Ganoderma sinensis strain and molecular marker identification method thereof

Abstract

The invention discloses a Ganoderma sinensis strain and a molecular marker identification method thereof, and relates to the technical field of microorganisms, the Ganoderma sinensis strain is preserved in China Center for Type Culture Collection, and the preservation number is CCT CC NO: M 20251554; the ITS sequence of the Ganoderma sinensis strain is as shown in SEQ ID NO: 15; according to the method, ganoderma foense 37 is successfully bred through a cross breeding technology, the strain has the comprehensive excellent characters that the yield is high, the polysaccharide content is remarkably increased, and the taste is close to that of wild ganoderma foense, the problems that an existing cultivated variety is reduced in active ingredient and poor in taste are solved, meanwhile, a set of molecular marker identification method based on seven groups of specific primers is established, and the method is suitable for large-scale popularization and application. The method can be used for rapidly, accurately and reliably identifying the ganoderma fosinense 37 and effectively distinguishing the ganoderma fosinense 37 from parents and other strains, and is simple and convenient to operate, good in repeatability and suitable for wide popularization and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to Ganoderma sinense and a molecular marker identification method thereof. BACKGROUND

[0002] Ganoderma sinense is a Chinese endemic rare edible and medicinal fungus variety, also known as black mushroom, Xuan mushroom and wood mushroom. It is recorded in the Shennong Herbal Classic in the Qin and Han Dynasties that Ganoderma sinense has the effects of treating deafness, benefiting joints, preserving spirit, benefiting essence and strengthening bones. Modern research shows that the main functional components in Ganoderma sinense are polysaccharides, triterpenes and alkaloids. Ganoderma sinense polysaccharides are important functional factors for immune regulation and anti-tumor effects. Research shows that Ganoderma sinense polysaccharides have the activities of anti-inflammatory analgesia, immune regulation, anti-tumor, antioxidant and intestinal microorganism regulation. Therefore, Ganoderma sinense has high medical value, and Ganoderma lucidum (G. lucidum and G. sinense) was included in the Chinese Pharmacopoeia as a legal Chinese medicinal material in 2000. With the opening of Ganoderma lucidum (G. lucidum and G. sinense) as medicine and food, the demand for Ganoderma sinense is also increasing.

[0003] The existing technology mainly focuses on the functional research of active substances, the separation of substances and the identification of substance structures, and there is little report on the genetic breeding research of Ganoderma sinense. Currently, the cultivated Ganoderma sinense strains are mainly wild isolated and domesticated, that is, the wild Ganoderma sinense strains are selected through evaluation experiments, and suitable planting varieties are screened out. For example, the earliest identified Ganoderma sinense variety "Wu 2" and the later selected varieties "Anhui Ganoderma sinense 4", "Anhui Ganoderma sinense 1" and "Anhui Ganoderma sinense 2" are all obtained through field collection and domestication screening. This breeding method belongs to the traditional early variety breeding method, which requires a long time and has narrow genetic resources and low overall breeding efficiency.

[0004] Due to long-term cultivation and a lack of scientific maintenance of the strains, currently cultivated Ganoderma lucidum varieties have experienced varying degrees of degeneration, particularly a significant decrease in the content of active ingredients, severely impacting their intrinsic quality. The polysaccharide and triterpenoid content of Ganoderma lucidum fruiting bodies is clearly defined in the Chinese Pharmacopoeia and is a crucial intrinsic indicator of Ganoderma lucidum. Furthermore, Ganoderma lucidum fruiting bodies possess a natural taste advantage, making them more readily accepted by consumers compared to other species in the Ganoderma genus such as Ganoderma lucidum var. rubrum, Ganoderma lucidum var. rubrum, and Ganoderma lucidum var. pineense. However, there are significant differences in taste between Ganoderma lucidum cultivated using different methods. Cultivated varieties, in particular, have a distinct "fungus flavor," while naturally wild-harvested Ganoderma lucidum almost entirely lacks this flavor. Wild Ganoderma lucidum generally has a better taste and commands a market price more than five times that of artificially cultivated greenhouse varieties and more than twice that of artificially cultivated forest-grown varieties. Meanwhile, due to long-term uncontrolled harvesting and the dwindling resources of tree species required for wild growth, such as maple deadwood, the number of wild Ganoderma lucidum species is decreasing. Therefore, this invention proposes a Ganoderma lucidum strain and its molecular marker identification method to solve the problems existing in the prior art. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to propose a Ganoderma lucidum strain and its molecular marker identification method, which, through hybridization breeding of Ganoderma lucidum, can selectively breed hybrids with higher yield and effective active ingredients, as well as better taste.

