Patent Application
20230265379
The Sequence Listing file 876090 listing sequence_ST25.txt having a file size of 3000 bytes and a creation date of Jul. 21, 2020, is incorporated herein by reference in its entirety.
The present invention relates to the development of a homokaryotic Agaricus bisporus (Lange) Imbach mushroom fungus line culture designated N-s34 and to cultures obtained, descended, or otherwise derived from line N-s34. More particularly, the present invention relates to cultures incorporating at least one set of chromosomes having a genotype present in the genotype of the chromosomes found in line N-s34. The present invention further relates to F1 hybrids, and to a particular F1 hybrid strain, designated LA3782, descended from N-s34. This particular strain indeed displays an excellent yield weight of the harvested crop, especially in the third-flush, and a very good shelf-life of the mushroom products. The invention additionally relates to progeny, lines and strains derived from or descended from, or otherwise developed or obtained from, line N-s34 and from said hybrid strain LA3782. The invention further relates to methods of use of the cultures described hereinabove.
The edible mushroom Agaricus bisporus (Lange) Imbach var. bisporus, a microorganism belonging to the basidiomycete fungi, is widely cultivated around the world. In Europe and North America, it is the most widely cultivated mushroom species. According to recent market data, “Volume of sales of the 2017-2018 United States mushroom crop totaled 917 million pounds . . . . Value of sales for the 2017-2018 United States mushroom crop was $1.23 billion . . . ” (USDA NASS, 2019). “In 2019, production and sales of cultivated Agaricus bisporus mushrooms in Europe totaled 1,700,000 metric tons, with a market value of approximately 2.5 billion Euros. About 12%, or 200,000 metric tons, of that crop were brown-capped mushrooms.” (Sylvan, Inc, internal market analysis).
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.
Cultures are the means by which the mushroom strain developers prepare, maintain, and propagate their industrial microorganisms. Cultures of Agaricus, like those of other microorganisms, are prepared, maintained, propagated and stored on sterile media using various microbiological laboratory methods and techniques 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’ and ‘casing inoculum’ are 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.
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. Compost prepared from lignocellulosic material such as straw, augmented with nitrogenous material, is finished and pasteurized within a suitable facility. Mushroom spawn, which comprises a sterilized friable ‘carrier substrate’ onto which a pure culture of one mushroom strain has been aseptically incorporated via inoculum and then propagated, is mixed with the pasteurized compost and is incubated for approximately 13 to about 19 days at a controlled temperature, during which time the mycelium of the mushroom culture colonizes the entire mass of compost and begins to digest it. A non-nutritive ‘casing layer’ of material such as peat is then placed over the compost to a depth of from about 1.5 to about 2 inches. Additional ‘casing inoculum’ incorporating the same mushroom culture may be incorporated into the casing layer to accelerate the formation and harvesting of mushrooms, and to improve uniformity of the distribution of mycelium and mushrooms in and on the casing surface. Environmental conditions, including temperature and humidity, within the cropping facility are then carefully managed to promote and control the transition of the culture from vegetative to reproductive growth at the casing/air interface. In a further about 13 to about 18 days after casing, mushrooms will have developed to the correct stage for harvest and sale.
A first flush of mushrooms comprising the original culture will be picked over a 3- to 5-day period. Additional flushes of mushrooms appear at about weekly intervals. Commercially, two or three flushes of mushrooms are produced and harvested before the compost is removed and replaced in the cropping facility. 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), and market segment (for example white-capped vs. brown-capped, or closed-capped vs. open-capped) of the product.
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 post-harvest (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 post-harvest weight, or other element of product quality, for a longer time during post-harvest storage.
There is a need for more diverse, more versatile, and more profitable Agaricus bisporus mushroom strains. To meet this need for improved, diverse Agaricus bisporus 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.
Novel strains are most preferably and successfully developed from new hybridizations (fusions) between haploid homokaryotic lines, including novel lines. Thus, the need continues to exist for new lines that can be used to produce new hybrid strains of Agaricus bisporus mushroom cultures and microorganisms that in turn provide improved and/or novel combinations of characteristics for producer profitability and for improved mushroom products over other previous strains of Agaricus bisporus.
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. Successful outcomes are rare and generally unpredictable, and rely in part on the serendipitous identification of breeding stocks and lines that are discovered to demonstrate an increased ability or tendency to produce one or more commercially acceptable strains via application of strain development techniques. 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. Therefore, any novel breeding stock or line with the ability to produce acceptable new commercial strains via the application of strain development techniques is of great value to the mushroom strain developer and to the mushroom industry.
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.
Thus, there is a general need for commercially acceptable A. bisporus strains with different, diverse, novel genotypes, relative to other commercially produced strains, for three reasons:
First, strains vegetatively incompatible with other strains in commercial production are known to retard the spread of viral diseases between cultivated strains, due to an inability, or limited ability, of incompatible strains to anastomose (=physically fuse) with each other and exchange cytoplasm. The incompatibility phenotype can be assessed using techniques that are well known in the art. Alternating or rotating the use of incompatible strains within a facility can improve harvest yields immediately, by sharply reducing the transmission/infection rate, while reducing viral disease reservoirs and pressure over a period of weeks or months. Thus there is a need for commercially acceptable mushroom strains that are genetically distinct from and vegetatively incompatible with the other commercial strains now in use, specifically, the brown-capped B14528/Tuscan and BR06/Heirloom strains. The strain B14528 has been deposited under the Budapest Treaty governing the deposit of microorganisms at the Agricultural Research Services Culture Collection (NRRL), Peoria, Ill., USA under NRRL Accession Number 50900. The strain BR06 has been deposited under Budapest Treaty governing the deposit of organisms at the American Type Culture Collection (ATCC), Rockville, Md., USA, under ATCC accession number PTA-6876.
Second, 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.
Third, 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.
Thus, any commercially acceptable hybrid strain, or breeding line, 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. The use of novel lines that incorporate DNA from non-cultivar stocks meets the need of providing important genetic diversification of the strain pool used to produce crops of cultivated A. bisporus mushrooms. There is an even greater need for diverse and novel breeding lines capable of being used to produce diverse, novel commercially acceptable hybrid strains via strain development techniques. There is a correspondingly great need for the novel hybrid strains so produced in such usage. Every 1% of observed genotypic difference between two strains represents approximately 120 functional genes which may be different.
Most commercial production of brown-capped Agaricus bisporus mushrooms today employs either of only two strains: BR06/Heirloom (PTA-6876) or B14528/Tuscan (NRRL 50900). 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 BR06/“Heirloom” or B14528/“Tuscan”, (2) on a desirable commercial production schedule, in other words an harvest schedule that minimizes costs and maximizes crop value, more evenly than the Heirloom strain, while (3) also producing mushrooms of good appearance and high quality for the consumer, and which retain more of their initial weight, compared to the BR06/Heirloom strain, over an extended period of days in the post-harvest sales chain. There is a corresponding need for mushroom lines that can transmit genetic material capable of providing these traits, in addition to other commercially acceptable characteristics, in their hybrid descendent strains.
The present invention fulfills this need by providing new lines and 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.
The present invention is directed generally to an Agaricus bisporus culture comprising at least the set of chromosomes of the Agaricus bisporus line N-s34, 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 Jun. 30, 2020, under the CNCM Accession Number I-5528, wherein said set of chromosomes comprises preferably the sequence-characterized allelic markers listed in Table I. It is further directed to a culture as described above, characterized in that it is selected from the group consisting of: (a) the line N-s34, a representative culture of same having been deposited under the CNCM Accession Number I-5528 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on Jun. 30, 2020, and (b) F1 hybrid strains produced by mating the line N-s34 to a second line, and (c) homokaryons of said F1 hybrid strains defined in (b), preferably characterized in that said second line is an homokaryon obtained from strain BP-1, and more preferably characterized in that it is the strain LA3782, a representative culture of said strain having been deposited under the CNCM Accession Number I-5527 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on Jun. 30, 2020.
Another aspect of the invention relates to an Agaricus bisporus mushroom culture comprising at least one haploid set of chromosomes of the strain LA3782, a representative culture of said strain having been deposited under the CNCM Accession Number I-5527 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on Jun. 30, 2020, said set of chromosomes preferably comprising the sequence-characterized allelic markers listed in Table II, more preferably characterized in that it is selected from the group consisting of: (a) an homokaryon of the strain LA3782, a representative culture of said strain having been deposited under the CNCM Accession Number I-5527 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on Jun. 30, 2020, and (b) F2 hybrids produced by mating said homokaryon (a) with a second line.
Another aspect of the invention relates to an Agaricus bisporus mushroom strain culture of the F2, F3, F4, or F5 generation, descended from the F1 hybrid defined above, and preferably from the F1 hybrid LA3782, or from a strain derived from strain LA3782, 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 Agaricus bisporus strain LA3782, 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 Jun. 30, 2020, under the CNCM Accession Number I-5527.
