Bovine liver mushroom and molecular marker identification method thereof

Abstract

The invention provides bovine liver mushroom and a molecular marker identification method thereof, and belongs to the technical field of breeding. The beef liver shiitake mushroom J3 is preserved in the China Center for Type Culture Collection on May 21, 2025, and the preservation number of the beef liver shiitake mushroom J3 is CCTCC NO: M 20251141. According to the method, the beef liver mushroom J3 with unique characters is successfully cultivated by combining a mushroom cross breeding technology with precise screening after fruiting. The sporocarp of the beef liver shiitake mushroom J3 inherits the advantage of high vigor of two parent hyphae, and obtains the characters different from the two parent sporocarp: the stem is thick, the cap is not easy to open, the meat quality is thick, the whole sporocarp looks like bolete without opening, the meat quality is fleshy, the taste is crisp and tender, the edibility of the sporocarp is high, and the development and application prospects are very good.

Description

Technical Field

[0001] This invention relates to the field of breeding technology, and in particular to a method for identifying *Lentinula edulis* and its molecular markers. Background Technology

[0002] Shiitake mushrooms, a renowned edible fungus, are widely cultivated and consistently rank first in production volume among cultivated edible fungi in China, holding an irreplaceable core position in the edible fungi production system. Meanwhile, after decades of rapid development, the edible fungi industry has achieved a leapfrog improvement, with its total output already ranking first in the world, demonstrating its strong industrial strength.

[0003] However, in recent years, affected by multiple factors such as global economic fluctuations and the continuous expansion of the industry, the edible fungi industry is facing severe structural challenges—the contradiction of supply exceeding demand in the overall market is becoming increasingly prominent, and competition among enterprises has become fierce. Among them, the problems faced by major cultivated varieties, represented by shiitake mushrooms, are even more prominent: the phenomenon of variety homogenization is becoming increasingly serious, which has led to fierce "internal friction" and seriously restricts the high-quality development of the industry and the improvement of market competitiveness.

[0004] Despite the long history of shiitake mushroom cultivation, the current main cultivated varieties also have some problems. Taking the "808" variety, which has been the main cultivated variety for the past 20 years, as an example, although it has advantages such as strong mycelial activity, round and umbrella-shaped fruiting bodies, thick and brown caps, short stems and high yield, and is a medium- and low-temperature adaptable variety, its long-term single promotion has led to a decrease in its market recognition and a gradual weakening of its competitive advantage. "Yuhua No. 2", which was promoted more than 10 years ago (Wang Jiandang, Hou Yanping, Han Jiandong, et al. Selection of suitable shiitake mushroom varieties, liquid seed production and high-efficiency cultivation technology in Weihai area [J]. Shandong Agricultural Sciences, 2014, 46(08):125-126+131.), even though it has the characteristics of good mycelial activity, early fruiting, whiter caps, easy cracking and high yield, it is also difficult to meet the current market demand for differentiated and high-quality shiitake mushrooms due to its long promotion period and the increase of similar substitute varieties.

[0005] Variety is the core competitiveness of industrial development. Against the backdrop of increasingly prominent homogenization of shiitake mushroom varieties, cultivating new varieties with both excellent traits and market recognition has become an urgent need to break through the bottleneck of industrial development and enhance market competitiveness. Summary of the Invention

[0006] The purpose of this invention is to provide a Boletus edulis and its molecular marker identification method. The fruiting body of Boletus edulis J3 resembles an unopened Boletus edulis, with a thick stem, a cap that is not easily opened, thick flesh, a crisp and tender texture, and high edibility. It has a good prospect for development and application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a porcini mushroom ( Lentinula edodes J3, the aforementioned Boletus edulis J3 was deposited on May 21, 2025 at the China Center for Type Culture Collection, located at Wuhan University, with accession number CCTCC NO:M20251141.

[0008] The present invention also provides a protoplast produced from the above-mentioned *Lentinula edodes* J3.

[0009] The present invention also provides spores produced by the above-mentioned Boletus edulis J3.

[0010] The present invention also provides a mycelium produced from the above-mentioned Boletus edulis J3.

[0011] The present invention also provides a fruiting body produced by the above-mentioned Boletus edulis J3.

[0012] The present invention also provides a shiitake mushroom substrate comprising the above-mentioned *Porcini shiitake* J3.

[0013] This invention also provides the application of the above-mentioned Boletus edulis J3 in shiitake mushroom breeding.

