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 relates generally to a method for eliminating invasive 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 technology]

[0002] The present invention follows the earlier release of a white hybrid mushroom strain developed by Sylvan America, Inc., designated J15987 (NRRL Accession No. 67646), as disclosed in U.S. Pat. No. 10,440,930, the disclosure of which is incorporated herein by reference. Strain J15987 has a higher yield than currently dominant white cultivars and produces high-quality mushrooms, i.e., mushrooms with thicker cap flesh and less red coloring compared to standard commercial white mushrooms obtained from a commercially accepted strain designated A-15. Initial testing of strain J15987 proved very satisfying for customers with its powerful combination of yield and quality, and these mushrooms and the strain were initially very commercially accepted. However, a problem arose. It has been discovered that the J15987 strain contains an unprecedented phenotype or trait referred to as "Aggressive Incompatibility," which creates farm-level problems when the A-15 or similar strains are also used on mushroom farms. Thus, there is a need for methods to eliminate the "Aggressive Incompatibility" (as defined below) phenotype or trait from J15987 and similar white mushroom strains, and for developing similar white mushroom strains that retain or otherwise provide the favorable traits of the J15987 strain (e.g., high yield and / or high quality, etc.) while eliminating the Aggressive Incompatibility (AI) trait.

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

[0004] Agaricus cultures, like other microorganisms, are prepared, maintained, grown, and stored in sterile media. Pure cultures are manipulated in sterile areas using sterile tools and techniques for various purposes, including spore germination, new hybridization, and inoculum production. Commercially available media are "seeds" and can be of several different recipes, most commonly sterilized millet or rye. Seed production is generally performed on a large scale. For example, one liter of pure Agaricus inoculum can be used to produce 14,0000 liters of sterile seed. End-user mushroom farms receive the pure seed cultures in sterile packaging.

[0005] Commercial mushroom cultivation occurs in dedicated mushroom farm structures. While there are many variations in the method, a typical one is described below. Compost is prepared from waste lignocellulosic materials, such as wheat straw, enriched with nitrogenous materials, and finished. It is then transferred to specific conditions (aerobic, at temperatures between 40°C and 50°C) and pasteurized, thereby creating a substrate favorable for Agaricus colonization. Mushroom spawn is added, and the substrate is allowed to colonize at controlled temperatures for a period of 11 to 17 days. Once colonization, or "hyphal growth," is complete, a non-nutritive layer of saturated peat (adjusted to a pH between 8 and 8.5 using sugar beet lime) is applied to the surface of the compost in an approximately 5 cm layer. Additionally, casing spawn can be added to the peat or casing layer to accelerate mycelial growth. Once colonization has occurred, environmental conditions are adjusted to lower the temperature to approximately 18-19°C and reduce CO2 to approximately 1300 ppm in a process called "flushing." Mushrooms emerge in the mushroom house 13-18 days after application of the casing soil. Mushrooms are harvested over a 3-4 day period. An additional flush or break in production occurs, and after harvest, the compost is removed and replaced at the mushroom farm.

[0006] In the United States, 72% of the total mushroom harvest in 2020-2021 will be white Agaricus, with the remainder being brown Agaricus varieties. The total value of the mushroom harvest in the United States during that same period exceeds $1 billion. In other regions, there is a stronger preference for white mushrooms. Market demand for white mushrooms in the United States and elsewhere is limited and strict with regard to phenotypic traits, such as size, shape, whiteness, and firmness, 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 / firmness, and individual weight. Some examples of commercial targets for new white mushroom strains are increased crop yield, altered yield distribution between production flushes, disease resistance, insect resistance, improved shelf life, reduced spoilage, ease of management, suitability for mechanical harvesting, differential response to stressors (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 distinguish and classify strains from one another. Strains may have different ancestry, which is directly reflected in their genotypes and phenotypes.

[0007] The first two true hybrid strains of A. bisporus were developed in a laboratory in Horst, The Netherlands, around 1980. These two "Horst" strains, designated U1 and U3, are closely related hybrids developed by crossing two existing 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. Over time, hybrid U1 became the dominant white genotype in the market.

[0008] Homokaryons of strain U1 were recovered by deheterokaryosis of the cultures. The parent of PHSW is H39, and the parent of PHOW is H97. H97 was deposited by A. Sonnenberg at the Fungal Genetics Stock Center, Kansas, USA, under accession number 10389, and also at the American Type Culture Collection under accession number MYA-4626. The H97 genome was sequenced and made publicly available by the Joint Genome Initiative (California, USA), and this genome has proven to be extremely 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, have been developed from spores or tissues derived from the original (i.e., first) strain and meet the criteria for U1 Essentially Derived Varieties (EDV). A group of strains developed from a single ancestor, either by somatic selection or spore culture, or by related methods of "essential derivation," as discussed below, is referred to as a derived family. Other than relatively minor genetic differences, all developed white strains within the U1 derived family 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. Like other fungi, reproductive propagules are spores. Agaricus produces spores by meiosis in meiotic sporangia known as basidiophores. In the first life cycle, A. bisporus spores each receive only one haploid postmeiotic nucleus; these spores are capable of mating but are incapable of producing mushrooms. These haploid spores germinate to produce homokaryon progeny or lineages, which can then be crossed with other compatible homokaryons to produce novel hybrid heterokaryons capable of producing mushrooms. Heterokaryons generally exhibit a much lower mating ability than homokaryons. This life cycle is called heteromixis and is similar to outbreeding. This life cycle is functional but generally not dominant in strains of Agaricus bisporus var. bisporus.

[0011] The second uniparental life cycle, termed intramixis, is similar to a form of inbreeding and predominates in the majority of strains of Agaricus bisporus var. bisporus. The majority of spores receive two nuclei after meiosis, and the majority of such pairs consist of non-sister nuclear pairs (NSNPs) with heteroallelic genotypes at most or all centromeric-linked loci, including the MAT locus. The MAT genotype determines the heterokaryotic phenotype of these progeny, which are fertile and give rise to mushroom crops. Unusually among eukaryotes, relatively few chromosomal crossovers have been observed in A. bisporus's meiotic progeny, and empirically, very little heteroallelicity (similar to heterozygosity) is lost between heterokaryotic intramixed progeny of heterokaryotic strains.

[0012] In both life cycles, the spore is the cell that is part of the mushroom, and that cell is the propagule, always having as its single parent the mushroom and the culture that gives rise to that mushroom.

[0013] With respect to spores, live spores are dormant heterokaryons or homokaryons. Spores are part of a mushroom organism culture, incorporating only the genetic material of the single culture from which they arise (often conveniently referred to as the "parent"). Other parts of the culture include the cap, stalk, gills, cells (defined as the hyphal compartment incorporating the nucleus, mitochondria, cytoplasm, protoplasts, RNA, DNA, proteins, cell membranes, and cell walls, including their transverse walls), hyphae, and mycelium. Spores can be aseptically collected on sterile substrates, suspended in sterile water at various dilutions, and plated onto sterile agar growth media to produce germinated spores and incorporate the culture into the spores. A preferred technique is to place live Agaricus cultures in sealed Petri plates, which can stimulate spore germination by diffusing volatile pheromones. Germinated spores can be isolated onto new nutrient agar plates using a sterile microtool such as a steel needle under a microscope. This method can be used to obtain heterokaryotic and homokaryotic progeny of strains containing spores of the strains and the cultures incorporated within the spores.

[0014] The development of novel hybrid varieties through heteromixis involves the controlled physical association and mating of two compatible cultures to obtain novel heterokaryon cultures. Homokaryons (i.e., "lines") are the preferred starting cultures for mating because they have the greatest ability to anastomose with other cultures and achieve cytoplasmic fusion. Heterokaryons can also be brought into physical contact, but the probability of a mating resulting in the successful formation of a novel heterokaryon is unreasonably low. Compatibility is determined by genotype at the MAT locus; two homokaryons with the same MAT allele cannot establish heterokaryons after anastomosis, thus homokaryon compatibility becomes a genetic difference. In a defined mating program, homokaryon lines are obtained and related in predetermined pairwise combinations. In one method, homokaryon pairs are placed in close proximity on the surface of nutrient agar in a Petri dish and allowed to grow together (in physical association) until anastomosis occurs between the two cultures. Successful mating results in heterokaryons. Novel hybrid heterokaryons can be obtained by transferring mycelium from the fusion zone of the dish.

[0015] There is a clear need for commercially acceptable white Agaricus bisporus strains with distinct genotypes compared to the U1 derivative lineage for two reasons. First, strains that are vegetatively incompatible with strains in the U1 derivative lineage have been shown to slow the spread of viral diseases between strains, and such diseases have been shown to be a major cause of crop losses in the commercial mushroom industry. Second, it is well known that the agricultural industry relies extensively on crops of a single genetic lineage (e.g., white A. bisporus in most parts of the world), increasing the risk of catastrophic crop failures on an entire facility or even an entire industry scale. Therefore, from a food security and risk management perspective, it is highly desirable to simultaneously provide genetic diversification with commercially acceptable performance and crop characteristics.

[0016] As noted above, this invention is a direct result of the commercial release of hybrid J15987 (NRRL Accession No. 67646) (U.S. Pat. No. 10,440,930), a white hybrid strain. Pre-commercial testing demonstrated that J15987 had thicker cap flesh and thicker stalks compared to other white cultivars, such as those derived from the A-15 strain. Furthermore, color analysis of J15987 using a Minolta Chroma Meter demonstrated less redness in the mushroom caps after harvest. The commercial potential was considered significant, and the strain was grown in the United States shortly after the patent was filed. The strain successfully met all of the original crop production standards. However, a significant problem emerged. In some mushroom-growing "houses" (or rooms), particularly those producing mushrooms derived from the A-15 strain, a lack of mushroom growth was observed in large areas of the compost, both in the compost and casing layers. It became clear that this observation was unusual and out of the ordinary.

