Suppressed Recombination and a Pairing Anomaly on the Mating-Type Chromosome of Neurospora tetrasperma

Corresponding author: Donald O. Natvig, Department of Biology, University of New Mexico, Albuquerque, NM 87131., dnatvig{at}unm.edu (E-mail)

Communicating editor: R. H. DAVIS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, MERINO et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.

IN 1927, SHEAR and DODGE described the ascomycete genus Neurospora using a combination of morphological and genetic features. One of the species described, Neurospora crassa, for several decades an important experimental organism in genetics and biochemistry, possesses a life cycle that typifies that of heterothallic perithecial ascomycetes. Each ascus produces eight homokaryotic, self-sterile ascospores (sexual progeny) that represent the products of a single meiosis followed by a mitotic division before ascospore delimitation. Each ascospore is either mating type A or a, depending on inheritance at the mat (mating-type) locus. Sequences at matA and mata are not homologous, and they have been termed idiomorphs (reviewed in GLASS and KULDAU 1992 ). The mat locus is located on the largest of seven chromosomes (linkage group I). We refer to this chromosome as the mating-type chromosome and to the remaining six chromosomes as autosomes (MERINO et al. 1996 ).

In contrast with the eight-spored N. crassa, asci of N. tetrasperma produce four binucleate, dual-mating-type (A+a) ascospores per ascus (DODGE 1927 ). Individuals resulting from such ascospores, although haploid, are heterokaryotic for mating type and thereby self-fertile. This reproductive system is referred to as pseudohomothallism or secondary homothallism. The self-fertility of N. tetrasperma results from programmed ascus development that includes segregation of mating-type idiomorphs at the first division of meiosis and overlapping nuclear spindles at subsequent meiotic and mitotic divisions (Figure 1; see RAJU 1992 ; RAJU and PERKINS 1994 ).

View larger version (48K): In this window In a new window Download PPT slide   Figure 1. Ascospore formation and tetrad analysis in N. tetrasperma. (A) In N. tetrasperma, the first-division segregation of mating types, programmed spindle alignment, and nuclear movement during meiosis and a subsequent mitosis lead to delimitation of four heterokaryotic ascospores (diagrammatic chromosomes represent different nuclear types). The ascospores exist in two pairs, with the two members of a pair being genetically identical to one another. A crossover of the type shown, between the mating-type locus (A) and a distal locus such as cyt-21 (C), therefore results in four different nuclear types (a tetratype ascus) but only two different ascospore types. (B) Tetrad analysis is therefore possible when homokaryotic strains are obtained after ascospore germination. This is true even when only a subset of the possible homokaryotic progeny is recovered from a given ascus. The analysis of cyt-21 is simplified by the fact that crossing over is suppressed between the mating-type locus and the centromere. As an example, the results obtained with ascus 36 (Table 3) are illustrated here. Homokaryotic strains representing both nuclear types (mating types A and a) were recovered from each of two ascospores (141, 142), a single homokaryotic strain (mating type a) was recovered from a third ascospore (143), and no homokaryotic strains were recovered from the fourth ascospore (144). The association of two different cyt-21 alleles (illustrated C1 and C2) with the three mating-type a homokaryons from this ascus indicates a tetratype ascus that has resulted from a crossover between cyt-21 and the mating-type locus.

Developmental and life cycle differences aside, N. tetrasperma and N. crassa are clearly closely related. Chromosomes of the two species are essentially indistinguishable cytologically, and gene order appears to be conserved (HOWE and HAYSMAN 1966 ; PERKINS 1985 ). In addition, a few viable ascospores are produced when homokaryotic strains of N. tetrasperma are crossed with N. crassa (METZENBERG and AHLGREN 1973 ), further attesting to the close relationship between the two species.