[0006] To achieve the objective of this invention, the invention is implemented through the following technical solution: a Ganoderma lucidum strain, the Ganoderma lucidum strain being classified and named Fu Zi Zhi 37, is deposited at the China Center for Type Culture Collection with accession number CCTCC NO:M20251554, and the ITS sequence of the Ganoderma lucidum strain is shown in SEQ ID NO:15.

[0007] A molecular marker identification method for a Ganoderma lucidum strain, comprising the following steps:

[0008] Step 1: Cultivate and collect the hyphae of the test strain, and extract genomic DNA;

[0009] Step 2: Perform PCR amplification on the extracted genomic DNA using at least one set of specific primers;

[0010] Step 3: Perform electrophoresis on the PCR amplification products to determine whether the test strain is Ganoderma lucidum 37 by checking for the presence of specific DNA fragments corresponding to Ganoderma lucidum 37.

[0011] A further improvement is made in step two, where the specific primers consist of seven sets, namely:

[0012] The first group of primers consists of forward primer S37P1A with sequence as shown in SEQ ID NO: 1 and reverse primer S37P1B with sequence as shown in SEQ ID NO: 2;

[0013] The second group of primers consists of forward primer S37P2A with sequence as shown in SEQ ID NO: 3 and reverse primer S37P2B with sequence as shown in SEQ ID NO: 4;

[0014] The third group of primers consists of forward primer S37P3A with sequence as shown in SEQ ID NO: 5 and reverse primer S37P3B with sequence as shown in SEQ ID NO: 6;

[0015] The fourth group of primers consists of forward primer S37P4A with sequence as shown in SEQ ID NO: 7 and reverse primer S37P4B with sequence as shown in SEQ ID NO: 8;

[0016] The fifth group of primers consists of forward primer S37P5A with sequence as shown in SEQ ID NO: 9 and reverse primer S37P5B with sequence as shown in SEQ ID NO: 10;

[0017] The sixth group of primers consists of forward primer S37P6A with sequence as shown in SEQ ID NO: 11 and reverse primer S37P6B with sequence as shown in SEQ ID NO: 12;

[0018] The seventh group of primers consists of forward primer S37P7A with sequence as shown in SEQ ID NO: 13 and reverse primer S37P7B with sequence as shown in SEQ ID NO: 14.

[0019] Further improvement lies in that in the step two, the reaction system of the PCR amplification is: 1-1.5 μl of DNA containing 80-150 ng, Premix Taq 12.5 μL, 1 μL of forward primer and reverse primer with a concentration of 10 μmol / L, respectively, 9-9.5 μL of ddH2O, and the total volume is 25 μL.

[0020] Further improvement lies in that in the step two, the reaction procedure of the PCR amplification is: 94℃ pre-denaturation for 5 min, followed by 35 cycles, each cycle including 98℃ denaturation for 10 sec, 55-61℃ annealing for 30 sec, 72℃ extension for 45 sec to 1 min 25 sec, and finally 72℃ extension for 10 min.

[0021] Further improvement lies in that in the step three, the electrophoresis detection is performed under the condition that 5 muL of the PCR product is electrophoresed in 0.5 x TBE buffer by using 1.2% agarose gel at a voltage of 5 V / cm, and the PCR product is mixed with a loading buffer containing 0.1% bromophenol blue and 40% sucrose before loading.

