Five strains of Boletus sinensis and their identification by SSR molecular markers

Abstract

The present application discloses a method for identifying five strains of Boletus sinensis and their SSR molecular markers, relating to the technical field of boletes. The strains are Boletus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005; ZJHJSQ001 has a deposit number of CCTCC NO: M 20242071, and its ITS sequence is shown in SEQ NO. 1; ZJHJSQ002 has a deposit number of CCTCC NO: M 20242070, and its ITS sequence is shown in SEQ NO. 2. The combination of SSR molecular marker primers obtained by this method can quickly and easily distinguish Boletus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005, achieving the advantages of cost savings, improved efficiency, convenient operation, and accurate results.

Description

Technical Field

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

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

[0003] While a few varieties are poisonous or bitter and therefore inedible, most boletus species are edible. They typically have large fruiting bodies, thick flesh, and robust stalks. They are sweet and delicious, rich in nutrients, and are renowned worldwide as edible mushrooms. Boletus contains eight essential amino acids, as well as alkaloids such as adenine, choline, and putrescine. They also have medicinal properties, including relief of lower back and leg pain, numbness in the hands and feet, limb convulsions, and abnormal vaginal discharge in women.

[0004] Boletes, a type of ectomycorrhizal fungus, are difficult to grow on artificial culture media. Even when they do grow, culturing their fruiting bodies in vitro is difficult, a global challenge. Because mycorrhizal structures are the product of long-term co-evolution, the unclear ecological conditions, nutritional pathways, and conditions for fruiting body differentiation and development make the artificial domestication of many ectomycorrhizal edible fungi, including boletes, extremely challenging. Successful domestication will undoubtedly improve the utilization of natural resources while promoting the rapid and healthy growth of forests. Producing fungi from forests and promoting forest growth through fungi creates economic value while protecting 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 genus, but the wild yield is extremely rare, far from enough to meet market demand, which fully reflects the necessity of artificial cultivation. For example, a Chinese saprophytic boletus strain is disclosed in CN202110631616.9, and the preservation number of the strain 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 their 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 strains are isolated, the yield of artificial cultivation is low and uneven. Therefore, it is extremely important to obtain high-quality Chinese saprophytic boletus strains and develop an accurate and effective strain identification system using molecular biology techniques.

[0008] The information disclosed in the background section is only intended to enhance understanding of the overall background of the invention and should not be considered as an admission or any form of suggestion that the information constitutes the prior art already known to a person of ordinary skill 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, the application discloses five strains of Chinese saprophytic boletus with neat fruiting, thick stipes, high yield and good resistance.

[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;

[0011] 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. No. 19; the Boletus144-F sequence is shown in SEQ No. 20; the Boletus144-R sequence is shown in SEQ No. 21; the Boletus191-F sequence is shown in SEQ No. 22; and the Boletus191-R sequence is shown in SEQ No. 23.

[0012] Preferably, the cultivation method of the five strains of Boletus sinensis comprises the following steps:

[0013] 1) Inoculating the culture medium in a container filled with culture medium with a liquid culture solution of at least one of five strains of Boletus sinensis at an inoculum volume of at least 10 ml per container to obtain an inoculated culture device;

[0014] 2) Cultivate the inoculated culture container in the dark at 23-28°C and 50-60% humidity for 40-55 days, and then obtain the transplant culture container;

[0015] 3) Open one end of the transplanting culture container and cover it with planting soil. After 10-15 days of fruiting, when the fruiting bodies grow to 9-12 cm in height, increase the humidity to 90-95%. Continue to cultivate under 120-180 lux for 5-7 days until the mushrooms are mature and can be picked.

[0016] 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.

[0017] Preferably, the cultivation material is a mixture of the following components in parts 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; and the moisture content of the cultivation material is 50-65%.

[0018] 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.

[0019] Another aspect of the present application further provides a method for identifying the five aforementioned strains of Boletus sinensis using SSR molecular markers, comprising the following steps:

[0020] Step S1: transferring the mycelia or fruiting body of the tested Boletus sinensis onto a potato dextrose agar solid medium, culturing at 25° C. for 15 days, and then collecting the mycelia;

[0021] Step S2: extracting genomic DNA from 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;

[0022] Step S3: using the above-mentioned SSR molecular markers to perform PCR amplification of the SSR-marked products from the total genomic DNA obtained in step 2;

[0023] 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 based on 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 ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains based on the obtained SSR allelic fragment number combination.

