MUSHROOM VARIETIES

Abstract

New and unique brown varieties of the mushroom Agaricus bisporus Imbach were produced by crossing the progeny of brown mushrooms and off-white hybrid mushrooms to bridging cross strains. The resultant hybrids, named RO10425, RO14176, and RO12756, exhibit distinct and stable characteristics. Hybrid RO10425 exhibits a lighter cap color, thicker cap flesh, and rounder cap, especially when compared to available brown mushroom varieties. RO10425 also exhibits fast mushroom development and high yield. Hybrid RO14176 exhibits a light cap color, thicker cap flesh, rounder cap, faster mushroom development, and higher yield, especially when compared to available brown mushroom varieties. Hybrid RO12756 exhibits a light cap color, thicker cap flesh, rounder cap, faster mushroom development, and high yield, especially when compared to available brown mushroom varieties.

Claims

1. A hybrid Agaricus bisporus mushroom strain selected from the group consisting of RO10425, RO14176, and RO12756, representative culture of which having been deposited under ATCC Nos. PTA-127856, PTA-127855, and PTA-127857 respectively.

2. A hybrid mushroom produced by the hybrid Agaricus bisporus mushroom strain of claim 1.

3. A mushroom part from the hybrid mushroom of claim 2.

4. The mushroom part of claim 3, wherein the mushroom part is a fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, protoplast, gill, cell, or product produced from the mushroom.

5. The hybrid mushroom of claim 2, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of: lighter cap color as exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; higher yield; rounder cap; faster time to pinset, first break, and/or second break; thicker cap flesh as exhibited by thicker cap apex flesh and thicker cap edge flesh; and faster percent harvested speed, when compared to the corresponding physical characteristics of BR06, BR06 having representative sample accessible under ATCC under Accession No. PTA-6876.

6. The hybrid mushroom of claim 2, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of: light brown cap color as exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; as high or higher tray coverage; thicker cap flesh as exhibited by thicker cap apex flesh and thicker cap edge flesh; and rounder cap, when compared to the corresponding physical characteristics of B14528, B14528 having representative sample accessible under NRRL Accession No. 50900.

7. A method of producing a hybrid mushroom culture of Agaricus bisporus, the method comprising the steps of: (a) providing a first homokaryon selected from a wild strain, designated AA-0096, or a progeny thereof, wherein said wild strain AA-0096 is available from ATCC under Accession No. PTA-6903; (b) providing a second homokaryon selected from a commercial off-white hybrid strain, which is compatible with first homokaryon; (c) crossing the first homokaryon with the second homokaryon to form a first culture of intermediate hybrid homokaryons; (d) backcrossing the first culture to the second homokaryon to form a second culture of intermediate hybrid homokaryons; and (e) crossing the second culture to a bridging cross strain to form the hybrid mushroom culture, designated RO10425, a representative culture of which having been deposited under ATCC Accession No. PTA-127856.

8. The method of claim 7, the method further comprising: providing the hybrid 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; or: providing the hybrid mushroom culture in derived cultures selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue explants cultures, protoplasts, dormant spores, germinating spores, inbred descendants, transgenic cultures, and cultures having a genome incorporating a single locus conversion.

9. The method of claim 7, wherein the bridging cross strain is 429, representative sample of 429 being available under ATCC Accession No. PTA-6877.

10. The method of claim 7, wherein the commercial off-white hybrid strain is derived from a U1 mushroom strain.

11. The method of claim 7, the method further comprising steps of producing at least one hybrid mushroom, said steps comprising: (f) inoculating a mushroom growth medium with the hybrid mushroom culture; (g) maintaining said inoculated growth medium under conditions conducive to mushroom fruiting; and (h) collecting the at least one hybrid mushroom from said growth medium.

12. A hybrid mushroom produced by the method of claim 11, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of: lighter cap color as exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; higher yield; rounder cap; faster time to pinset, first break, and/or second break; thicker cap flesh as exhibited by thicker cap apex flesh and thicker cap edge flesh; and faster percent harvested speed, when compared to the corresponding physical characteristics of BR06, BR06 having representative sample accessible under ATCC under Accession No. PTA-6876.

13. A hybrid mushroom produced by the method of claim 11, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of: light brown cap color as exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; as high or higher tray coverage; thicker cap flesh as exhibited by thicker cap apex flesh and thicker cap edge flesh; and rounder cap, when compared to the corresponding physical characteristics of B14528, B14528 having representative sample accessible under NRRL Accession No. 50900.

14. A hybrid mushroom produced by the method of claim 11.

15. A mushroom part from the hybrid mushroom of claim 14, wherein the mushroom part is a fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, protoplast, gill, cell, or product produced from the mushroom.

16. The method of claim 7, the method further comprising a step (f): providing (i) a homokaryon of the hybrid mushroom culture and (ii) a third homokaryon selected from the cross of a second bridging cross strain and a commercial off-white hybrid strain, which is compatible with the homokaryon of the hybrid mushroom culture; and crossing the homokaryon of the hybrid mushroom culture with the third homokaryon to form an additional hybrid mushroom culture, designated RO12756, a representative culture of which having been deposited under ATCC Accession No. PTA-127857; or allowing the hybrid mushroom culture to undergo plasmogamy; and selecting a heterokaryotic single spore isolate from the result of the plasmogamy, said heterokaryotic single spore isolate designated RO14176, a representative culture of which having been deposited under ATCC Accession No. PTA-127855.

17. The method of claim 16, the method further comprising: providing the additional hybrid mushroom culture or a culture produced by the heterokaryotic single spore isolate 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; or providing the additional hybrid mushroom culture in derived cultures selected from the group consisting of homokaryons, heterokaryons, aneuploids, somatic subcultures, tissue explants cultures, protoplasts, dormant spores, germinating spores, inbred descendants, transgenic cultures, and cultures having a genome incorporating a single locus conversion.

18. The method of claim 16, wherein the bridging cross strain is 429, representative sample of 429 being available under ATCC Accession No. PTA-6877.

19. The method of claim 16, wherein the commercial off-white hybrid strain is derived from a U1 mushroom strain.

20. The method of claim 16, further comprising steps of producing at least one hybrid mushroom, said steps comprising: (h) inoculating a mushroom growth medium with the hybrid mushroom culture; (i) maintaining said inoculated growth medium under conditions conducive to mushroom fruiting; and (j) collecting the at least one hybrid mushroom from said growth medium.

21. A hybrid mushroom produced by the method of claim 20, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of: lighter cap color as exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; higher yield; rounder cap; faster time to pinset, first break, and/or second break; thicker cap flesh as exhibited by thicker cap apex flesh and thicker cap edge flesh; and faster percent harvested speed, when compared to the corresponding physical characteristics of BR06, BR06 having representative sample accessible under ATCC under Accession No. PTA-6876.

22. A hybrid mushroom produced by the method of claim 20, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of: light brown cap color as exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; as high or higher tray coverage; thicker cap flesh as exhibited by thicker cap apex flesh and thicker cap edge flesh; and rounder cap, when compared to the corresponding physical characteristics of B14528, B14528 having representative sample accessible under NRRL Accession No. 50900.

23. A hybrid mushroom produced by the method of claim 20.

24. A mushroom part from the hybrid mushroom of claim 23, wherein the mushroom part is a fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, protoplast, gill, cell, or product produced from the mushroom.

25. Use of (i) a spore, protoplast, homokaryon, or tissue culture of RO14176; (ii) a spore, protoplast, homokaryon, or tissue culture of RO10425; or (iii) a spore, protoplast, homokaryon, or tissue culture of RO12756, said use comprising isolation of the spore, protoplast, homokaryon, or tissue culture, wherein: representative sample of RO10425 having been deposited under ATCC Accession No. PTA-127856; representative sample of RO14176 having been deposited under ATCC Accession No. PTA-127855; and representative sample of RO12756 having been deposited under ATCC Accession No. PTA-127857.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fec.

[0059] FIG. 1 shows a representative comparison of Agaricus bisporus variety BR06 (left) and variety RO12756 (right) tray coverage at first break. BR06 displays lower tray coverage, and RO12756 displays higher tray coverage.

[0060] FIG. 2 shows a longitudinal slice of a mushroom of variety RO14176 with markings added to illustrate the calculation of Cap Edge Flesh Thickness, where Cap Edge Flesh Thickness is calculated by ((LG.sub.LCS.sub.L)+(CS.sub.RRG.sub.R))/CS.sub.D. LG.sub.L=left-most x-coordinate of the left gill, CS.sub.L=left-most x-coordinate of the cross-section, CS.sub.R=right-most x-coordinate of the cross-section, RG.sub.R=right-most x-coordinate of the right gill, and CS.sub.D=diameter of the cross-section.

[0061] FIG. 3 shows a longitudinal slice of a mushroom of variety RO14176 with markings added to illustrate the calculation of Cap Apex Flesh Thickness, where Cap Apex Flesh Thickness is calculated by (CS.sub.T((LG.sub.T+RG.sub.T)/2))/CS.sub.H. CS.sub.T=the top-most y-coordinate of the cross-section, LG.sub.T=the top-most y-coordinate of the left gill, RG.sub.T=the top-most y-coordinate of the right gill, and CS.sub.H=the height of the cross-section.

[0062] FIG. 4 shows a representative comparison of cap colors for variety BR06 (left), variety B14528 (center), and variety RO12756 (right) cap colors based on Cap L* Value, Cap a* Value, and Cap b* Value at first break. Mushrooms pictured were grown in the same room, under the same environmental conditions, and were harvested from the same trial in May 2025. All photos have the same scale. Scale bar=1 cm.

[0063] FIG. 5 shows representative comparisons of cap height, relative cap height, and relative cap flesh thickness for variety B14528 (left), variety BR06 (center), and variety RO12756 (right) at first break. Mushrooms pictured were grown in the same room, under the same environmental conditions, and were harvested from the same trial in May 2025. All photos have the same scale. Scale bar=1 cm.

[0064] FIG. 6 shows comparisons between mushrooms of variety RO14176 (top row) and variety RO10425 (bottom row). The top left panel shows caps of 10 representative mushrooms from variety RO14176 and their average L*, a*, and b* values. The top right panel shows a longitudinal slice of each mushroom cap in the top left panel with the 10 mushrooms' average cap diameter, cap height, cap flesh thickness, and stipe diameters reported in millimeters. The bottom left panel shows caps of 10 representative mushrooms from variety RO10425 and their average L*, a*, and b* values. The bottom right panel shows a longitudinal slice of each mushroom cap in the bottom left panel with the 10 mushrooms' average cap diameter, cap height, cap flesh thickness, and stipe diameters reported in millimeters. Mushrooms pictured were grown in the same room, under the same environmental conditions, and were harvested from the same trial in April 2025. All photos have the same scale. Scale bar=1 cm.

[0065] FIG. 7 shows multiple mushroom caps of variety RO14176 from a top-down view.

[0066] FIG. 8 shows multiple longitudinal slices of mushrooms of variety RO14176.

[0067] FIG. 9 shows multiple mushroom caps of variety RO10425 from a top-down view.

[0068] FIG. 10 shows multiple longitudinal slices of mushrooms of variety RO10425.

DETAILED DESCRIPTION OF THE INVENTION

[0069] It is an object of the disclosure to provide brown varieties of Agaricus bisporus mushroom with improved commercial characteristics relative to existing brown commercial mushrooms.

[0070] The term break as used herein may refer to a flush of fruiting bodies, or a length of time attributed to a period of mushroom growth and harvesting.

[0071] Agaricus bisporus mushrooms to be marketed as cremini or baby bellas are generally considered to be of harvestable maturity when the caps have reached approximately 3-5 cm in diameter and the veil tissue covering the gills has not yet opened.

[0072] The present disclosure of hybrid mushrooms arose from a breeding program that crossed compatible homokaryons collected from wild brown strains, sometimes a bridging cross strain, and a commercial white mushroom strain. One bridging cross strain was produced by crossing compatible homokaryons from a commercial strain of Agaricus bisporus and a commercial off-white hybrid mushroom strain. Furthermore, subsequent hybrid strains can be produced by crossing cultures of compatible homokaryons selected from these commercial brown mushroom strains, to induce and/or eliminate certain parental characteristics in the hybrid strains. The specific wild mushroom strain of A. bisporus used to provide the desired genetic and phenotypical characteristics is known as AA-0096. The wild strain AA-0096 described in the scientific literature has unique genetic characteristics. Strain AA-0096, also known as BP-1 and ARP-023, is available from the American Type Culture Collection (ATCC) under the accession number 76562 as a non-patent deposit. This strain was re-deposited by the current inventors under the Budapest Treaty governing the deposit of organisms for patent purposes at the American Type Culture Collection, Rockville, MD, USA, under ATCC Accession No. PTA-6903. Hybrid strains obtained by crossing intermediate hybrid strains (or bridging cross strains) of A. bisporus and commercial off-white hybrid strains, and successive hybrids obtained by crossing such hybrids with other commercial off-white hybrid strains, can be cultivated to have at least one of the improved genetic and phenotypic characteristics described herein by beneficially using the procedures described herein.

[0073] The description of the varied embodiments of this disclosure presented herein, and the availability of the aforementioned strains from their deposits or in the commercial marketplace, enable one skilled in the art to practice the current disclosure using standard methods of mushroom breeding and/or production. In one embodiment of the present disclosure, cloning processes that produce genetically identical crops of mushroom, and crossing processes that produce non-genetically identical progeny, described herein, facilitate the implementation of embodiments of this disclosure and the production of hybrid mushrooms. Further progeny (as well as later crosses derived from these progeny) can be cultivated with desirable characteristics using general breeding techniques.

Breeding Techniques

[0074] Methods of the production of mushroom strains, either as direct progeny (clones) of a given strain or as hybrid progeny by crossing with a second strain, are well known. Patent Application No. US 2015/0216127 A1 Mushroom Line JIQ102-S69 and Methods and Uses Thereof, Patent Application No. US 2015/0216128 A1 titled Hybrid Mushroom Strain JI 1500 and Descendants Thereof, U.S. Pat. No. 9,017,988 B1 entitled Hybrid Mushroom. Strain B14528 and Descendants Thereof, U.S. Pat. No. 5,304,721, entitled Method for the Production of High Proportions of Homokaryons in Breeding Stock of the Mushroom Agaricus bisporus, U.S. Pat. No. 4,996,390 entitled Novel Interspecific Mushroom Strains, publications in the scientific literature such as An Efficient Protoplasting/Regeneration System for Agaricus bisporus and Agaricus bitorquis. Curr. Microbiolo., 17:285-291, 1988, by Sonnenberg et al., Protoplast Production and Regeneration from Mycorrhizal Fungi and Their Use for Isolation and Mutants, Can. J, Microbiol, 34:157-161, 1988 by Herbraud et al., DNA Polymorphisms in Commercial and Wild Strains of the Cultivated Mushroom, Agaricus bisporus Theor. Appl. Genet., 76:712-718, 1988 by Loftus et al., The Genetics and the Breeding of Species Agaricus by Elliott, Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (=A. bisporus), Appl. Environ. Microbiol. 54:1643-1648, 1988, by Castle et al., and books like The Biology and Technology of the Cultivated Mushroom, by Flegg et al., John Wiley and Sons, 1985, pp. 111-139.

[0075] Given the availability of mushroom breeding techniques, a person skilled in the art of mushroom production can practice the disclosure and the production of brown hybrid mushrooms and descendants thereof.

