Method for eliminating aggressive incompatibility traits from strains of Agaricus bisporus and related strains and lines

Abstract

A method for eliminating the aggressive incompatibility (AI) trait from an Agaricus bisporus mushroom strain, the method comprising the steps of crossing a culture of a hybrid mushroom strain designated B18287-s82, a representative culture of which is deposited under NRRL accession number 68168, with a culture of a white mushroom strain designated WBL-s290 to obtain an F1 hybrid mushroom strain designated J19109, a representative culture of the F1 strain being deposited under NRRL accession number 68163. When a culture of the new F1 strain designated J19109 is induced to form fruiting bodies and homokaryotic spores derived therefrom are obtained, a culture of a homokaryotic strain is obtained from the homokaryotic spores derived from the F1 strain J19109, and a culture of the homokaryotic strain derived from the F1 strain J19109 is selected. The culture of the homokaryotic strain derived from the F1 strain J19109 is crossed with a culture of a mushroom strain designated J11500-s80 to obtain an F2 hybrid mushroom strain. The culture of the F2 hybrid mushroom strain is tested to determine the presence or absence of the AI trait, wherein if the AI trait is absent, the AI trait has been eliminated from the F2 hybrid mushroom strain.

Description

Technical Field

[0001] The present invention generally relates to a method for eliminating aggressive incompatibility traits (also referred to as AI traits) from strains of Agaricus bisporus. Various new strains and lines related to this method are also disclosed.

Background Art

[0002] The present invention follows the release of the white hybrid mushroom strain designated J15987 (NRRL Deposit No. 67646), developed by Sylvan America, Inc., which is disclosed in U.S. Patent No. 10,440,930, the disclosure of which is incorporated herein by reference. The J15987 strain has a higher yield than currently dominant white cultivars and produces high-quality mushrooms, i.e., mushrooms with a thicker cap flesh and less red coloration compared to standard commercially available white mushrooms obtained from the commercially accepted strain designated A-15. In initial tests of the J15987 strain, customers were very satisfied with this powerful combination of yield and quality, and these mushrooms and this strain were initially very commercially accepted. However, problems arose. The J15987 strain was found to contain a previously uncharacterized phenotype or trait called "Aggressive Incompatibility" that causes farm-level problems when A-15 or similar strains are also used on a mushroom farm. Accordingly, there is a need to develop methods for eliminating the phenotype or trait of "Aggressive Incompatibility" (as defined below) from white mushroom strains similar to J15987, and to develop similar white mushroom strains that retain the good traits of the J15987 strain (e.g., high yield and / or high quality, etc.) or provide them in other ways while eliminating the Aggressive Incompatibility (AI) trait.

[0003] The edible mushroom Agaricus bisporus (Lange) Imbach var. bisporus, which is a basidiomycete, is cultivated worldwide. It is the most widely cultivated mushroom species in Europe and the United States. The value of the annual harvest in the United States in 2020 - 2021 corresponded to 1 billion US dollars (National Agricultural Statistics Service / USDA data).

[0004] Cultures of Agaricus are prepared, maintained, propagated, and stored in a sterile medium, like other microorganisms. For various purposes, including spore germination, creation of new hybrids, and creation of inoculation materials, manipulations on pure cultures are performed using sterile tools and sterile techniques within a sterile area. Commercially available media are "seed spawn", which can be of several different recipes, most commonly of sterilized millet or rye. The production of seed spawn is generally carried out on a large scale. For example, 1 liter of pure Agaricus inoculation material can be used for 140,000 liters of sterilized seed spawn. The end - user, the mushroom farm, receives the aseptically packaged pure seed spawn culture.

[0005] The commercial cultivation of mushrooms is carried out within dedicated structures of mushroom farms. There are many variations in the method, but a typical one is described below. Compost is prepared from waste lignocellulosic materials such as wheat straw, fortified with nitrogen materials, completed, transferred under specific conditions (aerobic, temperature between 40°C and 50°C) for pasteurization, thereby creating a substrate favorable for colonization by Agaricus. Mushroom spawn is added, and the substrate is allowed to colonize over a period of 11 to 17 days at a controlled temperature. Once colonization, i.e., "mycelial elongation" is complete, a non-nutritive layer of saturated peat (adjusted to a pH between 8 and 8.5 using calcium carbonate) is applied to the surface of the compost to a layer approximately 5 cm thick. Additionally, casing spawn can be added to the peat or casing layer to accelerate mycelial growth. Once colonization occurs, the environmental conditions are adjusted in a process called "flushing", reducing the air temperature to approximately 18 - 19°C and reducing CO2 to approximately 1300 ppm. On the 13th to 18th day after the application of the casing soil, mushrooms appear in the mushroom house. The mushrooms are harvested over a period of 3 to 4 days. Additional flushes or breaks in production are caused, and after harvesting, the compost is removed and replaced at the mushroom farm.

[0006] In the United States, 72% of the total harvest from 2020 to 2021 was white Agaricus, with the remainder being brown Agaricus varieties. The total value of mushroom harvests in the United States during the same period exceeded $1 billion. In other regions, there is a stronger preference for white mushrooms. The market demand for white mushrooms in the United States and other regions is limited, and it is strict with respect to phenotypic traits such as size, shape, whiteness, hardness, and related traits such as extended shelf life. Therefore, new white Agaricus strains should also meet a specific set of market requirements. Strains can be classified based on several traits such as cap shape (e.g., cap roundness, flesh thickness, stalk thickness), color (whiteness), density / hardness, and single weight. Some examples of commercial targets for new white mushroom strains are increased crop yields, altered yield distribution between production flushes, disease resistance, pest resistance, improved shelf life, reduced bruising, ease of management, suitability for mechanical harvesting, different responses to stress factors (e.g., temperature or substrate changes), seasonal effects, farm practices, and strain incompatibility. DNA marker techniques such as single nucleotide polymorphisms (SNPs) can also be used to discriminate and classify strains from each other. Strains can have different ancestries, which are directly reflected in their genotypes and phenotypes.

[0007] Around 1980, the first two true hybrid strains of A. bisporus were developed at a research institute in Horst, the Netherlands. These two "Horst" strains, designated U1 and U3, are closely related hybrids developed by crossing two existing groups of white strains, pre-hybrid smooth white (PHSW) and pre-hybrid off white (PHOW), as described in M. Imbernon et al., Mycologia, 88(5), 749-761 (1996), which is incorporated herein by reference. Gradually, the hybrid strain U1 became the dominant white genotype in the market.

[0008] The parental homokaryon of the U1 strain was recovered by curing the culture of heterokaryons. The parent of PHSW is H39, and the parent of PHOW is H97. H97 was deposited by A. Sonnenberg with the Fungal Genetics Stock Center in Kansas, USA, under accession number 10389, and also with the American Type Culture Collection under accession number MYA-4626. The H97 genome was sequenced by the Joint Genome Initiative (California, USA) and made publicly available, and it has proven to be very useful as a reference for single nucleotide polymorphism (SNP) detection (see below).

[0009] The U1 strain is the ancestor of all white strains currently cultivated in North America and Europe. Many mushroom strains, such as A-15 and S-130, for example, have been developed from spores or tissue from the original (i.e., first) strain and meet the criteria for essentially derived varieties (EDV) of U1. A group of strains developed either by somatic selection or spore culture, or by methods related to "essential derivation" as considered below, from a single ancestor is referred to as a derived line group. Except for relatively minor genetic differences, all white strains developed within the U1 derived line group share a single complex N+N heterokaryon genotype with the original U1 strain. With the notable exception of China, modern white mushroom cultivation is monoculture.

[0010] Agaricus bisporus has a reproductive syndrome known as amphithallism, in which two distinct life cycles function simultaneously. As with other fungi, the reproductive propagules are spores. Agaricus produces spores by meiosis in basidia, which are known as spore sacs of meiosis. In the first life cycle, A. bisporus spores each receive only one haploid post-meiotic nucleus; these spores are capable of mating but not of producing mushrooms. These haploid spores germinate to produce homokaryotic progeny or lines, and these homokaryotic progeny or lines can be mated with other compatible homokaryons to produce new hybrid heterokaryons capable of producing mushrooms. Heterokaryons generally exhibit much lower mating ability than homokaryons. This life cycle is called heteromixis and is similar to outcrossing. This life cycle functions but is generally not dominant in strains of Agaricus bisporus var. bisporus.

[0011] The second parental life cycle is called intramixis and is similar to the form of inbreeding and is dominant in the majority of strains of Agaricus bisporus var. bisporus. Most spores receive two post-meiotic nuclei, and most of such nuclear pairs consist of non-sister nuclear pairs (NSNPs) with heterozygous genotypes at most or all kinetochore-linked loci, including the MAT locus. The MAT genotype determines the heterokaryotic phenotypic expression of these progeny that are reproductively capable and produce mushroom crops. It is recognized that relatively little chromosomal recombination occurs in the post-meiotic progeny of A. bisporus, which is rare among eukaryotes. Empirically, very little heterozygosity (similar to heterozygosity) is lost among the intramixis progeny of heterokaryotic strains.

[0012] In either life cycle, the spore is a cell that is part of the mushroom, and that cell is a propagule that always has as a single parent the mushroom and the culture from which the mushroom arises.

[0013] With respect to spores, viable spores are heterokaryotic or homokaryotic in a dormant state. Spores are part of the mushroom organism culture, and only the genetic material of the single culture (often referred to as the "parent" for convenience) that gives rise to them is incorporated. Other parts of the culture include the cap, stalk, gills, cells (defined as hyphal compartments incorporating cell walls including nuclei, mitochondria, cytoplasm, protoplasts, RNA, DNA, proteins, cell membranes, and transverse walls), hyphae, and mycelia. Spores can be aseptically collected onto sterile material, suspended in sterile water at various dilutions to produce germinated spores, and plated onto a sterile agar growth medium to incorporate the culture into the spores. A preferred technique is to place a living Agaricus culture in a sealed Petri plate that can stimulate spore germination by diffusing volatile pheromones. Germinated spores can be isolated onto a new nutrient agar plate using sterile microtools such as a steel needle under a microscope. Using this method, progeny of the heterokaryotic and homokaryotic strains containing the spores of the above strains and the culture incorporated into the spores can be obtained.

[0014] The development of new hybrid varieties by heteromixis involves physically associating and mating two compatible cultures under control to obtain a new heterokaryotic co - existent culture. Homokaryons (i.e., "strains") are preferred starting cultures for mating because they have the greatest ability to anastomose and achieve cytoplasmic fusion with other cultures. Heterokaryons can also be physically contacted, but the probability of mating resulting in the successful formation of new heterokaryons is unreasonably low. Compatibility is determined by the genotype at the MAT locus; two homokaryons with the same MAT allele cannot establish a heterokaryon after anastomosis, so homokaryon compatibility becomes a genetic difference. In a defined mating program, homokaryotic strains are obtained and associated in a given pair combination. In one method, homokaryon pairs are placed very close to each other on the surface of a nutrient agar medium in a Petri dish and grown together (physically associated) until anastomosis occurs between the two cultures. A successful result is that mating results in a heterokaryon. A new hybrid heterokaryon can be obtained by transferring mycelium from the fusion zone of the dish.

[0015] For two reasons, there is a clear need for commercially recognized white Agaricus bisporus strains with genotypes different from the U1 - derived strain group. First, strains that are not nutritionally compatible with U1 - derived strain group strains have been found to slow the spread of viral diseases among strains, and such diseases are known to be a major cause of crop losses in the commercial mushroom industry. Second, the agricultural industry widely relies on crops of a single genetic strain (e.g., white A. bisporus in most of the world), and it is well - known that the risk of catastrophic crop failures on a facility - wide or industry - wide scale is increasing. Therefore, from the perspectives of food security and risk management, it is highly desirable to simultaneously provide genetic diversity and commercially recognized performance and crop characteristics.

[0016] As described above, the present invention is a direct result of the commercial release of the hybrid J15987 strain (NRRL Deposit No. 67646) (U.S. Patent No. 10,440,930), a white hybrid strain. Prior to commercialization testing, it was demonstrated that the J15987 strain has a thicker cap flesh and a thicker stalk compared to other white cultivated varieties such as those derived from the A-15 strain. Additionally, color analysis using a Minolta Chroma Meter on J15987 demonstrated that the harvested mushroom caps have less redness. Commercial potential was considered important, and the strain was grown in the United States immediately after the patent was filed. The strain successfully met all of the original crop production criteria. However, an important problem emerged. In several mushroom growing "houses" (or rooms), particularly those producing mushrooms derived from the A-15 strain, a lack of mushroom growth was observed in extensive areas of the compost, both in the compost and the casing layer. This observation was found to be abnormal and different from normal.

