A kind of ganoderma lucidum and its specific molecular marker and application

Abstract

The present invention relates to the technical field of ganoderma lucidum, and in particular to a ganoderma lucidum and its specific molecular marker and application. The present invention provides a hybrid strain ganoderma lucidum GZJ10, which has a sterile pore structure on the lower surface of the pileus, does not produce spore powder, has different properties from both parents, has a fast growth rate, high fresh weight of fruiting bodies, high biological conversion rate of fruiting bodies, and large flower shape. No spore powder is produced during the growth process, and it can be used as an excellent variety for cultivating ganoderma lucidum fruiting bodies. The present invention also provides an identification and verification method for hybrid strain specific molecular markers, and the identification and verification method is accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of ganoderma lucidum, and in particular to ganoderma lucidum and its specific molecular marker and application. Background Art

[0002] Ganoderma lucidum is one of the origins of the precious Chinese medicinal material Ganoderma lucidum. The Pharmacopoeia of the People's Republic of China (2020 edition) stipulates that Ganoderma lucidum is the dried fruiting body of the Polyporaceae fungus Ganoderma lucidum (Leyss. exFr.) Karst. or Ganoderma sinense Zhao, Xu et Zhang. Modern pharmacological studies have shown that Ganoderma lucidum has anti-cancer, antiviral, antioxidant, antibacterial, cardiovascular disease prevention and treatment and anti-epileptic effects. The biological activity of Ganoderma lucidum is closely related to the various functional substances or trace elements it contains. Its main functional substances include polysaccharides, triterpenes, sterols and other chemical components. Under normal circumstances, Ganoderma lucidum will eject reproductive cells, namely basidiospores, in the later stages of its growth and development. In very rare cases, Ganoderma lucidum fruiting bodies may only eject a small number of spores or even no spores. Producers can select such strains as fruiting body-specific varieties to improve economic benefits.

[0003] Since the germination rate of Ganoderma lucidum spores in nature and under normal experimental conditions is extremely low, the progress of Ganoderma lucidum hybridization experiments is slow. Therefore, it is of great significance to accelerate Ganoderma lucidum breeding through protoplast uninucleation hybridization technology. Protoplast uninucleation hybridization refers to the use of monokaryon strains obtained by protoplast uninucleation for hybrid breeding. Protoplast uninucleation hybridization technology has multiple advantages: protoplast uninucleation does not undergo meiosis and is directly derived from vegetative hyphae, which is conducive to the preservation and stable expression of the excellent traits of the parents; monokaryons can be directly obtained from the parent mycelium through experimental means, shortening the breeding time; many wild germplasm resources that cannot form fruiting bodies under artificial conditions can use protoplast uninucleation technology to obtain monokaryon strains for subsequent breeding work, broadening the genotypes of hybrid parents.

[0004] The specific molecular markers of Ganoderma lucidum strains are authentic, reliable, and clear at a glance. They have smaller errors than classic morphological identification, are not affected by environmental conditions, and are more intuitive.

[0005] The formation of Ganoderma spores has a complex mechanism. The mycelium nutrients of sporeless Ganoderma varieties are all supplied to the growth of fruiting bodies. Whether the content of active ingredients in their fruiting bodies is higher than that of Ganoderma varieties with spore-forming ability, and whether the lack of spore-forming ability has an impact on other physiological and biochemical traits, there is no clear conclusion and the mechanism is not clear. Currently, "cultivating" Ganoderma varieties are very scarce, with only a few such as "Longzhi No. 2", and most of them are low-yield or extremely low-yield Ganoderma varieties, which seriously restricts the development of the industry. Therefore, it is urgent to cultivate special Ganoderma that does not produce spores as a high-quality resource for studying the mechanism of Ganoderma spore formation. Summary of the invention

[0006] The present invention provides a ganoderma lucidum and its specific molecular marker and application. The ganoderma lucidum is also called 'Liaozhi No. 1', which is a new strain of "cultivating type" red ganoderma lucidum with no sterile holes and no spore powder. The strain has a fast growth rate and high yield, and does not produce spore powder during the growth process. It can be used as a good strain for factory production, and the strain can be used as a good material for analyzing the genes and metabolic pathways involved in the key stage of ganoderma spore formation through research methods such as metabolomics and transcriptomics.