In a population study of N. tetrasperma isolates using RFLP markers, MERINO et al. 1996 found that sibling A and a strains derived from wild-type heterokaryons are highly heteroallelic with respect to sequences on the mating-type chromosome. The region examined extended from the mating-type locus on the left arm to arg-13 on the right arm (Figure 2). In contrast with sequences on the mating-type chromosome, sequences on autosomes were highly homoallelic. These results led to the hypothesis that autosomal sequences are homoallelic as a result of repeated selfing combined with crossing over, whereas sequences on the mating-type chromosomes are heteroallelic as a result of suppressed crossing over maintained over evolutionary time (MERINO et al. 1996 ).

View larger version (11K): In this window In a new window Download PPT slide   Figure 2. Comparative linkage maps of the mating-type chromosome for N. crassa and N. tetrasperma (maps drawn approximately to scale). (A) N. crassa. Numbers along the linkage group are distances between N. crassa markers, expressed as map units. This map is based on PERKINS et al. 1982 , PERKINS 1962 , and D. J. JACOBSON (unpublished data). (B) N. tetrasperma. Recombination rates are expressed as the ratio of number of recombinants over total tested for a sample of random progeny (see text). Lines and ratios in boldface are data from outcross P556A x P581a (top thick line) and selfing cross P556A x P556a (). Other ratios are from previous studies of N. tetrasperma [data of HOWE and HAYSMAN 1966 and D. D. PERKINS (unpublished data)]. Note the greatly reduced recombination frequencies relative to N. crassa for genes in the regions to the right of nit-2. In contrast, the interval between nit-2 and cyt-21 (*) indicates 50% recombination; tetrad analysis revealed that 100% of meioses contained a crossover in this interval (see text and Table 3).

The goal of this study was to experimentally test the hypothesis of suppressed recombination on the N. tetrasperma mating-type chromosome. This was done by following segregation of alleles in selfing and nonselfing crosses. The results support earlier inferences that crossing over is suppressed over a region of this chromosome estimated to exceed 100 map units. We also discovered a region on the far left arm where a crossover is, surprisingly, obligate. By examining meiotic chromosome pairing microscopically in various laboratory and wild-collected N. tetrasperma strains, we found that the recombination block is correlated with an anomalous unpaired segment measuring up to half of the length of this chromosome. The unpaired segment is interstitial, and pairing is apparently normal at both ends.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains:
Wild-collected strains of N. tetrasperma were chosen on the basis of expectations that scorable, natural polymorphic markers could be identified and that sexual crosses would produce viable ascospores. A total of 22 homokaryotic strains derived from 11 wild-type heterokaryons from diverse geographic regions had been characterized previously for relatedness (MERINO et al. 1996 ) and sexual compatibility (JACOBSON 1995 ). From this collection, two closely related, but genetically distinct heterokaryotic strains, P556 and P581, were chosen. Homokaryotic, single-mating-type sibling strains, representing the component A and a nuclei from these heterokaryons, are designated P556A, P556a, P581A, and P581a (FGSC numbers 2510, 2511, 2508, and 2509, respectively).

Identification and analysis of genetic markers:
Three genes in linkage group I (cyt-21, nit-2, and al-1) were chosen for genetic analysis of the presumed recombination block on the basis of locations relative to each other in N. crassa, the region examined in the previous population study (MERINO et al. 1996 ), and the mating locus (Figure 2A). In addition, two genes on other linkage groups, nit-3 (linkage group IV) and frq (linkage group VII), were examined.

Noncoding regions were targeted for marker development because preliminary studies demonstrated that such sequences were more likely to be polymorphic. Polymerase chain reaction (PCR) primers for initial sequence analyses were designed on the basis of published N. crassa gene sequences. For each gene, we amplified a 500- to 1500-bp fragment from each of four parental strains (P556A, P556a, P581A, and P581a), sequenced each parental fragment to identify polymorphic regions, and then designed allele-specific primers to characterize progeny using PCR. Strain-specific PCR primer pairs were designed such that target DNA from a given parental strain could be amplified using one primer pair but not the other (Table 1 and Figure 3). All strains were examined using both primer pairs for a given locus to identify which allele was inherited (Figure 3). DNAs from parental strains were used as positive and negative controls in all PCR screenings. When strain-specific PCR gave ambiguous results, alleles were confirmed by direct sequencing of a larger PCR fragment that included the polymorphic region.

