Novel non-sporeless Pleurotus genus strains

Abstract

Offering a sporeless Pleurotus strain with a flatter cap and a very short stipe compared to the Spoppo and Allerpo strains. [Solution] The present invention relates to the development of a novel non-sporeless strain of the genus Pleurotus named FiBABH-334, the representative culture of which was deposited on March 21, 2023, with the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, under CNCM accession number I-5939. This non-sporeless strain of Pleurotus yields very high harvest yields and has a very long shelf life for mushroom products. The present invention also relates to cultures obtained from this strain, or its offspring, or cultures induced by other means. More specifically, the present invention relates to a culture containing at least one haploid chromosome set of the strain, in particular a mononucleus thereof, or an F1 hybrid produced by crossing this mononucleus with another strain of the genus Pleurotus. The present invention further relates to methods of using the above-mentioned culture.

Description

Technical Field

[0001] The present invention relates to the development of a new non-sporulating strain of the genus Pleurotus named FiBABH-334, and a representative culture thereof was deposited on March 21, 2023, at the National Collection of Microorganisms (CNCM), Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, Paris, France, under the CNCM deposit number I-5939. This non-sporulating strain of the genus Pleurotus has a very high harvest yield and a very long shelf life for mushroom products. The present invention also relates to cultures obtained from this strain, or its descendants, or induced by other means. More specifically, the present invention relates to a culture containing at least one haploid chromosome set of the above-mentioned strain, particularly its monokaryon, or an F1 hybrid produced by mating this monokaryon with another strain of the genus Pleurotus. The present invention further relates to a method of using the above-mentioned cultures.

Background Art

[0002] The genus Pleurotus is an indispensable mushroom commercially and is widely cultivated around the world. The production volume of Pleurotus mushrooms accounts for about 二十五% of the total cultivated mushrooms in the world. In the United States and Europe, Pleurotus is regarded as a special mushroom, but in Korea, its cultivation is economically profitable and it is one of the mushrooms with high consumption. Pleurotus is rich in protein, dietary fiber, essential amino acids, carbohydrates, water-soluble vitamins, and minerals. These mushrooms contain abundant functional bioactive molecules that affect health. Pleurotus mushrooms have found unique uses such as spices, fragrances, and excellent preservation properties. In addition to its unique uses, Pleurotus is known as a unique delicacy with high nutritional value and medicinal effects. Furthermore, the genus Pleurotus can quickly establish itself in various agricultural environments and has a biological efficiency of 100%, so it can be easily cultivated (Raman et al, 2021).

[0003] It should be noted that the "二十五%" in the English translation of the relevant part in the original text should be the correct percentage number in Chinese characters. If you can provide the accurate number, I can further optimize the translation.One of the main problems in cultivating Pleurotus genus fungi is the occurrence of respiratory illnesses among harvesters and handlers. The symptoms of this disease are identical to those of "mushroom worker lung," a more general term also used to identify many other respiratory illnesses: exogenous allergic alveolitis. Several researchers have reported that the occurrence of this disease is associated with exposure to Pleurotus spores (Baars JJP et al, 2000). Pleurotus strains are enormous in spore production. Conventional and existing strains produce 200 million to 660 million spores per gram of tissue per 24 hours. Spore production in "low-spore-forming" or "low-spore-forming" strains is about one-tenth of that (Sonnenberg et al, 1996), but it is still enough to produce spore concentrations far above the threshold for inducing symptoms of exogenous allergic alveolitis (Baars JJP et al, 2000).

[0004] In this context, non-sporeless strains of the genus Pleurotus are needed to improve the health of mushroom growers.

[0005] Several non-sporeless species of Pleurotus ostreatus are already being produced. Somycel strains 3200 and 3210 (US plant patent US4,658,083), reported in the 1980s, are asporeless Pleurotus species with typical decurrent lamellae on most of the stipe tip and more developed lamellae than other prior art strains. However, these strains have a cornet-shaped, trumpet-shaped, or curled shape, which impairs ease of packaging. Furthermore, they have very long stipes (US4,658,083).

[0006] The most well-known non-sporeless strain of the genus Pleurotus is the so-called "Spoppo" strain, described in plant patent application US2006 / 0010549. Like strain Somycel3210, it possesses non-sporeless basidia, but unlike Somycel3210, it is a mushroom with a well-developed, fleshy, heavy, husk-like cap. The husk-like cap is large (up to 15 cm in diameter), usually curved, and eccentrically attached to the stipe. The stipe is very long (average 54.8 mm, US2006 / 0010549), and the overall height of the mushroom is approximately 64.6 mm (after second flash, this result). Yields are very good.

[0007] Recently, a non-sporeless strain of the genus Pleurotus, known as "Allerpo," has been introduced (US plant patent USPP32,864). This strain's morphology is similar to the Spoppo strain, and both strains originate from the same breeding program. In particular, the cap of the Allerpo strain is darker and browner than that of the Spoppo strain. The stipe is very long (average 51.5 mm, USPP32,864), and the overall height of the plant is approximately 65.2 mm (first flush, this result). The yield is the same as that of the Spoppo strain. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, these three sporeless Pleurotus species from prior art are morphologically characterized by curved caps and long stipes, resulting in a total height exceeding 50 mm, with the stipe accounting for the majority of the height. The tall stipe and curved cap shape affect the packaging of the mushrooms. Since mushrooms are a highly perishable product, they must be processed and transported appropriately and carefully to maintain quality, extend shelf life, and ensure good marketability.

[0009] In these circumstances, there is a need to provide sporeless mushroom strains with flatter caps and smaller stems so that the final mushroom product can be stored and transported more easily in large quantities (without taking up much space), the edible portion of the mushroom can be optimized, and waste after the stems are removed can be reduced (i.e., the product can be made more valuable). [Means for solving the problem]

[0010] This invention addresses this need by providing a strain of non-sporeless oyster mushroom (Pleurotus) that has a flatter cap and a much shorter stem than the Spoppo and Allerpo strains. These two interesting characteristics facilitate the packaging and transport of the mushrooms (allowing for more mushrooms to be packaged in the same volume without affecting the quality of the edible portion) and also help reduce waste (see examples below). Importantly, this novel strain has a yield comparable to the Spoppo and Allerpo strains (see examples below), which are known for their superior yields. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a photograph comparing the mushroom of the present invention with the Spoppo strain mushroom. [Figure 2] Figure 2 shows how to measure the diameter of the mushroom cap and the height of the mushroom. [Modes for carrying out the invention]

[0012] The present invention's strain of oyster mushrooms This invention proposes a novel strain of oyster mushroom (Pleurotus ostreatus) that has significant economic value due to its advantages of not releasing spores, high yield, flat cap, and short stem.

[0013] This strain has been named "FiBABH-334". Its representative culture was deposited on March 21, 2023, with CNCM accession number I-5939 at the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, Paris.

[0014] This strain was obtained by backcrossing the TPLO4 strain (PVR 20173194) with the ATTC-58937 mutant. TPLO4 was partially restored by two consecutive backcrosses.

[0015] The mushrooms produced by the FiBABH-334 strain account for approximately 23% of the total weight (standard deviation ±2.45) over two flashes (calculated based on the number of mushrooms (kg) / 100 kg of substrate). They are relatively short in size (27.5 mm in the first flash and 36.4 mm in the second flash, about half the size of mushrooms from the Spoppo strain; see the examples below).

[0016] The cap color of 30 mushrooms of the FiBABH-334 strain was measured using a Minolta chromatometer, and the Lab color values ​​were L: 69.3 (SD ± 4.1), a: 1.9 (SD ± 0.8), and b: 14.1 (SD ± 1.8).

