Hybrid mushroom strain J15987 and derivatives thereof

Abstract

A hybrid mushroom culture of Agaricus bisporus, designated as strain J15987, includes a representative culture of the strain, which has been deposited under NRRL Accession No. 67646.

Claims

1. A hybrid mushroom culture of Agaricus bisporus comprising: a culture designated as strain J15987, a representative culture of the strain having been deposited under NRRL Accession No. 67646.

2. An Essentially Derived Variety of the hybrid mushroom culture designated as strain J15987 of claim 1, wherein said Essentially Derived Variety is a culture of a strain derived from an initial culture of strain J15987, wherein a culture of the strain has been deposited under NRRL Accession No. 67646, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of strain J15987.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Initially, in order to provide clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

(2) Allele: One or two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome; a heritable unit of the genome at a defined locus, ultimately identified by its DNA sequence (or by other means).

(3) Amphithallism: A reproductive syndrome in which heteromixis and intramixis are both active.

(4) Anastomosis: Fusion of two or more hyphae that achieves cytoplasmic continuity.

(5) Basidiomycete: A monophyletic group of fungi producing meiospores on basidia; a member of a corresponding subdivision of Fungi such as the Basidiomycetales or Basidiomycotina.

(6) Basidium: The meiosporangial cell, in which karyogamy and meiosis occur, and upon which the basidiospores are formed.

(7) 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.

(8) Breeding: Development of strains, lines or varieties using methods that emphasize sexual mating.

(9) Cap: Pileus; part of the mushroom, the gill-bearing structure.

(10) 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.

(11) Carrier substrate: A medium having both nutritional and physical properties suitable for achieving both growth and dispersal of a culture; examples are substrates that are formulated for mushroom spawn, casing inoculum, and other inoculum.

(12) 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.

(13) Casing inoculum (CI): A formulation of inoculum material incorporating a mushroom culture, typically of a defined heterokaryotic strain, suitable for mixing into the casing layer.

(14) Cloning: Somatic propagation without selection.

(15) Combining ability: The capacity of an individual to transmit superior performance to its offspring. General combining ability is an average performance of an individual in a particular series of matings.

(16) Compatibility: See heterokaryon compatibility, vegetative compatibility, and/or sexual compatibility; incompatibility is the opposite of compatibility.

(17) Culture: The tangible living organism; the organism propagated on various growth media and substrates; a portion of, or the entirety 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.

(18) Derivation: Development from a strain; see Essentially Derived Variety (EDV).

(19) Derived lineage group: The set of EDVs derived from a single initial strain or variety.

(20) Descent: Genealogical descent over a limited number (e.g., 10 or fewer) of generations.

(21) Diploid: Having two haploid chromosomal complements within a single nuclear envelope.

(22) Directed mutagenesis: A process of altering the DNA sequence of at least one specific gene locus.

(23) 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.

(24) Essentially Derived Variety (EDV): A congruent, brief, practical definition of an EDV is a culture derived from an initial culture such that the resulting culture has present at least 75% of the genome or genotype of the initial strain or culture. (Supplemental to the definition of an EDV, it is illustrative to note here that an EDV culture having most or all, but at least 75%, of its genome or genotype present in the genome or genotype of an initial strain or culture may be derived from an initial strain or culture by using a method selected from a group of methods comprising: (a) somatic selection, (b) tissue culture selection, selfing including (c) mating of sibling offspring lines and (d) intramictic reproduction via single or multiple spores, (e) repeated back-mating to the initial line, strain or culture, (f) mutagenesis including induced, directed and targeted mutagenesis (g) genetic transformation (h) a process of single-locus trait conversion, (i) a process of deheterokaryotization, (j) isolation of spontaneous mutants, to produce a culture of an EDV of an initial culture.)

(25) 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.

(26) 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.

(27) Fungus: An organism classified as a member of the Kingdom Fungi.

(28) Genealogical relationship: A familial relationship of descent from one or more progenitors, for example that between parents and offspring.

(29) Genetic identity: The genetic information that distinguishes an individual, including representations of said genetic information such as, and including: genotype, genotypic fingerprint, genome sequence, genetic marker profile; genetically identical=100% genetic identity, X % genetically identical=having X % genetic identity etc.

(30) Genotypic fingerprint: A description of the genotype at a defined set of marker loci; the known genotype.

(31) Gill: Lamella; part of the mushroom, the hymenophore- and basidium-bearing structure.

(32) Haploid: Having only a single complement of nuclear chromosomes; see homokaryon.

(33) Heteroallelic: Having two different alleles at a locus; analogous to heterozygous.

(34) Heteroallelism: Differences between homologous chromosomes in a heterokaryotic genotype; analogous to heterozygosity.

(35) 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 reproductively competent (in the absence of any rare interfering genetic defects at loci other than the Mat locus), and which exhibits vegetative incompatibility reactions with other heterokaryons; also called a strain or stock in the strain development context.

(36) Heterokaryon compatibility: The absence of antagonism observed during physical proximity or contact between two heterokaryons that are not genetically identical; see Heterokaryon Incompatibility.

(37) 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; i.e., a genetic system that allows one heterokaryon culture to discriminate and recognize another culture as being either self or non-self, that operates in basidiomycete heterokaryons to limit anastomosis (hyphal fusion) and cytoplasmic contact; vegetative incompatibility.

(38) Heterokaryotic: Having the character of a heterokaryon.

(39) Heteromixis: Life cycle involving mating between two different non-sibling haploid individuals or gametes; outbreeding.

(40) 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.

(41) 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.

(42) Homokaryotic: Having the character of a homokaryon; haploid.

(43) Hybrid: Of biparental origin, usually applied to heterokaryotic strains and cultures produced in controlled matings.

