Early-matured yellow-flavor enoki mushroom variety and application thereof in enoki mushroom breeding

Abstract

The application discloses an early-maturing yellow Flammulina velutipes variety and application thereof in Flammulina velutipes breeding, and belongs to the technical field of Flammulina velutipes molecular breeding. The application creates a new germplasm by using CRISPR-Cas9 gene editing technology, and finally changes the fruiting phenotype of the strain by changing a key gene by using the gene editing technology to create a new variety "Shangjin No. 3". The new variety has at least the following advantages: high yield; long stem, golden yellow color, and better appearance quality; the content of up to 19 kinds of amino acids is improved, especially the content of lysine, asparagine and glutamine is extremely significantly improved, and the content of polysaccharide and linoleic acid is also obviously improved, and the nutritional value is very high; short growth cycle, and early-maturing advantage. The application establishes a domestic leading edible mushroom precision breeding technology platform, and has great 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 yellow 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 content of amino acids, polysaccharides, and cis-linolenic acid in existing main cultivated varieties, and the lack of yellow enoki mushroom varieties on the market. It provides an early-maturing yellow enoki mushroom (… Flammulina filiformis The "Shangjin No. 3" variety and its application in enoki mushroom breeding and cultivation. The "Shangjin No. 3" variety has a high yield, a short growth period, a long stem, and a golden color. It also boasts high levels of up to 19 amino acids, as well as high levels of polysaccharides and cis-linolenic acid, making it highly valuable 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 yellow enoki mushroom (… Flammulina filiformis The variety has the accession number GDMCC No: 67008.

[0006] Early-maturing enoki mushrooms in this invention Flammulina filiformis The “Shangjin No. 3” 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: 67008.

[0007] Furthermore, the early-maturing yellow enoki mushroom variety was obtained by hybridizing a gene-mutated monokaryotic strain transformant with a protoplast monokaryotic cell of the "J4137" strain.

[0008] The parent variety “J4137” used in this invention is a wild-domesticated and selected variety from the Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences, with the preservation number GDMCC No: 61530.

[0009] 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.

[0010] Furthermore, the gene-mutated mononuclear strain transformants were obtained using CRISPR-Cas9 gene editing technology.

[0011] Furthermore, the gene-mutated mononuclear strain transformants are transformants with loss of function of the FfCry-DASH gene.

[0012] 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.

[0013] The second objective of this invention is to provide the application of the aforementioned early-maturing yellow enoki mushroom variety in food production.

[0014] Furthermore, the food contains any one or more of the following: lysine, asparagine, glutamine, alanine, glutamic acid, aspartic acid, valine, arginine, leucine, isoleucine, serine, threonine, tyrosine, phenylalanine, glycine, tryptophan, proline, methionine, citrulline, enoki mushroom polysaccharide, and cis-linoleic acid.

[0015] The third objective of this invention is to provide the application of the aforementioned early-maturing yellow enoki mushroom variety in enoki mushroom breeding or cultivation.

[0016] Furthermore, the applications include the cultivation or planting of new strains of yellow enoki mushrooms, high-yield enoki mushrooms, early-maturing enoki mushrooms, and long-stemmed enoki mushrooms.

[0017] 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. 3". Furthermore, the core advantage of RNP+DonorDNA technology is that it does not introduce any exogenous fragments, achieving truly traceless editing. The new variety possesses at least the following advantages: higher yield; longer stipes, golden color, and superior appearance; increased content of up to 19 amino acids, especially lysine, asparagine, and glutamine, with significant increases; and also noticeable increases in polysaccharide and cis-linoleic acid content, resulting in very high nutritional value; short growth cycle and early maturity. This invention establishes a leading domestic precision breeding technology platform for edible fungi, which is of great strategic significance for enhancing the core competitiveness of my country's edible fungi industry.

[0018] Certificate of Preservation for "Shangjin No. 3": Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; Accession number: GDMCC No: 67008; Deposit date: September 22, 2025; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Taxonomic nomenclature: Flammulina filiformis .

[0019] Certificate of Preservation for “J4137”: Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; Accession number: GDMCC No: 61530; Date of deposit: February 8, 2021; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Taxonomic nomenclature: Flammulina filiformis . Attached Figure Description

[0020] Figure 1 The images show the morphological characteristics of the fruiting bodies of "Shangjin No. 3", the parent "J4137", and the control sample on day 10 of culture in Example 3 of this invention.

[0021] Figure 2 The images show the fruiting body morphology of "Shangjin No. 3", its parent "J4137", and the control sample during the harvesting period in Example 3 of this invention.

[0022] Figure 3 This is a comparison of the single-bottle yield, cap diameter, and stipe length of "Shangjin No. 3" in Example 3 of the present invention with the parent "J4137" and the control sample. Detailed Implementation

[0023] 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.

[0024] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0025] 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.

[0026] 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.

[0027] 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).

[0028] 4. PEG-mediated protoplast transformation 30 µL of protoplasts (10 6 10 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).

[0029] 5. Screening and identification of transformants White, normally growing hyphae of the transformants were selected for colony PCR detection. Using primers FfCry1F / 1R, FfpyrG-4F / 4R, and FfCry-4F / 4R, the pyrG fragment and FfCry-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.

[0030] 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.

[0031] 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).

