Copyright 0 1996 by the Genetics Society of America

Correlation of Genetic and Physical Maps at the A Mating-Type Locus of Cop;nuS cinereus

Lewis Lukens,* Huang Yicunt and Georgiana May*

*Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108 and tCentre for Natural Product Research, Institute of Molecular and Cell Biology, National University of Singapore, Singapore 11 9260

Manuscript received May 17, 1996 Accepted for publication September 14, 1996

ABSTRACT The A mating type locus of Copnnw cinereus is remarkable for its extreme diversity, with over 100

different alleles in natural populations. Classical genetic studies have demonstrated that this hypervaria- bility arises in part from recombination between two subloci of A, alpha and beta, although more recent population genetic data have indicated a third segregating sublocus. In this study, we characterized the molecular basis by which recombination generates nonparental A mating types. We mapped the fre- quency and location of all recombination events in two crosses and correlated the genetic and physical maps of A. We found that all recombination events were located in 6 kb of noncoding DNA between the alpha and beta subloci and that the rate of recombination in this noncoding region matched that generally observed for this genome. No recombination within gene clusters or within coding regions was observed, and the two alpha and beta subloci described in genetic analyses correlated with the previously characterized alpha and beta gene clusters. We propose that pairs of genes constitute both the sex determining and the hereditary unit of A

T HE extreme diversity of fungal and plant mating types found in natural populations has long fasci-

nated evolutionary biologists (WRIGHT 1939, 1960, 1964) and geneticists (EMERSON 1938, 1939; RAPER 1966). As in plant S loci, basidiomycete fungi have com- patibility loci encoding a very large number of mating types. For the Copn'nus cinereus A mating type locus, over 100 types are estimated to occur, and any two different mating types are sufficient to allow a compatible mating between two haploid cells (WHITEHOUSE 1949; RAPER

1966). With the presence of so many mating types in natural populations, the chance of two haploids meet- ing with common and incompatible mating types be- comes very low (IWASA and SASAKI 1987). Although re- combination within the sex determining loci of algae (FERRIS and GOODENOUGH 1994), animals (CHARLES WORTH 1994), plants (CLARK and KAO 1991; Boms and NASRALLAH 1993), and filamentous ascomycete fungi (GLASS et al. 1990; STABEN and YANOFSKY 1990) is not observed, it has long been recognized as an important process generating variation at the A mating type locus of basidiomycetes (RAPER 1966).

The correspondence of the genetic and physical maps for the A locus has not been established for any agaric Basidiomycete and therefore we have a limited understanding of how variation is generated. For C. cinereus, the role of recombination in generating new mating types was first recognized when DAY (1960)

Corresponding authm: Georgiana May, Department of Plant Biology, 1445 Gortner Ave., University of Minnesota, St. Paul, MN 55108-1095. E-mail: gmay@maroon.tc.umn.edu

Genetics 144: 1471-1477 (Decernher, 1996)

mapped two closely linked, segregating subloci within A, which he termed alpha and beta. Using recombi- nants, DAY showed that a genetic difference in either region was sufficient for compatibility. Recent molecu- lar analyses have demonstrated that A is composed of several gene pairs clustered in two regions. These re- gions have been termed the alpha and beta gene clus- ters to correspond to DAY'S description of the locus. An allelic difference at any gene pair between two mating strains is sufficient for compatibility (MAY et al. 1991; KUES et al. 1992). However, a view of the A locus as having two segregating subloci conflicts with results of population genetic analyses, which have demonstrated that three subloci segregate in natural populations. Each segregating sublocus corresponds to a gene pair, and each pair exhibits moderate levels of allelic varia- tion. Thus, the hypervariabiltiy of A is accounted for by different combinations of three, variable subloci (MAY and MATZKE 1995).

Because of the discrepancy between genetic and pop- ulation genetic analyses, we mapped the location and determined the frequency of recombination events rel- ative to the gene pairs and to the alpha and beta gene clusters within the A locus. We used the method of DAY (1960) to first select for recombinants in the chromo- somal region surrounding the A locus and then to screen for recombination events within the A locus, which generated new A mating types. We recovered recombinant A mating type loci from two different crosses and calculated recombination frequencies. The positions of recombination events were then mapped using probes derived from the A mating type region

1472 L. Lukens, H. Yicun and G. May

(MAY et al. 1991). All recombination events occurred in only one, noncoding region of the A mating type locus between the alpha and beta gene clusters. Recombina- tion within the beta gene cluster, as demonstrated in population genetic analyses, must occur quite rarely.