View larger version (70K): In this window In a new window Download PPT slide   Figure 3. Agarose gel analysis of cyt-21 strain-specific PCR. Lane 1 is a size standard. Lanes 2–5 are parental controls using (a) a primer pair specific for the cyt-21 allele from strain P581a and (b) a primer pair specific for the cyt-21 allele from strain P556A (see text and Table 1). Note the absence of a band for the first primer pair (a) with the P556A parent (lane 2) and the second primer pair (b) for the P581a parent (lane 5). Lanes 6–13 include PCR screenings of ascospore progeny 68, 69, 70, and 71 (see Table 3). Progeny 68 and 70 inherited the cyt-21 allele from P581a, whereas progeny 69 and 71 inherited the cyt-21 allele from P556A.

Genomic DNA was prepared with a PureGene DNA isolation kit (Genetra Systems, Inc.). Agarose gel electrophoresis, PCR, and automated sequence analysis were performed as described previously (SKUPSKI et al. 1997 ).

Crosses:
Cross P556A x P556a represents a normal selfing of a heterokaryotic wild strain of N. tetrasperma. Such crosses have limitations for studies employing natural polymorphism, however, because sequence polymorphism is detected only in regions of suppressed crossing over on linkage group I. This was predicted on the basis of our previous studies with restriction-fragment length polymorphism (MERINO et al. 1996 ), and it was confirmed here in preliminary analyses. To examine segregation of heteroallelic markers on autosomes and crossover-competent regions of linkage group I, we employed the cross P556A x P581a. Natural polymorphic markers between these two strains have been found for all chromosomes examined (see below).

Crosses were made on synthetic cross (SC) medium in 9-cm Petri dishes (DAVIS and DE SERRES 1970 ). The protoperithecial parent (either P556a or P581a) was grown for 2 days at 25° and was then fertilized with mass conidia from P556A in both crosses. The selfing cross P556A x P556a was allowed to discharge ascospores onto the petri dish lid, where they aged for 10–14 days. Single, random ascospores were isolated to 13 x 75-mm tubes containing 1 ml of SC, in which they were heat shocked at 60° for 40 min, and then incubated at 25° for at least 14 days.

For the outcross P556A x P581a, the products of single asci, which represented unordered tetrads, were collected as groups of four ascospores forcibly shot from perithecia onto a block of agar (PERKINS 1974 ). The spores were aged for 10–14 days on agar medium containing 100 mM Tris, pH 8.0, and 0.5 mM EDTA (METZENBERG 1988 ). Ascospores from 50 asci, 200 total progeny, were then separated, germinated, and incubated as described above.

Because N. tetrasperma naturally produces dual-mating-type progeny, the progeny from the selfing cross P556A x P556a had to be separated into single-mating-type components to score genetic markers. This was accomplished by isolating single-conidium cultures from each progeny and testing for self-sterility and for mating type (RAJU 1992 ; JACOBSON 1995 ). At least one arbitrary A or a component from each progeny was chosen for marker scoring. The same procedure was used to separate single-mating-type components from a few selected self-fertile progeny from cross P556A x P581a. This was not necessary for most progeny from P556A x P581a, because this outcross exhibits a type of sexual dysfunction characterized by a large proportion of single-mating-type, self-sterile progeny (JACOBSON 1995 ).

