AU2015212394B2 - Mushroom line J10102-s69, hybrid mushroom strain J11500, descendants thereof, and methods and uses therefor - Google Patents

Abstract

An

Description

MUSHROOM LINE J10102-s69, HYBRID MUSHROOM STRAIN J11500,

DESCENDANTS THEREOF, AND METHODS AND USES THEREFOR TECHNICAL FIELD

[0001]This invention relates generally to the field of microorganism strain

development and more particularly, to the development of homokaryotic lines and

heterokaryotic strains of mushroom fungus. More specifically, the present invention

relates to the development of a homokaryotic Agaricus bisporus mushroom fungus

line designated J10102-s69 and to an Agaricus bisporus hybrid strain designated

J11500, as well as to cultures descended or derived from line J10102-s69 or strain

J11500, and to methods of making and using said hybrid cultures.

BACKGROUND OF THE INVENTION

[0002]The edible mushroom Agaricus bisporus (Lange) Imbach var. bisporus, a

microorganism belonging to the basidiomycete fungi, is widely cultivated around the

world. In Europe and North America, it is the most widely cultivated mushroom

species. The value of the annual Agaricus bisporus mushroom crop in the United

States was about $1,110,000,000 in 2012-2013, according to the National

Agricultural Statistics Service, Agricultural Statistics Board, U.S. Department of

Agriculture (August 20, 2013). Accordingly, development of novel hybrid mushroom

strains or lines of this mushroom fungus is seen as highly desirable to the cultivated

mushroom industry, particularly if those novel strains or lines can be developed to

provide various desirable traits within a single strain, culture, hybrid or line. Most

cultures and strains of A. bisporus are unsuitable for commercial cultivation, and the

development of successful new hybrid strains is challenging and only rarely results in

a useful new strain. The problem facing mushroom strain developers and commercial mushroom spawn producers is therefore to develop and identify the rare useful lines, strains and cultures having commercial value.

[0003]Thus, various entities within the mushroom industry, including Sylvan

America, Inc., have set up mushroom strain development programs. The goal of a

mushroom strain development program is to combine, in a single strain, culture,

hybrid, or line, various desirable traits. Strains currently available to the mushroom

industry allow growers to produce crops of mushrooms successfully and profitably.

Several factors exist that influence the degree of success and profitability achieved.

Characteristics of strains that are factors that can improve producer profitability

include increased productivity (higher yield or shorter cycle time), accelerated

revenue capture (earlier harvest), reduced costs (for example, greater ease and

speed of harvesting), reduced shrinkage (pre-sale weight loss), reduced

overweighting of product in packages (extra weight of product packaged, due to

particular sizes of individual mushrooms), improved consistency of crop performance

responses to variations in raw materials, growing conditions and practices, superior

crop performance in particular facilities, regions, etc., reduced losses to diseases

including viral, bacterial and fungal disease agents, and/or reduced losses to insect

and nematode pests of the crop. There also exist improvable properties of the

mushroom product that increase demand in the distribution chain, and thus sales

volume and/or sales price, such as improved visual appeal (more desirable

coloration, shape, size, or surface texture), improved or distinct flavor characteristics,

improved keeping qualities (longer persistence of desirable visual attributes), etc.

Still other improvements may enhance the suitability of the mushroom crop for

mechanical harvesting, canning, and/or food processing. Thus there are many

characteristics by which a novel strain might be judged as superior in a particular

production facility or sales market, or in the industry regionally or globally.

[0004] All of these characteristics can be assessed using techniques that are well

known in the art. Novel strains are most preferably and successfully developed from

unique hybridizations between homokaryotic lines, including novel lines. Thus, in the

cultivated mushroom industry with its diverse, dynamic and evolving raw materials

characteristics, availabilities and costs, technical capabilities, economic framework

including labor availability and costs, and consumer and market preferences, the

need continues to exist for new hybrid strains, and for new lines that can be used to

produce new hybrid strains, of Agaricus bisporus that provide for flexibility of

operations, for improved characteristics for producer profitability and for improved

mushroom products over other previous strains of Agaricus bisporus.

[0005]There is also a need for commercially acceptable A. bisporus strains with

different genotypes, relative to the U1 derived lineage group, for two reasons. First,

strains incompatible with strains of the U1 derived lineage group are known to retard

the spread of viral diseases between cultivated strains. The incompatibility

phenotype can be assessed using techniques that are well known in the art and are

detailed below. Second, it is well understood that when an agricultural crop industry

relies extensively on a single genetic lineage (i.e., creates a commercial monoculture

as now exists for the white-capped U1 lineage of A. bisporus), there is an increased

risk of unpredictable, catastrophic crop failure on a facility-wide or even industry-wide

scale, for example upon the emergence of a new pathogen. Therefore from a risk

management and food security perspective, it is highly desirable to simultaneously

provide both genetic diversification and commercially acceptable performance and

crop characteristics. The use of novel lines that incorporate DNA from non-cultivar

stocks provides important genetic diversification of the strain pool used to produce

crops of cultivated A. bisporus mushrooms.

[0006]Cultures are the means by which mushroom strain developers prepare,

maintain, and propagate their microorganisms. Cultures of Agaricus, like those of

other microorganisms, are prepared, maintained, propagated and stored on sterile

media using various microbiological laboratory methods and techniques. Sterile

tools and aseptic techniques are used within clean rooms or sterile transfer hoods to

manipulate cells of pure cultures for various purposes including clonal propagation

and for the development of new strains using diverse techniques. Commercial

culture inocula including mushroom 'spawn' and 'casing inoculum' are also prepared

using large-scale microbiological production methods (e.g., from 1 to 14,000 liters

per batch), and are provided to the end user as pure cultures contained within sterile

packaging.

[0007]One use of such cultures is to produce mushrooms. Mushrooms are

cultivated commercially within purpose-built structures on dedicated farms. While

there are many variations on methods, the following description is typical. Compost

prepared from lignocellulosic material such as straw, augmented with nitrogenous

material, is finished and pasteurized within a suitable facility. Mushroom spawn,

which comprises a sterilized friable 'carrier substrate' onto which a pure culture of

one mushroom strain has been aseptically incorporated via inoculum and then

propagated, is mixed with the pasteurized compost and is incubated for

approximately 13 to about 19 days at a controlled temperature, during which time the

mycelium of the mushroom culture colonizes the entire mass of compost and begins

to digest it. A non-nutritive 'casing layer' of material such as peat is then placed over

the compost to a depth of from about 1.5 to about 2 inches. Additional 'casing

inoculum' incorporating the same mushroom culture may be incorporated into the

casing layer to accelerate the formation and harvesting of mushrooms, and improve

uniformity of the distribution of mycelium and mushrooms in and on the casing surface. Environmental conditions, including temperature and humidity, in the cropping facility are then carefully managed to promote and control the transition of the culture from vegetative to reproductive growth at the casing/air interface. In a further about 13 to about 18 days after casing, mushrooms will have developed to the correct stage for harvest and sale. A flush of mushrooms comprising the original culture will be picked over a 3 to 4 day period. Additional flushes of mushrooms appear at about weekly intervals. Commercially, two or three flushes of mushrooms are produced and harvested before the compost is removed and replaced in the cropping facility.

[0008]Seventy to ninety-five percent of the Agaricus mushrooms cultivated in the

United States, Europe, and elsewhere have a white pileus color, in accordance with

consumer preferences. Market requirements for white mushrooms in the USA,

Europe and elsewhere are narrow and precise for many observable phenotypic traits

such as size, shape, color, color retention, firmness, and related traits such as shelf

life. Consequently, genetically different strains of commercially successful white

Agaricus bisporus mushrooms are not easily differentiated on the basis of

appearance of the mushrooms, which must conform to the relatively strict market

requirements. Strains may be, in particular instances, differentiated on the basis of

traits associated with the mushroom, such as mushroom size, mushroom shape

(e.g., cap roundness, flesh thickness), color (i.e., white cap versus brown cap),

surface texture (e.g., cap smoothness), tissue density and/or firmness, delayed

maturation, basidial spore number greater than two, sporelessness, increased dry

matter content, improved shelf life, and reduced bruising, as well as traits associated

with the culture itself, and/or products incorporating the culture, and/or crops

incorporating the culture, including increased crop yield, altered distribution of yield

over time, decreased spawn to pick interval, resistance to infection by, symptoms of, or transmission of bacterial, viral or fungal diseases, insect resistance, nematode resistance, ease of crop management, suitability of crop for mechanical harvesting, and behavioral responses to environmental conditions including stressors, nutrient substrate composition, seasonal influences, farm practices, self/non-self interactions

(compatibility or incompatibility) with various mushroom strains, to give some

examples. Strains may also be differentiated based on their genotypic fingerprint

(presence of specific alleles at defined marker loci in the nuclear or mitochondrial

genome). Strains may have different ancestry, which will be reflected directly by the

genotype, and indirectly, in some cases, by the phenotype.

[0009]Circa 1980, the first two white hybrid strains of A. bisporus, developed by a

laboratory at Horst, the Netherlands, were introduced into commercial cultivation.

These two "Horst" strains, called Ul and U3, are closely related hybrid strains

produced by matings between two pre-existing white cultivated strains, as per M.

Imbernon et al., Mycologia, 88(5), 749-761 (1996). The two parents of Ul and U3

are commercial strains belonging to two longstanding categorical types of strains

known as the 'smooth-white' (SW) strains and the 'off-white' (OW) strains. The

original homokaryons (or 'lines') obtained from the SW and OW strains, and used in

the hybridization that produced the Ul strain, were designated H39 and H97

respectively; these cultures may no longer exist (A. Sonnenberg, pers. comm.).

However, a number of laboratories have deheterokaryotized the Ul strain and

obtained neohaplont cultures incorporating one or the other nuclear type

corresponding to those contributed by H39 or H97, as well as the mitochondrial type

of U1. These two types of neohaplonts of Ul are referred to categorically as the

SWNC and OWNC lines or homokaryons, respectively. An OWNC line designated

'H97' was deposited in the public culture collection of the Fungal Genetics Stock

Center of Kansas, USA, by A. Sonnenberg, under the number 10389, and in the public collection of the American Type Culture Collection of Maryland, USA, under the number MYA-4626. The genome of H97 was sequenced and placed in the public domain by the Joint Genome Institute of California, USA (Morin et al. 2012).

[0010]The Ul strain is thought to be the direct progenitor of all other white A.

bisporus mushrooms currently cultivated in most regions of the world. Many

commercial mushroom strains developed from U, such as A15 and S130, meet the

criteria for Essentially Derived Varieties (as the term is applied to plant varieties, and

extended to apply to mushroom varieties or strains, in conformity with statutory

frameworks including the US PVPA (2014)) of Ul, having been developed from

spores of the initial strain which retain the great majority of the parental genotype

(this behavior was shown by R. W. Kerrigan et al. in Genetics, 133, 225-236 (1993)).

A group of strains developed either by cloning or by spore culture, or by any other

method of 'essential derivation' as discussed below, from a single progenitor (as

opposed to outbreeding between two different progenitors) is called a derived

lineage group. Except for relatively minor acquired genetic differences all white

strains developed within the Horst Ul derived lineage group share a single

composite N+N heterokaryotic genotype, or a subset of that genotype, with the

original Ul strain. For this reason, modern white Agaricus mushroom cultivation is

effectively a monoculture.

[0011] Agaricus bisporus has a reproductive syndrome known as amphithallism, in

which two distinct life cycles operate concurrently. As in other fungi, the reproductive

propagule is a spore. Agaricus produces spores meiotically, on a meiosporangium

known as a basidium. In a first life cycle, A. bisporus spores each receive a single

haploid postmeiotic nucleus; these spores are competent to mate but not competent

to reproduce mushrooms. These haploid spores germinate to produce homokaryotic

offspring or lines which can mate with other compatible homokaryons to produce novel hybrid heterokaryons that are competent to produce mushrooms.

Heterokaryons generally exhibit much less ability to mate than do homokaryons.

This lifecycle is called heteromixis, or more commonly, outbreeding. This life cycle

operates but typically does not predominate in strains of Agaricus bisporus var.

bisporus.

[0012] A second, inbreeding life cycle called intramixis predominates in most strains

of Agaricus bisporus var. bisporus. Most spores receive two post-meiotic nuclei, and

most such pairs of nuclei consist of Non-Sister Nuclear Pairs (NSNPs) which have a

heteroallelic genotype at most or all centromeric-linked loci including the MAT locus.

That MAT genotype determines the heterokaryotic phenotype of these offspring,

which are reproductively competent and can produce a crop of mushrooms.

Unusually among eukaryotes, relatively little chromosomal crossing-over is observed

to have occurred in postmeiotic offspring of A. bisporus var. bisporus; empirically,

very little heteroallelism (analogous to heterozygosity) is lost among heterokaryotic

offspring of a heterokaryotic strain. Consequently, parental and offspring

heterokaryotic genotypes and phenotypes tend to closely resemble each other, as

noted above; for this reason, essential derivation, e.g., the production of Essentially

Derived Varieties (EDVs), is a familiar strain development technique among

commercial mushroom spawn producers. In statutory frameworks, an EDV is

subject to the rights and protections granted to the rightsholders of the initial strain

from which the EDV is derived. For all of these reasons a need exists to develop

novel hybrid strains incorporating novel combinations of genetic material, i.e., novel

compositions of matter, from more than one parental strain, and which are

consequently not EDVs.

SUMMARY OF THE INVENTION

[0013]The advantages of the present invention over existing prior art relating to

Agaricus bisporus mushrooms and cultures, which shall become apparent from the

description which follows, are accomplished by the invention as hereinafter

described and claimed.

[0014] The present invention is directed generally to a new and distinct

homokaryotic line of Agaricus bisporus designated J10102-s69, to a new and distinct

Agaricus bisporus hybrid strain designated J11500, to lines and strains derived or

descended from J10102-s69 or J11500 including Essentially Derived Varieties

(EDVs) of line J10102-s69 or strain J11500, to cultures of each of the foregoing, and

to processes for the production of cultures of each of the foregoing as well as

methods for using the line designated J10102-s69 or the strain J11500 or lines or

strains derived or descended from J10102-s69 or J11500 or cultures thereof.

[0015] In accordance with the present invention, there is disclosed an Agaricus

bisporus culture designated as Agaricus bisporus line J10102-s69, a representative

culture of the line having been deposited under NRRL Accession No. 50893. A

deposit of a representative culture of the Agaricus bisporus line J10102-s69, as

disclosed herein, has been made with the Agricultural Research Services Culture

Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604 USA. The

date of deposit was January 15, 2014. The culture deposited was taken from the

same culture maintained by Sylvan America, Inc., Kittanning, Pennsylvania, USA,

the assignee of record, since prior to the filing date of this application. All restrictions

upon the deposit have been removed, and the deposit is intended to meet all deposit

requirements of all patent offices throughout the world, including the U.S. Patent and

Trademark Office, and all deposit requirements under the Budapest Treaty. The

NRRL Accession No. is 50893. The deposit will be maintained in the depository for

Q a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.

The culture will be irrevocably and without restriction or condition released to the

public upon the filing of the patent application or upon the issuance of a patent,

whichever is required by the applicable patent laws.

