MUSHROOM LINE J10102-S69, HYBRID MUSHROOM STRAIN J11500, DESCENDANTS THEREOF, AND METHODS AND USES THEREFOR

TECHNICAL FIELD

[0001] 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.

BACKGROUND OF THE INVENTION

[0002] The edible mushroom Agaricus bisporus (Lange) Imbach var. bisporus, a microorganism belonging to the basidiomycete fungi, is widely cultivated around the world. In Europe and North America, it is the most widely cultivated mushroom species. The value of the annual Agaricus bisporus mushroom crop in the United States was about $1 , 110,000,000 in 2012-2013, according to the National Agricultural Statistics Service, Agricultural Statistics Board, U.S. Department of Agriculture (August 20, 2013). Accordingly, development of novel hybrid mushroom strains or lines of this mushroom fungus is seen as highly desirable to the cultivated mushroom industry, particularly if those novel strains or lines can be developed to provide various desirable traits within a single strain, culture, hybrid or line. Most cultures and strains of A. bisporus are unsuitable for commercial cultivation, and the development of successful new hybrid strains is challenging and only rarely results in a useful new strain. The problem facing mushroom strain developers and l commercial mushroom spawn producers is therefore to develop and identify the rare useful lines, strains and cultures having commercial value.

[0003] Thus, various entities within the mushroom industry, including Sylvan America, Inc., 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. There also exist improvable properties of the mushroom product that increase demand in the distribution chain, and thus sales volume and/or sales price, such as improved visual appeal (more desirable coloration, shape, size, or surface texture), improved or distinct flavor characteristics, improved keeping qualities (longer persistence of desirable visual attributes), etc. Still other improvements may enhance the suitability of the mushroom crop for mechanical harvesting, canning, and/or food processing. Thus there are many characteristics by which a novel strain might be judged as superior in a particular production facility or sales market, or in the industry regionally or globally. [0004] All of these characteristics can be assessed using techniques that are well known in the art. Novel strains are most preferably and successfully developed from unique hybridizations between homokaryotic lines, including novel lines. Thus, in the cultivated mushroom industry with its diverse, dynamic and evolving raw materials characteristics, availabilities and costs, technical capabilities, economic framework including labor availability and costs, and consumer and market preferences, the need continues to exist for new hybrid strains, and for new lines that can be used to produce new hybrid strains, of Agaricus bisporus that provide for flexibility of operations, for improved characteristics for producer profitability and for improved mushroom products over other previous strains of Agaricus bisporus.

[0005] There is also a need for commercially acceptable A. bisporus strains with different genotypes, relative to the U1 derived lineage group, for two reasons. First, strains incompatible with strains of the U1 derived lineage group are known to retard the spread of viral diseases between cultivated strains. The incompatibility phenotype can be assessed using techniques that are well known in the art and are detailed below. Second, it is well understood that when an agricultural crop industry relies extensively on a single genetic lineage (i.e., creates a commercial monoculture as now exists for the white-capped U1 lineage of A. bisporus), there is an increased risk of unpredictable, catastrophic crop failure on a facility-wide or even industry-wide scale, for example upon the emergence of a new pathogen. Therefore from a risk management and food security perspective, it is highly desirable to simultaneously provide both genetic diversification and commercially acceptable performance and crop characteristics. The use of novel lines that incorporate DNA from non-cultivar stocks provides important genetic diversification of the strain pool used to produce crops of cultivated A. bisporus mushrooms. [0006] 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.

[0007] One use of such cultures is to produce mushrooms. 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. In a further about 13 to about 18 days after casing, 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. Commercially, two or three flushes of mushrooms are produced and harvested before the compost is removed and replaced in the cropping facility.

[0008] Seventy to ninety-five percent of the Agaricus mushrooms cultivated in the United States, Europe, and elsewhere have a white pileus color, in accordance with consumer preferences. Market requirements for white mushrooms in the USA, Europe and elsewhere are narrow and precise for many observable phenotypic traits such as size, shape, color, color retention, firmness, and related traits such as shelf life. Consequently, genetically different strains of commercially successful white Agaricus bisporus mushrooms are not easily differentiated on the basis of appearance of the mushrooms, which must conform to the relatively strict market requirements. 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.

[0009] Circa 1980, the first two white hybrid strains of A. bisporus, developed by a laboratory at Horst, the Netherlands, were introduced into commercial cultivation. These two "Horst" strains, called U1 and U3, are closely related hybrid strains produced by matings between two pre-existing white cultivated strains, as per M. Imbernon et al., Mycologia, 88(5), 749-761 (1996). The two parents of U1 and U3 are commercial strains belonging to two longstanding categorical types of strains known as the 'smooth-white' (SW) strains and the Off-white' (OW) strains. The original homokaryons (or 'lines') obtained from the SW and OW strains, and used in the hybridization that produced the U1 strain, were designated H39 and H97 respectively; these cultures may no longer exist (A. Sonnenberg, pers. comm.). However, a number of laboratories have deheterokaryotized the U1 strain and obtained neohaplont cultures incorporating one or the other nuclear type corresponding to those contributed by H39 or H97, as well as the mitochondrial type of U1. These two types of neohaplonts of U1 are referred to categorically as the SWNC and OWNC lines or homokaryons, respectively. An OWNC line designated Ή97' 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).

[0010] 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.

[0011] 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,

[0012] 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. bisporus; empirically, very little heteroallelism (analogous to heterozygosity) is lost among heterokaryotic offspring of a heterokaryotic strain. Consequently, parental and offspring heterokaryotic genotypes and phenotypes tend to closely resemble each other, as noted above; for this reason, essential derivation, e.g., the production of Essentially Derived Varieties (EDVs), is a familiar strain development technique among commercial mushroom spawn producers. In statutory frameworks, an EDV is subject to the rights and protections granted to the rightsholders of the initial strain from which the EDV is derived. For all of these reasons a need exists to develop novel hybrid strains incorporating novel combinations of genetic material, i.e., novel compositions of matter, from more than one parental strain, and which are consequently not EDVs. SUMMARY OF THE INVENTION

[0013] The advantages of the present invention over existing prior art relating to Agaricus bisporus mushrooms and cultures, which shall become apparent from the description which follows, are accomplished by the invention as hereinafter described and claimed.

