Tricholoma matsutake mushroom and molecular marker identification method thereof

Abstract

The invention discloses a tricholoma matsutake mushroom, which is classified and named as tricholoma matsutake mushroom 11, and is preserved in China Center for Type Culture Collection on March 24, 2025 with the preservation number of CCTCC NO: M 2025577. The molecular marker identification method comprises culture and collection of hyphae, extraction of genome DNA, DNA fingerprint PCR amplification and electrophoresis detection of PCR products. According to the method, the tricholoma matsutake mushroom 11 which inherits the advantage of high thermotype of two parents, is good in hypha temperature tolerance and high in vigor, is similar to tricholoma matsutake in sporocarp shape and is not opened is obtained by carrying out crossbreeding of the mushrooms and screening after mushroom cultivation and fruiting, and the tricholoma matsutake mushroom 11 has good development and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of shiitake mushrooms, in particular to a shiitake mushroom and a molecular marker identification method thereof. Background Art

[0002] Shiitake mushrooms are a famous edible fungus in my country. They are widely cultivated and their output ranks first among the cultivated edible fungi in my country all year round. They occupy an important position in China's edible fungus production. After decades of development, my country's edible fungus industry has developed rapidly, and its output has long ranked first in the world.

[0003] However, with the changes in the global economic situation in recent years, the overall supply of my country's edible fungi industry exceeds demand, and competition among enterprises is becoming increasingly fierce. In particular, edible fungi species with large cultivation volumes are facing serious domestic problems caused by increasingly serious variety homogeneity. This urgently requires the cultivation of new varieties to improve market competitiveness. Therefore, the present invention proposes a matsutake mushroom and a molecular marker identification method thereof to solve the problems existing in the prior art. Summary of the Invention

[0004] In response to the above problems, the purpose of the present invention is to propose a matsutake mushroom and a molecular marker identification method thereof. By carrying out hybrid breeding of shiitake mushrooms and screening after mushrooms are cultivated, a matsutake mushroom is obtained that inherits the advantages of higher temperature types of the two parents, has good mycelium temperature resistance, high vitality, and fruiting bodies that are shaped like matsutake mushrooms and do not open.

[0005] To achieve the purpose of the present invention, the present invention is implemented through the following technical scheme: a matsutake mushroom, the matsutake mushroom is classified and named matsutake mushroom 11, which has been preserved in the China Center for Type Culture Collection on March 24, 2025, with the preservation number CCTCC NO: M2025577.

[0006] The above-mentioned Matsutake Mushroom 11 is bred by hybridization of Mushroom "Nongxiang 17" and Mushroom "L238" as parents; "Nongxiang 17" is a new variety of Mushroom bred by the inventor, and has obtained the national plant variety right authorization (variety right number: CNA20221002740). It is a medium-high temperature variety with good flower shape, high yield, short cultivation cycle and other characteristics. Its fruiting body opens normally; Mushroom "L238" is a wide temperature variety cultivated in the market in recent years. The variety also has the characteristics of good flower shape, high yield, and short cultivation cycle, and its fruiting bodies open normally; Matsutake Mushroom 11 inherits the advantages of higher temperature types of its two parents. Its mycelium has good temperature resistance and high vitality, and it also produces specific characteristics different from the two parents: the fruiting body flower shape is similar to Matsutake, and it does not open. In addition, Matsutake Mushroom 11 is a medium-temperature type, and mushrooms are produced at 95 days of age. The mushrooms are concentrated, have a crisp and tender taste, and the fruiting bodies are highly edible. It has good development and application prospects.

[0007] The mother culture medium of Matsutake mushroom 11 is potato dextrose medium (PDA): 200g potato, 20g glucose, 20g agar strip, 1000mL water; the culture conditions are conventional mushroom culture.

[0008] A molecular marker identification method for Lentinus edodes, comprising culturing and collecting mycelium, extracting genomic DNA, amplifying DNA fingerprints by PCR, and detecting the PCR products by electrophoresis;

[0009] The DNA fingerprint PCR amplification specifically involves designing 8 sets of primer pairs to perform PCR amplification on the DNA of Matsutake mushroom 11 to obtain amplified products, wherein the 8 sets of primer pairs are forward primers LP1A to LP8A and reverse primers LP1B to LP8B, and the sequences are shown in SEQ ID No. 1 to SEQ ID No. 16 respectively;

[0010] Electrophoresis detection of the PCR product: After electrophoresis detection, only one fragment appeared in one lane, and the fragment size was consistent with the designed corresponding primer, which is the molecular marker of Matsutake mushroom 11.

