Sporless pleurotus strain F2-18 and cultivation method and application thereof

Abstract

The application discloses a sporeless pleurotus ostreatus strain F2-18 and a cultivation method and application thereof, relates to the technical field of edible fungus hybridization and breeding and molecular marker assisted breeding, and belongs to the technical field of edible fungus hybridization and breeding and molecular marker assisted breeding. The strain F2-18 has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 42585. In the breeding process of the strain, a molecular marker based on an MSH4 gene is combined to perform auxiliary screening, and a target strain with no spore and excellent agronomic characters is obtained. Through cultivation test verification, the fruiting body rate of the strain is 100%, the phenotype is stable, the yield is significantly improved compared with a sporeless parent, and compared with a spore parent, the strain avoids the harm caused by spore release, and has a potential for factory cultivation application.

Description

Technical Field

[0001] This invention relates to the field of edible fungi breeding and cultivation technology, specifically to a spore-free oyster mushroom strain F2-18 and its cultivation method and application. The spore-free oyster mushroom strain F2-18 is a spore-free oyster mushroom strain obtained based on hybridization breeding combined with molecular marker-assisted screening. Background Technology

[0002] Oyster mushroom ( Pleurotus ostreatus As an important type of edible fungus, oyster mushrooms are widely cultivated due to their strong adaptability, high yield, and rich nutritional value. However, the existing main cultivated oyster mushroom varieties produce a large number of spores during the production process. The release of spores not only pollutes the production environment but may also adversely affect the respiratory health of workers. At the same time, it can easily lead to equipment contamination and strain degradation, which is not conducive to industrialized and large-scale production.

[0003] To address the aforementioned issues, existing research has attempted to breed sporophyllous or oyster mushroom varieties with few or no sporulations. However, compared to sporulated varieties, existing sporophyllous varieties generally suffer from lower yields, looser flower shapes, and other insufficient commercial traits or poor trait stability, making it difficult to simultaneously meet the production demands for both sporophyllous characteristics and high yield and quality. Therefore, it remains necessary to further breed a new oyster mushroom variety that combines stable sporophyllous characteristics with excellent agronomic traits to meet the needs of the modern oyster mushroom industry. Summary of the Invention

[0004] The purpose of this invention is to provide a spore-free oyster mushroom strain and its cultivation method and application, so as to overcome the problems of low yield, insufficient commercial characteristics and stability of existing spore-free oyster mushroom varieties. The spore-free oyster mushroom strain is named F2-18, which has stable spore-free characteristics and its comprehensive characteristics are superior to those of the spore-free parent.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a sporozoan-free oyster mushroom strain F2-18, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42585. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is March 6, 2026, and it is classified and named *Pleurotus ostreatus*. Pleurotus ostreatus It is abbreviated as F2-18.

[0006] The second aspect of the present invention provides a mushroom mycelium, which is obtained by growing and developing the non-spore-forming mushroom strain F2-18 described in the first aspect above.

[0007] The third aspect of the present invention provides a oyster mushroom fruiting body, which is obtained by growth and development of the sporeless oyster mushroom strain F2-18 described in the first aspect above.

[0008] The fourth aspect of the present invention provides the above-described spore-free oyster mushroom strain F2-18 of the first aspect, the oyster mushroom mycelium of the second aspect, or the oyster mushroom fruiting body of the third aspect in at least one of the following applications (A) to (D): (A) Its application as a parent in hybridization breeding; (B) Application in strain propagation; (C) Application in sub-entity production; (D) Applications in food processing.

[0009] The fifth aspect of this invention provides a method for cultivating the above-mentioned asporophytic oyster mushroom strain F2-18, comprising the following steps: S1: The non-spore-forming oyster mushroom strain F2-18 was activated and cultured on PDA or PDB medium to prepare the mother culture; S2: The mother seed is inoculated into the branch seed culture medium to obtain the branch seed; S3: Inoculate the original branch spawn into the cultivation bag substrate and cultivate at room temperature in the dark, with a relative humidity of 60%-70% and a carbon dioxide concentration of less than 0.3%. Fruiting can begin after the mycelium has fully grown into the cultivation bag. S4: During the fruiting stage, the temperature is 816℃, the relative humidity is not less than 95%, the light intensity is 100-300 lx, and the carbon dioxide concentration is less than 0.1%.

