Copyright 0 1988 by the Genetics Society of America

Nuclear Migration and Mitochondrial Inheritance in the bitorquis

William E. A. Hintz, James B. Anderson and Paul A. Horgen Mushroom Research Group, Center for Plant Biotechnology, Department of Botany, University of Toronto, Erindale Campus, Mississauga, Ontario, Canada L5L I C6 Manuscript received October 21, 1987 Revised copy accepted January 28, 1988

ABSTRACT Mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs) were used as genetic markers for following mitochondrialinheritance in themushroom Agaricus bitorquis. In many basidiomycetes, bilateral nuclear migration between paired homokaryotic mycelia gives rise to two discrete dikaryons which have identical nuclei but different cytoplasms. Although nuclear migration is rare in A. bitorquis, unidirectional nuclear migration occurred when a nuclear donating strain (8- l), was paired with anuclear recipient strain (34-2). The dikaryonrecovered over the nuclear recipient mate (Dik D) contained nuclei from both parents but only mitochondria from the recipient mate; thus nuclei of 8-1, but not mitochondria, migrated through the resident hyphae of 34-2 following hyphal anastomosis. The two mitochondrial types present in a dikaryon recovered at the junction of the two cultures (Dik A) segregated during vegetative growth. Dikaryotic cells having the 34-2 mitochondrial type grew faster than cellswith the 8-1 mitochondrial type. Fruitbodies, derived from a mixed population of cells having the same nuclearcomponents but different cytoplasms, were chimeric for mitochondrial type. The transmission of mitochondria was biased in favor of the 8-1 type in the spore progeny of the chimeric fruitbody. Protoplasts of dikaryon (Dik D), which contained both nuclear types but only the 34-2 mitochondrial type, were regenerated and homokaryons containing the 8-1 nuclear type and the 34-2 mitochondrial type were recovered.

HE field mushroom, Agaricus bitorquis, is heter- tion of nuclei occurs independently of any movement T othallic with mating types controlled by a single of organelles (CASSELTONand CONDIT 1972). Al- multiallelic incompatibility locus (RAPER1976). The though nuclear migration is rare in A. bitorpis, one fruitbodies produce four-spored basidia and single- stock (8-1) has been identified, which when paired spore isolates are homokaryotic with haploid nuclei. with a compatible mate (34-2), donates nuclei which A compatiblemating of two homokaryotic isolates migrate unilaterally through the resident mycelium results in the presence of a raised line of dikaryotic of the recipient mate (ANDERSONet al. 1984). mycelium at the confluence of the paired cultures Extensive mt DNA restriction pattern heteroge- (RAPER and KAYE1978; ANDERSONet al. 1984). Clamp neity exists between isolates of A. bitorquis (HINTZet connections, often a feature of dikaryons within the al. 1985). The main purpose of this study was to Hymenomycetes, are absent in A. bitorpis. Dikaryosis develop RFLP markers to follow the transmission of can be confirmed visually with a microscope or by specific mitochondrial and nuclear types after hyphal fruitbody formation. Central to any study of breedinganastomosis, nuclear migration, fruitbody formation biology is the transmission of organellar genomes as and sporulation. We have shown thatthere is no well as the transmission of nuclear traits. We were movement of mitochondria associated with the mi- interested in determining the mitochondrial inherit- gration of nuclei following hyphal anastomosis. A ance pattern in crosses of A. bitorquis and assessing second purpose of this study was to resolve a dikar- the mitochondrial contribution to the phenotype of yon, of known nuclear and mitochondrial genotypes, vegetative cultures. into homokaryons, of predicted genotypes, by regen- In another basidiomycete, Coprinus cinereus, bipar- erating protoplasts. This allowed the reassociation of ental inheritance of mitochondria results from events the 34-2 mitochondrial genotype with the 8-1 nuclear directly following hyphal anastomosis in a pairing of genotype which resulted in a new homokaryon, with- homokaryons.Donor nuclei migrate bilaterally out sexual fusion of the nuclei in the dikaryon. through the established cells of each recipient mate, resulting in two discrete dikaryons, having identical MATERIALS AND METHODS pairs of nuclei but different cytoplasms (BAPTISTA- Growth and maintenance of strains: Working cultures FERREIRA,ECONOMOU and CASSELTON1983). Migra- of A. bitorquis (8-1, 34-2) were maintained on CYM agar

