Mushroom line J10102-s69 and methods and uses therefor

Abstract

A culture comprising at least one set of the chromosomes of the 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 I. Methods are provided for introducing a desired trait into a culture of Agaricus bisporus line J10102-s69, as well as methods and processes for producing hybrid mushroom cultures. A method of mushroom strain development is further provided.

Claims

1. A 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 I.

2. The culture of claim 1, 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.

3. A part of the culture of claim 1 selected from the group consisting of hyphae, mushrooms, spores, cells, and protoplasts.

4. A part of the F1 hybrid mushroom culture of claim 2 selected from the group consisting of hyphae, spores, cells, and protoplasts.

5. A product incorporating the culture of claim 1, the product selected from the group consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed mushrooms, mushroom pieces, and colonized substrates including grain, compost, and friable particulate matter.

6. A product incorporating the F1 hybrid mushroom culture of Agaricus bisporus of claim 2, the product selected from the group consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed mushrooms, mushroom pieces, colonized substrates, grain, compost, and friable particulate matter.

7. A mushroom produced by growing a crop of mushrooms from the culture of claim 1.

8. A mushroom produced by growing a crop of mushrooms from the F1 hybrid Agaricus bisporus mushroom culture of claim 2.

9. An Essentially Derived Variety of the culture of claim 1, wherein the Essentially Derived Variety is a culture derived from a single initial culture of line J10102-s69, wherein a culture of the line has been deposited under NRRL Accession No. 50893, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line J10102-s69.

10. An Essentially Derived Variety of the F1 hybrid Agaricus bisporus mushroom culture of claim 2, wherein the Essentially Derived Variety is a culture derived from a single initial culture of the F1 hydrid Agaricus bisporus mushroom such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture.

11. A process for introducing a desired trait into a culture of Agaricus bisporus line J10102-s69, a culture of the line J10102-s69 having been deposited under NRRL Accession Number 50893, comprising the steps of: (1) mating the culture of Agaricus bisporus 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 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 described in Table I, from the subsequent hybrid of step (4).

12. A process of producing a hybrid mushroom culture, comprising: mating a first mushroom culture with a second mushroom culture, wherein at least one of the first and second mushroom cultures is an Agaricus bisporus culture having all of the physiological and morphological characteristics of line J10102-s69, wherein the culture of said line J10102-s69 was deposited under the NRRL Accession Number 50893.

13. A hybrid culture produced by the process of claim 12.

14. A part of the hybrid culture of claim 12, selected from the group consisting of hyphae, spores, cells, and protoplasts.

15. A hybrid mushroom produced by growing a crop of mushrooms from said hybrid culture of claim 12.

16. A product incorporating the hybrid culture of claim 12, the product selected from the group consisting of mycelium, spawn, inoculum, casing inoculum, fresh mushrooms, processed mushrooms, mushroom pieces, colonized substrates, grain, compost, and friable particulate matter.

17. An Agaricus bisporus culture having all of the physiological and morphological characteristics of line J10102-s69, wherein the culture of said line J10102-s69 was deposited under the NRRL Accession Number 50893.

18. The Agaricus bisporus culture of claim 17, further comprising a marker profile in accordance with the marker profile of line J10102-s69 shown in Table I.

19. A cell of the Agaricus bisporus culture of claim 17.

20. The cell of claim 19, further comprising a marker profile in accordance with the profile of line J10102-s69 shown in Table I.

21. A spore comprising the cell of claim 19.

22. The Agaricus bisporus culture of claim 9 further defined as having a genome comprising a single locus trait conversion.

23. The Agaricus bisporus culture of claim 22, 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.

24. A method of producing a mushroom culture comprising the steps of: (a) growing a progeny culture produced by mating the culture of claim 17 with a second Agaricus bisporus culture; (b) mating the progeny culture with itself or a different culture to produce a progeny culture of a subsequent generation; (c) growing a progeny culture of a subsequent generation and mating the progeny culture of a subsequent generation with itself or a different culture; and (d) repeating steps (b) and (c) for an additional 0-5 generations to produce a mushroom culture.