[0017] The culture was returned to the test facility where the suspected antagonistic interaction between strain J15987 and other white cultivated varieties was investigated in more detail. The genetic characteristics of the interaction between strains are governed by self / non-self recognition systems, known either as nutritional incompatibility or as heterokaryon incompatibility. When strain J15987 was brought into contact with other dependent varieties (EDV) of the U1 family, such as A-15, it was found to have a very aggressive and unusual antagonistic reaction. When the inoculum was mixed at a low rate of 1% J15987 to 99% A-15, the Agaricus mycelium died rapidly and the A-15 genotype was ultimately replaced by J15987. This interaction was named "Aggressive Incompatibility", abbreviated as "AI" for this interaction, and the phenotype was defined as the "AI" trait. Specifically, unrelated strains of basidiomycetes, including Agaricus bisporus, usually show incompatibility reactions, which have been exploited for virus control, but the AI trait is a much more aggressive, extreme and problematic manifestation of this well-known phenomenon. This trait is presumed to be under genetic control, as is generally understood in the case of nutritional incompatibility in basidiomycetes.

Summary of the Invention

Problems to be Solved by the Invention

[0018] Conventional mixing equipment in mushroom farms tends to operate in such a way that some mixing of different strains occurs between the substrates being inoculated. Such events are generally minor and have little negative impact. Since these areas are small, the impact on total yield is minimal and has been traditionally tolerated. However, the effects of larger and more extreme AI interactions are unacceptably large commercially, and customer evaluations were that strain J15987 could not be handled profitably in farm operations that also cultivated strain A-15. Therefore, there is a need for strains that are commercially desirable or have the positive traits of strain J15987 while lacking the AI trait.

Means for Solving the Problems

[0019] To understand and overcome (eliminate) the underlying genetic basis involved in the manifestation of the AI trait in the J15987 strain, a series of crosses were carried out and the resulting hybrids were analyzed. The crossing scheme is described in Scheme I below. Essentially, a wild-type variation strain (i.e., B18287-s82, NRRL Deposit No. 68168) was combined with one of the homokaryotic parent strains of the J15987 strain (i.e., WBL-s290, NRRL Deposit No. 68167) to generate a new F1 hybrid strain (i.e., J19109, NRRL Deposit No. 68163). Subsequently, the homokaryon derived from that hybrid strain (e.g., J19109-s40 strain, NRRL Deposit No. 68165) was crossed with the other homokaryotic parent strain of the J15987 strain (i.e., J11500-s80, NRRL Deposit No. 68164) to yield a new F2 hybrid strain (e.g., J20176, NRRL Deposit No. 68166). Comparisons were made using the F2 hybrid strains to demonstrate the presence or absence of the AI trait. Based on these comparisons, it was revealed that the crossing scheme was successful and that the majority of the new hybrids did not have the AI trait. Further development of a model for genetically controlling / eliminating the AI trait was possible from the pattern of inheritance.

[0020]

Chemical formula

[0021] First, it was necessary to develop a homokaryon (B18287-s82 strain) obtained from a hybrid strain developed by crossing two homokaryons, namely W01-s1 and So76-s12b. This homokaryotic strain, B18287-s82, was developed from the wild strain W01 collected by R. Kerrigan in North America in the 1990s and the parental homokaryon obtained from the pre-hybrid off-white strain So76. DNA tests showed that W01 was a true wild strain and not a commercial strain that had escaped. Furthermore, W01 had growth requirements very similar to those of current white and brown strains.

[0022] Accordingly, one aspect of the present invention generally relates to a new and distinct homokaryotic coexistence system of Agaricus bisporus designated B18287-s82, and processes using the strain designated B18287-s82. In at least one embodiment, a culture is provided that includes at least one set of chromosomes of the Agaricus bisporus B18278-s82 strain, where the at least one set of chromosomes includes all of the alleles of the B18278-s82 strain at marker loci characterized by the sequences listed in Table I below. A deposit of a culture of the Agaricus bisporus B18287-s82 strain disclosed herein was made with the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA and was accepted. The deposit date was June 10, 2022. The deposited culture was obtained from the same culture maintained by Sylvan, Inc., Kittanning, Pennsylvania, USA, the record assignee since before the filing date of this application. All restrictions upon deposit have been removed, and the deposit is intended to meet all of the deposit requirements of the U.S. Patent and Trademark Office, including 37 C.F.R. Secs. 1.801-1.809, and all of the deposit requirements under the Budapest Treaty. The NRRL accession number is 68168. The deposit will be maintained at the depository institution for 30 years, or five years after the last request, or for the longest of these periods, and will be replaced as necessary during that period. The culture will be made available to the public without change, without restriction, or subject to the conditions required by any applicable patent law at the time of filing of this patent application or at the time of issuance of the patent.

[0023] In one embodiment, a progeny variety (EDV) of the B18287-s82 line may also be provided. Such an EDV may include a culture derived from an original culture, where the original culture is a culture of the B18287-s82 line, and thus at least 75% of its genome or genotype is present in the genome or genotype of the original culture of the B18287-s82 line. In other embodiments, at least 90%, or 95% or 98% or 99% of its genome or genotype is present in the genome or genotype of the original culture of the B18287-s82 line. In other embodiments, such an EDV may include a culture derived directly from or only from the original culture, and thus all of its genome or genotype is present in the genome or genotype of the original culture of the B18287-s82 line.

[0024] The B18287-s82 isogenic coexistents were crossed with WBL-s290. Note that this line is one of the parental isogenic coexistents currently found for the J15987 strain (the other is J11500-s80, which was also used in the present invention). The resulting F1 heterokaryotic coexistent strain was named J19109.

[0025] Accordingly, another aspect of the present invention generally relates to a new and distinct homokaryotic coexistence system of Agaricus bisporus designated as WBL-s290, and processes using the strain designated as WBL-s290. In at least one embodiment, a culture is provided that contains at least one set of chromosomes of the Agaricus bisporus WBL-s290 strain, where the at least one set of chromosomes contains all of the alleles of the WBL-s290 strain at marker loci characterized by the sequences listed in Table I below. A deposit of a culture of the Agaricus bisporus WBL-s290 strain disclosed herein was made with and accepted by the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The deposit date was June 10, 2022. The deposited culture was obtained from the same culture maintained by Sylvan, Inc., Kittanning, Pennsylvania, USA, the record assignee since before the filing date of this application. All restrictions at the time of deposit are removed, and the deposit is intended to meet all of the deposit requirements of the U.S. Patent and Trademark Office, including 37 C.F.R. Sec. 1.801-1.809, and all of the deposit requirements under the Budapest Treaty. The NRRL accession number is 68167. The deposit will be maintained at the depository institution for 30 years, or for 5 years after the last request, or for the longest of the term of the patent, and will be replaced as necessary during that period. The culture will be made available to the public without change and without restriction or condition, as required by any applicable patent law at the time of filing of this patent application or at the time of issuance of the patent.

[0026] In one embodiment, a dependent variety (EDV) of the WBL-s290 line may also be provided. Such an EDV may include a culture derived from the first culture, where the first culture is a culture of the WBL-s290 line, and thus at least 75% of its genome or genotype is present in the genome or genotype of the first culture of the WBL-s290 line. In other embodiments, at least 90%, or 95% or 98% or 99% of its genome or genotype is present in the genome or genotype of the first culture of the WBL-s290 line. In other embodiments, such an EDV may include a culture derived directly from the first culture or derived only from the first culture, and thus all of its genome or genotype is present in the genome or genotype of the first culture of the WBL-s290 line.

[0027] Another aspect of the present invention generally relates to a new distinct Agaricus bisporus mushroom strain culture designated J19109, which is a hybrid strain obtained by the directed mating of two homokaryotic co-cultures, B18287-s82 and WBL-s290. The deposit of the J19109 strain culture was made with and accepted by the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The deposit date is June 10, 2022. The deposited culture was obtained from the same culture maintained by Sylvan Inc., Kittanning, Pennsylvania, USA, which was the record assignee prior to the filing date of this application. All restrictions upon deposit are removed, and the deposit is intended to meet all the deposit requirements of the U.S. Patent and Trademark Office, including 37 C.F.R. Sec. 1.801-1.809, and all the deposit requirements under the Budapest Treaty. The NRRL accession number is 68163. The deposit will be maintained at the depository institution for 30 years, or for 5 years after the last request, or for the longer of the two periods during the term of the patent, and will be replaced as necessary during that period. The culture will be made available to the public without change and without restriction or condition, in accordance with patent law, at the time of filing of the priority application or at the time of issuance of the patent for this strain.

[0028] In one embodiment, an essentially derived variety (EDV) strain of J19109 may also be provided. Such an EDV may include a culture of Agaricus bisporus derived from the first culture, where the first culture is the culture of the J19109 strain, and thus at least 75% of its genome or genotype is present in the genome or genotype of the first culture of the J19109 strain. Alternatively, other EDVs may include cultures that are directly derived from the first culture of the J19109 strain, and thus all of their genome or genotype is present in the genome or genotype of the first culture of the J19109 strain.

[0029] Upon repetition, a culture of a hybrid mushroom strain designated B18287-s82, a representative culture of which is deposited under NRRL accession number 68168, was mated with a culture of a white mushroom strain designated WBL-s290, a representative culture of which is deposited under NRRL accession number 68167, to obtain an F1 hybrid mushroom strain designated J19109, a representative culture of which is deposited under NRRL accession number 68163. Subsequently, fruiting body formation of the culture of the F1 strain designated J19109 was carried out to obtain homokaryotic spores derived therefrom, and a homokaryotic strain was obtained from the homokaryotic spores. Subsequently, a culture of the homokaryotic strain derived from the F1 strain J19109 was selected. In particular, the homokaryotic strain derived from the F1 strain J19109 lacks alleles at the centromere-linked loci of the WBL-s290 strain on chromosomes 4, 7, and 9.

[0030] Once a culture of the homokaryotic strain derived from the F1 strain J19109 was selected, the culture was mated with a culture of a mushroom strain designated J11500-s80. Note that J11500-s80 is the other parental homokaryon currently found with respect to the J15987 strain. An F2 hybrid mushroom strain resulting from the mating was obtained, and the culture of the resulting F2 hybrid mushroom strain was tested to determine the presence or absence of the AI trait. Here, if the AI trait is absent, the AI trait has been eliminated from the F2 hybrid mushroom strain.

[0031] Accordingly, another aspect of the present invention generally relates to a new and distinct homokaryotic coexistence system of Agaricus bisporus designated J11500-s80, and processes using the strain designated J11500-s80. In at least one embodiment, a culture is provided that contains at least one set of chromosomes of the Agaricus bisporus J11500-s80 strain, where the at least one set of chromosomes contains all of the alleles of the J11500-s80 strain at marker loci characterized by the sequences listed in Table I below. A deposit of a culture of the Agaricus bisporus J11500-s80 strain disclosed herein was made with and accepted by the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The deposit date was June 10, 2022. The deposited culture was obtained from the same culture maintained by Sylvan, Inc., Kittanning, Pennsylvania, USA, the record assignee, prior to the filing date of this application. All restrictions upon deposit have been removed, and the deposit is intended to meet all of the requirements of the U.S. Patent and Trademark Office, including 37 C.F.R. Sec. 1.801-1.809, and all of the requirements under the Budapest Treaty. The NRRL accession number is 68164. The deposit will be maintained at the depository institution for 30 years, or five years after the last request, or for the longest of the three periods, and will be replaced as necessary during that period. The culture will be made available to the public without change, without restriction, or subject to the conditions required by any applicable patent law at the time of filing of this patent application or at the time of issuance of the patent.

[0032] In one embodiment, a progeny variety (EDV) of the J11500-s80 line may also be provided. Such an EDV may include a culture derived from the first culture, where the first culture is a culture of the J11500-s80 line, and thus at least 75% of its genome or genotype is present in the genome or genotype of the first culture of the J11500-s80 line. In other embodiments, at least 90%, or 95% or 98% or 99% of its genome or genotype is present in the genome or genotype of the first culture of the J11500-s80 line. In other embodiments, such an EDV may include a culture derived directly from the first culture or derived only from the first culture, and thus all of its genome or genotype is present in the genome or genotype of the first culture of the J11500-s80 line.