Genetics 119: 35-41 (May, 1988). 36 W. E. A. Hintz, J. B. Anderson and P. A. Horgen (STEVENS1981) at 23". Mating was done by pairing two 1 mm' plugs of homokaryotic cultures at a distance of 1.0 cm on CYM agar plates (ANDERSONet al. 1984). Plugs of dikaryotic mycelium were removed from the interface and also distal to the interface of the two homokaryons and transferred to CYM agar plates. For fruiting trials sterile grain (1.0 g CaC0'; 100 g rye seed; 120 ml H20) (CHANG and HAYES1978) was inoculated with dikaryotic mycelium and incubated for 21 days at 23". The "spawn" was cased with water-saturated calcined earth (Turface, International Minerals and Chemical Corp. Mundelein, Illinois) mixed with, 5.0% (w/w) CaCOJ and 1.0% (w/w) Hydrogel (Viterra Planta-gel, Nepera Chemical Co., Harriman,Sew York) (SANA~TONIO 1971) and transferred to an incubator with 85% relative humidity at 21". Spore prints were prepared frommature fruiting bodies and single germlings were isolated following dilution plating of spores on CYM agar. Protoplasts were isolated from dikarvotic mycelium and homokaryons were recovered as described by CASTLE,HOR- GEN and ANDERSON(1987). Mycelium for DNA isolations was grown in CYM liquid medium in petri dishes with no agitation. FIGLKE1 .-Suclear migration in Agaricus. An A. bitorpi.\ nu- Source of genetic markers: For the generation of nuclear clear donating strain (X- l, Ipf)was paired with a sexually compatible DNA markers, A. bitorpis (8- 1) nuclear DNA was separated nuclear accepting strain (34-2, right) on CYM agar. Following from mt DKA by centrifugation in bis-benzimide (Hoescht hyphal anastomosis and nuclear migration, plugs of dikaryotic 33258, Sigma Chemical Co.) cesium chloridegradients mycelium were recovered from the junction of the two parental (HUDSPETHet al. 1980). Purified nuclear DNA was digested cultures (Dik A, open circle) and from the recipient mate (Dik D, with the restriction enzyme EcoRI (Pharmacia, Uppsala, closed circle). Sweden) and ligated to similarly digested bacterial plasmid vector pBR 325 (MANIATIS,FRITSCH and SAMBROOK1982). Several clones, containing 2.0-7.0 kilo-basepair (kbp) in- serts, were chosen at random for use as nuclear markers. r! Mitochondrial DNA markers were generated by cloning BamHI digested A. brunnescens mt DNA into the polylinker region of the bacterial plasmid vector pUC 18. The cloned mt DNAs used in this study were between 6.0 and 10.0 kbp in length. Restriction analysis: Whole-cell DNA was isolated from freeze-dried mycelium according tothe procedureof ZOLAN and PUKKILA(1986). DNA samples (ca. 5 pg) were digested with BamHI and subjected to electrophoresis on 15 X 18 cm, 0.7% agarose gels in TBE (89 mM Tris-HCI; 89 mM boric acid; 2.0 mM EDTA, pH 8.0) at 30 V for 16 hr. Bacteriophage lambda DNA, digested with both EcoRI and HindIII, was used as a size standard. The digested DNA I was transferred from the gel onto Genescreen Plus mem- brane(Dupont NEN Research Products, Boston, Massa- chusetts) by capillary blotting according to manufacturer's directions. Plasmid DNAs (ca. 1 kg) were labeled with [a- "PIdCTP (3000 Ci/mrnol; Dupont NEN) using the Be- thesda Research Laboratories (Gaithersburg, Maryland) nick-translation kit. On average, specific activity of 10' cpm/kg DNA was obtained. Hybridization and wash con- ditions were as described by HINTZet al. (1985). Autora- diography was performed using a Dupont NEN intensify- ing screen and Kodak XAR-5 film. ~I~LKCr.