25. A method for developing a second culture in any generation in a mushroom strain development program comprising: applying any mushroom strain development technique that results in the development of a second culture, to a first mushroom culture, or parts thereof, wherein said first mushroom culture is selected from the group consisting of (1) a culture comprising at least one set of chromosomes of an Agaricus bisporus line J10102-s69, (2) an F1 hybrid Agaricus bisporus mushroom culture produced by mating a culture of the line J10102-s69 with a different Agaricus bisporus culture, (3) an Essentially Derived Variety culture derived from a single initial culture of line J10102-s69, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of line J10102-s69, (4) an Essentially Derived Variety culture derived from a single initial culture of an F1 hydrid Agaricus bisporus mushroom, such that at least 75% of its genome or genotype is present in the genome or genotype of the initial culture of the F1 hybrid Agaricus bisporus mushroom, and (5) a culture having all of the physiological and morphological characteristics of line J10102-s69, wherein the culture of said line J10102-s69 was deposited under the NRRL Accession Number 50893, to provide the second culture.

26. The method for developing a mushroom culture in a mushroom strain development program of claim 25 wherein the mushroom strain development technique is selected from the group consisting of inbreeding, outbreeding, selfing, repeated mating back to an initial culture, introgressive trait conversions, essential derivation, pedigree-assisted breeding, marker assisted selection, mutagenesis, and transformation.

27. A method of mushroom strain development comprising the steps of: (a) obtaining a molecular marker profile of Agaricus bisporus 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 mushroom culture of claim 10 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.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) 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.

(2) Allele: A heritable unit of the genome at a defined locus, ultimately identified by its DNA sequence (or by other means).

(3) Amphithallism: A reproductive syndrome in which heteromixis and intramixis are both active.

(4) Anastomosis: Fusion of two or more hyphae that achieves cytoplasmic continuity.

(5) Basidiomycete: A monophyletic group of fungi producing meiospores on basidia; a member of a corresponding subdivision of Fungi such as the Basidiomycetales or Basidiomycotina.

(6) Basidium: The meiosporangial cell, in which karyogamy and meiosis occur, and upon which the basidiospores are formed.

(7) 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.

(8) Breeding: Development of strains, lines or varieties using methods that emphasize sexual mating.

(9) Cap: Pileus; part of the mushroom, the gill-bearing structure.

(10) 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.

(11) Carrier substrate: A medium having both nutritional and physical properties suitable for achieving both growth and dispersal of a culture.

(12) 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.

(13) Casing inoculum (CI): A formulation of inoculum material incorporating a mushroom culture, typically of a defined heterokaryotic strain, suitable for mixing into the casing layer.

(14) Cloning: Somatic propagation without selection.

(15) 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).

(16) Compatibility: See heterokaryon compatibility.

(17) 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, protoplasts, nuclei, mitochondria, cytoplasm, DNA, RNA, and proteins, cell membranes and cell walls.

(18) Derivation: Development from a strain; see Essentially Derived Variety (EDV).

(19) Derived lineage group: The set of EDVs derived from a single initial strain or variety.

(20) Descent: Genealogical descent over a limited number (e.g., 10 or fewer) of generations.

(21) Diploid: Having two haploid chromosomal complements within a single nuclear envelope.

(22) 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.

(23) Essentially Derived Variety (EDV): (Note: EDV definitions incorporate elements of (1) relatedness, (2) methods of derivation, (3) and empirical tests.) In general, a variety that is predominantly derived from an initial variety or from an EDV of an initial variety, and which conforms to 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.

(24) 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.

(25) 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.

(26) Fungus: An organism classified as a member of the Kingdom Fungi.

(27) Genotypic fingerprint: A description of the genotype at a defined set of marker loci; the known genotype.

(28) Gill: Lamella; part of the mushroom, the hymenophore- and basidium-bearing structure.

(29) Haploid: Having only a single complement of nuclear chromosomes; see homokaryon.

(30) Heteroallelic: Having two different alleles at a locus; analogous to heterozygous.

(31) Heteroallelism: Differences between homologous chromosomes in a heterokaryotic genotype; analogous to heterozygosity.

(32) 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.

(33) Heterokaryon compatibility: The absence of antagonism observed during physical proximity or contact between two heterokaryons that are not genetically identical; see Heterokaryon Incompatibility.

(34) 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.

(35) Heterokaryotic: Having the character of a heterokaryon.