[0076] In some embodiments, a culture of selected homokaryons collected from a brown commercial strain of A. bisporus is first crossed with compatible homokaryons from an off-white commercial white strain to form a first hybrid strain, also referred to as an intermediate or a bridging cross strain. In some embodiments, the off-white commercial mushroom strain is derived from a U1 mushroom strain, referring to cultivar line Horst U1 developed in 1980. The homokaryons are usually collected from the natural process of spore formation, but they are also obtained through a technique called protoplasting. Homokaryons or heterokaryons collected from spores are called Single Spore Isolates (SSI or ssi), and those created from protoplasting are called Protoplasts (p). The fundamental difference between these two types of homokaryons is that the SSIs have gone through a meiosis step, and protoplasts have not. SSIs are collected from spore prints, and protoplasts are created from the enzymatic removal of the mycelial fungal cell wall. In some embodiments, SSIs are labeled slow growers (sg). In some embodiments, SSIs are utilized for breeding of hybrid mushrooms. In some embodiments, protoplasts are utilized for breeding of hybrid mushrooms.

[0077] In some embodiments, spores from wild mushroom strain designated AA-0096 are used. In some embodiments, spores from at least one commercial off-white hybrid variety are used.

Essentially Derived Varieties

[0078] Heterokaryotic spores of initial parental strain retain the great majority of the parental genotype (Kerrigan et. al., Genetics, 133, 225-236, 1993). A group of strains either developed by cloning or by spore culture, or by any other means of essential derivation, from a single progenitor, as opposed to outbreeding, is called a derived lineage group. In general, an Essentially Derived Variety (EDV) is a variety that is predominantly derived from an initial variety or from an EDV of an initial variety and retains the essential characteristics of the initial variety except where the differences that arise are solely from such a derivation. EDVs incorporate elements of (1) relatedness, (2) methods of derivation, (3) and empirical tests. In the art of mushroom strain development, a strain or culture predominantly or entirely derived from a single initial strain or culture, or from the modification of an initial culture using methods including somatic selection, tissue culture selection, selfing, mating among sibling lines, including intramixis (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 remains predominantly identical to the initial variety, is an EDV. In one or more embodiments, an Essentially Derived Variety of A. bisporus strain or intermediate strain or hybrid strain may be used. It is to be noted that the usage of the term derived in many techniques and processes described herein is not in any way congruent with Essentially Derived Variety.

Novelty of the New Hybrid Strains

[0079] In addition to novel pedigree lines, unique genetics help differentiate the brown hybrid mushrooms of this breeding program from existing commercial mushrooms. New or improved phenotypic traits also help distinguish these brown hybrids from other commercial brown or white type mushrooms.

[0080] The new hybrid strains described herein possess several traits that give them marketable advantages over other commercial brown strains, including the following: [0081] 1. Higher yield at later breaks, which enable growers to achieve higher yield per unit area compared to other commercial brown mushrooms. [0082] 2. Faster developmental timing, which enables growers to increase their efficiency by allowing them to pick off mushrooms before the end of the growing cycle. Faster strains might also allow for more cropping cycles per year. Traits herein related to developmental timing include Days to Pinset, Days to 1st Break, Days to 2nd Break, and Percent Harvested Speed. [0083] 3. Thick-fleshed morphology, which reflects higher quality than other commercial brown mushrooms. This morphology remains more consistent at later breaks for RO10425 when compared to other commercial varieties, and this increases visual appeal of sliced product. Pre-sliced button mushrooms have been growing in popularity over the last 10+ years; a 10-year report on mushroom sales trends demonstrated that sliced product has eclipsed whole product fresh mushroom sales, encompassing over 50% of fresh brown mushrooms sold in 2023 in the United States (Roerink, A. (2023 October). Ten-year Trends in Fresh Mushroom Sales. Mushroom Council. Available at www[dot]mushroomcouncil[dot]org/wp-content/uploads/2023/10/Ten-Year-Fresh-Mushroom-Trends[dot]pdf).

Genetics of the New Hybrid Strains

[0084] Mushrooms within the scope of the present disclosure that are called clones, can be prepared by any of the known cloning processes (as well as those that may be discovered in the future) from a mushroom of the disclosure. These clones are prepared without a sexual crossing process and have the same genetic and physical characteristics as their parents. A genetic trait is any property of the genetic material of a mushroom strain (usually a gene sequence) that can be measured by a conventional analytical technique. Examples of genetic traits include RAPD, RFLP, AFLP, SNPs, or SCAR bands, as they appear on gels using standard analytical techniques. These well-known analytical techniques are described in numerous scientific publications, including the following: [0085] SCAR: Paran, I. and R. W. Michelmore (1993). Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor. Appl. Genet. 85:985-993. [0086] RAPD: Khush, R. S., Becker, E. and M. Wach (1992). DNA Amplification Polymorphisms of the cultivated mushroom Agaricus bisporus. Appl. Env Microbiol 59:2971-2977. [0087] RFLP: Castle, A J., P. A. Horgen and J. B Anderson 1987. Restriction fragment length polymorphisms in the mushrooms. Agaricus brunnescens and Agaricus bitorquis. Appl Env Microbiol 53:816-822 [0088] AFLP: Mueller U G and Wolfenbarger L L (1999) AFLP genotyping and fingerprinting. Trends Ecol Evol 14:389-394. [0089] SNP: Wakui M. (2013) [Analysis of single nucleotide polymorphisms (SNPs)]. Rinsho Byori. 2013 November; 61 (11): 1008-17. Japanese. PMID: 24450106.

[0090] Heterokaryotic spore collection and analysis is another valuable resource for genetic analysis, as this allows the examination of spores that are likely very similar to clones described above, due to their unusual meiosis undertaken.

Superiority of the New Hybrid Strains

[0091] Following the methods described herein, strains derived herein exhibit improved characteristics over the commercially available brown varieties BR06 and/or B14528. These improvements may include thicker cap flesh, faster timing to reach pinset and harvestable maturity, higher tray coverage, a unique brown cap color, softer cap firmness, thicker stipes and cap flesh relative to cap diameter, higher compost colonization levels, faster development of harvestable mushrooms, and higher overall yield.

[0092] These improvements are due to the introduction of a wild parent, AA-0096, to commercial off-white hybrid strains and subsequent crosses and selection, as well as the inclusion of both commercial and wild brown mushroom strains. These improvements are in addition to the many traits that have made U1 derived off-white hybrids successful commercial mushroom strains. Mushrooms with improved cap color, yield, and percent harvested speed will attract a higher price than conventional brown mushroom strains.

Mushroom Breeding and Hybridization

[0093] In mushroom breeding, mycelial (=vegetative) cultures of two compatible progenitors (typically these are haploid homokaryotic strains called homokaryons) must come into physical contact so that one or more fusion zones can occur between the progenitors. Within those fusion zones nuclei from the two progenitors become associated. In Agaricus bisporus, a novel, hybrid mycelium ultimately containing two compatible haploid nuclear types (one from each of the two progenitors) plus one mitochondrial type (from either one of the progenitors) emerges. This novel hybrid mycelium can be isolated and propagated to provide the new hybrid culture, which can be further subdivided and propagated for commercial or other purposes.

[0094] It should be recognized that the mushrooms of the present disclosure are hybrids (equivalent to crosses), as they are formed by the hybridization of at least two independently derived strains, since they were formed by hybridizing the wild strain AA-0096 with a second, commercialized strain of A. bisporus (with the second strain, in some embodiments, being itself a cross between at least two other identified strains of A. bisporus). The term hybrid mushroom may be used herein to represent the product of a cross between a wild A. bisporus strain and a commercial strain, often including a cross between a brown mushroom strain and an off-white hybrid variety. Differences in terminology (and in the specific techniques used alongside the terminology) are merely indicative of the variety of terminology and techniques used in the mushroom production field.

[0095] After parental homokaryotic spores germinated into mycelia, single spore isolates (SSIs) were selected, preferentially those exhibiting slow mycelial growth. The slowly growing mycelia of SSIs usually contain one nuclear type, are thus homokaryotic, and are suitable for crossing, and thus for breeding. Slow growers are denoted as sg. Some homokaryons were obtained through protoplasting; this technique involves treating the vegetative mycelia of the heterokaryons to damage the fungal cell wall, then selecting colonies of protoclones that are each derived from a single, regenerated protoplast. All crosses were initially cultivated on a small scale, and spores were collected for the parents of the crosses to produce the hybrids described herein.

[0096] One useful feature in the breeding programs for these novel mushroom varieties is the use of a bridging cross strain, which is a hybrid variety produced from crossing a commercial brown variety with a commercial white variety. The particular bridging cross strain of note in these breeding programs is known as the 429 strain. Strain 429 has been previously deposited under the provisions of the Budapest Treaty with the American Type Culture Collection, Rockville, Md., USA, ATCC accession No. PTA-6877 (deposited on Jul. 20, 2005). Strain 429 is the product of crossing a commercial brown strain with a commercial white strain. The 29 of this strain is a unique homokaryon collected from an off-white hybrid strain.

[0097] The varieties, mushrooms, strains, and cultures disclosed herein are understood to comprise parts of mushrooms, such as the fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, protoplast, gill, cell, or product produced from the mushroom. In some embodiments, the mushroom part from the hybrid mushroom produced by any method disclosed herein is a spore. In some embodiments, the mushroom part produced from a mushroom strain disclosed herein is a spore. In some embodiments, RO14176 is a mushroom part produced from the hybrid mushroom of RO10425. In some embodiments, the mushroom part produced from the hybrid mushroom of RO10425 is RO14176.

EXAMPLES

Example 1: Characterization of RO10425

[0098] The following example describes the characterization of strain RO10425 compared to a related variety and commercial varieties.

Methodology

[0099] In the descriptions and tables that follow, numerous traits are calculated/measured as follows in Table 1.

[0100] Visual depictions of the measurements used for calculating Cap Apex Flesh Thickness are shown in FIG. 3. Visual depictions of the measurements used for calculating Cap Edge Flesh Thickness are shown in FIG. 2.

[0101] Mushrooms were harvested at Stage 3 as described by Hammond & Nichols (Hammond, J. B. W., & Nichols, R. (1976). Carbohydrate metabolism in Agaricus bisporus (Lange) Sing.: changes in soluble carbohydrates during growth of mycelium and sporophore. Microbiology, 93(2), 309-320), when cap diameters were approx. 3-5 cm with the veil still intact. To reduce confounding effects introduced by different trials, developmental timing and yield traits were compared only from trials where all strains being compared were present together.

[0102] Images of mushroom caps and longitudinal cross-sections were captured using a Canon Rebel SL1 camera (Canon, Inc., Tokyo, Japan) in a custom-built photo box with standardized lighting conditions. The camera was calibrated for color consistency using a ColorChecker gray balance card (Calibrite LLC, Wilmington, DE). L*, a* and b* values were captured across the surface of each mushroom cap and cross-section using ImageJ (Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671-675). Images captured in 2024 and 2025 were used for analysis. Extreme outliers were excluded from the data, defined as those falling 3 times the interquartile range (IQR) above the 75th percentile for that trait or more than 3 times the IQR below the 25th percentile for that trait.

[0103] All statistical analyses were carried out in the R programming language (R Core Team, (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. www[dot]R-project[dot]org/). Levene's test was performed using the car package (Fox, J., & Weisberg, S. (2018). An R companion to applied regression. SAGE Publications), Shapiro-Wilk tests and Tukey HSD were performed using the stats package (R Core Team (2013)), and standard ANOVAs, Kruskal-Wallis tests and Dunn's tests were performed using the rstatix package (Kassambara, A (2023). rstatix: Pipe-Friendly Framework for Basic Statistical Tests. R package version 0.7.2, rpkgs[dot]datanovia[dot]com/rstatix/).

TABLE-US-00001 TABLE 1 Trait Tool Measurement Scale Timing Cap Color Darkness visually 0 to 5 (from white to dark brown), tray- 1st break**, 2nd assessed level measurement break** Cap Color a* Value image analysis 128 to +127 (from pure green to pure 1st break**, 2nd (Green-Red) red), individual mushroom-level break** measurement Cap Color b* Value image analysis 128 to +127 (from pure blue to pure 1st break**, 2nd (Blue-Yellow) yellow), individual mushroom-level break** measurement Cap Color L* Value image analysis 0 to 100 (from pure black to pure white), 1st break**, 2nd (Dark-Light) individual mushroom-level measurement break** Cap Diameter image analysis millimeters (mm), individual mushroom- 1st break**, 2nd level measurement break* Cap Firmness manual 1 to 5 (from very soft to very firm), tray- 1st break**, 2nd squeeze test level measurement break** Cap Apex Flesh calculated distance from top of gills to top of cap of 1st break**, 2nd Thickness ratio and mushroom, relative to cap height, break* image analysis individual mushroom-level measurement Cap Edge Flesh calculated sum of distances from outer edge of each 1st break**, 2nd Thickness ratio and gill to outer edges of cap of mushroom, break** image analysis relative to cap diameter, individual mushroom-level measurement Cap Flesh Thickness hand- millimeters (mm), distance from top of 1st break**, 2nd measurement gill-stipe connection (at center of stipe) to break** top of cap, individual mushroom-level measurement Cap Height image analysis millimeters (mm), distance from base of 1st break**, 2nd cap to top of cap, individual mushroom- break** level measurement Cap Roundness image analysis Unitless ratio (0-1), 1st break**, 2nd (Calculated) calculated as 4 * CS.sub.A/( * CS.sub.D.sup.2), where break** CS.sub.A = area of the cross-section, and CS.sub.D = diameter of the cross-section; assessed with image analysis, individual mushroom-level measurement Cap Roundness visually 1 to 5 (from very flat to spherical), tray- 1st break**, 2nd (Visual) assessed level measurement break** Compatibility calculated pounds per square foot (lb/ft.sup.2) and 1st break** ratio kilograms per square meter (kg/m.sup.2), weight of 1st break mushrooms yielded relative to area of tray, tray-level measurement Compost Colonization visually 0 to 5 (from no colonization to robust before 1st break assessed colonization), tray-level measurement (10 days after spawning) Days to 1st Break visually number of days elapsed after casing at 1st break** assessed which at least half of tray is of harvestable maturity for 1st break, tray-level measurement Days to 2nd Break visually number of days elapsed after casing at 2nd break** assessed which at least half of tray is of harvestable maturity for 2nd break, tray-level measurement Days to Pinset visually number of days elapsed after casing at before 1st break assessed which pins first reach 5 mm, tray-level measurement Percent Harvested calculated % of yield harvested by a designated date 17 days after Speed ratio within break, casing (1st yield at date within break/total yield for break), 24 days break, trial-level measurement after casing (2nd break), 32 days after casing (3rd break) Relative Cap Flesh calculated Cap Flesh Thickness Value/Cap Diameter 1st break**, 2nd Thickness ratio Value, individual mushroom-level break** measurement Relative Cap Height calculated Cap Height Value/Cap Diameter Value, 1st break**, 2nd ratio individual mushroom-level measurement break** Relative Stipe calculated Stipe Diameter Value/Cap Diameter 1st break**, 2nd Diameter ratio Value, individual mushroom-level break** measurement Stipe Diameter hand- millimeters (mm), stipe diameter at base of 1st break**, 2nd measurement cap, individual mushroom-level break** measurement Tray Coverage visually 0 to 5 (from no coverage to full coverage), 1st break**, 2nd assessed tray-level measurement break** Yield (Each Break) weighing kg/m.sup.2, tray-level measurement; or Assessed lb/ft.sup.2, tray-level measurement cumulatively throughout 1st, 2nd, and 3rd breaks Yield (Total) weighing kg/m.sup.2, tray-level measurement; or Assessed lb/ft.sup.2, tray-level measurement cumulatively across 1st, 2nd, and 3rd breaks **= at date when at least half of tray is of harvestable maturity in break

Results

Objective Description of the Variety RO10425

[0104] The following detailed description sets forth the breeding procedures and the characteristics of new cultivar designated RO10425. The strain is maintained and propagated as vegetative mycelium. Spawn (inoculum for the substrate compost) is prepared from the vegetative mycelium. The variety has shown uniformity and stability for the traits, within the limits of expected environmental influence for the traits.

[0105] All homokaryons utilized are strains of Agaricus bisporus.