Yet another aspect of the invention relates to an Agaricus bisporus mushroom culture that is derived from an initial culture, wherein said initial culture is chosen in the group consisting of: (a) the strain LA3782, a representative culture of said strain having been deposited under the CNCM Accession Number I-5527 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on Jun. 30, 2020, (b) the Agaricus bisporus line N-s34, 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 Jun. 30, 2020, under the CNCM Accession Number I-5528, and (c) 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 203 sequence-characterized allelic markers of N-s34 listed in Table 1 or of LA3287 listed in Table 11, 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 N-s34 listed in Table 1 or of LA3287 listed in Table II.
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 a BR06/Heirloom strain of Agaricus bisporus, (b) a third-flush yield of the crops of said culture significantly exceeds that of the BR06/Heirloom strain, and (c) the mushroom product of the crops of said culture retains more weight after a number of days of post-harvest storage at 4 degrees Celsius than does the mushroom product of the BR06/Heirloom strain, the number of days selected from the group comprising 3, 4, 5, 6, 7, and 8 days.
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.
The present invention also relates to a method for developing a new Agaricus bisporus culture, said method comprising applying at least one mushroom strain development technique to the homokaryon line N-s34, 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 Jun. 30, 2020, under the CNCM Accession Number I-5528, or to an homokaryon of the strain LA3782, 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 Jun. 30, 2020, under the CNCM Accession Number I-5527, 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 is equal to or exceeds the yield performance of crops of a BR06/Heirloom strain of Agaricus bisporus, (b) a third-flush yield of the crops of said culture significantly exceeds that of the BR06/Heirloom strain, and (c) the mushroom product of the crops of said culture retains more weight after a number of days of post-harvest storage at 4 degrees Celsius than does the mushroom product of the BR06/Heirloom strain, the number of days selected from the group comprising 3, 4, 5, 6, 7, and 8 days. In a preferred embodiment, said new culture is an F2, F3, F4, or F5 hybrid descended from an F1 hybrid of N-s34, or from a strain derived from the strain LA3782, 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 203 sequence-characterized allelic markers of N-s34 listed in Table 1 or of LA3782 listed in Table 11; 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 the Agaricus bisporus line N-s34, 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 Jun. 30, 2020, under the CNCM Accession Number I-5528.
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.
Mushroom-forming fungi exhibit an alternation of generations, from heterokaryotic (N+N, with two haploid nuclei, functionally like the 2N diploid state) to homokaryotic (1N) and further upon mating to become heterokaryotic again. In most eukaryotes, a parent is conventionally considered to be either diploid or heterokaryotic. The haploid ‘generation’ is often, but not always, termed a gamete (e.g., pollen, sperm). In fungi, which are microorganisms, the haploid generation can live and grow indefinitely and independently, for example in laboratory cell culture; while these haploid homokaryons function as gametes in matings, they are equivalent to inbred lines (e.g., of plants) and are more easily referred to as 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’.
Now, with respect to the invention and as noted hereinabove, the present invention relates to at least a homokaryotic line, and more specifically, a culture comprising at least one set of chromosomes of an Agaricus bisporus line designated N-s34, and methods for using the line designated N-s34. The N-s34 line is a homokaryon and its genome and genotype are haploid and thus is entirely homoallelic (although some limited regions of duplicated DNA may be present in its genome).
In a first aspect, the present invention is directed to an Agaricus bisporus culture comprising at least the set of chromosomes of the Agaricus bisporus line N-s34, a representative culture of said line having been deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, rue du Docteur Roux, 75724 PARIS Cedex 15, on Jun. 30, 2020, under the CNCM Accession Number I-5528.
The deposit of a culture of the Agaricus bisporus line N-s34, 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 Jun. 30, 2020. 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.
Agaricus bisporus mushroom line N-s34 is, based on whole-genome sequencing, a haploid, homokaryotic filamentous basidiomycete culture which in vegetative growth produces a branching network of hyphae, i.e., a mycelium. Growth can produce an essentially two-dimensional colony on the surface of solidified (e.g., agar-based) media, or a three-dimensional mass in liquid or solid-matrix material.
A culture comprising at least one set of chromosomes of an Agaricus bisporus line designated N-s34 may be either a homokaryon or a heterokaryon. It may be (a) line N-s34 itself, (b) a culture having full genotypic identity with N-s34, in agreement with the allelic genotype of N-s34 presented in Table I, (c) a culture having at least one set of genotypic markers which are a subset of those of the genotype of N-s34 representing at least 65%, 70%, 75%, or 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, of the markers present in N-s34, or comprising at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100 markers out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I (d) a culture having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% genotypic identity with N-s34, and (e) an F1 hybrid having N-s34 as a direct parent, said hybrid displaying all the allelic markers listed in Table I (on at least one of its two alleles).
Cultures of the invention include a culture having at least one genealogical relationship with the culture N-s34, wherein the genealogical relationship is selected from the group of consisting of (1) identity: i.e., self, clone, subculture, (2) descent: i.e., inbred descendent, outbred descendent, back-bred descendent, F1 hybrid, F2 hybrid, F3 hybrid, F4 hybrid, F5 hybrid, and (3) derivation: i.e., derived culture, somatic selection, tissue selection, mutagenized culture, transformed culture. Note that when a relationship involves descent solely from a single parent, the resultant cultures can also be considered to have been ‘derived’ from that parental culture.
In a preferred embodiment, the Agaricus bisporus culture of the invention is selected from the group consisting of:
The present invention also targets the homokaryons of said F1 hybrid strains defined in (b), the genome of said homokaryons containing at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, of the markers present in N-s34, among which at least one marker is present in N-s34 but is absent from the second line.
In a preferred embodiment, said second line is an homokaryon obtained from strain BP-1 (also known as AA0096 or ARP023 or PTA-6903).
LA3782 is one example of an F1 hybrid heterokaryon strain having outbred descent from the homokaryotic line N-s34. It is also called Tuscan820. More precisely, it has been obtained by mating the line N-s34 with an homokaryon of strain BP-1 also known as AA-0096 and ARP-023. This strain BP-1 has been deposited under Budapest Treaty governing the deposit of organisms at the American Type Culture Collection (ATCC), Rockville, Md., USA, under ATCC Accession Number PTA-6903.
Mushrooms produced in crops by strain LA3782 are about 39 kg/m2 (S.D.±1.98) over 3 flushes in phase 3 system, and typically each weigh about 20-45 grams for medium size mushrooms. Cap color measurements on mushrooms of LA3782 produced L-a-b color of L:71,49 (S.D±2,9) a:7,12 (S.D.±1,02) b:23,28 (S.D.±1,28) when measurements were taken on 30 mushrooms using a Minolta Chromameter.
In a preferred embodiment, the culture of the invention is the strain LA3782, a representative culture of said strain having been deposited under the CNCM Accession Number I-5527 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, on Jun. 30, 2020. The deposit of a culture of the Agaricus bisporus strain LA3782, 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 of the Budapest Treaty. The date of deposit was Jun. 30, 2020. 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.
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.
In Inventor's experience in evaluating whole genome sequences of many dozens of diverse Agaricus bisporus lines and strains, a typical number of SNP markers distinguishing any two unrelated homokaryons is roughly 300,000. This means that transmission of even 1% of a set of chromosomes or genotypic markers in a genealogy still represents about 3,000 distinctive identifying markers for a relationship to N-s34. 200 markers, or even 6 highly polymorphic ones, can establish identity, paternity, and derivation among cultures beyond question, while many thousands of available SNP markers can cumulatively provide a robustly-supported method for establishing genealogical relatedness over multiple generations.
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).
The whole genomic sequence of line N-s34 has been obtained and, consequently, about 95% (about 30.2 Mb) of the entire DNA sequence genotype of line N-s34 is known to the Assignee with certainty. The total number of SNP markers distinguishing the reference genome H97 from line N-s34, and which are known to the Assignee, is at least 141,923. That number is expected to be higher when distinguishing N-s34 from other homokaryons. A brief excerpt of the genotype of line N-s34 and strain LA3782 at numerous sequence-characterized marker loci distributed at intervals along each of the 13 chromosomes of N-s34 and LA3782 is provided in Tables I and II. Only for information, the sequences of the same marker loci are provided for the homokaryotic line J147566s3 disclosed in WO2018/102990.
