Five boletus sinensis strains and SSR molecular marker identification method thereof

Abstract

The invention discloses five boletus sinensis strains and an SSR (simple sequence repeat) molecular marker identification method thereof, and relates to the technical field of boletus, and the five boletus sinensis strains are boletus sinensis strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005. The preservation number of the ZJHJSQ001 is CCTCC (China Center For Type Culture Collection) NO: M 20242071, and the ITS sequence of the ZJHJSQ001 is as shown in SEQ NO.1; the preservation number of the ZJHJSQ002 is CCTCC (China Center For Type Culture Collection) NO: M 20242070, and the ITS sequence of the ZJHJSQ002 is as shown in SEQ NO. 2. The combination of the SSR molecular marker primers obtained by the method can conveniently and quickly distinguish boletus sinensis strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005, and has the advantages of saving cost, improving efficiency, being convenient to operate and accurate in result.

Description

Technical Field

[0001] The present application relates to the technical field of boletus, and in particular to five Chinese saprophytic boletus strains and a method for identifying the same using SSR molecular markers. Background Art

[0002] Boletus sinensis ( Buchwaldoboletus xylophilus (Petch) Both&B. Ortiz ), which belongs to the kingdom Fungi fungi , Basidiomycota Basidiomycota , Agaricomycetes Agaricomycotina , Agaricus Agaricomycetes Agaricus Agaricomycetidae , Boletales Boletales , Boletaceae Boletaceae , Saprophytic Boletus Buchwaldoboletus .

[0003] Except for a few species that are poisonous or bitter and cannot be eaten, most species of Boletus are edible. They are usually large in fruiting body, thick in flesh, thick in stalk, sweet and delicious, and rich in nutrition. Boletus contains 8 essential amino acids for the human body, as well as alkaloids such as adenine, choline and putrescine. Boletus has medicinal value and plays a role in treating back and leg pain, numbness of hands and feet, limb convulsions, abnormal vaginal discharge in women, etc.

[0004] Boletus is an ectomycorrhizal fungus that is difficult to grow on artificial culture media. Even if it grows, it is difficult to culture its fruiting bodies in vitro, which is a global problem. Since the mycorrhizal structure is the result of long-term co-evolution, some unclear ecological conditions, nutritional methods, and conditions for the differentiation and development of fruiting bodies make the artificial domestication of many ectomycorrhizal edible fungi, including boletus, very difficult. Once domesticated, it will surely improve the utilization rate of natural resources while also promoting the rapid and healthy growth of forests. Producing fungi from forests and promoting forests with fungi not only creates economic value but also protects the ecological environment.

[0005] Currently, the only boletus that can be cultivated artificially is the dark brown-veined boletus (commonly known as black boletus), but its yield from artificial cultivation is not high and it has a slightly sour and earthy smell when eaten.

[0006] Chinese saprophytic boletus belongs to the Boletus family, but the wild yield is extremely rare, far from enough to meet market demand, which fully reflects the necessity of artificial cultivation. For example, CN202110631616.9 discloses a Chinese saprophytic boletus strain, the deposit number of which is: CGMCC No.21959. When the strain is cultivated for mushroom production, the mycelium growth rate is 8.43mm / d, the mycelium full bag time is 54d, the bag contamination rate is 3.74%, and the fresh mushroom yield is 114.81g / bag. However, the strain does not provide a strain identification method and its indicators, and it is impossible to accurately and reliably monitor and distinguish a variety of Chinese saprophytic boletus. At the same time, the application does not disclose the cultivation materials used in the mushroom bags, the artificial cultivation conditions and the operating methods, and it is impossible to determine which specific planting method should be used to achieve the above-mentioned effects.

[0007] However, due to the scarcity of wild resources, even if wild fungus species are isolated, the yield of artificial cultivation is low and uneven. Therefore, it is extremely important to develop high-quality Chinese saprophytic boletus strains and use molecular biology technology to develop an accurate and effective strain identification system.

[0008] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0009] In response to the above technical problems, the present application provides five strains of Chinese saprophytic boletus and a method for identifying them by SSR molecular markers. The method can accurately identify the five strains of Chinese saprophytic boletus. At the same time, five strains of Chinese saprophytic boletus with uniform mushroom production, thick stipes, high yield and good resistance are disclosed.

[0010] The present application provides ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains of Boletus sinensis; the deposit number of ZJHJSQ001 is CCTCC NO: M 20242071, and the ITS sequence is shown in SEQ NO.1; the deposit number of ZJHJSQ002 is CCTCC NO: M 20242070, and the ITS sequence is shown in SEQ NO.2; the deposit number of ZJHJSQ003 is CCTCC NO: M 20242069, and the ITS sequence is shown in SEQ NO.3; the deposit number of ZJHJSQ004 is CCTCC NO: M 20242068, and the ITS sequence is shown in SEQ NO.4; the deposit number of ZJHJSQ005 is CCTCC NO: M20242067, and the ITS sequence is shown in SEQ NO. Shown in NO.5; The SSR molecular markers used to identify the five strains of Chinese saprophytic boletus were as follows: Boletus022-F sequence is shown in SEQ NO.6; Boletus022-R sequence is shown in SEQ NO.7; Boletus034-F sequence is shown in SEQ NO.8; Boletus034-R sequence is shown in SEQ NO.9; Boletus035-F sequence is shown in SEQ NO.10; Boletus035-R sequence is shown in SEQ NO.11; Boletus057-F: sequence is shown in SEQ NO.12; Boletus057-R sequence is shown in SEQ NO.13; Boletus084-F sequence is shown in SEQ NO.14; Boletus084-R sequence is shown in SEQ NO.15; Boletus093-F sequence is shown in SEQ NO.16; Boletus093-R sequence is shown in SEQ NO.17; Boletus110-F sequence is shown in SEQ NO.18; Boletus110-R sequence is shown in SEQ NO. NO.19; Boletus144-F sequence is shown in SEQ NO.20; Boletus144-R sequence is shown in SEQ NO.21; Boletus191-F sequence is shown in SEQ NO.22; Boletus191-R sequence is shown in SEQ NO.23.

[0011] Preferably, the cultivation method of five strains of Boletus sinensis comprises the following steps: 1) Inoculating at least one of five strains of Chinese saprophytic boletus into the cultivation material of a single container with a liquid culture solution of at least 10 ml / single container to obtain an inoculated cultivation device; 2) After dark culturing the inoculated culture container for 40 to 55 days at 23 to 28°C and 50 to 60% air humidity, a transplanting culture container is obtained; 3) After opening one end of the transplanting culture container and covering the open end with planting soil, after 10-15 days of fruiting management, when the fruiting body grows to 9-12 cm in height, increase the environmental humidity to 90-95%, and continue to cultivate under 120-180 lux for 5-7 days until it matures and can be picked.

[0012] Preferably, the planting soil is prepared by mixing 1 ton of soil and 4 to 6 kg of formaldehyde, stirring the mixture evenly, fumigating the mixture for at least 3 days, and emitting formaldehyde for 1 day before use.

[0013] Preferably, the cultivation material is mixed by weight: 45-55 parts of sawdust, 25-35 parts of cottonseed hulls, 12-14 parts of wheat bran, 4-6 parts of soil, 0.2-0.7 parts of gypsum, 0.2-0.7 parts of lime, and 0.1 parts of potassium dihydrogen phosphate; the moisture content of the cultivation material is 50-65%.

[0014] Preferably, the mushroom production management is to increase the air humidity to 85-90%, the temperature to 23-28°C, and continue culturing in the dark for 10-15 days.

[0015] Another aspect of the present application also provides a method for identifying the five Chinese saprophytic boletus strains as described above using SSR molecular markers, comprising the following steps: Step S1: transferring the mycelium or fruiting body of the tested Boletus sinensis onto a potato dextrose agar solid medium, and collecting the mycelium after culturing at 25° C. for 15 days; Step S2: extracting the genomic DNA of the obtained mycelium, and using a biological spectrophotometer to detect whether the purity and concentration of the obtained total genomic DNA meet the detection requirements, if so, proceed to step S3, if not, increase the mass of the mycelium sample and repeat steps S1-2; Step S3: using the SSR molecular markers as described above to perform PCR amplification of the SSR-marked products from the total genomic DNA obtained in step 2; Step S4: Use capillary electrophoresis to detect the amplification product obtained in step S3, obtain the number of allelic fragments and the molecular weight of each fragment, determine the SSR allelic fragment number combination of the sample to be tested according to the number of allelic fragments and the molecular weight of each fragment, and judge whether the strain to be tested is at least one of the Chinese saprophytic boletus strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 according to the obtained SSR allelic fragment number combination.