[0106] Two initial homokaryotic strains were used for breeding. Wild brown strain AA-0096 was crossed with a commercial off-white hybrid mushroom strain.

[0107] More specifically, in a first cross, an AA-0096 slow growing SSI (sg) was crossed with a commercial off-white hybrid protoplast (p).

[0108] A progeny homokaryon resultant from this cross was then selected and backcrossed to the commercial off-white hybrid parent strain. A homokaryon produced by the backcrossing was then selected and crossed to a homokaryon of a bridging cross strain. This cross produced RO10425

[0109] There are multiple characteristics of RO10425 distinguishable from other varieties, such as a different cap color, rounder cap shape, thicker cap apex flesh, thicker cap edge flesh, and higher or as high yield at second break compared to other available brown commercial strains. Other characteristics of RO10425 distinguishable from other varieties are described below (compared to commercial line BR06, commercial line B14528, and related variety RO14176).

[0110] FIG. 9 and FIG. 10 show mushroom caps and longitudinal slices, respectively, of strain RO10425.

Comparison to Other Mushroom Varieties

Initial Trials

Tray-Level Comparisons

[0111] In further evidence of the distinctness and superiority of mushroom strain RO10425, provided below are the results of trials conducted across multiple strains' yielded trays of mushrooms. These tray trials compare yield at each break. Characteristics were measured from 15 trays of strain RO10425 mushrooms and 23 trays of strain BR06 mushrooms (Table 2A and 2B), and corresponding measurements were taken from 24 trays of strain RO14176 mushrooms (Table 3A and 3B).

[0112] Table 2A below provides qualitative comparisons between mushroom strain RO10425 and strain BR06. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO10425.

TABLE-US-00002 TABLE 2A Characteristic RO10425 BR06 First Break Yield (lb/ft.sup.2) Lower Higher Second Break Yield (lb/ft.sup.2) Similar Similar Third Break Yield (lb/ft.sup.2) Higher Lower All Breaks Combined Yield (lb/ft.sup.2) Similar Similar

[0113] Table 2B below provides quantitative comparisons between mushroom strain RO10425 and strain BR06. Column 1 lists the tray-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO10425, column 3 shows the standard deviation of the characteristic for mushroom strain RO10425, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00003 TABLE 2B RO10425 BR06 Characteristic Average St. Dev. Average St. Dev. p-value First Break Yield (lb/ft.sup.2) 1.61 0.64 2.54 1.29 1.33E02 Second Break Yield (lb/ft.sup.2) 1.69 0.48 1.47 0.51 1.92E01 Third Break Yield (lb/ft.sup.2) 1.32 0.41 0.75 0.50 8.12E04 All Breaks Combined Yield (lb/ft.sup.2) 4.62 0.61 4.76 1.24 6.79E01

[0114] Table 3A below provides qualitative comparisons between mushroom strain RO10425 and strain RO14176. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to RO14176, and column 3 shows the qualitative descriptor for mushroom strain RO14176 relative to RO10425.

TABLE-US-00004 TABLE 3A Characteristic RO10425 RO14176 First Break Yield (lb/ft.sup.2) Lower Higher Second Break Yield (lb/ft.sup.2) Similar Similar Third Break Yield (lb/ft.sup.2) Higher Lower All Breaks Combined Yield (lb/ft.sup.2) Lower Higher

[0115] Table 3B below provides quantitative comparisons between mushroom strain RO10425 and strain BR06. Column 1 lists the tray-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO10425, column 3 shows the standard deviation of the characteristic for mushroom strain RO10425, column 4 shows the average value of the characteristic for mushroom strain RO14176, column 5 shows the standard deviation of the characteristic for mushroom strain RO14176, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00005 TABLE 3B RO10425 RO14176 Characteristic Average St. Dev. Average St. Dev. p-value First Break Yield (lb/ft.sup.2) 1.61 0.64 2.54 0.83 7.01E04 Second Break Yield (lb/ft.sup.2) 1.69 0.48 1.91 0.52 1.91E01 Third Break Yield (lb/ft.sup.2) 1.32 0.41 0.92 0.38 3.41E03 All Breaks Combined Yield (lb/ft.sup.2) 4.62 0.61 5.37 1.08 1.94E02

Individual Mushroom-Level Comparisons

[0116] In additional evidence of the distinctness and superiority of mushroom strain RO10425, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Diameter, Cap Height, Cap Flesh Thickness, Stipe Diameter, Relative Cap Height, Relative Cap Flesh Thickness, and Relative Stipe Diameter (as described in Table 1). Measurements from 49 first break and 37 second break RO10425 mushrooms were compared to 80 first break and 40 second break BR06 mushrooms (Tables 4A and 4B).

[0117] Table 4A below provides qualitative comparisons between mushroom strain RO10425 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO10425.

TABLE-US-00006 TABLE 4A Characteristic RO10425 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Similar Similar Cap Height Similar Similar Cap Flesh Thickness Similar Similar Stipe Diameter Larger Smaller Relative Cap Height Similar Similar Relative Cap Flesh Thicker Thinner Thickness Relative Stipe Diameter Larger Smaller Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Larger Smaller Cap Height Taller Shorter Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Similar Similar Relative Cap Flesh Similar Similar Thickness Relative Stipe Diameter Similar Similar

[0118] Table 4B below provides quantitative comparisons between mushroom strain RO10425 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO10425, column 3 shows the standard deviation of the characteristic for mushroom strain RO10425, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00007 TABLE 4B RO10425 BR06 St. St. Characteristic Average Dev. Average Dev. p-value First Break Cap L* Value (dark-light) 65.84 5.15 62.20 5.58 3.22E04 Cap a* Value (green-red) 7.51 0.99 9.43 1.15 5.94E17 Cap b* Value (blue-yellow) 26.03 1.29 28.67 1.63 7.91E17 Cap Diameter 43.45 3.41 44.05 3.80 3.70E01 Cap Height 27.09 1.99 27.45 1.99 3.27E01 Cap Flesh Thickness 15.03 1.42 14.57 1.46 8.09E02 Stipe Diameter 21.66 2.08 20.03 2.08 3.18E05 Relative Cap Height 0.62 0.03 0.62 0.04 9.82E01 Relative Cap Flesh 0.35 0.04 0.33 0.03 1.61E02 Thickness Relative Stipe Diameter 0.56 0.05 0.53 0.03 1.02E03 Second Break Cap L* Value (dark-light) 64.44 2.79 62.19 3.79 4.12E03 Cap a* Value (green-red) 7.05 0.79 7.99 1.07 3.86E05 Cap b* Value (blue-yellow) 26.48 1.15 27.01 1.06 3.64E02 Cap Diameter 45.20 3.51 41.98 4.43 7.39E04 Cap Height 28.78 2.27 26.81 2.89 1.42E03 Cap Flesh Thickness 15.19 1.61 14.30 1.53 1.54E02 Stipe Diameter 20.90 2.02 18.74 3.24 8.38E04 Relative Cap Height 0.64 0.04 0.64 0.03 8.37E01 Relative Cap Flesh 0.34 0.04 0.34 0.02 5.52E01 Thickness Relative Stipe Diameter 0.46 0.04 0.45 0.05 8.97E02

[0119] Additional analyses were performed on the measurements taken across individual mushrooms of each variety at multiple breaks, in order to compare more generalized mushroom traits irrespective of whether the mushrooms belonged to the first vs. second break of harvesting. The same characteristics described above (see Table 1) were measured from 86 mushrooms of strain RO10425 across the first and second break, and corresponding measurements were taken from 150 mushrooms of strain BR06 across the first and second break (Table 5A and 5B).

[0120] Table 5A below provides qualitative comparisons between mushroom strain RO10425 and strain BR06 made irrespective of break. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO10425.

TABLE-US-00008 TABLE 5A Characteristic RO10425 BR06 Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Larger Smaller Cap Height Taller Shorter Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Taller Shorter Relative Cap Flesh Thicker Thinner Thickness Relative Stipe Diameter Larger Smaller

[0121] Table 5B below provides quantitative comparisons between mushroom strain RO10425 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO10425, column 3 shows the standard deviation of the characteristic for mushroom strain RO10425, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00009 TABLE 5B RO10425 BR06 St. St. Characteristic Average Dev. Average Dev. p-value Cap L* Value (dark-light) 65.24 4.33 62.86 4.95 2.49E04 Cap a* Value (green-red) 7.31 0.93 8.77 1.31 3.90E17 Cap b* Value (blue-yellow) 26.22 1.24 28.31 1.61 5.99E21 Cap Diameter 44.20 3.55 42.84 4.00 9.33E03 Cap Height 27.82 2.26 26.53 2.59 1.55E04 Cap Flesh Thickness 15.10 1.50 14.01 1.68 1.12E06 Stipe Diameter 21.33 2.07 18.70 2.98 6.06E12 Relative Cap Height 0.63 0.03 0.62 0.04 4.84E02 Relative Cap Flesh 0.34 0.04 0.33 0.03 9.34E04 Thickness Relative Stipe Diameter 0.48 0.04 0.44 0.05 1.96E12

[0122] Tables 6A and 6B compare instant strain RO10425 and related strain RO14176. Table 6A below provides qualitative comparisons between mushroom strain RO10425 and strain RO14176. Measurements from 49 first break and 37 second break RO10425 mushrooms were compared to 80 first break and 40 second break RO14176 mushrooms (Tables 6A and 6B). Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to RO14176, and column 3 shows the qualitative descriptor for mushroom strain RO14176 relative to RO10425.

TABLE-US-00010 TABLE 6A Characteristic RO10425 RO14176 First Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Similar Similar Cap b* Value (blue-yellow) Similar Similar Cap Diameter Similar Similar Cap Height Shorter Taller Stipe Diameter Larger Smaller Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Larger Smaller Second Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Redder Greener Cap b* Value (blue-yellow) Yellower Bluer Cap Diameter Larger Smaller Cap Height Similar Similar Stipe Diameter Larger Smaller Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Thinner Thicker Relative Stipe Diameter Similar Similar

[0123] Table 6B below provides quantitative comparisons between mushroom strain RO10425 and strain RO14176. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO10425, column 3 shows the standard deviation of the characteristic for mushroom strain RO10425, column 4 shows the average value of the characteristic for mushroom strain RO14176, column 5 shows the standard deviation of the characteristic for mushroom strain RO14176, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00011 TABLE 6B RO10425 RO14176 St. St. Characteristic Average Dev. Average Dev. p-value First Break Cap L* Value (dark-light) 65.84 5.15 65.91 4.79 9.38E01 Cap a* Value (green-red) 7.51 0.99 7.49 1.05 9.32E01 Cap b* Value (blue-yellow) 26.03 1.29 26.35 1.35 1.82E01 Cap Diameter 43.45 3.41 43.53 3.27 9.01E01 Cap Height 27.09 1.99 27.89 2.12 3.55E02 Cap Flesh Thickness 15.03 1.42 15.33 1.36 2.42E01 Stipe Diameter 21.66 2.08 19.99 1.90 7.06E06 Relative Cap Height 0.62 0.03 0.64 0.03 2.43E03 Relative Cap Flesh 0.35 0.04 0.35 0.03 3.25E01 Thickness Relative Stipe Diameter 0.50 0.04 0.46 0.04 4.71E07 Second Break Cap L* Value (dark-light) 64.44 2.79 65.61 3.67 1.24E01 Cap a* Value (green-red) 7.05 0.79 6.36 0.76 2.32E04 Cap b* Value (blue-yellow) 26.48 1.15 25.05 1.46 1.07E05 Cap Diameter 45.20 3.51 41.88 2.68 1.23E05 Cap Height 28.78 2.27 28.22 2.12 2.65E01 Cap Flesh Thickness 15.19 1.61 15.75 1.61 1.31E01 Stipe Diameter 20.90 2.02 19.16 2.29 6.92E04 Relative Cap Height 0.64 0.04 0.67 0.04 7.23E05 Relative Cap Flesh 0.34 0.04 0.38 0.04 1.79E05 Thickness Relative Stipe Diameter 0.46 0.04 0.46 0.05 6.21E01

[0124] FIG. 6 shows representative caps and longitudinal sections of caps from mushrooms of strain RO10425 compared to mushrooms of strain RO14176.

[0125] Additional analysis was conducted for measurements taken across individual mushrooms of each variety at multiple breaks, in order to compare more generalized mushroom traits irrespective of whether the mushrooms belonged to the first vs. second break of harvesting. The same characteristics described above (see Table 1) were measured from 86 mushrooms of strain RO10425 across the first and second break, and corresponding measurements were taken from 170 mushrooms of strain RO14176 across the first and second break (Table 7A and 7B). Table 7A below provides qualitative comparisons between mushroom strain RO10425 and strain RO14176. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to RO14176, and column 3 shows the qualitative descriptor for mushroom strain RO14176 relative to RO10425.

TABLE-US-00012 TABLE 7A Characteristic RO10425 RO14176 Cap L* Value (dark-light) Darker Lighter Cap a* Value (green-red) Redder Greener Cap b* Value (blue-yellow) Similar Similar Cap Diameter Larger Shorter Cap Height Taller Shorter Cap Flesh Thickness Similar Similar Stipe Diameter Larger Smaller Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Larger Smaller

[0126] Table 7B below provides quantitative comparisons between mushroom strain RO10425 and strain RO14176. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO10425, column 3 shows the standard deviation of the characteristic for mushroom strain RO10425, column 4 shows the average value of the characteristic for mushroom strain RO14176, column 5 shows the standard deviation of the characteristic for mushroom strain RO14176, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00013 TABLE 7B RO10425 RO14176 St. St. Characteristic Average Dev. Average Dev. p-value Cap L* Value (dark-light) 65.24 4.33 67.15 4.61 1.60E03 Cap a* Value (green-red) 7.31 0.93 6.93 1.09 6.45E03 Cap b* Value (blue-yellow) 26.22 1.24 26.44 1.59 2.60E01 Cap Diameter 44.20 3.55 42.36 3.30 5.22E05 Cap Height 27.82 2.26 27.14 2.37 2.88E02 Cap Flesh Thickness 15.10 1.50 14.70 1.84 7.93E02 Stipe Diameter 21.33 2.07 18.53 2.68 1.68E15 Relative Cap Height 0.63 0.03 0.64 0.04 2.54E02 Relative Cap Flesh 0.34 0.04 0.35 0.04 3.75E01 Thickness Relative Stipe Diameter 0.48 0.04 0.44 0.05 2.98E11

Additional Trials

Tray-Level Comparisons

[0127] Several additional trials were conducted for characterizing RO10425. Table 8 below provides qualitative comparisons between mushroom strain RO10425 and strain BR06. Traits of particular interest are bolded. Column 1 lists the tray-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO10425.

TABLE-US-00014 TABLE 8 Characteristic RO10425 BR06 Before First Break Days to Pinset Similar Similar First Break Days to 1st Break Similar Similar Percent Harvested Speed Similar Similar Yield (kg/m.sup.2) Similar Similar Second Break Days to 2nd Break Similar Similar Yield (kg/m.sup.2) Higher Lower Third Break Yield (kg/m.sup.2) Similar Similar All Breaks Combined Yield (kg/m.sup.2) Similar Similar

[0128] The calculations that informed these qualitative descriptors are shown in Table 10.

[0129] Table 9 below provides qualitative comparisons between mushroom strain RO10425 and strain B14528. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO10425.

TABLE-US-00015 TABLE 9 Characteristic RO10425 B14528 Before First Break Days to Pinset Similar Similar First Break Days to 1st Break Similar Similar Percent Harvested Speed Similar Similar Yield (kg/m.sup.2) Similar Similar Second Break Days to 2nd Break Similar Similar Yield (kg/m.sup.2) Similar Similar Third Break Yield (kg/m.sup.2) Similar Similar All Breaks Combined Yield (kg/m.sup.2) Similar Similar

[0130] The calculations that informed these qualitative descriptors are shown in Table 10.