[0011] A further improvement is that the DNA fingerprint PCR amplification reaction system comprises 1 to 1.5 μl of 80 to 150 ng of DNA, 12.5 μl of Premix Taq (with dye), 1 μl of 10 μmol / L forward primers LP1A to LP8A, 1 μl of 10 μmol / L reverse primers LP1B to LP8B, 9 to 9.5 μl of ddH2O, and a total volume of 25 μl.

[0012] The reaction procedure was pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 63-65°C for 30 sec, extension at 72°C for 1-3 min, 35 cycles; and extension at 72°C for 10 min.

[0013] A further improvement is that the electrophoresis detection conditions of the PCR product are as follows: 5 μL of the PCR product is mixed with 1 μL of loading buffer, spotted on a 1.2% agarose gel, and electrophoresed in 0.5XTBE buffer at a voltage of 5 V / cm; wherein the loading buffer contains 0.1% bromophenol blue and 40% sucrose.

[0014] The beneficial effects of the present invention are as follows: the fruiting bodies of the matsutake mushroom 11 obtained by hybridization and breeding of the present invention inherit the advantages of higher temperature types of the two parents, the mycelium has good temperature resistance and high vitality, and at the same time, the fruiting bodies have the specific characteristics of being shaped like matsutake mushrooms and not opening umbrellas, and have good development and application prospects; the molecular marker identification method of the matsutake mushroom 11 is authentic and reliable, with small identification errors, and the method is simple to operate and suitable for promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1This is a morphological diagram of the fruiting bodies of the present invention's Matsutake mushroom 11 and its parents "Nongxiang 17" and "L238".

[0016] Figure 2 This is a characteristic diagram of the fruiting body of Matsutake mushroom 11 of the present invention.

[0017] Figure 3 This is the electrophoresis diagram of the PCR product of the present invention. DETAILED DESCRIPTION

[0018] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0019] In the following examples, the tested shiitake mushroom strain “Nongxiang 17” is a new variety of shiitake mushroom bred by the inventors, which has obtained the national plant variety right authorization (variety right number: CNA20221002740), and the shiitake mushroom “L238” was purchased from the market.

[0020] Example 1

[0021] This embodiment provides a breeding method for the matsutake mushroom "Matsutake mushroom 11".

[0022] Creation of breeding materials: "Nongxiang 17" and "L238" were used as breeding parents. After fruiting tests, spores of the two parents were ejected when their fruiting bodies matured, and spore germination was carried out. The germinated spores were picked up under a microscope and confirmed by lock-like union. Monosporic strains without lock-like union were selected as breeding materials. Finally, 20 monosporic strains of "Nongxiang 17" and 25 monosporic strains of "L238" were obtained.

[0023] Hybrid pairing: After subculture of the monosporic strains of "Nongxiang 17" and "L238", 13 monosporic strains with better growth of "Nongxiang 17" were selected for double-single pairing with "L238", and 7 hybrids were obtained; 14 monosporic strains with better growth of "L238" were selected for single-single pairing with the monosporic strain LH17-1 of "Nongxiang 17", and 14 hybrids were obtained; a total of 21 hybrids were used for subsequent screening.

[0024] Hybrid screening: 21 hybrids were used to make spawn, and according to the conventional method of making and cultivating shiitake mushroom spawn, fruiting test was carried out. The fruiting of each hybrid was observed visually. It was found that the fruiting body morphology of one of the hybrids numbered 64 was obviously different from that of the two parents, as shown in the attached manual. Figure 1 In order to verify the stability of its characteristics, further fruiting observation is needed.

