Early-maturing flammulina velutipes variety and application thereof in flammulina velutipes breeding

Abstract

The invention discloses an early-maturing flammulina velutipes variety and application thereof in flammulina velutipes breeding, and belongs to the technical field of flammulina velutipes molecular breeding. According to the invention, a new germplasm is created by using a CRISPR-Cas9 gene editing technology, and the fruiting phenotype of the strain is finally changed by changing a key gene by using the gene editing technology, so that a new variety 'Shangjin No.2' is created. The new variety at least has the following advantages: the yield is high; stipes are long, pilei are internally buckled and thick, and the appearance quality is good; the health-care food has the characteristics of high asparagine content, high glutamine content, high polysaccharide content and high cis-linoleic acid content, and is high in nutritional value; the method has the advantages of short growth period and prematurity. According to the invention, a domestic leading edible mushroom precise breeding technology platform is established, and the method has important strategic significance for improving the core competitiveness of the edible mushroom industry in China.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology of enoki mushrooms, and particularly relates to an early-maturing enoki mushroom variety and its application in enoki mushroom breeding. Background Technology

[0002] Enoki mushrooms are not only nutritious, with a crisp and tender texture and delicious taste, but they also possess medicinal value, such as promoting intellectual development and anti-tumor properties. However, the high degree of homogenization in enoki mushroom products has led to a gradual decline in profitability, thus limiting the industry's development. Enoki mushroom breeding methods typically involve protoplast monokaryotic hybridization, but due to the severe homogenization of existing enoki mushroom varieties, it is difficult to find strains with significant genetic differences for hybridization. Therefore, traditional single-strain hybridization yields enoki mushroom varieties with little difference from their parents, making it difficult to develop new strains with significant breakthroughs in phenotype and genetic background.

[0003] Genetic modification of existing germplasm resources using genetic engineering techniques to obtain new enoki mushroom germplasm, followed by hybridization using this new germplasm as a parent, is an effective way to create new enoki mushroom varieties. However, research on this technology in the breeding of new enoki mushroom varieties is still limited. Summary of the Invention

[0004] Based on the above situation, this invention addresses the existing problems of long growth cycle and high production capacity consumption of enoki mushrooms in industrialized production, the low amino acid content of existing main cultivated varieties, and the defects of enoki mushroom varieties on the market such as easy cap opening and thin caps. It provides an early-maturing enoki mushroom (… Flammulina filiformis The “Shangjin No. 2” variety and its application in the breeding and cultivation of enoki mushrooms. “Shangjin No. 2” has a high yield, a short growth period, a long stipe, and a thick, inwardly curled cap. It also has high levels of asparagine and glutamine, making it a valuable variety for applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide an early-maturing enoki mushroom (… Flammulina filiformis The variety has the accession number GDMCC No: 67007.

[0006] Early-maturing enoki mushrooms in this invention Flammulina filiformis The “Shangjin No. 2” variety was deposited on September 22, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology, with accession number GDMCC No: 67007.

[0007] Furthermore, the early-maturing enoki mushroom variety was obtained by hybridizing a gene-mutated monokaryotic strain transformant with a protoplast monokaryotic cell of the "Shangyan No. 1" strain.

[0008] Furthermore, the hybridization includes the steps of single-to-single hybridization pairing of protoplast mononuclear cells and microscopic examination, wherein the strains with clamp connections are the successfully hybridized strains.

[0009] Furthermore, the gene-mutated mononuclear strain transformants were obtained using CRISPR-Cas9 gene editing technology.

[0010] Furthermore, the gene-mutated mononuclear strain transformants are transformants with loss of function of the FfCry-DASH gene.

[0011] Furthermore, the gene-mutated mononuclear strain transformant was obtained by knocking out the FfCry-DASH gene in the *Flammulina velutipes* genome using the pyrG-deficient Dan3 strain via RNP+DonorDNA. The core advantage of RNP+DonorDNA technology is that it does not introduce any exogenous fragments, achieving truly seamless editing.

[0012] The second objective of this invention is to provide the application of the aforementioned early-maturing enoki mushroom variety in food production.

[0013] Furthermore, the food contains any one or more of asparagine, glutamine, enoki mushroom polysaccharide, and cis-linoleic acid.

[0014] The third objective of this invention is to provide the application of the aforementioned early-maturing enoki mushroom variety in enoki mushroom breeding or cultivation.