[0131] The first part of analyzing trait measurements in the additional trials herein included making comparisons between all three of instant strain RO10425, comparative strain BR06, and comparative strain B14528. For these three strains, a Shapiro-Wilk test and a Levene test were performed first to assess if the data satisfy the assumptions of normality and equal variances among groups, respectively, of a standard ANOVA. The Shapiro-Wilk test and Levene test determined that most of these traits satisfied neither assumption. Therefore, a Kruskal-Wallis test (Kruskal, W. H., & Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. Journal of the American statistical Association, 47(260), 583-621), a non-parametric variation of ANOVA that does not assume normality, equal variances among groups, or equal sample sizes among groups, was performed. Medians and interquartile ranges (IQR) were reported, as these metrics are robust to non-normal distributions. Means are also reported for consistency with normally-distributed traits.

[0132] The Shapiro-Wilk test and Levene test determined that most developmental timing traits (Days to Pinset, Days to 1st Break, and Days to 2nd Break) and yield traits were not normally distributed and variances among groups were not equal. The Kruskal-Wallis test and Dunn post-hoc test (Dunn's Test; Dunn, O. J. (1964). Multiple comparisons using rank sums. Technometrics, 6(3), 241-252) were used in these cases. When the Kruskal-Wallis test results were significant based on a threshold of p<0.05, Dunn's test was performed post-hoc to make pairwise strain comparisons (Dunn, 1964). P-values were adjusted using the Bonferroni correction to minimize risk of Type 1 error (false significance). An exception to this trend was Percent Harvested Speed, which was normally distributed and exhibited equal variance for all strains. For assessing Percent Harvested Speed, a standard ANOVA was used and a Tukey Honestly Significant Difference (HSD) test was performed post-hoc for pairwise strain comparisons. Standard deviations multiplied by 100 for consistency alongside percentages (SD*100) were reported for this trait. This testing was performed on groups of three strains, as displayed in Table 10, but the other results herein using this test show a pairwise subset of the tests for consistency with the initial trials.

[0133] In Table 10, column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test. Traits of particular interest are bolded.

TABLE-US-00016 TABLE 10 Significance Number Group Variety Mean Median IQR of Trials (Dunn's Test) Before First Break Days to Pinset RO10425 12.00 12.00 2.00 4 a B14528 12.00 12.00 1.75 4 a BR06 12.40 12.00 1.00 4 a First Break Days to 1st Break RO10425 18.00 18.00 1.00 4 a B14528 17.60 18.00 1.75 4 a BR06 18.10 18.00 1.00 4 a Yield (kg/m.sup.2) RO10425 10.64 10.64 1.76 3 a B14528 9.81 9.52 4.44 3 a BR06 10.30 8.35 5.57 3 a Second Break Days to 2nd Break RO10425 24.90 25.00 1.75 4 a B14528 24.80 25.00 1.00 4 a BR06 25.30 26.00 1.00 4 a Yield (kg/m.sup.2) RO10425 8.74 8.10 3.27 3 a B14528 7.13 6.93 1.51 3 ab BR06 6.20 5.86 1.61 3 a Third Break Yield (kg/m.sup.2) RO10425 3.66 3.52 0.78 3 ab B14528 5.81 5.42 1.81 3 b BR06 2.64 1.95 2.10 3 a All Breaks Combined Yield (kg/m.sup.2) RO10425 23.05 23.14 4.25 3 a B14528 22.75 22.61 2.29 3 a BR06 19.14 18.70 5.52 3 a Significance Percent Harvested Speed Group (measured in First Break) SD * 100 (Tukey HSD) RO10425 36.7% 34.8% 32.70 3 a B14528 36.2% 45.2% 25.00 3 a BR06 28.9% 12.9% 37.70 3 a

Individual Mushroom-Level Comparisons

[0134] In additional evidence of the distinctness and superiority of mushroom strain RO10425, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Apex Flesh Thickness, Cap Edge Flesh Thickness, and Cap Roundness (Calculated) (as described in Table 1). Measurements were taken for hundreds of mushrooms per trait, noted as sample size (n) in Tables 11B and 12B.

[0135] Table 11A below provides qualitative comparisons between mushroom strain RO10425 and strain BR06. Traits of particular interest are bolded. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO10425.

TABLE-US-00017 TABLE 11A Characteristic RO10425 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter

[0136] Table 11B shows additional quantitative comparisons between mushroom strain RO10425 and strain BR06. Column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test.

TABLE-US-00018 TABLE 11B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO10425 65.66 65.22 7.34 209 a BR06 63.30 62.74 9.83 797 b Cap a* Value (green-red) RO10425 7.35 7.43 1.97 209 a BR06 8.67 8.96 2.58 787 b Cap b* Value (blue-yellow) RO10425 28.43 28.58 3.10 209 a BR06 28.72 29.04 3.17 776 b Cap Apex Flesh Thickness RO10425 0.55 0.55 0.04 166 a BR06 0.52 0.52 0.05 541 b Cap Edge Flesh Thickness RO10425 0.30 0.31 0.05 166 a BR06 0.21 0.21 0.09 537 b Cap Roundness (Calculated) RO10425 0.64 0.63 0.05 166 a BR06 0.61 0.61 0.04 541 b Second Break Cap L* Value (dark-light) RO10425 68.82 69.47 8.20 157 a BR06 65.68 65.49 5.90 340 b Cap a* Value (green-red) RO10425 6.15 6.26 2.55 157 a BR06 7.21 7.31 2.42 340 b Cap b* Value (blue-yellow) RO10425 27.69 27.46 2.54 157 a BR06 28.15 28.49 2.86 340 b Cap Apex Flesh Thickness RO10425 0.55 0.55 0.04 157 a BR06 0.51 0.52 0.07 362 b Cap Edge Flesh Thickness RO10425 0.32 0.32 0.05 157 a BR06 0.23 0.23 0.09 362 b Cap Roundness (Calculated) RO10425 0.61 0.61 0.05 157 a BR06 0.60 0.59 0.06 363 b

[0137] Table 12A below provides qualitative comparisons between mushroom strain RO10425 and strain B14528. Traits of interest are bolded in Tables 12A and 12B. Column 1 lists the individual mushroom-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO10425.

TABLE-US-00019 TABLE 12A Characteristic RO10425 B14528 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter Second Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Similar Similar Cap b* Value (blue-yellow) Similar Similar Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter

[0138] Table 12B below provides quantitative comparisons between mushroom strain RO10425 and strain B14528, which resulted in the qualitative descriptors of Table 12A. Column 1 shows each individual mushroom-level trait (as described in Table 1) and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of mushrooms assessed (n), and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test.

TABLE-US-00020 TABLE 12B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO10425 65.66 65.22 7.34 209 a B14528 64.06 64.13 6.56 340 c Cap a* Value (green-red) RO10425 7.35 7.43 1.97 209 a B14528 8.26 8.31 2.13 340 c Cap b* Value (blue-yellow) RO10425 28.43 28.58 3.10 209 a B14528 30.31 30.16 3.39 340 c Cap Apex Flesh Thickness RO10425 0.55 0.55 0.04 166 a B14528 0.47 0.46 0.06 387 c Cap Edge Flesh Thickness RO10425 0.30 0.31 0.05 166 a B14528 0.22 0.22 0.08 387 b Cap Roundness (Calculated) RO10425 0.64 0.63 0.05 166 a B14528 0.61 0.61 0.05 389 b Second Break Cap L* Value (dark-light) RO10425 68.82 69.47 8.20 157 a B14528 68.56 69.02 6.54 170 a Cap a* Value (green-red) RO10425 6.15 6.26 2.55 157 a B14528 6.09 5.96 2.05 170 a Cap b* Value (blue-yellow) RO10425 27.69 27.46 2.54 157 a B14528 27.37 27.78 3.22 170 a Cap Apex Flesh Thickness RO10425 0.55 0.55 0.04 157 a B14528 0.46 0.46 0.05 198 c Cap Edge Flesh Thickness RO10425 0.32 0.32 0.05 157 a B14528 0.23 0.23 0.06 198 b Cap Roundness (Calculated) RO10425 0.61 0.61 0.05 157 a B14528 0.60 0.60 0.05 198 b

[0139] As conducted in initial trials, comparisons were made between instant strain RO10425 and related strain RO14176. Tables 13A below provides qualitative comparisons between mushroom strain RO10425 and strain RO14176. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO10425 relative to RO14176, and column 3 shows the qualitative descriptor for mushroom strain RO14176 relative to RO10425.

TABLE-US-00021 TABLE 13A Characteristic RO10425 RO14176 First Break Cap L* Value (dark-light) Darker Lighter Cap a* Value (green-red) Redder Greener Cap b* Value (blue-yellow) Similar Similar Cap Diameter Similar Similar Cap Height Similar Similar Cap Flesh Thickness Similar Similar Stipe Diameter Larger Smaller Relative Cap Height Similar Similar Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Larger Smaller Second Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Similar Similar Cap b* Value (blue-yellow) Similar Similar Cap Diameter Similar Similar Cap Height Similar Similar Cap Flesh Thickness Similar Similar Stipe Diameter Larger Smaller Relative Cap Height Similar Similar Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Larger Smaller

[0140] Table 13B below provides quantitative comparisons between mushroom strain RO10425 and strain RO14176 that informed the qualitative comparisons of Table 13A. In Table 13B, Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO10425, column 3 shows the standard deviation of the characteristic for mushroom strain RO10425, column 4 shows the average value of the characteristic for mushroom strain RO14176, column 5 shows the standard deviation of the characteristic for mushroom strain RO14176, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00022 TABLE 13B RO10425 RO14176 St. St. Characteristic Average Dev. Average Dev. p-value First Break Cap L* Value (dark-light) 65.17 5.27 66.97 4.84 1.18E05 Cap a* Value (green-red) 7.55 1.68 6.89 1.63 6.95E07 Cap b* Value (blue-yellow) 28.52 2.09 28.46 2.07 6.92E01 Cap Diameter 43.18 3.81 43.25 3.94 8.21E01 Cap Height 27.67 2.65 27.87 2.19 3.44E01 Cap Flesh Thickness 15.26 1.61 15.19 1.73 6.44E01 Stipe Diameter 21.66 2.08 19.92 2.43 1.94E06 Relative Cap Height 0.65 0.04 0.65 0.04 3.41E01 Relative Cap Flesh 0.35 0.03 0.35 0.05 1.66E01 Thickness Relative Stipe Diameter 0.50 0.04 0.45 0.05 1.50E09 Second Break Cap L* Value (dark-light) 68.82 5.25 69.32 4.59 3.11E01 Cap a* Value (green-red) 6.15 1.63 6.02 1.64 4.26E01 Cap b* Value (blue-yellow) 27.69 1.91 27.50 2.11 3.19E01 Cap Diameter 43.19 4.08 42.56 3.62 1.08E01 Cap Height 26.65 2.49 26.28 2.29 1.31E01 Cap Flesh Thickness 14.51 1.34 14.24 1.60 6.04E02 Stipe Diameter 20.90 2.02 17.80 2.58 4.81E11 Relative Cap Height 0.62 0.04 0.62 0.05 3.57E01 Relative Cap Flesh 0.34 0.03 0.34 0.04 9.52E01 Thickness Relative Stipe Diameter 0.46 0.04 0.42 0.05 1.84E06

[0141] One of the advantages of the strain RO10425 is its incompatibility with other commercial strains. Table 14 below provides results of compatibility tests between strain RO10425 and commercial strain BR06, commercial strain B14528, related strain RO12756, and related strain RO14176. These results indicate that strain RO10425 is incompatible with strain BR06, strain B14528, and related strain RO12756, while strain RO10425 is compatible with related strain RO14176. This was true regardless of which tested strain served as the compost variety and which tested strain served as the casing variety.

TABLE-US-00023 TABLE 14 Strains (compost variety/ Mean Median SD Significance casing variety) lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 Trials Group RO10425/RO10425 2.71 13.23 2.86 13.94 0.50 2.44 3 a RO12756/RO12756 2.62 12.79 2.61 12.74 0.41 2.00 3 a RO14176/RO14176 2.66 12.99 2.41 11.77 0.61 2.98 3 a BR06/BR06 2.62 12.79 2.46 11.99 1.15 5.61 2 a B14528/B14528 2.93 14.31 2.92 14.23 0.34 1.66 1 a RO10425/RO14176, 2.43 11.86 2.75 13.40 0.77 3.76 1 a RO14176/RO10425 RO10425/B14528, 0.71 3.47 0.65 3.15 0.17 0.83 1 b B14528/RO10425 RO12756/RO10425, 0.91 4.44 1.04 5.08 0.35 1.71 1 b RO10425/RO12756 RO10425/BR06, 0.80 3.91 0.92 4.47 0.51 2.49 1 b BR06/RO10425

Example 2: Characterization of RO14176

[0142] The following example describes the characterization of strain RO14176 compared to commercial varieties.

Methodology

[0143] The measurements and calculations were conducted as in Example 1.

Results

Objective Description of the Variety RO14176

[0144] The following detailed description sets forth the breeding procedures and the characteristics of new cultivar designated RO14176. The strain is maintained and propagated as vegetative mycelium. Spawn (inoculum for the substrate compost) is prepared from the vegetative mycelium. The variety has shown uniformity and stability for the traits, within the limits of environmental influence for the traits.

[0145] All homokaryons utilized are strains of Agaricus bisporus.

[0146] RO14176 was produced as a heterokaryon SSI derived from strain RO10425.

[0147] RO14176 sets pins and produces harvestable mushrooms at first break and second break as fast or faster than available brown commercial strains. RO14176 additionally exhibits higher yield at third break, and higher overall yield, than available brown commercial strains. RO14176 also exhibits rounder cap shape, thicker cap apex flesh, thicker cap edge flesh, and a new cap color compared to available brown commercial strains. RO14176 additionally exhibits Percent Harvested Speed faster or as fast as other available brown commercial strains.

[0148] Other characteristics of RO14176 distinguishable from other varieties are described below (compared to commercial line BR06 and commercial line B14528).

[0149] FIG. 7 and FIG. 8 show mushroom caps and longitudinal slices, respectively, of strain RO14176.

Comparisons to Commercial Mushroom Varieties

Initial Trials

Tray-Level Comparisons

[0150] In further evidence of the distinctness and superiority of mushroom strain RO14176, provided below are the results of trials conducted across multiple strains' yielded trays of mushrooms. These trials compare timing for trays to reach pinset and harvestable maturity, tray coverage, cap color darkness, cap firmness, cap roundness (visual), and yield at each break. Characteristics were measured from 46 trays of strain RO14176 mushrooms for yield traits and from 26 trays of strain RO14176 mushrooms for other traits. Corresponding measurements were taken from 38 trays of strain BR06 mushrooms for yield traits and 19 trays of strain BR06 mushrooms for other traits (Table 15A and 15B), and corresponding measurements were taken from 17 trays of strain B14528 mushrooms (Table 16A and 16B).

