Lentinula edodes strain fx85 and breeding method and application thereof

Abstract

The application discloses a lentinula edodes strain FX85 and a breeding method and application thereof, the lentinula edodes strain FX85 has been preserved in the China Center for Type Culture Collection on December 3, 2024, and the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20242713. The average yield of fresh mushrooms of the FX85 obtained by the breeding is 912 g / stick, which is 8.07 % higher than that of a control variety new 808, the polysaccharide content of the strain is increased by 8.75 % compared with that of the strain new 808, the protein content is increased by 21.57 %, the cap and handle ratio is increased by 13.77 % compared with that of the strain new 808, and the agronomic characters and yield are more advantageous than those of the strain new 808, and the high-yield lentinula edodes variety obtained by the application has popularization value. The high-yield lentinula edodes variety obtained by the application can enrich the existing lentinula edodes germplasm resource bank, help to adjust the lentinula edodes planting structure and improve the lentinula edodes planting benefit.

Description

Technical Field

[0001] This invention belongs to the field of mushroom technology, specifically relating to the shiitake mushroom strain FX85 and its breeding methods and applications. Background Technology

[0002] mushroom( Lentinus edodes (Berk.) sing) belongs to the class Basidiomycetes in taxonomy ( Basidaiomycetes ), Agaricalales ( Agaricales ), Small Bark Ferns ( Marasmiaceae ), Lentinus genus ( Lentinula It is the world's second largest edible fungus, with a wide cultivation area, rich nutrition, delicious taste, and high medicinal and economic value. It is a highly valuable medicinal and edible fungus.

[0003] Currently, there are many known varieties of shiitake mushrooms, such as Shiitake 9608, Shiitake K5, Shiitake 939, Shiitake 808, Shiitake A6, Shiitake 238, Shiitake 241-4, Shiitake 303, Luyu No. 6 Shiitake, Huxiang F2, Shenxiang 1504 and Shenxiang 215. Different varieties of shiitake mushrooms have different mushroom shapes and nutritional contents.

[0004] Breeding high-yield, high-nutritional-value shiitake mushroom strains can enrich the shiitake mushroom germplasm resource bank, help adjust the shiitake mushroom industry structure, and improve planting efficiency.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide the FX85 strain of shiitake mushroom, its breeding method and application. This strain has the characteristics of high yield and large cap-to-stem ratio, which can enrich the existing shiitake mushroom germplasm resource bank, help adjust the shiitake mushroom planting structure and improve the shiitake mushroom planting benefits.

[0007] To achieve the above objectives, the present invention provides a shiitake mushroom strain ( Lentinus edodes FX85, the shiitake mushroom strain FX85, was deposited on December 3, 2024 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242713.

[0008] The mycelium of the shiitake mushroom FX85 of this invention is dense and vigorous, pure white in color, and its optimal growth temperature is 25 ℃. It exhibits strong heat resistance and contamination resistance, with early and uniform color change, and a deeper color change in the substrate. The primordia development time is 4-6 days earlier than the control variety, Xin 808. The fruiting bodies of shiitake mushroom FX85 are medium to large in size, hemispherical, and relatively round. The average yield of fresh mushrooms from strain FX85 is 912 g / substrate, which is 8.07% higher than the control variety Xin 808. The cap is brown, convex in longitudinal section, darker in the center and lighter at the edge. The cap diameter of strain FX85 is 4.55±0.09 cm, convex, and 1.58±0.06 cm thick, brown in color, with moderate firmness; dark scales are evenly distributed on the cap edge, and the flesh is white, possessing the characteristic aroma of shiitake mushrooms. The gills are off-white. The stipe is 3.38±0.25 cm long and 1.63±0.08 cm thick, white, and of uniform thickness from top to bottom, nearly cylindrical. The cap-to-stem ratio (the ratio of cap diameter to stipe diameter) of FX85 is 2.81±0.11, while that of Xin 808 is 2.47±0.24. FX85's cap-to-stem ratio is 13.77% higher than Xin 808's. In summary, FX85 and Xin 808 differ in yield, cap thickness, and stipe length-to-diameter ratio. FX85 has a larger cap-to-stem ratio and higher yield, reflecting the specificity of the FX85 strain.