[0016]In accordance with a further aspect of the present invention, there is disclosed

an Agaricus bisporus culture comprising at least one set of chromosomes of an

Agaricus bisporus line J10102-s69, the culture of the line B10102-s69 having been

deposited under the NRRL Accession Number 50893, wherein said chromosomes

comprise all of the alleles of the line J10102-s69 at the sequence-characterized

marker loci listed in Table II. In accordance with yet a further aspect of the present

invention, there is disclosed an F1 hybrid Agaricus bisporus culture designated as

strain J11500, a representative culture of the strain having been deposited under

NRRL Accession No. 50895. Another aspect of the present invention provides an F1

hybrid Agaricus bisporus culture produced by mating the Agaricus bisporus culture of

line J10102-s69 with a different Agaricus bisporus culture. In one embodiment, it will

be appreciated that the invention may be achieved by a method for producing a

hybrid mushroom culture of Agaricus bisporus that includes the step of mating a

homokaryotic line J10102-s69, a culture of which was deposited under NRRL

Accession No. 50893 as above, with a homokaryotic line OWNC, a culture of which

was deposited with the Agricultural Research Services Culture Collection, 1815

North University Street, Peoria, Illinois 61604 USA under NRRL Accession No.

50894. The date of deposit of line OWNC was January 15, 2014. This culture

deposited was taken from the same culture maintained by Sylvan America, Inc.,

Kittanning, Pennsylvania, USA, the assignee of record, since prior to the filing date

of this application. All restrictions upon the deposit have been removed, and the deposit is intended to meet all deposit requirements of all patent offices throughout the world, including the U.S. Patent and Trademark Office, and all deposit requirements under the Budapest Treaty. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period. The culture will be irrevocably and without restriction or condition released to the public upon the filing of the patent application or upon the issuance of a patent, whichever is required by the applicable patent laws.

1n

[0017] Such a mating of line J10102-s69 and line OWNC provides an F1 hybrid

Agaricus bisporus culture designated as strain J11500, a deposit of a representative

culture of the Agaricus bisporus strain J11500, as disclosed herein, having been

made with the Agricultural Research Services Culture Collection (NRRL), 1815 North

University Street, Peoria, Illinois 61604 USA. The date of deposit was January 15,

2014. The culture deposited was taken from the same culture maintained by Sylvan

America, Inc., Kittanning, Pennsylvania, USA, the assignee of record, since prior to

the filing date of this application. All restrictions upon the deposit have been

removed, and the deposit is intended to meet all deposit requirements of all patent

offices throughout the world, including the U.S. Patent and Trademark Office, and all

deposit requirements under the Budapest Treaty. The NRRL Accession No. is

50895. The deposit will be maintained in the depository for a period of 30 years, or 5

years after the last request, or for the effective life of the patent, whichever is longer,

and will be replaced as necessary during that period. The culture will be irrevocably

and without restriction or condition released to the public upon the filing of the patent

application or upon the issuance of a patent, whichever is required by the applicable

patent laws.

[0018] The present invention further encompasses a culture that is an Essentially

Derived Variety (EDV), as defined herein, of an initial culture, wherein the initial

culture is a culture of line J10102-s69 as above, an F1 hybrid Agaricus bisporus

culture produced by mating the Agaricus bisporus culture of line J10102-s69 with a

different Agaricus bisporus culture as above, or the F1 hybrid strain J11500 as

above. Further discussion of EDVs is set forth hereinbelow. In one embodiment, an

Agaricus bisporus culture produced by essential derivation has at least one of the

essential characteristics of strain J11500, for example the same heterokaryon compatibility phenotype, and/or the further characteristics of cap roundness, flesh thickness, yield performance, and yield timing relative to commercial strain A-15.

[0019]The present invention further encompasses an Agaricus bisporus mushroom

culture including at least one set of chromosomes of any of the cultures of line

J10102-s69 above, hybrid strain J11500 above, or EDVs of the cultures above,

wherein said chromosomes comprise all of the alleles of the culture above at the

sequence-characterized marker loci listed in the appropriate column of Table I or

appropriate row of Table II below. In one embodiment, an Essentially Derived

Variety of the culture of line J10102-s69 is produced. In other embodiments, the

culture above may be an F1 hybrid Agaricus bisporus mushroom culture produced

by mating the culture of the line J10102-s69 or an EDV of J10102-s69, or of a line

obtained from strain J11500 or an EDV of strain J11500, with a different Agaricus

bisporus culture.

[0020] In accordance with yet a further aspect of the invention, there is disclosed a

mushroom culture of Agaricus bisporus having a genotypic fingerprint which has

characters at each of the marker loci in Table II, wherein all of the characters of said

fingerprint are also present in the genotypic fingerprint of either line J10102-s69,

representative culture of the line having been deposited under NRRL Accession No.

50893, or strain J11500, a representative culture of the strain having been deposited

under NRRL Accession No. 50895 at the same marker loci and wherein the

mushroom culture has the essential physiological and morphological characteristics

of line J10102-s69 or strain J11500. In accordance with yet a further aspect of the

invention, there is disclosed a mushroom culture of Agaricus bisporus having at least

one set of chromosomes comprising the chromosomes of line J10102-s69 or strain

J11500, wherein the at least one set of chromosomes has characters at each of the

marker loci in Table II, wherein all of the characters of said fingerprint are also present in the genotypic fingerprint of either line J10102-s69, representative culture of the line having been deposited under NRRL Accession No. 50893, or strain

J11500, a representative culture of the strain having been deposited under NRRL

Accession No. 50895 at the same marker loci. Genotypic fingerprints are

descriptions of the genotype at defined loci, where the presence of characterized

alleles is recorded. Such fingerprints provide powerful and effective techniques for

recognizing clones and all types of EDVs of an initial strain, as well as for

recognizing ancestry within outbred lineages. Many techniques are available for

defining and characterizing loci and alleles in the genotype. The

most detailed approach is provided by whole-genome sequencing

19n

(WGS), which allows for direct characterization and comparison of DNA sequences

across the entire genome. Using this approach to generate robust genotypic

fingerprints incorporating large numbers of marker loci, it is possible to establish the

nature of the relationship between two strains, including strains related by

genealogical descent over several generations. Sylvan America, Inc. has tracked

genetic markers through four to six generations of its breeding pedigrees. If a

sufficient number of rare markers are present in an initial strain or line, it will be

possible to identify descent from an initial strain or line after several outbred

generations without undue experimentation. In a hypothetical example, the mean

expectation for genomic representation of an initial haploid line after 4 outbred

generations is 3.1% in an F4 hybrid, which corresponds to ca. 1Mb of the nuclear

genomic DNA of A. bisporus. Based on Sylvan America's analyses, that amount of

DNA from each of two unrelated strains of A. bisporus may typically contain from

about 10,000 to about 20,000 single nucleotide polymorphisms (SNPs), any one of

which may provide a distinguishing marker linking the F4 hybrid to the initial line. By

using multiple independent markers, ancestors of a strain can be identified with a

very high probability of success and with high confidence.

[0021] In the embodiment described above, characters at at least two marker loci

are selected. It will be appreciated that in other embodiments, characters at at least

three, four, five or six marker loci may be selected. It is noted that prior art patents

have used from one to four marker loci.

[0022] One trait of biological and commercial interest is heterokaryon incompatibility.

The genetics of these self/non-self recognition systems are not well elucidated in

basidiomycete fungi such as Agaricus, but are known in other genera to involve

multiple alleles at multiple independent loci. Differences in the presumed genotype

at the incompatibility loci prevent successful anastomoses and cytoplasmic continuity among physical mixtures of two or more heterokaryons. One consequence of such antagonistic responses is a retardation of growth and development, and a reduction of crop yield; this sort of partial crop failure is well known and evident to the experienced grower. Another consequence of heterokaryon incompatibility is restriction on the opportunity for endocellular viruses to move freely throughout or among mycelial networks. Virus diseases such as those caused by the LIV or MVX viruses can have severe negative impacts on facility productivity and must be remediated using hygiene practices which can be assisted by strain rotation. A method of improving mushroom farm hygiene called 'virus-breaking' is carried out by replacing cropping material (compost, spawn, casing inoculum) incorporating an initial strain with inoculum and cropping material incorporating another different strain that is incompatible with the initial strain. In the most effective implementation of the virus-breaking method, all biological material of the initial strain at a mushroom farm is replaced with biological material of the second, incompatible strain. Strain incompatibility creates an effective if not absolute barrier to movement of virus from biological reservoirs within a facility into new crops. Rotating cultivation usage among mushroom strains of different genotypes may also interrupt infection and infestation cycles of exogenous pests and pathogens. Accordingly, in at least one embodiment of the present invention, any of the above cultures exhibit heterokaryon incompatibility toward heterokaryon strains in the U1 derived lineage group. The observable heterokaryon incompatibility demonstrates the genetic distinctness of strain J11500 relative to strains like A-15 that belong to the U1 derived lineage group.

[0023] In one or more embodiments, the Agaricus bisporus cultures of the present

invention have all of the physiological and morphological characteristics of line

J10102-s69, wherein the culture of line J10102-s69 has been deposited under the

NRRL Accession Number 50893, or strain J11500, wherein a culture of strain

J11500 has been deposited under NRRL Accession No. 50895.

[0024] The present invention also includes methods of production of any of the

cultures above, including the culture of line J10102-s69, the culture of strain J11500,

EDVs of J10102-s69, or EDVs of strain J11500, cultures that exhibit heterokaryon

incompatibility as above, and cultures that have a genotypic fingerprint as described

above or all of the physiological and morphological characteristics of the cultures

above. In accordance with yet a further aspect of the present invention, there is

disclosed a method for producing a hybrid mushroom culture of Agaricus bisporus

comprising mating a first parental Agaricus bisporus mushroom culture with a

second parental Agaricus bisporus mushroom culture, wherein at least one of the

first and second parental Agaricus bisporus mushroom cultures is a culture having

the essential physiological and morphological characteristics of line J10102-s69,

wherein the culture of said line J10102-s69 was deposited under the NRRL

Accession Number 50893.

[0025] In one or more embodiments, the method above further includes providing a

mushroom culture, as produced above, in mushroom products selected from the

group consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms,

processed mushrooms, parts of mushrooms, mushroom extracts and fractions,

mushroom pieces, and colonized substrates selected from grain, compost, and

friable particulate matter. In other embodiments, the method may include providing

the mushroom culture in derived or descended cultures selected from the group

consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue

explants cultures, protoplasts, dormant spores, germinating spores, inbred

descendents and outbred descendents, transgenic cultures, and cultures having a

genome incorporating a single locus conversion.

1;

[0026]In one or more embodiments, a cell may be obtained from any of the cultures

above or any of the methods for producing the cultures as noted above. In one or

more embodiments, the cell above may further include a marker profile having

characters at least two marker loci selected from the markers provided in the

appropriate column of Table I or appropriate row of Table II, wherein all of the

characters of said marker profile are also present in the marker profile of either line

J10102-s69, representative culture of the line having been deposited under NRRL

Accession No. 50893, or strain J11500, a representative culture of the strain having

been deposited under NRRL Accession No. 50895. Again, it will be appreciated that

in other embodiments, characters at at least three, four, five or six marker loci may

be selected as discussed above.

[0027] In other embodiments, a spore may comprise the cells above. In other

embodiments, the hybrid culture above may be further defined as having a genome

including a single locus trait conversion. In further embodiments, the locus above

may be selected from the group consisting of a dominant allele and a recessive

allele. In one or more other embodiments, the locus above may confer a trait

selected from the group consisting of mushroom size, mushroom shape, mushroom

cap roundness, mushroom flesh thickness, mushroom color, mushroom surface

texture, mushroom cap smoothness, tissue density, tissue firmness, delayed

maturation, basidial spore number greater than two, sporelessness, increased dry

matter content, increased shelf life, reduced brusing, increased yield, altered

distribution of yield over time, decreased spawn to pick interval, resistance to

infection by symptoms of or transmission of bacterial, viral or fungal disease or

diseases, insect resistance, nematode resistance, ease of crop management,

suitability of crop for mechanical harvesting, canning and/or processing, desired

1A behavioral response to environmental conditions, to stressors, to nutrient substrate composition, to seasonal influences, and to farming practices.

[0028] In accordance with yet a further aspect of the present invention, there is

disclosed a process for introducing a desired trait into a culture of Agaricus bisporus

line J10102-s69 comprising the steps of: (1) mating the culture of line J10102-s69 to

a second culture of Agaricus bisporus having the desired trait, to produce a hybrid;

(2) obtaining an offspring that carries at least one gene that determine the desired

trait from the hybrid produced above; (3) mating the offspring of the hybrid with the

culture of line J10102-s69 to produce a new hybrid; (4) repeating the steps of (2)

obtaining and (3) mating at least once to produce a subsequent hybrid; (5) obtaining

a homokaryotic line carrying at least one gene that determines the desired trait and

comprising at least 75% of the alleles of line J10102-s69, at sequence-characterized

marker loci selected from the markers loci described in Tables I and II, from the

subsequent hybrid of step (4). That is, step (4) may be repeated up to any of 1, 2, 3,

4, 5, 6, 7, 8, 9 and 10 times. In other embodiments, repeating steps (2) and (3) may

occur more than 10 times. In one embodiment, the homokaryotic line obtained may

comprise 80% of the alleles of line J10102-s69 at the sequence-characterized

marker loci described in Tables I and II. In other embodiments, the homokaryotic

line obtained may comprise 85%, 90%, 95%, 96%, 97%, 98%, 99% or may be

comprise essentially 100% of the alleles of line J10102-s69 at the sequence

characterized marker loci described in Tables I and II.

[0029] Still one or more other aspects of the present invention may be provided by

a method of producing a mushroom culture. The method includes (a) growing a first

hybrid culture produced by mating any of the above cultures or cultures produced

from the methods above, with a first different Agaricus bisporus culture; (b) mating a

first homokaryotic progeny line of the first hybrid culture with the first or a second different culture to produce a second hybrid culture of a subsequent descendant generation; (c) optionally, growing a second homokaryotic progeny line culture of the subsequent generation and mating the second homokaryotic progeny line of the

17n second hybrid culture of the subsequent descendant generation with the first or the second or a third different Agaricus bisporus culture; and (d) repeating steps (b) and

(c) for an additional 0, 1, 2, 3, 4 or 5 (i.e., 0-5) generations to produce a mushroom

culture. In one embodiment, the produced mushroom culture above is an inbred

culture. In another embodiment the produced mushroom culture is an outbred

culture. In one or more other embodiments, the method above may further include

the step of mating the inbred culture with a second, distinct culture to produce an F1

hybrid culture.

[0030] Yet one or more other aspects of the present invention may be provided by a

method for developing a second culture in a mushroom strain development program.

Such a method includes applying mushroom strain development techniques to a first

mushroom culture, or parts thereof, wherein the first mushroom culture is any of the

above cultures or cultures produced from the methods above. It is the application of

the mushroom strain development techniques that results in the development of the

second culture. Such known mushroom strain development techniques are selected

from the group consisting of inbreeding, back-mating, outbreeding, selfing,

introgressive trait conversions, essential derivation, pedigree-assisted breeding,

marker assisted selection, and transformation.

