EP3099778A2 - Lignée de champignon j10102-s69, souche de champignon hybride j11500, leurs descendants ainsi que procédés et utilisations associés - Google Patents

Lignée de champignon j10102-s69, souche de champignon hybride j11500, leurs descendants ainsi que procédés et utilisations associés

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Publication number
EP3099778A2
EP3099778A2 EP15742750.1A EP15742750A EP3099778A2 EP 3099778 A2 EP3099778 A2 EP 3099778A2 EP 15742750 A EP15742750 A EP 15742750A EP 3099778 A2 EP3099778 A2 EP 3099778A2
Authority
EP
European Patent Office
Prior art keywords
culture
mushroom
line
strain
hybrid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP15742750.1A
Other languages
German (de)
English (en)
Other versions
EP3099778A4 (fr
Inventor
Richard W. Kerrigan
Mark P. Wach
Michelle E. Schultz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sylvan America Inc
Original Assignee
Sylvan America Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US14/169,578 external-priority patent/US9648812B2/en
Priority claimed from US14/169,658 external-priority patent/US9622428B2/en
Application filed by Sylvan America Inc filed Critical Sylvan America Inc
Publication of EP3099778A2 publication Critical patent/EP3099778A2/fr
Publication of EP3099778A4 publication Critical patent/EP3099778A4/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H15/00—Fungi; Lichens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • This invention relates generally to the field of microorganism strain development and more particularly, to the development of homokaryotic lines and heterokaryotic strains of mushroom fungus. More specifically, the present invention relates to the development of a homokaryotic Agaricus bisporus mushroom fungus line designated J10102-s69 and to an Agaricus bisporus hybrid strain designated J11500, as well as to cultures descended or derived from line J10102-s69 or strain J11500, and to methods of making and using said hybrid cultures.
  • mushroom strain development programs have set up mushroom strain development programs.
  • the goal of a mushroom strain development program is to combine, in a single strain, culture, hybrid, or line, various desirable traits. Strains currently available to the mushroom industry allow growers to produce crops of mushrooms successfully and profitably. Several factors exist that influence the degree of success and profitability achieved.
  • Characteristics of strains that are factors that can improve producer profitability include increased productivity (higher yield or shorter cycle time), accelerated revenue capture (earlier harvest), reduced costs (for example, greater ease and speed of harvesting), reduced shrinkage (pre-sale weight loss), reduced overweighting of product in packages (extra weight of product packaged, due to particular sizes of individual mushrooms), improved consistency of crop performance responses to variations in raw materials, growing conditions and practices, superior crop performance in particular facilities, regions, etc., reduced losses to diseases including viral, bacterial and fungal disease agents, and/or reduced losses to insect and nematode pests of the crop.
  • Cultures are the means by which mushroom strain developers prepare, maintain, and propagate their microorganisms. Cultures of Agaricus, like those of other microorganisms, are prepared, maintained, propagated and stored on sterile media using various microbiological laboratory methods and techniques. Sterile tools and aseptic techniques are used within clean rooms or sterile transfer hoods to manipulate cells of pure cultures for various purposes including clonal propagation and for the development of new strains using diverse techniques.
  • Commercial culture inocula including mushroom 'spawn' and 'casing inoculum' are also prepared using large-scale microbiological production methods (e.g., from 1 to 14,000 liters per batch), and are provided to the end user as pure cultures contained within sterile packaging.
  • large-scale microbiological production methods e.g., from 1 to 14,000 liters per batch
  • Mushrooms are cultivated commercially within purpose-built structures on dedicated farms. While there are many variations on methods, the following description is typical.
  • Compost prepared from lignocellulosic material such as straw, augmented with nitrogenous material is finished and pasteurized within a suitable facility.
  • Mushroom spawn which comprises a sterilized friable 'carrier substrate' onto which a pure culture of one mushroom strain has been aseptically incorporated via inoculum and then propagated, is mixed with the pasteurized compost and is incubated for approximately 13 to about 19 days at a controlled temperature, during which time the mycelium of the mushroom culture colonizes the entire mass of compost and begins to digest it.
  • a non-nutritive 'casing layer' of material such as peat is then placed over the compost to a depth of from about 1.5 to about 2 inches.
  • Additional 'casing inoculum' incorporating the same mushroom culture may be incorporated into the casing layer to accelerate the formation and harvesting of mushrooms, and improve uniformity of the distribution of mycelium and mushrooms in and on the casing surface.
  • Environmental conditions, including temperature and humidity, in the cropping facility are then carefully managed to promote and control the transition of the culture from vegetative to reproductive growth at the casing/air interface.
  • mushrooms will have developed to the correct stage for harvest and sale.
  • a flush of mushrooms comprising the original culture will be picked over a 3 to 4 day period. Additional flushes of mushrooms appear at about weekly intervals.
  • two or three flushes of mushrooms are produced and harvested before the compost is removed and replaced in the cropping facility.
  • Strains may be, in particular instances, differentiated on the basis of traits associated with the mushroom, such as mushroom size, mushroom shape (e.g., cap roundness, flesh thickness), color (i.e., white cap versus brown cap), surface texture (e.g., cap smoothness), tissue density and/or firmness, delayed maturation, basidial spore number greater than two, sporelessness, increased dry matter content, improved shelf life, and reduced bruising, as well as traits associated with the culture itself, and/or products incorporating the culture, and/or crops incorporating the culture, including increased crop yield, altered distribution of yield over time, decreased spawn to pick interval, resistance to infection by, symptoms of, or transmission of bacterial, viral or fungal diseases, insect resistance, nematode resistance, ease of crop management, suitability of crop for mechanical harvesting, and behavioral responses to environmental conditions including stressors, nutrient substrate composition, seasonal influences, farm practices, self/non-self interactions (compatibility or incompatibility) with various mushroom strains, to give some examples.
  • Strains may also be differentiated based on their genotypic fingerprint (presence of specific alleles at defined marker loci in the nuclear or mitochondrial genome). Strains may have different ancestry, which will be reflected directly by the genotype, and indirectly, in some cases, by the phenotype.
  • An OWNC line designated ⁇ 97' was deposited in the public culture collection of the Fungal Genetics Stock Center of Kansas, USA, by A. Sonnenberg, under the number 10389, and in the public collection of the American Type Culture Collection of Maryland, USA, under the number MYA-4626.
  • the genome of H97 was sequenced and placed in the public domain by the Joint Genome Institute of California, USA (Morin et al. 2012).
  • the U1 strain is thought to be the direct progenitor of all other white A. bisporus mushrooms currently cultivated in most regions of the world.
  • Many commercial mushroom strains developed from U1 such as A15 and S130, meet the criteria for Essentially Derived Varieties (as the term is applied to plant varieties, and extended to apply to mushroom varieties or strains, in conformity with statutory frameworks including the US PVPA (2014)) of U1 , having been developed from spores of the initial strain which retain the great majority of the parental genotype (this behavior was shown by R. W. Kerrigan et al. in Genetics, 133, 225-236 (1993)).
  • a group of strains developed either by cloning or by spore culture, or by any other method of 'essential derivation' as discussed below, from a single progenitor (as opposed to outbreeding between two different progenitors) is called a derived lineage group. Except for relatively minor acquired genetic differences all white strains developed within the Horst U 1 derived lineage group share a single composite N+N heterokaryotic genotype, or a subset of that genotype, with the original U1 strain. For this reason, modern white Agaricus mushroom cultivation is effectively a monoculture.
  • Agaricus bisporus has a reproductive syndrome known as amphithallism, in which two distinct life cycles operate concurrently. As in other fungi, the reproductive propagule is a spore. Agaricus produces spores meiotically, on a meiosporangium known as a basidium. In a first life cycle, A. bisporus spores each receive a single haploid postmeiotic nucleus; these spores are competent to mate but not competent to reproduce mushrooms. These haploid spores germinate to produce homokaryotic offspring or lines which can mate with other compatible homokaryons to produce novel hybrid heterokaryons that are competent to produce mushrooms.
  • Heterokaryons generally exhibit much less ability to mate than do homokaryons. This lifecycle is called heteromixis, or more commonly, outbreeding. This life cycle operates but typically does not predominate in strains of Agaricus bisporus var. bisporus,
  • a second, inbreeding life cycle called intramixis predominates in most strains of Agaricus bisporus var. bisporus. Most spores receive two post-meiotic nuclei, and most such pairs of nuclei consist of Non-Sister Nuclear Pairs (NSNPs) which have a heteroallelic genotype at most or all centromeric-linked loci including the MAT locus. That MAT genotype determines the heterokaryotic phenotype of these offspring, which are reproductively competent and can produce a crop of mushrooms. Unusually among eukaryotes, relatively little chromosomal crossing-over is observed to have occurred in postmeiotic offspring of A. bisporus var.
  • NNPs Non-Sister Nuclear Pairs
  • the present invention is directed generally to a new and distinct homokaryotic line of Agaricus bisporus designated J10102-s69, to a new and distinct Agaricus bisporus hybrid strain designated J11500, to lines and strains derived or descended from J10102-s69 or J1 1500 including Essentially Derived Varieties (EDVs) of line J10102-s69 or strain J11500, to cultures of each of the foregoing, and to processes for the production of cultures of each of the foregoing as well as methods for using the line designated J10102-s69 or the strain J11500 or lines or strains derived or descended from J 10102-s69 or J 11500 or cultures thereof.
  • EDVs Essentially Derived Varieties
  • one aspect of the present invention provides for an Agaricus bisporus culture designated Agaricus bisporus line J10102-s69.
  • a deposit of a representative culture of the Agaricus bisporus line J10102-s69, as disclosed herein, has been made with the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit was January 15, 2014. The culture deposited was taken from the same culture maintained by Sylvan America, Inc., Kittanning, Pennsylvania, USA, the assignee of record, since prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to meet all deposit requirements of all patent offices throughout the world, including the U.S.
