Method for excluding aggressive incompatible traits from agaricus bisporus strains and related strains and strains

Abstract

The invention relates to a method for excluding aggressive incompatibility (AI) characters from an Agaricus bisporus mushroom strain. Wherein the method comprises mating a culture of a hybrid mushroom line named B18287-s82, a representative culture of the line having been preserved in NRRL accession number 68168, with a culture of a white mushroom line named WBL-s290 to obtain an F1 hybrid mushroom strain named J19109 (a representative culture of the F1 strain having been preserved in NRRL accession number 68163). When a culture of a new F1 strain named J19109 is fruited and a homonuclear spore is obtained therefrom, a homonuclear culture is obtained from the homonuclear spore of the F1 strain J19109, and the homonuclear culture is selected from the F1 strain J19109. And mating the homonuclear line culture from the F1 strain J19109 with the culture of the mushroom line named as J11500-s80 to obtain the F2 hybrid mushroom strain. The culture of the F2 hybrid mushroom strain is tested to determine the presence or absence of the AI trait, where the AI trait has been excluded from the F2 hybrid mushroom strain in the absence of the AI trait.

Description

Technical Field

[0001] The present invention generally relates to a method for eliminating the aggressive incompatibility trait (also known as the AI ​​trait) from Agaricus bisporus strains. Also disclosed are a variety of novel strains and strains associated with the method. Background Art

[0002] The present invention follows the release of an earlier white hybrid mushroom strain designated J15987 (NRRL Deposit No. 67646) developed by Sylvan America, Inc. as disclosed in U.S. Patent No. 10,440,930, the disclosure of which is incorporated herein by reference. Strain J15987 has a higher yield and produces higher quality mushrooms than the current dominant white cultivars, meaning that these mushrooms have a thicker cap flesh and less red color than the standard commercial white mushrooms obtained from the commercially acceptable strain designated A-15. In early testing of strain J15987, consumers were very pleased with the strong combination of yield and quality, which made these mushrooms and this strain initially very popular commercially. However, a problem arose. It was discovered that strain J15987 contained an unprecedented phenotype or trait known as "aggressive incompatibility" that caused problems at the mushroom farm level when these farms also used strain A-15 or similar strains. Therefore, there is a need for a method to exclude the phenotype or trait of "aggressive incompatibility" (as defined below) from white mushroom strains similar to J15987, and to develop such similar white mushroom strains that retain or otherwise provide the desirable traits (such as high yield and / or high quality) of strain J15987 while excluding the aggressive incompatibility (AI) trait.

[0003] The edible mushroom, Agaricus bisporus (Lange) Imbach var. bisporus, is a basidiomycete fungus that is cultivated worldwide. In Europe and America, it is the most widely cultivated mushroom species. The crop is valued at approximately $1 billion per year in the United States in 2020-21 (National Agricultural Statistics Service / USDA data).

[0004] Agaricus cultures are prepared, maintained, propagated, and stored on sterile media in a manner similar to other microorganisms. Pure cultures for a variety of purposes, including spore germination, preparation of new hybrids, and preparation of inocula, are manipulated using sterile tools and aseptic techniques within a sterile field. Commercial media are "spawn," which can have a number of different formulations, the most common being sterile millet or rye. Production of spawn is typically large-scale. For example, 1 liter of pure Agaricus inoculum can be used for 14,0000 liters of sterilized spawn. The end user, the mushroom farm, receives the pure spawn culture in sterile packaging.

[0005] Mushrooms are grown commercially in specially constructed structures at mushroom farms. Although there are many variations in the method, a typical method is described below. Compost is prepared from waste lignocellulosic materials, such as wheat straw, nitrogen-containing materials are added, finished and transferred to specific conditions (aerobic, temperature between 40 and 50°C) for pasteurization, thereby creating a substrate that is conducive to the colonization of Agaricus. Mushroom spawn is added and colonized in the substrate for 11 to 17 days at controlled temperature. Once colonization or "spawn run" is completed, a non-nutritious saturated peat layer (pH adjusted to between 8 and 8.5 with beet lime) is applied to the surface of the compost to a layer of about 5 cm. In addition, a covering spawn can be added to the peat or cover layer to accelerate mycelial growth. Once colonization occurs, environmental conditions are adjusted in a process called "mushroom boom", where the air temperature is reduced to about 18-19°C and CO2 is reduced to about 1300ppm. The mushrooms will be fruiting in the mushroom house on the 13th to 18th day after the application of the cover soil (covering the soil). The mushrooms are harvested within a 3 to 4 day period. Additional production flushes or breaks are produced and harvested before removing and replacing the compost on the mushroom farm.

[0006] In the United States, 72% of the total harvest in 2020-21 were white Agaricus, and the rest were brown Agaricus varieties. During the same period, the total value of the U.S. mushroom crop exceeded $1 billion. In other regions, people have a stronger preference for white mushrooms. In the United States and elsewhere, for white mushrooms, the market requirements for phenotypic traits such as size, shape, whiteness, firmness, and related traits such as extended shelf life are narrow and precise. Therefore, new white Agaricus strains should meet a specific set of market requirements. Strains can be isolated based on some traits, such as cap shape (e.g., cap roundness, flesh thickness, stipe thickness), color (whiteness), density / firmness (firmness), and piece weight. Some examples of commercialization goals for new white button mushroom strains are increased crop yield, altered yield distribution throughout the production boom, disease resistance, insect resistance, improved shelf life, reduced crushing, ease of management, suitability for mechanical harvesting, differential responses to stressors (e.g., temperature or substrate changes), seasonal effects, farm practices, and strain incompatibility. DNA marker technology, such as single nucleotide polymorphisms (SNPs), can also be used to distinguish and separate strains from each other. Strains can have different ancestries, which will directly reflect the genotype and phenotype.

[0007] Around 1980, the first two true hybrids of A. bisporus were developed in a laboratory in Horst, the Netherlands. The two "Horst" strains, called U1 and U3, were closely related hybrids produced by mating two existing white strains, the prehybridized smooth white strain (PHSW) and the prehybridized gray-white strain (PHOW), as described in M. Imbernon et al., Mycologia, 88(5), 749-761 (1996), which is incorporated herein by reference. Over time, the hybrid strain U1 became the predominant white genotype on the market.

[0008] The parent homokaryon of strain U1 is recovered by making the culture go heterokaryon. The PHSW parent is H39, and the PHOW parent is H97. H97 was deposited at the Fungal Genetics Stock Center in Kansas, USA, with accession number 10389 by A. Sonnenberg, and was also deposited at the American Type Culture Collection with accession number MYA-4626. The Joint Genome Project (California, USA) has sequenced the H97 genome and placed it in the public domain, and the genome has been shown to be very valuable as a reference for single nucleotide polymorphism (SNP) detection (see below).

[0009] Strain U1 is the ancestor of all white strains currently cultivated in North America and Europe. Several mushroom strains, such as A-15 and S-130, meet the criteria for substantially derived varieties (EDVs) of U1, which are developed from spores or tissue of the original (i.e., initial) strain. Groups of strains developed from a single ancestor by somatic selection or spore culture, or related methods of "substantial derivation" as discussed below, are called derived lineage groups. In addition to relatively minor genetic differences, all white strains developed within the U1 derived lineage group share a single composite N+N heterokaryotic genotype with the original strain U1. With the notable exception of China, modern white mushroom cultivation is a monoculture.

[0010] Agaricus bisporus has a reproductive syndrome called amphithallism, in which two different life cycles proceed simultaneously. As in other fungi, the reproductive propagules are spores. Agaricus produces spores by meiosis on thick sporangiums called basidia. In the first life cycle, Agaricus bisporus spores each receive a single haploid postmeiotic nucleus; these spores are able to mate, but cannot produce mushrooms. These haploid spores germinate to produce homokaryotic progeny or lines, which can mate with other compatible (compatible) homokaryons to produce new hybrid heterokaryons capable of producing mushrooms. Heterokaryons usually show much poorer mating ability than homokaryons. This life cycle is called heterothallic, which is similar to outcrossing. This life cycle works, but is usually not dominant in strains of Agaricus bisporus var. bisporus.

[0011] The second life cycle is called intramixis, which is similar to a form of inbreeding and is dominant in most bisporus varieties of Agaricus bisporus. Most spores receive two post-meiotic nuclei, and most of these nuclei are composed of non-sister nuclei (NSNP), which have heteroallelic genotypes at most or all centromere-linked loci including the MAT locus. The MAT genotype determines the heterokaryotic phenotype of these offspring, which have reproductive capacity and can produce mushroom crops. Unusually, in eukaryotes, relatively few chromosome crossings have been observed in the meiotic progeny of Agaricus bisporus; according to experience, in the heterokaryotic intramictic progeny of heterokaryotic strains, there are few heteroallelic (similar to heterozygosity) losses.

[0012] In both life cycles, the spore is the cell that is part of the mushroom; the cell is the reproductive body that always has the mushroom as its single parent and produces the culture of mushrooms.

[0013] In the case of spores, live spores are heterokaryons or homokaryons in a dormant state. Spores are part of a culture of a mushroom organism; they incorporate only the genetic material (hereditary material) of the single culture from which they were produced (often referred to as the "parent" for convenience). Other parts of the culture include the cap, stipe, gills, cells (defined as the hyphal compartment that incorporates the nucleus, mitochondria, cytoplasm, protoplasts, RNA, DNA, proteins, cell membranes, and cell walls (including cross-walls), hyphae, and mycelium. Spores can be aseptically collected on sterile material, suspended in sterile water at various dilutions, and plated onto sterile agar growth medium to produce germinated spores and cultures incorporating spores. The preferred technique would be to have a live Agaricus culture in a closed petri dish, which can stimulate spore germination by diffusion of volatile pheromones. The germinated spores can be isolated under a microscope using sterile microscopic tools, such as steel pins, onto fresh nutrient agar plates. Using this method, heterokaryotic and homokaryotic progeny of the strain containing spores can be obtained, as well as cultures incorporated into the spores of the strain.

[0014] The development of novel hybrid varieties by heterothallic mating includes controlled physical combination and mating of two compatible (affinity) cultures to obtain novel heterokaryon cultures. Homokaryons (= "lines") are preferred starting cultures for mating because they have the greatest ability to join with other cultures and achieve cytoplasmic fusion. Heterokaryons can also be placed in physical contact, but the probability of mating that results in the successful formation of novel heterokaryons is unreasonably low. Compatibility (affinity) is determined by the genotype at the MAT locus; two homokaryons with the same MAT alleles cannot establish heterokaryons after joining, so homokaryon compatibility represents genetic heterogeneity. In a determined mating program, homokaryon lines are obtained and combined in predetermined paired combinations. In one method, homokaryon pairs can be placed in close proximity on the surface of a nutrient agar medium in a culture dish and grown together (in physical combination), when point anastomoses occur between the two cultures. The successful result is the mating of heterokaryons. By transferring mycelia from the fusion zone of the culture dish, novel hybrid heterokaryons can be obtained.

[0015] There is a clear need for commercially acceptable white Agaricus bisporus strains having different genotypes relative to the U1-derived lineage group for two reasons. First, strains that are nutritionally incompatible with strains of the U1-derived lineage group are known to slow the spread of viral diseases between strains, and these diseases are known to be a major cause of crop losses in the commercial mushroom industry. Second, it is well known that when an agricultural industry is extensively dependent on crops with a single genetic lineage, as is the case with white Agaricus bisporus in most of the world, the risk of catastrophic crop failures occurring across a facility or industry is increased. Therefore, from a food security and risk management perspective, it is highly desirable to provide both genetic diversity and commercially acceptable performance and crop characteristics.

[0016] As mentioned above, the present invention is a direct result of the commercial release of hybrid strain J15987 (NRRL deposit number 67646), which is a white hybrid strain (U.S. Patent No. 10,440,930). Pre-commercial testing showed that strain J15987 had a thicker cap flesh and thicker stipe than other white cultivars from strain A-15. In addition, color analysis of J15987 using a Minolta colorimeter showed less red on the mushroom cap after harvest. The commercial potential was considered significant and the strain was planted in the United States directly after the patent application. It successfully met all the production standards of the original crop. However, a significant problem occurred. It was observed that in some mushroom growing "houses" (or rooms), especially where mushrooms from strain A-15 were produced, there were large areas of compost without mushroom growth in both the compost and the cover layer. Obviously, this observation was abnormal and unusual.