[0151] Table 15A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00024 TABLE 15A Characteristic RO14176 BR06 Before First Break Days to Pinset Faster Slower First Break Days to 1st Break Faster Slower Tray Coverage Higher Lower Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Rounder Flatter Yield (lb/ft.sup.2) Similar Similar Second Break Days to 2nd Break Similar Similar Tray Coverage Similar Similar Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Flatter Rounder Yield (lb/ft.sup.2) Higher Lower Third Break Yield (lb/ft.sup.2) Similar Similar All Breaks Combined Yield (lb/ft.sup.2) Higher Lower

[0152] Table 15B below provides initial quantitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the tray-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00025 TABLE 15B RO14176 BR06 St. St. Characteristic Average Dev. Average Dev. p-value Before First Break Days to Pinset 11.23 0.82 12.68 1.42 8.25E05 First Break Days to 1st Break 17.46 1.03 18.53 1.71 1.27E02 Tray Coverage 3.88 0.91 3.16 1.30 3.27E02 Cap Color Darkness 3.85 0.37 4.21 0.42 3.46E03 Cap Firmness 3.69 0.47 4.68 0.48 1.57E08 Cap Roundness (Visual) 3.38 0.50 2.95 0.23 9.07E04 Yield (lb/ft.sup.2) 2.47 0.80 2.16 1.36 2.00E01 Second Break Days to 2nd Break 24.42 0.86 24.95 0.91 5.48E02 Tray Coverage 3.81 0.49 3.42 1.22 1.49E01 Cap Color Darkness 3.58 0.50 4.05 0.23 4.15E04 Cap Firmness 3.81 0.40 4.44 0.51 3.61E05 Cap Roundness (Visual) 2.69 0.47 2.94 0.24 4.24E02 Yield (lb/ft.sup.2) 1.93 0.42 1.47 0.63 1.68E04 Third Break Yield (lb/ft.sup.2) 1.00 0.33 0.87 0.47 1.66E01 All Breaks Combined Yield (lb/ft.sup.2) 5.39 0.92 4.51 1.44 9.79E04

[0153] Table 16A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00026 TABLE 16A Characteristic RO14176 B14528 Before First Break Days to Pinset Similar Similar First Break Days to 1st Break Similar Similar Tray Coverage Similar Similar Cap Color Darkness Similar Similar Cap Firmness Similar Similar Cap Roundness (Visual) Rounder Flatter Yield (lb/ft.sup.2) Similar Similar Second Break Days to 2nd Break Faster Slower Tray Coverage Higher Lower Cap Color Darkness Similar Similar Cap Firmness Similar Similar Cap Roundness (Visual) Similar Similar Yield (lb/ft.sup.2) Similar Similar Third Break Yield (lb/ft.sup.2) Similar Similar All Breaks Combined Yield (lb/ft.sup.2) Similar Similar

[0154] Table 16B below provides initial quantitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the tray-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain B14528, column 5 shows the standard deviation of the characteristic for mushroom strain B14528, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00027 TABLE 16B RO14176 B14528 St. St. Characteristic Average Dev. Average Dev. p-value Before First Break Days to Pinset 11.23 0.82 11.41 0.87 4.92E01 First Break Days to 1st Break 17.46 1.03 17.47 0.51 9.73E01 Tray Coverage 3.88 0.91 3.59 0.87 2.94E01 Cap Color Darkness 3.85 0.37 3.82 0.39 8.49E01 Cap Firmness 3.69 0.47 3.88 0.33 1.56E01 Cap Roundness (Visual) 3.38 0.50 2.88 0.33 7.05E04 Yield (lb/ft.sup.2) 2.47 0.80 2.39 0.54 7.11E01 Second Break Days to 2nd Break 24.42 0.86 25.12 0.86 1.30E02 Tray Coverage 3.81 0.49 3.47 0.51 3.67E02 Cap Color Darkness 3.58 0.50 3.76 0.56 2.60E01 Cap Firmness 3.81 0.40 3.82 0.39 8.99E01 Cap Roundness (Visual) 2.69 0.47 2.82 0.39 3.47E01 Yield (lb/ft.sup.2) 1.93 0.42 1.74 0.38 1.09E01 Third Break Yield (lb/ft.sup.2) 1.00 0.33 1.18 0.38 6.16E02 All Breaks Combined Yield (lb/ft.sup.2) 5.39 0.92 5.31 0.76 7.45E01

Individual Mushroom-Level Comparisons

[0155] In additional evidence of the distinctness and superiority of mushroom strain RO14176, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Diameter, Cap Height, Cap Flesh Thickness, Stipe Diameter, Relative Cap Height, Relative Cap Flesh Thickness, and Relative Stipe Diameter (as described in Table 1). Measurements from 210 first break and 130 second break RO14176 mushrooms were compared to 350 first break and 130 second break BR06 mushrooms (Tables 17A and 17B) and 90 first break and 40 second break B14528 mushrooms (Tables 18A and 18B).

[0156] Table 17A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00028 TABLE 17A Characteristic RO14176 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Larger Smaller Cap Height Taller Shorter Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Taller Shorter Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Similar Similar Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Smaller Larger Cap Height Similar Similar Cap Flesh Thickness Thicker Thinner Stipe Diameter Smaller Larger Relative Cap Height Taller Shorter Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Smaller Larger

[0157] Table 17B below provides initial quantitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00029 TABLE 17B RO14176 BR06 St. St. Characteristic Average Dev. Average Dev. p-value First Break Cap L* Value (dark-light) 65.73 4.23 63.49 6.52 1.00E05 Cap a* Value (green-red) 7.64 1.00 8.53 1.85 2.45E10 Cap b* Value (blue-yellow) 26.84 1.43 28.15 2.48 7.83E12 Cap Diameter 43.93 3.61 42.78 4.41 1.45E03 Cap Height 28.15 2.18 25.75 2.35 1.27E29 Cap Flesh Thickness 15.37 1.96 13.25 1.46 1.88E44 Stipe Diameter 19.92 2.43 19.25 2.47 1.19E03 Relative Cap Height 0.64 0.03 0.60 0.04 4.25E33 Relative Cap Flesh 0.35 0.04 0.31 0.03 3.34E39 Thickness Relative Stipe Diameter 0.45 0.05 0.45 0.06 5.18E01 Second Break Cap L* Value (dark-light) 68.87 3.95 64.27 3.81 5.37E20 Cap a* Value (green-red) 6.66 0.95 8.20 1.14 1.67E27 Cap b* Value (blue-yellow) 26.77 1.64 28.31 1.65 1.08E13 Cap Diameter 42.13 3.35 43.26 4.15 1.32E02 Cap Height 26.36 2.27 25.80 2.61 5.55E02 Cap Flesh Thickness 14.15 1.77 13.46 1.52 6.12E04 Stipe Diameter 17.80 2.58 18.97 3.67 2.54E03 Relative Cap Height 0.63 0.05 0.60 0.04 1.01E07 Relative Cap Flesh 0.34 0.04 0.31 0.03 1.18E07 Thickness Relative Stipe Diameter 0.42 0.05 0.44 0.08 3.93E02

[0158] Table 18A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00030 TABLE 18A Characteristic RO14176 B14528 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Similar Similar Cap Height Taller Shorter Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Taller Shorter Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Larger Smaller Second Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Smaller Larger Cap Height Similar Similar Cap Flesh Thickness Thicker Thinner Stipe Diameter Similar Similar Relative Cap Height Taller Shorter Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Similar Similar

[0159] Table 18B below provides initial quantitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO14176, column 3 shows the standard deviation of the characteristic for mushroom strain RO14176, column 4 shows the average value of the characteristic for mushroom strain B14528, column 5 shows the standard deviation of the characteristic for mushroom strain B14528, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00031 TABLE 18B RO14176 B14528 St. St. Characteristic Average Dev. Average Dev. p-value First Break Cap L* Value (dark-light) 65.73 4.23 64.48 5.25 2.98E02 Cap a* Value (green-red) 7.64 1.00 8.03 1.65 1.30E02 Cap b* Value (blue-yellow) 26.84 1.43 29.38 1.50 3.68E34 Cap Diameter 43.93 3.61 43.84 3.80 8.49E01 Cap Height 28.15 2.18 26.60 1.97 1.91E08 Cap Flesh Thickness 15.37 1.96 12.91 0.93 2.05E27 Stipe Diameter 19.92 2.43 17.58 1.44 3.66E17 Relative Cap Height 0.64 0.03 0.61 0.03 1.59E15 Relative Cap Flesh 0.35 0.04 0.30 0.03 2.71E28 Thickness Relative Stipe Diameter 0.45 0.05 0.40 0.04 2.36E18 Second Break Cap L* Value (dark-light) 68.87 3.95 68.52 4.46 6.36E01 Cap a* Value (green-red) 6.66 0.95 7.88 1.49 7.36E09 Cap b* Value (blue-yellow) 26.77 1.64 28.55 1.65 1.30E08 Cap Diameter 42.13 3.35 44.86 3.59 1.67E05 Cap Height 26.36 2.27 26.29 1.72 8.51E01 Cap Flesh Thickness 14.15 1.77 12.60 1.32 8.12E07 Stipe Diameter 17.80 2.58 18.11 3.29 5.33E01 Relative Cap Height 0.63 0.05 0.59 0.04 1.36E05 Relative Cap Flesh 0.34 0.04 0.28 0.03 9.66E13 Thickness Relative Stipe Diameter 0.42 0.05 0.40 0.07 9.03E02

Additional Trials

Tray-Level Comparisons

[0160] Several additional trials were conducted for characterizing RO14176. In further evidence of the distinctness and superiority of mushroom strain RO14176, provided below are the results of trials conducted across multiple strains' yielded trays of mushrooms. These trials compare timing for trays to reach pinset and harvestable maturity, and yield at each break.

[0161] Table 19A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Traits of particular interest are bolded. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00032 TABLE 19A Characteristic RO14176 BR06 Before First Break Days to Pinset Faster Slower First Break Days to 1st Break Faster Slower Percent Harvested Speed Faster Slower Yield (kg/m.sup.2) Similar Similar Second Break Days to 2nd Break Faster Slower Yield (kg/m.sup.2) Similar Similar Third Break Yield (kg/m.sup.2) Higher Lower All Breaks Combined Yield (kg/m.sup.2) Higher Lower

[0162] Table 19B below provides further quantitative comparisons between mushroom strain RO14176 and strain BR06.

[0163] The first part of analyzing trait measurements in the additional trials herein included making comparisons between all three of instant strain RO14176, comparative strain BR06, and comparative strain B14528. For these three strains, a Shapiro-Wilk test and a Levene test were performed first to assess if the data satisfy the assumptions of normality and equal variances among groups, respectively, of a standard ANOVA. The Shapiro-Wilk test and Levene test determined that most of these traits satisfied neither assumption. Therefore, a Kruskal-Wallis test (Kruskal, W. H., & Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. Journal of the American statistical Association, 47(260), 583-621), a non-parametric variation of ANOVA that does not assume normality, equal variances among groups, or equal sample sizes among groups, was performed. Medians and interquartile ranges (IQR) were reported, as these metrics are robust to non-normal distributions. Means are also reported for consistency with normally-distributed traits.

[0164] The Shapiro-Wilk test and Levene test determined that most developmental timing traits (Days to Pinset, Days to 1st Break, and Days to 2nd Break) and yield traits were not normally distributed and variances among groups were not equal. The Kruskal-Wallis test and Dunn post-hoc test (Dunn's Test; Dunn, O. J. (1964). Multiple comparisons using rank sums. Technometrics, 6(3), 241-252) were used in these cases. When the Kruskal-Wallis test results were significant based on a threshold of p<0.05, Dunn's test was performed post-hoc to make pairwise strain comparisons (Dunn, 1964). P-values were adjusted using the Bonferroni correction to minimize risk of Type 1 error (false significance). An exception to this trend was Percent Harvested Speed, which was normally distributed and exhibited equal variance for all strains. For assessing Percent Harvested Speed, a standard ANOVA was used and a Tukey Honestly Significant Difference (HSD) test was performed post-hoc for pairwise strain comparisons. Standard deviations multiplied by 100 for consistency alongside percentages (SD* 100) were reported for this trait. This testing was performed on groups of three strains, but the results show a pairwise subset of the tests for consistency with the initial trials.

[0165] In Table 19B, column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test. Traits of particular interest are bolded.

TABLE-US-00033 TABLE 19B Significance Number Group Variety Mean Median IQR of Trials (Dunn's Test) Before First Break Days to Pinset RO14176 11.4 12.0 1.00 7 a BR06 12.7 12.5 1.00 7 b First Break Days to 1st Break RO14176 17.7 18.0 1.00 7 a BR06 18.8 18.0 2.00 7 b Yield (kg/m.sup.2) RO14176 12.40 12.25 4.69 6 a BR06 10.99 11.03 8.10 6 a Second Break Days to 2nd Break RO14176 25.3 25.0 2.00 7 a BR06 25.9 26.0 2.00 7 b Yield (kg/m.sup.2) RO14176 8.45 8.50 3.52 6 a BR06 7.37 6.59 5.13 6 a Third Break Yield (kg/m.sup.2) RO14176 4.20 3.71 2.69 6 a BR06 3.27 2.34 3.03 6 b All Breaks Combined Yield (kg/m.sup.2) RO14176 25.05 23.88 3.66 6 a BR06 21.58 22.02 4.39 6 b Significance Percent Harvested Speed Group (measured in First Break) SD * 100 (Tukey HSD) RO14176 38.7% 39.9% 13.10 5 b BR06 14.5% 12.9% 16.90 5 a

[0166] Table 20A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Traits of particular interest are bolded. Column 1 lists the tray-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00034 TABLE 20A Characteristic RO14176 B14528 Before First Break Days to Pinset Similar Similar First Break Days to 1st Break Similar Similar Percent Harvested Speed Similar Similar Yield (kg/m.sup.2) Similar Similar Second Break Days to 2nd Break Similar Similar Yield (kg/m.sup.2) Similar Similar Third Break Yield (kg/m.sup.2) Similar Similar All Breaks Combined Yield (kg/m.sup.2) Similar Similar

[0167] Table 20B below provides additional quantitative comparison between mushroom strain RO14176 and strain B14528. Column 1 shows the strains compared for each trait, column 2 shows the mean value for that strain's measurements, column 3 shows the median value of the same measurements, column 4 shows either the interquartile range (IQR) or the standard deviation multiplied by 100 for consistency alongside percentages (SD*100) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to either Dunn's Test or Tukey HSD.

TABLE-US-00035 TABLE 20B Significance Number Group Characteristic Mean Median IQR of Trials (Dunn's Test) Before First Break Days to Pinset RO14176 11.4 12.0 1.00 7 a B14528 11.2 11.0 1.50 7 a First Break Days to 1st Break RO14176 17.7 18.0 1.00 7 a B14528 17.6 18.0 1.00 7 a Yield (kg/m.sup.2) RO14176 12.40 12.25 4.69 6 a B14528 11.91 12.21 4.35 6 a Second Break Days to 2nd Break RO14176 25.3 25.0 2.00 7 a B14528 25.5 25.0 1.00 7 ab Yield (kg/m.sup.2) RO14176 8.45 8.50 3.52 6 a B14528 7.47 7.76 2.93 6 a Third Break Yield (kg/m.sup.2) RO14176 4.20 3.71 2.69 6 a B14528 4.49 3.95 2.59 6 a All Breaks Combined Yield (kg/m.sup.2) RO14176 25.05 23.88 3.66 6 a B14528 23.88 23.97 6.69 6 a Percent Harvested Significance Speed (measured Group in First Break) Mean Median SD * 100 (Tukey HSD) RO14176 38.7% 39.9% 13.10 5 b B14528 41.8% 40.3% 7.60 5 b

Individual Mushroom-Level Comparisons

[0168] In additional evidence of the distinctness and superiority of mushroom strain RO14176, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Apex Flesh Thickness, Cap Edge Flesh Thickness, and Cap Roundness (Calculated) (as described in Table 1). Measurements were taken for hundreds of mushrooms per trait, noted as sample size (n) in Tables 21B and 22B.

[0169] Table 21A below provides qualitative comparisons between mushroom strain RO14176 and strain BR06. Traits of particular interest are bolded. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO14176.

TABLE-US-00036 TABLE 21A Characteristic RO14176 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter

[0170] Table 21B below provides additional quantitative comparisons between mushroom strain RO14176 and strain BR06. Column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of mushrooms assessed (n), and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test.