(44) Hybridizing: Physical association, for example on a petri dish containing a sterile agar-based nutrient medium, of two cultures, usually homokaryons, in an attempt to achieve anastomosis, plasmogamy, and formation of a sexual heterokaryon (=mating); succeeding in the foregoing.

(45) Hyphae: Threadlike elements of mycelium, composed of cell-like compartments.

(46) Inbreeding: Matings that include sibling-line matings, back-matings to parent lines or strains, and intramixis; reproduction involving parents that are genetically related.

(47) Incompatibility: See heterokaryon incompatibility.

(48) Induced mutagenesis: A non-spontaneous process of altering the DNA sequence of at least one gene locus.

(49) Initial culture: A culture which is used as starting material in a strain development process; more particularly a strain from which an Essentially Derived Variety is obtained.

(50) 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.

(51) Intramixis: A uniparental sexual life cycle involving formation of a complementary mated pair of postmeiotic nuclei within the basidium or individual spore.

(52) 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.

(53) Lamella: see gill.

(54) 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.

(55) Lineage group: see derived lineage group. The set of EDVs derived from a single initial strain or variety.

(56) Locus: A defined contiguous part of the genome, homologous although often varying among different genotypes; plural: loci.

(57) Marker assisted selection: Using linked genetic markers including molecular markers to track trait-determining loci of interest among offspring and through pedigrees.

(58) MAT: The mating-type locus, which determines sexual compatibility and the heterokaryotic state.

(59) Mating: The sexual union of two cultures via anastomosis and plasmogamy; methods of obtaining matings between mushroom cultures are well known in the art.

(60) Mycelium: The vegetative body or thallus of the mushroom organism, comprised of threadlike hyphae.

(61) Mushroom: The reproductive structure of an agaric fungus; an agaric; a cultivated food product of the same name.

(62) Neohaplont: A haploid culture or line obtained by physically deheterokaryotizing (reducing to haploid components) a heterokaryon; a somatically obtained homokaryon.

(63) 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.

(64) Outbreeding: Mating among unrelated or distantly related individuals.

(65) 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.

(66) Pedigree-assisted breeding: The use of genealogical information to identify desirable combinations of lines in controlled mating programs.

(67) Phenotype: Observable characteristics of a strain or line as expressed and manifested in an environment.

(68) Plasmogamy: Establishment, via anastomosis, of cytoplasmic continuity leading to the formation of a sexual heterokaryon.

(69) Progenitor: Ancestor, including parent (the direct progenitor).

(70) Selfing: Mating among sibling lines; also intramixis.

(71) Sexual compatibility: A condition among different lines of allelic non-identity at the Mat locus, such that two lines are able to mate to produce a stable and reproductively competent heterokaryon. The opposite condition, sexual incompatibility, occurs when two lines have the same allele at the Mat locus.

(72) Somatic: Of the vegetative mycelium.

(73) Spawn: 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.

(74) Spore: Part of the mushroom, the reproductive propagule.

(75) Stem: Stipe; part of the mushroom, the cap-supporting structure.

(76) 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.

(77) Stipe: see stem.

(78) Strain: A heterokaryon with defined characteristics or a specific identity or ancestry; equivalent to a variety.

(79) Targeted mutagenesis: A process of altering the DNA sequence of at least one specific gene locus.

(80) Tissue culture: A de-differentiated vegetative mycelium obtained from a differentiated tissue of the mushroom.

(81) Trait conversion: A method for the 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.

(82) 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.

(83) Vegetative compatibility: The absence of the phenomenon of antagonism observed during physical proximity or contact between two heterokaryons that are not genetically identical, determined by a multilocus self/non-self recognition system that operates in basidiomycete heterokaryons to limit anastomosis (hyphal fusion) and cytoplasmic contact; Heterokaryon compatibility; the opposite of Vegetative incompatibility.

(84) Vegetative incompatibility: The phenomenon of antagonism observed during physical proximity or contact between two heterokaryons that are not genetically identical, determined by a multilocus self/non-self recognition system that operates in basidiomycete heterokaryons to limit anastomosis (hyphal fusion) and cytoplasmic contact; Heterokaryon incompatibility.

(85) 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.

(86) Yield: The net fresh weight of the harvest crop, normally expressed in pounds per square foot.

(87) 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.

(88) With respect to the definition of homokaryon above, it is noted that 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. In practical terms, for fungal strain development purposes, 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. Also, 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. Therefore, in the present application, the use of the term homoallelic to characterize a line includes entirely or predominately homoallelic lines, and cultures described in this way are functional homokaryons, are putatively homokaryotic, and are all defined as homokaryons in the present application.

(89) Now, with respect to the invention and as noted hereinabove, the present invention relates to cultures of the hybrid Agaricus bisporus strain J15987 and to cultures derived or descended from J15987. Such cultures are used to produce mushrooms and parts of mushrooms. Thus, the present invention further relates to methods of making and using the strain J15987 and Essentially Derived Varieties (EDVs) of the strain J15987.

(90) The morphological and physiological characteristics of strain J15987 in culture on Difco brand PDA medium, which is a standard culture medium, are provided as follows. Strain J15987 growing on PDA medium in an 8.5 cm diameter Petri dish produced a white or light brown-yellow or tan colored irregularly lobate colony with a roughly circular overall outline that increased in diameter by (1.11-1.53) 1.54 (1.55-2.15) mm/day during dynamic equilibrium-state growth between days 10 and 34 after inoculation using a 3.3-3.5 mm diameter circular plug of the culture on PDA as inoculum. Hyphae of the culture on Difco PDA were irregular and about cylindrical, with an internodal measurement of (53) 61-92 (105) um, and exhibited a wide range of branching angles from about 10 to 90 degrees off the main hyphal axis. The strain 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 strain J15987.