--~eg~egauor~ UI IIULIC~Idllu lllllucllulluIldl IIMI~.FIJ during fruiting. EamHI digested DSAs from A. biforguk parental homokaryotic strains 8-1, 34-2 (lanes 1 and2 respectively), the RESULTS dikaryon recovered at the junction of the homokaryons (Dik A, Two bitorpis lane 3), the dikaryon recovered over the nuclear recipient mate homokaryotic isolates of A. (8-1, 34- (Dik D, lane 4). homokaryotic spore progeny of Dik A (AISI, 2), having unique mt DNA restriction patterns, were AlS6, A2S5, A2S6; lanes 54,and homokaryotic spore progeny paired on CY M agar at a distance of 1 cm. After 2 1 of Dik D (D2S6, D2S7, D3S6,D3S7; lanes 9-12) were electropho- days of growth, fluffy mycelium, distinctive of dikar- resed on a 0.74 agarose gel, transferred to a Genescreen Plus yon formation, appeared at the junction of the two filter, and hybridized with radiolabeled nuclear DNA marker p8- In3 (A) or mt DNA marker p5Om9 (B). The sizes of strongly homokaryotic strains. During the next 14 days, the hybridizing fragments are indicated at the right of the figure zone ofdikaryotization migrated unilaterally over the (kbp). Weakly hybridizing fragments or partial digestion products resident mycelium of strain 34-2 (Figure 1). Plugs of ,L'PVP nnt ;ncl>nA~Ain the ~n~lwcic Mitochondrial Inheritance in Agaricus 37

TABLE 1 Genotypes of A. bitorquis strains

Nuclear probes Mitochondrial probes Mating Strain p8- I n3 p8- 1n7 p8- 1 n33 p50m9 p5OrnlO p50m4b

8-1 a( A1 a(13.5)a(15.6)a(l1.813.7) a(5.0/4.4/4.1)a(17.1) 1 0.4)a 34-2 A2 b(17.6/13.2) b(5.5/4.7/3.8) b(8.4l1.7) b(4.6) b(19.2) b(18.2) b(19.2) b(4.6) b(8.4l1.7) b(5.5/4.7/3.8)b(17.6/13.2) A2 34-2 Dik A A1/A2 ab ab ab ab ab ab b a AlSl A2 b a a a a b b AIS6 AI b b a a a a a A2S5 A2 a a a a a A2S6 AI a a b b a a Dik D A l/A2 ab ab ab b b b b b D2S6 A2 b b b b b D2S7 AI b b a b b b D3S6 A2 b b b b b b D3S7 A1 a a b b b b

Sizes of restriction fragment(s) (kbp) hybridizing to the probe in parentheses.

dikaryotic mycelium were excised from the zone of heritance was observed. Markers,which distinguished initial culture contact (Dik A) and also from distal to the parental mitochondrial types, were selected from the zone of contact over the recipient mate (Dik D) the A. bmnnescens mt DNA library. The mt DNA (Figure1). These dikaryons were allowed to fruit marker p50m9, hybridized to two 8-1 mt DNA frag- and single-spore isolates were recovered from several ments, 11.8 kbp and 3.7 kbp in length, and a single fruitbodies of each dikaryon. For the RFLP analysis, 34-2 mt DNA fragment, 4.6 kbp in length (Figure DNA was isolated from the parental homokaryons 2B, lanes 1 and 2). The dikaryon (Dik D), collected (8-1, 34-2), the two dikaryons described in Figure 1 from a site distal to the junction of the two cultures, (Dik A, Dik D), four single-spore progeny of Dik A carried only the 34-2 mitochondrial type (Figure 2B, (A1S1, AlS6, A2S5, A2S6), andfour single-spore lane 4). This indicated that nuclei, but no mitochon- progeny of Dik D (D2S6, D2S7, D3S6, D3S7). Seg- dria, migrated through the resident hyphae of the regation of nuclearmarkers in the spore progeny nuclearrecipient mate (34-2) following hyphal an- was shown by hybridization experiments with cloned astomosis. All of the spore progeny of Dik D con- nuclear DNA probes. The hybridization pattern of tained the 34-2 mitochondrial type (Figure 2B, lanes the nuclear marker p8-In3 is shown in Figure 2A. 9-12). The dikaryon (Dik A), from the interface of The two parental nuclear types were distinguished the paired homokaryons, initially contained both the by p8-In3 hybridizing to a single 10.4-kbp fragment 8-1 and 34-2 mt types (Figure 2B, lane 3). During of 8-1 DNA and two fragments (13.2 and 17.6 kbp) the routine subculturing of Dik A, in which plugs of of 34-2 DNA (Figure 2A, lanes 1 and 2). The dikar- mycelium were removed from the leading edge of a yons, Dik A and Dik D, contained nuclei from both mature colony and transplanted to a fresh CYM agar parents as was clearly demonstrated by the presence plate, only one mitochondrial type (34-2) could be of both parental hybridization patterns (Figure 2A, detected in the culture after a few transfers. The 8- lanes 3 and 4). The single-spore progeny were hom- 1 mitochondrial type was lost as a result of serial okaryotic and had either the 8-1 pattern or the 34-2 transfer. To determine howquickly the two mito- pattern in approximatelya 1 : 1ratio (Figure 2A, chondrial types segregated in culture,a junction lanes 5-12). The blot was probed with two additional dikaryon was transferred to CYM agar and grown nuclear markers (p8- ln7, p8- ln33) and the sizes of for 14 days. Seven plugs of mycelium were removed homologous restriction fragments determined (Table randomlyfrom the center of the colony outward, 1). The mating types A1 and A2 served as an addi- and the mitochondrial types determined (Figure 3). tional marker locus for the spore progeny (Table 1). Certain sectors of the colony contained dikaryotic Each of the four markers (p8-ln3, p8-ln7, p8-ln33, cells containing the 8-1 mitochondrial type (Figure mating type) segregated in a pattern typical of Men- 3, lanes 3 and 4) while other sectors of the colony delian inheritance. Recombinant nuclear genotypes contained the 34-2 mitochondrial type. Mycelium at were found among theprogeny of the dikaryons, but the leading edge of the colony (Figure 3, lanes 5-7) the number of progenyexamined was too low to consistently carried the34-2 mitochondrial type while detect linkage. both mitochondrial types were present in the center When the DNAs were hybridized with the cloned of the colony (Figure 3, lanes 1 and 2). Cells contain- mt DNA markers, a non-Mendelian pattern of in- ingthe 34-2 mitochondrialtype had outcompeted 38 W. E. A. Hintz, J. B. Anderson and P. A. Horgen 5678 the 8-1 mitochondrial type after 2.0 cmof radial growth. The junction dikaryon (Dik A), composed of a mixture of dikaryotic cells having either the 8-1 or - 11.8 34-2 mitochondrial types, was fruitedand spore progeny recovered. All of the Dik A spore progeny contained the 8-1 mitochondrial type (Figure 2B, lanes 5-8; Table 1). This was confirmed by analyzing 4 h DNA from five single-spore cultures, from each of two additional fruiting bodies. These tenDik A spore - 37 progeny also contained only the 8-1 mitochondrial type. The 34-2 mitochondrial type was not found in any of the Dik A spore progeny and recombinant FICVRF.3.-Segregation of mitochondrial types during vegeta- mitochondrial genomes were not detected. It could tive growth. A plug of dikaryotic mycelium recovered from the junction of the two cultures was transferred to a fresh CYM agar be argued that the junctionfruitbody was composed plate. After 14 days of growth, seven plugs of mycelium were exclusively of dikaryotic cells having the 8-1 mito- randomly sampled from the center of the colony (lane 1) to the chondrial type. An explant was therefore taken from edge of the colony (lane 7) and DNA was extracted. Hybridization the cap tissue of the Dik A fruitbody and transferred with the mt DNA marker p5Om9 demonstrated that the two most onto CYM agar. This explant was subcultured twice central samples (lanes 1 and 2) contained both 8-1 and 34-2 mt DSA, two sectors contained 8-1 mt DSA (lanes 3 and 4) while the before DNAwas extracted so that dikaryotic cells, three outermost samples (lanes 5-7) contained only 34-2 mt DNA. containing the 34-2 mitochondrial type, could dom- An explant of mycelium was taken from a junction fruitbody,and inate the culture. Only the 34-2 mitochondrial type serially transferred twice before DNAwas extracted. The trans- was detected in the serially transferred cap mycelium ferred explant contained 34-2 mt DNA (lane 8). (Figure3, lane 8). From a single fruitbody,both mitochondrial types were recovered, indicating that 123456789 the fruitbody derived from the junction of 8-1 and 34-2 was chimeric for mitochondria. A To determine theeffect of mitochondrial genotype onculture morphology,a new strain, which had nuclei from 8-1 but mitochondriafrom 34-2, was constructed. Protoplasts of Dik D were allowed to . 