Cytological examination:
The 11 selfings and two outcrosses examined are listed in Table 2. Note that the strains included were those used for progeny analysis and those used in other recent studies of the reproductive genetics of N. tetrasperma (JACOBSON 1995 ; MERINO et al. 1996 ). A and a strains were mated as described above. At 3–4 days after fertilization, strips of agar bearing developing perithecia (unfixed) were hydrolyzed and stained with the DNA-specific fluorochrome acriflavin (RAJU 1986 ). For cytological observation, 10–20 perithecia were dissected on a microscope slide, and the rosettes of asci were squashed in a drop of water under a cover glass. The edges of the cover glass were sealed with melted dental wax. The preparations were examined at x1250 with an epifluorescence microscope (excitation ~450 nm and emission 540 nm). Photomicrographs were made using Ilford XP-2 film (ISO 400) with exposures determined empirically.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We studied the segregation of selected markers in two different types of N. tetrasperma crosses. One of these, a "self-cross," employed homokaryotic strains P556A and P556a, which were derived from the same wild-type parent, P556A + a. A second cross, an "outcross," employed homokaryotic strains P556A and P581a, derived from wild-type strains P556A + a and P581A + a, respectively. P556A + a and P581A + a were collected from different sites on the Hawaiian island of Kauai (PERKINS et al. 1976 ). They are closely related but not genetically identical (MERINO et al. 1996 ). In previous studies, crosses between P556A and P581a were observed to exhibit a mild form of sexual dysfunction, characterized by heterokaryon breakdown (JACOBSON 1995 ).

The P556A x P581a cross was used in part to facilitate the identification of molecular polymorphisms that could be employed as genetic markers. MERINO et al. 1996 had shown that wild-type N. tetrasperma heterokaryons rarely exhibit heteroallelism for autosomal loci. This is in contrast to loci on the mating-type chromosome, which are frequently heteroallelic, an observation that led to the supposition that crossing over is suppressed on this chromosome. On the basis of these earlier observations, we inferred that molecular markers would be difficult or impossible to identify for the strains employed in a self-cross, except in the case of genes within the region of suppressed crossing over. Sequencing efforts of the current study proved this supposition correct.

Therefore, we employed the P556A x P556a self-cross to confirm suppressed crossing over among selected loci on the mating-type chromosome, whereas the P556A x P581a outcross was employed to examine markers on autosomes and on regions of the mating-type chromosome that are crossover competent. Together, these crosses provided a means to test the limits of suppressed crossing over with respect to both linkage group I and the remainder of the genome.

P556A x P556a:
In total, 50 random progeny from the P556A x P556a self-cross were germinated, and all were self-fertile as expected. Single-conidium isolations confirmed these self-fertile ascospore cultures as true A + a heterokaryons. A single-mating component from each of 40 of these progeny was scored for the mating-type chromosome markers nit-2, mat, and al-1. No crossovers were detected in the interval between the mat and al-1 loci. Two crossovers were detected between nit-2 and mat (Figure 2). These were confirmed as true crossovers rather than gene conversion events by assaying both nuclear components (A and a) of the ascospores; the nuclei in each ascospore showed reciprocal crossovers between nit-2 and mat.

P556A x P581a:
The parental strains in the P556A x P581a outcross were polymorphic for all markers tested, indicating the usefulness of this cross for the study of segregation. Because this cross was reported to exhibit mild sexual dysfunction (JACOBSON 1995 ), the experiment was designed to evaluate if the dysfunction might influence segregation. Ascospores shot as groups of 4 spores were collected from 50 separate asci (200 spores total). A total of 121 ascospores (60.5%) germinated and formed viable colonies. Of these 121, 98 (81%) were self-sterile and single-mating type, indicating that heterokaryotic ascospores separated into single-mating-type mycelia upon germination, often with only one mating-type component surviving. The inference that most or all ascospores from this cross begin as heterokaryons derives in part from the fact that frequently both mating types can be recovered, albeit separately, from conidia derived from self-fertile ascospores (see JACOBSON 1995 ). The levels of viability and heterokaryon breakdown observed here were equivalent to those described previously for this cross.