[0016] Preferably, the strain that meets the first group of SSR allele number combinations: 2 / (2+3) / (2+3+5) / (3+5) / (4+5) / (2+5) / (2+4+5) / (1+2) / (1+3) in step S4 is the Chinese saprophytic boletus ZJHJSQ001 strain; In the first group of SSR allelic fragment number combinations 2 / (2+3) / (2+3+5) / (3+5) / (4+5) / (2+5) / (2+4+5) / (1+2) / (1+3), "2" is the allelic fragment number 2 of SEQ ID NO: 6-7, with a molecular weight of 188-180.99 bp; "(2+3)" is allelic fragment numbers 2 and 3 of SEQ ID NOs: 8 to 9, the molecular weight of allelic fragment number 2 is 265 to 265.99 bp, and the molecular weight of allelic fragment number 3 is 268 to 268.99 bp; "(2+3+5)" is allelic fragment numbers 2, 3, and 5 of SEQ ID NOs: 10 to 11, the molecular weight of allelic fragment number 2 is 119 to 119.99 bp, the molecular weight of allelic fragment number 3 is 120 to 120.99 bp, and the molecular weight of allelic fragment number 5 is 126 to 126.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 12 to 13, wherein the molecular weight of allelic fragment number 3 is 123 to 123.99 bp, and the molecular weight of allelic fragment number 5 is 130 to 130.99 bp; "(4+5)" refers to allelic fragment numbers 4 and 5 of SEQ ID NOs: 14 to 15, wherein the molecular weight of allelic fragment number 4 is 179 to 179.99 bp, and the molecular weight of allelic fragment number 5 is 185 to 185.99 bp; “ (2+5) ” refers to allelic fragment numbers 2 and 5 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 2 is 212 to 212.99 bp, and the molecular weight of allelic fragment number 5 is 226 to 226.99 bp; “ (2+4+5) ” refers to allelic fragment numbers 2, 4, and 5 of SEQ ID NOs: 18 to 19, wherein the molecular weight of allelic fragment number 2 is 140 to 140.99 bp, the molecular weight of allelic fragment number 4 is 147 to 147.99 bp, and the molecular weight of allelic fragment number 5 is 149 to 149.99 bp; "(1+2)" is allelic fragment numbers 1 and 2 of SEQ ID NOs: 20-21, the molecular weight of allelic fragment number 1 is 229-229.99 bp, and the molecular weight of allelic fragment number 2 is 232-232.99 bp; “ (1+3) ” refers to allelic fragment numbers 1 and 3 of SEQ ID NOs: 22 to 23, wherein the molecular weight of allelic fragment number 1 is 240 to 240.99 bp, and the molecular weight of allelic fragment number 3 is 257 to 257.99 bp; The strain that meets the second group of SSR allele number combination: 3 / (1+3) / (3+6) / (1+4) / 2 / (4+5) / (1+3) / (3+5) / (2+4) in step S4 is the Chinese saprophytic boletus ZJHJSQ002 strain; The second group of SSR allelic fragment number combinations 3 / (1+3) / (3+6) / (1+4) / 2 / (4+5) / (1+3) / (3+5) / (2+4) in which "3" is the allelic fragment number 3 of SEQ ID NO: 6-7, with a molecular weight of 186-186.99 bp; "(1+3)" is allelic fragment numbers 1 and 3 of SEQ ID NOs: 8 to 9, the molecular weight of allelic fragment number 1 is 262 to 262.99 bp, and the molecular weight of allelic fragment number 3 is 268 to 268.99 bp; "(3+6)" refers to allelic fragment numbers 3 and 6 of SEQ ID NOs: 10 to 11, the molecular weight of allelic fragment number 3 is 120 to 120.99 bp, and the molecular weight of allelic fragment number 6 is 127 to 127.99 bp; "(1+4)" refers to allelic fragment numbers 1 and 4 of SEQ ID NOs: 12 to 13, wherein the molecular weight of allelic fragment number 1 is 112 to 112.99 bp, and the molecular weight of allelic fragment number 4 is 124 to 124.99 bp; "2" is the allele number 2 of SEQ ID NO: 14-15, and the molecular weight of the allele number 2 is 173-173.99 bp; "(4+5)" refers to allelic fragment numbers 4 and 5 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 4 is 220 to 220.99 bp, and the molecular weight of allelic fragment number 5 is 226 to 226.99 bp; “ (1+3)” is the allelic fragment numbers 1 and 3 of SEQ ID NOs: 18 to 19, the molecular weight of the allelic fragment number 1 is 134 to 134.99 bp, and the molecular weight of the allelic fragment number 3 is 143 to 143.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 20 to 21, the molecular weight of allelic fragment number 3 is 234 to 234.99 bp, and the molecular weight of allelic fragment number 5 is 270 to 270.99 bp; "(2+4)" refers to allelic fragment numbers 2 and 4 of SEQ ID NOs: 22 to 23, wherein the molecular weight of allelic fragment number 2 is 255 to 255.99 bp, and the molecular weight of allelic fragment number 4 is 272 to 272.99 bp.

[0017] Preferably, the strain that meets the third group of SSR allele number combination: 2 / (2+3) / (3+5) / (3+5) / (4+5) / (2+5) / (2+5) / (1+2) / (1+3) in step S4 is the Chinese saprophytic boletus ZJHJSQ003 strain; The third group of SSR allelic fragment number combinations: 2 / (2+3) / (3+5) / (3+5) / (4+5) / (2+5) / (2+5) / (1+2) / (1+3), in which "2" is the allelic fragment number 2 of SEQ ID NO: 6-7, with a molecular weight of 188-180.99 bp; "(2+3)" refers to allelic fragment numbers 2 and 3 of SEQ ID NOs: 8 to 9, wherein the molecular weight of allelic fragment number 2 is 265 to 265.99 bp, and the molecular weight of allelic fragment number 3 is 268 to 268.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 10 to 11, wherein the molecular weight of allelic fragment number 3 is 120 to 120.99 bp, and the molecular weight of allelic fragment number 5 is 126 to 126.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 12 to 13, wherein the molecular weight of allelic fragment number 3 is 123 to 123.99 bp, and the molecular weight of allelic fragment number 5 is 130 to 130.99 bp; "(4+5)" refers to allelic fragment numbers 4 and 5 of SEQ ID NOs: 14 to 15, wherein the molecular weight of allelic fragment number 4 is 179 to 179.99 bp, and the molecular weight of allelic fragment number 5 is 185 to 185.99 bp; "(2+5)" refers to allelic fragment numbers 2 and 5 of SEQ ID NOs: 16 to 17, wherein the molecular weight of allelic fragment number 2 is 212 to 212.99 bp, and the molecular weight of allelic fragment number 5 is 226 to 226.99 bp; "(2+5)" refers to allelic fragment numbers 2 and 5 of SEQ ID NOs: 18 to 19, wherein the molecular weight of allelic fragment number 2 is 140 to 140.99 bp, and the molecular weight of allelic fragment number 5 is 149 to 149.99 bp; "(1+2)" is allelic fragment numbers 1 and 2 of SEQ ID NOs: 20-21, the molecular weight of allelic fragment number 1 is 229-229.99 bp, and the molecular weight of allelic fragment number 2 is 232-232.99 bp; "(1+3)" refers to allelic fragment numbers 1 and 3 of SEQ ID NOs: 22 to 23, wherein the molecular weight of allelic fragment number 1 is 240 to 240.99 bp, and the molecular weight of allelic fragment number 3 is 257 to 257.99 bp.