[0031]Still another aspect of the present invention may be provided by a method of

mushroom strain development. This method includes obtaining a molecular marker

profile of Agaricus bisporus mushroom line J10102-s69, a culture of which was

deposited under the NRRL Accession Number 50893. Another step of the method

includes obtaining an F1 hybrid culture, for which the deposited mushroom culture of

the Agaricus bisporus mushroom line J10102-s69 is a parent. Once the F1 hybrid

culture is obtained, the selection of homokaryotic progeny, based upon their

genotypes, for lines that possess characteristics of the molecular marker profile of line J10102-s69 as above may be conducted to obtain a culture of a desirable selected line. Finally, a further step of mating a culture of the selected line as set forth above with a different mushroom culture is employed. Once this is done, it may optionally be repeated. In one embodiment, it is not repeated. In other embodiments, it is repeated 1, 2, 3, 4 or 5 times.

[0032] Other embodiments of the foregoing may include the production of hybrid

mushroom cultures incorporating the line J10102-s69, the production of mushrooms

from cultures incorporating line J10102-s69, the production of mushroom parts from

cultures incorporating line J10102-s69. Still other uses include processes for making

a mushroom culture that comprise mating homokaryotic Agaricus bisporus line

J10102-s69 with another mushroom culture and processes for making a mushroom

culture containing in its genetic material one or more traits introgressed into line

J10102-s69 through introgressive trait conversion or transformation, and to the

mushroom cultures, mushrooms, and mushroom parts produced by such

introgression. Further, the invention may include a hybrid mushroom culture,

mushroom, mushroom part, including a spore, or culture part produced by mating the

homokaryotic line J10102-s69, or an introgressed trait conversion of line J10102

s69, with another mushroom culture. Still other uses of the present invention include

the production of homokaryotic mushroom lines derived from mushroom line J10102

s69, as well as the processes for making other homokaryotic mushroom lines

derived from mushroom line J10102-s69, and to the production of the inbred

mushroom lines and their parts derived by the use of those processes.

[0033] Cultures of strain J11500 are noted to produce mushrooms, parts of

mushrooms, parts of the culture, and strains and lines descended or derived from

such cultures. Thus, the present invention encompasses strain J11500, Essentially

Derived Varieties of strain J11500, more particularly EDVs incorporating at least

75% of the genetic material of strain J11500, dormant or active growing cultures

present in dormant or germinating spores of strain J11500, and cultures descended

from and incorporating the genetic material of strain J11500. The present invention

is also directed towards methods of making and using strain J11500. Uses of

J11500 include methods for producing mushrooms and parts of mushrooms

including spores, for improving farm hygiene, for producing offspring from

homokaryotic and heterokaryotic spores, for producing hybrid descendents via

outcrossing with a second line or strain, and for producing EDVs by any means

known in the art.

[0034] With respect to spores, living spores are heterokaryons or homokaryons in a

dormant state. Spores are one part of the mushroom organism. Other parts include

caps, stems, gills, cells (defined as hyphal compartments incorporating nuclei,

mitochondria, cytoplasm, a cell membrane, and a cell wall including crosswalls),

hyphae, and mycelium. Spores may be aseptically collected on sterile material,

suspended in sterile water at various dilutions, and plated onto sterile agar growth

media in order to produce germinated spores and the cultures incorporated within

the spores. A preferred technique is to have within the enclosed petri plate a living

Agaricus culture which may stimulate spore germination via the diffusion of a volatile

pheromone. Germinated spores may be isolated under a microscope using sterile

microtools such as steel needles, onto fresh nutrient agar plates. Using this method,

cultures of heterokaryotic and homokaryotic offspring of a heterokaryotic strain

comprising the spores and the cultures incorporated within the spores of the

heterokaryotic strain may be obtained.

[0035] Development of novel hybrid varieties via heteromixis comprises the

controlled association and mating of two compatible cultures to obtain a novel

heterokaryon culture. Homokaryons (= 'lines') are the preferred starting cultures for making matings as they have maximal ability to anastomose and achieve plasmogamy with other cultures. Heterokaryons may also be confronted but with commercially unreasonably low probabilities of a mating resulting in successful formation of a novel heterokaryon. Compatibility is determined by the genotype at the MAT locus; two homokaryons with the same MAT allele cannot establish a heterokaryon after anastomosis. In a defined mating program, homokaryotic lines are obtained and are associated in predetermined pairwise combinations. In one method, homokaryon pairs may be placed in close proximity on the surface of a nutrient agar medium in a petri dish and allowed to grow together (in a physical association), at which point anastomoses between the two cultures occur. A successful outcome is a mating. The novel hybrid heterokaryon may be obtained by transferring mycelium from the fusion zone of the dish. Such a paired mating method was used to develop hybrid heterokaryotic strains from line J10102-s69, and from lines obtained from J11500 and from other descendants of J10102.

[0036]In contrast, EDVs are most often derived directly (otherwise predominantly)

from a single initial culture (e.g., strain); all such derivations produce EDVs. There is

no universally accepted definition of an EDV; one example of a definition applicable

to plant varieties is provided by the US Plant Variety Protection Act (revised edition,

February 2006). The definition employed herein is congruent with the term as it is

widely understood. 'Essential derivation' methods of obtaining cultures which are by

definition consequently EDVs of a single initial culture of A. bisporus include somatic

selection, tissue culture selection, single spore germination, multiple spore

germination, selfing, repeated mating back to the initial culture, mutagenesis, and

transformation, to provide some examples. DNA-mediated transformation of A.

bisporus has been reported by Velcko, A. J. Jr., Kerrigan, R. W., MacDonald, L. A.,

Wach, M. P., Schlagnhaufer, C., and Romaine, C. P. 2004, Expression of novel genes in Agaricus bisporus using an Agrobacterium-mediated transformation technique. Mush. Sci. 16: 591-597, and references therein. Transformation may introduce a single new gene or allele into the genome of an initial culture.

[0037] Although in statutory frameworks EDVs are defined primarily by the methods

used to produce them, it is also true that EDVs are inherently unambiguously

recognizable by their genotype, which will be entirely or predominantly (75% or

greater) a subset of that of the single initial culture. Percentages of the initial

genotype that will be present in Agaricus bisporus EDVs range from almost 100% in

the case of somatic selections, to 99.x% in the case of strains modified by DNA

mediated transformation, to 90-100% in the case of single or multiple spore

selections or some mutagenesis, including instances where no heteroallelism is lost

during meiotic internuclear reassociation of homologous chromosomes, to an

average of from about 75% to about 85% in the case of sibling-offspring matings (=

selfing), to about 75% on average in a first generation of back-mating, increasingly

approaching 99.x% with each successive generation of back-mating. Many methods

of genotype determination, including methods described below, and others well

known in the art, may be employed to determine the percentage of DNA of an initial

culture that is present in another culture.

[0038] Repeated mating back to the initial culture to introgress a single trait into the

genetic background of an initial culture is called introgressive trait conversion, and

according to accepted definitions of EDVs, also produces an EDV of the initial

culture. In a hypothetical example, in the first successive repetition of this process a

resultant strain of this generation will have on average about 75% of the DNA of the

initial strain while about 25% of the DNA will have been contributed by a second

strain or line; as this process is repeated the DNA representation of the initial strain

will increase, approaching 97% on average after 3 further successive repetitions.

There is no universally accepted quantitative threshold for the proportion of DNA

contributed by an initial culture in an EDV of an initial culture; from the foregoing it is

apparent that approximately 75-100% genotype identity with an initial culture is

indicative of status as an EDV of an initial culture, with 75%, being a minimum

threshold. It is also established that an EDV of an EDV is also an EDV of an initial

strain. Finally, because Agaricus bisporus alternates generations between

heterokaryotic strains and homokaryotic lines, the criteria for essential derivation

apply equally to cultures of both strains and lines.

[0039] As noted above, hybrid mushroom strain producers are always looking for

hybrid strains that allow growers to produce crops of mushrooms successfully and

profitably. In the case of strain J11500 and strains derived or descended from that

strain, positive attributes documented thus far include a rounder cap shape and

thicker cap flesh, both of which appeal to consumers, than existing successful

commercial strain A-15, and a total harvested yield that may exceed that of strains

like A-15, and yield timing that is accelerated as compared to strain A-15, a trait that

is particularly suitable for certain segments of the market, and which tends to

accelerate revenue capture and decrease crop cycle time (potentially allowing

greater throughput).

[0040] In addition, and as noted above, strain J11500 has a different genotype from

the U1 derived lineage group. Accordingly, strain J11500 is incompatible with strains

of the U1 derived lineage group, which is a characteristic known to retard the spread

of viral diseases between strains. Thus, strain J11500 confers a potential benefit in

strain rotation programs designed to manage facility hygiene. Strain J11500 has

been found to simultaneously provide both genetic diversification and commercially

acceptable performance and crop characteristics.

[0041] It will be appreciated that, in one or more embodiments, a part of any of the

cultures above or any cultures produced from the methods above may be selected

from the group consisting of hyphae, spores, and cells and parts of cells, including,

nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA, proteins, cell membranes

and cell walls, each part being present in either the vegetative mycelium of the

culture or in mushrooms produced by the culture, or both. The parts may be present

in both the vegetative mycelium of the culture and in mushrooms produced by the

cultures above. The spores may be dormant or germinated spores, and may include

heterokaryons and homokaryons incorporated therein.

[0042] Further, in other embodiments, any of the cultures above or any cultures

produced from the methods above may be incorporated into products selected from

mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed

mushrooms, mushroom extracts and fractions, mushroom pieces, and colonized

substrates including grain, compost, and friable particulate matter. It will be

appreciated that mushroom pieces refer to stems, pilei, and other larger portions of

the mushroom itself. In other embodiments, the F1 hybrid mushroom culture of

Agaricus bisporus above may be processed into one or more products selected from

the group consisting of mycelium, spawn, inoculum, casing inoculum, fresh

mushrooms, processed mushrooms, mushroom extracts and fractions, mushroom

pieces, and colonized substrates including grain, compost, and friable particulate

matter. In other embodiments, a mushroom may be produced by growing a crop of

mushrooms from any of the cultures above. In other embodiments, a mushroom

may be produced by growing a crop of mushrooms from the F1 hybrid mushroom

culture above. In still other embodiments, an Essentially Derived Variety of the F1

hybrid mushroom culture above is produced.

[0043]Thus, in one or more embodiments, a method for producing a hybrid

mushroom culture of Agaricus bisporus may includes the step of mating a

homokaryotic line J10102-s69, a culture of which was deposited under NRRL

Accession No. 50893, with a homokaryotic line OWNC, a culture of which was

deposited under NRRL Accession No. 50894. Such a mating provides the hybrid

mushroom culture J11500, which exhibits antagonism toward heterokaryon strains in

the U1 derived lineage group. The observable heterokaryon incompatibility

demonstrates the genetic distinctness of strain J11500 relative to strains like A-15

that belong to the U1 derived lineage group. In one or more embodiments, the

method further includes providing a mushroom culture of the invention in mushroom

products selected from the group consisting of mycelium, spawn, inoculum, casing

inoculum, fresh mushrooms, processed mushrooms, parts of mushrooms,

mushroom extracts and fractions, mushroom pieces, and colonized substrates

selected from grain, compost, and friable particulate matter. In other embodiments,

the method may include providing the mushroom culture in derived or descended

cultures selected from the group consisting of homokaryons, heterokaryons,

aneuploids, somatic subcultures, tissue explants cultures, protoplasts, dormant

spores, germinating spores, inbred descendents and outbred descendents,

transgenic cultures, and cultures having a genome incorporating a single locus

conversion.

[0044]In other embodiments, a cell or a culture including the cell, is produced by the

method(s) above. Thus, one or more embodiments may include a method further

including the step of growing the hybrid mushroom culture to produce hybrid

mushrooms and parts of mushrooms. Other embodiments may provide for methods

wherein the hybrid mushroom culture produced, or the cell, includes a marker profile

having characters at at least two (or three, or four, or five, or six) marker loci ITS, p1n150-G3-2, MFPC-1-ELF, AN, AS, and FF, wherein all of the characters of said marker profile are also present in the marker profile of either line J10102-s69 or strain J11500. Still other embodiments may provide for methods wherein the hybrid mushroom culture produced, or the cell, includes a marker profile having characters at at least two (or three, or four, or five, or six) marker loci described in Tables I orII, wherein all of the characters of said marker profile are also present in the marker profiles of either line J10102-s69 orstrain J11500.

[0045] Finally, another aspect of the present invention may be accomplished by

various methods that use any of the culture above for various uses. In one

embodiment, the method further includes producing or otherwise growing a crop of

edible mushrooms by carrying out the steps described hereinabove. In another

embodiment, the method may include the cultures above in crop rotation to reduce

pathogen pressure and pathogen reservoirs in mushroom growing facilities as

described hereinabove. In yet another embodiment, the method includes using the

cultures above to produce offspring as described hereinabove.

DETAILED DESCRIPTION OF THE INVENTION

[0046]Initially, in order to provide clear and consistent understanding of the

specification and claims, including the scope to be given such terms, the following

definitions are provided.

[0047] Allele: A heritable unit of the genome at a defined locus, ultimately identified

by its DNA sequence (or by other means); in a genotype, an allelic character.

[0048] Amphithallism: A reproductive syndrome in which heteromixis and intramixis

are both active.

[0049] Anastomosis: Fusion of two or more hyphae that achieves cytoplasmic

continuity.

[0050] Basidiomycete: A monophyletic group of fungi producing meiospores on

basidia; a member of a corresponding subdivision of Fungi such as the

Basidiomycetales or Basidiomycotina.

[0051] Basidium: The meiosporangial cell, in which karyogamy and meiosis occur,

and upon which the basidiospores are formed.

[0052] Bioefficiency: For mushroom crops, the net fresh weight of the harvested

crop divided by the dry weight of the compost substrate at the time of spawning, for

any given sampled crop area or compost weight.

[0053] Breeding: Development of strains, lines or varieties using methods that

emphasize sexual mating; see Descent.

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

[0055] Cap Roundness: Strictly, a ratio of the maximum distance between the

uppermost and lowermost parts of the cap, divided by the maximum distance across

the cap, measured on a longitudinally bisected mushroom; typically averaged over

many specimens; subjectively, a 'rounded' property of the shape of the cap.

[0056] Carrier substrate: A medium having both nutritional and physical properties

suitable for achieving both growth and dispersal of a culture.

[0057] Casing layer, casing: A layer of non-nutritive material such as peat or soil

that is applied to the upper surface of a mass of colonized compost in order to permit

development of the mushroom crop.

[0058] Casing inoculum (Cl): A formulation of inoculum material incorporating a

mushroom culture, typically of a defined heterokaryotic strain, suitable for mixing into

the casing layer.

[0059] Cloning: Somatic propagation without selection.

[0060] Combining ability: The capacity of an individual to transmit traits or superior

performance to its offspring (known and available methods of assessment vary by

trait).

[0061] Compatibility: See heterokaryon compatibility.

[0062] Culture: The tangible living organism; the organism propagated on various

growth media and substrates; one instance of one physical strain, line, homokaryon

or heterokaryon; the sum of all of the parts of the culture, including hyphae,

mushrooms, spores, cells, nuclei, mitochondria, cytoplasm, protoplasts, DNA, RNA,

proteins, cell membranes and cell walls.

[0063] Derivation: Development of a strain or culture from a single initial strain, or

predominantly from a single initial strain, in contrast to descent via sexual mating

between two parental strains; see Essentially Derived Variety (EDV).

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

from that single initial strain or variety.

[0065] Descent: The production of offspring from two parents, and/or four

grandparents, and/or additional progenitors, via sexual mating; in contrast to

derivation from a single initial strain.

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

nuclear envelope.

