restriction fragment length polymorphisms in the mushrooms

Vol. 53, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1987, p. 816-8220099-2240/87/040816-07$02.00/0Copyright C 1987, American Society for Microbiology

Restriction Fragment Length Polymorphisms in the MushroomsAgaricus brunnescens and Agaricus bitorquisALAN J. CASTLE,* PAUL A. HORGEN, AND JAMES B. ANDERSON

Department ofBotany, University of Toronto, Erindale Campus, Mississauga, Ontario, Canada L5L IC6

Received 14 August 1986/Accepted 8 January 1987

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species),were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates ofboth species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in SouthernDNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wildtype, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species.There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescensstrains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA fromheterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A.brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplastregenerates from one commercial strain and two strains collected in the wild.

Efforts to breed new strains of the cultivated mushroomAgaricus brunnescens Peck [A. bisporus (Lange) Imbach]have been hampered by the rarity of the genetic markers thatare necessary for a controlled breeding program. The recov-ery of mutant strains is difficult because of the life cycle ofthis organism. Fruit-bodies of A. brunnescens producemostly two-spored basidia. Each basidiospore is binucleateand on germination forms hyphae with multinucleate cells.Haploid, monokaryotic propagules are nonexistent. As aresult, the standard genetic techniques of induction andisolation of recessive markers are prohibitively inefficientprocesses. For example, Raper et al. (27) recovered only twonovel auxotrophic mutants from a sample of over 18,000UV- or x-ray-irradiated hyphal tips.Spontaneous variants have supplied the majority of traits

commonly used in mushroom breeding programs (26). Thesemutants have exhibited alterations in sporocarp morphologyand color, colonial growth characteristics, and yield (9, 14,15, 25). Recently, other naturally occurring variations, en-zyme polymorphisms, have been used as markers for abreeding program (19, 29). Examination of isozymes inparental strains and progeny provides an unequivocal dem-onstration of hybrid production since these markers arecodominant.DNA restriction fragment length polymorphisms (RFLPs)

have been used as genetic markers in a wide variety oforganisms, including humans (3), plants (4, 11, 28), fungi (20,31, 33), and protozoa (13). Like isozymes, these charactersare codominant and can be used to identify hybrids. Inaddition, RFLPs may be observed with either coding ornoncoding DNAs. As most DNA in eucaryotes is not ex-pressed (noncoding), a vast array of traits independent ofcomplications arising from differential gene expression andperhaps selection may be obtained from a library of clonedfragments.The main purpose of this study was to develop RFLPs as

genetic markers in A. brunnescens. Two points were impor-tant in the application of these polymorphisms to a breeding

* Corresponding author.

program. First, how many cloned DNA fragments must beexamined before a polymorphism is identified? If mostfragments were conserved between several strains, then thecost in time and effort of a search for RFLPs might beprohibitive. Second, can various commercial strains betyped according to RFLP patterns? Royse and May (29)were able to separate 34 commercial strains into only fivedistinct groups based on isozyme characteristics. Theirresults indicate that there are few genetic differences be-tween commercial varieties. RFLPs might reveal furtherdistinctions among these strains or may substantiate theconclusion reached in the isozyme work.A second purpose of this study was to identify probes that

distinguish A. brunnescens from the related species A.bitorquis. Probes of this type would allow identification ofthe hybrids that are formed between these two species. A.bitorquis is a highly variable species (10), and formation ofA. brunnescens x A. bitorquis hybrids, if possible, mighteventually provide a means of introducing novel genotypesinto commercial strains.A third purpose was to use RFLPs to identify homokary-

otic isolates recovered from protoplasts of heterokaryoticstrains of A. brunnescens. If DNAs from homokaryoticisolates have less complex patterns than heterokaryoticstrains in Southern DNA-DNA hybridizations, then thischaracteristic could be used to confirm the recovery ofhomokaryons from heterokaryons. Crosses betweenhomokaryons could be a rapid method of forming strainswith novel genotypes (26). Previously, homokaryons wereisolated by micromanipulation of basidiospores from rarefour-spored basidia (6), or by screening randommonosporous cultures for the inability to fruit, both of whichare time-consuming processes.To answer these questions, 11 A. brunnescens strains

were surveyed for RFLPs with 10 probes. Two homokary-otic strains were included as indicators of segregation; theremaining nine were heterokaryotic. Of these, two strainswere collected in the wild, and seven were from commercialstocks. Seven isolates of A. bitorquis were also examinedwith the same set of probes.