[0024] 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;

[0025] 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 NOs: 6-7, with a molecular weight of 188-180.99 bp;

[0026] "(2+3)" represents allelic fragment numbers 2 and 3 of SEQ ID NOs: 8-9, wherein the molecular weight of allelic fragment number 2 is 265-265.99 bp, and the molecular weight of allelic fragment number 3 is 268-268.99 bp;

[0027] "(2+3+5)" represents allelic fragment numbers 2, 3, and 5 of SEQ ID NOs: 10 to 11, wherein 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;

[0028] "(3+5)" represents 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;

[0029] "(4+5)" represents 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;

[0030] “ (2+5) ” represents allelic fragment numbers 2 and 5 of SEQ ID NOs: 16-17, wherein the molecular weight of allelic fragment number 2 is 212-212.99 bp, and the molecular weight of allelic fragment number 5 is 226-226.99 bp;

[0031] “(2+4+5)” represents allelic fragment numbers 2, 4, and 5 of SEQ ID NOs: 18-19, wherein the molecular weight of allelic fragment number 2 is 140-140.99 bp, the molecular weight of allelic fragment number 4 is 147-147.99 bp, and the molecular weight of allelic fragment number 5 is 149-149.99 bp;

[0032] "(1+2)" represents allelic fragment numbers 1 and 2 of SEQ ID NOs: 20-21, wherein 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;

[0033] “ (1+3) ” represents allelic fragment numbers 1 and 3 of SEQ ID NOs: 22-23, wherein 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;

[0034] The strain that meets the second group of SSR allele number combinations in step S4: 3 / (1+3) / (3+6) / (1+4) / 2 / (4+5) / (1+3) / (3+5) / (2+4) is the Chinese saprophytic boletus ZJHJSQ002 strain;

[0035] 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 allelic fragment number 3 of SEQ ID NO: 6-7, with a molecular weight of 186-186.99 bp;

[0036] "(1+3)" represents allelic fragment numbers 1 and 3 of SEQ ID NOs: 8 to 9, wherein 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;

[0037] "(3+6)" refers to allelic fragment numbers 3 and 6 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 6 is 127 to 127.99 bp;

[0038] "(1+4)" represents 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;

[0039] "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;

[0040] "(4+5)" represents allelic fragment numbers 4 and 5 of SEQ ID NOs: 16-17, wherein the molecular weight of allelic fragment number 4 is 220-220.99 bp, and the molecular weight of allelic fragment number 5 is 226-226.99 bp;

[0041] "(1+3)" represents allelic fragment numbers 1 and 3 of SEQ ID NOs: 18-19, wherein the molecular weight of allelic fragment number 1 is 134-134.99 bp, and the molecular weight of allelic fragment number 3 is 143-143.99 bp;

[0042] "(3+5)" represents allelic fragment numbers 3 and 5 of SEQ ID NOs: 20-21, wherein the molecular weight of allelic fragment number 3 is 234-234.99 bp, and the molecular weight of allelic fragment number 5 is 270-270.99 bp;

[0043] "(2+4)" refers to allelic fragment numbers 2 and 4 of SEQ ID NOs: 22-23. The molecular weight of allelic fragment number 2 is 255-255.99 bp, and the molecular weight of allelic fragment number 4 is 272-272.99 bp.

[0044] Preferably, the strain that meets the third group of SSR allele number combinations: 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;

[0045] 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 allelic fragment number 2 of SEQ ID NO: 6-7, with a molecular weight of 188-180.99 bp;

[0046] "(2+3)" represents allelic fragment numbers 2 and 3 of SEQ ID NOs: 8-9, wherein the molecular weight of allelic fragment number 2 is 265-265.99 bp, and the molecular weight of allelic fragment number 3 is 268-268.99 bp;

[0047] "(3+5)" represents 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;

[0048] "(3+5)" represents 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;

[0049] "(4+5)" represents allelic fragment numbers 4 and 5 of SEQ ID NOs: 14-15, wherein the molecular weight of allelic fragment number 4 is 179-179.99 bp, and the molecular weight of allelic fragment number 5 is 185-185.99 bp;

[0050] "(2+5)" represents allelic fragment numbers 2 and 5 of SEQ ID NOs: 16-17, wherein the molecular weight of allelic fragment number 2 is 212-212.99 bp, and the molecular weight of allelic fragment number 5 is 226-226.99 bp;

[0051] "(2+5)" represents allelic fragment numbers 2 and 5 of SEQ ID NOs: 18-19, wherein the molecular weight of allelic fragment number 2 is 140-140.99 bp, and the molecular weight of allelic fragment number 5 is 149-149.99 bp;

[0052] "(1+2)" represents allelic fragment numbers 1 and 2 of SEQ ID NOs: 20-21, wherein 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;

[0053] "(1+3)" represents 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.