[0017] The cultures were returned to the testing facility where suspected antagonistic interactions between strain J15987 and other white cultivars were studied in more detail. The genetics of strain-strain interactions are controlled by a self / non-self recognition system, which is also known as vegetative incompatibility, or heterokaryon incompatibility. Strain J15987 was found to have a very aggressive, unusual antagonistic response when in contact with other U1 family EDVs, such as A-15. When strains were mixed in ratios as low as 99% A-15 to 1% J15987, the Agaricus mycelium died rapidly and the A-15 genotype was eventually replaced by J15987. This interaction is referred to as "aggressive incompatibility" and the interaction is abbreviated as "AI" and the phenotype is defined as the "AI" trait. To be clear, unrelated basidiomycete strains, including A. bisporus, often exhibit incompatibility responses that have been exploited for virus control, but the AI ​​trait is a more aggressive, extreme, and problematic manifestation of a more common phenomenon. Presumably, this trait is under genetic control, as is generally understood for nutritional incompatibility in basidiomycetes.

[0018] Conventional mixing equipment on mushroom farms tends to operate in a manner that allows a certain degree of mixing between the inoculation substrates of different strains. These events are usually small and have a moderate negative impact. Because these areas are small, their impact on total yield is minimal and is traditionally tolerable. However, the impact of larger and more extreme AI interactions is so large that it cannot be tolerated commercially, and the evaluation of consumers (clients) is that strain J15987 cannot be advantageously processed in farm operations where strain A-15 is also cultivated. Therefore, it is necessary to have the positive attributes of strain J15987 while also lacking one or more commercially desirable strains of the AI ​​trait. Summary of the invention

[0019] In order to understand and overcome (eliminate) the potential genetic basis that causes AI trait performance in strain J15987, a series of matings were developed and the hybrid produced was analyzed. Mating schemes are described in the following scheme 1. In essence, wild variant lines (i.e. B18287-s82, NRRL preserving number 68168) are combined with one of the homonuclear parents of strain J15987 (i.e. WBL-s290, NRRL preserving number 68167) to produce new F1 hybrid strains (i.e. J19109, NRRL preserving number 68163). Then, the homonuclear body from the hybrid strain (e.g., J19109-s40, NRRL preserving number 68165) is mated with another homonuclear parent of strain J15987 (i.e. J11500-s80, NRRL preserving number 68164) to provide new F2 hybrid strains (e.g., J20176, NRRL preserving number 68166). The F2 hybrids were confronted to confirm the presence or absence of the AI ​​trait. Based on these confrontations, it was clear that the mating scheme was successful and most of the new hybrids were free of the AI ​​trait. The inheritance pattern allowed for further development of a genetic control / elimination model for the AI ​​trait.

[0020] Solution 1

[0021]

[0022] First, it was revealed that a homokaryon (line B18287-s82) obtained from a hybrid strain produced by mating two homokaryons (i.e., W01-s1 and So76-s12b) was necessary. This homokaryon line B18287-s82 was developed from a parent homokaryon obtained from wild strain W01 collected by R. Kerrigan in North America in the 1990s and pre-hybridized gray-white strain So76. DNA testing showed that W01 was a true wild strain, not an escaped commercial strain. In addition, W01 had very similar growth requirements to the current white and brown strains.

[0023] Thus, one aspect of the present invention generally relates to a new unique homokaryotic line of Agaricus bisporus designated B18287-s82, and methods of using the line designated B18287-s82. In at least one embodiment, a culture comprising at least one set of chromosomes of Agaricus bisporus line B18278-s82 is provided, wherein the at least one set of chromosomes comprises all alleles of line B18278-s82 at the marker locus characterized by the sequences listed in Table I below. As disclosed herein, the deposit of the culture of Agaricus bisporus line B18287-s82 has been made and accepted by the Agricultural Research Agency Culture Collection (NRRL) (1815 North University Street, Peoria, Illinois 61604 USA). The deposit date is June 10, 2022. The deposited culture is taken from the same culture preserved by Sylvan, Inc., Kittanning, Pennsylvania, USA (registered assignee) before the filing date of the present application. All restrictions on the deposit have been removed, and the deposit is intended to satisfy all deposit requirements of the U.S. Patent and Trademark Office, including 37 C.FR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL deposit number is 68168. The deposit will remain in the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be renewed as necessary during that time. Upon filing of a patent application or issuance of a patent, the culture will be irrevocably and without restriction or conditional release to the public, as required by applicable patent law.

[0024] In one embodiment, an Essentially Derived Variety (EDV) of line B18287-s82 can be provided. The EDV can include a culture derived from an initial culture, wherein the initial culture is a culture of line B18287-s82, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line B18287-s82. 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 line B18287-s82. In other embodiments, the EDV can include a culture directly or solely derived from an initial culture, such that its genome or genotype is entirely present in the genome or genotype of the initial culture of line B18287-s82.

[0025] The B18287-s82 homokaryon was mated with WBL-s290. It should be noted that this line is one of the parental homokaryons currently present in strain J15987 (the other is J11500-s80, which is also used in this invention). The resulting F1 heterokaryon strain was named J19109.

[0026] Thus, another aspect of the invention generally relates to a new unique homokaryotic line of Agaricus bisporus designated WBL-s290, and methods of using the line designated WBL-s290. In at least one embodiment, a culture comprising at least one set of chromosomes of Agaricus bisporus line WBL-s290 is provided, wherein the at least one set of chromosomes comprises all alleles of line WBL-s290 at the marker loci characterized by the sequences listed in Table I below. As disclosed herein, a deposit of a culture of Agaricus bisporus line WBL-s290 has been made and accepted by the Agricultural Research Service Culture Collection (NRRL) (1815 North University Street, Peoria, Illinois 61604 USA). The deposit date is June 10, 2022. The deposited culture is taken from the same culture deposited by Sylvan, Inc., Kittanning, Pennsylvania, USA (registered assignee) prior to the filing date of the present application. All restrictions on the deposit have been removed, and the deposit is intended to satisfy all deposit requirements of the U.S. Patent and Trademark Office, including 37 C.FR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL deposit number is 68167. The deposit will remain in the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be renewed as necessary during that period. Upon filing of a patent application or issuance of a patent, the culture will be irrevocably and without restriction or conditional release to the public, as required by applicable patent law.

[0027] In one embodiment, a substantially derived variety (EDV) of WBL-s290 can be provided. The EDV can include a culture derived from an initial culture, wherein the initial culture is a culture of WBL-s290, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of WBL-s290. 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 WBL-s290. In other embodiments, the EDV can include a culture directly or solely derived from an initial culture, such that its genome or genotype is entirely present in the genome or genotype of the initial culture of WBL-s290.

[0028] Another aspect of the invention generally relates to a new and unique Agaricus bisporus strain culture designated J19109; it is a hybrid strain obtained by direct mating of two isokaryotic cultures, B18287-s82 and WBL-s290. A deposit of a culture of strain J19109 has been made and accepted by the Agricultural Research Service Culture Collection (NRRL) (1815 North University Street, Peoria, Illinois 61604 USA) . The deposit date is June 10, 2022. The deposited culture is taken from the same culture deposited by Sylvan, Inc., Kittanning, Pennsylvania, USA (registered assignee) prior to the filing date of the present application. All restrictions on the deposit have been removed, and the deposit is intended to meet all deposit requirements of the United States Patent and Trademark Office, including 37 C.FR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL deposit number is 68163. The deposit will be maintained at the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be replaced as needed during this period. Under the Patent Act, upon filing of a priority application or issuance of a patent for the strain, the culture will be irrevocably and without restriction or conditionally released to the public.

[0029] In one embodiment, a substantially derived variety (EDV) of strain J19109 can be provided. The EDV can include a culture of Agaricus bisporus derived from an initial culture, wherein the initial culture is a culture of strain J19109, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of strain J19109. Alternatively, other EDVs can include a culture directly derived from an initial culture of strain J19109, such that its entire genome or genotype is present in the genome or genotype of the initial culture of strain J19109.

[0030] To reiterate, a culture of a hybrid mushroom line designated B18287-s82 (a representative culture of which has been deposited under NRRL deposit number 68168) was mated with a culture of a white mushroom line designated WBL-s290 (a representative culture of which has been deposited under NRRL deposit number 68167) to obtain an F1 hybrid mushroom strain designated J19109 (a representative culture of which has been deposited under NRRL deposit number 68163). Then, the culture of the F1 strain designated J19109 was fruited to obtain homokaryotic spores therefrom, thereby obtaining a homokaryotic line. Then, a homokaryotic line culture from the F1 strain J19109 was selected. Notably, the homokaryotic line from the F1 strain J19109 lacked alleles at the centromere-linked loci of the line WBL-s290 on chromosomes 4, 7, and 9.

[0031] After selecting an isokaryotic line culture from the F1 strain J19109, the culture was mated with a culture of a mushroom line designated J11500-s80, which was indicated as another parental isokaryon currently present in strain J15987. From the mating, a resulting F2 hybrid mushroom strain was obtained, and the culture of the resulting F2 hybrid mushroom strain was tested to determine the presence or absence of the AI ​​trait, wherein in the absence of the AI ​​trait, the AI ​​trait had been excluded from the F2 hybrid mushroom strain.

[0032] Thus, another aspect of the invention generally relates to a new unique homokaryotic line of Agaricus bisporus designated J11500-s80, and methods of using the line designated J11500-s80. In at least one embodiment, a culture comprising at least one set of chromosomes of Agaricus bisporus line J11500-s80 is provided, wherein the at least one set of chromosomes comprises all alleles of line J11500-s80 at the marker loci characterized by the sequences listed in Table I below. As disclosed herein, a deposit of a culture of Agaricus bisporus line J11500-s80 has been made and accepted by the Agricultural Research Service Culture Collection (NRRL) (1815 North University Street, Peoria, Illinois 61604 USA). The deposit date is June 10, 2022. The deposited culture is taken from the same culture stored by Sylvan, Inc., Kittanning, Pennsylvania, USA (registered assignee) prior to the filing date of the present application. All restrictions on the deposit have been removed, and the deposit is intended to satisfy all deposit requirements of the U.S. Patent and Trademark Office, including 37 C.FR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL deposit number is 68164. The deposit will remain in the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be renewed as necessary during that period. Upon filing of a patent application or issuance of a patent, the culture will be irrevocably and without restriction or conditional release to the public, as required by applicable patent law.

[0033] In one embodiment, a substantially derived variety (EDV) of line J11500-s80 can be provided. The EDV may include a culture derived from an initial culture, wherein the initial culture is a culture of line J11500-s80, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line J11500-s80. 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 line J11500-s80. In other embodiments, the EDV may include a culture directly or solely derived from an initial culture, such that its genome or genotype is entirely present in the genome or genotype of the initial culture of line J11500-s80.

[0034] It will be further understood that in one or more embodiments, the F2 hybrid strain excluding the AI ​​trait will retain at least two beneficial traits present in strain J15987. These beneficial traits can be selected from: a cap shape that is as round as the cap shape of strain J15987, a stipe that is as thick as the stipe of strain J15987, a flesh that is as thick as the flesh of strain J15987, and a redness (a) value within 10% or 5% or 2.5% or 1% or 0.5% of the redness (a) value of strain J15987 on the Lab color measurement scale. It will be understood that the terms "as round as..." and "as thick as..." ("as thick as...") mean that the cap, stipe and / or flesh of the mushrooms produced by the F2 hybrid strain are within a statistically significant range of the roundness or thickness (thickness) of the cap, stipe or flesh of the mushrooms produced by strain J15987 as measured. In alternative embodiments, the roundness of the cap shape of the mushroom of the F2 hybrid strain may be within 20% or 15% or 10% or 5% of the roundness of the cap shape of the mushroom of strain J15987. In other alternative embodiments, the thickness of the stipe of the mushroom of the F2 hybrid strain may be within 20% or 15% or 10% or 5% of the thickness of the stipe of the mushroom of strain J15987. In other alternative embodiments, the thickness of the flesh of the mushroom of the F2 hybrid strain may be within 20% or 15% or 10% or 5% of the thickness of the flesh of the mushroom of strain J15987.