17.6 regenerateand colonies were transferredto CYM - 13.2 agar. The recovery rate for viable protoplasts was on - 10.4 theorder of 0.01%. Homokaryons were initially identified by their slower growth rate on CYM agar. Of the 36 protoplasts regenerated, 15 were sexually compatible with 34-2 and none with 8-1. The other 17 protoplast derived cultures were dikaryotic. Hy- B bridization with the nuclear DNA probe p8-ln3 confirmed the nuclear statusof six homokaryotic and dikaryotic protoplast regenerates. Protoplasts 2 1, 29, - 11.8 and 15 were homokaryotic, having the 8-1 nuclear type, while protoplasts 23, 2, and 26 were dikaryotic having both the 8-1 and 34-2 nuclear types (Figure 4A). Probing with the other two nuclear DNA probes - 4.6 (p8-ln7, p8-ln33) gave the same result. No recorn- . 3.7 binant nuclear genotypes were detected. All of the protoplastderived cultures carried only the 34-2 mitochondrial type (Figure 4B). Thus a new homo- karyotic strain (8n34m), carryingthe 8-1 nuclear type FICXRE4.-Dedikaryotization of A. bitorpis. BamHI digested DNAs fromparental homokaryons 8-1, 34-2 (lanes 1 and 2, and the 34-2 mitochondrial type, was constructed. respectively), the dikaryon Dik D (lane 3) and six derived protoplast When 8n34mwas paired with 34-2, nuclear migration cultures; pp21, pp29, ppl5, pp23, pp2, pp26 (lanes 4-9 respec- towards 34-2 was observed. Hyphae of the parental tively) were electrophoresed on a 0.7% agarose gel, transferred to strain 8-1 grew closely appressed to the surface of a Genescreen Plus filter and hybridized with radiolabeled nuclear the agar and the colony appeared flat while hyphae DNA marker p8-11-13(A) or mt DNA marker p5Om9 (B).The sizes of the hybridizing fragments are indicated atthe right of the of 8n34m grew aerially andthe colony appeared figure(kbp). Protoplasts pp21,pp29, and ppl5 have the same fluffier (Figure 5). The growth rate of 8n34m was nuclear type as 8-1 but carry the mitochondrial type of 34-2. the same as that of 8-1. Mitochondrial Inheritance in Agaricus 39

FIGURE5.-Effect of mitochondrial type on homokaryotic growth. Strain 8-1 (Itft) grew closely appressed to the CYXl agar surface while 8n34m (right) had a more aerial growth pattern and the colony appeared fluffier.

DISCUSSION When thereciprocal expertiment is performed (trans- fer of a nuclear accepting strain to a lawn of macer- Within the basidiomycetes, mitochondrial inherit- ated 8-1 mycelium), the dikaryotic mycelium does ance has been most closely studied in C. cinereus. not spread (ANDERSONet al. 1984). The rate of nuclear Reciprocal nuclear migration occurs at the junction migration is greater than can be accounted for by of two mating colonies as donor nuclei migrate simple out-growth of the dikaryon. A dikaryon (Dik through the resident cells of each recipient mono- D), collected over-the recipient mate away from the karyon (CASSELTONand CONDIT1972). Because mi- junction of the two cultures, contained both nuclear tochondria remain immobile, one consequence of a types but only the 34-2 mt type. This clearly dem- reciprocal exchange of migrating nuclei is the estab- onstrated that 8-1 nuclei, but not mitochondria, mi- lishment of two distinct dikaryons, each carrying a grated through theresident hyphae of 34-2 following different mitochondrial type. Following nuclear mi- hyphal anastomosis. As a direct consequence of uni- gration, the vast majority of dikaryotic cells contain directional nuclear migration, a single mitochondrial bothparental nuclei buteither one or the other type (342) was present in the majority of the dikar- parentalmitochondrial type. There is little oppor- yotic cells resulting from this cross. The other mito- tunity for recombination of mitochondrial genomes chondrial type (8-1) was found in dikaryons only as this can occur only in the anastomosed cells at the from the junctionof the cultures. This could ensure junction of thetwo mates (BAPTISTA-FERREIRA,ECON- that more fruiting bodies are derived from the di- OMOU and