TABLE-US-00037 TABLE 21B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO14176 65.76 65.82 5.82 469 a BR06 63.30 62.74 9.83 797 b Cap a* Value (green-red) RO14176 7.45 7.47 1.58 469 a BR06 8.67 8.96 2.58 787 b Cap b* Value (blue-yellow) RO14176 28.17 28.03 2.64 469 a BR06 28.72 29.04 3.17 776 b Cap Apex Flesh Thickness RO14176 0.54 0.54 0.06 453 a BR06 0.52 0.52 0.05 541 b Cap Edge Flesh Thickness RO14176 0.27 0.26 0.07 449 a BR06 0.21 0.21 0.09 537 b Cap Roundness (Calculated) RO14176 0.64 0.64 0.05 453 a BR06 0.61 0.61 0.04 541 b Second Break Cap L* Value (dark-light) RO14176 68.92 68.86 6.39 263 a BR06 65.68 65.49 5.90 340 b Cap a* Value (green-red) RO14176 6.37 6.43 1.60 261 a BR06 7.21 7.31 2.42 340 b Cap b* Value (blue-yellow) RO14176 27.36 27.35 2.11 261 a BR06 28.15 28.49 2.86 340 b Cap Apex Flesh Thickness RO14176 0.54 0.54 0.04 235 a BR06 0.51 0.52 0.07 362 b Cap Edge Flesh Thickness RO14176 0.31 0.31 0.06 235 a BR06 0.23 0.23 0.09 362 b Cap Roundness (Calculated) RO14176 0.61 0.60 0.05 235 a BR06 0.60 0.59 0.06 363 b

[0171] Table 22A below provides qualitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO14176 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO14176.

TABLE-US-00038 TABLE 22A Characteristic RO14176 B14528 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter Second Break Cap L* Value (dark-light) Similar Similar Cap a* Value (green-red) Redder Greener Cap b* Value (blue-yellow) Similar Similar Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Rounder Flatter

[0172] Table 22B shows additional quantitative comparisons between mushroom strain RO14176 and strain B14528. Column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the characteristic, column 4 shows the interquartile range (IQR) for the characteristic, column 5 shows the number of mushrooms assessed (n), and column 6 shows the statistical significance group calculated according to Dunn's Test.

TABLE-US-00039 TABLE 22B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO14176 65.76 65.82 5.82 469 a B14528 64.06 64.13 6.56 340 c Cap a* Value (green-red) RO14176 7.45 7.47 1.58 469 a B14528 8.26 8.31 2.13 340 c Cap b* Value (blue-yellow) RO14176 28.17 28.03 2.64 469 a B14528 30.31 30.16 3.39 340 c Cap Apex Flesh Thickness RO14176 0.54 0.54 0.06 453 a B14528 0.47 0.46 0.06 387 c Cap Edge Flesh Thickness RO14176 0.27 0.26 0.07 449 a B14528 0.22 0.22 0.08 387 b Cap Roundness (Calculated) RO14176 0.64 0.64 0.05 455 a B14528 0.61 0.61 0.05 389 b Second Break Cap L* Value (dark-light) RO14176 68.92 68.86 6.39 263 a B14528 68.56 69.02 6.54 170 a Cap a* Value (green-red) RO14176 6.37 6.43 1.60 261 a B14528 6.09 5.96 2.05 170 c Cap b* Value (blue-yellow) RO14176 27.36 27.35 2.11 261 a B14528 27.37 27.78 3.22 170 a Cap Apex Flesh Thickness RO14176 0.54 0.54 0.04 235 a B14528 0.46 0.46 0.05 198 c Cap Edge Flesh Thickness RO14176 0.31 0.31 0.06 235 a B14528 0.23 0.23 0.06 198 b Cap Roundness (Calculated) RO14176 0.61 0.60 0.05 235 a B14528 0.60 0.60 0.05 198 b

[0173] One of the advantages of the strain RO14176 is its incompatibility with other commercial strains. Table 23 below provides results of compatibility tests between strain RO14176 and commercial strain BR06, commercial strain B14528, or related strain RO10425. These results indicate that strain RO14176 is incompatible with strain BR06 and strain B14528, and compatible with related strain RO10425. This was true regardless of which tested strain served as the compost variety and which tested strain served as the casing variety.

TABLE-US-00040 TABLE 23 Strains (compost variety/ Mean Median SD Significance casing variety) lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 Trials Group RO14176/RO14176 2.66 12.99 2.41 11.77 0.61 2.98 3 a RO10425/RO10425 2.71 13.23 2.86 13.94 0.50 2.44 3 a BR06/BR06 2.62 12.79 2.46 11.99 1.15 5.61 2 a B14528/B14528 2.93 14.31 2.92 14.23 0.34 1.66 1 a RO10425/RO14176, 2.43 11.86 2.75 13.40 0.77 3.76 1 a RO14176/RO10425 RO14176/BR06, 1.19 5.81 1.20 5.86 0.37 1.81 1 b BR06/RO14176 RO14176/B14528, 0.70 3.42 0.76 3.69 0.21 1.03 1 b B14528/RO14176

Example 3: Characterization of RO12756

[0174] The following example describes the characterization of strain RO12756 compared to commercial varieties.

Methodology

[0175] The measurements and calculations were conducted as in Example 1.

Results

Objective Description of the Variety RO12756

[0176] The following detailed description sets forth the breeding procedures and the characteristics of new cultivar designated RO12756. The strain is maintained and propagated as vegetative mycelium. Spawn (inoculum for the substrate compost) is prepared from the vegetative mycelium. The new variety was tested on a scale of hundreds of individual mushrooms. The variety has shown uniformity and stability for the traits, within the limits of environmental influence for the traits.

[0177] All homokaryons utilized are strains of Agaricus bisporus.

[0178] In a first cross, a bridging cross strain 429 slow grower (sg) was crossed with a commercial off-white hybrid strain protoplast.

[0179] RO12756 was produced through the cross of a homokaryon SSI of the first cross to a homokaryon SSI of strain RO10425.

[0180] RO12756 sets pins and produces harvestable mushrooms as fast or faster than available brown commercial strains. RO12756 also exhibits high tray coverage, high yield, a desirably light brown cap color, medium firmness, and thick stipes and cap flesh relative to cap diameter. RO12756 additionally exhibits rounder cap shape, thicker cap apex flesh, thicker cap edge flesh, and percent harvested speed faster or as fast as other available brown commercial strains.

[0181] Other characteristics of RO12756 distinguishable from other varieties are described below (compared to commercial line BR06 and commercial line B14528).

Comparisons to Commercial Mushroom Varieties

Initial Trials

Tray-Level Comparisons

[0182] In further evidence of the distinctness and superiority of mushroom strain RO12756, provided below are the results of trials conducted across multiple strains' yielded trays of mushrooms. These trials compare timing for trays to reach pinset and harvestable maturity, tray coverage, cap color darkness, cap firmness, cap roundness (visual), yield at each break, and percentage of each break's yield harvested by 17 days post-casing (1st break), 24 days post-casing (2nd break), and 32 days post-casing (3rd break). Characteristics were measured from 74-147 trays of strain RO12756 mushrooms with corresponding measurements taken of 94-166 trays of strain BR06 mushrooms (Table 24A and 24B) and 12-28 trays of strain B14528 mushrooms (Table 25A and 25B). Percent Harvest Speed characteristics were measured for 34-36 trials containing strain RO12756, 33-36 trials containing strain BR06, and 3-4 trials containing strain B14528.

[0183] In addition to the comparisons made below, FIGS. 1 and FIGS. 4-5 visually compare variety RO12756 to other varieties in tray coverage (FIG. 1), cap color (FIG. 4), and qualities regarding cap height and cap flesh thickness (FIG. 5).

[0184] Table 24A below provides qualitative comparisons between mushroom strain RO12756 and strain BR06. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00041 TABLE 24A Characteristic RO12756 BR06 Before First Break Days to Pinset Faster Slower First Break Days to 1st Break Faster Slower Tray Coverage Higher Lower Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Flatter Rounder Percent Harvested Speed Similar Similar Yield (lb/ft.sup.2) Similar Similar Second Break Days to 2nd Break Faster Slower Tray Coverage Similar Similar Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Flatter Rounder Percent Harvested Speed Higher Lower Yield (lb/ft.sup.2) Similar Similar Third Break Percent Harvested Speed Higher Lower Yield (lb/ft.sup.2) Higher Lower All Breaks Combined Yield (lb/ft.sup.2) Higher Lower

[0185] Table 24B below provides initial quantitative comparisons between mushroom strain RO12756 and strain BR06. Column 1 lists the tray-level or trial-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00042 TABLE 24B RO12756 BR06 Characteristic Average St. Dev. Average St. Dev. p-value Before First Break Days to Pinset 11.722 0.999 12.316 0.880 4.139E05 First Break Days to 1st Break 17.317 0.752 17.598 0.735 1.153E02 Tray Coverage 4.178 0.799 3.935 0.866 2.900E02 Cap Color Darkness 2.916 0.601 3.872 0.380 6.840E35 Cap Firmness 3.543 0.519 4.154 0.425 4.790E19 Cap Roundness (Visual) 2.848 0.387 2.984 0.127 2.930E04 Percent Harvested Speed 24.105% 23.663 16.868% 19.090 1.241E01 Yield (lb/ft.sup.2) 2.483 0.701 2.523 1.017 6.860E01 Second Break Days to 2nd Break 24.824 0.800 25.274 0.675 1.122E04 Tray Coverage 4.000 0.611 4.150 0.605 6.140E02 Cap Color Darkness 3.009 0.427 3.859 0.349 9.160E45 Cap Firmness 3.509 0.553 3.969 0.435 9.200E12 Cap Roundness (Visual) 2.634 0.484 2.992 0.154 1.030E13 Percent Harvested Speed 42.447% 25.152 25.151% 23.811 1.431E03 Yield (lb/ft.sup.2) 1.606 0.537 1.601 0.617 9.340E01 Third Break Percent Harvested Speed 63.200% 26.702 34.306% 32.035 2.320E05 Yield (lb/ft.sup.2) 1.038 0.313 0.747 0.371 9.180E13 All Breaks Combined Yield (lb/ft.sup.2) 5.127 0.792 4.871 0.918 9.080E03

[0186] Table 25A below provides qualitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00043 TABLE 25A Characteristic RO12756 B14528 Before First Break Compost Colonization Higher Lower First Break Days to 1st Break Similar Similar Tray Coverage Similar Similar Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Similar Similar Percent Harvested Speed Similar Similar Second Break Days to 2nd Break Similar Similar Tray Coverage Higher Lower Cap Color Darkness Lighter Darker Cap Firmness Softer Firmer Cap Roundness (Visual) Similar Similar Percent Harvested Speed Similar Similar

[0187] Table 25B below provides initial quantitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the tray-level or trial-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, column 4 shows the average value of the characteristic for mushroom strain B14528, column 5 shows the standard deviation of the characteristic for mushroom strain B14528, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00044 TABLE 25B RO12756 B14528 Characteristic Average St. Dev. Average St. Dev. p-value Before First Break Compost Colonization 4.350 0.737 3.643 1.026 6.920E05 First Break Days to 1st Break 17.317 0.752 17.000 0.649 8.599E02 Tray Coverage 4.178 0.799 4.000 0.471 2.630E01 Cap Color Darkness 2.916 0.601 4.000 0.272 4.050E16 Cap Firmness 3.543 0.519 3.786 0.418 2.400E02 Cap Roundness (Visual) 2.848 0.387 2.929 0.262 2.990E01 Percent Harvested Speed 24.105% 23.660 19.000% 11.247 4.392E01 Second Break Days to 2nd Break 24.824 0.800 24.417 0.669 9.848E02 Tray Coverage 4.000 0.611 3.250 0.550 1.150E06 Cap Color Darkness 3.009 0.427 3.929 0.378 2.670E19 Cap Firmness 3.509 0.553 3.857 0.448 2.470E03 Cap Roundness (Visual) 2.634 0.484 2.536 0.508 3.430E01 Percent Harvested Speed 42.447% 25.152 39.000% 34.438 8.380E01

Individual Mushroom-Level Comparisons

[0188] In additional evidence of the distinctness and superiority of mushroom strain RO12756, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Diameter, Cap Height, Cap Flesh Thickness, Stipe Diameter, Relative Cap Height, Relative Cap Flesh Thickness, and Relative Stipe Diameter (as described in Table 1). Measurements from 390 first break and 100 second break RO12756 mushrooms were compared to 400 first break and 120 second break BR06 mushrooms (Tables 26A and 26B) and 150 first break and 80 second break B14528 mushrooms (Tables 27A and 27B).

[0189] Table 26A below provides qualitative comparisons between mushroom strain RO12756 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00045 TABLE 26A Characteristic RO12756 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Smaller Larger Cap Height Shorter Taller Cap Flesh Thickness Similar Similar Stipe Diameter Smaller Larger Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Smaller Larger Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Similar Similar Cap Height Shorter Taller Cap Flesh Thickness Similar Similar Stipe Diameter Similar Similar Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Similar Similar

[0190] Table 26B below provides initial quantitative comparisons between mushroom strain RO12756 and strain BR06. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, column 4 shows the average value of the characteristic for mushroom strain BR06, column 5 shows the standard deviation of the characteristic for mushroom strain BR06, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00046 TABLE 26B RO12756 BR06 Characteristic Average St. Dev. Average St. Dev. p-value First Break Cap L* Value (dark-light) 74.237 3.524 64.335 5.707 5.943E128 Cap a* Value (green-red) 3.899 1.581 7.605 1.770 1.242E138 Cap b* Value (blue-yellow) 24.770 2.559 28.453 2.451 3.905E76 Cap Diameter 40.571 3.829 42.200 4.005 7.566E09 Cap Height 24.199 1.994 25.455 2.059 1.797E17 Cap Flesh Thickness 13.069 1.395 13.130 1.313 5.270E01 Stipe Diameter 18.258 2.139 19.546 2.585 1.130E13 Relative Cap Height 0.598 0.033 0.605 0.031 3.410E03 Relative Cap Flesh Thickness 0.323 0.028 0.312 0.026 3.060E08 Relative Stipe Diameter 0.451 0.040 0.464 0.054 9.166E05 Second Break Cap L* Value (dark-light) 73.318 3.497 66.428 4.274 1.03E28 Cap a* Value (green-red) 4.282 1.216 6.359 1.461 1.17E23 Cap b* Value (blue-yellow) 24.796 3.168 27.600 2.440 2.72E12 Cap Diameter 42.833 5.946 43.005 4.063 8.00E01 Cap Height 24.156 2.112 25.313 2.234 1.19E04 Cap Flesh Thickness 12.359 1.048 12.526 1.163 2.71E01 Stipe Diameter 16.115 1.904 15.878 2.022 3.75E01 Relative Cap Height 0.569 0.053 0.590 0.035 6.90E04 Relative Cap Flesh Thickness 0.293 0.040 0.292 0.025 9.06E01 Relative Stipe Diameter 0.379 0.038 0.370 0.035 5.72E02

[0191] Table 27A below provides qualitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00047 TABLE 27A Characteristic RO12756 B14528 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Smaller Larger Cap Height Shorter Taller Cap Flesh Thickness Similar Similar Stipe Diameter Larger Smaller Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Thicker Thinner Relative Stipe Diameter Larger Smaller Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Diameter Similar Similar Cap Height Shorter Taller Cap Flesh Thickness Thicker Thinner Stipe Diameter Larger Smaller Relative Cap Height Shorter Taller Relative Cap Flesh Thickness Similar Similar Relative Stipe Diameter Larger Smaller

[0192] Table 27B below provides quantitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the average value of the characteristic for mushroom strain RO12756, column 3 shows the standard deviation of the characteristic for mushroom strain RO12756, column 4 shows the average value of the characteristic for mushroom strain B14528, column 5 shows the standard deviation of the characteristic for mushroom strain B14528, and column 6 shows the p-values of two-sample, two-sided t-tests. Bolded p-values indicate p<0.05.