[0010] For example, in step S3, the relative humidity can be 62%, 65%, 67%, or 69%, or any of the above specific humidity ranges; the carbon dioxide concentration can be 0.29%, 0.25%, 0.2%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, etc., or any of the above specific carbon dioxide concentration ranges.

[0011] For example, in step S4, the temperature during the fruiting stage can be 9℃, 10℃, 12℃, 14℃, 15℃, etc., or any of the above-mentioned specific humidity ranges; the relative humidity can be 95%, 96%, 97%, 98%, or 99%, or any of the above-mentioned specific humidity ranges; the light intensity can be 102lx, 120lx, 150lx, 180lx, 200lx, 220lx, 250lx, 270lx, 290lx, or 300lx, etc., or any of the above-mentioned specific light intensity ranges; the carbon dioxide concentration can be 0.09%, 0.07%, 0.05%, 0.02%, 0.01%, 0.005%, etc., or any of the above-mentioned specific carbon dioxide concentration ranges.

[0012] Furthermore, based on the amount of raw materials added per liter of water, the PDA culture medium comprises: 200g potato, 20g glucose, 1L water, 20g agar, pH natural. Furthermore, based on the amount of raw materials added per liter of water, the PDB culture medium comprises: 200g of potato, 20g of glucose, 1L of water, and natural pH. The pH mentioned above naturally refers to the preparation of the two culture media without any additional pH adjustments.

[0013] Further, according to the mass percentage, the original seed culture medium of the branches includes the following raw materials: 80-90% wood branches, 8-15% wheat bran, 1-2% gypsum, and water accounts for 60% to 65% of the mass percentage of the raw materials; preferably, 89% wood branches, 10% wheat bran, 1% gypsum, and water accounts for 60% to 65% of the mass percentage of the raw materials.

[0014] Further, according to the mass percentage, the cultivation substrate consists of: 25-35% cottonseed hulls, 40-55% corn cobs, 15-20% wheat bran, 1-3% soybean meal, 1-2% gypsum, 1-3% lime, and water accounts for 60%-65% of the mass percentage of the raw materials; preferably, it consists of 30% cottonseed hulls, 48% corn cobs, 18% wheat bran, 2% soybean meal, 1% gypsum, 1% lime, and water accounts for 63% of the mass percentage of the raw materials.

[0015] The sixth aspect of the present invention provides a primer combination for detecting different phenotypes of sporulation traits in the *Pleurotus ostreatus* strain, wherein the *Pleurotus ostreatus* strain varieties are CCMSSC01366, CCMSSC00406, the above-mentioned sporozoid *Pleurotus ostreatus* strain F2-18 or its progeny population, and the primer combination is a primer pair consisting of the nucleotide sequences shown in Sequence 1 and Sequence 2 in the sequence listing, namely MSH4JGY-F and MSH4JGY-R.

[0016] MSH4JGY-F 5'-GTAGGTTTGGCAGCCCTAGATAAG-3' (sequence 1) MSH4JGY-R 5'-TTCATCAAGGTTTTGAGCAACTTG-3' (Sequence 2) The seventh aspect of the present invention provides the application of the above primer combination in the identification and / or breeding of oyster mushroom varieties, wherein the oyster mushroom varieties are CCMSSC01366, CCMSSC00406, the oyster mushroom strain F2-18 of claim 1 or its progeny population, and in the identification or breeding of oyster mushroom varieties, the primer combination identifies the phenotype of oyster mushroom in different sporulation traits.

[0017] The eighth aspect of the present invention provides a method for breeding the sporozoanoid oyster mushroom strain F2-18, comprising the following steps: S1: Protoplast karyotype preparation Using the sporeless oyster mushroom cultivar CCMSSC01366 (1366) as the parent, protoplasts of this cultivar were first prepared. Through protoplast monokaryotic technology, two protoplast monokaryotic strains with different nuclei of this cultivar were obtained, one of which was 1366-104 and the other was 1366-3.