[0014] The present invention also provides the application of the above-mentioned shiitake mushroom strain in the preparation of shiitake fruiting bodies, shiitake mycelia, protoplasts and / or shiitake spores.

[0015] The present invention also provides a primer set for identifying J3 molecular markers of *Lentinula zedoaria*, characterized in that the primer set for identifying J3 molecular markers of *Lentinula zedoaria* includes the primer sequences shown in SEQ ID NO.1 to SEQ ID NO.14.

[0016] This invention also provides a molecular marker identification method for *Lentinula zedoaria* based on the above-mentioned J3 molecular marker identification primer set, characterized by comprising the following steps: (1) Culture, collection and extraction of mycelium and genomic DNA; (2) DNA fingerprint PCR amplification based on the primer sequences shown in SEQ ID NO.1~SEQ ID NO.14; (3) Electrophoretic detection of PCR products.

[0017] The beneficial effects of this invention compared to the prior art are as follows: (1) This invention discloses a type of *Lentinula edulis* J3, which was deposited at the China Center for Type Culture Collection on May 21, 2025, with accession number CCTCC NO:M 20251141. This invention successfully cultivated *Lentinula edulis* J3 with unique traits by combining shiitake mushroom hybridization breeding technology with precise screening after fruiting. The fruiting body of *Lentinula edulis* J3 inherits the advantages of high mycelial vigor of the two parent species, while also acquiring traits different from the fruiting bodies of the two parent species: thick stem, cap that is not easy to open, thick flesh, the overall fruiting body shape resembles an unopened *Lentinula edulis*, thick flesh, crisp and tender taste, high edibility of the fruiting body, and has good prospects for development and application.

[0018] (2) This invention also discloses a molecular marker identification method for *Lentinula zedoaria* J3, which involves the identification of *Lentinula zedoaria* J3 through mycelial culture and collection, extraction of genomic DNA, DNA fingerprint PCR amplification, and electrophoretic detection of PCR products. The *Lentinula zedoaria* J3 identified by this invention is authentic and reliable, with minimal identification error. Furthermore, the method is simple to operate and suitable for widespread application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The images show the morphological characteristics of the fruiting bodies of *Lentinula edulis* J3 and its parents "808" and "Yuhua 2" in Embodiment 1 of this invention. Figure 2 This is a feature diagram of the J3 fruiting body of *Lentinula edulis* in Embodiment 1 of the present invention; Figure 3 This is an electrophoresis diagram of the PCR product in Example 3 of the present invention. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The reagents and instruments used in the following examples are shown below: CTAB extraction solution: 100 mmol / L Tris-HCl, 2.0% CTAB, 20 mmol / L EDTA, 1.4 mol / L NaCl, pH 8.0; CTAB precipitation solution: 50 mmol / L Tris-HCl, 1.0% CTAB, 10 mmol / L EDTA, pH 8.0; CTAB / NaCl: 0.7mol / L NaCl, 10%CTAB; Chloroform to isoamyl alcohol: the volume ratio of chloroform to isoamyl alcohol is 24:1; TE buffer: 10 mmol / L TrisHCl, 1 mmol / L EDTA; 0.5×TBE: 44.5mmol / L Tris, 50mmol / L HBO3, 1mmol / L EDTA; Premix Taq (Ex Taq Version 2.0) with dyes and PCR amplification reagents were purchased from Dalian Takara Bio Co., Ltd. Potato glucose agar (PDA): 200g potato, 20g glucose, 20g agar strips, 1000mL water.

[0027] SW-CJ-1FB Single-Person Horizontal and Vertical Dual-Use Cleanroom Workbench: Suzhou Cleanroom Equipment Co., Ltd. Sterilization equipment: Shanghai Shenan LDZM-60L-Ⅰ; Refrigerated centrifuge: Sigma 3K30; PCR amplification instrument: Eppendorf AG22331 Hamburg; Palm-sized centrifuge: Lx-100 palm-sized centrifuge from Qilin Medical Instrument Factory, Haimen City, Jiangsu Province; SDC-6 Energy-Saving Intelligent Thermostatic Bath: Ningbo Xinzhi Biotechnology Co., Ltd.; Gel imaging system: TANON-MINI Space 1000.