[0033] Furthermore, in one or more embodiments, it will be understood that the F2 hybrid strains in which the AI trait is eliminated retain at least two beneficial traits found in the J15987 strain. Such beneficial traits can be selected from the group consisting of an umbrella shape with a roundness similar to that of the J15987 strain, a stem thickness similar to that of the J15987 strain, a flesh thickness similar to that of the J15987 strain, and an a (redness) value on the L-a-b color measurement scale within 10%, or 5%, or 2.5%, or 1%, or 0.5% of the a (redness) value of the 15987 strain. The terms "similar roundness" and "similar thickness" are understood to mean that the measured umbrella, stem, and / or flesh of the mushroom fruited by the F2 hybrid strain fall within the range of statistical significance relative to the roundness or thickness of the umbrella, stem, or flesh of the mushroom fruited by the J15987 strain. In alternative embodiments, the roundness of the umbrella shape of the mushroom of the F2 hybrid strain can be within 20%, or 15%, or 10%, or 5% of the roundness of the umbrella shape of the mushroom of the J15987 strain. In other alternative embodiments, the thickness of the stem of the mushroom of the F2 hybrid strain can be within 20%, or 15%, or 10%, or 5% of the thickness of the stem of the mushroom of the J15987 strain. In still other alternative embodiments, the thickness of the flesh of the mushroom of the F2 hybrid strain can be within 20%, or 15%, or 10%, or 5% of the thickness of the flesh of the mushroom of the J15987 strain.

[0034] In one embodiment, the homokaryotic line selected from the F1 strain J19109 can be the homokaryotic line designated J19109-s40. It will be understood that the culture of the line designated J19109-s40 is crossed with a mushroom line designated J11500-s80, where the resulting F2 hybrid strain is designated J20176. The hybrid J20176 strain has been found to not have the AI trait.

[0035] Accordingly, another aspect of the present invention generally relates to a new and distinct homokaryotic coexistence system of Agaricus bisporus designated J19109-s40, and processes using the strain designated J19109-s40. In at least one embodiment, a culture is provided that contains at least one set of chromosomes of the Agaricus bisporus J19109-s40 strain, where the at least one set of chromosomes contains all of the alleles of the J19109-s40 strain at marker loci characterized by the sequences listed in Table I below. A deposit of a culture of the Agaricus bisporus J19109-s40 strain disclosed herein was made with and accepted by the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The deposit date was June 10, 2022. The deposited culture was obtained from the same culture maintained by Sylvan, Inc., Kittanning, Pennsylvania, USA, the record assignee, prior to the filing date of this application. All restrictions upon deposit have been removed, and the deposit is intended to meet all of the deposit requirements of the U.S. Patent and Trademark Office including 37 C.F.R. Sec. 1.801-1.809, and all of the deposit requirements under the Budapest Treaty. The NRRL accession number is 68165. The deposit will be maintained at the depository institution for 30 years, or five years after the last request, or for the longest of the term of the patent, and replaced as necessary during that period. The culture will be made available to the public without change, without restriction or condition, at the time required by any applicable patent law at the time of filing of this patent application or at the time of issuance of the patent.

[0036] In one embodiment, a progeny variety (EDV) of the J19109-s40 line may also be provided. Such an EDV may include a culture derived from the original culture, where the original culture is a culture of the J19109-s40 line, and thus at least 75% of its genome or genotype is present in the genome or genotype of the original culture of the J19109-s40 line. In other embodiments, at least 90%, or 95%, or 98%, or 99% of its genome or genotype is present in the genome or genotype of the original culture of the J19109-s40 line. In other embodiments, such an EDV may include a culture derived directly from the original culture or derived only from the original culture, and thus all of its genome or genotype is present in the genome or genotype of the original culture of the J19109-s40 line.

[0037] Another aspect of the present invention generally relates to a new distinct Agaricus bisporus mushroom strain culture designated J20176, which is a hybrid strain obtained by the directed mating of two homokaryotic co-cultures, J11500-s80 and J19109-s40. A culture of the J20176 strain was deposited with the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA and was accepted. The deposit date was June 10, 2022. The deposited culture was obtained from the same culture maintained by Sylvan Inc., Kittanning, Pennsylvania, USA, the record assignee, prior to the filing date of this application. All restrictions upon deposit are removed, and the deposit is intended to meet all the deposit requirements of the U.S. Patent and Trademark Office, including 37 C.F.R. Sec. 1.801-1.809, and all the deposit requirements under the Budapest Treaty. The NRRL accession number is 68166. The deposit will be maintained at the depository institution for 30 years, or for 5 years after the last request, or for the longest of these periods which is the duration of the patent, and will be replaced as necessary during that period. The culture will be made available to the public without change and without restriction or condition, in accordance with the patent law, at the time of filing of the priority application or at the time of issuance of the patent with respect to this strain.

[0038] In one embodiment, an essentially derived variety (EDV) of the J20176 strain may also be provided. Such an EDV may include a culture of Agaricus bisporus derived from the original culture, where the original culture is a culture of the J20176 strain, and thus at least 75% of its genome or genotype is present in the genome or genotype of the original culture of the J20176 strain. Alternatively, other EDVs may include cultures that are directly derived from the original culture of the J20176 strain, and thus all of their genome or genotype is present in the genome or genotype of the original culture of the J20176 strain.

[0039] Furthermore, in one or more embodiments, it will be understood that the F2 hybrid strain designated J20176 has the AI trait eliminated and retains at least two beneficial traits found in the J15987 strain. Such beneficial traits can be selected from the group consisting of an umbrella shape with a roundness similar to that of the J15987 strain, a stalk thickness similar to that of the J15987 strain, a flesh thickness similar to that of the J15987 strain, and a redness (a) value within 10%, or 5%, or 2.5%, or 1%, or 0.5% of the redness (a) value of the J15987 strain in the L-a-b color measurement scale. The terms "similar roundness" and "similar thickness" are understood to mean that the umbrella, stalk, and / or flesh of the mushroom formed by the F2 hybrid J20176 strain falls within the range of statistical significance with respect to the roundness or thickness of the umbrella, stalk, or flesh of the mushroom formed by the J15987 strain. In an alternative embodiment, the roundness of the umbrella shape of the mushroom of the F2 hybrid J20176 strain can be within 20%, or 15%, or 10%, or 5% of the roundness of the umbrella shape of the mushroom of the J15987 strain. In other alternative embodiments, the thickness of the stalk of the mushroom of the F2 hybrid J20176 strain can be within 20%, or 15%, or 10%, or 5% of the thickness of the stalk of the mushroom of the J15987 strain. In yet other alternative embodiments, the thickness of the flesh of the mushroom of the F2 hybrid J20176 strain can be within 20%, or 15%, or 10%, or 5% of the thickness of the flesh of the mushroom of the J15987 strain.

[0040] By the method of the present invention, it can be seen that the AI trait is advantageously eliminated from Agaricus bisporus strains, including at least the J20176 strain. More specifically, by this method, the aggressive incompatibility (AI) trait can be eliminated from Agaricus bisporus mushroom strains that are descendants of both the J10165 strain (also referred to as WBL) and the J11500 strain (disclosed in U.S. Patent No. 9,622,428), where a culture of a white mushroom line designated WBL-s290 is crossed with a culture of a mushroom line designated J11500-s80, resulting in a hybrid mushroom strain designated J15987 having the AI trait, and a representative culture of said strain is deposited under NRRL accession number 67646.

[0041] Regarding obtaining cultures of homokaryotic systems from homokaryotic spores derived from the F1 strain J19109, when the J19109 heterokaryon was induced to form fruit bodies and F2 spores were collected, a total of 52 initial homokaryons were obtained, 16 of which are described in some of the tables below. All 52 homokaryons were crossed with J11500-s80, the second parent of J15987, and the resulting strains were screened extensively for commercial potential. From the data collected and shown below, the beneficial traits of J15987 were maintained, and more importantly, the majority of the new J19109×J11500-s80 hybrids did not have the AI trait. One hybrid, the J20176 strain, was selected for controlled testing. This strain functions robustly and is thus a strong candidate for commercialization.

[0042] The entire pool of homokaryotic systems of the J19109 strain shows similar potential to J19109-s40 and is considered a beneficial breeding line with the potential to produce high-quality mushroom hybrids without the AI trait.

[0043] One of the uses of the hybrids derived from J19109 is the production of edible mushroom crops for sale. Thus, mushrooms obtained from cultures of any of the strains or lines specified above are part of the present invention. Another use is the improvement of facility hygiene by strain rotation and the "virus break" effect. A further use is to incorporate the genetic material of the J19109 strain into cultures that are descendants and derivatives or progeny, including dormant spores, germinated spores, and protoplasts. There are additional uses as described above.

[0044] The unusual biological properties of the fungus, particularly intramyxixis in Agaricus bisporus, make it easy and straightforward to derive virtual copies of cultures with essentially the same phenotype. In plant breeding, such copies are known as dependent varieties, or EDVs. Some examples of methods for obtaining cultures that are EDVs as a result of the definition of a single original culture of A. bisporus include somatic selection, tissue culture selection, single spore germination, multiple spore germination, selfing, repeated backcrossing to the original culture, mutagenesis, and transformation. DNA-mediated transformation of A. bisporus is 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 Agrobacterium-mediated transformation technique. Mush. Sci. 16:591-597, and references therein, which are incorporated herein by reference. Transformation can introduce a single new gene or allele into the genome of the original culture. Additionally, recent reports on other closely related fungi have raised the possibility of CRISPR gene editing.

[0045] EDVs are clearly recognizable by their genotype, being mainly or additionally or even completely a subset of a single original culture. The percentage of the first genotype present in Agaricus bisporus EDVs ranges from 100% or virtually 100% in the case of single spore cultures and somatic selections, up to 99.x% in the case of strains modified by DNA-mediated transformation, 90 - 99.x% in the case of some single or multiple spore selections or some mutagenesis, and on average at least 75 - 85% in the case of sibling progeny mating (= selfing) and backcrossing to the original culture. To determine the percentage of the DNA of the original culture present in another culture and to make a clear determination for any method used to manipulate or exploit the two cultures and their relatedness and the original culture, many genotyping methods can be used, including the methods described below and other methods well known in the art.

[0046] Repeated backcrossing to the original culture also results in the EDV of the original culture. In a hypothetical example, in the first successive iteration of this process, the resulting strain of this generation has on average about 75% of the DNA of the first strain, while about 25% of the DNA is due to the contribution of a second strain or line. As this process is repeated, the DNA corresponding to the first strain increases and approaches 97% on average after three further successive iterations. In general, it will be understood that any culture having a genotype identity of 75 - 100% to the original culture exhibits the EDV of the original culture. The EDV of the EDV is also established as being the EDV of the first strain. Finally, since Agaricus bisporus alternates between heterokaryotic and homokaryotic systems, the criteria for essential derivation apply equally to both strains and cultures of lines.

[0047] A genetic fingerprint is a description of the genotype at defined loci, where the presence of the characterized alleles is recorded. Such fingerprints provide a powerful and effective technique for recognizing the clones of the original strain and all types of EDVs, as well as for recognizing ancestors that fall within the range of outbred lines. Many techniques are available for defining and characterizing the loci and alleles of the genotype. The most detailed approach is provided by whole-genome sequencing (WGS), which enables the direct characterization and comparison of DNA sequences across the entire genome. By using this approach to generate robust genetic fingerprints incorporating a large number of marker loci, it is possible to establish the nature of the relatedness between two strains, including strains associated by phylogenetic descent over several generations. The applicant tracked genetic markers over four to six generations of the strain development pedigree. If a sufficient number of differential markers are present in the original strain or line, it becomes possible to identify the descent from the original strain or line after several outbred generations without excessive experimentation. In a hypothetical example, at the fourth outbred generation, the average expected value of the genome corresponding to the first haploid line in the F4 hybrid is 3.1% (50% / 2 4 ), which corresponds to approximately 1 Mb of the nuclear genomic DNA of A. bisporus. Based on the applicant's analysis, the amount of DNA derived from each of two unrelated strains of A. bisporus may generally contain from about 10,000 to about 20,000 single nucleotide polymorphisms (SNPs), any one of which may provide a discriminative marker linking the F4 hybrid to the original line. By using a large number of independent markers, the original strain of the line can be identified with a very high probability of success and with reasonable confidence.

[0048] One of the biologically and commercially interesting traits is heterokaryon incompatibility. The genetics of these self / non-self recognition systems in basidiomycetes such as Agaricus are not fully understood, but in other groups of fungi, it has been found that multiple alleles at multiple independent loci are involved. Heterokaryon incompatibility occurs in the majority of fungi and it should be noted that it is quite distinct from the Aggressive Incompatibility trait reported herein. The "normal" incompatibility is usually a hyphal interaction in a local area where growth is attenuated between two genotypes. This function is thought to prevent the transmission of viruses between different genotypes of the same species.