[0014] 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.

[0015] More specifically, 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. Patent and Trademark Office, and all deposit requirements under the Budapest Treaty. The NRRL Accession No. is 50893. 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.

[0016] 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. In one embodiment, it will be appreciated that 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. The date of deposit of line OWNC was January 15, 2014. This 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 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. [0017] 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. 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.

[0018] 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. In one embodiment, 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.

[0019] 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. In one embodiment, an Essentially Derived Variety of the culture of line J10102-s69 is produced. In other embodiments, 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.

[0020] 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. If a sufficient number of rare markers are present in an initial strain or line, it will be possible to identify descent from an initial strain or line after several outbred generations without undue experimentation. In a hypothetical example, 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. Based on Sylvan America's analyses, 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. By using multiple independent markers, ancestors of a strain can be identified with a very high probability of success and with high confidence.

[0021] In the embodiment described above, 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.

[0022] One trait of biological and commercial interest is heterokaryon incompatibility. The genetics of these self/non-self recognition systems are not well elucidated in basidiomycete fungi such as Agaricus, but are known in other genera to involve multiple alleles at multiple independent loci. Differences in the presumed genotype at the incompatibility loci prevent successful anastomoses and cytoplasmic continuity among physical mixtures of two or more heterokaryons. One consequence of such antagonistic responses is a retardation of growth and development, and a reduction of crop yield; this sort of partial crop failure is well known and evident to the experienced grower. Another consequence of heterokaryon incompatibility is restriction on the opportunity for endocellular viruses to move freely throughout or among mycelial networks. 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. In the most effective implementation of the virus-breaking method, 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.

[0023] In one or more embodiments, 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.

[0024] 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. In one embodiment 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.

[0025] In one or more embodiments, 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. In other embodiments, 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.

[0026] In one or more embodiments, a cell may be obtained from any of the cultures above or any of the methods for producing the cultures as noted above. In one or more embodiments, 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. Again, it will be appreciated that in other embodiments, characters at at least three, four, five or six marker loci may be selected as discussed above.

[0027] In other embodiments, a spore may comprise the cells above. In other embodiments, the hybrid culture above may be further defined as having a genome including a single locus trait conversion. In further embodiments, the locus above may be selected from the group consisting of a dominant allele and a recessive allele. In one or more other embodiments, 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.

[0028] 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. That is, 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. Upon completion of step (4), 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). In one embodiment, 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. In other embodiments, 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.

[0029] 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. In one embodiment, 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.

[0030] Yet one or more other aspects of the present invention may be provided by a method for developing a second culture in a mushroom strain development program. Such a method 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.

[0031] 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. Finally, 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.

[0032] 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. Further, 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.

[0033] 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. Thus, 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. 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.

[0034] With respect to 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.

[0035] Development of novel hybrid varieties via heteromixis comprises the controlled association and mating of two compatible cultures to obtain a novel heterokaryon culture. Homokaryons (= 'lines') are the preferred starting cultures for making matings as they have maximal ability to anastomose and achieve plasmogamy with other cultures. Heterokaryons may also be confronted but with commercially unreasonably low probabilities of a mating resulting in successful formation of a novel heterokaryon. Compatibility is determined by the genotype at the MAT locus; two homokaryons with the same MAT allele cannot establish a heterokaryon after anastomosis. In a defined mating program, homokaryotic lines are obtained and are associated in predetermined pairwise combinations. In one method, 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.

[0036] In contrast, EDVs are most often derived directly (otherwise predominantly) from a single initial culture (e.g., strain); all such derivations produce EDVs. There is no universally accepted definition of an EDV; one example of a definition applicable to plant varieties is provided by the US Plant Variety Protection Act (revised edition, February 2006). The definition employed herein is congruent with the term as it is widely understood. '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. DNA-mediated transformation of A. bisporus has been reported by Velcko, A. J. Jr., Kerrigan, R. W., MacDonald, L. A., Wach, M. P., Schlagnhaufer, C, and Romaine, C. P. 2004, Expression of novel genes in Agaricus bisporus using an Agro bacterium-mediated transformation technique. Mush. Sci. 16: 591-597, and references therein. Transformation may introduce a single new gene or allele into the genome of an initial culture.

[0037] Although in statutory frameworks 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. x% in the case of strains modified by DNA- mediated transformation, to 90-100% in the case of single or multiple spore selections or some mutagenesis, .including instances where no heteroallelism is lost during meiotic internuclear reassociation of homologous chromosomes, to an average of from about 75% to about 85% in the case of sibling-offspring matings (= selfing), to about 75% on average in a first generation of back-mating, increasingly approaching 99.x% with each successive generation of back-mating. 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.

[0038] Repeated mating back to the initial culture to introgress a single trait into the genetic background of an initial culture is called introgressive trait conversion, and according to accepted definitions of EDVs, also produces an EDV of the initial culture. In a hypothetical example, in the first successive repetition of this process a resultant strain of this generation will have on average about 75% of the DNA of the initial strain while about 25% of the DNA will have been contributed by a second strain or line; as this process is repeated the DNA representation of the initial strain will increase, approaching 97% on average after 3 further successive repetitions. There is no universally accepted quantitative threshold for the proportion of DNA contributed by an initial culture in an EDV of an initial culture; from the foregoing it is apparent that approximately 75-100% genotype identity with an initial culture is indicative of status as an EDV of an initial culture, with 75%, being a minimum threshold. It is also established that an EDV of an EDV is also an EDV of an initial strain. Finally, because Agaricus bisporus alternates generations between heterokaryotic strains and homokaryotic lines, the criteria for essential derivation apply equally to cultures of both strains and lines.

[0039] As noted above, hybrid mushroom strain producers are always looking for hybrid strains that allow growers to produce crops of mushrooms successfully and profitably. In the case of strain J1 1500 and strains derived or descended from that strain, 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).

[0040] In addition, and as noted above, 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. [0041] It will be appreciated that, in one or more embodiments, 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.

