Morchella M6288 and identification method and application thereof

Abstract

The present application relates to a Morchella eximia strain M6288 (Morchella eximia), the preservation number is GDMCC No:66623. The Morchella eximia strain screened scientifically has the advantages of short total growth cycle, relatively consistent cultivation time, high fruiting body uniformity, concentrated harvesting time, short stipe, no need to cut stipe after harvesting, high nutrient content and high disease resistance. The present application also relates to a specific identification method and specific primer of the Morchella eximia strain M6288, the identification method is simple to operate, does not need to use fruiting body after fruiting, can be identified only by using mycelium, and the identification time is only 2-3 hours, so that the breeding efficiency is improved, and the cost is greatly saved.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically to a morel mushroom strain M6288, its identification method, and its applications. Background Technology

[0002] Morel mushrooms, as a rare edible and medicinal fungus, have become a hot topic in the development of high-value-added specialty agriculture and functional foods due to their unique flavor and rich nutrition, which have led to a growing market demand. In recent years, significant progress has been made in the artificial domestication and breeding of morel mushrooms, with several cultivated varieties gradually achieving large-scale application. Currently, 90% of the commercially available artificially cultivated morel mushrooms on the market are *Morchella esculenta*. However, existing morel strains still face challenges in cultivation, including long growth periods, unstable yields, poor fruiting uniformity, and susceptibility to diseases (especially white mold during the fruiting stage), hindering the development of large-scale production. Therefore, it is necessary to select morel mushrooms with good stability, disease resistance, excellent marketability, and high levels of active ingredients. Furthermore, given the difficulty in distinguishing morel strains by appearance, the market is flooded with counterfeit new varieties, necessitating a rapid and accurate method for variety identification. Summary of the Invention

[0003] The purpose of this invention is to provide a morel strain M6288 (Morchella eximia), with accession number GDMCC No:66623.

[0004] Another object of the present invention is to provide the application of the morel strain of the present invention in the cultivation and breeding of morel mushrooms.

[0005] Another object of the present invention is to provide specific primer combinations, which are any one or a combination of primer combination 1, primer combination 2, primer combination 3, primer combination 4.

[0006] The primer combination 1 includes an upstream primer F with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO.2.

[0007] The primer combination 2 includes an upstream primer F with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO.4.

[0008] The primer combination 3 includes an upstream primer F with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO.6.

[0009] The primer combination 4 includes an upstream primer F with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO.8.

[0010] Another object of the present invention is to provide the application of the specific primer combinations of the present invention in the cultivation and breeding of morel mushrooms.

[0011] Another objective of this invention is to provide a method for identifying morel strain M6288 (Morchella eximia), with accession number GDMCC No:66623, which specifically includes the following steps:

[0012] (1) Using the DNA of the mycelium to be tested as a PCR template, perform a PCR reaction using any one or a combination of primer combination 1, primer combination 2, primer combination 3, primer combination 4, or other primer combinations.

[0013] (2) Detect whether the amplification product shows bands, and identify whether it is morel M6288 based on the number of bands;

[0014] Primer combination 1 includes upstream primer F with the nucleotide sequence shown in SEQ ID NO.1 and downstream primer R with the nucleotide sequence shown in SEQ ID NO.2.

[0015] Primer combination 2 includes upstream primer F with the nucleotide sequence shown in SEQ ID NO.3 and downstream primer R with the nucleotide sequence shown in SEQ ID NO.4.

[0016] Primer combination 3 includes upstream primer F with the nucleotide sequence shown in SEQ ID NO. 5 and downstream primer R with the nucleotide sequence shown in SEQ ID NO. 6.

[0017] Primer combination 4 includes upstream primer F with nucleotide sequence as shown in SEQ ID NO.7 and downstream primer R with nucleotide sequence as shown in SEQ ID NO.8.

[0018] In a preferred embodiment of the present invention, in step (1) of PCR amplification, each 20.0 μL amplification reaction system includes: 10 μL of PCR amplification enzyme premix, 8 μL of sterile distilled water, 0.5 μL each of upstream and downstream primers, and 1 μL of DNA template.