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RFLPs IN A. BRUNNESCENS AND A. BITORQUIS 817

TABLE 1. Origin of strains used in this study

Toronto Sourcea Other designationdesignation and characteristics

A. brunnescensAgl-l ATCC ATCC 24662; homokaryonAgl-2 ATCC ATCC 24663; homokaryonAg2 ATCC ATCC 24558; heterokaryonAg6 Leaver Swayne 21; heterokaryonAg7 Leaver Swiss American A3.2; heterokaryonAg27 Leaver MC-1; heterokaryonAg33 Leaver Amycel 220; heterokaryonAg37 Leaver Amycel U3; heterokaryonAg52 Saltfleet Lambert 81; heterokaryonAg83 RWK Wild-type isolate; heterokaryonAg84 RWK Wild-type isolate; heterokaryon

A. bitorquisAg4 ATCC ATCC 24666; heterokaryonAg9 JBA Wild-type isolate; heterokaryonAgll JBA Wild-type isolate; heterokaryonAgl2 JBA Wild-type isolate; heterokaryonAgl7 JBA Wild-type isolate; heterokaryonAgl8 JBA Wild-type isolate; heterokaryonAgl9 JBA Wild-type isolate; heterokaryori

aAbbreviations: ATCC, American Type Culture Collection, Rockville,Md.; Leaver, Leaver Mushrooms, Ltd., Campbellville, Ontario; Saltfleet,Saltfleet Mushrooms, Ltd., Binbrook, Ontario; RWK, R. W. Kerrigan, Uni-versity of California, Santa Barbara, Calif.; JBA, J. B. Anderson, Universityof Toronto.

MATERIALS AND M1ETHODS

Strains. The various A. brunnescens and A. bitorquisisolates used in this study are listed in Table 1. With theexception of the strains obtained from the American TypeCulture Collection (Rockville, Md.), all cultures were de-rived frort fruit-body tissue. The conditions for growth andmaintenance of these strains were as described previously(1). For experiments with protoplasts, cultures were grownin MPFYE medium (K. Dahlberg, personal communication)both before and after digestion. MPFYE contains 20 g offructose, 20 g of malt extract, 2 g of yeast extract, 2 g ofpeptone, 0.5 g of MgSO4 7H20, 1 g of K2HPO4, 0.46 g ofKH2PO4, 0.2 ml of Vogel trace elements solution, and waterto 1 liter. Vogel trace elements solution was prepared by

mixing 5 g of citric acid monohydrate, 5 g of ZnSO4 - 7H20,1 g of Fe (NH4)2(SO4)2 - 6H20, 0.25 g of CuS04 - 5H20, 0.05g of MnSO4 H20, 0.05 g of H3BO3, 0.05 g ofNaMoO4- 2H20, and water to 100 ml. Chloroform (1 ml)was added as a preservative.DNA isolation. DNA was isolated from mycelium that was

grown in liquid culture, harvested, washed with distilledwater, and freeze-dried. Nuclear DNA was used for cloningand was extracted by the procedure of Mohan et al. (22).Nuclear DNA was separated from mitochondrial DNA bybis-benzimide cesium chloride gradient centrifugation (12)and quantified by UV spectrometry. Total mycelial DNAwas used for blotting and was extracted by the method ofMurray and Thompson (23), as modified previously (35).DNA isolated by this procedure was quantified, after restric-tion endonuclease digestion, on 0.7% agarose gels by com-

parison with known quantities of salmon sperm DNA. Theelectrophoresis buffer was TBE (0.089 M Tris base, 0.089 MH3BO3, 0.002 M EDTA).DNA cloning. Nuclear DNA from strains Ag33 and Ag4

were digested with the enzyme EcoRI (Pharmacia, Uppsala,Sweden) and ligated to plasmid vector pUC18 (32). Follow-ing transformation of the bacterial strain JM83, clones werepurified and the recombinant plasmids were isolated fromeach clone by the boiling lysis mini-prep method (18). Sizesof the cloned fragments were determined by digestion of therecombinant plasmid with EcoRI and electrophoresis on0.7% agarose gels (32). The average size of the 10 clonedfragments used in this survey was 2,590 base pairs (bp).

