INTRODUCTION

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Pleurotus ostreatus ("oyster mushroom") is an edible basidiomycete of increasing biotechnological interest due to its ability to degrade both wood (10, 19) and chemicals related to lignin degradation products (1). Furthermore, this fungus produces secondary metabolites with pharmaceutical applications (2, 3, 14) and some proteins of potential industrial use (33, 34, 40). This biotechnological interest in P. ostreatus has fueled research on different aspects of its molecular biology. Genetic studies on it are limited, however, by the lack of knowledge about the details of the organization of its genetic material.

Fungal cytogenetics has been hampered by the small size of the chromosomes, the lack of known sexual stages in many medically and industrially important species, and the occurrence of endomitosis (4). The chromosomes of most fungi, however, are small enough to be separated by using pulsed-field gel electrophoresis (PFGE) (see reference 21 for a review). The molecular karyotype of Coprinus cinereus, obtained by PFGE, has been correlated with its genetic map by the use of cloned gene probes and genetic studies (26, 27). Electrophoretic karyotyping of different fungi has shown that chromosome length polymorphisms (CLPs) are a common and prominent feature of these organisms (see reference 42 for a review). Two mechanisms have been proposed for the generation of these polymorphisms: increasing the copy numbers of particular sequences, such as the ribosomal DNA (rDNA) (27, 36) and subtelomeric repeats (9, 17), and mitotic and meiotic recombination processes (42, 43). In many cases, however, these CLPs seem to have minor genetic consequences, since many different karyotypes are found in a given species. However, some reduction in spore viability has been observed in outcrosses between C. cinereus strains with differences in their chromosome sizes (25) and in crosses between Saccharomyces cerevisiae strains with pronounced CLPs (42). For some species, it has been reported that CLPs within a given strain will eventually disappear by chromosome recombination (43).

The molecular karyotype of P. ostreatus has not been fully clarified yet. Several authors have reported different chromosome numbers and genome sizes by using various analytical techniques, including microscopy, restriction fragment length polymorphism (RFLP) analysis, and PFGE (4, 23, 31); however, the results are sometimes contradictory. By using PFGE experiments, Sagawa and Nagata (31) reported six chromosomes and a total genome size of 20.8 Mbp with chromosome sizes ranging from 2.1 to 5.2 Mbp per chromosome. Peberdy et al. (23) identified nine chromosomes by PFGE with a total genome size of 31.3 Mbp and chromosome sizes ranging from 1.1 to 5.7 Mbp per chromosome. Microscopy studies, on the other hand, have been hampered by the small size of P. ostreatus chromosomes. Notwithstanding, chromosome numbers ranging from 6 to 10 have been reported (4). The optimization of PFGE separation of fungal chromosomes (36, 37, 43) allowed the study of the molecular karyotype of Agaricus bisporus and the assignment of genes to chromosomes in C. cinereus. In this study, we used this technique to clarify the genomic structure of P. ostreatus. These data, together with a genetic linkage map of this P. ostreatus strain (16), will help in the design of breeding and cloning strategies in this mushroom.

	MATERIALS AND METHODS

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Microbial strains and culture conditions. P. ostreatus N001 is the dikaryotic fungal commercial strain used in this work (15, 24). Vegetative cultures of monokaryotic and dikaryotic mycelia were grown on solid Eger medium (20 g of malt extract, 15 g of agar, 1 liter of H₂O) (7) at 24°C in the dark.