[0017] Cultures were returned to the laboratory, where the suspected antagonistic interaction between J15987 and other white cultivars was investigated in more detail. The genetic characteristics of the inter-strain interaction are governed by a self / non-self recognition system known as vegetative incompatibility or heterokaryon incompatibility. J15987 was found to exhibit a highly aggressive and unusual antagonistic response when in contact with other subordinate varieties (EDVs) of the U1 family, such as A-15. When the inoculum was mixed at a low ratio of 99% A-15 to 1% J15987, the Agaricus mycelium rapidly died, and the A-15 genotype was eventually replaced by J15987. This interaction was termed "Aggressive Incompatibility," abbreviated as "AI," and the phenotype was defined as the "AI" trait. Specifically, while unrelated strains of basidiomycetes, including Agaricus bisporus, typically exhibit an incompatibility response that has been exploited for virus control, the AI ​​trait is a much more aggressive, extreme, and problematic manifestation of this well-known phenomenon, and is presumed to be under genetic control, similar to what is commonly understood to be the case for vegetative incompatibility in basidiomycetes. Summary of the Invention [Problem to be solved by the invention]

[0018] Conventional mushroom farm mixing equipment tends to be operated in a way that results in some degree of intermixing of different strains among inoculated substrates. Such events are generally minor and have no significant negative impact. Because these areas are small, their impact on overall yield is minimal and has traditionally been tolerated. However, the impact of larger, more extreme AI interactions can be commercially unacceptable, and customer evaluation has shown that J15987 cannot be profitably handled in farm operations that also grow A-15. Therefore, there is a need for a commercially desirable strain or strains that possess the positive attributes of J15987 while lacking the AI ​​traits. [Means for solving the problem]

[0019] To understand and overcome (eliminate) the underlying genetic basis responsible for the manifestation of the AI ​​trait in strain J15987, a series of crosses were conducted and the resulting hybrids were analyzed. The crossing scheme is described below in Scheme I. Essentially, a wild-type variation line (i.e., B18287-s82, NRRL accession number 68168) was combined with one of the homokaryonous parents of strain J15987 (i.e., WBL-s290, NRRL accession number 68167) to generate a new F1 hybrid strain (i.e., J19109, NRRL accession number 68163). The homokaryons from that hybrid (e.g., line J19109-s40, NRRL accession number 68165) were then crossed with the other homokaryon parent of strain J15987 (i.e., J11500-s80, NRRL accession number 68164) to produce a new F2 hybrid (e.g., J20176, NRRL accession number 68166). Contrasts using the F2 hybrids were performed to demonstrate the presence or absence of the AI ​​trait. Based on these contrasts, it was clear that the breeding scheme was successful and that the majority of the new hybrids did not possess the AI ​​trait. The pattern of inheritance allowed for further development of a model for genetic control / elimination of the AI ​​trait.

[0020] [ka]

[0021] First, a homokaryon (line B18287-s82) derived from a hybrid developed by crossing two homokaryons, W01-s1 and So76-s12b, was developed. This homokaryon line, B18287-s82, was developed from parental homokaryons derived from the wild-type strain W01, collected by R. Kerrigan in North America in the 1990s, and the prehybrid off-white strain So76. DNA testing demonstrated that W01 was a true wild-type strain, not a commercial escapee. Furthermore, W01 had very similar growth requirements to the current white and brown strains.

[0022] Thus, one aspect of the present invention is directed to a new, distinct homokaryonous strain of Agaricus bisporus generally designated B18287-s82, and processes for using the strain designated B18287-s82. In at least one embodiment, a culture is provided comprising at least one set of chromosomes of Agaricus bisporus strain B18278-s82, wherein the at least one set of chromosomes comprises all of the alleles of strain B18278-s82 at the marker loci characterized by the sequences listed in Table I below. A culture of Agaricus bisporus strain B18287-s82 disclosed herein has been deposited with and received at the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, USA. The date of deposit is June 10, 2022. The deposited cultures were obtained from cultures maintained by the assignee of record, Sylvan, Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions on the deposit have been removed, and the deposit is intended to comply with all deposit requirements of the US Patent and Trademark Office, including 37 CFR Secs. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL Accession Number is 68168. The deposit will be maintained in the depository for 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and will be replaced as needed during that period. The cultures will be made available to the public irrevocably and without restriction or conditions upon the filing of this patent application or the issuance of a patent, whichever is required by applicable patent law.

[0023] In one embodiment, a dependent variety (EDV) of the B18287-s82 strain may also be provided. Such an EDV may include a culture derived from an initial culture, where the initial culture is a culture of the B18287-s82 strain, and thus at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of the B18287-s82 strain. 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 initial culture of the B18287-s82 strain. In other embodiments, such an EDV may include a culture derived directly or exclusively from the initial culture, and thus all of its genome or genotype is present in the genome or genotype of the initial culture of the B18287-s82 strain.

[0024] The B18287-s82 homokaryon was crossed with WBL-s290. Note that this line is one of the parent homokaryons currently found for strain J15987 (the other being J11500-s80, which was also used in this study). The resulting F1 heterokaryon strain was designated J19109.

[0025] Thus, another aspect of the present invention is generally directed to a new, distinct homokaryonous strain of Agaricus bisporus designated WBL-s290, and processes using the strain designated WBL-s290. In at least one embodiment, a culture is provided that includes at least one set of chromosomes of Agaricus bisporus strain WBL-s290, wherein the at least one set of chromosomes includes all of the alleles of strain WBL-s290 at the marker loci characterized by the sequences listed in Table I below. A culture of Agaricus bisporus strain WBL-s290 disclosed herein has been deposited with and received at the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit is June 10, 2022. The deposited cultures were obtained from cultures maintained by the assignee of record, Sylvan, Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions on the deposit have been removed, and the deposit is intended to comply with all deposit requirements of the US Patent and Trademark Office, including 37 CFR Secs. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL Accession Number is 68167. The deposit will be maintained in the depository for 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and will be replaced as needed during that period. The cultures will be made available to the public irrevocably and without restriction or conditions upon the filing of this patent application or the issuance of a patent, whichever is required by applicable patent law.

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

[0027] Another aspect of the present invention is generally directed to a new and distinct Agaricus bisporus mushroom strain culture designated J19109, which is a hybrid strain obtained by directional mating of two homokaryon cultures, B18287-s82 and WBL-s290. A deposit of a culture of strain J19109 has been made and deposited with the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, USA. The date of deposit is June 10, 2022. The deposited culture was obtained from the same culture maintained by Sylvan Inc., Kittanning, Pennsylvania, USA, the assignee of record, prior to the filing date of this application. All restrictions on the deposit are removed, and the deposit is intended to satisfy all deposit requirements of the US Patent and Trademark Office, including 37 CFR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL Accession Number is 68163. The deposit will be maintained at the depository for 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and will be replaced as needed during that period. The culture will be made available to the public irrevocably and without restriction or with conditions, in accordance with patent laws, upon the filing of a priority application or the issuance of a patent for this strain.

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

[0029] To reiterate, a culture of a hybrid mushroom line designated B18287-s82, a representative culture of which has been deposited under NRRL Accession No. 68168, was crossed with a culture of a white mushroom line designated WBL-s290, a representative culture of which has been deposited under NRRL Accession No. 68167, to obtain an F1 hybrid mushroom line designated J19109, a representative culture of which has been deposited under NRRL Accession No. 68163. A culture of the F1 line designated J19109 was then subjected to fruiting to obtain homokaryon spores from which homokaryon lineages were obtained. Cultures of homokaryon lineages derived from the F1 line J19109 were then selected. Notably, the homokaryon lines derived from the F1 strain J19109 lack alleles of the centromeric linkage loci on chromosomes 4, 7, and 9 of the WBL-s290 line.

[0030] Once a culture of a homokaryon line derived from F1 strain J19109 was selected, that culture was crossed with a culture of a mushroom line designated J11500-s80. Note that J11500-s80 is the other parent homokaryon currently found for strain J15987. The resulting F2 hybrid mushroom line was obtained from the cross, and the resulting F2 hybrid mushroom line culture was tested to determine the presence or absence of the AI ​​trait. If the AI ​​trait is absent, the AI ​​trait has been eliminated from the F2 hybrid mushroom line.

[0031] Thus, another aspect of the present invention is generally directed to a new, distinct homokaryonous strain of Agaricus bisporus designated J11500-s80, and processes using the strain designated J11500-s80. In at least one embodiment, a culture is provided that includes at least one set of chromosomes of Agaricus bisporus strain J11500-s80, wherein the at least one set of chromosomes includes all of the alleles of strain J11500-s80 at the marker loci characterized by the sequences listed in Table I below. A culture of Agaricus bisporus strain J11500-s80 disclosed herein has been deposited with and received at the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit is June 10, 2022. The deposited cultures were obtained from cultures maintained by the assignee of record, Sylvan, Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions on the deposit have been removed, and the deposit is intended to comply with all deposit requirements of the US Patent and Trademark Office, including 37 CFR Secs. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL Accession Number is 68164. The deposit will be maintained in the depository for 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and will be replaced as needed during that period. The cultures will be made available to the public irrevocably and without restriction or conditions upon the filing of this patent application or the issuance of a patent, whichever is required by applicable patent law.

[0032] In one embodiment, a dependent variety (EDV) of the J11500-s80 strain may also be provided. Such an EDV may include a culture derived from an initial culture, where the initial culture is a culture of the J11500-s80 strain, and thus at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of the J11500-s80 strain. 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 initial culture of the J11500-s80 strain. In other embodiments, such an EDV may include a culture derived directly or solely from the initial culture, and thus all of its genome or genotype is present in the genome or genotype of the initial culture of the J11500-s80 strain.

[0033] Furthermore, in one or more embodiments, it will be understood that the F2 hybrid strain from which the AI ​​trait is eliminated retains at least two beneficial traits found in the J15987 strain. Such beneficial traits can be selected from the group consisting of a cap shape that is similar in roundness to that of the J15987 strain, a stalk that is similar in thickness to that of the J15987 strain, flesh that is similar in thickness to that of the J15987 strain, and a redness (a) value on the Lab color measurement scale that is within 10%, or 5%, or 2.5%, or 1%, or 0.5% of the redness (a) value of the J15987 strain. It will be understood that the terms "similar in roundness" and "similar in thickness" mean that the measured cap, stalk, and / or flesh of a mushroom fruited by the F2 hybrid strain falls within a statistically significant range relative to the roundness or thickness of the cap, stalk, or flesh of a mushroom fruited by the J15987 strain. In alternative embodiments, the roundness of the mushroom cap shape of the F2 hybrid strain may be within 20%, or within 15%, or within 10%, or within 5% of the roundness of the mushroom cap shape of the J15987 strain. In other alternative embodiments, the thickness of the mushroom stalk of the F2 hybrid strain may be within 20%, or within 15%, or within 10%, or within 5% of the thickness of the mushroom stalk of the J15987 strain. In yet other alternative embodiments, the thickness of the mushroom flesh of the F2 hybrid strain may be within 20%, or within 15%, or within 10%, or within 5% of the thickness of the mushroom flesh of the J15987 strain.