Southern blots and nick translation. Total Agaricus DNA(5 ,ug) was digested with EcoRI (5 U/,ug of DNA) andelectrophoresed on 0.7% agarose gels at 1.7 V/cm for 15 to16 h or at 6.7 V/cm for 3 to 4 h. Fragnments produced by thedigestion of bacteriophage lambda DNA with the enzymeHindIll were used as molecular size markers. The gels werestained with 0.5 p,g of ethidium bromide per ml and photo-graphed. DNA was transferred from the gel to Gene ScreenPlus (Du Pont NEN Research Products, Boston, Mass.) bythe capillary method recommended by the manufacturer ofthe membrane. Probes were prepared by radioactively label-ing recombinant plasmids with [a-32P]dCTP or [a-32P]dATP(3,000 Ci/mmol; Dupont NEN) with a nick-translation kitfrom Bethesda Research Laboratories (Gaithersburg, Md.).On average, specific activity of 5 x 107 cpm/,ug ofDNA was

recovered. Hybridization of the probe to the blot was also

TABLE 2. A. brunnescens EcoRI restriction fragments homologous to probes containing Ag33 DNA

Fragment size (kbp) of the following strains:Probe

Agl-1 Agl-2 Ag2 Ag6 Ag7 Ag27 Ag33 Ag37 Ag52 Ag83 Ag84

pAg33nll 2.9 2.9 2.9 2.9 2.9 2.9 2.9a NTb 2.9 2.9 2.9

pAg33n25 2.4 1.4 2.4 1.4 1.4 1.4 1.4 1.4 1.4 1.4 1.41.0 1.0 1.0 1.0a 1.0

pAg33nlO 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.76.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3

1.7 1.7 1.7 1.7 1.7 1.7 1.7 1.71.4 1.4 1.4 1.4 1.4 1.4 1.4a 1.4 1.4 1.4

pAg33nl8 1.6 1.6 1.61.2 1.2 1.2 1.2 1.2 1.2a 1.2 1.2 1.2 1.2

1.0 1.0 1.0

pAg33nl3 3.9 3.9 3.9 3.9 3.93.7 3.7 3.7 3.7 3.7 3.7 3.7a 3.7 3.7 3.7 3.7

a The size of the cloned fragment as determined by electrophoresis of EcoRI-digested recombinant plasmids.b NT, Not tested.

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APPL. ENVIRON. MICROBIOL.

TABLE 3. A. bitorquis EcoRI restriction fragments homologous to probes containing Ag33 DNA


Ag4 Ag9 Agll Agl2 Agl7 Agl8 Agl9

pAg33nll 15.8 15.8 8.9 15.8 15.8 15.88.3 8.3 8.3 8.3

6.52.4

pAg33n25 1.9 1.9 1.9 1.9 1.9 1.91.6 1.6 1.6

pAg33nlO 15.9 15.9 15.9 15.9 15.9 15.9 15.911.2 11.2 11.2 11.2 11.2

4.9 4.9 4.93.5

pAg33nl8 1.2 1.2 1.2 1.2 1.2 1.2 1.2

pAg33nl3 4.6 4.54.1 NTa 4.1 4.1 NT 4.1

3.1a NT, Not tested.

carried out according to the directions of the membrane incubated for 2 to 4 days under the same conditions. Thesemanufacturer. Briefly, blots were prehybridized with 50% cultures were harvested by centrifugation (10,000 X g, 10formamide-1 M NaCl-1% sodium dodecyl sulfate-10% dex- min) and washed with sterile 0.01 M potassium phosphatetran sulfate at 42°C for 6 h. Approximately 107cpm of labeled buffer (pH 6.9) followed by another centrifugation. Theprobe was added to the prehybridization solution along with pellet was weighed and suspended in filter-sterilized100 ,ug of denatured salmon sperm DNA per ml, and Novozym 234 (10 mg/ml; Novo Laboratories, Wilton,hybridization was allowed to proceed overnight. The Conn.) in 0.6 M MgSO4-10 mM sodium citrate (pH 5.0). Aposthybridization wash sequence included two 5-min washes sample of 50 to 60 mg of Novozym 234 was used per g ofin 2 x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium mycelium. Digestion of cell walls was allowed to proceed forcitrate) at room temperature, two 30-min washes in 2 x 4 h at room temperature. Every hour the cultures wereSSC-1% sodium dodecyl sulfate at 65°C, and two 30-min shaken vigorously but otherwise were kept still. Hyphalwashes in 0.lx SSC at room temperature. After washing, fragments were removed by filtration of the digestion mix-blots were exposed to X-Omat film (Eastman Kodak Co., ture through 3 layers of cheesecloth and one layer of glassRochester, N.Y.) with a Cronex Lightning-Plus intensifying wool packed up to the 10-ml mark of a 50-ml syringe.screen at -70°C. Protoplasts were collected from the effluent by centrifuga-