[0042] Further, in other embodiments, 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. It will be appreciated that mushroom pieces refer to stems, pilei, and other larger portions of the mushroom itself. In other embodiments, 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. In other embodiments, a mushroom may be produced by growing a crop of mushrooms from any of the cultures above. In other embodiments, a mushroom may be produced by growing a crop of mushrooms from the F1 hybrid mushroom culture above. In still other embodiments, an Essentially Derived Variety of the F1 hybrid mushroom culture above is produced. [0043] Thus, in one or more embodiments, 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. Such 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. In one or more embodiments, 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. In other embodiments, 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.

[0044] In other embodiments, a cell or a culture including the cell, is produced by the method(s) above. Thus, 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.

[0045] Finally, another aspect of the present invention may be accomplished by various methods that use any of the culture above for various uses. In one embodiment, the method further includes producing or otherwise growing a crop of edible mushrooms by carrying out the steps described hereinabove. In another embodiment, the method may include the cultures above in crop rotation to reduce pathogen pressure and pathogen reservoirs in mushroom growing facilities as described hereinabove. In yet another embodiment, the method includes using the cultures above to produce offspring as described hereinabove.

DETAILED DESCRIPTION OF THE INVENTION

[0046] 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.

[0047] 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.

[0048] Amphithallism: A reproductive syndrome in which heteromixis and intramixis are both active.

[0049] Anastomosis: Fusion of two or more hyphae that achieves cytoplasmic continuity. [0050] Basidiomycete: A monophyletic group of fungi producing meiospores on basidia; a member of a corresponding subdivision of Fungi such as the Basidiomycetales or Basidiomycotina.

[0051] Basidium: The meiosporangial cell, in which karyogamy and meiosis occur, and upon which the basidiospores are formed.

[0052] 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.

[0053] Breeding: Development of strains, lines or varieties using methods that emphasize sexual mating; see Descent.

[0054] Cap: Pileus; part of the mushroom, the gill-bearing structure.

[0055] 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.

[0056] Carrier substrate: A medium having both nutritional and physical properties suitable for achieving both growth and dispersal of a culture.

[0057] 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.

[0058] 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.

[0059] Cloning: Somatic propagation without selection. [0060] 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).

[0061] Compatibility: See heterokaryon compatibility.

[0062] 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.

[0063] Derivation: Development of a strain or culture from a single initial strain, or predominantly from a single initial strain, in contrast to descent via sexual mating between two parental strains; see Essentially Derived Variety (EDV).

[0064] Derived lineage group: An initial strain or variety and the set of EDVs derived from that single initial strain or variety.

[0065] 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.

[0066] Diploid: Having two haploid chromosomal complements within a single nuclear envelope.

[0067] 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.

[0068] Essentially Derived Variety (EDV): (Note: 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 having 75% to 99.99999% genetic identity with an initial strain or variety, or to 100% in a heterokaryon with internuclear reassociation of chromosomes. In general, 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. In the art of mushroom strain development, 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).

[0069] 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'. [0070] 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.

[0071] Fungus: An organism classified as a member of the Kingdom Fungi.

[0072] 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.

[0073] Genotypic fingerprint: A description of the genotype at a defined set of marker loci; the known genotype.

[0074] Gill: Lamella; part of the mushroom, the hymenophore- and basidium- bearing structure.

[0075] Haploid: Having only a single complement of nuclear chromosomes; see homokaryon.

[0076] Heteroallelic: Having two different alleles at a locus; analogous to heterozygous.

[0077] Heteroallelism: Differences between homologous chromosomes in a heterokaryotic genotype; analogous to heterozygosity.

[0078] 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. [0079] Heterokaryon compatibility: The absence of antagonism observed during physical proximity or contact between two heterokaryons that are not genetically identical; see Heterokaryon Incompatibility.

[0080] 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.

[0081] Heterokaryotic: Having the character of a heterokaryon.

[0082] Heteromixis: Life cycle involving mating between two different non-sibling haploid individuals or gametes; outbreeding.

[0083] 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.

[0084] 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.

[0085] Homokaryotic: Having the character of a homokaryon; haploid.

[0086] Hybrid: Of biparental origin, usually applied to heterokaryotic strains and cultures produced in controlled matings.

[0087] 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.

[0088] Hyphae: Threadlike elements of mycelium, composed of cell-like compartments or 'cells'.

[0089] Inbreeding: Matings that include sibling-line matings, back-matings to parent lines or strains, and intramixis; reproduction involving parents that are genetically related.

[0090] Incompatibility: See heterokaryon incompatibility.

[0091] 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.

[0092] Intramixis: A uniparental sexual life cycle involving formation of a complementary 'mated' pair of postmeiotic nuclei within the basidium or individual spore.

[0093] 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.

[0094] Lamella: see 'gill'.

[0095] 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.

[0096] 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. [0097] Locus: A defined contiguous part of the genome, homologous although often varying among different genotypes; plural: loci.

[0098] Marker assisted selection: Using linked genetic markers including molecular markers to track trait-determining loci of interest among offspring and through pedigrees.

[0099] MAT: The mating-type locus, which determines sexual compatibility and the heterokaryotic state.

[0100] Mating: The sexual union of two cultures via anastomosis and plasmogamy; methods of obtaining matings between mushroom cultures are well known in the art.

[0101] Mycelium: The vegetative body or thallus of the mushroom organism, comprised of threadlike hyphae.

[0102] Mushroom: The reproductive structure of an agaric fungus; an agaric; a cultivated food product of the same name.

[0103] Neohaplont: A haploid culture or line obtained by physically deheterokaryotizing (reducing to haploid components) a heterokaryon; a somatically obtained homokaryon.

[0104] 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.

[0105] Outbreeding: Mating among unrelated or distantly related individuals.

[0106] 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. [0107] 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.

[0108] Pedigree-assisted breeding: The use of genealogical information to identify desirable combinations of lines in controlled mating programs.

[0109] Phenotype: Observable characteristics of a strain or line as expressed and manifested in an environment.

[0110] Plasmogamy: Establishment, via anastomosis, of cytoplasmic continuity leading to the formation of a sexual heterokaryon.

[0111] Progenitor: Ancestor, including parent (the direct progenitor).

[0112] 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.

[0113] Selfing: Mating among sibling lines; also intramixis.

[0114] Somatic: Of the vegetative mycelium.

[0115] 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.