[0018] Preferably, the strain conforming to the fourth group of SSR allele number combination: 1 / 3 / 3 / (2+4) / (1+3) / (1+3) / (3+6) / 2 / (3+5) in step S4 is the Chinese saprophytic boletus ZJHJSQ004 strain; In the fourth group of SSR allelic fragment number combinations 1 / 3 / 3 / (2+4) / (1+3) / (1+3) / (3+6) / 2 / (3+5), "1" is the allelic fragment number 1 of SEQ ID NO: 6-7, with a molecular weight of 178-178.99 bp; "3" is the molecular weight of allele number 3 of SEQ ID NO: 8-9, and allele number 3 is 268-268.99 bp; "3" is the allele number 3 of SEQ ID NO: 10-11, and the molecular weight of the allele number 3 is 120-120.99 bp; "(2+4)" refers to allelic fragment numbers 2 and 4 of SEQ ID NOs: 12 to 13, wherein the molecular weight of allelic fragment number 2 is 117 to 117.99 bp, and the molecular weight of allelic fragment number 4 is 124 to 124.99 bp; "(1+3)" is allelic fragment numbers 1 and 3 of SEQ ID NOs: 14 to 15, the molecular weight of allelic fragment number 1 is 165 to 165.99 bp, and the molecular weight of allelic fragment number 3 is 176 to 176.99 bp; "(1+3)" is allelic fragment numbers 1 and 3 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 1 is 204 to 204.99 bp, and the molecular weight of allelic fragment number 3 is 217 to 217.99 bp; "(3+6)" refers to allelic fragment numbers 3 and 6 of SEQ ID NOs: 18 to 19, wherein the molecular weight of allelic fragment number 3 is 143 to 143.99 bp, and the molecular weight of allelic fragment number 6 is 152 to 152.99 bp; "2" is the allele number 2 of SEQ ID NO: 20-21, and the molecular weight of the allele number 2 is 232-232.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 22 to 23, wherein the molecular weight of allelic fragment number 3 is 257 to 257.99 bp, and the molecular weight of allelic fragment number 5 is 287 to 287.99 bp.

[0019] Preferably, the strain that meets the fifth group of SSR allele number combinations: (2+4) / (1+4) / (2+4) / 4 / 5 / (4+6) / (3+4) / (2+4) / (4+6) in step S4 is the Chinese saprophytic boletus ZJHJSQ005 strain; The fifth group of SSR allele number combinations (2+4) / (1+4) / (2+4) / 4 / 5 / (4+6) / (3+4) / (2+4) / (4+6) "(2+4)" is allelic fragment numbers 2 and 4 of SEQ ID NOs: 6 to 7, with a molecular weight of 188 to 180.99 bp, and allelic fragment number 4 has a molecular weight of 238 to 238.99 bp; "(1+4)" is allelic fragment numbers 1 and 4 of SEQ ID NOs: 8 to 9, the molecular weight of allelic fragment number 1 is 262 to 262.99 bp, and the molecular weight of allelic fragment number 4 is 271 to 271.99 bp; "(2+4)" is allelic fragment numbers 2 and 4 of SEQ ID NOs: 10 to 11, the molecular weight of allelic fragment number 2 is 119 to 119.99 bp, and the molecular weight of allelic fragment number 4 is 123 to 123.99 bp; "4" is the allele number 4 of SEQ ID NO: 12-13, and the molecular weight of the allele number 4 is 124-124.99 bp; "5" is the allele number 5 of SEQ ID NO: 14-15, and the molecular weight of the allele number 5 is 185-185.99 bp; "(4+6)" refers to allelic fragment numbers 4 and 6 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 4 is 220 to 220.99 bp, and the molecular weight of allelic fragment number 6 is 227 to 227.99 bp; "(3+4)" refers to allelic fragment numbers 3 and 4 of SEQ ID NOs: 18 to 19, wherein the molecular weight of allelic fragment number 3 is 143 to 143.99 bp, and the molecular weight of allelic fragment number 4 is 147 to 147.99 bp; "(2+4)" refers to allelic fragment numbers 2 and 4 of SEQ ID NOs: 20 to 21, the molecular weight of allelic fragment number 2 is 232 to 232.99 bp, and the molecular weight of allelic fragment number 4 is 240 to 240.99 bp; "(4+6)" refers to allelic fragment numbers 4 and 6 of SEQ ID NOs: 22 to 23, wherein the molecular weight of allelic fragment number 4 is 272 to 272.99 bp, and the molecular weight of allelic fragment number 6 is 297 to 297.99 bp.

[0020] The beneficial effects of this application include: 1) The five strains of Chinese saprophytic boletus and their SSR molecular marker identification method provided in this application, the combination of SSR molecular marker primers obtained by the method can easily and quickly distinguish Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains, achieve the advantages of cost saving, efficiency improvement, easy operation and accurate results, and have good application prospects.

[0021] 2) The five strains of Chinese saprophytic boletus and their SSR molecular marker identification method provided in this application can prevent the selected excellent strains of Chinese saprophytic boletus from being mixed with other similar varieties during production, operation and market circulation, thereby effectively protecting their intellectual property rights. It is of great significance for the authenticity identification of Chinese saprophytic boletus varieties during the production process.

[0022] The new strains of Boletus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 were deposited in China Center for Type Culture Collection (CCTCC) on September 25, 2024, address: Wuhan University, Wuhan, China; postal code: 430072, telephone: (027) 68754052; the classification name of Boletus sinensis ZJHJSQ001 is Boletus sinensis ZJHJSQ001 Buchwaldoboletus xylophilus ZJHJSQ001; the classification name of Chinese saprophytic boletus ZJHJSQ002 is Chinese saprophytic boletus ZJHJSQ002 Buchwaldoboletus xylophilus ZJHJSQ002; the classification name of Chinese saprophytic boletus ZJHJSQ003 is Chinese saprophytic boletus ZJHJSQ003 Buchwaldoboletus xylophilus ZJHJSQ003; the classification name of Chinese saprophytic boletus ZJHJSQ004 is Chinese saprophytic boletus ZJHJSQ004 Buchwaldoboletus xylophilus ZJHJSQ004; the classification name of Chinese saprophytic boletus ZJHJSQ005 is Chinese saprophytic boletus ZJHJSQ005 Buchwaldoboletus xylophilus ZJHJSQ005; The deposit number of ZJHJSQ001 is CCTCC NO: M 20242071, and the ITS sequence is shown in SEQ NO.1; The deposit number of ZJHJSQ002 is CCTCC NO: M 20242070, and the ITS sequence is shown in SEQ NO.2; The deposit number of ZJHJSQ003 is CCTCC NO: M 20242069, and the ITS sequence is shown in SEQ NO.3; The deposit number of ZJHJSQ004 is CCTCC NO: M 20242068, and the ITS sequence is shown in SEQ NO.4; The deposit number of ZJHJSQ005 is CCTCC NO: M 20242067, and the ITS sequence is shown in SEQ NO.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Phylogenetic tree of the Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains provided in this application; Figure 2 The relative molecular weight peak graph of the allele sites detected by the primer Boletus022 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Figure 3 The relative molecular weight peak graph of the allele sites detected by the primer Boletus034 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Figure 4 The relative molecular weight peak graph of the allele sites detected by the primer Boletus035 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Figure 5 The relative molecular weight peak graph of the allele sites obtained by sequentially detecting the primer Boletus057 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Figure 6 The relative molecular weight peak graph of the allele sites detected by the primer Boletus084 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Figure 7 The relative molecular weight peak graph of the allele sites detected by the primer Boletus093 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Figure 8 The relative molecular weight peak graph of the allele sites detected by the primer Boletus110 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Fig. 9The relative molecular weight peak graph of the allele sites detected by the primer Boletus144 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Fig.10 The relative molecular weight peak graph of the allele sites detected by the primer Boletus191 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005; Fig.11 The UPGMA clustering tree of genetic distances of five Chinese saprophytic boletus strains constructed based on SSR molecular markers provided in this application; Fig.12 The photos of artificially cultivated Boletus sinensis provided for this application are: a) is an enlarged view of a relatively small fruiting body; b) is a photo of fruiting bodies growing out of a fungus bag; c) is a photo of larger fruiting bodies growing out of a fungus bag; d) is a photo of a single larger fruiting body being weighed, showing that its weight is 161.2 g. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0025] Example Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0026] Example 1 Obtaining the strains of Boletus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 The specific steps are as follows: (1) The fruiting bodies of Chinese saprophytic boletus ZJHJSQ001 and ZJHJSQ002 were collected in Sanya, Hainan on May 10, 2020; the fruiting bodies of Chinese saprophytic boletus ZJHJSQ003 were collected in Sanya, Hainan on June 10, 2020; the fruiting bodies of Chinese saprophytic boletus ZJHJSQ004 were collected in Dongfang City, Hainan on April 10, 2020; the fruiting bodies of Chinese saprophytic boletus ZJHJSQ005 were collected in Pu'er, Yunnan on September 12, 2024; the obtained fruiting bodies were numbered, cleaned, and placed in an ultra-clean workbench; (2) Use a clean scalpel to cut the inside of the fruiting body into small pieces, pick out a piece, and inoculate it into a sterilized PDA plate; the composition of the PDA plate culture medium is 20g potato, 20g glucose, 20g agar powder, 1g peptone, and 1g agar powder.