CASSELTON1983). The normal pattern for karyon having the 34-2 mitochondrial type. Hypo- basidiomycetes appears to be the equalbiparental thetically, spores with the mitochondrial type of the transmission of mitochondria to the spore progeny. nuclear recipient would then be preferentially dis- We have observed variationa of this pattern in crosses persed within an interfertile population of A. bitorquis of sexually compatible strains of A. bitorquis. Cloned in the wild. This assumes that there is no biasin nuclear and mtDNA markers were used to determine mitochondrial transmission during fruitbody forma- the fate of the parental nuclear and mitochondrial tion and sporulation. A second consequence of uni- types following hyphal anastomosis, nuclear migra- directionalnuclear migration is that nuclei of the tion, and sporulation. When homokaryotic strains 8- donor strain (8-1) are brought intocombination with 1 and 34-2 were paired, a dikaryon was formed at a wide variety of mitochondrial genomes whenever the interface of the two cultures, which then migrated compatiblea mating occurs. Nuclear migration, unidirectionally over the resident mycelium of 34-2. therefore, promotesmixing of nuclear and mito- This was either due to the intrusive growth of 8-1 chondrial genotypes. hyphaethrough the resident 34-2 hyphae, or the Unidirectional migration of 8- 1 nuclei accounts for migration of 8-1 nuclei through the resident cells of the clonal propogation of the 34-2 mt type, but how 34-2. When inoculum of strain 8-1 is transferred to is the transmission of the 8-1 mt type affected by the a lawn of macerated mycelium of a nuclear accepting lack of reciprocal migration by 34-2 nuclei? At the strain, the spread of dikaryotized mycelium is rapid; junction of homokaryotic hyphae of 8-1 and 34-2, muchmore so thanthe growth of thedikaryon. anastomosis resulted in the mixing of nuclei and 40 W. E. A. Hintz, J. B. Andersonand P. A. Horgen mitochondria of both mating types in a single fused drial type to the spores. The bias in mitochondrial cell. It is only in these fused cells that mitochondrial transmission may dependon the timing of spore genomerecombination is possible. In most fungi, maturation and release. Late release of spores with persistentheteroplasmons are rare, and organelles the 34-2 mitochondrial type would result in an ap- segregate rapidly duringmitotic divisions subsequent parent bias in mitochondrial transmission. A third to fusion. This probably invokes an element of ran- possibility is that the presence of the 8-1 mitochon- domness in the physical partitioning of organelles drial type in achimeric fruitbody suppresses the (BIRKY1978; TREAT-CLEMMONSand BIRKY 1983). development of basidia and having the Segregation of mitochondria from the anastomosed 34-2 mitochondrial type. Microdissection and culture A. bitorquis cellsgave rise to two dikaryons, having of immature spores and gilltissues in conjunction thesame nuclear components but different mito- with mt DNA analysis will resolve this question. chondria. Recombinant mitochondrial genomes were The growth rate of the dikaryon, and the trans- not detected by RFLPs. Since dikaryons having either mission of mitochondria to the spore progeny, were the 8-1 or 34-2 mitochondrial types were recovered bothinfluenced by mitochondrialgenotype. Does mitochondrialinheritance was biparental. The two mitochondrial genotype influence the phenotype of cell types segregated upon vegetative growth, and the vegetative homokaryons? In order to answer this cells having the 34-2 mitochondrial type eventually question we selected homokaryotic strains having outcompeted cells having the 8-1 mitochondrial type. identical nuclei butdifferent cytoplasms. This is During sporocarp development one,or a very few, possible for fungi with an extended dikaryotic stage germ cells proliferate to give rise to the gills, basidia, prior to sexual fusion of the nuclei provided that: and eventually the spores. At thejunction oftwo (1) the two mitochondrial types segregate in a