Protoplast production and regeneration. The techniques tion (750 x g, 1 h). The pellet was suspended in 0.5 ml of 0.6used for protoplast production and regeneration with A. M MgSO4-40 mM sodium citrate (pH 5.0), and the protoplastbrunnescens have been described previously (1) but were concentration was determined with a hemacytometer. Colo-modified as suggested by K. Dahlberg and C. Emmons of the nies derived from regenerated protoplasts were obtained byCampbell Institute for Research and Technology, Napoleon, plating the suspension on nutritive medium containing 0.6 MOhio (personal communication). Strains were grown in liq- mannitol as an osmotic stabilizer. Osmotic fragility of theuid shake culture (120 rpm, 25°C) for 3 to 4 weeks, and then protoplasts was tested in each experiment by plating frac-the mycelium was macerated in a Waring blender and tions of the suspension on nutritive medium lacking man-

TABLE 4. A. brunnescens EcoRI restriction fragments homologous to probes containing Ag4 DNA


Agl-l Agl-2 Ag2 Ag6 Ag7 Ag27 Ag33 Ag37 Ag52 Ag83 Ag84

pAg4nl2 8.0 8.03.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.7 3.71.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

pAg4n7 1.1 1.1 1.1 1.1 1.1 1.1 1.1 NTa 1.1 1.1 1.1

pAg4n9 1.4 1.4 1.4 1.4 1.4 1.4 1.4 NT 1.4 1.4 1.4

pAg4n6 10.88.1 8.1 8.1 8.1 8.1 8.1 8.1 8.1 8.1 8.1

5.8 5.8 6.9 6.9 6.9 6.9 6.9 6.9 6.9

pAg4n27 3.5 3.5 3.5 3.5 3.5 3.53.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3 3.3

2.2 2.2a NT, Not tested.

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TABLE 5. A. bitorquis EcoRI restriction fragments homologous to probes containing Ag4 DNA

Fragment size (kbp) in the following strains:Probe

Ag4 Ag9 Agll Agl2 Ag17 Agl8 Agl9

pAg4nl2 5.6a 5.6 5.6 5.6 5.6 5.6 5.64.8 4.8 4.8 3.4 4.8 4.8

pAg4n7 .la 1.1 1.1 1.1 1.1 1.1 1.1

pAg4n9 1.9 1.9 1.9 1.9 1.9 1.91.7a 1.7

pAg4n6 16.7 9.4 9.44.2 4.2 4.2 4.2 4.2

3.9a 3.9 3.9 3.9 3.9 3.9 3.9

pAg4n27 3.3a 3.3 3.3 3.3 3.3 3.3 3.3

a The size of the cloned fragment as determined by electrophoresis of EcoRI-digested recombinant plasmids.

nitol. Plates were incubated at 25°C for 2 to 4 weeks beforecolonies were observed and transferred. Each isolated col-ony was then grown in liquid medium for 2 weeks, har-vested, and freeze-dried. Total DNA was extracted, andSouthern blots were prepared and hybridized as describedabove.

RESULTS

Strain and species identification with RFLPs. Five recom-binant clones carrying DNA from A. brunnescens Ag33 andfive clones with A. bitorquis Ag4 DNA were used as probesin the survey for restriction fragment polymorphisms (Tables2 to 5). Several hybridization patterns were evident. Onlyone probe, pAg4n7 (Tables 4 and 5), showed a singleconserved band of 1.1 kbp in all isolates of both species.Probes pAg4n27 (Tables 4 and 5) and pAg33nl8 (Tables 2and 3) produced bands that were common to both speciesand that were polymorphic within A. brunnescens. Theremaining seven probes distinguished all A. brunnescensstrains from all A. bitorquis strains. Most cloned fragmentswere polymorphic within both species. The exceptions wereprobes pAg4n9 and pAg33nll (Tables 2 to 5), which werenot polymorphic within A. brunnescens. Overall, 9 of 10probes revealed RFLPs in one species or the other; 7 of 10probes were polymorphic in A. brunnescens and 7 of 10 werepolymorphic in A. bitorquis. On average, each probe hybrid-ized with 1.7 bands in each heterokaryotic A. brunnescensisolate, 1.25 bands in each homokaryotic isolate, and 1.5bands in each A. bitorquis isolate.