[0028] This invention provides a molecular marker identification method for *Lentinula zedoaria* based on the J3 molecular marker identification primer set, comprising the following steps: (1) Culture, collection and extraction of mycelium and genomic DNA; (2) DNA fingerprint PCR amplification based on the primer sequences shown in SEQ ID NO.1~SEQ ID NO.14; The DNA fingerprint PCR amplification includes: designing 7 sets of primer pairs to perform PCR amplification on the DNA of Boletus edulis J3 to obtain amplification products, wherein the 7 sets of primer pairs are forward primers J3P1A~J3P7A and reverse primers J3P1B~J3P7B, and the sequences are shown as SEQ ID No.1~SEQ ID No.14 respectively; The DNA fingerprint PCR amplification reaction system consists of 1-1.5 μL of DNA containing 80-150 ng, 12.5 μL of PremixTaq, 1 μL of forward primers J3P1A-J3P7A at a concentration of 10 μmol / L, 1 μL of reverse primers J3P1B-J3P7B at a concentration of 10 μmol / L, 9-9.5 μL of ddH2O, and a total volume of 25 μL. The reaction program was as follows: 94℃ pre-denaturation for 5 min; 98℃ denaturation for 10 sec, Tm annealing for 30 sec, 72℃ extension for X, 35 cycles; 72℃ extension for 10 min. The primers J3P1A and J3P1B were annealed at 62℃ and extended at 72℃ for 1 min 30 sec; primers J3P2A and J3P2B, and primers J3P3A and J3P3B were annealed at 65℃ and extended at 72℃ for 1 min 30 sec; primers J3P4A and J3P4B, and primers J3P5A and J3P5B were annealed at 62℃ and extended at 72℃ for 1 min; primers J3P6A and J3P6B were annealed at 62℃ for 30 sec and extended at 72℃ for 40 sec; and primers J3P7A and J3P7B were annealed at 62℃ and extended at 72℃ for 1 min 30 sec.

[0029] (3) Electrophoretic detection of PCR products; Electrophoresis analysis revealed only one fragment per lane, with the fragment size matching the designed primers, indicating it was a molecular marker for *Lentinula edodes* J3. Specifically, primers J3P1A / J3P1B produced a 1437 bp marker fragment, primers J3P2A / J3P2B produced a 1243 bp marker fragment, primers J3P3A / J3P3B produced a 1004 bp marker fragment, primers J3P4A / J3P4B produced an 875 bp marker fragment, primers J3P5A / J3P5B produced a 992 bp marker fragment, primers J3P6A / J3P6B produced a 548 bp marker fragment, and primers J3P7A / J3P7B produced a 1400 bp marker fragment.

[0030] Example 1 Embodiment 1 of the present invention provides a method for breeding "Boletus edulis J3", the specific steps of which are as follows: (1) Preparation of breeding materials Using shiitake mushroom '808' as the breeding parent, after fruiting trials, ejected spores were collected when the fruiting bodies of '808' matured, and spore germination was then carried out. After germination, the germinated spores were picked up under a microscope and observed for clamp connections. Single-spore strains without clamp connections were selected as breeding materials, resulting in 35 single-spore strains.

[0031] Hybridization: 35 "808" single-spore strains were passaged, of which 13 showed abnormal or slow growth and were discarded. 22 single-spore strains with good growth were selected and paired with "Yuhua No. 2" shiitake mushrooms in a double-to-single pairing to obtain 14 hybrids.

[0032] (2) Hybridization screening: The 14 hybrids were prepared and cultivated using conventional shiitake mushroom spawn production and cultivation methods. The fruiting of each hybrid was observed using a visual method.

[0033] Observation revealed that the fruiting body morphology of one of the 14 hybrids, numbered J3, was significantly different from that of its two parents. Its fruiting body had a thick stalk, a hemispherical cap, and was difficult to open. Figure 1 As shown. Further observation of fruiting stages verified the stability of the J3 trait.

[0034] (3) Stability test: Fruiting was conducted on J3 for three consecutive years in 2022, 2023, and 2024, and its characteristics were found to be stable, with strong mycelial vigor. The fruiting bodies resembled the unopened cap of the famous wild porcini mushroom. Figure 2As shown: the stipe is thick, the cap does not open easily and is hemispherical. Furthermore, it produces fruiting bodies with good uniformity, high edibility, and a crisp, tender texture; the stipe is also edible, unlike regular shiitake mushrooms where the stipe is discarded before consumption. Therefore, this hybrid was named *Lentinula edulis* J3.

[0035] The Boletus edulis J3 specimen was deposited on May 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, with accession number CCTCC NO:M 20251141.