[0009] A second objective of this invention is to provide a method for breeding the aforementioned shiitake mushroom strain FX85, the method comprising the following steps:

[0010] 1) Dilute the spores of the first flush of superior fruiting bodies of Shenxiang 215 and Huxiang F2 with sterile water, take the spore suspension and spread it evenly on PDA medium plates, and incubate at a constant temperature of 25±2 ℃. Select monokaryotic mycelia without clamp connections; wherein, the superior fruiting body spores are uniform in shape and size, smooth in surface, without wrinkles or spots, and the spores are free from deformities and defects;

[0011] 2) The selected monokaryotic mycelia of Shenxiang 215 and Huxiang F2 were inoculated onto PDA plates with a 3 cm gap between the two monokaryotic mycelia and cultured at 25±2 ℃. When a fusion line was formed between the two colonies, the mycelia in the middle of the fusion line were taken for microscopic examination and the binkaryotic mycelia with clamp connections were selected for culture.

[0012] 3) After the heterozygotes are converted into the controlled original strain, they are placed in the mushroom bags for fruiting experiments to screen out hybrid strains with good mushroom shape and dense fruiting; good mushroom shape means that the shiitake mushrooms are of uniform size and free from damage, defects and deformities.

[0013] 4) Conduct fruiting tests on the screened hybrid strains, collect the first flush of fruiting bodies and test the specificity, uniformity and stability of the control shiitake mushrooms, and screen out the target dominant strains.

[0014] Preferably, in step 1), the spore content in the spore suspension is 10. 5 per mL.

[0015] Preferably, in step 3), the formulation of the mushroom bag contains the following components by weight percentage: 78% hardwood sawdust, 20% wheat bran, 1% white sugar and 1% gypsum.

[0016] Preferably, in step 3), the fruiting experiment involves placing the mushroom bags in a dark cultivation room at 24±2 ℃ and 70% relative humidity after inoculation with the original spawn. When the mycelium has covered the entire mushroom bag and irregular bulges appear, the bags are transferred to an outdoor greenhouse, perforated, and pressed to separate the bags, allowing the culture medium inside to fully contact the air. During the day, the temperature is maintained at 22~24 ℃, and the diffused light and ventilation in the greenhouse are increased to create an alternating dry and wet environment. Temperature and humidity differences stimulate the aerial mycelium on the surface of the mushroom tubes to collapse, secrete pigments, and form a reddish-brown glossy film. After the color change is complete, the bags are removed and the mushrooms are harvested.

[0017] A third objective of this invention is to provide the fruiting body of the aforementioned shiitake mushroom strain FX85.

[0018] A fourth objective of this invention is to provide spores of the aforementioned shiitake mushroom strain FX85.

[0019] The fifth objective of this invention is to provide the mycelium of the aforementioned shiitake mushroom strain FX85.

[0020] The sixth objective of this invention is to provide a spawn of the aforementioned shiitake mushroom strain FX85.

[0021] The seventh objective of this invention is to provide the application of the aforementioned shiitake mushroom strain FX85 in food or as a parent in hybridization breeding.

[0022] The shiitake mushroom strain FX85, its breeding method, and its application of the present invention have the following advantages:

[0023] The FX85 strain bred in this invention has a predominantly brown cap with white, velvety scales along the edge. The average yield of fresh mushrooms from strain FX85 is 912 g / stick, which is 8.07% higher than the control variety Xin 808. The polysaccharide content of strain FX85 is 8.75% higher than Xin 808, the protein content is 21.57% higher, and the cap-to-stem ratio is 13.77% higher. It exhibits superior agronomic traits and yield compared to strain Xin 808, making it worthy of widespread application. This high-yielding shiitake mushroom variety bred in this invention can enrich the existing shiitake germplasm resource bank, help adjust the shiitake mushroom cultivation structure, and improve the economic benefits of shiitake mushroom cultivation. Attached Figure Description

[0024] Figure 1 This is a diagram showing the mycelial growth of FX85 in this invention.