[0035] In one embodiment, the homokaryotic line selected from the F1 strain J19109 may be the homokaryotic line designated J19109-s40. It will be appreciated that the line culture designated J19109-s40 was mated with the mushroom line designated J11500-s80, wherein an F2 hybrid strain designated J20176 was produced. The hybrid strain J20176 was found to not have the AI ​​trait.

[0036] Thus, another aspect of the invention generally relates to a new unique homokaryotic line of Agaricus bisporus designated J19109-s40, and methods of using the line designated J19109-s40. In at least one embodiment, a culture comprising at least one set of chromosomes of Agaricus bisporus line J19109-s40 is provided, wherein the at least one set of chromosomes comprises all alleles of line J19109-s40 at the marker loci characterized by the sequences listed in Table I below. As disclosed herein, a deposit of a culture of Agaricus bisporus line J19109-s40 has been made and accepted by the Agricultural Research Service Culture Collection (NRRL) (1815 North University Street, Peoria, Illinois 61604 USA). The deposit date is June 10, 2022. The deposited culture is taken from the same culture stored by Sylvan, Inc., Kittanning, Pennsylvania, USA (registered assignee) prior to the filing date of the present application. All restrictions on the deposit have been removed, and the deposit is intended to satisfy all deposit requirements of the U.S. Patent and Trademark Office, including 37 C.FR Sec. 1.801-1.809, and all deposit requirements under the Budapest Treaty. NRRL deposit number is 68165. The deposit will remain in the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be renewed as necessary during that period. Upon filing of a patent application or issuance of a patent, the culture will be irrevocably and without restriction or conditional release to the public, as required by applicable patent law.

[0037] In one embodiment, a substantially derived variety (EDV) of line J19109-s40 can be provided. The EDV may include a culture derived from an initial culture, wherein the initial culture is a culture of line J19109-s40, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line J19109-s40. 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 line J19109-s40. In other embodiments, the EDV may include a culture directly or solely derived from an initial culture, such that its genome or genotype is entirely present in the genome or genotype of the initial culture of line J19109-s40.

[0038] Another aspect of the invention generally relates to a new and unique Agaricus bisporus strain culture designated J20176; it is a hybrid strain obtained by direct mating of two isokaryotic cultures, J11500-s80 and J19109-s40. A deposit of a culture of strain J20176 has been made and accepted by the Agricultural Research Service Culture Collection (NRRL) (1815 North University Street, Peoria, Illinois 61604 USA). The deposit date is June 10, 2022. The deposited culture is taken from the same culture deposited by Sylvan, Inc., Kittanning, Pennsylvania, USA (registered assignee) prior to the filing date of the present application. All restrictions on the deposit have been removed, and the deposit is intended to meet all deposit requirements of the United States Patent and Trademark Office, including 37 C.FR Sec. 1.801-1.809, and all deposit requirements set forth in the Budapest Treaty. The NRRL deposit number is 68166. The deposit will be maintained at the depository for 30 years, or 5 years after the last request, or for the life of the patent, whichever is longer, and will be renewed as needed during this period. Under the Patent Act, upon the filing of a priority application or the issuance of a patent for the strain, the culture will be irrevocably and without restriction or conditionally released to the public.

[0039] In one embodiment, a substantially derived variety (EDV) of strain J20176 can be provided. The EDV can include a culture of Agaricus bisporus derived from an initial culture, wherein the initial culture is a culture of strain J20176, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of strain J20176. Alternatively, other EDVs can include a culture directly derived from an initial culture of strain J20176, such that its entire genome or genotype is present in the genome or genotype of the initial culture of strain J20176.

[0040] It will be further understood that in one or more embodiments, the F2 hybrid strain designated as J20176 excludes the AI ​​trait and retains at least two beneficial traits present in strain J15987. These beneficial traits can be selected from: a cap shape that is as round as the cap shape of strain J15987, a stipe that is as thick as the stipe of strain J15987, a flesh that is as thick as the flesh of strain J15987, and a redness (a) value within 10% or 5% or 2.5% or 1% or 0.5% of the redness (a) value of strain J15987 on the Lab color measurement scale. It will be understood that the terms "as round as..." and "as thick as..." ("as thick as...") mean that the cap, stipe and / or flesh of mushrooms produced by the F2 hybrid strain J20176 are within a statistically significant range of the roundness or coarseness (thickness) of the cap, stipe or flesh of mushrooms produced by strain J15987. In alternative embodiments, the roundness of the cap shape of the mushrooms of the F2 hybrid strain J20176 may be within 20% or 15% or 10% or 5% of the roundness of the cap shape of the mushrooms of the strain J15987. In other alternative embodiments, the thickness (thickness) of the stipes of the mushrooms of the F2 hybrid strain J20176 may be within 20% or 15% or 10% or 5% of the thickness of the stipes of the mushrooms of the strain J15987. In other alternative embodiments, the thickness of the flesh of the mushrooms of the F2 hybrid strain J20176 may be within 20% or 15% or 10% or 5% of the thickness of the flesh of the mushrooms of the strain J15987.

[0041] It can be seen that the method described in the present invention advantageously eliminates the AI ​​trait from Agaricus bisporus strains, including at least strain J20176. More specifically, the method is capable of eliminating the aggressive incompatibility (AI) trait from Agaricus bisporus strains derived from (derived from) strain J10165 (also known as WBL) and strain J11500 (as disclosed in U.S. Pat. No. 9,622,428), wherein it is known that mating a culture of the white mushroom line designated WBL-s290 with a culture of the mushroom line designated J11500-s80 provides a hybrid mushroom strain designated J15987 having the AI ​​trait, a representative culture of which has been deposited under NRRL deposit number 67646.

[0042] With respect to obtaining homokaryotic cultures from homokaryotic spores from F1 strain J19109, it will be appreciated that when J19109 heterokaryons are fruited and F2 spores are collected, 52 homokaryons are initially obtained in total, and 16 of these are described in several tables in the following tables. All 52 homokaryons were mated with the second parent J11500-s80 of J15987, and the commercialization potential of the resulting strains was extensively screened. The collected data, as shown below, indicate that the beneficial traits of J15987 are maintained, and more importantly, most of the new J19109×J11500-s80 hybrids do not have the AI ​​trait. Select a hybrid (strain J20176) for controlled testing. The strain is robust and therefore a strong candidate for commercialization.

[0043] The entire pool of isokaryotic lines of strain J19109 showed similar potential to J19109-s40, and all are considered valuable breeding lines with the potential to produce high quality mushroom hybrids lacking the AI ​​trait.

[0044] One of the uses of the hybrids derived from J19109 is to produce edible mushroom crops for sale. Therefore, mushrooms obtained from cultures of any of the strains or strains specified above are part of the present invention. Another use is to improve facility hygiene through strain rotation and "virus destruction" effects. Other uses are to incorporate genetic material of strain J19109 into progeny and derived or subsequent generations of cultures, including dormant and germinated spores and protoplasts. As mentioned above, there are other uses.

[0045] The unusual biology of fungi, and specifically endogenous hybridization in Agaricus bisporus, makes it easy and straightforward to derive virtual copies of cultures with essentially identical phenotypes. In plant breeding, these copies are referred to as substantially derived varieties or EDVs. Methods for obtaining cultures (thus, by definition, EDVs of a single initial culture of Agaricus bisporus) include, by way of example, somatic selection, tissue culture selection, monospore germination, polyspore germination, selfing, repeated backcrossing to the initial culture, mutagenesis, and transformation. DNA-mediated transformation of Agaricus bisporus has been reported in Velcko, AJ Jr., Kerrigan, RW, MacDonald, LA, Wach, MP, Schlagnhaufer, C., and Romaine, CP 2004, Expression of novel genes in Agaricus bisporus using an Agrobacterium-mediated transformation technique. Mush. Sci. 16: 591-597, and references therein, which are incorporated herein by reference. Transformation can introduce a single new gene or allele into the genome of the initial culture. In addition, recent reports on other closely related fungi have raised the possibility of gene editing by CRISPR.

[0046] EDVs are clearly distinguishable by their genotype, which is predominantly or even exclusively a subset of a single original culture. The percentage of the original genotype present in the Agaricus bisporus EDV ranges from 100% or almost 100% in the case of monospore culture and somatic selection, to 99.x% in the case of strains modified by DNA-mediated transformation, to 90-99.x% in the case of some monospore or polyspore selection or some mutagenesis, to an average of at least 75-85% in the case of sib-progeny mating (= selfing) and backcrossing to the original culture. A variety of genotype determination methods, including those described below and others well known in the art, can be used to determine the percentage of DNA of an original culture present in another culture and to clearly determine the relationship between the two cultures as well as any method for manipulating or utilizing the original culture.

[0047] Repeated backcrossing to the initial culture also produces the EDV of the initial culture. In a hypothetical example, in the first consecutive repetition of the process, the resulting strain of this generation will have an average of about 75% of the DNA of the initial strain, while about 25% of the DNA will be provided (contributed) by the second strain or strain; when the process is repeated, the DNA representation of the initial strain will increase, averaging close to 97% after a further 3 consecutive repetitions. Generally, it will be understood that any culture with 75-100% genotypic identity to the initial culture is an indication of the EDV of the initial culture. It is also established that the EDV of the EDV is also the EDV of the initial strain. Finally, since Agaricus bisporus alternates generations between heterokaryotic strains and homokaryotic strains, the criteria of substantial derivation apply equally to cultures of both strains and strains.

[0048] A genotypic fingerprint is a description of the genotype at a defined locus, in which the presence of the characterized allele is recorded. These fingerprints provide a powerful and efficient technique for identifying all types of EDVs of clones and initial strains and for identifying ancestors in outbred pedigrees. A variety of techniques are available for defining and characterizing (identifying) loci and alleles in a genotype. Whole genome sequencing (WGS) provides the most detailed method, which allows direct characterization and comparison of DNA sequences of entire genomes. Using this method to generate robust genotypic fingerprints that introduce a large number of marker loci, it is possible to establish (establish) relationship properties between two strains, including strains associated with pedigree progeny over multiple generations. The applicant has tracked genetic markers through four to six generations of its strain development pedigree. If a sufficient number of unique markers are present in the initial strain or strain, it is possible to identify progeny from the initial strain or strain after several outbred generations without excessive experiments. In a hypothetical example, in an F4 hybrid, after 4 outbred generations, the average expected value of the genome representation of the initial haploid strain is 3.1% (50% / 2 4 ), which corresponds to about 1 Mb of nuclear genomic DNA of Agaricus bisporus. Based on the applicant's analysis, the amount of DNA from each of two unrelated Agaricus bisporus strains can typically contain about 10,000 to about 20,000 single nucleotide polymorphisms (SNPs), any of which can provide a distinguishing marker linking the F4 hybrid to the original strain. By using multiple independent markers, the ancestors of the strains can be identified with a very high probability of success and reasonable confidence.

[0049] One trait of biological and commercial value (commercial interest) is heterokaryon incompatibility. The genetics of these self / non-self recognition systems have not been well elucidated in basidiomycete fungi, such as Agaricus, but are known to involve multiple alleles at multiple independent loci in other fungal groups. Note that heterokaryon incompatibility occurs in most fungi and is very different from the aggressive incompatibility traits reported herein. "Normal" incompatibility is typically a localized area of ​​mycelial interaction where growth between two genotypes is attenuated. It is believed that this function will prevent viruses from being transferred between different genotypes of the same species.

[0050] It should be noted that there are at least two double-stranded RNA viruses that affect mushroom crops, namely La France disease and Mushroom virus X. Heterokaryon incompatibility will prevent successful conjugation and cytoplasmic continuity in a physical mixture of two or more heterokaryons, thereby producing a "virus-break". On a mushroom farm, virus-break is performed by replacing crop materials (compost, spawn, cover inoculum) that incorporate the initial strain with inoculum and crop materials that incorporate another different strain that is incompatible with the initial strain. In the most effective implementation of the virus-break method, all biological materials of the initial strain of the mushroom farm are replaced with biological materials of the second incompatible strain. Strain incompatibility creates an effective (if not absolute) barrier to the movement of viruses from the biorepository within the facility to the new crop. Rotational cultivation use between mushroom strains with different genotypes can also interrupt the infection and infestation cycle of exogenous pests and pathogens.

