WO2001012850A1

WO2001012850A1 - Non-sporulating basidomycetes

Info

Publication number: WO2001012850A1
Application number: PCT/NL2000/000568
Authority: WO
Inventors: Thomas Stefan Peter Mikosch; Antonius Silvester Maria Sonnenberg
Original assignee: Proefstation Voor De Champignoncultuur
Priority date: 1999-08-18
Filing date: 2000-08-14
Publication date: 2001-02-22
Also published as: AU6600200A

Abstract

The invention relates to the field of molecular biology as well as to the field of breeding methods for mushrooms, in particular cultivatable mushrooms. In particular the invention relates to the use of diagnostic methods derived from the field of molecular biology to be applied in breeding programmes that select mushrooms on genetic traits that improve their commercial value. The invention provides methods localising, identifying or marking genes or alleles or genetic trait loci, in particular but not restricted to those corresponding to a mushroom gene related to spore production, in samples such as but not limited to mycelium, spores or fruit bodies of mushrooms, by allowing for specific hybridisation with or amplification of genomic fragments of those genes or alleles or genetic trait loci of mushrooms.

Description

Title: Non-sporulating Basidomycetes

The invention relates to the field of molecular biology as well as to the field of breeding methods for mushrooms, in particular cultivable mushrooms. In particular the invention relates to the use of diagnostic methods derived from the field of molecular biology to be applied in breeding programmes that select mushrooms on genetic traits that improve their commercial value.

There are 50,000-200,000 different species of fungi in the world, depending on the reference. Their taxonomy is based on different systems and undergoes constant revision. The word mushroom refers mainly to macrofungi with edible fruiting bodies, while toadstool refers to those with poisonous fruiting bodies and other minor macrofungi. The macrofungi, with bolets and agarics as major classes, mostly belong to the Basidiomycotina, fungi producing basidiospores . Consequently, mushrooms and macrofungi are often called "basidiomycetes" , the word constituting a class description. However, mushrooms should not be limited to this one group, as morels, for example, such as true morel (Morchella esculenta) , belong to Ascomycotina: fungi producing ascospores . Mushroom is not a suitable designation for bracket fungi (Polyporaccae) , puff balls (Lycoperdacceae) or jelly fungi (Tremellales) , all of which are woodland macrofungi spreading abundant basidiospores. One of the major problems in cultivating oyster mushrooms is the abundant production of spores . Many workers in the production of mushrooms develop an allergy with symptoms similar to an "extrinsic allergic alveolytis (EAA) (Sonnenberg et al . , De champignon cultuur 40(7) :269- 272, 1996) . Especially mushroom worker's lung, a type III IgG mediated hypersensitivity pneumonitis or extrinsic allergic alveolitis is mentioned (Kurup et al . , Mycopathologia 98:91-99, 1989). In the mid-seventies Schulz et al . (Lancet 1, 29, 1974) described such an illness in Bavarian mushroom workers, and proposed Pleurotus florida spores to be the cause. The patients had an immediate skin reaction to an extract of Pleurotus spores and serum precipitins to Pleurotus antigen on analysis with Ouchterlony ' s gel diffussusion.

At the same time Stewart & Pickering (Lancet, p317, February 23, 1974) reported respiratory symptoms in workers cultivating Agaricus hortensis . These workers had precipitins to Agaricus hortensis but not to thermophilic actinomycetes . Skin and provocation tests to both these species were negative.

In the cultivation of oyster mushrooms, Pleurotus ostreatus, a similar disease occurs. In the Netherlands, four workers were reported to have developed EAA after working with oyster mushrooms. After inhalation provocation with the basidiospores of this mushroom the patients showed the symptoms of the disease (Cox et al . , Eur. Resp. J. 1:466-468, 1988) .

Also in the cultivation of Shii-take, Lentinus edodes, symptoms of EAA are commonly seen in workers . Five workers involved in picking and complaining of respiratory problems were subjected to inhalation provocation with basidiospores of Lentinus endodes ; all five developed specific symptoms of EAA (Cox et al . , Atemw. und Lungenkrankh 15:233-234, 1989) .

Furthermore, mushroom spores are often vectors that can transmit (pathogenic) viruses from one mushroom culture to another, thereby transmitting viral disease among mushrooms (A. Dieleman-van Zaayen, Mushroom Science VIII, London, 1972, pl31-154), and thereby endangering profitable mushroom culture. Also, the uncontrollable spore propagation of species, such as Pleurotus and Lentinus species, which decompose wood can attack woods of any kind and can thus cause considerable damage, especially to buildings neighbouring mushroom farms. In the light of above, it would be desirable in commercial mushroom farming to cultivate spore-less mushrooms . First and foremost , mushroom workers working with spore- less mushrooms only, would not be prone to spore- induced disease. Secondly, the mushroom cultures themselves would stand less risk to acquire viral infections, and thirdly, there would be less risk for fungal -growth at places where that is not desired.

Sonnenberg et al . , (ibid) measured the production of spores by fruit bodies of several commercial lines of so- called "traditional" and "poorly-sporing" strains of P. ostreatus and P. pulmonarius and estimated the number of spores that might be present in the air in growing rooms (Table 1) . It was found that the number of spores produced mainly depends on the weight of the fruit bodies, and particular of the head, hence the surface area of tissue that produces spores. An almost linear correlation was found between the weight of the head of the fruit bodies and the number of spores produced. This suggests that the developmental state of the fruit bodies has only a minor influence on spore production. Spore production by present "traditional" strains varies between 200 and 660 million spores per gram tissue per 24 hours. Spore production by the so called "poorly sporing" strains were in general ten times lower, varying from 21 to 60 million spores per gram tissue per 24 hours. Assuming that approximately 3 kilograms of fruit bodies are produced per m² and the ventilation in a growing room is 10 m³ per m² of cultivating area, spore concentration in the air might increase to 10¹⁰ spores per m³ and, when growing "poorly-sporing" strains, 10⁹ spores per m³. Even the latter number of spores is much higher than the threshold values for the induction of symptoms of extrinsic allergic alveolitis (EAA) , illustrating that in essence spore-less mushrooms are needed in mushroom cultivation to solve the spore- associated problems .