[0007] The present invention provides a Ganoderma lucidum GZJ10, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, a deposit date of November 7, 2022, and a deposit number of CGMCC No.40361.

[0008] The colony morphology of this strain on PDA solid medium is relatively regular, basically a perfect circle. The hyphae are white, sparser than the parent, short and velvety, with aerial hyphae but not lush, and mature and leathery with light yellow secretions. The growth rate of this strain at 20℃ and 30℃ is faster than that of its two parent strains. The average daily growth rate of the hyphae of this strain measured and calculated after 6 days of dark culture at 20℃ is 7.33mm / d, and the average daily growth rate of the hyphae of this strain measured and calculated after 6 days of dark culture at 30℃ is 14.35mm / d.

[0009] The present invention also provides a product, which is prepared from the Ganoderma lucidum GZJ10.

[0010] Preferably, the product is Ganoderma lucidum fruiting body, food, health care product or medicine.

[0011] Preferably, the product comprises the Ganoderma lucidum GZJ10 water extract.

[0012] Preferably, the Ganoderma lucidum fruiting body is obtained by cultivating the Ganoderma lucidum GZJ10 strain.

[0013] The present invention also provides a method for preparing ganoderma lucidum, using "Shanghai Agricultural Lingzhi No. 1" and "Xin Da Pian" as starting strains, and obtaining the target strain through a protoplast mononuclear hybridization method.

[0014] Preferably, the starting strains are hybridized, and the hybrid strains are subjected to antagonistic reactions with the two parents, and the hybrid strains that have antagonistic reactions with both parents are subjected to cultivation tests.

[0015] Preferably, the present invention uses two Ganoderma lucidum (Leyss.ex Fr.) Karst. varieties, "Shanghai Nong Lingzhi No. 1" and "Xin Da Pian", as starting strains, and prepares monokaryotic strains by protoplast monokaryotic method. The prepared monokaryotic strains of the two varieties are hybridized, and the hybrid strains are subjected to antagonistic reactions with the two parents. The hybrid strains that have antagonistic reactions with both parents are subjected to cultivation tests, and it is found that there are obvious differences in the mycelium and fruiting body morphology with the two parents, and this specific trait of the hybrid strain remains stable in multiple cultivation tests.

[0016] Preferably, the preparation method is used to prepare the Ganoderma lucidum GZJ10.

[0017] The invention also provides a matrix for cultivating ganoderma lucidum. The formula of the matrix comprises 76-80% sawdust, 12-14% wheat bran, 6-8% corn flour, 0.7-1.3% sucrose and 0.7-1.3% gypsum. The water content of the matrix is ​​55-60%.

[0018] Preferably, the formula comprises 78% sawdust, 13% wheat bran, 7% corn flour, 1% sucrose and 1% gypsum.

[0019] Preferably, the cultivation method is used to cultivate the Ganoderma lucidum GZJ10.

[0020] The invention also provides a method for cultivating ganoderma lucidum, which utilizes the matrix for cultivating ganoderma lucidum.

[0021] The present invention also provides the use of the Ganoderma lucidum GZJ10 and the product in any of the following items:

[0022] 1) Preparation of food, health products, medicines or cosmetics;

[0023] Preferably, the food is a beverage;

[0024] Preferably, the drug has the effects of improving human immunity, inhibiting tumors, antibacterial, anti-oxidative or anti-inflammatory;

[0025] Preferably, the cosmetic has anti-inflammatory or anti-oxidative effects;

[0026] 2) Used to extract biologically active substances;

[0027] Preferably, the bioactive substances are polysaccharides, triterpenes and sterols.

[0028] The present invention also provides a marker, comprising any one of the following SNP molecular markers and / or InDel markers; the markers are all located on chromosome JABWUW010000051.1;

[0029] 1) SNP molecular marker at position 1019034, where the base is A or G;

[0030] Preferably, the base at this position is A and is identified as Ganoderma lucidum GZJ10;

[0031] More preferably, the base is G and identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film";

[0032] 2) SNP molecular marker at position 1019048, where the base is C or T;

[0033] Preferably, the base is C and identified as Ganoderma lucidum GZJ10;

[0034] More preferably, the base is T and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0035] 3) SNP molecular marker at position 1019062, where the base is C or T;

[0036] Preferably, the base is C and identified as Ganoderma lucidum GZJ10;