[0022] Further improvement lies in that in the step three, the electrophoresis detection is performed under the condition that 5 muL of the PCR product is electrophoresed in 0.5 x TBE buffer by using 1.2% agarose gel at a voltage of 5 V / cm, and the PCR product is mixed with a loading buffer containing 0.1% bromophenol blue and 40% sucrose before loading.

[0023] Further improvement lies in that in the step three, the electrophoresis detection is performed under the condition that 5 muL of the PCR product is electrophoresed in 0.5 x TBE buffer by using 1.2% agarose gel at a voltage of 5 V / cm, and the PCR product is mixed with a loading buffer containing 0.1% bromophenol blue and 40% sucrose before loading.

[0024] The present application has the advantages that the present application successfully breeds the Ganoderma lucidum 37 by hybridization breeding technology, and the Ganoderma lucidum 37 has the advantages of high yield, significantly improved polysaccharide content, and close taste to wild Ganoderma lucidum, and solves the problems of existing cultivated varieties, such as decreased active ingredients and poor taste, and the present application establishes a molecular marker identification method based on seven specific primers, which can quickly, accurately and reliably identify the Ganoderma lucidum 37 and effectively distinguish it from parents and other strains, and the method is simple in operation, good in repeatability, and suitable for wide application. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0026] Figure 1 is a schematic diagram of a fruiting body of the Ganoderma lucidum 37 of the present application in growth;

[0027] Figure 2 is a schematic diagram of the Ganoderma lucidum 37 (S37) and the parent G.s0007, G.s0014 of the present application in the case of out of Ganoderma;

[0028] Figure 3 is a schematic diagram of the Ganoderma lucidum 37 (S37) and the parent G.s0007, G.s0014 of the present application in the case of out of Ganoderma;

[0029] Figure 4 is an electropherogram of the PCR product of the present application. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] Fuzizhi 37 is obtained by hybridization and selection from Ganoderma lucidum 'G.s0007' and Ganoderma lucidum 'G.s0014'. 'G.s0014', i.e., 'Wuzhi No. 2', is a variety identified by the Fujian Crop Variety Approval Committee in March 2012, which is suitable for under-forest cultivation in a wild-like manner. Ganoderma lucidum 'G.s0007' was introduced from the Yatai Mushroom Research Institute of Jinzhou, Liaoning Province in 2008, and its properties are unknown. The strain may have been severely degraded due to long-term storage, and no fruiting bodies were produced during the out-of-fruit test. The yield of Fuzizhi 37 is higher than that of the parent strains, and the polysaccharide content is also significantly higher than that of 'Wuzhi No. 2' which can produce fruiting bodies. In addition, the taste is basically similar to that of wild Ganoderma lucidum, i.e., it has the characteristics of yield, active ingredient content, and taste, and has a good development and application prospect.

[0032] The mother strain culture medium of Fuzizhi 37 is potato dextrose agar (PDA): 200 g of potato, 20 g of glucose, 20 g of agar strips, and 1000 mL of water; and the culture conditions are conventional Ganoderma lucidum culture.

[0033] The specific primers used in this example are 7 groups, which are as follows:

[0034] The first group of primers consists of a forward primer S37P1A with a sequence as shown in SEQ ID NO: 1 and a reverse primer S37P1B with a sequence as shown in SEQ ID NO: 2;

[0035] S37P1A: 5'-CGATCTGAGCACTGGATTAG-3'

[0036] S37P1B: 5'-CGACGTGAAAGCACTATTCC-3'

[0037] The second group of primers consists of a forward primer S37P2A with a sequence as shown in SEQ ID NO: 3 and a reverse primer S37P2B with a sequence as shown in SEQ ID NO: 4;

[0038] S37P2A: 5'-CGCACCGGGAGACAAGATTC-3'

[0039] S37P2B: 5'-CCTTCGCGACCCAAACAAAC-3'