[0027] (3) The plates were kept at a constant temperature of 25°C and away from light for 14 days to obtain Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005.

[0028] Example 2 Molecular Identification of Boletus sinensis Strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 The ITS sequences of “Sinophytic Boletus sinensis strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005” were obtained, identified and preserved according to the following steps: (1) DNA extraction The test strain was transferred to potato dextrose agar solid medium (20 g potatoes, 20 g glucose, 20 g agar powder, 1 g peptone, 1 g agar powder), and cultured at 25°C for 15 days, then the mycelium was collected; the genomic DNA of the mycelium was extracted using the Qingke TSP101-200 kit, and the purity and concentration of the obtained DNA were detected by agarose gel electrophoresis and biospectrophotometer.

[0029] (2) ITS amplification and detection PCR amplification was performed using ITS universal primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3' sequence as shown in SEQ NO.25) and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3' sequence as shown in SEQ NO.24). The 25μL amplification system included: 20ul of Gold Mix (green, Qingke TSE101), 0.5μmol / L upstream and downstream primers, and 50ng of genomic template DNA. The PCR amplification program was: 94℃2 minutes; 94℃15s, 60℃30s, 72℃60s, 30 cycles; 72℃10 minutes. After the PCR was completed, 5μL of 6× loading buffer was added and mixed, and the results were detected by 1.0% agarose gel electrophoresis. After the test was qualified, sequencing was performed.

[0030] (3) Sequence comparison and phylogenetic analysis The obtained ITS sequences were compared with the NCBI database using BLAST software, and the identities of the corresponding sequences of various strains of Chinese saprophytic boletus in the database were all over 97%. At the same time, the obtained "Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains" were deposited in the China Center for Type Culture Collection (CCTCC) on September 25, 2024.

[0031] The deposit number of strain ZJHJSQ001 is CCTCC NO: M 20242071, and the ITS sequence is shown in SEQ NO.1: gcctttgcct tagtcctcgt tctcttttct cttttgcgtc gatccgttct ctcacacacc 60 tgtgcacctg ttgtaggttt tctcgagaga ggaaacctat gtttttcata aacacctttg 120 tatgtctata gaatgtctat gttgacgtcg accactgggc gacgttaaac aaaactatga 180 acaactttca gcaacggatc tcttggctct cgcatcgatg aagaacgcag cgaattgcga 240 taagtaatgt gaattgcaga ttttcagtga atcatcgaat ctttgaacgc accttgcgct 300 ccttggtatt ccgaggagca tgcctgtttg agtgtcattg aattctcaac catgtcttga 360 tgtcgtcgag cgcatggctt ggatgtgggg gttgctggcg gcgcgagctg tcggctctcc 420 ttaaatgcat tagcaaaggg cgagcctttc gtgcacggcc ttcgacgtga taatgatcgt 480 cgtggctgga gcggaaggca tgaatcgtcg ttgcttctaa tctctgatcc ttt 533 The preservation number of ZJHJSQ002 is CCTCC NO: M 20242070, and its ITS sequence is shown in SEQNO.2: aggggggagt cgagcgggcg aagtttggtc gaggttgtcg ctggcccttc ggggcatgtt 60 gcacgccttt gccttagtcc tcgttctctt ttctcttttg cgtcgatccg ttctctcaca 120 cacctgtgca cctgttgtag gttttctcga gagaggaaac ctatgttttt cataaacacc 180 tttgtatgtc tatagaatgt ctatgttgac gtcgaccact gggcgacgtt aaacaaaact 240 atgaacaact ttcagcaacg gatctcttgg ctctcgcatc gatgaagaac gcagcgaatt 300 gcgataagta atgtgaattg cagattttca gtgaatcatc gaatctttga acgcaccttg 360 cgctccttgg tattccgagg agcatgcctg tttgagtgtc attgaattct caaccatgtc 420 ttgatgtcgt cgagcgcatg gcttggatgt gggggttgct ggcggcgcga gctgtcggct 480 ctccttaaat gcattagcaa agggcgagcc tttcgtgcac ggccttcgac gtgataatga 540 tcgtcgtggc tggagcggaa ggcatgaatc gtcgttgctt ctaatctctg atccttttgg 600 gatcgctttc gaaacttgac ctcaaatcag gtaggactac ccgctgaact taagcatatc 660 The deposit number of ZJHJSQ003 is CCTCC NO: M 20242069, and the ITS sequence is shown in SEQ NO.3: gcctttgcct tagtcctcgt tctcttttct cttttgcgtc gatccgttct ctcacacacc 60 tgtgcacctg ttgtaggttt tctcgagaga ggaaacctat gtttttcata aacacctttg 120 tatgtctata gaatgtctat gttgacgtcg accactgggc gacgttaaac aaaactatga 180 acaactttca gcaacggatc tcttggctct cgcatcgatg aagaacgcag cgaattgcga 240 taagtaatgt gaattgcaga ttttcagtga atcatcgaat ctttgaacgc accttgcgct 300 ccttggtatt ccgaggagca tgcctgtttg agtgtcattg aattctcaac catgtcttga 360 tgtcgtcgag cgcatggctt ggatgtgggg gttgctggcg gcgcgagctg tcggctctcc 420 ttaaatgcat tagcaaaggg cgagcctttc gtgcacggcc ttcgacgtga taatgatcgt 480 cgtggctgga gcggaaggca tgaatcgtcg ttgcttctaa tctctgatcc ttttggggatc 540 gct 543 The deposit number of ZJHJSQ004 is CCTCC NO: M 20242068, and the ITS sequence is shown in SEQ NO.4: aggggggaag tcgagcgggc gaagtttggt cgaggttgtc gctggccctt cggggcatgt 60 tgcacgcctt tgccttagtc ctcgttctct tttctctttt gcgtcgatcc gttctctcac 120 acacctgtgc acctgttgta ggttttctcg agagaggaaa cctatgtttt tcataaacac 180 ctttgtatgt ctatagaatg tctatgttga cgtcgaccac tgggcgacgt taaacaaaac 240 tatgaacaac tttcagcaac ggat 264 The deposit number of ZJHJSQ005 is CCTCC NO: M 20242067, and its ITS sequence is shown in SEQNO.5: cgcctttgcc ttagtcctcg ttctcttttc tcttttgcgt cgatccgttc tctcacacac 60 ctgtgcacct gttgtaggtt ttctcgagag aggaaaccta tgtttttcat aaacaccttt 120 gtatgtctat agaatgtcta tgttgacgtc gaccactggg cgacgttaaa caaaactatg 180 aacaactttc agcaacggat ctcttggctc tcgcatcgat gaagaacgca gcgaattgcg 240 ataagtaatg tgaattgcag attttcagtg aatcatcgaa tctttgaacg caccttgcgc 300 tccttggtat tccgaggagc atgcctgttt gagtgtcatt gaattctcaa ccatgtcttg 360 atgtcgtcga gcgcatggct tggatgtggg ggttgctggc ggcgcgagct gtcggctctc 420 cttaaatgca ttagcaaagg gcgagccttt cgtgcacggc cttcgacgtg ataatgatcg 480 tcgtggctgg agcggaaggc atgaatcgtc gttgcttcta atctctgatc cttttgggat 540 At the same time, the saprophytic boletus species and wood-growing microsaprophytic boletus ( Buchwaldoboletus lignicola ) and used MEGA7 software to construct the NJ phylogenetic tree with default parameters and 1000 Bootstrap tests to obtain the phylogenetic tree results.