[0049] It should be noted that there are at least two double-stranded RNA viruses that affect mushroom crops, La France disease and Mushroom Virus X. Heterokaryon incompatibility prevents anastomosis and the successful continuity of cytoplasm between physical mixtures of two or more heterokaryons, thereby resulting in "virus break". In mushroom farms, virus break is achieved by replacing the planting materials (compost, spawn, casing inoculum) incorporated into the first strain with inoculum and planting materials incorporated into a different strain that is incompatible with the first strain. The most effective implementation of the virus break method is to replace all the biological materials of the first strain in the mushroom farm with the biological materials of a second incompatible strain. Although strain incompatibility is not an absolute barrier to the movement of viruses from the biological reservoir within the facility into new crops, it creates an effective barrier. Rotating the usage methods for cultivation between mushroom strains with different genotypes can also interfere with the infection and invasion cycles of exogenous pests and pathogens.

[0050] As described above, hybrid mushroom strain producers are constantly seeking hybrid strains that enable growers to produce crops successfully and profitably. In the case of J19109 and new hybrids derived from or descended from strains derived therefrom, positive traits that have been demonstrated to date include an attractive appearance (round caps, thick stems compared to market leader A-15, good resistance to being damaged, all of which are appealing to customers), and a total yield comparable to or exceeding that of strains such as A-15. In the examples of the present applicant, mushroom growers can obtain high-yield crops of high-quality mushrooms by using the new hybrids described herein. Furthermore, these strains have the notable advantage of being comparable in quality to the previous strain J15987 developed by the present applicant and not having an Aggressive Incompatibility phenotype.

[0051] The strains currently available in the mushroom industry enable growers to produce mushroom crops successfully and usually profitably. There are several factors that affect the degree of success and profitability achieved. For example, the strain must be able to produce at least an equivalent crop yield over two to three breaks or flushes compared to the strains currently sold and commercially grown. Also, some physical characteristics of the resulting mushrooms, such as the category of cap color, as well as general size and dimensions such as cap diameter, enable the mushrooms to be sold in the category of accessible products.

[0052] The J19109 SSI×J11500-s80 hybrid meets these market needs and solves the current problem of the unavailability of such strains in the market. Furthermore, this new group of strains has a different genotype compared to other current white cultivated varieties (see Tables I and II below), addressing and solving potential problems related to the reliability of monoculture crops.

[0053] One or more aspects of the present invention can be realized by hybrid mushroom cultures of Agaricus bisporus developed by the present invention. Accordingly, products incorporating any of the above strains or cultures are encompassed by the present invention. Such products include mycelium, inoculum, raw mushrooms or processed mushroom products, mushroom spores, mushroom seeds, mushroom preparations and extracts and fractions, mushroom pieces, mushroom inoculation materials, casing inoculation materials, casing seeds, casing soil, inoculated compost, colonized compost, post-cultivation compost, and friable particulate matter.

[0054] Furthermore, various portions of the cultures of any of the above strains or cultures may have value and commercial utility. Accordingly, a portion selected from the group consisting of mycelium, mushrooms, spores, cells, nuclei, and protoplasts is contemplated. In yet another one or more embodiments, single cell microorganisms are contemplated. Accordingly, cells of any of the above strains and cultures are provided.

[0055] Note that the J20176 strain, a representative culture of which has been deposited under NRRL accession number 68166, was deposited to provide a suitable example of a strain that may meet the requirements of the present invention. Accordingly, the J20176 strain encompasses various portions of the culture, including mycelium, spores, and cells, as well as portions of cells including nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes, and cell walls, which portions are present in both the vegetative mycelium of the culture and the mushrooms produced by the culture. The spores can be dormant or germinated spores and can contain heterokaryons and homokaryons incorporated therein.

[0056] One or more products can be made that incorporate a hybrid mushroom culture of Agaricus bisporus designated as a strain that is a descendant of J19109. Such products include mycelium, inoculum, inoculation material, casing inoculation material, raw mushrooms, processed mushroom products, mushroom extracts and fractions, mushroom pieces, and colonized substrates selected from grains, compost, and friable particulate materials. It will be understood that mushroom pieces refer to the stalk, cap, and other larger parts of the mushroom itself.

[0057] One or more other aspects of the invention can be realized by an essentially derived variety (EDV) of a hybrid mushroom culture of a strain such as J19109. In one or more embodiments, an Agaricus bisporus culture resulting from essential derivation has at least one of the essential characteristics of a hybrid derived from J19109. For the purposes of the present invention, as an example of an elite strain that is a direct result of the invention described herein, one hybrid known as J20176, J19109-s40×J11500-s80 was deposited. J20176 is a strain that does not have the AI trait and has further characteristics of the roundness of the cap, flesh thickness, yield performance, and production timing of J15987 compared to U1 EDVs such as A-15.

[0058] Other aspects of the invention can be realized by a method for producing a hybrid Agaricus bisporus culture comprising the step of crossing a homokaryon such as J19109-s40 (deposited under NRRL accession number 68165) with a second homokaryon. In one embodiment, the second homokaryon, the J11500-s80 strain, has a culture deposited under NRRL accession number 68164. Such crossing results in a mushroom culture, J20176, that exhibits a normal pattern of antagonism against a panel of commercial cultivars based on U1 EDV, e.g., A-15. This antagonism demonstrates that the J20176 strain is genetically distinct. In one or more embodiments, the method further comprises providing a mushroom culture of the invention comprising a culture substrate selected from mycelium, inoculum, inoculation material, casing inoculation material, raw mushrooms, processed mushrooms, parts of mushrooms, mushroom extracts and fractions, mushroom pieces, and grains, compost, and friable particulate matter. In other embodiments, the method may comprise providing the mushroom culture as a derived culture selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue explant cultures, protoplasts, dormant spores, germinating spores, inbred and non-inbred progeny, transgenic cultures, gene-edited cultures, and cultures having a genome with a single locus conversion.

[0059] Yet another aspect of the present invention can be realized by a hybrid mushroom culture of Agaricus bisporus having a genotypic fingerprint with specific traits at marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AF, and FF. In one or more embodiments, the culture has a genotypic fingerprint with specific traits at the marker loci described in Table II, where all of the traits of the fingerprint are present in the genotypic fingerprint. Specifically, meiosis and random combinations ensure that each homokaryotic spore, which is a descendant of J19109, has a unique pattern of markers. It is easy for those skilled in the art to infer that the J19109 homokaryotic breeding strain is a descendant based on genetic DNA markers.

[0060] One or more further aspects of the present invention can be realized by a culture, cell, or culture containing such cells produced by the above method. Thus, one or more embodiments can include a method further comprising growing a hybrid mushroom culture to yield hybrid mushrooms and parts of the mushrooms. Other embodiments can provide a method in which the produced hybrid mushroom culture, or cell, includes a marker profile with specific traits at marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AF, and FF, and all of the traits of the marker profile are also present in the marker profiles of the J20176 and J19109 strains. Still other embodiments can provide a method in which the produced hybrid mushroom culture, or cell, includes a marker profile with specific traits at the marker loci described in Table I, and all of the traits of the marker profile are also present in the marker profile of the J20176 strain.

[0061] These and other advantages of the present invention over existing prior art with respect to Agaricus bisporus mushrooms and cultures will become apparent from the following description and are realized by the present invention as described and claimed below.

DETAILED DESCRIPTION OF THE INVENTION

[0062] First, in order to provide a clear and consistent understanding of this specification and the claims, the following explanations are provided, including the scope in which such terms are indicated.

[0063] Allele: One or more alternative forms of a gene that occur by mutation and are found at the same location on a chromosome; a genetic unit of the genome at a specifically prepared locus that is ultimately identified by its DNA sequence (or by other means).

[0064] Aggressive Incompatibility: An interaction between two heterokaryons in which the two cultures show strong antagonism towards each other and the reaction is much more severe than a typical heterokaryon incompatibility reaction. At the mushroom farm level, in both the compost and the casing layer, large areas where the mycelium has died are observed, resulting in a yield loss of at least 15%, more commonly at least 50%. In the laboratory, the presence of just 1% of J15987 (a culture with the Aggressive Incompatibility (AI) trait) can kill U1 EDVs such as A-15. Generally, as described above, if more than 15% of A-15 is killed by J15987 or another strain, it can be said that the J15987 or other strain has the AI trait. Over time, the strain with the AI trait replaces the U1 EDV and becomes the only genotype present.

[0065] Amphithallism: A reproductive behavior pattern in which both heteromixis and intramixis are active.

[0066] Anastomosis: The fusion of two or more hyphae that realizes cytoplasmic continuity.

[0067] Basidiomycetes: A monophyletic group of fungi that produce meiospores in basidia; members of the corresponding subphylum of fungi such as Basidiomycetales or Basidiomycotina.

[0068] Basidium: A meiosporangium cell in which karyogamy and meiosis occur, resulting in the formation of basidiospores.

[0069] 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 inoculation with spawn, for any given sampled growing area or compost weight.

[0070] Breeding: The development of strains, lines or varieties using methods that emphasize sexual mating.

[0071] Cap: Pileus; A part of the mushroom, a structure with folds.

[0072] Cap flatness: A criterion for evaluating the shape or thickness of the cap of a mature, open mushroom.

[0073] Cap roundness: Strictly speaking, the ratio obtained by bisecting the mushroom vertically and measuring the maximum distance between the top and bottom of the cap, divided by the maximum distance across the cap; generally, the average over many specimens; subjectively, the "rounded" characteristic of the cap shape.

[0074] Carrier substrate: A medium having both nutritional and physical properties suitable for achieving both growth and dispersion of a culture; examples are substrates formulated for mushroom spawn, casing inoculum, and other inoculum materials.

[0075] Casing layer, casing: A layer of non-nutritive material such as peat or soil applied to the upper surface of a mass of colonized compost to enable the development of mushroom crops.

[0076] Casing inoculum (CI): A formulation of an inoculum incorporating a generally specially prepared heterokaryotic strain of mushroom culture suitable for mixing into the casing layer.

[0077] Cloning: somatic cell propagation without selection.

[0078] Combining ability: the ability of an individual to transmit superior performance to its progeny. General combining ability is the average performance of an individual in a specific set of matings.

[0079] Compatibility: see heterokaryon compatibility, nutritional compatibility, sexual compatibility; incompatibility is the reverse of compatibility.

[0080] CRISPR: (Clustered Regularly Interspaced Short Palindromic Repeat) a genetic engineering technique for modifying the genome of a living organism.

[0081] Culture: a living organism with substance; an organism grown on various growth media and substrates; part or all of one physical strain, line, homokaryon or heterokaryon; a collection of all parts of a culture, including hyphae, mushrooms, spores, cells, nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes and cell walls.

[0082] Cultivar: a variety or strain cultivated commercially

[0083] Derivation: the generation or acquisition of a culture from or mainly from only the first strain or culture; see EDV. The terms "deriving" and "derived" refer to this process or its result.

[0084] Derived line group: a set of derived EDVs of a single first strain, including the first strain.

[0085] Pedigree: a phylogenetic pedigree over a limited number (e.g., 10 or less) of generations.

[0086] Diploid: having two haploid chromosome sets within a single nuclear membrane.

[0087] Directed variation induction: The process of altering the DNA sequence at at least one specific genetic locus.

[0088] EDV (Essential Derivative Variety): A culture derived only from or mainly from the original strain or culture; a culture in which more than 75% of its genotype is that of the original strain and the situation is the result of such derivation. When directly derived from or obtained alone from the original strain or culture, the culture is likely to have all of the genotype of the original culture.

[0089] Meat thickness: The ratio obtained by measuring the mushroom longitudinally bisected and dividing the maximum distance between the upper end of the stalk and the topmost part of the cap by the maximum distance across the cap; generally the average over many specimens; subjectively referred to as "thick-fleshed".

[0090] Flush: The period during which mushrooms occur within a cropping cycle, separated by intervals during which no mushrooms occur; the term "flush" encompasses the terms "break" and "wave" and can be read as either of these terms.

[0091] Fungus: A microorganism classified as a member of the kingdom Fungi.

[0092] Gene editing: Generally, the process of altering the sequence of a functional gene to inactivate the gene, or of altering a specific gene by a CRISPR-Cas9 or similar enzyme system. In other uses, new sequences (including genes) can be introduced into the genome.