MATERIALS AND METHODS

Mating system: In the dikaryon, the A locus controls the synchronized division of parental nuclei and the initiation of the clamp cell (SWIEZYNSKI and DAY 1960a). Only one gene pair in A must be different from its homologue's counterpart for sexual reproduction to proceed (MAY et al. 1991; KUES et al. 1992). Fully compatible matings and complete sexual development require that the two mating haploids must also differ at B, which controls clamp cell fusion and nuclear mi- gration. Fully compatible interactions result in a dikaryon in which each cell contains the two parental nuclei and is joined to the neighboring cell by the clamp connection. When test- ing for compatibility at A, we made crosses such that B was also compatible and used the presence of fused clamp connec- tions as a marker for a compatible interaction.

Crosses and fruiting: Haploids were crossed by placing blocks of inocula 3 mm apart on complete YMG/T media and incubating at 37" until dikaryotic cells with fused clamp connections were produced (YMG/T media: 0.2% yeast ex- tract, 0.5% malt extract, 0.2% glucose, 0.01% tryptophan, 1.6% agar). Resulting dikaryons produced fruit bodies and haploid basidiospores after 1-2 wk at a 24" 12-hr light/lBhr dark cycle. Spores were removed from the fruit bodies by vortexing small pieces of fruit body in YMG/T liquid media. A hemocytometer was used to determine spore concentration.

DNA manipulations: DNAs were extracted from all strains as described by ZOIAN et al. (1986). Approximately 500 ng of each DNA sample were digested with the restriction endonu- clease enzymes necessary for mapping, and fragments were size fractionated in 0.8% agarose gels in l x TAE with -0.1 ng of 1 kb ladder (Gibbco/BRL Life Sciences) as a size marker. The DNA was then transferred to MagnaGraph nylon membranes (Micron Separations Inc.) as previously described (MAY and MATZKE 1995).

Probes used in mapping experiments span the A locus and have been described (MAY and MATZKE 1995). Probes were labeled with EJ2P]dCTP as per FEINBERG and VOGELSTEIN (1983) except that the labeling reaction proceeded for 10 hr at 21". Hybridization of probes to sequences bound to nylon membranes proceeded for 2.5 hr at 65" in Rapid Hybridiza- tion Buffer (Amersham Life Sciences, Amersham, UK). Blots were washed with 2X SSC (0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS) for 15 min at 65", followed by a 30-min 24" wash in 2~ SSC, and a 30-min wash at 65" in 0.2 X SSC/O.l% SDS. The blots were then exposed to Kodak X-omat film for 12- 72 hr prior to developing.

RESULTS

Selecting recombinants in the A chromosomal re- gion: Because 0.07 map units separate the alpha and beta subloci of A, we reasoned that we should recover recombinant A mating type loci in only seven of 10,000 spores (DAY 1960). Our strategy was to use DAY'S ap- proach, which was to first select for recombinants in the A chromosomal region and then to mate these re- combinants with parental A testers to screen for recom- binant A loci with nonparental A types. To select for

TABLE 1

Strains used in crosses and in determining A mating types of haploid spores

Strain name Genotype Origin

5037"' Aq3 Bd3 pabl-1 T. KAMADA, Okayama

5132" A ; B; ade8-1 T. KAMADA

128" Bq2 ade8-1 MAY lab H001" A , Bqj tester MAY lab H005"sb A43 B7 tester MAY lab

University, Japan

P1h AI2 B7 tester MAY lab

"These strains were used as parentals and/or testers in

These strains were used as parentais and/or testers in cross cross No. 1 (5037 X 5132).

No. 2 (5037 X 128).

recombinants in the A chromosomal region, we crossed parental strains with flanking auxotrophic markers. One parent carried pub 1-1, which confers para-amino- benzoic acid auxotrophy and is located 0.5 map units proximal to the A locus. The other parent carried a& 8-1, which confers adenine auxotrophy and is 1.3 map units distal to the A locus (Table 1, Figures 1 and 2). Recombination events in the pnbl-ade8 interval result in Pab+ Ade+ spores and the reciprocal, Pab- Ade- spores. To select the Pab+ Ade' recombination prod- ucts, spores were spread on minimal media (MOORE and PUKKILA 1985) where only Pab+ Ade+ colonies grew vigorously. Other haploid colonies, assumed parental auxotrophs and Pab- Ade- recombinants, grew very poorly and were not recovered by this selection. Conse- quently, all recombination events were mapped using the Pab+ Ade' recombinants.