[0025] Stability test: Because the fruiting body morphology of hybrid 64 is obviously different from that of conventional shiitake mushrooms, we have been producing mushrooms for three consecutive years and found that its properties are stable, the mycelium is very active, and the fruiting body is similar to the well-known wild edible mushroom, Tricholoma matsutake, in its unopened state. Figure 2 As shown, the stem is long and the cap does not open. Furthermore, the mushrooms are uniform during fruiting, exhibit good resistance to stress, and are highly edible (common shiitake mushrooms have their stems discarded when consumed). The fruiting body is crispy and tender, and can be eaten similarly to pan-fried matsutake mushrooms. Therefore, this hybrid was named Matsutake Mushroom 11.

[0026] Matsutake mushroom 11 was deposited in the China Center for Type Culture Collection on March 24, 2025, address: Wuhan University, Wuhan, with the deposit number CCTCC NO: M 2025577.

[0027] Example 2

[0028] This embodiment provides a productive cultivation method of pine mushroom "pine mushroom 11".

[0029] Production of 11 strains of Matsutake and Shiitake mushrooms

[0030] The matsutake mushroom 11 strain is activated twice on a PDA culture medium, transferred to a conventional culture medium for shiitake mushroom original species, and cultured at 23°C to 25°C to produce original species and cultivated species; liquid strains can also be produced to obtain liquid strains of matsutake mushroom 11.

[0031] Management of bag preparation, cultivation, bag color change, and fruiting of Matsutake Mushroom 11

[0032] The selection of materials, formulation of various ingredients, bagging, and sterilization procedures for cultivating matsutake mushrooms 11 are the same as for conventional shiitake mushrooms. A certain proportion of pine sawdust may also be added. Use polypropylene bags measuring 15cm x 55cm, and do not use a water-retaining film when filling. After approximately 95 days of inoculation, transfer the mushrooms to the fruiting shed for fruiting management. To ensure fruiting quality, the temperature should not exceed 20°C, and the humidity should be controlled between 88% and 95%. When the buds emerge, cut the small buds for fruiting, limiting the number of buds to 12 to 15. Low humidity can cause the cap to dry out and crack, resulting in "flowery mushrooms," and the stems to become rough and hairy.

[0033] After the mushrooms of Matsutake and Lentinus edodes have grown, 400-500g of fresh mushrooms are harvested from each bag in the first and second waves. The morphological characteristics are shown in the attached instructions. Figure 2 As shown, the fruiting bodies are primarily scattered and do not open. The cap is round, convex, smooth, and reddish-brown or light brown, with a diameter of 4 to 5 cm and a thickness of 2 to 2.4 cm. The flesh is firm and white, and the stipe is columnar, uniform in thickness, and 4 to 6 cm long. When cultivated in dry conditions, it may have cilia on its surface. The stipe is edible and can be fried or grilled like matsutake, with a taste similar to or similar to that of matsutake.

[0034] The cultivation cycle of Matsutake Mushroom 11 is short, the mycelium is vigorous, the mushrooms are neatly produced, the yield is high, the fruiting bodies have a good taste, and the stipes of fresh mushrooms are edible, which increases the edible rate of the mushroom fruiting bodies (at least half of the stipes of fresh mushrooms should be discarded, or almost all of the stipes should be discarded when drying), and has good development and application prospects.

[0035] Example 3

[0036] This embodiment provides a molecular marker identification method for the pine mushroom "pine mushroom 11".

[0037] 1. The genomic DNA of Matsutake Mushroom 11 and its parents "Nongxiang 17" and "L238" was extracted by CTAB method. The specific extraction steps were carried out according to the method of Tao Yongxin et al. (Tao Y, Xie B, Yang Z, et al. Identification and expression analysis of a new glycoside hydrolase family 55exo-β-1,3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development[J]. Gene, 2013, 527(1):154-160.). The extracted DNA was stored at -20°C for later use.

[0038] 2. The genomic DNA of strains such as Matsutake Mushroom 11, "Nongxiang 17," and "L238" were sent to Wuhan Fraser Gene Information Co., Ltd. for whole-genome second-generation sequencing. The sequencing data volume was 3 to 4G of clean data. Bioinformatics analysis was performed on these genomic data to identify genes unique to the hybrid Matsutake Mushroom 11 and its parents, "Nongxiang 17," and "L238." Primers were then designed. The information for the eight pairs of primers designed is as follows:

[0039] Primers LP1A and LP1B can amplify a 1795bp marker fragment from the genomes of "L238" and Matsutake mushroom 11, while "Nongxiang 17" does not have this marker fragment.

