New shiitake ( lentinula edodes) strain with increased properties

Abstract

The present invention relates to the development of a new Shiitake (Lentinula edodes) strain designated ST428, a representative culture thereof having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number I-5946. This Shiitake strain displays an excellent yield weight of the harvested crop, a dark brown cap color and a very good shelf-life of the mushroom products. The present invention also relates to cultures obtained, descended, or otherwise derived from this strain. More particularly, the present invention relates to cultures comprising at least one haploid set of chromosomes of said strain, especially to monokaryons thereof or to F1 hybrids produced by mating this monokaryon with a second Shiitake line. The invention further relates to methods of use of the cultures described hereinabove.

Description

[0001] NEW SHIITAKE (Lentinula edodes) STRAIN WITH INCREASED PROPERTIES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the development of a new Shiitake (Lentinula edodes) strain designated ST428, a representative culture thereof having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number I- 5946. This Shiitake strain displays an excellent yield weight of the harvested crop, a dark brown cap color and a very good shelf-life of the mushroom products. The present invention also relates to cultures obtained, descended, or otherwise derived from this strain. More particularly, the present invention relates to cultures comprising at least one haploid set of chromosomes of said strain, especially to monokaryons thereof or to Fl hybrids produced by mating this monokaryon with a second Shiitake line. The invention further relates to methods of use of the cultures described hereinabove.

[0004] BACKGROUND OF THE INVENTION

[0005] The edible mushroom Shiitake, a microorganism belonging to the basidiomycete fungi, is widely cultivated around the world. Shiitake mushroom market size is projected to grow from 0.74 billion $ in 2023 to 1.2 billion $ by 2032, exhibiting a compound annual growth rate of 6.56% between 2023 and 2032. Many aesthetic applications, as well as high nutritional and therapeutic value are the key market drivers enhancing market growth. Shiitake products are popular around the world, owing their rich savoury flavour and several health advantages (they containg high concentration of vitamins, antioxidants, and are low in calories and almost fat-free). These mushrooms can be consumed fresh or in processed forms such as dried form. In 2022, fresh mushrooms held an 89.5% revenue share of the mushroom market. Fresh form distribution is difficult for producers or distributors because of their limited shelf life. Development of novel hybrid mushroom strains or lines of this valuable mushroom fungus is seen as highly desirable to the cultivated mushroom industry, in general to improve genetic diversification of the crop, and particularly if those novel strains or lines can be developed to provide various desirable traits, or novel combinations of traits, within a single strain, culture, hybrid or line.

[0006] Cultures are the means by which the mushroom strain developers prepare, maintain, and propagate their industrial microorganisms. Cultures of mushrooms, like those of other microorganisms, are prepared, maintained, propagated and stored on sterile media using various microbiological laboratory methods and techniques known in the art. Sterile tools and aseptic techniques are used within clean rooms or sterile transfer hoods to manipulate cells of pure cultures for various purposes including clonal propagation and for the development of new strains using diverse techniques. Commercial culture inocula including mushroom 'spawn' is also prepared using large-scale microbiological production methods and are provided to the end user as pure cultures on substrate media contained within sterile packaging.

[0007] One use of such cultures is to produce mushrooms for sale and consumption. Mushrooms are cultivated commercially within purpose-built structures on dedicated mushroom farms. While there are many variations on methods, and no single standard cultivation method, the following description represents a typical method. Traditionally shiitake has been cultivated on natural wood log. This method of culture has been replaced in Asia by cultivation in sterilized substrate for a better biological efficiency and a shorter cycle of culture. Another way of culture would be the cultivation on pasteurized substrate. This alternative remains the most accessible since straw is easily accessible, and pasteurization would require less energy. This methodology remains dominant in Europe.

[0008] Concerning sterile cultivation, substrate is generally composed by sawdust supplemented by bran, gypsum and CaCO3, sucrose can sometime be added. Sterilization is done by autoclaving.

[0009] Trough spawning is preferably used in the US while localized spawning at the surface or in an inoculation hole is commonly used in Asia. Inoculated substrate is incubated between 21 and 27°C (temperature in the substrate) at high Relative humidity (95 -100%) and high CO2 level (>10000 ppm) for 10 to 12 weeks (depending on the performance of the strain). At the end of the incubation, we observe formation of blisters, and the surface of the substrate turns brown. When incubation is completed, bags are removed, and fructification is induced by lowering the temperature to 17°C, removing CO2. Humidity will be maintained high during primordia formation and then reduced during mushroom development. Harvest can last for 12 to 16 weeks, along different flushes.

[0010] For pasteurized cultivation, wheat straw is shredded into small pieces, it is soaked in water for 6 to 12 hours. And then mixed with gypsum. Generally, it is supplemented to provide enough nutriment for mycelium growth and to decrease Trichoderma contamination. The straw is then pasteurized with steam at 65°C for 12 to 24 hours.

[0011] Mushroom spawn, which comprises a sterilized friable 'carrier substrate' onto which a pure culture of one mushroom strain has been aseptically incorporated via inoculum is mixed to the pasteurized substrate at rate 5-7% (w / w). Mixture is is placed into perforated plastic bags and incubated at 25°C (temperature in the substrate) for 4 to 8 weeks (depending on the performance of the strain). At the end of the incubation, like for culture with sterile substrate, bags are removed when we observe formation of blister and a brown surface and fructification is induced by lowering the temperature to 17°C, a relative humidity of 90% and light cycle 12 hours light: 12 hours dark. Humidity will be maintained high during primordia formation and then reduced during mushroom development. Harvest can last for 12 to 16 weeks, along different flushes.