Screening for A locus recombinants: To recover non- parental A mating types, we crossed all Pab+ Ade' pro- totrophic strains with testers for the parental A mating types (Table 1). To ensure correct scoring of A mating types, test crosses were made such that the B mating type of the tester differed from that of the putative A recombinant. We scored A mating types as different and thus compatible if clamp connections on the outer margins of the mating colonies were observed (CASSEL- TON 1978).

Because strain background may influence recombi- nation at A, two crosses were made to reduce the chance that our results were strain specific. Strain 5037 (A43 B43pabl-1) was a parent in both crosses and was paired with either strain 5132 (A7 B7 ade8-1) or strain 128 (A42 B42 ade8-1; Table 1). We used 5037 in both crosses because all mating type genes have been mapped (KUES et al. 1994b) and because cloned fragments along the entire length enabled molecular mapping of recombi- nation events. Restriction site differences in A between these parents allowed us to map recombination events using restriction fragment length polymorphisms (RFLPS) .

Genetic and Physical Maps o f A

SB p8 893

A43 P5 H3 H4 B H S H B KH S K S G H H H

I I I I I I t

P2

pab 1-1'' 7.0 I ! ! 5.7 5.7 3.8 '0.9 I 1 5 I

a7 a2 -4 bl

a gene cluster - f3 gene cluster

1 4 Z

A 7 S H H KH S KGHS H

10.2 '0.8' 2.8 ' 2.0 ' 2.3 I 8.0 ade 8- 1

I I

31% 3% 62% 3% - 1 5 kb

FIGURE I.-Mapping recombination events in cross 1: ,443 X A7. Physical locations of recombination events rcsdting i n nonparental A mating types were mapped using restriction site polymorphisms across the A locus and the probe DNAs d i a - grammed at the top of the figure. The bar below the A i map denotes the different regions t o which recombination events w r c mapped: the 0.Skb IfindlII f'ragment in A7 (grey filled box); the 0.9-kb i n t e n d betwen a Hind111 site i n A7and a BnntHI sitr in tt4? (open box); the 3.9-kb intend between a BnmHl site i n A43 and a Hindlll site in A i (f0nvard slash box); and the O.fi kb intenal between a IfindIlI site in A7and a Snrl site in A43 (solid filled box). Of the 32 events analyzed, the percentage of each class of recombination events is shown below the bar. Fragment sizes refer t o IfindlII fragments only, and Ifindlll sitcs are shown in bold. Restriction sites: R, RnmHI; G, 13gilI; H, Ifindlll; K, K p l ; S, Snd. Map orientation: the centromere is Ieft\vard (proximal) and the telomere is rightward (distal) to the A locus as shown here.

In the first experiment, we crossed 5037 (A43 R43 i )nbl - l ) with5132 (A7117ndr8 - I ) . Strain 5132wasused because the A43 and the A i loci do not share alleles at any of the gene pairs. Thus any recombination event within A would yield a new mating type and be detected in our screen. The Hind111 restriction site map of 5132 had previously been determined (MAY and MATZKF. 1995). In the second experiment, we crossed 5037 with 128 (A42 1142 a d d - I ) . Strain 128 was constructed from a cross of 5132 and Java6 (A42 R42) and was used be- cause the A42 mating type locus is well characterized and has previously been compared with the A43 locus (K~:I.:s r / nl. 1994b). However, the A42 mating type shares the d l - I allele with the A43 mating type, and any recombination events that occurred between the kgene pair and the d l gene would not have been detected as new, nonparental mating types.

After isolating prototrophic colonies demonstrating nonparental A mating types, we mapped restriction sites in A, compared them with sites unique for each parent, and determined the region in which recombination had occurred (Figures 1 and 2). M'e aligned the maps of different A loci at the Hind111 site spanned by probe P8 because it is consenred in most A loci, including A 7, A42 and A43 (MAY and MATZKE 1995). In an average of 10% of all prototrophs, RFLP patterns of both parcn- t a l A loci were identified. These were scored as hetero- karyons, and we excluded these progeny from further analyses. The results of subsequent mapping experi- mentq described below confirmed that all of the strains reported here are haploid, prototrophic strains re- sulting from recombination events occurring between the pnhl-I and the n&8-1 auxotrophic markers. M7e re- covered 32 prototrophic isolates with recombinant A

mating types from the first cross, A43 X A i , and 12 isolates with recombinant A mating types from the scc- ond cross, A43 X A42.