[0037] More preferably, the base is T and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0038] 4) SNP molecular marker at position 1019075, where the base is T or C;

[0039] Preferably, the base at this position is T and is identified as Ganoderma lucidum GZJ10;

[0040] More preferably, the base is C and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0041] 5) InDel marker at position 1019091, where the base is T, TACCTTCCG or TGCCTTCCG;

[0042] Preferably, the base at this position is T and is identified as Ganoderma lucidum GZJ10;

[0043] Or, the base is TACCTTCCG and identified as "Shanghai Agricultural Lingzhi No. 1";

[0044] Alternatively, the base at this location is TGCCTTCCG and is identified as a "new blockbuster".

[0045] 6) InDel marker at position 1019101, where the base is C or CG;

[0046] Preferably, the base is C and identified as Ganoderma lucidum GZJ10;

[0047] Or, the base is CG and identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film";

[0048] 7) SNP molecular marker at position 1019110, where the base is T or C;

[0049] Preferably, the base at this position is T and is identified as Ganoderma lucidum GZJ10;

[0050] More preferably, the base is C and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0051] 8) SNP molecular marker at position 1019114, where the base is T or C;

[0052] Preferably, the base at this position is T and is identified as Ganoderma lucidum GZJ10;

[0053] More preferably, the base is C and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0054] 9) SNP molecular marker at position 1019144, where the base is T or C;

[0055] Preferably, the base at this position is T and is identified as Ganoderma lucidum GZJ10;

[0056] More preferably, the base is C and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0057] 10) SNP molecular marker at position 1019148, where the base is G or T;

[0058] Preferably, the base is G and identified as Ganoderma lucidum GZJ10;

[0059] More preferably, the base is T and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0060] 11) SNP molecular marker at position 1019157, where the base is G or C;

[0061] Preferably, the base is G and identified as Ganoderma lucidum GZJ10;

[0062] More preferably, the base is C and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0063] 12) SNP molecular marker at position 1019170, where the base is C or A;

[0064] Preferably, the base is C and identified as Ganoderma lucidum GZJ10;

[0065] More preferably, the base is A and identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film";

[0066] 13) SNP molecular marker at position 1019172, where the base is C or T;

[0067] Preferably, the base is C and identified as Ganoderma lucidum GZJ10;

[0068] More preferably, the base is T and identified as "Shanghai Nong Lingzhi No. 1" or "Xin Da Pian";

[0069] 14) InDel marker at position 1019183, the base at this position is C or CAACTT; preferably, the base at this position is C and is identified as Ganoderma lucidum GZJ10;

[0070] Or, the base at this location is identified as "Shanghai Agricultural Lingzhi No. 1" or "New Big Film" by CAACTT;

[0071] 15) InDel marker at position 1019189, where the base is G or GA;

[0072] Preferably, the base is G and identified as Ganoderma lucidum GZJ10;

[0073] Or, the base is GA and it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film".

[0074] 16) InDel marker 1019123, the base at this position is ACTG or A;

[0075] Preferably, the base is ACTG and identified as Ganoderma lucidum GZJ10;

[0076] Or, the base at that location is A and it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film".

[0077] The present invention also provides a primer for amplifying the marker;

[0078] Or, comprising the nucleotide sequence shown in SEQ ID No.1-2.

[0079] The present invention also provides a method for identifying Ganoderma lucidum, using the marker or the primer;

[0080] Preferably, 1) extracting genomic DNA of the Ganoderma lucidum to be tested;

[0081] 2) using the genomic DNA of the Ganoderma lucidum to be tested as a template and using the primers to perform a PCR amplification reaction;

[0082] 3) Analyze PCR amplification products;

[0083] Preferably, it is used to identify one or more of Ganoderma lucidum GZJ10, "Shanghai Agricultural Lingzhi No. 1" or "New Big Film";

[0084] Further preferably, a PCR amplification reaction is performed using the nucleotide sequence shown in SEQ ID No. 1-2, and if the product is 651 bp, it is identified as Ganoderma lucidum GZJ10;

[0085] Or, if the product is 660 bp, it is identified as "Shanghai Agricultural Lingzhi No. 1" or "New Big Film".