[0017] As described later in the examples, the FiBABH-334 strain exhibits excellent yield (23.26% total yield), similar to the commercially dominant Spoppo strain. Furthermore, the FiBABH-334 strain has a significantly lower waste rate (ratio of cut weight to uncut weight of mushrooms), making it more economically valuable than the Spoppo strain.

[0018] The Pleurotus strain of the present invention can be considered a sporeless strain because it does not produce visible spores on the spore print for 24 hours. In fact, the amount of spores produced is at least 60.106 times less than that of the parent strain TPLO4. Nevertheless, the Pleurotus strain of the present invention can produce and germinate 20 to 50 spores on the spore print in 24 hours.

[0019] In a first embodiment, the present invention relates to a sporeless Pleurotus strain containing at least one haploid chromosome set of strain FiBABH-334, a representative culture of which was deposited on March 21, 2023, with the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, under CNCM accession number I-5939. The deposit of the culture of Pleurotus strain FiBABH-334 disclosed herein was made to the National Microbial Collection (CNCM) by Somycel (4 Rue Carnot - ZI Sud, 37130 Langeais).

[0020] The deposited culture is identical to the culture maintained by the assignee, Somycel, in Lange, France, prior to the filing date of this application, and the inventor and assignee have permission to refer to this deposited biological material in any patent application. All restrictions on the deposit have been lifted, and the deposit is intended to satisfy all deposit requirements of the Budapest Convention. The deposit date is March 21, 2023. The deposited material will be maintained at the depositary for 30 years, or 5 years from the last request, or the term of the patent, whichever is longer, and will be replaced as needed during this period. The deposited culture will be made publicly available irrevocably and without limitation at the time required by applicable patent law, either at the time of filing the patent application or at the time of issuance of the patent.

[0021] Mushroom cultivation is most reliably identified by genotyping, as market demands that superior cultivars conform to a narrow phenotypic range. Genotyping is characterized by genetic marker profiles, allowing for the identification of systematically related cultivars, including isolates (clones or subculturings) of the same lineage, strain, or culture, or cultivars that are offspring or completely derived from the original cultivar. Furthermore, it can be used to determine or verify the evolutionary lineage of strains over multiple generations.

[0022] In the experience of the inventors evaluating the whole-genome sequences of dozens of various strains and lines of the genus Pleurotus, the typical number of SNP markers that distinguish two unrelated monokaryons is about 300,000. This means that even if 1% of the set of chromosomal or genotype markers in the phylogenetic tree is transmitted, there are about 3,000 discriminant markers indicating the relationship with a specific monokaryon. With 200 - 300 markers, the identity, origin, and derivation among cultures can be established without doubt. On the other hand, by stacking thousands of available SNP markers, a reliable method for establishing the relatedness of the phylogenetic tree over multiple generations can be provided.

[0023] In addition to whole-genome sequencing (WGS), methods for obtaining gene marker profiles using various techniques such as single nucleotide polymorphism (SNP) marking and sequence characterized amplified region (SCAR) marking are well-known in the art. Since any of these techniques can analyze the sequences of specific loci, the same results can be obtained for any locus (however, in dikaryon analysis, WGS provides more detailed information about the distribution of SNPs in the haploid sequence and is effective for confirming allelic sequences).

[0024] The whole-genome sequence of the FiBABH-334 strain has been obtained, and as a result, about 95% (about 34.4 - 35.6 Mb) of the whole DNA sequence genotype of the FiBABH-334 strain is surely known to the licensee.

[0025] Tables 1 - 8 show a concise extract of the genotypes at a number of marker loci characterized by sequences, which are distributed at intervals on each of the 11 chromosomes of the FiBABH-334 strain. For reference, the sequences of the same marker loci of the PC15 strain, Spoppo strain, and Allerpo strain are shown. This table shows that the strain of the present invention differs from the Spoppo strain by at least 298 SNP markers and from the Allerpo strain by at least 298 SNP markers.

[0026]

Table 1

[0027] Table 2

[0028] Table 3

[0029] Table 4

[0030] Table 5

[0031] Table 6

[0032] Table 7

[0033] Table 8

[0034] Tables 1-8 consist of SNP marker sets described as 9-mers. Positional information refers to 11 of the 12 substantial contigs in the PC15 genome sequence assembly (JGI). Since dikaryotic organisms contain two sets of chromosomes, one from each haploid parent, each marker locus in FiBABH-334 has two allele copies (two characteristics or elements of the genotype). IUPAC nucleotide codes and so-called "ambiguity" codes (see also nucleotide and amino acid symbols listed in Annex C, Appendix 2, Table 1, and Standard ST) are listed in Standard ST. In Tables 1-8, heteroallelic codes, used in Chapter 25 of the Handbook of Industrial Property Information and Literature (WIPO) (December 2009) to actually represent dikaryotic or diploid genotypes, are used to represent heteroallelic DNA sequence locations in the above 9-base DNA marker sequences representing genotype marker loci, where each of the two alleles incorporates a different nucleotide at a specific position. The identity of each marker locus was identified by scaffold and SNP position information obtained from the PC15 V2.0 standard reference genome sequence published by the U.S. Department of Energy Joint Genome Laboratory (Alfaro et al. 2016), and is incorporated herein by reference.

[0035] However, it will be understood that any suitable pair of polymerase chain reaction (PCR) primers surrounding a defined marker region can be used for allele identification using appropriate PCR primer design and usage methods known in the art. Methods for determining lineage relationships between cultures using these and other markers are described below.

[0036] Fungi that form mushrooms undergo alternation of generations, progressing from dikaryotic (N+N, possessing two haploid nuclei, functionally equivalent to a 2N diploid state) to monokaryotic (1N), and then returning to dikaryotic through mating. In most eukaryotes, the parents are conventionally considered to be diploid or dikaryotic. The haploid "generation" is often called gametes (e.g., pollen, sperm). In fungi, which are microorganisms, the haploid generation can survive and grow indefinitely and independently, for example, in laboratory cell cultures. These haploid monokaryotic cells (monokaryoplasms) function as gametes in mating, but are equivalent to inbred lines (e.g., plants) and are more readily accepted as lineages (or the "monokaryoplasmic parent" of a hybrid). Here, the term "parent," when used alone, refers, depending on the context, to the dikaryotic culture that is the direct ancestor of the haploid lineage culture, or to the once-removed ancestor of a strain belonging to a subsequent dikaryotic generation obtained from the cross of at least one such lineage. Thus, the term "line" narrowly refers to a haploid (N) homoallelic culture within its life cycle. Cultures that include N+N dikaryotes, seedlings, or cultures used for mushroom production resulting from crosses may be called "strains." Thus, the term "line" narrowly refers to a haploid (N) homoallelic culture within its life cycle. Cultures that include N+N dikaryotes, seedlings, or cultures used for mushroom production resulting from crosses may be called "strains."

[0037] In a preferred embodiment, the strain of the present invention is as follows: (a) F1 hybrid (crossbreed) produced by crossing a single nucleus derived from FiBABH-334 with a second lineage. (b) FiBABH-334 strain, a representative culture of the aforementioned strain, deposited on March 21, 2023, with CNCM accession number I-5939, at the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, Paris, and (c) Derivatives of strain (b), wherein cells obtained from the FiBABH-334 strain by somatic cell selection, tissue culture selection, single spore germination, multiple spore germination, mating between mycelia obtained from mononuclear or dinuclear spores of these hybrids, self-pollination, repeated mating to initial cultures, mutagenesis, transformation, gene transfer transformation, inbreeding, or transformation. It is selected from the group consisting of the following.

[0038] In some embodiments, the strains of the present invention can be obtained by using at least one strain development method selected from the group consisting of inbreeding, outcrossing, i.e., self-pollination, backcrossing, gene transfer transformation, induction, somatic cell selection, tissue selection, monospore selection, polyspore selection, pedigree-assisted breeding, marker-assisted selection, mutagenesis, and transformation, and by applying the at least one strain development method to a first strain, or a portion thereof, which includes at least one set of chromosomes from one nucleus of strain FiBABH-334.