TABLE-US-00048 TABLE 27B RO12756 B14528 Characteristic Average St. Dev. Average St. Dev. p-value First Break Cap L* Value (dark-light) 74.237 3.524 67.579 5.925 1.560E47 Cap a* Value (green-red) 3.899 1.581 5.987 1.854 3.850E34 Cap b* Value (blue-yellow) 24.770 2.559 27.637 2.363 3.510E29 Cap Diameter 40.571 3.829 43.218 3.827 6.590E13 Cap Height 24.199 1.994 26.568 1.872 2.430E33 Cap Flesh Thickness 13.069 1.395 13.057 1.012 9.210E01 Stipe Diameter 18.258 2.139 17.787 2.018 1.790E02 Relative Cap Height 0.598 0.033 0.616 0.030 2.260E09 Relative Cap Flesh Thickness 0.323 0.028 0.303 0.024 3.050E14 Relative Stipe Diameter 0.451 0.040 0.413 0.042 1.900E21 Second Break Cap L* Value (dark-light) 73.318 3.497 68.288 5.217 8.20E13 Cap a* Value (green-red) 4.282 1.216 5.256 1.241 3.51E07 Cap b* Value (blue-yellow) 24.796 3.168 26.285 2.185 4.48E04 Cap Diameter 42.833 5.946 41.712 3.417 1.35E01 Cap Height 24.156 2.112 25.062 2.039 4.14E03 Cap Flesh Thickness 12.359 1.048 11.868 1.370 7.02E03 Stipe Diameter 16.115 1.904 14.725 1.442 2.04E07 Relative Cap Height 0.569 0.053 0.602 0.033 4.09E06 Relative Cap Flesh Thickness 0.293 0.040 0.286 0.036 2.14E01 Relative Stipe Diameter 0.379 0.038 0.355 0.044 1.48E04

Additional Trials

Tray-Level Comparisons

[0193] Table 28A below provides qualitative comparisons between mushroom strain RO12756 and strain BR06. Traits of particular interest are bolded. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00049 TABLE 28A Characteristic RO12756 BR06 Before First Break Days to Pinset Faster Slower First Break Days to 1st Break Faster Slower Percent Harvested Speed Faster Slower Yield (kg/m.sup.2) Similar Similar Second Break Days to 2nd Break Faster Slower Yield (kg/m.sup.2) Higher Lower Third Break Yield (kg/m.sup.2) Higher Lower All Breaks Combined Yield (kg/m.sup.2) Higher Lower

[0194] Table 28B below provides quantitative comparisons between mushroom strain RO12756 and strain BR06.

[0195] The first part of analyzing trait measurements in the additional trials herein included making comparisons between all three of instant strain RO12756, comparative strain BR06, and comparative strain B14528. For these three strains, a Shapiro-Wilk test and a Levene test were performed first to assess if the data satisfy the assumptions of normality and equal variances among groups, respectively, of a standard ANOVA. The Shapiro-Wilk test and Levene test determined that most of these traits satisfied neither assumption. Therefore, a Kruskal-Wallis test (Kruskal, W. H., & Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. Journal of the American statistical Association, 47(260), 583-621), a non-parametric variation of ANOVA that does not assume normality, equal variances among groups, or equal sample sizes among groups, was performed. Medians and interquartile ranges (IQR) were reported, as these metrics are robust to non-normal distributions. Means are also reported for consistency with normally-distributed traits.

[0196] The Shapiro-Wilk test and Levene test determined that most developmental timing traits (Days to Pinset, Days to 1st Break, and Days to 2nd Break) and yield traits were not normally distributed and variances among groups were not equal. The Kruskal-Wallis test and Dunn post-hoc test (Dunn's Test; Dunn, O. J. (1964). Multiple comparisons using rank sums. Technometrics, 6(3), 241-252) were used in these cases. When the Kruskal-Wallis test results were significant based on a threshold of p<0.05, Dunn's test was performed post-hoc to make pairwise strain comparisons (Dunn, 1964). P-values were adjusted using the Bonferroni correction to minimize risk of Type 1 error (false significance). An exception to this trend was Percent Harvested Speed, which was normally distributed and exhibited equal variance for all strains. For assessing Percent Harvested Speed, a standard ANOVA was used and a Tukey Honestly Significant Difference (HSD) test was performed post-hoc for pairwise strain comparisons. Standard deviations multiplied by 100 for consistency alongside percentages (SD* 100) were reported for this trait. This testing was performed on groups of three strains, but the results show a pairwise subset of the tests for consistency with the initial trials.

[0197] In Table 28B, column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test. Traits of particular interest are bolded.

TABLE-US-00050 TABLE 28B Significance Number of Group Variety Mean Median IQR Trials (Dunn's Test) Before First Break Days to Pinset RO12756 11.4 11.0 1.00 8 a BR06 12.6 13.0 1.00 8 b First Break Days to 1st Break RO12756 17.5 17.0 1.00 8 a BR06 18.5 18.0 1.00 8 b Yield (kg/m.sup.2) RO12756 11.91 12.40 4.88 8 a BR06 11.96 12.69 9.52 8 a Second Break Days to 2nd Break RO12756 24.8 25.0 2.00 7 a BR06 25.5 25.0 1.00 7 b Yield (kg/m.sup.2) RO12756 8.54 8.15 2.64 8 a BR06 7.37 5.96 4.39 8 b Third Break Yield (kg/m.sup.2) RO12756 4.78 4.83 1.86 8 a BR06 3.66 2.78 2.78 8 b All Breaks Combined Yield (kg/m.sup.2) RO12756 25.29 24.95 4.15 8 a BR06 23.00 23.48 5.81 8 b Significance Percent Harvested Speed Group (measured in First Break) SD * 100 (Tukey HSD) RO12756 48.2% 50.9% 16.30 8 b BR06 28.6% 27.4% 20.90 8 a

[0198] Table 29A below provides qualitative comparisons between mushroom strain RO12756 and strain B14528. Traits of particular interest are bolded. Column 1 lists the tray-level or trial-level characteristic (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00051 TABLE 29A Characteristic RO12756 B14528 Before First Break Days to Pinset Similar Similar First Break Days to 1st Break Similar Similar Percent Harvested Speed Similar Similar Yield (kg/m.sup.2) Similar Similar Second Break Days to 2nd Break Similar Similar Yield (kg/m.sup.2) Similar Similar Third Break Yield (kg/m.sup.2) Similar Similar All Breaks Combined Yield (kg/m.sup.2) Similar Similar

[0199] Table 29B below provides additional quantitative comparison between mushroom strain RO12756 and strain B14528. Column 1 shows the strains compared for each trait, column 2 shows the mean value for that strain's measurements, column 3 shows the median value of the same measurements, column 4 shows either the interquartile range (IQR) or the standard deviation multiplied by 100 for consistency alongside percentages (SD*100) for the same measurements, column 5 shows the number of trials conducted, and column 6 shows the statistical significance group calculated for that strain for that trait according to either Dunn's Test or Tukey HSD.

TABLE-US-00052 TABLE 29B Significance Group Number of (Dunn's Characteristic Mean Median IQR Trials Test) Before First Break Days to Pinset RO12756 11.40 11.00 1.00 8 a B14528 11.40 11.00 1.00 8 a First Break Days to 1st Break RO12756 17.50 17.00 1.00 8 a B14528 17.50 17.00 1.00 8 a Yield (kg/m.sup.2) RO12756 11.91 12.40 4.88 8 a B14528 12.55 12.25 5.27 8 a Second Break Days to 2nd Break RO12756 24.80 25.00 2.00 7 a B14528 25.00 25.00 0.50 7 a Yield (kg/m.sup.2) RO12756 8.54 8.15 2.64 8 a B14528 8.45 8.01 2.39 8 a Third Break Yield (kg/m.sup.2) RO12756 4.78 4.83 1.86 8 a B14528 5.52 5.57 2.88 8 a All Breaks Combined Yield (kg/m.sup.2) RO12756 25.29 24.95 4.15 8 a B14528 26.51 27.10 5.76 8 a Percent Harvested Speed Significance (measured in Group First Break) Mean Median SD * 100 (Tukey HSD) RO12756 48.2% 50.9% 16.30 8 b B14528 53.5% 55.7% 14.00 8 b

Individual Mushroom-Level Comparisons

[0200] In additional evidence of the distinctness and superiority of mushroom strain RO12756, provided below is a summary of precise quantitative measurements of individual mushrooms at first and second breaks. These data compare Cap L* Value, Cap a* Value, and Cap b* Values in the International Commission on Illumination (CIELAB) color space, Cap Apex Flesh Thickness, Cap Edge Flesh Thickness, and Cap Roundness (Calculated) (as described in Table 1). Measurements were taken for hundreds of mushrooms per trait, noted as sample size (n) in Tables 30B and 31B.

[0201] Table 30A below provides qualitative comparisons between mushroom strain RO12756 and strain BR06. Traits of particular interest are bolded. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to BR06, and column 3 shows the qualitative descriptor for mushroom strain BR06 relative to RO12756.

TABLE-US-00053 TABLE 30A Characteristic RO12756 BR06 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Similar Similar Cap Roundness (Calculated) Similar Similar Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Flatter Rounder

[0202] Table 30B below provides additional quantitative comparisons between mushroom strain RO12756 and strain BR06. Column 1 shows each trait and the strains compared for said trait, column 2 shows the mean value for that strain's measurements of that trait, column 3 shows the median value of the same measurements, column 4 shows the interquartile range (IQR) for the same measurements, column 5 shows the number of mushrooms assessed (n), and column 6 shows the statistical significance group calculated for that strain for that trait according to Dunn's Test.

TABLE-US-00054 TABLE 30B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO12756 72.12 72.29 4.81 580 a BR06 63.30 62.74 9.83 797 b Cap a* Value (green-red) RO12756 5.62 5.66 1.88 580 a BR06 8.67 8.96 2.58 787 b Cap b* Value (blue-yellow) RO12756 26.98 27.06 4.11 580 a BR06 28.72 29.04 3.17 776 b Cap Apex Flesh Thickness RO12756 0.55 0.55 0.06 575 a BR06 0.52 0.52 0.05 541 b Cap Edge Flesh Thickness RO12756 0.22 0.23 0.10 569 a BR06 0.21 0.21 0.09 537 a Cap Roundness (Calculated) RO12756 0.61 0.61 0.04 579 a BR06 0.61 0.61 0.04 541 a Second Break Cap L* Value (dark-light) RO12756 73.02 72.87 4.35 200 a BR06 65.68 65.49 5.90 340 b Cap a* Value (green-red) RO12756 4.84 4.95 1.64 200 a BR06 7.21 7.31 2.42 340 b Cap b* Value (blue-yellow) RO12756 26.55 26.83 3.24 200 a BR06 28.15 28.49 2.86 340 b Cap Apex Flesh Thickness RO12756 0.54 0.54 0.04 198 a BR06 0.51 0.52 0.07 362 b Cap Edge Flesh Thickness RO12756 0.27 0.27 0.10 199 a BR06 0.23 0.23 0.09 362 b Cap Roundness (Calculated) RO12756 0.56 0.56 0.05 199 a BR06 0.60 0.59 0.06 363 b

[0203] Table 31A below provides qualitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1), column 2 shows the qualitative descriptor for the characteristic for mushroom strain RO12756 relative to B14528, and column 3 shows the qualitative descriptor for mushroom strain B14528 relative to RO12756.

TABLE-US-00055 TABLE 31A Characteristic RO12756 B14528 First Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Similar Similar Cap Roundness (Calculated) Similar Similar Second Break Cap L* Value (dark-light) Lighter Darker Cap a* Value (green-red) Greener Redder Cap b* Value (blue-yellow) Bluer Yellower Cap Apex Flesh Thickness Thicker Thinner Cap Edge Flesh Thickness Thicker Thinner Cap Roundness (Calculated) Flatter Rounder

[0204] Table 31B shows additional quantitative comparisons between mushroom strain RO12756 and strain B14528. Column 1 lists the mushroom-level characteristics (as described in Table 1) and each strain, column 2 shows the mean value of the characteristic, column 3 shows the median value of the characteristic, column 4 shows the interquartile range (IQR) for the characteristic, column 5 shows the number of mushrooms assessed (n), and column 6 shows the statistical significance group calculated according to Dunn's Test.

TABLE-US-00056 TABLE 31B Number of Significance Mushrooms Group Variety Mean Median IQR (n) (Dunn's Test) First Break Cap L* Value (dark-light) RO12756 72.12 72.29 4.81 580 a B14528 64.06 64.13 6.56 340 b Cap a* Value (green-red) RO12756 5.62 5.66 1.88 580 a B14528 8.26 8.31 2.13 340 c Cap b* Value (blue-yellow) RO12756 26.98 27.06 4.11 580 a B14528 30.31 30.16 3.39 340 c Cap Roundness (Calculated) RO12756 0.61 0.61 0.04 579 a B14528 0.61 0.61 0.05 389 a Cap Apex Flesh Thickness RO12756 0.55 0.55 0.06 575 a B14528 0.47 0.46 0.06 387 c Cap Edge Flesh Thickness RO12756 0.22 0.23 0.10 569 a B14528 0.22 0.22 0.08 387 a Second Break Cap L* Value (dark-light) RO12756 73.02 72.87 4.35 200 a B14528 68.56 69.02 6.54 170 c Cap a* Value (green-red) RO12756 4.84 4.95 1.64 200 a B14528 6.09 5.96 2.05 170 c Cap b* Value (blue-yellow) RO12756 26.55 26.83 3.24 200 a B14528 27.37 27.78 3.22 170 c Cap Roundness (Calculated) RO12756 0.56 0.56 0.05 199 a B14528 0.60 0.60 0.05 198 b Cap Apex Flesh Thickness RO12756 0.54 0.54 0.04 198 a B14528 0.46 0.46 0.05 198 c Cap Edge Flesh Thickness RO12756 0.27 0.27 0.10 199 a B14528 0.23 0.23 0.06 198 b

[0205] One of the advantages of the strain RO12756 is its incompatibility with other commercial strains. Table 32 below provides results of compatibility tests between strain RO12756 and commercial strain BR06, commercial strain B14528, or related strain RO10425. These results indicate that strain RO12756 is incompatible with strain BR06, strain B14528, and related strain RO10425. This was true regardless of which tested strain served as the compost variety and which tested strain served as the casing variety.

TABLE-US-00057 TABLE 32 Strains (compost variety/ Mean Median SD Significance casing variety) lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 lb/ft.sup.2 kg/m.sup.2 Trials Group RO12756/RO12756 2.62 12.79 2.61 12.74 0.41 2.00 3 a RO10425/RO10425 2.71 13.23 2.86 13.94 0.50 2.44 3 a BR06/BR06 2.62 12.79 2.46 11.99 1.15 5.61 2 a B14528/B14528 2.93 14.31 2.92 14.23 0.34 1.66 1 a RO12756/BR06, 0.70 3.42 0.62 3.03 0.25 1.22 2 b BR06/RO12756 RO12756/B14528, 0.68 3.32 0.60 2.91 0.43 2.10 1 b B14528/RO12756 RO12756/RO10425, 0.91 4.44 1.04 5.08 0.35 1.71 1 b RO10425/RO12756

DEPOSIT INFORMATION

Hybrid mushroom variety RO10425

[0206] A deposit of the hybrid mushroom variety RO10425 is maintained by Amycel, LLC, having an address of 260 Westgate Drive, Watsonville CA, 95076. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed by affording access to a deposit of at least 25 cryopreserved or freeze-dried vials from same harvest (0.5 ml per vial) of the same variety made according to the Budapest Treaty in the American Type Culture Collection, (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA.

[0207] The hybrid mushroom variety RO10425 was deposited on Apr. 18, 2025, according to the Budapest Treaty in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA. The deposit has been assigned ATCC number PTA-127856. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed.

[0208] The deposit will be maintained in the ATCC depository, which is a public depository, for a period of at least 30 years, or at least 5 years after the most recent request for a sample of the deposit, or for the effective life of the patent, whichever is longer, and will be replaced if a deposit becomes nonviable during that period.