[0012] Following harvest, mushrooms are graded, sorted, weighed, packed and shipped under refrigeration. Profitability associated with a strain is dependent upon (1) the yield weight of the harvested crop, net of losses from disease, damage and post-harvest weight loss, (2) variable labor and other costs of harvesting or processing mushrooms of different sizes, weights, spacing / timing behaviors, and types or grades, and (3) crop value based on the quality and marketability of the mushroom product as determined by appearance, physical characteristics, condition during post-harvest storage and marketing (i.e., "shelf life" effects).

[0013] For many producers, having a steady harvest of mushrooms from day to day or week to week would solve costly problems in harvest- and packing-labor scheduling and management, and also related problems with product inventory, storage and delivery. High yield combined with more balanced yield between flushes is desirable for many growers. While steady production, which is largely a biological trait of individual strains, mitigates some of these costly issues, a further solution to the problem of declines in postharvest (or 'shelf-life') quality and value, including loss of salable weight (due to evaporation and respiration), is desired in the form of a strain which retains more postharvest weight, or other element of product quality, for a longer time during post-harvest storage.

[0014] There is a need for more diverse, more versatile, and more profitable Shiitake mushroom strains. To meet this need for improved, diverse Shiitake mushroom strains, various entities within the mushroom industry have set up mushroom strain development programs. The goal of a mushroom strain development program is to combine, in a single strain, culture, hybrid, or line, various desirable traits. Strains currently available to the mushroom industry allow growers to produce crops of mushrooms successfully and profitably. There are many characteristics by which a novel strain might be judged as improved over existing strains, or more suitable, in a particular production facility or sales market, or in the industry regionally or globally. Such characteristics can be assessed using techniques that are well known in the art.

[0015] In Assignee's aggregate operating experience of almost 100 years of mushroom strain development in Assignee's research centers, it has been extremely difficult to develop more than a handful of strains which are acceptable with respect to all necessary commercial characteristics. While many traits could cause a strain not to be commercially acceptable, three of the foremost qualifying traits are crop yield, crop timing, and appearance / "quality" of the mushrooms produced.

[0016] Market conditions change over time. Consumer preferences shift and evolve. New pathogens emerge. Raw materials fluctuate in price, composition and availability. Therefore, spawn producers and mushroom producers need access to diversified commercially acceptable strains that present different, alternative combinations of characters that allow for flexible and effective responses to changing market or production conditions, including challenges which may be unforeseeable (e.g., pathogens, agricultural chemical regimens, altered availability or year-to-year properties of particular compost raw materials, etc.). Genetic diversity is responsible for diversity of phenotypic characters including both evident characteristics and others which might not become evident or explicitly valuable except under changed or unpredictable conditions.

[0017] Thus, there is a general need for commercially acceptable Shiitake strains with different, diverse, novel genotypes, relative to other commercially produced strains, for at least two reasons:

[0018] First, it is well understood that when an agricultural crop industry relies extensively on a single, or only two, genetic lineage(s) (i.e., creates a near-monoculture situation as now exists in most countries for brown-capped mushrooms), there is an increased risk of unpredictable, catastrophic crop failure on a facility-wide or even industry-wide scale, due to emerging diseases or other conditions. Therefore, from a risk management and food security perspective, it is highly desirable to simultaneously provide both genetic diversification and commercially acceptable performance and crop characteristics in an expanded range of commercially available strains.

[0019] Second, it is understood that flavor ("taste") is perceived by different persons in highly individual ways. Both untrained and trained tasters register idiosyncratic preferences for mushrooms produced by different strains; there is no single "best-tasting" mushroom strain, but rather a diverse collection of individual preferences. Preferences for cap color are also diverse and idiosyncratic. Providing genetically diverse offerings of mushrooms provides the consumer with more options and a better chance of finding a mushroom that may become a personal "favorite". Increased consumer choice and satisfaction supports increased sales pricing and volume and is beneficial to all parties.

[0020] Thus, any commercially acceptable hybrid strain with a novel genotype is useful and advantageous in overcoming the industry-scale problem of limited genetic diversity and global crop resilience, and also the problem of limited options for crop rotation and facility hygiene management, while increasing the prospects for broader consumer acceptance and satisfaction. Most commercial production of brown-capped Shiitake mushrooms today employs the Sylvan strains (Sylvan 4325, 4080, 4312, 4320), Mycelia strains (M3782 for example) or strains used by QiHe Biotech for their synthetic logs. For all the reasons mentioned above, the mushroom industry has need of other strains that (1) produce an acceptable yield of mushrooms, for example a yield of at least 95%, and preferably of at least 100%, of current commercial strains such as Sylvan4325, (2) have a good appearance and in particular a desirable dark brown color, and (3) produce mushrooms of high quality for the consumer, and which retain more of their initial weight over an extended period of days in the postharvest sales chain, compared to the Sylvan4325 strain.

[0021] The present invention fulfills this need by providing new strains that are genetically distinct from all the prior art strains and which meet the desires of mushroom producers, marketers and consumers, including commercially acceptable strains having the specific performance and shelf-life improvements noted above.