Protoplast preparation and mycelium regeneration. The medium used for the production of P. ostreatus protoplasts was MMP (36): 10 g of malt extract, 5 g of mycological peptone (Oxoid, Hampshire, United Kingdom), and 1 liter of 10 mM 3'-(N-morpholino)-propanesulfonic acid (MOPS) adjusted to pH 7.0 with KOH. Solid medium agar-MMP was made by adding 15 g of agar per liter of MMP. Ten petri dishes containing agar-MMP covered with uncoated cellophane were each inoculated with 5 plugs of mycelium and incubated at 24°C in the dark. The mycelia were removed from the cellophane after 5 days of incubation and fragmented in liquid MMP with a Waring blender for 15 s at low speed. The suspension was used to inoculate three 2-liter Fernbach flasks (150 ml of MMP per flask) and incubated for 2 days at 24°C in the dark without shaking. The mycelium was collected by filtration through a nylon cloth (300-µm mesh) and rinsed with sterile water. The washed mycelium was removed from the nylon cloth and transferred to a clean sterile tube which was filled up to 40 ml with sterile water. Then 40 ml of 2× protoplasting medium (3 mg of Trichoderma harzianum cell wall lytic enzymes per ml [37, 38], 1.2 M sucrose, and 10 mM MOPS [pH 6.0] adjusted with KOH and sterilized by filtration through a 0.45-µm-pore-size membrane) was added. The suspension was mixed, transferred to a Fernbach flask, and incubated for 1 h at 24°C under gentle shaking. The mixture of hyphal fragments and protoplasts was filtered through nylon cloths of 1,000-, 500-, and 150-µm mesh equilibrated with 0.6 M sucrose and through a glass wool column (total column volume, 50 ml) equilibrated with 0.6 M sucrose, to remove cellular debris. Protoplasts were collected by centrifugation (15 min, 500 × g, 10°C) and washed twice with a 0.6 M sucrose solution. After suspension of the final pellet in 0.6 M sucrose, the protoplast concentration was determined by using a hemocytometer.

Incompatibility tests. The mating genotype of the monokaryotic protoclones isolated in this work was determined by mating experiments with the four incompatibility testers derived from P. ostreatus N001 (15). The mating trials were made by inoculating the protoclone and the corresponding tester, placed 4 cm apart in the center of a petri dish containing solid Eger medium. The plate was then incubated at 24°C in the dark for approximately 18 days, until the two mycelia formed a large contact zone. A piece of mycelium was cut off from the contact zone, placed on a new culture plate, allowed to grow for some days, and examined under the microscope for the presence of true clamp connections (dikaryons), unfused (false) clamp connections (common B heterokaryons), or the absence of clamp connections (common A heterokaryons) (8).

DNA manipulation. For DNA isolation, 100 ml of liquid SMY culture medium (10 g of sucrose, 10 g of malt extract, 4 g of yeast extract, 1 liter H₂O [pH 5.6]) was inoculated with either dikaryotic or monokaryotic mycelium and incubated at 24°C in the dark without shaking. Mycelia were collected by vacuum filtration and frozen in liquid nitrogen until used. DNA from 2 g of mycelium was purified by using the protocol described by Dellaporta et al. (5) with minor modifications: the frozen mycelium was ground to a fine powder with a mortar, resuspended in 15 ml of extraction buffer (100 mM Tris, 50 mM EDTA, 500 mM NaCl, 10 mM -mercaptoethanol [pH 8.0]), and gently shaken. Then 10 ml of a solution of sodium dodecyl sulfate, containing 200 g of H₂O per liter, was added to the suspension, and the mixture was incubated at 65°C for 10 min. Most of the proteins and polysaccharides were removed by adding 5 ml of 5 M potassium acetate followed by an incubation at 0°C for 20 min and removal of the precipitate by centrifugation. General molecular biology protocols were used as described by Sambrook et al. (32) and Dieffenbach and Dveksler (6).