[0018] S2: Generation of F1 hybrids The monokaryotic strain 1366-104 was hybridized with the basidiospore monokaryotic strain BPC067 of the cultivated oyster mushroom CCMSSC00406 (406) to successfully obtain the F1 hybrid BPC067-104 (F1 generation). Among them, the parent strain 406 has the superior traits such as brown cap and stable characteristics.

[0019] S3: Screening and testcrossing of F1 generation hybrid progeny strains First, for the F1 generation hybrids, spores were spread on plates and individual basidiospore strains were selected. Microscopic examination of clamp connections was then performed to obtain mononuclear basidiospore strains as the segregating population. Then, using the primer combination described in section six above, the mononuclear strains in the segregating population were screened to select mononuclear basidiospore strains exhibiting asporosis. These strains were then testcrossed with another protoplast-monukaryotic strain, 1366-3, of the asporosis strain 1366.

[0020] S4: Obtain F2-18 varieties After testcrosses, the resulting population of strains underwent cultivation trials to verify the sporozoic trait. Simultaneously, comprehensive traits (including inoculation-to-harvest time (PIH), cap length (CL), cap width (CW), stipe length (SL), and stipe diameter (SD)) were measured, and new hybrid strains with comprehensive traits no less than existing commercial strains were screened. Based on the three F2 generation strains (F2-18, F2-212, and F2-192) with cultivation potential, further verification was conducted through expanded cultivation trials, successfully obtaining the new sporozoic variety F2-18 with good comprehensive traits. This variety exhibits stable sporozoic traits, high yield, and other excellent agronomic traits, demonstrating strong adaptability and suitability for widespread application under various cultivation conditions.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The new sporozoic strain F2-18 provided by this invention has been verified through multiple fruiting trials to have stable sporozoic traits. Its comprehensive traits are superior to those of the sporozoic parent strain, and its yield is significantly higher than that of the sporozoic parent strain. Moreover, compared with the sporulated parent strain, it avoids the harm caused by sporulation and has the potential for industrialized cultivation. Through farm-scale cultivation trials, compared with two parent strains, namely the sporozoic commercial strain and the sporulated commercial strain, the new hybrid strain F2-18 has a fruiting rate of 100%, stable phenotype, and consistent traits under different cultivation conditions, and has good potential for industrialized cultivation.

[0022] (2) This new oyster mushroom variety can be used as a parent for subsequent hybridization breeding, as well as for strain propagation or fruiting body production and food processing.

[0023] (3) Compared with traditional breeding, the method for selecting sporozoic strains can identify sporozoic genotypes at an early stage based on molecular markers. Screening with the aid of MSH4 molecular markers can significantly improve screening efficiency by at least 50%, avoiding large-scale fruiting experiments and arduous manual phenotypic screening, thereby shortening the breeding cycle and improving the selection efficiency.

[0024] In summary, the spore-free oyster mushroom strain F2-18 described in this invention is of great significance for enriching the resources of spore-free oyster mushroom varieties, improving the competitiveness of the edible fungi industry, and promoting the industrialized production of oyster mushrooms. Attached Figure Description

[0025] Figure 1 This is a roadmap for hybrid breeding of oyster mushrooms without spores.

[0026] Figure 2 The image shows the electrophoresis results of screening the F1 generation segregating population using molecular markers. af represents the electrophoresis results of different strains in the population, where M is the molecular weight marker, and the numbers above the image are the identification numbers of different strains in the segregating population.

[0027] Figure 3 This is a morphological diagram of the fruiting body of the sporeless strain F2-18.

[0028] Figure 4 Morphological diagram of the fruiting body of the sporeless parent 1366.

[0029] Figure 5 Morphological diagram of the fruiting body of the spore-bearing parent 406.

[0030] Figure 6 This describes the method for measuring the color of the mushroom cap.

[0031] Figure 7 The distribution of cap color for different strains.

[0032] Figure 8The fruiting body development of different strains.