  • Another aspect of the present invention provides an F1 hybrid Agaricus bisporus culture produced by mating the Agaricus bisporus culture of line J 10102- s69 with a different Agaricus bisporus culture.
  • the invention may be achieved by a method for producing a hybrid mushroom culture of Agaricus bisporus that includes the step of mating a homokaryotic line J10102-s69, a culture of which was deposited under NRRL Accession No. 50893 as above, with a homokaryotic line OWNC, a culture of which was deposited with the Agricultural Research Services Culture Collection, 1815 North University Street, Peoria, Illinois 61604 USA under NRRL Accession No. 50894.
  • Such a mating of line J10102-s69 and line OWNC provides an F1 hybrid Agaricus bisporus culture designated as strain J1 1500, a deposit of a representative culture of the Agaricus bisporus strain J11500, as disclosed herein, having been made with the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit was January 15, 2014.
  • NRRL Agricultural Research Services Culture Collection
  • the culture deposited was taken from the same culture maintained by Sylvan America, Inc., Kittanning, Pennsylvania, USA, the assignee of record, since prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to meet all deposit requirements of all patent offices throughout the world, including the U.S. Patent and Trademark Office, and all deposit requirements under the Budapest Treaty.
  • the NRRL Accession No. is 50895.
  • the deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.
  • the culture will be irrevocably and without restriction or condition released to the public upon the filing of the patent application or upon the issuance of a patent, whichever is required by the applicable patent laws.
  • the present invention further encompasses a culture that is an Essentially Derived Variety (EDV), as defined herein, of an initial culture, wherein the initial culture is a culture of line J10102-s69 as above, an F1 hybrid Agaricus bisporus culture produced by mating the Agaricus bisporus culture of line J10102-s69 with a different Agaricus bisporus culture as above, or the F1 hybrid strain J1 1500 as above. Further discussion of EDVs is set forth hereinbelow.
  • EDV Essentially Derived Variety
  • an Agaricus bisporus culture produced by essential derivation has at least one of the essential characteristics of strain J11500, for example the same heterokaryon compatibility phenotype, and/or the further characteristics of cap roundness, flesh thickness, yield performance, and yield timing relative to commercial strain A-15.
  • the present invention further encompasses an Agaricus bisporus mushroom culture including at least one set of chromosomes of any of the cultures of line J10102-s69 above, hybrid strain J11500 above, or EDVs of the cultures above, wherein said chromosomes comprise all of the alleles of the culture above at the sequence-characterized marker loci listed in the appropriate column of Table I or appropriate row of Table II below.
  • an Essentially Derived Variety of the culture of line J10102-s69 is produced.
  • the culture above may be an F1 hybrid Agaricus bisporus mushroom culture produced by mating the culture of the line J10102-s69 or an EDV of J10102-s69, or of a line obtained from strain J1 1500 or an EDV of strain J11500, with a different Agaricus bisporus culture.
  • the invention also includes a mushroom culture of Agaricus bisporus having a genotypic fingerprint which has characters at at least two marker loci selected from the markers provided in the appropriate column of Table I or appropriate two of Table II, wherein all of the selected characters of said fingerprint are present in the genotypic fingerprint of either line J10102-s69, representative culture of the line having been deposited under NRRL Accession No. 50893, or strain J1 1500, a representative culture of the strain having been deposited under NRRL Accession No. 50895.
  • Genotypic fingerprints are descriptions of the genotype at defined loci, where the presence of characterized alleles is recorded.
  • Such fingerprints provide powerful and effective techniques for recognizing clones and all types of EDVs of an initial strain, as well as for recognizing ancestry within outbred lineages. Many techniques are available for defining and characterizing loci and alleles in the genotype. The most detailed approach is provided by whole-genome sequencing (WGS), which allows for direct characterization and comparison of DNA sequences across the entire genome. Using this approach to generate robust genotypic fingerprints incorporating large numbers of marker loci, it is possible to establish the nature of the relationship between two strains, including strains related by genealogical descent over several generations. Sylvan America, Inc. has tracked genetic markers through four to six generations of its breeding pedigrees.
  • the mean expectation for genomic representation of an initial haploid line after 4 outbred generations is 3.1 % in an F4 hybrid, which corresponds to ca. 1 Mb of the nuclear genomic DNA of A. bisporus.
  • that amount of DNA from each of two unrelated strains of A. bisporus may typically contain from about 10,000 to about 20,000 single nucleotide polymorphisms (SNPs), any one of which may provide a distinguishing marker linking the F4 hybrid to the initial line.
  • SNPs single nucleotide polymorphisms
  • characters at at least two marker loci are selected. It will be appreciated that in other embodiments, characters at at least three, four, five or six marker loci may be selected. It is noted that prior art patents have used from one to four marker loci.
  • Virus diseases such as those caused by the LIV or MVX viruses can have severe negative impacts on facility productivity and must be remediated using hygiene practices which can be assisted by strain rotation.
  • a method of improving mushroom farm hygiene called 'virus-breaking' is carried out by replacing cropping material (compost, spawn, casing inoculum) incorporating an initial strain with inoculum and cropping material incorporating another different strain that is incompatible with the initial strain.
  • cropping material compound, spawn, casing inoculum
  • cropping material incorporating another different strain that is incompatible with the initial strain.
  • all biological material of the initial strain at a mushroom farm is replaced with biological material of the second, incompatible strain.
  • Strain incompatibility creates an effective if not absolute barrier to movement of virus from biological reservoirs within a facility into new crops. Rotating cultivation usage among mushroom strains of different genotypes may also interrupt infection and infestation cycles of exogenous pests and pathogens. Accordingly, in at least one embodiment of the present invention, any of the above cultures exhibit heterokaryon incompatibility toward heterokaryon strains in the U1 derived lineage group. The observable heterokaryon incompatibility demonstrates the genetic distinctness of strain J11500 relative to strains like A-15 that belong to the U1 derived lineage group.
  • the Agaricus bisporus cultures of the present invention have all of the physiological and morphological characteristics of line J10102-s69, wherein the culture of line J10102-s69 has been deposited under the NRRL Accession Number 50893, or strain J1 1500, wherein a culture of strain J11500 has been deposited under NRRL Accession No. 50895.
  • the present invention also includes methods of production of any of the cultures above, including the culture of line J10102-s69, the culture of strain J11500, EDVs of J10102-S69, or EDVs of strain J11500, cultures that exhibit heterokaryon incompatibility as above, and cultures that have a genotypic fingerprint as described above or all of the physiological and morphological characteristics of the cultures above.
  • the method for producing a hybrid mushroom culture of Agaricus bisporus includes mating a first parental mushroom culture with a second parental mushroom culture, wherein at least one of the first and second parental mushroom cultures is one of the cultures above or a line obtained from one of the cultures above.
  • the method above further includes providing a mushroom culture, as produced above, in mushroom products selected from the group consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed mushrooms, parts of mushrooms, mushroom extracts and fractions, mushroom pieces, and colonized substrates selected from grain, compost, and friable particulate matter.
  • the method may include providing the mushroom culture in derived or descended cultures selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue explants cultures, protoplasts, dormant spores, germinating spores, inbred descendents and outbred descendents, transgenic cultures, and cultures having a genome incorporating a single locus conversion.
  • a cell may be obtained from any of the cultures above or any of the methods for producing the cultures as noted above.
  • the cell above may further include a marker profile having characters at at least two marker loci selected from the markers provided in the appropriate column of Table I or appropriate row of Table II, wherein all of the characters of said marker profile are also present in the marker profile of either line J10102-s69, representative culture of the line having been deposited under NRRL Accession No. 50893, or strain J1 1500, a representative culture of the strain having been deposited under NRRL Accession No. 50895.
  • characters at at least three, four, five or six marker loci may be selected as discussed above.
  • a spore may comprise the cells above.
  • the hybrid culture above may be further defined as having a genome including a single locus trait conversion.
  • the locus above may be selected from the group consisting of a dominant allele and a recessive allele.
  • the locus above may confer a trait selected from the group consisting of mushroom size, mushroom shape, mushroom cap roundness, mushroom flesh thickness, mushroom color, mushroom surface texture, mushroom cap smoothness, tissue density, tissue firmness, delayed maturation, basidial spore number greater than two, sporelessness, increased dry matter content, increased shelf life, reduced brusing, increased yield, altered distribution of yield over time, decreased spawn to pick interval, resistance to infection by symptoms of or transmission of bacterial, viral or fungal disease or diseases, insect resistance, nematode resistance, ease of crop management, suitability of crop for mechanical harvesting, canning and/or processing, desired behavioral response to environmental conditions, to stressors, to nutrient substrate composition, to seasonal influences, and to farming practices.
  • One or more other aspects of the present invention may be provided by a process for introducing a desired trait into a culture of Agaricus bisporus line J 10102- s69.
  • Such a process may be initiated by (1) mating the culture of line J10102-s69 to a second culture of Agaricus bisporus having the desired trait, to produce a hybrid.
  • the process further proceeds by (2) obtaining an offspring that carries at least one gene that determine the desired trait from the hybrid produced above.
  • the process further includes (3) mating the offspring of the hybrid with the culture of line J 10102- s69 to produce a new hybrid and (4) repeating the steps of (2) obtaining and (3) mating at least once to produce a subsequent hybrid.
  • step (4) may be repeated up to any of 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10 times. In other embodiments, repeating steps (2) and (3) may occur more than 10 times.
  • the process then provides (5) obtaining a homokaryotic line carrying at least one gene that determines the desired trait and comprises at least 75% of the alleles of line J10102-s69 at the sequence-characterized marker loci described in Tables I and II, from the subsequent hybrid of step (4).
  • the homokaryotic line obtained may comprise 80% of the alleles of line J10102-s69 at the sequence- characterized marker loci described in Tables I and II.
  • the homokaryotic line obtained may comprise 85%, 90%, 95%, 96%, 97%, 98%, 99% or may be comprise essentially 100% of the alleles of line J10102-s69 at the sequence- characterized marker loci described in Tables I and II.