[0015] Furthermore, the applications include the cultivation or planting of new strains of high-yield enoki mushrooms, early-maturing enoki mushrooms, long-stemmed enoki mushrooms, enoki mushrooms with inward-curling caps, and enoki mushrooms with thick caps.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes CRISPR-Cas9 gene editing technology to create a new germplasm and, by altering a key gene, ultimately changes the fruiting phenotype of the strain, creating the new variety "Shangjin No. 2". The core advantage of the RNP+DonorDNA technology used is that it does not introduce any exogenous fragments, achieving truly traceless editing. The new variety possesses at least the following advantages: high yield; long stipe, thick and inwardly curled cap, and excellent appearance; high asparagine, glutamine, polysaccharide, and cis-linoleic acid content, resulting in high nutritional value; and a short growth cycle, exhibiting early maturity. This invention establishes a leading precision breeding technology platform for edible fungi in China, which is of significant strategic importance for enhancing the core competitiveness of my country's edible fungi industry.

[0017] Certificate of Preservation for "Shangjin No. 2": Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; Accession number: GDMCC No: 67007; Deposit date: September 22, 2025; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Taxonomic nomenclature: Flammulina filiformis .

[0018] Certificate of Preservation for "Shangyan No. 1": Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; Accession number: GDMCC No: 61490; Date of deposit: January 22, 2021; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Taxonomic nomenclature: Flammulina filiformis . Attached Figure Description

[0019] Figure 1 This is a comparison of the growth of "Shangjin No. 2" with its parent and control samples at 22 days of cultivation in Example 3 of the present invention.

[0020] Figure 2 This is a visual comparison of the appearance, stem, and cap of "Shangjin No. 2" with its parent and control samples in Example 3 of the present invention.

[0021] Figure 3 The ratio of yield, stipe length, and cap length of "Shangjin No. 2" to its parent and control samples in Example 3 of this invention. Detailed Implementation

[0022] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. Unless otherwise specified, the products and equipment used in the following embodiments are commercially available, and the methods used are consistent with conventional methods unless otherwise specified.

[0023] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0024] Example 1: Obtaining mononuclear KO transformants Using the existing pyrG-deficient Dan3 strain (named ΔDan3) in the laboratory, the FfCry-DASH gene in the *Flammulina velutipes* genome was knocked out using CRISPR-Cas9 gene editing technology via RNP+DonorDNA, resulting in a transformant (named KO) with FfCry-DASH gene loss of function. The specific preparation process is as follows: 1. Design of Donor DNA To achieve the simultaneous knockout of the FfCry-DASH gene and the reinstatement of the pyrG gene via homologous recombination, a DonorDNA design is required. Specifically, this design involves adding 1000 bp homologous arms of the FfCry-DASH gene upstream and downstream of the pyrG gene. These FfCryDASH gene homologous arms are sequences located upstream and downstream of the sgRNA target site.

[0025] 2. Preparation of liquid bacterial strains Take the pyrG-deficient Dan3 strain of *Flammulina velutipes* (named ΔDan3) stored at 4℃, place it in a 25℃ incubator for 24 h to recover and culture, discard the front tissue block (approximately 50 mm) of the pyrG-deficient strain mother culture, and take a 0.5 cm piece. 2 Inoculate the mycelial blocks onto PDA agar plates (100 mg / L uridine, 50 mg / L uracil) and incubate at 25°C until the mycelium completely covers the PDA agar plate. Take the activated mycelial blocks from the plates, punch holes with a 5 mm punch, and inoculate the punched mycelial blocks into 9 cm diameter PDA agar plates (100 mg / L uridine, 50 mg / L uracil). Incubate at 25°C for ten days. Remove the original mycelial blocks from the center, and pick out the remaining mycelial blocks to homogenize them in a homogenizer (pre-fill the homogenizer with 100 ml of PDB agar) to prepare liquid inoculum. Inoculate 10 mL of the liquid inoculum into 100 mL of PDB agar and incubate at 25°C, shaking manually three times a day (morning, noon, and evening). After 3 days, homogenize again and inoculate 10 mL of the homogenate into 100 mL of PDB agar.

[0026] 3. Preparation of protoplasts Collect the liquid mycelia from the previous step, wash with 0.6 M mannitol, and digest with 2% (w / v) lysozyme for 90 minutes. After filtering out the incompletely digested mycelia, resuspend the protoplasts in 1× MTC buffer (0.6 M mannitol, 100 mM CaCl2, 100 mM Tris-HCl, pH 7.5).