[0033] Figure 9 Microscopic examination of sporulation of different strains. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The operation steps or processes not described in detail in the following embodiments are conventional techniques in the field. You can refer to relevant professional books or manuals, such as "Edible Fungi Genetics and Breeding", "Edible Fungi Cultivation (Volume 1)" (2nd Edition) and "Modern Edible Fungi Production Technology" (2nd Edition); you can also refer to relevant product instructions.

[0036] Example 1: Breeding of the non-spore-forming oyster mushroom strain F2-18 See the breeding roadmap for the spore-free oyster mushroom strain F2-18. Figure 1 The details are as follows: (1) Selection of parental strains: CCMSSC01366 (hereinafter referred to as 1366) Oyster mushroom ( Pleurotus ostreatus The variety is a commercially available, non-spore-forming strain from abroad (preserved in the China National Standard Collection of Edible Fungi Strains, CCMSSC), but compared to commercially available spore-forming strains, its commercial characteristics (such as yield and mushroom shape) are relatively inferior. (See [link to relevant documentation]). Figure 4 To further improve the yield and other commercial traits of oyster mushrooms without spores, the dark-colored and commercially viable oyster mushroom variety CCMSSC00406 (hereinafter referred to as 406, preserved in the National Edible Fungi Standard Strain Bank CCMSSC) was selected. Pleurotus cornucopiae Hybrid breeding was carried out using *Pleurotus ostreatus* strain 1366 and asporanic commercial *Pleurotus ostreatus* strain 406 as parents. See fruiting bodies for details. Figure 5 .

[0037] (2) Preparation of protoplast mononuclearization: (a) Take strain 1366 and activate it on PDA medium plates. Inoculate pea-sized pieces of bacteria onto fresh PDA plates with a diameter of 90 mm, seal them and place them in a constant temperature incubator at 25℃ for static culture.

[0038] (b) Weigh 0.1 g of lysozyme, dissolve it in 5 ml of mannitol solution (0.6 M) to prepare a 2% lysozyme solution, sterilize it through a 0.22 μm filter, and then dispense the sterile solution into three 10 ml centrifuge tubes.

[0039] (c) Scrape the mycelia obtained after static culture in step (a) and loosen them as much as possible. Transfer them to a 10 mL centrifuge tube, add the 2% lysozyme solution obtained in step (b), and incubate at 30℃ for 3.5-4 h. Invert and shake for 2 min every 30 min to promote protoplast release. After each shaking, take a small amount of solution and drop it into a hemocytometer in a clean bench for observation under a microscope. Filter the mycelial mixture solution after enzymatic hydrolysis through a syringe to remove residual mycelia, obtain a protoplast suspension, and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 2500 rpm for 10 min, discard the supernatant, and resuspend in an equal volume of osmotic buffer (0.6 M mannitol solution).

[0040] (d) Take a new centrifuge tube, add 900 μL of mannitol solution, and dilute with 100 μL of the resuspended protoplast suspension to obtain a concentration gradient of 10. ⁻¹ The protoplast solution. A concentration gradient of 10... ⁻¹ The protoplast solution was spread onto regeneration medium plates, 100-150 μL per plate. After incubation at 25℃ for 3-5 days, colony growth was observed, and single colonies were transferred to fresh PDA medium for purification. Slides were prepared for microscopic examination, and the presence or absence of clamp connections determined whether the strain was binucleate or monokaryotic. Several monokaryotic strains were randomly selected from the collected strains for antagonism experiments. Monokaryotic strains of the same strain were inoculated onto PDA medium and cultured in confrontation at 25℃. After the hyphae came into contact, hyphae at the contact site were picked and slides were prepared, and the presence of clamp connections was examined under a microscope. Finally, protoplast monokaryotic strains with two different nuclei were obtained and named 1366-104 and 1366-3, respectively.

[0041] (3) Construction of parental strain hybridization and collection of basidiospores A 5 mm diameter mycelium block of monokaryotic strain 1366-104 and a basidiospore monokaryotic strain BPC067 (Horticulturae 2023, 9(11), 1238; https: / / doi.org / 10.3390 / horticulturae 9111238) of *Pleurotus ostreatus* CCMSSC00406(406) were inoculated onto a 9.0 cm diameter PDA plate, with the two strains spaced 2 cm apart, and cultured at 25°C. Three days after the mycelia of the two strains came into contact, mycelia were collected by punching holes on both sides of the colony and at the junction, and clamp connections were observed under a 40x microscope. The presence of clamp connections indicated successful hybridization, and the F1 hybrid strain BPC067-104 was obtained.