[0025] Figure 2 The FX85 and control group culture sticks of this invention are shown.

[0026] Figure 3 This is a morphological diagram of the FX85 sub-entity of the present invention.

[0027] Figure 4 The following are cross-sectional views of the FX85 sub-entity of the present invention: (a) cross section; (b) side section.

[0028] Figure 5 This is the FX85 phylogenetic tree of the present invention.

[0029] Figure 6 This is the identification diagram of the FX85 ISSR of the present invention.

[0030] Figure 7 This is a cluster diagram of the genetic relationships of FX85 in this invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0032] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0034] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0035] In the quantitative experiments in the following examples, three replicate experiments were set up, and the results were averaged.

[0036] Experimental Example 1: High-yielding shiitake mushroom strain ( Lentinus edodes Obtaining the FX85

[0037] The strains of this invention were obtained through single-spore hybridization and systematic selection of Shenxiang 215 and Huxiang F2. Shenxiang 215 is a strain with large and thick mushrooms, firm caps, and excellent marketability. It has shown good adaptability and stability in demonstration and promotion in multiple locations. Huxiang F2 is a variety with significantly increased yield, a high proportion of high-quality mushrooms, and strong resistance to adverse conditions. It has demonstrated outstanding economic value and production potential in scientific and technological cooperation applications. Therefore, these two strains were selected as the parent materials of this invention.

[0038] The specific steps of hybridization breeding are as follows:

[0039] (1) Preparation of single spores

[0040] Spores were collected from the first flush of superior fruiting bodies of *Houtianthus fragrans* var. *huxiang* F2 and *Shenxiang* 215, and diluted with sterile water to 100%. 5 50 μL of spore suspension was transferred to a PDA medium plate and spread evenly. The plate was then incubated at 25°C for 3–5 days. The newly germinated star-shaped colonies were examined under a microscope. The monokaryotic mycelia of Huxiang F2 and Shenxiang 215 without clamp connections were selected for further culture.

[0041] (2) Mating type identification

[0042] The monokaryotic mycelia obtained above were hybridized and their genotypes were identified. The genotype results were represented by ITS and ISSR.

[0043] (3) Hybrid pairing

[0044] Under aseptic conditions, monokaryotic mycelia of both parents were taken onto a PDA using a 0.8 cm diameter punch. The culture blocks were spaced 3 cm apart and incubated at 25°C for 3–5 days. When a fusion line appeared between the two colonies, the mycelia in the middle of the fusion line were examined under a microscope. If clamp connections were formed, it indicated that the mycelia were dikaryotic and the hybridization was successful.

[0045] (4) Heterozygous mycelial growth and fruiting experiment

[0046] The heterozygotes were transferred to tubes, and mycelial growth was observed. A fruiting experiment was conducted using the heterozygotes. The culture medium formula (by weight percentage) was: 78% hardwood sawdust, 20% wheat bran, 1% sugar, and 1% gypsum. 17 cm × 33 cm spawn bags were used for cultivation, with 20 bags per strain, each bag weighing approximately 1 kg. The bags were steam-sterilized at 108–110 °C for 5 h. After inoculation, the bags were placed in the dark on shelves in a cultivation room at 24 ± 2 °C and 70% relative humidity. When the mycelium completely covered the bag and irregular bulges appeared, the bags were transferred to an outdoor greenhouse. Approximately 30 holes were punctured in each bag, and the bags were gently pressed to separate the substrate from the culture medium, ensuring sufficient contact between the culture medium and air. During the day, the temperature was maintained at around 23 ℃. Increased diffused light inside the greenhouse, enhanced ventilation, and the creation of an alternating wet and dry environment stimulated by temperature and humidity differences. This promoted the collapse of aerial mycelium on the surface of the mushroom logs, causing them to secrete pigments and form a glossy reddish-brown film. After color change, the mushrooms were removed from the bags and allowed to grow. Fruiting trials were conducted under the same conditions, and strains with good mushroom shape, dense or relatively dense fruiting, and high yield were selected for the next round of screening, as shown in Table 1.