(91) Hybrid strain J15987 is the product of 7 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, the subject matter of U.S. Pat. No. 9,648,812, issued May 16, 2017, hereby incorporated by reference, and a descendent of the first hybrid (and of other hybrids produced by Sylvan America, Inc.), was mated with line OWNC to produce the novel hybrid strain J11500, the subject matter of U.S. Pat. No. 9,622,428, issued Apr. 18, 2017, hereby incorporated by reference. In a subsequent generation, homokaryon s-80 from strain J11500 was mated with a second homokaryon (=line), called s-290, from a proprietary experimental breeding stock, to produce the seventh generation hybrid heterokaryon J15987.

(92) Cultures of strain J15987 produce commercially acceptable and desirable crops of white mushrooms. Table I presents yield data as pounds per square foot, in three independent crop tests with internal replication. As shown in Table I, productivity of strain J15987 is comparable to and often greater than the productivity of the A-15 strain, with mean total (2-flush) yields for strain J15987 and the A-15 control strain shown.

(93) TABLE-US-00001 TABLE I Yield of strain J15987 vs A-15, in pounds per square foot, over two flushes, in three tests with two to four replicates for each treatment Total yield Test ID J15987 A-15 16-261 4.70 4.64 17-07 5.09 5.78 17-159 6.15 5.72

(94) Table I demonstrates that the yield of strain J15987 is comparable to, and can exceed, that of the commercial standard A-15 strain. This is very uncommon for genetically distinct hybrid strains that produce high-quality white mushrooms.

(95) Strain J15987 has an attractive and commercially acceptable appearance, i.e., color and proportions, as can be seen from the data in the following two tables. Mushroom cap surface color is an important element of how the fresh white button mushroom product is perceived, evaluated and valued by all parties including consumers. Color components can be measured objectively by using a Minolta CR-200 Chromameter. Color measurement was measured according to the L-a-b color-space measurement framework. The a measurement component is positively correlated with redness. The smaller the a value, the whiter the mushroom appears and therefore also appears fresher to the human eye. This is an important trait.

(96) Table II reports mean values for color measurement a measured from mushrooms from strains J15987 and A-15 from first and second flush from the same test crop. In addition, p-values from general t-tests of the raw data are displayed. For Table II, medium-sized, closed cap mushrooms were harvested from the first heavy pick day of first flush and of second flush. A total of thirty chromameter readings were taken from individual mushrooms for each strain. Color measurements were taken on the top of the mushroom cap.

(97) TABLE-US-00002 TABLE II Mushroom cap surface redness expressed as a values, for mushrooms of strains J15987 and A-15 Strain Flush 1 Flush 2 J15987 0.48 0.76 A-15 0.49 0.18 p-value (t-test): 2.2e6 4.1e9

(98) Data in Table II therefore shows a highly significant difference between J15987 and A-15 for cap redness; J15987 mushroom caps were significantly less red, with a lower a value.

(99) Data in Table III, below, summarize the means of morphometric measurements taken on thirty first-flush mushrooms from each strain. Equal numbers of mushrooms, from the first heavy pick day, of both strains, grown at the same time in the same environment and conditions, were measured. Proportional measures (ratios of two direct measurements) were calculated since absolute dimensions vary widely among mushrooms of any strain and are influenced by cultural factors. (1) Cap Diameter (CD) is defined here as the greatest horizontal distance between two vertical lines tangential to either side of the cap. (2) Cap Flesh Thickness (CFT) is the vertical distance from the top of the lamellae (i.e. gills) adjacent to the stipe, to the surface of the pileus directly above. (3) Stem Thickness (ST) is the greatest horizontal distance across the stem. Two ratios, ST:CD and CFT:CD were compared statistically using a t-test.

(100) TABLE-US-00003 TABLE III Mushroom proportions of strains J15987 and A-15 Strain ST CD ST:CD CFT CFT:CD J15987 15.16 41.70 0.36 15.35 0.37 A-15 13.01 39.26 0.33 13.30 0.34

(101) The p-values obtained from a t-test of the raw data showed that strain J15987 had a significantly thicker stem (p=1.7e-3) and a significantly thicker cap flesh (p=8.8e-4) than the A-15 strain.

(102) Cross-strain incompatibility can also be a useful commercial mushroom trait. Strain J15987 is incompatible with strain A-15, a proxy for the U1 derived lineage group.

(103) A test of compatibility of strain J15987 with the strain A-15, a member of the U1 lineage, was performed in a spawn-mixing experiment; the results are shown in TABLE IV.

(104) TABLE-US-00004 TABLE IV Compatibility of J15987 with A-15 in Spawn-Mixing Experiment Spawn Strain: 100% J15987 99.5% A-15; 0.5% J15987 100% A-15 Casing Strain: 100% J15987 100% A-15 100% A-15 Rep 1 2.26 lbs. 0.00 lbs. 1.76 lbs. Rep 2 2.17 lbs. 0.00 lbs. 1.74 lbs. Rep 3 2.16 lbs. 0.00 lbs. 1.73 lbs. Avg. 2.20 lbs. 0.00 lbs. 1.74 lbs.

(105) Table IV shows that in this test, in the presence of 0.5% (by weight) spawn of strain J15987, mushroom production (i.e., yield) of the A-15 strain was completely prevented, indicating a strong incompatibility reaction between these two strains. This phenomenon tends to prevent anastomosis and viral infection. If fruiting of virus-infected strains in the U1 lineage is also prevented, then virus infection reservoirs of such strains will diminish rapidly in commercial facilities introducing the use of J15987.

(106) The incompatibility phenotype of a strain is known to be routinely transmitted into spores and thus is ordinarily inherited by EDVs derived from spores.

(107) Given that strain J15987 has multiple non-cultivar progenitors, and that considerable genetic diversity exists among strains, the genotypic fingerprint of strain J15987, as expected, shows numerous differences with that of the commercial-standard U1 lineage group. A unique fingerprint allows strain J15987 (and its Essentially Derived Varieties and descendants) to be unambiguously identified. Agronomically, 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. Strain J15987 meets those criteria.