Distinct genotypes could be defined by this set of probes.For example, strains Ag2 and Ag52 appeared as separategenotypes based on probes pAg33n25, pAg33nl0, pAg4nl2,pAg4n6, and pAg4n27 (Tables 2 and 4). In this regard, all A.bitorquis isolates could be distinguished from one another aswell as from all A. brunnescens strains. Similarly, most A.brunnescens strains appeared to be unique genetically. Atleast two and usually four or five probes indicated differ-ences between any two strains. There was an obviousexception to this pattern. Despite the presence of extensivepolymorphism within this group, commercial strains Ag6,Ag7, Ag27, and Ag33 exhibited identical patterns with all 10probes. The two wild-type isolates Ag83 and Ag84 showedhybridization patterns similar to those of the commercialstrains, thus confirming that they were more similar tocultivated A. brunnescens than any of the A. bitorquisisolates, yet Ag83 and Ag84 had genotypes that were dif-ferent from those of any of the other strains. In particular,

Ag84 showed a 10.8-kbp band that was not present in anyother strain when probed with pAg4n6 (Table 4).The RFLP patterns seen in the two homokaryotic strains

Agl-1 and Agl-2 were distinctly less complex than patternsin the heterokaryotic strains. That is, the homokaryoticstrains lacked one or more of the bands seen inheterokaryotic stocks when probed with clones pAg33n25,pAg33nlO, pAg33nl8, pAg33nl3 (Table 2), pAg4n6, orpAg4n27 (Table 4).Homokaryon isolation. Protoplasts (8.6 x 104) were recov-

ered from 3.4 g (wet weight) of Ag84 mycelium and 4.6 x 104protoplasts were plated on MPFYE containing 0.6 M man-nitol. Only 11 colonies were recovered. No colonies wererecovered from 2.6 x 104 protoplasts plated on MPFYElacking mannitol. Results of analysis of the 11 recoveredcolonies for restriction fragment patterns suggested that oneisolate, Ag84-7P, was homokaryotic (Fig. 1). One of the twobands present in the parental heterokaryon was missing inthe clone from the recovered protoplast. A similar result wasobtained with a second probe, thus providing more evidencethat a simpler nuclear type was recovered in this isolate.

Digestion of 2.86 g of Ag83 mycelium with Novozym 234resulted in the recovery of 1.3 x 107 protoplasts, and 30colonies grew on the MPFYE plus mannitol plates that wereinoculated with a total of 4.3 x 106 protoplasts. One colonyfrom 4.3 x 106 protoplasts was observed on the plateslacking the osmotic stabilizer mannitol. Eleven of the regen-erated colonies were tested with two cloned fragments.Results obtained with both probes suggested that 1 colony,Ag83-28P, was homokaryotic, while the remaining 10 ap-peared to be heterokaryotic (Fig. 2).

A 1 2 3 4 5 6 78 91011 1213146.7

5.8 -0.:F

FIG. 1. EcoRI-digested DNA from homokaryotic standardstrains Agl-1 and Agl-2 (lanes 1 and 2, respectively), parental strainAg84 (lane 3), and regenerated Ag84 protoplast isolates (lanes 4 to14). Probes were 3 2P-labeled pAg33nlO (A) and pAg4n6 (B). Lane 10shows loss of restriction fragments. The numbers along the siderefer to DNA fragment sizes, in kilobase pairs.

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a6.8192-'W

FIG. 2. EcoRI-digested DNA from homokaryotic standardstrains Agl-1 and Agl-2 (lanes 1 and 2, respectively), parental strainAg83 (lane 3), and regenerated protoplast isolates from Ag83 (lanes4 to 13). Probes were 32P-labeled pAg33nlO (A) and pAg4n6 (B).Restriction fragment sizes are given along the side, in kilobase pairs.