[0032] Depend on Figure 1 It can be seen that the ITS sequences of strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 and Boletus sinensis are clustered into one branch with a support rate of 100%.

[0033] Based on the above results, it can be concluded that strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 belong to the Boletaceae family ( Boletaceae ), Buchwaldoboletus xylophilus of the genus Buchwaldoboletus.

[0034] Example 3 Identification of ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains using SSR molecular markers On August 22, 2024, the gene sequences SEQ NO.1~5 and their biological samples were submitted to Wuhan Tianyi Huayu Gene Technology Co., Ltd. (Address: 3F-4F, Building C12, Phase 3, Biomedical Enterprise Accelerator, No. 388, Gaoxin 2nd Road, Jiangxia District, Wuhan) for SSR genetic diversity analysis. The analysis was performed using a 96-channel fully automatic ABI 3730xL genetic analyzer, and 9 pairs of SSR molecular markers were screened: Boletus022-F sequence is shown in SEQ NO.6; Boletus022-R sequence is shown in SEQ NO.7; Boletus034-F sequence is shown in SEQ NO.8; Boletus034-R sequence is shown in SEQ NO.9; Boletus035-F sequence is shown in SEQ NO.10; Boletus035-R sequence is shown in SEQ NO.11; Boletus057-F: sequence is shown in SEQ NO.12; Boletus057-R sequence is shown in SEQ NO. NO.13; Boletus084-F sequence is shown in SEQ NO.14; Boletus084-R sequence is shown in SEQ NO.15; Boletus093-F sequence is shown in SEQ NO.16; Boletus093-R sequence is shown in SEQ NO.17; Boletus110-F sequence is shown in SEQ NO.18; Boletus110-R sequence is shown in SEQ NO.19; Boletus144-F sequence is shown in SEQ NO.20; Boletus144-R sequence is shown in SEQ NO.21; Boletus191-F sequence is shown in SEQ NO.22; Boletus191-R sequence is shown in SEQ NO.23, and the primer sequence information is shown in Table 1.

[0035] The screening experiment process is the same as the existing operation, including the following steps: 1. Sample sorting and verification; 2. Nucleic acid extraction and testing; 3. Synthesis of typing primers; 4. Fluorescence PCR amplification; 5. Primer polymorphism screening; 6. Preliminary experiment of population typing; 7. Population typing test; 8. Result analysis; The specific steps include: (1) Simplify genome development and SSR markers A library with an insert size of 400 was constructed. Paired-end (PE) sequencing was performed on five saprophytic boletus strains, ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005, using the next-generation sequencing technology (NGS) based on the Illumina NovaSeq sequencing platform and the double-end PE500 sequencing scheme. 1G of raw data was sequenced for each strain. The library construction and sequencing was in double-end sequencing mode. The data of all samples were merged into popA, and then FLASH (version 1.2.11) (Magoc and Salzberg2011) was used to overlap the sequences into one high-quality read. The parameters were set as follows: (1) Min overlap: 10; (2) Max mismatch density: 0.2; (3) Allow "outie" pairs: false; According to the above parameters, the sequences are merged to obtain the sequence results.

[0036] Microsatellite identification tool (MISA) (http: / / pgrc.ipk-gatersleben.de / misa / ) was used to search for SSR loci in all sequences. The search parameters used were as follows: mono-10, di-6, tri-5, Tetra-5, penta-5, hexa-5, and the maximum interval allowed between two different SSRs in the composite sequence was set to 100 bp.

[0037] The data of five Boletus saprophyticus strains were merged into popA. A total of 40,810 SSRs were found in 9,356,338 sequences. Among all the sequences with SSRs, 991 sequences contained more than one SSR. The number of SSRs in a composite form was 947. Through the SSR locus resequence type, number of repetitions, and polymorphism in the population, 192 SSR loci shared by the five strains and with polymorphism were screened as candidate SSR loci for the development of polymorphic markers.

[0038] Through fluorescence capillary electrophoresis technology, the linker method was used to verify and screen the candidate sites and obtain 9 polymorphic markers for variety differentiation; the specific information of the 9 SSR sites is shown in the following table: Table 1 SSR marker primer information Example 4 Identification method of Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains This embodiment sets 5 treatments, and the tested strains of each treatment are Boletus sinensis ZJHJSQ001~5: 1. Experimental Methods (1) DNA extraction The tested strains were transferred to potato dextrose agar solid medium and cultured at 25°C for 15 days before collecting mycelium. The genomic DNA of the mycelium was extracted using the Qingke TSP101-200 kit, and the purity and concentration of the obtained DNA were detected by agarose gel electrophoresis and biospectrophotometer at a wavelength of 280 nm. The DNA of each strain was used for PCR amplification in step (2).

[0039] (2) PCR amplification Using the 9 pairs of SSR molecular markers obtained in Example 3, respectively, the DNA of each strain extracted in step (1) was subjected to PCR amplification of the SSR markers to obtain amplified products; The PCR amplification system was: total volume 20ul, including: Qingke TSE101-Gold Medal Mix (green) 16.45ul, 10 μMTag DNA enzyme 1.2ul, 10umol / L SSR marker forward primer and reverse primer 1.2uL each, template DNA 1uL. PCR reaction conditions: 98℃ 2 minutes; 98℃ 10s, 60℃ 10s, 72℃ 10s, 35 cycles; 72℃ 5 minutes. To ensure the accuracy of identification, a total of three repeated experiments were performed.

[0040] (3) Detect amplification products by capillary electrophoresis: ABI HiDi Formamide buffer and GeneScan 500LIZ Size Standard internal standard reagent were mixed at a volume ratio of 130:1 to prepare a mix; the mix was dispensed into a 96-well reaction plate, and 10ul of the mix was added to each well; 0.5ul of sample template (the sample was the amplified product) was added to the 96-well plate, and the plate was centrifuged at 4000 rpm and then stopped; the mixing plate was pre-denatured at 95°C for 5 minutes using a metal bath heater, and immediately placed at -20°C after being taken out; after cooling, the plate was taken out, centrifuged at 4000 rpm, thawed, and mixed to obtain the sample to be tested; a 3730 sequencer was used for capillary tube electrophoresis, with 2ul of sample and 6ul of bromophenol blue added, a voltage of 300V, and a time of 12 minutes.

[0041] The amplification product detection results of Chinese saprophytic boletus ZJHJSQ001~5 under 9 pairs of SSR molecular markers: Boletus022, Boletus034, Boletus035, Boletus057, Boletus084, Boletus093, Boletus110, Boletus144, Boletus191 were obtained respectively. The results are shown in the relative molecular weight peak diagram of the allele site. Figures 2~10 shown.

[0042] 2. Result analysis: (1) Comparative analysis of peak graphs of capillary electrophoresis fluorescence detection By comparing Figures 2~10 It can be seen that the peaks of each SSR fluorescent marker of the five bacterial species are clearly identifiable. Figure 2 The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 181, 187, 181, 179, 181+239, respectively; Figure 3 The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 266+269, 263+269, 266+269, 269, 263+272, respectively; Figure 4 The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 120+121+127, 121+128, 121+127, 121, 118+124, respectively; Figure 5 The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 124+131, 113+125, 124+131, 118+125, 125, respectively; Figure 6The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 180+186, 174, 180+186, 166+177 and 186, respectively; Figure 7 The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 213+227, 221+227, 213+227, 205+218, 221+228, respectively; Figure 8 The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 141+148+150, 135+144, 141+150, 144+153 and 144+148, respectively; Fig. 9 The results of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022, the sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 230+233, 235+271, 230+233, 233, 233+241, respectively; Fig.10 This is the result of amplifying Boletus sinensis ZJHJSQ001~5 with primer Boletus022. The fragment sizes corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 241+258, 256+273, 241+258, 258+288 and 273+298, respectively.

[0043] Figures 2~10 The results show that the 9 pairs of SSR molecular marker primer combinations provided by the present invention can accurately distinguish the ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains.