pre- cultures,one would expectto occasionally recover dictable way and (2) the hyphaecan be dedikaryotized chimericfruitbodies, composed of dikaryotic cells and homokaryons recovered. Nuclear migration pro- having different cytoplasms. This is aptly illustrated vided a mechanism for the prediction of mitochon- by the pale-gill variant of Coprinw lugopus described drial type, hence a dikaryon, (Dik D), carrying both by DAY (1959). This is a cytoplasmically inheritable parental nuclei (8-1,34-2) but only one mitochondrial conditionwhere the density of tetradson the gill type(34-2), was chosen for dedikaryotization.Pro- surface is greatly reduced, hence the fruit bodies are toplasts containing single nuclei, or multiple nuclei pale in color. Pairing of the pale-gill homokaryon of the same type, gave rise to homokaryotic cultures with anormal stock results in certainfruitbodies uponregeneration. Almost 50% of theprotoplast having sectored, rather than uniformlypale or dark, regenerates were homokaryotic and contained nuclei gills. This probably indicates segregation of the two from 8-1 and mitochondriafrom 34-2. Thisstrain cytoplasmic types even during growth and differen- was called 8n34m to indicate the source of its genetic tiation of the fruitbody (FINCHAM,DAY and RADFORD components. No homokaryonscontaining nuclei 1979). Asimilar result was expected forthe A. bitorquis from 34-2 were recovered. Asymmetry in the recov- dikaryon collected at the junctionof the two cultures. ery of homokaryons has been observed in other Certain of the germ line cells should contain the 8-1 basidiomycetes and has beencorrelated to mating cytoplasm whereas others shouldcontain the 34-2 type in Schizophyllum (RAPER 1985). For this reason cytoplasm with bothcontributing to the fruitbody the reciprocal experiment (the recovery of homokar- and spores. Fruiting of the junction dikaryon (Dik yons containing 34-2 nuclei and 8-1 mitochondria A) revealed a biaswhich favoredthe transmission from Dik A) was not possible. When8n34m was the 8-1 mitochondrial type to the prog- paired with 34-2, nuclear migration toward 34-2 was eny despite the junctionfruitbody being chimericfor again observed. Nuclear migrationwas unaffected by mitochondrialtype. We expectedthat spores with mitochondrial genotype and must therefore be de- either parental mitochondrial type would be equally pendent on the 8-1nucleus. Back-crossing the spore recovered from the chimeric fruitbody; however, the progenyto the parental stocks, gave inconclusive transmission of mitochondria to the spore progeny results for the transmission of the nuclear migration was biased in favor of the nuclear donating strain (8- trait (not shown). This was in part dueto the difficulty 1). Whether this is ageneral feature of nuclear in effectively scoring this trait. Nuclear migration is donating strains, or is unique to 8-1, remains to be probably an elaborate process requiring the interac- determined. Spore inviability, conferred by the 34-2 tion of many nuclear gene products. We have shown, mitochondrialgenotype, could explain this bias in however, that nuclear migration was not dependent mitochondrial transmission. This is not the case, on the mitochondrial genome. As in the dikaryon, however, as spore progeny of the dikaryon collected the mitochondrialgenotype of the homokaryons distal to the junction zone (Dik D), carried the 34-2 influenced vegetative growth. Unmodified strain mitochondrial type. There was no physiological bar- 8-1 grew closely appressedto the agar surface rier preventing the transmission of either mitochon- while the hyphae of 8n34m grew more aerially. The M itochond rial InheritanceMitochondrial in Agaricus 41 two cultures were easily distinguished by colony BAFTISTA-FERREIRA,J. L. C., S. ECONOMOUand L. A. CASSELTON, morphology. 1983Mitochondrial geneticsof Coprinus: Recombination of Mitochondrial genotype had an obvious effect on mitochondrial genomes. Curr. Genet. 