[0040] Primers LP2A and LP2B can amplify a 2487bp marker fragment from the genomes of "L238" and Matsutake mushroom 11, while "Nongxiang 17" does not have this marker fragment.

[0041] Primers LP3A and LP3B can amplify a 1954bp marker fragment from the genomes of "L238" and Matsutake mushroom 11, while "Nongxiang 17" does not have this marker fragment.

[0042] Primers LP4A and LP4B can amplify a 1175bp marker fragment from the genomes of "L238" and Matsutake mushroom 11, while "Nongxiang 17" does not have this marker fragment.

[0043] Primers LP5A and LP5B can amplify a 1908bp marker fragment from the genomes of "L238" and Matsutake mushroom 11, while "Nongxiang 17" does not have this marker fragment.

[0044] Primers LP6A and LP6B can amplify a 2969bp marker fragment from the genomes of Nongxiang 17 and Matsutake 11, while "L238" does not have this marker fragment.

[0045] Primers LP7A and LP7B can amplify a 2912bp marker fragment from the genomes of "Nongxiang 17" and Matsutake 11, while "L238" does not have this marker fragment.

[0046] Primers LP8A and LP8B can amplify a 1886bp marker fragment from the genomes of Nongxiang 17 and Matsutake 11, while "L238" does not have this marker fragment.

[0047] The sequences of the eight designed primer pairs and the sizes of the marker fragments obtained by amplifying the DNA of Matsutake and Lentinus edodes 11 using these eight primer pairs are shown in Table 1.

[0048] Table 1 Specific marker amplification information

[0049]

[0050] 3. PCR amplification of specific fragments

[0051] PCR amplification reaction system: 1.2 μl of 100 ng DNA, 12.5 μl of Premix Taq (with dye), 1 μl of 10 μmol / L forward primer, 1 μl of 10 μmol / L reverse primer, 9.3 μl ddH2O;

[0052] The PCR amplification reaction program for primers LP1A and LP1B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 63°C for 45 sec, extension at 72°C for 2 min, 35 cycles; and extension at 2°C for 10 min.

[0053] The PCR amplification reaction program for primers LP2A and LP2B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 64°C for 45 sec, extension at 72°C for 2 min 30 sec, 35 cycles; extension at 72°C for 10 min.

[0054] The PCR amplification reaction program for primers LP3A and LP3B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 63°C for 45 sec, extension at 72°C for 2 min, 35 cycles; and extension at 72°C for 10 min.

[0055] The PCR amplification reaction program for primers LP4A and LP4B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 65°C for 45 sec, extension at 72°C for 1 min 15 sec, 35 cycles; extension at 72°C for 10 min.

[0056] The PCR amplification reaction program for primers LP5A and LP5B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 64°C for 45 sec, extension at 72°C for 2 min, 35 cycles; and extension at 72°C for 10 min.

[0057] The PCR amplification reaction program for primers LP6A and LP6B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 63°C for 45 sec, extension at 72°C for 3 min, 35 cycles; and extension at 72°C for 10 min.

[0058] The PCR amplification reaction program for primers LP7A and LP7B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 64°C for 45 sec, extension at 72°C for 3 min, 35 cycles; and extension at 72°C for 10 min.

[0059] The PCR amplification reaction program for primers LP8A and LP8B was as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 65°C for 45 sec, extension at 72°C for 2 min, 35 cycles; and extension at 72°C for 10 min.

[0060] 4. Electrophoresis detection of PCR products

[0061] Take 5μl of PCR product and spot it on 1.2% agarose gel. Use DNA MARK of DL2000 as the reference of fragment size. Electrophoresis is carried out in 0.5×TBE buffer at 5V / cm for 30-40min. After electrophoresis, take a picture on gel imager. The electrophoresis diagram is as attached in the manual. Figure 3 As shown, primers LP1 to LP5 are shared by Matsutake Lentinus edodes 11 and L238, distinguishing them from Nongxiang 17. Primers P6 to P8 are shared by Matsutake Lentinus edodes 11 and Nongxiang 17, distinguishing them from L238. These fragments are markers for Matsutake Lentinus edodes 11. M represents a DNAMARK with a DNA marker size of DL5000. Lane marker 1 represents the parent L238, lane 2 represents Matsutake Lentinus edodes 11, and lane 3 represents Nongxiang 17.