[0086] The present invention also provides the use of the marker, the primer or the method for identifying Ganoderma lucidum in any of the following:

[0087] (1) Application in identifying phenotypes of Ganoderma lucidum varieties;

[0088] (2) Application in identification, improvement or marker-assisted breeding of Ganoderma lucidum strains;

[0089] (3) Application in early prediction of Ganoderma lucidum traits;

[0090] (4) Application in screening or creating Ganoderma lucidum with sterile holes or spore-free properties;

[0091] (5) Application in Ganoderma lucidum genotyping.

[0092] Beneficial effects of the present invention:

[0093] The strain has a fast growth rate, high content of medicinal ingredients, high fresh weight of fruiting bodies, high biological conversion rate of fruiting bodies, and large flower shape. It does not produce spore powder during the growth process and can be used as a special high-quality variety for cultivating Ganoderma lucidum fruiting bodies.

[0094] At present, domestic edible fungi factory cultivation facilities often use air conditioning units as cooling (heating) equipment, and the spores ejected during the growth of Ganoderma lucidum fruiting bodies can easily cause blockage of temperature control air conditioners. Since this strain does not produce spores, it is more suitable for annual cultivation in domestic edible fungi factory facilities than general Ganoderma lucidum varieties.

[0095] The contents of polysaccharide, triterpenoid and sterol of the Ganoderma lucidum GZJ10 of the present invention are far superior to the standards specified in the pharmacopoeia.

[0096] The specific molecular marker provided by the present invention can accurately identify Ganoderma lucidum GZJ10, and in particular can identify Ganoderma lucidum GZJ10 and its parents. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0098] Figure 1 This is a diagram of the antagonistic reaction between the GZJ10 strain of the present invention and its two parents.

[0099] Figure 2 This is a mycelium morphology diagram of the GZJ10 strain of the present invention.

[0100] Figure 3 This is a morphological diagram of the fruiting body of the GZJ10 strain of the present invention when it is opened.

[0101] Figure 4 This is a statistical box plot of the fruiting body traits of the GZJ10 strain and its parents in Example 4 of the present invention.

[0102] Figure 5 This is a graph showing the growth of the GZJ10 strain of the present invention.

[0103] Figure 6 This is a morphological diagram of the fruiting body (stem) of the GZJ10 strain of the present invention.

[0104] Figure 7 This is a growth diagram of the "Shanghai Agricultural Ganoderma Lucidum No. 1" of the present invention.

[0105] Figure 8 This is a morphological diagram of the fruiting body (stem) of "Shanghai Nong Lingzhi No. 1" of the present invention.

[0106] Fig. 9 This is a growth diagram of the "new large piece" of the present invention.

[0107] Fig.10 This is a morphological diagram of the fruiting body (stem) of the "New Large Slice" strain of the present invention. DETAILED DESCRIPTION

[0108] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0109] The present invention provides a hybrid strain Ganoderma lucidum GZJ10, which is obtained by a protoplast mononuclear hybridization method, wherein the mononuclear strain obtained by protoplast mononuclearization retains two mating types in the parents, and the obtained hybrid strain has stable genetic traits, does not have the phenomenon of offspring trait separation, and accelerates the breeding efficiency. The hybrid strain obtained by the present invention does not produce spore powder, has no fungal pores, and has different traits from the two parents, which may be due to the fact that during the protoplast extraction process, due to external environmental factors, the cells further undergo heritable stable mutations.

[0110] The present invention also provides a method for identifying and verifying hybrid strain-specific molecular markers

[0111] 1) The hybrid strain and the parent strain were resequenced at a depth of 100× and compared with the reference genome (GenBank assembly accession: GCA_019426095.1) to identify specific molecular markers of the hybrid strain.

[0112] 2) The specific molecular markers of the hybrid strain obtained in step 1) were further verified by PCR amplification and Sanger sequencing, and the results were accurate.

[0113] The Ganoderma lucidum GZJ10 of the present invention has been deposited on November 7, 2022 in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and the classification name is Ganoderma lucidum and the preservation number is CGMCC No.40361.

[0114] Example 1 Preparation method of hybrid strain of the present invention

[0115] 1) Slant culture: The original strains of the parent strain of Ganoderma lucidum were inoculated on potato dextrose agar (PDA) solid culture medium under sterile conditions and cultured at 28°C in the dark for about 7 days. Mycelium was visible covering the entire slant.