[0039] In a preferred embodiment, the strain of the present invention is a sporeless Pleurotus ostreatus strain of the F2 or F3 generation derived from the FiBABH-334 strain, preferably comprising at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 30%, at least 20%, or at least 10% (preferably at least 99%, at least 98%, at least 95%, at least 92%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70%) of the single nucleotide polymorphisms (SNPs) present in the genome of the FiBABH-334 strain. March 21, 2023, Pasteur Institute, Paris, 25 rue du Docteur Roux, 75724 PARIS, Cedex A representative culture of the strain deposited with 15 National Microbial Collections (CNCM) under CNCM accession number I-5939; or a representative culture of the strain containing 80%-100%, 60%-80%, 40-60%, 20-40%, 10-20%, 10-15%, or 4-10% (preferably including 80%-100% or 60-80%) of the single nucleotide polymorphisms (SNPs) present in the genome of the FiBABH-334 strain, deposited with 15 National Microbial Collections (CNCM) under CNCM accession number I-5939 on March 21, 2023.

[0040] In another preferred embodiment, the strain of the present invention is a strain derived from the FiBABH-334 strain, or a strain derived from the FiBABH-334 strain, and includes at least about 280, at least about 270, at least about 260, at least about 250, at least about 240, at least about 230, at least about 220, at least about 210, at least about 200, at least about 180, at least about 170, at least about 150, at least about 130, at least about 100, at least about 75, at least about 65, at least about 50, or at least about 25 allele markers from the 298 sequences characterized by the FiBABH-334 strain listed in Tables 1 to 8.

[0041] In another preferred embodiment, the strain of the present invention is a strain derived from the FiBABH-334 strain, or a strain derived from the FiBABH-334 strain, comprising at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 30%, at least 20%, or at least 10% (preferably at least 99%, at least 98%, at least 95%, at least 92%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70%) of the single nucleotide polymorphisms (SNPs) present in the genome of the FiBABH-334 strain. March 21, 2023, Pasteur Institute, Paris, 25 rue du Docteur Roux, 75724 PARIS, Cedex Representative cultures of the strain deposited with 15 National Microbial Collections (CNCM) under CNCM accession number I-5939; or strains containing 80%-100%, 60%-80%, 40-60%, 20-40%, 10-20%, 10-15%, or 4-10% (preferably including 80%-100% or 60-80%) of the allele marker characterized by the sequence of the FiBABH-334 strain listed in Tables 1-8.

[0042] In another embodiment, the present invention relates to a sporeless mushroom strain of the genus Pleurotus derived from the initial strain FiBABH-334, a representative culture of which was deposited on March 21, 2023, with the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, under CNCM accession number I-5939.

[0043] Preferably, the derivative strain contains at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 30%, at least 20%, or at least 10% (preferably at least 99%, at least 98%, at least 95%, at least 92%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70%) of the single nucleotide polymorphisms (SNPs) present in the genome of the FiBABH-334 strain. March 21, 2023, Pasteur Institute, Paris, 25 rue du Docteur Roux, 75724 PARIS, Cedex Representative cultures of the strain deposited with 15 National Microbial Collections (CNCM) under CNCM accession number I-5939; or strains containing 80%-100%, 60%-80%, 40-60%, 20-40%, 10-20%, 10-15%, or 4-10% (preferably including 80%-100% or 60-80%) of the allele marker characterized by the sequence of the FiBABH-334 strain listed in Tables 1-8.

[0044] The strain culture of the present invention is different from the parent strain, TPLO4. In preferred embodiments, the strain of the present invention differs from TPLO4 in at least 10%, preferably at least 20%, of allele markers. In other words, the strain of the present invention does not have more than 90%, preferably more than 80%, of identity with TPLO4.

[0045] In another embodiment, the present invention relates to cultures of any of the above-described mushroom strains.

[0046] In this specification, "mushroom culture" refers to mushroom microorganisms grown on various growth media and substrates. This includes physical strains, lineages, mononuclear or dinuclear bodies. Once a culture is obtained, it can be propagated vegetatively in the laboratory.

[0047] The cultured product of the present invention has the following characteristics: (a) The total yield of the culture is at least equivalent to the yield of the Pleurotus genus Spoppo crop, (b) The average total height (including stem and cap) of the mushrooms obtained was significantly lower than the average height of mushrooms of the genus Spoppo. (c) The average height of the mushroom stalks obtained was significantly lower than the average height of the stalks of Spoppo mushrooms, and (d) The caps of the obtained mushrooms are flatter than the caps of mushrooms of the genus Spoppo. It has.

[0048] In particular, the total yield of the cultured product according to the present invention is approximately 23.26% after two flushes (see the examples described later).

[0049] Furthermore, the average total height of the mushroom (including the cap and stem) is preferably in the range of 25 mm to 40 mm. Typically, for mushrooms with a cap diameter of 85 mm or less, the average total height of the mushroom (including the cap and stem) does not exceed 50 mm.

[0050] Mushrooms of the genus Pleurotus In this book, the term "mushroom" refers, depending on the context, to the reproductive organs of fungi of the genus Pleurotus, or to the cultivated food of the same name. The genus Pleurotus consists of three distinct parts: - A fleshy shell or a spatula-shaped cap (cap (pileus)), - A short or long eccentric or central axis called a stalk, and - Long ridges and grooves under the cap called gills or lamellae It is a genus of mushrooms that possesses gills. The gills extend from the edge of the cap to the stem and contain spores.

[0051] A "spore" is the reproductive propagule of a mushroom. A living spore is a dormant dinuclear or mononuclear body. Other parts of a mushroom include the cap, stipe, gills, cells (defined as compartments of the hyphae including the nucleus, mitochondria, cytoplasm, cell membrane, and cell wall containing the crossing wall), hyphae, and mycelium.

[0052] In this specification, the term "monokaryon" refers to a culture containing a single type (or somatic cell lineage) of haploid nuclei (cytogenetically denoted as n). Monokaryons are typically non-fertile (i.e., do not form fruiting bodies), but may function as gametes in sexually complementary anastomoses. Despite being haploid, monokaryons are also called "lineages" because they transmit a uniform genotype to their offspring.

[0053] On the other hand, a "dikaryon" refers to a culture that has two complementary haploid nuclei within a common cytoplasm. Therefore, it is functionally and physiologically similar to a diploid individual (however, cytogenetically it is represented as n+n rather than 2n). Unlike a mononuclear organism, it is fertile (i.e., it forms fruiting bodies). In the context of plant breeding, a dikaryon is also called a "strain." In this invention, a dikaryon strain produced by controlled crossbreeding is also called a "hybrid."

[0054] In this specification, the term "anastomosis" refers to the fusion of two or more hyphae to achieve cytoplasmic continuity. The term "plasmogamy" refers to the formation of a sexual dinucleus, which involves the exchange of nuclei, mitochondria, cytoplasm, and other intracellular components, after cytoplasmic continuity has been established through anastomosis. The sexual joining of two cultures via anastomosis and plasmogamy is referred to herein as "mating."

[0055] Reproduction of the genus Pleurotus Like other fungi, the genus Pleurotus exhibits different reproductive processes: vegetative reproduction (mitosis), sexual reproduction (meiosis), and sexual reproduction (other phenomena). Pleurotus exhibits a typical life cycle of basidiomycetes. First, spores germinate, and monokaryotic mycelia (N) are formed. When two compatible monokaryotic mycelia (at different loci, Mat A and Mat B) come into contact, fertile dikaryotic mycelia (N+N) are formed through hyphal or cytoplasmic fusion.