The number of protoplasts recovered from commercialstrain Ag37 could not be determined, as numerous cell wallfragments, not removed by filtration, prevented an accuratecount. These fragments were observed in three separateprotoplast trials and appeared to be a characteristic uniqueto this strain. Only six colonies were produced duringregeneration (none on control plates); but of these isolates,one, Ag37-4P, lost 1.7- and 6.9-kbp fragments, as determinedwith two probes (Fig. 3).With strains Ag84 and Ag37, homokaryotic colonies could

be recognized by colony morphology, as they producedappressed colonies with few aerial hyphae, as opposed to thevigorous heterokaryotic colonies, which produced abundantaerial hyphae. These isolates grew more slowly than eitherthe parental strains or the heterokaryotic regenerates. Thehomokaryon recovered from Ag83 also grew more slowlyand thus could be distinguished from the parentalheterokaryon. Colony morphology, however, was not areliable indicator of nuclear status with this particular strain.Among the group of colonies from regenerated protoplastswere vigorous types, as well as slow-growing colonies thatwere morphologically distinct from the parental Ag83heterokaryon. Both of these types subsequently proved tobe heterokaryotic when tested for RFLP patterns.

DISCUSSION

A high degree of restriction fragment polymorphism wasobserved in both A. brunnescens and A. bitorquis. Thefrequency of polymorphic fragments was comparable with, ifnot greater than, a similar analysis of domestic cultivars ofmaize (Zea mays), in which more than 50% of uniquesequence cloned fragments were polymorphic (4). This spe-cies was considered to be highly variable. The observedfrequencies in both maize and the two mushroom specieswere substantially higher than those in similar tests withtomato (Lycopersicon esculentum) (11), lettuce (Lactucasativa) (16), and humans (2, 3, 34). A library of randomlycloned fragments, therefore, should provide a very largeselection of genetic markers which can be recovered quiteeasily. The most striking result from this study was that mostof the heterokaryotic A. brunnescens strains wereheteromorphic for most polymorphisms. This observationagreed with previous findings of high frequencies ofheterozygosity for mating type (6, 14, 27) and for variousisozyme loci (19, 29) in commercial strains.

There appeared to be fewer different genotypes thanexpected in the sample of commercial A. brunnescensstrains, considering the high incidence of polymorphism.Earlier studies with isozyme (29) and auxotrophic markers(26) produced si milar results. Most of the strains currentlyavailable for research and commercial use are thought tohave originated from one or very few common ancestors(26). A more intensive screening involving many moremarkers might turn up differences between these strains.Even if differences are observed eventually, the near homo-geneity of the commercial strains stresses the need tointroduce new germplasm into these stocks.The most efficient way of generating new genotypes in A.

brunnescens would be to isolate homokaryons from differentstrains and to cross them with each other. Homokaryonscould be obtained from monosporous progeny (14), particu-larly those progeny from rare four-spored basidia (6). Be-cause of the infrequent production of this type of basidium,as well as the low rate of basidiospore germination, this is avery tedious process (7). Isolation of the nuclear compo-nents of a constructed heterokaryon of this species fromcolonies of regenerated protoplasts was shown previously tobe feasible (1). In this study it was possible to recover one ofthe two nuclear types from each of three strains. Thesimplicity of RFLP patterns observed in Agl-1 and Agl-2(Tables 2 and 4) provided the basis for verification ofhomokaryosis. The loss of DNA heterogeneity that wasobserved in three of the regenerated protoplast colonies(Fig. 1 to 3) was considered to be due to the loss of a singlenuclear type in these isolates. Other explanations such asaneuploidy or mitotic recombination are unlikely as nuclearfusion in vegetative hyphae is a very rare event (8). Thepossibility that these three isolates might have nuclear ratiosdifferent from the normal 1:1 ratio in the parental strains wasexcluded by the absence of any indication of the missingfragments in x-ray films that were overexposed to theSouthern blots. The screening process was efficient, asputative homokaryons were isolated from all three strains,despite the very low recovery of regenerated colonies. Itmay be possible to separate the nuclear components ofseveral commercial strains. The initial screening process

6.3-

1.7-1.4-

8.1-6.9

FIG. 3. EcoRl-digested DNA from parental strain Ag37 (lane 1)and six regenerated protoplast isolates (lanes 2 to 7) probed with32P-labeled pAg33nlO (A) and pAg4n6 (B). Restriction fragmentssizes are given along the side, in kilobase pairs.