[0116] Spore: Part of the mushroom, the reproductive propagule.

[0117] Stem: Stipe; part of the mushroom, the cap-supporting structure.

[0118] 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.

[0119] Stipe: see 'stem'.

[0120] Strain: A heterokaryon with defined characteristics or a specific identity or ancestry; equivalent to a variety.

[0121] 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.

[0122] Tissue culture: A de-differentiated vegetative mycelium obtained from a differentiated tissue of the mushroom.

[0123] 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'.

[0124] 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.

[0125] 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.

[0126] Yield: The net fresh weight of the harvest crop, normally expressed in pounds per square foot. [0127] 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.

[0128] 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, 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.

[0129] Now, with respect to the invention and as noted hereinabove, 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.

[0130] 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. 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.

[0131] 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. Among the remaining 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.

[0132] 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.

[0133] In light of the usefulness of line J10102-s69 to produce hybrid cultures, it is at least a further embodiment of the invention to provide an F1 hybrid Agaricus bisporus culture by mating the Agaricus bisporus culture of line J10102-s69 with a different Agaricus bisporus culture of another line. Thus, any cultures derived or descended from line J10102-s69 may be a part of the invention.

[0134] In one embodiment, 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.

[0135] It will be appreciated that 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. Thus, the present invention further relates to methods of making and using the strain J1 1500 and EDVs of the strain J1 1500.

[0136] 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. [0137] 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. In most eukaryotes, a parent is conventionally considered to be either diploid or heterokaryotic. The haploid 'generation' is often, but not always, termed a gamete (e.g., pollen, sperm). In fungi, which are microorganisms, 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). Herein, 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'.

[0138] If one parental line carries allele 'p' at a particular locus, and the other parental line carries allele 'q', the F1 hybrid resulting from a mating of these two lines will carry both alleles, and the genotype can be represented as 'p/q' (or 'pq', or 'p+q'). Sequence-characterized markers are co-dominant and both alleles will be evident when an appropriate sequencing protocol is carried out on cellular DNA of the hybrid. The profile of line J10102-s69 can therefore be used to identify hybrids comprising line J10102-s69 as a parent line, since such hybrids will comprise two sets of alleles, one of which sets will be from, and match that of, line J10102-s69. 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.

[0139] Means of obtaining genetic marker profiles using diverse techniques including whole genome sequencing are well known in the art. For the purpose of providing a detailed embodiment of this invention, the whole genomic sequence of strain J11500 and of the cultures of its parent lines, including J10102-s69 and OWNC, and of selected EDVs of J1 1500 have been obtained and provided 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 -80C, and were lyophilized until dry. Cap tissue was obtained from mushrooms produced by cultures of the heterokaryotic J1 1500 (and EDV) strains, and was frozen and lyophilized. DNA was extracted 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 lllumina MiSeq technology. Assemblies of the sequencing reads into genomic sequence using the public-domain reference genome sequence of H97 were performed by Sylvan America, Inc. Consequently about 93% to about 95% of the entire genotype of line J10102-s69, of strain J11500 and of three EDVs of strain J1 1500 are known to Sylvan America, Inc with certainty. 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.

[0140]

TABLE I

Scaffold Position of SNP [H97 Culture:

V2.0 ref. coords.] J10102-S69 OWNC J11500 J11500-ms2

1 99995 CTACGTTGA CTACATTGA CTACrTTGA CTACrTTGA

1 349966 AAGGCGGTT AAGGTGGTT AAGGyGGTT AAGGyGGTT

1 600059 TTTT TTT4 1 1 1 1 1 1 1 l -C 1 1 1 \ yJJ[-/A] 1 1 1 \ y [-/A]