Microsatellite analysis. Microsatellite analysis was performed using the sequence 5'-GACA GACA GACA-3' as an oligonucleotide. A PCR was performed in a reaction volume of 25 µl containing 100 ng of mycelial DNA as a template, 2 mM MgCl₂, 67 mM Tris HCl (pH 8.8), 16 mM (NH₄)₂SO₄, 0.1 g of Tween 20 per liter, a 200 µM concentration of each of the four nucleotide triphosphates (ATP, CTP, GTP, and TTP), 1.9 to 2.5 µM concentrations of the primer oligonucleotide, and 1 U of Taq polymerase (Eurobiotaq; Ecogen, Barcelona, Spain). The amplification reactions were performed in a Perkin-Elmer Cetus DNA cycler (The Perkin-Elmer Corporation, Norwalk, Conn.). The amplification reactions were performed for 35 cycles with the following touchdown (11) cycle profile: a 1-min DNA denaturation step at 94°C, a 2-min annealing step (see below), and a 3-min extension step at 72°C. The annealing temperature in the first two cycles was 65°C; it was subsequently reduced each cycle by 2°C for the next 11 cycles and was continued at 40°C for 22 cycles. The reaction was finished with a 7-min-long extension step at 72°C. Amplification products were analyzed by electrophoresis in 1.5% (wt/vol) agarose gels in TAE buffer (400 mM Tris, 200 mM sodium acetate, 20 mM EDTA [pH 8.3]) and stained with ethidium bromide.

Chromosome-sized DNA preparations. The isolation of genomic DNA suitable for chromosome separation by PFGE was performed as described by Sonnenberg et al. (36): protoplasts were diluted with 2% (wt/vol) InCert agarose (FMC Corporation, Rockland, Maine) kept at 42°C to a final concentration of 0.7% (wt/vol) agarose and 10⁹ protoplasts per ml. After transfer to a prewarmed mold (40°C), the agarose was allowed to solidify on ice for 30 min. The resulting plugs were incubated in NDS buffer (0.5 M EDTA, 0.01 M Tris-HCl [pH 9.5], 1% [wt/vol] N-laurylsarcosine) containing 1 mg of proteinase K per ml for 25 h at 50°C. The plugs were washed three times in 50 mM EDTA (pH 8.0) at room temperature and stored at 4°C in 50 mM EDTA containing 0.2% (wt/vol) NaN₃ until they were used.

Pulsed-field electrophoretic separation of chromosomes. Pulsed-field electrophoresis conditions were optimized for the separation of P. ostreatus chromosomes. The modifications were based on the method previously described by Sonnenberg et al. (37). The gels were run at 14°C in 0.8% (wt/vol) agarose (SeaKem; FMC Corporation) in 0.5× TBE-cytidine buffer (1× TBE-cytidine is 0.089 M Tris-borate, 0.0025 M EDTA, 1 mM cytidine [pH 8.3]) with a CHEF-DR II apparatus (Bio-Rad, Hercules, Calif.). The electrophoretic parameters used had three ramped switching intervals: (i) from 600 to 800 s at 100 V during 48 h, (ii) from 2,500 to 3,000 s at 50 V during 48 h, and (iii) from 3,300 to 3,600 s at 50 V during 96 h. The electrophoretic buffer was replaced once after 96 h. The chromosomes were visualized by ethidium bromide staining by using a solution containing 0.5 µg of ethidium bromide per ml in 1 mM cytidine. The chromosomes of Hansenula wingei and of Schizosaccharomyces pombe (Bio-Rad) were used as size standards.

Identification of chromosome-specific DNA probes. Two different kinds of chromosome-specific DNA tags were used: probes for functional genes and anonymous DNA sequences (Table 1). The anonymous probes were generated as described by Williams et al. (41) and Larraya et al. (15): PCRs for the generation of randomly amplified polymorphic DNA (RAPD) markers were performed in a reaction volume of 25 µl containing 100 ng of mycelial DNA as a template, 2 mM MgCl₂, 67 mM Tris HCl (pH 8.8), 16 mM (NH₄)₂SO₄, 0.1 g of Tween 20 per liter, a 200 µM concentration of each of the four nucleotide triphosphates (ATP, CTP, GTP, and TTP), 1.9 to 2.5 µM concentrations of the primer oligonucleotide, and 1 U of Taq polymerase (Eurobiotaq; Ecogen). The oligonucleotides used as primers for the reaction were 10-mers belonging to the S, L, and R Operon series (Operon Technologies Inc., Alameda, Calif.). Amplification reactions were performed in a PTC-200 (Peltier thermal cycler; MJ Research, Watertown, Mass.) by using the following program: a 1-min denaturation at 94°C, a 1-min annealing at 37°C, and a 1.3-min extension at 72°C, for 39 cycles. Amplification products were analyzed by electrophoresis in 1.5% (wt/vol) agarose gels in TAE buffer (400 mM Tris, 200 mM sodium acetate, 20 mM EDTA [pH 8.3]) and stained with ethidium bromide. For molecular size markers, HindIII-EcoRI-digested  DNA was used (32). The appropriate RAPD markers were extracted from the agarose gels and cloned into pGEM-T (Promega, Southampton, United Kingdom) so they could be used as chromosome-specific probes.