(36) Heteromixis: Life cycle involving mating between two different non-sibling haploid individuals or gametes; outbreeding.

(37) 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.

(38) 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.

(39) Homokaryotic: Having the character of a homokaryon; haploid.

(40) Hybrid: Of biparental origin, usually applied to heterokaryotic strains and cultures produced in controlled matings.

(41) 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.

(42) Hyphae: Threadlike elements of mycelium, composed of cell-like compartments.

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

(44) Incompatibility: See heterokaryon incompatibility.

(45) Inoculum: A culture in a form that permits transmission and propagation of the culture, for example onto new media; specialized commercial types of inoculum include spawn and CI.

(46) Intramixis: A uniparental sexual life cycle involving formation of a complementary mated pair of postmeiotic nuclei within the basidium or individual spore.

(47) 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.

(48) Lamella: see gill.

(49) 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.

(50) Lineage group: see derived lineage group. The set of EDVs derived from a single initial strain or variety.

(51) Locus: A defined contiguous part of the genome, homologous although often varying among different genotypes; plural: loci.

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

(53) MAT: The mating-type locus, which determines sexual compatibility and the heterokaryotic state.

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

(55) Mycelium: The vegetative body or thallus of the mushroom organism, comprised of threadlike hyphae.

(56) Mushroom: The reproductive structure of an agaric fungus; an agaric; a cultivated food product of the same name.

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

(58) Offspring: Descendents, 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.

(59) Outbreeding: Mating among unrelated or distantly related individuals.

(60) 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.

(61) Pedigree-assisted breeding: The use of genealogical information to identify desirable combinations of lines in controlled mating programs.

(62) Phenotype: Observable characteristics of a strain or line as expressed and manifested in an environment.

(63) Plasmogamy: Establishment, via anastomosis, of cytoplasmic continuity leading to the formation of a sexual heterokaryon.

(64) Progenitor: Ancestor, including parent (the direct progenitor).

(65) Selfing: Mating among sibling lines; also intramixis.

(66) Somatic: Of the vegetative mycelium.

(67) 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.

(68) Spore: Part of the mushroom, the reproductive propagule.

(69) Stem: Stipe; part of the mushroom, the cap-supporting structure.

(70) 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.

(71) Stipe: see stem.

(72) Strain: A heterokaryon with defined characteristics or a specific identity or ancestry; equivalent to a variety.

(73) Tissue culture: A de-differentiated vegetative mycelium obtained from a differentiated tissue of the mushroom.

(74) 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.

(75) 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.

(76) 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.

(77) Yield: The net fresh weight of the harvest crop, normally expressed in pounds per square foot.

(78) 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.

(79) 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. 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, and cultures described in this way are functional homokaryons, are putatively homokaryotic, and are all defined as homokaryons in the present application.

(80) Now, with respect to the invention and as noted hereinabove, the present invention relates 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, Ill. 61604 USA (NRRL) as Accession No. 50893.

(81) 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)(4.5) 68 (10) urn, and exhibited a wide range of branching angles from about 10 to 90 degrees off the main hyphal axis.

(82) Line J10102-s69 can be used to produce hybrid cultures with desirable productivity, timing, appearance, and other agronomic traits as is required of successful commercial mushroom strains, while also providing more diversified, non-cultivar germplasm. Line J10102-s69 has been found to have an advantageous genotype for mating to produce commercially useful hybrid strains. Several useful stocks have contributed to the genome of line J10102-s69. Line J10102-s69 has, for example, a mating-type allele 2 on scaffold 1 contributed by the traditional smooth-white stock, and a white color determining allele, as reported by allele E1 at the MFPC-1-ELF marker locus on scaffold 8, contributed by the traditional off-white hybrid stock. Among the remaining genomic scaffolds are at least three (i.e., scaffolds 2, 9 and 10) contributed by other, wild stocks in the pedigree. In combination, these diverse genetic contributions were observed to have combined to produce a superior line with excellent combining ability in matings.

(83) 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.

(84) 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 can be used to determine or validate a pedigree.