(108) For the purpose of this invention, the whole genomic DNA sequence of strain J15987 and of the cultures of its parent lines have been obtained by Sylvan America Inc. using the following method. The homokaryotic parent line cultures were grown in sterile broth growth medium after maceration. After 2-4 weeks, hyphal cells were collected by filtration, were frozen at 80 C, and were lyophilized until dry. Cap tissue was obtained from mushrooms produced by cultures of the heterokaryotic J15987 strain, and was frozen and lyophilized. DNA was extracted from the lyophilized samples using a CTAB protocol followed by RNAse treatment and gel purification. A contractor, SeqWright, prepared DNA libraries from the DNA of each culture, and sequenced the libraries using Illumina MiSeq technology. Assemblies of the reads into genomic sequence using the public-domain reference genome sequence of H97 was performed by the Applicant. Consequently about 93% to about 95% of the entire genotype of strain J15987 and of its parental homokaryons are known to Sylvan America, Inc. with certainty. The total number of markers distinguishing strain J15987 that are known to the assignee is about 325,000. A brief excerpt of the genotype of J15987 and related cultures at numerous sequence-characterized marker loci distributed at intervals along each of the 19 H97 V2.0 reference scaffolds larger than 100 Kb in length is provided in Table V.

(109) TABLE-US-00005 TABLEV Positionof SNP[H97 V2.0ref. Culture: Scaffold coords.] H97 J10102-s69 J11500 J11500-s80 Lines-290 J15987 1 115817 CCGAGCGCA CCGA CGCA CCGA CGCA CCGA CGCA CCGAGCGCA CCGA CGCA 1 349966 AAGGTGGTT AAGG GGTT AAGG GGTT AAGG GGTT AAGGTGGTT AAGG GGTT 1 600145 GTTGGATTA GTTG ATTA GTTG ATTA GTTG ATTA GTTGGATTA GTTG ATTA 1 850014 CCTTTTCAC C TTTTC C C TTTTC C C TTTTC C CCTTTTCAC C TTTTC C 1 1099971 GTCGACACC GTCG CACC GTCG CACC GTCG CACC GTCGACACC GTCG CACC 1 1350278 GGAGAGTCG GGAG TCG GGAG TCG GGAG TCG GGAGAGTCG GGAG TCG 1 1599956 AATAAGCGC AATA GCGC AATA GCGC AATA GCGC AATAAGCGC AATA GCGC 1 1850032 CGAGTAATT CGAG AATT CGAG AATT CGAG AATT CGAGTAATT CGAG AATT 1 2119049 ACAATCCAA ACAA CAA ACAA CAA ACAA CAA ACAATCCAA ACAA CAA 1 2360610 TTCTACCAC TTCT CCAC TTCT CCAC TTCT CCAC TTCTACCAC TTCT CCAC 1 2612870 AATAGGAGT AATA GAGT AATA GAGT AATA GAGT AATAGGAGT AATA GAGT 1 2804522 GAAGACGAC GAAG GAC GAAG GAC GAAG GAC GAAGACGAC GAAG GAC 1 2858975 GCCGTTCTT GCCG TCTT GCCG TCTT GCCG TCTT GCCGTTCTT GCCG TCTT 1 3069801 CCAAACGCG CCAA CGCG CCAA CGCG CCAA CGCG CCAAACGCG CCAA CGCG 1 3256057 TATCTGTTT TATC GTTT TATC GTTT TATC GTTT TATCTGTTT TATC GTTT 2 101776 TTACTGCTC TTACTGCTC TTACTGCTC TTACTGCTC TTAC GCTC TTAC GCTC 2 101820 ATTAAAGAT ATTA GAT ATTA GAT ATTAAAGAT ATTAAAGAT ATTAAAGAT 2 235195 TTAAATACA TTAA ACA TTAA ACA TTAAATACA TTAAA ACA TTAAA ACA 2 350156 TCGGGGGTG TCGG GGTG TCGG GGTG TCGGGGGTG TCGGGGGTG TCGGGGGTG 2 600112 ATGTATACG ATGT TACG ATGT TACG ATGTATACG ATGTATACG ATGTATACG 2 850338 TGGTGCTAA TGGT CTAA TGGT CTAA TGGTGCTAA TGGTGCTAA TGGTGCTAA 2 1099413 CCTGACTCA CCTG CTCA CCTG CTCA CCTGACTCA CCTGACTCA CCTGACTCA 2 1349512 CTCAGCAGT CTCA CAGT CTCA CAGT CTCAGCAGT CTCAGCAGT CTCAGCAGT 2 1600085 CACAATGCC CACA TGCC CACA TGCC CACAATGCC CACAATGCC CACAATGCC 2 1690101 ACTTGACAA ACTTGAC A ACTTGAC A ACTTGACAA A TT ACAA A TT ACAA 2 1902928 GATGGATGT GATG ATGT GATG ATGT GATGGATGT GATGGATGT GATGGATGT 2 1956830 ATTCCTCAT ATTC TCAT ATTC CAT ATTCCTCAT ATTC TCAT ATTC CAT 2 2150201 GTCGTAGGT GTCG AGGT GTCG AGGT GTCGTAGGT GTCGTAGGT GTCGTAGGT 2 2320631 GTGACGTTG GTGAC TTG GTGAC TTG GTGACGTTG GTGA TTG GTGA TTG 2 2400354 CAGAGTCGC CAGA TCGC CAGA TCGC CAGAGTCGC CAGAGTCGC CAGAGTCGC 2 2650136 ATAATTCCT ATAA TCCT ATAA TCCT ATAATTCCT ATAATTCCT ATAATTCCT 2 2903045 AGAAATAGA AGAA TAGA AGAA TAGA AGAAATAGA AGAAATAGA AGAAATAGA 2 3047973 TGACTTCTC TGACTTCTC