Somatic recombination in the cultivated mushroom Agaricus bisporus

Mycof. Res. 100 (2): 188-192 (1996) Printed in Great Britain

188

Somatic recombination in the cultivated mushroom Agaricus bisporus

JIANPING XU, PAUL A. HORGEN A N D JAMES B. ANDERSON* Department of Botany, University of Toronto, Mississauga, Ontario L5L 1C6, Canada

Agaricus bisporus, the cultivated button mushroom, has a mostly secondarily homothallic life cycle. This mode of sexual reproduction could limit outcrossing and recombination among homokaryons in natural populations and also creates difficulties in mushroom breeding. An alternative source of recombinant genotypes is from somatic pairings of heterokaryons. In this study, two homokaryon x heterokaryon and three heterokaryon x heterokaryon pairings were made to examine the possibility of somatic recombination. Four subcultures from the confrontation zone of each pairing were taken for analysis of restriction fragment length polymorphisms at 18 nuclear loci representing seven linkage groups and two regions of mitochondria1 DNA. A strikingly high frequency of somatic recombination was observed. Five of the eight subcultures from the two homokaryon x heterokaryon pairings and five of the 12 subcultures from the three heterokaryon x heterokaryon pairings were recombinant. No loss of marker alleles was detected in any of the self-self pairings of the six original strains. No recombination was observed between the two regions of mtDNA examined in this study. The recombination among nuclear loci involves nuclear reassortment, exchange of genetic material between nuclei, and, in one case, crossing over between markers located on the same chromosome. While the mechanism of somatic recombination in A. bisporus is not known, heterokaryons might be used in pairings with other heterokaryons or with hornokaryons to produce abundant recombinant genotypes for mushroom breeding.

In the majority of basidiomycete fungi, genetic individuals originate with the mating of two genetically non-identical gametic nuclei that remain associated with one another in a heterokaryotic mycelium, but d o not fuse until just prior to meiosis in the basidia of fruit-bodies. Genetic exchange and recombination, however, are not necessarily limited to the sexual cycle. A low frequency of exchange and recombination may occur between the component nuclei of unpaired heterokaryons (Parag, 1962). Furthermore, when a heterokaryon contributes gametic nuclei to a homokaryon in a process known as the Buller phenomenon (Raper, 1966), some of the fertilizing nuclei contributed by the heterokaryon are of recombinant genotypes (for a recent review, see Carvalho, Smith & Anderson, 1995). There is also evidence that genetic exchange can occur between heterokaryons. In Heterobasidion annosurn, pairings of heterokaryons yield subcultures that are nonparental with respect to somatic incompatibility reactions (Hansen, Stenlid & Johansson, 1993), a recognition response that distinguishes most mycelia of independent origin and is never expressed in pairings of genetically identical mycelia (Rayner, 1991). Furthermore, in the cultivated button mushroom Agaricus bisporus (Lange) Imbach, Raper, Raper & Miller, (1972) observed that pairings of heterokaryons that were homoallelic for complementary auxotrophic mutations

Corresponding author

produced stable, prototrophic heterokaryons when nutritional selection was applied. In this study, we asked whether or not somatic recombination in A . bisporus occurs frequently enough in pairings of heterokaryons with heterokaryons and in pairings of heterokaryons with homokaryons to be observed without selection. The sexual system and patterns of genetic recombination in A. bisporcls are unusual among basidiomycetes. Most basidia have two spores, each of which receives two of the four haploid products of meiosis. The packaging of nuclei in spores is not random. Most often, a basidiospore receives two nonsister nuclei after the second meiotic division (Summerbell et al., 1989). Furthermore, many genetic markers show close linkage to their respective centromeres and therefore usually segregate at meiosis I (Summerbell ef al., 1989; Kerrigan et al., 1993b). The net effect of this system is that over 90% of the basidiospores produced remain heterozygous at loci for which they were heterozygous in the parental heterokaryon (Royse & May, 1982; Summerbell ef al., 1989; Allen, Moore & Elliott, 1992). Also, most spores germinate to ~ r o d u c eheterokaryotic mycelia that are heteroallelic at the mating-type locus and are self-fertile (Raper, Miller & Raper, 1972). This reproductive cycle therefore results in a relatively low frequency of genotypic change, which is evident as loss of heterozygosity. There is, however, some opportunity for crossing between unrelated gametic nuclei of A. bisporus. A minority of basidia have three of four spores, some of which are homokaryotic