TTGA
TTGA
TTGA
ACAT
ACAT
ACAT
GGTT
GGTT
GGTT
ATGA
ATGA
ATGA
AGGA
AGGA
AGGA
TTTT
TTTT
TTTT
AAAC
AAAC
AAAC
ATCG
ATCG
ATCG
ACTGGT
ACTGG
ACTGGT
CACC
CACC
CACC
ACTA
ACTA
ACTA
GCGC
GCGC
GCGC
AATT
AATT
AATT
CAA
CAA
CAA
AAAT
TTTG
TTTG
TTTG
GAC
GAC
GAC
TCTT
TCTT
TCTT
GTTT
GTTT
GTTT
GACCAGG
GA
AGG
GACCAGG
GGTG
TACG
CTAA
CTCA
CCAA
TGTT
CAGT
TTCA
C
AGAT
TGCC
TCTT
TCTT
TCTT
AATT
AATT
AATT
AGGG
TCAA
TCAA
TCAA
AC
C
CAA
AC
C
AC
C
TCCT
TCCT
AGGT
GCTT
GCTT
CAGC
CAGC
TCCT
TTTC
G
AGAG
GGAA
TCGT
TTTC
GGCG
CCTT
TCAT
GATT
TGGG
AGCC
AAGA
AAGC
CCAC
ATA
TAAC
GCGT
AAT
AAGC
AACT
GGAC
GA
CAAT
CGAA
TACT
CGG
GTA
AGAA
GACC
GACC
GACC
ACTG
ACTG
ACTG
AGGG
AGGG
AGGG
GAAT
GAAT
GAAT
GCCC
GCCC
GCCC
TTCA
TTCA
TTCA
TCT
TCT
TCT
GACA
GACA
GACA
TCC
TCC
TCC
CCTT
CCTT
CCTT
CTCC
AGTC
AGTC
AGTC
AAAA
AAAA
AAAA
CTTG
GGTT
ATTT
AGGA
ATTG
TAG
GATT
ATAA
TCAG
GTTT
ATTA
CATA
ACCT
GAAG
GCAA
CACC
CGCG
CGCG
TTCT
TTCT
TTGA
TTGA
CAAC
GCAG
GACG
GACG
ATCT
ATCT
TGCA
TGCA
TACT
GCCT
GCCT
AGTA
GTTG
GTTG
TTCT
TTCT
ATAC
ATAC
ATAC
GTATCCC
GT
TCCC
TCCC
TGTAATC
TG
ATC
TGTAATC
ACGT
ACGT
ACGT
GTGGTGA
GTGA
GGGA
GGGA
GGGA
GACA
GACA
CACC
C
CC
GTTT
GTTT
GTTT
CATC
CATC
TGG
TGG
TGG
ATCG
TCAA
TCAA
ATCA
ATCA
AAAG
AAAG
AATT
TTT
TCAT
TCAT
CAAA
CCTT
CGTC
CTGT
GGGG
GCGC
GCGC
GCCC
GCCC
TCA
CACC
CACC
TG
CACG
T
CCACG
ATTC
TACA
TACA
CACC
TGCT
TGCT
TGCT
CG
CG
CG
AGCC
GGTC
GGTC
TTCT
TTCT
TTCT
GAGG
AGGA
AGGA
ACTG
ACTG
CTGC
CTGC
GTCA
GTCA
TCAG
TCAG
AATA
CCTT
GCCT
G
AA
TT
GAAA
ATGG
ATGG
AGAT
AGAT
CCGT
CCTA
CCTA
GGAA
GGAA
GGAA
TCTG
TCTG
TCTG
TACG
TTCA
AACG
AACG
AACG
AC
A
AC
A
AC
A
C
TG
TGGG
TCCG
TCCG
GCAA
TGTT
Tables I and II comprise sets of SNP markers present in N-s34 and LA3782, respectively, described as 9-mers. Positional information refers to the 17 substantial contigs of the H97 V. 2.0 genome sequence assembly (JGI). 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 LA3782. 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 Tables I and II 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 scaffold and SNP position information derived from the H97 V2.0 standard reference genome sequence published by the U.S. Department of Energy Joint Genome Institute (Morin et al. 2012), incorporated herein by reference.
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. Distinctions between the homoallelic genotypes of line N-s34 and line H97 are evident, as is the composite nature of the example heteroallelic genotype of F1 hybrid strain LA3782, in which the presence of the genome of N-s34 is evident, as expected, by virtue of perfect conformity, with no conflicts, with the presence of the alleles known to be evident in LA3782. The genotype of strain LA3782 is a composite of those of line N-s34 and the BP-1 homokaryon, and demonstrates that the N-s34 chromosome set can be observed within the F1 hybrid genotype. Methods employing these and other markers to determine genealogical relationships between cultures are provided below.
Alternatively, one can use the six SCAR marker loci p1 n150, ITS, MFPC-1-ELF, AS, AN, and FF as described in U.S. Pat. Nos. 7,608,760, 9,017,988 and below. Each have approximately 10 (or more) known alleles, so that the number of heterokaryotic genotypes possible is on the order of one trillion (1012). These six markers are the six most commonly referenced marker loci in the industry and are considered art standard designations in that all six of the marker loci have been used, in one form or another, to characterize the genotype of Agaricus strains in at least one public source publication. Brief descriptions of relevant alleles at these six unlinked marker loci are provided in Table Ill. Genotypes at these six loci were determined both by Whole Genome Sequencing and by SCAR-PCR, as described in the experimental part below.
The markers of Tables I to III 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 Agaricus bisporus. 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, and especially the 6 highly polymorphic markers of table 1111, 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, F1 hybrid, and subsequent generations of hybrids, with high statistical confidence.
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, said first culture comprising at least one set of chromosomes of an Agaricus bisporus line N-s34.
If one parental line carries allele ‘p’ at a particular locus, and the other parental line carries allele ‘q’, the F1 hybrid resulting from a mating of these two lines will carry both alleles, and the genotype at that locus can be represented as ‘p/q’ (or ‘pq’, or ‘p+q’). Sequence-characterized markers are ordinarily codominant and both alleles will be evident when an appropriate sequencing protocol is carried out on cellular DNA of the hybrid. After determining the genotypic profile of a strain or hybrid, reference to the genotypic profile of line N-s34 can therefore be used to identify hybrids comprising line N-s34 as a parent, or parental, line, since such hybrids will comprise two sets of alleles, one of which sets will be from, and will match that of, line N-s34. The match can be demonstrated by subtraction of the second allele from the genotype, leaving the N-s34 allele evident at every locus. A refinement of this approach is possible with hybrids of Agaricus bisporus as a consequence of the heterokaryon (N+N) condition existing in hybrids. The two (pre-meiotic, non-recombinant) haploid nuclei can be physically isolated by various known techniques (e.g., protoplasting) into viable ‘neohaplont’ subcultures, and each may then be characterized independently. One of the two neohaplont nuclear genotypes from the F1 hybrid will be that of line N-s34, demonstrating its prior use in the mating step of the method, and its presence in the hybrid. Obtaining deheterokaryotized neohaplont homokaryons from a heterokaryon, including a heterokaryotic culture of the invention, by repartitioning individual haploid nuclei using protoplasting, fragmentation, hyphal tip excision, or other technique, is one method of culture derivation.
As described in the experimental part below, LA3782 has an improved yield, a more balanced yield due to improved third-break yield, and mushrooms with improved keeping qualities, compared to a leading commercial strain, Heirloom/BR06. It achieves these improvements by virtue of a novel genotype which is more than 30% different from other known brown-capped strains (see table VI). That genotype also confers a phenotype that is incompatible with other leading brown-capped strains, providing a barrier to infection by endogenous viruses, a trait which can be exploited by farm hygiene regimens. 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 Agaricus bisporus mushrooms are grown and sold.
In a preferred embodiment of the invention, the strain culture of the invention is characterized in that the total yield performance of the crops of said culture are equal to or exceed the total yield performance of crops of a BR06/Heirloom or J15051 strain of Agaricus bisporus. Total yield performance can be measured as defined below in large scale trials. During such trials, incubation period can be for example of 18 days in bulk phase III tunnel, spawning rate can be 8 litres/ton of compost phase II. Trays can be filled with 135 kg incubated compost with a filling rate of 90 kg/m2. Mc substrate supplement can be added at the rate of 1.33 kg/m2. Carbo 9 casing from supplier Euroveen can be applied with 1200 g/m2 compost casing, premixed. Airing can start on day 4 after casing. To collect yield, mushrooms can be picked and weighed daily, on at least three replicates. Data can be collected over several flushes. Total yields should be compared on the same number of flushes.
In another embodiment of the invention, the strain culture of the invention is characterized in that the third-flush yield of the crops of said culture significantly exceeds that of the BR06/Heirloom strain. Yield performance can be measured as defined above. Data are preferably collected over at least three flushes. In a preferred embodiment, the third-flush yield of the strain of the invention exceeds the BR06/Heirloom third-flush yield by more than 15%, preferably by more than 20%, more preferably by more than 30% when cultured and picked in the same conditions. The examples below demonstrate that the third-flush yield of LA3782 is also higher than the third-flush yield measured for two other strains of the prior art, namely Tuscan and J15051 (Table VIII). In a preferred embodiment, the third-flush yield of the strain of the invention exceeds the Tuscan and the J15051 third-flush yields by more than 15%, preferably by more than 20%, more preferably by more than 30% when cultured and picked in the same conditions.
In another embodiment, the culture of the invention is a strain of Agaricus bisporus that produces mushrooms which have a significantly higher piece weight than do mushrooms produced by BR06/Heirloom. This trait can be assessed during the first and second flush of mushroom production, on several medium size mushrooms (typically 4-5 cm in diameter). Each replicate is individually weighed. In a preferred embodiment, the mushroom piece weight of the strain of the invention after the first flush exceeds the BR06/Heirloom and Tuscan mushroom piece weight by more than 10%, preferably by more than 20%, more preferably by more than 30% when cultured and picked in the same conditions (Table X below).
In another embodiment, the mushroom product of the crops of said culture retains more weight after a number of days of post-harvest storage at 4 degrees Celsius than does the mushroom product of the BR06/Heirloom strain, the number of days selected from the group comprising 3, 4, 5, 6, 7, and 8 days. 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 8 days 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.