TGACTTCTC TGACTTCTC TGAC TCTC TGAC CTC 2 3048019 GTCCGCTGC GTCC CTGC GTCC CTGC GTCCGCTGC GTCCGCTGC GTCCGCTGC 3 65650 GGCGCTTTT GGCG TTTT GGCG TTTT GGCGCTTTT GGCGCTTTT GGCGCTTTT 3 119281 TTTATACTC TTTA ACTC TTTA ACTC TTTATACTC TTTATACTC TTTATACTC 3 249570 GTATTATGT GTATTATGT GTATTATGT GTATTATGT GTATTATGT GTATTATGT 3 500000 GTATACCAA GTATACCAA GTATACCAA GTATACCAA GTATACCAA GTATACCAA 3 750000 GTCCGGCCA GTCCGGCCA GTCCGGCCA GTCCGGCCA GTCCGGCCA GTCCGGCCA 3 1000000 CACGCGACG CACGCGACG CACGCGACG CACGCGACG CACGCGACG CACGCGACG 3 1250000 TTTTTCCGG TTTTTCCGG TTTTTCCGG TTTTTCCGG TTTTTCCGG TTTTTCCGG 3 1500000 GTCTGGACA GTCTGGACA GTCTGGACA GTCTGGACA GTCTGGACA GTCTGGACA 3 1750000 ACGCCTGAC ACGCCTGAC ACGCCTGAC ACGCCTGAC ACGCCTGAC ACGCCTGAC 3 2000000 GTCTCAGGG GTCTCAGGG GTCTCAGGG GTCTCAGGG GTCTCAGGG GTCTCAGGG 3 2250000 CGTGGCGAT CGTGGCGAT CGTGGCGAT CGTGGCGAT CGTGGCGAT CGTGGCGAT 3 2500000 AATCCTCAC AATCCTCAC AATCCTCAC AATCCTCAC AATCCTCAC AATCCTCAC 4 100004 GAGTGATAA GAGT AT A GAGT AT A GAGTGATAA GAGT AT A GAGT AT A 4 383799 CAGCCAGAC CAGC AGAC CAGC AGAC CAGCCAGAC CAGC AGAC CAGC AGAC 4 598147 GATCGACAG GATC ACAG GATC ACAG GATCGACAG GATC ACAG GATC ACAG 4 852119 CGAATATTC CGAA A TC CGAA A TC CGAATATTC CGAA A TC CGAA A TC 4 1100085 GATGCCGAA GATG CGAA GATG CGAA GATGCCGAA GATG CGAA GATG CGAA 4 1350536 CGAACTCGG CGAA CGG CGAA CGG CGAACTCGG CGAA CGG CGAA CGG 4 1599885 GATACTTGC GATA TTGC GATA TTGC GATACTTGC GATA TTGC GATA TTGC 4 1850288 ATTCGTGTA ATTC GTA ATTC GTA ATTCGTGTA ATTC GTA ATTC GTA 4 2100356 TCAGAGACC TCAG GACC TCAG GACC TCAG GACC TCAGAGACC TCAG GACC 4 2284257 TCTGGACTG TCTG ACTG TCTG ACTG TCTG ACTG TCTGGACTG TCTG ACTG 5 100211 TCCTTGAAT TCCT GAAT TCCT GAAT TCCT GAAT TCCTTGAAT TCCT GAAT 5 350872 GGCGTGCCC GGCG GCCC GGCG GCCC GGCG GCCC GGCGTGCCC GGCG GCCC 5 599922 CGTCATTCA CGTC TTCA CGTC TTCA CGTC TTCA CGTCATTCA CGTC TTCA 5 851262 TAATTCTCT TAAT TCT TAAT TCT TAAT TCT TAATTCTCT TAAT TCT 5 1099776 ACATTGACA ACAT GACA ACAT GACA ACAT GACA ACATTGACA ACAT GACA 5 1352539 TTGTGATCC TTGT TCC TTGT TCC TTGT TCC TTGTGATCC TTGT TCC 5 1599904 AACTTCCTT AACT CCTT AACT CCTT AACT CCTT AACTTCCTT AACT CCTT 5 1851458 AAATAATCC AAAT TCC AAAT TCC AAAT TCC AAATAATCC AAAT TCC 5 2100025 CCCTTAGTC CCCT AGTC CCCT AGTC CCCT AGTC CCCTTAGTC CCCT AGTC 5 2278878 GGTCGAAAA GGTC AAAA GGTC AAAA GGTC AAAA GGTCGAAAA GGTC AAAA 6 106294 GCCATCTCG GCCA CTC GCCA CTC GCCA CTC GCCATCTCG GCCA CTC 6 350337 CATTTGGTT CATT GGTT CATT GGTT CATT GGTT CATTTGGTT CATT GGTT 6 600047 GGAGCATTT GGAG ATTT GGAG ATTT GGAG ATTT GGAGCATTT GGAG ATTT 6 849990 AGTTCAGGA AGTT AGGA AGTT AGGA AGTT AGGA AGTTCAGGA AGTT AGGA 6 1098535 CAAAGATTG CAAA ATTG CAAA ATTG CAAA ATTG CAAAGATTG CAAA ATTG 6 1349453 TGTCGGTAG TGTC TAG TGTC TAG TGTC TAG TGTCGGTAG TGTC TAG 6 1600000 AAACCTGGA AAACCTGGA AAACCTGGA AAACCTGGA AAACCTGGA AAACCTGGA 6 1764645 AACCGGATT AACC GATT AACC GATT AACC GATT AACCGGATT AACC GATT 6 2000087 GATTTTGCG GATT TGCG GATT TGCG GATT TGCG GATTTTGCG GATT TGCG 6 2252662 GGGTTGGTA GGGT GGTA GGGT GGTA GGGT GGTA GGGTTGGTA GGGT GGTA 7 227441 ACACATACT ACAC TACT ACAC TACT ACACATACT ACAC TACT ACAC TACT 7 350044 ATATTCTTT ATAT CTTT ATAT CTTT ATATTCTTT ATAT CTTT ATAT CTTT 7 600111 CAATTATTA CAAT ATTA CAAT ATTA CAATTATTA CAAT ATTA CAAT ATTA 7 850516 TGACGCATA TGAC CATA TGAC CATA TGACGCATA TGAC CATA TGAC CATA 7 1100248 TCACGGAAG TCAC GAAG TCAC GAAG TCACGGAAG TCAC GAAG TCAC GAAG 7 1350089 CTTTTCCCC CTTT CCCC CTTT CCCC CTTTTCCCC CTTT CCCC CTTT CCCC 7 1605047 ATACTTGGC ATAC TG C ATAC TG C ATACTTGGC ATAC TG C ATAC TG C 7 1850000 GAGATACT GAGATACT GAGATACT GAGATACT GAGATACT GAGATACT 7 1898793 TCCGCATAA TCCG AT A TCCG AT A TCCGCATAA TCCG AT A TCCG AT A 7 1991505 TCTACGGTT TCTA GTT TCTA GTT TCTACGGTT TCTA GTT TCTA GTT 8 350000 ATTGACGCG ATTGACGCG ATTGACGCG ATTGACGCG ATTGACGCG ATTGACGCG 8 600000 CATTGACGG CATTGACGG CATTGACGG