10° to 20° C, in practice often at around 12° to 18° C. At temperatures around 10-12° to 18-20° C, the time for the formation of fruit bodies is in general the least whereas the yield is the most. Furthermore, at those temperatures emerging fruit bodies of mushrooms in general are less susceptible for and thus better protected against infections with bacteria or fungi, furthermore, better quality and more fruit bodies can develop that keep longer, and less heating is needed for the cultivation process; all desirable characteristics when cultivating mushrooms for commerce. Consequently, fructification temperatures of around 10-12° to 18-20° C are in general considered optimal for commercial mushroom cultivation. P. ostreatus 42 X 11 has been used several times in mushroom breeding programmes to cross with normal spore-producing strains to obtain commercially more viable strains with a spore-less character. In one example (US 4658083) Pleurotus strains are obtained that are poorly spore-producing. These spores can germinate and homokaryotic mycelium issuing from the spores bears the gene responsible for the poor spore- production. However, besides the fact that these strains cannot be considered essentially spore-less (see for example also Table 1, Som 3200 and Som 3300, that still produce more spores than desired) they still have other disadvantages as well. For one, their need for light is greater than that of the known commercial strains, but in intensity rather than in time of exposure. Also, these descendants of P. ostreatus 42 X 11 again have various undesirable morphological characteristics which make them less commercially attractive, for example: spore-less lamellae are decurrent over the major part of the height of the foot, and the lamellae part of the head is more developed than that of the other strains of Pleurotus, and, again, these strains in general also have the above described undesirable trumpet shape. The invention provides methods localising, identifying or marking genes or alleles or genetic trait loci, in particular but not restricted to those corresponding to a mushroom gene related to reduced spore production, in samples such as but not limited to mycelium, spores or fruit bodies of mushrooms, by allowing for specific hybridisation with or amplification of genomic fragments of those genes or alleles or genetic trait loci of mushrooms. The invention thus provides access to molecular markers and marker assisted selection of mushrooms and strains thereof that is based upon genetic variation that exists within mushroom genes that influence a production trait directly or indirectly. One of the methods that the invention provides is a method that identifies or marks loci or genes and that can distinguish between characteristics of alleles of those genes which characteristics serve as markers in selection programmes for mushrooms and strains thereof with specific versions of those genes that are directly linked with improved production traits.

The invention further provides a method wherein markers such as polymorphic restriction sites or microsatellite sequences associated with or within functional genes and thus different alleles of those genes are identified by allowing for specific hybridisation with or amplification of genomic fragments of those genes, in particular by allowing for specific amplification of fragments of a gene related to spore production. Hybridisation or amplification (the best known being PCR) are methods that are well known in the art.

A short description of the PCR and other hybridisation techniques used herein is given herein. Other nucleic acid sequences, primers, enzymes or conditions can of course be applied. After hybridisation and/or amplification suitable methods of identifying wanted alleles, nucleic acid sequences and molecular markers such as micro-satelites or polymorphic sites are known in the art, e.g. in Sambrook , Fritsch and Maniatis (Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989) .

By a method as provided by the invention large numbers of mushrooms and strains thereof can be rapidly genotyped for studies in which genotypic variation can be associated with growth characteristics and other production or performance traits of mushrooms, and a method as provided by the invention allows to select or obtain a mushroom or strain thereof having a desired genetic trait. For example, from cultures of basidiomycetes such as Agaricus species such as Agaricus bisporus, Lentinus species such as Lentinus edodes, and Pleurotus species such as Pleurotus ostreatus, but also from less cultivated species such as Auricularia, Volvariella and Flammulina species, those mushrooms can now, using marker assisted selection, be selected or obtained having a desired genetic trait, for example related to fructification requirements, production yields, colour, size, or any other commercially attractive trait.

In a preferred embodiment, the invention provides marker assisted selection from cultures of basidiomycetes such as Agaricus species such as Agaricus bisporus, Lentinus species such as Lentinus edodes, and Pleurotus species such as Pleurotus ostreatus, but also from less cultivated species such as Auricularia, Volvariella and Flammulina species, on a genetic trait related to spore production, for example by using a method as provided by the invention for localising, identifying or marking an allele of a mushroom, whereby said localisation, identification or marking is carried out using a nucleic acid molecule or fragment thereof is derived from a gene related to reduced spore production of said mushroom, for example wherein said nucleic acid molecule hybridises with or comprises a nucleotide sequence as listed in figure 1, 2 or 3 or a complementary sequence or the RNA equivalent thereof .