[0036] Example 2 Embodiment 2 of the present invention provides a method for the productive cultivation of Boletus edulis J3, the specific steps of which are as follows: (1) Preparation of J3 strain of Boletus edulis Take the J3 strain of Boletus edulis from step (1), activate it twice on PDA medium, transfer it to the conventional medium of shiitake mushroom spawn, and prepare the spawn and cultivation spawn at 23℃~25℃.

[0037] (2) Cultivation Using standard shiitake mushroom culture medium, 15cm × 55cm polypropylene plastic bags were selected. A moisture-retaining film was not required when filling the bags. *Pleurotus ostreatus* J3 was inoculated to obtain spawn bags. After approximately 110 days of cultivation, the spawn bags were transferred to a fruiting shed for fruiting management. To ensure the quality of the fruiting bodies, the temperature should not exceed 20℃, and the humidity should be controlled at 80%–95%. When the mushroom buds appear, incisions were made at the small buds to allow fruiting, controlling the number of buds to approximately 10. Results were as follows... Figure 2 As shown.

[0038] Figure 2 The results show that the fruiting bodies of *Lentinula edodes* J3 are scattered, clustered, or grouped, and do not easily open. The caps are hemispherical, reddish-brown or light brown, thick, with firm, white flesh. The stems can be over 3 cm thick, and a single fruiting body weighs 30-60 grams. The fruiting bodies have a good taste, and the fresh mushroom stems are edible, increasing the edible rate of *Lentinula edodes* fruiting bodies (at least half of the stem should be removed from fresh *Lentinula edodes*, or almost the entire stem should be removed when dried), demonstrating excellent potential for development and application.

[0039] (3) Detection of stipe size Thirty mature fruiting bodies were randomly selected, and the diameter of the stipe was measured. At the same time, the diameter of the stipe of the same number of mature fruiting bodies of parent 808 and Yuhua No. 2 was measured. In addition, the diameter of the stipe of the fruiting bodies of shiitake mushrooms for sale on the shelf of Yonghui Supermarket in Huai'an Peninsula, Cangshan District, Fuzhou City was randomly measured. The results are shown in Table 1.

[0040] Table 1. Diameter of the stipe of shiitake mushroom fruiting bodies

[0041] As can be seen from Table 1, the fruiting body of "Boletus japonicus J3" has the thickest stipe, with an average of 33.76 mm, which is much larger than the stipe diameter of the two parent varieties, and also much larger than the stipe diameter of the fruiting bodies of shiitake mushrooms sold in the market.

[0042] Example 3 Example 3 of this invention provides a molecular marker identification method for Leucospira foetida J3, the specific steps of which are as follows: (1) DNA extraction Genomic DNA was extracted from lentil J3 and its parents “808” and “Yuhua 2” using the CTAB method (refer to 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), and stored at -20℃ for later use.

[0043] (2) Primer design Genomic DNA from strains such as Boletus edulis J3, parent strains "808", and "Yuhua 2" was sent to Wuhan Fraser Gene Information Co., Ltd. for whole-genome next-generation sequencing. The sequencing data volume was 3-4G of clean data. Bioinformatics analysis was performed on these genomic data to identify genes unique to the hybrid Boletus edulis J3 compared to the parent strains "808" and "Yuhua 2". Primers were designed, and the information of the 7 pairs of primers and the length of the amplified fragments are shown in Table 2.

[0044] Table 2 Specific marker amplification information

[0045] In Table 2: Primers J3P1A and J3P1B were able to amplify a 1437 bp marker fragment in the genomes of '808' and 'Boletus edulis J3', while 'Yuhua 2' did not have this marker fragment.

[0046] Primers J3P2A and J3P2B were able to amplify a 1243 bp marker fragment in the genomes of '808' and 'Boletus edulis J3', while 'Yuhua 2' did not have this marker fragment.

[0047] Primers J3P3A and J3P3B were able to amplify a 1004 bp marker fragment in the genomes of '808' and 'Boletus edulis J3', while 'Yuhua 2' did not have this marker fragment.

[0048] Primers J3P4A and J3P4B were able to amplify an 875 bp marker fragment in the genomes of 'Yuhua 2' and 'Boletus japonicus J3', while '808' did not have this marker fragment.

[0049] Primers J3P5A and J3P5B were able to amplify a 992 bp marker fragment in the genomes of 'Yuhua 2' and 'Boletus japonicus J3', while '808' did not have this marker fragment.