CATTGACGG CATTGACGG CATTGACGG 8 850000 AAATCGCTT AAATCGCTT AAATCGCTT AAATCGCTT AAATCGCTT AAATCGCTT 8 1100000 CATACGATC CATACGATC CATACGATC CATACGATC CATACGATC CATACGATC 8 1350000 AGCTTAACA AGCTTAACA AGCTTAACA AGCTTAACA AGCTTAACA AGCTTAACA 8 1600100 CTGAACCCT CTGAACCCT CTGAACCCT CTGAACCCT CTGAACCCT CTGAACCCT 9 100105 CTCAACCGA CTCA CCGA CTCA CCGA CTCAACCGA CTCAACCGA CTCAACCGA 9 352455 AGTCCTCCA AGTC CCA AGTC CCA AGTCCTCCA AGTCCTCCA AGTCCTCCA 9 599918 TATCTCCAC TATCTCCAC TATCTCCAC TATCTCCAC TATC CCAC TATC CCAC 9 599950 TGGTATCCC TGGT TCCC TGGT TCCC TGGTATCCC TGGTATCCC TGGTATCCC 9 798851 CTTCGATGC CTTC ATGC CTTC ATGC CTTCGATGC CTTC ATGC CTTC ATGC 9 800528 TCGACGACC TCGA GACC TCGA GACC TCGACGACC TCGACGACC TCGACGACC 9 1010845 GGGTGGTGA GGGT GTGA GGGT GTGA GGGTGGTGA GGGTGGTGA GGGTGGTGA 9 1050049 ATCTTTGAT ATCT TGAT ATCT TGAT ATCTTTGAT ATCT TGAT ATCT TGAT 9 1250269 CTGTCTTGG CTGTCTTGG CTGTCTTGG CTGTCTTGG CTGT TTGG CTGT TTGG 9 1335069 ATTTGCTTC ATTT CTTC ATTTr TTC ATTTGCTTC ATTTGCTTC ATTTGCTTC 9 1656962 TATCTACTG TATC ACTG TATC ACTG TATCTACTG TATCTACTG TATCTACTG 10 100438 AATTAATTT AATT ATTT AATT ATTT AATT ATTT AATTAATTT AATT ATTT 10 299994 CGCGGGGGC CGCGGGGGC CGCGGGGGC CGCGGGGGC CGCG GGGC CGCG GGGC 10 352915 GCGTTCGTG GCGT CGTG GCGT CGTG GCGT CGTG GCGTTCGTG GCGT CGTG 10 550293 CGGCTCGGC CGGCTCGGC CGGCTCGGC CGGCTCGGC CGGC CGGC CGGC CGGC 10 600032 TTACACTGG TTAC CTGG TTAC CTGG TTAC CTGG TTACACTGG TTAC CTGG 10 860249 CCGCAAATT CCGC AAATT CCGC AAATT CCGC AAATT CCGCAAATT CCGC AAATT 10 1000344 ATTATGACA ATTA GACA ATTA GACA ATTA GACA ATTA GACA ATTA GACA 10 1110433 GGAAGACAA GGAA ACAA GGAA ACAA GGAA ACAA GGAAGACAA GGAA ACAA 10 1303902 TGATTTACT TGAT TACT TGAT TACT TGAT TACT TGATTTACT TGAT TACT 10 1330031 GGATCTGTA GGAT TGTA GGAT TGTA GGAT TGTA GGAT TGTA GGAT TGTA 10 1490452 AATCAGATG AATC GATG AATC GATG AATC GATG AATCAGATG AATC GATG 11 104770 AATGAGAGG AATGAGAGG AATGAGAGG AATGAGAGG AATG GAGG AATG GAGG 11 349990 GACGGCTTC GACGGCTTC GACGGCTTC GACGGCTTC GACG CTTC GACG CTTC 11 600001 TGGGCGCGC TGGGCGCGC TGGGCGCGC TGGGCGCGC TGGG GCGC TGGG GCGC 11 908344 TAGAAAGAA TAGAAAGAA TAGAAAGAA TAGAAAGAA TAGA AGAA TAGA AGAA 11 1100296 TTCTAAAAT TTCTAAAAT TTCTAAAAT TTCTAAAAT TTCT AAAT TTCT AAAT 11 1239957 GCTTACTGC GCTTACTGC GCTTACTGC GCTTACTGC GCTT CTGC GCTT CTGC 12 88001 ACGACAGAG ACGACAGAG ACGACAGAG ACGACAGAG ACGA A AG ACGA A AG 12 100000 CCTTCTAGT CCTTCTAGT CCTTCTAGT CCTTCTAGT CCTTCTAGT CCTTCTAGT 12 439214 CACGATGAT CACGATGAT CACGATGAT CACGATGAT CACG TGAT CACG TGAT 12 700059 GCTGCCATG GCTGCCATG GCTGCCATG GCTGCCATG GCTG CATG GCTG CATG 12 1000000 CGAGGAGGA CGAGGAGGA CGAGGAGGA CGAGGAGGA CGAGGAGGA CGAGGAGGA 12 1000704 TTCTGGTGC TTCTGGTGC TTCTGGTGC TTCTGGTGC TTCT GTGC TTCT GTGC 13 100697 ACGTCTTTA ACGT TTTA ACGT TTTA ACGT TTTA ACGTC TTA ACGT TTA 13 370946 AATCTACAA AATC A AA AATC A AA AATC A AA AATC AC A AATC A A 13 604345 CTTCAGCAT CTTC GCAT CTTC GCAT CTTC GCAT CTTCAGCAT CTTC GCAT 13 850249 GGCTAGTAA GG T GT A GG T GT A GG T GT A GG T GT A GG T GT A 14 113109 AGGGAAATA AGGG AATA AGGG AATA AGGGAAATA AGGG AATA AGGG AATA 14 372086 CGATCCCTT CGAT C TT CGAT C TT CGATCCCTT CGAT C TT CGAT C TT 14 725684 ATGAGTTCG ATGA TT G ATGA TT G ATGAGTTCG ATGA TT G ATGA TT G 15 97145 TGACGTTTT TGACGTTTT TGACGTTTT TGACGTTTT TGAC TTTT TGAC TTTT 15 449866 GAATTTCGG GAAT TCGG GAAT TCGG GAAT TCGG GAAT TCGG GAAT TCGG 16 208609 CACATGCAC CACA GCAC CACA GCAC CACA GCAC CACATGCAC CACA GCAC 16 463539 TCGTTCACC TCGT CACC TCGT CACC TCGT CACC TCGTTCACC TCGT CACC 17 119990 AAAATTGCG AAAATTGCG AAAATTGCG AAAATTGCG AAAA TGCG AAAA TGCG 17 338415 TGAGAAGCC TGAG AGCC TGAG AGCC TGAG AGCC TGAGAAGCC TGAG AGCC 17 449833 ATCAGACAA ATCA AC A ATCA AC A ATCA AC A ATCA AC A ATCA AC A 18 101884 ATTACGGAC ATTA GGAC ATTA GGAC ATTACGGAC ATTA GGAC ATTA GGAC 19 98377 GCTATTGGG GCTA TGGG GCTA TGGG GCTATTGGG GCTA TGGG GCTA TGGG