Probe labeling. Appropriate DNA probes were labeled with digoxigenin according to the supplier's specifications (Boehringer Mannheim, Mannheim, Germany) (13). PFGE-separated chromosomes were blotted onto the appropriate membranes and hybridized with the digoxigenin-labeled probes.

Biological material accession numbers. The two protoclones produced by dedikaryotization of P. ostreatus N001 described in this paper have been deposited in the Spanish Type Culture Collection under the accession numbers in parentheses: PC9 (CECT20311) and PC15 (CECT20312).

Nucleotide sequence accession numbers. The chromosome-specific DNA sequences described in this paper have been deposited in the EMBL and GenBank sequence databases under the accession numbers in parentheses: vmh1 (AJ238147), Rib (AJ242635), S11₉₀₀ (AJ242636), L12₁₈₄₁ (AJ242637), L15₆₁₄ (AJ242640), L16₈₇₇ (AJ242641), R8₃₀₃ (AJ242642), L3₁₃₃₈ (AJ242643), and vmh3 (AJ238148).

	RESULTS

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Protoclone isolation. In most fungi, the constituent nuclei of the dikaryon do not fuse during vegetative growth. Protoplasting of the mycelium and isolation of colonies derived from single regenerated protoplasts occasionally results in monokaryons. These monokaryons are called protoclones (35), and their nuclear constitution is identical to either one of the two parental nuclei present in the dikaryon from which they derived. In contrast to the parental monokaryons used to construct a dikaryon by mating, the protoclones share the cytoplasm of the dikaryon they were produced from.

Electrophoretic karyotype of P. ostreatus N001. The chromosomes of P. ostreatus N001 (dikaryon) and of the protoclones (monokaryons) PC9 and PC15, each of the latter bearing one of the two nuclei present in the dikaryon, were separated by PFGE (Fig. 1). The number of bands that could be resolved differs for the two protoclones: nine different bands could be identified in PC9, whereas eight bands appeared in the lane corresponding to PC15. Ethidium bromide staining of the chromosome bands revealed two in PC9 (4.7 and 2.9 Mbp) and three in PC15 (4.7, 3.4, and 2.9 Mbp) which were stained more intensely than the others, suggesting that each one of them could correspond to two different chromosomes of approximately the same size. The lane corresponding to the dikaryon, on the other hand, showed all of the bands that appeared in each one of the two protoclones, as expected. The sizes of the P. ostreatus N001 chromosomes varied between 1.4 and 4.7 Mbp (Table 1). Moreover, chromosome sizes in the two protoclones were different, indicating CLPs within P. ostreatus N001.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   Molecular karyotype of P. ostreatus N001. (A) PFGE separation of the chromosomes from the dikaryon N001 and the two protoclones PC9 and PC15. (B) Idiotype of the chromosomes present in the protoclones PC9 and PC15.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   Specific probe assignment to the different chromosomes of P. ostreatus. (A) PFGE separation of the chromosomes present in the protoclones PC9 and PC15. (B) Southern blot analysis of the hybridization of chromosome-specific probes selected among the RFLP probes used to construct the linkage map of P. ostreatus.