[0093] Phylogenetic relatedness: Descent from one or more ancestors, e.g., the genealogical relatedness between parent and offspring.

[0094] Genetic identity: Genetic information for identifying an individual, including, for example, the display of such genetic information, and including genotype, genotype fingerprint, genomic sequence, gene marker profile; "genetically identical" = 100% genetic identity, "X% genetically identical" = having X% genetic identity, etc.

[0095] Genotype fingerprint: Description of the genotype in a specially prepared set of marker loci; known genotype.

[0096] Gill: Lamella; a part of a mushroom, a structure having a hymenium and basidia.

[0097] Haploid: Having only a single set of nuclear chromosomes; see homokaryon.

[0098] Heteroallelic: Having two different alleles at a locus; similar to heterozygosity.

[0099] Heteroallelism: Difference between homologous chromosomes in a heterokaryotic genotype; similar to heterozygosity.

[0100] Heterokaryon: As a technical term, a sexual heterokaryon: having two complementary (i.e., always heteroallelic at the Mat locus) types of haploid nuclei in a common cytoplasm, and thus functionally and physiologically similar to a diploid individual (however, cytogenetically represented as N+N rather than 2N), fertile (in the absence of any rare interfering genetic defects at loci other than Mat), and referring to a culture that shows a nutritional incompatibility reaction to other heterokaryons; also referred to as a strain or stock in the context of strain development.

[0101] Heterokaryon compatibility: The absence of antagonism observed in a state of physical proximity or contact between two genetically non-identical heterokaryons; see heterokaryon incompatibility.

[0102] Heterokaryon incompatibility: The phenomenon of antagonism observed in a physical proximity or contact state between two heterokaryons that are not genetically identical; a multi-locus self / non-self recognition system; that is, a genetic system that enables one heterokaryon culture to discriminate and recognize another culture as either self or non-self in order to restrict anastomosis (hyphal fusion) and cytoplasmic contact in basidiomycete heterokaryons; nutritional incompatibility.

[0103] Heterokaryosis: Having the trait of a heterokaryon.

[0104] Heteromixis: A life cycle involving mating between two different non-sibling haploid individuals or gametes; similar to outcrossing.

[0105] Homoallelism: Not having more than one allele at a locus. The equivalent term for a diploid organism is "homozygosity". Haploid lines are, by definition, completely homoallelic at all non-duplicate loci.

[0106] Homokaryon: A haploid culture having a single type (or somatic line) of haploid nuclei (represented cytogenetically as N), usually infertile, not showing the typical self / non-self incompatibility reaction to heterokaryons, and capable of functioning as a gamete in sexually complementary anastomoses; a "line" that transmits a uniform genotype to its progeny, similar to an inbred plant line; predominantly homoallelic lines that mate well and have insufficient fruiting body formation are presumed homokaryons for strain development; see the discussion below.

[0107] Homokaryosis: Having the trait of a homokaryon; haploid.

[0108] Hybrid: Of biparental origin and usually applied to heterokaryon strains and cultures produced by controlled mating.

[0109] Hybridize: To attempt to effect anastomosis, cytoplasmic fusion, and the formation of a sexual heterokaryon (= mating) between two cultures, usually homokaryons, for example, by physical association on a Petri dish containing a sterile agar-based nutrient medium; the success of the foregoing.

[0110] Hyphae: Thread-like elements of the mycelium, composed of cell-like compartments.

[0111] Inbreeding: Mating that includes sib-mating, backcrossing to parental lines or strains, and intramixis; reproduction involving genetically related parents.

[0112] Incompatibility: See heterokaryon incompatibility.

[0113] Induced mutagenesis: A non-spontaneous process that alters the DNA sequence of at least one genetic locus.

[0114] Primary culture: A culture used as starting material in a strain development process; more specifically, a strain from which a variety is obtained.

[0115] Inoculum: A culture in a form that allows for transfer and growth, e.g., in a new medium; spawn and CI are examples of specialized commercial forms of inoculum.

[0116] Intramixis: A uniparental sexual life cycle involving the formation of complementary "mated" pairs of post-meiotic nuclei within a basidium or individual spore.

[0117] Backcross transformation: Crossing the progeny of a hybrid to a parental line or strain, and thus introducing a desired trait from one strain into the dominant genetic background of another parental line or strain.

[0118] Lamella: See "gill".

[0119] Strain: A culture used in mating to produce hybrid strains; usually a homokaryon and thus homozygous or, if not, a highly homozygous non-heterokaryon (non-NSNPP) culture; in practice, a functionally homokaryotic and completely or mainly homozygous culture; similar to a mainly or completely homozygous inbred line in plant breeding.

[0120] System group: See "derived system group". A set of EDV derivatives from a single original strain or variety.

[0121] Locus: A specifically prepared continuous part of the genome that is homologous but often varies between different genotypes; plural: loci.

[0122] Marker-assisted selection: Using linked genetic markers including molecular markers to track the trait-determining locus among progeny and through pedigrees.

[0123] MAT: Mating type locus that determines sexual compatibility and heterokaryosis.

[0124] Mating: Sexual union by anastomosis and cytoplasmic fusion of two cultures; methods for obtaining mating between mushroom cultures are well known in the art.

[0125] Mycelium: The vegetative or thalloid body of the mushroom organism, composed of thread-like hyphae.

[0126] Mushroom: The reproductive structure of fungi of the family Agaricaceae; agaric; a cultivated food of the same name.

[0127] Neohaplont: A haploid culture or strain obtained by physically deheterokaryotizing a heterokaryon (reducing it to haploid components); a homokaryon obtained somatically.

[0128] Progeny: Offspring within a single generation, e.g., of a parental heterokaryon; often used to describe a culture obtained from spores of a single mushroom of a single strain.

[0129] Outcrossing: Mating between unrelated or distantly related individuals; similar to heteromixis in mushrooms.

[0130] Parent: The most recent ancestor of an individual; the parental strain is a heterokaryon and the parental line is a homokaryon; a heterokaryon can be the parent of an F1 heterokaryon through an intermediate parental line.

[0131] Pedigree stock development: Identifying desirable combinations of lines in a controlled mating program using phylogenetic information.

[0132] Phenotype: Observable characteristics of a strain or line that are expressed and manifested in an environment.

[0133] Cytoplasmic fusion: Establishment of cytoplasmic continuity by anastomosis leading to the formation of a sexual heterokaryon.

[0134] Ancestor: An ancestor including the parent (direct ancestor).

[0135] Selfing: Mating between sibling lines; similar to intramixis.

[0136] Sexual compatibility: A state in which two different lines with non-identical alleles at the Mat locus can mate to produce a stable and fertile heterokaryon. The opposite state of sexual incompatibility occurs when two lines have the same allele at the Mat locus.

[0137] Somatic cell: A cell of a vegetative mycelium.

[0138] Spawn: A mushroom culture, generally a sterile substrate of easily crushed and dispersible particulate matter, and in some cases grains, on which a pure culture of a heterokaryon is generally grown; a commercial inoculation material for compost; reference to spawn includes reference to a culture on a substrate.

[0139] Spore: A part of a mushroom, a reproductive propagule.

[0140] Stem: The stipe; a part of a mushroom, the structure that supports the cap.

[0141] Axenic growth medium: A nutrient medium that supports the growth of organisms and has been sterilized by autoclaving or other methods; examples include agar-based solid nutrient media such as potato dextrose agar (PDA), nutrient broth, and many other materials.

[0142] Stipe: See "stem".

[0143] Strain: A heterokaryon having specially prepared characteristics or a specific identity or ancestry; similar to a variety.

[0144] Targeted mutagenesis: A process of altering the DNA sequence at at least one specific genetic locus.

[0145] Tissue culture: Dedifferentiated vegetative hyphae obtained from differentiated tissues of a mushroom.

[0146] Transformation: A method for selectively introducing the genetic determinants of one or more desired traits (single-locus transformation) into the genetic background of the first strain while retaining the majority of the genetic background of the first strain. See "gene transfer transformation" and "transformation".

[0147] Transformation: A process by which the genetic material of individual cells is altered by incorporating foreign (exogenous) DNA into its genome; a method for obtaining transformation including single-locus transformation.

[0148] Nutritional compatibility: The absence of the phenomenon of antagonism observed in a state of physical proximity or contact determined by a multi-locus self / non-self recognition system that operates to limit anastomosis (hyphal fusion) and cytoplasmic contact in basidiomycete heterokaryons between two genetically non-identical heterokaryons; heterokaryon compatibility; the reverse of nutritional incompatibility.

[0149] Nutritional incompatibility: A phenomenon of antagonism observed in a state of physical proximity or contact between two genetically non-identical heterokaryons, determined by a multi-locus self / non-self recognition system that operates to restrict anastomosis (hyphal fusion) and cytoplasmic contact in basidiomycete heterokaryons; heterokaryon incompatibility.

[0150] Virus break: The continuous use of multiple incompatible strains, i.e., strains showing heterokaryon incompatibility, in a programmed strain rotation in a mushroom production facility to reduce the transmission of viruses from the on-site virus reservoir to newly planted crops.

[0151] Whole genome sequence (WGS): The DNA sequence of an organism such as Agaricus bisporus

[0152] Yield: The net fresh weight of the harvested crop, usually expressed in pounds per square foot.

[0153] Yield pattern: The distribution of yield within each flush and between all flushes; affects the size, quality, harvesting cost, and relative disease pressure of the crop and product.

[0154] Regarding the above definition of homokaryons, homokaryons and homozygous lines are the subject of technical and practical considerations: Note that a homokaryon is a haploid culture that is clearly completely homozygous in classical terms. In practice, with respect to the purpose of developing fungal strains, this definition is somewhat broadened to accommodate both technical limitations and cytological variations, mainly by treating all homozygous lines as homokaryons. As a technical limitation, the fact that the genome contains duplicated DNA regions with repeated elements such as transposons, and may also contain large duplications of chromosomal segments due to past transposition events, and such regions may appear non-homozygous by most genotyping methods, can be cited. Two different A. bisporus genomes sequenced by the Joint Genome Institute, a US federal government agency, differ by an estimated length of 4.4% and gene number of 8.2%, suggesting a significant amount of DNA duplication or rearrangement within different strains of the species. The currently available A. bisporus genomes cannot fully account for the physical arrangement of such elements and transpositions, and thus the assembled genome sequences of haploid lines may have regions that appear heterozygous using currently available genotyping methods. Cytologically, homokaryon progeny usually become spores that receive one haploid post-meiotic nucleus. However, spores that receive two third-division nuclei from the basidium become genetically equivalent to homokaryons. Spores that receive second-division "sister" post-meiotic nuclei become functional homokaryons, although there may be some terminal "islands" of heterozygosity due to crossovers during meiosis. Also, in meiosis where homologs segregate asymmetrically, there may be spores that are aneuploid functional homokaryons with extra chromosomes that give rise to regions of heterozygosity. All of these cultures are highly homozygous and all function as homokaryons. Due to scientific and technical limitations, it becomes impossible to distinguish such cultures, and it also becomes impossible to exclude DNA segment duplications as an explanation for limited isolated regions of genome sequence assemblies that appear heterozygous.Accordingly, in the present application, the use of the term "homoallelism" to characterize a strain encompasses entirely or mainly homozygous strains, and cultures so described are functional homokaryons, presumably homozygous, and all are defined as homokaryons in the present application.

[0155] Note that cultures of strains that are descendants of the B18287-s82 and / or WBL-s290 strains, such as J19109, give rise to mushrooms, parts of mushrooms, parts of cultures, as well as strains and lines that are descendants or derived from such cultures. Accordingly, the present invention encompasses cultures and parts of cultures of the B18287-s82 and WBL-s290 strains, as well as the F1 hybrid J19109 strain, mushrooms and parts of mushrooms, including spores, that result therefrom. Further, EDVs and cultures derived only or mainly from the first cultures derived from the F1 progeny of B18287-s82 and / or WBL-s290, cultures that are dormant or actively growing present in the dormant or germinated spores of the J19109 strain, and cultures into which the genetic material of the F1 progeny of the J19109 strain has been incorporated.

[0156] The present invention further relates to methods for the production and use of the J19109 strain and dependent varieties (EDVs) of the J19109 strain. The use of cultures derived from J19109 and the other cultures described above includes their incorporation into commercial products such as mushroom spawn and casing inoculation materials, as well as mushroom production, the development of additional new cultures of A. bisporus, and farm hygiene including crop diversification and "virus breaking".