Two separate samples of these progeny, random progeny and tetrads, were scored for the available markers. Progeny from a given ascus are not independent, as they are products of the same meiosis. Therefore, a sample of 43 single-mating-type progeny (including 4 derived from self-fertile progeny) was obtained by selecting one strain per ascus. This random sample was used to determine overall linkage of markers in the cross. If at least 3 progeny of the same mating type survived from a single ascus, tetrad analysis could be performed with the missing markers being derived on the basis of the alleles present (Figure 1B). The sample of progeny used for tetrad analysis consisted of 67 single-mating-type strains from 19 asci (Table 3). One progeny from each ascus was also represented in the sample of 43 random strains. Of 50 total asci examined, 4 produced no viable progeny.

The allele ratios in the set of random progeny were skewed in some instances (e.g., 29 mat-A:17 mat-a, 18 frq⁵⁵⁶:18 frq⁵⁸¹, 27 nit-3⁵⁵⁶:14 nit-3⁵⁸¹). However, when unlinked markers were considered in pairwise combinations, ratios of parental to nonparental genotypes were not significantly different from 1:1 (data not shown), indicating no segregation distortion. Independent segregation of the markers in the outcross P556A x P581a therefore suggests that the sexual dysfunction observed for this cross does not affect segregation, supporting the value of this cross for segregation studies in N. tetrasperma.

Results obtained with cross P556A x P581a confirmed the region of blocked recombination on the mating-type chromosome (Figure 2), and they provided a surprising result with respect to a locus, cyt-21, outside the recombination block on the left arm. In the sample of random progeny, cyt-21 showed 51% recombination with markers within the recombination block (Figure 2). This result would be expected with genes on separate linkage groups undergoing independent assortment. However, cyt-21 maps to the far left end of linkage group I in N. crassa (KUIPER et al. 1988 ; NELSON et al. 1998 ), where it is closely linked to ro-10, with ~1% recombination (Figure 2; D. J. JACOBSON, unpublished results). Our analysis of N. tetrasperma tetrads confirmed linkage between cyt-21 and other linkage group I markers, with a single obligate crossover between nit-2 and cyt-21 outside the recombination block accounting for the high level of recombination observed for cyt-21. This conclusion stems from the fact that in all 19 tetrads examined cyt-21 segregated at the second division with respect to the linkage group I recombination block markers: 19 tetratype, 0 parental ditype, 0 nonparental ditype (Table 3). In contrast, markers on other linkage groups, nit-3 (linkage group IV) and frq (linkage group VII), segregated independently with respect to the linkage group I recombination block: 6 tetratype, 10 parental ditype, 3 nonparental ditype tetrads, and 6 tetratype, 5 parental ditype, 8 nonparental ditype tetrads (Table 3), respectively. These results indicate clearly that cyt-21 is linked to, but is outside, the linkage group I recombination block and that a single obligate crossover takes place between nit-2 and cyt-21 in 100% of meioses. Whether crossing over is blocked in the right arm, in regions distal to arg-13, remains to be determined.

Chromosome pairing at pachytene:
Homologous chromosomes are paired intimately at the pachytene stage of meiosis, and this stage has been used extensively to detect pairing anomalies in rearrangement strains (see PERKINS and BARRY 1977 ). Pachytene karyotypes of N. tetrasperma are comparable generally to those of N. crassa. Because we were interested in chromosome pairing behavior and any correlation with the observed recombination block in N. tetrasperma, we limited our observations to the pachytene stage. To ensure that observed chromosome pairing was generally indicative of the species and that there was no difference between selfing and outcrossing, 11 self-crosses and 2 outcrosses were observed (Table 2). These included the crosses examined genetically in this study and those where previous evidence of a recombination block was found (MERINO et al. 1996 ).

The rosettes examined on each microscope slide provided hundreds of asci at the desired stage. While the general chromosome pairing can be observed in most of these asci, specific pairing anomalies can best be detected in relatively rare pachytene spreads where the individual bivalents are well separated. In the analyzable spreads (10–20 nuclei for each cross), all but the longest chromosome showed intimate homologous pairing along the entire length. No difference in the pairing of autosomes was apparent when comparing self- vs. outcrosses. The longest chromosome (linkage group I, the mating-type chromosome) often showed a long unpaired segment in the middle, with normal pairing at both ends (Figure 4, A–C). This observation is consistent in all 13 matings examined in this study. However, in the eight-spored sibling species N. crassa and N. intermedia, where there is no recombination block on the mating-type chromosome, all seven chromosomes showed complete homologous pairing (Figure 4D and Figure E).