[0027] 4. PEG-mediated protoplast transformation 30 µL of protoplasts (10 610 µL RNPs complex (300 nM), 5 µL Donor DNA, 1 µL Triton X-100 [final conversion concentration 0.01% (w / v)], 10 µL 10 × Cas9 nuclease reaction buffer, 31.5 µL 2 × MTC buffer, and 12.5 µL PTC buffer (60% polyethylene glycol (PEG) 4000, 100 mM CaCl2, and 10 mM Tris-HCl; pH 7.5) were mixed. The mixture was placed on ice for 20 minutes, then 500 μL of PTC buffer was added, and the mixture was incubated at 20 °C for 90 minutes. Then, 1 mL of 1 × MTC buffer and 1.6 mL of resuscitation medium (200 g / L potato starch, 20.0 g / L glucose, 109.3 g / L mannitol) were added, and the mixture was incubated at 20 °C for 24 h. Finally, 3.6 mL of the upper culture medium (200 g / L potato starch, 20.0 g / L glucose, 20.0 g / L low melting point agarose, 109.3 g / L mannitol) was added to the prepared lower culture medium (200 g / L potato starch, 20.0 g / L glucose, 20.0 g / L agarose, 109.3 g / L mannitol).

[0028] 5. Screening and identification of transformants Hyphae of transformants with clean white color and normal growth were selected for colony PCR detection. Using FfCry1F / 1R, FfpyrG-4F / 4R, and FfCry-4F / 4R as primers, the pyrG fragment and Cry-DASH fragment containing the target site in all transformants were amplified by PCR. The amplified products were detected by 1% agarose gel electrophoresis. The PCR amplified products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were compared.

[0029] Sequencing results showed that, compared with the control, the FfCry-DASH gene of one transformant was cleaved at the target site, with the last 98 bases missing. At the same time, the pyrG gene sequencing results showed that the pyrG gene of the transformant had been replenished. The transformant was named KO strain.

[0030] The above information can be found in the Master's thesis of Shanghai Ocean University: Study on FfCry-DASH gene of blue light receptor cryptochrome in enoki mushroom, by Li Jingting, or in patent: 202210466588.4 (including the relevant sequences used).

[0031] Example 2: "Shangjin No. 2" was obtained by hybridization of KO with the protoplast mononuclear cells of "Shangyan No. 1". Twenty monokaryotic strains in the laboratory were hybridized with KO (a gene mutant strain obtained by knocking out the FfCry-DASH gene from monokaryotic Dan3 strain through CRISPR-Cas9 gene editing technology) and Dan3 monokaryotic strains that had not undergone gene editing. The hybridized strains were examined under a microscope, and those with clamp connections were considered to have successfully hybridized. A total of 9 pairs of hybrids were successfully hybridized.

[0032] Nine pairs of hybrid strains were used for fruiting experiments in bag cultivation. Each bag contained approximately 950 g of wet substrate with a moisture content of around 65% and a natural pH. Forty-eight bags of each strain were cultivated. During the mycelium growth stage, the bags were incubated in the dark at a temperature of 14-19℃ and humidity maintained above 85%. After mycelium removal, the bags were transferred to a fruiting cultivation room for fruiting management, and fruiting characteristics and yield statistics were recorded. The fruiting experiment was repeated three times.

[0033] The fruiting phenotypes of the nine hybrid combinations showed significant differences. Among them, the combinations that were hybridized with the protoplast mononuclear strain of "Shangyan No. 1" (KO×Shangyan No. 1 and Dan3×Shangyan No. 1) showed the greatest differences (measured in terms of primordia emergence time, stipe growth rate, harvest time, stipe length, etc.). KO×Shangyan No. 1 was named "Shangjin No. 2", and Dan3×Shangyan No. 1 was named the control sample.

[0034] Example 3: Quality Inspection of "Shangjin No. 2" 1. Comparison of growth of "Shangjin No. 2" with its parents and control samples at 22 days of culture. Figure 1 As shown, a direct comparison of the appearance, stem, and cap of "Shangjin No. 2" with its parent and control samples is presented. Figure 2 As shown, the yield, stipe length, and cap length of "Shangjin No. 2" are compared with those of its parents and control samples. Figure 3 As shown, it is clear that "Shangjin No. 2" has a higher yield. At the same time, "Shangjin No. 2" has a significantly longer stem, a thicker, inward-curving cap, and a better appearance.