[0044] (2) Analysis of band combinations amplified by 9 pairs of SSR primers The 9 pairs of SSR primers were used to amplify allelic fragments in the tested Boletus sinensis ZJHJSQ001-5, which were encoded according to the molecular weight and numbered.

[0045] The strain that meets the first group of SSR allele number combination of 2 / (2+3) / (2+3+5) / (3+5) / (4+5) / (2+5) / (2+4+5) / (1+2) / (1+3) is the Chinese saprophytic boletus ZJHJSQ001 strain; The strain that meets the second group of SSR allele number combination of 3 / (1+3) / (3+6) / (1+4) / 2 / (4+5) / (1+3) / (3+5) / (2+4) is the Chinese saprophytic boletus ZJHJSQ002 strain; The strain that met the third group SSR allele number combination of 2 / (2+3) / (3+5) / (3+5) / (4+5) / (2+5) / (2+5) / (1+2) / (1+3) was the Chinese saprophytic boletus ZJHJSQ003 strain; The strain that meets the fourth group SSR allele number combination of 1 / 3 / 3 / (2+4) / (1+3) / (1+3) / (3+6) / 2 / (3+5) is the Chinese saprophytic boletus ZJHJSQ004 strain; The strain that meets the fifth group SSR allele number combination of (2+4) / (1+4) / (2+4) / 4 / 5 / (4+6) / (3+4) / (2+4) / (4+6) is the Chinese saprophytic boletus ZJHJSQ005 strain.

[0046] The information on the number of allele fragments and molecular weight of the above 9 pairs of SSR primers is shown in Table 2: Table 2 (3) Cluster analysis The genetic diversity of five Chinese saprophytic boletus strains was analyzed based on the polymorphic fragments amplified by 9 pairs of SSRs. Based on Nei's genetic distance, the UPGMA clustering tree of the five Chinese saprophytic boletus strains was constructed ( Fig.11 ). Specific method: Get the similarity matrix or distance matrix in the similarity module of NTSYS software, and then get the clustering tree in the clustering module. Fig.11 It can be seen that the 9 pairs of primers provided by the present invention clustered the Chinese saprophytic boletus ZJHJSQ003 and ZJHJSQ001 into one group, the other ZJHJSQ002 and ZJHJSQ004 strains were clustered into one group respectively, and the Chinese saprophytic boletus ZJHJSQ005 strain was a separate branch and was completely distinguished from the other strains.

[0047] It can be seen that the 9 pairs of SSR primer combinations provided by the present invention can be applied to the identification of Chinese saprophytic boletus ZJHJSQ001-5 and can accurately distinguish Chinese saprophytic boletus ZJHJSQ001-5.

[0048] Based on the above content, it can be seen that the SSR molecular markers provided by the present invention have high polymorphism and good stability, can be used for the identification of Boletus sinensis ZJHJSQ001-5, population genetic diversity analysis, etc., and have good application value.

[0049] Example 5 Bag material cultivation method of the Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains This embodiment sets 5 treatments, and the liquid bacteria used in each treatment are Boletus sinensis ZJHJSQ001-5.

[0050] 1) Prepare the cultivation material (parts by weight): weigh the raw materials according to the cultivation material ratio of 50 parts of sawdust, 30 parts of cottonseed hulls, 13.9 parts of wheat bran, 5 parts of soil, 0.5 parts of gypsum, 0.5 parts of lime, and 0.1 parts of potassium dihydrogen phosphate, add water and mix well to make the water content 55%; 2) Sterilization of material loading: Put the prepared culture material into a culture bag (specification 15*30*5 silk), sterilize with high pressure steam (sterilize at 121℃ for 15min) and cool to room temperature; 3) Inoculation: The inoculation volume of the liquid bacteria is 10 ml / bag. The liquid bacteria are inoculated into a cultivation bag cooled to room temperature. Under the conditions of 25°C and air humidity of 55%, dark culture is carried out for 45 days to obtain a transplanting bacteria bag; Preparation of liquid culture: Take the plate strain and inoculate it into a culture solution of 20 g / L potato, 20 g / L glucose, 3 g / L peptone, 1 g / L yeast powder, 1 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate, and culture it at 25°C for 4 days to obtain the liquid culture.

[0051] 4) Covering with soil: Mix the soil with formaldehyde water (mix 1 ton of soil with 5 kg of formaldehyde) and fumigate for 3 days in advance. Emit formaldehyde for 1 day before use to get planting soil. Open one end of the transplanting fungus bag covered with mycelium and cover the surface of the open end with 1 cm of planting soil. 5) Management of mushroom production: Increase the air humidity to 89%, the temperature to 25°C, provide adequate ventilation, and continue to cultivate in the dark for 13 days; 6) When the fruiting bodies of Boletus sinensis grow to 10cm in height, increase the humidity to 95% and continue to cultivate under 160lux for 6 days until they are mature and can be harvested. Fig.12As shown in a) to d). As can be seen from the photos, the strain has good growth, thick stipes, uniform mushroom production and high yield. The single weight of the largest fruiting body can reach 161.2g.

[0052] After harvest, statistics showed that the biological efficiency of ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 were 46%, 49%, 47%, 48% and 50%, respectively.

[0053] Example 6 Bottle cultivation method of Boletus sinensis strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 The difference from Example 5 is: Prepare 0.08 parts by weight of potassium dihydrogen phosphate in the cultivation material, add water and mix well to make the water content 50%; Sterilization of material loading: put the prepared culture material into the culture bottle (1100ml), sterilize it with high pressure steam and cool it to room temperature; According to statistics, the biological efficiency of ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 were 46%, 48%, 46%, 47% and 49% respectively.

[0054] Example 7 The difference from Example 5 is: Prepare the cultivation material (parts by weight): weigh the raw materials according to the cultivation material ratio of 45 parts of sawdust, 25 parts of cottonseed hulls, 12 parts of wheat bran, 4 parts of soil, 0.2 parts of gypsum, 0.2 parts of lime, and 0.1 parts of potassium dihydrogen phosphate, add water and mix well to make the water content 50%; The inoculum volume was 20 ml per container.

[0055] In step 3), dark culture was performed at 23°C and 50% humidity for 40 days to obtain a transplanting fungus bag; Step 4) Mix soil with formaldehyde water (mix 1 ton of soil with 4 kg of formaldehyde); Step 5) Fruiting management: Increase the air humidity to 85%, the temperature to 23°C, provide adequate ventilation, and continue to cultivate in the dark for 10 days; Step 7) When the fruiting bodies of Boletus sinensis grow to 9 cm in height, increase the environmental humidity to 90% and continue to culture under light for 5 days until they are mature and can be picked.

[0056] Example 8 The difference from Example 5 is: Prepare the cultivation material (parts by weight): weigh the raw materials according to the cultivation material ratio of 55 parts of sawdust, 35 parts of cottonseed hulls, 14 parts of wheat bran, 6 parts of soil, 0.7 parts of gypsum, 0.7 parts of lime, and 0.1 parts of potassium dihydrogen phosphate, add water and mix well to make the water content 65%; The inoculum volume was 20 ml per container.

[0057] In step 3), the transplanting bacteria bag is obtained by dark culturing at 28°C and 60% air humidity for 55 days; Step 4) Mix soil with formaldehyde water (mix 1 ton of soil with 6 kg of formaldehyde); Step 5) Fruiting management: Increase the air humidity to 90%, the temperature to 28°C, provide adequate ventilation, and continue to cultivate in the dark for 15 days; Step 7) When the fruiting bodies of Boletus sinensis grow to 12 cm in height, increase the environmental humidity to 95% and continue to culture under light for 7 days until they are mature and can be picked.