[0040] Third set of primers consisting of forward primer S37P3A of sequence as shown in SEQ ID NO: 5 and reverse primer S37P3B of sequence as shown in SEQ ID NO: 6;

[0041] S37P3A: 5'-AGGGCGTGCTTGCTTTCATC-3'

[0042] S37P3B: 5'-CACCTGAGCCTGCAGAGAAC-3'

[0043] Fourth set of primers consisting of forward primer S37P4A of sequence as shown in SEQ ID NO: 7 and reverse primer S37P4B of sequence as shown in SEQ ID NO: 8;

[0044] S37P4A: 5'-TTCGCGTTTGTCGGACTATC-3'

[0045] S37P4B: 5'-GACACCATCGGTAGCATTTC-3'

[0046] Fifth set of primers consisting of forward primer S37P5A of sequence as shown in SEQ ID NO: 9 and reverse primer S37P5B of sequence as shown in SEQ ID NO: 10;

[0047] S37P5A: 5'-GAGTACAGAGGGTCGTACAC-3'

[0048] S37P5B: 5'-CTAGCAAGCGAGACTATGTG-3'

[0049] Sixth set of primers consisting of forward primer S37P6A of sequence as shown in SEQ ID NO: 11 and reverse primer S37P6B of sequence as shown in SEQ ID NO: 12;

[0050] S37P6A: 5'-GGGCTTATGTCTCCCATTTG-3'

[0051] S37P6B: 5'-AAACAGCGGTAGTGACCAAC-3'

[0052] Seventh group of primers: forward primer S37P7A with a sequence as shown in SEQ ID NO: 13 and reverse primer S37P7B with a sequence as shown in SEQ ID NO: 14;

[0053] S37P7A: 5'-CTCGAACCTGAAGGCCCTAC-3'

[0054] S37P7B: 5'-CAAGCCACCTCGCTAGAGAC-3'

[0055] The ITS sequence of the Ganoderma lucidum strain of the present example is shown in SEQ ID NO: 15.

[0056] Example 1

[0057] Reference is made to Figure 1 , Figure 2 The present example provides a breeding method for Ganoderma lucidum "Fu Ganoderma 37", which comprises the following steps:

[0058] Breeding material creation: Ganoderma lucidum G.s0007, G.s0011 and G.s0012 were used as breeding maternal parent, and protoplasts were prepared according to existing conventional preparation technology, and regenerated strains were obtained after protoplast regeneration, and lock-like association was observed, and regenerated strains without observed lock-like association were left, i.e. these strains were mononucleated strains (breeding haploid).

[0059] Hybridization pairing: after the mononucleated strains were placed at room temperature and away from light for 1 month, they were transferred again, and the single nucleated strains with good growth of each regenerated strain of the parent were selected as breeding haploid, and were respectively labeled as S07-1, S11-1 and S12-1, and were respectively paired with another parent for double-single pairing, and the specific pairing information is shown in Table 1 below, and 12 combinations were paired, and all were hybridized, i.e. 12 hybrids were obtained.

[0060] Table 1 Ganoderma lucidum hybridization pairing information

[0061] No. Parent Monokine strain Cross No. 1 Gs.0011 S07-1 S34 2 Gs.0012 S07-1 S35 3 Gs.0013 S07-1 S36 4 Gs.0014 S07-1 S37 5 Gs.0007 S11-1 S47 6 Gs.0012 S11-1 S48 7 Gs.0013 S11-1 S49 8 Gs.0014 S11-1 S50 9 Gs.0007 S12-1 S60 10 Gs.0011 S12-1 S61 11 Gs.0013 S12-1 S62 12 Gs.0014 S12-1 S63

[0062] Hybridization screening: the 12 hybrids were made into strains, and were cultivated according to the conventional Ganoderma lucidum strain preparation and bag cultivation method, and the cultivation formula was as follows: sawdust 78%, wheat bran 13%, corn meal 7%, sucrose 1%, gypsum 1%, water content 55-60%, and the bag size was 170mm x 350mm, and the fruiting test was carried out, and the mushroom fruiting of each hybrid was observed by using the direct observation method. It was found that one of the hybrids with the number S37 had the best consistency in fruiting, and the flower shape was also relatively beautiful, and the other hybrids had poor uniformity.