1 850014 C7TTTTCGC CCI 1 1 I CAC Cy l 1 1 I CrC Cyl 1 1 I CrC

1 1099971 GTCGGCACC GTCGACACC GTCGrCACC GTCGrCACC

1 1350278 GGAGG7TCG GGAGAGTCG GGAGr/fTCG GGAGr/fTCG

1 1599956 AATAGGCGC AATAAGCGC AATArGCGC AATArGCGC

1 1850032 CGAGCAATT CGAGTAATT CGAGyAATT CGAGyAATT

1 2119049 ACAACTCAA ACAATCCAA ACAAyyCAA ACAAyyCAA

1 2400243 ACTTGATGA ACTTCATGA ACTTsATGA ACTTsATGA

1 2612870 AATAAGAGT AATAGGAGT AATArGAGT AATArGAGT

1 2858975 GCCGCTCTT GCCGTTCTT GCCGyTCTT GCCGyTCTT

1 2804522 GAAGGGGAC GAAGACGAC GAAGrsGAC GAAGrsGAC

1 3047987 AAGG4GGGG AAGGGGGGG AAGGrGGGG AAGGrGGGG

1 3164166 ATAATCGGG ATAAGGGGG ATAA/rsGGG ATAA/rsGGG

1 3256057 TATCCGTTT TATCTGTTT TATCyGTTT TATCyGTTT

2 101820 ATTACGGAT ATTAAAGAT ATTAmrGAT ATTAmrGAT

2 350156 TCGG4GGTG TCGGGGGTG TCGGrGGTG TCGGrGGTG

2 600112 ATGTGTACG ATGTATACG ATGTrTACG ATGTrTACG

2 850338 TGGTTCTAA TGGTGCTAA TGGT/fCTAA TGGT/fCTAA

2 1099413 CCTGGCTCA CCTGACTCA CCTGrCTCA CCTGrCTCA

2 1349512 CTCAACAGT CTCAGCAGT CTCArCAGT CTCArCAGT

2 1600085 CACA7TGCC CACAATGCC CACAwTGCC CACAwTGCC

2 1901773 ACTC4AATT ACTCGAATT ACTCrAATT ACTCrAATT

2 2150201 GTCG4AGGT GTCGTAGGT GTCGwAGGT GTCGwAGGT

2 2400281 TCAACACTC TCAAAACCC TCAAmACyC TCAAmACyC

2 2650136 ATAAATCCT ATAATTCCT ATAAwTCCT ATAAwTCCT

2 2903593 ACTATAGGA ACTAAAAGA ACTAwArGA ACTAwArGA

2 3048019 GTCC4CTGC GTCCGCTGC GTCCrCTGC GTCCrCTGC

3 65650 GGCGGTTTT GGCGC I 1 1 1 GGCGs l 1 1 1 GGCGs l 1 1 1

3 119281 TTTACACTC TTTATACTC TTTAyACTC TTTAyACTC

3 249570 GTATTATGT GTATTATGT GTATTATGT GTATTATGT

3 750000 GTCCGGCCA GTCCGGCCA GTCCGGCCA GTCCGGCCA

3 1250000 1 1 1 1 TCCGG 1 1 1 1 TCCGG 1 1 1 1 TCCGG TTTT TCCGG

3 1750000 ACGC TGAC ACGCCTGAC ACGCCTGAC ACGCCTGAC

3 2250000 CGTGGCGAT CGTGGCGAT CGTGGCGAT CGTGGCGAT 3 2520748 TAATTCCAC TAATGCCAC TAAT/fCCAC TAAT/fCCAC

4 100004 GAG AATGA GAGTGATAA GAGTrATrA GAGTrATrA

4 340893 AGG4GGTAC AGGTGGTAT AGGrGGTAy AGGrGGTAy

4 598147 GATC4ACAG GATCGACAG GATCrACAG GATCrACAG

4 852119 CGAACA TC CGAATATTC CGAAyAyTC CGAAyAyTC

4 1100085 GATG4CGAA GATGCCGAA GATGmCGAA GATGmCGAA

4 1350536 CGAAACCGG CGAACTCGG CGAAmyCGG CGAAmyCGG

4 1599885 GATAATTGC GATACTTGC GATAmTTGC GATAmTTGC

4 1850288 ATTC4CGTA ATTCGTGTA ATTCryGTA ATTCryGTA

4 2100356 TCAGGGACC TCAGAGACC TCAGrGACC TCAGrGACC

4 2284257 TCTG4ACTG TCTGGACTG TCTGrACTG TCTGrACTG

5 100211 TCCTCGAAT TCCTTGAAT TCCTyGAAT TCCTyGAAT

5 350872 GGCGCGCCC GGCGTGCCC GGCGyGCCC GGCGyGCCC

5 599922 CGTCGTTCA CGTCATTCA CGTCrTTCA CGTCrTTCA

5 851262 TAATCGTCT TAATTCTCT TAATysTCT TAATysTCT

5 1099776 ACATCGACA ACATTGACA ACATyGACA ACATyGACA

5 1352539 TTGTTGTCC TTGTGATCC TTGT/rrTCC TTGT/rrTCC

5 1599904 AACTCCCTT AACTTCCTT AACTyCCTT AACTyCCTT

5 1851458 AAATTCTCC AAATAATCC AAATwmTCC AAATwmTCC

5 2100025 CCCTCAGTC CCCTTAGTC CCCTyAGTC CCCTyAGTC

5 2278878 GGTC4AAAA GGTCGAAAA GGTCrAAAA GGTCrAAAA

6 106294 GCCACCTC4 GCCATCTCG GCCAyCTCr GCCAyCTCr

6 350337 CATTCGGTT CATTTGGTT CATTyGGTT CATTyGGTT

6 600047 GGAGTATTT GGAGCATTT GGAGyATTT GGAGyATTT

6 849990 AGTTTAGGA AGTTCAGGA AGTTyAGGA AGTTyAGGA

6 1098535 CAAAAATTG CAAAGATTG CAAArATTG CAAArATTG

6 1349453 TGTDMTAG TGTCGGTAG TGTCrrTAG TGTCrrTAG

6 1600000 AAACCTGGA AAACCTGGA AAACCTGGA AAACCTGGA

6 1764645 AACC4GATT AACCGGATT AACCrGATT AACCrGATT

6 2000087 GATT TGCG GA I 1 1 I GCG GATTyTGCG GATTyTGCG

6 2252662 GGGTCGGTA GGGTTGGTA GGGTyGGTA GGGTyGGTA

7 100284 GAAACTCAG GAAATTCAG GAAAyTCAG GAAAyTCAG

7 350044 ATATCCTTT ATATTCTTT ATATyCTTT ATATyCTTT

7 600111 CAATCATTA CAATTATTA CAATyATTA CAATyATTA

7 850516 TGAC4CATA TGACGCATA TGACrCATA TGACrCATA

7 1100248 TCAC4GAAG TCACGGAAG TCACrGAAG TCACrGAAG

7 1350089 CTTTCCCCC C I 1 1 I CCCC CTTTyCCCC CTTTyCCCC

7 1605047 ATACGTG4C ATACTTGGC ATAC/fTGrC ATAC/fTGrC

7 1850000 GAGATACT GAGATACT GAGATACT GAGATACT

7 1898793 TCCGTATGA TCCGCATAA TCCGyATrA TCCGyATrA

7 1991505 TCTA/V»GTT TCTACGGTT TCTAmrGTT TCTAmrGTT

8 350000 ATTG4CGCG ATTG4CGCG ATTG4CGCG ATTG4CGCG

8 600000 CATTGACGG CATTGACGG CATTGACGG CATTGACGG

8 1100000 CATACGATC CATACGATC CATACGATC CATACGATC

8 1350000 AGCTTAACA AGCTTAACA AGCTTAACA AGCTTAACA

8 1600100 CTGAACCCT CTGAACCCT CTGAACCCT CTGAACCCT

9 100105 CTCAGCCGA CTCAACCGA CTCArCCGA CTCArCCGA

9 352455 AGTCTCCCA AGTCCTCCA AGTCyyCCA AGTCyyCCA 9 599950 TGGTGTCCC TGGTATCCC TGGTrTCCC TGGTrTCCC

9 1010845 GGGT4GTGA GGGTGGTGA GGGTrGTGA GGGTrGTGA

9 1244202 GATGGAGAT GATGAAGAT GATGrAGAT GATGrAGAT

9 1504476 TAC ACC TACTGTACC TACTrTACC TACTrTACC

9 1656962 TATCCACTG TATCTACTG TATCyACTG TATCyACTG

10 100438 AATTCATTT AATTAATTT AATTmATTT AATTmATTT

10 350030 GCGG7TCAA GCGGCTCAA GCGG VTCAA GCGGVTCAA

10 600032 TTACGCTGG TTACACTGG TTACrCTGG TTACrCTGG