[0067] Essential derivation: A process by which an Essentially Derived Variety is

obtained from an initial variety or strain or from an EDV of an initial variety or strain;

modification of an initial culture using methods including somatic selection, tissue

culture selection, selfing including intramictic reproduction via single spores and

multiple spores and mating of sibling offspring lines, back-mating to the initial variety,

or mutagenesis and/or genetic transformation of the initial variety to produce a distinct culture in which the genotype of the resulting culture is predominantly that of the initial culture.

[0068] Essentially Derived Variety (EDV): (Note: EDV definitions, for example, as

applied to plants in the US PVPA, incorporate elements of (1) relatedness, (2)

methods of derivation, (3) and empirical tests.) A variety having 75% to 99.99999%

genetic identity with an initial strain or variety, or to 100% in a heterokaryon with

internuclear reassociation of chromosomes. In general, a variety that is entirely or

predominantly derived from an initial variety or from an EDV of an initial variety, and

which conforms to specified or "essential" characteristics of the initial variety except

for distinguishing differences resulting from the act of derivation, is an EDV of the

initial variety. In the art of mushroom strain development, a strain or culture

predominantly or entirely derived from a single initial strain or culture, thus having

most or all, but at least 75%, of its genome or genotype present in the genome or

genotype of the initial strain or culture; a strain or culture obtained from an initial

strain or culture by somatic selection, tissue culture selection, selfing including

mating of sibling offspring lines and intramictic reproduction via single or multiple

spores, back-mating to the initial strain or culture, or mutagenesis and/or genetic

transformation of the initial strain or culture; a strain or culture reconstituted from

neohaplonts derived from an initial strain or culture, whether or not the haploid lines

have been passed into or out of other heterokaryons; a strain or culture with the

same essential phenotype as that of an initial strain or culture; in contrast to descent

(via sexual mating between two parental strains).

[0069] Flesh Thickness: A ratio of the maximum distance between the top of the

stem and the uppermost part of the cap, divided by the maximum distance across

the cap, measured on a longitudinally bisected mushroom; typically averaged over

many specimens; subjectively called 'meatiness'.

[0070] Flush: A period of mushroom production within a cropping cycle, separated

by intervals of non-production; the term flush encompasses the terms 'break' and

'wave' and can be read as either of those terms.

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

[0072] Genealogical descent: Descent from progenitors, including parents, over a

limited number (e.g., 10 or fewer) of typically outcrossed generations; in contrast to

derivation from a single initial strain.

[0073] Genotypic fingerprint: A description of the genotype at a defined set of

marker loci; the known genotype.

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

bearing structure.

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

homokaryon.

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

heterozygous.

[0077] Heteroallelism: Differences between homologous chromosomes in a

heterokaryotic genotype; analogous to heterozygosity.

[0078] Heterokaryon: As a term of art this refers to a sexual heterokaryon: a culture

which has two complementary (i.e., necessarily heteroallelic at the MAT locus) types

of haploid nuclei in a common cytoplasm, and is thus functionally and physiologically

analogous to a diploid individual (but cytogenetically represented as N+N rather than

2N), and which is potentially reproductively competent, and which exhibits self/non

self incompatibility reactions with other heterokaryons; also called a strain or stock in

the breeding context.

[0079] Heterokaryon compatibility: The absence of antagonism observed during

physical proximity or contact between two heterokaryons that are not genetically

identical; see Heterokaryon Incompatibility.

[0080] Heterokaryon incompatibility: The phenomenon of antagonism observed

during physical proximity or contact between two heterokaryons that are not

genetically identical; a multilocus self/non-self recognition system that operates in

basidiomycete heterokaryons to regulate contact through anastomosis.

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

[0082] Heteromixis: Life cycle involving mating between two different non-sibling

haploid individuals or gametes; outbreeding.

[0083] Homoallelic: Having not more than one allele at a locus. The equivalent

term in a diploid organism is 'homozygous'. Haploid lines are by definition entirely

homoallelic at all non-duplicated loci.

[0084] Homokaryon: A haploid culture with a single type (or somatic lineage) of

haploid nucleus (cytogenetically represented as N), and which is ordinarily

reproductively incompetent, and which does not exhibit typical self/non-self

incompatibility reactions with heterokaryons, and which may function as a gamete in

sexually complementary anastomoses; a 'line' which, as with an inbred plant line,

transmits a uniform genotype to offspring; a predominantly homoallelic line that

mates well and fruits poorly is a putative homokaryon for strain development

purposes; see discussion below.

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

[0086] Hybrid: Of biparental origin, usually applied to heterokaryotic strains and

cultures produced in controlled matings.

[0087] Hybridizing: Physical association, for example on a petri dish containing a

sterile agar-based nutrient medium, of two cultures, usually homokaryons, in an attempt to achieve anastomosis, plasmogamy, and formation of a sexual heterokaryon (= mating); succeeding in the foregoing.

[0088] Hyphae: Threadlike elements of mycelium, composed of cell-like

compartments or'cells'.

[0089] Inbreeding: Matings that include sibling-line matings, back-matings to parent

lines or strains, and intramixis; reproduction involving parents that are genetically

related.

[0090] Incompatibility: See heterokaryon incompatibility.

[0091] Inoculum: A culture in a form that permits transmission and propagation of

the culture, for example onto new media; specialized commercial types of inoculum

include spawn and Cl; plural: inocula.

[0092] Intramixis: A uniparental sexual life cycle involving formation of a

complementary 'mated' pair of postmeiotic nuclei within the basidium or individual

spore.

[0093] Introgressive trait conversion: mating offspring of a hybrid to a parent line or

strain such that a desired trait from one strain is introduced into a predominating

genetic background of the other parent line or strain.

[0094] Lamella: see'gill'.

[0095] Line: A culture used in matings to produce a hybrid strain; ordinarily a

homokaryon which is thus homoallelic, otherwise a non-heterokaryotic (non-NSNPP)

culture which is highly homoallelic; practically, a functionally homokaryotic and

entirely or predominantly homoallelic culture; analogous in plant breeding to an

inbred line which is predominantly or entirely homozygous.

[0096] Lineage group: see 'derived lineage group'. The set of EDVs derived from a

single initial strain, line or variety, plus the initial strain, line or variety.

[0097] Locus: A defined contiguous part of the genome, homologous although often

varying among different genotypes; plural: loci.

[0098] Marker assisted selection: Using linked genetic markers including molecular

markers to track trait-determining loci of interest among offspring and through

pedigrees.

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

heterokaryotic state.

[0100] Mating: The sexual union of two cultures via anastomosis and plasmogamy;

methods of obtaining matings between mushroom cultures are well known in the

art.

[0101] Mycelium: The vegetative body or thallus of the mushroom organism,

comprised of threadlike hyphae.

[0102] Mushroom: The reproductive structure of an agaric fungus; an agaric; a

cultivated food product of the same name.

[0103] Neohaplont: A haploid culture or line obtained by physically

deheterokaryotizing (reducing to haploid components) a heterokaryon; a somatically

obtained homokaryon.

[0104] Offspring: Descendants, for example of a parent heterokaryon, within a

single generation; most often used to describe cultures obtained from spores from a

mushroom of a strain.

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

[0106] OW-type strain: A category of cultivar strains traditionally called 'Off-white'

strains, comprising an initial strain and its derived lineage group, exemplified by

strain Somycel 76; OW strain, OW.

[0107] Parent: An immediate progenitor of an individual; a parent strain is a

heterokaryon, a parent line is a homokaryon; a heterokaryon may be the parent of an

F1 heterokaryon via an intermediate parent line.

[0108] Pedigree-assisted breeding: The use of genealogical information to identify

desirable combinations of lines in controlled mating programs.

[0109] Phenotype: Observable characteristics of a strain or line as expressed and

manifested in an environment.

[0110] Plasmogamy: Establishment, via anastomosis, of cytoplasmic continuity

leading to the formation of a sexual heterokaryon.

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

[0112] Progeny: In Agaricus bisporus, strictly speaking, new heterothallic or

homothallic individuals (cultures, mycelia, etc.) produced by an initial heterokaryotic

individual via meiosis and sporulation, and, ultimately, germination and growth, i.e.,

single-spore isolates or SSIs; broadly speaking, offspring, sometimes used to

encompass individuals of the first hybrid generation of heterokaryotic descendants of

an initial individual.

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

[0114] Somatic: Of the vegetative mycelium.

[0115] Spawn: A mushroom culture, typically a pure culture of a heterokaryon,

typically on a sterile substrate which is friable and dispersible particulate matter, in

some instances cereal grain; commercial inoculum for compost; reference to spawn

includes reference to the culture on a substrate.

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

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

[0118] Sterile Growth Media: Nutrient media, sterilized by autoclaving or other

methods, that support the growth of the organism; examples include agar-based solid nutrient media such as Potato Dextrose Agar (PDA), nutrient broth, and many other materials.

[0119] Stipe: see'stem'.

[0120] Strain: A heterokaryon with defined characteristics or a specific identity or

ancestry; equivalent to a variety.

[0121] SW-type strain: A category of cultivar strains traditionally called 'Smooth

white' strains, comprising an initial strain and its derived lineage group, exemplified

by strain Somycel 53; SW strain, SW.

[0122] Tissue culture: A de-differentiated vegetative mycelium obtained from a

differentiated tissue of the mushroom.

[0123] Trait conversion: Selective introduction of the genetic determinants of one (a

single-locus conversion) or more desirable traits into the genetic background of an

initial strain while retaining most of the genetic background of the initial strain. See

'Introgressive trait conversion' and 'Transformation'.

[0124] Transformation: A process by which the genetic material carried by an

individual cell is altered by the incorporation of foreign (exogenous) DNA into its

genome; a method of obtaining a trait conversion including a single-locus

conversion.

[0125] Virus-breaking: Using multiple incompatible strains, i.e. strains exhibiting

heterokaryon incompatibility, successively in a program of planned strain rotation

within a mushroom production facility to reduce the transmission of virus from on-site

virus reservoirs into newly planted crops.

[0126] Yield: The net fresh weight of the harvest crop, normally expressed in

pounds per square foot.

[0127] Yield pattern: The distribution of yield within each flush and among all

flushes; influences size, quality, picking costs, and relative disease pressure on the

crop and product.

[0128] With respect to the definition of homokaryon above, it is noted that

homokaryons and homoallelic lines are subject to technical and practical

considerations: A homokaryon in classical terms is a haploid culture which is

axiomatically entirely homoallelic. In practical terms, for fungal strain development

purposes, the definition is broadened somewhat to accommodate both technical

limitations and cytological variation, by treating all predominately homoallelic lines as

homokaryons. Technical limitations include the fact that genomes contain duplicated

DNA regions including repeated elements such as transposons, and may also

include large duplications of chromosomal segments due to historical translocation

events; such regions may appear not to be homoallelic by most genotyping

methods. Two different A. bisporus genomes sequenced by the Joint Genome

Institute, a U.S. federal facility, differ in estimated length by 4.4%, and in gene

numbers by 8.2%, suggesting a considerable amount of DNA duplication or

rearrangement within different strains of the species. No presently available genome

of A. bisporus can completely account for the physical arrangement of such

elements and translocations, and so the assembled genome sequences of haploid

lines may have regions that appear to be heteroallelic using currently available

genotyping methods. Cytologically, a homokaryotic offspring will ordinarily be a

spore that receives one haploid, postmeiotic nucleus. However, a spore receiving

two third-division nuclei from the basidium will be genetically equivalent to a

homokaryon. A spore receiving two second-division 'sister' postmeiotic nuclei will be

a functional homokaryon even though some distal 'islands' of heteroallelism may be

present due to crossovers during meiosis. Also, a meiosis that has an asymmetrical separation of homologues can produce an aneuploid, functionally homokaryotic spore in which an extra chromosome, producing a region of heteroallelism, is present. All of these cultures are highly homoallelic and all function as homokaryons. Technological limitations make it impractical to distinguish among such cultures, and also to rule out DNA segment duplication as an explanation for limited, isolated regions of the genome sequence assembly that appear to be heteroallelic. Therefore, in the present application, the use of the term 'homoallelic' to characterize a line includes entirely or predominately homoallelic lines, regardless of the presence of regions of genome duplication, or of aneuploidy, and cultures described in this way are functional homokaryons, are putatively homokaryotic, and are all defined as homokaryons in the present application.

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

invention relates initially to a homokaryotic line, and more specifically, a line of

Agaricus bisporus designated J10102-s69, and methods for using the line

designated J10102-s69. A culture of the line designated J10102-s69 has been

deposited with the Agricultural Research Services Culture Collection (NRRL) 1815

North University Street, Peoria, Illinois 61604 USA ("NRRL") as Accession No.

50893.

[0130] Agaricus bisporus mushroom line J10102-s69 is a haploid filamentous

basidiomycete culture which in vegetative growth produces a branching network of

hyphae, i.e. a mycelium. Growth can produce an essentially two-dimensional colony

on the surface of solidified (e.g., agar-based) media, or a three-dimensional mass in

liquid or solid-matrix material. The morphological and physiological characteristics of

line J10102-s69 in culture on Difco brand PDA medium are provided as follows. Line

J10102-s69 growing on PDA medium in a 10 cm diameter Petri dish produced a light

brown-yellow or 'tan' colored irregularly lobate colony with a roughly circular overall outline that increased in diameter by (0.3-0.4-) 0.7 (-0.8-1.4) mm/day during dynamic equilibrium-state growth between days 12 and 26 after inoculation using a 6.5-7 mm diameter circular plug of the culture on PDA as inoculum. Hyphae of the culture on

Difco PDA were irregular and about cylindrical, measured (12-) 41-71 (-99) x (4.5-)

6-8 (-10) um, and exhibited a wide range of branching angles from about 10 to 90

degrees off the main hyphal axis.

[0131]Line J10102-s69 can be used to produce hybrid cultures with desirable

productivity, timing, appearance, and other agronomic traits as is required of

successful commercial mushroom strains, while also providing more diversified, non

cultivar germplasm. Line J10102-s69 has been found to have an advantageous

genotype for mating to produce commercially useful hybrid strains. Several useful

stocks have contributed to the genome of line J10102-s69. Line J10102-s69 has, for

example, a mating-type allele 2 on scaffold 1 contributed by the traditional smooth

white stock, and a 'white' color determining allele, as reported by allele El at the

MFPC-1-ELF marker locus on scaffold 8, contributed by the traditional off-white

hybrid stock. Among the remaining genomic scaffolds are at least three (i.e.,

scaffolds 2, 9 and 10) contributed by other, wild stocks in the pedigree. In

combination, these diverse genetic contributions were observed to have combined to

produce a superior line with excellent combining ability in matings.

[0132] The J10102-s69 line is haploid and thus is entirely homoallelic (although

some limited regions of duplicated DNA may be present in its genome). The line has

shown uniformity and stability in culture. The line has been increased by transfer of

pure inocula into larger volumes of sterile culture media. No variant traits have been

observed or are expected in line J10102-s69.

[0133] In light of the usefulness of line J10102-s69 to produce hybrid cultures, it is at

least a further embodiment of the invention to provide an F1 hybrid Agaricus bisporus culture by mating the Agaricus bisporus culture of line J10102-s69 with a different Agaricus bisporus culture of another line. Thus, any cultures derived or descended from line J10102-s69 may be a part of the invention.