(85) Means of obtaining genetic marker profiles using diverse techniques including whole genome sequencing are well known in the art. The whole genomic sequence of line J10102-s69 has been obtained by Sylvan America, Inc., and consequently, about 93%-95% (about 28 Mb) of the entire genotype of line J10102-s69 is known to the Assignee with certainty. The total number of markers distinguishing line J10102-s69 that are known to the assignee is about 300,000. A brief excerpt of the genotype of line J10102-s69 at numerous sequence-characterized marker loci distributed at intervals along each of the 13 chromosomes is provided in Table I.

(86) TABLE-US-00001 TABLEI Position ofSNP [H97V2.0 ref. Culture: Scaffold coords.] J10102-s69 OWNC J11500 1 99995 CTAC TTGA CTACATTGA CTAC TTGA 1 349966 AAGG GGTT AAGGTGGTT AAGG GGTT 1 600059 TTTT TTT TTTTTTTT-C TTTT TT[-/A] 1 850014 C TTTTC C CCTTTTCAC C TTTTC C 1 1099971 GTCG CACC GTCGACACC GTCG CACC 1 1350278 GGAG TCG GGAGAGTCG GGAG TCG 1 1599956 AATA GCGC AATAAGCGC AATA GCGC 1 1850032 CGAG AATT CGAGTAATT CGAG AATT 1 2119049 ACAA CAA ACAATCCAA ACAA CAA 1 2400243 ACTT ATGA ACTTCATGA ACTT ATGA 1 2612870 AATA GAGT AATAGGAGT AATA GAGT 1 2858975 GCCG TCTT GCCGTTCTT GCCG TCTT 1 2804522 GAAG GAC GAAGACGAC GAAG GAC 1 3047987 AAGG GGGG AAGGGGGGG AAGG GGGG 1 3164166 ATAA GGG ATAAGGGGG ATAA GGG 1 3256057 TATC GTTT TATCTGTTT TATC GTTT 2 101820 ATTA GAT ATTAAAGAT ATTA GAT 2 350156 TCGG GGTG TCGGGGGTG TCGG GGTG 2 600112 ATGT TACG ATGTATACG ATGT TACG 2 850338 TGGT CTAA TGGTGCTAA TGGT CTAA 2 1099413 CCTG CTCA CCTGACTCA CCTG CTCA 2 1349512 CTCA CAGT CTCAGCAGT CTCA CAGT 2 1600085 CACA TGCC CACAATGCC CACA TGCC 2 1901773 ACTC AATT ACTCGAATT ACTC AATT 2 2150201 GTCG AGGT GTCGTAGGT GTCG AGGT 2 2400281 TCAA AC C TCAAAACCC TCAA AC C 2 2650136 ATAA TCCT ATAATTCCT ATAA TCCT 2 2903593 ACTA A GA ACTAAAAGA ACTA A GA 2 3048019 GTCC CTGC GTCCGCTGC GTCC CTGC 3 65650 GGCG TTTT GGCGCTTTT GGCG TYTT 3 119281 TTTA ACTC TTTATACTC TTTA ACTC 3 249570 GTAT ATGT GTAT ATGT GTAT ATGT 3 750000 GTCC GCCA GTCC GCCA GTCC GCCA 3 1250000 TTTT CCGG TTTT CCGG TTTT CCGG 3 1750000 ACGC TGAC ACGC TGAC ACGC TGAC 3 2250000 CGTG CGAT CGTG CGAT CGTG CGAT 3 2520748 TAAT CCAC TAATGCCAC TAAT CCAC 4 100004 GAGT AT A GAGTGATAA GAGT AT A 4 340893 AGG GGTA AGGTGGTAT AGG GGTA 4 598147 GATC ACAG GATCGACAG GATC ACAG 4 852119 CGAA A TC CGAATATTC CGAA A TC 4 1100085 GATG CGAA GATGCCGAA GATG CGAA 4 1350536 CGAA CGG CGAACTCGG CGAA CGG 4 1599885 GATA TTGC GATACTTGC GATA TTGC 4 1850288 ATTC GTA ATTCGTGTA ATTC GTA 4 2100356 TCAG GACC TCAGAGACC TCAG GACC 4 2284257 TCTG ACTG TCTGGACTG TCTG ACTG 5 100211 TCCT GAAT TCCTTGAAT TCCT GAAT 5 350872 GGCG GCCC GGCGTGCCC GGCG GCCC 5 599922 CGTC TTCA CGTCATTCA CGTC TTCA 5 851262 TAAT TCT TAATTCTCT TAAT TCT 5 1099776 ACAT