KR101834565B1 - CAPS Marker for Differentiation of Shiitake Mushroom Cultivar Sanmaru-1 and use thereof - Google Patents

Abstract

The present invention provides discriminating kit and method capable of quickly and easily discriminating shiitake mushroom cultivar Sanmaru-1. The discriminating kit and method for shiitake mushroom cultivar Sanmaru-1 of the present invention have advantages in that cost and time for cultivar discrimination can be reduced and the results are easily interpreted.

Description

{CAPS Marker for differentiation of Shiitake Mushroom Cultivar Sanmaru-1 and use thereof}

The present invention relates to a CAPS marker for discriminating mushroom cultivar ridge No. 1 and its use.

Globally, the mushroom industry has increased rapidly in the late 1990s, and mushrooms, mushrooms, mushrooms, mushrooms, and top mushrooms account for about 95% of the world's mushroom production, , Which are commonly grown in Asian countries, account for about 17%. In Korea, the production and production of shiitake mushrooms account for about 97% of domestic mushroom production in 2016, and the preference of mushrooms in Korea is 20.6% for shiitake mushroom, 13.4% for shiitake mushroom, 12.9% for oyster mushroom, Followed by mushroom mushroom (11.1%) and top mushroom (8.9%). As such, altitude has a very important place in domestic mushroom market and short-term income forest products.

Korea joined the UPOV for the 50th time in 2002, and since 2008, shiitake mushrooms have been designated as crop varieties under the seed industry law. At present, when cultivating shiitake mushroom cultivars in Korea, we distinguish among cultivars according to the investigation method of shiitake mushroom characteristics produced by Korea Forest Service. However, it would be more efficient to use molecular markers other than morphological markers. The adoption of the Nagoya Protocol by the 10th Conference of the Parties to the Convention on Biological Diversity in October 2010 is expected to strengthen the protection of existing genetic resources. Therefore, it is very important to distinguish domestic shiitake mushroom cultivars distributed in Korea as molecular markers, as well as to protect the genetic resources of Korean mushroom genomes and to differentiate the imported cultivars.

Molecular markers have the advantage that they are stable regardless of developmental stages and can be detected in all tissues because they target DNA sequence differences, which is the essence of genetic phenomena. The types of molecular markers developed so far include AFLPs (Restriction Fragment Length Polymorphisms), RFLPs (Restriction Fragment Length Polymorphisms), Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSRs), Sequence Tagged Sites And Cleaved Amplification Polymorphic Sequence (CAPS). Double CAPS is a method to identify the size difference of DNA fragment according to the cleavage site by treating the amplified PCR product with a restriction enzyme. It is a molecule that can predict the mutation of single nucleotide polymorphism (SNP) It is a marker. The CAPS marker-based assay has the advantage that it is cost-effective because it is simple and requires no expensive equipment.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a method for quickly and accurately discriminating the mushroom cultivar Rimaru 1. As a result, specific SNP (Single Nucleotide Polymorphism) of mushroom variety Ridge 1 was identified, and cleaved amplification polymorphic sequence (CAPS) marker, which can distinguish it, was used for polymerase chain reaction and restriction enzyme treatment. 1 of the present invention can be discriminated.

Accordingly, an object of the present invention is to provide a kit for discriminating mushrooms of a mushroom variety.

Another object of the present invention is to provide a method for distinguishing the mushroom cultivar Rimaru 1.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a nucleic acid molecule comprising 10-100 consecutive nucleotide sequences comprising a single nucleotide polymorphism site having thymine (T) at the 120 th nucleotide position of the first sequence of the sequence listing There is provided a kit for discriminating a mushroom cultivar Rimaru 1 comprising a primer or a probe specifically binding thereto.

The present inventors have sought to develop a method for quickly and accurately discriminating the mushroom cultivar Rimaru 1. As a result, specific SNP (Single Nucleotide Polymorphism) of mushroom variety Ridge 1 was identified, and cleaved amplification polymorphic sequence (CAPS) marker, which can distinguish it, was used for polymerase chain reaction and restriction enzyme treatment. 1).

The kit for discriminating mushroom cultivar 1 of the present invention comprises a primer or a probe specifically binding to 10-100 consecutive nucleotide sequences comprising a single nucleotide polymorphic site having a thymine at the 120th nucleotide position of the first sequence of the sequence listing .

As used herein, the term " nucleotide "is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

As used herein, the term " single nucleotide polymorphism (SNP) " refers to a DNA sequence that occurs when a single base (A, T, C or G) in the genome is different between members of a species or between individual chromosomes . Single bases can be changed (substituted), removed (deleted), or added (inserted) to the polynucleotide sequence. The SNP can cause a change in the translation frame. Single nucleotide polymorphisms can be included in coding sequences of genes, non-coding regions of genes, or in intergenic regions between genes. SNPs in the coding sequence of a gene do not necessarily cause changes in the amino acid sequence of the target protein due to the degeneracy of the genetic code. SNPs that form the same polypeptide sequence are called synonymous (also called silent mutations) and SNPs that form other polypeptide sequences are said to be non-synonymous. Non-consensual SNPs can be missense or nonsense, and mismatch changes produce other amino acids while nonsense changes form non-mature termination codons. SNPs that are not in the protein-coding region can induce gene silencing, transcription factor binding, or non-coding RNA sequences.

As used herein, the term " primer " refers to an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, i.e. the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis under conditions of temperature and pH. Specifically, the primer is a deoxyribonucleotide and a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.

In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or And may include an intercalating agent.

As used herein, the term " probe " means a linear oligomer having a natural or modified monomer or linkage comprising a deoxyribonucleotide and a ribonucleotide that can hybridize to a particular nucleotide sequence. Specifically, probes are single strands for maximum efficiency in hybridization. The probe is specifically a deoxyribonucleotide.