[0022] DETAILLED DESCRIPTION OF THE INVENTION

[0023] The shiitake (also called shitake) is an edible mushroom of the Lentinula edodes species. Native to East Asia, it is now cultivated and consumed around the globe. It is considered a medicinal mushroom in some forms of Asian traditional medicine.

[0024] Shiitake mushrooms are also commonly called "sawtooth oak mushrooms", "black forest mushrooms", "black mushrooms", "golden oak mushrooms", or "oakwood mushrooms", as they were traditionally cultivated on the decaying wood of deciduous trees, particularly shii and other chinquapins, chestnut, oak, maple, beech, sweetgum, poplar, hornbeam, ironwood, and mulberry trees.

[0025] Traditionally producing shiitake mushrooms on wood logs induce three specific and straightforward phases: fruiting, harvesting, and storage.

[0026] Temperature and moisture changes trigger the shiitake fungus to produce mushrooms. Therefore, log shocking (or soaking) is often used for "forced fruiting" and is done at a time planned by the grower. Keep in mind that forced fruiting can reduce the production life of the log. Shocking can be done by various methods, such as using stock tanks. Water should be clean and have a significantly different temperature compared to ambient temperatures. The length of soak depends on the air and water temperatures, the log's age, and the log's bark thickness. As a rule, the closer the air (log) temperature is to the water temperature, the longer the soak. For example, soak time in the summer is usually six to 24 hours, while soak time is usually two to three days in the spring or fall. Older logs and thin-barked logs absorb water quickly and do not require soaking times as long as younger or thick-barked logs. After logs are shocked, they are stacked for fruiting, normally using the high A-frame or lean-to configuration. The stacking arrangement should make it easy to access all sides of each log for harvest. Logs stacked outdoors can use a protective covering to minimize wind and rain, stabilize temperatures, and localize humidity around the logs. Burlap and plastic should be used with care and are recommended only for experienced growers. Fruiting (humidity) blankets, which are a better choice for the grower, are porous, white, synthetic, felted materials that hold water, allow air movement, and provide some insulation.

[0027] Shiitake logs are ready to fruit when the shiitake has colonized the outer cylinder of available sapwood. At six to 24 months post-inoculation, the mycelium has stored enough nutrients to form mushrooms.

[0028] "Pinning" refers to early mushroom development. It often occurs as the logs dry three to five days after shocking but can also occur naturally after a week. At this stage, mushrooms are highly vulnerable to cold and windy conditions, which may inhibit growth. Whereas well-colonized soaked logs can have moisture contents as high as 80-90%, pinning requires a log moisture content of 35% to an optimum of 60% . Optimum temperatures for pinning are between 13 and 18 °C with relative humidity of at least 85% .

[0029] Once the "pins" have emerged, the protective covering or fruiting blanket over the logs can be removed. Exposing more of each log produces dry, firm mushrooms. This typically takes seven to 10 days following shocking. Depending on the temperature and shiitake strain, the fruiting period usually lasts from one to two weeks. A grower can extend the natural outdoor fruiting season by using different combinations of strains (cold weather, warm weather, and wide-range).

[0030] Shiitake should be picked when the cap is opened approximately 50-75% . At this stage, the gills that are exposed by the cap edges are still rolled under the cap. Harvesting mature mushrooms with the cap 100% opened can lead to reduced shelf life, a longer delay before the next flush, and increased pest problems. Shiitakes are picked by grasping the lower portion of the stem and pulling the mushroom from the log with a slight twisting motion. Shiitake can also be harvested by cutting them as close as possible to the log surface with a sharp knife or pair of scissors. Since bruises on the caps and gills will discolor rapidly, only the stems should be touched during picking. Picking mushrooms from the bottom of the log and working up can minimize the accumulation of bark flecks and other debris on unpicked mushrooms. Also, after shiitakes are picked, the stems can be trimmed to remove debris. Picked mushrooms can be put into a basket, box, paper bag, or other suitable container. Plastic bags should be avoided, as they can hasten mushroom decomposition. Air vents in the container are recommended so the shiitake can be cooled rapidly. To prevent bruising and promote rapid cooling, containers should not be filled more than 4-6 inches deep with shiitake mushrooms.

[0031] The immediate objective after picking is to cool the shiitake as rapidly as possible. The use of plastic crates or baskets that are slatted on all sides are recommended for refrigerator storage. Mushroom shelf life is reduced dramatically by using containers that do not allow rapid cooling. Also, frost-free refrigerators tend to dry mushrooms excessively. The storage life of shiitakes is similar to that of Agaricus bisporus and is highly dependent on temperature.

[0032] Cultivation process has evolved, with dedicated wood or straw substrates instead of real tree logs.

[0033] Inoculated substrates can be incubated between 4 weeks (pasteurized substrate) to 12 weeks (sterile substrate) at a temperature between 21 to 25°c and a Relative Humidity at 80-85%. Standard light cycle 12h dark:12h light can be used but incubation can also be performed in the dark. When substrate bags are fully colonized and turned brown, plastic can be removed to provide oxygen for the development of the mushroom. Primordias can be induced by decreasing temperature (17°C in the room), and enhancing the relative humidity to 90-95% with a standard 12:12h lightdark cycle and a CO2 level inferior or equal to 1000 ppm.. During the flush and mushroom development CO2 level can be set between 1000 and 2000 ppm.