GACA ACATTGACA ACAT GACA 5 1352539 TTGT TCC TTGTGATCC TTGT TCC 5 1599904 AACT CCTT AACTTCCTT AACT CCTT 5 1851458 AAAT TCC AAATAATCC AAAT TCC 5 2100025 CCCT AGTC CCCTTAGTC CCCT AGTC 5 2278878 GGTC AAAA GGTCGAAAA GGTC AAAA 6 106294 GCCA CTC GCCATCTCG GCCA CTC 6 350337 CATT GGTT CATTTGGTT CATT GGTT 6 600047 GGAG ATTT GGAGCATTT GGAG ATTT 6 849990 AGTT AGGA AGTTCAGGA AGTT AGGA 6 1098535 CAAA ATTG CAAAGATTG CAAA ATTG 6 1349453 TGTC TAG TGTCGGTAG TGTC TAG 6 1600000 AAAC TGGA AAAC TGGA AAAC TGGA 6 1764645 AACC GATT AACCGGATT AACC GATT 6 2000087 GATT TGCG GATTTTGCG GATT TGCG 6 2252662 GGGT GGTA GGGTTGGTA GGGT GGTA 7 100284 GAAA TCAG GAAATTCAG GAAA TCAG 7 350044 ATAT CTTT ATATTCTTT ATAT CTTT 7 600111 CAAT ATTA CAATTATTA CAAT ATTA 7 850516 TGAC CATA TGACGCATA TGAC CATA 7 1100248 TCAC GAAG TCACGGAAG TCAC GAAG 7 1350089 CTTT CCCC CTTTTCCCC CTTT CCCC 7 1605047 ATAC TG C ATACTTGGC ATAC TG C 7 1850000 GAGA ACT GAGA ACT GAGA ACT 7 1898793 TCCG AT A TCCGCATAA TCCG AT A 7 1991505 TCTA GTT TCTACGGTT TCTA GTT 8 350000 ATTG CGCG ATTG CGCG ATTG CGCG 8 600000 CATT ACGG CATT ACGG CATT ACGG 8 1100000 CATA GATC CATA GATC CATA GATC 8 1350000 AGCT AACA AGCT AACA AGCT AACA 8 1600100 CTGA CCCT CTGA CCCT CTGA CCCT 9 100105 CTCA CCGA CTCAACCGA CTCA CCGA 9 352455 AGTC CCA AGTCCTCCA AGTC CCA 9 599950 TGGT TCCC TGGTATCCC TGGT TCCC 9 1010845 GGGT GTGA GGGTGGTGA GGGT GTGA 9 1244202 GATG AGAT GATGAAGAT GATG AGAT 9 1504476 TACT TACC TACTGTACC TACT TACC 9 1656962 TATC ACTG TATCTACTG TATC ACTG 10 100438 AATT ATTT AATTAATTT AATT ATTT 10 350030 GCGG TCAA GCGGCTCAA GCGG TCAA 10 600032 TTAC CTGG TTACACTGG TTAC CTGG 10 850000 TCGG CGGA TCGG CGGA TCGG CGGA 10 860249 CCGC AAATT CCGCAAATT CCGC AAATT 10 1109960 AGGA ATGA AGGAAATGA AGGA ATGA 10 1303902 TGAT TACT TGATTTACT TGAT TACT 10 1490452 AATC GATG AATCAGATG AATC GATG 11 100000 TATT TTAG TATT TTAG TATT TTAG 11 350000 GTCA CAAG GTCA CAAG GTCA CAAG 11 600000 ATGG CGCG ATGG CGCG ATGG CGCG 11 850000 CTTC CCAT CTTC CCAT CTTC CCAT 11 1100000 TTAC GTTG TTAC GTTG TTAC GTTG 11 124000 AGCC AGTA AGCC AGTA AGCC AGTA 12 100000 CCTT TAGT CCTT TAGT CCTT TAGT 12 1000000 CGAG AGGA CGAG AGGA CGAG AGGA 13 100697 ACGT TTTA ACGTCTTTA ACGT TTTA 13 370521 TTTG GTCA TTTGAGTCA TTTG GTCA 13 604345 CTTC GCAT CTTCAGCAT CTTC GCAT 13 850249 GG T GT A GGCTAGTAA GG T GT A 14 113109 AGGG AATA AGGGAAATA AGGG AATA 14 372086 CGAT C TT CGATCCCTT CGAT C TT 14 725684 ATGA TT G ATGAGTTCG ATGA TT G 15 150013 GTGG CCGT GTGGCCCGT GTGG CCGT 15 449866 GAAT TCGG GAATTTCGG GAAT TCGG 16 208609 CACA GCAC CACATGCAC CACA GCAC 16 400000 CCTC GATT CCTC GATT CCTC GATT 17 120000 TATT TTCA TATT TTCA TATT TTCA 17 338415 TGAG AGCC TGAGAAGCC TGAG AGCC 17 449833 ATCA AC A ATCAGACAA ATCA AC A 18 101884 ATTA GGAC ATTACGGAC ATTA GGAC 19 98377 GCTA TGGG GCTATTGGG GCTA TGGG