  • Still one or more other aspects of the present invention may be provided by a method of producing a mushroom culture.
  • the method includes (a) growing a first hybrid culture produced by mating any of the above cultures or cultures produced from the methods above, with a first different Agaricus bisporus culture; (b) mating a first homokaryotic progeny line of the first hybrid culture with the first or a second different culture to produce a second hybrid culture of a subsequent descendant generation; (c) optionally, growing a second homokaryotic progeny line culture of the subsequent generation and mating the second homokaryotic progeny line of the second hybrid culture of the subsequent descendant generation with the first or the second or a third different Agaricus bisporus culture; and (d) repeating steps (b) and (c) for an additional 0, 1 , 2, 3, 4 or 5 (i.e., 0-5) generations to produce a mushroom culture.
  • the produced mushroom culture above is an inbred culture. In another embodiment the produced mushroom culture is an outbred culture. In one or more other embodiments, the method above may further include the step of mating the inbred culture with a second, distinct culture to produce an F1 hybrid culture.
  • a method for developing a second culture in a mushroom strain development program includes applying mushroom strain development techniques to a first mushroom culture, or parts thereof, wherein the first mushroom culture is any of the above cultures or cultures produced from the methods above. It is the application of the mushroom strain development techniques that results in the development of the second culture.
  • Such known mushroom strain development techniques are selected from the group consisting of inbreeding, back-mating, outbreeding, selfing, introgressive trait conversions, essential derivation, pedigree-assisted breeding, marker assisted selection, and transformation.
  • Still another aspect of the present invention may be provided by a method of mushroom strain development.
  • This method includes obtaining a molecular marker profile of Agaricus bisporus mushroom line J10102-s69, a culture of which was deposited under the NRRL Accession Number 50893.
  • Another step of the method includes obtaining an F1 hybrid culture, for which the deposited mushroom culture of the Agaricus bisporus mushroom line J10102-s69 is a parent. Once the F1 hybrid culture is obtained, the selection of homokaryotic progeny, based upon their genotypes, for lines that possess characteristics of the molecular marker profile of line J10102-s69 as above may be conducted to obtain a culture of a desirable selected line.
  • a further step of mating a culture of the selected line as set forth above with a different mushroom culture is employed. Once this is done, it may optionally be repeated. In one embodiment, it is not repeated. In other embodiments, it is repeated 1 , 2, 3, 4 or 5 times.
  • Other embodiments of the foregoing may include the production of hybrid mushroom cultures incorporating the line J10102-s69, the production of mushrooms from cultures incorporating line J10102-s69, the production of mushroom parts from cultures incorporating line J10102-s69.
  • Still other uses include processes for making a mushroom culture that comprise mating homokaryotic Agaricus bisporus line J10102-s69 with another mushroom culture and processes for making a mushroom culture containing in its genetic material one or more traits introgressed into line J10102-s69 through introgressive trait conversion or transformation, and to the mushroom cultures, mushrooms, and mushroom parts produced by such introgression.
  • the invention may include a hybrid mushroom culture, mushroom, mushroom part, including a spore, or culture part produced by mating the homokaryotic line J10102-s69, or an introgressed trait conversion of line J 10102- s69, with another mushroom culture.
  • Still other uses of the present invention include the production of homokaryotic mushroom lines derived from mushroom line J 10102- s69, as well as the processes for making other homokaryotic mushroom lines derived from mushroom line J10102-s69, and to the production of the inbred mushroom lines and their parts derived by the use of those processes.
  • strain J11500 Cultures of strain J11500 are noted to produce mushrooms, parts of mushrooms, parts of the culture, and strains and lines descended or derived from such cultures.
  • the present invention encompasses strain J11500, Essentially Derived Varieties of strain J11500, more particularly EDVs incorporating at least 75% of the genetic material of strain J11500, dormant or active growing cultures present in dormant or germinating spores of strain J11500, and cultures descended from and incorporating the genetic material of strain J11500.
  • the present invention is also directed towards methods of making and using strain J1 1500.
  • J11500 Uses of J11500 include methods for producing mushrooms and parts of mushrooms including spores, for improving farm hygiene, for producing offspring from homokaryotic and heterokaryotic spores, for producing hybrid descendents via outcrossing with a second line or strain, and for producing EDVs by any means known in the art.
  • spores living spores are heterokaryons or homokaryons in a dormant state. Spores are one part of the mushroom organism. Other parts include caps, stems, gills, cells (defined as hyphal compartments incorporating nuclei, mitochondria, cytoplasm, a cell membrane, and a cell wall including crosswalls), hyphae, and mycelium. Spores may be aseptically collected on sterile material, suspended in sterile water at various dilutions, and plated onto sterile agar growth media in order to produce germinated spores and the cultures incorporated within the spores.
  • a preferred technique is to have within the enclosed petri plate a living Agaricus culture which may stimulate spore germination via the diffusion of a volatile pheromone. Germinated spores may be isolated under a microscope using sterile microtools such as steel needles, onto fresh nutrient agar plates. Using this method, cultures of heterokaryotic and homokaryotic offspring of a heterokaryotic strain comprising the spores and the cultures incorporated within the spores of the heterokaryotic strain may be obtained.
  • homokaryon pairs may be placed in close proximity on the surface of a nutrient agar medium in a petri dish and allowed to grow together (in a physical association), at which point anastomoses between the two cultures occur.
  • a successful outcome is a mating.
  • the novel hybrid heterokaryon may be obtained by transferring mycelium from the fusion zone of the dish.
  • Such a paired mating method was used to develop hybrid heterokaryotic strains from line J10102-s69, and from lines obtained from J11500 and from other descendants of J 10102.
  • EDVs are most often derived directly (otherwise predominantly) from a single initial culture (e.g., strain); all such derivations produce EDVs.
  • 'Essential derivation' methods of obtaining cultures which are by definition consequently EDVs of a single initial culture of A. bisporus include somatic selection, tissue culture selection, single spore germination, multiple spore germination, selfing, repeated mating back to the initial culture, mutagenesis, and transformation, to provide some examples.
  • EDVs are defined primarily by the methods used to produce them, it is also true that EDVs are inherently unambiguously recognizable by their genotype, which will be entirely or predominantly (75% or greater) a subset of that of the single initial culture. Percentages of the initial genotype that will be present in Agaricus bisporus EDVs range from almost 100% in the case of somatic selections, to 99.
  • Many methods of genotype determination including methods described below, and others well known in the art, may be employed to determine the percentage of DNA of an initial culture that is present in another culture.
  • hybrid mushroom strain producers are always looking for hybrid strains that allow growers to produce crops of mushrooms successfully and profitably.
  • positive attributes documented thus far include a rounder cap shape and thicker cap flesh, both of which appeal to consumers, than existing successful commercial strain A-15, and a total harvested yield that may exceed that of strains like A-15, and yield timing that is accelerated as compared to strain A-15, a trait that is particularly suitable for certain segments of the market, and which tends to accelerate revenue capture and decrease crop cycle time (potentially allowing greater throughput).
  • strain J11500 has a different genotype from the U1 derived lineage group. Accordingly, strain J1 1500 is incompatible with strains of the U1 derived lineage group, which is a characteristic known to retard the spread of viral diseases between strains. Thus, strain J1 1500 confers a potential benefit in strain rotation programs designed to manage facility hygiene. Strain J11500 has been found to simultaneously provide both genetic diversification and commercially acceptable performance and crop characteristics.
  • a part of any of the cultures above or any cultures produced from the methods above may be selected from the group consisting of hyphae, spores, and cells and parts of cells, including, nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes and cell walls, each part being present in either the vegetative mycelium of the culture or in mushrooms produced by the culture, or both.
  • the parts may be present in both the vegetative mycelium of the culture and in mushrooms produced by the cultures above.
  • the spores may be dormant or germinated spores, and may include heterokaryons and homokaryons incorporated therein.
  • any of the cultures above or any cultures produced from the methods above may be incorporated into products selected from mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed mushrooms, mushroom extracts and fractions, mushroom pieces, and colonized substrates including grain, compost, and friable particulate matter.
  • mushroom pieces refer to stems, pilei, and other larger portions of the mushroom itself.
  • the F1 hybrid mushroom culture of Agaricus bisporus above may be processed into one or more products selected from the group consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed mushrooms, mushroom extracts and fractions, mushroom pieces, and colonized substrates including grain, compost, and friable particulate matter.
  • a mushroom may be produced by growing a crop of mushrooms from any of the cultures above.
  • a mushroom may be produced by growing a crop of mushrooms from the F1 hybrid mushroom culture above.
  • an Essentially Derived Variety of the F1 hybrid mushroom culture above is produced.
  • a method for producing a hybrid mushroom culture of Agaricus bisporus may includes the step of mating a homokaryotic line J10102-s69, a culture of which was deposited under NRRL Accession No. 50893, with a homokaryotic line OWNC, a culture of which was deposited under NRRL Accession No. 50894.
  • a mating provides the hybrid mushroom culture J11500, which exhibits antagonism toward heterokaryon strains in the U1 derived lineage group.
  • the observable heterokaryon incompatibility demonstrates the genetic distinctness of strain J11500 relative to strains like A-15 that belong to the U1 derived lineage group.
  • the method further includes providing a mushroom culture of the invention in mushroom products selected from the group consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed mushrooms, parts of mushrooms, mushroom extracts and fractions, mushroom pieces, and colonized substrates selected from grain, compost, and friable particulate matter.
  • the method may include providing the mushroom culture in derived or descended cultures selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue explants cultures, protoplasts, dormant spores, germinating spores, inbred descendents and outbred descendents, transgenic cultures, and cultures having a genome incorporating a single locus conversion.
  • a cell or a culture including the cell is produced by the method(s) above.
  • one or more embodiments may include a method further including the step of growing the hybrid mushroom culture to produce hybrid mushrooms and parts of mushrooms.
