertility of other crosses between t ox l and TOXl strains (YODER and GRACEN 1975; YODER 1976; BRONSON 1988; BRON- SON, TAGA and YODER 1990). Low fertility is common in crosses heterozygous for a reciprocal translocation; up to 67% of asci may abort four spores. Two of the four viable spores contain normal chromosomes and two contain translocated chromosomes (PERKINS 1974). The high frequencies of asci containing two TOXl and two toxl viable progeny observed in this and other crosses (TAGA, BRONSON and YODER 1985; BRONSON 1988) could be due to the tight linkage of Toxl to the translocation breakpoint.

The region around Toxl may have a high concen- tration of repetitive DNA; if so, similarity between nonhomologous chromosomes could have increased the probability of a chromosome rearrangement event. Of six RFLPs mapping less than 4 cM from Toxl , three were detected by probes known to hybridize to repetitive DNA; this compares to five out of 1 13 RFLPs elsewhere in the genome. Recombination between repeated genes on nonhomologous chromosomes has been shown to cause chromosome translocations UINKS-ROBERTSON and PETES 1986). Translo- cations also have been reported to occur commonly in centromeric regions and in heterochromatic DNA (GILL et al. 1980), both of which contain highly repetitive sequences.

Although the hybridization of probes to chromosomal DNAs demonstrates that the strains used to make the map differ by a reciprocal translocation, it is not clear that a simple translocation event can explain all the characteristics of the region around Toxl. An unusual number of RFLPs mapped in this region; six RFLPs mapped less than 4 cM from Toxl. By comparison, linkages among a comparable number of markers elsewhere in the genome averaged about 75 cM. There are two possible explanations for this anomaly, a high frequency of polymorphism near


T o x l and reduced recombination during meiosis, either of which could be caused by poor structural or sequence homology. Further suggestion that there may be poor homology in the region around Toxl comes from estimates of the amount of DNA in the chromosomes involved in the translocation. In this study, the total DNA in these chromosomes was about 900 kb more in the TOXl strain than in the toxl strain. Because the two strains were unrelated and showed numerous chromosome size polymorphisms, this difference may not have any particular meaning. How- ever, based on migration rates of chromosomes in pulsed field gel electrophoresis, H.-R. CHANG, L. K. LYNGHOLM and C. R. BRONSON (unpublished results) estimated that chromosomes of TOXl C-strains of C. heterostrophus may have an extra 1.2 Mb of DNA in the region around Toxl compared to closely related toxl C-strains. If there are structural and/or sequence differences in addition to the translocation, it is not clear how or when they arose relative to the translocation.

Chromosome size polymorphisms, in addition to those associated with the reciprocal translocation, were abundant between the two parental strains and the progeny (Figures 3 and 4). The deletions or insertions identified between B30.A3.R.45 and Hm540 may have contributed to differences in chromosome size. However, many of the differences can not be accounted for by known differences in chromosome arrangement. This may be because of the probes used. The probe libraries were constructed only from the C-strains and were screened to remove most, of the probes that hybridized to repetitive DNA. Therefore, any sequences present in Hm540, but not B30.A3.R.45, and any differences in the amount and location of repetitive DNA may not have been detected. New chromosome sizes were seen in the progeny, possibly due to recombination between homologous chromosomes with more than one structural difference (ONO and ISHINO-ARAO 1988). Similar chromosome size variation has been observed among other isolates of C. heterostrophus (H.-R. CHANG, L. K. LYNGHOLM and C. R. BRONSON, unpublished results), and among a variety of fungal species, for example, Ustilago maydis (KINSCHERF and LEONG 1988), S. cerevisiae (CARLE and OLSON 1985; JOHNSTON and MOR- TIMER 1986; ONO and ISHINO-ARAO 1988), Candida (MAGEE and MACEE 1987), and M . grisea (M. J. O R - BACH, personal communication).

The ratio of physical distance to genetic distance in C. heterostrophus is low. The total map distance of C. heterostrophus was estimated to be, at a minimum, 1501 cM. Based on pulsed field gel electrophoresis, the physical genome size was estimated to be about 35 Mb. Therefore, in the cross used to make the map, the kb/cM ratio is roughly 23. Because the strains

used to make the map were selected for higher than average differences in DNA sequence, this ratio may be higher than would be expected in crosses among more closely related strains. In tomato, recombination in regions heterozygous for introgressed chromosome segments is suppressed, causing increases in the ratio of physical to genetic distance (MESSGUER et al. 199 1 ; M. GANAL, personal communication). The kb/cM ratio for C. heterostrophus is in the range reported for a wide variety of fungi, for example, yeast (10 kb/cM) (FINCHAM 1983), N. crassa (about 43 kb/cM) (M. J. ORBACH, personal communication), Bremia lactucae (25 kb/cM) (HULBERT et al. 1988), and Phanerochaete chrysosporium (59 kb/cM) (RAEDER, THOMPSON and BRODA 1989).

This map of the nuclear genome of C. heterostrophus should facilitate the study of fungal pathogenesis to plants. The map should permit the isolation of genes by various marker-based cloning strategies, such as chromosome walking and jumping, and by complementation from chromosome and chromosome fragment specific libraries. Although at present only a single pathogenicity locus, T o x l , has been mapped, efforts to identify additional loci by mutation of C- strains are in progress (P. R. THORSON, L. K. LYNG- HOLM and C. R. BRONSON, unpublished results); genes identified will be mapped by crossing to Hm540. The map may also aid in the study of pathogen populations. For example, strains may be characterized by their RFLPs, chromosome size polymorphisms, differences in chromosome arrangement and the presence or absence of the B chromosome. These differences may be useful as markers for the study of pathogen spread and evolution in natural populations. The efficiency in characterizing strains and cloning genes provided by this map should encourage increased research into the complex biological and evolutionary interactions between plants and their pathogens.

Cultures of the parents, progeny and probes used to prepare the map and a list of markers in progeny are available on request from C. R. BRONSON.

We thank B. G. TURCEON and 0. C. YODER for fungal strains and cloned genes, C. S. LEVINCS I l l and Lubrizol Genetics, Inc. for plasmid pATH13, R. W. DAVIS for blueprints for the CHEF apparatus, and R. L. KEIL, S. HENIKOFF and J.-B. FAN for cloned genes. We also wish to express gratitude to D. M. BERNARD for technical assistance, to H.-R. CHANG for the preparation of Figure 2, and to D. D. PERKINS for his critical reading of the manuscript. Special thanks go to M. J. ORBACH for technical advice. This research was supported by grants 87-CRCR-1-2343 from the U.S. Department of Agriculture and PH-86-4 from the Iowa State University Agri- cultural Biotechnology Council to C.R.B. and C.F.F. Journal Paper No. 5-14469 of the Iowa Agriculture and Home Economics Exper- iment Station, Ames, Iowa, Project No. 2855.

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a restriction fragment length polymorphism map and electrophoretic

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