[0034] In a first aspect, the present invention is directed to a Shiitake culture comprising at least one haploid set of chromosomes of the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, said set of chromosomes preferably comprising the sequence-characterized allelic markers listed in Table I, more preferably characterized in that it is selected from the group consisting of: (a) an homokaryon of the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, and (b) F2 hybrids produced by mating said homokaryon (a) with a second line.

[0035] In one embodiment, the Shiitake culture comprises at least one haploid set of chromosomes of the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, the chromosomes of said culture / mushroom comprising at least 50 out of the 398 sequence-characterized allelic markers listed in Table I.

[0036] Another aspect of the invention relates to a Shiitake mushroom strain culture of the Fl, F2, F3, or F4 generation, descended from the hybrid ST428, or from a strain derived from strain ST428, and comprising respectively at least 40-60%, at least 20-30%, at least 10-15%, or at least 4-8% of the Single-Nucleotide Polymorphisms (SNPs) present in the genome of the Shiitake strain ST428, a representative culture of said line having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number 1-5946. Yet another aspect of the invention relates to a Shiitake mushroom culture that is derived from an initial culture, wherein said initial culture is chosen in the group consisting of: (a) the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Micro or ganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, and (b) any culture that is defined hereinabove as a culture of the invention; and which may be characterized in that it comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100 markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, or in that it comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence-characterized allelic markers of ST428 listed in Table

[0037] I.

[0038] In a preferred embodiment of the invention, the culture of the invention as described hereinabove is characterized in that: (a) the yield performance of the crops of said culture are equal to or exceed the yield performance of crops of the Sylvan4325 strain of Shiitake, (b) a darker brown color of the cap of the mushroom, as compared with the cap color of Sylvan4325 Shiitake mushrooms, and (c) the mushroom product of the crops of said culture retains more weight after a number of weeks of post-harvest storage at 4 degrees Celsius than does the mushroom product of the Sylvan4325 strain of Shiitake, the number of weeks being selected from the group comprising 2, 3, and 4 weeks.

[0039] In a more preferred embodiment of the invention, the culture of the invention as described hereinabove is the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023. Mushrooms produced in crops by strain ST428 are about 23 % (w / w) (S.D. ±2) in one flush. Cap color measurements on mushrooms of ST428 produced L-a-b color of L:31.8 (S.D ± 3,8) a:11.2 (S.D. ±1,3) b:14.7 (S.D. ± 1,5) when measurements were taken on 50 mushrooms using a Minolta Chromameter.

[0040] The invention also relates to cells, hyphae, mycelium, mushrooms, germinated spores, ungerminated spores, homokaryons, and heterokaryons including SNPs, NSNPs, and aneuploids obtained from a culture of the invention as well as a product incorporating the culture of the invention, including spawn, inoculum, mushrooms, mushroom parts, mushroom pieces, processed foods. In a preferred embodiment, the invention relates to liquid formulations containing the strain of the invention, or to inoculated sterile or pasteurized substrates containing the strain of the invention.

[0041] The present invention also relates to a method for developing a new Shiitake culture, said method comprising applying at least one mushroom strain development technique to an homokaryon of the strain ST428, a representative culture of which having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number 1-5946, or to a progeny thereof, to provide a new culture. Preferably, said new culture is characterized in that: (a) the yield performance of the crops of said culture are equal to or exceed the yield performance of crops of the Sylvan4325 strain of Shiitake, (b) a darker brown color of the cap of the mushroom, as compared with the cap color of Sylvan4325 Shiitake mushrooms, and (c) the mushroom product of the crops of said culture retains more weight after a number of weeks of post-harvest storage at 4 degrees Celsius than does the mushroom product of the Sylvan4325 strain of Shiitake, the number of weeks being selected from the group comprising 2, 3, and 4 weeks. In a preferred embodiment, said new culture is an F2, F3, F4, or F5 hybrid descended from a strain derived from the strain ST428, and has a genotype that comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100 markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I; or has a genotype that comprises at least 40-60%, at least 20-30%, at least 10-15%, or at least 4-8% of the Single-Nucleotide Polymorphisms (SNPs) present in the genome of ST428, a representative culture of said line having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number I- 5946.

[0042] The deposit of a culture of the Shiitake hybrid called "ST428", as disclosed herein, has been made by Somycel, 4 Rue Carnot - ZI Sud, 37130 Langeais, with the Collection Nationale de Cultures de Microorganismes (CNCM). The culture deposited was taken from the same culture maintained by Somycel, Langeais, France, the assignee, since prior to the filing date of this application, and the inventors and assignee have received authorization to refer to this deposited biological material in any and all patent applications. All restrictions upon the deposit have been removed, and the deposit is intended to meet all deposit requirements under the Budapest Treaty. The date of deposit was April 13, 2023. Moreover, the deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of any patent, whichever is longer, and will be replaced as necessary during this period. The culture of this deposit will be irrevocably and without restriction of condition released to the public upon the filing of the patent application or upon the issuance of a patent, whichever is required by the applicable patent laws.