[0116] The preparation method of PDA solid culture medium is as follows: weigh 46g of PDA powder, add appropriate amount of deionized water to completely dissolve it, add 10g of agar powder to increase its coagulation degree, adjust the volume to 1L, and sterilize it by high pressure steam at 121°C for 20min.

[0117] 2) Primary seed culture: The strains cultured in step 1) were inoculated into liquid culture medium under sterile conditions, protected from light at 28° C., 50%-70% humidity, and cultured in a shaking incubator at 220 rpm for 5-7 days to obtain primary seeds.

[0118] 3) Secondary seed culture: Take about 5 ml of the cultured bacteria in step 2) and transfer it to a new liquid culture medium. Add an appropriate amount of sterile glass beads and culture it in a shaking incubator at 220 rpm. Maintain the culture at 28° C. and humidity of 50%-70% for 3-5 days to obtain secondary seeds.

[0119] The liquid culture medium used in steps 2) and 3) is prepared by weighing 4 g of Yeast extract, 10 g of Malt Extract, and 4 g of glucose, distilled water to 1 L, and sterilized by high-pressure steam at 121° C. for 20 min.

[0120] 4) Protoplast preparation: filter the liquid mycelia of step 3) with 10cm diameter funnel (4 layers of non-woven fabrics), wash 2-3 times with sterile distilled water, wash 1-2 times with 0.6mol / L mannitol, transfer the washed mycelia to a 50ml centrifuge tube, 4 ℃, 6000rpm, 2730×g, centrifugal 5min. Remove supernatant, add lysozyme by the standard of 1mL 2% lysozyme added to every 300mg wet mycelia, put into a shaking table at 31 ℃, 40rpm, shake up occasionally, after enzymolysis 2.5-3h, filter the enzymolysis solution with 6cm diameter funnel (6 layers of non-woven fabrics), rinse the filter screen with 0.6mol / L mannitol, collect the filtrate in a 50ml centrifuge tube, 4080rpm, 1260×g, centrifugal 5min, abandon supernatant, and obtain protoplasts. Take an appropriate amount of 0.6 mol / L mannitol to wash the precipitate 1-2 times, resuspend it in 1 mL of 0.6 mol / L mannitol solution, count it on a blood cell counting plate using a microscope, and dilute the suspension to the required concentration (usually 10 5 / ml).

[0121] 5) Protoplast regeneration: Take 150 μL of the diluted suspension obtained in step 4) and mix it evenly in the regeneration medium. After culturing in the dark at 26° C. for 6-7 days, carefully pick small colonies with pointed tweezers every day and transfer them to PDA solid medium until no new colonies appear or they are too overgrown to be picked.

[0122] The preparation method of the regeneration medium is as follows: 4 g of Yeast extract, 10 g of Malt Extract, 4 g of glucose, 10 g of agar, distilled water to 1 L, and sterilized by high-pressure steam at 121°C for 20 minutes.

[0123] 6) Selection and identification of monokaryotic strains: When the diameter of the colony in step 5) reaches 0.5 cm or more, place the culture dish under an inverted microscope to observe the hyphae morphology at the edge of the colony. If the hyphae morphology is mostly slender, smooth, with few branches and no lock-like union, it can be determined to be a monokaryotic hyphae.

[0124] 7) Hybridization of monokaryotic strains and culture of hybrids: Inoculate monokaryotic hyphae of two different parents in PDA culture medium, 1 cm apart, and culture at 26°C for 8-10 days. Antagonistic lines appear in the opposing culture areas, indicating successful hybridization. Use an inoculation knife to cut 4-5 pieces of 0.5 mm thick 1 cm on both sides of the antagonistic line and on the edges of the two parent colonies away from the antagonistic line. 2 Pick out the bacterial blocks, transfer them to new PDA culture medium, culture them at 26℃ for 7-8 days, and examine them under an inverted microscope. If there is a lock-like union, it is identified as a heterozygous strain.

[0125] 8) Identification of hybrid strains: The hybrid strain identified in step 7) is subjected to an antagonism test with the parent strain, and hybrid strains that have antagonistic lines with both parents are selected for cultivation tests.