By “significantly”, it is herein meant that the third-flush yield/mushroom weight of the strain of the invention is superior to the yield/mushroom weight of the reference strain with a probability/p-value inferior or equal to 0.05 or less, according to a t-test or other parametric statistical test that compares a series of quantitative results from two or more treatments.
In another embodiment, the strain culture of the invention is able to produce a mushroom whose cap-color is similar to the one of LA3782, as described in Table XI below.
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 a BR06/Heirloom strain of Agaricus bisporus, (b) a third-flush yield of the crops of said culture significantly exceeds that of the BR06/Heirloom strain, and (c) the mushroom product of the crops of said culture retains more weight after a number of days of post-harvest storage at 4 degrees Celsius than does the mushroom product of the BR06/Heirloom strain, the number of days selected from the group comprising 3, 4, 5, 6, 7, and 8 days. The strain BR06/Heirloom is the one that has been deposited under Budapest Treaty governing the deposit of organisms at the American Type Culture Collection (ATCC), Rockville, Md., USA, under ATCC accession number PTA-6876.
Another genetically-determined phenomenon exhibited by Agaricus bisporus and other basidiomycete fungi is vegetative incompatibility. Empirically, it is regularly observed that, in physical contact, a first strain is unable to fuse (anastomose) freely and grow together with any other genetically distinct strain, in other words, with any other strain having less than complete genetic identity with a first strain. The genetics are only partially understood for ‘model’ basidiomycetes, but are known to involve multiple genes and alleles, providing such a large number of combinations that, for practical purposes, each genotype (and each independent strain, including wild strains, cultivars, and hybrids) is extremely unlikely to reoccur in a second strain, and therefore, is effectively unique. The vegetative incompatibility phenotype has two significant commercial and technical implications. First, by using protocols that pair two strains in cropping tests and assess their interaction, it provides a practical test of identity or non-identity between pairs of strains, independent of ‘genetic fingerprinting’. Second, vegetative incompatibility between non-identical strains retards or even prevents the transmission of detrimental viruses between different strains, which can improve facility hygiene and profitability.
In a preferred embodiment of the invention, the culture of the invention is vegetatively incompatible with the strains of the prior art, in particular with the strain BR06/Heirloom or B14528/Tuscan, as shown below.
In a preferred embodiment, the culture of the invention is a culture of a strain of Agaricus bisporus that has less than 99%, 98%, 97%, 96%, 95%, 90%, 80%, 75%, 70%, or 60% genetic similarity to BR06/Heirloom and B14528/Tuscan, and preferably, to a group of any brown-capped strains having both a history of commercial sales and a presence in the record of patent cases in the prior art, the group specifically comprising S600/X618, Bs526, Fr24, Brawn, J15051, BR06/Heirloom and B14528/Tuscan.
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 line/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, F1 hybrid, F2 hybrid, F3 hybrid, F4 hybrid, F5 hybrid, and (3) derivation, i.e., derived culture.
LA3782 is an F1 hybrid strain having N-s34 as one parent and a homokaryon from strain BP-1 as a second parent. In strains of the F1 generation incorporating a set of chromosomes and genotypic markers from N-s34, by virtue of direct descent from the N-s34 parent, 50% of the heterokaryotic strain's genotypic markers will be those of the set from N-s34. An F2 hybrid in this genealogy descending from N-s34 will have on average 25%, and typically about 20-30%, of its genotypic markers from those of N-s34. An F3 hybrid in this genealogy descending from N-s34 will have on average 12.5%, and typically about 10-15%, of its genotypic markers from those of N-s34. An F4 hybrid in this genealogy descending from N-s34 will have on average 6.25%, and typically about 4-8%, of its genotypic markers from those of N-s34. An F5 hybrid in this genealogy descending from N-s34 will have on average 3.13%, and typically about 1.5-4.5%, of its genotypic markers from those of N-s34. In other words, the F1 offspring of N-s34 will comprise about 100 out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I, the F2 offspring will comprise about 50 out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I, the F3 offspring of N-s34 will comprise about 25 out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I, and the F4 offspring will comprise about 10 out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I,
The culture of the invention is a strain of Agaricus bisporus that has a genealogical relationship of identity, descent, or derivation from (a) line N-s34 or from (b) strain LA3782. More precisely, the culture of the invention may have, as the initial culture from which it is derived, one of the following cultures: an Agaricus bisporus haploid line culture N-s34, a haploid line culture comprising at least one set of chromosomes of an Agaricus bisporus line N-s34, a hybrid heterokaryotic culture obtained by mating N-s34 with a second culture to produce an F1 generation, any culture of generation F2, F3, F4, F5, inclusive, that is obtained from the F1 generation of the invention, a culture obtained from line N-s34 by using at least one strain development technique, an inbred descendent of N-s34, an outcrossed descendent of N-s34, and a derived variety of any culture that was obtained from N-s34 by using at least one strain development technique.
In a particular aspect, the present invention relates to an Agaricus bisporus mushroom strain culture of the F2, F3, F4, or F5 generation, descended from the F1 hybrid as defined above, and preferably from the F1 hybrid LA3782, or from a strain derived from strain LA3782. 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 Agaricus bisporus strain LA3782, 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 Jun. 30, 2020, under the CNCM Accession Number I-5527.
More precisely, the F1 offspring of LA3782 (F2 offspring of N-s34) will comprise at least about 100 allelic markers out of the 203 sequence-characterized allelic markers of LA3782 listed in Table II and at least about 50 allelic markers out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I; the F2 offspring will comprise at least about 50 out of the 203 sequence-characterized allelic markers of LA3782 listed in Table II and at least about 25 out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I; and the F3 offspring of LA3782 will comprise at least about 25 out of the 203 sequence-characterized allelic markers of LA3782 listed in Table II and at least about 10 out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I,
In other words, the strain culture of the invention preferably comprises at least about 100 out of the 203 sequence-characterized allelic markers of LA3782 listed in Table II (F1 offspring of LA3782), at least about 50 out of the 203 sequence-characterized allelic markers of LA3782 listed in Table II (F2 offspring of LA3782) or at least about 25 out of the 203 sequence-characterized allelic markers of LA3782 listed in Table II (F3 offspring of LA3782).
The strain culture of the invention is not the strain BP-1 having been deposited under Budapest Treaty governing the deposit of organisms at the American Type Culture Collection (ATCC), Rockville, Md., USA, under ATCC Accession Number PTA-6903. In a preferred embodiment, the strain culture of the invention differs from BP-1 on at least 10%, 20%, 30%, 40%, or at least 50% of its allelic markers. In other words, the strain culture of the invention does not have more than 90%, 80%, 70%, 60% or 50% of identity with BP-1.
In one embodiment, the culture of the invention has a set of chromosomes having at least 65%, at least 70%, or at least 75% genotypic and genomic identity with the chromosomes of the culture of line N-s34, preferably with the culture of the F1 hybrid produced by mating line N-s34 with a second, different Agaricus bisporus culture, more preferably with the strain LA3782.
In a particular embodiment, the strain of the invention is an F2 hybrid having the F1 hybrid heterokaryon culture LA3782 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 F1 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; an F5 hybrid having said F4 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 F4 hybrid.
The SNPs present in the genome of the Agaricus bisporus line N-s34 can be easily identified by whole genome sequencing or by using conventional markers such as those described in U.S. Pat. No. 7,608,760 or 9,017,988. Table I gives a number of useful sequences that characterize the line N-s34 of the invention. Any other SNP can however be used to identify progenies of the lines of the invention.
The SNPs present in the genome of the Agaricus bisporus strain LA3782 can be easily identified by whole genome sequencing or by using conventional markers such as those described in U.S. Pat. No. 7,608,760 or 9,017,988. Table II and Table III give a number of useful sequences that characterize the strain LA3782 of the invention. Any other SNP can however be used to identify progenies of the strains of the invention.
In a preferred embodiment, the Agaricus bisporus mushroom strain culture of the invention descends from line N-s34 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 Agaricus bisporus line N-s34, preferably of the SNPs disclosed in Table I. In another preferred embodiment, the Agaricus bisporus mushroom strain culture of the invention descends from line N-s34 and contains at least about 100, between 50 and 100, between 25 and 50 or between 10 and 25 allelic markers out of the 203 sequence-characterized allelic markers of N-s34 listed in Table I.
In a preferred embodiment, the Agaricus bisporus mushroom strain culture of the invention descends from the strain LA3782 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 Agaricus bisporus strain LA3782, preferably of the SNPs disclosed in Table II or Table III. In another preferred embodiment, the Agaricus bisporus mushroom strain culture of the invention descends from the strain LA3782 and contains at least about 100, between 50 and 100, between 25 and 50 or between 10 and 25 allelic markers out of the 203 sequence-characterized allelic markers of LA3782 listed in Table II,
The term “approximately” or “about” herein inculcates a range of plus or minus 20% above or below the stated value.
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 %.
In another embodiment, the culture of the invention comprises at least one set of chromosomes having at least 65%, 70%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% genetic identity with the chromosomes of N-s34. In a further embodiment, the culture of the invention comprises at least one set of chromosomes having a genotype with at least 65%, 70%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% representation of the markers present on the chromosomes of N-s34.