[0134] In one embodiment, an F1 hybrid mushroom culture of Agaricus bisporus can

be produced by mating the homokaryotic line J10102-s69, a culture of which was

deposited under NRRL Accession No. 50893 as above, with another homokaryotic

line, OWNC, a culture of which was deposited with the Agricultural Research

Services Culture Collection, 1815 North University Street, Peoria, Illinois 61604 USA

under NRRL Accession No. 50894. The mating of these two lines results in the

production of the F1 hybrid strain J11500, a culture of which was deposited with the

Agricultural Research Services Culture Collection, 1815 North University Street,

Peoria, Illinois 61604 USA under NRRL Accession No. 50895.

[0135] It will be appreciated that the present invention further relates to not only to

cultures of the F1 hybrid strain J11500, but also to Essentially Derived Varieties

(EDVs) of the strain J11500, as well as to cultures derived or descended from strain

J11500 and EDVs of strain J11500. Such cultures are used to produce mushrooms

and parts of mushrooms. Thus, the present invention further relates to methods of

making and using the strain J11500 and EDVs of the strain J11500.

[0136] Mushroom cultures are most reliably identified by their genotypes, in part

because successful cultivar strains are required by the market to conform to a

narrow phenotypic range. The genotype can be characterized through a genetic

marker profile, which can identify isolates (subcultures) of the same line, strain or

variety, or a related variety including a variety derived entirely from an initial variety

(i.e., an Essentially Derived Variety), or from an EDV of an initial variety, or can be

used to determine or validate a pedigree.

[0137] Mushroom-forming fungi exhibit an alternation of generations, from

heterokaryotic (N+N, with two haploid nuclei, functionally like the 2N diploid state) to

homokaryotic (1N) and further upon mating to become heterokaryotic again. Inmost

eukaryotes, a parent is conventionally considered to be either diploid or

heterokaryotic. The haploid 'generation' is often, but not always, termed a gamete

(e.g., pollen, sperm). In fungi, which are microorganisms, the haploid generation can

live and grow indefinitely and independently, for example in laboratory cell culture;

while these haploid homokaryons function as gametes in matings, they are

equivalent to inbred lines (e.g., of plants) and are more easily referred to as parents

(of hybrids). Herein, the term 'parent' refers to the culture that is a, or the, direct

progenitor of another culture within the alternating generations of the sexual lifecycle.

The term 'line' refers more narrowly to a haploid (N) homoallelic culture within the

lifecycle. The N+N heterokaryon resulting from a mating, or comprising a breeding

stock, or comprising a culture used to produce a crop of mushrooms, may be called

a 'strain'.

[0138] If one parental line carries allele 'p' at a particular locus, and the other

parental line carries allele 'q', the F1 hybrid resulting from a mating of these two lines

will carry both alleles, and the genotype can be represented as 'p/q' (or 'pq', or

'p+q'). Sequence-characterized markers are co-dominant and both alleles will be

evident when an appropriate sequencing protocol is carried out on cellular DNA of

the hybrid. The profile of line J10102-s69 can therefore be used to identify hybrids

comprising line J10102-s69 as a parent line, since such hybrids will comprise two

sets of alleles, one of which sets will be from, and match that of, line J10102-s69.

The match can be demonstrated by subtraction of the second allele from the

genotype, leaving the J10102-s69 allele evident at every locus. A refinement of this

approach is possible with hybrids of Agaricus bisporus as a consequence of the heterokaryon (N+N) condition existing in hybrids. The two haploid nuclei can be physically isolated by various known techniques (e.g., protoplasting) into

'neohaplont' subcultures, and each may then be characterized independently. One

of the two neohaplont nuclear genotypes from the F1 hybrid will be that of line

J10102-s69, demonstrating its use in the mating and its presence in the hybrid.

[0139] Means of obtaining genetic marker profiles using diverse techniques

including whole genome sequencing are well known in the art. For the purpose of

providing a detailed embodiment of this invention, the whole genomic sequence of

strain J11500 and of the cultures of its parent lines, including J10102-s69 and

OWNC, and of selected EDVs of J11500 have been obtained and provided by

Sylvan America Inc. using the following method. The homokaryotic parent line

cultures were grown in sterile broth growth medium after maceration. After 2-4

weeks, hyphal cells were collected by filtration, were frozen at -80C, and were

lyophilized until dry. Cap tissue was obtained from mushrooms produced by cultures

of the heterokaryotic J11500 (and EDV) strains, and was frozen andlyophilized.

DNA was extracted using a CTAB protocol followed by RNAse treatment and gel

purification. A contractor, SeqWright, prepared DNA libraries from the DNA of each

culture, and sequenced the libraries using Illumina MiSeq technology. Assemblies of

the sequencing reads into genomic sequence using the public-domain reference

genome sequence of H97 were performed by Sylvan America, Inc. Consequently

about 93% to about 95% of the entire genotype of line J10102-s69, of strain J11500

and of three EDVs of strain J11500 are known to Sylvan America, Inc with

certainty. The OWNC line "H97" was sequenced and resequenced by the Joint

Genome Institute and was placed in the public domain, thus its genome is known

with about 100% certainty. The total number of markers distinguishing either line

J10102-s69 or strain J11500 that are known to the assignee is about 300,000. A brief excerpt of the genotypes of line J10102-s69, of the OWNC line, of J11500, and of the EDV J11500-ms2 at numerous sequence-characterized marker loci distributed at intervals along each of the 19 H97 V2.0 reference chromosomal scaffolds larger than 100 Kb in length is provided in Table 1.

[0140]

TABLE I Scaffold Position of SNP [H97 Culture: V2.0 ref. coords.] J10102-s69 OWNC J11500 J11500-ms2 1 99995 CTACGTTGA CTACATTGA CTACrTTGA CTACrTTGA 1 349966 AAGGCGGTT AAGGTGGTT AAGGyGGTT AAGGyGGTT 1 600059 TTTTCTTTA TTTTTTTT-C TTTTyTT[-/A] TTTTyTT[-/A] 1 850014 CTTTTCGC CCTTTTCAC CyTTTTCrC CyTTTTCrC 1 1099971 GTCGGCACC GTCGACACC GTCGrCACC GTCGrCACC 1 1350278 GGAGGTTCG GGAGAGTCG GGAGrkTCG GGAGrkTCG 1 1599956 AATAGGCGC AATAAGCGC AATArGCGC AATArGCGC 1 1850032 CGAGCAATT CGAGTAATT CGAGyAATT CGAGyAATT 1 2119049 ACAACTCAA ACAATCCAA ACAAyyCAA ACAAyyCAA 1 2400243 ACTTGATGA ACTTCATGA ACTTsATGA ACTTsATGA 1 2612870 AATAAGAGT AATAGGAGT AATArGAGT AATArGAGT 1 2858975 GCCGCTCTT GCCGTTCTT GCCGyTCTT GCCGyTCTT 1 2804522 GAAGGGGAC GAAGACGAC GAAGrsGAC GAAGrsGAC 1 3047987 AAGGAGGGG AAGGGGGGG AAGGrGGGG AAGGrGGGG 1 3164166 ATAATCGGG ATAAGGGGG ATAAksGGG ATAAksGGG 1 3256057 TATCCGTTT TATCTGTTT TATCyGTTT TATCyGTTT 2 101820 ATTACGGAT ATTAAAGAT ATTAmrGAT ATTAmrGAT 2 350156 TCGGAGGTG TCGGGGGTG TCGGrGGTG TCGGrGGTG 2 600112 ATGTGTACG ATGTATACG ATGTrTACG ATGTrTACG 2 850338 TGGTTCTAA TGGTGCTAA TGGTkCTAA TGGTkCTAA 2 1099413 CCTGGCTCA CCTGACTCA CCTGrCTCA CCTGrCTCA 2 1349512 CTCAACAGT CTCAGCAGT CTCArCAGT CTCArCAGT 2 1600085 CACATTGCC CACAATGCC CACAwTGCC CACAwTGCC 2 1901773 ACTCAAATT ACTCGAATT ACTCrAATT ACTCrAATT 2 2150201 GTCGAAGGT GTCGTAGGT GTCGwAGGT GTCGwAGGT 2 2400281 TCAACACTC TCAAAACCC TCAAmACyC TCAAmACyC 2 2650136 ATAAATCCT ATAATTCCT ATAAwTCCT ATAAwTCCT 2 2903593 ACTATAGGA ACTAAAAGA ACTAwArGA ACTAwArGA 2 3048019 GTCCACTGC GTCCGCTGC GTCCrCTGC GTCCrCTGC 3 65650 GGCGGTTTT GGCGCTTTT GGCGsTTTT GGCGsTTTT 3 119281 TTTACACTC TTTATACTC TTTAyACTC TTTAyACTC 3 249570 GTATTATGT GTATTATGT GTATTATGT GTATTATGT 3 750000 GTCCGGCCA GTCCGGCCA GTCCGGCCA GTCCGGCCA 3 1250000 TTTTTCCGG TTTTTCCGG TTTTTCCGG TTTTTCCGG 3 1750000 ACGCCTGAC ACGCCTGAC ACGCCTGAC ACGCCTGAC 3 2250000 CGTGGCGAT CGTGGCGAT CGTGGCGAT CGTGGCGAT

3 2520748 TAATTCCAC TAATGCCAC TAATkCCAC TAATkCCAC 4 100004 GAGTAATGA GAGTGATAA GAGTrATrA GAGTrATrA 4 340893 AGGAGGTAC AGGTGGTAT AGGrGGTAy AGGrGGTAy 4 598147 GATCAACAG GATCGACAG GATCrACAG GATCrACAG 4 852119 CGAACACTC CGAATATTC CGAAyAyTC CGAAyAyTC 4 1100085 GATGACGAA GATGCCGAA GATGmCGAA GATGmCGAA 4 1350536 CGAAACCGG CGAACTCGG CGAAmyCGG CGAAmyCGG 4 1599885 GATAATTGC GATACTTGC GATAmTTGC GATAmTTGC 4 1850288 ATTCACGTA ATTCGTGTA ATTCryGTA ATTCryGTA 4 2100356 TCAGGGACC TCAGAGACC TCAGrGACC TCAGrGACC 4 2284257 TCTGAACTG TCTGGACTG TCTGrACTG TCTGrACTG 5 100211 TCCTCGAAT TCCTTGAAT TCCTyGAAT TCCTyGAAT 5 350872 GGCGCGCCC GGCGTGCCC GGCGyGCCC GGCGyGCCC 5 599922 CGTCGTTCA CGTCATTCA CGTCrTTCA CGTCrTTCA 5 851262 TAATCGTCT TAATTCTCT TAATysTCT TAATysTCT 5 1099776 ACATCGACA ACATTGACA ACATyGACA ACATyGACA 5 1352539 TTGTTGTCC TTGTGATCC TTGTkrTCC TTGTkrTCC 5 1599904 AACTCCCTT AACTTCCTT AACTyCCTT AACTyCCTT 5 1851458 AAATTCTCC AAATAATCC AAATwmTCC AAATwmTCC 5 2100025 CCCTCAGTC CCCTTAGTC CCCTyAGTC CCCTyAGTC 5 2278878 GGTCAAAAA GGTCGAAAA GGTCrAAAA GGTCrAAAA 6 106294 GCCACCTCA GCCATCTCG GCCAyCTCr GCCAyCTCr 6 350337 CATTCGGTT CATTTGGTT CATTyGGTT CATTyGGTT 6 600047 GGAGTATTT GGAGCATTT GGAGyATTT GGAGyATTT 6 849990 AGTTTAGGA AGTTCAGGA AGTTyAGGA AGTTyAGGA 6 1098535 CAAAAATTG CAAAGATTG CAAArATTG CAAArATTG 6 1349453 TGTCAATAG TGTCGGTAG TGTCrrTAG TGTCrrTAG 6 1600000 AAACCTGGA AAACCTGGA AAACCTGGA AAACCTGGA 6 1764645 AACCAGATT AACCGGATT AACCrGATT AACCrGATT 6 2000087 GATTCTGCG GATTTTGCG GATTyTGCG GATTyTGCG 6 2252662 GGGTCGGTA GGGTTGGTA GGGTyGGTA GGGTyGGTA 7 100284 GAAACTCAG GAAATTCAG GAAAyTCAG GAAAyTCAG 7 350044 ATATCCTTT ATATTCTTT ATATyCTTT ATATyCTTT 7 600111 CAATCATTA CAATTATTA CAATyATTA CAATyATTA 7 850516 TGACACATA TGACGCATA TGACrCATA TGACrCATA 7 1100248 TCACAGAAG TCACGGAAG TCACrGAAG TCACrGAAG 7 1350089 CTTTCCCCC CTTTTCCCC CTTTyCCCC CTTTyCCCC 7 1605047 ATACGTGAC ATACTTGGC ATACkTGrC ATACkTGrC 7 1850000 GAGATACT GAGATACT GAGATACT GAGATACT 7 1898793 TCCGTATGA TCCGCATAA TCCGyATrA TCCGyATrA 7 1991505 TCTAAAGTT TCTACGGTT TCTAmrGTT TCTAmrGTT 8 350000 ATTGACGCG ATTGACGCG ATTGACGCG ATTGACGCG 8 600000 CATTGACGG CATTGACGG CATTGACGG CATTGACGG 8 1100000 CATACGATC CATACGATC CATACGATC CATACGATC 8 1350000 AGCTTAACA AGCTTAACA AGCTTAACA AGCTTAACA 8 1600100 CTGAACCCT CTGAACCCT CTGAACCCT CTGAACCCT 9 100105 CTCAGCCGA CTCAACCGA CTCArCCGA CTCArCCGA 9 352455 AGTCTCCCA AGTCCTCCA AGTCyyCCA AGTCyyCCA

9 599950 TGGTGTCCC TGGTATCCC TGGTrTCCC TGGTrTCCC 9 1010845 GGGTAGTGA GGGTGGTGA GGGTrGTGA GGGTrGTGA 9 1244202 GATGGAGAT GATGAAGAT GATGrAGAT GATGrAGAT 9 1504476 TACTATACC TACTGTACC TACTrTACC TACTrTACC 9 1656962 TATCCACTG TATCTACTG TATCyACTG TATCyACTG 10 100438 AATTCATTT AATTAATTT AATTmATTT AATTmATTT 10 350030 GCGGTTCAA GCGGCTCAA GCGGYTCAA GCGGYTCAA 10 600032 TTACGCTGG TTACACTGG TTACrCTGG TTACrCTGG 10 850000 TCGGTCGGA TCGGTCGGA TCGGTCGGA TCGGTCGGA 10 860249 CCGCGAAATT CCGCAAATT CCGCrAAATT CCGCrAAATT 10 1109960 AGGAGATGA AGGAAATGA AGGArATGA AGGArATGA 10 1303902 TGATCTACT TGATTTACT TGATyTACT TGATyTACT 10 1490452 AATCTGATG AATCAGATG AATCwGATG AATCwGATG 11 100000 TATTCTTAG TATTCTTAG TATTCTTAG TATTCTTAG 11 350000 GTCAGCAAG GTCAGCAAG GTCAGCAAG GTCAGCAAG 11 600000 ATGGGCGCG ATGGGCGCG ATGGGCGCG ATGGGCGCG 11 850000 CTTCCCCAT CTTCCCCAT CTTCCCCAT CTTCCCCAT 11 1100000 TTACAGTTG TTACAGTTG TTACAGTTG TTACAGTTG 11 124000 AGCCAAGTA AGCCAAGTA AGCCAAGTA AGCCAAGTA 12 100000 CCTTCTAGT CCTTCTAGT CCTTCTAGT CCTTCTAGT 12 1000000 CGAGGAGGA CGAGGAGGA CGAGGAGGA CGAGGAGGA 13 100697 ACGTATTTA ACGTCTTTA ACGTmTTTA ACGTmTTTA 13 370521 TTTGTGTCA TTTGAGTCA TTTGwGTCA TTTGwGTCA 13 604345 CTTCCGCAT CTTCAGCAT CTTCmGCAT CTTCmGCAT 13 850249 GGTTGGTGA GGCTAGTAA GGyTrGTrA GGyTrGTrA 14 113109 AGGGGAATA AGGGAAATA AGGGrAATA AGGGrAATA 14 372086 CGATTCTT CGATCCCTT CGATyCyTT CGATyCyTT 14 725684 ATGAATTTG ATGAGTTCG ATGArTTyG ATGArTTyG 15 150013 GTGGACCGT GTGGCCCGT GTGGmCCGT GTGGmCCGT 15 449866 GAATCTCGG GAATTTCGG GAATyTCGG GAATyTCGG 16 208609 CACACGCAC CACATGCAC CACAyGCAC CACAyGCAC 16 400000 CCTCGGATT CCTCGGATT CCTCGGATT CCTCGGATT 17 120000 TATTCTTCA TATTCTTCA TATTCTTCA TATTCTTCA 17 338415 TGAGGAGCC TGAGAAGCC TGAGrAGCC TGAGrAGCC 17 449833 ATCAAACTA ATCAGACAA ATCArACwA ATCArACwA 18 101884 ATTATGGAC ATTACGGAC ATTAyGGAC ATTAyGGAC 19 98377 GCTACTGGG GCTATTGGG GCTACTGGG GCTACTGGG