  • Other embodiments may provide for methods wherein the hybrid mushroom culture produced, or the cell, includes a marker profile having characters at at least two (or three, or four, or five, or six) marker loci ITS, p1 n150-G3-2, MFPC-1-ELF, AN, AS, and FF, wherein all of the characters of said marker profile are also present in the marker profile of either line J10102-s69 or strain J11500.
  • Still other embodiments may provide for methods wherein the hybrid mushroom culture produced, or the cell, includes a marker profile having characters at at least two (or three, or four, or five, or six) marker loci described in Tables I or II, wherein all of the characters of said marker profile are also present in the marker profiles of either line J10102-s69 or strain J11500.
  • the method further includes producing or otherwise growing a crop of edible mushrooms by carrying out the steps described hereinabove.
  • the method may include the cultures above in crop rotation to reduce pathogen pressure and pathogen reservoirs in mushroom growing facilities as described hereinabove.
  • the method includes using the cultures above to produce offspring as described hereinabove.
  • Allele A heritable unit of the genome at a defined locus, ultimately identified by its DNA sequence (or by other means); in a genotype, an allelic character.
  • Amphithallism A reproductive syndrome in which heteromixis and intramixis are both active.
  • Anastomosis Fusion of two or more hyphae that achieves cytoplasmic continuity.
  • Basidiomycete A monophyletic group of fungi producing meiospores on basidia; a member of a corresponding subdivision of Fungi such as the Basidiomycetales or Basidiomycotina.
  • Basidium The meiosporangial cell, in which karyogamy and meiosis occur, and upon which the basidiospores are formed.
  • Bioefficiency For mushroom crops, the net fresh weight of the harvested crop divided by the dry weight of the compost substrate at the time of spawning, for any given sampled crop area or compost weight.
  • Cap Pileus; part of the mushroom, the gill-bearing structure.
  • Cap Roundness Strictly, a ratio of the maximum distance between the uppermost and lowermost parts of the cap, divided by the maximum distance across the cap, measured on a longitudinally bisected mushroom; typically averaged over many specimens; subjectively, a 'rounded' property of the shape of the cap.
  • Carrier substrate A medium having both nutritional and physical properties suitable for achieving both growth and dispersal of a culture.
  • Casing layer, casing A layer of non-nutritive material such as peat or soil that is applied to the upper surface of a mass of colonized compost in order to permit development of the mushroom crop.
  • Casing inoculum A formulation of inoculum material incorporating a mushroom culture, typically of a defined heterokaryotic strain, suitable for mixing into the casing layer.
  • Cloning Somatic propagation without selection.
  • Combining ability The capacity of an individual to transmit traits or superior performance to its offspring (known and available methods of assessment vary by trait).
  • Culture The tangible living organism; the organism propagated on various growth media and substrates; one instance of one physical strain, line, homokaryon or heterokaryon; the sum of all of the parts of the culture, including hyphae, mushrooms, spores, cells, nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes and cell walls.
  • Derived lineage group An initial strain or variety and the set of EDVs derived from that single initial strain or variety.
  • Descent The production of offspring from two parents, and/or four grandparents, and/or additional progenitors, via sexual mating; in contrast to derivation from a single initial strain.
  • Diploid Having two haploid chromosomal complements within a single nuclear envelope.
  • Essential derivation A process by which an Essentially Derived Variety is obtained from an initial variety or strain or from an EDV of an initial variety or strain; modification of an initial culture using methods including somatic selection, tissue culture selection, selfing including intramictic reproduction via single spores and multiple spores and mating of sibling offspring lines, back-mating to the initial variety, or mutagenesis and/or genetic transformation of the initial variety to produce a distinct culture in which the genotype of the resulting culture is predominantly that of the initial culture.
  • EDV Essentially Derived Variety
  • EDV definitions for example, as applied to plants in the US PVPA, incorporate elements of (1) relatedness, (2) methods of derivation, (3) and empirical tests.
  • a variety that is entirely or predominantly derived from an initial variety or from an EDV of an initial variety, and which conforms to specified or "essential" characteristics of the initial variety except for distinguishing differences resulting from the act of derivation is an EDV of the initial variety.
  • a strain or culture predominantly or entirely derived from a single initial strain or culture, thus having most or all, but at least 75%, of its genome or genotype present in the genome or genotype of the initial strain or culture; a strain or culture obtained from an initial strain or culture by somatic selection, tissue culture selection, selfing including mating of sibling offspring lines and intramictic reproduction via single or multiple spores, back-mating to the initial strain or culture, or mutagenesis and/or genetic transformation of the initial strain or culture; a strain or culture reconstituted from neohaplonts derived from an initial strain or culture, whether or not the haploid lines have been passed into or out of other heterokaryons; a strain or culture with the same essential phenotype as that of an initial strain or culture; in contrast to descent (via sexual mating between two parental strains).
  • Flesh Thickness A ratio of the maximum distance between the top of the stem and the uppermost part of the cap, divided by the maximum distance across the cap, measured on a longitudinally bisected mushroom; typically averaged over many specimens; subjectively called 'meatiness'.
  • Flush A period of mushroom production within a cropping cycle, separated by intervals of non-production; the term flush encompasses the terms 'break' and 'wave' and can be read as either of those terms.
  • Fungus An organism classified as a member of the Kingdom Fungi.
  • Genealogical descent Descent from progenitors, including parents, over a limited number (e.g., 10 or fewer) of typically outcrossed generations; in contrast to derivation from a single initial strain.
  • Genotypic fingerprint A description of the genotype at a defined set of marker loci; the known genotype.
  • Haploid Having only a single complement of nuclear chromosomes; see homokaryon.
  • Heteroallelic Having two different alleles at a locus; analogous to heterozygous.
  • Heteroallelism Differences between homologous chromosomes in a heterokaryotic genotype; analogous to heterozygosity.
  • Heterokaryon As a term of art this refers to a sexual heterokaryon: a culture which has two complementary (i.e., necessarily heteroallelic at the MAT locus) types of haploid nuclei in a common cytoplasm, and is thus functionally and physiologically analogous to a diploid individual (but cytogenetically represented as N+N rather than 2N), and which is potentially reproductively competent, and which exhibits self/non- self incompatibility reactions with other heterokaryons; also called a strain or stock in the breeding context.
  • Heterokaryon compatibility The absence of antagonism observed during physical proximity or contact between two heterokaryons that are not genetically identical; see Heterokaryon Incompatibility.
  • Heterokaryon incompatibility The phenomenon of antagonism observed during physical proximity or contact between two heterokaryons that are not genetically identical; a multilocus self/non-self recognition system that operates in basidiomycete heterokaryons to regulate contact through anastomosis.
  • Heterokaryotic Having the character of a heterokaryon.
  • Heteromixis Life cycle involving mating between two different non-sibling haploid individuals or gametes; outbreeding.
  • Homoallelic Having not more than one allele at a locus.
  • the equivalent term in a diploid organism is 'homozygous'.
  • Haploid lines are by definition entirely homoallelic at all non-duplicated loci.
  • Homokaryon A haploid culture with a single type (or somatic lineage) of haploid nucleus (cytogenetically represented as N), and which is ordinarily reproductively incompetent, and which does not exhibit typical self/non-self incompatibility reactions with heterokaryons, and which may function as a gamete in sexually complementary anastomoses; a 'line' which, as with an inbred plant line, transmits a uniform genotype to offspring; a predominantly homoallelic line that mates well and fruits poorly is a putative homokaryon for strain development purposes; see discussion below.
  • Homokaryotic Having the character of a homokaryon; haploid.
  • Hybrid Of biparental origin, usually applied to heterokaryotic strains and cultures produced in controlled matings.
  • Hyphae Threadlike elements of mycelium, composed of cell-like compartments or 'cells'.
  • Inbreeding Matings that include sibling-line matings, back-matings to parent lines or strains, and intramixis; reproduction involving parents that are genetically related.
  • Inoculum A culture in a form that permits transmission and propagation of the culture, for example onto new media; specialized commercial types of inoculum include spawn and CI; plural: inocula.
  • Intramixis A uniparental sexual life cycle involving formation of a complementary 'mated' pair of postmeiotic nuclei within the basidium or individual spore.
  • Introgressive trait conversion mating offspring of a hybrid to a parent line or strain such that a desired trait from one strain is introduced into a predominating genetic background of the other parent line or strain.
  • Lamella see 'gill'.
  • Line A culture used in matings to produce a hybrid strain; ordinarily a homokaryon which is thus homoallelic, otherwise a non-heterokaryotic (non-NSNPP) culture which is highly homoallelic; practically, a functionally homokaryotic and entirely or predominantly homoallelic culture; analogous in plant breeding to an inbred line which is predominantly or entirely homozygous.
  • non-NSNPP non-heterokaryotic
  • Lineage group see 'derived lineage group'. The set of EDVs derived from a single initial strain, line or variety, plus the initial strain, line or variety.
  • Locus A defined contiguous part of the genome, homologous although often varying among different genotypes; plural: loci.
  • Marker assisted selection Using linked genetic markers including molecular markers to track trait-determining loci of interest among offspring and through pedigrees.
  • MAT The mating-type locus, which determines sexual compatibility and the heterokaryotic state.
  • Mating The sexual union of two cultures via anastomosis and plasmogamy; methods of obtaining matings between mushroom cultures are well known in the art.
  • Mycelium The vegetative body or thallus of the mushroom organism, comprised of threadlike hyphae.
  • Mushroom The reproductive structure of an agaric fungus; an agaric; a cultivated food product of the same name.
  • Neohaplont A haploid culture or line obtained by physically deheterokaryotizing (reducing to haploid components) a heterokaryon; a somatically obtained homokaryon.
  • Offspring Descendants, for example of a parent heterokaryon, within a single generation; most often used to describe cultures obtained from spores from a mushroom of a strain.