More precisely, the Agaricus bisporus mushroom culture of the invention can be derived from the initial culture chosen in the group consisting of:
Preferably, said culture is characterized in that it comprises at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the sequence-characterized allelic markers of N-s34 listed in Table I or of LA3782 listed in Table II.
In another aspect, the present invention relates to cells, hyphae, mycelium, mushrooms, germinated spores, ungerminated spores, homokaryons, and heterokaryons including SNPs, NSNPs, and aneuploids obtained from the progeny and derived culture described above.
The present invention also relates to methods for producing the lines and strains of the invention. In particular, the present invention relates to a method for developing a new Agaricus bisporus culture, said method comprising applying at least one mushroom strain development technique to the homokaryon line N-s34, 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 Jun. 30, 2020, under the CNCM Accession Number I-5528 or to a progeny thereof, to provide a new culture.
Also, the present invention relates to a method for developing a new Agaricus bisporus culture, said method comprising applying at least one mushroom strain development technique to an homokaryon of the strain LA3782, 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 Jun. 30, 2020, under the CNCM Accession Number I-5527, or to a progeny thereof, to provide a new culture.
Preferably, said new culture will have any of the features described above for the strains of the invention.
Specifically, said new culture will preferably have any of the following desired traits: (a) an enhanced total yield performance, (b) an enhanced third-flush yield, (c) a good weight, and/or (d) a brown color.
In a preferred embodiment, said new culture will have:
These features have been described in details above. The strain BR06/Heirloom is the one that has been deposited under Budapest Treaty governing the deposit of organisms at the American Type Culture Collection (ATCC), Rockville, Md., USA, under ATCC accession number PTA-6876.
In a particularly preferred embodiment, this new culture will be the F2, F3, F4, or F5 generation descended from the F1 hybrid LA3782, or from a strain derived from strain LA3782. As such, it may comprise 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 Agaricus bisporus strain LA3782, 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 Jun. 30, 2020, under the CNCM Accession Number I-5527.
Preferably, said SNPs are the complete set of SNPs of said hybrid, or a subset thereof, for example the subset disclosed in Table II or Table III.
A number of strain development techniques are known in the art. Some of them are detailed below, in the definition part of the application. Any known technique can be used.
Introducing a desired trait into a culture, for example into Agaricus bisporus line N-s34, can comprise the steps of: (1) physically mating the culture of Agaricus bisporus line N-s34 to a second resultant culture of Agaricus bisporus having the desired trait, to produce a hybrid; (2) obtaining an offspring that carries at least one gene that determines the desired trait from the hybrid; (3) mating said offspring of the hybrid with the culture of Agaricus bisporus line N-s34 to produce a new hybrid; (4) repeating steps (2) and (3) at least once to produce a subsequent hybrid; (5) obtaining a homokaryotic line carrying at least one gene that determines the desired trait and comprising at least 75% of the alleles of line N-s34, for example at the sequence-characterized marker loci described in Table I, from the subsequent hybrid of step (4).
The number “75% of parental DNA in a back-mating (backcross) is an approximation because in the meiosis occurring in the F1 hybrid, random assortment of recombined or unrecombined chromosomes will result in haploid/homokaryotic nuclei having more or less DNA from each of the two parents, balanced around a mean value of 50% (which becomes a mean of 25% in the back-mating).
In another aspect, the present invention relates to a method of producing a mushroom culture comprising the steps of:
In a particular embodiment, said method comprises the steps of:
In another aspect, the present invention relates to a method of producing edible mushrooms, including the step of inoculating compost with a heterokaryotic culture of the invention to produce a crop of mushrooms. A yet further embodiment of the invention is a method of improving farm hygiene, including the step of inoculating compost with the culture of the invention. Yet another embodiment of the invention is a method of crop diversification, including the step of inoculating compost with a culture of the invention.
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 the “definitions” below.
Initially, in order to provide clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Flush: A period of mushroom production within a cropping cycle, separated by intervals of non-production; the term flush encompasses the terms ‘break’ and ‘wave’ and can be read as either of those terms.
Heterokaryon: As a term of art this refers to a sexual heterokaryon: a culture which has two complementary (i.e., necessarily heteroallelic at the MAT locus) types of haploid nuclei in a common cytoplasm, and is thus functionally and physiologically analogous to a diploid individual (but cytogenetically represented as N+N rather than 2N), and which is reproductively competent (in the absence of any rare interfering genetic defects at loci other than MAT), and which exhibits vegetative incompatibility reactions with other heterokaryons; also called a strain or stock in the strain development context.
With respect to the definition of homokaryon above, it is noted that homokaryons and homoallelic lines are subject to technical and practical considerations: A homokaryon in classical terms is a haploid culture which is axiomatically entirely homoallelic. In practical terms, for fungal strain development purposes, the definition is broadened somewhat to accommodate both technical limitations and cytological variation, by treating all predominately homoallelic lines as homokaryons. Technical limitations include the fact that genomes contain duplicated DNA regions including repeated elements such as transposons, and may also include large duplications of chromosomal segments due to historical translocation events. Two different A. bisporus genomes sequenced by the Joint Genome Institute (JGI), a U.S. federal facility, differ in estimated length by 4.4%, and in gene numbers by 8.2%, suggesting a considerable amount of DNA duplication or rearrangement within different strains of the species. No presently available genome of A. bisporus can completely account for the physical arrangement of such elements and translocations, and so the assembled genome sequences of haploid lines may have regions that appear to be heteroallelic using currently available genotyping methods. Cytologically, a homokaryotic offspring will ordinarily be a spore that receives one haploid, postmeiotic nucleus. However, a spore receiving two third-division nuclei from the basidium will be genetically equivalent to a homokaryon. A spore receiving two second-division ‘sister’ postmeiotic nuclei will be a functional homokaryon even though some distal ‘islands’ of heteroallelism may be present due to crossovers during meiosis. Also, a meiosis that has an asymmetrical separation of homologues can produce an aneuploid, functionally homokaryotic spore in which an extra chromosome, producing a region of heteroallelism, is present. All of these cultures are highly homoallelic and all function as homokaryons. Technological limitations make it impractical to distinguish among such cultures, and also to rule out DNA segment duplication as an explanation for limited, isolated regions of the genome sequence assembly that appear to be heteroallelic. Therefore, in the present application, the use of the term ‘homoallelic’ to characterize a line includes entirely or predominately homoallelic lines, and cultures described in this way are functional homokaryons, are putatively homokaryotic, and are all defined as homokaryons in the present application.
Agaricus bisporus has a reproductive syndrome known as amphithallism, in which two distinct life cycles, namely heteromixis and intramixis, operate concurrently. As in other fungi, the reproductive propagule is a spore. Agaricus produces spores meiotically, on a meiosporangium known as a basidium. In a first life cycle, A. bisporus spores each receive a single haploid postmeiotic nucleus; these spores are competent to mate but are not competent to produce mushrooms. These haploid spores germinate to produce homokaryotic offspring or lines which can mate with other sexually compatible homokaryons to produce novel hybrid heterokaryons that are competent to produce mushrooms. Heterokaryons generally exhibit much less ability to mate than do homokaryons. This lifecycle is called heteromixis, or more commonly, outbreeding. This life cycle, which may be carried out to obtain new hybrid strains in strain development programs, operates but typically does not predominate in strains of Agaricus bisporus var. bisporus.
A second, inbreeding life cycle called intramixis predominates in most strains of Agaricus bisporus var. bisporus. Most spores, typically 90%-99.9%, receive two post-meiotic nuclei, and most such pairs of nuclei, typically at least 90%, consist of Non-Sister Nuclear Pairs (NSNPs) which have a heteroallelic genotype at most or all centromeric-linked loci including the MAT (=mating type) locus. That MAT genotype determines the expression of the heterokaryotic phenotype of these offspring, which are reproductively competent strains and can produce a crop of mushrooms. Unusually among eukaryotes, relatively lower amounts of chromosomal crossing-over (3.9 crossovers per haploid offspring per generation with the U1 strain as the parent, per Wei Gao, 2014) is observed to have occurred in postmeiotic offspring of Agaricus bisporus; empirically, very little heteroallelism (analogous to heterozygosity), usually not more than 1% on average, per Sonnenberg et al. (2011) is lost among heterokaryotic offspring of a heterokaryotic strain. Consequently, parental and heterokaryotic offspring genotypes and phenotypes tend to closely resemble each other, as noted above. In other words, heterokaryotic offspring of Agaricus bisporus are usually functionally equivalent to, and ordinarily indistinguishable from, their parent, although trivial genetic rearrangements of the parental genome may be present.