Traditionally, breeding programmes have selected for phenotypic characteristics or production traits of mushrooms and strains thereof, and by selecting mushrooms and strains thereof on their breeding value calculated mainly from phenotypic measurements of production traits, in several instances breeding has greatly improved the genotype for production traits of mushrooms. Selection for phenotypic characteristics entails mainly selection of the offspring of the mushrooms to be selected whereas selection of specific genotypic characteristics as provided by the invention characteristics allows for earlier and specific detection of mushrooms and strains thereof of interest. In particular, in a method as provided by the invention for selection of specific genotypic characteristics nucleic acid related to said genotypic characteristics is used. For example, the invention provides an isolated and/or recombinant nucleic acid or fragment thereof comprising a nucleic acid hybridising with a nucleic acid derived from a mushroom gene related to reduced spore production. Such a nucleic acid or fragment thereof according to the invention comprises a nucleotide sequence having at least 50% homology with a nucleotide sequence as listed in figure 1, 2 or 3 or a complementary sequence or the RNA equivalent thereof. Furthermore is provided such a nucleic acid according to the invention wherein said homology is at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%. Preferably, such a nucleic acid is derived from a mushroom where it is not or only weakly expressed in vegetative mycelium (expression as detected by Northern hybridisation) , and preferentially or even only expressed in lamellae, preferably specifically expressed in basidia and not or only aspecifically or little in other cell types of lamellae. Within methods that select on specific genotypic characteristics, the invention provides methods that detect genetic variation in genes or genetic trait loci that are associated with production traits of mushrooms and strains thereof as well as methods that detect genetic variation in functional genes that directly influence those production traits. One of the former methods as provided by the invention is a marker assisted selection wherein polymorphisms, microsatelites , or other markers identified in a random manner are associated with production traits. One of the latter methods as provided by the invention comprises using nucleic acid derived from said gene or functional homologous to select desired mushrooms. For example, the invention provides a method for selecting a mushroom and strains thereof for its potential sporelessness comprising testing a sample from said mushroom for the presence of a nucleic acid sequence located at genetic trait locus marking a gene related to spore production. Furthermore, the invention provides an essentially spore-less mushroom obtainable or selectable by a method according to the invention, for example selected or obtained from cultures of basidiomycetes such as Agaricus species such as Agaricus bisporus, Lentinus species such as Lentinus edodes, and Pleurotus species such as Pleurotus ostreatus, but also from less cultivated species such as Auricularia, Volvariella and Flammulina species. Essentially spore-less herein means a spore production as for example seen with Pleurotus that is generally less than about ten, preferably less than about five spored basidia per lamella. For example the invention provides an essentially spore-less mushroom or strains thereof selected from a cultivatable basidiomycete showing optimal fructification at around 10° to 20° C, such as a spore-less oyster mushroom derived from a Pleurotus species. Also provided is an essentially spore-less mushroom having a relatively small stipe and a relatively well developed head instead of the undesirable trumpet shape, in particular when derived from a Pleurotus species. The invention furthermore provides a mushroom or strains thereof, herein also called a knockout mushroom, provided with a functional deletion in a gene related to spore-production. In a knockout mushroom the expression of a gene (or more genes) is at least partly prevented or hampered by functionally deleting said gene. Standard methods to transform (with poly-ethylene-glycol , electroporation, biolistics, Agrobacterium) are well established with various organisms including fungi. Strategies to silence a gene are also well established and numerous (gene-replacement, antisense RNA, ribozyme, etc.), as are the methods to achieve knock-out etc. (restriction enzyme mediated integration (REMI) , linearised plasmid, fragments, etc.) . In particular, the invention provides a mushroom or strain thereof wherein said gene or part thereof hybridises with a nucleic acid or part thereof comprising a nucleic acid hybridising with a nucleic acid derived from a mushroom gene related to spore production, in particularly a gene related to the number of basidia bearing (fertile) spores or frequency of basidia bearing (fertile) spores versus basidia bearing infertile or no spores . Such a nucleic acid or fragment thereof according to the invention comprises a nucleotide sequence having at least 50% homology with a nucleotide sequence as listed in figure 1, 2 or 3 or a complementary sequence or the RNA equivalent thereof. In a preferred embodiment, the invention provides a knockout mushroom according to the invention which is essentially spore-less, for example derived by functionally deleting a gene of a mushroom from cultures of basidiomycetes such as Agaricus species such as Agaricus bisporus, Lentinus species such as Lentinus edodes, and Pleurotus species such as Pleurotus ostreatus, but also from less cultivated species such as Auricularia, Volvariella and Flammulina species.

The invention finds its use in cultivation of mushroom whereby an essentially sporeless character is desirable.

Cultivation of mushrooms is done for various purposes, e.g. for consumption because of their gourmet, nutritional, nutraceutical , medicinal or herbal characteristics, for bioremediation, for mycorrhization of trees, and so on, and, considering that essentially sporeless musrooms of all kinds, and in particular of Basidomycetes, are provided, such essentially spore-less cultures are provided as well. The invention provides use and/or cultivation of basidiomycetes for purposes such as consumption, bioremediation, mycorrhization of trees and their use as nutri- and nutraceuticals and as source as pharmaceutical compositions. As said, many fungi in this Class of Holobasidiomycetes produce spores that are known to be allergenic (Latge & Paris, 1991. The fungal spore: reservoir of allergens, pp 379-401. In G.T.Cole and H.C. Hoch (ed.), The fungal spore and disease initiation in plants and animals. Plenum Press, NY. Horner, Helbling, Salvaggio & Lehrer, 1995. Fungal allergens. Clinical Microbiology Reviews, vol 8 (2) , pp 161-179) and many basidiomycetes are known for their therapeutic effect (Wasser & Weis, 1999. Therapeutic effect of substances occurring in higher Basidiomycetes mushrooms : a modern perspective. Critical Review in Immunology 19(1), pp65- 96) . Many of these mushrooms are cultivated (Chang & Miles, 1989. Edible mushrooms and their cultivation. CRC Press) . Most of these species produce spores that are allergenic (Horner, Helbling & Lehrer, 1993. Basidiomycete allergens: comparison of three Ganoderma species. Allergy 48(2), 110- 116. Horner, Helbling, Salvaggio & Lehrer, 1995. Fungal allergens. Clinical Microbiology Reviews, vol 8 (2), pp 161-179. Van Loon, Cox, Wuisman, Burgers & Van Griensven, 1992. Mushroom worker's lung. Detection of antibodies against Shii-take (Lentinus edodes) spore antigens in Shiitake workers. J Occup Med, 34(11): 1097-1101), and the invention now provides esentially spore-less production of these mushrooms. Also, mushrooms produced as mycorrhiza's in plantations can cause allergic reactions (Chavasco et al . , 1997. Evaluation of the allergenicity of spore and mycelia extracts of Pisolithus tinctorius. Rev. Inst . Med. Trop. Sao Paulo, 39(5): 245-252.) .The inhibition or elimination of spore production also leads to an inhibition of the maturation of fruit bodies. The ultimate goal of a mushroom is to produce and distribute spores. During maturation of fruit bodies, tissue is degraded and used as substrate to produce spores (Umar & Van Griensven, 1997. Morphological studies on the life span, developmental stages, senescent and death of fruit bodies of Agaricus bisporus. Mycol . Res. 101 (12): 1409-1422). The essential elimination of spore production, as provided herein, therefore, provides a longer shelf life of picked mushrooms as well .

The invention is further described in the detailed description without limiting the invention.

Detailed description.

Pleurotus ostreatus fruit bodies produce very high amounts of spores, starting already early in the development of mushrooms . Very often growing rooms appear foggy because of the enormous number of spores in the air, reaching concentrations of up to 10¹⁰ spores per m³. Therefore, appropriate face masks have to be worn in growing rooms and workers handling the mushrooms after they are harvested also have to be protected against spore inhalation, i. e. by handling mushrooms in an exhaust hood. The ultimate solution to meet the health risks caused by Pleurotus spores is to dramatically reduce spore production or even prevent sporulation completely.