(87) Table I presents a fingerprint excerpted from the SNP (Single Nucleotide Polymorphism) genotype of the entire genome sequences of line J10102-s69, of line OWNC, and of the F1 hybrid J11500 strain obtained from the mating of lines J10102-s69 and OWNC. 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 F1 hybrids. The IUPAC nucleotide and ambiguity codes are used to represent the observed 9-base DNA marker sequences reported above, each of which represents a genotypic marker locus. The identity of each marker locus is specified by the scaffold and SNP position information derived from the H97 V2.0 reference genome sequence published by the U.S. Department of Energy Joint Genome Institute (Morin et al. 2012). Distinctions between the homoallelic genotypes of line J10102-s69 and line OWNC are evident, as is the composite relationship of the example heteroallelic genotype of strain J11500 to those of its two parental lines.

(88) Genotype data for six additional marker loci is provided in TABLE II.

(89) TABLE-US-00002 TABLE II Alleles of J10102-s69 at 6 marker loci, for lines J10102-s69, OWNC, and SWNC Marker: Line ITS p1n150 MFPC-1-ELF AN AS FF J10102-S69 4 2 E1 N5 SC FF1 OWNC 1 1T E1 N1 SD FF1 SWNC 2 2 E2 N2 SC FF2
OWNC and SWNC are two lines derived from two traditional white-capped cultivar stocks, as described in the concurrently filed patent application entitled Hybrid Mushroom Strain J11500 and Descendants Thereof, the disclosure of which is incorporated by reference. Each is genotypically distinct, as shown in Table II.

(90) Line J10102-s69 can be identified through its molecular marker profile as shown in Tables I and II. A culture or product incorporating the genetic marker profile shown in Tables I and II is an embodiment of the invention. Another embodiment of this invention is an Agaricus bisporus line or its parts comprising the same alleles as the line J10102-s69 for at least 75% of the loci listed in Tables I and II. In other embodiments, this line or its parts comprises the same alleles as the line J10102-s69 for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or essentially 100% of the loci listed in Tables I and II.

(91) A cell comprising the same alleles as a cell of line J10102-s69 for at least 75% of the loci listed in Tables I and II is also an embodiment of this invention. In other embodiments, cells comprising the same alleles as a cell of line J10102-s69 for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or essentially 100% of the loci listed in Tables I and II, are provided. Also encompassed within the scope of the invention are cultures substantially benefiting from the use of line J10102-s69 in their development, such as hybrid offspring having line J10102-s69 as a parent, and line 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.

(92) 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. In most eukaryotes, a parent is conventionally considered to be either diploid or heterokaryotic. The haploid generation is often, but not always, termed a gamete (e.g., pollen, sperm). In fungi, which are microorganisms, the haploid generation can live and grow indefinitely and independently, for example in laboratory cell culture; while these haploid homokaryons function as gametes in matings, they are equivalent to inbred lines (e.g., of plants) and are more easily referred to as parents (of hybrids). Herein, the term parent refers to the culture that is a, or the, direct progenitor of another culture within the alternating generations of the sexual lifecycle. The term line refers more narrowly to a haploid (N) homoallelic culture within the lifecycle. The N+N heterokaryon resulting from a mating, or comprising a breeding stock, or comprising a culture used to produce a crop of mushrooms, may be called a strain.