[0047] Table 1 Initial screening results of dominant strains

[0048]

[0049] Note: New 808 refers to the existing shiitake mushroom variety L808; mushrooms with the FX number are strains selected in the first round of screening that have good mushroom shape, dense or relatively dense fruiting, and high yield.

[0050] (5) Further screening of dominant strains

[0051] Using the mushroom bags and formula from step (4), further fruiting experiments were conducted on the hybrid strains screened in the previous step. First flush mushroom fruiting bodies were collected for agronomic trait recording, crude polysaccharide and crude protein detection, and the target dominant strain FX85 was further screened out.

[0052] (6) Stability cultivation experiment

[0053] Using the mushroom bags and formula from step (4), a strain stability cultivation experiment was conducted to observe its genetic stability. The purified strain material, namely the target dominant strain FX85, was obtained by isolating the first flush of mushroom tissue.

[0054] Identification of strain in Experiment Example 2

[0055] I. Morphological characteristics

[0056] 1. Mycelial morphological characteristics

[0057] The mycelium of strain FX85 of this invention is dense and vigorous, initially pure white, and turns slightly light brown after a period of culture, such as... Figure 1 As shown.

[0058] 2. Morphological characteristics of mushroom logs

[0059] Depend on Figure 2 It can be seen that (the mushroom log with the number FX in the figure is the mushroom obtained from the fruiting experiment in Experiment Example 1), the color of the mushroom log is darker than that of the new 808, and the color is dark brown.

[0060] 3. Morphological characteristics of sub-entities

[0061] The fruiting body of *Lentinula edodes* FX85 is medium to large in size, hemispherical, and relatively round. The cap is brown, darker in the center and lighter at the edges, and convex in longitudinal section. Dark scales are evenly distributed along the cap edge. The cap diameter is 4.55±0.09 cm, and the cap thickness is 1.58±0.06 cm. The flesh is white and possesses the characteristic aroma of shiitake mushrooms. The gills are off-white. The stipe is 3.38±0.25 cm long and 1.63±0.08 cm thick, white, and of uniform thickness from top to bottom, nearly cylindrical. See below for the fruiting body morphology and its cross-section. Figure 3 , Figure 4 .

[0062] II. Molecular biological characteristics

[0063] This invention utilizes a rapid fungal DNA extraction kit to extract DNA and amplify and sequence the ITS (intracytoplasmic sperm injection) of the fruiting bodies of strain FX85 obtained through hybridization. The sequenced ITS sequence is SEQ ID NO.1. A phylogenetic tree was constructed based on the identification results, as follows: Figure 5 As shown.

[0064] The specific details of SEQ ID NO.1 are as follows:

[0065] .

[0066] Based on the above morphological characteristics and ITS sequence classification and identification studies, it has been determined that the strain FX85 of this invention belongs to the Basidiomycetes class (Basidiomycetes). Basidaiomycetes ), Agaricalales ( Agaricales ), Small Bark Ferns ( Marasmiaceae ), Lentinus genus ( Lentinula This strain Lentinus edodes FX85 was deposited on December 3, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242713.