[0141] Table I presents a 'fingerprint' excerpted from the SNP (Single Nucleotide

Polymorphism) marker genotype of the entire genome sequences of line J10102

s69, of line OWNC, of the F1 hybrid J11500 strain obtained from the mating of lines

J10102-s69 and OWNC, and of the J11500-ms2 EDV of strain J11500. The IUPAC

nucleotide and ambiguity codes are used to represent the observed 9-base DNA marker sequences reported above, each of which represents one or two allelic characters at a genotypic marker locus, with, for example, the code "y" indicating the presence of two alleles, one with a "t" and the other with a "c", at that position. The identity of each marker locus is uniquely and unambiguously specified by the scaffold and SNP position information derived from the H97 V2.0 archival reference genome sequence published by the U.S. Department of Energy Joint Genome

Institute (Morin et al. 2012).

[0142] That is, it will be appreciated that every nucleotide in the nuclear genome of

Agaricus bisporus has a unique and specific identity, specified by the scaffold

number and nucleotide position number of that nucleotide within the art-standard

reference sequence (Version 2.0) of A. bisporus line H97, as determined by and

placed into the public domain by The U.S. D.O.E. Joint Genome Institute and the

Agaricus Genome Consortium, as described in the publication by Morin et al.,

"Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms

governing adaptation to a humic-rich ecological niche." Proc. Nat'l Acad. Sci. USA

109: 17501-17506 (2012). As known in the art, any genetic marker or marker locus

in the A. bisporus genome may be identified by specifying the positional information

from the H97 reference sequence. For example, the first marker listed in Table I

occurs at 1:101993 (i.e., scaffold 1: position 101993). As a convenience only, Table

I also provides short sequences flanking the SNP marker nucleotide position; in the

first example at 1:101993, the provided sequence is GAAGnACAT, where "n"

represents the position of the variable nucleotide at 1:101993 which constitutes the

informative genetic marker. The amount of flanking sequence that is shown is

arbitrary and is provided only as an aid in confirming the correct 'look-up' of the

marker in the reference genome sequence.

[0143] It is evident that a composite relationship of the heteroallelic genotype of

strain J11500 exists with respect to the homoallelic genotypes of its two parental

lines, namely line J10102-s69 and line OWNC, and further that the two parental lines

are very distinct, genotypically. It is further evident that the heterokaryon genotype

of the example EDV J11500-ms2 matches that of its initial strain, J11500. It will be

appreciated that the use of J10102-s69 in conjunction with line OWNC to provide

strain J11500 is but one example of the F1 hybrid generation, it being noted that

J10102-s69 has been used in at least 71 matings with other lines of Agaricus

bisporus to produce other F1 hybrids.

[0144] Genotype data for six additional marker loci is provided in TABLE || and in

the following text. Marker loci and allelic characters are specified hereinbelow.

[0145]

TABLE II

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

Marker: ITS p1n150 MFPC-1-ELF AN AS FF

Line/strain

J10102-s69 14 2 El N5 SC FF1

OWNC 11 1T El N1 SD FF1

SWNC 12 2 E2 N2 SC FF2

J11500 11/14 1T/2 El/El N1/N5 SC/SD FF1/FF1

[0146] Line J10102-s69 and strain J11500 can be identified through their molecular

marker profiles, i.e., their genotypic fingerprints, as shown in Tables I and II. OWNC

and SWNC are two lines derived from two traditional white-capped cultivar stocks, as

described hereinabove. Each is genotypically distinct, as shown in Table 1l.

[0147] A brief description of the genotype of strain J11500, in the context of its

pedigree including progenitors J10102, line J10102-s69, OW heterokaryon Somycel

76, and line OWNC, and in comparison to other white strains, at a further six

unlinked marker loci is provided below. Because the J11500 heterokaryon

incorporates two sets of chromosomes, there are two allelic copies (two characters

or elements of the genotype) at each marker locus. The brief genotype excerpt

provided below therefore consists of either 6 or 12 characters or elements,

respectively, for lines or strains, as also presented in Table 1l. The brief genotype

was prepared by the assignee of record using targeted Polymerase Chain Reactions

to amplify genomic regions bracketing the markers, as unambiguously defined

below, from each of the culture DNAs. Any suitable PCR primers that bracket the

defined marker regions may be used for this purpose; methods of designing suitable

primers, for example from the H97 reference genome sequence, are well known in

the art. The amplified PCR product DNA was sequenced by a contractor, Eurofins,

using methods of their choice, and the genotypes were determined by direct

inspection of these sequences in comparison to Sylvan America's database of

reference marker/allele sequences.

Description of the p1n150-G3-2 marker:

[0148]The 5' end of this marker segment begins at position 1 with the first "[" in the

sequence TCCCAAGT, corresponding to H97 JGI V2.0 Scaffold 1 position 868615

(Morin et al. 2012) and extending in a reverse orientation (relative to the scaffold

orientation) for ca. 600 nt in most alleles; an insertion in the DNA of allele 1T has

produced a longer segment. At present, 9 alleles incorporating at least 30

polymorphic positions have been documented from diverse strains in Sylvan

America's breeding collection.

[0149] Alleles present in the J10102-s69 and J11500 pedigree over three

generations are alleles 1T, 2, 3, 4, and 9, characterized as follows (using the format:

nucleotide base character @ alignment position, based on alignment of alleles 2, 3,

and 4, and the alignable portions of allele 1T) :

[0150] Allele 1T: 'C' @ 193; insertion of Abr1 transposon of 320 nt @ 206A207; 'T'

@ 327;'C'@ 374; 'G'@ 378; 'G'@ 422; 'C'@ 431; 'G'@ 472; etc.

[0151] Allele 2: no Abr1 insertion; 'C'@ 193; 'C'@ 327, 'C'@ 374; 'C'@ 378; 'G'@

422; 'T'@ 431; 'G'@ 472; etc.

[0152] Allele 3: no Abr1 insertion; 'C' @ 193; 'T' @ 327, 'G' @ 374; 'C' @ 378; 'G' @

422; 'T'@ 431; 'A'@ 472; etc.

[0153] Allele 4: no Abr1 insertion; 'C'@ 193; 'T'@ 327, 'C'@ 374; 'C'@ 378; 'A'@

422; 'T'@ 431; 'G'@ 472; etc.

[0154] Allele 9: no Abr1 insertion; 'G' @ 193; 'C' @ 327, 'C' @ 374; 'C' @ 378; 'G'

@ 422; 'T'@ 431; 'G'@ 472; etc.

[0155] Because of linkage to the MAT locus, which is obligately heteroallelic in fertile

heterokaryons, genotypes of all known and expected heterokaryons at p1n150-G3-2

are also heteroallelic.

[0156] The J10102 heterokaryon has an'1T/2' heteroallelic genotype.

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

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

genotype.

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

genotype.

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

[0161] The genotype of the J11500 heterokaryon atthe p1n150-G3-2 marker'locus'

is '1T/2' (heteroallelic), designating the presence of alleles 1T and 2. Allele 1T was contributed by the OWNC line. Allele 2 was transmitted from the J10102 heterokaryon via the J10102-s69 homokaryon. The '1T/2' genotype distinguishes

J11500 from many other heterokaryons including from all of its own grandparents,

although not from the U1 strain family.

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

[0162] The ITS segment is part of the nuclear rDNA region, which is a cassette that

is tandemly repeated up to an estimated 100 times in the haploid genome of A.

bisporus. Therefore there is no single precise placement of this sequence in the

assembled H97 genome, and in fact it is difficult or impossible to precisely assemble

the sequence over all of the tandem repeats. Three cassette copies were included

on scaffold 10 of the H97 JGI V2.0 assembly, beginning at position 1612110; a

partial copy is also assembled into scaffold 29 (Morin et al. 2012). The 5' end of this

marker segment begins at position 1 with the first "G" in the sequence GGAAGGAT,

and extending in a forward orientation (relative to the scaffold orientation) for ca.

703-704 nt in most alleles. At present, more than 9 alleles incorporating at least 11

polymorphic positions have been documented from diverse strains in Sylvan's

breeding collection.

[0163] Alleles present in the J10102-s69 and J11500 immediate pedigree are alleles

11, 12, and 14, characterized as follows (using the format: nucleotide base character

@ alignment position, based on alignment of 9 alleles).

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

[0165] Allele 12: 'T' @ 52; 'T' @ 461; 'T' @ 522; 'T' @ 563; etc.

[0166] Allele 14: 'C' @ 52; 'A' @ 461; 'C' @ 522; 'C' @ 563; etc.

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

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

[0169] The genotype of the J11500 heterokaryon at the ITS marker 'locus' is '11/14'

(heteroallelic), designating the presence of alleles 11 and 14. Allele 11 was

contributed by the OWNC line. Allele 14 was transmitted from the J10102

heterokaryon via the J10102-s69 homokaryon. This distinguishes J11500 from the

U1 strain family, which has an '11/12' genotype.

Description of the MFPC-1-ELF marker:

[0170]The 5' end of this marker segment begins at position 1 with the first "G" in the

sequence GGGAGGGT, corresponding to H97 JGI V2.0 Scaffold 8 position 829770

(Morin et al. 2012) and extending in a forward orientation (relative to the scaffold

orientation) for ca. 860 nt in most alleles. At present, at least 7 alleles incorporating

at least 40 polymorphic positions have been documented from diverse strains in

Sylvan's breeding collection.

[0171] Alleles present in the J10102-s69 and J11500 immediate pedigree, are

alleles El, E2, and E8, characterized as follows (using the format: nucleotide base

character @ alignment position, based on alignment of 8 alleles).

[0172] Allele El: 'A' @ 77; 'A' @ 232; 'A' @ 309; 'T' @ 334; 'A' @ 390; 'A' @ 400;

'T' @ 446, 'A'@ 481; etc.

[0173] Allele E2: 'G' @ 77; 'A' @ 232; 'G' @ 309; 'T' @ 334; 'G' @ 390; 'G' @ 400;

'C' @ 446, 'G' @ 481; etc.

[0174] Allele E8: 'A' @ 77; 'G' @ 232; 'G' @ 309; 'A' @ 334; 'A' @ 390; 'A' @ 400;

'C' @ 446, 'G' @ 481; etc.

[0175] The J10102 heterokaryon has an 'E/E8' heteroallelic genotype.

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

[0177] The genotype of the J11500 heterokaryon at the MFPC-1-ELF marker'locus'

is 'E/E', designating the presence of two copies of alleles El. One copy of allele

El was contributed by the OWNC line; a second copy of allele El was transmitted

from the J10102 heterokaryon via the J10102-s69 homokaryon. This homoallelic

genotype distinguishes J11500 from the predominant Ul-type of commercial cultivar,

which has an 'E/E2' genotype.

Description of the AN marker:

[0178] The 5' end of this marker segment begins at position 1 with the first "G" in the

sequence GGGTTTGT, corresponding to H97 JGI V2.0 Scaffold 9 position 1701712

(Morin et al. 2012) and extending in a forward orientation (relative to the scaffold

orientation) for ca. 1660 nt (in the H97 genome) to 1700 nt (in the alignment space)

in known alleles; several insertions/deletions have created length polymorphisms

which, in addition to point mutations of individual nucleotides, characterize the

alleles. At present, 5 alleles incorporating more than 70 polymorphic positions have

been documented from diverse strains in Sylvan's breeding collection.

[0179] Alleles present in the J10102-s69 and J11500 immediate pedigree are alleles

N1, N2 and N5, characterized in part as follows (using the format: nucleotide base

character @ alignment position, based on alignment of alleles N1 through N5):

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

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

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

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

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

[0185] The genotype of the J11500 heterokaryon at the AN marker'locus' is 'N1/N5'

(heteroallelic), designating the presence of alleles N1 and N5. Allele N1 was

contributed by the OWNC line. Allele N5 was transmitted from the J10102

heterokaryon via the J10102-s69 homokaryon.

[0186] The 'N1/N5' genotype at the AN marker locus distinguishes J11500 from

commercial strains U1 and A-15, which have an 'N1/N2' genotype. This element of

the genotype fingerprint can also distinguish J11500 from among many other strains.

Description of the AS marker:

[0187] The 5' end of this marker segment begins at position 1 with the first "G" in the

sequence GG(T/N)GTGAT, corresponding to H97 JGI V2.0 Scaffold 4 position

752867 (Morin et al. 2012) and extending in a forward orientation (relative to the

scaffold orientation) for ca. 1620 nt (in the H97 genome) to 1693 nt (in the alignment

space) in known alleles; several insertions/deletions have created length

polymorphisms which, in addition to point mutations of individual nucleotides,

characterize the alleles. At present, 7 alleles incorporating more than 80

polymorphic positions have been documented from diverse strains in Sylvan's

breeding collection.

[0188] Alleles present in the J10102-s69 and J11500 immediate pedigree are alleles

SC and SD, characterized in part as follows (using the format: nucleotide base

character @ alignment position, based on alignment of alleles SA through SG) :

[0189] Allele SC: 'T' @ 28; 'GATATC' @ 258-263; 'G' @ 275;

[insertion]+'TTTCCAGC'+[insertion] @ 309-249; 'C'@ 404, etc.

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

'T' @ 404, etc.

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

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

[0193] The genotype of the J11500 heterokaryon at the AS marker 'locus' is 'SC/SD'

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

[0194] The 'SC/SD' genotype at the AS marker locus is also shared by commercial

strains U1 and A-15. While this element of the genotype fingerprint distinguished

J11500 from among many other strains, it does not distinguish J11500 from the U1

strain family.