Genetic mapping of the A43 X A7 cross: In the first cross, we selected 81 Pab' Ade' recombinants from 21,000 spores and calculated a map distance of 0.77 map units between f ~ n h l and nrk.8. This distance is con- siderably less than the 1.81 map units estimated hy DAY (1960), although in DAY'S experiments, distances varied from 1.04 to 1.85 cM. Five additional selection experi- ments recovered a total of 302 Pab' Ade' prototrophs from which 32 recombinant A mating types (10.6%) were identified by crosses with parental A types. Al- though the rate of recovering prototrophs was approxi- mately half that calculated by DAY, the rate of recovering new A mating types was approximately hvice that calcu- lated by DAY, and we thus obtained a map distance within A of 0.077 cM. This frequency is very similar to that reported by DAY, who estimated 0.068-0.076 cM in reciprocal crosses. To obtain greater map resolution for recombination events within the j)nhl-ohP8 intend, we analyzed an additional 22 Pab'Ade' recombinants by Southern blot analysis of Hind111 digested genomic DNA. Using a 50-kb cosmid clone, Pd11, which covers the region j)nhl to A (courtesy Dr. I,. C,\ssl:.f:ros; Ox- ford University) as a probe, we detected no rccombi- nants within this in tend . Hybridizing a cosmid clone covering the entire A mating type locus and flanking regions (C20SE12; MAY r / nl. 1991 ) to the same blot revealed that three prototrophs (14%) recombined within the A loc~w. We inferred that the remaining 19 (86%) recombined in the intewal A to 011~8. M'e had expected to obtain approximately 6 recornbinants in the ~INI)I-A intenal (0.5 map units pnbl-A/1.81 map

1474 L. Lukens, H. Yicun and G. May

SB P8 893 P5

H4 H3

A43 B H S H B KH S K S G H P2 H H

I I I I I I I I I pab 1-1' 7.0 I ! ,12 I 5.7 I 5.7 3.13 ' 0.9 15 I

a1 a2 L - w bZ

a gene cluster - p gene cluster

A42 H B H H H H H H H I I I . I A

9.0 I 4.5 I 1.8 ' 2.9 '0.6' 3.7 I 3.8 0.9 14.5 adeb- 1

a2_

100% - 1 5 kb

FIGURE 2.-Mapping recombination events in cross 2: A43 X A42. Recombination events resulting in nonparcntal A mating types were mapped as in cross 1 using the same set of probes derived from the A43 locus. Twelve recombination evcnts were mapped to the noncoding region (grey filled box). Fragment sizes refer to Hind111 fragments only and Hind111 sites arc shown in hold. Restriction sites: R, BrtmHI; H, HindIII; G, BgllI; K, KpnI; S, Sad. Map orientation: the centromere is leftward (prosimal) and the telomere is rightward (distal) to the A locus as shown here.

units pnhl-adP8 X 22 Pab' Ade' recombinants). The skewed recovery of recombinants suggests that linkage distances between the Pab and Ade markers differ be- tween DAY'S parental strains and those used in this study.

We next determined if recombination events oc- curred in only one region within the A locus. We rea- soned that if crossing over had occurred in only one location between two subloci ( . g . , the alpha and beta gene clusters), we would recover only one, nonparental A mating type for all prototrophs tested. If crossing over had occurred in two locations to yield three segregating subloci, as inferred from population genetic studies, we would recover two nonparental A mating types (WY and MATZKE 1995). All isolates with nonparental A mat- ing types were crossed against each other such that the mated strains both had recombinant A mating types and different R mating types. All 32 recombinant, non- parental A loci exhibited the same A mating type and thus were incompatible with each other at A. This result is consistent with a single crossover region between two subloci.