[0019] In the preferred embodiment of the present invention, the PCR amplification program in step (1) is as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 40 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 30 cycles; 72℃ final extension for 5 minutes.

[0020] In a preferred embodiment of the present invention, the method for detecting the amplification product in step (2) is agarose gel electrophoresis.

[0021] In a preferred embodiment of the present invention, when PCR reaction is performed with primer combination 1, M6288 cannot amplify a band, while the other strains all have two bands.

[0022] In a preferred embodiment of the present invention, when PCR reaction is performed with primer combination 2, M6288 has only one band, while the other strains all have two bands.

[0023] In a preferred embodiment of the present invention, when PCR is performed using primer combination 3, the band of M6288 is slightly larger than that of other strains.

[0024] In a preferred embodiment of the present invention, when PCR is performed using primer combination 4, M6288 shows no target band, while the other strains show one band.

[0025] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0026] The present invention employs the following detection method:

[0027] 1. Amino acids: GB 5009.124-2016; Protein: GB 5009.5-2016 Method I; Fat: GB 5009.6-2016 Method I; Determination of total sugar content: GB / T 15672-2009; Determination of dietary fiber content: GB / T 22224-2008.

[0028] 2. The drying rate refers to the percentage of the weight of dried mushrooms to the weight of fresh mushrooms after drying. The calculation formula is: Drying rate = Weight of dried mushrooms ÷ Weight of fresh mushrooms × 100%.

[0029] Compared with the prior art, the beneficial technical effects of the present invention include:

[0030] 1. This invention utilizes wild morel germplasm resources and, through multispore self-pollination and artificial cultivation screening, successfully obtained morel strain M6288, which has the following advantages: First, the morel strain has good fruiting body shape and a high cap ratio, eliminating the need to cut the stem after harvesting, saving more than 10% in losses, resulting in a better overall taste and a high dry weight ratio, which can increase the proportion of nutrients such as amino acids in the fruiting body, showing significant quality advantages in both dried and fresh consumption; Second, it has good stability, can stably produce fruiting bodies in plains and plateaus, and has wide temperature difference adaptability and dual tolerance to low and high temperature fluctuations, with stable yield and traits, making it suitable for morel cultivation and breeding; Third, it has strong disease resistance, with no symptoms of white mold during the fruiting period; Fourth, it has a short total growth cycle, relatively consistent fruiting time, high uniformity of fruiting bodies, and concentrated harvesting time, enabling short-cycle synchronous fruiting and intensive harvesting, thereby reducing costs.

[0031] 2. This invention presents a method for identifying strain M6288 using PCR with specific primer combinations 1-4. The four primer combinations can be used individually or in combination, providing a molecular-level technical tool for strain identification. This identification method is highly specific, simple to operate, and requires no fruiting bodies after fruiting; identification can be performed using only mycelium. The identification time is only 2-3 hours (significantly shorter than the 1-2 weeks of traditional mycelial antagonism tests), improving breeding efficiency while significantly reducing costs.

[0032] Preservation of biological materials

[0033] The strain described in this invention is named M6288 (Morchella eximia), and was deposited on July 2, 2025, with accession number: GDMCC No: 66623. The depository is Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province. Attached Figure Description

[0034] Figure 1 The resistance of morel strains to white mold infection in this invention: Left: M6288, Right: Control strain;

[0035] Figure 2 DNA extraction and electrophoresis detection results;

[0036] Figure 3 Electrophoretic detection of PCR products with different primer sets. Detailed Implementation

[0037] The present invention will be described below with reference to the embodiments. However, the present invention is not limited to the embodiments.

[0038] PDA medium: 200g potato, 20g glucose, 20g agar, 1L water.

[0039] Original culture medium: Soak sawdust in water for 1 day and then take it out. Soak wheat in water for 2-3 days and then take it out. Mix them in a volume ratio of 35% soil, 60% sawdust and 5% wheat, and control the moisture content to 50-55% to obtain the original culture medium.

[0040] Cultivation medium: Soak wheat in water for 2-3 days in advance, and soak rice husks and sawdust in water for 1 day in advance. Mix wheat, soil, rice husks, sawdust and lime in a volume ratio of 30%, 8.5%, 20%, 40%, and 1.5%, and control the moisture content to 50-60% to obtain the cultivation medium.