A heterokaryotic selfed offspring of an F1 hybrid that itself has a ‘p/q’ genotype at a hypothetical locus will in the example have a genotype of ‘pip’, ‘q/q’, or 134. Two types of selfing lead to differing expectations about representation of alleles of line N-s34 present in the F1 hybrid in the next heterokaryotic generation obtained from a mating of N-s34. When two randomly obtained haploid offspring from the same F1 hybrid, derived from individual spores of different meiotic tetrads, are mated (i.e., in inter-tetrad selfing), representation of the line N-s34 marker profile in each recombined haploid parental line and in each sib-mated heterokaryon will be 50% on average, and slightly more than 75% (to about 85%) of heteroallelism present in the F1 hybrid will on average be retained in the sib-mated heterokaryon (note that the expectation over 75% is due to the mating-compatibility requirement for heteroallelism at the mating type locus (MAT) on the large Chromosome 1, which comprises about 10% of the nuclear genome). Distinctively, in addition, Agaricus bisporus regularly undergoes a second, characteristic, spontaneous intra-tetrad form of selfing called intramixis, producing heterokaryotic postmeiotic spores carrying two different recombined haploid nuclei almost always having complementary, heteroallelic MAT alleles. An offspring developing from any one of these spores is a postmeiotic self-mated heterokaryon with ca. 100% retention of the heteroallelism present in the single F1 parent around all 13 pairs of centromeres. In theory this value would decrease to an average of 66.7% retention of F1 heteroallelism for distal markers unlinked to their centromeres; however empirical observations suggest higher rates of retention even for such distal markers, in conjunction with limited amount of crossing-over. Applicant typically observes 95%-100% retention of heteroallelism in such heterokaryotic offspring; Sonnenberg et al. (2011) reported an average of 99% retention among such offspring of the U1 strain. Transmission of the line N-s34 marker profile in such selfed offspring may be incomplete by a small percentage (typically 0-5%) due to the effects of infrequent meiotic crossovers however while DNA (and genotypic markers) from N-s34 will still represent 50% on average of the resulting heterokaryotic genome. Both types of selfed offspring are considered to be derived strains from the initial F1 hybrid, and the latter type comprises most (often [95-] 99 [-100]%) of the initial genotype of the F1 hybrid, and may express a very similar phenotype to that of the F1 hybrid, and be functionally equivalent to it.
When the relationship is one of inbred descent from a heterokaryon, via offspring homokaryons, the two cultures will have a degree of genetic identity with on average about 85% representation and 100% commonality of origin (with respect to the parental culture). When the relationship is one of intramictic inbred descent from a heterokaryon, via a single heterokaryotic spore, the two cultures will have a degree of genetic identity with on average about 95%-99%-100% representation and 100% commonality of origin (with respect to the parental culture). When the relationship is one of back-bred descent from an F1 heterokaryon, via mating an offspring homokaryon to a parental homokaryon such as N-s34, the representation and commonality of origin of the parental homokaryon genotype in the back-bred heterokaryon will both be roughly 75% on average. Somatic selection cultures and tissue selection cultures will effectively have 100% genetic identity with the initial culture, possibly with epigenetic alterations, or rearrangements, or rare mutations, often present at the same rate as in unselected clonal subcultures, and which are virtually impossible to detect. Mutagenized cultures will similarly have effectively 100% genetic identity with their initial culture, except for one or more random point mutations that are impractical to detect. Transformed cultures will typically have at least 99.99% to 100% genetic identity with their initial culture, plus one small piece of exogenous DNA which may or may not be integrated into the Agaricus genome.
A. Distinction of the Lines/Strains of the Invention from Known Brown Prior Art Strains
The LA3782 strain is substantially different from other brown-capped Agaricus bisporus strains in the prior art.
To demonstrate this, here are provided the allelic genotype data for six standard markers that were previously reported as SCAR markers (U.S. Pat. Nos. 7,608,760, 9,017,988, and subsequent). Brief descriptions of relevant alleles at these six unlinked marker loci are provided below. Genotypes at these six loci were determined both by Whole Genome Sequencing and by SCAR-PCR, as described below.
For the SCAR-PCR method, the amplified PCR product DNA was sequenced by a contractor, Eurofins, using methods of their choice, and the genotypes were determined by direct inspection of these sequences followed by SNP analysis and comparison to Applicant's database of reference marker/allele sequences.
These 6 markers are defined as follows:
The “p1n150-3G-2” marker is a refinement of the p1n150 marker reported on Chromosome 1 by Kerrigan, R. W., et al. “Meiotic behavior and linkage relationships in the secondarily homothallic fungus Agaricus bisporus.” Genetics 133, 225-236 (1993), and shown to be linked to the MAT (mating type) locus by Xu et al., “Localization of the mating type gene in Agaricus bisporus.” App. Env. Microbiol. 59(9): 3044-3049 (1993) and has also been used in other published studies. While several different primers can be and have been used to amplify segments of DNA in which the p1n150-3G-2 marker is present and from which it can be sequenced, digested, electrophoretically characterized, or otherwise analyzed, the primer sequences employed by the inventors for the development of the disclosed data were: Forward: 5′-aggcrycccatcttcasc-3′ (SEQ ID NO:1); Reverse: 5′-gttcgacgacggactgc-3′ (SEQ ID NO:2), with 35 PCR cycles, 56° C. anneal temperature, 1 min. extension time.
The “ITS” marker has been adopted as the official ‘barcode’ sequence for all fungi (Schoch et al., Fungal Barcoding Consortium, “Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.” Proc. Nat. Acad. Sci. <www.pnas.org/cgi/content/short/1117018109> (2012)), and has been used in innumerable publications, including Morin et al., “Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.” Proc. Nat'l Acad. Sci. USA 109: 17501-17506 (2012) on the complete A. bisporus genome sequence. White et al. (1990), Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to methods and applications. (Innis M A, Gelfand D H, Sninsky J J, White T J, eds). Academic Press, New York, USA: 315-322, published many primer sequences for the ITS marker, of which the inventors used primers ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′ (SEQ ID NO:3) and ITS4: 5′-TCCTCCGCTTATTGATATGC-3′ (SEQ ID NO:4), with 35 PCR cycles, 56° C. anneal temperature, 1 min. extension time.
The “MFPC-1-ELF” marker is derived from a sequence mapped by Marie Foulongne-Oriol et al., “An expanded genetic linkage map of an intervarietal Agaricus bisporus var. bisporus-A. bisporus var. burnettii hybrid based on AFLP, SSR and CAPS markers sheds light on the recombination behaviour of the species.” Fungal Genetics and Biology 47: 226-236 (2010) that is linked to the PPC-1 locus described by Callac et al., “Evidence for PPC1, a determinant of the pilei-pellis color of Agaricus bisporus fruit bodies.” Fungal Genet. Biol. 23, 181-188 (1998). An equivalent linked marker has been used as described in Loftus et al., “Use of SCAR marker for cap color in Agaricus bisporus breeding programs.” Mush. Sci. 15, 201-205 (2000). While several different primers can be and have been used to amplify segments of DNA in which the MFPC-1-ELF marker is present and from which it can be sequenced, digested, electrophoretically characterized, or otherwise analyzed, the primer sequences employed by the inventors for the development of the disclosed data were: Forward: 5′-aytcrcaamaacataccttcaac-3′ (SEQ ID NO:5); reverse: 5′-cattcggcgattttctca-3′ (SEQ ID NO:6), with 35 PCR cycles, 55° C. anneal temperature, 0.5 min. extension time.
The AN, AS, and FF markers were designed from sequences obtained from PCR products produced by the use of primers disclosed by Robles et al., U.S. Pat. No. 7,608,760, and/or from contiguous or overlapping genome sequences, to improve upon the performance, reliability, and consistency of results, as compared to the markers as originally described by Robles et al.; they are genotypically and genomically equivalent. While several different primers can be and have been used to amplify segments of DNA in which either the AN, AS, or FF marker is present and from which it can be sequenced, digested, electrophoretically characterized, or otherwise analyzed, the primer sequences employed by the inventors for the development of the disclosed data were:
All the brown strains commercially available in the prior art (Heirloom, Tuscan, S-600, Bs526, Fr24 and Brawn) have been compared to show that the strains of the invention are different. The brown prior art mushroom strain J15051 (NRRL accession number 67316) disclosed in WO2018102290 was also included.
Table IV below summarizes the allelic markers at these 6 loci for the cultures of the invention and for a number of other prior art strains.
Whole-genome sequences were aligned by contigs with reference to the H97 V2.0 reference sequence, using the Seqman NGen module of the Lasergene software package (DNAStar, Inc.). By inspecting the aligned sequences of two or more cultures, SNPs at individual loci have been determined and compared directly.
Tables V and VI below show the genotypes of the relevant strains at the 203 SNP marker loci used in Tables I and II, and also the overall genetic similarity calculation between each strain and LA3782.