[0051] As mentioned above, hybrid mushroom strain producers are constantly searching for hybrid strains that will allow growers to successfully and profitably produce crops. In the case of the new hybrids derived from J19109 and derivatives or progeny thereof, the positive attributes documented to date include attractive appearance (rounded caps, thick stipes, good bruise resistance compared to market leader A-15, all of which are attractive to consumers), and overall yields that can match or exceed strains such as A-15. In our example, mushroom growers can obtain high-yield crops of high-quality mushrooms by using the new hybrids described herein. In addition, these strains match the high quality of J15987, an earlier strain developed by the applicants of the present invention, with the significant advantage of lacking an aggressive incompatibility phenotype.

[0052] The strains currently available to the mushroom industry enable growers to produce mushroom crops successfully and generally profitably. Several factors influence the degree of success and profitability achieved. For example, the strain must be able to produce at least comparable crop yields in two to three batches or two to three fruiting waves relative to strains currently marketed and grown commercially. In addition, some physical properties of the mushrooms produced, such as cap color categories, and conventional sizes and dimensions, such as cap diameter, allow the mushrooms to be marketed in familiar product categories.

[0053] The J19109 SSI x J11500-s80 hybrid meets those market needs and addresses the current unavailability of such strains on the market. Additionally, this new set of strains is genotypically distinct when compared to other existing white cultivars (see Tables I and II below), which addresses potential issues associated with reliance on monocultures.

[0054] One or more aspects of the present invention may be achieved by hybrid mushroom cultures of Agaricus bisporus developed by the present invention. Thus, the present invention encompasses products incorporating any culture of the above strains or strains. These products may include mycelium, spawn, fresh or processed mushrooms, mushroom spores, mushroom spawn, mushroom preparations and extracts and fractions, mushroom pieces, mushroom inoculum, casing inoculum, casing spawn, casing soil, inoculated compost, colonized compost, post-cropped compost, and friable particulate matter.

[0055] In addition, various parts of the culture of any of the above strains or strains may have value and commercial applications. Thus, parts selected from mycelium, mushrooms, spores, cells, nuclei and protoplasts are contemplated. Further, in one or more embodiments, unicellular microorganisms are contemplated. Thus, cells of any culture of the above strains or strains are provided.

[0056] It should be noted that strain J20176 has been deposited (a representative culture of which has been deposited under NRRL deposit number 68166) to provide a suitable example of a possible strain that meets the requirements of the present invention. Thus, strain J20176 may include multiple parts of the culture, including hyphae, spores, and cells and cell parts, including nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes, and cell walls, which are present in the vegetative mycelium of the culture and in the mushrooms produced by the culture. The spores may be dormant or germinated spores and may include heterokaryons and homokaryons incorporated therein.

[0057] One or more products of the hybrid mushroom culture of Agaricus bisporus incorporating the progeny strain designated as J19109 may be produced. These products include mycelium, spawn, inoculum, overlay inoculum, fresh mushrooms, processed mushrooms, mushroom extracts and fractions, mushroom pieces and colonization substrates selected from grains, compost and loose granules. It will be understood that mushroom pieces refer to the stipe, cap and other larger parts of the mushroom itself.

[0058] One or more other aspects of the invention may be achieved by a substantially derived variety (EDV) of a hybrid mushroom culture of a strain, such as J19109. In one or more embodiments, the Agaricus bisporus culture produced by substantial derivation has at least one of the essential characteristics of the J19109 derived mating partner. For the present invention, as an example of a superior strain as a direct result of the invention described herein, we have deposited a mating partner, J19109-s40×J11500-s80, which is referred to as J20176. J20176 is a strain lacking the AI ​​trait and has other characteristics of J15987 such as cap roundness, flesh thickness, yield performance and yield time relative to a U1 EDV, such as A-15.

[0059] Other aspects of the invention can be achieved by a method for producing a hybrid culture of Agaricus bisporus, the method comprising the step of mating a homokaryon, such as J19109-s40 (deposited under NRRL accession number 68165) with a second homokaryon. In one embodiment, the second homokaryon is J11500-s80, a culture of which is deposited under NRRL accession number 68164. This mating provides a mushroom culture J20176, which demonstrates a normal antagonistic pattern to a group of commercial cultivars based on U1 EDV, such as A-15. This antagonism confirms the genetic uniqueness of strain J20176. In one or more embodiments, the method further comprises providing a mushroom culture of the present invention, the culture consisting of mycelium, spawn, inoculum, cover inoculum, fresh mushrooms, processed mushrooms, mushroom parts, mushroom extracts and fractions, mushroom pieces, and a colonization substrate selected from grains, compost, and loose particles. In other embodiments, the method may include providing the mushroom culture in a derivative culture selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic cell subcultures, tissue explant cultures, protoplasts, dormant spores, germinated spores, inbred and outbred progenies, transgenic cultures, gene-edited cultures, and cultures with a single locus converted genome.

[0060] Other aspects of the invention can be achieved by a hybrid mushroom culture of Agaricus bisporus having a genotypic fingerprint having characteristics at marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AF and FF. In one or more embodiments, the culture has a genotypic fingerprint having the characteristics described in Table II at the marker loci, wherein all characteristics of the fingerprint are present in the genotypic fingerprint. It should be understood that meiosis and random assortment will ensure that each homokaryotic spore of the J19109 progeny will have a unique marker pattern. Based on the inherited DNA markers, one skilled in the art will directly infer that the J19109 homokaryotic breeding stocks are progeny.

[0061] One or more other aspects of the invention may be achieved by a culture, cell, or culture comprising the cell produced by the above method. Thus, one or more embodiments may include a method further comprising the step of growing a hybrid mushroom culture to produce hybrid mushrooms and mushroom parts. Other embodiments may provide such methods, wherein the hybrid mushroom culture or cell produced comprises a marker spectrum having characteristics at marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AF, and FF, wherein all features of the marker spectrum are also present in the marker spectrum of strains J20176 and J19109. Still other embodiments may provide such methods, wherein the hybrid mushroom culture or cell produced comprises a marker spectrum having characteristics at the marker loci described in Table I, wherein all features of the marker spectrum are also present in the marker spectrum of strain J20176.

[0062] These and other advantages of the present invention over the prior art relating to Agaricus bisporus and cultures will become apparent from the following description and are achieved by the invention hereinafter described and claimed. DETAILED DESCRIPTION

[0063] Initially, in order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0064] Allele: One or two or more alternative forms of a gene resulting from mutation and found at the same location on a chromosome; a heritable unit of the genome located at a defined locus and ultimately identified by its DNA sequence (or by other means).

[0065] Aggressive Incompatibility: An interaction between two heterokaryons in which the two cultures show strong antagonism towards each other, a reaction much more severe than typical heterokaryon incompatibility reactions. At the mushroom farm level, large areas of dead mycelium have been observed on both compost and mulch, with 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 U1 EDVs, such as A-15. Generally, as mentioned above, if more than 15% of A-15 is killed by J15987 or other strains, it is said to have the AI ​​trait. Over time, strains with the AI ​​trait will replace the U1 EDV and will become the only genotype present.

[0066] Amphithallism: A reproductive syndrome in which both heterothallic hybridization and intramixis are active.

[0067] Conjugation: The fusion of two or more hyphae that achieves cytoplasmic continuity.

[0068] Basidiomycete: A monophyletic group of fungi that produce meiospores on a basidium; members of the corresponding divisions of fungi such as the order Basidiomycetales or the subdivision Basidiomycotina.

[0069] Basidiosome: The meiosporangial cell in which karyogamy and meiosis occur and on which basidiospores are formed.

[0070] Biological efficiency: For mushroom crops, for any given sampled crop area or compost weight, the net fresh weight of the harvested crop divided by the dry weight of the compost substrate at the time of spawn inoculation.

[0071] Breeding: The development of strains, lines, or varieties using methods that emphasize sexual mating.

[0072] Pileus: The part of a mushroom that contains the gills.

[0073] Cap Flatness: A measure of the shape or thickness of a mature, open mushroom cap.

[0074] Cap roundness: Strictly, the ratio of the maximum distance between the uppermost and lowermost parts of the cap divided by the maximum distance through the cap, measured on a mushroom cut in half lengthwise; usually averaged over several specimens; subjectively, the "round" quality of the cap shape.

[0075] Carrier Substrate: A medium of nutritional and physical properties suitable for achieving growth and dispersion of a culture; examples are substrates formulated for mushroom spawn, overlay inoculants, and other inoculants.

[0076] Mulch: A layer of non-nutrient material, such as peat or soil, applied to the upper surface of a mass of colonized compost to allow a mushroom crop to develop.

[0077] Casing inoculum (CI): A preparation of inoculum material incorporating a mushroom culture (usually a defined heterokaryotic strain), suitable for mixing into a covering layer.

[0078] Cloning: Unselected reproduction of somatic cells.

[0079] Combining ability: The ability of an individual to pass on superior performance to its offspring. General combining ability is the average performance of an individual in a particular series of matings.

[0080] Compatibility (affinity): See heterokaryon compatibility, nutritional compatibility, sexual compatibility; incompatibility is the opposite of compatibility.

[0081] CRISPR: (Clustered Regularly Interspaced Short Palindromic Repeats) A technique in genetic engineering whereby the genome of a living organism can be modified.

[0082] Culture: A tangible living organism; an organism propagated on a variety of growth media and substrates; part or all of a physical strain, strain, homokaryon or heterokaryon; the sum of all parts of a culture, including mycelium, fungus, spores, cells, nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes and cell walls.

[0083] Cultivar: A commercially cultivated variety or strain.

[0084] Derivation: The development or acquisition of a culture solely or predominantly from an initial strain or culture; see EDV. The terms "derivation" and "derived" denote the process or the result thereof.

[0085] Derived lineage group: Group of EDVs derived from a single original strain, and including the original strain.

[0086] Progeny: The descendants of a pedigree over a finite number (e.g., 10 or fewer) generations.

[0087] Diploid: There are two haploid chromosome complements within a single nuclear envelope.

[0088] Site-directed mutagenesis: The process (method) of altering the DNA sequence of at least one specific genetic locus.

[0089] EDV (Essentially Derived Variety): A culture derived solely or predominantly from an initial strain or culture; a culture having 75% or more of its genotype present in the genotype of the initial strain, which condition is the result of its derivation. In the case of direct or sole derivation from an initial strain or culture, the culture is likely to have all of the genotypes of the initial culture.

[0090] Flesh thickness: the ratio of the maximum distance between the top of the stipe and the top of the cap, measured on a mushroom cut in half lengthwise, divided by the maximum distance through the cap; usually averaged over multiple specimens; subjectively, called "fleshyness".

[0091] Boom: Periods of mushroom production separated by intervals of non-production within a boom harvest cycle; the term boom encompasses the terms "break" and "wave" and may be interpreted as either of these terms.

[0092] Fungi: Microorganisms classified as members of the kingdom Fungi.

[0093] Gene Editing: The process of altering a specific gene, usually by CRISPR-Cas9 or a similar enzyme system, whereby the functional gene sequence is altered to render it inactive. Among other uses, new sequences (including genes) can be introduced into the genome.

[0094] Genealogical Relationship: The familial relationship of descendants from one or more ancestors, for example, the familial relationship between parents and children.

[0095] Genetic Identity: Genetic information that distinguishes individuals, including representations of said genetic information, such as and including: genotype, genotypic fingerprint, genome sequence, genetic marker spectrum; "genetically identical" = 100% genetic identity, "X% genetically identical" = having X% genetic identity, etc.

[0096] Genotypic fingerprint: A description of the genotype at a defined set of marker loci; a known genotype.

[0097] Gills: Gills (Lamella); the part of a mushroom that contains the hymenium and basidium structures.