	DISCUSSION

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The experiments described in this study allowed the identification of 11 chromosomes per haploid genome in this strain of P. ostreatus. This number also corresponds to the number of linkage groups identified in P. ostreatus N001 (16). Additionally, each chromosome was individualized by chromosome-specific probes which allowed the clarification of ambiguities due to CLPs. Chromosome sizes cover all the ranges described by other authors for this species. The optimization of the pulse conditions allowed the reproducible resolution of chromosomes which, otherwise, would have comigrated in complex bands. The total haploid genome length for P. ostreatus is, on average, 35.0 Mbp. This value is slightly higher than that published by Peberdy et al. (23) (31.3 Mbp).

CLPs in fungi have been reported by several authors (see reference 42 for a review). Our data indicate that CLPs occur also in P. ostreatus, even within a single dikaryon. The fate of these polymorphic chromosomes during the meiotic process has not been investigated.

Table 1 shows the relative size differences between the two members of each pair of homologous chromosomes. Chromosomes II, VI, and VII show the major relative differences (i.e., more than 10% of their size). The rest of the chromosome size variations fall in the range of 3 to 9%, with the exception of chromosome I which was, essentially, of the same size in both protoclones. Interestingly, the size difference for the whole genome between the two protoclones is rather small (0.7 Mbp, 2% of the total size). This suggests that the total amount of genetic information present in each one of the two protoclones is similar, although it is differently organized. In this context, translocations have been demonstrated in C. cinereus (43) that support different organizations of the same genetic information in these fungi.

The size difference for chromosome II between protoclones PC9 and PC15 is 0.7 Mbp. This is about 15% of its total size. This chromosome carries the genomic region hybridizing to the rDNA probe. CLPs in chromosomes carrying rDNA genes have been reported by other authors in A. bisporus (36), Candida albicans (12, 29), Ustilago hordei (20), Leptosphaeria maculans (22), S. cerevisiae (29), and Cladosporium fulvum (39). According to Pukkila and Skrzynia (27) and Sonnenberg et al. (36), this polymorphism in C. cinereus and A. bisporus can be due to differences in the copy numbers of the rDNA genes. In the case of P. ostreatus, however, we could not find differences in the rDNA gene copy numbers between the two homologous chromosomes. This suggests that either the polymorphism detected in chromosome II is not due to the copy number of the rDNA genes or that the copy number polymorphism falls outside of the region detected by the probe used. It is interesting, however, to point out that a positive correlation between the growth rate and rDNA copy number has been reported in different fungi, such as Kluyveromyces lactis (18), Neurospora crassa (28), C. albicans, and S. cerevisiae (30). In this context, it is worth mentioning that protoclone PC9 (which carries a larger chromosome II) grows faster than protoclone PC15 (which carries a smaller chromosome II) (unpublished results). The sizes of chromosome VI differ by 0.4 Mbp (12% of its total size) when the homologous chromosomes in protoclones PC9 and PC15 are compared. It is relevant to point out that the two laccase genes probed in these experiments (POX1 and POX2) (10) hybridize to this chromosome and that chromosome VI was larger in protoclone PC9 (a fast-growing monokaryon) than in PC15 (a slow-growing monokaryon). The sizes of chromosome VII differ by 0.3 Mbp (9% of its total size) when the homologous chromosomes in protoclones PC9 and PC15 are compared. Finally, chromosome IX shows a small CLP (0.2 Mbp, corresponding to 7% of its total length). It is interesting to point out that RFLP probes found to be genetically linked to the B type incompatibility locus hybridize to this chromosome (16).

The CLPs detected in P. ostreatus N001 could reflect the hybrid nature of this commercial strain, which is vegetatively propagated by mushroom producers without going through sexual stages that would normalize chromosome sizes as predicted by the model of Zolan et al. (42, 43).

In summary, the results presented here clarify the chromosome number in P. ostreatus, demonstrate the existence of CLPs within a strain, and raise questions about the fate of chromosomes during meiosis in this fungus. These results will complement those of the P. ostreatus linkage map (16) and will help in the genetic manipulation of this fungus for breeding and biotechnological purposes.