[0056] Cultivation methods for the genus Pleurotus In a further embodiment, the present invention relates to mushrooms produced by cultivating mushrooms from the strain of the present invention or from the culture of the present invention. This also includes parts and fragments of mushrooms.

[0057] The present invention also relates to products incorporating the strain or culture of the present invention, such as starter cultures, inoculum, processed foods, grains, substrates, and easily breakable particulate matter.

[0058] Furthermore, the present invention relates to a method for producing mushrooms, the method comprising the following steps: a) A step of inoculating a non-sporeless strain of the genus Pleurotus of the present invention or a culture of the present invention into a mushroom growing medium, b) A step of maintaining the inoculated growth medium under conditions suitable for the formation of fruiting bodies of mushrooms, and c) The process of harvesting mushrooms from the growth medium. Includes.

[0059] Pleurotus genus fungi can be cultivated on substrates prepared from agricultural by-products and waste (e.g., straw). Because it is easy to cultivate on a large scale, selecting the appropriate substrate and mycelial growth period is readily possible for those skilled in the art. In commercial production, substrates made from broadleaf trees, sawdust, and straw, with added nutrients, are commonly used. Artificial substrates need to be pre-treated in a clean environment, primarily to remove contaminants. Pleurotus genus fungi can establish and produce mycelium on pre-treated coniferous (pine) wood chips. However, unpre-treated coniferous wood chip substrates inhibit mycelial establishment due to the presence of inhibitory components. Pleurotus genus fungi can also promote economic growth and save forest ecosystems by utilizing wood waste and unused wood residues.

[0060] Spawn (mycelium) is the seed of a mushroom and is used to grow the mycelium of the mushroom on a designated solid substrate during cultivation. The temperature of the spawning chamber should preferably be maintained at 25-30°C. The run period and primordial initiation of the spawn are usually observed for 24-30 days. Light is not essential in the spawning running room. Spawning is performed in a clean spawning area within the bulk chamber. For example, the preparation of oyster mushroom spawn is described in Raman et al., 2021.

[0061] Pleurotus genus are microorganisms that can be handled in the laboratory under sterile conditions (laminar hood and / or clean area, sterile culture media, equipment, apparatus, packaging, etc.) using conventional microbiological methods. The optimal conditions are a temperature of approximately 24°C and a pH of approximately 7. Mycelium or spores can be stored in a cool, dark place (e.g., 2-4°C). Suitable culture media include malt extract agar, potato dextrose agar, potato dextrose yeast agar, and Pleurotus genus-specific substrate agar.

[0062] The sown substrate, culture medium, stalk, and rod (via mycelial bits / mycelium) can be inoculated into cultivation substrates (perforated polyethylene bags, polypropylene bottles, trays). Various cultivation methods are employed for the production of Pleurotus ostreatus, including wall-mounted, shelf-type, tray-type, jar-type, bag-type, bottle-type, and grid-frame-type cultivation. The most common cultivation methods are shelf-type, bag-type, and bottle-type. Shelf-type (row / bed) cultivation is considered a low-cost, high-productivity technique. In bag-type cultivation, the spawn (mycelium) is tied to the top and arranged horizontally. The advantage of plastic bottle cultivation is that it allows for efficient cultivation with high yields even in limited land areas.

[0063] Culture bags and bottles should be placed vertically or horizontally to allow the spawn to spread, and the spawn culture chamber should preferably be maintained at 24-25°C and 90% relative humidity. Spreading of Pleurotus ostreatus spawn takes approximately 12-26 days at 24±1°C in the substrate. Bags / bottles inoculated with spawn form colonies (the outside turns white) and are eventually transferred to the culture chamber. For commercial production, the chamber should be maintained at 15-23°C and 85-95% relative humidity, with a day length of 12 hours and a light density of 15-350 lux. To improve the yield of Pleurotus ostreatus, external nitrogen supply to the substrate is recommended. Cultivation of Pleurotus ostreatus has been tested with various bagging systems. Plastic bags have been shown to yield higher yields than other bagging systems such as trays, racks, and cylindrical containers. Hanging plastic bags in rows reduces contamination levels and allows for good air circulation (Raman et al., 2021).

[0064] Primordial development is usually observed in 16-27 days. Mature mushrooms are typically observed within 3-4 days after pinhead formation. Mature mushrooms are harvested before watering. The entire cultivation process is completed in 50-55 days.

[0065] After picking or harvesting mushrooms, other mushrooms begin to grow one after another at intervals of about a week. The period in the cultivation cycle between periods of mushroom production and periods of no production is called a "flush." ​​If flashes continue, yields generally decrease. Currently, 2-3 flashes are common in industrial systems, but more occur in traditional systems.

[0066] Finally, the crops are removed, the used substrate is discarded, the facility is cleaned, and a new crop cycle begins.

[0067] In this specification, yield is defined as the weight of picked (or harvested) mushrooms / weight of picked (or harvested) / weight of pasteurized substrate, i.e., as a percentage.

[0068] Mushroom processing after harvesting For fresh markets, mushrooms are hand-picked. Hand-picking requires a harvesting period of 3 to 5 days (with intervals between harvests). This is achieved through a smooth, or even faster, transition from the vegetative (mycelial) growth phase to the reproductive growth phase, which correlates with the number and size of the mushrooms.

[0069] Fresh mushrooms spoil very easily and cannot be preserved without proper handling. The decline in quality and value during storage is mainly due to weight loss, shrinkage, browning, and spore formation. Post-harvest processing techniques extend the shelf life, and mushroom quality plays a crucial role in commercialization.

[0070] The shelf life of freshly harvested oyster mushrooms is reported to be 8-14 days at 0°C and 1-2 days at 20°C. Controlled packaging is an alternative technique to extend the shelf life of mushrooms without chemical treatment (such as low temperature or controlled atmosphere packaging).

[0071] Raising offspring In a further embodiment, the present invention relates to cells, spores, hyphae and mycelium obtained from the strain of the present invention or from the culture of the present invention.

[0072] The present invention also relates to a single nucleus of the bacterial strain of the present invention. In fact, although the strain of the present invention is characterized as "sporeless," it does produce some spores. These very few spores can be isolated on the spore print (never more than 50 spores on the entire spore print in 24 hours, preferably 30 or fewer). Furthermore, a single nucleus of the strain of the present invention can also be isolated by protoplastation or debinucleation. SNPs present in the genome of the single nucleus of the present invention can be easily identified by whole-genome sequencing or by using conventional markers such as those described in U.S. Patent No. 7,608,760 or No. 9,017,988. Tables 1-8 show some useful sequences that can characterize the single nucleus of the present invention. However, other SNPs can also be used to identify the single nucleus of the present invention. In preferred embodiments, the mononuclear cells of the present invention are derived from the FiBABH-334 strain and contain at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 30%, at least 20%, at least 10%, or 80%-100%, 60%-80%, 40-60%, 20-40%, 10-20%, 10-15%, or 4-10% of the SNPs present in the genome of the FiBABH-334 strain, preferably those disclosed in Tables 1-8. In another preferred embodiment, the mononuclear body of the present invention is derived from strain FiBABH-334 and comprises at least about 280, at least about 270, at least about 260, at least about 250, at least about 240, at least about 230, at least about 220, at least about 210, at least about 200, at least about 180, at least about 170, at least about 150, at least about 130, at least about 100, at least about 75, at least about 65, at least about 50, or at least about 25 allele markers from among the 298 allele markers characterized by the sequences of the FiBABH-334 strain listed in Tables 1 to 8.

[0073] To calculate the percentage of SNPs between two strains, compare the composite 9-mer genotypes at each locus, assigning 1 for a perfect match and 0 for a less-than-perfect match. Then, sum these values ​​for all loci in the pairwise comparison between each strain and divide by the total number of loci compared. The resulting decimal can finally be converted to a percentage.