  • OW-type strain A category of cultivar strains traditionally called 'Off-white' strains, comprising an initial strain and its derived lineage group, exemplified by strain Somycel 76; OW strain, OW.
  • Parent An immediate progenitor of an individual; a parent strain is a heterokaryon, a parent line is a homokaryon; a heterokaryon may be the parent of an F1 heterokaryon via an intermediate parent line.
  • Pedigree-assisted breeding The use of genealogical information to identify desirable combinations of lines in controlled mating programs.
  • Phenotype Observable characteristics of a strain or line as expressed and manifested in an environment.
  • Plasmogamy Establishment, via anastomosis, of cytoplasmic continuity leading to the formation of a sexual heterokaryon.
  • Progenitor Ancestor, including parent (the direct progenitor).
  • Progeny In Agaricus bisporus, strictly speaking, new heterothallic or homothallic individuals (cultures, mycelia, etc.) produced by an initial heterokaryotic individual via meiosis and sporulation, and, ultimately, germination and growth, i.e., single-spore isolates or SSIs; broadly speaking, offspring, sometimes used to encompass individuals of the first hybrid generation of heterokaryotic descendants of an initial individual.
  • a mushroom culture typically a pure culture of a heterokaryon, typically on a sterile substrate which is friable and dispersible particulate matter, in some instances cereal grain; commercial inoculum for compost; reference to spawn includes reference to the culture on a substrate.
  • Stem Stipe; part of the mushroom, the cap-supporting structure.
  • Sterile Growth Media Nutrient media, sterilized by autoclaving or other methods, that support the growth of the organism; examples include agar-based solid nutrient media such as Potato Dextrose Agar (PDA), nutrient broth, and many other materials.
  • PDA Potato Dextrose Agar
  • Strain A heterokaryon with defined characteristics or a specific identity or ancestry; equivalent to a variety.
  • SW-type strain A category of cultivar strains traditionally called 'Smooth- white' strains, comprising an initial strain and its derived lineage group, exemplified by strain Somycel 53; SW strain, SW.
  • Tissue culture A de-differentiated vegetative mycelium obtained from a differentiated tissue of the mushroom.
  • Trait conversion Selective introduction of the genetic determinants of one (a single-locus conversion) or more desirable traits into the genetic background of an initial strain while retaining most of the genetic background of the initial strain. See 'Introgressive trait conversion' and 'Transformation'.
  • Transformation A process by which the genetic material carried by an individual cell is altered by the incorporation of foreign (exogenous) DNA into its genome; a method of obtaining a trait conversion including a single-locus conversion.
  • Virus-breaking Using multiple incompatible strains, i.e. strains exhibiting heterokaryon incompatibility, successively in a program of planned strain rotation within a mushroom production facility to reduce the transmission of virus from on-site virus reservoirs into newly planted crops.
  • Yield The net fresh weight of the harvest crop, normally expressed in pounds per square foot.
  • Yield pattern The distribution of yield within each flush and among all flushes; influences size, quality, picking costs, and relative disease pressure on the crop and product.
  • homokaryons and homoallelic lines are subject to technical and practical considerations:
  • a homokaryon in classical terms is a haploid culture which is axiomatically entirely homoallelic.
  • the definition is broadened somewhat to accommodate both technical limitations and cytological variation, by treating all predominately homoallelic lines as homokaryons.
  • Technical limitations include the fact that genomes contain duplicated DNA regions including repeated elements such as transposons, and may also include large duplications of chromosomal segments due to historical translocation events; such regions may appear not to be homoallelic by most genotyping methods. Two different A.
  • bisporus genomes sequenced by the Joint Genome Institute, a U.S. federal facility differ in estimated length by 4.4%, and in gene numbers by 8.2%, suggesting a considerable amount of DNA duplication or rearrangement within different strains of the species.
  • No presently available genome of A. bisporus can completely account for the physical arrangement of such elements and translocations, and so the assembled genome sequences of haploid lines may have regions that appear to be heteroallelic using currently available genotyping methods. Cytologically, a homokaryotic offspring will ordinarily be a spore that receives one haploid, postmeiotic nucleus. However, a spore receiving two third-division nuclei from the basidium will be genetically equivalent to a homokaryon.
  • a spore receiving two second-division 'sister' postmeiotic nuclei will be a functional homokaryon even though some distal 'islands' of heteroallelism may be present due to crossovers during meiosis.
  • a meiosis that has an asymmetrical separation of homologues can produce an aneuploid, functionally homokaryotic spore in which an extra chromosome, producing a region of heteroallelism, is present. All of these cultures are highly homoallelic and all function as homokaryons. Technological limitations make it impractical to distinguish among such cultures, and also to rule out DNA segment duplication as an explanation for limited, isolated regions of the genome sequence assembly that appear to be heteroallelic.
  • the use of the term 'homoallelic' to characterize a line includes entirely or predominately homoallelic lines, regardless of the presence of regions of genome duplication, or of aneuploidy, and cultures described in this way are functional homokaryons, are putatively homokaryotic, and are all defined as homokaryons in the present application.
  • the present invention relates initially to a homokaryotic line, and more specifically, a line of Agaricus bisporus designated J10102-s69, and methods for using the line designated J10102-s69.
  • a culture of the line designated J10102-s69 has been deposited with the Agricultural Research Services Culture Collection (NRRL) 1815 North University Street, Peoria, Illinois 61604 USA (“NRRL”) as Accession No. 50893.
  • NRRL Agricultural Research Services Culture Collection
  • Agaricus bisporus mushroom line J10102-s69 is a haploid filamentous basidiomycete culture which in vegetative growth produces a branching network of hyphae, i.e. a mycelium. Growth can produce an essentially two-dimensional colony on the surface of solidified (e.g., agar-based) media, or a three-dimensional mass in liquid or solid-matrix material.
  • solidified e.g., agar-based
  • the morphological and physiological characteristics of line J10102-s69 in culture on Difco brand PDA medium are provided as follows.
  • Line J10102-s69 growing on PDA medium in a 10 cm diameter Petri dish produced a light brown-yellow or 'tan' colored irregularly lobate colony with a roughly circular overall outline that increased in diameter by (0.3-0.4-) 0.7 (-0.8-1.4) mm/day during dynamic equilibrium-state growth between days 12 and 26 after inoculation using a 6.5-7 mm diameter circular plug of the culture on PDA as inoculum.
  • Hyphae of the culture on Difco PDA were irregular and about cylindrical, measured (12-) 41-71 (-99) x (4.5-) 6-8 (-10) urn, and exhibited a wide range of branching angles from about 10 to 90 degrees off the main hyphal axis.
  • Line J10102-s69 can be used to produce hybrid cultures with desirable productivity, timing, appearance, and other agronomic traits as is required of successful commercial mushroom strains, while also providing more diversified, non- cultivar germplasm.
  • Line J10102-s69 has been found to have an advantageous genotype for mating to produce commercially useful hybrid strains.
  • Several useful stocks have contributed to the genome of line J10102-s69.
  • Line J10102-s69 has, for example, a mating-type allele 2 on scaffold 1 contributed by the traditional smooth- white stock, and a 'white' color determining allele, as reported by allele E1 at the MFPC-1-ELF marker locus on scaffold 8, contributed by the traditional off-white hybrid stock.
  • genomic scaffolds are at least three (i.e., scaffolds 2, 9 and 10) contributed by other, wild stocks in the pedigree. In combination, these diverse genetic contributions were observed to have combined to produce a superior line with excellent combining ability in matings.
  • the J10102-s69 line is haploid and thus is entirely homoallelic (although some limited regions of duplicated DNA may be present in its genome).
  • the line has shown uniformity and stability in culture. The line has been increased by transfer of pure inocula into larger volumes of sterile culture media. No variant traits have been observed or are expected in line J10102-s69.
  • an F1 hybrid mushroom culture of Agaricus bisporus can be produced by mating the homokaryotic line J10102-s69, a culture of which was deposited under NRRL Accession No. 50893 as above, with another homokaryotic line, OWNC, a culture of which was deposited with the Agricultural Research Services Culture Collection, 1815 North University Street, Peoria, Illinois 61604 USA under NRRL Accession No. 50894.
  • the mating of these two lines results in the production of the F1 hybrid strain J1 1500, a culture of which was deposited with the Agricultural Research Services Culture Collection, 1815 North University Street, Peoria, Illinois 61604 USA under NRRL Accession No. 50895.
  • the present invention further relates to not only to cultures of the F1 hybrid strain J11500, but also to Essentially Derived Varieties (EDVs) of the strain J11500, as well as to cultures derived or descended from strain J11500 and EDVs of strain J11500. Such cultures are used to produce mushrooms and parts of mushrooms.
  • the present invention further relates to methods of making and using the strain J1 1500 and EDVs of the strain J1 1500.
  • Mushroom cultures are most reliably identified by their genotypes, in part because successful cultivar strains are required by the market to conform to a narrow phenotypic range.
  • the genotype can be characterized through a genetic marker profile, which can identify isolates (subcultures) of the same line, strain or variety, or a related variety including a variety derived entirely from an initial variety (i.e., an Essentially Derived Variety), or from an EDV of an initial variety, or can be used to determine or validate a pedigree.
  • Mushroom-forming fungi exhibit an alternation of generations, from heterokaryotic (N+N, with two haploid nuclei, functionally like the 2N diploid state) to homokaryotic (1 N) and further upon mating to become heterokaryotic again.
  • N+N heterokaryotic
  • haploid 'generation' is often, but not always, termed a gamete (e.g., pollen, sperm).
  • the haploid generation can live and grow indefinitely and independently, for example in laboratory cell culture; while these haploid homokaryons function as gametes in matings, they are equivalent to inbred lines (e.g., of plants) and are more easily referred to as parents (of hybrids).
  • the term 'parent' refers to the culture that is a, or the, direct progenitor of another culture within the alternating generations of the sexual lifecycle.
  • the term 'line' refers more narrowly to a haploid (N) homoallelic culture within the lifecycle.