Although basidiomycetes, like Pleurotus ostreatus have developed a very special life cycle, which differs in many points from the life cycles of other fungi and multicellular organisms, some features of sexual reproduction are unique to all life forms with a sexual phase, i.e. meiosis. The process by which fertile haploid gametes (ova, sperm, pollen, spores) or vegetative haploid cells are formed by diploid cells is called meiosis and this process is inevitably coupled with a sexual phase. This is also the case for basidiomycetes, here karyogamy - the formation of a diploid nucleus - , meiosis and spore formation are located within a special cell type, the basidia, and are intimately connected: meiosis obviously requires karyogamy, and spore formation is prevented if meiosis is blocked completely. Due to this relation between meiosis and sporulation and because meiosis is well studied in a number of organisms, we have focused on meiosis as a target for genetic engineered or marker selected spore-less Pleurotus strains. One characteristic feature of meiosis is pairing of homologous chromosomes and exchange of homologous sequences by recombination. Common to all models of genetic recombination is a homologous alignment and DNA strand exchange step. In prokaryotes the central role of Escherichia coli RecA protein homologues in homologous recombination is well established. Similar proteins, with a high degree of homology to RecA proteins exist in eukaryotes . Phylogenetic analyses of eukaryotic recA homologues reveal a gene duplication early in eukaryotic evolution which gave rise to two (rad51 and dmcl in S . cerevisiae) putatively monophyletic groups of recA-like genes. Members of the rad51 group are involved in recombination and recombinational repair, mutants in these genes are sensitive to DNA-damaging agents and ionising radiation, and are defective in meiosis. Members of the dmcl group are required for meiosis and sporulation but seem not to be involved in somatic DNA repair. Unlike rad51 mutant yeast cells which undergo meiosis and sporulation to some extend, yeast cells lacking Dmcl arrest in meiotic prophase and do not sporulate. The Rad51 and Dmcl polypeptide sequences share about 50 % identity (Diener & Fink) .

We focused on the isolation of a recA-like gene from Pleurotus ostreatus belonging to the dmcl group and to functionally characterise a dmcl homologous gene from Pleurotus on a molecular level, by comparing expression levels and location of such a gene in a commercial and a non sporulating mutant strain of Pleurotus ostreatus. So far a dmcl homologous gene has not been found in a member of the basidiomycota .

In addition to the isolation and characterisation of a meioses specific gene (dmcl) we used cDNA subtraction hybridisation to clone genes that are differentially expressed during meiose/sporulation of Pleurotus .

Subtractive hybridisation of cDNA sequences is a powerful method to isolate genes that are differentially .expressed in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. To isolate genes differentially expressed during meiose/sporulation of Pleurotus we took advantage of an existing mutant strain of Pleurotus ostreatus with a sporeless phenotype . Pleurotus ostreatus ATCC 58937 is a spontaneous mutant resulting from mating compatible offspring of a wildtype strain. Using cDNA from a sporulating commercial strain (Somycel 3015) and the sporeless strain for subtractive hybridisation we were able to isolate a number of expressed sequence tags (EST) differentially expressed during meiosis/spore formation. These ESTs have been characterised by northern hybridisation and expression was located using in si tu hybridisation's. At least two of these ESTs are clearly not expressed in the spore-less strain. Both sequences do not show significant homology to known protein encoding sequences, and our results show that these genes are involved in meiosis/sporulation.

The mutant strain ATCC 58937 has been used in the past for traditional strain improvement by crossing the constituent homokaryons of the mutant with compatible commercial strains. Somycel developed a number of strains. The common features of all the strains developed by Somycel using ATCC 58937 for breeding are:

- none of the commercially attractive strains are completely or even essentially spore-less (Sonnenberg et al.)

- all strains bear morphological characteristics of the mutant strain, i.e. they produce trumpet like instead of oyster caps

Especially the latter makes these strains unattractive for potential customers. Therefore, none of the "spore-less" strains developed by Somycel became a commercial success. A spore-less strain of Pleurotus thus has to meet at least three criteria: sporelessness, attractive for consumers and good production yield. One way to create such a strain is to identify one or more genes in the mutant

ATCC 58937 responsible for the spore-less phenotype and to use genetic engineering to inactivate one or more of these genes in a commercial strain of a mushroom such as Pleurotus . Since such genes as for example disclosed herein are involved in the formation of spores they can be used as targets to switch off sporulation using genetic transformation. One way to generate a spore-less mushroom is by genetic modification of for example a commercial strain. Genetic modification in this context means: preventing (silencing) the expression of a gene (or more genes) , and thus the translation of functionally active gene products thereof, by functionally deleting said gene or fragments thereof. In general standard methods to transform (PEG, electroporation, biolistics, Agrobacterium) are well established with various organisms including f ngi. Strategies to silence a gene are also well established and numerous (knock-out, antisense RNA, ribozyme, etc.), as are the methods to achieve silencing etc. (REMI, linearised plasmid, fragments, etc.) .

An alternative way of making a spore-less Pleurotus strain of commercial interest is classical breeding, helped by marker-assisted selection to detect spore-less strains with a lack of expression or reduced expression of these sequences, if needed supplemented by the use of mutagens (chemical and physical) to generate strains with reduced expression. Furthermore, the invention provides a combination of genetic manipulation and breeding.

The two homokaryons which make up a mushroom such as ATCC 58937 can be isolated by protoplasting and crossed with compatible homokaryons of a commercial strains. The spore-less phenotype seems to be linked to some extent with unfavoured morphological characteristics of the spore-less strain (see above) . Therefore, it is advantageous to have genetic markers linked to sporulation. A preselection of offspring using these markers limits the number of cultures that have to be tested on substrate and enhances the likelihood of obtaining a commercially successful sporeless strain.

Material and Methods :

Fungal strains and growth conditions: The Pleurotus ostreatus strain Somycel 3015 and the sporeless mutant P. ostreatus ATCC 58937 where used for all experiments and the following media have been used: MMP-medium (1% malt extract, 0.5 % mycological peptone, 10 mM KMOPS (3- [N-morpholino] propansulfonic acid) pH 7.0, 1.5 % Agar) was used for general growth and maintenance of mycelium. For DNA and RNA isolation mycelium was grown on MMP-plates covered with cellophane disks. All incubations have been performed at 24 °C .