[0126] Example 2 Cultivation method of hybrid strain and parent strain of the present invention

[0127] 1) Mother strain culture: The original strains of the Ganoderma lucidum hybrid strain and the parent strain were inoculated on potato dextrose agar (PDA) solid culture medium under sterile conditions, and cultured in the dark at 28°C for about 7 days, and hyphae were visible covering the entire slope;

[0128] 2) The formula of the original seed matrix and the cultivation matrix is: 78% sawdust, 13% wheat bran, 7% corn flour, 1% sucrose, 1% gypsum, and 55-60% water content; bag planting, single head inoculation method, vertical placement. The raw materials and production process should comply with the requirements of NY / T 528-2002 Chapter 3, Article 7, Chapter 4, Articles 2 to 7.

[0129] 3) Initial screening of strains: Use 14×28cm polypropylene bags, the quality of which should meet the requirements of GB / T1003-2008. Seal one end, sterilize by high pressure, and inoculate aseptically. Inoculate 40 bags of each strain, and inoculate two parent strains as controls at the same time, and culture at 26±2℃.

[0130] The greenhouse is used to grow mushrooms, and the temperature in the greenhouse is maintained at 26±2℃, the relative humidity of the air is 75-95%, the carbon dioxide concentration is below 600mg / L, and the sunlight is natural. The mushrooms are grown at one end, and the mushroom growing time, powder spraying time, maturity time, fruiting body shape, color, mushroom uniformity, resistance to impurities, disease resistance, yield, biological efficiency, quality, etc. are recorded. The selection index is set according to the breeding goal, and the target traits are initially selected to exceed the control strain, or the strains with equivalent yield and better target traits than the control are selected as the target strains for rescreening.

[0131] 4) Strain rescreening: Rescreening requires 2-3 production cycles, and 100 bags are inoculated for each strain. The target traits of the target strains screened initially are identified for specificity, consistency, and stability. For strains with better target traits than the control strains and good specificity, consistency, and stability, production screening tests are conducted.

[0132] 5) Production screening: The production screening test is arranged for 1-2 production cycles, and the number of cultivation for each strain is 500 bags, so as to produce the main cultivated variety and the control variety. The target traits of the strains entering the production screening test are identified for specificity, consistency and stability, and the strains with better target traits than the control strains and good specificity, consistency and stability are selected as excellent strains.

[0133] Example 3 Screening and identification method of hybrid strain-specific molecular markers of the present invention

[0134] 1) Preparation of DNA samples of hybrid strains and parents: The mycelium of the hybrid strains and parent strains to be tested was activated and cultured, and the genomic DNA was extracted. The DNA concentration was detected by agarose gel electrophoresis and Nanodrop.

[0135] 2) Whole genome resequencing: Using MGISEQ-2000 gene sequencer or DNBSEQ-T7 gene sequencer, using the double-end sequencing method, a small fragment library (350bp) is constructed for double-end sequencing to obtain whole genome resequencing data. Each DNA sample sequencing generates 4Gb raw data (sequencing data output ≥95% is qualified and no retesting is required), Q30 ≥80%, and the total sequencing volume is 12Gb raw data.

[0136] 3) Off-machine data quality control: remove adapters, contamination sequences and low-quality reads pairs based on raw data; generate statistical data; perform quality control processing such as statistical error rate, quality value, GC content, etc.

[0137] 4) Bioinformatics analysis: The sequencing data were compared with the reference genome (GenBank assembly accession: GCA_019426095.1), and the alignment rate, coverage depth, genome coverage and other information were statistically analyzed; based on the gene structure annotation file, SNP detection, SNP annotation, and statistics on the results of SNP detection and annotation were completed; based on the gene structure annotation file, InDel detection and annotation, and statistics on the distribution of InDel lengths in coding regions were performed.

[0138] 5) Identification and verification of specific molecular markers: Analyze the results in step 4) to screen the SNP and InDel molecular marker sites that are different between the hybrid strain and both parents. Design primers based on the screened hybrid strain specific molecular marker sites, and further verify by PCR amplification and Sanger sequencing to obtain 15 accurate SNP and InDel homozygous specific molecular marker sites.

[0139] Example 4 Analysis of phenotypic characteristics and application advantages of the GZJ10 strain of the present invention

[0140] 1) Analysis of fruiting body characteristics of strain GZJ10 (Table 1, Figure 4 ): Compared with the two parents, the more obvious characteristics of GZJ10 fruiting body are that it does not spray spore powder, the mushroom flesh is not obviously layered, and there are many ring patterns on the upper surface of the cap. Further LSD multiple comparison analysis method was used to find that the fresh weight of GZJ10 single fruiting body was significantly higher than that of the two starting strains, Shanghai Nong Lingzhi No. 1 and Xinda Pian, and the cap size and stipe length were significantly higher than the two parents, and the cap was significantly thinner than the two parents.