[0054] Preferably, the strain that meets the fourth group of SSR allele number combinations: 1 / 3 / 3 / (2+4) / (1+3) / (1+3) / (3+6) / 2 / (3+5) in step S4 is the Chinese saprophytic boletus ZJHJSQ004 strain;

[0055] 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 allelic fragment number 1 of SEQ ID NO: 6-7, with a molecular weight of 178-178.99 bp;

[0056] "3" is the molecular weight of allele number 3 of SEQ ID NO: 8-9, and allele number 3 is 268-268.99 bp;

[0057] "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;

[0058] "(2+4)" represents 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;

[0059] "(1+3)" represents allelic fragment numbers 1 and 3 of SEQ ID NOs: 14-15, wherein the molecular weight of allelic fragment number 1 is 165-165.99 bp, and the molecular weight of allelic fragment number 3 is 176-176.99 bp;

[0060] "(1+3)" represents allelic fragment numbers 1 and 3 of SEQ ID NOs: 16-17, wherein the molecular weight of allelic fragment number 1 is 204-204.99 bp, and the molecular weight of allelic fragment number 3 is 217-217.99 bp;

[0061] "(3+6)" represents allelic fragment numbers 3 and 6 of SEQ ID NOs: 18-19, wherein the molecular weight of allelic fragment number 3 is 143-143.99 bp, and the molecular weight of allelic fragment number 6 is 152-152.99 bp;

[0062] "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;

[0063] "(3+5)" refers to allelic fragment numbers 3 and 5 of SEQ ID NOs: 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.

[0064] 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;

[0065] The fifth group of SSR allele number combinations (2+4) / (1+4) / (2+4) / 4 / 5 / (4+6) / (3+4) / (2+4) / (4+6)

[0066] "(2+4)" refers to allelic fragments 2 and 4 of SEQ ID NOs: 6 to 7, with molecular weights ranging from 188 to 180.99 bp, and allelic fragment 4 with a molecular weight of 238 to 238.99 bp;

[0067] "(1+4)" represents allelic fragment numbers 1 and 4 of SEQ ID NOs: 8 to 9, wherein 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;

[0068] "(2+4)" represents allelic fragment numbers 2 and 4 of SEQ ID NOs: 10 to 11, wherein 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;

[0069] "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;

[0070] "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;

[0071] "(4+6)" refers to allelic fragment numbers 4 and 6 of SEQ ID NOs: 16 to 17, wherein 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;

[0072] "(3+4)" represents allelic fragment numbers 3 and 4 of SEQ ID NOs: 18-19, wherein the molecular weight of allelic fragment number 3 is 143-143.99 bp, and the molecular weight of allelic fragment number 4 is 147-147.99 bp;

[0073] "(2+4)" represents allelic fragment numbers 2 and 4 of SEQ ID NOs: 20-21, wherein the molecular weight of allelic fragment number 2 is 232-232.99 bp, and the molecular weight of allelic fragment number 4 is 240-240.99 bp;

[0074] "(4+6)" refers to allelic fragment numbers 4 and 6 of SEQ ID NOs: 22-23. The molecular weight of allelic fragment number 4 is 272-272.99 bp, and the molecular weight of allelic fragment number 6 is 297-297.99 bp.

[0075] The beneficial effects of this application include:

[0076] 1) The present application provides a method for identifying five strains of Chinese saprophytic boletus and their SSR molecular markers. The combination of SSR molecular marker primers obtained by this method can quickly and easily distinguish Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains, thereby achieving the advantages of cost savings, improved efficiency, convenient operation and accurate results, and has good application prospects.

[0077] 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. This is of great significance for the authenticity identification of Chinese saprophytic boletus varieties during production.

[0078] The new strains of Boletus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 were deposited in the 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;

[0079] The deposit number of ZJHJSQ001 is CCTCC NO: M 20242071, and the ITS sequence is shown in SEQ NO.1;

[0080] The deposit number of ZJHJSQ002 is CCTCC NO: M 20242070, and the ITS sequence is shown in SEQ NO. 2;

[0081] The deposit number of ZJHJSQ003 is CCTCC NO: M 20242069, and the ITS sequence is shown in SEQ NO. 3;

[0082] The deposit number of ZJHJSQ004 is CCTCC NO: M 20242068, and the ITS sequence is shown in SEQ NO. 4;

[0083] 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

[0084] Figure 1 Phylogenetic tree of the Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains provided in this application;

[0085] Figure 2 The relative molecular weight peaks of the alleles obtained by sequentially detecting the primer Boletus022 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0086] Figure 3 The relative molecular weight peak graphs of the allele sites obtained by sequentially detecting the primer Boletus034 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0087] Figure 4 The relative molecular weight peak graphs of the allele sites obtained by sequentially detecting the primer Boletus035 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0088] Figure 5 The relative molecular weight peak graphs 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;