Bacterial strains and growth conditions:

Escherichia coli strain DH5α was used for cloning, amplification and maintenance of plasmids. E. coli DH5α was grown in LB-medium ( 10 g/1 casein, 5 g/1 yeast extract, 10 g/1 NaCl) for LB-plates medium was supplemented with 15 g/1 Agar. E. coli strain LE 392 was used as host strain for bacteriophage λ-EMBL3. E. coli LE392 was grown on LB-plates for general purposes, for bacteriophage λ-EMBL3 infection E. coli LE392 was grown in LM-medium (LB-medium supplemented with 0.2 % maltose and 10 mM MgSO for LM- plates medium was supplemented with 15 g/1 Agar. E. coli strain XLl-Blue MRF^" was used as host strain for bacteriophage λ-Uni-ZAP XR. E. coli XLl-Blue MRF^" was grown on LB-medium supplemented with 12.5 μg/ l tetracycline for general purposes, for bacteriophage λ-Uni-ZAP XR infection E. coli XLl-Blue MRF^" was grown in LM-medium. Isolation of genomic DNA from Pleurotus mycelium

100 mg freeze dried mycelium was grounded with 50 mg sand and suspended in 500 μl TES (100 mM Tris/HCl, pH 8.0 , 10 mM EDTA, 2 % (v/w) SDS) then 10 μl proteinase K (1 mg in 100 μ 1 TES) was added. The suspension was incubated 1 h at 60 °C. Subsequently 200 μl 5 M NaCl and 70 μl 10 % (v/w) CTAB (Cetyltrimethylammonium bromide) were added and incubated for 10 min at 65 °C . Then 780 μl Cl (Chloroform/Isoamylalcohol , 24:1) was added and incubated for 30 min on ice. Following centrifugation for 10 min at 4 °C the upper phase was pipetted into a new tube and 245 μl 5 M NH₄HAc (ammonium acetate) was added and incubated for 1 h on ice. Following centrifugation for 30 min at 4°C, the supernatant was pipetted into a new tube, 0.55 volumes of isopropanol was added and incubated for 30 min on ice. After centrifugation as above the sediment was washed with 70 % (v/v) ethanol. The dried sediment was suspended in 50 μl H₂0.

PCR with degenerated oligonucleotide primers:

For amplification of the recA-like sequences in P. ostreatus, a 25 μl PCR reaction contained 0.3 μg of P. ostreatus (Somycel 3015) genomic DNA, 5 μM of primers A and B, 0.2 mM deoxyribonucleotide triphosphates and 0.3 units Super TAQ (HC) (HT Biotechnology LTD) in buffer prepared as recommended by HT Biotechnology LTD. The first PCR consisted of an initial denaturation step for 5 min at 94 °C, followed by a touch-down PCR with following parameters: denaturation for 1 min at 94 °C, annealing starting at 65 °C with an decrease of 2 °C after every second cycle, amplification for 2 min at 72 °C . After the touch-down PCR reached an annealing temperature of 43 °C a PCR for 10 cycles under the following conditions was performed: 1 min 94 °C, 2 min 43 °C, 2 min 72 °C. The final cycle was followed by an additional 5 min at 72 °C . For a second amplification 5 μl of the first PCR where used as template for a PCR under the same conditions as described above. Primer A is the oligonucleotide 5 ' -GGNGARTTYMGNWSNGGNAAR-3 ' and primer B is 5 ' -YTCNCCTCKNCCTSWRWARTC-3 ' , where N is A, C, G or T; K is G or T; M is A or C; R is A or G; S is C or G; W is A or T; Y is C or T. Primer A is the sense strand and primer B is the antisense strand primer.

Cloning of PCR products :

PCR products where cloned in the vector pGEM-T (Promega) under conditions recommended by Promega. From the resulting recombinant clones six where chosen for sequencing. Sequence analysis using the program BLAST indicated that two of the clones have significant homology with RAD51- and DMCl-like genes. Of these two clones the one with a length of ~380 bp showed a higher homology to RAD51 encoding genes this clone was assigned pms8 and the one with a length of ~620 bp showed higher homology to DMCl encoding genes this clone was named pms21. Clone pms21 has been used to screen a genomic and a cDNA λ-library of Pleurotus ostreatus in order to isolate the complete coding sequence of the two genes .

Northern blot analysis:

Total RNA was isolated from tissue or mycelium after freezing in liquid nitrogene and grinding the sample using a morter and pestil. About 100 mg of the powdered samples where transferred to a tube containing 3 ml extraction buffer (100 mM Tris/HCl pH 9.0, 10 mM EDTA, 1 % (w/v) SDS). Subsequently the samples where extracted once with PCI (Phenol/Chloroform/Isoamylalcohol, 25:24:1) and two times with Cl (Chloroform/ Isoamylalcohol , 24:1) for phase separation the tubes where centrifuged at 15 000 g, 4 °C for 10 min. The RNA was precipitated with LiCl at a final concentration of 2M LiCl at 4 °C overnight. RNA was sedimentet by centrifugation at 15 000 g, 4 °C for 30 min and washed twice with 80 % (v/v) Ethanol. The isolated RNA was disolved in H₂0 and stored in portions of 30 μg at -20 °C. For gel electrophoreses the RNA was denaturated with glyoxal/DMSO (1M glyoxal , 45 % (v/v) DMSO, 30 mM Bis-Tris,

10 mM PIPES, 1 mM EDTA, 0.1 % (w/v) bromphenolblue, pH 6.5) for 1 h at 55 °C and separated in a 1 % BTPE-agarose gel (30 mM Bis-Tris, 10 mM PIPES, 1 mM EDTA, pH 6.5) . The RNA was blotted onto Hybond N nylon membranes by capillar transfer using 20 x SSC (3 M NaCl, 0.3 M sodium citrat, pH 7.0) . Blots where cross linked 5 rain with UV-light and used for hybridisations after baking for 2 h at 80 °C. Northern hybridization was performed according to the standard protocol of the Boehringer-Mannheim DIG (Digoxygenine) System for filter hybridisations using DIG-labeled RNA probes . Single strand probes were generated from cDNA containing plasmids using T7 and T3 RNA-polymerases and the Boehringer Mannheim DIG RNA Labeling Mix. To generate run- off transcripts the plasmids were digested on one side of the cDNA inserts prior to in vitro transcription according to the manufacturer's protocol. The coding strands of cDNA's were used as negative controls (e.g. sense probe).