[0157] Here, with respect to the present invention and as described above, the present invention further relates to the F1 strain J19109, its isogenic lines, and its F2 hybrid derivatives, for example, hybrids (i.e., crossbreeds) created between J19109 SSI isogenic line descendants and other white isogenic lines, such as J11500-s80. Further, the present invention encompasses cultures derived from or descendants of the J19109 strain. Such cultures can be used to produce mushrooms and parts of mushrooms.

[0158] Details of the mating are described herein with respect to J19109. Specifically, one particular J19109-s40 line is mated with J11500-s80 to yield the hybrid J20176 strain. A representative culture of the strain, J20176, is deposited under the NRRL accession number ^^^^^. In one embodiment, the method further includes growing an edible mushroom crop by performing the steps described above herein. In another embodiment, the method can include using the J20176 strain or a derivative variety of the J20176 strain in crop rotation to reduce pressure from pathogens and pathogen reservoirs in a mushroom growing facility as described above herein. In yet another embodiment, the method includes using the J20176 strain and the EDV of the J20176 strain to produce descendants as described above herein.

[0159] Strains that are descendants of the J19109-s40 and J11500-s80 lines, such as the culture of J20176, are noted to give rise to mushrooms, parts of mushrooms, parts of the culture, as well as strains and lines that are descendants of or derived from such cultures. Thus, the present invention encompasses the J19109-s40 and J11500-s80 lines, as well as cultures and parts of cultures of the hybrid J20176 strain, parts of mushrooms including the mushrooms and spores resulting therefrom. Further, EDV and cultures derived only from or mainly from the first culture derived from descendants of J10109-s40 and / or J11500-s80, cultures that are dormant or actively growing present in the dormant or germinated spores of the J20176 strain, and cultures into which the genetic material of descendants of the J20176 strain has been incorporated.

[0160] The present invention further relates to methods for the creation and use of the J20176 strain and Deposited Derivative Varieties (EDV) of the J20176 strain. The use of cultures derived from J20176 and the other cultures described above includes incorporation into commercial products such as mushroom spawn and inoculation materials for casing, as well as mushroom production, the development of additional new cultures of A. bisporus, and farm sanitation including crop diversification and "virus breaking".

[0161] The morphological and physiological characteristics of the J20176 strain cultured in Difco brand PDA medium, a standard culture medium, are presented as follows. The J20176 strain grown in PDA medium in a Petri dish with a diameter of 8.5 cm was irregularly leaf-shaped, white or light tea-yellow or "tan", with an overall nearly circular outline, and during the growth in a dynamic equilibrium state between the 8th and 26th days after inoculating the PDA with a circular plug with a diameter of 3.5 mm using the culture as the inoculation material, it produced colonies that increased in diameter at a rate of (1.32~1.48~)1.49(~1.50~1.57) mm / day. The strain was increased by transferring a pure inoculation material to a larger volume of sterile culture medium. There are no variant traits observed or predicted in the J20176 strain.

[0162] Methods for obtaining, manipulating, and crossing cultures of the present invention for creating progeny, inoculum, products, and crops of the present invention, for improving mushroom farm hygiene using a strain rotation program, and for obtaining genotype fingerprints of mushroom cultures are described herein and are well known to those skilled in the art. Although the present invention has been described with respect to specific embodiments in this application, those skilled in the art can, in light of the teachings herein, generate additional embodiments and modifications without departing from the spirit of the claimed invention and without exceeding its scope. Accordingly, it is understood that the description herein is provided merely to facilitate understanding of the present invention and should not be construed as limiting the scope of the present invention.

[0163] The J20176 hybrid strain is a hybrid created between J19109-s40 and J11500-s80. The J2076 hybrid does not surely have the AI trait (see Table IV below). Furthermore, the J20176 strain has a strong combination of desirable traits derived from the J15987 pedigree chart. When the strain 20176 was grown in pre-commercialization tests in North America, Europe, and China, it had strong commercial potential.

[0164] The crossing of the Agaricus bisporus cultures of the present invention can be achieved by growing two different cultures, one of which is the genetic lineage present in the spores of the J19109 strain, preferably on a sterile medium, in a very close proximity to each other until anastomosis (i.e., hyphal or cell fusion) occurs. In a successful mating, the resulting fused culture is a first-generation non-inbred hybrid culture incorporating the genome derived from the lineage obtained from the J19109 strain. This lineage may be obtained from the mushroom spores of J19109, which is part of one embodiment of the present invention. Alternatively, this lineage may be obtained from protoplasts derived from the basidia, or other parts of the organism of J19109, which is another part of one embodiment of the present invention.

[0165] Regarding the object of the present invention, the whole-genome DNA sequences of the J19109 strain and its constitutive homokaryons (B18287-s82 and WBL-s290) were obtained. Also, the genomic DNA sequences of the J11500-s80 line and the J19109-s40 line (the parent of J20176) were obtained. To prepare DNA, the cultures were grown in a sterile broth growth medium after dissociation. After 2 to 4 weeks, the hyphal cells were collected by filtration, frozen at -80°C, and freeze-dried until dry. The cap tissue was obtained from the mushrooms resulting from the cultures of the heterokaryon (such as J20176), frozen, and freeze-dried. From the freeze-dried samples, DNA was extracted using the CTAB protocol, followed by ribonuclease (RNAse) treatment and gel purification. A DNA library was prepared from the DNA of each culture by the contractor Genewiz (New Brunswick, New Jersey), and sequencing of the library using Illumina technology was performed. The assembly of reads into genomic sequences was carried out by Sylvan, Inc. using the public domain reference genomic sequence of H97 version 2.0 (Morin et al. 2012; PNAS 109(43):17501, incorporated herein by reference). As a result, Sylvan, Inc. knows with certainty about approximately 93% to approximately 95% of the entire genotype of the J20176 strain and its parental homokaryons.

[0166] Although the understanding in the industry regarding the genetic control of heterokaryon incompatibility is limited, it is known to be separated from the well-known mating type gene(s), and that more than one species, for example, 4 or 5 genes in Coprinopsis, are involved and the locations on the chromosomes are not defined.

[0167] In the 2010s, powerful techniques based on SNP analysis became available. Each SNP marker represents a difference in a single DNA building block called a nucleotide. For example, in one mutation, an "A" can become a "G". Furthermore, whole-genome sequencing (WGS) technology, especially using devices manufactured by Illumina, can obtain large-scale genome coverage exceeding 50-fold. These "Illumina reads" can then be aligned to a reference genome using appropriate software. The reference genome is important because it can numerically define the exact location of each SNP within the genome using base pair positions.

[0168] Table I presents a comparison based on SNPs of the genotypes of the cultures used to create the invention described herein. To obtain the whole-genome sequences, Illumina 250bp reads aligned to the H97 version 2.0 reference genome using DNASTAR's Lasergene version 18 were utilized. The genotypes of the J19109 strain and J20176 strain and their parental lines B18287-s82 and WBL-s290 (for the J19109 strain) and J11500-s80 and J19109-240 (for the J20176 strain) are presented in Table I at marker loci characterized by a number of sequences distributed at intervals along each of 19 H97 Version 2.0 reference scaffolds larger than 100Kbp in length.

[0169]

Table 1

[0170] The SNP pattern of each strain and line is unique, and thus it can be clearly confirmed that it is a robust form of genetic identification. Furthermore, any person skilled in the art can verify the SNP markers described in Table I.

[0171] The data in Table I are shown as 9mers, and in most cases, the central base (position 5) gives rise to the SNP. Robust markers were selected at positions aligned across each of the first 19 scaffolds. The majority of the markers in Table II are standard markers used in other patent cases, e.g., US9,017,988 and US10,440,930. The SNP alleles at those positions were determined for the homokaryotic parents (J11500-s80 and J19109-s40) of J20176, in addition to B18287-s82, WBL-s290, J19109 heterokaryon, and J11500-s80.

[0172]

Table 2

[0173] Table II reports data from six genetic loci that are standardly reported for the same panel of strains used in Table I, e.g., US9,017,988 and US10,440,930. As in Table II, for one heterokaryotic strain (J19109), there are two alleles at each marker locus, and for the six homokaryotic cultures, there is a single allele per locus. Data were generated by using targeted polymerase chain reaction (PCR) to amplify genomic regions spanning specially prepared markers from each of the culture DNAs. Data were generated using PCR primers flanking the specially prepared marker regions at the locations indicated by the position information presented below; methods for designing appropriate primers are well known in the art. From the amplified PCR products, DNA sequencing was performed by the contractor Eurofins (Louisville, Kentucky) using a selected method, and the genotypes were determined by directly comparing these sequences to the Sylvan database of reference marker / allele sequences. In most cases, the sequences were further confirmed by directly examining the corresponding whole-genome sequences for those cultures.

[0174] Description of p1n150 - G3 - 2 Marker: The 5' end of this marker segment starts from the first 'T' of the sequence TCCCAAGT corresponding to position 1868615 of H97 JGI V2.0 scaffold 1 (Morin et al. 2012), in the reverse direction (relative to the orientation of the scaffold), and spans approximately 600 nt in most alleles; an insertion into the DNA of allele 1T results in a longer segment. Currently, 9 alleles incorporating at least 30 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain collection. Since this marker is closely associated with the MAT locus, it is an important part of the Agaricus breeding program. MAT is a complex of genes that controls mating between homokaryons.

[0175] As described above, allele 1T contains a 320 - base - pair transposon. All other known alleles of p1n150 lack this intron.

[0176] Analysis using p1n150 of a panel of 88 J19109 - derived homokaryotic SSI identified 52 matches of p1n150 alleles for Mat - 1, and a second cohort of 36 strains containing a different mating - type allele was identified and tentatively named "Mat - X".

[0177] To generate new hybrids, a complete set of Mat - X homokaryons was crossed with a panel of J11500 SSI homokaryons known to be MAT - 2. Surprisingly, none of these hybrids were successful, and it was concluded that MAT - X is functionally identical to MAT - 2. Since the other parental homokaryon of B18287, So76 - s12b, is known to be MAT - 1, the MAT - 2 allele in J19109 must be a descendant of the wild - type W01 - s1 homokaryon.

[0178] The next step was to align the DNA from the p1n150 amplicon derived from W01-s1, B18187-s82, and J11500-s80 to examine whether the p1n50 sequence of MAT-2 was a perfect match. The data showed a difference between the sequence of J11500-s80 MAT-2 and the sequences that W01-s1 and B18187-s82 had. Therefore, the new allele of p1n150 discovered in W01-s1 and B18287-s82 was named "2A".

[0179] Allele 1T: Insertion of a 320-nt ABR transposon @206^207, "A" @321; "T" @327; "C" @374; "G" @378; "G" @422; etc.

[0180] Allele 2: No Abr1 insertion; "A" @321; "C" @327, "C" @374; "C" @378; "G" @422; etc

[0181] Allele 2A: No Abr1 insertion; "A" @321; "C" @327, "T" @374; "C" @378; "T" @422; etc

[0182] Description of the ITS (= ITS 1+2 region) marker: The ITS segment is part of the nuclear rDNA region located on chromosome 9 (scaffold 10 of JGI H97 V2.0). rDNA is a cassette that is tandemly repeated up to an estimated 100 times in the haploid genome of A. bisporus. Therefore, there is no single exact positioning of this sequence in the assembled H97 genome, and in fact, it is difficult or impossible to accurately assemble the sequence across all tandem repeats. On scaffold 10 of the H97 JGI V2.0 assembly, three cassette copies starting at position 1,612,110 were included; partial copies were also assembled onto scaffold 29 (Morin et al. 2012). The 5' end of this marker segment starts with the first "G" of the sequence GGAAGGAT at position 1 and extends forward (relative to the orientation of the scaffold) for approximately 703 - 704 nt in the majority of alleles. To date, more than nine alleles incorporating at least 11 polymorphic positions have been demonstrated from diverse strains in Sylvan's microbial strain collection.

[0183] The alleles present in this application are I1, I2, and I4. They are characterized as follows (format: using nucleotide base symbols @ alignment positions, based on the alignment of nine alleles).

[0184] Allele I1: "C" @ 52; "T" @ 461; "T" @ 522; "T" @ 563; etc.

[0185] Allele I2: "T" @ 52; "T" @ 461; "T" @ 522; "T" @ 563; etc.

[0186] Allele I4: "C" @ 52; "A" @ 461; "C" @ 522; "C" @ 563; etc.

[0187] Description of the MFPC-1-ELF marker: The 5' end of this marker segment starts with the first "G" of the sequence GGGAGGGT, corresponding to position 8829770 of the H97 JGI V2.0 scaffold (Morin et al. 2012) from position 1, and extends forward (relative to the orientation of the scaffold) for approximately 860 nt in the majority of alleles. Currently, at least 7 alleles incorporating at least 40 polymorphic positions have been demonstrated from diverse strains in Sylvan's microbial strain collection.