[0074] The terms "approximately" or "about" used here mean a range of plus or minus 10% from the stated value.

[0075] In another embodiment, the culture of the present invention comprises at least one pair of chromosomes having at least 65%, 70%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% genetic identity with the mononuclear chromosome of the present invention. In yet another embodiment, the culture of the present invention comprises at least one pair of chromosomes having a genotype having at least 65%, 70%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% phenotype of markers present on the mononuclear chromosome of the present invention.

[0076] In a further embodiment, the present invention relates to a method for developing a novel culture of the non-sporeless genus Pleurotus, the method comprising applying at least one mushroom strain development technique, the representative culture being strain FiBABH-334, deposited on March 21, 2023, at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, under CNCM accession number I-5939, or its offspring, to provide a novel culture.

[0077] Furthermore, the present invention relates to a method for developing a novel non-sporeless Pleurotus genus culture, which includes providing a novel culture by applying at least one mushroom strain development technique to a single nucleus (strain) of the present invention.

[0078] In particular, the method may include crossing a single nucleus or strain of the present invention with a second single or double nucleus of the genus Pleurotus.

[0079] Preferably, the novel culture has any of the characteristics described above for the strain of the present invention. Specifically, the novel culture has the following desired traits: (a) The total yield of the culture is at least equivalent to the yield of the Pleurotus genus Spoppo crop, (b) The average total height (including stem and cap) of the mushrooms obtained was significantly lower than the average height of mushrooms of the genus Spoppo. (c) The average height of the mushroom stalks obtained was significantly lower than the average height of the stalks of Spoppo mushrooms, and (d) The caps of the obtained mushrooms are flatter than the caps of mushrooms of the genus Spoppo. It is preferable that it has one of the following:

[0080] These characteristics were described in detail above. The Spoppo strain is specifically described in plant patent application US2006 / 0010549.

[0081] In a particularly preferred embodiment, this new culture is the F1, F2, F3, or F4 generation derived from the FiBABH-334 strain. Therefore, it may contain at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 30%, at least 20%, at least 10%, or 80%-100%, 60%-80%, 40-60%, 20-40%, 10-20%, 10-15%, or 4-10%, as well as preferably at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, or at least 60% of the allele markers characterized by the sequences of the FiBABH-334 strain listed in Tables 1-8.

[0082] In another preferred embodiment, the new culture contains at least about 280, at least about 270, at least about 260, at least about 250, at least about 240, at least about 230, at least about 220, at least about 210, at least about 200, at least about 180, at least about 170, at least about 150, at least about 130, at least about 100, at least about 75, at least about 65, at least about 50, or at least about 25 allele markers from the 298 allele markers characterized by the FiBABH-334 strain listed in Tables 1-8.

[0083] In another embodiment, the present invention involves the following steps: (a) A step of growing a progeny culture produced by crossing the culture of the present invention with a second culture of the genus Pleurotus, (b) A process of producing a next-generation offspring culture by crossing the offspring culture with itself or a different culture, (c) A step of growing the next generation of offspring cultures and crossbreeding those offspring cultures with themselves or other cultures, (d) A process to produce a mushroom culture by repeating steps (b) and (c) for 0 to 5 more generations. This relates to a method for producing mushroom cultures, including [specific components / products].

[0084] In a particular embodiment, the method comprises the following steps: (a) Steps to obtain the molecular marker profile of the FiBABH-334 strain, (b) A step of obtaining a culture of an F1 hybrid containing at least one set of chromosomes of the FiBABH-334 strain, (c) A step of crossing a culture obtained from the F1 hybrid culture of step (b) with a different mushroom culture, and (d) Step of selecting offspring having the characteristics of the molecular marker profile of the FiBABH-334 strain. Includes.

[0085] definition To provide a clear and consistent understanding of this specification and the claims, including the scope to which terms are given, the following definitions are provided:

[0086] Allele: One of two or more alternative forms of a gene that arises from a mutation and resides at the same location on a chromosome. It is a heritable unit of the genome located at a defined locus and is ultimately identified by DNA sequencing (or other means).

[0087] Anastomosis: The fusion of two or more fungal hyphae to achieve cytoplasmic continuity.

[0088] Basidiomycetes: A monophyletic group of fungi that form meiotic spores on basidia. They belong to the corresponding subphyla of fungi, such as basidiomycetales and basidiomycotina.

[0089] Basidium: These are meiosporangial cells in which nuclear fusion and meiosis occur, forming basidiospores.

[0090] Bioefficiency: For mushroom crops, this is the value obtained by dividing the net fresh weight of the harvested crop by the dry weight of the compost substrate at the time of inoculation with the spawn (any area of ​​harvested crop or weight of compost).

[0091] Breeding: The development of fungal strains, lines, or cultures using methods that emphasize sexual mating.

[0092] Cap: A part of a mushroom, a structure with gills.

[0093] Carrier substrate: A culture medium possessing nutritional and physical properties suitable for achieving both growth and dispersion of cultures.

[0094] Cloning: Somatic cell proliferation without selection. It produces clones, which are a category of phylogenetic relationships (i.e., "identity").

[0095] Breeding ability: The ability of an individual to pass on superior traits to its offspring. General breeding ability refers to the average ability of individuals in a particular breeding line.

[0096] Culture: A physical organism, an organism that grows in various growing media or substrates, a single physical strain, lineage, part or whole of a mononuclear or dinuclear body, hyphae, mushrooms, spores, cells, protoplasm, nucleus, mitochondria, cytoplasm, DNA, RNA, proteins, cell membrane, cell wall, etc., the sum of all parts of the culture.

[0097] Dinuclearization: The transformation of a two-nucleus cell into a single-nucleus cell pair.

[0098] Derivation: Generally refers to the development of strains, lines, or cultures using methods other than sexual mating, and / or development from a single or primarily early strain or culture. See Derived strains and Derived cultures.

[0099] Derived culture: A culture obtained from a derivative as defined above. Examples include "derived strain" and "derived line," but it is not limited to these. It is one category of relationships that indicate lineage.

[0100] Derived lineage group: A collection of strains or cultures that are derived solely from a single initial strain or culture (the oldest member of the group).

[0101] Derived strains / lines: Strains / lines derived from a single parent strain / lineage, either alone or primarily derived from it. Methods for obtaining strains / lines derived from a parent strain / lineage include somatic cell selection, tissue culture selection, monospore germination, polyspore germination, self-pollination, repeated crosses with the parent strain, mutagenesis, and transformation. In oyster mushrooms (Pleurotus ostreatus), a characteristic of derived strains is their high fidelity to the genotype and phenotype of the parent strain.

[0102] In somatic cell selection and tissue culture selection, the induced culture may be a clone, or virtually a clone, of the original culture, and, as with mutagenesis, it may not be practical to identify the actual differences from the original strain. Measurable genetic identity with the original strain can reach 100%. In transformed induced strains, more than 99.99% of the genetic makeup is from the original strain, and only a small portion of the introduced DNA is usually identifiable.

[0103] In both monospore and polyspore germination, the genetic makeup of the derived strain is 100% that of the original strain. Descent: A lineage (genealogical descent) that spans a limited number of sexual generations (e.g., 10 generations or less). A category of phylogenetic relationships.

[0104] In the art to which this invention belongs, a dikaryon refers to a sexual dikaryon, a culture having two complementary haploid nuclei (i.e., necessarily heteroalleles at the MAT locus) within a common cytoplasm, and is therefore functionally and physiologically similar to a diploid individual (though cytogenetically represented as N+N rather than 2N), is fertile (provided there are no rare inhibitory genetic defects at loci other than MAT), and exhibits nutrient incompatibility with other dikaryons. In the context of lineage development, it is also called a strain or stock.