Physical mapping of A43 X A7 recombinants We mapped the physical location of recombination events in all 32 nonparental A recombinants from the first cross. Starting at the proximal end of the locus, we worked toward the distal end using successive and over- lapping probe DNAs (Figure 1 ) . Nonparental frag- ments indicated a recombination event and were justi- fied to the parental maps. M'e improved on the previously reported restriction map for A7 (strain 5132; WY and MATZKE 1995) by mapping additional sites for Sod and KpnI and calculating the size of a small region within the a-gene pair that does not hybridize to avail- able probes. We report most recombinant events rela- tive to mapped Hind111 fragment sizes, and these are represented in bold in Figure 1.

Ten of the 32 events (31.2%) initiated in the 0 . 8 4 ~ HindIII fragment of A7 (grey fill box, Figure 1). The following experimenh established this result. Probe P.5 hybridized to the 10.2-kb HindIII A i fragment and probe H4 hybridized to the same 10.2-kb fragment and a 3.6-kb nonparental Hind111 fragment (0.8 + 2.8 kb) . Probe H3 hybridized to the 5.7-kb Hind111 fragment characteristic of the A43 parental type. T ~ u s , the 3.6- kb fragment must have resulted from a crossover event within the 0.8-kb fragment of A7 (grey fill box, Figure 1 ) . Results with probe P8 confirmed this result, as P8 hybridized to both the H4-hybridizing fragment (the 3.6-kb recombinant fragment) and the HS-hybridizing fragment (the A43 5.7-kb Hind111 fragment). The 0.8- kb Hind111 A 7fragment was not present in these recom- binant A loci; thus, crossover to A43 must have occurred prior to the Hind111 site on the right of this fragment. Subsequent hybridization experiments with distal probes Bg3 and P2 revealed only the A43 fragment patterns.

Twenty of the 32 recombination events (62.5%) oc- curred within the region spanning the 2.8- and 2.0-kb Hind111 fragments of A 7 (forward slash box, Figure 1). Probe H4 showed that the 2.8-kb HindIII fragment of A7 was intact, and probe H3 demonstrated a 5.7-kb Hind111 fragment of the same size as the H3 fragment in A43. However, because the HindIII site spanned b y probe P8 is apparently conserved in thesc strains, cross- over events initiating either to the left or the right of this site would still produce the 5.7-kb HindIII fragment observed. Probe P8 confirmed the presence of both the 2 .8 and 5.7-kb Hind111 fragments. Subsequent mapping of Sac1 sites followed by hybridization of Snd/HindIII genomic digests confirmed that all these recombination events were leftward of both the Sac1 site just within the h l gene of A43 and the HindIII site that forms the right boundary of the 2.0 khfragment. In all crossovers of

Genetic and Physical Maps of A 1475

this class, the left boundary of recombination events was established as the BamHI site in the H4 hybridizing fragment of A43 by probing BamHI and BamHI/KpnI digests with probe H4. The BamHI site of A43 was not present in these recombinants. Further refinement of the recombination map for this region was precluded by the high conservation of restriction sites between these two strains in this region. Thus, the recombina- tion interval for this class of events was mapped between the BamHI and HindIII sites as shown in Figure 1 (for- ward slash box). One recombinant of 32 did contain both the BamHI site in H4 (A43) and the 0.8-kb HindIII fragment of A7 (Figure 1). These observations placed the crossover event between the HindIII and BamHI sites as shown in Figure 1 (open box).

One complex recombination event occurred distally to those reported above but exhibited the same recom- binant A mating type as determined by compatibility tests (solid fill box, Figure 1). Using the H4 and H3 probes as above, we detected the 0.8-, the 2.8- and a 2.0- kb HindIII fragments of the A7 parental mating type. However, probe H3 did not detect an A72.3-kb HindIII fragment but did hybridize to a new 3.8-kb HindIII frag- ment in genomic digests. Using BglII and BglII/HindIII digests, probe H3 revealed a 2.5-kb BglII fragment and confirmed that a crossover from A7 to A43 occurred distal to the 2.0-kb HindIII fragment and within the 2.3- kb region of A7 (Figure 1). Using probe P8 on Sad/ HindIII digests to confirm that A7 fragments were in- herited in the region proximal to this crossover, we detected the expected A7 fragments, 0.7- and 1.5-kb SacI/HindIII and the proximal 2.8-kb HindIII fragment. However, an additional 1.9-kb nonparental band was observed and we interpret this to be the product of an unequal exchange event within the 1.8-kb Sac1 fragment of A43. Outside of the region covered by the P8 and H3 probes, proximal and distal probes revealed only A7 and A43 bands respectively. Consequently, more than one exchange event must have occurred in this region.