[0188] Note that MFPC-ELF is associated with the PPC-1 locus on scaffold 8, which has been found to be a major factor controlling the color of the cap. Mushrooms with a white cap have the E1 allele in MFPC-ELF.

[0189] Allele E1: "A" @ 63; "A" @ 77; "A" @ 232; "A" @ 309; "T" @ 334; "A" @ 390; "A" @ 400; "T" @ 446, "A" @ 481; etc.

[0190] All strains in Table II shared the E1 allele in MFPC-ELF.

[0191] Description of the AN marker: The 5' end of this marker segment starts with the first "G" of the sequence GGGTTTGT, corresponding to position 1701712 of the H97 JGI V2.0 scaffold 9 (Morin et al. 2012) from position 1, and extends forward (relative to the orientation of the scaffold) for approximately 1660 (H97 genome) - 1700 nt (alignment interval) in known alleles; several insertions / deletions have created length polymorphisms that characterize alleles in addition to single nucleotide point mutations. Currently, at least 5 alleles incorporating more than 70 polymorphic positions have been demonstrated from diverse strains in Sylvan's microbial strain collection.

[0192] There is only one allele, N1, present in the strain used in this application (format: using nucleotide base symbols @ alignment positions, based on the alignment from alleles N1 to N4):

[0193] Allele N1: "G" @ 640; [Deletion] @ 844 - 846; "C" @ 954; "T" @ 882; "A" @ 954, etc.

[0194] All strains in this test shared the same N1 allele in scaffold 9.

[0195] Description of the AS marker: The 5' end of this marker segment starts from position 1 with the first "G" of the sequence GG(T / N)GTGAT corresponding to position 4752867 of H97 JGI V2.0 scaffold 4 (Morin et al. 2012), and extends forward (relative to the orientation of the scaffold) over approximately 1620 (H97 genome) - 1693 nt (alignment interval) in known alleles; several insertions / deletions have created length polymorphisms that, in addition to point mutations of individual nucleotides, characterize the alleles. Currently, 7 alleles incorporating over 80 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain collection.

[0196] The alleles present in the B18287 / J19109 phylogenetic tree are alleles SC and SD, and are characterized in part as follows (format: using nucleotide base symbols @ alignment positions, based on the alignment from alleles SA to SG):

[0197] Allele SC: "T" @ 28; "GATATC" @ 258 - 263; "G" @ 275; [Insertion] + "TTTC T CAGC" + [Insertion] @ 309 - 249; "C" @ 404, etc.

[0198] Allele SD: "C" @ 28; [Deletion] @ 258 - 263; "T" @ 275; [Deletion] @ 309 - 249; "T" @ 275; [Deletion] @ 309 - 349, "T" @ 404, etc.

[0199] Description of the FF marker: The 5'-end of this marker segment starts with the first "T" of the sequence TTCGGGTG, corresponding to position 12281999 of the H97 JGI V2.0 scaffold (Morin et al. 2012) from position 1, and extends forward (relative to the orientation of the scaffold) for approximately 570 nt in the majority of alleles. Currently, 7 alleles incorporating at least 20 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain collection.

[0200] Two alleles are found in the strain used to create the present invention; FF1 and FF2.

[0201] Allele FF1: "CCG" @ 48 - 50, "C" @ 91, etc.

[0202] Allele FF2: "TTC" @ 48 - 50, "C" @ 91, etc.

[0203] The FF1 genotype is found in white strains such as So76 and U1, as well as related EDVs, e.g., A15. In this test, this marker was inherited by one of the breeding strains, So76-s12b.

[0204] Having the FF2 genotype enables the identification of W0-s01, B18287-s82, WBL-s290, J19109, and J11500-s80 (and related EDVs and direct descendants).

[0205] As described above, one of the objectives of the J19109 and J20176 series of diagrams was to develop a method for eliminating the Aggressive Incompatibility (AI) trait while preserving as many of the positive traits seen in J15987 as possible. To test this concept, a test protocol was designed to measure the effect of mixing a small amount of inoculum from an F2 hybrid strain having a J19109-derived line as one of the homokaryotic parental strains and J11500-s80 as the second homokaryotic parental strain, or from a control strain, with compost inoculated with the commercially available A-15 strain. Within the scope of this experimental design, normal incompatibility interactions are not thought to interfere with the overall mushroom yield, but strains having the antagonistic trait of J15987 are thought to significantly reduce, and even zero, the yield of A-15.

[0206] Sixteen F2 strains were selected for this test (see Table III below), and J15987 and A15 were used as positive and negative controls, respectively. The yield of A-15 was obtained in a replicated paired-plot test. In the normal cultivation of Agaricus bisporus, the first break of mushrooms is harvested over a period of 3 to 4 days. Skilled harvesters collect the earliest mushrooms and leave the space filled with mushrooms as the crop grows. In contrast, when the AI trait is expressed, very few mushrooms are produced. The vast mushroom beds are barren, and there are also some areas where a few mushrooms may appear. In other words, the AI trait results in a catastrophic reduction in mushroom yield. Generally, the yield reduction due to heterokaryotic incompatibility or nutritional incompatibility can be 10% or less, or in most cases 5% or less. However, the AI characteristic is catastrophic enough that a reduction of more than 50% is not uncommon. However, for the purposes of the present invention, the AI trait is defined as having a post-test yield reduction of 15% or less of the A15 control yield.

[0207] The next step was to search for correlations between Aggressive Incompatibility and the chromosomes present in strains #1, #16 and J15987. To determine the genotype of the J19109 homokaryon, which is the parent of each strain, one amplicon per chromosome was amplified using PCR. These markers were located as close as possible to the center of each chromosome. Note that the chromosomes of A. bisporus are very generally inherited in non-recombinant units and can be tracked to a first approximation by a single marker located at the center. In Table III below, the nine chromosomes were characterized according to which parent (B18287-s82 or WBL-s290) contributed each chromosome.

[0208] Agaricus bisporus has 13 chromosomes per homokaryon genome (Foulogne-Oriol et al. 2010). Four chromosomes were not included in the analysis of this study: 1) Chromosome 1 contains the MAT locus that controls mating in Agaricus bisporus. A cohort of the J19109 test strains was pre-screened for the MAT-1 allele. This is because 50% of the remaining strains obtained were not compatible with J11500-s80. 2) Chromosome 3 was 100% biased towards the allele of WBL-s290. 3) Chromosome 6 was 100% biased towards the allele of WBL-s290. 4) Chromosome 12 was 100% biased towards the allele of B18287-s82.

[0209] Such extreme bias, such as in the latter three chromosomes, is likely to be caused by the influence of deleterious alleles, but in any case, segregation at that locus was virtually not observed, and thus, analysis of the correlation between genotype and phenotype was impossible. However, since both marker segregation and phenotypic segregation were observed in the analysis performed, the four chromosomes listed above do not appear to play a major role or any role at all in conferring the AI trait phenotype.

[0210] Therefore, for this test, there was a panel of 9 informative chromosomes.

[0211]

Table 3

[0212] Table III shows the yields and genetic alleles for hybrid strains obtained by individually crossing J11500-s80 from 16 different J19109 isogenic lines to form F2 hybrid strains. This list includes J20176. In this test: For each strain, three 2.76 square foot containers (「tubs」) were each inoculated with 55 lb of Phase II mushroom compost. Yield was the sum of the three tubs.

[0213] For each treatment in the table, including J20176, 1 wt% of the relevant strain inoculum was thoroughly mixed with 99% A15 inoculum, and the mixed inoculum was thoroughly combined with the compost. Mycelial growth was completed on day 15, and a casing layer was applied. For each treatment in Table III, casing soil inoculated with A15 was added (at a rate of 1.5% calculated using the dry weight of the casing soil).

[0214] Mushrooms were allowed to emerge to a harvestable state from day 16 to day 20, and on day 20, only the first break of production was harvested, ending the test.

[0215] The yield ratio is shown in the third column. This number was calculated by dividing the total yield of each treatment by the A15 control yield.

[0216] Finally, the other columns in Table III show the allelic inheritance of 9 Agaricus chromosomes, indicating whether the allele was inherited from one or the other of the parents (B18287-s82 and WBL-s290) of J19109. Note that the parental isogenic lines in the table are abbreviated as 290 for WBL-s290 and s82 for B18287-s82 to fit the table.

[0217] In the presence of strain #1, strain #16, and J15987, it was clearly observed that the yield of A15 crops was much lower than when any of the other strains were present. In other words, these strains meet the defined test criteria for the manifestation of the AI trait in the test using A15. By using the yield ratio data, it can be estimated that the yield ratio was 0.15 or less for all of the strain #1, strain #16, and J15987 test treatments. The strain closest to strain #16 was strain #5, and its yield ratio was 0.53. However, as described above, this strain was severely affected by Trichoderma contamination, resulting in a decrease in yield. Therefore, it was proposed that a practical definition of the AI trait in this test protocol include a yield reduction of 15% or less of the A15 control yield.

[0218] Furthermore, when testing the pattern of chromosomal inheritance, it was revealed that all of strain #1, strain #16, and J15987 share the same allele of WBL-s290 on chromosomes 4, 7, and 9. None of the other strains in this test share this haplotype, strongly suggesting a correlation with the aggressive incompatibility trait.

[0219] The probability (chance) that the AI trait occurring randomly with chromosomes 4, 7, and 9 is completely consistent can be calculated using Bernoulli's classical binomial probability formula, P = n × q x (1 - q) n-x (where P is the binomial probability, n is the total number of strains tested, x is the observed number of strains matching a specific genotype, and q is the probability that a specific genotype exists per sample).

[0220] Out of a total of 17 strains tested, 3 strains (2 from the test cohort plus J15987) that match a specific genotype were used. For each strain, the probability that the genotype exists was 0.125 (no significant deviation from the theoretical probability of 0.5 per allele was observed in the observed segregation; with 3 alleles, 0.5 3Using (p = 0.125), the binomial probability calculated for this scenario is 0.0154, that is, a probability (chance) of 1.54%. In other words, the p - value for the possibility that the correlation between the phenotype and the haplotype was due to chance was 0.0154. Conventionally, a p - value less than 0.05 is regarded as strong evidence for rejecting random chance.

[0221] However, the probability of 0.5 for each allele used above assumes Mendel's 50:50 ratio.

[0222] Using the data in Table III, the theoretical probability of 0.5 can be made more precise. The allele of s290 was present at 8 / 17 (= 0.47:0.53) for chromosome 4, 7 / 17 (= 0.41:0.59) for chromosome 7, and 10 / 17 (= 0.41:0.59) for chromosome 9. For such a small sample size, the observed ratio between each allele and its alternative matches well with the expected value of the ideal 50:50 ratio. Thus, overall for all three chromosomes it is 25 / 51, which is 0.49:0.51 (very close to the 50:50 ratio).

[0223] Furthermore, since J15987 is part of another cohort, the binomial probability could be calculated without using the data of J15987, that is, calculated only for J20157 and J20209. As a result, the sample size decreased from 17 to 16. The binomial probability in this case is 7.03%, which is still good odds (93%) that the association between the chromosome and the AI trait is real.

[0224] Overall, statistical analysis indicated that Aggressive Incompatibility is highly likely to be significantly correlated with and thus explained by the effect of incompatibility factors on three specific chromosomes derived from the WBL - s290 parent.

[0225] In conclusion, these data demonstrate that the AI trait is under genetic control and that it is possible to create new hybrids that do not have the AI trait despite retaining the gene structure (and phenotype) present in J15987 in a significant percentage. Finally, there is robust and statistically supported segregation data strongly suggesting that AI is controlled by factors on three separate Agaricus chromosomes, enabling the claimed method to be used with a high degree of predictive confidence.

[0226]

Table 4

[0227]

Table 5

[0228] The data in Tables IV and V above show the productivity of 12 different J19109 SSI isogenic coexistents × J11500-s80 hybrids grown in two separate tests. It is notable that all of this new group of hybrids had higher yields than the A15 test control and were similar in performance to the J15987 control.

[0229] Furthermore, the first:second break ratios were also calculated for both tests. It is clear that J15987 is severely biased towards the first break. The J19109 hybrids also tend to be biased towards the first break, although to varying degrees.

[0230] One of the objectives of the present invention was to demonstrate that the high yield of J15987 is reproduced in hybrids derived from J19109. This is important because this range of productivity is required for the commercial success of a strain. The data in Tables IV and V clearly show that this has been achieved.