[0141] Table 1 Comparative analysis of fruiting body traits between GZJ10 and its parents

[0142]

[0143]

[0144] The color of the lower surface of the cap is yellow during the growing period and yellow-red during the mature period. The color of the two parents during the growing period is milky white and light yellow during the mature period. The color of general red ganoderma varieties is milky white to light yellow, which is significantly different from the two parents and other red ganoderma.

[0145] 2) Pore characteristics of GZJ10 strain ( Figure 5-10 ): There are significant differences in the fruiting body characteristics of the GZJ10 strain and its two parent strains, Shanghai Lingzhi No. 1 and Xinda Pian. Shanghai Lingzhi No. 1 and Xinda Pian have obvious fungus pores, but the GZJ10 strain has no fungus pores.

[0146] 3) Determination of the content of polysaccharides, triterpenes and sterols in GZJ10 (Table 2): The method in the Pharmacopoeia of the People's Republic of China (2020 edition, Volume 1) was used to extract bioactive substances from the fruiting bodies of the GZJ10 strain. It was found that the content of polysaccharides, triterpenes and sterols was far superior to that of the two parental starting strains, and the polysaccharides, triterpenes and sterols met the standards of the Pharmacopoeia of the People's Republic of China (2020 edition, Volume 1).

[0147] Table 2 Determination of polysaccharide and triterpenoid contents in GZJ10

[0148]

[0149] 4) Analysis of the application advantages of GZJ10 strain: From steps 1), 2), and 3), it can be seen that compared with "Longzhi No. 2" and other "cultivating" Ganoderma lucidum varieties that spray a small amount of spore powder, GZJ10 does not produce spore powder at all due to its aseptic hole characteristics. It is easy to manage during greenhouse cultivation, and the ventilation equipment will not be blocked by the diffusion of spore powder. It can also save the cost of removing spore powder on the surface of the fruiting body. In addition, although the appearance of the GZJ10 strain is different from that of the two parent strains, its polysaccharide and triterpene content is far superior to the two parent strains. Therefore, using GZJ10 as a raw material for processing "cultivating" Ganoderma lucidum can increase production efficiency.

[0150] Example 5 Specific molecular markers of the GZJ10 strain of the present invention

[0151] By analyzing the resequencing results of the GZJ10 strain and the two parental strains, a large number of SNP and InDel molecular marker sites that are different from both parents were screened in the GZJ10 strain. Primers were further designed based on the unique molecular marker sites of the screened GZJ10 strain (Table 3), and further verified by PCR amplification and Sanger sequencing methods. The sequencing results are shown in SEQID No.3, SEQ ID No.4, and SEQ ID No.5. 16 specific SNP and InDel homozygous molecular markers (Table 4) and 16 heterozygous molecular marker sites different from the parents were identified in the GZJ10 strain.

[0152] Table 3 Specific molecular marker primer sequences and amplified specific fragment information

[0153]

[0154] Table 4 16 SNPs and InDel homozygous specific molecular marker sites of GZJ10 strain verified by sequencing

[0155]

[0156] Table 5 16 SNP and InDel heterozygous molecular marker sites of GZJ10 strain that are different from the parent and verified by sequencing

[0157]

[0158]

[0159] >SEQ ID No.3GZJ10

[0160]

[0161]

[0162] >SEQ ID No.4 Shanghai Agricultural Ganoderma Lucidum No. 1

[0163]

[0164] >SEQ ID No.5 new blockbuster

[0165]

[0166]

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Lingzhi ( Ganoderma lucidum ) GZJ10, characterized in that, It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.40361.

2. A product, characterized in that: It is prepared from the Ganoderma lucidum GZJ10 described in claim 1.

3. The product according to claim 2, characterized in that: The product is a ganoderma lucidum fruiting body or a medicine.

4. The product according to claim 3, characterized in that: The Ganoderma lucidum fruiting body is obtained by cultivating the Ganoderma lucidum GZJ10 strain according to claim 1.