[0042] The F1 generation hybrid strain was subjected to fruiting treatment, and the basidiospores released from the mature fruiting bodies were collected. The basidiospores were isolated by dilution and coating method to obtain a population of basidiospore mononuclear strains containing 200 strains.

[0043] (4) DNA extraction and MSH4 molecular marker amplification Basidiospore monokaryotic isolates from the F1 generation hybrid strains and mycelia of the parental strains were separately placed into sterile 2 ml centrifuge tubes and rapidly ground in liquid nitrogen. Genomic DNA was extracted using a DNA extraction kit provided by TIANGEN, following the manufacturer's instructions. To ensure the accuracy of DNA extraction and eliminate false negative results due to extraction failure, all basidiospore monokaryotic isolates were also subjected to ITS testing as an internal control marker to verify whether the extracted DNA could be used for subsequent analysis.

[0044] To identify the sporotypic genotype of the F1 generation strain population at an early stage, PCR amplification and screening were performed using the extracted DNA as a template and specific primer pairs developed based on the MSH4 gene. The primer pairs used are as follows: MSH4JGY-F: 5'-GTAGGTTTGGCAGCCCTAGATAAG-3' (sequence 1); MSH4JGY-R: 5'-TTCATCAAGGTTTTGAGCAACTTG-3' (Sequence 2).

[0045] MSH4JGY-PCR amplification reaction system: 25 μL of 2×Phanta Max Master Mix, 2 μL of primer MSH4JGT-F, 2 μL of primer MSH4JGY-R, 2 μL of DNA template, 19 μL of ddH2O, and a total volume of 50 μL.

[0046] MSH4JGY-PCR amplification reaction program: Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 2 min, 34 cycles; extension at 72℃ for 5 min, final temperature 4℃.

[0047] The amplification products were detected by agarose gel electrophoresis, and the bands were observed under a UV imaging system.

[0048] Specific primers designed based on the conserved domains of the MSH4 gene (3485 bp in length, available at: https: / / mycocosm.jgi.doe.gov / cgi-bin / dispGeneModel?db=PleosPC15_2&id=1101251) were used to amplify PCR in 125 randomly selected basidiospore isolates, screening for strains lacking the MSH4 gene (MSH4 gene deletion corresponds to an acellular phenotype and the absence of a 3485 bp PCR band). Agarose gel electrophoresis was performed on the amplification products of the MSH4JGY primer pair, and the results are as follows: Figure 2 The results showed that 53 strains failed to amplify the MSH4 gene, suggesting that these basidiospore-bearing strains may have the potential for sporulation-free traits. These 53 strains were used as candidate strains for subsequent testcrosses.

[0049] (5) Test cross and phenotypic analysis Candidate strains obtained from the previous step based on MSH4 amplification screening, namely sporolytic mononuclear strains, were selected and testcrossed with testcross strain 1366-3. A total of 19 strains were successfully testcrossed. The successfully testcrossed strains were first subjected to a laboratory-scale fruiting experiment, and phenotypic traits were recorded. The specific procedures are as follows: Nineteen F2 strains that successfully underwent testcrosses were transferred to 90 mm PDA plates for activation, and then transferred 3-5 times to prepare inoculation bottles.

[0050] For micro-fruiting in the laboratory, PC plastic bottles (diameter = 70 mm, height = 90 mm, 280 mL) were used for cultivation experiments, with three cultivation bottles for each strain. The culture medium formula was: 94% cottonseed hulls, 5% wheat bran, 1% gypsum, with a moisture content of approximately 65%. After the culture medium was prepared, it was filled into the cultivation bottles, with a wet weight of 180 g in each bottle, and sterilized for 2 hours (0.15 MPa, 126℃). Mycelium was taken from the culture medium using a 1 cm inner diameter punch, and three pieces were inoculated into each cultivation bottle. The bottles were then incubated at 25℃ in the dark until the mycelium completely covered the cultivation bottle. The cultivation bottles that had completed mycelial growth were transferred to an intelligent fruiting box, with the environmental conditions set as follows: temperature 15-18℃, air humidity maintained at 85%-95%, light intensity 300-500 lux, light duration 12 hours per day, ventilation 3-4 times a day, and carbon dioxide concentration kept below 0.1%.