[0034] In one embodiment, the homokaryon line selected from F1 strain J19109 can be the homokaryon line designated J19109-s40. It will be understood that the line culture designated J19109-s40 is crossed with a mushroom line designated J11500-s80, where the resulting F2 hybrid line is designated J20176. The hybrid J20176 line has been found to lack the AI ​​trait.

[0035] Thus, another aspect of the present invention is generally directed to a new, distinct homokaryonous strain of Agaricus bisporus designated J19109-s40, and processes using the strain designated J19109-s40. In at least one embodiment, a culture is provided that includes at least one set of chromosomes of Agaricus bisporus strain J19109-s40, wherein the at least one set of chromosomes includes all of the alleles of strain J19109-s40 at the marker loci characterized by the sequences listed in Table I below. A culture of Agaricus bisporus strain J19109-s40 disclosed herein has been deposited with and received at the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The date of deposit is June 10, 2022. The deposited cultures were obtained from cultures maintained by the assignee of record, Sylvan, Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions on the deposit have been removed, and the deposit is intended to comply with all deposit requirements of the US Patent and Trademark Office, including 37 CFR Secs. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL Accession Number is 68165. The deposit will be maintained in the depository for 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and will be replaced as needed during that period. The cultures will be made available to the public irrevocably and without restriction or conditions upon the filing of this patent application or the issuance of a patent, whichever is required by applicable patent law.

[0036] In one embodiment, a dependent variety (EDV) of the J19109-s40 strain may also be provided. Such an EDV may include a culture derived from an initial culture, where the initial culture is a culture of the J19109-s40 strain, and thus at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of the J19109-s40 strain. 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 initial culture of the J19109-s40 strain. In other embodiments, such an EDV may include a culture derived directly or exclusively from the initial culture, and thus all of its genome or genotype is present in the genome or genotype of the initial culture of the J19109-s40 strain.

[0037] Another aspect of the present invention is generally directed to a new and distinct Agaricus bisporus mushroom strain culture designated J20176, which is a hybrid strain obtained by directional mating of two homokaryon cultures, J11500-s80 and J19109-s40. A deposit of a culture of strain J20176 has been made and deposited with the Agricultural Research Services Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, USA. The date of deposit is June 10, 2022. The deposited culture was obtained from a culture maintained by the assignee of record, Sylvan Inc., Kittanning, Pennsylvania, USA, prior to the filing date of this application. All restrictions on the deposit are removed, and the deposit is intended to satisfy all deposit requirements of the US Patent and Trademark Office, including 37 CFR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL Accession Number is 68166. The deposit will be maintained at the depository for 30 years, or five years after the last request, or for the life of the patent, whichever is longer, and will be replaced as needed during that period. The culture will be made available to the public irrevocably and without restriction or with conditions, in accordance with patent laws, upon the filing of a priority application or the issuance of a patent for this strain.

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

[0039] It will further be appreciated that in one or more embodiments, the F2 hybrid strain designated J20176 eliminates the AI ​​trait and retains at least two beneficial traits found in the J15987 strain. Such beneficial traits may be selected from the group consisting of a rounded cap shape similar to that of the J15987 strain, a stalk thickness similar to that of the J15987 strain, meat thickness similar to that of the J15987 strain, and a redness (a) value on the Lab color measurement scale that is within 10%, or within 5%, or within 2.5%, or within 1%, or within 0.5% of the redness (a) value of the J15987 strain. The terms "similar roundness" and "similar thickness" will be understood to mean that the cap, stalk, and / or flesh of a mushroom fruited by the F2 hybrid J20176 strain falls within a statistically significant range of the roundness or thickness of the cap, stalk, or flesh of a mushroom fruited by the J15987 strain. In alternative embodiments, the roundness of the cap shape of the F2 hybrid J20176 strain may be within 20%, or within 15%, or within 10%, or within 5% of the roundness of the cap shape of the J15987 strain. In other alternative embodiments, the thickness of the stalk of the F2 hybrid J20176 strain may be within 20%, or within 15%, or within 10%, or within 5% of the thickness of the stalk of the J15987 strain. In still other alternative embodiments, the thickness of the mushroom flesh of the F2 hybrid J20176 strain may be within 20%, or within 15%, or within 10%, or within 5% of the thickness of the mushroom flesh of the J15987 strain.

[0040] The method of the present invention has been found to advantageously eliminate the AI ​​trait from Agaricus bisporus strains, including at least strain J20176. More particularly, the method has been found to be capable of eliminating the invasive incompatibility (AI) trait from Agaricus bisporus mushroom strains that are descendants of both strain J10165 (also referred to as WBL) and strain J11500 (disclosed in U.S. Pat. No. 9,622,428), wherein crossing a culture of a white mushroom line designated WBL-s290 with a culture of a mushroom line designated J11500-s80 has been found to result in a hybrid mushroom line designated J15987 having the AI ​​trait, a representative culture of which has been deposited under NRRL Accession No. 67646.

[0041] Regarding obtaining cultures of homokaryon lines from homokaryon spores derived from the F1 strain J19109, J19109 heterokaryons were allowed to fruit and F2 spores were harvested, resulting in an initial total of 52 homokaryons, 16 of which are listed in some of the tables below. All 52 homokaryons were crossed with J11500-s80, the second parent of J15987, and the resulting strains were extensively screened for commercial potential. The collected data, shown below, demonstrate that the beneficial traits of J15987 were maintained, and more importantly, the majority of the new J19109 x J11500-s80 hybrids did not possess AI traits. One hybrid, strain J20176, was selected for controlled testing. This strain performed robustly and is therefore a strong candidate for commercialization.

[0042] The entire pool of homokaryon lines of strain J19109 showed similar potential as J19109-s40, and all are considered to be valuable breeding lines with the potential to produce high-quality mushroom hybrids without the AI ​​trait.

[0043] One use of hybrids derived from J19109 is the production of commercial edible mushroom crops. Thus, mushrooms obtained from cultures of any of the above-specified lines or strains are part of this invention. Another use is the improvement of facility hygiene through strain rotation and the "virus-busting" effect. A further use is the incorporation of genetic material from the J19109 strain into progeny and derived or descendent cultures, including resting and germinating spores and protoplasts. Additional uses exist as noted above.

[0044] The unusual biological properties of fungi, particularly intramixis in Agaricus bisporus, make it easy and simple to derive virtual copies of cultures with essentially the same phenotype. In plant breeding, such copies are called dependent varieties, or EDVs. Methods for obtaining cultures that are EDVs by definition from a single initial culture of A. bisporus include somatic selection, tissue culture selection, single-spore germination, multiple-spore germination, selfing, repeated backcrossing to the initial culture, mutagenesis, and transformation, to name a few. DNA-mediated transformation of A. bisporus has been reported by Velcko, AJ Jr., Kerrigan, RW, MacDonald, LA, Wach, MP, Schlagnhaufer, C., and Romaine, CP2004, Expression of novel genes in Agaricus bisporus using an Agrobacterium-mediated transformation technique. Mush. Sci. 16:591-597, which is incorporated herein by reference, and references therein. Transformation can introduce a single new gene or allele into the genome of the initial culture. Furthermore, recent reports on other closely related fungi raise the possibility of CRISPR gene editing.

[0045] EDVs are clearly recognizable by their genotype, which is primarily or even entirely a subset of a single initial culture. The percentage of the initial genotype present in Agaricus bisporus EDV ranges from 100% or virtually 100% for single-spore cultures and somatic selection, to up to 99.x% for strains modified by DNA-mediated transformation, to 90-99.x% for some single- or multiple-spore selections or some mutagenesis, to an average of at least 75-85% for sib-offspring matings (=selfing) and backcrosses to the initial culture. Many genotyping methods, including those described below and others known in the art, can be used to determine the percentage of DNA from the initial culture present in another culture and to make unequivocal determinations about the relatedness of the two cultures and any methods used to manipulate or exploit the initial culture.

[0046] Repeated backcrossing to the initial culture also yields the EDV of the initial culture. In a hypothetical example, in the first successive iterations of this process, the resulting strains of this generation will have, on average, about 75% of the DNA of the initial strain, while about 25% of the DNA will be contributed by the second strain or lineage. As this process is repeated, the DNA corresponding to the initial strain increases, approaching 97% on average after three additional successive iterations. In general, it will be understood that any culture with 75-100% genotypic identity to the initial culture will exhibit the EDV of the initial culture. It will also be established that the EDV of the EDV is also the EDV of the initial strain. Finally, because Agaricus bisporus undergoes generational alternation between heterokaryotic and homokaryotic strains, the essential criteria of derivation apply equally to cultures of both strains and lineages.

[0047] A genotypic fingerprint is a description of the genotype at a defined locus, recording the presence of characterized alleles. Such fingerprints provide a powerful and effective technique for recognizing clones and all types of EDV from the original strain, as well as for recognizing ancestors within outbred lineages. Many techniques are available for defining and characterizing genotypic loci and alleles. The most detailed approach is provided by whole genome sequencing (WGS), which allows for the direct characterization and comparison of DNA sequences across the entire genome. Using this technique to generate robust genotypic fingerprints incorporating multiple marker loci, it is possible to establish the nature of the relationship between two strains, including strains related by phylogenetic lineage over several generations. Applicant has tracked genetic markers over four to six generations of strain development pedigrees. If a sufficient number of differential markers are present in the original strain or lineage, it becomes possible to identify lineages from the original strain or lineage after several outbred generations without undue experimentation. In a hypothetical example, at the fourth outbred generation, the average expected genome proportion corresponding to the original haploid line in the F4 hybrid is 3.1% (50% / 2 4 ), which corresponds to approximately 1 Mb of A. bisporus nuclear genomic DNA. Based on Applicant's analysis, the amount of DNA 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 an identification marker linking the F4 hybrid to the original lineage. By using a large number of independent markers, the origin of a strain can be identified with a very high success rate and a reasonable degree of confidence.