View larger version (94K): In this window In a new window Download PPT slide   Figure 4. Neurospora wild-type chromosomes at pachytene (acriflavin staining; x4000). (A–C) N. tetrasperma. (A) Outcross P556A x P581a. (B) Self-cross P505A x P505a. (C) Self-cross P535A x P535a. Note the incomplete pairing of the longest chromosome (arrows). This pairing disturbance correlates with the observed recombination block in the mating-type chromosome. Pairing is normal in all other chromosomes. (D and E) N. crassa (FGSC 3250) x N. intermedia (FGSC 2316) (from RAJU 1986 ). Note complete pairing along the entire length of the longest chromosome (arrows). The karyotypes of N. crassa and N. intermedia are similar and show complete pairing in both conspecific and interspecific crosses. The chromosomes are better spread in the interspecific cross photographed.

The cytologically detectable pairing anomaly in N. tetrasperma is restricted to the mating-type chromosome and involves only a portion of the chromosome. The pairing anomaly roughly corresponds to the position expected from the genetically described recombination block. To our knowledge, this is the first report that shows a correlation between a localized recombination block and a corresponding pairing anomaly in wild-type strains. The visual extent of the pairing anomaly emphasizes that the recombination block extends well beyond what is needed for the segregation of mating types at the first division of meiosis. Pairing of both distal regions suggests that crossing over may also occur in the distal region of the right arm of linkage group I.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Suppressed crossing over on a large portion of the mating-type chromosome:
This study genetically defined a region of reduced recombination on the mating-type chromosome of wild strains of N. tetrasperma, spanning the region from nit-2, located on the left arm, to at least al-1 approximately midway along the right arm. In the portion of this region from mat (left arm) to al-1, no crossovers were observed among 131 progeny examined from selfing and nonselfing crosses; this included samples of random progeny (Figure 2) and tetrads (Table 3). In the portion of this region from mat to nit-2 (left of mat), 2 of 40 progeny (5%) from the selfing cross exhibited crossovers, while no crossovers were observed in the same interval among progeny scored from the nonselfing cross. The observation of crossovers between mat and nit-2 suggests the possibility that recombination is suppressed less stringently in this region than in the region from mat to al-1. This is additionally supported by the fact that nit-2 appears to be homoallelic in strain P581 (data not shown). Sequences within the recombination block tend to exhibit high levels of heteroallelism when sibling a and A mating-type chromosomes are compared in wild strains of N. tetrasperma (MERINO et al. 1996 ). In any event, it is clear that crossing over in the region between nit-2 and mat in N. tetrasperma is still extremely low compared to the same region in N. crassa and in the region to the left of nit-2 in N. tetrasperma.

There is some evidence that the amount of recombination in the suppressed region varies among N. tetrasperma isolates. Employing marked strains derived from wild-type 85, HOWE and HAYSMAN 1966 detected limited apparent second-division segregation of single N. tetrasperma markers mapped to linkage group I (a maximum of 2 of 29 asci). Although the map positions of these markers [rib(125), cyh(114), lys(122), and acr(111)] have not been determined in relation to mat or the centromere, most are likely within the region of suppressed recombination. One of the markers, rib(125), apparently has no homolog in N. crassa linkage group I from which to postulate map location. Significantly, no second-division segregation was detected by HOWE and HAYSMAN 1966 for mat (0 of 474 asci) or al (0 of 27 asci). We have detected similar levels of second-division segregation in crosses involving the strains used by Howe (D. J. JACOBSON, unpublished data).