RACE PCR

5 μg total RNA from lamellae of Somycel 3015 was used as starting material. For first strand syntheses Superscript

11 (Gibco BRL, Life Technologies) was used as recommended by the manufacturer. Following first strand syntheses 1 μl

RNase A (10 μg/μl) was added to the reaction mix and incubated for 30 min at room temperature. Subsequently the sample was extracted once with PCI

(Phenol/Chloroform/Isoamylalcohol, 25:24:1) and two times with Cl (Chloroform/ Isoamylalcohol, 24:1) for phase separation the tubes were centrifuged at 15 000 g, 4 °C for 10 min. Following ethanol precipitation the DNA/RNA hybrids were suspended in 7 μl H₂0 and ligated into pBluescript SK+ linearised with the restriction enzyme EcoRV using T4 DNA ligase from Promega under conditions recommended by Promega. 1 μl of the ligation products were amplified using one gene specific primer and the M13 forward or reverse universal sequencing primers with Platinum Taq DNA polymerase (Gibco BRL, Life Technologies) as recommended by the manufacturer. PCR conditions: 5 min 94 °C and 30 cycles: 1 min 94 °C, 2 min 45 °C, 2 min 72 °C . PCR products were cloned in the vector pGEM-T (Promega) under conditions recommended by Promega.

Universal sequencing primers were : forward 5 ' GTAAAACGACGGCCAGT, reverse 5 ' CAGGAAACAGCTATGAC . Gene specific primer and first strand syntheses primer was 5 ' TGAGTAGTTGACGACGC .

In situ hybridisation of Pleurotus ostreatus tissue with RNA probes

Fruit body tissue was fixed in 4 % (w/v) paraformaldehyde, 0.25 % (v/v) glutaraldehyde in 0.1 M potassium phosphate buffer pH 7.4 and sections were embedded in paraffin. Subsequently, 4 μm thick sections were made on a paraffin microtome. Proteinase K (1 μg/ml in 100 mM Tris/HCl pH 8.0 , 50 mM EDTA) treatment was carried out at 37 °C and acetylation was performed for 10 min in 0.25 % (v/v) acetic anhydrid, 0.1 M triethanolamine pH 8.0. Prehybridisation buffer contained 50 % (v/v) deionised formamide, 300 mM NaCl, 1 % (w/v) Boehringer Mannheim blocking reagent, 0.15 mg/ml yeast tRNA, 1 mM EDTA and 10 mM Tris/HCl pH 8.0. For hybridisation (45 °C) , the prehybridisation buffer was suplemented with 5 % (w/v) dextran sulphat and 50 ng probe per slide. Single strand probes were generated from cDNA containing plasmids using the Boehringer Mannheim DIG RNA Labeling Mix according to the manufacturer's protocol. The coding strands of cDNA's were used as negative controls. After hybridisation, slides were washed subsequently in 2 x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) for 1 h at stringent temperatures, rinsed in NTE (500 mM NaCl, 10 mM Tris/HCl pH 8.0 , 0.1 mM EDTA) and incubated in NTE with 5 μg/ml RNase A at room temperature for 30 min, washed 1 h in 1 x SSC and 30 min in 0.5 x SSC at stringent temperatures . Blocking was performed for 4 h with 1 % (w/v) Boehringer-Mannheim blocking reagent in maleat buffer (150 mM NaCl, 100 mM maleic acid pH 7.5) and overnight with 10 % (w/v) BSA. Detection of hybrids was carried out with anti-digoxygenin-alkaline-phosphatase conjugate (Boehringer-Mannheim) and NBT (nitroblue tetrazolium chloride) according to the manufacturer's instructions. Sections were viewed by light microscopy.

Crossing experiments

For crossing experiments compatible homokaryons of Pleurotus ostreatus were used.

Mycelium samples of strains with opposite mating types were placed on a MMP agar plate (1% malt extract, 0.5 % mycological peptone, 10 mM KMOPS (3- [N-morpholino] propansulfonic acid) pH 7.0 , 1.5 % Agar) about 1 cm away from each other, and incubated at 24 °C until mating was detectable. For confirmation of mating (plasmogamy) mycelium samples were checked for clamp connection formation using light microscopy. Dikaryotic mycelium, resulting from successful crossing experiments, was used to inoculate 750 g sterilised straw- substratum. After colonisation of the substrate at 24 °C in the dark, induction of fruit body formation was performed at 18 °C in constant light. Fruit bodies were harvested and lamellae sections were checked for spore production using light microscopy. For the isolation of single spore cultures (offspring) fruit bodies were placed above a petri dish for 24 h. Subsequently spores were collected with sterile water from the petri dish and plated onto MMP agar plates (1% malt extract, 0.5 % mycological peptone, 10 mM KMOPS (3- [N-morpholino] propansulfonic acid) pH 7.0, 1.5 % Agar) in different dilutions. Homokaryons were selected based on slow growth and absence of clamp connections. Compatible homokaryotic offspring (single spore isolates) were used for further crossing experiments as described above .

Results

Identification of a DMCl-like sequence in Pleurotus ostreatus

To identify a gene in P. ostreatus related by homology to the meiosis specific genes of the DMCl-protein family, we amplified genomic DNA of P. ostreatus using oligonucleotides degenerated for codons of amino acid sequences conserved between members of the DMCl and RAD51 gene families of different organisms.

After amplification, cloning and sequencing of the PCR products, we have been able to identify a sequence of ~620 bp that was likely to encode for a protein with strong homology to known DMCl proteins, this clone was assigned pms21. Comparison of the predicted amino acid sequence of pms21 with the amino acid sequence of known protein encoding sequences by using the sequence analysis program BLAST, we have been able to identify pms21 as a possible member of the DMCl encoding gene family. The pms21 sequence could encode a polypeptide that is about 70 % identical to the coding sequence of the Sac char omyces cerevisiae DMCl. As expected pms21 also showed some homology to coding sequences of the RAD51-protein family. To the RAD51 coding sequence of S . cerevisiae pms21 is about 55 % identical, a level of identity shared by Saccharomyces DMCl and RAD51.

Characterisation of the coding sequence of DMCl in P. ostreatus

For further characterisation of the DMCl-like sequence pms21, we isolated clones from a P. ostreatus genomic and cDNA library. We screened a genomic λ-library of P. ostreatus using pms21, and isolated two clones of the DMC1- like gene, overlapping each other and covering a 3239 bp region of the locus that contains the complete coding region of the DMCl-like sequence. The coding part of this sequence shares 69 % and 63 % amino acid identity with the DMCl homologue DLH1 of Candida albicans and DMCl of S. cerevisiae respectively, but only 51 % identity with RAD51 from Saccharomyces . Indicating that this sequence belongs to the DMCl encoding meiosis specific gene family, we therefore designated the sequence Pleurotus DMCl (DMClp) . In order to get more information on the organisation of the gene we isolated two cDNA clones from a λ library using pms21 as probe. After sequencing we aligned the cDNA and the genomic sequences, and have been able to determine the exact position of introns within the genomic sequence. Because the cDNA clones did not cover the full length of the corresponding mRNA, we used a 5 ' RACE PCR to get the missing sequence information.