[0098] Haploid: Having only a single complement of nuclear chromosomes; see homokaryon.

[0099] Heteroallele: Having two different alleles at a single locus; similar to a heterozygote.

[0100] Heteroallele: Differences between homologous chromosomes in a heterokaryotic genotype; similar to heterozygosity.

[0101] Heterokaryon: As a term in the art, it refers to a 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 instead of 2N), and that has reproductive capacity (absence of any rare disruptive genetic defects at loci other than Mat), and that exhibits nutritional incompatibility reactions with other heterokaryons; also called strains or stocks in the context of strain development.

[0102] Heterokaryon Compatibility: No antagonism is observed during physical proximity or contact between two genetically distinct heterokaryons; see Heterokaryon Incompatibility.

[0103] Heterokaryotic incompatibility: Antagonism observed between two genetically distinct heterokaryons during physical proximity or contact; Multilocus self / non-self recognition system; i.e., a genetic system that allows one heterokaryotic culture to distinguish and recognize another culture as self or non-self, which functions in basidiomycete heterokaryons to restrict conjugation (hyphal fusion) and cytoplasmic contacts; Vegetative incompatibility.

[0104] Heterokaryosis: Having the characteristics of heterokaryons.

[0105] Heterothallic mating: A life cycle involving mating between two different non-sibling haploid individuals or gametes; similar to outcrossing.

[0106] Isoallelic: Having no more than one allele at a locus. The equivalent term in diploid organisms is "homozygous." By definition, a haploid system is completely isoallelic at all non-replicated loci.

[0107] Homokaryon: A haploid culture (cytogenetically indicated as N) with a single type (or somatic cell lineage) of haploid nuclei and which is generally sterile and does not exhibit typical self / non-self incompatibility reactions with heterokaryons and can function as a gamete in sexual complementary conjugation; a "line" that transmits a uniform genotype to progeny, as inbred plant lines; major isoallele lines that mate well but have poor results are putative homokaryons for strain development purposes; see discussion below.

[0108] Homokaryotic: Having homokaryotic characteristics; haploid.

[0109] Hybrid: Of biparental origin, usually applied to heterokaryotic strains and cultures produced in controlled mating.

[0110] Hybridization: Physical joining of two cultures (usually homokaryons) in an attempt to achieve conjugation, plasmogamy and formation of sexual heterokaryons (=mating), e.g. on a petri dish containing a sterile, agar-based nutrient medium; successful achievement of the above processes.

[0111] Hyphae: The filamentous component of the mycelium, composed of cell-like compartments.

[0112] Inbreeding (Inbreeding): Mating, including mating between sibling lines, backcrossing to a parental line or strain, and endogenous hybridization; involving reproduction between genetically related parents.

[0113] Incompatibility: See heterokaryotic incompatibility.

[0114] Induced mutagenesis: A non-spontaneous process that alters the DNA sequence of at least one genetic locus.

[0115] Initial Culture: The culture used as the starting material in a strain development process; more specifically, the strain from which a substantially derived species is obtained.

[0116] Inoculum: A culture in a form that allows it to be propagated and multiplied, e.g., on new media; specialized commercial inoculum types include spawn and CI.

[0117] Endogenous hybridization: The sexual life cycle of a single parent involving the formation of complementary "mating" pairs in post-meiotic nuclei within a basidium or individual spore.

[0118] Introgression trait conversion: Mating the progeny of a hybrid to a parental line or strain, thereby introducing a desired trait from one strain into the dominant genetic background of the other parental line or strain.

[0119] Lamella: See “lamella”.

[0120] Line: A culture used in mating to produce a hybrid strain; usually homokaryotic and therefore isoallelic, otherwise a highly isoallelic non-heterokaryotic (non-NSNPP) culture; in fact, a culture that is functionally homokaryotic and completely or predominantly isoallelic; analogous to a predominantly or completely homozygous inbred line in plant breeding.

[0121] Phylogeny: See “derived phylogeny.” Group of EDVs derived from a single original strain or variety.

[0122] Locus: A defined contiguous portion of a genome that is homologous, although often differing between different genotypes; plural: loci.

[0123] Marker Assisted Selection: The use of linked genetic markers, including molecular markers, to trace the locus of a determinant trait of interest in progeny and through pedigrees.

[0124] MAT: a mating type locus that determines sexual compatibility and heterokaryotic status.

[0125] Mating: The sexual union of two cultures by conjugation and cytoplasmic fusion; methods of achieving mating between mushroom cultures are well known in the art.

[0126] Mycelium: The vegetative body or thallus of a mushroom organism, composed of filamentous hyphae.

[0127] Mushroom: The reproductive structure of the agaric fungus; the toadstool; the cultivated food of the same name.

[0128] Neohaplont: A haploid culture or strain obtained by physically de-heterokaryonizing (reducing to haploid components) a heterokaryon; a homokaryon obtained from a somatic cell.

[0129] Progeny: The offspring of a single generation, e.g., a parent heterokaryon; most often used to describe cultures obtained from spores of a strain of mushroom.

[0130] Outbreeding: Mating between unrelated or distantly related individuals; similar to heterothallic mating in mushrooms.

[0131] Parent: The direct ancestor of an individual; the parent strain is a heterokaryon, the parent line is a homokaryon; a heterokaryon can be the parent of an F1 heterokaryon through an intermediate parent line.

[0132] Pedigree-assisted strain development: Use of pedigree information to identify desired strain combinations for controlled mating programs.

[0133] Phenotype: Observable characteristics of a strain or strain that behave and appear in the environment.

[0134] Cytoplasmic fusion: The establishment of cytoplasmic continuity by conjugation, which results in the formation of sexual heterokaryons.

[0135] Ancestors: Ancestors, including parents (direct ancestors).

[0136] Selfing: Mating between sibling lines; see also endogenous hybridization.

[0137] Sexual compatibility: The situation between different lines with allele non-identity at the Mat locus, which allows the two lines to mate to produce stable and reproductively competent heterokaryons. The opposite situation, sexual incompatibility, occurs when the two lines have identical alleles at the Mat locus.

[0138] Somatic: Vegetative mycelium.

[0139] Spawn: A mushroom culture, usually a pure culture of heterokaryons, usually on a sterile substrate of loose (friable) and dispersible particles, in some cases on grains; a commercial inoculant used in composting; reference to spawn includes reference to culture on substrate.

[0140] Spore: The part of a mushroom that is the reproductive propagule.

[0141] Stipe: The part of a mushroom that supports the cap.

[0142] Sterile Growth Medium: A nutrient medium that supports the growth of organisms that has been sterilized by autoclaving or other methods; examples include agar-based solid nutrient media, such as potato dextrose agar (PDA), nutrient broth, and a variety of other materials.

[0143] Stipe: See stipe.

[0144] Strain: A heterokaryon with defined characteristics or a specific identity or ancestry; analogous to a variety.

[0145] Targeted mutagenesis: The process (method) of altering the DNA sequence of at least one specific locus.

[0146] Tissue culture: Dedifferentiated vegetative mycelium obtained from the differentiated tissue of mushrooms.

[0147] Trait conversion: A method of selectively introducing the genetic determinants of one (single locus conversion) or more desired traits into the genetic background of an initial strain while retaining most of the genetic background of the initial strain. See "introgression trait conversion" and "transformation".

[0148] Transformation: The process of changing the genetic material (hereditary material) carried by an individual cell by incorporating foreign (exogenous) DNA into its genome; methods for obtaining trait conversion, including single locus conversion.

[0149] Vegetative compatibility: No antagonism is observed during physical proximity or contact between two genetically distinct heterokaryons, which is determined by a multilocus self / non-self recognition system that functions in basidiomycete heterokaryons to restrict conjugation (hyphal fusion) and cytoplasmic contacts; heterokaryotic compatibility; the opposite of nutritional incompatibility.

[0150] Vegetative incompatibility: Antagonism observed during physical proximity or contact between two genetically distinct heterokaryons, determined by a multilocus self / non-self recognition system that functions in basidiomycete heterokaryons to restrict conjugation (hyphal fusion) and cytoplasmic contacts; heterokaryotic incompatibility.

[0151] Virus destruction: The sequential use of multiple incompatible strains, i.e., strains that exhibit heterokaryotic incompatibility, in a planned strain rotation program within a mushroom production facility to reduce virus transmission from in situ virus reservoirs to newly planted crops.

[0152] Whole genome sequence (WGS): The DNA sequence of an organism, such as Agaricus bisporus.

[0153] Yield: Net fresh weight of harvested crop, usually expressed in pounds per square foot.

[0154] Yield pattern: Distribution of yield within each wave and across all waves; affects crop and product size, quality, picking costs, and relative disease pressure.

[0155] Regarding the above definition of homokaryons, it should be noted that homokaryons and isoallelic lines are subject to technical and practical considerations: a homokaryon in classical terms is a haploid culture that is axiomatically completely isoallelic. In practical terms, for the purposes of fungal strain development, the definition is somewhat extended to accommodate technical limitations and cytological variations by considering all major isoallelic lines as homokaryons. Technical limitations include the fact that genomes contain duplicated DNA regions, including repetitive elements such as transposons, and may also include large duplications of chromosome segments due to historical translocation events; these regions may not appear as isoallelic by most genotyping methods. The Joint Genome Institute, a US federal agency, sequenced two different Agaricus bisporus genomes and estimated that they differed by 4.4% in length and 8.2% in gene number, indicating that there are extensive DNA duplications or rearrangements within different strains of the species. The currently available Agaricus bisporus genomes cannot fully account for the physical arrangement and translocation of these elements, and therefore the assembled genome sequences of haploid lines may have regions that appear as heteroallelic using currently available genotyping methods. Cytologically, homokaryotic progeny will usually be spores that receive a haploid nucleus after meiosis. However, spores that receive two third-division nuclei from basidium are genetically equivalent to homokaryons. Spores that receive two second-division "sister" nuclei after meiosis will be functional homokaryons, although due to the crossover during meiosis, some distal "islands" with heteroallelicity may exist. In addition, meiosis with asymmetric separation of homologs can produce aneuploid, functionally homokaryotic spores, in which there are extra chromosomes that produce heteroallelic regions. All of these cultures are highly homoallelic, and all act as homokaryons. Technical limitations make it impractical to distinguish these cultures, and it is also impractical to exclude DNA segment replication, which explains the limited, isolated regions of genomic sequence assembly that are manifested as heteroallelic genes. Thus, in this application, use of the term "isoallelic" to characterize strains includes strains that are completely or predominantly isoallelic, and cultures described in this manner are functionally homokaryotic, putatively homokaryotic, and are defined as homokaryotic in this application.

[0156] It is noted that cultures of progeny strains of line B18287-s82 and / or WBL-s290 (such as J19109) produce mushrooms, mushroom parts, culture parts, and progeny or derived strains and lines of these cultures. Thus, the present invention encompasses cultures and culture parts of line B18287-s82 and WBL-s290, and the F1 hybrid strain J19109, mushrooms and mushroom parts produced therefrom, including spores. Additionally, EDVs and cultures are derived solely or primarily from the following: initial cultures derived from F1 progeny of B18287-s82 and / or WBL-s290, dormant or actively growing cultures present in dormant or germinating spores of strain J19109, and cultures incorporating genetic material of F1 progeny of strain J19109.

[0157] The invention further relates to methods of making and using strain J19109 and substantially derived varieties (EDVs) of strain J19109. Uses of cultures derived from J19109 and the other cultures mentioned above include their incorporation into commercial products such as mushroom spawn and overlay inoculants, as well as for the production of mushrooms, the development of other novel Agaricus bisporus cultures and for crop diversification and farm sanitation, including "virus destruction".

[0158] Now, for the purposes of the present invention and as mentioned above, the present invention also relates to F1 strain J19109, its homokaryotic lines and its F2 hybrid derivatives, such as crosses (i.e. matings) made between J19109 SSI homokaryotic progeny and other white homokaryotic progeny (such as J11500-s80). In addition, the present invention includes cultures derived from strain J19109 or cultures of progeny of strain J19109. These cultures can be used to produce mushrooms and mushroom parts.