(93) 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 codominant 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.

(94) A heterokaryotic selfed offspring of an F1 hybrid that itself has a IA 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. 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 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 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.

(95) Essentially Derived Varieties are most often derived directly 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 of methods that are well known in the art. Repeated mating back to the initial culture to introgress a single trait into the genetic background of an initial variety or strain is called introgressive trait conversion, and produces an EDV of the initial strain. 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, herein incorporated by reference. Transformation may introduce a single new gene or allele into the genome of an initial variety.

(96) Therefore, in accordance with the above, one or more embodiments of this invention include a line J10102-s69 progeny mushroom culture, culture part, mushroom, or mushroom part that is a first-generation (F1) heterokaryotic hybrid mushroom culture comprising two sets of alleles, wherein one set of alleles is the same as line J10102-s69 at all of the marker loci listed in Table I. A mushroom cell or hyphal element wherein one set of the alleles is the same as line J10102-s69 at all of the marker loci listed in Table I is also an embodiment of the invention. This mushroom cell or hyphal element may be a part of a culture, a commercial inoculum or spawn product, a mushroom, or a part of a mushroom produced by mating line J10102-s69 with another mushroom culture. Further embodiments of this invention may include an essentially derived variety of the F1 hybrid, produced by inter-tetrad or intra-tetrad selfing of the F1 hybrid, or by modification of the F1 culture, and more specifically by somatic selection, tissue culture selection, single spore germination, multiple spore germination, selfing, repeated mating back to the initial culture, mutagenesis, and transformation.

(97) While many types of molecular markers are known, and can be used, all of these ultimately derive from the primary DNA sequence of the genome. The essential genotype of a line or strain is embodied in its genomic DNA sequence. The marker profile presented in Table I represents selected short segments of the genome sequence of line J10102-s69, at loci which are known to have differing sequences among other lines and strains, selected at widely spaced intervals spanning the entire nuclear genome. Commercial sequencing providers and commercial technologies such as Illumina MiSeq, among others, may be used to obtain whole-genome sequences from total cellular DNA preparations. Other techniques for obtaining genotype profiles may also be used as appropriate.

(98) Line J10102-s69 and its presence in cultures, culture parts, hybrids, mushrooms and mushroom parts can be identified through a molecular marker profile. A mushroom culture cell or hyphal element having the marker profile shown in Table I is an embodiment of the invention. Such a mushroom cell or hyphal element may be heterokaryotic.

(99) Line J10102-s69 represents a new base genetic line into which a new locus or trait may be introgressed. Direct transformation and inbreeding represent two useful methods that can be applied to accomplish such an introgression. Introgression producing a trait conversion comprises the step of mating line J10102-s69 to a second strain, and then mating progeny of that mating with line J10102-s69, repetitively, until a derived variant of line J10102-s69 incorporating an introduced gene determining a novel trait is obtained. Strains and lines produced by this method may have, for example, in the range of 75, 80, 85, 90, 95, 96, 97, 98, 99, or 99.9% of the DNA of line J10102-s69, and are therefore Essentially Derived Varieties of line J10102-s69, and are an embodiment of the invention.

(100) In order to demonstrate practice of the present invention, 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 in Sylvan America, Inc.'s corresponding patent application filed the same day and entitled Hybrid Mushroom Strain J11500 and Descendants Thereof, herein incorporated by reference. No previous line has ever been observed to produce the combinations of desirable traits observed among hybrids incorporating line J10102-s69.

(101) As previously discussed, the results in Table I compare a specific hybrid mushroom culture strain, namely a strain designated J11500, which has been deposited with the NRRL as Accession No. 50895 and which is the subject of a concurrently filed patent application entitled Hybrid Mushroom Strain J11500 and Descendants Thereof, the disclosure of which is incorporated by reference, for which line J10102-s69 is a parent with other hybrids. The results show that homokaryotic line J10102-s69 shows good combining ability.

(102) 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 progeny of the first generation is designated F1. F1 hybrids may be useful as new commercial varieties for mushroom production, or as starting material for the production of inbred offspring and/or EDVs, or as parents of the next generation of haploid lines for producing subsequent hybrid strains.

(103) 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, 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.

(104) 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.

(105) 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.

(106) 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.