An estimate of the size of the region of suppressed crossing over in N. tetrasperma can be made by using genetic data from N. crassa as a reference and by assuming that gene order and distances between genes are comparable between species. Estimates of map distance are imprecise in N. crassa because genetic background affects recombination rate greatly and genes have been mapped using strains of numerous backgrounds. However, linkage group I is by far the best characterized chromosome and provides a good estimate of the scale of the recombination block. In a study of >1200 tetrads, PERKINS 1962 placed the distance from cr-1 to al-1 in N. crassa at 45 map units. The distance from cr-1 to nit-2 appears to be at least 45 map units on the basis of published recombination rates for markers within this interval (PERKINS et al. 1982 ). Combining the intervals gives an estimated distance from nit-2 to al-1 of at least 90 map units. The distance encompassed by the region of suppressed recombination totals at least 120 map units when extended to arg-13, which can be assigned to the region tentatively based on the RFLP data of MERINO et al. 1996 . Given the total estimated size for linkage group I of 200 map units (PERKINS 1962 ), the recombination block in N. tetrasperma involves at least 50–60% of the linkage group and 10% of the genome, estimated at 1000 map units for N. crassa (PERKINS and BARRY 1977 ). This estimate correlates well with our cytological observations of the region in question.

MERINO et al. 1996 proposed that suppressed recombination on the mating-type chromosome of N. tetrasperma is adaptive toward the goal of preserving self-fertility. Given the nuclear events associated with ascus development in this species, a crossover between the mating-type locus and the centromere would result in homokaryotic, self-sterile ascospores. The region examined previously was largely to the right of the mating-type locus. Our current results expand the region of suppressed recombination to include the nit-2 locus located substantially left of the mating-type locus. It is unclear why the linkage group I recombination block includes regions substantially left of the mating-type locus and right of the centromere, because crossing over in these regions will not result in ascospores homokaryotic for mating type.

Obligate crossing over distal to the recombination block:
A surprising result of our study is that cyt-21 recombined with markers within the recombination block with a frequency of ~50%, a frequency that would be expected as a result of independent assortment. Tetrad analysis, however, showed clearly that the cyt-21 results do not reflect independent assortment (Table 3). Independent assortment would result, not exclusively in tetratypes, but in all three tetrad classes: tetratype, parental ditype, and nonparental ditype. Both frq and nit-3, located on autosomal chromosomes, displayed all three tetrad classes with respect to the mat locus. However, only tetratype tetrads were evident for cyt-21 with respect to mat. These results indicate that there is an obligate crossover between cyt-21 and its centromere and that this crossover occurs, at least in the tetrads assayed, in the region between cyt-21 and nit-2 (Figure 2).

Obligate single crossovers have been observed or inferred to occur in two other pseudohomothallic ascomycetes, Podospora anserina and Gelasinospora tetrasperma, both in the same ascomycetous family as Neurospora, the Sordariaceae. Like N. tetrasperma, these species produce ascospores that are heterokaryotic for mating type, but such ascospores result from a very different program of ascus development, with mating type segregating at the second division of meiosis (see RAJU and PERKINS 1994 ). An obligate crossover between the mating-type locus and the centromere is required to guarantee second-division segregation of opposite mating-type alleles and to enclose them in the same ascospore (MARCOU et al. 1979 ). This is in contrast with N. tetrasperma, in which the formation of heterokaryotic ascospores depends on first-division segregation of mating type ensured by suppressed recombination between mat and the centromere. The observed obligate crossover at one end of the N. tetrasperma mating-type chromosome does not affect the assortment of mating type, and most likely serves a different purpose (see below).

Cytology:
The extent and position of the large unpaired region of the mating-type chromosome in N. tetrasperma suggest that the lack of pairing is causally related to the recombination block. Since prepachytene behavior in Neurospora does not lend itself to observations with the light microscope, we are uncertain if the unpaired region of the mating-type chromosome is deficient in presynaptic alignment or, instead, entire homologs align but subsequently fail to synapse and recombine except at the ends.