Description of the FF marker:

[0195] The 5' end of this marker segment begins at position 1 with the first "T" in the

sequence TTCGGGTG, corresponding to H97 JGI V2.0 Scaffold 12 position 281999

(Morin et al. 2012) and extending in a forward orientation (relative to the scaffold

orientation) for ca. 570 nt in most alleles. At present, 7 alleles incorporating at least

20 polymorphic positions have been documented from diverse strains in Sylvan's

breeding collection.

[0196] Alleles present in the J10102-s69 and J11500 immediate pedigree are Alleles

FF1 and FF2, characterized as follows (using the format: nucleotide base character

@ alignment position, based on alignment of alleles 1 and 2):

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

[0198] Allele FF2: 'TTC'@ 48-50

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

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

[0201] The genotype of the J11500 heterokaryon at the FF marker 'locus' is

'FF1/FF1' (homoallelic), designating the presence of two copies of allele FF-1,

contributed by both the OWNC line and the J10102-s69 homokaryon. This

distinguishes J11500 from the predominant Ul-type of commercial cultivar, which has an 'FF1/FF2' genotype. This element of the genotype fingerprint can also distinguish J11500 from among many other strains.

[0202] By using the foregoing markers, or any combination of many other available

markers such as those in Table 1, the uniqueness of the genotypes of line J10102

s69 and strain J11500 is evident. Given that strain J11500 has 4 non-cultivar

progenitors and that considerable genetic diversity exists among strains, the

genotypic fingerprint of strain J11500 shows numerous differences with that of the

U1 lineage group. A unique fingerprint allows strain J11500 (and its Essentially

Derived Varieties and descendents) to be unambiguously identified. Agronomically,

genetic diversity among cultivated strains is a desirable objective because it is well

established that genetic monocultures among agricultural crop species can lead to

disastrous failures due to particular disease, pest, or environmental pressures. Any

otherwise desirable commercial strain with genetic novelty is therefore valuable.

Vegetative incompatibility between genetically distinct cultivated strains is also

economically valuable in addressing virus control and farm hygiene. Strain J11500

meets those criteria.

[0203] A culture or product incorporating the genetic marker profile shown in the

respective column of Table I or row of Table || labeled J10102-s69 or J11500 is an

embodiment of the invention. Another embodiment of this invention is an Agaricus

bisporus line or strain or its parts comprising at least 75% of the same alleles as the

line J10102-s69 or the strain J11500 for the loci listed in the respective column of

Table I and/or row of Table 1l. In other embodiments, this line or strain or its parts

comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or essentially 100%

of the same alleles as the line J10102-s69 or the strain J11500 for the loci listed in

the respective column of Table I and/or row of Table 1l.

[0204] A cell having at least 75% of the same alleles as a cell of line J10102-s69 or

a cell of strain J11500 for the loci listed in the respective column of Table I and/or

row of Table II is also an embodiment of this invention. In other embodiments, cells

having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or essentially 100% of

the same alleles as a cell of line J10102-s69 or a cell of strain J11500 for the loci

listed in the respective column of Table 1, and/or row of Table 1l, are provided. Also

encompassed within the scope of the invention are cultures substantially benefiting

from the use of line J10102-s69 or strain J11500 in their development, such as

hybrid offspring having line J10102-s69 or a line obtained from strain J11500 as a

parent, and line derived from J10102-s69 having a trait introduced through

introgressive matings of offspring back to line J10102-s69, or through transformation.

Similarly, an embodiment of this invention is an Agaricus bisporus heterokaryon

comprising at least one allele per locus that is the same allele as is present in the

J10102-s69 line for at least 75% of the marker loci listed in Tables I and II. In other

embodiments, heterokaryons comprising at least one allele per locus that is the

same allele as is present in the J10102-s69 line for at least 80%, 85%, 90%, 95%,

96%, 97%, 98%, 99% or essentially 100% of the marker loci listed in Tables I and II,

are provided. More particularly, the heterokaryon may be a hybrid descendent of

line J10102-s69. Another embodiment of this invention is a culture of a strain having

a genotype which is a complete or partial subset of the genotype of strain J11500.

[0205] Hybrid strain J11500 is the product of 6 generations of controlled line matings

by Sylvan America, Inc. The original mating was made between line JB 137-s8 and

line SWNC. In the sixth generation, line J10102-s69, a descendent of the first hybrid

(and of other hybrids produced by Sylvan, Inc.), was mated with line OWNC to

produce the novel hybrid strain J11500.

[0206] Cultures of strain J11500 produce commercially acceptable and desirable

crops of white mushrooms. Table Ill presents yield data as pounds per square foot,

in three independent crop tests with internal replication. As shown in Table Ill,

productivity of J11500 is comparable to and often greater than the productivity of the

A15 strain, with total (3-flush) yield averaging 101.3% of the A-15 control and

ranging as high as 106.8% under standard growing conditions for A-15. Distribution

of the crop over the three-flush harvest period is relatively accelerated, meaning that

more of the crop is picked during first flush, when disease pressure and incidence

are lowest and product quality may be correspondingly higher. In a general t-test on

this small data set, first break yield differences between J11500 and A-15

approached statistical significance (p = 0.057).

[0207]

TABLE Ill

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

J11500 A-15 J11500 A-15 J11500 A-15

12-108 2.87 2.27 4.50 4.04 5.02 4.70

12-119 2.47 2.15 3.73 3.81 4.34 4.61

12-146 2.57 2.39 3.92 3.71 4.60 4.47

Averages 2.63 2.27 4.05 3.85 4.65

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

[0208] Within the first flush, yield is also accelerated. Over the four productive days

of the first flush, the cumulative daily yield data in Table IV, reporting averages of the same three tests, shows that the harvest of strain J11500 is accelerated over that of the A-15 control.

[0209]

TABLE IV

Day (after casing): 14 15 16 17

Cumulative daily yield:

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

[0210] Timing to harvest is about equivalent to that of commercial strain A15 (both

about 13 to 19 days), and sometimes may be slightly faster, which can be

economically advantageous. Table V shows that in the same crop tests, on average,

strain J11500 began to produce its crop 0.43 days before A-15, and the peak of

production in the first flush was 0.24 days earlierfor strain J11500.

[0211]

TABLE V

Test ID Days to first pick Peak first flush pick day

J11500 A-15 J11500 A-15

12-108 14.0 15.3 14.7 15.3

12-119 14.0 14.0 14.0 14.3

12-146 14.0 14.0 15.0 14.8

Averages 14.0 14.43 14.56 14.8

Days gained +0.43 +0.24

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

to be desirable commercial mushroom traits. J11500 typically produces mushrooms

with caps having thicker flesh, and which are subjectively rounder, than those of

A15; objectively, the following physical measurement ratios demonstrate the shape

differences of J11500 compared to A15.

[0213] Cap roundness, expressed as cap height / cap diameter (CH/CD) is an

economically important trait reflecting a consumer preference for rounder

mushrooms. Measurements were made on samples of 10 first break mushrooms of

equivalent maturity from both J11500 and the commercial control A-15. J11500 was

rounder (0.68) compared to the control A-15 (0.60), and this difference was

significant (t-test, p = 9.15E-07).

[0214] Similarly, cap 'meatiness', expressed as flesh thickness / cap diameter

(FT/CD) is an economically important trait reflecting a consumer preference for

thicker-fleshed mushrooms. Measurements were made on samples of 10 first break

mushrooms of equivalent maturity from both J11500 and the commercial control A

15. J11500 was meatier (0.36) compared to the control A-15 (0.33), and this

difference was significant (t-test, p = 0.0054).

[0215] Cross-strain incompatibility can also be a useful commercial mushroom trait.

J11500 is incompatible with A-15, a proxy for the U1 derived lineage group. When

casing material incorporating inoculum of J11500 is placed over compost colonized

with A-15, or conversely when A-15 is placed over J11500, i.e., in non-self pairings,

a partial crop failure ensues, demonstrating incompatibility as shown by the yield

data in Table VI:

TABLE VI

Spawn strain Casing strain Identity First flush yield

J11500 J11500 Self 2.47 lbs.

A-15 A-15 Self 2.03 lbs.

J11500 A-15 Non-self 0.50 lbs.

A-15 J11500 Non-self 0.17 lbs.

[0216] A heterokaryotic selfed offspring of an F1 hybrid that itself has a 'p/q'

genotype will in the example have a genotype of 'p/p', 'q/q', or 'p/q'. Two types of

selfing lead to differing expectations about representation of alleles of line J10102

s69 and of the F1 hybrid in the next heterokaryotic generation. When two randomly

obtained haploid offspring from the same F1 hybrid, derived from individual spores of

different meiotic tetrads, are mated (i.e., in inter-tetrad selfing), representation of the

line J10102-s69 marker profile in each recombined haploid parental line and in each

sib-mated heterokaryon will be 50% on average, and slightly more than 75% (to

about 85%) of heteroallelism present in the F1 hybrid will on average be retained in

the sib-mated heterokaryon (the expectation over 75% is due to the mating

requirement for heteroallelism at the mating type locus (MAT) on Chromosome 1).

Distinctively, in addition, Agaricus bisporus regularly undergoes a second,

characteristic, spontaneous intra-tetrad form of selfing called intramixis, producing

heterokaryotic postmeiotic spores carrying two different recombined haploid nuclei

having complementary, heteroallelic MAT alleles. An offspring developing from any

one of these spores is a postmeiotic self-mated heterokaryon with ca. 100%

retention of the heteroallelism present in the single F1 parent around all 13 pairs of

centromeres, due to the association of non-sister (first division) postmeiotic nuclei

enforced by the requirement for sexual complementation at the centromerically

linked MAT locus. In theory this value decreases to an average of 66.7% retention

of F1 heteroallelism for distal markers unlinked to their centromeres; however

empirical observations suggest higher rates of retention above 90% and approaching

99%-100% even for such distal markers. Transmission of the line J10102-s69

marker profile in such selfed offspring may be incomplete by a small percentage

(typically 0-10%) due to the effects of infrequent odd-numbered meiotic crossovers, while representing 50% on average of the resulting heterokaryotic genome. Both types of selfed offspring are considered to be Essentially Derived Varieties (EDVs) of the initial F1 hybrid, and the latter type comprises most (often 95-100%) of the genotype of the F1, and may express a very similar phenotype to that of the F1 hybrid.

[0217] The heterokaryon, or vegetative, incompatibility of J11500 with A-15, a

phenotype evidently determined by the genotype, is transmitted into heterokaryotic

spores along with most or all of the parental genotype, and thus is inherited by EDVs

derived from spores, as shown by the yield data in TABLE VII. A deposit of a culture

of an example of an EDV, namely strain J11500-ms2, obtained from hybrid strain

J11500, as disclosed herein, has also been made with the Agricultural Research

Services Culture Collection (NRRL) 1815 North University Street, Peoria, Illinois

61604 USA. The date of deposit was January 15, 2014. The culture deposited was

taken from the same culture maintained by Sylvan America, Inc., Kittanning, Pa., the

assignee of record, since prior to the filing date of this application. All restrictions

upon the deposit have been removed, and the deposit is intended to meet all deposit

requirements of the U.S. Patent and Trademark Office, including 37 C.F.R. Sec.

1.801-1.809, and all deposit requirements under the Budapest Treaty. The NRRL

Accession No. is 50896. The deposit will be maintained in the depository for a

period of 30 years, or 5 years after the last request, or for the effective life of the

patent, whichever is longer, and will be replaced as necessary during that period.

The culture will be irrevocably and without restriction or condition released to the

public upon filing of a priority application or upon the issuance of a patent according

to the patent laws.

[0218]

TABLE VII

Spawn strain Casinq strain Identity First flush yield

A-15 A-15 Self 1.74 lbs.

A-15 J11500-ms2 Non-self 0.58 lbs.

A-15 J11500-ms3 Non-self 0.63 lbs.

A-15 J11500-ms4 Non-self 0.58 lbs.

A-15 J11500-ms5 Non-self 0.44 lbs.

A-15 J11500-mslO Non-self 0.53 lbs.

[0219] A test of compatibility of an EDV of strain J11500 (designated J11500-ms2)

with the strain J11500 itself was performed and the results are shown in TABLE VIl.

[0220]

TABLE VIII

Spawn strain Casing strain Identity First flush

J11500 J11500 Self 1.95 lbs.

J11500 J11500-ms2 Self: EDV 2.69 lbs.

J11500 J11500-ms2 Self: EDV 3.13 lbs.

[0221] Table VIII shows that in test 13-177, the EDV strain designated J11500-ms2

was completely compatible with the initial strain J11500, and in fact demonstrated

higher first break yield than strain J11500 as opposed to a partial crop failure that

would have indicated incompatibility.

[0222] One use of the culture of strain J11500 is the production of crops of edible

mushrooms for sale. Another use is for the improvement of facility hygiene via strain

rotation and a 'virus-breaking' effect. A third use is to incorporate the genetic

material of strain J11500 into offspring and derived or descended cultures including

dormant and germinating spores and protoplasts. Additional uses also exist as

noted above.

[0223] Hybridization of Agaricus bisporus cultures of the invention may be

accomplished by allowing two different cultures, one of which is a genetic line

present in a spore of J11500, to grow together in close proximity, preferably on

sterile media, until anastomosis (i.e., hyphal or cell fusion) occurs. In a successful

mating, the resultant fusion culture is a first-generation outbred hybrid culture

incorporating a genetic line present in a mushroom spore which is one part of one

embodiment of the present invention. Protoplasts derived from basidia or other parts

of the organism are another part of the J11500 mushroom that may be used to

transmit genetic material of J11500 into new cultures.

[0224] Methods for obtaining, manipulating, and mating cultures of the present

invention, for producing offspring, inoculum, products, and crops of the current

invention, for using a strain rotation program to improve mushroom farm hygiene,

and for obtaining the genotypic fingerprint of mushroom cultures, are described

hereinabove and are also well known to practitioners of the art.

[0225] In order to demonstrate practice of the present invention at it relates to line

10102-s69, the line J10102-s69 was compared to other lines. J10102-s69 is a line

selected from among haploid progeny of a 5th generation in a hybrid pedigree

initiated by Sylvan America, Inc. in 1993. This line, within a suitable heterokaryotic

genetic background, recessively confers a white cap color trait upon heterokaryotic

offspring; cap color is determined primarily by recessive alleles at the Ppc-1 locus on

Chromosome 8. Line J10102-s69 has the Mat-2 mating type genotype and

behavioral phenotype. It also contributes to and supports several commercially

desirable traits in hybrid offspring, including crop timing and productivity, and

mushroom size, appearance and general retail appeal. Because line J10102-s69 is

a haploid line, it is incapable of producing a crop of mushrooms, and consequently

no "J10102-s69 mushroom" is obtainable and no direct characterization of a crop or

product phenotype is possible. Therefore most selection criteria applied to haploid

lines including line J10102-s69 are determined empirically by evaluating a series of

matings which share a common parent such as line J10102-s69. In effect, this

'combining ability', i.e., the ability to mate successfully and produce a high proportion

of interesting and useful novel hybrids in strain development programs, is applied

using qualitative, quantitative, objective and subjective criteria. Line J10102-s69 is

among the top-ranked haploid lines discovered from among its cohort of sibling lines.

No previous hybrid, prior to creation of hybrids using line J10102-s69, had the

particular combination of desirable traits (including specific details of its rounder cap,

thicker flesh, and accelerated cropping, plus a particular novel incompatibility

phenotype) seen among hybrids incorporating line J10102-s69, as described herein.