[0048] One trait of biological and commercial interest is heterokaryon incompatibility. While the genetics of these self / nonself recognition systems in Basidiomycetes such as Agaricus are not fully understood, other groups of fungi are known to involve multiple alleles at multiple independent loci. Note that heterokaryon incompatibility occurs in the majority of fungi and is entirely separate from the aggressive incompatibility trait reported herein. "Normal" incompatibility is usually a localized area of ​​hyphal interaction between two genotypes, resulting in attenuated growth. 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 successful anastomosis and cytoplasmic continuity between physical mixtures of two or more heterokaryons, thereby resulting in "virus breakthrough." In mushroom farms, virus breakthrough is achieved by replacing the planting material (compost, seed culture, casing inoculant) incorporated into the original strain with inoculum and planting material incorporated into another, different strain that is incompatible with the original strain. The most effective implementation of the virus breakthrough method is to replace all biological material of the original strain in a mushroom farm with biological material of a second, incompatible strain. Strain incompatibility creates an effective, if not absolute, barrier to the transfer of viruses from the biological reservoir within the facility into new crops. Cultivation rotation between genotypically distinct mushroom strains can also disrupt the infection and invasion cycles of exogenous pests and pathogens.

[0050] As noted above, hybrid mushroom strain producers are constantly searching for hybrid strains that will enable growers to successfully and profitably produce crops. In the case of new hybrids derived from J19109 and strains derived or descended from it, positive attributes that have been demonstrated include attractive appearance (rounder caps, thicker stalks, and better resistance to bruising compared to market leader A-15, all of which are appealing to customers) and total yields that match or exceed those of strains such as A-15. In Applicant's example, mushroom growers can obtain high-yielding crops of high-quality mushrooms by using the new hybrids described herein. Furthermore, these strains match the high quality of J15987, an earlier strain developed by Applicant, and have the notable advantage of not having the aggressive incompatibility phenotype.

[0051] The strains currently available to the mushroom industry allow growers to successfully and usually profitably produce mushroom crops. Several factors influence the degree of success and profitability achieved. For example, the strain must be capable of producing crop yields at least equivalent to those currently marketed and commercially grown over two to three breaks or flushes. Also, some physical characteristics of the resulting mushrooms, such as cap color categories and typical sizes and dimensions, such as cap diameter, allow the mushrooms to be sold in familiar product categories.

[0052] The J19109 SSI x J11500-s80 hybrid meets these market needs and solves the current problem of no such lines being available in the market. Furthermore, this new group of lines is genotypically distinct compared to other current white cultivars (see Tables I and II below), addressing and resolving potential issues related to monoculture reliability.

[0053] One or more aspects of the present invention may be realized by the hybrid mushroom cultures of Agaricus bisporus developed by the present invention. Accordingly, products incorporating any of the above-mentioned lineage or strain cultures are encompassed by the present invention. Such products may include mycelium, spawn, fresh or processed mushrooms, mushroom spores, mushroom spawn, mushroom preparations and extracts and fractions, mushroom pieces, mushroom inoculants, casing inoculants, casing spawn, casing soil, inoculated compost, colonized compost, post-cultivation compost, and friable particulate matter.

[0054] Furthermore, various parts of the culture of any of the above strains or lines may have value and commercial use. Thus, parts selected from the group consisting of mycelia, mushrooms, spores, cells, nuclei, and protoplasts are contemplated. In yet another embodiment, a single-cell microorganism is contemplated. Thus, cells of any of the cultures of the above strains and lines are provided.

[0055] It is noted that strain J20176, a representative culture of which has been deposited under NRRL Accession No. 68166, was deposited to provide a suitable example of a strain that may meet the requirements of the present invention. Thus, strain J20176 encompasses various portions of the culture, including mycelia, spores, and cells, as well as cellular parts, including nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes, and cell walls, present in both the vegetative mycelium of the culture and the resulting mushrooms. The spores may be resting or germinated spores, and may contain heterokaryons and homokaryons incorporated therein.

[0056] One or more products can be made incorporating hybrid mushroom cultures of Agaricus bisporus, designated a strain descended from J19109. Such products include mycelium, starter cultures, inoculants, casing inoculants, raw mushrooms, processed mushroom products, mushroom extracts and fractions, mushroom pieces, and colonized substrates selected from grains, compost, and friable particulate matter. Mushroom pieces will be understood to refer to the stalk, cap, and other larger parts of the mushroom itself.

[0057] One or more other aspects of the present invention may be realized by a subordinate 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 possesses at least one essential characteristic of a hybrid derived from J19109. One hybrid, J19109-s40 x J11500-s80, known as J20176, has been deposited with the present invention as an example of a superior strain that is a direct result of the invention described herein. J20176 is a strain that lacks the AI ​​trait and possesses the additional characteristics of cap roundness, flesh thickness, yield performance, and yield timing of J15987 compared to a U1 EDV such as A-15.

[0058] Another aspect of the present invention may be realized by a method for generating a culture of hybrid Agaricus bisporus, comprising crossing a homokaryon, such as J19109-s40 (deposited under NRRL Accession No. 68165), with a second homokaryon. In one embodiment, the second homokaryon is strain J11500-s80, a culture of which has been deposited under NRRL Accession No. 68164. Such a cross results in mushroom culture J20176, which exhibits a typical pattern of antagonism against a panel of commercial cultivars based on U1 EDV, e.g., A-15. This antagonism demonstrates that strain J20176 is genetically distinct. In one or more embodiments, the method further comprises providing a mushroom culture of the invention comprising mycelium, seed culture, inoculum, casing inoculum, raw mushrooms, processed mushroom products, mushroom parts, mushroom extracts and fractions, mushroom pieces, and a culture substrate selected from grains, compost, and friable particulate matter. In other embodiments, the method may comprise providing the mushroom culture as a derivative culture selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue explant cultures, protoplasts, resting spores, germinating spores, inbred and outbred progeny, transgenic cultures, gene-edited cultures, and cultures having a genome with a single locus transposition.

[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 the 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 listed in Table II, where all of the traits in the fingerprint are present in the genotypic fingerprint. Specifically, meiosis and random assortment ensure that each homokaryon spore descended from J19109 has a unique pattern of markers. One skilled in the art can easily infer the ancestry of J19109 homokaryon breeding strains based on genetic DNA markers.

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

[0061] These and other advantages of the present invention over the existing prior art relating to Agaricus bisporus mushrooms and cultures will become apparent from the following description and are realized by the invention as hereinafter described and claimed. DETAILED DESCRIPTION OF THE INVENTION

[0062] To initially provide a clear and consistent understanding of the specification and claims, including the scope to which such terms are to be given, the following explanations are provided.

[0063] Allele: One or more alternative forms of a gene that arise by mutation and are found at the same location on a chromosome; a genetic unit of the genome at a specially reserved 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 exhibit strong antagonism toward each other, a reaction much more severe than a typical heterokaryon incompatibility reaction. At the mushroom farm level, large areas of mycelial death are observed in both the compost and casing layers, resulting in yield losses of at least 15%, more commonly at least 50%. In the laboratory, the presence of as little as 1% of J15987 (a culture with the Aggressive Incompatibility (AI) trait) can kill a U1 EDV such as A-15. Generally, as noted above, if more than 15% of A-15 is killed by J15987 or another strain, then that J15987 or other strain is said to have the AI ​​trait. Over time, the strain with the AI ​​trait replaces the U1 EDV and becomes the only genotype present.

[0065] Amphitheaterism: a mode of reproductive behavior in which both heteromixis and intramixis are active.

[0066] Anastomosis: the fusion of two or more hyphae, achieving cytoplasmic continuity.

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

[0068] Basidium: Meiosporangial cell in which karyofusion and meiosis occur, resulting in the formation of basidiospores.

[0069] Bioefficiency: For mushroom crops, the net fresh weight of the harvested crop for any given sampled area or compost weight divided by the dry weight of the compost substrate at the time of inoculation.

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

[0071] Cap: Fungal pileus; part of a mushroom, a structure with gills.

[0072] Cap Flatness: A measure of the shape or thickness of the cap of a mature, open mushroom.

[0073] Cap roundness: strictly speaking, the ratio of the maximum distance between the top and bottom of the cap, measured by bisecting the mushroom lengthwise, to the maximum distance across the cap; generally averaged over many specimens; subjectively, the "rounded" character of the cap shape.

[0074] Carrier Substrate: A medium having both nutritional and physical properties suitable for both the growth and dispersion of cultures; examples are substrates formulated for mushroom spawn, casing inoculants, and other inoculants.

[0075] Casing layer, casing: A layer of non-nutritive material such as peat or soil applied to the top surface of a colonized compost mass to allow the emergence of a mushroom crop.

[0076] Casing inoculant (CI): An inoculant formulation suitable for mixing into the casing layer, generally incorporating specially prepared heterokaryonous mushroom cultures.

[0077] Cloning: somatic cell propagation without selection.

[0078] Combining ability: the ability of an individual to transmit superior performance to its offspring. Generally, combining ability is the average performance of an individual in a particular series of matings.

[0079] Compatibility: See heterokaryon compatibility, vegetative compatibility, sexual compatibility; incompatibility is the opposite of compatibility.

[0080] CRISPR: (Clustered Regularly Interspaced Short Palindromic Repeats) A genetic engineering technique by which the genome of living organisms is altered.

[0081] Culture: a substantial living organism; an organism grown in various growth media and substrates; part or all of one physical strain, lineage, homokaryon, or heterokaryon; the 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: commercially grown variety or strain

[0083] Derived: The development or acquisition of a culture solely or primarily from an original strain or culture; see EDV. The terms "derive" and "derived" refer to this process or its result.

[0084] Derived clade: A set of EDVs derived from a single original strain, including the original strain.

[0085] Pedigree: genealogical lineage spanning a limited number of generations (e.g., 10 or fewer).