The open reading frame (ORF) of the Pleurotus DMCl gene is interrupted by 17 intervening sequences (IVSs) , all IVSs are bounded by splice-site consensus sequences. The length of the IVSs varies between 47 and 67 bp . Within the 5' non- translated region we have been able to identify a core promoter element (TATA) and within the 3 ' non-translated part of the gene we have been able to identify a polyadenylation signal (AATA) , in the cDNA clones the poly (A) tail was located 214 bp downstream of the TAA stop codon. A compilation of the sequence and organisation of the Pleurotus DMCl gene is given in figure 1.

Expression patterns of DMCl in P. ostreatus Our preliminary designation based on the extent of amino acid identity was supported by the RNA expression patterns of DMClp. RNA was prepared from different fruit body tissues and from vegetative grown mycelium of P. ostreatus Somycel 3015 and the spore-less mutant ATCC 58937. No expression of DMClp was detected in vegetative grown mycelium or in stipe tissue of both strains. Expression was only detected in lammellar tissue of the normal sporulating strain, but not in lammellar tissue of the spore-less mutant ATCC 58937 (Fig 5) . These results indicate that DMClp is contributing to the non-sporulating phenotype of P. ostreatus ATCC 58937. For further analysis of DMClp expression we performed in si tu northern hybridisations on gill tissue sections of both Pleurotus strains using a labelled cDNA coding for DMClp as probe. As expected from the results gained by northern blot analysis expression of DMClp was not detectable in lammellar sections of the spore-less strain. In the normal sporulating strain P. ostreatus Somycel 3015 expression of

DMClp is located in the basidia. These results indicate that DMClp is expressed only in basidia of P. ostreatus and is involved in meiosis/spore formation.

Isolation and cloning of ESTs using cDNA subtraction hybridisation

Using mRNA isolated from lamellae of the commercial strain (Somycel 3015) and the spore-less mutant strain of Pleurotus ostreatus (ATCC 58937) we performed a PCR select cDNA subtraction according to the manufacturers protocol (Clontech) . The resulting subtractive EST library consists of about 180 plasmid clones. After pre-screening for differential expressed clones we identified two clones with a very clear pattern of differential expression. Expression of the ESTs assigned 1A1 and 1B3 was analysed by northern hybridisation. For both clones expression was found only in RNA isolated from lamellae of the commercial strain and in contrast no expression was detectable in RNA isolated from lamellae of the spore-less strain. For RNA isolated from fruit body stipes or from vegetative mycelium no expression was found in the commercial strain and the spore-less strain (Fig 6) .

Expression of clones 1A1 and 1B3 has been further characterised by in si tu hybridisation (Fig 7 and 8) . Using gill tissue section of the normal sporulating strain (Somycel 3015) and the spore-less strain we have been able to show that expression of both ESTs is located in the basidia of the sporulating strain and is completely absent in the non-sporulating strain. The results of the northern and the in si tu hybridisation indicate that both EST clones are differentially expressed during meiose/sporulation in a normal sporulating Pleurotus strain, and are not expressed in a essentially spore-less strain. Based on these results we do conclude that both ESTs (1A1 and 1B3) are involved in meiosis and/or spore formation of Pleurotus ostreatus .

Sequence analysis did not reveal a significant homology with a known protein encoding sequence for both ESTs.

Characterisation of a sporeless Pleurotus strain and it's constituting homokaryons by crossing experiments.

When using above identified methods in identifying or detecting essentially spore less mushrooms, interesting observations are made. Whereas in US patent 4,242,832, the genetic components causing the sporeless trait is considered to be restricted to the "42" nucleus in the "42 x 11" strain (as also described by Leal-Lara ( Leal-Lara H. Mushroom Science X. Proceedings of the Tenth International Congress on the Science and Cultivation of Edible Fungi, pp 145-155; 1978)), our crossing experiments and offspring analysis indicate that at least one genetic component of each nucleus ("42" and "11") is necessary for obtaining a sporeless phenotype. When "42" is crossed with a monokaryon of a sporulating commercial strain, all fruit bodies of the resulting dikaryon produce normal numbers of spores. When the offspring of these fruit bodies (isolated as single sporecultures) are crossed with the other nuclear component of the sporeless strain (i.e. strain "11") 50% of the resulting dikaryons produce sporulating fruit bodies and 50% produce sporeless fruit bodies. When the reverse cross is done, i.e. "11" crossed with a monokaryon of a sporulating commercial strain, also all fruit bodies of the resulting dikaryon produce spores. Crossing of the offspring, isolated as single spore isolates, with the "42" monokaryon results also in 50% dikaryons with sporulating fruit bodies and 50% dikaryons with sporeless fruit bodies. These experiments clearly indicate that at least one genetic component of each nuclear type of the "42 x 11" strain is needed to obtain a sporeless phenotype. The here in described experiments have also shown that the undesired shape of fruit bodies (trumpet cap, more stipe than cap) of the "42 x 11" strain is not completely linked to the sporeless phenotype, i.e. we have found individuals in the described experiments that produced sporeless fruit bodies that had essentially the same shape as fruit bodies produced by commercial strains. This shows that markers linked to the sporeless trait can be used to produce a sporeless strain with normal fruit bodies, for example using marker assisted breeding or selection as provided herein (Fig 4) . References

- Latge, J.P. & Paris, S. 1991. The fungal spore: reservoir of allergens, p. 379-401. In G.T. Cole & H.C. Hoch (ed.), The fungal spore and disease initiation in plants and animals. Plenum Press, New York.

- Horner, W.E., Helbling, A., Salvaggio, J.E. & Lehrer, S.B. 1995. Fungal Allergens. Clinical Microbiology Reviews Vol 8(2), 161-179.

- Wasser, S.P. & Weis, A.L. 1999. Therapeutic effects of substances occurring in higher Basidiomycetes mushrooms: a modern perspective. Crit . Rev. Immunol. 19(1), 65-96.

- Chang, S-T. & Miles, P.G. 1989. Edible Mushrooms and their Cultivation. CRC Press, Boca Raton, Florida.

- Horner, W.E., Helbling, A. & Lehrer, S.B. 1993. Basidiomycete allergens : comparison of three Ganoderma species. Allergy 110(6): 110-116.