J. Xu, P. A. Horgen and J. B. Anderson and self-sterile (Elliott, 1972; Raper ef al., 1972; Kerrigan ef al., 1993 b and references therein). The actual proportion of matings of unrelated homokaryons in wild populations is unknown. In the laboratory, breeding of commercial mushroom strains has relied heavily on matings among homokaryons of independent origin (Fritsche, 1983). but this proven method of crossing is laborious and time-consuming. Since most basidiospores in A. bisporus are heterokaryotic, and the possibility of heterokaryotic mycelia existing in nature for extended periods of time is high, we hypothesized that it would be more likely for a given heterokaryon to encounter another heterokaryon than a homokaryon in the wild. By similar reasoning, confrontations of different homokaryons would be even less frequent. In this study, we therefore asked whether somatic recombination could potentially be important in what are likely to be the two most frequent types of interactions in A. bisporus: pairings of heterokaryons with heterokaryons and heterokaryons with homokaryons. Our expectation was that, in the absence of meiosis, somatic recombination would be infrequent and therefore probably not detectable in the small sample of subcultures analysed in this study. Contrary to expectations, we observed a strikingly high frequency of somatic recombination in pairings. One recombinant even showed unambiguous evidence for crossing over between loci that show genetic and physical linkage.

MATERIALS A N D METHODS For pairings, two homokaryons, one (Ag89-65) derived from a wild-collected heterokaryon, and the other (Ag90-30) derived from a cultivated strain and four heterokaryons, two cultivated (Ag2 and Ag50), one wild-collected (Ag89), and one an artificially constructed hybrid (Ag93b), were used. With the exception of one of the nuclear types of the heterokaryon Ag89, the mating types for all nuclei are known (Table 2, Xu ef al., unpublished). Pairings were made by placing blocks of inoculum 2 cm apart on diluted Complete Yeast Medium (CYM) amended with compost extract (Xu, Horgen & Anderson, 1993). After 4 wk of incubation at room temperature, four subcultures were taken from the region where the two mycelia met one another in each of the pairings. In order to minimize the chance of sampling a mixture of hyphae of different genotypes, each explant was subcultured three times by transferring mycelium from the margin of a 3-wk-old colony as described by Xu ef al. (1993). The third subculture was used to inoculate a Petri dish containing liquid CYM. After 3 wk incubation at room temperature, the mycelium was harvested by filtration. DNA extraction and Southern hybridization of EcoR I digested genomic DNA with cloned probes were done exactly as described by Kerrigan ef al. (1993 b). For each locus scored, the sizes of the restriction fragments corresponding to the alleles are listed in Table I. Linkage relationships among 13 of the 18 nuclear loci had been determined previously by analysis of haploid offspring of meiosis, and by hybridization of cloned probe DNAs to chromosome-sized DNAs resolved by CHEF electrophoresis (Kerrigan ef al., 1993b). For loci 4n6, 4n14, 4n27/1, 4n27/2,

189 Table 1. Allelic interpretation of restriction fragments (in kb) hybridizing to probes used in this study

Locus

Allele L~nkage group 0

I

2

3

I I I I I1 I1 Ill

v v VI X XI1 XI1 ? ? 7 ? ?

Null

Null

mtDNA mtDNA

33115, 33n10/1, 33n10/2, 33n18 and 33n25, fragment sizes, but not allelic designations, correspond to Kerrigan, Horgen & Anderson (1993a). Since the remaining five loci did not segregate in the haploid population used to infer the genetic linkage map of A. bisporus, the chromosomal locations were not known for these loci. The two mitochondria1 RFLP loci utilized were those described in the studies of Jin & Horgen (1993) and Hintz, Anderson & Horgen (1988). Each recombinant subculture was compared to the phenotypically more similar of the two strains that were originally confronted. The potential number of loci at which the genotype of the subculture was different was thereby minimized. All subculture genotypes were stable for at least six months, the duration of our genetic analysis. In addition, each of the original two homokaryotic and four heterokaryotic strains was paired with itself and subcultured exactly as in the other pairings.