5. A method for identifying Ganoderma lucidum, characterized in that: The following SNP molecular markers and InDel markers were used in combination; the markers were all located on chromosome JABWUW010000051.1; 1) SNP molecular marker at position 1019034, where the base is A or G; 2) SNP molecular marker at position 1019048, where the base is C or T; 3) SNP molecular marker at position 1019062, where the base is C or T; 4) SNP molecular marker at position 1019075, where the base is T or C; 5) InDel marker at position 1019091, where the base is T, TACCTTCCG or TGCCTTCCG; 6) InDel marker at position 1019101, where the base is C or CG; 7) SNP molecular marker at position 1019110, where the base is T or C; 8) SNP molecular marker at position 1019114, where the base is T or C; 9) SNP molecular marker at position 1019144, where the base is T or C; 10) SNP molecular marker at position 1019148, where the base is G or T; 11) SNP molecular marker at position 1019157, where the base is G or C; 12) SNP molecular marker at position 1019170, where the base is C or A; 13) SNP molecular marker at position 1019172, where the base is C or T; 14) InDel marker at position 1019183, where the base is C or CAACTT; 15) InDel marker at position 1019189, where the base is G or GA; 16) InDel marker 1019123, the base here is ACTG or A; The position of the marker is referenced to the genome GenBank assembly accession: GCA_019426095.

1.

6. The method for identifying Ganoderma lucidum according to claim 5, characterized in that: 1) The base at this position is A and is identified as Ganoderma lucidum GZJ10; Or, if the base is G, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 2) The base at this position is C and it is identified as Ganoderma lucidum GZJ10; Or, if the base is T, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 3) The base at this position is C and it is identified as Ganoderma lucidum GZJ10; Or, if the base is T, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 4) The base at this position is T and is identified as Ganoderma lucidum GZJ10; Or, if the base is C, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 5) The base at this position is T and is identified as Ganoderma lucidum GZJ10; Or, the base is TACCTTCCG and identified as "Shanghai Agricultural Lingzhi No. 1"; Or, the base at this position is TGCCTTCCG and is identified as a "new blockbuster"; 6) The base at this position is C and it is identified as Ganoderma lucidum GZJ10; Or, the base is CG and identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 7) The base at this position is T and is identified as Ganoderma lucidum GZJ10; Or, if the base is C, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 8) The base at this position is T and is identified as Ganoderma lucidum GZJ10; Or, if the base is C, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 9), the base at this position is T and identified as Ganoderma lucidum GZJ10; Or, if the base is C, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 10), the base is G and it is identified as Ganoderma lucidum GZJ10; Or, if the base is T, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 11), the base is G and it is identified as Ganoderma lucidum GZJ10; Or, if the base is C, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 12), the base at this position is C and identified as Ganoderma lucidum GZJ10; Or, if the base is A, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 13), the base at this position is C and identified as Ganoderma lucidum GZJ10; Or, if the base is T, it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 14), the base at this position is C and identified as Ganoderma lucidum GZJ10; Or, the base at that location is identified as "Shanghai Agricultural Lingzhi No. 1" or "New Big Film" by CAACTT; 15), the base here is G and it is identified as Ganoderma lucidum GZJ10; Or, the base is GA and it is identified as "Shanghai Nong Lingzhi No. 1" or "New Big Film"; 16), the base at this position is ACTG and is identified as Ganoderma lucidum GZJ10; Or, the base at that location is A and it is identified as "Shanghai Agricultural Lingzhi No. 1" or "New Big Film".

7. The method for identifying Ganoderma lucidum according to claim 5, characterized in that: A PCR amplification reaction is performed using the nucleotide sequence shown in SEQ ID No. 1-2. If the product is 651 bp, it is identified as Ganoderma lucidum GZJ10; Or, if the product is 660 bp, it is identified as "Shanghai Agricultural Lingzhi No. 1" or "New Big Film".

8. Use of the method for identifying Ganoderma lucidum according to any one of claims 5 to 7 in any one of the following: (1) Application in identifying the phenotypes of Ganoderma lucidum varieties; (2) Application in identification, improvement or marker-assisted breeding of Ganoderma lucidum strains; (3) Application in early prediction of Ganoderma lucidum traits; (4) Application in screening or creating Ganoderma lucidum with sterile holes or non-spore powder characteristics; (5) Application in Ganoderma lucidum genotyping.

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