[0105] Dikaryotic: Having the characteristics of a dikaryotic body: There are two haploid nuclei within a common cytoplasm. This is usually interpreted as meaning two sexually complementary nuclei, but there are exceptions.

[0106] Diploid: Has two haploid chromosome pairs within a single nuclear membrane.

[0107] Directed mutagenesis: A process that alters the DNA sequence of at least one specific gene locus.

[0108] Flush: A period in the crop cycle during which mushroom production is interrupted by a non-productive period. The term "flush" encompasses both "break" and "wave" and can be interpreted as either term.

[0109] Foot: Synonymous with stem or stalk.

[0110] Fungi: Microorganisms classified in the kingdom of fungi.

[0111] Genealogical relationship: For example, the relationship between parent and child, a family relationship that is related by identity, lineage, or derivation from one or more ancestors.

[0112] Genetic identity refers to the genetic information that distinguishes an individual, including genotype, genotypic fingerprint, genome sequence, and genetic marker profile, as well as the phenotype of that genetic information. "Genetically identical" means 100% genetic identity, "X% genetic identity" means having X% genetic identity, etc. If the percentage is less than 100%, % genetic similarity may be used instead of % genetic identity.

[0113] Genotypic fingerprint: A description of genotypes within a defined set of marker gene loci. Known genotypes.

[0114] Genetic similarity (or genotype similarity): A term used to describe the degree to which one set of genetic markers (i.e., one genotype) is similar to another set of genetic markers. Any representative set of genetic markers, such as SNP markers, can be used. The percentage of markers common to the genotypes of two individuals or cultures can be used to quantify the similarity between two cultures and serves as an inverse measure of the uniqueness of the two cultures. These terms are used interchangeably with (percent) genetic identity or genotype identity. The percentage of similarity can be calculated based on the genotypes of any set of markers.

[0115] Gills: Part of the plate-like tissue of a mushroom, a structure containing a hymenophore and basidia.

[0116] Haploid: A cell with only one nuclear chromosome. See "monokaryon".

[0117] Heteroallelic: The presence of two different alleles at a single gene locus. Similar to heterozygosity.

[0118] Heteroallelism: Differences between homologous chromosomes in dinuclear genotypes. Similar to heterozygosity.

[0119] Heteromixis: A life cycle involving the mating of two different haploid individuals or gametes that are not siblings; in other words, outcrossing.

[0120] Homoallelic: Having no alleles at a single gene locus. In diploid organisms, the synonym is "homozygosity." By definition, haploid lineages are completely homoallelic at all non-overlapping gene loci.

[0121] Hybrid: A hybrid resulting from two parents, usually applied to dikaryotic strains or cultures produced through controlled crossbreeding.

[0122] Hybridization: The process of physically joining two cultures, normally mononuclear, on a petri dish containing sterile agar, for example, to attempt anastomosis, cytoplasmic fusion, and the formation of a sexually dinuclear (mating). The goal is to achieve these results.

[0123] Hyphae: The thread-like elements of a mycelium, composed of cell-like compartments.

[0124] Inbreeding: Mating between siblings ("selfing"), backcrossing to a parent's lineage or strain, and mating that involves reproduction with genetically related parents.

[0125] Induced mutation: An involuntary process that alters the DNA sequence at at least one gene locus.

[0126] Initial strain, initial culture: A strain or culture used alone or as the primary starting material in the strain induction process. More specifically, a strain or culture from which derivative strains or derivative cultures are obtained. The oldest member of a derived lineage group.

[0127] Inoculum: A form of culture that allows for transmission and proliferation, such as a culture on a new substrate. Special commercially available inoculums include sprouted cells and CIs, and the culture resides on a carrier substrate.

[0128] Intramixis: A uniparental sexual reproduction life cycle involving the formation of a complementary "mating" pair of post-meiotic nuclei within a basidia or individual spore. Superficially, it resembles the process of asexual reproduction.

[0129] Transformation by gene transfer: This involves crossing the offspring of a hybrid with a parent line or parent plant to introduce desirable traits from one line into the dominant genetic background of the other parent line or parent plant.

[0130] Lamella: See folds.

[0131] Line: A culture used to produce hybrid strains through crossbreeding. Usually mononuclear and therefore homoallelic, or if not, a non-dinuclear culture and therefore highly homoallelic. In practice, a functionally mononuclear and completely or predominantly homoallelic culture. Similar to a dominant or completely isozygous inbred line in plant breeding.

[0132] Lineage group: See "derived lineage group". A collection of lineages or cultures that are derived from (or originate from) only a single lineage or culture.

[0133] Locus: A defined, continuous region of the genome. While often different between different genotypes, they exhibit homology. Plural form: loci.

[0134] Low-spore-producing strains: A strain is considered a low-spore-producing strain if its spore production is 10 times lower than that of conventional strains, for example, if it produces approximately 10⁴ to 10⁸ spores per spore cluster in 24 hours.

[0135] Marker-assisted selection: Using linkage gene markers, including molecular markers, to track the locus that determines the desired trait through offspring and lineage.

[0136] MAT: Two unrelated (linked) mating loci, mat A and mat B. These determine sex compatibility and dinuclear status.

[0137] Mating: Sexual fusion of two cultures by anastomosis and plasmogamy. Methods for obtaining controlled mating between mushroom cultures are well known in the art.

[0138] Monokaryon: A haploid culture possessing a single type (or somatic cell lineage) of haploid nuclei (denoted as N in cytogenetics). It is typically infertile, does not exhibit the typical self / incompatible reaction with dinuclei, and may function as a gamete in sexually complementary anastomoses. Similar to inbred plant lines, it is a "lineage" that transmits a uniform genotype to its offspring. Mainly homoallelic lines that breed well but have low fruiting body formation rates are considered presumably monokaryons for the purpose of cultivar development. See the explanation below.

[0139] Monokaryotic: Possesses the characteristics of a single nucleus. A haploid.

[0140] Mycelium: The vegetative body or thallus of mushrooms, composed of filamentous hyphae.

[0141] Mushroom: The reproductive tissue of fungi belonging to the genus Pleurotus. Also known as a cultivated food.

[0142] Neohaplont: A haploid culture or strain obtained by physically denucleating a dinucleus (reducing it to haploid components). Also known as a somatically obtained mononuclear cell or derived mononuclear cell.

[0143] Offspring: For example, the offspring of a single generation of a dikaryotic parent organism. This term is most commonly used to refer to a culture obtained from the spores of a particular mushroom strain.

[0144] Outbreeding: Mating between unrelated or distantly related individuals.

[0145] Parent: The direct ancestor of the individual. The parent plant is dikaryotic, while the parent lineage is monokaryotic. Dikaryotic plants can become F1 dikaryotic parents through the descendants of intermediate parent lineages / monokaryotic plants.

[0146] Pedigree-assisted breeding: Using lineage information to identify desirable lineage combinations in a controlled breeding program.

[0147] Phenotype: Observable characteristics that are expressed and revealed in the environment of a strain or lineage.

[0148] Cytoplasmic fusion (plasmogamy): The establishment of cytoplasmic continuity through anastomosis, leading to the formation of a sexually reproductive binucleus.

[0149] For strains with low spore-forming ability, see Low-Spore-Forming Strains.

[0150] Ancestors: This includes ancestors, including parents (i.e., direct ancestors).

[0151] Progeny: See offspring.

[0152] Selfing: Crossbreeding between sibling lines. See also intramixis.

[0153] Sexual compatibility: A condition in which different strains have mismatched alleles at different mat A and mat B loci. This condition allows the two strains to cross and produce stable, fertile dikaryotic bodies. The opposite condition, sexual incompatibility, occurs when two strains have identical alleles at their respective mat A and mat B loci.

[0154] In somatic cells: vegetative mycelium.