[0043] As described in the experimental part below, ST428 has a good yield, and mushrooms with improved keeping qualities, compared to a leading commercial strain, Sylvan4325. It achieves these improvements by virtue of a novel genotype which is different from other known brown-capped strains (see table I). Further, the genetic distinctness provides genetic diversification of the global mushroom crop, which will provide new opportunities to meet existing and emerging challenges in the diverse markets in which edible Shiitake mushrooms are grown and sold. In some embodiments, the culture of the invention may be obtained using at least one strain development technique selected from the group consisting of inbreeding, including intramixis, outbreeding, i.e., heteromixis, selfing, backmating, introgressive trait conversion, derivation, somatic selection, tissue selection, single-spore selection, multispore selection, pedigree-assisted breeding, marker assisted selection, mutagenesis and transformation, and applying said at least one strain development technique to a first mushroom culture, or parts thereof.

[0044] In other embodiments, the culture of the invention results from a strain development technique and is a culture derived, descended, or otherwise obtained from the strain culture of the invention. The resulting culture thus has at least one genealogical relationship with the initial culture, wherein that genealogical relationship is selected from the group consisting of (1) identity, i.e., self, clone, subculture, (2) descent, i.e., inbred descendent, outcrossed descendent, backcrossed descendent, Fl hybrid, F2 hybrid, F3 hybrid, F4 hybrid, F5 hybrid, and (3) derivation, i.e., derived culture. In a particular aspect, the present invention relates to a Shiitake mushroom strain culture of the Fl, F2, F3, or F4 generation, descended from the hybrid ST428 or from a strain derived from strain ST428. Said strain preferably comprises respectively at least 40-60%, at least 20-30%, at least 10- 15%, or at least 4-8% of the Single-Nucleotide Polymorphisms (SNPs) present in the genome of the Shiitake strain ST428, a representative culture of said line having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number 1-5946.

[0045] More precisely, the Fl offspring of ST428 will comprise at least about 200 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I; the F2 offspring will comprise at least about 100 out of the 398 sequence-characterized allelic markers of ST428 listed in Table I; the F3 offspring will comprise at least about 50 out of the 398 sequence-characterized allelic markers of ST428 listed in Table I; and the F4 offspring of ST428 will comprise at least about 25 out of the 398 sequence-characterized allelic markers of ST428 listed in Table I.

[0046] In other words, the strain culture of the invention preferably comprises at least about 200 out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, at least about 100 out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, at least about 50 out of the 398 sequence-characterized allelic markers of ST428 listed in Table I or at least about 25 out of the 398 sequence-characterized allelic markers of ST428 listed in Table I.

[0047] In a particular embodiment, the strain of the invention is a Fl hybrid having the heterokaryon culture ST428 as at least one parent, and having at least one haploid chromosome set comprising 50% of the allelic markers present in the genotype of the ST428; an F2 hybrid having said Fl hybrid as at least one parent, and having at least one haploid chromosome set comprising 50% of the allelic markers present in the genotype of the Fl hybrid; an F3 hybrid having said F2 hybrid as at least one parent, and having at least one haploid chromosome set comprising 50% of the allelic markers present in the genotype of the F2 hybrid; an F4 hybrid having said F3 hybrid as at least one parent, and having at least one haploid chromosome set comprising 50% of the allelic markers present in the genotype of the F3 hybrid.

[0048] The SNPs present in the genome of the Shiitake strain of the invention can be easily identified by whole genome sequencing Table I gives a number of useful sequences that characterize the strain of the invention. Any other SNP can however be used to identify progenies of the strains of the invention.

[0049] In a preferred embodiment, the Shiitake mushroom strain culture of the invention descends from the strain ST428 and contains approximately 50%, approximately 25%, approximately 12.5%, approximately 6.25%, or approximately 3.13% of the SNPs present in the genome of the Shiitake strain ST428, preferably of the SNPs disclosed in Table I. In another preferred embodiment, the Shiitake mushroom strain culture of the invention descends from the strain ST428 and contains at least about 200, between 100 and 200, between 50 and 100 or between 25 and 50 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I.

[0050] In a preferred embodiment of the invention, the strain culture of the invention is characterized in that the first flush yield performance of the crops of said culture are equal to or exceed the total yield performance of crops of a Sylvan4325 strain of Shiitake. First flush yield performance can be measured as defined below in large scale trials. During such trials, incubation period can be for example of 7 week of incubation, spawning rate can be 8 % (w / w). Bags can be filled with 12 kg of inoculated pasteurized substrate. To collect yield, mushrooms can be picked and weighed daily, on at least four replicates. Data can be collected over several flushes.

[0051] In another embodiment, the mushroom product of the crops of said culture retains more weight after a number of weeks of post-harvest storage at 4 degrees Celsius than does the mushroom product of the Sylvan4325 strain, the number of weeks selected from the group comprising 1, 2, 3 and 4 weeks. This measurement can be done as disclosed in the experimental part below. Piece weight collection can be carried out as disclosed in the example part below. Piece weight is preferably evaluated in Flush 1, for example for three to five replicate styrofoam tills per strain. Briefly, the weight of the empty till is recorded, then a define number mushrooms are placed into each till, spaced enough to not touch each other. They are placed with the stem up, and immediately weighed. This weight corresponds to the "initial weight". Then the tills are placed at 4°C for 1, 2, 3, 4 weeks in a walk-in cooler. The till weights are recorded each day. After subtracting the weight of the empty till, percentage of weight retention can be calculated.

[0052] In another embodiment, the strain culture of the invention is able to produce a mushroom whose cap-color is browner than the cap-color of Sylvan4325, as described in Table III below.