[0089] Figure 6 The relative molecular weight peaks of the allele sites obtained by sequentially detecting the primer Boletus084 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0090] Figure 7 The relative molecular weight peak graphs of the allele sites obtained by sequentially detecting the primer Boletus093 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0091] Figure 8 The relative molecular weight peak graphs of the allele sites obtained by sequentially detecting the primer Boletus110 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0092] Figure 9The relative molecular weight peaks of the alleles obtained by sequentially detecting the primers Boletus144 provided in the present application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0093] Figure 10 The relative molecular weight peaks of the alleles obtained by sequentially detecting the primer Boletus191 provided in this application in the selected Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005;

[0094] Figure 11 The UPGMA cluster tree of genetic distances of five Chinese saprophytic boletus strains constructed based on SSR molecular markers provided in this application;

[0095] Figure 12 Photos of artificially cultivated Boletus sinensis mushrooms provided for this application: a) is an enlarged view of a relatively small fruiting body; b) is a photo of a fruiting body growing out of a fungus bag; c) is a photo of a larger fruiting body 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

[0096] The present invention will be further described in detail below with reference to 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 scope of protection of the present invention.

[0097] Example

[0098] 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.

[0099] Example 1 Obtaining the strains of Boletus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005

[0100] The specific steps are as follows:

[0101] (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 a clean bench;

[0102] (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 PDA plate culture medium consists of 20g potatoes, 20g glucose, 20g agar powder, 1g peptone, and 1g agar powder.

[0103] (3) The plates were kept in a constant temperature and dark place at 25°C for 14 days to obtain Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005.

[0104] Example 2 Molecular Identification of Boletus sinensis Strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005

[0105] The ITS sequences of the Chinese saprophytic boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 strains were obtained, identified and deposited according to the following steps:

[0106] (1) DNA extraction

[0107] 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), cultured at 25°C for 15 days, and then the mycelia were collected. The genomic DNA of the mycelia was extracted using the Qingke TSP101-200 kit, and the purity and concentration of the obtained DNA were detected by agarose gel electrophoresis and biospectrophotometry.

[0108] (2) ITS amplification and detection

[0109] PCR amplification was performed using the universal ITS primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3' sequence, as shown in SEQ NO. 25) and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3' sequence, as shown in SEQ NO. 24). A 25 μL amplification system consisted of 20 μL of Gold Mix (green, Qingke TSE101), 0.5 μmol / L upstream and downstream primers, and 50 ng of genomic template DNA. The PCR amplification program was as follows: 94°C for 2 minutes, followed by 30 cycles of 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 60 seconds, and finally 72°C for 10 minutes. After PCR completion, 5 μL of 6x loading buffer was added and mixed, and the sample was checked by electrophoresis on a 1.0% agarose gel. After passing the test, sequencing was performed.

[0110] (3) Sequence comparison and phylogenetic analysis

[0111] The obtained ITS sequences were compared with the NCBI database using BLAST software, and their identities with the corresponding sequences of various strains of Boletus sinensis in the database reached over 97%. The resulting strains, Boletus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005, were deposited with the China Center for Type Culture Collection (CCTCC) on September 25, 2024.

[0112] The deposit number of strain ZJHJSQ001 is CCTCC NO: M 20242071, and the ITS sequence is shown in SEQ NO.1:

[0113] gcctttgcct tagtcctcgt tctcttttct cttttgcgtc gatccgttct ctcacacacc 60

[0114] tgtgcacctg ttgtaggttt tctcgagaga ggaaacctat gtttttcata aacacctttg 120

[0115] tatgtctata gaatgtctat gttgacgtcg accactgggc gacgttaaac aaaactatga 180

[0116] acaactttca gcaacggatc tcttggctct cgcatcgatg aagaacgcag cgaattgcga 240

[0117] taagtaatgt gaattgcaga ttttcagtga atcatcgaat ctttgaacgc accttgcgct 300

[0118] ccttggtatt ccgaggagca tgcctgtttg agtgtcattg aattctcaac catgtcttga 360

[0119] tgtcgtcgag cgcatggctt ggatgtgggg gttgctggcg gcgcgagctg tcggctctcc 420

[0120] ttaaatgcat tagcaaaggg cgagcctttc gtgcacggcc ttcgacgtga taatgatcgt 480

[0121] cgtggctgga gcggaaggca tgaatcgtcg ttgcttctaa tctctgatcc ttt 533

[0122] The preservation number of ZJHJSQ002 is CCTCC NO: M 20242070, and the ITS sequence is shown in SEQNO.2:

[0123] aggggggagt cgagcgggcg aagtttggtc gaggttgtcg ctggcccttc ggggcatgtt 60

[0124] gcacgccttt gccttagtcc tcgttctctt ttctcttttg cgtcgatccg ttctctcaca 120

[0125] cacctgtgca cctgttgtag gttttctcga gagaggaaac ctatgttttt cataaacacc 180

[0126] tttgtatgtc tatagaatgt ctatgttgac gtcgaccact gggcgacgtt aaacaaaact 240

[0127] atgaacaact ttcagcaacg gatctcttgg ctctcgcatc gatgaagaac gcagcgaatt 300

[0128] gcgataagta atgtgaattg cagattttca gtgaatcatc gaatctttga acgcaccttg 360

[0129] cgctccttgg tattccgagg agcatgcctg tttgagtgtc attgaattct caaccatgtc 420

[0130] ttgatgtcgt cgagcgcatg gcttggatgt gggggttgct ggcggcgcga gctgtcggct 480

[0131] ctccttaaat gcattagcaa agggcgagcc tttcgtgcac ggccttcgac gtgataatga 540

[0132] tcgtcgtggc tggagcggaa ggcatgaatc gtcgttgctt ctaatctctg atccttttgg 600

[0133] gatcgctttc gaaacttgac ctcaaatcag gtaggactac ccgctgaact taagcatatc 660

[0134] The deposit number of ZJHJSQ003 is CCTCC NO: M 20242069, and its ITS sequence is shown in SEQNO.3:

[0135] gcctttgcct tagtcctcgt tctcttttct cttttgcgtc gatccgttct ctcacacacc 60

[0136] tgtgcacctg ttgtaggttt tctcgagaga ggaaacctat gtttttcata aacacctttg 120

[0137] tatgtctata gaatgtctat gttgacgtcg accactgggc gacgttaaac aaaactatga 180

[0138] acaactttca gcaacggatc tcttggctct cgcatcgatg aagaacgcag cgaattgcga 240

[0139] taagtaatgt gaattgcaga ttttcagtga atcatcgaat ctttgaacgc accttgcgct 300

[0140] ccttggtatt ccgaggagca tgcctgtttg agtgtcattg aattctcaac catgtcttga 360

[0141] tgtcgtcgag cgcatggctt ggatgtgggg gttgctggcg gcgcgagctg tcggctctcc 420

[0142] ttaaatgcat tagcaaaggg cgagcctttc gtgcacggcc ttcgacgtga taatgatcgt 480

[0143] cgtggctgga gcggaaggca tgaatcgtcg ttgcttctaa tctctgatcc ttttgggatc 540

[0144] gct 543

[0145] The preservation number of ZJHJSQ004 is CCTCC NO: M 20242068, and its ITS sequence is as shown in SEQNO.4:

[0146] aggggggaag tcgagcgggc gaagtttggt cgaggttgtc gctggccctt cggggcatgt 60

[0147] tgcacgcctt tgccttagtc ctcgttctct tttctctttt gcgtcgatcc gttctctcac 120

[0148] acacctgtgc acctgttgta ggttttctcg agagaggaaa cctatgtttt tcataaacac 180

[0149] ctttgtatgt ctatagaatg tctatgttga cgtcgaccac tgggcgacgt taaacaaaac 240

[0150] tatgaacaac tttcagcaac ggat 264

[0151] The preservation number of ZJHJSQ005 is CCTCC NO: M 20242067, and its ITS sequence is shown in SEQNO.5:

[0152] cgcctttgcc ttagtcctcg ttctcttttc tcttttgcgt cgatccgttc tctcacacac 60

[0153] ctgtgcacct gttgtaggtt ttctcgagag aggaaaccta tgtttttcat aaacaccttt 120

[0154] gtatgtctat agaatgtcta tgttgacgtc gaccactggg cgacgttaaa caaaactatg 180

[0155] aacaactttc agcaacggat ctcttggctc tcgcatcgat gaagaacgca gcgaattgcg 240

[0156] ataagtaatg tgaattgcag attttcagtg aatcatcgaa tctttgaacg caccttgcgc 300

[0157] tccttggtat tccgaggagc atgcctgttt gagtgtcatt gaattctcaa ccatgtcttg 360

[0158] atgtcgtcga gcgcatggct tggatgtggg ggttgctggc ggcgcgagct gtcggctctc 420

[0159] cttaaatgca ttagcaaagg gcgagccttt cgtgcacggc cttcgacgtg ataatgatcg 480

[0160] tcgtggctgg agcggaaggc atgaatcgtc gttgcttcta atctctgatc cttttgggat 540

[0161] At the same time, the species of the genus Saprophytic Boletus and the wood-growing Microsaprophytic Boletus ( Buchwaldoboletus lignicola ) ITS sequences, and MEGA7 software was used to construct the NJ phylogenetic tree, using default parameters and 1000 Bootstrap tests to obtain the phylogenetic tree results.