7: 405-407. BIRKY,C. W. Jr. 1978Transmission genetics of mitochondria the phenotypic expression of both the dikaryotic and and chloroplasts. Ann. Rev. Genet. 21: 471-512. the homokaryotic stages in the life history of A. CASSELTON,L. A,, and A. CONDIT,1972 A mitochondrial mutant bitorpis. Two strategies can be taken by basidiomy- of Coprinus lagopus. J. Gen. Microbiol. 72: 521-527. cetes for the transmission of mitochondria. Bidirec- CASTLE,A. J., P. A. HORCEN andJ. B. ANDERSON. 1987Restriction tional nuclear migration results in the equal clonal fragment length polymorphisms in the Agaricus brunnescens and Agaricus bitorquis. Appl.Environ. Microbiol. propagation of the mitochondrial genotypes of both 53: 816-822. mates. Specific mitochondrial types can be traced CHANG,S. T., and W. A. HAYES,1978 The Biology and Cultivation back through the pedigree of an individual as mito- of Edible Mushrooms. Academic Press, New York. chondrial inheritance is clonal and biparental (TAY- DAY,P. R. 1959 A cytoplasmically controlled abnormality of the Coprinus lagqus. 13: LOR 1986). When there is no nuclear migration, the tetrads of Heredity 81-87. FISCHAM,J. R. S., P. R. DAYand A. RADFORD,1979 Botanical parental mitochondrial genotypes segregate fromthe Monographs. IV. (Fourth Edition) Fungal Genetics. University of anastomosed cells and there is a much greater op- California Press, Berkeley, Calif. portunity for mitochondrial genome recombination. HINTZ,W. E., M. MOHAN,J. B. ANDERSONand P. A. HORGEN. Mitochondrial inheritance could be either biparental 1985 The mitochondrial DNAs of Agaricus: heterogeneity in or uniparental, depending on the bias in the trans- A. bitorquis and homogeneity in A. bmnnescens. Curr. Genet. 9: 127-132. mission of mitochondria via the basidiospore prog- HUDSPETH,M. E., D. S. SHUYARD,K. M. TA~Iand L. I. GROSSMAN. eny. Recombination of different mt DNAs prevents 1980 Rapid purification of yeast mitochondrial DNA in high the tracing of specific mitochondrial types through a yield. Biochim. Biophys. Acta 610: 221-228. pedigree; however, mitochondrial mutations can still MANIATIS,T., E. F. FRITSCHand J. SAYBROOK,1982 Molecular betraced. Unilateral nuclear migration combined Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. elements of both of these strategies. Transmission of RAPER,C. A,, 1976 Sexuality and the life cycle of the edible, wild the parental mitochondrial types to the dikaryon was Agaricus bitorquis. J. Gen. Microbiol. 95: 54-66. biparental but unequal, favoring that of the nuclear RAPER,C. A,,1985 B-Mating-type genesinfluence survival of recipient mate. Alternatively, the biasin mitochon- nuclei separatedfrom heterokaryons of Schizophyllum. Exp. drial transmission via the basidiospore progeny fa- Mycol. 9: 149-160. vored the mitochondrial type of the nuclear donor. RAPER,C. A,, and G. KAYE, 1978 Sexual and other relationships in the genus Agaricus. J. Gen. Microbiol. 105: 135-151. This compensatoryfactor could ensurethat both SANANTONIO, J. P., 1971A laboratorymethod to obtain fruit mitochondrial types are maintained within a breeding from cased grain spawn of the cultivated mushroom, Agaricus population of A. bitorpis. bisporus. Mycologia 63: 16-21. STEVENS,R. B., 1981 Guidebook, Edited by R. B. STEVENS. Thisstudy was supported by StrategicGrant GI540 and by University of Washington Press, Seattle, Wash. Cooperative Research DevelopmentGrant CRD-8616 fromthe TAYLOR,J. T., 1986 Fungal evolutionary biology and mitochon- Natural Sciences and Engineering Research Council of Canada. drial DNA. Exp. Mycol. 10: 259-269. We thank A. CASTLEfor his help in protoplast production. TREAT-CLEMMONS,L. G., and W. BIRKY,JR., 1983 Persistent LITERATURECITED heteroplasmic cells for mitochondrial genes in Saccharomyces cerevisiae. Curr. Genet. 7: 489-492. ANDERSON,J. B., D. M. PETSCHE,F. B. HERR andP. A. HORGEN, ZOLAN, M., and P. PUKKILA, 1986 Inheritanceof DNA methyla- 1984 Breeding relationships among several species of Agar- tion in Coprinus cinereus. Mol. Cell. Biol. 6: 195-200. icus. Can. J. Bot. 62: 1884-1889. Communicating editor: R. L. METZENBERC