[0231] Regarding the data in Table VI and Table VII, significance is indicated by asterisks. For p = 0.05 or less, *; for p = 0.01 or less, **; for p = 0.001 or less, ***; for p = 0.0001 or less, ****.

[0232]

Table 6

[0233] It was further demonstrated that the overall quality of the mushroom shape and color of J15987 was reproduced in the hybrid strains derived from J19109. It is clearly shown from the data in Table VI that this was achieved.

[0234] The measurements of the mushroom caps were obtained using a Storm 3C301 digital caliper. A sample size of 20 medium-sized mushrooms at commercial maturity (diameter 35 - 40 mm, with the veil closed) was harvested and measured to obtain the values of the cap diameter and cap height. Then, the mushrooms were cut in half longitudinally to measure the flesh thickness and the stalk width. The ratios between these values were calculated to obtain the cap roundness (cap height / cap diameter), flesh thickness (flesh thickness / cap height), and stalk thickness (stalk width / cap diameter).

[0235] The cap roundness is an approximate measure of how spherical the mushroom cap appears. A larger value indicates a more rounded mushroom, which is preferred by growers and customers based on visual appeal. A smaller value indicates a flatter mushroom. It is clearly demonstrated that all of the hybrid strains derived from J19109 recorded in Table VI fall within the continuum around J15987 and this trait is retained.

[0236] The thickness of the stalk is the ratio of the width of the stalk of the mushroom to the diameter of its cap. The larger the value, the thicker the stalk. Different markets have their own preferences regarding the thickness of the stalk, and depending on the needs of the market, some prefer a thicker stalk while others prefer a thinner one. Almost all of the hybrid strains derived from J19109 recorded in Table VI showed a slightly thinner stalk than J15987, but this is neither a universally positive nor negative quality.

[0237] The flesh thickness is a rough estimate of what volume of the mushroom cap is composed of flesh tissue rather than stalk or pleat tissue. The larger the value, the higher the ratio of flesh tissue, which is preferred by growers as an indicator of quality. All of the hybrid strains derived from J19109 recorded in Table VI fall within the continuum around J15987, clearly demonstrating that this trait is retained.

[0238] When considering these mushroom shape data together, the shape of J15987 was clearly retained.

[0239]

Table 7

[0240] The color of the mushroom cap was measured using a Minolta Chroma Meter CR - 200. The sample size was 20 medium - sized mushrooms with a diameter of 30 - 40 mm. The L*a*b color space system was used. Here, "L" is the evaluation criterion for lightness, where 100 is pure white and 0 is pure black. For the other two measured values, "a" is the green / red axis and "b" is the yellow / blue axis. For "a", red values are on the positive side of the common axis and green values are on the negative side. Similarly, for the "b" axis, yellow values are positive and blue values are negative.

[0241] The L value is an objective evaluation criterion for how white the mushroom cap appears in an undamaged state. For white mushrooms, a higher value is desirable. It is clearly demonstrated that all of the hybrid strains derived from J19109 recorded in Table VII fall within the continuum around J15987 and this trait is retained. In fact, some strains recorded significantly higher L values than J15987, suggesting that the trait was not merely retained but improved.

[0242] The readings of the "a" value and "b" value contribute to the color shade of the mushroom. None of the hybrids derived from J19109 recorded in Table VII were significantly different from J15987, demonstrating that the color of this strain was retained in these hybrids.

[0243] [Table 8]

[0244] Table VIII shows the yields obtained from the first pre - commercialization test of J20176 conducted using the A - 15 control. The growth conditions were typical of European commercial growers using the "Dutch" system with large amounts of Phase I and Phase II composting, and large amounts of mycelial elongation (Phase III). The A - 15 control was flushed in the chamber using industry - standard conditions.

[0245] In the first break, the yield of J20176 was higher than the control, but this difference did not meet the significance level. Only the observation of the third break reached significance, with the third - break yield of A - 15 being higher than that of J20176. The comparison of total yields showed no significant difference between these two strains. These data demonstrate that even under commercial growth conditions not optimized for the requirements of J20176, the yield potential of J20176 was shown and it had substantially the same yield as the A - 15 strain. Generally, to obtain good yields, the process must be improved over the long term. Observationally, the J20176 mushrooms were rounder and whiter than the A - 15 control.

[0246] In this test, special attention was paid to searching for evidence of Aggressive Incompatibility, assuming that J20176 and A-15 exist in adjacent regions in situ. Typical inter-strain interactions were observed in the areas where colonized compost or casing was mixed, and typical incompatibility reactions were seen where growth was not observed in a very narrow area when the A-15 and J20176 strains grew together. The AI trait phenomenon was not induced by J20176 under commercial conditions. These data indicate the market suitability and potential of J20176.

[0247] Although the present invention has been described with respect to specific embodiments in this application, those skilled in the art can generate additional embodiments and modifications without departing from the spirit of the claimed invention and without exceeding its scope, in light of the teachings herein. Therefore, it is understood that the description herein is provided merely to facilitate the understanding of the present invention and should not be construed as limiting the scope of the present invention.

Claims

**Claim 1** A method for eliminating the aggressive incompatibility (AI) trait from Agaricus bisporus mushroom strains, comprising crossing a culture of a white mushroom strain designated WBL-s290, a representative culture of which is deposited under NRRL Accession No. 68167, with a culture of a mushroom strain designated J11500-s80, a representative culture of which is deposited under NRRL Accession No. 68164, to yield a hybrid mushroom strain designated J15987 having the AI trait, a representative culture of which is deposited under NRRL Accession No. 67646, wherein the method further comprises crossing a culture of a hybrid mushroom strain designated B18287-s82, a representative culture of which is deposited under NRRL Accession No. 68168, with a culture of a white mushroom strain designated WBL-s290 to obtain an F1 hybrid mushroom strain designated J19109, a representative culture of which is deposited under NRRL Accession No. 68163; fruiting a culture of the new F1 strain designated J19109 to obtain homokaryotic spores derived therefrom; obtaining and selecting a culture of a homokaryotic strain from the homokaryotic spores derived from the F1 strain J19109; crossing a culture of the homokaryotic strain derived from the F1 strain J19109 with a culture of a mushroom strain designated J11500-s80 to obtain an F2 hybrid mushroom strain; and testing a culture of the F2 hybrid mushroom strain to determine the presence or absence of the AI trait, wherein if the AI trait is absent, the AI trait has been eliminated from the F2 hybrid mushroom strain. A method comprising the above steps. **Claim 2** The method of claim 1, wherein the homokaryotic strain derived from the F1 strain J19109 lacks centromere-linked alleles of the white mushroom WBL-s290 strain on chromosomes 4, 7, and 9. **Claim 3** The method according to claim 1, wherein the F2 hybrid strain eliminates the AI trait and provides at least two beneficial traits found in the J15987 strain, including an umbrella shape with the same roundness as that of the J15987 strain, a stalk with the same thickness as that of the J15987 strain, flesh with the same thickness as that of the J15987 strain, and a redness (a) value within 10% of the redness (a) value of the J15987 strain in the L-a-b color measurement scale.

4. The method according to claim 1, wherein the culture of the isonuclear coexistence system derived from the F1 strain J19109 is a strain culture called J19109-s40, and a representative culture of the strain is deposited under the NRRL accession number 68165.

5. The method according to claim 4, wherein the step of crossing the culture of the isonuclear coexistence system derived from the F1 strain J19109 with the culture of the mushroom strain called J11500-s80 includes crossing the strain culture called J19109-s40 with the mushroom strain called J11500-s80, resulting in an F2 hybrid strain called J20176, and a representative culture of the strain is deposited under the NRRL accession number 68166.

6. The method according to claim 5, wherein the F2 hybrid strain called J20176 does not have the AI trait and retains at least two beneficial traits found in the J15987 strain, including an umbrella shape with the same roundness as that of the J15987 strain, a stalk with the same thickness as that of the J15987 strain, flesh with the same thickness as that of the J15987 strain, and a redness (a) value within 10% of the redness (a) value of the J15987 strain in the L-a-b color measurement scale.

7. A culture comprising at least one set of chromosomes of the Agaricus bisporus B18278-s82 strain, wherein the culture of the B18278-s82 strain is deposited under the NRRL accession number 68168, and the chromosomes contain all the alleles of the B18278-s82 strain at the marker loci characterized by the sequences listed in Table I.

8. A culture derived from an original culture, wherein the original culture is the culture according to claim 6, at least 75% of whose genome or genotype is present in the genome or genotype of the original culture of the B18287-s82 strain, and the culture of the original strain is deposited under NRRL accession number 68168, or the culture is directly derived from the original culture and all of its genome or genotype is present in the genome or genotype of the original culture of the B18287-s82 strain. **Claim 9** A culture comprising at least one set of chromosomes of the Agaricus bisporus WBL-s290 strain, wherein the culture of the WBL-s290 strain is deposited under NRRL accession number 65167, and the chromosomes contain all of the alleles of the WBL-s290 strain at marker loci characterized by the sequences listed in Table I. **Claim 10** A culture derived from an original culture, wherein the original culture is the culture according to claim 8, at least 75% of whose genome or genotype is present in the genome or genotype of the original culture of the WBL-s290 strain, and the culture of the original strain is deposited under NRRL accession number 68167, or the culture is directly derived from the original culture and all of its genome or genotype is present in the genome or genotype of the original culture of the WBL-s290 strain. **Claim 11** A culture comprising at least one set of chromosomes of the Agaricus bisporus J11500-s80 strain, wherein the culture of the J11500-s80 strain is deposited under NRRL accession number 65164, and the chromosomes contain all of the alleles of the J11500-s80 strain at marker loci characterized by the sequences listed in Table I. **Claim 12** A culture derived from an original culture, wherein the original culture is the culture according to claim 8, at least 75% of its genome or genotype is present in the genome or genotype of the original culture of the J11500-s80 strain, the original culture of the strain is deposited under the NRRL accession number 68164, or the culture is directly derived from the original culture and all of its genome or genotype is present in the genome or genotype of the original culture of the J11500-s80 strain.

13. A culture containing at least one set of chromosomes of the Agaricus bisporus J19109-s40 strain, wherein the J19109-s40 strain culture is deposited under the NRRL accession number 68165, and the chromosomes contain all alleles of the J19109-s40 strain at marker loci characterized by the sequences listed in Table I.

14. A culture derived from an original culture, wherein the original culture is the culture according to claim 10, at least 75% of its genome or genotype is present in the genome or genotype of the original culture of the J19109-s40 strain, the original culture of the strain is deposited under the NRRL accession number 68165, or the culture is directly derived from the original culture and all of its genome or genotype is present in the genome or genotype of the original culture of the J19109-s40 strain.

15. A hybrid mushroom culture of Agaricus bisporus designated as the J19109 strain, wherein a representative culture of the strain is deposited under the NRRL accession number 68163.

16. A culture of Agaricus bisporus derived from an original culture, wherein the original culture is the culture according to claim 12, at least 75% of its genome or genotype is present in the genome or genotype of the original culture of the J19109 strain, the culture of the strain is deposited under the NRRL accession number 68163, or the culture is directly derived from the original culture and all of its genome or genotype is present in the genome or genotype of the original culture of the J19109 strain.

17. A hybrid mushroom culture of Agaricus bisporus designated as strain J20176, wherein a representative culture of said strain has been deposited under NRRL accession number 68166.

18. A culture of Agaricus bisporus derived from a first culture, wherein the first culture is the culture according to claim 15, at least 75% of its genome or genotype being present in the genome or genotype of the first culture of strain J20176, and the culture of said strain having been deposited under NRRL accession number 68166, or the culture being directly derived from the first culture and all of its genome or genotype being present in the genome or genotype of the first culture of strain J20176.

19. A mushroom obtained from the culture according to any one of claims 13 to 16.

20. A product incorporating the culture according to any one of claims 7 to 16, the product being selected from the group consisting of mycelium, inoculum, raw mushroom or processed mushroom product, mushroom spore, mushroom inoculum, mushroom preparation and extract and fraction, mushroom piece, mushroom inoculation material, casing inoculation material, casing inoculum, casing soil, colonized compost, colonized compost, post-cultivation compost, and friable particulate matter.

21. A part of the culture according to any one of claims 7 to 16, selected from the group consisting of mycelium, mushroom, dormant spore, germinating spore, homokaryon, heterokaryon, cell, nucleus, and protoplast.

22. Any cell of the culture according to any one of claims 7 to 16.

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