[0162] Depend on Figure 1 It can be seen that the ITS sequences of strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 and those of Boletus sinensis clustered into one branch with a support rate of 100%.

[0163] Based on the above results, it can be concluded that the strains ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 belong to the Boletaceae family ( Boletaceae ), Buchwaldoboletus xylophilus of the genus Buchwaldoboletus.

[0164] Example 3 Identification of ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 Strain of Boletus sinensis Using SSR Molecular Markers

[0165] 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, Biopharmaceutical 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 and obtained: 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. The sequence of Boletus191-F is shown in SEQ NO. 22; the sequence of Boletus191-R is shown in SEQ NO. 23. The primer sequence information is shown in Table 1.

[0166] The screening experiment process is the same as the existing operation and includes the following steps:

[0167] 1. Sample preparation and verification; 2. Nucleic acid extraction and testing; 3. Synthesis of typing primers; 4. Fluorescence PCR amplification; 5. Primer polymorphism screening; 6. Preliminary group typing experiment; 7. Group typing test; 8. Result analysis;

[0168] The specific steps include:

[0169] (1) Simplify genome development of SSR markers

[0170] A library with 400 inserts was constructed and paired-end (PE) sequencing was performed on five strains of Boletus saprophyticus, ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005, using next-generation sequencing (NGS) technology on the Illumina NovaSeq platform and a paired-end PE500 sequencing protocol. One gigabyte of raw data was sequenced for each strain. This library construction and sequencing was performed in paired-end mode, merging data from all samples into popA. Sequences were then aligned using FLASH (version 1.2.11) (Magoc and Salzberg 2011) to create a single high-quality read based on overlap. The following parameters were used:

[0171] (1) Min overlap: 10;

[0172] (2) Max mismatch density: 0.2;

[0173] (3) Allow "outie" pairs: false;

[0174] According to the above parameters, the sequences are merged to obtain the sequence results.

[0175] The 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, and hexa-5. The maximum interval allowed between two different SSRs in the composite sequence was set to 100 bp.

[0176] Data from five Boletus saprophyticus strains were merged into popA, and a total of 40,810 SSRs were identified in 9,356,338 sequences. Of all the sequences with SSRs, 991 contained more than one SSR, and 947 SSRs were present in complex form. Based on the type of SSR loci, the number of repeats, and polymorphism within the population, 192 SSR loci were identified. These polymorphic SSR loci shared by the five strains were selected as candidate SSR loci for the development of polymorphic markers.

[0177] By using fluorescence capillary electrophoresis technology and the linker method, 9 polymorphic markers for variety differentiation were obtained through verification and screening of candidate sites. The specific information of the 9 SSR loci is shown in the following table:

[0178] Table 1 SSR marker primer information

[0179]

[0180] Example 4 Identification Method of Chinese Saprophytic Boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 Strains

[0181] This example sets up 5 treatments, and the test strains of each treatment are respectively Chinese saprophytic boletus ZJHJSQ001~5:

[0182] 1. Experimental Methods

[0183] (1) DNA extraction

[0184] The strains to be tested were transferred to potato dextrose agar solid medium and cultured at 25°C for 15 days before the mycelia were collected. The genomic DNA of the mycelia was extracted using the Qingke TSP101-200 kit, and the purity and concentration of the obtained DNA were detected by agarose gel electrophoresis and biospectrophotometry at a wavelength of 280 nm. The DNA of each strain was used for PCR amplification in step (2).

[0185] (2) PCR amplification

[0186] The 9 pairs of SSR molecular markers obtained in Example 3 were used to perform PCR amplification of the SSR markers on the DNA of each strain extracted in step (1) to obtain amplified products;

[0187] The PCR amplification system used in a total volume of 20 μl included: 16.45 μl of Qingke TSE101-Gold Mix (green), 1.2 μl of 10 μM Tag DNase, 1.2 μl each of 10 μmol / L SSR marker forward and reverse primers, and 1 μl of template DNA. PCR reaction conditions were: 98°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 10 seconds, and 72°C for 5 minutes. To ensure identification accuracy, the experiment was repeated three times.