  • the N+N heterokaryon resulting from a mating, or comprising a breeding stock, or comprising a culture used to produce a crop of mushrooms, may be called a 'strain'.
  • the match can be demonstrated by subtraction of the second allele from the genotype, leaving the J10102-s69 allele evident at every locus.
  • a refinement of this approach is possible with hybrids of Agaricus bisporus as a consequence of the heterokaryon (N+N) condition existing in hybrids.
  • the two haploid nuclei can be physically isolated by various known techniques (e.g., protoplasting) into 'neohaplont' subcultures, and each may then be characterized independently.
  • One of the two neohaplont nuclear genotypes from the F1 hybrid will be that of line J10102-s69, demonstrating its use in the mating and its presence in the hybrid.
  • DNA was extracted using a CTAB protocol followed by RNAse treatment and gel purification.
  • the OWNC line "H97" was sequenced and resequenced by the Joint Genome Institute and was placed in the public domain, thus its genome is known with about 100% certainty.
  • the total number of markers distinguishing either line J10102-S69 or strain J1 1500 that are known to the assignee is about 300,000.
  • a brief excerpt of the genotypes of line J10102-s69, of the OWNC line, of J1 1500, and of the EDV J11500-ms2 at numerous sequence-characterized marker loci distributed at intervals along each of the 19 H97 V2.0 reference chromosomal scaffolds larger than 100 Kb in length is provided in Table I.
  • Table I presents a 'fingerprint' excerpted from the SNP (Single Nucleotide Polymorphism) marker genotype of the entire genome sequences of line J 10102- s69, of line OWNC, of the F1 hybrid J1 1500 strain obtained from the mating of lines J10102-S69 and OWNC, and of the J11500-ms2 EDV of strain J11500.
  • SNP Single Nucleotide Polymorphism
  • the lUPAC nucleotide and ambiguity codes are used to represent the observed 9-base DNA marker sequences reported above, each of which represents one or two allelic characters at a genotypic marker locus, with, for example, the code "y” indicating the presence of two alleles, one with a "t” and the other with a "c", at that position.
  • the identity of each marker locus is uniquely and unambiguously specified by the scaffold and SNP position information derived from the H97 V2.0 archival reference genome sequence published by the U.S. Department of Energy Joint Genome Institute (Morin et al. 2012).
  • every nucleotide in the nuclear genome of Agaricus bisporus has a unique and specific identity, specified by the scaffold number and nucleotide position number of that nucleotide within the art-standard reference sequence (Version 2.0) of A. bisporus line H97, as determined by and placed into the public domain by The U.S. D.O.E. Joint Genome Institute and the Agaricus Genome Consortium, as described in the publication by Morin et al., "Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.” Proc. Nat'l Acad. Sci. USA 109: 17501-17506 (2012).
  • any genetic marker or marker locus in the A. bisporus genome may be identified by specifying the positional information from the H97 reference sequence.
  • the first marker listed in Table I occurs at 1 :101993 (i.e., scaffold 1 : position 101993).
  • Table I also provides short sequences flanking the SNP marker nucleotide position; in the first example at 1 :101993, the provided sequence is GAAGnACAT, where "n" represents the position of the variable nucleotide at 1 : 101993 which constitutes the informative genetic marker.
  • the amount of flanking sequence that is shown is arbitrary and is provided only as an aid in confirming the correct 'look-up' of the marker in the reference genome sequence.
  • Genotype data for six additional marker loci is provided in TABLE II and in the following text. Marker loci and allelic characters are specified hereinbelow.
  • Line J10102-s69 and strain J1 1500 can be identified through their molecular marker profiles, i.e., their genotypic fingerprints, as shown in Tables I and II.
  • OWNC and SWNC are two lines derived from two traditional white-capped cultivar stocks, as described hereinabove. Each is genotypically distinct, as shown in Table II.
  • a brief description of the genotype of strain J1 1500, in the context of its pedigree including progenitors J 10102, line J10102-s69, OW heterokaryon Somycel 76, and line OWNC, and in comparison to other white strains, at a further six unlinked marker loci is provided below.
  • the brief genotype excerpt provided below therefore consists of either 6 or 12 characters or elements, respectively, for lines or strains, as also presented in Table II.
  • the brief genotype was prepared by the assignee of record using targeted Polymerase Chain Reactions to amplify genomic regions bracketing the markers, as unambiguously defined below, from each of the culture DNAs. Any suitable PCR primers that bracket the defined marker regions may be used for this purpose; methods of designing suitable primers, for example from the H97 reference genome sequence, are well known in the art.
  • the amplified PCR product DNA was sequenced by a contractor, Eurofins, using methods of their choice, and the genotypes were determined by direct inspection of these sequences in comparison to Sylvan America's database of reference marker/allele sequences.
  • the 5' end of this marker segment begins at position 1 with the first "T” in the sequence TCCCAAGT, corresponding to H97 JGI V2.0 Scaffold 1 position 868615 (Morin et al. 2012) and extending in a reverse orientation (relative to the scaffold orientation) for ca. 600 nt in most alleles; an insertion in the DNA of allele 1T has produced a longer segment.
  • 9 alleles incorporating at least 30 polymorphic positions have been documented from diverse strains in Sylvan America's breeding collection.
  • Alleles present in the J10102-s69 and J11500 pedigree over three generations are alleles 1T, 2, 3, 4, and 9, characterized as follows (using the format: nucleotide base character @ alignment position, based on alignment of alleles 2, 3, and 4, and the alignable portions of allele 1T) :
  • Allele 1T 'C @ 193; insertion of Abr1 transposon of 320 nt @ 206 ⁇ 207; T @ 327;'C @ 374; 'G' @ 378; 'G' @ 422; 'C @ 431 ; 'G' @ 472; etc.
  • Allele 2 no Abr1 insertion; 'C @ 193; 'C @ 327, 'C @ 374; 'C @ 378; 'G' @ 422; T @ 431 ; 'G' @ 472; etc.
  • Allele 3 no Abr1 insertion; 'C @ 193; T @ 327, 'G' @ 374; 'C @ 378; 'G' @ 422; T @ 431 ; ⁇ ' @ 472; etc.
  • Allele 4 no Abr1 insertion; 'C @ 193; T @ 327, 'C @ 374; 'C @ 378; ⁇ ' @ 422; T @ 431 ; 'G' @ 472; etc.
  • Allele 9 no Abr1 insertion; 'G' @ 193; 'C @ 327, 'C @ 374; 'C @ 378; 'G' @ 422; T @ 431 ; 'G' @ 472; etc.
  • the J 10102 heterokaryon has an '1T/2' heteroallelic genotype.
  • the U1 heterokaryon has an '1T/2' heteroallelic genotype.
  • 'Off-White' heterokaryons such as Somycel 76 have a '1T/3' heteroallelic genotype.
  • 'Smooth- White' heterokaryons such as Somycel 53 have a '2/3' heteroallelic genotype.
  • the J9277 heterokaryon has a '1T/4' heteroallelic genotype.
  • the genotype of the J11500 heterokaryon at the p1 n150-G3-2 marker 'locus' is '1T/2' (heteroallelic), designating the presence of alleles 1T and 2. Allele 1T was contributed by the OWNC line. Allele 2 was transmitted from the J 10102 heterokaryon via the J10102-s69 homokaryon. The '1T/2' genotype distinguishes J11500 from many other heterokaryons including from all of its own grandparents, although not from the U1 strain family.
  • the ITS segment is part of the nuclear rDNA region, which is a cassette that is tandemly repeated up to an estimated 100 times in the haploid genome of A. bisporus. Therefore there is no single precise placement of this sequence in the assembled H97 genome, and in fact it is difficult or impossible to precisely assemble the sequence over all of the tandem repeats.
  • Three cassette copies were included on scaffold 10 of the H97 JGI V2.0 assembly, beginning at position 16121 10; a partial copy is also assembled into scaffold 29 (Morin et al. 2012).
  • the 5' end of this marker segment begins at position 1 with the first "G" in the sequence GGAAGGAT, and extending in a forward orientation (relative to the scaffold orientation) for ca. 703-704 nt in most alleles. At present, more than 9 alleles incorporating at least 11 polymorphic positions have been documented from diverse strains in Sylvan's breeding collection.
  • Alleles present in the J10102-s69 and J11500 immediate pedigree are alleles 11 , I2, and I4, characterized as follows (using the format: nucleotide base character @ alignment position, based on alignment of 9 alleles).
  • Allele 11 'C @ 52; T @ 461 ; T @ 522; T @ 563; etc.
  • Allele I2 T @ 52; T @ 461 ; T @ 522; T @ 563; etc.
  • the J10102 heterokaryon has an '11/14' heteroallelic genotype.
  • the U1 heterokaryon has an '11/12' heteroallelic genotype.
  • the genotype of the J11500 heterokaryon at the ITS marker 'locus' is '11/14' (heteroallelic), designating the presence of alleles 11 and I4. Allele 11 was contributed by the OWNC line. Allele I4 was transmitted from the J 10102 heterokaryon via the J10102-s69 homokaryon. This distinguishes J11500 from the U1 strain family, which has an '11/12' genotype.
  • the 5' end of this marker segment begins at position 1 with the first "G” in the sequence GGGAGGGT, corresponding to H97 JGI V2.0 Scaffold 8 position 829770 (Morin et al. 2012) and extending in a forward orientation (relative to the scaffold orientation) for ca. 860 nt in most alleles.
  • At present, at least 7 alleles incorporating at least 40 polymorphic positions have been documented from diverse strains in Sylvan's breeding collection.
  • Alleles present in the J10102-s69 and J11500 immediate pedigree are alleles E1 , E2, and E8, characterized as follows (using the format: nucleotide base character @ alignment position, based on alignment of 8 alleles).
  • Allele E1 'A' @ 77; 'A' @ 232; 'A' @ 309; T @ 334; 'A' @ 390; 'A' @ 400; T' @ 446, 'A' @ 481 ; etc.