[0058] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Five strains of Chinese saprophytic boletus, characterized in that: The strains are ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 of Boletus sinensis; the deposit number of ZJHJSQ001 is CCTCC NO: M 20242071, and the ITS sequence is shown in SEQ NO.1; the deposit number of ZJHJSQ002 is CCTCC NO: M 20242070, and the ITS sequence is shown in SEQ NO.2; the deposit number of ZJHJSQ003 is CCTCC NO: M 20242069, and the ITS sequence is shown in SEQ NO.3; the deposit number of ZJHJSQ004 is CCTCC NO: M 20242068, and the ITS sequence is shown in SEQ NO.4; the deposit number of ZJHJSQ005 is CCTCC NO: M 20242067, and the ITS sequence is shown in SEQ NO. Shown in NO.5; The SSR molecular markers used to identify the five strains of Boletus sinensis were as follows: Boletus022-F sequence is shown in SEQ NO.6; Boletus022-R sequence is shown in SEQ NO.7; Boletus034-F sequence is shown in SEQ NO.8; Boletus034-R sequence is shown in SEQ NO.9; Boletus035-F sequence is shown in SEQ NO.10; Boletus035-R sequence is shown in SEQ NO.11; Boletus057-F: sequence is shown in SEQ NO.12; Boletus057-R sequence is shown in SEQ NO.13; Boletus084-F sequence is shown in SEQ NO.14; Boletus084-R sequence is shown in SEQ NO.15; Boletus093-F sequence is shown in SEQ NO.16; Boletus093-R sequence is shown in SEQ NO.17; Boletus110-F sequence is shown in SEQ NO.18; Boletus110-R sequence is shown in SEQ NO.

19. NO.19; Boletus144-F sequence is shown in SEQ NO.20; Boletus144-R sequence is shown in SEQ NO.21; Boletus191-F sequence is shown in SEQ NO.22; Boletus191-R sequence is shown in SEQ NO.

23.

2. The five strains of Boletus sinensis according to claim 1, characterized in that: The cultivation method of five Chinese saprophytic boletus strains comprises the following steps: 1) Inoculating at least one of five strains of Chinese saprophytic boletus into the cultivation material of a single container with a liquid culture solution of at least 10 ml / single container to obtain an inoculated cultivation device; 2) After dark culturing the inoculated culture container at 23-28°C and 50-60% air humidity for 40-55 days, a transplanting culture container is obtained; 3) After opening one end of the transplanting culture container and covering the open end with planting soil, after 10-15 days of fruiting management, when the fruiting body grows to 9-12 cm in height, increase the environmental humidity to 90-95%, and continue to cultivate under 120-180 lux for 5-7 days until it matures and can be picked.

3. The five strains of Chinese saprophytic boletus according to claim 2, characterized in that: Planting soil is prepared by mixing 1 ton of soil and 4-6 kg of formaldehyde, stirring evenly, fumigating for at least 3 days, and emitting formaldehyde for 1 day before use.

4. The five strains of Boletus sinensis according to claim 2, characterized in that: The cultivation material is mixed by weight: 45-55 parts of sawdust, 25-35 parts of cottonseed hulls, 12-14 parts of wheat bran, 4-6 parts of soil, 0.2-0.7 parts of gypsum, 0.2-0.7 parts of lime, and 0.1 parts of potassium dihydrogen phosphate; the moisture content of the cultivation material is 50-65%.

5. The five strains of Chinese saprophytic boletus according to claim 2, characterized in that: The mushroom production management is to increase the air humidity to 85-90%, the temperature to 23-28°C, and continue to cultivate in the dark for 10-15 days.

6. A method for identifying five strains of Boletus sinensis by SSR molecular markers according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step S1: transferring the mycelium or fruiting body of the tested Boletus sinensis to a potato dextrose agar solid medium, and collecting the mycelium after culturing at 25° C. for 15 days; Step S2: extracting the genomic DNA of the obtained mycelium, and using a biological spectrophotometer to detect whether the purity and concentration of the obtained total genomic DNA meet the detection requirements, if so, proceed to step S3, if not, increase the mass of the mycelium sample and repeat steps S1-2; Step S3: using SSR molecular markers to perform PCR amplification of the SSR-marked products from the total genomic DNA obtained in step 2; Step S4: Use capillary electrophoresis to detect the amplification product obtained in step S3, obtain the number of allelic fragments and the molecular weight of each fragment, determine the SSR allelic fragment number combination of the sample to be tested according to the number of allelic fragments and the molecular weight of each fragment, and judge whether the strain to be tested is at least one of the Chinese saprophytic boletus strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 according to the obtained SSR allelic fragment number combination.

7. The method according to claim 6, characterized in that The strain that meets the first group of SSR allele number combinations in step S4: 2 / (2+3) / (2+3+5) / (3+5) / (4+5) / (2+5) / (2+4+5) / (1+2) / (1+3) is the Chinese saprophytic boletus ZJHJSQ001 strain; In the first group of SSR allelic fragment number combinations 2 / (2+3) / (2+3+5) / (3+5) / (4+5) / (2+5) / (2+4+5) / (1+2) / (1+3), "2" is the allelic fragment number 2 of SEQ ID NO: 6-7, with a molecular weight of 188-180.99 bp; "(2+3)" is allelic fragment numbers 2 and 3 of SEQ ID NOs: 8 to 9, the molecular weight of allelic fragment number 2 is 265 to 265.99 bp, and the molecular weight of allelic fragment number 3 is 268 to 268.99 bp; "(2+3+5)" is allelic fragment numbers 2, 3, and 5 of SEQ ID NOs: 10 to 11, the molecular weight of allelic fragment number 2 is 119 to 119.99 bp, the molecular weight of allelic fragment number 3 is 120 to 120.99 bp, and the molecular weight of allelic fragment number 5 is 126 to 126.99 bp; "(3+5)" is allelic fragment numbers 3 and 5 of SEQ ID NOs: 12 to 13, the molecular weight of allelic fragment number 3 is 123 to 123.99 bp, and the molecular weight of allelic fragment number 5 is 130 to 130.99 bp; "(4+5)" is allelic fragment numbers 4 and 5 of SEQ ID NOs: 14 to 15, the molecular weight of allelic fragment number 4 is 179 to 179.99 bp, and the molecular weight of allelic fragment number 5 is 185 to 185.99 bp; " (2+5) " is allelic fragment numbers 2 and 5 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 2 is 212 to 212.99 bp, and the molecular weight of allelic fragment number 5 is 226 to 226.99 bp; " (2+4+5) " is allelic fragment numbers 2, 4, and 5 of SEQ ID NOs: 18 to 19, the molecular weight of allelic fragment number 2 is 140 to 140.99 bp, the molecular weight of allelic fragment number 4 is 147 to 147.99 bp, and the molecular weight of allelic fragment number 5 is 149 to 149.99 bp; "(1+2)" is the allelic fragment numbers 1 and 2 of SEQ ID NO: 20-21, the molecular weight of the allelic fragment number 1 is 229-229.99 bp, and the molecular weight of the allelic fragment number 2 is 232-232.99 bp; " (1+3) " is allelic fragment numbers 1 and 3 of SEQ ID NO: 22-23, the molecular weight of allelic fragment number 1 is 240-240.99 bp, and the molecular weight of allelic fragment number 3 is 257-257.99 bp; The strain that meets the second group of SSR allele number combination: 3 / (1+3) / (3+6) / (1+4) / 2 / (4+5) / (1+3) / (3+5) / (2+4) in step S4 is the Chinese saprophytic boletus ZJHJSQ002 strain; The second group of SSR allelic fragment number combinations 3 / (1+3) / (3+6) / (1+4) / 2 / (4+5) / (1+3) / (3+5) / (2+4) in which "3" is the allelic fragment number 3 of SEQ ID NO: 6-7, with a molecular weight of 186-186.99 bp; "(1+3)" is the allelic fragment numbers 1 and 3 of SEQ ID NOs: 8 to 9, the molecular weight of the allelic fragment number 1 is 262 to 262.99 bp, and the molecular weight of the allelic fragment number 3 is 268 to 268.99 bp; "(3+6)" is the allelic fragment numbers 3 and 6 of SEQ ID NOs: 10 to 11, the molecular weight of the allelic fragment number 3 is 120 to 120.99 bp, and the molecular weight of the allelic fragment number 6 is 127 to 127.99 bp; "(1+4)" is the allelic fragment numbers 1 and 4 of SEQ ID NOs: 12 to 13, the molecular weight of the allelic fragment number 1 is 112 to 112.99 bp, and the molecular weight of the allelic fragment number 4 is 124 to 124.99 bp; "2" is the allele number 2 of SEQ ID NO: 14-15, and the molecular weight of the allele number 2 is 173-173.99 bp; "(4+5)" is allelic fragment numbers 4 and 5 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 4 is 220 to 220.99 bp, and the molecular weight of allelic fragment number 5 is 226 to 226.99 bp; "(1+3)" refers to allelic fragment numbers 1 and 3 of SEQ ID NOs: 18 to 19, wherein the molecular weight of allelic fragment number 1 is 134 to 134.99 bp, and the molecular weight of allelic fragment number 3 is 143 to 143.99 bp; "(3+5)" is allelic fragment numbers 3 and 5 of SEQ ID NOs: 20 to 21, the molecular weight of allelic fragment number 3 is 234 to 234.99 bp, and the molecular weight of allelic fragment number 5 is 270 to 270.99 bp; "(2+4)" refers to allelic fragment numbers 2 and 4 of SEQ ID NOs: 22 to 23, wherein the molecular weight of allelic fragment number 2 is 255 to 255.99 bp, and the molecular weight of allelic fragment number 4 is 272 to 272.99 bp.