[0062] 5. Reagents and instruments

[0063] CTAB extraction solution: 100 mmol / L Tris-HCl, 2.0% CTAB, 20 mmol / L EDTA, 1.4 mol / L NaCl, pH 8.0;

[0064] CTAB precipitation solution: 50 mmol / L Tris-HCl, 1.0% CTAB, 10 mmol / L EDTA, pH 8.0;

[0065] CTAB / NaCl: 0.7mol / L NaCl, 10% CTAB;

[0066] Chloroform-isoamyl alcohol: the volume ratio of chloroform to isoamyl alcohol is 24:1;

[0067] TE buffer: 10 mmol / L TrisHCl, 1 mmol / L EDTA;

[0068] 0.5×TBE: 44.5mmol / L Tris, 50mmol / L HBO3, 1mmol / L EDTA;

[0069] Premix Taq (Ex Taq Version 2.0) with dye and PCR amplification reagents were purchased from Dalian Takara Biotechnology Co., Ltd.

[0070] Main instruments:

[0071] SW-CJ-1FB single-person horizontal and vertical dual-purpose clean bench: Suzhou Purification Equipment Co., Ltd.

[0072] Sterilizer: Shanghai Shenan LDZM-60L-Ⅰ;

[0073] Refrigerated centrifuge: Sigma3K30;

[0074] PCR amplifier: Eppendorf AG22331 Hamburg;

[0075] Palm-type centrifuge: Lx-100 palm-type centrifuge produced by Qilin Medical Instrument Factory, Haimen City, Jiangsu Province;

[0076] SDC-6 energy-saving intelligent constant temperature bath: Ningbo Xinzhi Biotechnology Co., Ltd.

[0077] Gel imaging system: TANON-MINISpace 1000.

[0078] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A matsutake mushroom, characterized by: The classification name of the matsutake mushroom is matsutake mushroom 11, which was deposited in the China Center for Type Culture Collection on March 24, 2025, with the preservation number CCTCC NO: M 2025577.

2. The molecular marker identification method for mushrooms according to claim 1, characterized in that: It includes the cultivation and collection of mycelium, extraction of genomic DNA, DNA fingerprint PCR amplification and electrophoresis detection of PCR products; The DNA fingerprint PCR amplification specifically involves designing 8 sets of primer pairs to perform PCR amplification on the DNA of Matsutake mushroom 11 to obtain amplified products, wherein the 8 sets of primer pairs are forward primers LP1A to LP8A and reverse primers LP1B to LP8B, and the sequences are shown in SEQ ID No. 1 to SEQ ID No. 16 respectively; Electrophoresis detection of the PCR product: After electrophoresis detection, only one fragment appeared in one lane, and the fragment size was consistent with the designed corresponding primer, which is the molecular marker of Matsutake mushroom 11.

3. The molecular marker identification method for shiitake mushrooms according to claim 2, characterized in that: The DNA fingerprint PCR amplification reaction system is 1-1.5 μl of 80-150 ng of DNA, 12.5 μl of Premix Taq, 1 μl of 10 μmol / L forward primers LP1A-LP8A, 1 μl of 10 μmol / L reverse primers LP1B-LP8B, 9-9.5 μl of ddH2O, and a total volume of 25 μl. The reaction procedure was pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 sec, annealing at 63-65°C for 30 sec, extension at 72°C for 1-3 min, 35 cycles; and extension at 72°C for 10 min.

4. The molecular marker identification method for Matsutake mushrooms according to claim 2, characterized in that: The electrophoresis detection conditions of the PCR product are as follows: 5 μL of the PCR product is mixed with 1 μL of loading buffer, spotted on a 1.2% agarose gel, and electrophoresed in 0.5X TBE buffer at 5 V / cm; The loading buffer contained 0.1% bromophenol blue and 40% sucrose.

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