[0041] Nutrient conversion bag (polypropylene corner-folded bag with specifications of 12 mm × 24 mm): Soak wheat in water for 2-3 days in advance and take it out. Soak rice husks in water for 1 day in advance and take it out. Mix wheat and rice husks in each bag at a volume ratio of 95% and 5% respectively, and control the moisture content to 50-55% to obtain nutrient conversion bag (350-380g / bag).

[0042] Example 1: Screening of Morel strain M6288 of the present invention

[0043] Morel specimens were collected from the wild in Yajiang County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province in 2024. Ascospores were collected, and multispore self-pollination was performed, followed by purification to obtain pure strains. The ITS sequence of the strain was amplified using ITS4 and ITS5 primers, and the ITS sequence of Morchella eximia (accession number KM587970.1) was matched with that of Morchella eximia in NCBI, with a sequence identity of 99.43%.

[0044] Molecular identification has led to the naming of this strain as M6288 (Morchella eximia). It was deposited on July 2, 2025, with accession number GDMCC No: 66623. The depository is Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province.

[0045] Example 2: Production test study of the morel strain M6288 of the present invention

[0046] From November 2024 to January 2025, a production trial of strain M6288 was conducted in Anzhou District, Mianyang City, Sichuan Province (cultivation site altitude approximately 700 meters). The control strain was the conventionally cultivated *Morchella esculenta* spawn provided by Mianyang Haotian Agricultural Technology Co., Ltd. The specific production steps were as follows:

[0047] (1) The morel strain of Example 1 was inoculated onto PDA solid medium and cultured at 25°C for 10 days to obtain a vigorous morel mother culture; about 10 pieces (1 cm × 1 cm) of fungal blocks were cut with a sterile scalpel and inoculated onto the original culture medium and cultured at 22°C for 15 days to obtain the morel original culture.

[0048] (2) Under aseptic conditions, take about 10g of the morel spawn from step (1) and inoculate it into a sterilized culture medium bag. Incubate at 22℃ for 10 days to obtain morel spawn.

[0049] (3) Morel cultivation and fruiting experiment steps: Spread quicklime on the experimental land and plow it. Make ridges with a width of 100cm, unlimited length, and 40cm interval as the standard. Crush the spawn in step (2) mechanically and sow it on the ridge surface. Sow 150kg of spawn per mu. Then cover it with 2-3cm of soil and control the soil moisture at about 20%. About 10 days after sowing, mycelial frost will form and the soil surface will turn white. At this time, place nutrient bags evenly on the ridge surface. The side of the nutrient bag that is in contact with the ground needs to be cut open with a knife so that the mycelium can use nutrients. About 1000kg of nutrient bags are needed per mu. After the nutrient bags are placed, build a small arched shed 50cm high on the ridge and cover it with black plastic film. About 30 days after sowing, the nutrient bags will start to shrivel and the primordia will start to form. Water them to promote fruiting and keep the temperature and humidity constant. The fruiting bodies will grow one after another and be harvested after maturity.

[0050] The results showed that the strain of this invention has an early fruiting period: the spawn can be obtained in about 10 days, the time from sowing to the appearance of the first fruiting body is about 30-40 days, the time for the fruiting body to reach the harvest standard is about 40 days, the total production cycle is about 90 days, and the average yield per mu (667 square meters) can reach 650 kg. The fruiting time is relatively consistent, the fruiting bodies are highly uniform, and the harvesting time is concentrated, which can save at least 60% of labor costs and is suitable for large-scale production and harvesting.

[0051] The morel mushroom strain of this invention exhibits excellent traits such as short stipes and good mushroom shape. The cap / stipe ratio of this morel mushroom is approximately 6, and the cap weight accounts for approximately 90%, which is superior to the control strain (cap / stipe ratio approximately 2.2). The morel mushroom cap has a crisp and tender texture and a rich flavor; an increased cap ratio results in a better overall taste and higher commercial value.