[0159] The details of the mating are described herein with respect to J19109. Specifically, a specific line J19109-s40 was mated with J11500-s80 to produce a hybrid strain J20176. A representative culture of strain J20176 is deposited under NRRL accession number ^^^^^. In one embodiment, the method further comprises planting an edible mushroom crop by implementing the steps described above. In another embodiment, the method may include using strain J20176 or a substantially derivative variety of strain J20176 in crop rotation to reduce pathogen pressure and pathogen reservoirs in a mushroom cultivation facility as described above. In yet another embodiment, the method comprises using strain J20176 and an EDV of strain J20176 to produce progeny as described above.

[0160] It is noted that cultures of descendant strains of strains J19109-s40 and J11500-s80 (such as J20176) produce mushrooms, mushroom parts, culture parts, and descendant strains and strains of these cultures or derived strains and strains. Therefore, the present invention covers cultures and culture parts of strains J19109-s40 and J11500-s80, as well as hybrid strain J20176, mushrooms and mushroom parts produced therefrom, including spores. In addition, EDVs and cultures are derived solely or primarily from the following: initial cultures derived from descendants of J10109-s40 and / or J11500-s80, dormant or actively growing cultures present in dormant or germinating spores of strain J20176, and cultures incorporating genetic material of descendants of strain J20176.

[0161] The invention further relates to methods of making and using strain J20176 and substantially derived varieties (EDVs) of strain J20176. Uses of cultures derived from J20176 and the other cultures mentioned above include their incorporation into commercial products such as mushroom spawn and overlay inoculants, as well as for the production of mushrooms, the development of other novel Agaricus bisporus cultures, and for crop diversification and farm hygiene, including "virus destruction".

[0162] The morphological and physiological characteristics of strain J20176 cultured on Difco brand PDA medium (which is a standard medium) are provided below. Strain J20176 grown on PDA medium in a culture dish of 8.5 cm in diameter produces irregular lobed colonies of white or light brown-yellow or "tan (tan)", which have a roughly circular overall outline, and during the dynamic equilibrium state growth period between the 8th day to the 26th day after inoculation using a circular culture plug (plug) of 3.5 mm in diameter on PDA as an inoculum, the diameter of the bacterium colony increases by (1.32-1.48-)1.49 (-1.50-1.57) mm / day. Strain is increased by transferring pure inoculum to a larger volume of sterile medium. Variant traits are not observed or expected in strain J20176.

[0163] Methods for obtaining, manipulating and mating the cultures of the present invention, methods for producing progeny, inoculants, products and crops of the present invention, methods for using strain rotation programs to improve mushroom farm sanitation, and methods for obtaining genotypic fingerprints of mushroom cultures have been described above and are also well known to practitioners in the art. Although the present invention has been described in conjunction with specific embodiments in the present application, other embodiments and modifications can be generated by those skilled in the art based on the teachings herein without departing from the spirit of the claimed invention or exceeding its scope. Therefore, it should be understood that the description herein is provided only to assist in understanding the present invention and should not be regarded as limiting its scope.

[0164] The J20176 hybrid is a mating formed between J19109-s40 and J11500-s80. The J2076 hybrid verifiably lacks the AI ​​trait (see Table IV below). In addition, strain J20176 has a strong combination of desired traits derived from the J15987 lineage. Strain 20176 has been pre-commercially tested in North America, Europe, and China and has strong commercial potential.

[0165] The hybridization of the Agaricus bisporus cultures of the present invention can be accomplished by growing two different cultures (one of which is the genetic line present in the spores of strain J19109) together, preferably on sterile medium, in close proximity until conjugation (i.e., hyphal or cell fusion) occurs. In a successful mating, the resulting fused culture is a first generation outcross hybrid culture that incorporates the genome of the line obtained from strain J19109. The line can be obtained from the mushroom spores of J19109, which is part of one embodiment of the present invention. Alternatively, the line can be obtained from protoplasts derived from the basidia of J19109 or other parts of the organism of J19109, which is another part of one embodiment of the present invention.

[0166] For the purposes of the present invention, the complete genomic DNA sequences of strain J19109 and its constituent homokaryons (B18287-s82 and WBL-s290) have been obtained. In addition, the genomic DNA sequences of strains J11500-s80 and J19109-s40 (parents of J20176) have also been obtained. For DNA preparation, after maceration, the cultures were grown in sterile broth growth medium. After 2-4 weeks, the mycelial cells were collected by filtration, frozen at -80°C, and lyophilized until dry. Cap tissue was obtained from mushrooms produced from cultures of heterokaryotic strains (such as J20176), frozen and lyophilized. DNA was extracted from the lyophilized samples using the CTAB procedure, followed by RNase treatment and gel purification. Contractor Genewiz (New Brunswick, New Jersey) prepared DNA libraries from the DNA of each culture and sequenced the libraries using Illumina technology. Sylvan, Inc. performed assembly of reads into the genome sequence using the public domain reference genome sequence of H97 version 2.0 (Morin et al. 2012; PNAS 109(43):17501, incorporated herein by reference). Thus, about 93% to about 95% of the entire genotype of strain J20176 and its parental homokaryotes was known with certainty to Sylvan, Inc.

[0167] Industry understanding of the genetic control of heterokaryon incompatibility is limited; however, it is known to be separate from the well-known mating type gene or genes and to involve more than one gene, for example, 4 or 5 genes in Coprinopsis, of uncertain chromosomal location.

[0168] 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 a mutation, an "A" can be changed to a "G". In addition, large-scale genome coverage of more than 50 times can be obtained through whole genome sequencing (WGS) technology, most notably using equipment manufactured by Illumina. These "Illumina reads" can then be compared to a reference genome using appropriate software. The reference genome is important because the exact position of each SNP in the genome can be defined by a number using base pair positions.

[0169] In Table I, a SNP-based comparison of the genotypes of cultures used to practice the invention described herein is provided. To generate whole genome sequences, we used DNASTAR's Lasergene version 18 to align with the H97 version 2.0 reference genome using Illumina 250 bp reads. Table I provides the genotypes of strains J19109 and J20176 and their parental lines B18287-s82 and WBL-s290 (for strain J19109) and J11500-s80 and J19109-240 (for strain J20176) at multiple sequence-characterized marker loci at intervals greater than 100 Kbp along each of the 19 H97 version 2.0 reference backbones.

[0170]

[0171]

[0172]

[0173]

[0174] It can be clearly seen that the SNP pattern of each strain and strain is unique and is therefore a robust form of genetic identification. In addition, those skilled in the art will be able to validate the SNP markers described in Table 1.

[0175] The data in Table I are shown as 9-mers, and in most cases, the middle base (position 5) provides SNP. We selected robust markers at the loci compared between each of the first 19 skeletons. Most of the markers in Table II are standard markers used in other patent cases, such as US 9,017,988 and US10,440,930. Except for B18287-s82, WBL-s290, J19109 heterokaryon and J11500-s80, we determined the SNP alleles of the homokaryon parents (J11500-s80 and J19109-s40) of J20176 at these loci.

[0176]

[0177] Table II reports data from 6 loci, which are standardly reported for the same set of strains used in Table I, such as US 9,017,988 and US10,440,930. As shown in Table II, for one heterokaryotic strain (J19109), each marker locus has two alleles, while for 6 homokaryotic cultures, each locus has a single allele. Data were prepared by the applicant using targeted polymerase chain reaction (PCR) to amplify the genomic region covering the defined marker from each culture DNA. PCR primers that bracket the defined marker region are used to generate data at the position indicated by the position information provided below; methods for designing suitable primers are well known in the art. Based on the amplified PCR products, DNA was sequenced by contractor Eurofins (Louisville, Kentucky) using their selected method and compared with Sylvan's reference marker / allele sequence database to determine genotype by directly examining these sequences. In most cases, the sequence was further confirmed by direct examination of the corresponding whole genome sequence of that culture.

[0178] Description of p1n150-G3-2 marker:

[0179] The 5' end of the marker segment begins at position 1 of the first "T" in the sequence TCCCAAGT, which corresponds to H97JGI V2.0 backbone 1 position 868615 (Morin et al. 2012) and extends for about 600nt in the opposite orientation (relative to the backbone orientation) in most alleles; insertions in the DNA of allele 1T produce longer segments. Currently, 9 alleles incorporating at least 30 polymorphic positions have been recorded from different strains in the Sylvan culture collection. This marker is an important part of Agaricus breeding programs because it is tightly linked to the MAT locus. MAT is a gene complex that controls mating between homokaryons.

[0180] As mentioned above, allele 1T contains a 320 base pair transposon. All other known alleles of p1n150 lack this intron.

[0181] Analysis of a panel of 88 homokaryotic SSIs from J19109 with p1n150 identified 52 matches to the p1n150 allele of Mat-1 and a second group of 36 strains containing a different mating type allele (tentatively designated "Mat-X").

[0182] To generate new crosses, the entire set of Mat-X homokaryons was crossed with a set of J11500 SSI homokaryons known as MAT-2. To our surprise, none of these crosses were successful, and we concluded that MAT-X is functionally identical to MAT-2. The MAT-2 alleles in J19109 must be descendants of wild-type W01-s1 homokaryons, since the other parental homokaryon in B18287, So76-s12b, is known to be MAT-1.

[0183] The next step was to align the DNA from the p1n150 amplicons of W01-s1, B18187-s82, and J11500-s80 to see if the p1n50 sequence of MAT-2 was an exact match. The data showed differences between the J11500-s80 MAT-2 sequence and the sequences possessed by W01-s1 and B18187-s82. Therefore, the new p1n150 allele found in W01-s1 and B18287-s82 was named "2A".

[0184] Allele 1T: 320nt ABR transposon insertion @206^207, 'A' @321; 'T' @327; 'C' @374; 'G' @378; 'G' @422; etc.

[0185] Allele 2: no Abr1 insertion; ‘A’@321; ‘C’@327, ‘C’@374; ‘C’@378; ‘G’@422; etc.

[0186] Allele 2A: no Abr 1 insertion; 'A'@321; 'C'@327, 'T'@374; 'C'@378; 'T'@422; etc.

[0187] Description of ITS (=ITS1+2 region) markers:

[0188] The ITS segment is part of a nuclear rDNA region located on chromosome 9 (backbone 10 in JGI H97 v2.0). The rDNA is a cassette that is repeated in tandem up to an estimated 100 times in the haploid genome of Agaricus bisporus. Therefore, in the assembled H97 genome, there is no single precise location for this sequence, and it is in fact difficult or impossible to accurately assemble this sequence on all tandem repeats. Three copies of the cassette are included on backbone 10 of the H97 JGI V2.0 assembly (assembly), starting at positions 1612110; partial copies are also assembled into backbone 29 (Morin et al. 2012). The 5' end of this marker segment starts at position 1 of the first "G" in the sequence GGAAGGAT and extends for about 703-704 nt in a forward orientation (relative to the backbone orientation) in most alleles. Currently, more than 9 alleles incorporating at least 11 polymorphic positions have been recorded from different strains in the Sylvan culture collection.

[0189] The alleles present in the present application are I1, I2 and I4. The features are as follows (using the format: nucleotide base feature @ alignment position, based on alignment of 9 alleles).

[0190] Allele I1: 'C'@52; 'T'@461; 'T'@522; 'T'@563; etc.

[0191] Allele I2: 'T'@52; 'T'@461; 'T'@522; 'T'@563; etc.

[0192] Allele I4: ‘C’@52; ‘A’@461; ‘C’@522; ‘C’@563; etc.

[0193] Description of MFPC-1-ELF marker:

[0194] The 5' end of this marker segment begins at position 1 of the first "G" in the sequence GGGAGGGT, which corresponds to H97JGI V2.0 backbone 8 position 829770 (Morin et al. 2012) and extends for approximately 860 nt in the forward orientation (relative to the backbone orientation) in most alleles. Currently, at least 7 alleles incorporating at least 40 polymorphic positions have been documented from different strains in the Sylvan culture collection.

[0195] Note that MFPC-ELF is linked to the PPC-1 locus on backbone 8, which is known to be the major factor controlling cap color. White-capped mushrooms have the E1 allele at MFPC-ELF.