The relationship between presynaptic alignment (chromosome pairing), synapsis (indicated by synaptonemal complex), and recombination appears to vary among species. For example, synapsis is a prerequisite for crossing over in Drosophila (MCKIM et al. 1998 ) and Caenorhabditis elegans (DERNBURG et al. 1998 ). In Saccharomyces cerevisiae, however, crossing over is required for synapsis, but synapsis is not required for crossing over (reviewed in ROEDER 1997 ). Moreover, high levels of recombination in the absence of synaptonemal complexes in Schizosaccharomyces pombe and Aspergillus nidulans suggest that the yeast model may hold for other fungi as well (BAHLER et al. 1993 ; OLSON et al. 1978 ; EGEL-MITANI et al. 1982 ).

Chiasma formation is usually required for consistent chromosome disjunction at meiosis I (reviewed in ROEDER 1997 ), although certain nonrecombinant chromosomes (for example, chromosome 4 of Drosophila; HAWLEY and THEURKAUF 1993 ; DERNBURG et al. 1996 ) possess other mechanisms to ensure fidelity in chromosome segregation. Without at least one chiasma, the mating-type chromosome of N. tetrasperma might be subject to nondisjunction. The pairing at both ends observed here and the obligate crossover between cyt-21 and nit-2 likely reflect the need to maintain normal disjunction.

Why the single obligate crossover in linkage group IL?
It is reasonable to hypothesize that at least one chiasma per bivalent is necessary to ensure proper disjunction at anaphase I. The obligate exchange we have observed in the distal region between nit-2 and cyt-21 would provide such assurance. The question arises why one and only one exchange occurs between nit-2 and cyt-21, making this interval 50 map units long in N. tetrasperma, whereas it is only 26 map units long in N. crassa (Figure 2). The results suggest complete chiasma interference, although the underlying mechanism remains unknown. A comparable situation has been described in P. anserina for the region between the centromere and the mating-type locus, which shows 98% second-division segregation as the result of the obligate exchange, which is not localized at an invariant hotspot, but may occur at more than one site within the region (MARCOU et al. 1979 ).

It remains to be determined in N. tetrasperma if crossing over occurs to the right of the region of suppressed recombination, as might be expected from cytologically observed pachytene pairing, or if recombination there shows anomalies of high interference or chiasma localization, as has been shown for the left end.

Evolution of sex chromosomes and N. tetrasperma:
Animal sex chromosomes are commonly assumed to have evolved via a pathway that included an initial breakdown in recombination, followed by divergence between the chromosome responsible for the heterogametic sex (usually designated the Y chromosome) and that responsible for the homogametic sex (usually designated the X chromosome; CHARLESWORTH 1978 ; RICE 1987A , RICE 1987B , RICE 1994 ). It has been proposed that mammalian sex chromosomes, for example, evolved from autosomes that were homologous, with the exception of one small sex-determining region (GRAVES and FOSTER 1994 ). The pseudoautosomal region of the Y chromosome appears to be a relic of this homology. Furthermore, the pseudoautosomal region is assumed to be essential for proper chromosome segregation at meiosis I. The mating-type chromosome of N. tetrasperma therefore appears to share features of this proposed evolutionary pathway, insofar as there is suppression of recombination over a large segment, with adjacent regions that exhibit normal pairing and recombination.

	FOOTNOTES

¹ Present address: Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195.
² Present address: Celera Genomics, 45 W. Gude Dr., Rockville, MD 20850.

	ACKNOWLEDGMENTS

We express our sincere thanks to David Perkins and Robert Metzenberg for helpful advice and for sharing unpublished information. This work was supported by grants from the National Science Foundation to D.O.N. (MCB-9603902) and D.J.J. (MCB-9713015). Alena Gallegos was supported in part by the Minority Biomedical Research Support program of the University of New Mexico supported by the National Institutes of Health (GM-52576). N. B. Raju was supported in part by National Science Foundation grant MCB-9728675 to David Perkins.

Manuscript received August 16, 1999; Accepted for publication October 6, 1999.

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