[0051] The inoculation-to-harvest time (PIH) for each strain in the cultivation bottle was recorded, typically when the fruiting bodies were about 80% mature. Multiple PIH records for each strain were statistically analyzed using Genstat software. For cap color evaluation, analysis was performed immediately after harvest using a CM-700d spectrophotometer (Konica Minolta, Japan), following the instrument's operating instructions. The color parameters of the oyster mushroom caps were recorded. Color analysis generated three parameters: L, a, and b, which were then converted to a whiteness index (WI) using the formula WI = L - 3b. A higher WI value indicates a lighter cap color. The average of three measurements for each strain was used for quantitative and statistical analysis of cap color. The three sampling locations were as follows: Figure 6 As shown.

[0052] The micro-fruiting experiment was only a preliminary evaluation of the fruiting traits of the hybrids, and yield data were not collected. The results are shown in Table 1.

[0053] Table 1. Statistical results of fruiting data of 19 testcross strains from generation F2 in the laboratory. Note: In the table above, the letters in parentheses after the data represent the significance analysis results compared with other strains. The same letter between two groups indicates no significant difference (P>0.05), and completely different letters between two groups indicate a significant difference (P<0.05).

[0054] As shown in Table 1, the fruiting cycles of several hybrid strains were significantly different from those of the non-spore-bearing parent 1366 (P<0.05), with significantly shorter fruiting cycles comparable to those of the spore-bearing parent 406. Examples include hybrid strains F2-7, F2-18, F2-212, and F2-192. Regarding cap color, some hybrid strains showed significantly different cap colors from both parents, such as F2-18, F2-121, and F2-182. Other hybrid strains showed significant differences in cap color only compared to either the non-spore-bearing parent 1366 or the spore-bearing parent 406. Statistical information on cap color is as follows: Figure 7 As shown in the histogram, the WI values ​​of the 19 hybrid fruiting bodies exhibit a continuous distribution, displaying a continuous gradient from dark brown to light brown. This result indicates that cap color is a quantitative trait controlled by multiple genes and segregates during progeny formation. In the F2 generation of hybrids, 5 strains showed overparental behavior, with cap colors darker than the parental strain 406 (WI = 7.04). Furthermore, 11 strains had colors between the two parents, while 3 strains had lighter colors than the parental strain 1366 (WI = 13.39). Figure 7 As can be seen from this, the fruiting body color (WI=20.00) of strain F2-18 screened in this invention is lighter than that of parent strain 1366.

[0055] (6) Obtain varieties with cultivation potential After testing the fruiting phenotypes of the 19 F2 generation strains obtained after testcrosses, three F2 generation hybrid strains with better overall traits and potential for cultivation were selected based on three main characteristics: normal fruiting body morphology, short fruiting cycle, and differences in fruiting body color. These three strains are F2-18, F2-212, and F2-192.

[0056] Example 2 Comparison of agronomic traits of sporozoans acellularis strains and their parents under commercial cultivation conditions (1) Test strains: Oyster mushroom strain F2-18 (non-spore-forming) Sporozoite-free parental strain CCMSSC01366 (1366); The sporulated parent strain CCMSSC00406 (406); Other representative F2 generation strains obtained through screening include F2-212 and F2-192.

[0057] (2) Strain cultivation and fruiting management: (a) Strain activation and mother culture preparation The test strains were inoculated onto PDA medium plates and activated by culturing at 25°C.

[0058] The formula for PDA mother culture medium is: 200 g potato, 20 g glucose, 1 L water, 20 g agar, pH natural.

[0059] PDB mother culture medium: 200 g potato, 20 g glucose, 1 L water, natural pH, for liquid culture.