[0053] In another aspect, the present invention relates to a method of producing a mushroom culture comprising the steps of:

[0054] (a) growing a progeny culture produced by mating the culture of the invention (typically ST428) with a second Shiitake culture;

[0055] (b) mating the progeny culture with itself or a different culture to produce a progeny culture of a subsequent generation;

[0056] (c) growing a progeny culture of a subsequent generation and mating the progeny culture of a subsequent generation with itself or a different culture; and

[0057] (d) repeating steps (b) and (c) for an additional 0-5 generations to produce a mushroom culture. In a particular embodiment, said method comprises the steps of:

[0058] (a) obtaining a molecular marker profile of Shiitake mushroom strain ST428;

[0059] (b) obtaining an Fl hybrid culture comprising at least one set of chromosomes of the strain ST428;

[0060] (c) mating a culture obtained from the Fl hybrid culture (b) with a different mushroom culture; and

[0061] (d) selecting progeny that possess characteristics of said molecular marker profile of strain ST428.

[0062] In another aspect, the present invention relates to a method of producing edible mushrooms, including the step of inoculating compost with the strain of the invention to produce a crop of mushrooms.

[0063] In another aspect, the present invention also relates to any product incorporating the culture of the invention, including spawn, inoculum, mushrooms, mushroom parts, mushroom pieces, processed foods. All these terms are defined in WO2022 / 023290, which is incorporated herein by reference.

[0064] DEFINITIONS

[0065] Genetic identity (e.g., genotype), genealogy, and pedigree are all inextricably interrelated in a strain development or breeding program, as in the cultures of the present invention. The following information on life cycles and heterokaryotic and homokaryotic genotypes, and on parents, offspring, hybrids and descended strains, and derived strains may help to clarify relationships and expectations.

[0066] Mushroom-forming fungi exhibit an alternation of generations, from heterokaryotic (N+N, with two haploid nuclei, functionally like the 2N diploid state) to homokaryotic (IN) and further upon mating to become heterokaryotic again. In most eukaryotes, a parent is conventionally considered to be either diploid or heterokaryotic. The haploid 'generation' is often, but not always, termed a gamete (e.g., pollen, sperm). In fungi, which are microorganisms, the haploid generation can live and grow indefinitely and independently, for example in laboratory cell culture; while these haploid homokaryons function as gametes in matings, they are equivalent to inbred lines (e.g., of plants) and are more easily referred to as lines (or 'homokaryon-parents' of hybrids). Herein, the standalone term 'parent' refers, depending on context, to the heterokaryotic culture that is either a, or the, direct progenitor of a haploid line culture, or else the progenitor-once-removed of a strain belonging to the subsequent heterokaryotic generation obtained from a mating of at least one such line. The term 'line' thus refers narrowly to a haploid (N) homoallelic culture within the lifecycle. The N+N heterokaryon resulting from a mating, or comprising a breeding stock, or comprising a culture used to produce a crop of mushrooms, may be called a 'strain'.

[0067] Mushroom cultures are most reliably identified by their genotypes, in part because successful cultivar strains are required by the market to conform to a narrow phenotypic range. The genotype can be characterized through a genetic marker profile, which can identify isolates (clones or subcultures) of the same line, strain or culture, or a genealogically related culture including a descendent or a culture derived entirely from an initial culture, or additionally can be used to determine or validate a strain development pedigree over generations.

[0068] Means of obtaining genetic marker profiles using diverse techniques including whole genome sequencing (WGS) plus Single Nucleotide Polymorphism (SNP) marking and Sequence Characterized Amplified Region (SCAR) marking are well known in the art. Since both approaches can analyze the sequences of specific loci, both provide identical results for any locus (note that in heterokaryon analysis, WGS provides more insight into the distribution of SNPs on the haploid sequences; i.e., confirmation of allelic sequences).

[0069] The whole genomic sequence of strain ST428 has been obtained and, consequently, about 99.5% (about 46.5 Mb) of its DNA sequence genotype is known to the Assignee with certainty. The total number of SNP markers distinguishing the reference genome of Sylvan4325 from the genome of ST428 is at least 141,923. A brief excerpt of the genotype of strain ST428 at numerous sequence-characterized marker loci is provided in Table I. Because a heterokaryon incorporates two sets of chromosomes, one from each haploid parent, there are two allelic copies (two characters or elements of the genotype) at each marker locus for ST428. The IUPAC nucleotide and so-called "ambiguity" codes, (also see Annex C, Appendix 2, Table 1, Nucleotide and Amino Acid Symbols as set forth in Standard ST. 25 of the Handbook on Industrial Property Information and Documentation (WIPO) (December 2009)) which are actually heteroallelism codes when used to represent a heterokaryon or diploid genotype, are used in Table I to represent heteroallelic DNA sequence positions, wherein each of two alleles incorporates a different nucleotide at a particular position, in the observed 9-base DNA marker sequences reported above, each of which represents a genotypic marker locus. The identity of each marker locus is specified by the chromosome and SNP position information derived from the Genome assembly TB_Sanjo_l reference genome sequence published by the National Center for Biotechnology Information U.S incorporated herein by reference.

[0070] It will be appreciated however that any suitable Polymerase Chain Reaction (PCR) primers that bracket the defined marker regions may be used for identifying the alleles, using methods of designing and using suitable PCR primers that are well known in the art.