No previous line has ever been observed to produce the combinations of desirable

traits observed among hybrids incorporating line J10102-s69.

[0226] In light of the foregoing, a single mushroom hybrid results from the mating of

two haploid, homoallelic lines, each of which has a genotype that complements the

genotype of the other. The hybrid descendant of the first generation is designated

Fl. F1 hybrids may be useful as new commercial varieties for mushroom

production, or as starting material for the production of inbred offspring and/or EDVs,

or as parents of the next generation of haploid lines for producing subsequent hybrid

strains. Line J10102-s69 may be used to produce hybrid mushroom cultures. One such embodiment is the method of mating homokaryotic line J10102-s69 with another homokaryotic mushroom line, to produce a first generation F1 hybrid culture.

The first generation culture, part, mushroom, and mushroom part produced by this

method is an embodiment of the invention. The first generation F1 culture will

comprise a complete set of the alleles of the homokaryotic line J10102-s69. The

strain developer can use either strain development records or molecular methods to

identify a particular F1 hybrid culture produced using line J10102-s69. Further, the

strain developer may also produce F1 hybrids using lines which are transgenic or

introgressive trait conversions ('narrow modifications') of line J10102-s69. Another

embodiment is the method of mating line J10102-s69, or a narrowly modified version

of that line, with a different, heterokaryotic culture of Agaricus bisporus. This latter

method is less efficient than mating using two homokaryotic lines, but can also result

in the production of novel hybrid cultures.

[0227] The development of a mushroom hybrid in a typical mushroom strain

development program involves many or all of the following steps: (1) the obtaining of

strains or stocks from various germplasm pools of the mushroom species for initial

matings; (2) matings between pairs of pure cultures on sterile microbiological growth

media such as potato dextrose agar (PDA); (3) the obtaining and use of promising

hybrid strains from matings to produce subsequent generations of homokaryotic

progeny lines, such as line J10102-s69, which are individually uniform; (4) the use of

those lines in matings with other lines or strains to produce a subsequent hybrid

generation; (5) repetition of steps (2-4) as needed; (6) obtaining of pre-commercial

hybrid strains and the use of essential derivation techniques such as selfing to

produce a final commercial strain. In one embodiment, the repetition of steps (2-4)

may be performed up to 5 times. In various other embodiments, steps (2) to (4) may

be repeated anywhere from 0 up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. The homokaryotic lines are not reproductively competent ('fertile'). Fertility, the ability to produce a crop of mushrooms, is restored in complementary matings with other haploid, or less commonly, heterokaryotic strains. An important consequence of the homoallelism and homogeneity of the homokaryotic line is that the hybrid between a defined pair of homokaryotic lines may be recreated indefinitely as long as the homokaryotic lines are preserved and/or propagated. In a mating attempt between a homokaryotic line and a heterokaryon, in the absence of somatic recombination, either or both of only two possible defined novel heterokaryotic genotypes may be obtained, each of which will comprise line J10102-s69.

[0228] Using line J10102-s69, specific application with repetition of the steps

described above can produce any pedigree structure from any arrangement of

stocks, lines and hybrids within that structure. A hybrid of the F1, F2, F3, F4, F5, F6,

F7, F8, F9, F10 or any subsequent hybrid generation can be produced from line

J10102-s69 using steps 1-6 described above.

[0229] In order to demonstrate practice of the invention as it relates to F1 hybrid

strain J11500, a subculture of strain J11500 was propagated as described above to

produce spawn and casing inocula, which were used to produce crops of white

mushrooms under standard commercial cultivation practices as described herein

above (see Background of Invention section). Commercial culture inocula including

mushroom 'spawn' and 'casing inoculum' were prepared using commercial large

scale microbiological production methods, namely, by aseptically introducing

inoculum of a pure culture of strain J11500 into from one to about 2,000 liters of

sterilized growth media under sterile conditions, and were disbursed into sterile

packaging for test purposes. The mushroom spawn was mixed with pasteurized

compost and incubated for 13 to 18 days. A non-nutritive peat-based casing layer

was placed over the compost as previously described and a casing inoculum was incorporated into the casing layer. Under controlled environmental conditions, the first mushrooms reached the correct stage of development in a further 14 days. The mushrooms were picked over a 3 to 4 day period. Three flushes of mushrooms were harvested before each test was concluded.

[0230]The mushrooms produced by strain J11500 have a white pileus color. As the

Royal Horticultural Society (RHS) color charts do not provide a reference standard

for the color "white", direct measurements of color of the strain J11500 mushroom

cap have been made using a Minolta Chromameter and the L-a-b color space

system. One measurement was made on the caps of each of ten first break

mushrooms grown in a testing facility. The mean values, plus or minus the standard

error, for the measured L, a, and b color components were as follows: L = 89.58±

0.11; a = -1.21 ±0.015; b = 8.12 ±0.088. Colors within or substantially coinciding

with color space described by these three parameter distributions are called "white"

according to standard and accepted practices of the commercial mushroom industry.

[0231] Strain J10102 is a heterokaryotic strain obtained in Sylvan America, Inc.'s

strain development program. It did not have the combination of characters needed

to be successful commercially; however its performance and physical characteristics

approached those criteria, and the strain was assessed as having some unknown

potential for further development and improvement. Consequently, J10102 was

used as a parent in 165 matings to several diverse lines of A. bisporus that, it was

believed, might have had some useful potential in mating combinations. Individual

outcomes were unpredictable and variable; it was hoped that the experiment might

produce a successful result but the overall likelihood of that was considered to be

low. Of the 165 novel hybrids obtained, only two were of potential commercial

interest, and only one, J11500, consistently met the target criteria for a successful commercial strain. It was later determined in the course of testing that strain J11500 had other beneficial attributes as well.

[0232] Essentially Derived Varieties of strain J11500 were obtained from single

spores, multiple spore mixtures, and from tissue and somatic selections, as

described hereinabove. Spores of strain J11500 were obtained and were

germinated and used to produce heterokaryotic and homokaryotic offspring, and

outbred descendants as described hereinabove. Homokaryotic offspring lines were

used to make matings to other lines, and further hybrids were obtained from these

matings. Spawn and casing inoculum of J11500 and A-15 were used in self/self and

self/non-self combinations in test crops to confirm the incompatibility of the two

strains, a prerequisite for use in virus-breaking strategies, all as described

hereinabove.

[0233] Although the invention has been described in terms of particular

embodiments in this application, one of ordinary skill in the art, in light of the

teachings herein, can generate additional embodiments and modifications without

departing from the spirit of, or exceeding the scope of, the claimed invention.

Accordingly, it is understood that the descriptions herein are proffered only to

facilitate comprehension of the invention and should not be construed to limit the

scope thereof.

[0234]Throughout the specification, unless the context requires otherwise, the word

"comprise" or variations such as "comprises" or "comprising", will be understood

to imply the inclusion of a stated integer or group of integers but not the exclusion

of any other integer or group of integers.

[0235]Each document, reference, patent application or patent cited in this text is

expressly incorporated herein in their entirely by reference, which means that it

should be read and considered by the reader as part of this text. That the document, reference, patent application, or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0236]Reference to cited material or information contained in the text should not be

understood as a concession that the material or information was part of the

common general knowledge or was known in Australia or any other country.

Claims (25)

CLAIMS: What is claimed is:

1. An Agaricus bisporus culture designated as Agaricus bisporus line J10102-s69, a

representative culture of the line having been deposited under NRRL Accession No. 50893.

2. An Agaricus bisporus culture comprising at least one set of chromosomes of an Agaricus

bisporus line J10102-s69, the culture of the line J10102-s69 having been deposited under the

NRRL Accession Number 50893, wherein said chromosomes comprise all of the alleles of the

line J10102-s69 at the sequence-characterized marker loci listed in Table 1l.

3. The Agaricus bisporus culture of claim 2, wherein said culture is an F1 hybrid Agaricus

bisporus mushroom culture produced by mating a culture of the line J10102-s69 with a different

Agaricus bisporus culture.

4. An F1 hybrid Agaricus bisporus culture designated as strain J11500, a representative

culture of the strain having been deposited under NRRL Accession No. 50895.

5. A culture that is an Essentially Derived Variety of an initial culture, wherein the initial

culture is a culture of any of the preceding claims.

6. A culture according to any of claims 3 to 5, wherein the culture exhibits heterokaryon

incompatibility toward heterokaryon strains in the U1 derived lineage group.

7. A mushroom culture of Agaricus bisporus having a genotypic fingerprint which has

characters at each of the marker loci in Table 1l, wherein all of the characters of said fingerprint

are also present in the genotypic fingerprint of either line J10102-s69, representative culture of

the line having been deposited under NRRL Accession No. 50893, or strain J11500, a

representative culture of the strain having been deposited under NRRL Accession No. 50895 at

the same marker loci and wherein the mushroom culture has the essential physiological and

morphological characteristics of line J10102-s69 or strain J11500.

8. A mushroom culture of Agaricus bisporus having at least one set of chromosomes

comprising the chromosomes of line J10102-s69 or strain J11500, wherein the at least one set of

chromosomes has characters at each of the marker loci in Table 1l, wherein all of the characters

of said fingerprint are also present in the genotypic fingerprint of either line J10102-s69,

representative culture of the line having been deposited under NRRL Accession No. 50893, or

strain J11500, a representative culture of the strain having been deposited under NRRL

Accession No. 50895 at the same marker loci.

9. A method of producing a hybrid mushroom culture of Agaricus bisporus, comprising:

mating a first parental Agaricus bisporus mushroom culture with a second parental

Agaricus bisporus mushroom culture, wherein at least one of the first and second parental

Agaricus bisporus mushroom cultures is a culture having the essential physiological and

morphological characteristics of line J10102-s69, wherein the culture of said line J10102-s69 was

deposited under the NRRL Accession Number 50893.

10. The method according to claim 9, further comprising: providing the mushroom culture in

mushroom products selected from the group consisting of mycelium, spawn, inoculum, casing

inoculum, fresh mushrooms, processed mushrooms, parts of mushrooms, mushroom extracts

and fractions, mushroom pieces, and colonized substrates including grain, compost, and friable

particulate matter.

11. The method according to claim 9, further comprising: providing the mushroom culture in

derived or descended cultures selected from the group consisting of homokaryons,

heterokaryons, aneuploids, somatic subcultures, tissue explants cultures, protoplasts, dormant

spores, germinating spores, inbred descendents and outbred descendents, transgenic cultures,

and cultures having a genome incorporating a single locus conversion.

12. A cell of the culture of any of claims 1 to 8 or produced by the method of claims 9 to 11.

13. The cell according to claim 12, further comprising a marker profile having characters at at

least two marker loci selected from the markers provided in Tables I and II, wherein all of the

characters of said marker profile are also present in the marker profile of either line J10102-s69,

representative culture of the line having been deposited under NRRL Accession No. 50893, or

strain J11500, a representative culture of the strain having been deposited under NRRL

Accession No. 50895.

14. A spore comprising the cell of claims 12 or 13.

15. The culture of claim 4 or produced by the method of claim 11, further defined as having a

genome comprising a single locus trait conversion.

16. The culture of claim 15, wherein the locus confers a trait selected from the group

consisting of mushroom size, mushroom shape, mushroom cap roundness, mushroom flesh

thickness, mushroom color, mushroom surface texture, mushroom cap smoothness, tissue density, tissue firmness, delayed maturation, basidial spore number greater than two, sporelessness, increased dry matter content,_increased shelf life, reduced bruising, increased yield, altered distribution of yield over time, decreased spawn to pick interval, resistance to infection by symptoms of or transmission of bacterial, viral or fungal disease or diseases, insect resistance, nematode resistance, ease of crop management, suitability of crop from mechanical harvesting, desired behavioral response to environmental conditions, to stressors, to nutrient substrate composition, to seasonal influences, and to farming practices.

17. A process for introducing a desired trait into a culture of Agaricus bisporus line J10102

s69 comprising the steps of:

(1) mating the culture ofAgaricusbisporus line J10102-s69 to a second culture ofAgaricus

bisporus having the desired trait, to produce a hybrid;

(2) obtaining an offspring that carries at least one gene that determines the desired trait

from the hybrid;

(3) mating said offspring of the hybrid with the culture of Agaricus bisporus line J10102

s69 to produce a new hybrid;

(4) repeating steps (2) and (3) at least once to produce a subsequent hybrid;

(5) obtaining a homokaryotic line carrying at least one gene that determines the desired

trait and comprising at least 75% of the alleles of line J10102-s69, at sequence-characterized

marker loci selected from the markers loci described in Tables I andII, from the subsequent hybrid

of step (4).

18. A method of producing a mushroom culture comprising the steps of:

(a) growing a first hybrid culture produced by mating a line of the culture of any of claims

1 to 8 and 15 to 16 or the culture produced by methods of any of claims 9 to 11, with a first different

Agaricus bisporus culture;

(b) mating a first homokaryotic progeny line of the first hybrid culture with the first or a

second different Agaricus bisporus culture to produce a second hybrid culture of a subsequent

descendant generation;

(c) optionally, growing the second hybrid culture of the subsequent descendant

generation and mating a second homokaryotic progeny line of the second hybrid culture of the

subsequent descendant generation with the first or the second or a third different Agaricus

bisporus culture; and

(d) repeating steps (b) and (c) for an additional 0-5 generations to produce a mushroom

culture.

19. A method for developing a second culture in a mushroom strain development program

comprising:

applying mushroom strain development techniques to a first mushroom culture, or parts

thereof, wherein said first mushroom culture is a culture of any of claims 1 to 8 or 15 to 16 or a

culture produced by the methods of any of claims 9 to 11 or 18.

20. The method for developing a mushroom culture in a mushroom strain development program

of claim 19 wherein mushroom strain development techniques are selected from the group

consisting of inbreeding, back-mating, outbreeding, selfing, introgressive trait conversions,

essential derivation, pedigree-assisted breeding, marker assisted selection, and transformation.

21. A method of mushroom strain development comprising the steps of:

(a) obtaining a molecular marker profile of Agaricusbisporus mushroom line J10102-s69,

a culture of said line having been deposited under the NRRL Accession Number 50893;

(b) obtaining an F1 hybrid culture for which the line culture of claim 1 is a parent;

(c) mating a culture obtained from the F1 hybrid culture with a different mushroom culture

and;

(d) selecting progeny that possess characteristics of said molecular marker profile of line

J10102-s69 .

22. A part of the culture of any of claims of claims 1 to 8 or 15 to 16 or the culture produced

by the methods of any of claims 9 to 11 or 18 to 21, selected from the group consisting of hyphae,

cells, nuclei, , and spores including dormant spores and germinated spores having heterokaryons

and homokaryons incorporated therein.

23. A product incorporating the culture of any of claims 1 to 8 to 15 to 16 or the culture

produced by the methods of any of claims 9 to 11 or 18 to 21, the product selected from the group

consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed

mushrooms, mushroom extracts and fractions, mushroom pieces, and colonized substrates

including grain, compost, and friable particulate matter.

24. A mushroom produced by growing a crop of mushrooms from the culture of any of claims

3 to 8 or 15 to 16 or the culture produced by methods of any of claims 9 to 11 or 18 to 21.

25. Use of the culture of any of claims 3 to 8 or 15 to 16 or the culture produced by methods

of any of claims 9 to 11 or 18 to 21, in crop rotation to reduce pathogen pressure and pathogen

reservoirs in mushroom growing facilities, to produce offspring, or to produce mushrooms.