[0063] Stability test: considering the stability of Ganoderma lucidum and its sensitivity to the environment, in order to avoid missing the better hybrid and observe the stability of the hybrid, 12 hybrids were observed again. The wall type cultivation was adopted, and it was found that the primordium of hybrid S37 was still neat, the fruiting body was faster, and the morphology was beautiful (as shown in Figure 1

[0064] Compared with the parent, the hybrid S37 and the parent Gs.0007 and Gs.0014 were vertically placed in the fungus bag to observe the fruiting body. It was found that the parent Gs.0007 was difficult to form primordium, and the fruiting body development was abnormal. The primordium of the other parent Gs.0014 was poor in neatness, as shown in Figure 2 The weight of a single bag of hybrid S37 and parent Gs.0014 was measured. The average single bag weight of S37 was 42.15 g, while the average single bag weight of parent Gs.0014 was 30.13 g. The yield of hybrid S37 was much higher than that of parent Gs.0014, showing great advantage. Therefore, this hybrid was named Fuzi 37.

[0065] Fuzi 37 was preserved in China Center for Type Culture Collection on May 21, 2025, address: Wuhan University, preservation number CCTCC NO: M 20251141.

[0066] Example 2

[0067] Referring to Figure 3 , this embodiment carries out antagonism test of Ganoderma lucidum S37 and parent, including the following steps:

[0068] Ganoderma lucidum S37 and parent G.s0007, G.s0014 were activated on mother culture for 2 times. After activation, the fungus block was taken with a 4mm puncher, and inoculated in a flat dish with a diameter of 90mm. Three fungus blocks were arranged in a “triangle” shape and placed in a constant temperature incubator at 25℃ for 10 days. The results showed (as shown in Figure 3 ) that there was antagonism reaction between the three Ganoderma lucidum strains, indicating that the genetic material of Ganoderma lucidum strain S37 was different from the parent G.s0007, G.s0014.

[0069] Example 3

[0070] This embodiment detects the polysaccharide and triterpene content of Ganoderma lucidum strain S37, including the following steps:

[0071] ​The harvested fruiting bodies S37 and the main cultivar G.s0014 were dried to constant weight in a drying oven, and the polysaccharide and triterpene contents of the two samples were detected according to the method in the first part of the Chinese Pharmacopoeia 2020 edition. The detection results are that the polysaccharide content of S37 is 3.06%, and the triterpene is 0.728%, and the polysaccharide content of G.s0014 is 2.74%, and the triterpene is 0.756%. The polysaccharide content of S37 is higher than that of G.s0014, and the triterpene content of the two is close.

[0072] Example 4

[0073] This embodiment provides a taste evaluation method of Ganoderma lucidum "Fuzizhi 37". The market greenhouse-grown Ganoderma lucidum, forest-grown Ganoderma lucidum, and wild Ganoderma lucidum are purchased and sensory evaluation is performed with artificially planted (mushroom bag cultivation) "Fuzizhi 37". The specific steps are as follows:

[0074] Referring to the method of Zhang Yanjun (Zhang Yanjun. Research on the browning reason and preservation technology of Pleurotus geesterum [D]. Fuzhou: Fujian Agriculture and Forestry University, 2013: 23-28.), a sensory evaluation group composed of 10 trained food professional students (aged 20-27 years old) performs sensory evaluation on the four kinds of Ganoderma lucidum. The sensory evaluation standard is shown in Table 2. The evaluation results are shown in Table 3. As can be seen from Table 3, the "mushroom taste" of the greenhouse-grown Ganoderma lucidum is heavy and slightly bitter; the "mushroom taste" of the forest-grown Ganoderma lucidum is lighter than that of the greenhouse-grown Ganoderma lucidum, and the taste is also improved a lot; the wild Ganoderma lucidum has almost no "mushroom taste", and the aftertaste is obvious, and the taste is the best, which also corresponds to the market price; the "mushroom taste" of "Fuzizhi 37" provided by the present application is also basically non-existent, and the aftertaste is also relatively obvious, and the overall taste is similar to that of the wild.