  • Allele E2 'G' @ 77; 'A' @ 232; 'G' @ 309; T @ 334; 'G' @ 390; 'G' @ 400; 'C @ 446, 'G' @ 481 ; etc.
  • Allele E8 'A' @ 77; 'G' @ 232; 'G' @ 309; 'A' @ 334; 'A' @ 390; 'A' @ 400; 'C @ 446, 'G' @ 481 ; etc.
  • the J 10102 heterokaryon has an 'E1/E8' heteroallelic genotype.
  • the U1 heterokaryon has an 'E1/E2' heteroallelic genotype.
  • the genotype of the J11500 heterokaryon at the MFPC-1-ELF marker 'locus' is 'E1/E1', designating the presence of two copies of alleles E1.
  • One copy of allele E1 was contributed by the OWNC line; a second copy of allele E1 was transmitted from the J10102 heterokaryon via the J10102-s69 homokaryon.
  • This homoallelic genotype distinguishes J11500 from the predominant U1-type of commercial cultivar, which has an 'E1/E2' genotype.
  • the 5' end of this marker segment begins at position 1 with the first "G” in the sequence GGGTTTGT, corresponding to H97 JGI V2.0 Scaffold 9 position 1701712 (Morin et al. 2012) and extending in a forward orientation (relative to the scaffold orientation) for ca. 1660 nt (in the H97 genome) to 1700 nt (in the alignment space) in known alleles; several insertions/deletions have created length polymorphisms which, in addition to point mutations of individual nucleotides, characterize the alleles. At present, 5 alleles incorporating more than 70 polymorphic positions have been documented from diverse strains in Sylvan's breeding collection.
  • Alleles present in the J10102-s69 and J11500 immediate pedigree are alleles N1 , N2 and N5, characterized in part as follows (using the format: nucleotide base character @ alignment position, based on alignment of alleles N1 through N5) :
  • Allele N2 ⁇ ' @ 640; [deletion] @ 844-846; T @ 882; ⁇ ' @ 994, etc.
  • Allele N5 ⁇ ' @ 640; 'ACG' @ 844-846; 'C @ 882; 'G' @ 994, etc.
  • the J10102 heterokaryon has an 'N1/N5' heteroallelic genotype.
  • the U1 heterokaryon has an 'N1/N2' heteroallelic genotype.
  • the genotype of the J11500 heterokaryon at the AN marker 'locus' is 'N1/N5' (heteroallelic), designating the presence of alleles N1 and N5. Allele N1 was contributed by the OWNC line. Allele N5 was transmitted from the J 10102 heterokaryon via the J10102-s69 homokaryon. [0186]
  • the 'N1/N5' genotype at the AN marker locus distinguishes J1 1500 from commercial strains U1 and A-15, which have an 'N1/N2' genotype. This element of the genotype fingerprint can also distinguish J1 1500 from among many other strains.
  • the 5' end of this marker segment begins at position 1 with the first "G” in the sequence GG(T/N)GTGAT, corresponding to H97 JGI V2.0 Scaffold 4 position 752867 (Morin et al. 2012) and extending in a forward orientation (relative to the scaffold orientation) for ca. 1620 nt (in the H97 genome) to 1693 nt (in the alignment space) in known alleles; several insertions/deletions have created length polymorphisms which, in addition to point mutations of individual nucleotides, characterize the alleles. At present, 7 alleles incorporating more than 80 polymorphic positions have been documented from diverse strains in Sylvan's breeding collection.
  • Alleles present in the J10102-s69 and J11500 immediate pedigree are alleles SC and SD, characterized in part as follows (using the format: nucleotide base character @ alignment position, based on alignment of alleles SA through SG) :
  • Allele SC T @ 28; 'GATATC @ 258-263; 'G' @ 275; [insertion]+'TTTCTCAGC'+[insertion] @ 309-249; 'C @ 404, etc.
  • the J10102 heterokaryon has an 'SC/SD' heteroallelic genotype.
  • the U1 heterokaryon has an 'SC/SD' heteroallelic genotype.
  • Allele SD was contributed by the OWNC line. Allele SC was transmitted from the J 10102 heterokaryon via the J10102-s69 homokaryon.
  • the 'SC/SD' genotype at the AS marker locus is also shared by commercial strains U1 and A-15. While this element of the genotype fingerprint distinguished J11500 from among many other strains, it does not distinguish J1 1500 from the U1 strain family.
  • the 5' end of this marker segment begins at position 1 with the first "T” in the sequence TTCGGGTG, corresponding to H97 JGI V2.0 Scaffold 12 position 281999 (Morin et al. 2012) and extending in a forward orientation (relative to the scaffold orientation) for ca. 570 nt in most alleles.
  • 7 alleles incorporating at least 20 polymorphic positions have been documented from diverse strains in Sylvan's breeding collection.
  • Alleles present in the J10102-s69 and J1 1500 immediate pedigree are Alleles FF1 and FF2, characterized as follows (using the format: nucleotide base character @ alignment position, based on alignment of alleles 1 and 2) :
  • the J10102 heterokaryon has an 'FF1/FF2' heteroallelic genotype.
  • the U1 heterokaryon has an 'FF1/FF2' heteroallelic genotype.
  • the genotype of the J1 1500 heterokaryon at the FF marker 'locus' is 'FF1/FF1 ' (homoallelic), designating the presence of two copies of allele FF-1 , contributed by both the OWNC line and the J10102-s69 homokaryon. This distinguishes J11500 from the predominant 111-type of commercial cultivar, which has an 'FF1/FF2' genotype. This element of the genotype fingerprint can also distinguish J11500 from among many other strains.
  • strain J11500 has 4 non-cultivar progenitors and that considerable genetic diversity exists among strains, the genotypic fingerprint of strain J1 1500 shows numerous differences with that of the U1 lineage group.
  • a unique fingerprint allows strain J1 1500 (and its Essentially Derived Varieties and descendents) to be unambiguously identified.
  • genetic diversity among cultivated strains is a desirable objective because it is well established that genetic monocultures among agricultural crop species can lead to disastrous failures due to particular disease, pest, or environmental pressures. Any otherwise desirable commercial strain with genetic novelty is therefore valuable. Vegetative incompatibility between genetically distinct cultivated strains is also economically valuable in addressing virus control and farm hygiene. Strain J11500 meets those criteria.
  • a culture or product incorporating the genetic marker profile shown in the respective column of Table I or row of Table II labeled J10102-s69 or J11500 is an embodiment of the invention.
  • Another embodiment of this invention is an Agaricus bisporus line or strain or its parts comprising at least 75% of the same alleles as the line J10102-s69 or the strain J11500 for the loci listed in the respective column of Table I and/or row of Table II.
  • this line or strain or its parts comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or essentially 100% of the same alleles as the line J10102-s69 or the strain J11500 for the loci listed in the respective column of Table I and/or row of Table II.
  • a cell having at least 75% of the same alleles as a cell of line J10102-s69 or a cell of strain J1 1500 for the loci listed in the respective column of Table I and/or row of Table II is also an embodiment of this invention.
  • cells having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or essentially 100% of the same alleles as a cell of line J10102-s69 or a cell of strain J11500 for the loci listed in the respective column of Table I, and/or row of Table II are provided.
  • cultures substantially benefiting from the use of line J10102-s69 or strain J11500 in their development such as hybrid offspring having line J10102-s69 or a line obtained from strain J1 1500 as a parent, and line derived from J10102-s69 having a trait introduced through introgressive matings of offspring back to line J10102-s69, or through transformation.
  • an embodiment of this invention is an Agaricus bisporus heterokaryon comprising at least one allele per locus that is the same allele as is present in the J10102-s69 line for at least 75% of the marker loci listed in Tables I and II.
  • heterokaryons comprising at least one allele per locus that is the same allele as is present in the J10102-s69 line for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or essentially 100% of the marker loci listed in Tables I and II, are provided. More particularly, the heterokaryon may be a hybrid descendent of line J10102-s69.
  • Another embodiment of this invention is a culture of a strain having a genotype which is a complete or partial subset of the genotype of strain J11500.
  • Hybrid strain J1 1500 is the product of 6 generations of controlled line matings by Sylvan America, Inc. The original mating was made between line JB 137-s8 and line SWNC. In the sixth generation, line J10102-s69, a descendent of the first hybrid (and of other hybrids produced by Sylvan, Inc.), was mated with line OWNC to produce the novel hybrid strain J11500. [0206] Cultures of strain J11500 produce commercially acceptable and desirable crops of white mushrooms. Table III presents yield data as pounds per square foot, in three independent crop tests with internal replication.
  • Timing to harvest is about equivalent to that of commercial strain A15 (both about 13 to 19 days), and sometimes may be slightly faster, which can be economically advantageous.
  • Table V shows that in the same crop tests, on average, strain J1 1500 began to produce its crop 0.43 days before A-15, and the peak of production in the first flush was 0.24 days earlier for strain J11500.
  • Cap roundness and relative flesh thickness are considered to be desirable commercial mushroom traits.
  • J1 1500 typically produces mushrooms with caps having thicker flesh, and which are subjectively rounder, than those of A15; objectively, the following physical measurement ratios demonstrate the shape differences of J1 1500 compared to A15.
  • Cross-strain incompatibility can also be a useful commercial mushroom trait.
  • J11500 is incompatible with A-15, a proxy for the U1 derived lineage group.
  • casing material incorporating inoculum of J1 1500 is placed over compost colonized with A-15, or conversely when A-15 is placed over J11500, i.e., in non-self pairings, a partial crop failure ensues, demonstrating incompatibility as shown by the yield data in Table VI:
  • a heterokaryotic selfed offspring of an F1 hybrid that itself has a 'p/q' genotype will in the example have a genotype of 'p/p', 'q/q', or 'p/q'.
  • Two types of selfing lead to differing expectations about representation of alleles of line J 10102- s69 and of the F1 hybrid in the next heterokaryotic generation.