[0050] Primers J3P6A and J3P6B can amplify a 548 bp marker fragment in the genomes of 'Yuhua 2' and 'Boletus edulis J3', while '808' does not have this marker fragment.

[0051] Primers J3P7A and J3P7B were able to amplify a 1400 bp marker fragment in the genomes of 'Yuhua 2' and 'Boletus japonicus J3', while '808' did not have this marker fragment.

[0052] (3) Specific fragment PCR amplification The DNA of *Lentinula edodes* J3, parental strains "808", and "Yuhua 2" was amplified by PCR using the seven primer pairs listed in Table 2. The amplification systems were as follows: 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, and 9.3 μL of ddH2O.

[0053] The amplification procedures for the 7 primer pairs are as follows: A. Primers J3P1A and J3P1B-PCR: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 62℃ for 30 sec, extension at 72℃ for 1 min 30 sec, 35 cycles; extension at 2℃ for 10 min.

[0054] B. Primers J3P2A and J3P2B-PCR: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 65℃ for 30 sec, extension at 72℃ for 1 min 30 sec, 35 cycles; extension at 72℃ for 10 min.

[0055] C. Primers J3P3A and J3P3B - PCR: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 65℃ for 30 sec, extension at 72℃ for 1 min 10 sec, 35 cycles; extension at 72℃ for 10 min.

[0056] D. Primers J3P4A and J3P4B-PCR: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 62℃ for 30 sec, extension at 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min.

[0057] E. Primers J3P5A and J3P5B-PCR: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 62℃ for 30 sec, extension at 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min.

[0058] F. Primers J3P6A and J3P6B-PCR: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 62℃ for 30 sec, extension at 72℃ for 40 sec, 35 cycles; extension at 72℃ for 10 min.

[0059] G. Primers J3P7A and J3P7B-PCR: Pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 62℃ for 30 sec, extension at 72℃ for 1 min 30 sec, 35 cycles; extension at 72℃ for 10 min.

[0060] (4) Electrophoretic detection of PCR products Take 5 μL of PCR product and spot it onto a 1.2% agarose gel. Use DL2000 DNA MARK as a fragment size reference. Electrophoresis is performed in 0.5×TBE buffer at 5 V / cm for 30–40 min. After electrophoresis, photograph the gel using a gel imaging system. The electrophoresis results are shown below. Figure 3 As shown.

[0061] Figure 3 The results show that primers P1-P3 are shared fragments between *Lentinula edodes* J3 and "808", distinguishing them from "Yuhua No. 2"; P4-P7 are shared sequences between *Lentinula edodes* J3 and "Yuhua No. 2", distinguishing them from "808". These fragments are markers for *Lentinula edodes* J3; M represents a DNA marker of DL5000. Lane 1 represents the parent "Yuhua No. 2", lane 2 represents *Lentinula edodes* J3, and lane 3 represents "808".

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of porcini mushroom ( Lentinula edodes J3, characterized in that, The aforementioned *Lentinula edodes* J3 was deposited on May 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, with accession number CCTCC NO: M20251141.

2. A protoplast produced from the *Lentinula zedoaria* J3 as described in claim 1.

3. A type of spore produced by the *Lentinula edulis* J3 as described in claim 1.

4. A mycelium produced from the *Lentinula edulis* J3 as described in claim 1.

5. A fruiting body produced from the Boletus edulis J3 as described in claim 1.

6. A mushroom substrate comprising the *Lentinula edodes* J3 as described in claim 1.

7. The application of the Boletus edulis J3 as described in claim 1 in shiitake mushroom breeding.

8. The use of the shiitake mushroom strain as described in claim 1 in the preparation of shiitake fruiting bodies, shiitake mycelia, protoplasts and / or shiitake spores.

9. A primer set for identifying J3 molecular markers in *Lentinula edodes*, characterized in that... The J3 molecular marker identification primer set for *Lentinula zedoaria* includes the primer sequences shown in SEQ ID NO.1 to SEQ ID NO.

14.

10. A method for molecular marker identification of *Lentinula zedoaria* based on the J3 molecular marker identification primer set of claim 9, characterized in that, Includes the following steps: (1) Culture, collection and extraction of mycelium and genomic DNA; (2) DNA fingerprint PCR amplification based on the primer sequences shown in SEQ ID NO.1~SEQ ID NO.14; (3) Electrophoretic detection of PCR products.

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