[0075] Table 2 Sensory evaluation standard of Ganoderma lucidum

[0076] Score 1 2 3 4 Sweetness Slight bitter None Slight sweetness Distinct sweetness Fungus taste Heavy taste Medium taste Light taste None

[0077] Table 3 Sensory evaluation results of Ganoderma lucidum

[0078]

[0079] Example 5

[0080] Reference Figure 4 This embodiment provides a molecular marker identification method of Ganoderma lucidum "Fuzizhi 37", which comprises the following steps:

[0081] 1. Genomic DNA was extracted from *Volvariella volvacea* 37 and its parents “G.s0007” and “G.s0014” using the CTAB method. The specific extraction procedure was performed according to the method of Tao Yongxin et al. (Tao Y, Xie B, Yang Z, et al. Identification and expression analysis of a new glycoside hydrolase family 55exo-β-1,3-glucanase-encoding gene in *Volvariella volvacea* suggests a role in fruitingbody development[J]. Gene, 2013, 527(1):154-160.). The extracted DNA was stored at -20℃ for later use.

[0082] 2. Genomic DNA from *Ganoderma lucidum* strain 37 and its parents, "G.s0007" and "G.s0014", was sent to Wuhan Fraser Gene Information Co., Ltd. for whole-genome next-generation sequencing. The sequencing data volume was 3-4 GB of clean data. Bioinformatics analysis was performed on these genomic data to identify genes unique to the hybrid *Ganoderma lucidum* strain 37 and its parents "G.s0007" and "G.s0014". Primers were designed, and the information for the 7 pairs of primers is as follows:

[0083] Primers S37P1A and S37P1B were able to amplify a 710bp marker fragment in the genomes of 'G.s0014' and 'Fuzizhi 37', while 'G.s0007' did not have this marker fragment;

[0084] Primers S37P2A and S37PP2B were able to amplify an 866bp marker fragment in the genomes of 'G.s0014' and 'Fuzizhi 37', while 'G.s0007' did not have this marker fragment;

[0085] Primers S37P3A and S37P3B were able to amplify a 1352bp marker fragment in the genomes of 'G.s0014' and 'Fuzizhi 37', while 'G.s0007' did not have this marker fragment;

[0086] Primers S37P4A and S37P4B were able to amplify a 742bp marker fragment in the genomes of 'G.s0007' and 'Fuzizhi 37', while 'G.s0014' did not have this marker fragment;

[0087] Primers S37P5A and S37P5B were able to amplify a 1002bp marker fragment in the genomes of 'G.s0007' and 'Fuzizhi 37', while 'G.s0014' did not have this marker fragment;

[0088] Primer S37P6A and S37P6B can amplify a 1763bp marker fragment from the genomes of 'G.s0007' and 'Fu Zizhi 37', but not from 'G.s0014';

[0089] Primer S37P7A and S37P7B can amplify a 1219bp marker fragment from the genomes of 'G.s0007' and 'Fu Zizhi 37', but not from 'G.s0014';

[0090] The sequences of the 7 pairs of primers designed and the sizes of the marker fragments amplified from the DNA of Fu Zizhi 37 using the 7 pairs of primers are shown in Table 4 below.

[0091] Table 4 Specific marker amplification information

[0092]

[0093] 3. Specific fragment PCR amplification

[0094] PCR amplification reaction system: 1.2μl of DNA containing 100ng, 12.5μl of S37 Premix Taq (with dye), 1μl of forward primer with a concentration of 10μmol / L, 1μl of reverse primer with a concentration of 10μmol / L, 9.3μl of ddH2O;

[0095] Primer S37P1A, S37P1B-PCR amplification reaction program: 94℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 55℃ annealing for 30sec, 72℃ extension for 45sec, 35 cycles; 2℃ extension for 10min.