[0086] Diploid: having two sets of haploid chromosomes within a single nuclear envelope.

[0087] Directed mutagenesis: The process of altering the DNA sequence at at least one specific gene locus.

[0088] EDV (Dependent Variety): A culture derived solely or primarily from an original strain or culture; a culture in which 75% or more of its genotypes have the genotype of the original strain and whose condition is the result of that derivation. If directly derived or obtained solely from an original strain or culture, the culture is likely to have all of the genotypes of that original culture.

[0089] Flesh thickness: the ratio of the maximum distance between the top of the stalk and the top of the cap, measured by dividing the mushroom in half lengthwise, to the maximum distance across the cap; generally an average over many specimens; subjectively referred to as "flesh thickness."

[0090] Flush: A period in a crop cycle during which mushrooms occur, separated by intervals during which mushrooms do not occur; the term flush encompasses the terms "break" and "wave" and can be read as either of these terms.

[0091] Fungi: microorganisms classified as members of the kingdom Fungi.

[0092] Gene editing: The process of altering specific genes, typically by CRISPR-Cas9 or similar enzyme systems, which alter the sequence of a functional gene, rendering it inactive. Other uses include introducing new sequences (including genes) into a genome.

[0093] Genealogical relatedness: descent from one or more ancestors, e.g., genealogical relationship between parents and descendants.

[0094] Genetic identity: Genetic information that identifies an individual, including, for example, a representation of that genetic information, including genotype, genotype fingerprint, genome sequence, and genetic marker profile; "genetically identical" = 100% genetic identity, "X% genetically identical" = having X% genetic identity, etc.

[0095] Genotypic fingerprint: description of a genotype at a specially prepared set of marker loci; a known genotype.

[0096] Gill: lamella; part of a mushroom, the structure containing the scutella and basidium.

[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] Heteroallelic: Difference between homologous chromosomes in heterokaryonous genotypes; similar to heterozygosity.

[0100] Heterokaryon: Technically, sexual heterokaryon: a culture that has two complementary (i.e., necessarily heteroallelic at the Mat locus) types of haploid nuclei in a common cytoplasm, and is therefore functionally and physiologically similar to a diploid individual (but cytogenetically represented as N+N rather than 2N), is fertile (in the absence of any rare interfering genetic defects at loci other than Mat), and exhibits a nutritionally incompatible response to other heterokaryons; in the context of strain development, it is also referred to as a strain or stock.

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

[0102] Heterokaryon incompatibility: the phenomenon of antagonism observed between two genetically non-identical heterokaryons in physical proximity or contact; multilocus self / nonself recognition system; i.e., a genetic system that operates to limit anastomosis (hyphal fusion) and cytoplasmic contact in basidiomycete heterokaryons, allowing one heterokaryon culture to discriminate and recognize another culture as either self or nonself; nutritional incompatibility.

[0103] Heterokaryosis: Possession of heterokaryon traits.

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

[0105] Homoallelic: Having no more than one allele at a locus. For diploid organisms, the equivalent term is "homozygous." Haploid strains are, by definition, completely homoallelic at all non-overlapping loci.

[0106] Homokaryon: a haploid culture that has a single type (or somatic lineage) of haploid nucleus (cytogenetically designated N), is normally sterile, does not exhibit the typical self / non-self incompatibility reaction to heterokaryons, and can function as a gamete in sexually complementary anastomoses; a "line" that transmits a uniform genotype to progeny, as in inbred plant lines; a predominantly homoallelic line that hybridizes well and produces poor fruiting bodies is a putative homokaryon for strain development; see discussion below.

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

[0108] Hybrid: applied to heterokaryonous strains and cultures of biparental origin, usually produced by controlled mating.

[0109] Hybridize: The physical association of two cultures, usually homokaryons, for example on a Petri dish containing a sterile agar-based nutrient medium, in an attempt to achieve anastomosis, cytoplasmic fusion, and the formation of sexual heterokaryons (=mating); as previously mentioned, success.

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

[0111] Inbreeding: matings that include sib-line matings, backcrossing to a parental line or strain, 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 gene locus.

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

[0115] Inoculum: A culture in a form that allows for the propagation and growth of a culture, e.g., in fresh media; specialized commercial types of inoculum include seeds and CIs.

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

[0117] Introgression transformation: crossing hybrid offspring with parental lines or strains, thus introducing a desired trait from one strain into the dominant genetic background of the other parental line or strain.

[0118] Lamella: See gill.

[0119] Line: a culture used in crossing to produce a hybrid strain; usually a homokaryon and therefore homoallelic, or otherwise a highly homoallelic non-heterokaryon (non-NSNPP) culture; in fact, a functionally homokaryonous and completely or predominantly homoallelic culture; similar to a predominantly or completely homozygous inbred line in plant breeding.

[0120] Clade: See "Derived Clade." A set of EDV derivatives derived from a single original strain or variant.

[0121] Locus: a specifically reserved, contiguous portion of the genome that is homologous but often varies among different genotypes; plural: loci.

[0122] Marker-assisted selection: The use of linked genetic markers, including molecular markers, to track trait-determining loci of interest among offspring and through pedigrees.

[0123] MAT: mating type locus determining sexual compatibility and heterokaryon status.

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

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

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

[0127] Neohaploid: A haploid culture or line obtained by physically deheterokaryonizing (reducing heterokaryons to haploid components); a somatically derived homokaryon.

[0128] Progeny: descendants of parent heterokaryons within a single generation; often used to describe cultures obtained from spores from one mushroom of one strain.

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

[0130] Parent: The immediate ancestor of an individual; parental strains are heterokaryons and parental lines are homokaryons; heterokaryons can be the parents of F1 heterokaryons via intermediate parental lines.

[0131] Pedigree-assisted strain development: The use of phylogenetic information to identify desirable strain combinations in controlled breeding programs.

[0132] Phenotype: The observable characteristics of a strain or lineage that are expressed and manifested in the environment.

[0133] Cytoplasmic fusion: establishment of cytoplasmic continuity by anastomosis, leading to the formation of sexual heterokaryons.

[0134] Ancestor: Ancestors, including parents (direct ancestors).

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

[0136] Sexual compatibility: the condition between different lineages with non-identical alleles at the Mat locus, in which the two lineages can mate to produce stable and fertile heterokaryons. The opposite condition, sexual incompatibility, occurs when two lineages have the same allele at the Mat locus.

[0137] Somatic cells: cells of the vegetative hyphae.

[0138] Seed: A mushroom culture, generally a pure culture of heterokaryons, on a sterile substrate, generally a friable, dispersible particulate material, in some cases a grain; a commercial inoculant for compost; a reference to seed includes a reference to the culture on the substrate.

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

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

[0141] Sterile growth medium: A nutrient medium, sterilized by autoclaving or other methods, that supports the growth of organisms; 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: heterokaryon having specially prepared characteristics or a specific identity or ancestry; similar to a variety.

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

[0145] Tissue culture: dedifferentiated vegetative mycelium obtained from differentiated tissues of a mushroom.

[0146] Transformation: A method for selectively introducing the genetic determinants of one or more desirable traits (single-locus transformation) into the genetic background of an original strain, while retaining the majority of the genetic background of the original strain. See "introgression transformation" and "transformation."

[0147] Transformation: The process of altering the genetic material of an individual cell by incorporating foreign (exogenous) DNA into its genome; methods for obtaining transformation, including single-locus transformation.

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

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

[0150] Viral breakthrough: The sequential use of multiple incompatible strains, i.e., strains exhibiting heterokaryon incompatibility, within a mushroom production facility in a planned program of strain rotation to reduce transmission of viruses from on-site virus reservoirs to newly planted crops.

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

[0152] Yield: Net fresh weight of a harvested crop, usually expressed in pounds per square foot.

[0153] Yield patterns: Distribution of yield within each flush and among all flushes; influences crop and product size, quality, harvesting costs, and relative disease pressure.

[0154] Regarding the above definition of homokaryons, the distinction between homokaryons and homoallelic lineages is subject to technical and practical considerations: note that homokaryons, in classical terms, are haploid cultures that are self-evidently completely homoallelic. In practical terms, for the purposes of fungal strain development, this definition is broadened somewhat to accommodate both technical limitations and cytological variation by treating all primarily homoallelic lineages as homokaryons. Technical limitations include the fact that genomes contain duplicated DNA regions, including repeated elements such as transposons, and may also contain large duplications of chromosomal segments due to past translocation events, and such regions do not appear homoallelic by most genotyping methods. Two distinct A. bisporus genomes sequenced by the Joint Genome Institute, a U.S. federal agency, differ in estimated length by 4.4% and gene number by 8.2%, suggesting substantial DNA duplication or rearrangement within different strains of the species. The currently available A. bisporus genome cannot fully account for the physical arrangement of such elements and translocations; therefore, assembled genome sequences of haploid strains may contain regions that appear heteroallelic using currently available genotyping methods. Cytologically, homokaryon progeny typically result in spores that receive a single haploid postmeiotic nucleus. However, spores that receive two third-division nuclei from the basidium become genetically equivalent to homokaryons. Spores that receive second-division "sister" postmeiotic nuclei become functional homokaryons, despite the presence of some terminal "islands" of heteroallelicity due to crossing over during meiosis. Furthermore, meiosis in which homologs segregate asymmetrically can result in aneuploid, functionally homokaryon spores with an extra chromosome that generates a heteroallelic region. All of these cultures are highly homoallelic, and all function as homokaryons. Scientific and technological limitations make it impractical to differentiate such cultures, and also to rule out duplication of DNA segments as an explanation for limited, isolated regions of genome sequence assemblies that appear heteroallelic.Thus, in this application, the use of the term "homoallelic" to characterize a lineage encompasses completely or predominantly homoallelic lineages, and cultures so described are functional homokaryons and putatively homokaryons, all of which are defined as homokaryons in this application.

[0155] It is noted that cultures of strains descended from the B18287-s82 and / or WBL-s290 strains, such as J19109, produce mushrooms, portions of mushrooms, portions of cultures, and descendants of such cultures or strains and lines derived therefrom. Thus, the present invention encompasses cultures and portions of cultures of the B18287-s82 and WBL-s290 strains, as well as the F1 hybrid J19109 strain, and mushrooms and portions of mushrooms produced therefrom, including spores. Additionally, EDVs and cultures derived solely or primarily from initial cultures derived from the F1 descendants of B18287-s82 and / or WBL-s290, dormant or actively growing cultures present in dormant or germinating spores of the J19109 strain, and cultures incorporating genetic material from the F1 descendants of the J19109 strain.