10 850000 TCGGTCGGA TCGGTCGGA TCGGTCGGA TCGGTCGGA

10 860249 CCGCGAAATT CCGCAAATT CCGCrAAATT CCGCrAAATT

10 1109960 AGGAGATGA AGGAAATGA AGGArATGA AGGArATGA

10 1303902 TGAT TACT TGATTTACT TGATyTACT TGATyTACT

10 1490452 AATCTGATG AATCAGATG AATCwGATG AATCwGATG

11 100000 TATT TTAG TATTCTTAG TATTCTTAG TATTCTTAG

11 350000 GTCAGCAAG GTCAGCAAG GTCAGCAAG GTCAGCAAG

11 600000 ATGGGCGCG ATGGGCGCG ATGGGCGCG ATGGGCGCG

11 850000 CTTCCCCAT CTTCCCCAT CTTCCCCAT CTTCCCCAT

11 1100000 TTAC4GTTG TTAC4GTTG TTAC4GTTG TTAC4GTTG

11 124000 AGCC4AGTA AGCC4AGTA AGCC4AGTA AGCC4AGTA

12 100000 CCTT TAGT CCTTCTAGT CCTTCTAGT CCTTCTAGT

12 1000000 CGAGGAGGA CGAGGAGGA CGAGGAGGA CGAGGAGGA

13 100697 ACG ATTTA ACGTCTTTA ACGTmTTTA ACGTmTTTA

13 370521 TTTGTGTCA TTTGAGTCA TTTGwGTCA TTTGwGTCA

13 604345 CTTCCGCAT CTTCAGCAT CTTCmGCAT CTTCmGCAT

13 850249 GG7TGGTGA GGCTAGTAA GGyTrGTrA GGyTrGTrA

14 113109 AGGGGAATA AGGGAAATA AGGGrAATA AGGGrAATA

14 372086 CGAT7C7TT CGATCCCTT CGATyCyTT CGATyCyTT

14 725684 ATGAATTTG ATGAGTTCG ATGArTTyG ATGArTTyG

15 150013 GTGG4CCGT GTGGCCCGT GTGGmCCGT GTGGmCCGT

15 449866 GAAT TCGG GAATTTCGG GAATyTCGG GAATyTCGG

16 208609 CACACGCAC CACATGCAC CACAyGCAC CACAyGCAC

16 400000 CCTCGGATT CCTCGGATT CCTCGGATT CCTCGGATT

17 120000 TATT TTCA TATTCTTCA TATTCTTCA TATTCTTCA

17 338415 TGAGGAGCC TGAGAAGCC TGAGrAGCC TGAGrAGCC

17 449833 ATCAAACTA ATCAGACAA ATCArACu A ATCArACwA

18 101884 ATTATGGAC ATTACGGAC ATTAyGGAC ATTAyGGAC

19 98377 GCTA TGGG GCTATTGGG GCTACTGGG GCTACTGGG

[0141] 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. 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).

[0142] That is, it will be appreciated that 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). As known in the art, any genetic marker or marker locus in the A. bisporus genome may be identified by specifying the positional information from the H97 reference sequence. For example, the first marker listed in Table I occurs at 1 :101993 (i.e., scaffold 1 : position 101993). As a convenience only, 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. [0143] It is evident that a composite relationship of the heteroallelic genotype of strain J1 1500 exists with respect to the homoallelic genotypes of its two parental lines, namely line J10102-s69 and line OWNC, and further that the two parental lines are very distinct, genotypically. It is further evident that the heterokaryon genotype of the example EDV J11500-ms2 matches that of its initial strain, J1 1500. It will be appreciated that the use of J10102-s69 in conjunction with line OWNC to provide strain J1 1500 is but one example of the F1 hybrid generation, it being noted that J10102-s69 has been used in at least 71 matings with other lines of Agaricus bisporus to produce other F1 hybrids.

[0144] 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.

[0145]

TABLE II

Alleles at 6 marker loci, for lines J10102-s69, OWNC, SWNC, and strain J11500

Marker: ITS Dl n150 MFPC-1-ELF AN AS FF

Line/strain

J10102-S69 I4 2 E1 N5 SC FF1

OWNC 11 1T E1 N 1 SD FF1

SWNC I2 2 E2 N2 SC FF2

J1 1500 11/14 1T/2 E1/E1 N1/N5 SC/SD FF1/FF1

[0146] 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. [0147] 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. Because the J11500 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 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.

Description of the p1 n150-G3-2 marker:

[0148] 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. [0149] 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) :

[0150] Allele 1T: 'C @ 193; insertion of Abr1 transposon of 320 nt @ 206 ^Λ 207; T @ 327;'C @ 374; 'G' @ 378; 'G' @ 422; 'C @ 431 ; 'G' @ 472; etc.

[0151] Allele 2: no Abr1 insertion; 'C @ 193; 'C @ 327, 'C @ 374; 'C @ 378; 'G' @ 422; T @ 431 ; 'G' @ 472; etc.

[0152] Allele 3: no Abr1 insertion; 'C @ 193; T @ 327, 'G' @ 374; 'C @ 378; 'G' @ 422; T @ 431 ; Ά' @ 472; etc.

[0153] Allele 4: no Abr1 insertion; 'C @ 193; T @ 327, 'C @ 374; 'C @ 378; Ά' @ 422; T @ 431 ; 'G' @ 472; etc.

[0154] Allele 9: no Abr1 insertion; 'G' @ 193; 'C @ 327, 'C @ 374; 'C @ 378; 'G' @ 422; T @ 431 ; 'G' @ 472; etc.

[0155] Because of linkage to the MAT locus, which is obligately heteroallelic in fertile heterokaryons, genotypes of all known and expected heterokaryons at p1 n150-G3-2 are also heteroallelic.

[0156] The J 10102 heterokaryon has an '1T/2' heteroallelic genotype.

[0157] The U1 heterokaryon has an '1T/2' heteroallelic genotype.

[0158] 'Off-White' heterokaryons such as Somycel 76 have a '1T/3' heteroallelic genotype.