[0096] Primer S37P2A, S37P2B-PCR amplification reaction program: 94℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 61℃ annealing for 30sec, 72℃ extension for 55sec, 35 cycles; 72℃ extension for 10min.

[0097] Primer S37P3A, S37P3B-PCR amplification reaction program: 94℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 61℃ annealing for 30sec, 72℃ extension for 1min25sec, 35 cycles; 72℃ extension for 10min.

[0098] Primer S37P4A, S37P4B-PCR amplification reaction program: 94℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 56℃ annealing for 30sec, 72℃ extension for 45sec, 35 cycles; 72℃ extension for 10min.

[0099] PCR amplification reaction procedure of primers S37P5A, S37P5B: pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 57℃ for 30 sec, extension at 72℃ for 1 min 10 sec, 35 cycles; extension at 72℃ for 10 min.

[0100] PCR amplification reaction procedure of primers S37P6A, S37P6B: pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 57℃ for 30 sec, extension at 72℃ for 1 min 50 sec, 35 cycles; extension at 72℃ for 10 min.

[0101] PCR amplification reaction procedure of primers S37P7A, S37P7B: pre-denaturation at 94℃ for 5 min; denaturation at 98℃ for 10 sec, annealing at 61℃ for 30 sec, extension at 72℃ for 1 min 25 sec, 35 cycles; extension at 72℃ for 10 min.

[0102] 4. Electrophoresis detection of PCR products

[0103] 5 μl of PCR products were spotted on a 1.2% agarose gel, using DL2000 DNA MARK as a reference for fragment size, and electrophoresed in 0.5×TBE buffer at 5 V / cm for 30-40 min. After electrophoresis, the gel was imaged and photographed. The electrophoresis chart is shown in Fig. 1. Primers P1-P3 are common fragments of Fuzhizhi 37 and "G.s 0014", which are different from "G.s 0007"; P4-P7 are common sequences of Fuzhizhi 37 and "G.s 0007", which are different from "G.s 0014", and these fragments are the markers of Fuzhizhi 37; M represents DL5000 DNA MARK. Lane mark 1 is the parent "G.s 0014", 2 represents Fuzhizhi 37, and 3 represents "G.s 0007". Figure 4

[0104] 5. Reagents and instruments

[0105] CTAB extraction solution: 100 mmol / L Tris-HCl, 2.0% CTAB, 20 mmol / L EDTA, 1.4 mol / L NaCl, pH 8.0;

[0106] CTAB precipitation solution: 50 mmol / L Tris-HCl, 1.0% CTAB, 10 mmol / L EDTA, pH 8.0;

[0107] CTAB / NaCl: 0.7 mol / L NaCl, 10% CTAB;

[0108] Chloroform isoamyl alcohol: the volume ratio of chloroform to isoamyl alcohol is 24:1; ​

[0109] TE buffer: 10 mmol / L TrisCI, 1 mmol / L EDTA;

[0110] 0.5xTBE: 44.5 mmol / L Tris, 50 mmol / L H3BO3, 1 mmol / L EDTA;

[0111] Premix Taq (Ex Taq Version2.0) with dye and PCR amplification reagent, both purchased from Dalian Baosheng Biotechnology Co., Ltd.

[0112] Main instruments:

[0113] SW-CJ-1FB type single horizontal and vertical dual-purpose purification workbench: Suzhou Purification Equipment Co., Ltd.;

[0114] Sterilization pot: Shanghai Shen'an LDZM-60L-I;

[0115] Refrigerated centrifuge: Sigma 3K30;

[0116] Gene amplifier: EJWJ2025166;

[0117] Handheld centrifuge: LX-200;

[0118] Gel imaging system: Tanon MINI Space 2000;

[0119] Gel imaging system: TANON-MINI Space 1000.