[0052] The morel mushroom strain of this invention produces fruiting bodies with high nutritional content: 10.55% protein, 13116 μg / g amino acids, 442.4 mg / g total sugar, 6.16% fat, and 7.89% dietary fiber. Due to its higher cap ratio and a dry weight ratio of approximately 7.5:1, this strain enhances the overall nutritional content of the fruiting bodies, especially the proportion of amino acids, exhibiting a significant quality advantage in both dried and fresh consumption.

[0053] The morel strain of this invention has strong disease resistance and shows no symptoms of white mold during the fruiting period. Figure 1 (Left), the product yield reached 100%, which is superior to the control strain ( Figure 1 (Right, the infection rate of white mold is about 5%).

[0054] Example 3: Production test study of the morel strain M6288 of the present invention

[0055] The strains isolated from the fruiting bodies cultivated in Example 2 were used for a second production trial (i.e., asexual propagation material) in Xiaojin County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province (cultivation site altitude of about 3100 meters) from late April to late July 2025. The specific production steps were the same as in Example 2.

[0056] The phenotypic expression in the two cultivation trials showed no significant changes, which meets the requirements of the People's Republic of China agricultural industry standard "Guidelines for Testing the Specificity, Uniformity and Stability of Plant Varieties: Morel" (NY / T4221-2022): "If the phenotypic expression of the next batch of asexual propagation material of a morel variety is not significantly different from that of the previously provided propagation material, then the variety can be determined to be stable." In other words, strain M6288 has excellent traits and can be stably inherited.

[0057] Example 4: Identification method of Morel strain M6288 of the present invention

[0058] Ten morel mushroom strains were isolated from Sichuan Edible Fungi Research Institute, Chengdu Kechuang Fungi Industry Co., Ltd., Tianfu Fungi Capital Trading Center in Jintang County, Sichuan Province, Morel Mushroom Trading Center in Xiangyang City, Hubei Province, Baijia Market in Chengdu City, Sichuan Province, and Agricultural Trade Market in Jianyang City, Sichuan Province. These morel mushroom strains are highly representative nationwide. The strain information is shown in Table 1.

[0059] Table 1. Strain Information

[0060] .

[0061] The genomes of the above-mentioned strains were resequencing and SNP and InDel site screening was performed to identify strain-specific sites for M6288. Four primer combinations were designed for genome screening, and their sequence information is shown in Table 2 below. The primer combinations were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0062] Table 2 Primer sequences protected by this invention ;

[0063] The identification steps are as follows:

[0064] First, each Morel strain listed in Table 1 was inoculated onto PDA medium and cultured at 25°C for 7 days to obtain mycelium. 100 mg of each mycelium was then used to extract DNA from each strain using a fungal DNA extraction kit (MolPure® Fungal DNA Kit, 18812ES50, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.). The extracted DNA was then subjected to 1% agarose gel electrophoresis. 1% agarose gel preparation: 0.5 g of agarose powder was weighed into an Erlenmeyer flask, 50 mL of 1×TAE buffer was added, and the solution was heated in a microwave oven until clear. 5 μL of nucleic acid dye was added, mixed thoroughly, and then poured onto a gel plate. The electrophoresis results are shown below. Figure 2 As shown (where M is the GoldBand 1 kb DNA ladder (10510ES60), purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; 1-11 are the morel strain numbers in Table 1), the DNA extraction was correct and can be used for subsequent experiments.

[0065] PCR amplification was performed using primer combinations 1 (SEQ ID NO.1 and SEQ ID NO.2), 2 (SEQ ID NO.3 and SEQ ID NO.4), 3 (SEQ ID NO.5 and SEQ ID NO.6), and 4 (SEQ ID NO.7 and SEQ ID NO.8), with the extracted DNA used as the PCR template. The PCR reaction system (20 μL) consisted of: 10 μL of PCR amplification enzyme premix (Taq PCR Mix, 2X, dye-free, B639293, purchased from Sangon Biotech (Shanghai) Co., Ltd.), 8 μL of RNase-free double-distilled water (B541018-0010, purchased from Sangon Biotech (Shanghai) Co., Ltd.), 0.5 μL each of forward and reverse primers, and 1 μL of DNA template. PCR reaction procedure: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 40 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for a total of 30 cycles; final extension at 72℃ for 5 minutes. PCR products were detected by 1% agarose gel electrophoresis.