[0159] 'Smooth- White' heterokaryons such as Somycel 53 have a '2/3' heteroallelic genotype.

[0160] The J9277 heterokaryon has a '1T/4' heteroallelic genotype.

[0161] 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.

Description of the ITS (= ITS 1 +2 region) marker:

[0162] 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.

[0163] 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).

[0164] Allele 11 : 'C @ 52; T @ 461 ; T @ 522; T @ 563; etc.

[0165] Allele I2: T @ 52; T @ 461 ; T @ 522; T @ 563; etc.

[0166] Allele I4: 'C @ 52; Ά' @ 461 ; 'C @ 522; 'C @ 563; etc.

[0167] The J10102 heterokaryon has an '11/14' heteroallelic genotype.

[0168] The U1 heterokaryon has an '11/12' heteroallelic genotype. [0169] 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.

Description of the MFPC-1-ELF marker:

[0170] 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.

[0171] 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).

[0172] Allele E1 : 'A' @ 77; 'A' @ 232; 'A' @ 309; T @ 334; 'A' @ 390; 'A' @ 400; T' @ 446, 'A' @ 481 ; etc.

[0173] Allele E2: 'G' @ 77; 'A' @ 232; 'G' @ 309; T @ 334; 'G' @ 390; 'G' @ 400; 'C @ 446, 'G' @ 481 ; etc.

[0174] Allele E8: 'A' @ 77; 'G' @ 232; 'G' @ 309; 'A' @ 334; 'A' @ 390; 'A' @ 400; 'C @ 446, 'G' @ 481 ; etc.

[0175] The J 10102 heterokaryon has an 'E1/E8' heteroallelic genotype.

[0176] The U1 heterokaryon has an 'E1/E2' heteroallelic genotype.

[0177] 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.

Description of the AN marker:

[0178] 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.

[0179] 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) :

[0180] Allele N1 : 'G' @ 640; [deletion] @ 844-846; T @ 882; Ά' @ 994, etc.

[0181] Allele N2: Ά' @ 640; [deletion] @ 844-846; T @ 882; Ά' @ 994, etc.

[0182] Allele N5: Ά' @ 640; 'ACG' @ 844-846; 'C @ 882; 'G' @ 994, etc.

[0183] The J10102 heterokaryon has an 'N1/N5' heteroallelic genotype.

[0184] The U1 heterokaryon has an 'N1/N2' heteroallelic genotype.

[0185] 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.

Description of the AS marker:

[0187] 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.

[0188] 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) :

[0189] Allele SC: T @ 28; 'GATATC @ 258-263; 'G' @ 275; [insertion]+'TTTCTCAGC'+[insertion] @ 309-249; 'C @ 404, etc.

[0190] Allele SD: 'C @ 28; [deletion] @ 258-263; T @ 275; [deletion] @ 309-249; T @ 404, etc.

[0191] The J10102 heterokaryon has an 'SC/SD' heteroallelic genotype.

[0192] The U1 heterokaryon has an 'SC/SD' heteroallelic genotype.

[0193] The genotype of the J1 1500 heterokaryon at the AS marker 'locus' is 'SC/SD'

(heteroallelic), designating the presence of alleles SC and SD. Allele SD was contributed by the OWNC line. Allele SC was transmitted from the J 10102 heterokaryon via the J10102-s69 homokaryon.

[0194] 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.

Description of the FF marker:

[0195] 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's breeding collection.

[0196] 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) :

[0197] Allele FF1 : 'CCG' @ 48-50

[0198] Allele FF2: TTC @ 48-50

[0199] The J10102 heterokaryon has an 'FF1/FF2' heteroallelic genotype.

[0200] The U1 heterokaryon has an 'FF1/FF2' heteroallelic genotype.

[0201] 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.

[0202] By using the foregoing markers, or any combination of many other available markers such as those in Table I, the uniqueness of the genotypes of line J 10102- s69 and strain J1 1500 is evident. Given that 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. 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. Vegetative incompatibility between genetically distinct cultivated strains is also economically valuable in addressing virus control and farm hygiene. Strain J11500 meets those criteria.

[0203] 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. In other embodiments, 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. [0204] 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. In other embodiments, 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. Also encompassed within the scope of the invention are 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. Similarly, 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. In other embodiments, 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.

[0205] 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. As shown in Table III, productivity of J1 1500 is comparable to and often greater than the productivity of the A15 strain, with total (3-flush) yield averaging 101.3% of the A-15 control and ranging as high as 106.8% under standard growing conditions for A-15. Distribution of the crop over the three-flush harvest period is relatively accelerated, meaning that more of the crop is picked during first flush, when disease pressure and incidence are lowest and product quality may be correspondingly higher. In a general t-test on this small data set, first break yield differences between J1 1500 and A-15 approached statistical significance (p = 0.057).

[0207]

TABLE III

Test ID 1 ^st flush yield 1 ^st & 2 ^nd flush yield Total yield

J11500 A-15 J1 1500 A-15 J1 1500 A-15

12-108 2.87 2.27 4.50 4.04 5.02

4.70

12-119 2.47 2.15 3.73 3.81 4.34

4.61

12-146 2.57 2.39 3.92 3.71 4.60

4.47

Averages 2.63 2.27 4.05 3.85 4.65

4.59 % gain +16% +5% +1 %

[0208] Within the first flush, yield is also accelerated. Over the four productive days of the first flush, the cumulative daily yield data in Table IV, reporting averages of the same three tests, shows that the harvest of strain J11500 is accelerated over that of the A-15 control.

[0209]

TABLE IV

Day (after casing): 14 15 16 17

Cumulative daily yield:

J11500 yield as a percent of A- 15 yield 181 % 139% 128% 116%

[0210] 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.

[0211]

TABLE V

Test I D Days to first pick Peak first flush pick day

J1 1500 A-15 J11500 A-15

12-108 14.0 15.3 14.7 15.3

12-119 14.0 14.0 14.0 14.3

12-146 14.0 14.0 15.0 14.8

Averages 14.0 14.43 14.56 14.8

Days gained +0.43 +0.24

[0212] Cap roundness and relative flesh thickness (i.e., 'meatiness') 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.