[0032] Example 2: "Shangjin No. 3" was obtained by hybridization of KO with the protoplast mononuclear strain of "J4137". Twenty existing monokaryotic strains of *Flammulina velutipes* in the laboratory were hybridized with KO (a gene mutant strain obtained by knocking out the FfCry-DASH gene from the monokaryotic Dan3 strain through CRISPR-Cas9 gene editing technology) and unedited Dan3 monokaryotic strains. 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 hybridized strains were successfully hybridized.

[0033] 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.

[0034] The fruiting phenotypes of the nine hybrid combinations showed significant differences. Among them, the combinations that were hybridized with the protoplast mononuclear strain of "J4137" (KO×J4137 and Dan3×J4137) showed the greatest differences (measured in terms of primordia emergence time, stipe growth rate, harvest time, stipe length, etc.). KO×J4137 was named Shangjin No. 3, and Dan3×J4137 was named the control sample.

[0035] Example 3: Quality Inspection of "Shangjin No. 3" 1. Morphological images of the fruiting bodies of “Shangjin No. 3”, its parent “J4137”, and control samples on day 10 of culture are shown in the figure. Figure 1 As shown, the fruiting body morphology of "Shangjin No. 3", its parent "J4137", and the control sample during the harvest period are illustrated in the following figures. Figure 2 As shown, the data on single-bottle yield, cap diameter, and stipe length of "Shangjin No. 3" are compared with those of its parent "J4137" and control samples. Figure 3 As shown.

[0036] The results showed that the average yield per bottle of "Shangjin No. 3" was 150.40 g higher than that of the parent strain and 171.59 g higher than that of the control sample. The stipe length of "Shangjin No. 3" was 8.06 cm longer than that of the parent strain and 9.14 cm longer than that of the control sample. The cap diameter of "Shangjin No. 3" was 0.39 cm larger than that of the parent strain and 0.005 cm smaller than that of the control sample. Furthermore, "Shangjin No. 3" had a golden-yellow color and significantly better appearance.

[0037] 2. The content of each amino acid in "Shangjin No. 3" and the control sample was detected, and the results are shown in Table 1.

[0038] Table 1. Amino acid content (ug / g)

[0039] The test results above show that the lysine content of "Shangjin No. 3" was 110.61 ug / g higher than the control sample, the asparagine content was 95.50 ug / g higher, the glutamine content was 84.15 ug / g higher, the alanine content was 83.37 ug / g higher, the glutamic acid content was 63.12 ug / g higher, the aspartic acid content was 62.30 ug / g higher, the valine content was 50.89 ug / g higher, the arginine content was 49.24 ug / g higher, the leucine content was 45.76 ug / g higher, the isoleucine content was 35.92 ug / g higher, the serine content was 31.95 ug / g higher, the threonine content was 23.54 ug / g higher, and the tyrosine content was 23.50 ug / g higher. The content of phenylalanine, glycine, tryptophan, proline, methionine, and citrulline was 11.44 ug / g higher than the control sample, 9.45 ug / g higher, 2.65 ug / g higher, and 1.053 ug / g higher than the control sample. The content of most amino acids in "Shangjin No. 3" was increased, especially lysine and asparagine.

[0040] 3. The polysaccharide content in "Shangjin No. 3" and the control sample was detected. The results showed that the polysaccharide content in "Shangjin No. 3" was 324.33 mg / g, while that in the control sample was 197.17 mg / g. The polysaccharide content in the new enoki mushroom variety "Shangjin No. 3" was 127.16 mg / g higher than that in the control sample.

[0041] 4. The content of each fatty acid in "Shangjin No. 3" and the control sample was tested, and the test results are shown in Table 2.

[0042] Table 2 Fatty acid content (%)

[0043] The test results above show that the cis-linoleic acid content in "Shangjin No. 3" is the most significantly increased, being 8.909% higher than that of the control sample.

[0044] 5. In addition, observation of the growth cycle of "Shangjin No. 3" revealed that it could be harvested on the 22nd day of cultivation, while the parent and control samples could only be harvested on the 28th day. Its growth cycle was 6 days shorter than that of the parent and control samples.

[0045] In summary, this invention utilizes gene editing technology to create a new germplasm and, through CRISPR-Cas9 gene editing technology, alters a key gene to ultimately change the fruiting phenotype of the strain, creating the new variety "Shangjin No. 3". This new variety possesses at least the following advantages: higher yield; longer stipe, golden color, and superior appearance; increased content of up to 19 amino acids, especially lysine, asparagine, and glutamine, with significant increases; simultaneously, the polysaccharide and cis-linoleic acid content are also significantly improved, resulting in very high nutritional value; and a short growth cycle with early maturity. This invention establishes a leading precision breeding technology platform for edible fungi in China, which is of great strategic significance for enhancing the core competitiveness of my country's edible fungi industry.

[0046] 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 yellow enoki mushroom ( Flammulina filiformis ) variety, characterized in that, Its accession number is GDMCC No: 67008. It was obtained by hybridizing the gene-mutated monokaryotic strain transformant with the protoplast monokaryotic body of the "J4137" strain. The gene-mutated monokaryotic strain transformant was obtained by knocking out the FfCry-DASH gene in the genome of *Flammulina velutipes* using the pyrG-deficient Dan3 strain in the RNP+DonorDNA manner.

2. The early-maturing yellow enoki mushroom variety according to claim 1, 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.

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

Images