[0060] (b) Preparation of original seed Formula for culturing raw materials for branches: 89% wood branches, 10% wheat bran, 1% gypsum, and water accounting for 60% to 65% of the three raw materials by mass; The formula for the cultivation substrate is as follows: cottonseed hulls 30%, corn cobs 48%, wheat bran 18%, soybean meal 2%, gypsum 1%, lime 1%, and water accounting for 63% of the above raw materials by mass.

[0061] The bacterial culture block of the test strain was inoculated into a 200 mL PDB conical flask and cultured at 25 °C and 180 rpm for 3 days.

[0062] The original seed culture medium of branches was placed in a high-pressure resistant polypropylene bag with a specification of 10cm×30cm and sterilized at 121℃ for 2h. After sterilization, the original seed culture medium of branches was placed in a clean place and allowed to cool to room temperature. The fully expanded liquid inoculum was then inoculated onto it. The inoculated original seed culture medium of branches was then placed at 25℃ and cultured in the dark.

[0063] The cultivation substrate was placed in 10cm×30cm high-pressure resistant polypropylene bags, each weighing 1500g±100g. The bags were sterilized at 121℃ for 2 hours. After cooling to room temperature, a cutting was inserted into each bag. Fifty bags were inoculated for each variety and then placed indoors at 25℃ in the dark. Once the mycelium had fully colonized the substrate, the bags were transferred to a fruiting shed. The fruiting temperature was 8-16℃, the relative humidity was above 95%, the light intensity was 100-300 lx, and the carbon dioxide concentration was below 0.1%.

[0064] (3) Agronomic trait phenotypic determination The first flush of mushrooms was harvested when they reached 80% maturity. After harvesting, six bags of fruiting bodies (one fruiting body per bag) were randomly selected from each strain, and each bag was weighed. The weight obtained was taken as the yield per bag. The average yield per bag was then calculated and statistically analyzed (Table 2). Regarding sporulation characteristics, spore collection and microscopic examination confirmed that all three hybrids were sporozoites-free. For fruiting body characteristics, two medium-sized fruiting bodies were selected from each of the six fruiting bodies for detailed measurements. Specifically, cap length (CL), cap width (CW), stipe length (SL), and stipe diameter (SD) were measured using calipers. The average values ​​of each strain's characteristics and the differences between strains were then statistically analyzed. The results are shown in Table 2.

[0065] The fruiting experiment results showed that the fruiting cycles of the three hybrid strains were similar, consistent with those obtained in Table 1. Their cap length (CL), cap width (CW), and stipe length (SL) showed no significant differences compared to the two parents. The stipe diameter (SD) of hybrid strains F2-18 and F2-212 was close to that of the non-spore-forming parent 1366, and significantly different from that of the spore-forming parent 406. However, among the three hybrid strains, the yield per bag of F2-18 was significantly higher than that of the non-spore-forming parent, i.e., significantly higher than that of the non-spore-forming commercial strain 1366 (P < 0.05), while the yield per bag of the other two hybrid strains was either significantly lower than that of the non-spore-forming parent or showed no significant difference from the non-spore-forming parent. Morphological observation showed that the fruiting shape of F2-18 was more compact than that of 1366.

[0066] Table 2 Statistical results of agronomic phenotypic traits Note: In the table above, the data are the mean ± standard deviation of the traits. The letters in parentheses represent the significance analysis results compared with the data of other strains. The same letter between two groups indicates no significant difference (P>0.05), and completely different letters between two groups indicate significant difference (P<0.05).

[0067] For the fruiting body morphology of the tested strains, please refer to [reference needed]. Figure 8 For the sub-entity morphology of F2-18, please refer to [link / reference]. Figure 3 The microscopic results of sporulation of the tested fungal strains are as follows: Figure 9 As can be seen from the table and attached figures, after farm cultivation trials, F2-18 exhibits superior overall traits compared to the sporulated parent 1366, with a significantly increased yield. Furthermore, compared to the sporulated parent, it maintains its compact fruiting shape, characteristic of commercially viable products. Multiple cultivation trials have verified that the fruiting rate is 100%, and the sporulation-free and other commercial traits remain stable.