[0196] Allele E1: 'A'@63; 'A'@77; 'A'@232; 'A'@309; 'T'@334; 'A'@390; 'A'@400; 'T'@446, 'A'@481; etc.

[0197] All strains in Table II share the E1 allele at MFPC-ELF.

[0198] Description of AN markers:

[0199] The 5' end of this marker segment begins at position 1 of the first "G" in the sequence GGGTTTGT, which corresponds to H97 JGI V2.0 backbone 9 position 1701712 (Morin et al. 2012) and extends from about 1660 (in the H97 genome) to 1700 nt (in alignment space) in the forward orientation (relative to the backbone orientation) in the known alleles; some insertions / deletions produce length polymorphisms, which identify alleles in addition to point mutations of single nucleotides. Currently, 5 alleles incorporating more than 70 polymorphic positions have been recorded from different strains in the Sylvan culture collection.

[0200] In the strain used in the present application, only one allele N1 is present (using the following format: nucleotide base signature @ alignment position, based on alignment of alleles N1 to N4):

[0201] Alleles N1: ‘G’@640; [deletion]@844-846; ‘C’@954; ‘T’@882; ‘A’@954, etc.

[0202] In this study, all strains shared the same N1 allele on backbone 9.

[0203] Description of AS markers:

[0204] The 5' end of this marker segment begins at position 1 of the first "G" in the sequence GG(T / N)GTGAT, which corresponds to H97 JGI V2.0 backbone 4 position 752867 (Morin et al. 2012) and extends from about 1620 (in the H97 genome) to 1693 nt (in alignment space) in the forward orientation (relative to the backbone orientation) in the known alleles; some insertions / deletions create length polymorphisms, which identify alleles in addition to point mutations of single nucleotides. Currently, 7 alleles incorporating more than 80 polymorphic positions have been documented from different strains in the Sylvan culture collection.

[0205] The alleles present in the direct lineage of B18287 / J19109 are alleles SC and SD, which are partially characterized as follows (using the following format: nucleotide base feature @ alignment position, based on alignment of alleles SA to SG):

[0206] Alleles SC: 'T'@28; 'GATATC'@258-263; 'G'@275; [insert] + 'TTTC T CAGC'+[insert]@309-249;'C'@404, etc.

[0207] Allele SD: ‘C’@28; [deletion]@258-263; ‘T’@275; [deletion]@309-249; ‘T’@275; [deletion]@309-349, ‘T’@404, etc.

[0208] Description of FF markers:

[0209] The 5' end of this marker segment begins at position 1 of the first "T" in the sequence TTCGGGTG, which corresponds to H97JGI V2.0 backbone 12 position 281999 (Morin et al. 2012) and extends for approximately 570 nt in the forward orientation (relative to the backbone orientation) in most alleles. Currently, 7 alleles incorporating at least 20 polymorphic positions have been documented from different strains in the Sylvan culture collection.

[0210] Two alleles were found in the strains used to develop the present invention; FF1 and FF2.

[0211] Alleles FF1: ‘CCG’@48-50, ‘C’@91, etc.

[0212] Alleles FF2: ‘TTC’@48-50, ‘C’@91, etc.

[0213] The FF1 genotype is present in white strains, such as So76 and U1, and related EDVs, such as A15. In this study, one of the breeding stocks, So76-s12b, inherited the marker.

[0214] Possession of the FF2 genotype enabled identification of W0-s01, B18287-s82, WBL-s290, J19109, and J11500-s80 (as well as related EDVs and direct descendants).

[0215] As mentioned above, one of the goals of the J19109 and J20176 lineages was to develop methods to exclude aggressive incompatibility (AI) traits while retaining as many of the positive traits observed in J15987 as possible. To test this concept, a test protocol was designed to measure the effect of mixing a small amount of inoculum from an F2 hybrid strain (which has a strain from J19109 as one isokaryotic parent and a strain from J11500-s80 as a second isokaryotic parent) or a control strain into compost produced by inoculation with the commercial strain A-15. In this experimental design, normal incompatible interactions will not interfere with the overall yield of the mushrooms, while strains with the antagonistic traits of J15987 will result in a significant reduction in the yield of A-15, or even zero.

[0216] 16 F2 strains were selected for this study (see Table III below), where J15987 and A15 were used as positive and negative controls, respectively. Yields of A-15 were obtained in repeated confrontation crop tests. In normal Agaricus bisporus crops, the first burst of mushrooms is harvested within a period of 3 to 4 days. Skilled pickers pick the earliest mushrooms and leave space for the mushrooms to fill in as the crop grows (develops). In contrast, when the AI ​​trait is expressed, very few mushrooms are produced. Large areas of mushroom beds are not produced, and there are also some areas where a small number of mushrooms can appear. In other words, the AI ​​trait causes a catastrophic reduction in mushroom yield. Typically, heterokaryon incompatibility or nutritional incompatibility can reduce yield by 10% or less, and in most cases usually by 5% or less. However, the AI ​​trait is catastrophic, making it not uncommon for reductions of more than 50%. However, for the purposes of the present invention, the AI ​​trait will be defined as a yield reduction of 15% or less of the A15 control yield after testing.

[0217] The next step will be to look for correlations between aggressive incompatibility and the chromosomes present in strain #1, strain #16, and J15987. To determine the genotype of the parental J19109 homokaryon of each strain, one amplicon of each chromosome was amplified using PCR. These markers were placed as close to the center of each chromosome as possible. Note that the chromosomes of Agaricus bisporus are very commonly inherited as unrecombined units, and can be roughly tracked by a single centrally located marker. In Table III below, we identify 9 chromosomes based on which parent (B18287-s82 or WBL-s290) contributed each chromosome.

[0218] Agaricus bisporus has 13 chromosomes per homokaryon genome (Foulogne-Oriol et al. 2010). Four chromosomes were not included in the analysis:

[0219] 1) Chromosome 1 contains the MAT locus that controls mating in Agaricus bisporus. A pre-screening of the J19109 tester line group was performed for the MAT-1 allele, as the other 50% of the lines obtained were incompatible with J11500-s80.

[0220] 2) Chromosome 3 is 100% skewed to the WBL-s290 allele.

[0221] 3) Chromosome 6 is 100% biased towards the WBL-s290 allele.

[0222] 4) Chromosome 12 is 100% biased towards the B18287-s82 allele.

[0223] As with the last three chromosomes, the extreme skew may be due to the influence of deleterious alleles, but in any case, virtually no segregation was observed at this locus, so it was not possible to analyze the correlation of genotype with phenotype. However, since both marker segregation and phenotypic segregation were observed in the analyses performed, it seems that the four chromosomes listed above do not play a major role or any role in conferring the AI ​​trait phenotype.

[0224] Therefore, for this study, there were informative sets of 9 chromosomes.

[0225]

[0226] Data are only for mushrooms harvested during the first outbreak, in total pounds.

[0227] Mating #5 was affected by occasional disease which artificially reduced its yield.

[0228] C refers to the number of chromosomes per marker

[0229] Table III shows the yield and inherited alleles of hybrids obtained from 16 different J19109 homokaryons that were mated with J11500-s80 to form F2 hybrids. This list includes J20176. In this test:

[0230] For each strain, three 2.76 sq. ft. containers ("buckets") were inoculated with 55 lbs of Stage II mushroom compost spawn. The yield was the sum of the three buckets.

[0231] In each treatment in the table, including J20176, 1% by weight of the relevant strain of spawn was thoroughly mixed with 99% A15 spawn, and the mixed spawn was thoroughly mixed with the compost. The spawn run was completed on day 15, and the cover layer was applied. For each treatment in Table III, cover soil inoculated with A15 was added (1.5% rate, calculated using cover soil dry weight).

[0232] From day 16 to day 20, the mushrooms appeared ready to harvest, and the test was ended on day 20, with only the first burst of production being harvested.

[0233] The yield ratio is shown in column 3. This value was calculated by dividing the total yield of each treatment by the yield of the A15 control.

[0234] Finally, the other columns in Table III show the allelic inheritance of the nine Agaricus chromosomes, including alleles inherited from one or the other of the parents of J19109 (B18287-s82 and WBL-s290). Note that the parental homokaryons in the table are abbreviated to fit the table; WBL-s290 is abbreviated as s290, and B18287-s82 is abbreviated as s82.

[0235] It can be clearly observed that in the presence of strain #1, strain #16, and J15987, the yield of the A15 crop was much lower than when any of the other strains were present in small amounts; in other words, these strains met the test criteria defined for performance of the AI ​​trait in the tests conducted with A15. By utilizing the yield ratio data, it can be inferred that strain #1, strain #16, and the J15987 test treatments all had yield ratios of 0.15 or less. The strain most closely associated with strain #16 was strain #5, which had a ratio of 0.53, however as mentioned above, this strain was severely affected by Trichoderma contamination, resulting in reduced yield. Therefore, a working definition of the AI ​​trait was proposed in this test protocol, including a reduction in yield to 15% or less of the A15 control yield.

[0236] Furthermore, when the chromosomal inheritance pattern was investigated, it was apparent that strain #1, strain #16, and J15987 all shared the same WBL-s290 allele on chromosomes 4, 7, and 9. No other strains in this study shared this haplotype, strongly suggesting an association with the aggressive incompatibility trait.

[0237] Bernoulli's classic binomial probability formula P = n × q can be used x (1-q) n-xto calculate the chance that chromosomes 4, 7, and 9 are exactly identical to the AI ​​trait occurring randomly, where P is the binomial probability, n is the total number of strains studied, x is the number of strains observed to match a particular genotype, and q is the probability of a particular genotype occurring in each sample.

[0238] Three strains matched a specific genotype (two from the test cohort, plus J15987), for a total of 17 strains studied, and the probability of each genotype occurring was 0.125 (the observed segregation did not deviate significantly from the theoretical probability of 0.5 for each allele; at three alleles, 0.5 3 =0.125), the calculated binomial probability in this case is 0.0154, or a 1.54% chance. In other words, the p-value for the likelihood that the association between the phenotype and the haplotype is due to chance is 0.0154. Generally, a p-value of less than 0.05 is considered a strong basis for rejecting a random chance effect.

[0239] However, a probability of 0.5 for each allele as used above assumes a Mendelian 50:50 ratio.

[0240] The theoretical probability of 0.5 can be further refined using the data in Table III. For chromosome 4, there are 8 / 17 (=0.47:0.53) s290 alleles, for chromosome 7, there are 7 / 17 (=0.41:0.59) s290 alleles, and for chromosome 9, there are 10 (=0.41:0.59) s290 alleles in 17. For such a small sample size, the observed ratio is very consistent with the expectation of the ideal 50:50 ratio between each allele and its alternative gene. Therefore, all three chromosomes add up to 25 / 51, i.e. 0.49:0.51 (very close to the ratio of 50:50).

[0241] Alternatively, since J15987 is part of a separate cohort, we can calculate the binomial probability without the J15987 data, in other words, only J20157 and J20209. This reduces the sample size from 17 to 16. In this case, the binomial probability is 7.03%, which is still a good chance (93%) that the chromosome association with the AI ​​trait is real.

[0242] Overall, the statistical analysis indicated a high probability that the aggressive incompatibility was significantly associated with the effects of the incompatibility factors on three specific chromosomes from the WBL-s290 parent and could be explained by this effect.

[0243] Together, these data demonstrate that the AI ​​trait is under genetic control and that it is possible to make new matings that retain a significant percentage of the genetic makeup (and phenotype) present in J15987, but without the AI ​​trait. Finally, the robust, statistically supported segregation data strongly suggest that AI is controlled by genetic factors on three different Agaricus chromosomes, enabling the use of the claimed approach with high predictive confidence.

[0244]

[0245] The values ​​in the table are in lbs. / sq.ft.

[0246] Each value is the average of 6 replicates per plant.

[0247]

[0248] The values ​​in the table are in lbs. / sq.ft.

[0249] Each value is the average of 6 replicates per plant.

[0250] The data in Tables IV and V above demonstrate the yield potential of 12 different J19109SSI homokaryons x J11500-s80 hybrids grown in two separate tests. Notably, this new set of hybrids all exceeded the yield of the A15 test control and were closer in performance to the J15987 control.