RESULTS A N D DISCUSSION Genotypes of the four subcultures removed from each of the four pairings are presented in Table 2. Subcultures from a fifth pairing, between the heterokaryons Ag50 and Ag89, showed no recombination for the loci tested and are therefore not listed in Table 2. The phenotypes of all subcultures from the six self pairings were identical to those of the original six strains listed in Table 2. In total, ten of 20 subcultures were phenotypically different from the original strains used in the five pairings (Table 2). Although the ploidy and number of nuclear types present in the recombinant subcultures are not known, the kinds of genetic changes observed in the recovered subcultures can be inferred from the restriction-fragment phenotypes. For example, the phenotype of subculture 3 from

Somatic recombination in Agaricw

190

Table 2. Phenotypes of subcultures from pairings of heterokaryons with heterokaryons and heterokaryons with homokaryons of Agarrcw bispoms Linkage groups and loci

I

I1

Mat

A

B

Ag90-30 (horn) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag93b (nucl) Ag93b (nuc2) Ag89-65 (horn) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag2 (nucl) Ag2 (nuc2)

C

Ag2 (nucl) Ag2 (nuc2) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag50 (nucl) Ag50 (nuc2)

D

Ag2 (nucl) Ag2 (nuc2) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag89 (nucl) Ag89 (nucl)

A3

A2

A5 A1

No. of subculturesb

v

VI

1

2

3

4

5

6

7

8

9

10

1

3 3 213 213 213 3 2

1 1

1

112 112 112 1 2

1 1 112 112 1/2 I 2

113 113 113 1 3

3 3 112 112 1/2

1 1 112 112 112 2

2 2/3 213 213 213 3 2

2 2 1/2 112 112

2 1/2 112 112 112 2 1

2 112 112 112 112 2 1

2 113 113 2/3 113 1 3

3 112 112 1/3 112 2 1

0

0

1

I

1

A1 A2

111

1

2

I 1 213 213 213 3 2

1

2 113 113 1/2 113 3 I

2 3 3 2/3 3 3 3

1

2

2 2 1/2 2 2 2

1

3

1 1 1 I

3 2/3

1

3 3

2 2 2 2 112 1 2

1

1

1

0

2

4

1 112 112 1/2 2

I

1

3

1

2

Linkage groups and loci X

XI1

Not mapped

Mit No. of

A

B

Ag90-30 (horn) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag93b (nucl) Ag93b (nuc2) Ag89-65 (horn) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag2 (nucl) Ag2 (nuc2)

C

Ag2 (nucl) Ag2 (nuc2) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag50 (nucl) Ag50 (nuc2)

D

Ag2 (nucl)

I 1 2 2 2 2 2

0 2 0 0 0 0 0

1

1

2 2 2 2 2 2

2 2 2 2 2 2

0 2 2

I/Z 112 112 112 1 2

0 0 2 2 2 0 2

1 1 112 112 112 1 2

3 3 2 2 2 2 2

0 0 0 0 0 0 0

1 I I I I 1 1

1 2 2 2 2 2

2

2 I 1

2 1 1 0

2 1 1 1/2 1 1 1

2 112 112 2 112 1 2

0

2

1

2 I 1 1/2 1 I 1

1

1 I 1 1 1 1 I

1

1

1

1

1

1

I

1

2

2

I 1

2 2

113 113 113 1 3

112 112 112 1 2

3 1 1

1/3 1

1

1

2 2 2 I 2

1 1

0 1 1

1

14

1 0 0

0

191

J. Xu, P. A. Horgen and J. B. Anderson Table

2. (cant.)

Linkage groups and loci --

X

XI1

11

12

13

14

15

16

17

18

19

20

locia

I 1 213

I 1 2

2

0

1

2

1

2 2

2

112

1

1

2

1

2

2 2 2

2

2

2

0

3

0 0

1

2

2 2 2

1/2

2

2

1 1 1

1 2 1 1 1

0 2 0 1

213 3 2

1 2

1 1 0 1 0

2 2

1

2 112 213 112 213

1 1

2

I 2

1

9

1

1

1

1

1

0

0

27

Not mapped

Mit No. of

Ag2 (nuc2) Subculture 1 Subculture 2 Subculture 3 Subculture 4 Ag89 (nucl) Ag89 (nuc2) No. of~subcultures"

1

0 0 0

Note: Allelic designations shown for the strains originally paired are considered to be genotypes. Allelic designations shown for subcultures represent phenotypes since the ploidy and number of nuclear types present are unknown. Alleles designated zero '0' are null and can only be inferred to be present in a given subculture only when dominant alleles are absent. Numbers in bold indicate loci at which the subculture is phenotypically dissimilar from the genetically more similar of the two paired strains. a Number of loci at which the subculture is phenotypically dissimilar from the genetically more similar of the two paired strains. "umber of subcultures at which the locus is phenotypically dissimilar from the genetically more similar of the two paired strains.