[0067] Experimental Example 3: High-yielding shiitake mushroom strain ( Lentinus edodes Comparative analysis of the basic traits of FX85 and the new 808

[0068] This experimental example examines the high-yielding shiitake mushroom strain provided by this invention from three aspects: basic characteristics of the strain, crude polysaccharide content, and protein content. Lentinus edodes FX85 and strain Xin 808 were compared and analyzed. The basic characteristics of the strain were determined according to the "Agricultural Industry Standard of the People's Republic of China NY / T 2560—2014, Guidelines for Testing the Distinctiveness, Uniformity and Stability of New Plant Varieties, Shiitake Mushroom". The crude polysaccharide of Shiitake Mushroom fruiting body was determined according to the sulfuric acid-phenol method, and the protein was determined using the Kjeldahl method.

[0069] The results are shown in Table 2. In comparison, the high-yielding shiitake mushroom strain ( Lentinus edodes The mycelium of FX85 grows rapidly; the fruiting body has a nearly white stipe and a predominantly brown cap. The average yield of fresh mushrooms from strain FX85 is 912 g / stick, which is 8.07% higher than the control variety Xin 808. The edge is covered with very small, nearly white, velvety scales, and the cap has a convex shape in longitudinal section. The stipe is of equal thickness from top to bottom, cylindrical in shape, and of moderate length. The crude polysaccharide content exceeds that of the main promoted variety Xin 808 by 8.75%; the crude protein content exceeds that of the main promoted variety Xin 808 by 21.57%; and the agronomic traits and yield are more advantageous than Xin 808.

[0070] Table 2 shows a comparison of the basic characteristics of FX85 and the control strain.

[0071]

[0072]

[0073] Experiment Example 4: Identification of Inter-regional Simple Repeat Sequence Markers (ISSRs) in Strain FX85

[0074] The strain was activated and cultured at 25℃ for 7 days. Inoculum blocks were picked and inoculated onto PDA medium, and cultured at 25℃ for 10 days. The hyphae were ground using liquid nitrogen and stored at -80℃ for later use. Mycelial DNA was extracted using the CTAB method. Amplification was performed on a BioRad C1000 PCR instrument. The reaction system consisted of: 10 μL of 2×T5 Super PCR MIX (Basic), 8 μL of ddH2O, 1 μL of primer (P1: 5′-AGAGAGAGAGAGAGAGT-3′), and 1 μL of template DNA, for a total of 20 μL. The amplification annealing temperature was 54℃. After electrophoretic separation and imaging, band statistics were performed. ISSR bands were counted as 1 for those present and 0 for those absent, constructing an initial data matrix. Cluster analysis was then performed using NTSYSpc2.1 professional software.

[0075] ISSR electrophoresis patterns Figure 6 As shown, the results indicate that New 808 has 5 bands from top to bottom, while FX85 has 4 bands. There are significant differences in band distribution, staining depth, and band width between FX85 and New 808. The first and second bands of FX85 are brighter and wider than those of New 808 and Shenxiang 215, and FX85 lacks the third band of New 808.

[0076] Genetic relationship clustering diagrams were plotted on the strip data. Figure 7 It can be seen that (the mushroom logs numbered FX in the figure are the mushrooms obtained from the fruiting experiment in Experiment Example 1) are divided into two major categories at a genetic similarity coefficient of 0.77, indicating that the shiitake mushroom strains FX85 and Xin 808 (X808 in the figure) have significant genetic differences.

[0077] In summary, the high-yielding new shiitake mushroom varieties selected in this invention can enrich the shiitake mushroom germplasm resource bank, help adjust the shiitake mushroom industry structure, and improve planting efficiency.

[0078] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Shiitake mushroom strains ( Lentinus edodes The FX85 is characterized by: The shiitake mushroom strain FX85 was deposited on December 3, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20242713.

2. The fruiting body of the shiitake mushroom strain FX85 as described in claim 1.

3. Spores of the shiitake mushroom strain FX85 as described in claim 1.

4. The mycelium of the shiitake mushroom strain FX85 as described in claim 1.

5. The application of the shiitake mushroom strain FX85 as described in claim 1 in food or as a parent in hybridization breeding.

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