[0188] (3) Detection of amplified products by capillary electrophoresis:

[0189] 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 10 μl of the mix was added to each well; 0.5 μl of sample template (the sample was the amplified product) was added to the corresponding 96-well plate, and the plate was centrifuged at 4000 rpm and then stopped; the mixing plate was heated at 95°C for pre-denaturation for 5 minutes using a metal bath heater, and immediately placed in -20°C after being removed; after cooling, the plate was removed, centrifuged at 4000 rpm, thawed, and mixed to obtain the sample to be tested; capillary tube electrophoresis was performed using a 3730 sequencer, with a sample volume of 2 μl of sample and 6 μl of bromophenol blue, a voltage of 300 V, and a time of 12 minutes.

[0190] The amplification product detection results of Chinese saprophytic boletus ZJHJSQ001~5 were obtained under 9 pairs of SSR molecular markers: Boletus022, Boletus034, Boletus035, Boletus057, Boletus084, Boletus093, Boletus110, Boletus144, and Boletus191. The results are shown in the relative molecular weight peak diagram of the allele sites. Figures 2 to 10 shown.

[0191] 2. Results Analysis

[0192] (1) Comparative analysis of capillary electrophoresis fluorescence detection peak graphs

[0193] By comparing Figures 2 to 10 It can be seen that the peaks of each SSR fluorescent marker of the five bacterial species are clearly distinguishable. Figure 2 This is the result of amplifying Boletus sinensis ZJHJSQ001~5 using primer Boletus022. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 are 181, 187, 181, 179, and 181+239, respectively;

[0194] Figure 3 This is the result of amplifying Boletus sinensis ZJHJSQ001-5 using primer Boletus034. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 are 266+269, 263+269, 266+269, 269, and 263+272, respectively.

[0195] Figure 4This is the result of amplifying Boletus sinensis ZJHJSQ001~5 using primer Boletus035. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 are 120+121+127, 121+128, 121+127, 121, and 118+124, respectively;

[0196] Figure 5 This is the result of amplifying Boletus sinensis ZJHJSQ001-5 using primer Boletus057. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 are 124+131, 113+125, 124+131, 118+125, and 125, respectively;

[0197] Figure 6 This is the result of amplifying Boletus sinensis ZJHJSQ001-5 using primer Boletus084. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 are 180+186, 174, 180+186, 166+177, and 186, respectively.

[0198] Figure 7 This is the result of amplifying Boletus sinensis ZJHJSQ001-5 using primer Boletus093. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 are 213+227, 221+227, 213+227, 205+218, and 221+228, respectively.

[0199] Figure 8 This is the result of amplifying Boletus sinensis ZJHJSQ001-5 using primer Boletus110. 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.

[0200] Figure 9 This is the result of amplifying Boletus sinensis ZJHJSQ001-5 using primer Boletus144. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 are 230+233, 235+271, 230+233, 233, and 233+241, respectively.

[0201] Figure 10 This is the result of amplifying Boletus sinensis ZJHJSQ001-5 using primer Boletus191. The sizes of the fragments corresponding to ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 are 241+258, 256+273, 241+258, 258+288 and 273+298, respectively.

[0202] Figures 2 to 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.

[0203] (2) Analysis of band combinations amplified by 9 pairs of SSR primers

[0204] Nine pairs of SSR primers were used to amplify allelic fragments in the tested Suillus sinensis ZJHJSQ001~5, which were coded according to molecular weight and numbered.

[0205] The strain that meets the first group of SSR allele number combinations 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;

[0206] The strain that met 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) was the Chinese saprophytic boletus ZJHJSQ002 strain;

[0207] 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 Suillus sinensis ZJHJSQ003;

[0208] The strain that met the fourth group SSR allele number combination of 1 / 3 / 3 / (2+4) / (1+3) / (1+3) / (3+6) / 2 / (3+5) was the Chinese saprophytic boletus ZJHJSQ004 strain;

[0209] 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.

[0210] The number of allelic fragments and molecular weight information of the above 9 pairs of SSR primers are shown in Table 2:

[0211] Table 2

[0212]

[0213] (3) Cluster analysis

[0214] 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 ( Figure 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. Figure 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.

[0215] It can be seen that the 9 pairs of SSR primer combinations provided by the present invention can be used to identify Chinese saprophytic boletus ZJHJSQ001-5 and can accurately distinguish Chinese saprophytic boletus ZJHJSQ001-5.

[0216] 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 Chinese saprophytic boletus ZJHJSQ001-5, population genetic diversity analysis, etc., and have good application value.

[0217] Example 5 Bag Cultivation Method for Chinese Saprophytic Boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004, and ZJHJSQ005 Strains

[0218] This embodiment sets 5 treatments, and the liquid bacterial strains used in each treatment are respectively Boletus sinensis ZJHJSQ001-5.