(110) Table V presents a fingerprint excerpted from the SNP (Single Nucleotide Polymorphism) marker genotype of the entire genome sequences of hybrid strain J15987 and of related cultures. The IUPAC nucleotide and ambiguity codes are used to represent the observed 9-base DNA marker sequences reported above, each of which represents a genotypic marker locus. The identity of each marker locus is specified by the scaffold and SNP position information derived from the H97 V2.0 reference genome sequence published by the U.S. Department of Energy Joint Genome Institute (Morin et al. 2012). It is evident that a composite relationship of the heteroallelic genotype of strain J15987 exists with respect to the homoallelic genotypes of its two parental lines, namely line J11500-s80 and line s-290.

(111) A brief description of the genotype of strain J15987 at further six unlinked marker loci is provided below. Because the J15987 heterokaryon incorporates two sets of chromosomes, there are two allelic copies (two characters or elements of the genotype) at each marker locus. The brief genotype excerpt provided below therefore consists of 12 characters or elements. The brief genotype was prepared by the assignee of record using targeted Polymerase Chain Reactions to amplify genomic regions bracketing the defined markers 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 are well known in the art. From 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. In most cases the sequence was further confirmed by direct inspection of the corresponding whole genome sequence for that culture.

(112) Description of the p1n150-G3-2 Marker:

(113) 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. At present, 9 alleles incorporating at least 30 polymorphic positions have been documented from diverse strains in Sylvan America's breeding collection.

(114) Alleles present in the J15987 pedigree over three generations are alleles 1T and 2, 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):

(115) Allele 1T: C @ 193; insertion of Abr1 transposon of 320 nt @ 206{circumflex over ()}207; T @ 327; C @ 374; G @ 378; G @ 422; C @ 431; G @ 472; etc.

(116) Allele 2: no Abr1 insertion; C @ 193; C @ 327, C @ 374; C @ 378; G @ 422; T @ 431; G @ 472; etc.\

(117) Because of linkage to the MAT locus, which is obligatively heteroallelic in fertile heterokaryons, genotypes of all known and expected heterokaryons at p1n150-G3-2 are also heteroallelic.

(118) The genotype of the J15987 heterokaryon at the p1n150-G3-2 marker locus is 1T/2 (heteroallelic), designating the presence of alleles 1T and 2. Allele 1T was contributed by line s-290. Allele 2 was transmitted from the J11500-s80 line. The 1T/2 genotype distinguishes J15987 from many other heterokaryons, although not from the U1 strain family.

(119) Description of the ITS (=ITS 1+2 Region) Marker:

(120) The ITS segment is part of the nuclear rDNA region which is located on chromosome 9 (Scaffold 10 in JGI H97 V2.0). The rDNA 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 the tandem repeats. Three cassette copies were included on scaffold 10 of the H97 JGI V2.0 assembly, beginning at position 1612110; 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 America's breeding collection.