Hybrid Mushroom Variety RO14176

[0209] A deposit of the hybrid mushroom variety RO14176 is maintained by Amycel, LLC, having an address of 260 Westgate Drive, Watsonville CA, 95076. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed by affording access to a deposit of at least 25 cryopreserved or freeze-dried vials from same harvest (0.5 ml per vial) of the same variety made according to the Budapest Treaty in the American Type Culture Collection, (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA.

[0210] The hybrid mushroom variety RO14176 was deposited on Feb. 6, 2025, according to the Budapest Treaty in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA. The deposit has been assigned ATCC number PTA-127855. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed.

[0211] The deposit will be maintained in the ATCC depository, which is a public depository, for a period of at least 30 years, or at least 5 years after the most recent request for a sample of the deposit, or for the effective life of the patent, whichever is longer, and will be replaced if a deposit becomes nonviable during that period.

Hybrid Mushroom Variety RO12756

[0212] A deposit of the hybrid mushroom variety RO12756 is maintained by Amycel, LLC, having an address of 260 Westgate Drive, Watsonville CA, 95076. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed by affording access to a deposit of at least 25 cryopreserved or freeze-dried vials from same harvest (0.5 ml per vial) of the same variety made according to the Budapest Treaty in the American Type Culture Collection, (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA.

[0213] The hybrid mushroom variety RO12756 was deposited on Feb. 13, 2025, according to the Budapest Treaty in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia, 20110, USA. The deposit has been assigned ATCC number PTA-127857. Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed.

[0214] The deposit will be maintained in the ATCC depository, which is a public depository, for a period of at least 30 years, or at least 5 years after the most recent request for a sample of the deposit, or for the effective life of the patent, whichever is longer, and will be replaced if a deposit becomes nonviable during that period.

STATEMENT OF EMBODIMENTS

[0215] Embodiment 1. A mushroom variety selected from the group consisting of RO10425, RO14176, and RO12756, representative culture of which having been deposited under ATCC Nos. PTA-127856, PTA-127855, and PTA-127857 respectively.

[0216] Embodiment 2. An Agaricus bisporus culture designated as Agaricus bisporus line RO10425, a representative culture of the line having been deposited under ATCC No. PTA-127856.

[0217] Embodiment 3. An Agaricus bisporus culture designated as Agaricus bisporus line RO12756, a representative culture of the line having been deposited under ATCC No. PTA-127857.

[0218] Embodiment 4. A new and distinct variety of Agaricus bisporus Imbach mushroom selected from the group consisting of RO10425, RO14176, and RO12756, as shown and described herein.

[0219] Embodiment 5. A new and distinct variety of Agaricus bisporus Imbach mushroom named RO10425, as illustrated and described.

[0220] Embodiment 6. A method of producing a hybrid mushroom culture of Agaricus bisporus, the method comprising the steps of: [0221] (a) providing a first homokaryon selected from a wild strain, designated AA-0096, or a progeny thereof, wherein said wild strain AA-0096 is available from ATCC under Accession No. PTA-6903; [0222] (b) providing a second homokaryon selected from a commercial off-white hybrid strain, which is compatible with first homokaryon; [0223] (c) crossing the first homokaryon with the second homokaryon to form a first culture of intermediate hybrid homokaryons; [0224] (d) backcrossing the first culture to the second homokaryon to form a second culture of intermediate hybrid homokaryons; and [0225] (e) crossing the second culture to a bridging cross strain to form the hybrid mushroom culture, designated RO10425, a culture of which has been deposited under ATCC Accession No. PTA-127856.

[0226] Embodiment 7. The method of embodiment 6, wherein the bridging cross strain is 429, representative sample of 429 being available under ATCC Accession No. PTA-6877.

[0227] Embodiment 8. The method of embodiment 6 or 7, wherein the commercial off-white hybrid strain is derived from a U1 mushroom strain.

[0228] Embodiment 9. The method according to embodiments 6-8, further including the steps of producing at least one hybrid mushroom, said steps comprising: [0229] (f) inoculating a mushroom growth medium with the hybrid mushroom culture; [0230] (g) maintaining said inoculated growth medium under conditions conducive to mushroom fruiting; and [0231] (h) collecting at least one hybrid mushroom from said growth medium.

[0232] Embodiment 10. A hybrid Agaricus bisporus mushroom strain designated RO10425, a culture of which having been deposited under ATCC Accession No. PTA-127856.

[0233] Embodiment 11. A hybrid Agaricus bisporus mushroom strain, wherein the hybrid strain is a cross of a parental hybrid Agaricus bisporus mushroom strain with an Agaricus bisporus bridging cross strain, wherein the parental hybrid strain is derived from wild strain AA-0096 and a commercial off-white hybrid strain, and wherein a representative culture of said wild strain AA-0096 is available from ATCC under Accession No. PTA-6903.

[0234] Embodiment 12. The hybrid mushroom strain of embodiment 11, wherein the commercial off-white hybrid strain is derived from a U1 mushroom strain.

[0235] Embodiment 13. A hybrid mushroom strain designated RO10425, representative culture of which having been deposited under ATCC Accession No. PTA-127856, wherein cells from the hybrid mushroom culture have at least one marker presented herein.

[0236] Embodiment 14. A hybrid mushroom produced by the method of embodiment 9 or the hybrid mushroom strain of any one of embodiments 10-13, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of darker cap color, higher yield, and thicker cap as compared to the corresponding physical characteristics of BR06, BR06 having representative sample accessible under ATCC under Accession No. PTA-6876.

[0237] Embodiment 15. A mushroom part from the hybrid mushroom produced by the method of embodiment 9 or 14, or produced by the hybrid mushroom strain of any one of embodiments 10-14.

[0238] Embodiment 16. The mushroom part of embodiment 15, wherein the mushroom part is a fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, or product produced from the mushroom.

[0239] Embodiment 17. A new and distinct variety of Agaricus bisporus Imbach mushroom named RO12756, as illustrated and described.

[0240] Embodiment 18. A method of producing a hybrid mushroom culture of Agaricus bisporus, the method comprising the steps of: [0241] (a) providing a first homokaryon selected from a hybrid mushroom strain designated RO10425, or a progeny thereof, representative sample of RO10425 having been deposited under ATCC under Accession No. PTA-127856; [0242] (b) providing a second homokaryon selected from the cross of a bridging cross strain and a commercial off-white hybrid strain, which is compatible with first homokaryon; and [0243] (c) crossing the first homokaryon with the second homokaryon to form the hybrid mushroom culture, designated RO12756, a culture of which has been deposited under ATCC Accession No. PTA-127857.

[0244] Embodiment 19. The method of embodiment 18, wherein the bridging cross strain is 429, representative sample of 429 being available under ATCC Accession No. PTA-6877.

[0245] Embodiment 20. The method of embodiment 18 or 19, wherein the commercial off-white hybrid is distinct from the hybrid mushroom strain RO10425.

[0246] Embodiment 21. The method of any one of embodiments 18-20, wherein the commercial off-white hybrid strain is derived from a U1 mushroom strain.

[0247] Embodiment 22. The method according to embodiments 18-21, further including the steps of producing at least one hybrid mushroom, said steps comprising: [0248] (d) inoculating a mushroom growth medium with the hybrid mushroom culture; [0249] (e) maintaining said inoculated growth medium under conditions conducive to mushroom fruiting; and [0250] (f) collecting at least one hybrid mushroom from said growth medium.

[0251] Embodiment 23. A hybrid Agaricus bisporus mushroom strain designated RO12756, a culture of which having been deposited under ATCC Accession No. PTA-127857.

[0252] Embodiment 24. A hybrid Agaricus bisporus mushroom strain, wherein the hybrid strain is a cross of a parental hybrid Agaricus bisporus mushroom strain with hybrid mushroom strain designated RO10425, wherein the parental hybrid strain is a cross of a bridging cross strain to a commercial off-white hybrid strain, and wherein a representative culture of said strain RO10425 is available from ATCC under Accession No. PTA-127856.

[0253] Embodiment 25. A hybrid Agaricus bisporus mushroom strain, wherein the hybrid strain is a cross of a parental hybrid Agaricus bisporus mushroom strain with hybrid mushroom strain designated RO10425, wherein the parental hybrid strain is derived from a U1 type off-white hybrid, and wherein a representative culture of said strain RO10425 is available from ATCC under Accession No. PTA-127856.

[0254] Embodiment 26. The hybrid mushroom strain of embodiment 25, wherein the commercial off-white hybrid strain is derived from a U1 mushroom strain.

[0255] Embodiment 27. A hybrid mushroom strain designated RO12756, representative sample of which having been deposited under ATCC Accession No. PTA-127857, wherein cells from the hybrid mushroom strain have at least one marker presented herein.

[0256] Embodiment 28. A hybrid mushroom produced by the method of embodiment 22 or produced by the hybrid mushroom strain of any one of embodiments 23-27, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of light brown cap color as exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; [0257] as high or higher tray coverage; [0258] softer cap firmness; [0259] shorter cap height relative to cap diameter; [0260] as thick or thicker cap flesh relative to cap diameter; and [0261] as thick or thicker stipes relative to cap diameter
when compared to the corresponding physical characteristics of B14528, B14528 having representative sample accessible under NRRL Accession No. 50900.

[0262] Embodiment 29. A hybrid mushroom produced by the method of embodiment 22 or produced by the hybrid mushroom strain of any one of embodiments 23-27, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of faster speed to reach pinset and harvestable maturity; [0263] as high or higher tray coverage; [0264] as high or higher percent of 2nd break yield harvested by 24 after casing; [0265] as high or higher 3rd break yield by 32 days after casing; [0266] higher overall 3rd break yield; [0267] higher overall yield across all breaks; [0268] light brown cap color exhibited by a higher L* (lighter) value, lower a* (greener) value, and lower b* (bluer) value; [0269] softer cap firmness; [0270] shorter cap height relative to cap diameter:
as thick or thicker cap flesh relative to cap diameter; and
as thick or thicker stipes relative to cap diameter
when compared to the corresponding physical characteristics of BR06, BR06 having representative sample accessible under ATCC under Accession No. PTA-6876.

[0271] Embodiment 30. A mushroom or part of a mushroom grown from the hybrid mushroom of embodiment 28 or embodiment 29.

[0272] Embodiment 31. The mushroom part of embodiment 30, wherein the mushroom part is a fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, or product produced from the mushroom.

[0273] Embodiment 32. Use of a spore, protoplast, heterokaryon of RO14176, or homokaryon of RO10425 or RO12756, said use comprising isolation of the spore, protoplast, heterokaryon, or homokaryon by any method disclosed herein, [0274] representative sample of RO10425 having been deposited under ATCC Accession No. PTA-127856; [0275] representative sample of RO14176 having been deposited under ATCC Accession No. PTA-127855; and [0276] representative sample of RO12756 having been deposited under ATCC Accession No. PTA-127857.

[0277] Embodiment 33. A new and distinct variety of Agaricus bisporus Imbach mushroom named RO14176, as illustrated and described.

[0278] Embodiment 34. A method of producing a heterokaryotic hybrid mushroom culture of Agaricus bisporus, the method comprising the steps of: [0279] (a) providing a first homokaryon selected from a wild strain, designated AA-0096, or a progeny thereof, wherein said wild strain AA-0096 is available from ATCC under Accession No. PTA-6903; [0280] (b) providing a second homokaryon selected from a commercial off-white hybrid strain, which is compatible with first homokaryon; [0281] (c) crossing the first homokaryon with the second homokaryon to form a first culture of intermediate hybrid homokaryons; [0282] (d) backcrossing the first culture to the second homokaryon to form a second culture of intermediate hybrid homokaryons; [0283] (e) crossing the second culture to a bridging cross strain to form a hybrid mushroom culture, designated RO10425, a culture of which has been deposited under ATCC Accession No. PTA-127856; [0284] (f) allowing the hybrid mushroom culture, designated RO10425, to undergo plasmogamy; and [0285] (g) selecting a heterokaryotic single spore isolate from the result of the plasmogamy, said heterokaryotic single spore isolate designated RO14176, thereby producing the heterokaryotic hybrid mushroom culture.

[0286] Embodiment 35. The method of embodiment 34, wherein the bridging cross strain is 429, representative sample of 429 being available under ATCC Accession No. PTA-6877.

[0287] Embodiment 36. The method of embodiment 34 or 35, wherein the commercial off-white hybrid strain is derived from a U1 mushroom strain.

[0288] Embodiment 37. The method according to embodiments 34-36, further including the steps of producing at least one hybrid mushroom, said steps comprising: [0289] (h) inoculating a mushroom growth medium with the hybrid mushroom culture; [0290] (i) maintaining said inoculated growth medium under conditions conducive to mushroom fruiting; and [0291] (j) collecting at least one heterokaryotic hybrid mushroom from said growth medium.

[0292] Embodiment 38. A hybrid Agaricus bisporus mushroom strain designated RO14176, a culture of which having been deposited under ATCC Accession No. PTA-127855.

[0293] Embodiment 39. A hybrid mushroom strain designated RO14176, representative culture of which having been deposited under ATCC Accession No. PTA-127855, wherein cells from the hybrid mushroom culture have at least one marker presented herein.

[0294] Embodiment 40. A hybrid mushroom produced by the method of embodiment 37 or the hybrid mushroom strain of any one of embodiments 38-39, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of faster timing to reach pinset and harvestable maturity, higher tray coverage, softer cap firmness, different cap color, higher yield, taller cap, and thicker cap flesh, as compared to the corresponding physical characteristics of BR06, BR06 having representative sample accessible under ATCC under Accession No. PTA-6876.

[0295] Embodiment 41. A mushroom part from the hybrid mushroom produced by the method of embodiment 37 or produced by the hybrid mushroom strain of any one of embodiments 38-39.

[0296] Embodiment 42. The mushroom part of embodiment 41, wherein the mushroom part is a fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, or product produced from the mushroom.

[0297] Embodiment 43. A method of producing a hybrid mushroom culture of Agaricus bisporus, the method comprising the steps of: [0298] (a) providing a homokaryon selected from a hybrid mushroom strain designated RO10425, or a progeny thereof, representative sample of RO10425 having been deposited under ATCC Accession No. PTA-127856; [0299] (b) producing at least one heterokaryotic single spore isolate (SSI) from the homokaryon; [0300] (c) selecting, from among the at least one SSI, a desirable single spore isolate; and [0301] (d) producing the hybrid mushroom culture from the selected single spore isolate, [0302] wherein the hybrid mushroom culture is designated RO14176, representative sample of RO14176 having been deposited under ATCC Accession No. PTA-127855.

[0303] Embodiment 44. The method according to embodiment 43, further including the steps of producing at least one hybrid mushroom, said steps comprising: [0304] (e) inoculating a mushroom growth medium with the hybrid mushroom culture; [0305] (f) maintaining said inoculated growth medium under conditions conducive to mushroom fruiting; and [0306] (g) collecting at least one hybrid mushroom from said growth medium.

[0307] Embodiment 45. A hybrid mushroom produced by the method of embodiment 34 or produced by the hybrid mushroom strain of any one of embodiments 38-39, wherein said hybrid mushroom has at least one physical characteristic selected from the group consisting of faster timing to reach second break, [0308] higher tray coverage, [0309] different cap color, [0310] larger stipe diameter, [0311] taller cap, and [0312] thicker cap flesh [0313] when compared to the corresponding physical characteristics of B14528, B14528 having representative sample accessible under NRRL Accession No. 50900.

[0314] Embodiment 46. A mushroom or part of a mushroom grown from the hybrid mushroom of embodiment 40 or embodiment 45.

[0315] Embodiment 47. The mushroom part of embodiment 46, wherein the mushroom part is a fruiting body, cap, stipe, basidium, spore, mycelium, mixed or unmixed spawn, inoculated substrate, or product produced from the mushroom.