[0213] Cap roundness, expressed as cap height / cap diameter (CH/CD) is an economically important trait reflecting a consumer preference for rounder mushrooms. Measurements were made on samples of 10 first break mushrooms of equivalent maturity from both J1 1500 and the commercial control A- 15. J11500 was rounder (0.68) compared to the control A-15 (0.60), and this difference was significant (t-test, p = 9.15E-07).

[0214] Similarly, cap 'meatiness', expressed as flesh thickness / cap diameter (FT/CD) is an economically important trait reflecting a consumer preference for thicker-fleshed mushrooms. Measurements were made on samples of 10 first break mushrooms of equivalent maturity from both J11500 and the commercial control A- 15. J11500 was meatier (0.36) compared to the control A-15 (0.33), and this difference was significant (t-test, p = 0.0054).

[0215] 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. When 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:

TABLE VI

Spawn strain Casing strain Identity First flush yield

J11500 J1 1500 Self 2.47 lbs.

A-15 A-15 Self 2.03 lbs.

J11500 A-15 Non-self 0.50 lbs.

A-15 J11500 Non-self 0.17 lbs. [0216] 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. When two randomly obtained haploid offspring from the same F1 hybrid, derived from individual spores of different meiotic tetrads, are mated (i.e., in inter-tetrad selfing), representation of the line J10102-s69 marker profile in each recombined haploid parental line and in each sib-mated heterokaryon will be 50% on average, and slightly more than 75% (to about 85%) of heteroallelism present in the F1 hybrid will on average be retained in the sib-mated heterokaryon (the expectation over 75% is due to the mating requirement for heteroallelism at the mating type locus (MAT) on Chromosome 1). Distinctively, in addition, 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. In theory this value decreases to an average of 66.7% retention of F1 heteroallelism for distal markers unlinked to their centromeres; however empirical observations suggest higher rates of retention above 90% and approaching 99%-100% even for such distal markers. Transmission of the line J10102-s69 marker profile in such selfed offspring may be incomplete by a small percentage (typically 0-10%) due to the effects of infrequent odd-numbered meiotic crossovers, while representing 50% on average of the resulting heterokaryotic genome. 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.

[0217] The heterokaryon, or vegetative, incompatibility of J1 1500 with A-15, a phenotype evidently determined by the genotype, is transmitted into heterokaryotic spores along with most or all of the parental genotype, and thus is inherited by EDVs derived from spores, as shown by the yield data in TABLE VII. A deposit of a culture of an example of an EDV, namely strain J1 1500-ms2, obtained from hybrid strain J11500, as disclosed herein, has also 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, 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. [0218]

TABLE VII

Spawn strain Casing strain Identity First flush yield

A-15 A-15 Self 1.74 lbs.

A-15 J1 1500-ms2 Non-self 0.58 lbs.

A-15 J1 1500-ms3 Non-self 0.63 lbs.

A-15 J1 1500-ms4 Non-self 0.58 lbs.

A-15 J1 1500-ms5 Non-self 0.44 lbs.

A-15 J1 1500-ms10 Non-self 0.53 lbs.

[0219] A test of compatibility of an EDV of strain J11500 (designated J11500-ms2) with the strain J1 1500 itself was performed and the results are shown in TABLE VIII.

[0220]

TABLE VIII

Spawn strain Casing strain Identity First flush yield

J11500 J11500 Self 1.95 lbs.

J1 1500 J11500-ms2 Self: EDV 2.69 lbs.

J1 1500 J11500-ms2 Self: EDV 3.13 lbs.

[0221] 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. [0222] 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.

[0223] 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. 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 J1 1500 mushroom that may be used to transmit genetic material of J11500 into new cultures.

[0224] 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.

[0225] In order to demonstrate practice of the present invention at it relates to line 10102-S69, the line J10102-s69 was compared to other lines. 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. It also contributes to and supports several commercially desirable traits in hybrid offspring, including crop timing and productivity, and mushroom size, appearance and general retail appeal. Because 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. In effect, this 'combining ability', i.e., the ability to mate successfully and produce a high proportion of interesting and useful novel hybrids in strain development programs, is applied using qualitative, quantitative, objective and subjective criteria. 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.

[0226] In light of the foregoing, 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.

[0227] 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. In one embodiment, the repetition of steps (2-4) may be performed up to 5 times. In various other embodiments, 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. In a mating attempt between a homokaryotic line and a heterokaryon, in the absence of somatic recombination, either or both of only two possible defined novel heterokaryotic genotypes may be obtained, each of which will comprise line J10102-s69.

[0228] Using line J10102-s69, specific application with repetition of the steps described above can produce any pedigree structure from any arrangement of stocks, lines and hybrids within that structure. A hybrid of the F1 , F2, F3, F4, F5, F6, F7, F8, F9, F10 or any subsequent hybrid generation can be produced from line J10102-s69 using steps 1-6 described above.

[0229] In order to demonstrate practice of the invention as it relates to F1 hybrid strain J1 1500, a subculture of strain J1 1500 was propagated as described above to produce spawn and casing inocula, which were used to produce crops of white mushrooms under standard commercial cultivation practices as described herein above (see Background of Invention section). Commercial culture inocula including mushroom 'spawn' and 'casing inoculum' were prepared using commercial large- scale microbiological production methods, namely, by aseptically introducing inoculum of a pure culture of strain J11500 into from one to about 2,000 liters of sterilized growth media under sterile conditions, and were disbursed into sterile packaging for test purposes. 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. Under controlled environmental conditions, 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.

[0230]The mushrooms produced by strain J1 1500 have a white pileus color. As the Royal Horticultural Society (RHS) color charts do not provide a reference standard for the color "white", direct measurements of color of the strain J11500 mushroom cap have been made using a Minolta Chromameter and the L-a-b color space system. One measurement was made on the caps of each of ten first break mushrooms grown in a testing facility. The mean values, plus or minus the standard error, for the measured L, a, and b color components were as follows: L = 89.58 ± 0.11 ; a = -1.21 ± 0.015; b = 8.12 ± 0.088. Colors within or substantially coinciding with color space described by these three parameter distributions are called "white" according to standard and accepted practices of the commercial mushroom industry.

[0231] 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.

[0232] 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.

[0233] 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.