[0156] The present invention further relates to methods for producing and using strain J19109 and its derivative varieties (EDVs). Uses of J19109-derived cultures and the other cultures described above include their incorporation into commercial products such as mushroom spawn and casing inoculants, as well as mushroom production, the development of additional new cultures of A. bisporus, and farm health, including crop diversification and "virus breaking."

[0157] As noted herein and as described above, the present invention further relates to the F1 strain J19109, its homokaryon lines, and its F2 hybrid derivatives, such as crosses (i.e., hybrids) produced between the J19109 SSI homokaryon progeny and other white homokaryons, such as J11500-s80. Additionally, the present invention encompasses cultures derived from or descended from the J19109 strain. Such cultures can be used to produce mushrooms and mushroom parts.

[0158] Details of the cross are described herein for J19109. Specifically, one particular J19109-s40 lineage was crossed with J11500-s80 to generate the hybrid strain J20176. A representative culture of the strain, J20176, is designated NRRL Accession No. 68166 The mushroom has been deposited under the terms of the present application. In one embodiment, the method further comprises growing an edible mushroom crop by performing the steps described above. In another embodiment, the method may comprise using strain J20176 or a descendant of strain J20176 in a crop rotation to reduce pathogen pressure and pathogen reservoirs in a mushroom growing facility as described above. In yet another embodiment, the method comprises using strain J20176 and the EDV of strain J20176 to generate progeny as described above.

[0159] It is noted that cultures of strains descended from the J19109-s40 and J11500-s80 strains, such as J20176, produce mushrooms, portions of mushrooms, portions of cultures, and strains and lines descended from or derived from such cultures. Thus, the present invention encompasses cultures and portions of cultures of the J19109-s40 and J11500-s80 strains, as well as hybrid J20176 strains, and portions of mushrooms, including mushrooms and spores produced therefrom. Additionally, EDVs and cultures derived solely or primarily from initial cultures derived from descendants of J10109-s40 and / or J11500-s80, dormant or actively growing cultures present on dormant or germinating spores of the J20176 strain, and cultures into which genetic material from descendants of the J20176 strain has been incorporated.

[0160] The present invention further relates to methods for producing and using strain J20176 and its derivative varieties (EDVs). Uses of cultures derived from J20176 and the other cultures described above include incorporation into commercial products such as mushroom spawn and casing inoculants, as well as mushroom production, development of additional new cultures of A. bisporus, and farm health, including crop diversification and "virus breaking."

[0161] The morphological and physiological characteristics of strain J20176 grown in standard culture medium, Difco brand PDA medium, are presented below. When grown in PDA medium in 8.5 cm diameter Petri dishes, strain J20176 was white, light brownish-yellow, or "yellowish-brown," irregularly lobed, and roughly circular in overall outline. During dynamic equilibrium growth between days 8 and 26 after inoculation of the culture into PDA using a 3.5 mm diameter circular plug, the colony grew at a rate of (1.32-1.48-1.49 (1.50-1.57) mm / day. The strain was expanded by transferring pure inoculum to larger volumes of sterile culture medium. No variant traits were observed or predicted in strain J20176.

[0162] Methods for obtaining, manipulating, and crossing the cultures of the present invention to generate the progeny, inocula, products, and crops of the present invention, to improve mushroom farm hygiene using strain rotation programs, and to obtain genotypic fingerprints of mushroom cultures have been described hereinabove and are well known to those skilled in the art. While the present invention has been described with respect to specific embodiments in this application, those skilled in the art will be able to generate additional embodiments and modifications in light of the teachings herein without departing from the spirit and beyond the scope of the claimed invention. Accordingly, it will be understood that the description herein is provided merely to facilitate understanding of the invention and should not be construed as limiting the scope of the invention.

[0163] The J20176 hybrid is a cross developed between J19109-s40 and J11500-s80. The J20176 hybrid does not possess the AI ​​trait (see Table IV below). Additionally, the J20176 strain possesses a strong combination of desirable traits derived from the J15987 pedigree. Strain 20176 has been grown in pre-commercial trials in North America, Europe, and China and has strong commercial potential.

[0164] Hybridization of Agaricus bisporus cultures of the present invention can be achieved by growing two different cultures, one of which is a genetic lineage present in a spore of strain J19109, together, preferably in axenic medium, in close proximity until anastomosis (i.e., mycelial or cell fusion) occurs. Upon successful hybridization, the resulting fusion culture is a first-generation outbred hybrid culture incorporating a genome from a lineage derived from strain J19109. This lineage may be derived from mushroom spores of J19109, which is one embodiment of the present invention. Alternatively, this lineage may be derived from protoplasts derived from basidium or other parts of the J19109 organism, which is another embodiment of the present invention.

[0165] For the purposes of this invention, we obtained the complete genomic DNA sequences of strain J19109 and its constitutive homokaryons (B18287-s82 and WBL-s290). We also obtained the genomic DNA sequences of strains J11500-s80 and J19109-s40 (parents of J20176). To prepare DNA, cultures were grown in sterile broth growth medium after dissociation. After 2–4 weeks, mycelial cells were collected by filtration, frozen at −80°C, and lyophilized to dryness. Cap tissue from mushrooms arising from cultures of heterokaryons (such as J20176) was obtained, frozen, and lyophilized. DNA was extracted from the lyophilized samples using the CTAB protocol, followed by ribonuclease (RNAse) treatment and gel purification. A DNA library was prepared from each culture by a contract supplier, Genewiz (New Brunswick, New Jersey), and the library was sequenced using Illumina technology. Assembly of the reads into genome sequence was performed by Sylvan, Inc. using the public domain reference genome sequence of H97 version 2.0 (Morin et al. 2012; PNAS 109(43):17501, incorporated herein by reference). As a result, Sylvan, Inc. has confidently determined approximately 93% to 95% of the overall genotype of strain J20176 and its parental homokaryons.

[0166] The genetic control of heterokaryon incompatibility is understood in the art to be unrelated to known mating type gene(s) and to involve more than one gene, e.g., four or five in Coprinopsis, with no defined chromosomal location.

[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, using equipment manufactured by Illumina, among others, allows for 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 the precise location of each SNP within the genome can be numerically defined using base pair positions.

[0168] Table I provides a SNP-based comparison of the genotypes of the cultures used to generate the invention described herein. To generate the whole genome sequence, Illumina 250 bp reads aligned to the H97 Version 2.0 reference genome using DNASTAR's Lasergene version 18 were utilized. Table I presents the genotypes of strains J19109 and J20176 and their parental lineages, B18287-s82 and WBL-s290 (for strain J19109) and J11500-s80 and J19109-240 (for strain J20176), at marker loci characterized by multiple sequences spaced along each of the 19 H97 Version 2.0 reference scaffolds greater than 100 Kbp in length.

[0169] [Table 1] TIFF2023250454000003.tif249162TIFF2023250454000004.tif249162TIFF2023250454000005.tif26161

[0170] It can be clearly seen that the SNP patterns of each lineage and strain are unique and therefore a robust form of genetic identification. Furthermore, anyone skilled in the art can verify the SNP markers listed in Table I.

[0171] The data in Table I are presented as 9-mers, and in most cases, the central base (position 5) results in a SNP. Robust markers were selected at loci aligned across each of the first 19 scaffolds. The majority of the markers in Table II are standard markers used in other patent cases, such as US9,017,988 and US10,440,930. SNP alleles at these loci were determined for B18287-s82, WBL-s290, J19109 heterokaryons, and J11500-s80, as well as the homokaryon parents of J20176 (J11500-s80 and J19109-s40).

[0172] [Table 2]

[0173] Table II reports data from six loci that are typically reported for the same panel of strains utilized in Table I, e.g., US9,017,988 and US10,440,930. As shown in Table II, for one heterokaryonous strain (J19109), two alleles are present at each marker locus, 5 For homokaryonous cultures of the species, a single allele is present per locus. Data were generated by using targeted polymerase chain reaction (PCR) to amplify genomic regions spanning specially designed markers from each of the culture DNAs. PCR primers flanking the specially designed marker regions at locations indicated by the location information provided below were utilized to generate the data; methods for designing appropriate primers are well known in the art. DNA was sequenced from the amplified PCR products by a contractor, Eurofins (Louisville, Kentucky), using selected methods, and genotypes were determined by directly interrogating these sequences against the Sylvan database of reference marker / allele sequences. In most cases, sequences were further confirmed by directly interrogating the corresponding whole genome sequence for that culture.

[0174] Description of p1n150-G3-2 marker: The 5' end of this marker segment begins at position 1 with the first "T" of the sequence TCCCAAGT, corresponding to position 868615 of H97 JGI V2.0 scaffold 1 (Morin et al. 2012), and spans approximately 600 nt in the reverse direction (relative to the scaffold orientation) in the majority of alleles; longer segments result from insertions of allele 1T into the DNA. Currently, nine alleles incorporating at least 30 polymorphic positions have been documented from diverse strains in the Sylvan microbial strain collection. This marker is an important part of Agaricus breeding programs because it is closely linked to the MAT locus. MAT is a gene complex that controls mating between homokaryons.

[0175] Allele 1T contains the 320 base pair transposon, as described above. All other known alleles of p1n150 lack this intron.

[0176] Analysis of a panel of 88 homokaryon SSIs derived from J19109 with p1n150 identified 52 matches to the p1n150 allele for Mat-1 and also identified a second cohort of 36 strains containing alternative mating-type alleles, tentatively designated "Mat-X."

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

[0178] The next step was to align DNA from the p1n150 amplicons from W01-s1, B18187-s82, and J11500-s80 to determine whether the MAT-2 p1n50 sequence was a perfect match. The data showed differences between the J11500-s80 MAT-2 sequence and those from W01-s1 and B18187-s82. Therefore, we designated the new p1n150 alleles discovered in W01-s1 and B18287-s82 as "2A."