[0251] In addition, the 1st:2nd burst ratio was calculated for both experiments. Clearly, J15987 is more significantly biased towards the first burst. The J19109 hybrids also tend to be biased towards the first burst to varying degrees.

[0252] One of the objectives of the present invention is to confirm that the high yield of J15987 is repeated in the hybrid derived from J19109. This is important because the yield potential in this range is required for the commercial success of the strain. The data in Table IV and Table V clearly show that this has been achieved.

[0253] For the data in Tables VI and VII, significance is indicated by asterisks: * for p = 0.05 or less, ** for p = 0.01 or less, *** for p = 0.001 or less, **** for p = 0.0001 or less.

[0254] Statistics are compared with the J15987 control by paired t-test. Statistical significance is marked with *

[0255]

[0256] It has further been demonstrated that the overall quality of mushroom shape and color in J15987 is replicated in the hybrid derived from J19109. The data in Table VI clearly show that this has been achieved.

[0257] Mushroom cap measurements were performed using a Storm 3C301 digital caliper. Sample sizes of 20 commercially mature medium-sized mushrooms (35-40 mm in diameter with a closed veil) were harvested and measured to obtain values ​​for cap diameter and cap height. The mushrooms were then cut in half vertically to measure flesh thickness and stem width. The ratios between these values ​​were calculated to obtain cap roundness (cap height / cap diameter), flesh thickness (flesh thickness / cap height), and stem thickness (stem width / cap diameter).

[0258] Cap roundness is an approximation of the degree of sphericity exhibited by a mushroom's cap. Larger values ​​indicate rounder mushrooms, which are preferred by growers and consumers based on visual appeal. Smaller values ​​indicate flatter mushrooms. All of the J19109-derived hybrids recorded in Table VI fall into a continuous series around J15987, clearly indicating that this trait has been retained.

[0259] Stipe thickness is the ratio of the width of a mushroom's stipe to the diameter of its cap. Larger values ​​indicate wider stipes. Different markets have their own preferences for stipe thickness, with some markets preferring wider or narrower stipes depending on their needs. Almost all of the J19109-derived hybrids recorded in Table VI exhibit slightly narrower stipes than J15987, but this is not a universally favorable or unfavorable quality.

[0260] Flesh thickness is an approximation of how much of the volume of a mushroom's cap is made up of flesh tissue as opposed to stipe or gill tissue. Larger values ​​indicate a higher proportion of flesh tissue, which is preferred by growers as a quality marker. All of the J19109-derived hybrids recorded in Table VI fall into a continuous series around J15987, clearly indicating that this trait has been retained.

[0261] When these mushroom shape data are considered together, the shape of J15987 is clearly preserved.

[0262]

[0263] Statistics were paired t-test comparisons with J15987 controls.

[0264] The color of mushroom caps was measured using a Minolta colorimeter CR-200. The sample size was 20 medium-sized mushrooms with a diameter of 30-40 mm. The L*a*b system was used, where "L" is a measure of brightness, where 100 is perfect whiteness and 0 is perfect blackness. For the other two measurements, "a" is the green / red axis and "b" is the yellow / blue axis. For "a", red values ​​are arranged on the positive side of the common axis and green values ​​are arranged on the negative side. In a similar manner, on the "b" axis, yellow values ​​are positive and blue values ​​are negative.

[0265] The L value is an objective measure of the degree of whiteness that a mushroom cap exhibits when undamaged. For white mushrooms, higher values ​​are more desirable. All of the J19109-derived hybrids recorded in Table VII fall into a continuous series around J15987, clearly indicating that this trait has been retained. In fact, some strains have L values ​​recorded that are significantly higher than J15987, indicating that the trait has been improved rather than simply retained.

[0266] The "a" and "b" value readings contribute to the hue of the mushroom. None of the J19109 derived hybrids reported in Table VII differed significantly from J15987, indicating that the color of the strains was preserved in these hybrids.

[0267]

[0268] There were 14 replicates per plant. Yield was in lbs. / sq.ft. Statistics were paired T test

[0269] Table VIII shows the yields obtained from the first pre-commercial trial of J20176, including the A-15 control. The growing conditions were typical for European commercial growers using the "Dutch" system, including full Phase I and Phase II composting and a full spawn run (Phase III). The room produced a fruiting flush for the A-15 control at industry standard conditions.

[0270] J20176 had a higher yield than the control in the 1st outbreak, although the difference did not reach significance. The only observation that reached significance was the 3rd outbreak, where A-15 had a higher 3rd outbreak yield than J20176. The total yield comparison did not show a significant difference between the two strains. These data confirm that the yield potential of J20176 has essentially the same yield as strain A-15, even under commercial growth conditions that are not optimized for the requirements of J20176. Typically, in order to obtain good yields, methods must be fine-tuned over a long period of time. It was observed that the J20176 mushrooms were rounder and whiter than the A-15 control.

[0271] Considering that J20176 and A-15 were present in adjacent areas of the same physical space, special attention was paid to looking for evidence of aggressive incompatibility in this test. We observed typical strain-strain interactions: In areas where the colonized compost or mulch was mixed together, a typical incompatibility reaction was observed, with only a small area of ​​no growth where strains A-15 and J20176 grew next to each other. Under commercial conditions, J20176 did not induce AI trait phenomena. These data are indicative of the market suitability and potential of J20176.

[0272] Although the present invention has been described in conjunction with the specific embodiments in the present application, those skilled in the art can produce other embodiments and modifications according to the teachings herein without departing from the spirit of the claimed invention or exceeding its scope. Therefore, it should be understood that the description provided herein is only to help understand the present invention, and the description should not be regarded as limiting its scope.

Claims

1. A method for eliminating the aggressive incompatibility (AI) trait from a strain of Agaricus bisporus, wherein a culture of a white mushroom line designated WBL-s290, a representative culture of which has been deposited under NRRL deposit number 68167, a culture of the mushroom line designated J11500-s80, a representative culture of the hybrid mushroom strain designated J15987, a representative culture of which has been deposited under NRRL deposit number 67646, is mated with a culture of a mushroom line designated J11500-s80 to provide a hybrid mushroom strain designated J15987 having the AI ​​trait, the method comprising: mating a culture of a hybrid mushroom line designated B18287-s82 with a culture of a white mushroom line designated WBL-s290 to obtain an F1 hybrid mushroom strain designated J19109, a representative culture of the hybrid mushroom line designated B18287-s82 having been deposited under NRRL deposit number 68168, and a representative culture of the F1 strain having been deposited under NRRL deposit number 68163; Fermenting a culture of the new F1 strain designated J19109 to obtain homokaryotic spores therefrom; obtaining a homokaryotic culture from homokaryotic spores of the F1 strain J19109, and selecting a homokaryotic culture from the F1 strain J19109; and mating a culture of said isokaryotic line from the F1 strain J19109 with a culture of a mushroom line designated J11500-s80 to obtain an F2 hybrid mushroom strain; A culture of the F2 hybrid mushroom strain is tested to determine the presence or absence of the AI ​​trait, wherein in the absence of the AI ​​trait, the AI ​​trait has been excluded from the F2 hybrid mushroom strain.

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

3. The method of claim 1, wherein the F2 hybrid eliminates the AI ​​trait and provides at least two beneficial traits present in strain J15987, the beneficial traits being selected from: a cap shape as round as that of strain J15987, a stipe as thick as that of strain J15987, flesh as thick as that of strain J15987, and a redness (a) value within 10% of the redness (a) value of strain J15987 on the Lab color measurement scale.

4. The method of claim 1, wherein the isokaryotic culture from the F1 strain J19109 is a culture of the strain designated J19109-s40, a representative culture of which has been deposited under NRRL deposit number 68165.

5. The method of claim 4, wherein the step of mating the isokaryotic line culture from the F1 strain J19109 with the culture of the mushroom line designated J11500-s80 comprises the step of mating the strain culture designated J19109-s40 with the mushroom line designated J11500-s80, wherein a resulting F2 hybrid strain designated J20176 is produced, a representative culture of which has been deposited under NRRL deposit number 68166.

6. The method of claim 5, wherein the F2 hybrid strain designated as J20176 does not have the AI ​​trait and retains at least two beneficial traits present in strain J15987, the beneficial traits being selected from: a cap shape as round as that of strain J15987, a stipe as thick as that of strain J15987, flesh as thick as that of strain J15987, and a redness (a) value within 10% of the redness (a) value of strain J15987 on the Lab color measurement scale.

7. A culture comprising at least one set of chromosomes of Agaricus bisporus line B18278-s82, a culture of line B18278-s82 having been deposited under NRRL deposit number 68168, wherein the chromosomes include all of the alleles of line B18278-s82 at the marker loci characterized by the sequences listed in Table I.

8. A culture derived from an initial culture, wherein the initial culture is the culture of claim 6, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line B18287-s82, wherein the culture of the initial line has been deposited under NRRL deposit number 68168, or wherein the culture is directly derived from the initial culture, such that all of its genome or genotype is present in the genome or genotype of the initial culture of line B18287-s82.

9. A culture comprising at least one set of chromosomes of Agaricus bisporus line WBL-s290, a culture of line WBL-s290 having been deposited under NRRL deposit number 65167, wherein the chromosomes include all of the alleles of line WBL-s290 at the marker loci characterized by the sequences listed in Table I.

10. A culture derived from an initial culture, wherein the initial culture is the culture of claim 8, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line WBL-s290, wherein the culture of the initial line has been deposited under NRRL deposit number 68167, or wherein the culture is directly derived from the initial culture, such that all of its genome or genotype is present in the genome or genotype of the initial culture of line WBL-s290.

11. A culture comprising at least one set of chromosomes of Agaricus bisporus line J11500-s80, a culture of line J11500-s80 having been deposited under NRRL deposit number 65164, wherein the chromosomes include all of the alleles of line J11500-s80 at the marker loci characterized by the sequences listed in Table I.

12. A culture derived from an initial culture, wherein the initial culture is the culture of claim 8, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line J11500-s80, wherein the culture of the initial line has been deposited under NRRL deposit number 68164, or wherein the culture is directly derived from the initial culture, such that all of its genome or genotype is present in the genome or genotype of the initial culture of line J11500-s80.

13. A culture comprising at least one set of chromosomes of Agaricus bisporus line J19109-s40, a culture of line J19109-s40 having been deposited under NRRL deposit number 68165, wherein the chromosomes comprise all of the alleles of line J19109-s40 at the marker loci characterized by the sequences listed in Table I.

14. A culture derived from an initial culture, wherein the initial culture is the culture of claim 10, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line J19109-s40, wherein the culture of the initial line has been deposited under NRRL deposit number 68165, or wherein the culture is directly derived from the initial culture, such that all of its genome or genotype is present in the genome or genotype of the initial culture of line J19109-s40.

15. A hybrid mushroom culture of Agaricus bisporus designated strain J19109, a representative culture of which has been deposited under NRRL accession number 68163.

16. A culture of Agaricus bisporus derived from an initial culture, wherein the initial culture is the culture of claim 12, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of strain J19109, wherein the culture of the strain has been deposited under NRRL deposit number 68163, or wherein the culture is directly derived from the initial culture, such that all of its 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 number 68166.

18. A culture of Agaricus bisporus derived from an initial culture, wherein the initial culture is the culture of claim 15, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of strain J20176, wherein the culture of the strain has been deposited under NRRL deposit number 68166, or wherein the culture is directly derived from the initial culture, such that all of its genome or genotype is present in the genome or genotype of the initial culture of strain J20176.

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

20. A product incorporating the culture of any one of claims 7 to 16, the product selected from the group consisting of mycelium, spawn, fresh or processed mushrooms, mushroom spores, mushroom spawn, mushroom preparations and extracts and fractions, mushroom chunks, mushroom inoculants, cover inoculants, cover spawn, cover soil, inoculated compost, colonized compost, post-tillage compost and loose pellets.

21. The part of the culture according to any one of claims 7 to 16, selected from hyphae, mushrooms, dormant spores, germinated spores, homokaryons, heterokaryons, cells, nuclei and protoplasts.

22. A cell of any one of the cultures of any one of claims 7 to 16.

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