[0155] Spawn: A culture of mushrooms. Typically a pure culture of dikaryotic cells. Typically cultured on a sterile substrate, such as a granular material that is easily broken and dispersible (e.g., grain). Commercially available compost inoculations. Spawn also includes those cultured on a substrate.

[0156] Spores: A part of the fungus, the reproductive organ (propagule).

[0157] Sporeless strains: If no visible spores are observed on the spore print after 24 hours, the mushroom strain is considered a "sporeless strain." In fact, the number of residual spores in these strains is very small, and spores cannot be detected under these conditions. However, these mushrooms produce only a small amount of spores, for example, 20 to 50 spores per spore print in 24 hours. These spores can be detected, for example, by culturing them in a suitable medium and separating them under a microscope.

[0158] Spore print: A pattern of mature spores attached to a suitable surface, such as paper or glass, from the gills of a mushroom.

[0159] Stem: A part of the mushroom that supports the cap.

[0160] Sterile growth media: Nutrient media that promote bacterial growth, sterilized by methods such as autoclaving. Examples include agar-based solid nutrient media such as potato dextrose agar (PDA), nutrient broth, and many other materials.

[0161] Pattern (stipe): See "Pattern (stem)".

[0162] Strain: A dinuclear organism with distinct traits, or a specific identity or ancestry.

[0163] Targeted mutagenesis: A process that modifies the DNA sequence of at least one specific gene locus.

[0164] Tissue culture: Dedifferentiated vegetative mycelium obtained from the proliferation of differentiated tissues of mushrooms.

[0165] Trait conversion: A method of selectively introducing the genetic determinants of one or more desirable traits (i.e., single-locus conversion) into the genetic background of the original strain while retaining most of the original strain's genetic background. See "Transfer Transformation" and "Transformation".

[0166] Transformation: The process of altering the genetic material of individual cells by incorporating foreign DNA into the genome or cytoplasm. This includes transformations involving single-gene locus transformations, or methods for acquiring novel traits.

[0167] Yield: Net fresh weight of harvested crop. Usually expressed as kilograms per 100 kg of substrate (as a percentage).

[0168] Harvesting pattern: The distribution of yield within each flash (occurrence) and across all flashes. This affects size, quality, harvesting costs, and relative disease pressure on crops and products.

[0169] Regarding the above definition of monokaryon, it should be noted that monokaryons and homoallelic lines are subject to technical and practical considerations. In the classical sense, a monokaryon is a haploid culture that is obviously completely homoallelic. In practical terms, for the purpose of fungal strain development, the definition has been somewhat expanded by treating all homoallelic lines as monokaryons to accommodate both technical constraints and cytological variability. Technical constraints include the presence of duplicated DNA regions containing repeating elements such as transposons in the genome, and the possibility of large-scale duplication of chromosomal segments due to past translocation events. Two genome references for Pleurotus ostreatus have been sequenced.

[0170] The genus Pleurotus is a heterothallic homobasidiomycete, and mating is controlled by a bifactorial tetrapolar gene system.

[0171] In the case of reverse-crossed strains, where one offspring from an F1 dinucleus is crossed with one parent, the phenotypic and origin commonality of the parent's single nucleus in the reverse-crossed dinucleus is, on average, about 75%. Somatic cell-selected cultures and tissue-selected cultures have virtually 100% genetic identity with the original culture, but may contain epigenetic changes, rearrangements, or rare mutations. These are often present at the same rate as unselected clonal subculturings and are virtually undetectable. Mutagenic cultures have virtually 100% genetic identity with the original culture, except for one or more random point mutations that are difficult to detect. Transformed cultures typically have at least 99.99% to 100% genetic identity with the original culture and may also contain a small fragment of exogenous DNA, although it is uncertain whether it will be incorporated into the Pleurotus genus genome. [Examples]

[0172] Example 1. Yield of FiBABH-334 strain

[0173] In this study, mushrooms were cultivated using fermented wheat straw as a substrate. The substrate was inoculated with 40 liters / ton (=2%) of mycelium and packed into 12 kg blocks with 24 holes (20 mm in diameter). The study was conducted in a greenhouse. The cultivation period was 18-20 days, and the substrate temperature was 26-30°C. The temperature was adjusted to 17-18°C and the relative humidity to 85-90%, while fresh air and light were introduced for 12 hours / 24 hours. The strains were divided into three growth levels, and the experiment was repeated six times. To summarize the yield, mushrooms were harvested daily, and the stems were cut and weighed. Data was collected over two flashes. The yield of the FiBABH-334 strain was found to be equivalent to that of the SPOPPO strain in the first and second flashes. As shown in Table 9, there was no significant difference in cumulative yield. However, in the first flash, the Spoppo strain reached its harvest peak earlier than FiBABH-334, but in the second flash, both strains reached their harvest peaks at similar times (Table 11).

[0174] We evaluated the yields of two other top-performing strains, Allerpo and Somycel 3200, and none outperformed Spoppo. Allerpo was comparable to Spoppo or lower than Somycel 3200. Based on these results, we can conclude that Allerpo and Somycel 3200 are not superior in yield to FiBABH-334.

[0175] [Table 9]

[0176] Table 9: First and second flash yields (kg mushrooms / kg wet substrate (w / w)) for FiBABH-334 and Spoppo. Standard cultivation and harvesting procedures were used. General t-test: The difference from FiBABH-334 is significant with a p-value ≤ 0.05.

[0177] [Table 10]

[0178] Table 10: Flash yields (kg mushroom / kg wet substrate (w / w)) of Spoppo and 3200(A), and Spoppo and Allerpo(B) after two flashes. Standard cultivation and harvesting procedures were used. General t-test: The difference with Spoppo is significant at p-value ≤ 0.05.

[0179] [Table 11]

[0180] Table 11: Harvest peaks for FiBABH-334 and Spoppo strains. Results are expressed in days after inoculation. General t-test: The difference with Spoppo strain is statistically significant at p-value ≤ 0.05.

[0181] Example 2. Umbrella color of FiBABH-334 strain The color of the mushrooms was measured using a Minolta Chromameter CR-200 (made in Japan). Thirty medium-sized mushrooms were collected from the test subjects, and L * a * The value of the b parameter was obtained. Chromameter measurements were randomly taken from mushroom caps. L * a * In the b system, "L" is a variable representing brightness, where 0 represents complete darkness and 100 represents complete whiteness. The "b" value represents bluishness (-300) / yellowishness (+299). In other words, the darker the color of the mushroom cap, the lower the L value, and the more yellowish the mushroom, the higher the b value.

[0182] In this case, the L value indicates that the umbrella color of the FiBABH-334 strain is darker (Table 12).

[0183] [Table 12]

[0184] Table 12: Chromameter values ​​L, a, and b for the SPOPPO strain and the FiBABH-334 strain. General t-test: The difference from the Spoppo strain is significant with a p-value of ≤0.05.

[0185] Example 3. Characteristics of the umbrella of the FiBABH-334 strain. Using a Mitutoyo electronic caliper, 50 to 60 mushrooms were collected in a single flash, and their morphology was measured.

[0186] As shown in Figure 2, the diameter and height of each mushroom cap were measured.

[0187] As shown in Table 13, for equivalent umbrella diameter sizes between Spoppo and FiBABH-334, the FiBABH-334 strain is flatter.

[0188] This characteristic solves, or at least improves, packaging problems by saving space and simplifies packaging. The reduced height is due to the short stem morphology of FiBABH-334. This characteristic also leads to a reduction in waste (see below).

[0189] [Table 13]

[0190] Table 13: Cap diameter and mushroom height (unit: mm) of FiBABH-334, Spoppo, and Allerpo strains. General t-test: The difference with SPOPPO is statistically significant with a p-value of ≤0.05.