(121) Alleles present in the J15987 immediate pedigree are alleles I1, I2, and I4, characterized as follows (using the format: nucleotide base character @ alignment position, based on alignment of 9 alleles).

(122) Allele I1: C @ 52; T @ 461; T @ 522; T @ 563; etc.

(123) Allele I2: T @ 52; T @ 461; T @ 522; T @ 563; etc.

(124) Allele 14: C @ 52; A @ 461; C @ 522; C @ 563; etc.

(125) The line J11500-s80 homokaryon has an I4 genotype.

(126) The line s-290 homokaryon has an I2 genotype.

(127) The genotype of the J15987 heterokaryon at the ITS marker locus is I2/I4 (heteroallelic), designating the presence of alleles I2 and I4. This distinguishes J15987 from the U1 strain family, which has an I1/I2 genotype, and from many other strains.

(128) Description of the MFPC-1-ELF Marker:

(129) 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 America's breeding collection.

(130) Allele E1: A @ 77; A @ 232; A @ 309; T @ 334; A @ 390; A @ 400; T @ 446, A @ 481; etc.

(131) Allele E2: G @ 77; A @ 232; G @ 309; T @ 334; G @ 390; G @ 400; C @ 446; G @ 481; etc.

(132) The homokaryon stocks (H97, J10102-s69, line J11500-s80 and line s-290 all have the E1 allele, which is correlated with white cap color.

(133) The Sylvan hybrid heterokaryon stocks J11500 and J15987 are both E1/E1. This homoallelic genotype distinguishes J15987 from the U1-derived commercial cultivar lineage, which has an E1/E2 genotype.

(134) Description of the AN Marker:

(135) 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 (in the H97 genome) to 1700 nt (in 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 America's breeding collection.

(136) Alleles present in the J15987 immediate pedigree are alleles N1 and N5, characterized in part as follows (using the format: nucleotide base character @ alignment position, based on alignment of alleles N1 through N5):

(137) Allele N1: G @ 640; [deletion] @ 844-846; T @ 882; A @ 994, etc.

(138) Allele N2: A @ 640; [deletion] @ 844-846; T @ 882; A @ 994, etc.

(139) Allele N5: A @ 640; ACG @ 844-846; C @ 882; G @ 994, etc.

(140) The line J11500-s80 homokaryon has an N1 genotype.

(141) The line s-290 homokaryon has an N1 genotype.

(142) The N1/N1 genotype of strain J15987 at the AN marker locus distinguishes J15987 from commercial strains U1 and derivatives including A-15, which have an N1/N2 genotype, and also from many other strains.

(143) Description of the AS Marker:

(144) 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 (in the H97 genome) to 1693 nt (in 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 America's breeding collection.

(145) Alleles present in the J15987 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):

(146) Allele SC: T @ 28; GATATC @ 258-263; G @ 275; [insertion]+TTTCTCAGC+[insertion] @ 309-249; C @ 404, etc.

(147) Allele SD: C @ 28; [deletion] @ 258-263; T @ 275; [deletion] @ 309-249; T @ 404, etc.

(148) The line J11500-s80 homokaryon has the SD genotype.

(149) The line s-290 homokaryon has the SC genotype.

(150) The J15987 heterokaryon has the SC/SD genotype.

(151) The SC/SD genotype at the AS marker locus is also shared by commercial strains U1 and A-15. This element of the genotype fingerprint distinguishes J15987 from among many strains other than the U1 strain family.

(152) Description of the FF Marker:

(153) 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. At present, 7 alleles incorporating at least 20 polymorphic positions have been documented from diverse strains in Sylvan America's breeding collection.

(154) Alleles present in the J15987 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):

(155) Allele FF1: CCG @ 48-50

(156) Allele FF2: TTC @ 48-50

(157) The line J11500-s80 homokaryon has an FF1 genotype.

(158) The line s-290 homokaryon has an FF2 genotype.

(159) The genotype of the J15987 heterokaryon at the FF marker locus is FF1/FF2 (heteroallelic). Commercial strains U1 and A15 also share the FF1/FF2 genotype.

(160) The genotype data for the six additional marker loci in standard use as discussed above are provided in Table VI.

(161) TABLE-US-00006 TABLE VI Genotypes of relevant cultures at six standard marker loci Markers: Strain p1n150/Mat ITS MFPC-ELF AN AS FF U1 1T/2 I1/I2 E1/E2 N1/N2 SC/SD FF1/FF2 H97 1T I1 E1 N1 SD FF1 J10102-s69 2 I4 E1 N5 SC FF1 J11500 1T/2 I1/I4 E1/E1 N1/N5 SC/CD FF1/FF1 J11500-s80 2 I4 E1 N1 SD FF1 Line s-290 1T I2 E1 N1 SC FF2 J15987 1T/2 I2/I4 E1/E1 N1/N1 SC/SD FF1/FF2

(162) One use of the culture of strain J15987 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 J15987 into offspring and derived or descended cultures including dormant and germinating spores and protoplasts. Additional uses also exist as noted above.

(163) 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 strain J15987, to grow together in close proximity, preferably on sterile media, until anastomosis (i.e., hyphal or cell fusion) occurs. In a successful mating, 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 J15987 mushroom that may be used to transmit genetic material of strain J15987 into new cultures.

(164) Methods for obtaining, manipulating, and mating cultures of the present invention, for producing offspring, inoculum, products, and crops of the current invention, for using a strain rotation program to improve mushroom farm hygiene, and for obtaining the genotypic fingerprint of mushroom cultures, are described hereinabove and are also well known to practitioners of the art.

(165) Although the invention has been described in terms of particular embodiments in this application, one of ordinary skill in the art, in light of the teachings herein, can generate additional embodiments and modifications without departing from the spirit of, or exceeding the scope of, the claimed invention. Accordingly, it is understood that the descriptions herein are proffered only to facilitate comprehension of the invention and should not be construed to limit the scope thereof.