The results of the mapping experiments correlated well with our genetic results and demonstrated that the A mating type locus has chiefly two segregating regions. Of the total of 32 recombinants, all recombined within the -6 kb of noncoding sequence between the alpha and beta gene clusters as designated by KUES et al. (1992). Within that region, one-third recombined within a 0.8-kb region immediately distal to the a2 gene, and two thirds recombined elsewhere. One event was apparently more complicated and involved an apparent duplication. This latter event was the only event docu- mented that could have extended into mating type gene coding regions. With a physical distance of 6 kb and a genetic distance of 0.077 cM, we calculated the relation- ship between the physical and genetic distance as 77.9 kb/cM.

Genetic mapping of the A43 X A42 cross: In the sec-

ond cross, our selection yielded 32 Pab’ Ade’ prototro- phic recombinants out of 57,000 spores plated. To cal- culate the map distance between the pabl-l and ade8-1 markers, we corrected for the low germination rate (33%) observed in spores from this cross and obtained a map distance of 0.34 map units. Additional selection experiments recovered a total of 123 total Pab’ Ade’ spores from which 12 recombinant A mating types were recovered (9.7% of prototrophic strains). As in the first recombination experiment, strains carrying recombi- nant A mating types were crossed among themselves. A single nonparental A type was detected, indicating that just two subloci segregated within A. The calculated distance between these subloci was 0.03 map units. However, this estimate may be unreliable because low and variable germination rates greatly hampered our ability to directly determine the total number of viable spores in the experiment.

Physical mapping of the A43 X A42 recombi- nants All 12 A recombinants from the second cross recombined within the region between the alpha2 and beta1 genes (Figure 2). For all recombinant A loci re- covered, we used the same method of Southern analysis as described above for cross A43 X A7. Probe SB de- tected 9- and 4.5-kb HindIII fragments characteristic of A42. Probe H4 detected the same 4.5-kb HindIII fragment. Probe H3 yielded the 5.7-kb HindIII frag- ment of the A43 locus, and probe P8 demonstrated that the 4.5- and 5.7-kb HindIII fragments were in fact adjacent to one another. Because of a paucity of dif- fering restriction sites in appropriate locations, we did not further map restriction sites to locate recombina- tion events more exactly. Similar to the first cross, the left boundary of the recombination interval was at the 3’ end of the a2 gene and the right boundary was at the 3’ end of the b l gene (Figure 2). All recombination events resolved in the noncoding region between the alpha and beta gene clusters.

DISCUSSION

Our analysis of recombination within the C. cinereus A mating type locus revealed two segregating regions within A; these two regions correspond to the alpha and beta gene clusters identified by KUES et al. (1992). The recombination frequencies obtained suggest that the alpha and beta gene clusters do, in fact, correspond to the alpha and beta “subunits” described by DAY (1960). Of equal interest is the lack of recombination events within the beta gene clusters and within gene coding regions. Moreover, recombination events within the pabl-ade8 interval, but outside of the region cur- rently characterized as the A locus, did not generate new A mating types. These results demonstrate that all of the genes encoding mating specificities within the A locus have been described.

The findings reported here contrast with the descrip-

1476 L. Lukens, H. Yicun and G. May

tion of three segregating regions corresponding to the a, b, and d gene pairs in natural populations (MAY and MATZKE 1995). In our current experiments, we did not recover recombination events between the b and d gene pairs within the beta cluster and predict that such events must occur rarely. Our working model is as follows. First, because of sequence dissimilarities, rates of re- combination are very low between the b and d gene pairs within the beta cluster. Second, frequency depen- dent selection will retain novel, nonparental A mating types generated by such recombination events when they occur in small, locally dispersed populations where parental A types may be common. This model could be tested by isolating spores until two different nonparen- tal A types are recovered and by determining if different A mating types from local populations are composed of the same set of alleles for the a, b, and d gene pairs but in different associations.