- Van Loon, P.C., Cox, A.L., Wuisman, O.P., Burgers, S.L. & Van Griensven, L.J.L.D. 1992. Mushroom worker's lung.

Detection of antibodies against Shii-take (Lentinus edodes) spore antigens in Shii-take workers. J. Occup. Med. 34 (11) : 1097-1101.

- Chavasco, J.K., Gambale, W. , de Siqueira, A.M., Fiorini, J.E., Portocarrero, M.C., Mendes Junior, L.G. & do

Nascimento, L.C. 1997. Evaluation of the allergenicity of spore and mycelia extracts of Pisolithus tinctorius. Rev. Inst. Med. Trop. Paolo 39(5): 245-252.

- Umar, M.H. & Van Griensven, L.J.L.D. 1997. Morphological studies on the life span, developmental stages, senescence and death of fruit bodies of Agaricus bisporus. Mycol . Res. 101(12): 1409-1422. Figure legends

Figures 1, 2 or 3. Nucleotide sequences of dmcl, 1A1 or 1B3 , respectively.

Figure 4. Breeding diagram for identifying molecular markers linked to a spore-less phenotype and subsequent breeding .

For example: Both constituent monokaryons of ATCC58937 have been isolated by protoplasting the vegetative mycelium and the two parental types were selected from the regenerants . Both monokaryons were crossed to a monokaryotic single spore isolate (SSI) of the commercial line Somycel 3015 (FI) . Many SSIs isolated from one fruit body of each hybrid were crossed with the constituent monokaryons of ATCC58937. Each cross was cultivated on a small scale and the fruit bodies examined for the production of spores. In each set of offspring fruit bodies either contained a normal level of sporulation or did not produce any spores at all

(indicated as + or - in F2A and F2B in fig 1) . Molecular markers, as detected by hybridisation and/or amplification, are linked to phenotype.

Figure 5 Northern hybridisation using dmc lp as probe: sp+

= sporulating strain, sp- = sporeless strain. Lane 1+2 RNA from lamellae, lane 3+4 RNA from fruit body stripes, lane 5+6 RNA from vegetative mycelium

Figure 6 Northern hybridisation using 1A1 and 1B3 as probe:

A 1A1, B B23, sp+ = sporulating, sp-=sporeless, lanes 1 and 2 lamellae, lanes 3 and 4 fruit body stipes, lane 5 and 6 vegetative mycelium Figure 7 In situ hybridisation of gill section using a 1A1 antisense RNA-probe :

3015=sporulating strain, ATCC 58937 = sporeless strain.

Figure 8 In situ hybridisation of gill sections using a 1B3 antisense RNA-probe: 3015 = sporulating, ATCC 58937 = sporeless.

Table 1: Spore production of P. Ostreatus en P. Pulmonarius varieties.

Variety Species spore St. production^* dev.^$

M 102 P. Ostreatus 279.8 90

M 111 P. Ostreatus 291.8 63

M 131 P. Ostreatus 17.3 5

M 299 P. Ostreatus 66 16

M 300 P. Ostreatus 52 16

M 301 P. Ostreatus 27 3

M 321 P. Ostreatus 279 167

M 403 P. Ostreatus 390 145

LC 010 P. Ostreatus 90 58

Som P. Ostreatus 390 60

3035

Som P. Ostreatus 84 35

3100

Som P. Ostreatus 59 24

3200

Som P. Ostreatus 21 7

3200

Som P. Ostreatus 64 14

3200

Som P. Ostreatus 1 0.5

3300

Som HK P. Ostreatus 418 80

35

Lc 029 P. Pulmonarius . 454 205

Som P. Pulmonarius . 413 157

3015

M 201 P. Pulmonarius . 494 135

M 205 P. Pulmonarius . 663 159

M 230 P. Pulmonarius . 474 128

M 28 P. Pulmonarius . 447 174 *: Spore production is expressed as number (xl0⁸⁾ per gram tissue per 24 hour. $: Standarddeviation: for each variety four fruit bodies were measured

Claims

1. A method for localising, identifying or marking an allele of a mushroom, whereby said localisation, identification or marking is carried out using a nucleic acid molecule or fragment thereof whereby an allele is localised, identified or marked that is associated with reduced spore production of mushrooms.

2. A method according to claim 1 whereby said allele is related to the number of spore producing basidae per lamellae.

3. A method according to claim 1 or 2 allowing selection of an essentially spore-less mushroom.

4. A method according to anyone of claims 1 to 3 whereby said nucleic acid molecule or fragment thereof is derived from a gene related to spore production of said mushroom.

5. A method according to claim 4 wherein said nucleic acid molecule hybridises with or comprises a nucleotide sequence as listed in figure 1, 2 or 3 or a complementary sequence or the RNA equivalent thereof .

6. An isolated and/or recombinant nucleic acid or fragment thereof comprising a nucleic acid hybridising with a nucleic acid derived from a mushroom gene related to spore production.

7. A nucleic acid or fragment thereof according to claim 6 comprising a nucleotide sequence having at least 50% homology with a nucleotide sequence as listed in figure 1, 2 or 3 or a complementary sequence or the RNA equivalent thereof .

8. A nucleic acid or fragment thereof according to claim 7 wherein said homology is at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%.

9. A nucleic acid or fragment thereof according to anyone of claims 6 to 8 derived from a mushroom.

10. A method for selecting a mushroom on its potential sporelessness comprising testing a sample from said mushroom for the presence of a nucleic acid sequence located at genetic trait locus marking a gene related to spore production.

11. An essentially spore-less mushroom obtainable or selectable by a method according to anyone of claims 1 to 5 or 10.

12. An essentially spore-less mushroom according to claim

11 showing optimal fructification at around 10° to 20° C.

13. An essentially spore-less mushroom according to claim

12 derived from a Pleurotus species .

14. An essentially spore-less mushroom according to claim

13 having a relatively small stipe and a relatively well developed head.

15. An essentially spore-less mushroom derived from a Pleurotus species having a relatively small stipe and a relatively well developed head obtainable or selectable by a method according to anyone of claims 1 to 10.

16. A mushroom provided with a functional deletion in a gene related to spore-production.

17. A mushroom according to claim 16 wherein said gene or part thereof hybridises with a nucleic acid according to anyone of claims 6 to 9.

18. A mushroom according to claim 16 or 17 which is essentially spore-less.