[0071] The markers of Table I can be used for example to empirically determine inclusion of a culture within the scope. Genotype analysis including either Polymerase Chain Reaction (PCR) based analysis of polymorphic regions, or whole genome sequencing, is routinely used to establish the degree and nature of genetic identity with an initial culture to define the class of cultures directly or indirectly derived therefrom in Shiitake. Either all markers in the derived strain or culture will correspond to markers in the initial strain or culture, or else representation of the markers will typically be higher than 90%, but not lower than 65 or 70%, preferably not lower than 75%, in the derived strain or culture. Using a sufficient number of genetic markers, the status of a derived strain or culture can be unambiguously determined, and statistically beyond challenge. Similar analyses can establish the nature of the relationship between two cultures, including self, clone, subculture, somatic selection, tissue selection, inbred descendent, outbred descendent, back-bred descendent, transformed culture, mutagenized culture, Fl hybrid, and subsequent generations of hybrids, with high statistical confidence. The term "approximately" or "about" herein inculcates a range of plus or minus 20% above or below the stated value.

[0072] To calculate the percentage of SNPs between two strains, one can compare the composite 9-mer genotype at each locus and assign a value if 1 for a perfect match, or a 0 for anything less than a perfect match. Then the values can be totaled for all loci in each pairwise comparison between strains, and divided by the total number of loci compared. The resulting decimal can be eventually converted to %.

[0073] FIGURE LEGENDS

[0074] Figure 1 shows a picture of the mushroom of the invention, as compared with Sylvan 4325 mushroom.

[0075] EXAMPLES

[0076] 1. Development of a new Shiitake strain called ST428

[0077] A new Shiitake strain has been developed. It was called ST428. This hybrid results from a cross between the monokaryon 4306 B2 and the monokaryon 4339 s35.

[0078] Representative culture of said parental monokaryon having been deposited under the CNCM Accession Number 1-6018 for 4306 B2 and 1-6019 for 4339 s35 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on November 30.

[0079] As other fungal specie, Lentinula edodes has different reproduction processes: vegetative reproduction (mitosis), sexual reproduction (meiosis) and parasexual reproduction (other phenomena). Lentinula edodes show the typical life cycle of Basidiomycete fungi. It will start with the germination of spores that will conduct to a monokaryotic mycelium (N). When two compatible monokaryotic mycelium (different loci Mat A and Mat B) are confronted, a fertile dikaryotic mycelium (N+N) is created by hyphal fusion or plasmogamy. Importantly, this Shiitake strain has been developed for and can be used for the sterile and pasteurized substrate market.

[0080] 2. ST428 has the same yield as Sylvan4325 on pasteurized substrate

[0081] Yield performance was measured in several trials. Trials were performed on 12 kg bag of pasteurized straw-based substrate, spawning rate was 8 litres / ton. In the growing room we tested strains with 6 replications distributed across 2 growing levels. Inoculated substrates were incubated between 49 and 55 days, temperature was set to reach 25°c in the substrate. Relative Humidity was set at 80-85% . Four first week were on dark and then with a standard 12:12h light: dark cycle to induce the browning of the substrate. Primordias were induced by decreasing temperature (17°C in the room), the relative humidity was set at 90- 95% with a standard 12:12h lightdark cycle and a CO2 level inferior or equal to 2000 ppm.

[0082] When substrate bags were fully colonized and turned brown, plastic were removed to provide oxygen for the development of the mushroom. During the flush and mushroom development CO2 level is set between 1000 and 2000 ppm. Data were collected during 1stflush.

[0083] 1st flush

[0084] ST428 yield 23% sd 2%

[0085] Sylvan

[0086] 4325 yield 26% sd 4% p value 0,488

[0087] Table II: Flush yield of ST428 and Sylvan4325 after one flush expressed kg of mushrooni / kg wet substrate (w / w). Standard cultivation and harvest procedures were used. General t-test analysis: the difference with Sylvan 4325 is significant at p-value a < 0.05. The mushroom crop yield of strain ST428 was found to be equivalent to Sylvan4325 during 1stflush as shown in Table II.

[0088] 3. ST428 is darker than Sylvan4325

[0089] The mushroom color was measured using a Minolta Chroma Meter CR-200 (mfd. Japan). Sample sizes of fifty medium sized mushrooms were harvested from the tests and measured to obtain values for the L*a*b parameters. The Chroma Meter readings were randomly taken on the mushroom caps. In the L*a*b system, "L" is a brightness variable with 0 representing complete darkness and 100 representing complete whiteness and "b" value represents blueness (-300) / yellowness (+299). In other words, the darker a mushroom cap color, the lower the L value, and the more yellow a mushroom cap color, the higher b value.

[0090] In this case, L value has shown a darker cap color for ST428 (Table III). Based on b value Sylvan4325 is more "yellowish".

[0091] Strains L value sd a value sd b value sd

[0092] ST428 31,8 3,8 11,2 1,3 14,7 1,5

[0093] Sylvan 39,2 5,8 10,3 1,8 17,4 2,1

[0094] 4325 p value <0,0001 0,004 <0,0001

[0095] Table III: Chromameter value L,a,b of Sylvan 4325 and ST428 strains. General t-test analysis: the difference with Sylvan 4325 is significative with p-value a < 0.05.

[0096] 4. ST428 has a better shelf life than Sylvan4325.