TTGA
TTGA
TTGA
TTGA
TTGA
TTGA
TTGA
TTGA
ACAT
ACAT
ACAT
ACAT
ACAT
GGTT
GGTT
GGTT
GGTT
GGTT
GGTT
GGTT
GGTT
ATGA
ATGA
ATGA
ATGA
ATGA
ATGA
ATGA
ATGA
AGGA
AGGA
AGGA
AGGA
AGGA
AGGA
TTTT
TTTT
TTTT
TTTT
TTTT
TTTT
TTTT
TTTT
TATT
TATT
AAAC
AAAC
AAAC
AAAC
AAAC
AAAC
AAAC
AAAC
GATG
GATG
GATG
GATG
GATG
ATCG
ATCG
ATCG
ATCG
ATCG
ATCG
ATCG
ACTGG
ACTGG
ACTGG
ACTGGT
ACTGGT
ACTGG
ACTGG
CACC
CACC
CACC
CACC
CACC
ACTA
ACTA
ACTA
ACTA
ACTA
ACTA
ACTA
ACTA
GCGC
GCGC
GCGC
GCGC
AATT
AATT
AATT
AATT
AATT
AATT
AATT
AATT
CAA
CAA
CAA
CAA
CAA
CAA
CAA
CAA
AAAT
AAAT
AAAT
AAAT
AAAT
AAAT
AAAT
TTTG
TTTG
TTTG
TTTG
TTTG
TTTG
TTTG
TTTG
GAC
GAC
GAC
GAC
GAC
GAC
GAC
GAC
TCTT
TCTT
TCTT
TCTT
TCTT
TCTT
TCTT
TCTT
GTTT
GTTT
GTTT
GTTT
GTTT
GTTT
GTTT
GTTT
GA
AGG
AGG
GA
AGG
GA
CAGG
CAGG
AGG
GA
AGG
ATGT
ATGT
G
ATGT
ATGT
ATGT
GGTG
GGTG
GGTG
GGTG
GGTG
GGTG
GGTG
GGTG
TACG
TACG
TACG
TACG
TACG
TACG
TACG
TACG
CTAA
CTAA
CTAA
CTAA
CTCA
CTCA
CTCA
CTCA
CTCA
CTCA
CTCA
CTCA
CCAA
CCAA
CCAA
CCAA
CCAA
CCAA
CCAA
CCAA
TGTT
TGTT
TGTT
TGTT
TGTT
TGTT
TGTT
TGTT
CAGT
C
GT
CAGT
C
GT
C
GT
C
GT
C
GT
CAGT
TTCA
TTCA
TTCA
TTCA
TTCA
TTCA
TTCA
TTCA
C
AGAT
C
AGAT
C
AGAT
C
AGAT
C
AGAT
C
AGAT
C
AGAT
C
AGAT
TGCC
TGCC
TGCC
TGCC
TCTT
TCTT
TCTT
TCTT
TCTT
TCTT
TCTT
AATT
AATT
AATT
AATT
AATT
AGGG
AGGG
AGGG
AGGG
TCAA
TCAA
TCAA
TCAA
TCAA
TCAA
TCAA
CAA
AC
C
ACCC
CAA
AC
C
AC
C
ACCC
ACCC
CAA
ACCC
CAA
AC
C
TCCT
TCCT
TCCT
TCCT
AGGT
AGGT
AGGT
AGGT
AGGT
GCTT
GCTT
GCTT
GCTT
GCTT
GCTT
GCTT
CAGC
CAGC
CAGC
CAGC
CAGC
CAGC
TCCT
TTTC
TTTC
TTTC
TTTC
TTTC
G
AGAG
G
AGAG
G
AGAG
G
AGAG
G
AGAG
G
AGAG
G
AGAG
G
AGAG
GGAA
GGA
GGAA
GGAA
GGAA
GGAA
GGAA
TCGT
TCTG
TC
G
TCTG
TTTC
TTTC
TTTC
TTTC
TTTC
TTTC
TTTC
GGCG
GGCG
GGCG
GGCG
GGCG
GGCG
GGCG
GGCG
CCTT
CCTT
CCTT
CCTT
CCTT
CCTT
CCTT
TCAT
TCAT
TCAT
TCAT
GATT
GATT
GATT
GATT
GATT
GATT
GATT
GATT
TGGG
TGGG
TGGG
TGGG
TGGG
TGGG
TGGG
AGCC
AGCC
AGCC
AGCC
AGCC
AGCC
AGCC
AGCC
AAGA
AAGA
AAGA
AAGA
AAGA
AAGA
AAGA
AAGC
AAGC
AAGC
AAGC
AAGC
AAGC
CCAC
CCAC
CCAC
CCAC
CCAC
CCAC
ATA
ATA
ATA
ATA
AT
A
AT
A
AT
A
TAAC
TAAC
TAAC
TAAC
TAAC
TAAC
TAAC
TAAC
GCGT
GCGT
GCGT
AAT
AAT
AAT
AAT
AAT
AAT
AAT
CCAA
CCAA
CCAA
CCAA
AAGC
AAGC
AAGC
AAGC
AAGC
AAGC
AAGC
AAGC
AACT
AACT
AACT
AACT
AACT
AACT
AACT
GGAC
GGAC
GGAC
GGAC
GGAC
GGAC
GGAC
GGAC
GA
CAAT
GA
CAAT
GA
CAAT
GA
CAAT
GA
CAAT
GA
CAAT
GA
CAAT
GA
CAAT
CGAA
CGAA
CGAA
CGAA
CGAA
CGAA
CGAA
CGAA
TACT
TACT
TACT
TACT
TACT
TACT
TACT
TACT
CGG
CGG
CGG
CGG
CGG
CGG
CGG
CGG
TTGC
TTGC
TTGC
TTGC
GTA
GTA
GTA
GTA
GTA
GTA
GTA
GTA
AGAA
AGAA
AGAA
AGAA
AGAA
AGAA
AGAA
GACC
GACC
GACC
GACC
GACC
ACTG
ACTG
ACTG
ACTG
ACTG
ACTG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
AGGG
GAAT
GAAT
GAAT
GAAT
GAAT
GAAT
GAAT
GCCC
GCCC
GCCC
GCCC
GCCC
TTCA
TTCA
TTCA
TTCA
TTCA
TCT
TCT
TCT
TCT
TCT
TCT
TCT
TCT
GACA
GACA
GACA
GACA
GACA
GACA
GACA
TCC
TCC
TCC
TCC
TCC
TCC
TCC
TCC
CCTT
CCTT
CCTT
CCTT
CCTT
CCTT
CCTT
CTCC
CTCC
CTCC
CTCC
AGTC
AGTC
AGTC
AGTC
AGTC
AGTC
AGTC
AAAA
AAAA
AAAA
AAAA
AAAA
CTTG
CTTG
CTTG
CTTG
CTTG
CTTG
CTTG
CTTG
GGTT
GGTT
GGTT
GGTT
GGTT
GGTT
GGTT
ATTT
ATTT
ATTT
ATTT
ATTT
ATTT
AGGA
AGGA
AGGA
AGGA
AGGA
AGGA
ATTG
ATTG
ATTG
ATTG
ATTG
ATTG
ATTG
ATTG
TAG
TAG
TAG
TAG
TAG
TAG
TGGA
GATT
GATT
GATT
GATT
GATT
GATT
GATT
GATT
TGCG
TGCG
TGCG
TGCG
ATAA
ATAA
ATAA
ATAA
ATAA
TCAG
TCAG
TCAG
TCAG
TCAG
TCAG
GTTT
GTTT
GTTT
GTTT
GTTT
GTTT
GTTT
ATTA
ATTA
ATTA
ATTA
ATTA
ATTA
ATTA
CATA
CATA
CATA
CATA
CATA
CATA
CATA
ACCT
ACCT
ACCT
ACCT
ACCT
ACCT
ACCT
ACCT
GAAG
C
GAAG
GAAG
GAAG
GAAG
GAAG
GAAG
AT
T
AAA
ATAT
AAA
ATAT
AT
T
GCAA
GCAA
GCAA
GCAA
CAA
GCAA
GCAA
CACC
CACC
CACC
ACC
CACC
CACC
CGCG
CGCG
CGCG
CGCG
CGCG
CGCG
CGCG
TTCT
TTCT
TTCT
TTCT
TTCT
TTCT
TTCT
TTGA
GA
GA
TTGA
TGA
TTGA
GA
GA
CAAC
CAAC
CAAC
CAAC
CAAC
GTGA
GCAG
GCAG
GCAG
GCAG
GCAG
GCAG
GATT
GACG
GACG
GACG
GACG
GACG
GACG
GACG
GACG
GAAG
C
GG
ATCT
ATCT
ATCT
ATCT
ATCT
ATCT
ATCT
ATCT
TGCA
TGCA
TGCA
TGCA
TGCA
TGCA
TGCA
TGCA
TACT
TACT
TACT
TACT
TACT
GCCT
G
T
G
T
G
T
G
T
GCCT
G
T
G
T
AGTA
AGTA
AGTA
AGTA
AGTA
AGTA
AGTA
GTTG
GTTG
GTTG
GTTG
GTTG
GTTG
GTTG
GTTG
TTCT
TTCT
TTCT
TTCT
TTCT
TTCT
TTCT
ATAC
ATAC
ATAC
ATAC
ATAC
GT
TCCC
TCCC
TCCC
TCCC
TCCC
TCCC
TCCC
TCCC
TG
ATC
TGTAATC
TG
ATC
TGTAATC
TGTAATC
TGTAATC
TGTAATC
TGTAATC
ACGT
AC
T
ACGT
ACGT
ACGT
ACGT
AC
T
AC
T
GTGGTGA
GT
GTGA
GTGA
GTGA
GTGA
GGGA
GGGA
GGGA
GGGA
GGGA
GGGA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
GACA
C
CC
CC
CC
CC
CC
CC
CC
CC
GTTT
GTTT
GTTT
GTTT
GTTT
GTTT
GTTT
CATC
CATC
CATC
CATC
CATC
CATC
CATC
CATC
T
TGG
TGG
TGG
TGG
TGG
GTGG
TGG
TGG
ATCG
ATCG
TCAA
TCAA
TCAA
TCAA
TCAA
TCAA
TCAA
TCAA
ATCA
ATCA
ATCA
ATCA
ATCA
ATCA
ATCA
ATCA
AAAG
AAAG
AAAG
AAAG
AAAG
AAAG
AAAG
AAAG
AATT
AATT
AATT
AATT
AATT
AAAG
AATT
AA
TTT
ATTT
AA
TTT
TTT
CCAC
TCAT
TCAT
TCAT
TCAT
TCAT
TCAT
TCAT
TCAT
CAAA
AT
CAAA
ATGCAAA
ATG
A
A
A
A
AT
CAAA
GGAG
GGAG
CCTT
CCTT
CCTT
CGTC
CGTC
CGTC
CTGT
CTGT
CTGT
GCGA
GCGA
GCGA
GCGA
GCGA
GGGG
GGGG
GGGG
GGGG
GGGG
GGGG
GCGC
GCGC
GCGC
GCGC
GCGC
GCGC
GCGC
GCGC
GCCC
GCCC
GCCC
GCCC
GCCC
GCCC
GCCC
GCCC
TCA
TCA
TCA
TCA
TCA
TCA
TCA
TCA
CACC
CACC
CACC
CACC
CACC
CACC
CACC
CACC
TG
CACG
TGCCACG
T
CCACG
T
CCACG
T
CCACG
T
CCACG
TG
CACG
ATTC
ATTC
ATTC
ATTC
ATTC
TACA
TACA
TACA
TACA
TACA
TACA
TACA
TACA
CACC
CACC
CACC
CACC
CACC
TGCT
TGCT
TGCT
TGCT
TGCT
TGCT
TGCT
TGCT
CG
CG
CG
CG
CG
CG
CG
CG
AGCC
AGCC
AGCC
AGCC
GGTC
GGTC
GGTC
GGTC
GGTC
GGTC
TTCT
TTCT
TTCT
TTCT
TTCT
TTCT
TTCT
TTCT
GAGG
GAGG
GAGG
AGG
AGG
GAGG
AGGA
AGGA
AGGA
AGGA
AGGA
AGGA
AGGA
TTTA
TTTA
ACTG
ACTG
ACTG
ACTG
ACTG
ACTG
CTGC
CTGC
CTGC
CTGC
CTGC
CTGC
CTGC
GTCA
GTCA
GTCA
GTCA
GCAT
TCAG
TCAG
TCAG
TCA
TCAG
TCAG
AATA
AATA
AATA
AATA
AATA
AATA
AATA
AATA
CCTT
CCTT
C
TT
C
TT
C
TT
C
TT
CCTT
CCTT
GCCT
GCCT
GCCT
GCCT
G
AA
G
AA
G
AA
TT
GAAA
TT
GAAA
TT
G
AA
G
AA
TT
GAAA
ATGG
ATGG
ATGG
ATGG
ATGG
ATGG
AGAT
AGAT
AGAT
AGAT
AGAT
CCGT
CCGT
CCGT
CCGT
CCTA
CCTA
CCTA
CCTA
CCTA
CCTA
CCTA
GGAA
GGAA
GGAA
GGAA
GGAA
GGAA
TCTG
TCTG
TCTG
TCTG
TCTG
TCTG
TCTG
TCTG
TACG
TACG
TACG
TACG
TACG
TACG
TTCA
TTCA
TTCA
TTCA
AACG
AACG
AACG
AACG
AACG
AC
A
AC
A
AC
A
AC
A
AC
A
AC
A
C
TG
C
TG
C
TG
C
TG
TCA
G
C
TG
C
TG
TGGG
TGGG
TGGG
TCCG
TCCG
TCCG
TCCG
TCCG
TCCG
TCCG
TCCG
GCAA
GCAA
GCAA
GCAA
GCAA
GCAA
GCAA
GCAA
TGTT
TGTT
TGTT
T
TT
T
TT
TGTT
TGTT
The use of these markers to determine calculated % genetic similarity (identity) between two heterokaryotic cultures or strains is presented in Table VI.