8. The method according to claim 6, characterized in that The strain that meets the third group of SSR allele number combinations in step S4: 2 / (2+3) / (3+5) / (3+5) / (4+5) / (2+5) / (2+5) / (1+2) / (1+3) is the Chinese saprophytic boletus ZJHJSQ003 strain; The third group of SSR allelic fragment number combinations: 2 / (2+3) / (3+5) / (3+5) / (4+5) / (2+5) / (2+5) / (1+2) / (1+3), in which "2" is the allelic fragment number 2 of SEQ ID NO: 6-7, with a molecular weight of 188-180.99 bp; "(2+3)" refers to allelic fragment numbers 2 and 3 of SEQ ID NOs: 8 to 9, wherein the molecular weight of allelic fragment number 2 is 265 to 265.99 bp, and the molecular weight of allelic fragment number 3 is 268 to 268.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 10 to 11, wherein the molecular weight of allelic fragment number 3 is 120 to 120.99 bp, and the molecular weight of allelic fragment number 5 is 126 to 126.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 12 to 13, wherein the molecular weight of allelic fragment number 3 is 123 to 123.99 bp, and the molecular weight of allelic fragment number 5 is 130 to 130.99 bp; "(4+5)" refers to allelic fragment numbers 4 and 5 of SEQ ID NOs: 14 to 15, wherein the molecular weight of allelic fragment number 4 is 179 to 179.99 bp, and the molecular weight of allelic fragment number 5 is 185 to 185.99 bp; "(2+5)" is allelic fragment numbers 2 and 5 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 2 is 212 to 212.99 bp, and the molecular weight of allelic fragment number 5 is 226 to 226.99 bp; "(2+5)" is allelic fragment numbers 2 and 5 of SEQ ID NOs: 18 to 19, the molecular weight of allelic fragment number 2 is 140 to 140.99 bp, and the molecular weight of allelic fragment number 5 is 149 to 149.99 bp; "(1+2)" is the allelic fragment numbers 1 and 2 of SEQ ID NO: 20-21, the molecular weight of the allelic fragment number 1 is 229-229.99 bp, and the molecular weight of the allelic fragment number 2 is 232-232.99 bp; "(1+3)" refers to allelic fragment numbers 1 and 3 of SEQ ID NOs: 22 to 23, the molecular weight of allelic fragment number 1 is 240 to 240.99 bp, and the molecular weight of allelic fragment number 3 is 257 to 257.99 bp.

9. The method according to claim 6, characterized in that The strain that meets the fourth group of SSR allele number combination: 1 / 3 / 3 / (2+4) / (1+3) / (1+3) / (3+6) / 2 / (3+5) in step S4 is the Chinese saprophytic boletus ZJHJSQ004 strain; The fourth group of SSR allelic fragment number combinations 1 / 3 / 3 / (2+4) / (1+3) / (1+3) / (3+6) / 2 / (3+5) in which "1" is the allelic fragment number 1 of SEQ ID NO: 6-7, with a molecular weight of 178-178.99 bp; "3" is the molecular weight of allele number 3 of SEQ ID NO: 8-9, and the molecular weight of allele number 3 is 268-268.99 bp; "3" is the allele number 3 of SEQ ID NO: 10-11, and the molecular weight of allele number 3 is 120-120.99 bp; "(2+4)" refers to allelic fragment numbers 2 and 4 of SEQ ID NOs: 12 to 13, wherein the molecular weight of allelic fragment number 2 is 117 to 117.99 bp, and the molecular weight of allelic fragment number 4 is 124 to 124.99 bp; "(1+3)" is the allelic fragment numbers 1 and 3 of SEQ ID NOs: 14 to 15, the molecular weight of the allelic fragment number 1 is 165 to 165.99 bp, and the molecular weight of the allelic fragment number 3 is 176 to 176.99 bp; "(1+3)" is the allelic fragment numbers 1 and 3 of SEQ ID NOs: 16 to 17, the molecular weight of the allelic fragment number 1 is 204 to 204.99 bp, and the molecular weight of the allelic fragment number 3 is 217 to 217.99 bp; "(3+6)" refers to allelic fragment numbers 3 and 6 of SEQ ID NOs: 18 to 19, wherein the molecular weight of allelic fragment number 3 is 143 to 143.99 bp, and the molecular weight of allelic fragment number 6 is 152 to 152.99 bp; "2" is the allele number 2 of SEQ ID NO: 20-21, and the molecular weight of the allele number 2 is 232-232.99 bp; "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NO: 22-23, the molecular weight of allelic fragment number 3 is 257-257.99 bp, and the molecular weight of allelic fragment number 5 is 287-287.99 bp.

10. The method according to claim 6, characterized in that The strain that meets the fifth group of SSR allele number combination: (2+4) / (1+4) / (2+4) / 4 / 5 / (4+6) / (3+4) / (2+4) / (4+6) in step S4 is the Chinese saprophytic boletus ZJHJSQ005 strain; The fifth group of SSR allele number combinations (2+4) / (1+4) / (2+4) / 4 / 5 / (4+6) / (3+4) / (2+4) / (4+6) "(2+4)" is allelic fragment number 2 and 4 of SEQ ID NO: 6 to 7, with a molecular weight of 188 to 180.99 bp, and allelic fragment number 4 has a molecular weight of 238 to 238.99 bp; "(1+4)" is the allelic fragment numbers 1 and 4 of SEQ ID NOs: 8 to 9, the molecular weight of the allelic fragment number 1 is 262 to 262.99 bp, and the molecular weight of the allelic fragment number 4 is 271 to 271.99 bp; "(2+4)" is allelic fragment numbers 2 and 4 of SEQ ID NOs: 10 to 11, the molecular weight of allelic fragment number 2 is 119 to 119.99 bp, and the molecular weight of allelic fragment number 4 is 123 to 123.99 bp; "4" is the allele number 4 of SEQ ID NO: 12-13, and the molecular weight of allele number 4 is 124-124.99 bp; "5" is the allele number 5 of SEQ ID NO: 14-15, and the molecular weight of the allele number 5 is 185-185.99 bp; "(4+6)" is allelic fragment numbers 4 and 6 of SEQ ID NOs: 16 to 17, the molecular weight of allelic fragment number 4 is 220 to 220.99 bp, and the molecular weight of allelic fragment number 6 is 227 to 227.99 bp; "(3+4)" is allelic fragment numbers 3 and 4 of SEQ ID NOs: 18 to 19, the molecular weight of allelic fragment number 3 is 143 to 143.99 bp, and the molecular weight of allelic fragment number 4 is 147 to 147.99 bp; "(2+4)" is allelic fragment numbers 2 and 4 of SEQ ID NOs: 20 to 21, the molecular weight of allelic fragment number 2 is 232 to 232.99 bp, and the molecular weight of allelic fragment number 4 is 240 to 240.99 bp; "(4+6)" refers to allelic fragment numbers 4 and 6 of SEQ ID NOs: 22 to 23, wherein the molecular weight of allelic fragment number 4 is 272 to 272.99 bp, and the molecular weight of allelic fragment number 6 is 297 to 297.99 bp.

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