  • Agaricus bisporus regularly undergoes a second, characteristic, spontaneous intra-tetrad form of selfing called intramixis, producing heterokaryotic postmeiotic spores carrying two different recombined haploid nuclei having complementary, heteroallelic MAT alleles.
  • An offspring developing from any one of these spores is a postmeiotic self-mated heterokaryon with ca. 100% retention of the heteroallelism present in the single F1 parent around all 13 pairs of centromeres, due to the association of non-sister (first division) postmeiotic nuclei enforced by the requirement for sexual complementation at the centromerically- linked MAT locus.
  • Both types of selfed offspring are considered to be Essentially Derived Varieties (EDVs) of the initial F1 hybrid, and the latter type comprises most (often 95-100%) of the genotype of the F1 , and may express a very similar phenotype to that of the F1 hybrid.
  • EDVs Essentially Derived Varieties
  • the culture deposited was taken from the same culture maintained by Sylvan America, Inc., Kittanning, Pa., the assignee of record, since prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to meet all deposit requirements of the U.S. Patent and Trademark Office, including 37 C.F.R. Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty.
  • the NRRL Accession No. is 50896.
  • the deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.
  • the culture will be irrevocably and without restriction or condition released to the public upon filing of a priority application or upon the issuance of a patent according to the patent laws.
  • Table VIII shows that in test 13-177, the EDV strain designated J11500-ms2 was completely compatible with the initial strain J1 1500, and in fact demonstrated higher first break yield than strain J1 1500 as opposed to a partial crop failure that would have indicated incompatibility.
  • One use of the culture of strain J11500 is the production of crops of edible mushrooms for sale. Another use is for the improvement of facility hygiene via strain rotation and a 'virus-breaking' effect.
  • a third use is to incorporate the genetic material of strain J11500 into offspring and derived or descended cultures including dormant and germinating spores and protoplasts. Additional uses also exist as noted above.
  • Hybridization of Agaricus bisporus cultures of the invention may be accomplished by allowing two different cultures, one of which is a genetic line present in a spore of J11500, to grow together in close proximity, preferably on sterile media, until anastomosis (i.e., hyphal or cell fusion) occurs.
  • the resultant fusion culture is a first-generation outbred hybrid culture incorporating a genetic line present in a mushroom spore which is one part of one embodiment of the present invention.
  • Protoplasts derived from basidia or other parts of the organism are another part of the J1 1500 mushroom that may be used to transmit genetic material of J11500 into new cultures.
  • J10102-s69 is a line selected from among haploid progeny of a 5th generation in a hybrid pedigree initiated by Sylvan America, Inc. in 1993. This line, within a suitable heterokaryotic genetic background, recessively confers a white cap color trait upon heterokaryotic offspring; cap color is determined primarily by recessive alleles at the Ppc-1 locus on Chromosome 8.
  • Line J10102-s69 has the Mat-2 mating type genotype and behavioral phenotype.
  • line J10102-s69 is a haploid line, it is incapable of producing a crop of mushrooms, and consequently no "J10102-s69 mushroom" is obtainable and no direct characterization of a crop or product phenotype is possible. Therefore most selection criteria applied to haploid lines including line J10102-s69 are determined empirically by evaluating a series of matings which share a common parent such as line J10102-s69.
  • Line J10102-s69 is among the top-ranked haploid lines discovered from among its cohort of sibling lines. No previous hybrid, prior to creation of hybrids using line J10102-s69, had the particular combination of desirable traits (including specific details of its rounder cap, thicker flesh, and accelerated cropping, plus a particular novel incompatibility phenotype) seen among hybrids incorporating line J10102-s69, as described herein. No previous line has ever been observed to produce the combinations of desirable traits observed among hybrids incorporating line J10102-s69.
  • a single mushroom hybrid results from the mating of two haploid, homoallelic lines, each of which has a genotype that complements the genotype of the other.
  • the hybrid descendant of the first generation is designated F1.
  • F1 hybrids may be useful as new commercial varieties for mushroom production, or as starting material for the production of inbred offspring and/or EDVs, or as parents of the next generation of haploid lines for producing subsequent hybrid strains.
  • Line J10102-s69 may be used to produce hybrid mushroom cultures.
  • One such embodiment is the method of mating homokaryotic line J10102-s69 with another homokaryotic mushroom line, to produce a first generation F1 hybrid culture.
  • the first generation culture, part, mushroom, and mushroom part produced by this method is an embodiment of the invention.
  • the first generation F1 culture will comprise a complete set of the alleles of the homokaryotic line J10102-s69.
  • the strain developer can use either strain development records or molecular methods to identify a particular F1 hybrid culture produced using line J10102-s69. Further, the strain developer may also produce F1 hybrids using lines which are transgenic or introgressive trait conversions ('narrow modifications') of line J10102-s69.
  • Another embodiment is the method of mating line J10102-s69, or a narrowly modified version of that line, with a different, heterokaryotic culture of Agaricus bisporus. This latter method is less efficient than mating using two homokaryotic lines, but can also result in the production of novel hybrid cultures.
  • the development of a mushroom hybrid in a typical mushroom strain development program involves many or all of the following steps: (1) the obtaining of strains or stocks from various germplasm pools of the mushroom species for initial matings; (2) matings between pairs of pure cultures on sterile microbiological growth media such as potato dextrose agar (PDA); (3) the obtaining and use of promising hybrid strains from matings to produce subsequent generations of homokaryotic progeny lines, such as line J10102-s69, which are individually uniform; (4) the use of those lines in matings with other lines or strains to produce a subsequent hybrid generation; (5) repetition of steps (2-4) as needed; (6) obtaining of pre-commercial hybrid strains and the use of essential derivation techniques such as selfing to produce a final commercial strain.
  • PDA potato dextrose agar
  • steps (2-4) may be performed up to 5 times.
  • steps (2) to (4) may be repeated anywhere from 0 up to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.
  • the homokaryotic lines are not reproductively competent ('fertile'). Fertility, the ability to produce a crop of mushrooms, is restored in complementary matings with other haploid, or less commonly, heterokaryotic strains. An important consequence of the homoallelism and homogeneity of the homokaryotic line is that the hybrid between a defined pair of homokaryotic lines may be recreated indefinitely as long as the homokaryotic lines are preserved and/or propagated.
  • the mushroom spawn was mixed with pasteurized compost and incubated for 13 to 18 days.
  • a non-nutritive peat-based casing layer was placed over the compost as previously described and a casing inoculum was incorporated into the casing layer.
  • the first mushrooms reached the correct stage of development in a further 14 days.
  • the mushrooms were picked over a 3 to 4 day period. Three flushes of mushrooms were harvested before each test was concluded.
  • Strain J10102 is a heterokaryotic strain obtained in Sylvan America, Inc.'s strain development program. It did not have the combination of characters needed to be successful commercially; however its performance and physical characteristics approached those criteria, and the strain was assessed as having some unknown potential for further development and improvement. Consequently, J 10102 was used as a parent in 165 matings to several diverse lines of A. bisporus that, it was believed, might have had some useful potential in mating combinations. Individual outcomes were unpredictable and variable; it was hoped that the experiment might produce a successful result but the overall likelihood of that was considered to be low. Of the 165 novel hybrids obtained, only two were of potential commercial interest, and only one, J11500, consistently met the target criteria for a successful commercial strain. It was later determined in the course of testing that strain J1 1500 had other beneficial attributes as well.
  • strain J11500 Essentially Derived Varieties of strain J11500 were obtained from single spores, multiple spore mixtures, and from tissue and somatic selections, as described hereinabove. Spores of strain J11500 were obtained and were germinated and used to produce heterokaryotic and homokaryotic offspring, and outbred descendants as described hereinabove. Homokaryotic offspring lines were used to make matings to other lines, and further hybrids were obtained from these matings. Spawn and casing inoculum of J1 1500 and A-15 were used in self/self and self/non-self combinations in test crops to confirm the incompatibility of the two strains, a prerequisite for use in virus-breaking strategies, all as described hereinabove.

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Abstract

L'invention concerne une culture d'Agaricus bisporus désignée par lignée d'Agaricus bisporus J10102-s69, une culture représentative de la lignée ayant été déposée sous le numéro d'accès NRRL No. 50893. Des cultures d'hybrides F1 peuvent être dérivées des cultures ou obtenues en tant que descendants de cette lignée par croisement de la lignée J10102-s69 avec une autre lignée d'Agaricus bisporus différentes, comme OWNC, une culture d'une nouvelle souche d'hybride F1 étant ainsi produite, comme la souche J11500, une culture représentative de la souche ayant été déposée sous le numéro d'accès NRRL No. 50895. L'invention concerne également ces lignées et des souches dérivées ou obtenues en tant que descendants de ces lignées ou souches et/ou des VED de ces lignées ou souches.
EP15742750.1A 2014-01-31 2015-03-20 Lignée de champignon j10102-s69, souche de champignon hybride j11500, leurs descendants ainsi que procédés et utilisations associés Pending EP3099778A4 (fr)

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US14/169,578 US9648812B2 (en) 2014-01-31 2014-01-31 Mushroom line J10102-s69 and methods and uses therefor
US14/169,658 US9622428B2 (en) 2014-01-31 2014-01-31 Hybrid mushroom strain J11500 and descendants thereof
PCT/IB2015/052067 WO2015114612A2 (fr) 2014-01-31 2015-03-20 Lignée de champignon j10102-s69, souche de champignon hybride j11500, leurs descendants ainsi que procédés et utilisations associés

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US11785913B2 (en) * 2016-12-01 2023-10-17 Sylvan America, Inc. Mushroom line J14756-s3 and methods and uses therefor
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WO2023250454A1 (fr) * 2022-06-24 2023-12-28 Sylvan Inc. Procédé pour exclure le caractère d'incompatibilité agressive de souches de agaricus bisporus et de souches et de lignées associées

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