The 9-mers containing the reported SNPs in the tables have been treated as composites of two unitary alleles (in heterokaryon comparisons).
The composite 9-mer genotype has been compared at each locus and assigned a value if 1 for a perfect match, or a 0 for anything less than a perfect match. Then the values were totaled for all loci in each pairwise comparison between strains, and divided by the total number of loci compared, and the resulting decimal was converted to %.
Results are similar for the full set of SNP markers (N=203) and for a smaller set (N=170) excluding the SNPs that define alleles at the six SCAR marker loci (and which have a shorter interval distribution). The highest % genetic similarity or identity observed for LA3782 compared to the heterokaryotic genotypes of seven other strains is 67%. Identity for two clones of LA3782 would be 100%.
B. Vegetative Incompatibility
Substantial genetic dissimilarity (i.e., 100%−% genetic identity) is known to be associated with heterokaryon or ‘vegetative’ incompatibility. Incompatibility interferes with anastomosis and with mushroom production. From the data in Table VI, it would be expected that LA3782 would be incompatible with the other leading commercial brown-capped Agaricus bisporus strains. Table VII demonstrates this empirically.
General t-test analysis on three replicates (a-c); the difference between compatible and incompatible combinations is significant with p-value a ≤0.05 in all cases. In each treatment, one of the three strains was inoculated into compost, and after colonization, casing soil inoculated with one of the three strains was applied over the compost. Cultivation containers with 0.07 square meters of surface were used in standard growing conditions. Note that only combinations scored as compatible (marked with *) produced mushrooms.
C. Crop Yield
Yield performance was measured in large-scale trials. During these trials, incubation period was 18 days in bulk phase III tunnel, spawning rate was 8 litres/ton of compost phase II. Trays were filled with 135 kg incubated compost with a filling rate of 90 kg/m2. Mc substrate supplement was added at the rate of 1.33 kg/m2. Carbo 9 casing from supplier Euroveen was applied with 1200 g/m2 compost casing, premixed. In the growing room we tested strains with 12 replications distributed across 5 growing levels.
Airing started on day 4 after casing. To collect yield, mushrooms were picked and weighted daily on 12 replicates. Data were collected over 3 flushes.
The mushroom crop yield of strain LA3782 was found to be greater (better) than that of the BR06/Heirloom strain on third flush and also when aggregated over flushes 1, 2 and 3, as shown in Table VIII.
From Table VIII, strain LA3782 is shown to be highly productive, and also to have an improved flush-yield balance due to the higher third-flush yield, as compared to all the prior art strains that have been tested.
D. Weight Retention
The mushrooms produced by strain LA3782 also have improved weight retention during post-harvest storage, compared to those of the Heirloom strain, as shown in Table IX.
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, three flushes occur at weekly intervals. The expression of the trait in Flush 1 was evaluated. During this test, five replicate styrofoam tills per strain were evaluated. A till is a tray that can hold over 1 kg. The weight of the empty till was recorded. Thirty mushrooms approximately 4-5 cm in diameter, with tightly closed veils were placed into each till. 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 4° C. for 8 days in a walk-in cooler. Filled till weights were recorded each day beginning on day 3. After subtracting the weight of the empty till, percentage of weight retention was calculated as described above.
E. Mushroom Piece Weight
Table X shows that the piece weight (mean individual harvested mushroom weight) in crops from LA3782 is significantly greater than that of the Heirloom or Tuscan strains, especially in first flush. A greater piece weight can reduce the costs of harvesting the crop.
Trait data collection was carried out by a method in which mushroom samples were collected during the first and second flush of mushroom production. The expression of the trait in Flush 1 and Flush 2 was evaluated. During this test, 20 replicate medium size mushrooms (4-5 cm in diameter) per strain over 4 different levels were evaluated. Each replicate was individually weighed.
The average piece weight of mushrooms in flush 1 and flush 2 expressed in grams. In a general t-test analysis, the differences with LA3782 were significant at a p a ≤0.05 threshold.
F. Cap Color
The mushroom color was measured using a Minolta Chroma Meter CR-200 (mfd. Japan). Sample sizes of thirty medium sized mushrooms at commercial maturity (with closed veils) were harvested from the tests and measured to obtain values for the L*a*b parameters. The Chroma Meter readings were randomly taken at the tops of 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.
Finally, it will be understood that any variations evident fall within the scope of the claimed invention and thus, the specific selection of characteristics, techniques, and sources of homokaryons and heterokaryons can be determined without departing from the spirit of the present invention herein disclosed and described. Further, it will be understood that the scope of the invention is not necessarily limited to methods that produce mushroom strains and cultures that have all of the characteristics set forth herein, but rather to those strains, lines, and cultures that are produced, descended or otherwise derived from cultures having at least one parent that is derived from line N-s34 or strain LA3782. Accordingly, the scope of the invention shall include all modifications and variations that may fall within the scope of the attached claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20305862.3 | Jul 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/070901 | 7/26/2021 | WO |