[0120] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Ganoderma lucidum strain, characterized in that: The classification name of the Ganoderma lucidum strain is Ganoderma lucidum 37, which is preserved in China Center for Type Culture Collection with the accession number CCTCC NO:M20251554, and the ITS sequence of the Ganoderma lucidum strain is shown as SEQ ID NO:

15.

2. A method for identifying Ganoderma lucidum strain by molecular marker, characterized in that, The method comprises the following steps: Step one: culturing and collecting mycelia of the test strain, and extracting genomic DNA; Step two: performing PCR amplification on the extracted genomic DNA using at least one set of specific primers; Step three: performing electrophoresis detection on the PCR amplification product, and determining whether the test strain is Ganoderma lucidum 37 by whether a specific DNA fragment corresponding to Ganoderma lucidum 37 appears.

3. The molecular marker identification method for Ganoderma lucidum strains according to claim 2, characterized in that: In the step two, the specific primers are seven groups, which are respectively: The first group of primers consists of a forward primer S37P1A with the sequence shown as SEQ ID NO:1 and a reverse primer S37P1B with the sequence shown as SEQ ID NO:2; The second group of primers consists of a forward primer S37P2A with the sequence shown as SEQ ID NO:3 and a reverse primer S37P2B with the sequence shown as SEQ ID NO:4; The third group of primers consists of a forward primer S37P3A with the sequence shown as SEQ ID NO:5 and a reverse primer S37P3B with the sequence shown as SEQ ID NO:6; The fourth group of primers consists of a forward primer S37P4A with the sequence shown as SEQ ID NO:7 and a reverse primer S37P4B with the sequence shown as SEQ ID NO:8; The fifth group of primers consists of a forward primer S37P5A with the sequence shown as SEQ ID NO:9 and a reverse primer S37P5B with the sequence shown as SEQ ID NO:10; The sixth group of primers consists of a forward primer S37P6A with the sequence shown as SEQ ID NO:11 and a reverse primer S37P6B with the sequence shown as SEQ ID NO:12; The seventh group of primers consists of a forward primer S37P7A with the sequence shown as SEQ ID NO:13 and a reverse primer S37P7B with the sequence shown as SEQ ID NO:

14.

4. The molecular marker identification method for Ganoderma lucidum strains according to claim 2, characterized in that: In the step two, the reaction system of the PCR amplification is as follows: 1-1.5 μl of DNA with a content of 80-150 ng, 12.5 μL of Premix Taq, 1 μL of each of forward primers and reverse primers with a concentration of 10 μmol / L, 9-9.5 μL of ddH2O, and a total volume of 25 μL.

5. The method according to claim 2, wherein the Ganoderma lucidum strain is characterized by: In the step two, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94℃ for 5 min, followed by 35 cycles, each cycle including denaturation at 98℃ for 10 sec, annealing at 55-61℃ for 30 sec, extension at 72℃ for 45 sec to 1 min 25 sec, and finally extension at 72℃ for 10 min.

6. The method according to claim 2, wherein the Ganoderma lucidum strain is characterized by: In step three, the electrophoresis detection condition is as follows: 5 μL of the PCR product is electrophoresed in 0.5×TBE buffer at a voltage of 5 V / cm using a 1.2% agarose gel, and the PCR product is mixed with a loading buffer containing 0.1% bromophenol blue and 40% sucrose before loading.

7. The molecular marker identification method for Ganoderma lucidum strains according to claim 2, characterized in that: In step three, electrophoresis detection shows that only one fragment appears in one lane, and the fragment size is consistent with the corresponding primer design, which is the molecular marker of Fuzizhi 37.

8. The molecular marker identification method for Ganoderma lucidum strains according to claim 3, characterized in that: In step three, the specific DNA fragment sizes are as follows: 710 bp for the first group of primers, 866 bp for the second group of primers, 1352 bp for the third group of primers, 742 bp for the fourth group of primers, 1002 bp for the fifth group of primers, 1763 bp for the sixth group of primers, and 1219 bp for the seventh group of primers.

Images