[0035] 2. The content of each amino acid in "Shangjin No. 2" and the control sample was detected, and the results are shown in Table 1.

[0036] Table 1. Amino acid content (ug / g)

[0037] The test results above show that the L-glutamine content in "Shangjin No. 2" was 893.3816 ug / g, which was 269.329 ug / g higher than the control sample; the L-asparagine content was 227.5989 ug / g, which was 61.83 ug / g higher than the control sample; the aspartic acid content was 192.1473 ug / g, which was 41.3478 ug / g higher than the control sample; the glutamic acid content was 382.8103 ug / g, which was 26.37851 ug / g higher than the control sample; and the alanine content was 149.3879 ug / g, which was 25.82 ug / g higher than the control sample.

[0038] 3. The polysaccharide content in "Shangjin No. 2" and the control sample was detected. The results showed that the polysaccharide content of "Shangjin No. 2" was 433.34 (mg / g), while the polysaccharide content of the control sample was 344.80 (mg / g), indicating that "Shangjin No. 2" had a significant effect in increasing the polysaccharide content.

[0039] 4. The content of each fatty acid in "Shangjin No. 2" and the control sample was tested, and the test results are shown in Table 2.

[0040] Table 2 Fatty acid content (%)

[0041] The test results above show that the cis-linoleic acid content in "Shangjin No. 2" was the most significantly increased, rising by 8.044% compared to the control sample. Cis-linoleic acid can promote the growth of edible fungi mycelium and significantly promote the synthesis of beneficial metabolites. Exogenous addition of cis-linoleic acid can promote the synthesis of intracellular polysaccharides in enoki mushrooms. The accumulation of cis-linoleic acid can regulate cell membrane fluidity.

[0042] 5. In addition, observation of the growth cycle of "Shangjin No. 2" revealed that it could be harvested on the 22nd day of cultivation, while the parent could be harvested on the 28th day and the control sample could be harvested on the 27th day. Its growth cycle was shortened by 6 days compared with the parent and 5 days compared with the control sample.

[0043] In summary, this invention utilizes gene editing technology to create a new germplasm and, by altering a key gene, ultimately changes the fruiting phenotype of the strain, creating the new variety "Shangjin No. 2". This new variety possesses at least the following advantages: higher yield; longer stipe, thicker, inwardly curled cap, and superior appearance; high asparagine, glutamine, polysaccharide, and cis-linoleic acid content, resulting in high nutritional value; and a short growth cycle, exhibiting early maturity. This invention establishes a leading precision breeding technology platform for edible fungi in China, which is of significant strategic importance for enhancing the core competitiveness of my country's edible fungi industry.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An early-maturing enoki mushroom ( Flammulina filiformis ) variety, characterized in that, Its accession number is GDMCCNo: 67007.

2. The early-maturing enoki mushroom variety according to claim 1, characterized in that, It was obtained by hybridizing the gene-mutated monokaryotic strain transformant with the protoplast monokaryotic body of the "Shangyan No. 1" strain.

3. The early-maturing enoki mushroom variety according to claim 2, characterized in that, The hybridization includes the steps of single-to-single hybridization pairing of protoplast mononuclear cells and microscopic examination, wherein the strains with clamp connections are the successfully hybridized strains.

4. The early-maturing enoki mushroom variety according to claim 3, characterized in that, The gene-mutated mononuclear strain transformants were obtained using CRISPR-Cas9 gene editing technology.

5. The early-maturing enoki mushroom variety according to claim 4, characterized in that, The gene-mutated mononuclear strain transformants are transformants with loss of function of the FfCry-DASH gene.

6. The early-maturing enoki mushroom variety according to claim 5, characterized in that, The gene-mutated mononuclear strain transformant was obtained by knocking out the FfCry-DASH gene in the genome of *Flammulina velutipes* using the pyrG-deficient Dan3 strain via RNP+DonorDNA.

7. The application of the early-maturing enoki mushroom variety according to claim 1 in food production.

8. The application according to claim 7, characterized in that, The food contains any one or more of asparagine, glutamine, enoki mushroom polysaccharide, and cis-linoleic acid.

9. The application of the early-maturing enoki mushroom variety according to claim 1 in enoki mushroom breeding or cultivation.

10. The application according to claim 9, characterized in that, The applications include the cultivation or planting of new strains of high-yield enoki mushrooms, early-maturing enoki mushrooms, long-stemmed enoki mushrooms, enoki mushrooms with inward-curling caps, and enoki mushrooms with thick caps.

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