[0097] Trait data collection was carried out by a method in which mushroom samples were collected on the day of peak harvest during a 'flush' of mushroom production. A flush lasts four or five days, often with peak production on the second day; typically. The expression of the trait in Flush 1 was evaluated. During this test, six replicate styrofoam tills per strain were evaluated. A till is a tray that can hold over 200 g. The weight of the empty till was recorded. They were spaced enough to not touch each other and placed with the stem up, they were immediately weighed. An initial weight was recorded. The tills were placed at

[0098] 4°C for 4 weeks in a walk-in cooler. Filled till weights were recorded after 1, 2, 3 and 4 weeks. After subtracting the weight of the empty till, percentage of weight loss was calculated as described above. Results have shown that ST428 has better shelf life than Sylvan4325 of storage at 4°C (Table IV). After 1 week, the weight loss is about 11% of fresh weight against 9% for ST428. The difference of fresh weight loss become more important every week. After 4 week of storage the loss weight for is about 41.1 % for Sylvan 4325, and only 29.9 % for ST428.

[0099] Table IV: Percentage of weight loss after one, two, three and four weeks of post-harvest storage at 4C. General t-test analysis: the difference with Sylvan4325 is significative with p-value a < 0.05.

Claims

CLAIMS1. A Shiitake culture comprising at least one haploid set of chromosomes of the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, the chromosomes of said culture comprising at least 50 out of the 398 sequence-characterized allelic markers listed in Table I.

2. The culture of claim 1, characterized in that it is a Fl hybrid produced by mating an homokaryon of the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, with a second line.

3. A Shiitake mushroom strain culture of the Fl, F2, F3, or F4 generation, descended from the strain ST428 of claim 1, or from a strain derived from said strain ST428, and comprising respectively at least 40-60%, at least 20-30%, at least 10-15%, or at least 4-8% of the Single- Nucleotide Polymorphisms (SNPs) present in the genome of the Shiitake strain ST428, a representative culture of said line having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number 1-5946.

4. The Shiitake mushroom strain culture of claim 3, descending from ST428, or from a strain derived from strain ST428, comprising at least about 300 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, at least about 200 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, at least about 100 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, at least about 50 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I.

5. The Shiitake mushroom strain culture of claim 4, descending from ST428 or from a strain derived from strain ST428, comprising at least 40-60%, at least 20-30%, at least 10-15%, or at least 4-8% of the sequence-characterized allelic markers of ST428 listed in Table I.

6. A Shiitake mushroom strain culture that is derived from an initial culture, wherein said initial culture is the strain ST428, a representative culture of said strain having been deposited under the CNCM Accession Number 1-5946 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023.

7. The Shiitake mushroom strain culture of claim 6, wherein it comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the sequence-characterized allelic markers of ST428 listed in Table I.

8. The Shiitake mushroom strain culture of claim 1-7, characterized in that:(a) the total yield performance of the crops of said culture is equal to or exceed the yield performance of crops of Sylvan4325 strain of Shiitake, and(b) the cap of the mushroom of the crops of said culture has a darker brown color as compared with the cap color of Sylvan4325 Shiitake mushrooms, and(c) the mushroom product of the crops of said culture retains more weight after a number of weeks of post-harvest storage at 4 degrees Celsius than does the mushroom product of the Sylvan4325 strain, the number of weeks selected from the group comprising 1, 2, 3 and 4 weeks.

9. Cells, hyphae, mycelium, mushrooms, germinated spores, ungerminated spores, homokaryons, and heterokaryons including SNPs, NSNPs, and aneuploids obtained from the culture of any one of claims 1-8.

10. A product incorporating the culture of any of claims 1-8, including spawn, inoculum, mushrooms, mushroom parts, mushroom pieces, processed foods.

11. A method for developing a new Shiitake culture, said method comprising applying at least one mushroom strain development technique to an homokaryon of the strain ST428, a representative culture of which having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number 1-5946, or to a progeny thereof, to provide a new culture.

12. The method of claim 11, wherein said new culture is characterized in that:(a) the total yield performance of the crops of said culture is equal to or exceed the yield performance of crops of Sylvan4325 strain of Shiitake, and(b) the cap of the mushroom of the crops of said culture has a darker brown color as compared with the cap color of Sylvan4325 Shiitake mushrooms, and(c) the mushroom product of the crops of said culture retains more weight after a number of weeks of post-harvest storage at 4 degrees Celsius than does the mushroom product of the Sylvan4325 strain, the number of weeks selected from the group comprising 1, 2, 3, 4, 5, 6, 7, and 8 weeks.

13. The method of claim 11 or 12, characterized in that said culture is the Fl, F2, F3, or F4 generation descended from ST428 or from a strain derived from strain ST428, and comprising respectively at least 40-60%, at least 20-30%, at least 10-15%, or at least 4-8% of the Single-Nucleotide Polymorphisms (SNPs) present in the genome of the Shiitake strain ST428, a representative culture of said line having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on April 13, 2023, under the CNCM Accession Number I- 5946.

14. The method of any of claims 11 to 13, characterized in that said culture contains at least about 300 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, at least about 200 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I, at least about 100 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I or at least about 50 allelic markers out of the 398 sequence-characterized allelic markers of ST428 listed in Table I.

15. The method of any of claims 11 to 13, characterized in that said culture contains at least 40-60%, at least 20-30%, at least 10-15%, or at least 4-8% of the sequence-characterized allelic markers of ST428 listed in Table I.

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