[0191] Example 4. Waste rate Table 14 below shows the discard rate (mushroom cut weight / uncut weight ratio) for the FiBABH-334 and SPOPPO strains. Mushroom strains are typically cut to separate the mushrooms and remove any contaminated portions that have come into contact with the substrate. General t-test: The difference with the FiBABH-334 strain was statistically significant (P-value ≤ 0.05).

[0192] [Table 14]

Claims

1. A strain of the genus Pleurotus that is non-spore-forming and contains at least one haploid chromosome set of strain FiBABH-334, a representative culture of said strain that was deposited on March 21, 2023, with CNCM accession number I-5939, at the Pasteur Institute in Paris, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15.

2. (a) F1 hybrid produced by crossing a mononuclear plant derived from the FiBABH-334 strain with a second line, (b) FiBABH-334 strain, a representative culture of the said strain was deposited on March 21, 2023, with CNCM accession number I-5939, in the National Microbial Collection (CNCM) at the Pasteur Institute, Paris, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, and (c) Derivatives of strain (b), the derivatives are cells obtained from the FiBABH-334 strain by somatic cell selection, tissue culture selection, single spore germination, multiple spore germination, mating between mycelia obtained from mononuclear or dinuclear spores derived from these hybrids, self-pollination, repeated mating to initial cultures, mutagenesis, transformation, gene transfer transformation, inbreeding, or transformation. A strain of the genus Pleurotus ostreatus according to claim 1, characterized by being selected from the group consisting of the following.

3. Derivatives of the strain defined in (b) above, wherein the derivatives are cells obtained from the FiBABH-334 strain by somatic cell selection, tissue culture selection, single spore germination, multiple spore germination, mating between mycelia obtained from mononuclear or dinuclear spores derived from these hybrids, self-pollination, repeated mating to initial cultures, mutagenesis, transformation, gene transfer transformation, inbreeding, or transformation, as shown in Tables 1-8. Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 A non-spore-forming strain of Pleurotus ostreatus according to claim 2, comprising at least 99%, at least 98%, at least 95%, at least 92%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 30%, at least 20%, or at least 10% of allele markers characterized by the sequences of the FiBABH-334 strain listed in Tables 1 to 8, preferably comprising at least 99%, at least 98%, at least 95%, at least 92%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70% of allele markers characterized by the sequences of the FiBABH-334 strain listed in Tables 1 to 8.

4. Derivatives of the strain defined in (b) above, wherein the derivatives are cells obtained from the FiBABH-334 strain by somatic cell selection, tissue culture selection, single spore germination, multiple spore germination, mating between mycelia obtained from mononuclear or dinuclear spores derived from these hybrids, self-pollination, repeated mating to initial cultures, mutagenesis, transformation, gene transfer transformation, inbreeding, or transformation, as shown in Tables 9-16. Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 A non-spore-forming strain of Pleurotus ostreatus according to claim 2, comprising 80% to 100%, 60% to 80%, 40% to 60%, or 20% to 40% of allele markers characterized by the FiBABH-334 sequence listed in Tables 9 to 16, preferably comprising 80% to 100%, or 60% to 80% of allele markers characterized by the FiBABH-334 sequence listed in Tables 9 to 16.

5. A non-spore-bearing strain of the genus Pleurotus, of the F2 or F3 generation, of a representative culture of the FiBABH-334 strain, which was deposited on March 21, 2023, at the Pasteur Institute in Paris, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, with CNCM accession number I-5939.

6. A strain derived from the FiBABH-334 strain, or a strain derived from a strain derived from the FiBABH-334 strain, as shown in Tables 17-24. Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 A non-spore-forming strain of the genus Pleurotus according to claim 5, comprising at least about 200 allele markers from among the 298 sequences of the FiBABH-334 strain listed above that characterize the allele markers.

7. A strain derived from the FiBABH-334 strain, or a strain derived from the FiBABH-334 strain, as shown in Tables 25-32. Table 25 Table 26 Table 27 Table 28 Table 29 Table 30 Table 31 Table 32 A non-spore-forming strain of Pleurotus ostreatus according to claim 5 or 6, comprising at least 99%, at least 98%, at least 95%, at least 92%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 30%, at least 20%, or at least 10% of allele markers characterized by the sequences of the FiBABH-334 strain listed in Tables 25 to 32, preferably comprising at least 99%, at least 98%, at least 95%, at least 92%, at least 90%, at least 85%, at least 80%, at least 75%, or at least 70% of allele markers characterized by the sequences of the FiBABH-334 strain listed in Tables 25 to 32.

8. A strain derived from the FiBABH-334 strain, or a strain derived from a strain derived from the FiBABH-334 strain, as shown in Tables 33-40. Table 33 Table 34 Table 35 Table 36 Table 37 Table 38 Table 39 Table 40 A non-spore-forming strain of Pleurotus ostreatus according to claim 5 or 6, comprising 80% to 100%, 60% to 80%, 40% to 60%, or 20% to 40% of allele markers characterized by the sequence of the FiBABH-334 strain listed in Tables 33 to 40, preferably comprising 80% to 100%, or 60% to 80% of allele markers characterized by the sequence of the FiBABH-334 strain listed in Tables 33 to 40.

9. This is a sporeless mushroom strain of the genus Pleurotus, derived from the initial strain FiBABH-334, and a representative culture of this strain was deposited on March 21, 2023, with CNCM accession number I-5939, at the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, Paris.

10. Tables 41-48 Table 41 Table 42 Table 43 Table 44 Table 45 Table 46 Table 47 Table 48 The non-spore-free oyster mushroom strain of the genus Pleurotus according to claim 9, characterized in that it contains at least 85% of the allele markers characterized by the sequence of the FiBABH-334 strain listed above.

11. A culture according to any one of claims 1 to 10, characterized by the following: (a) The total yield of the culture is at least equivalent to the yield of the Pleurotus genus Spoppo crop, (b) The average total height of the mushrooms obtained (including the stem and cap) was significantly lower than the average height of the mushrooms from the Spoppo strain. (c) The average height of the mushroom stalks obtained was significantly lower than the average height of the mushroom stalks of the Spoppo strain, and (d) The caps of the obtained mushrooms are flatter than the caps of the Spoppo strain mushrooms. Culture.

12. Mushrooms produced by cultivating mushrooms from a strain according to any one of claims 1 to 10, or from a culture according to claim 11.

13. Cells, spores, hyphae, or mycelium obtained from a strain according to any one of claims 1 to 10, or from a culture according to claim 11.

14. A mononucleus of the FiBABH-334 strain, the representative culture of which was deposited on March 21, 2023, with CNCM accession number I-5939, at the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, Paris.

15. A product containing a strain according to any one of claims 1 to 10 or a culture according to claim 11, comprising a substrate containing a starter culture, an inoculum, processed food, grain, a substrate, and easily breakable particulate matter.

16. The following steps: a) A step of inoculating a mushroom growing medium with a non-sporeless strain of the genus Pleurotus according to claims 1 to 10, or a culture according to claim 11. b) A step of maintaining the inoculated growth medium under conditions suitable for the formation of fruiting bodies of mushrooms, and c) Step of harvesting mushrooms from the growth medium. A method for producing oyster mushrooms, including those of the genus Pleurotus.

17. A method for developing novel non-sporeless Pleurotus ostreatus cultures, comprising applying at least one mushroom strain development technique, specifically strain FiBABH-334, whose representative culture was deposited on March 21, 2023, with CNCM accession number I-5939, in the National Microbial Collection (CNCM) at the Pasteur Institute, 25 rue du Docteur Roux, 75724 PARIS, Cedex 15, Paris, or its descendants.

18. The method according to claim 17, comprising crossing a mononuclear body described in claim 14 or a strain described in claims 1 to 10 with a second mononuclear or dinuclear strain of the genus Pleurotus.

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