[0066] The results are as follows Figure 3 The results show that (where M is the GoldBand DL2000 DNA Marker (10501ES60), purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; 1-11 are the Morel strain numbers in Table 1), when using primer combination 1, M6288 (i.e., strain 1) could not amplify a band, while the other strains all had 2 bands; when using primer combination 2, M6288 had only 1 band, while the other strains had 2 bands; when using primer combination 3, the band of M6288 was slightly larger than that of the other strains; when using primer combination 4, M6288 had no band, while the other strains had 1 band.

[0067] The identification method of the present invention is simple to operate, and can be detected using only mycelium; the detection time is only 2-3 hours, which improves detection efficiency and saves costs significantly.

[0068] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A morel strain M6288 (Morchella eximia), with accession number GDMCC No:66623.

2. Specific primer combination, which is any one or a combination of primer combination 1, primer combination 2, primer combination 3, primer combination 4, etc. The primer combination 1 includes an upstream primer F with the nucleotide sequence shown in SEQ ID NO.1 and a downstream primer R with the nucleotide sequence shown in SEQ ID NO.

2. The primer combination 2 includes an upstream primer F with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer R with the nucleotide sequence shown in SEQ ID NO.

4. The primer combination 3 includes an upstream primer F with the nucleotide sequence shown in SEQ ID NO. 5 and a downstream primer R with the nucleotide sequence shown in SEQ ID NO.

6. The primer combination 4 includes an upstream primer F with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer R with a nucleotide sequence as shown in SEQ ID NO.

8.

3. The application of the morel strain M6288 as described in claim 1 and / or the specific primer combination as described in claim 2 in the cultivation and breeding of morel mushrooms.

4. A method for identifying a morel strain M6288, wherein the morel strain M6288 (Morchella eximia), with accession number GDMCC No:66623, specifically includes the following steps: (1) Using the DNA of the mycelium to be tested as a PCR template, perform a PCR reaction using any one or a combination of primer combination 1, primer combination 2, primer combination 3, primer combination 4, or other primer combinations. (2) Detect whether the amplification product shows bands, and identify whether it is morel M6288 based on the number of bands; Primer combination 1 includes upstream primer F with the nucleotide sequence shown in SEQ ID NO. 1 and downstream primer R with the nucleotide sequence shown in SEQ ID NO.

2. Primer combination 2 includes upstream primer F with the nucleotide sequence shown in SEQ ID NO.3 and downstream primer R with the nucleotide sequence shown in SEQ ID NO.

4. Primer combination 3 includes upstream primer F with the nucleotide sequence shown in SEQ ID NO. 5 and downstream primer R with the nucleotide sequence shown in SEQ ID NO.

6. Primer combination 4 includes upstream primer F with nucleotide sequence as shown in SEQ ID NO.7 and downstream primer R with nucleotide sequence as shown in SEQ ID NO.

8.

5. The method as described in claim 4, wherein in step (1) of PCR amplification, each 20.0 μL amplification reaction system comprises: 10 μL of PCR amplification enzyme premix, 8 μL of sterile distilled water, 0.5 μL each of upstream and downstream primers, and 1 μL of DNA template. Preferably, the PCR amplification program in step (1) is as follows: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 40 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 30 cycles; 72℃ final extension for 5 minutes.

6. The method according to any one of claims 4-5, wherein the method for detecting the amplification product in step (2) is agarose gel electrophoresis.

7. The method according to any one of claims 4-6, wherein when PCR is performed with primer combination 1, M6288 cannot amplify a band, while the other strains all have 2 bands.

8. The method according to any one of claims 4-7, wherein when PCR is performed with primer combination 2, M6288 has only one band, while the other strains have two bands.

9. The method according to any one of claims 4-8, wherein when PCR is performed with primer combination 3, the band of M6288 is slightly larger than that of other strains.

10. The method according to any one of claims 4-9, wherein when PCR is performed with primer combination 4, M6288 shows no target band, while the other strains show one band.

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