Given that the A locus encompasses ca. 25 kb of cod- ing and noncoding sequence, it is striking to find that all recombination events mapped within the 6-7-kb noncoding region between gene clusters. For the first cross, one-third of the events could be further localized to an 800-bp region immediately beyond the 3‘ end of the a2 gene. For both crosses, most events mapped to the middle of the noncoding region and further toward the b l gene, part of the beta cluster. Although all re- combination events apparently occurred within the noncoding region of A, there is little evidence for a recombination hotspot in this area. Nonparental A al- leles were recovered from 0.08% of all spores; a fre- quency comparable with that reported by DAY (1960). A recombination frequency of 0.08% over the approxi- mate 6-kb noncoding region gives a ratio of genetic distance to physical distance of 77.9 kb/cM. Few studies have correlated the physical and genetic maps of basid- iomycetes. However, MUTASA et al. (1990) demonstrated a physical distance of 50 kb for the interval pub1 to A wherein DAY estimated a genetic distance of 0.5 map units (DAY 1960). Thus, although the distribution of recombination events over the entire A region is discon- tinuous, this result is not likely due to an enhanced recombination rate within the noncoding region.

Perhaps the most remarkable finding of this paper is that recombination events are so thoroughly precluded from the coding regions and 5’ regulatory regions of any A gene. In yeast (WU and LICHTEN 1994), recombi- nation events often initiate near or in the 5’ regulatory regions. In maize (XU et al. 1995), recombination events have been shown to resolve in 5’ regions of genes at a high frequency. Because the A genes are transcribed (KUES et al. 1992), the very low levels of intragenic re- combination cannot be related to inaccessible DNA structure. Rather, low rates of recombination may sim- ply be due to the fact that DNA sequence identity in the 5’ coding sequences and associated promoters can be as low as 50%. Apparently, recombination events

have homogenized the sequence of the noncoding re- gion while frequency dependent selection for new al- leles has driven coding regions to high levels of se- quence diversity. The larger evolutionary question remains; early in their evolutionary history, did alleles diverge so rapidly that they escaped recombination?

The mating compatibility loci of basidiomycete fungi and of many plants exhibit hypervariability at the molec- ular level (CLARK 1993; RIVERS et al. 1993; SPECHT et al. 1994; MAY and MATZKE 1995). However, recombination is not apparently a source of variation at S loci of plants (CLARK and KAO 1991; Boms and NMRALLAH 1993), while it plays a large role in generating the variation at A. Taken together, the results of population and molecular genetic analyses demonstrate that within A, recombination occurs between gene pairs but not be- tween members of the gene pair or within coding re- gions of these genes. Should recombination occur within the gene pair and lead to a new association of alleles, the resulting haploid would be self-compatible and constitutively express sexual developmental path- ways (KUES et al. 1994a). The fact that such recombi- nants are not recovered from natural populations sug- gests that a self-compatible haploid would be at a selective disadvantage (GILLISSEN et al. 1992; CHARLES WORTH 1994) and that recombination within gene pairs is extremely rare. Although we cannot exclude the pos- sibility that recombination within gene pairs occurred more frequently early in the evolutionary history of these loci (CHARLESWORTH 1994), the extreme se- quence divergence between alleles at any gene pair could well be the result of low recombination rates over evolutionary time. While maintenance of linkage groups involved in sex determination is a common theme in plant and fungal compatibility loci as well as in organisms with sex chromosomes, C. cinereus has evolved an unusual recombination system for generat- ing hypervariability at the A locus. In C. cinereus and other basidiomycetes, the necessary linkage group con- sists only of the members of a single gene pair.

We thank TAKASHI KAMADA for the generous donation of C. cinereus strains as listed in Table 1. We thank LIZ MATZKE and MARK DAHL QUIST for technical assistance and SHAWN WHITE for a critical reading of the manuscript. The work was supported by the following grants: National Science Foundation DEB49307591 to G.M. and a NSF-REU supplement to L.L. and G.M. H.Y. was supported by Academica Sinica (P. R. China) during the work described here.

LITERATURE CITED

BOYES, D. C., and J. B. NASRAIIAH, 1993 Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica okacea. Mol. Gen. Genet. 236: 369-373.

CASSEI.TON, L. A., 1978 Dikaryon formation in higher Basidiomy- cetes, pp. 275-297 in Filamentous Fungi, edited by J. E. SMITH and D. R. BERRY. John Wiley and Sons, New York.

CHARLESWORTH, B., 1991 The evolution of sex chromosomes. Sci- ence 251: 1030-1033.

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Genetic and Physical Maps of A 1477

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Communicating editor: M. E. ZOIAN