[0068] In conclusion, F2-18 (CGMCC No. 42585) is a sporozoan-free oyster mushroom variety with potential for industrial cultivation. The methods and techniques used in the breeding of this strain are of great significance for enriching breeding technology reserves, expanding the product categories in the edible fungi market, and enhancing the competitiveness of edible fungi germplasm.

Claims

1. A sporozoan oyster mushroom strain F2-18, characterized in that, The asporophyte-free oyster mushroom strain F2-18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42585 and is classified as *Pleurotus ostreatus*. Pleurotus ostreatus .

2. A type of oyster mushroom mycelium, characterized in that, It was obtained by growing and developing the sporeless oyster mushroom strain F2-18 as described in claim 1.

3. A fruiting body of *Pleurotus ostreatus*, characterized in that, It was obtained by growing and developing the sporeless oyster mushroom strain F2-18 as described in claim 1.

4. The use of the non-spore-forming oyster mushroom strain F2-18 of claim 1, the mycelium of claim 2, or the fruiting body of claim 3 in at least one of the following (A) to (D): (A) Its application as a parent in hybridization breeding; (B) Application in strain propagation; (C) Application in sub-entity production; (D) Applications in food processing.

5. The method for cultivating the aspora oyster mushroom strain F2-18 according to claim 1, characterized in that, Includes the following steps: S1: Activate the non-spore-forming oyster mushroom strain F2-18 on PDA medium or PDB liquid medium to prepare the mother culture; S2: The mother seed is inoculated into the branch seed culture medium to obtain the branch seed; S3: Inoculate the original spawn into the culture medium of the cultivation bag and cultivate it in the dark at room temperature, with a relative humidity of 60%-70% and a carbon dioxide concentration of less than 0.3%. Fruiting can begin after the mycelium has fully grown into the culture bag. S4: During the fruiting stage, the temperature is 816℃, the relative humidity is not less than 95%, the light intensity is 100-300 lx, and the carbon dioxide concentration is less than 0.1%.

6. The cultivation method according to claim 5, characterized in that: The PDA culture medium comprises, based on the amount of raw materials added per liter of water: 200g potato, 20g glucose, 1L water, and 20g agar. And / or, based on the amount of raw materials added per liter of water, the PDB liquid culture medium comprises: 200g of potato, 20g of glucose, and 1L of water; And / or, by weight percentage, the original seed culture medium of the branches comprises the following raw materials: 80-90% wood branches, 8-15% wheat bran, 1-2% gypsum, and water accounts for 60%-65% of the raw materials by weight. And / or, by weight percentage, the cultivation substrate comprises the following raw materials: 25-35% cottonseed hulls, 40-55% corn cobs, 15-20% wheat bran, 1-3% soybean meal, 1-2% gypsum, 1-3% lime, and water accounts for 60%-65% of the raw materials by weight. And / or, the room temperature is 25°C.

7. The cultivation method according to claim 6, characterized in that: According to the mass percentage, the original seed culture medium of the branches includes the following raw materials: 89% wood branches, 10% wheat bran, 1% gypsum, and water accounts for 60%-65% of the mass percentage of the raw materials; The cultivation substrate comprises the following raw materials by weight percentage: 30% cottonseed hulls, 48% corn cobs, 18% wheat bran, 2% soybean meal, 1% gypsum, and 1% lime, with water accounting for 63% of the raw materials by weight.

8. A primer combination for identifying different phenotypes of sporulation traits in *Pleurotus ostreatus* strains, characterized in that, The primer combination is a primer pair consisting of the nucleotide sequences shown in Sequence 1 and Sequence 2 in the sequence listing, and the oyster mushroom strain is CCMSSC01366, CCMSSC00406, the sporeless oyster mushroom strain F2-18 of claim 1, or its progeny population.

9. The application of the primer combination described in claim 8 in the identification or breeding of oyster mushroom varieties, characterized in that, The oyster mushroom varieties are CCMSSC01366, CCMSSC00406, the spore-free oyster mushroom strain F2-18 as described in claim 1, or their progeny populations. In oyster mushroom variety identification or oyster mushroom breeding, the primer combination is used to identify the phenotype of oyster mushrooms in different sporulation traits.

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