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could be facilitated by looking for unhealthy colonies, be-cause all three recovered homokaryons were less vigorousthan the parental heterokaryons. This trait may be valuableas a primary screening criterion for homokaryons.The homokaryons obtained from the wild-type heterokary-

otic isolates will provide new sources of genetic variation forincorporation into commercial stocks. The genotypes ofboth strains Ag83 and Ag84 were different from those of any

of the commercial strains (Tables 2 and 4). Crosses betweenthe wild-type homokaryons and the commercialhomokaryons Agl-1 and Agl-2 (Table 1) and Ag37-4P (ob-tained in this study) are being attempted. It will be possibleto confirm heterokaryon formation of any of these crosses

with the present set of probes. Similarly, all possible A.brunnescens x A. bitorquis hybrids could be identified withmost probes examined in this study. If these hybrids couldbe formed, then valuable traits such as mycovirus resistance(5) might be introduced into commercial strains.The lack of vigor exhibited by all three homokaryotic

isolates as compared with the parental heterokaryons sug-

gested that the two nuclei of a heterokaryon may comple-ment each other for deleterious recessive traits. A substan-tial genetic load may be carried by various commercial andwild-type isolates of A. brunnescens. In extreme cases, one

or both nuclei may be sufficiently genetically damaged suchthat the strain is an obligate heterokaryon.

Previous observations support this hypothesis. Basid-iospore germination is characteristically very low in thisspecies. Germination frequencies of 1% (24) and 4.5% (21)have been reported. At least some of this inviability might beattributed to recessive deleterious alleles or to chromosomalaberrations leading to duplications or deficiencies inbasidiospores. Cytological evidence of chromosome rear-

rangements has been reported. Evans (8) observed dicentricbridges accompanied by acentric fragments, suggesting thatthe strain studied was heteromorphic for a paracentricinversion.The observations summarized above are consistent with

the heavy genetic load in some strains and with a high degreeof heterokaryosis that may be maintained by complementa-tion of deleterious traits. If this hypothesis were true, thenthe implications for mushroom breeding and genetics are

great. First, to isolate a desirable trait from a heterokaryonthat is heterozygous for recessive lethals, recombinant via-ble homokaryons carrying the beneficial character wouldhave to be isolated from basidiospore progeny. Second,several mechanisms have been proposed for the packagingof two nuclei into each basidiospore following meiosis in A.brunnescens. These hypotheses include (i) normal meiosisand random nuclear assortment resulting in a 2:1heterozygote:homozygote ratio in the basidiospore progeny

(17); (ii) nonrandom nuclear distribution due to abnormalspindle arrangement resulting in up to 80% heterozygotes(8); and (iii) nonrandom nuclear packaging resulting in anincreased probability of sister chromatids ending up in thesame basidiospore. In this case, most basidiospores wouldbe homozygous for most genes, particularly those that are

closely linked to their centromeres (30). Regardless of whichhypothesis best explains meiosis in A. brunnescens, geneticload might be a factor in any given cross. Thus, any

basidiospore that is homozygous for any detrimental reces-

sive allele would be nonviable or reduced in vigor. Linkageof any observed marker such as mating type to the deleteri-ous trait would mean that heterozygotes would be favoredover homozygotes, producing deviations from the expectedsegregation ratios.

The observed restriction fragment length patterns in theheterokaryotic and homokaryotic strains of A. brunnescensand A. bitorquis indicate that RFLPs will provide valuablegenetic markers for several areas. In particular, it will bepossible to type strains according to RFLP patterns, pro-vided that sufficient differences exist between these strains.Novel genotypes can be confirmed with these markersfollowing putative hybrid formation between A. brunnescenshomokaryons or, perhaps, between A. brunnescens and A.bitorquis homokaryons. In addition, these cloned fragmentscan be used as genetic markers in meiotic analyses. Ifsufficient markers of any type (RFLPs, isozymes,auxotrophs, etc.) are obtained, then some of the uncertaintysurrounding meiosis in this organism may be resolved.

ACKNOWLEDGMENTSWe thank Rick Kerrigan for supplying strains Ag83 and Ag84;

Kurt Dahlberg and Cheryld Emmons for demonstrating their proce-dures for protoplast production; and Randy Irvin, Rob Meyer, andWill Hintz for critically reading the manuscript.

This study was supported by strategic grant G1540 and byCooperative Research Development Grant CRD-8616 from theNatural Sciences and Engineering Research Council of Canada toJ.B.A. and P.A.H. and by a grant from the Campbell Institute forResearch and Technology, Napoleon, Ohio.

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