[0219] 1) Prepare the cultivation material (parts by weight): Weigh the raw materials according to the following ratio: 50 parts sawdust, 30 parts cottonseed hulls, 13.9 parts wheat bran, 5 parts soil, 0.5 parts gypsum, 0.5 parts lime, and 0.1 parts potassium dihydrogen phosphate, add water and mix well to make the moisture content 55%;

[0220] 2) Sterilization: Place the prepared cultivation material into a cultivation bag (specifications: 15*30*5 wire), sterilize with high-pressure steam (sterilize at 121°C for 15 minutes) and cool to room temperature;

[0221] 3) Inoculation: The inoculum volume of the liquid culture is 10 ml / bag. The liquid culture is inoculated into a cultivation bag cooled to room temperature. The culture is dark-cultured at 25°C and 55% humidity for 45 days to obtain a transplanting bag.

[0222] Preparation of liquid culture: Take a plate strain and inoculate it into a culture medium containing 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 liquid culture.

[0223] 4) Covering with soil: Mix the soil with formaldehyde water (1 ton of soil: 5 kg of formaldehyde) and fumigate for 3 days in advance. Emit formaldehyde for 1 day before use to make planting soil. Open one end of the transplanted fungus bag covered with mycelium and cover the surface of the open end with 1 cm of planting soil.

[0224] 5) Fruiting management: Increase the air humidity to 89%, the temperature to 25°C, provide adequate ventilation, and continue cultivating in dark conditions for 13 days;

[0225] 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 picked. Figure 12 The photos show that the strain is growing well, with strong stems, uniform mushroom production, and high yield. The largest fruiting bodies can weigh up to 161.2g each.

[0226] After harvest, statistics showed that the biological efficiency of one crop of ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 were 46%, 49%, 47%, 48% and 50%, respectively.

[0227] Example 6 Bottle Cultivation Method of Chinese Saprophytic Boletus ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 Strains

[0228] The difference from Example 5 is:

[0229] 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%;

[0230] Sterilization of material: put the prepared culture material into the culture bottle (1100ml), sterilize it with high pressure steam and cool it to room temperature;

[0231] According to statistics, the biological efficiency of one crop of ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 and ZJHJSQ005 were 46%, 48%, 46%, 47% and 49% respectively.

[0232] Example 7

[0233] The difference from Example 5 is:

[0234] 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 moisture content 50%;

[0235] The inoculum volume was 20 ml per container.

[0236] Step 3): Cultivate in the dark at 23°C and 50% humidity for 40 days to obtain a transplanted fungus bag;

[0237] Step 4) Mix soil with formaldehyde water (mix 1 ton of soil with 4 kg of formaldehyde);

[0238] Step 5) Fruiting management: Increase the air humidity to 85%, the temperature to 23°C, provide adequate ventilation, and continue cultivating in the dark for 10 days;

[0239] Step 7) When the fruiting bodies of Boletus sinensis grow to 9 cm in height, increase the humidity to 90% and continue to cultivate under light for 5 days until they mature and can be harvested.

[0240] Example 8

[0241] The difference from Example 5 is:

[0242] 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 moisture content 65%;

[0243] The inoculum volume was 20 ml per container.

[0244] Step 3): incubate in the dark at 28°C and 60% humidity for 55 days to obtain a transplanted fungus bag;

[0245] Step 4) Mix soil with formaldehyde water (mix 1 ton of soil with 6 kg of formaldehyde);

[0246] Step 5) Fruiting management: Increase the air humidity to 90%, the temperature to 28°C, provide adequate ventilation, and continue cultivating in the dark for 15 days;

[0247] Step 7) When the fruiting bodies of Boletus sinensis grow to 12 cm in height, increase the humidity to 95% and continue to cultivate under light for 7 days until they are mature and can be picked.

[0248] 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 scope of protection of the present invention.

Claims

1. Five strains of Chinese saprophytic boletus ( Buchwaldoboletus xylophilus ) strain, characterized in that The strain is Suillus sinensis ZJHJSQ001, ZJHJSQ002, ZJHJSQ003, ZJHJSQ004 or 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: M20242068, 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.

2. A combination of SSR molecular markers for identifying the five strains of Boletus sinensis according to claim 1, characterized in that: The combination of the SSR molecular markers consists of: Boletus022, Boletus034, Boletus035, Boletus057, Boletus084, Boletus093, Boletus110, Boletus144, and Boletus191; The primer sequences of the combination of the SSR molecular markers are 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; the Boletus144-F sequence is shown in SEQ No. 20; the Boletus144-R sequence is shown in SEQ No. 21; the Boletus191-F sequence is shown in SEQ No. 22; and the Boletus191-R sequence is shown in SEQ No. 23.

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