[0179] Allele 1T: 320nt ABR transposon insertion @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] ITS (=ITS 1+2 region) marker description: The ITS segment is part of the nuclear rDNA region located on chromosome 9 (scaffold 10 in JGI H97 V2.0). The rDNA is a cassette that is tandemly repeated up to an estimated 100 times in the haploid genome of A. bisporus. Therefore, there is no single, precise location 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. Three copies of the cassette were included on scaffold 10 of the H97 JGI V2.0 assembly, starting at positions 1612110; a partial copy is also assembled on scaffold 29 (Morin et al. 2012). The 5' end of this marker segment begins at position 1 with the first "G" of the sequence GGAAGGAT and spans approximately 703–704 nt in the forward direction (relative to the orientation of the scaffold) in the majority of alleles. To date, more than nine alleles incorporating at least 11 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain collection.

[0183] The alleles present in this application are I1, I2 and I4, characterized as follows (format: using nucleotide base symbol @ alignment position, based on alignment of 9 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] MFPC-1-ELF marker description: The 5' end of this marker segment begins at position 1 with the first "G" of the sequence GGGAGGGT, corresponding to H97 JGI V2.0 scaffold 8 position 829770 (Morin et al. 2012), and spans approximately 860 nt in the forward direction (relative to the orientation of the scaffold) in the majority of alleles. To date, diverse strains in the Sylvan microbial strain repository have demonstrated at least seven alleles incorporating at least 40 polymorphic positions.

[0188] Note that MFPC-ELF is associated with the PPC-1 locus on scaffold 8, which is known to be a major factor controlling cap color. Mushrooms with white cap color 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] AN Marker Description: The 5' end of this marker segment begins at position 1 with the first "G" of the sequence GGGTTTGT, corresponding to positions 1701712 of the H97 JGI V2.0 scaffold 9 (Morin et al. 2012), and spans approximately 1660 (H97 genome) to 1700 nt (alignment interval) in the known alleles (relative to the orientation of the scaffold); several insertions / deletions have created polymorphisms of length that characterize the alleles in addition to point mutations at individual nucleotides. Currently, five alleles incorporating over 70 polymorphic positions have been documented across diverse strains in the Sylvan microbial strain repository.

[0192] Only one allele, N1, is present in the strain used in this application (format: use nucleotide base symbol @ alignment position, based on alignment of alleles N1 to N4):

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

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

[0195] AS Marker Description: The 5' end of this marker segment begins at position 1 with the first "G" of the sequence GG(T / N)GTGAT, corresponding to position 752867 of the H97 JGI V2.0 scaffold 4 (Morin et al. 2012), and spans approximately 1620 (H97 genome) to 1693 nt (alignment interval) in known alleles (relative to the orientation of the scaffold). Several insertions and deletions have created point mutations at individual nucleotides, as well as length polymorphisms that characterize the alleles. Currently, seven alleles incorporating over 80 polymorphic positions have been documented across diverse strains in the Sylvan microbial strain repository.

[0196] The alleles present in the B18287 / J19109 most recent pedigree are alleles SC and SD, characterized in part as follows (format: nucleotide base symbol @ alignment position, based on alignment of alleles SA through SG):

[0197] Allele SC: "T" @ 28; "GATATC" @ 258 ~ 263; "G" @ 275; [insert] + "TTTC" T CAGC" + [insert] @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] FF Marker Description: The 5' end of this marker segment spans approximately 570 nt in the forward direction (relative to the orientation of the scaffold) from position 1, starting with the first "T" of the sequence TTCGGGTG, which corresponds to H97 JGI V2.0 scaffold 12 position 281999 (Morin et al. 2012). To date, seven alleles incorporating at least 20 polymorphic positions have been demonstrated from diverse strains in the Sylvan microbial strain repository.

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

[0201] Allergy FF1: "CCG" @48~50, "C" @91, etc.

[0202] Allergy FF2: "TTC" @48~50, "C" @91, etc.

[0203] The FF1 genotype is found in white strains such as So76 and U1 and related EDVs such as A15. In this study, one of the breeding strains, So76-s12b, carried this marker.

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

[0205] As mentioned above, one of the goals of the J19109 and J20176 pedigrees was to develop a method for preserving as many of the positive traits found in J15987 as possible while eliminating aggressive incompatibility (AI) traits. To test this concept, a test protocol was designed to measure the effects of mixing small amounts of inoculum derived from F2 hybrids with a J19109-derived line as one homokaryon parent and J11500-s80 as the second homokaryon parent, or control lines, with compost inoculated with commercially available A-15. Within this experimental design, normal incompatible interactions would not interfere with overall mushroom yield, but lines with antagonistic traits to J15987 would significantly reduce, or even eliminate, A-15 yield.

[0206] Sixteen F2 lines were selected for this study (see Table III below), with J15987 and A15 used as positive and negative controls, respectively. A-15 yields were obtained in replicated control cropping experiments. In typical Agaricus bisporus crops, the first mushroom break is harvested over a 3-4 day period. Experienced harvesters collect the earliest mushrooms, leaving a space that fills with mushrooms as the crop develops. In contrast, when the AI ​​trait is expressed, very few mushrooms are produced. Large areas of the mushroom bed are barren, and some areas may have only a few mushrooms. In other words, the AI ​​trait results in a catastrophic reduction in mushroom yield. Generally, yield reductions due to heterokaryon incompatibility or vegetative incompatibility can be 10% or less, or in most cases, 5% or less. However, the AI ​​trait is so devastating that reductions of over 50% are not uncommon. However, for purposes of this invention, the AI ​​trait is defined as a post-test yield loss of 15% or less of the A15 control yield.

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

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

[0209] Extreme biases such as those observed for the latter three chromosomes are likely due to deleterious alleles, but in either case, virtually no segregation was observed at the loci, making genotype-phenotype correlation analysis impossible. However, because both marker and phenotypic segregation were observed in the analyses performed, the four chromosomes listed above appear to play little or no role in conferring the AI ​​trait phenotype.

[0210] Thus, for this study, there was an informative panel of nine chromosomes.

[0211] [Table 3]

[0212] Table III shows the yield and heritable alleles for hybrids obtained by individually crossing J11500-s80 with 16 other J19109 homokaryons to form F2 hybrids. This list includes J20176. In this study: For each strain, 55 lb of Phase II mushroom compost was inoculated into each of three 2.76 square foot containers ("tubs"). The yield was the total for the three tubs.

[0213] For each treatment in the table, including J20176, 1% by weight 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 of the treatments 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 ready for harvest from day 16 to day 20, at which point only the first break in production was harvested to terminate the trial.

[0215] The third column shows the yield ratio, which 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 the nine Agaricus chromosomes, indicating whether the allele was inherited from one or the other of J19109's parents (B18287-s82 and WBL-s290). Note that the parental homokaryons in the table have been abbreviated to 290 for WBL-s290 and s82 for B18287-s82 to fit the table.

[0217] It is clearly evident that the A15 crop yield was much lower in the presence of line #1, line #16, and J15987 than in the presence of any of the other lines. In other words, these lines meet the defined test criteria for manifestation of the AI ​​trait in tests with A15. Using the yield ratio data, it can be estimated that the yield ratios for line #1, line #16, and J15987 test treatments were all 0.15 or less. The line closest to line #16 was line #5, with a yield ratio of 0.53; however, as noted above, this line was severely affected by Trichoderma contamination, resulting in yield loss. Therefore, a working definition of the AI ​​trait in this test protocol was proposed to include a yield loss of 15% or less of the A15 control yield.

[0218] Furthermore, examination of chromosomal inheritance patterns revealed that strain #1, strain #16, and J15987 all share the same WBL-s290 allele on chromosomes 4, 7, and 9. No other strains in this study share this haplotype, strongly suggesting a correlation with the invasive incompatibility trait.

[0219] The probability of a perfect match between chromosomes 4, 7, and 9 and a randomly occurring AI trait is given by 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 particular genotype, and q is the probability of a particular genotype being present per sample.

[0220] Of the 17 strains tested, three strains (two from the test cohort plus J15987) matched to a particular genotype, with a probability of 0.125 for each strain (the observed segregation did not significantly deviate from the theoretical probability of 0.5 per allele; with three alleles, the probability was 0.5 3= 0.125), the calculated binomial probability for this scenario is 0.0154, or a 1.54% chance. In other words, the p-value for the possibility that the phenotype-haplotype correlation was due to chance was 0.0154. Conventionally, a p-value of less than 0.05 is considered strong evidence to reject random chance.

[0221] However, the 0.5 probability per allele used above assumes a Mendelian 50:50 ratio.

[0222] The theoretical probability of 0.5 can be further refined using the data in Table III. The s290 allele was present 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 closely matches the expected ideal 50:50 ratio. Thus, the overall ratio for all three chromosomes is 25 / 51, which is 0.49:0.51 (very close to the 50:50 ratio).

[0223] Furthermore, because J15987 is part of a separate cohort, it is not possible to calculate the binomial probability without using the data from J15987; in other words, Strain #1 and Strain #16 This reduced the sample size from 17 to 16. The binomial probability in this case was 7.03%, which is still good odds (93%) that the association between the chromosome and the AI ​​trait is real.

[0224] Taken together, the statistical analysis indicated that aggressive incompatibility was significantly correlated with, and likely explained by, the influence of incompatibility elements 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 new hybrids can be produced that retain a significant percentage of the genetic makeup (and phenotype) present in J15987 yet lack the AI ​​trait. Finally, there are robust, statistically supported segregation data that strongly suggest that AI is controlled by genetic elements on three separate Agaricus chromosomes, allowing 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 additional J19109 SSI homokaryons x J11500-s80 hybrids grown in two separate trials. It is noteworthy that this new group of hybrids all out-yielded the A15 trial control and performed closely to the J15987 control.

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

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

[0231] For the data in Tables VI and VII, significance is indicated by an asterisk: * 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] We further demonstrated that the overall mushroom shape and color qualities of J15987 were reproduced in hybrids derived from J19109. The data in Table VI clearly show that this was achieved.

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

[0235] Cap roundness is an approximation of how spherical the mushroom cap appears. Higher values ​​indicate rounder mushrooms, which are preferred by growers and customers based on visual appeal. Lower values ​​indicate flatter mushrooms. All of the hybrids derived from J19109 recorded in Table VI fall within the continuum around J15987, clearly demonstrating the retention of this trait.