the pairing of Ag89-65 with Ag2 is fully consistent with the reassociation of the nuclear types from the two paired strains, specifically the nuclear type from Ag89-65 with nuclear type 2 of Ag2 (Table 2), with the additional loss of allele 1 at locus 17. In contrast, subculture no. 1 from Ag90-30 x Ag93b, can only be the result of genetic exchange among all three types of nuclei from each of the two original paired strains. Furthermore, this recombinant must be the result of a crossover between locus 8 and locus 9, which show genetic and physical linkage in strain Ag93b (Kerrigan ef al., 1993 b). The eight remaining subcultures are best explained by the loss of one or more alleles from the genetically more similar of the two original paired strains. Whether or not this loss occurred subsequently to genetic exchange between nuclei either from the same or from different original strains cannot be known from the data available. In all recombinants, the sizes of the recombined chromosomal segments are unknown and may in many cases include the entire chromosome. In addition to nuclear markers, two mtDNA loci were assayed in the five pairings examined in this study. The mtDNA type of each subculture was identical to that of the genetically more similar of the two original strains. Further, no recombination was observed between the two mtDNA loci. At present, the genetic mechanisms responsible for somatic recombination in A. bisporus are unknown. Our working hypothesis is that somatic recombination occurs after the fusion of two nuclei following hyphal anastomosis after which crossing over and/or loss of chromosomes may occur during successive rounds of mitoses. We speculate that somatic recombination is facilitated by the presence of three or four different nuclear genotypes in the same cytoplasm. The fact that no loss of marker alleles was observed in any of the six self-self pairings of the isolates used in this study is consistent with this hypothesis. Hansen ef al. (1993) also speculated that somatic recombination might be more common in basidiomycetes in which the vegetative heterokaryon consists of multinucleate hyphal compartments rather than species with strictly dikaryotic hyphal compartments; this hypothesis is

consistent with the high frequency of somatic recombination in A. bisporus, a species in which the heterokaryotic vegetative cells are highly multinucleate and there is no clear physical pairing between the two gametic nuclear types. Despite numerous studies of somatic recombination associated with heterokaryon-homokaryon pairings, however, the possibility of somatic recombination in heterokaryon-heterokaryon pairings in species with strictly dikaryotic hyphal compartments has not to our knowledge been investigated. Somatic recombination in artificial pairings of A. bisporus is a surprising observation that raises the question of how frequently genetic exchange and recombination occur through interaction of vegetative heterokaryons in nature. Sexual interactions between homokaryons have generally been assumed to be central to the basidiomycete life cycle. Our results are part of a growing body of evidence showing that somatic recombination may play a larger than expected role in shaping the genetic structure of natural populations of basidiomycetes. The report of Hansen et a1. (1993) shows that the somatic incompatibility response that is so widespread in higher fungi is not a complete barrier to genetic exchange between unrelated basidiomycete heterokaryons. Unfortunately, the genetic consequences of genetic exchange and recombination between heterokaryons may be very similar to the more well understood process of sexual mating between homokaryons in natural populations. Distinguishing the two kinds of genetic exchange would probably require additional genetic markers covering more of the genome as well as a realistic model of population genetic structure from which to compare expectations against actual observations from natural populations. In addition to its possible biological importance in nature, somatic recombination in A. bisporus may have practical application in commercial breeding of this economically important, cultivated fungus. Somatic recombination in pairings of heterokaryons might produce a broad range of genotypes more quickly and with less labour than does the method of obtaining and crossing homokaryons.

Somatic recombination in Agaricus

192

This was supported by Sylvan Spawn Laboratory, Inc., the Canadian Mushroom Growers Association, and a grant from the Ontario University Research Incentive Fund' The first author is supported by a Post Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada.

the mitochondrial genome of the cultivated mushroom Agaricus brunnescens ( = A. bis~orus). Genetics 14, 43-49. Jin, T. & Horgen, P. A. (1993). Further characterization of a large inverted repeat in the mitochondrial genomes of Agaricus bisporus ( = A. brunnescens) ,d related species, Current Genetics 23, 228-233. Kerrigan, R. W., Horgen, P. A. & Anderson, J. B. (1993a). The California population of Agaricus bisporus comprises at least two ancestral elements.

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