[0236] Stalk thickness is the ratio of the mushroom's stalk width to the diameter of its cap. Higher values ​​indicate thicker stalks. Different markets have their own preferences for stalk thickness, some preferring thicker stalks and others preferring thinner stalks depending on the needs of the market. Nearly all of the hybrids derived from J19109 recorded in Table VI exhibited slightly thinner stalks than J15987, but this is not universally a positive or negative quality.

[0237] Flesh thickness is an approximation of how much of the mushroom cap's volume is made up of flesh tissue rather than stalk or gill tissue. Higher values ​​indicate a higher proportion of flesh tissue, which is preferred by growers as a sign of quality. All of the hybrids derived from J19109 recorded in Table VI fall within the continuum around J15987, clearly demonstrating the retention of this trait.

[0238] When the shape data of these mushrooms were examined together, the shape of J15987 was clearly preserved.

[0239] [Table 7]

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

[0241] The L value is an objective measure of how white the mushroom cap appears when intact. For white mushrooms, higher values ​​are desirable. All of the hybrids derived from J19109 recorded in Table VII fall within the continuum around J15987, clearly demonstrating that this trait has been retained. In fact, several strains recorded significantly higher L values ​​than J15987, suggesting that the trait has been improved rather than simply retained.

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

[0243] [Table 8]

[0244] Table VIII shows the A-15 control. compared to Yields from initial pre-commercial trials of J20176 are shown. Growth conditions were typical of European commercial growers using a "Dutch" system with heavy Phase I and Phase II composting and heavy mycelial elongation (Phase III). A-15 controls were flushed in the laboratory using industry standard conditions.

[0245] Although J20176 yielded more than the control in the first break, this difference did not reach significance. Only the third break observation reached significance, with A-15 yielding more in the third break than J20176. A comparison of total yields showed no significant difference between the two strains. These data demonstrate the yield potential of J20176, demonstrating that even under commercial growth conditions not optimized for J20176's requirements, it produced yields virtually identical to those of the A-15 strain. In general, process refinement over time is required to achieve good yields. Observations indicated that J20176 mushrooms were rounder and whiter than the A-15 control.

[0246] In this study, particular attention was paid to looking for evidence of aggressive incompatibility, given that J20176 and A-15 existed in adjacent sympatric areas. Typical strain interactions were observed in areas mixed with colonized compost or casing, and a typical incompatibility reaction was observed, with A-15 and J20176 strains growing alongside each other in only small areas where no growth was observed. No AI traits were induced by J20176 under commercial conditions. These data demonstrate the market suitability and potential of J20176.

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

Claims

1. 1. A method for eliminating the invasive incompatibility (AI) trait from an Agaricus bisporus mushroom strain, wherein it has been found that crossing a culture of a white mushroom strain designated WBL-s290, a representative culture of which has been deposited under NRRL Accession No. 68167, with a culture of a mushroom strain designated J11500-s80, a culture of which has been deposited under NRRL Accession No. 68164, results in a hybrid mushroom strain designated J15987 having the AI ​​trait, a representative culture of which has been deposited under NRRL Accession No. 67646, said method comprising: crossing a culture of a hybrid mushroom line designated B18287-s82, a representative culture of which has been deposited under NRRL Accession No. 68168, with a culture of a white mushroom line designated WBL-s290 to obtain an F1 hybrid mushroom line designated J19109, a representative culture of which has been deposited under NRRL Accession No. 68163; allowing a culture of the new F1 strain, designated J19109, to form fruiting bodies and obtaining homokaryon spores therefrom; obtaining cultures of homokaryon lines from homokaryon spores derived from F1 strain J19109 and selecting cultures of homokaryon lines derived from F1 strain J19109; crossing a culture of a homokaryon line derived from F1 strain J19109 with a culture of a mushroom line designated J11500-s80 to obtain an F2 hybrid mushroom line; 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:

2. 2. The method of claim 1, wherein the homokaryon line derived from the F1 strain J19109 lacks the centromere-linked alleles of the white mushroom WBL-s290 line on chromosomes 4, 7, and 9.

3. 2. The method of claim 1, wherein the F2 hybrid line eliminates the AI ​​trait and provides at least two beneficial traits found in strain J15987 selected from the group consisting of a rounded cap shape similar to that of strain J15987, a stalk similar in thickness to that of strain J15987, meat similar in thickness to that of strain J15987, and a redness (a) value on the L-a-b color measurement scale that is within 10% of the redness (a) value of strain J15987.

4. 2. The method of claim 1, wherein the culture of the homokaryon lineage derived from F1 strain J19109 is the lineage culture designated J19109-s40, and a representative culture of said lineage has been deposited under NRRL Accession No. 68165.

5. 5. The method of claim 4, wherein the step of crossing a culture of a homokaryon line derived from F1 strain J19109 with a culture of a mushroom line designated J11500-s80 comprises crossing the line culture designated J19109-s40 with the mushroom line designated J11500-s80, resulting in a resulting F2 hybrid strain designated J20176, a representative culture of which has been deposited under NRRL Accession No. 68166.

6. 6. The method of claim 5, wherein the F2 hybrid line, designated J20176, does not have the AI ​​trait and retains at least two beneficial traits found in strain J15987 selected from the group consisting of a rounded cap shape similar to that of strain J15987, a stalk similar in thickness to that of strain J15987, a meat similar in thickness to that of strain J15987, and a redness (a) value on the Lab color measurement scale that is within 10% of the redness (a) value of strain J15987.

7. 1. A culture comprising at least one set of chromosomes of Agaricus bisporus strain B18278-s82, said culture of strain B18278-s82 having been deposited under NRRL Accession No. 68168, said chromosomes comprising all of the alleles of said strain B18278-s82 at marker loci characterized by the sequences listed in Table I.

8. 10. A culture derived from an initial culture, wherein the initial culture is the culture of claim 7, and wherein at least 75% of the genome or genotype is present in the genome or genotype of the initial culture of strain B18287-s82, and the initial strain culture is deposited under NRRL Accession No. 68168; or wherein the culture is directly derived from the initial culture, and wherein all of the genome or genotype is present in the genome or genotype of the original culture of strain B18287-s82.

9. A culture comprising at least one set of chromosomes of Agaricus bisporus strain WBL-s290, said culture of WBL-s290 having been deposited under NRRL Accession No. 68167, said chromosomes comprising all of the alleles of said WBL-s290 at marker loci characterized by the sequences listed in Table I.

10. 10. A culture derived from an initial culture, wherein the initial culture is the culture of claim 9, and wherein at least 75% of the genome or genotype is present in the genome or genotype of the original culture of the WBL-s290 strain, and the original culture of the strain is deposited under NRRL Accession No. 68167; or wherein the culture is directly derived from the original culture, and wherein all of the genome or genotype is present in the genome or genotype of the original culture of the WBL-s290 strain.

11. 1. A culture comprising at least one set of chromosomes of Agaricus bisporus strain J11500-s80, said culture of strain J11500-s80 having been deposited under NRRL Accession No. 68164, said chromosomes comprising all of the alleles of said strain J11500-s80 at marker loci characterized by the sequences listed in Table I.

12. 12. A culture derived from an initial culture, wherein the initial culture is the culture of claim 11, and wherein at least 75% of the genome or genotype is present in the genome or genotype of the initial culture of strain J11500-s80, and the initial strain culture is deposited under NRRL Accession No. 68164; or wherein the culture is directly derived from the initial culture, and wherein all of the genome or genotype is present in the genome or genotype of the original culture of strain J11500-s80.

13. A culture comprising at least one set of chromosomes of Agaricus bisporus strain J19109-s40, said J19109-s40 strain culture having been deposited under NRRL Accession No. 68165, said chromosomes comprising all of the alleles of said J19109-s40 strain at marker loci characterized by the sequences listed in Table I.

14. 14. A culture derived from an initial culture, wherein the initial culture is the culture of claim 13, and wherein at least 75% of the genome or genotype is present in the genome or genotype of the initial culture of strain J19109-s40, and the initial strain culture is deposited under NRRL Accession No. 68165; or wherein the culture is directly derived from the initial culture, and wherein all of the genome or genotype is present in the genome or genotype of the original culture of strain J19109-s40.

15. A hybrid mushroom culture of Agaricus bisporus designated strain J19109, a representative culture of said strain being deposited under NRRL Accession No. 68163.

16. 16. A culture of Agaricus bisporus derived from an initial culture, wherein the initial culture is the culture of claim 15, and wherein at least 75% of the genome or genotype is present in the genome or genotype of the initial culture of strain J19109, the culture of which is deposited under NRRL Accession No. 68163; or wherein the culture is directly derived from the initial culture, and wherein all of the genome or genotype is present in the genome or genotype of the initial culture of strain J19109.

17. A hybrid mushroom culture of Agaricus bisporus designated strain J20176, a representative culture of which has been deposited under NRRL Accession No. 68166.

18. 18. A culture of Agaricus bisporus derived from an initial culture, wherein the initial culture is the culture of claim 17, and wherein at least 75% of the genome or genotype is present in the genome or genotype of the initial culture of strain J20176, and wherein the culture of the strain is deposited under NRRL Accession No. 68166; or wherein the culture is directly derived from the initial culture and wherein all of the genome or genotype is present in the genome or genotype of the initial culture of strain J20176.

19. Mushrooms obtained from the culture of any of claims 13 to 18.

20. 20. A product incorporating the culture of any of claims 7 to 18, wherein the product is selected from the group consisting of mycelium, spawn, fresh or processed mushrooms, mushroom spores, mushroom spawn, mushroom preparations and extracts and fractions, mushroom pieces, mushroom inoculant, casing inoculant, casing spawn, casing soil, inoculated compost, colonized compost, post-cultivation compost, and friable particulate matter.

21. 19. A part of the culture described in any one of claims 7 to 18 selected from the group consisting of mycelia, mushrooms, resting spores, germinating spores, homokaryons, heterokaryons, cells, nuclei, and protoplasts.

22. 19. A cell of any of the cultures of any of claims 7 to 18.

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