As the primer or probe used in the present invention, a perfectly complementary sequence may be used for the sequence including the SNP, but a sequence substantially complementary to the specific sequence does not interfere with the specific hybridization May be used. Specifically, the probe used in the present invention includes a sequence that can be hybridized to a sequence comprising 10 to 30 contiguous nucleotide residues including the SNP of the present invention. More specifically, the 3'-end or the 5'-end of the probe has a base complementary to the SNP base. Generally, the stability of the duplex formed by hybridization tends to be determined by the agreement of terminal sequences, so that in a probe having a base complementary to the SNP base at the 3'-terminal or 5'-terminal, If the part is not hybridized, such a duplex can be disassembled under stringent conditions.

Suitable conditions for hybridization include, but are not limited to, Nucleic Acid Hybridization, A Practical Approach, Hayes, BD, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, IRL Press, Washington, DC (1985). ≪ / RTI > The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

As used herein, the term " complementary " means that under certain annealing or hybridization conditions, the primer or probe is sufficiently complementary to hybridize selectively to the target nucleic acid sequence and is substantially complementary and fully complementary perfectly complementary, meaning that it is completely complementary. As used herein, the term " substantially complementary sequence " as used in connection with a primer sequence is intended to encompass a complete sequence as well as a sequence that is comparable to that of the sequence to be compared, Inconsistent sequences are also included.

The kits for discriminating the mushroom cultivar Ridge mushroom 1 of the present invention can be applied by various methods known in the art used for identifying specific sequences. For example, techniques that may be applied to the present invention include fluorescence in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-strand conformational analysis (SSCA, Orita et al., PNAS, USA 86: 2776 (1989)), RNase protection analysis (Finkelstein et al., Genomics, 7: 167 (1990)), dot blot analysis, denaturing gradient gel electrophoresis (DGGE, Wartell et al., Nucl. Acids Res. (Modrich, Ann. Rev. Genet., 25: 229-253 (1991)) using a protein recognizing a nucleotide mismatch (e.g., mutS protein of E. coli) But are not limited to, allelic-specific PCR.

According to another aspect of the present invention, the present invention provides a primer set comprising (a) a primer set consisting of a nucleotide sequence of a second sequence and a third sequence; And (b) a restriction enzyme that recognizes a 4-20 consecutive nucleotide sequence comprising the 120th nucleotide of the first sequence of SEQ ID NO: 1.

According to one embodiment of the present invention, the restriction enzyme is TspR I, TscA I or Bts I Mut I.

The restriction enzyme TspR I or TscA I recognizes the nucleotide sequence of CASTG (S is G or C) and the restriction enzyme Bts I Mut I recognizes the nucleotide sequence of CASGTG. TscR I, TscA I or Bts I Mut I in the case where the 120th nucleotide position of the first sequence of the sequence listing is guanine (G) or cytosine (C), and adenine (A) or thymine It is not cut. That is, the mushroom cultivar ridge No. 1 can not be cleaved by the restriction enzyme because the 120th nucleotide position of the first sequence of the sequence listing is thymine, and thus it can be distinguished from the mushroom of another variety.

The above kit for discriminating the mushroom cultivar ridge line 1 can be carried out by the gene amplification method.

The term "amplification" as used herein in describing a gene amplification kit refers to a reaction to amplify a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP439,182), transcription-mediated amplification (TMA, WO 88/10315) (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (PCR), and the like. The term " amplification of target polynucleotide sequences " (CP-PCR), U.S. Patent No. 4,437,975) (AP-PCR), U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA), U.S. Patent No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21,22), and loop-mediated isothermal amplification. (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications.

For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, SpringerVerlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

When the kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include reagents necessary for PCR amplification, such as a buffer, a DNA polymerase, a DNA polymerase joiner, and dNTPs. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention, including the "Clenow" fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Specifically, the polymerase is a thermostable DNA polymerase from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. To provide joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{+ 2} to the reaction mixtures to have a desired degree of amplification can be achieved is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants. In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

The nucleotide sequence of the thus amplified marker of the present invention is subjected to restriction enzyme treatment to distinguish the mushroom cultivars by comparing the sizes of the products. For example, the size of the marker of the present invention is examined by performing gel electrophoresis on the result of amplification reaction described above, and observing and analyzing the resulting band.

The SNPs located in the restriction enzyme sites used in the present specification are referred to as CAPS markers.

In the present invention, the " CAPS marker " is a marker capable of analyzing a change in a site, such as SNP, which is changed by one base sequence or by a restriction enzyme generated by InDel. CAPS markers are PCR-amplified primers specific to loci, followed by restriction enzyme digestion and polymorphism analysis.

According to yet another aspect of the present invention, there is provided a method for differentiating a mushroom cultivar Ridgeline I comprising the following steps:

(a) separating nucleic acid molecules from shiitake;

(b) detecting a base type of single nucleotide polymorphism (SNP) of the nucleic acid molecule, wherein the SNP is a SNP located at a 120 th nucleotide position of the first sequence of the sequence listing; And

(c) The step of discriminating the mushroom cultivar ridge No. 1 from the base type of the detected SNP position. When the base at the SNP position is thymine (T), the mushroom cultivar Ridge No. 1 is judged.

As used herein, the term " nucleic acid molecule " is intended to encompass DNA (gDNA and cDNA) and RNA molecules in a comprehensive sense. The nucleotide, which is a basic constituent unit in a nucleic acid molecule, includes not only natural nucleotides, (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

According to one embodiment of the present invention, the nucleic acid molecule is genomic DNA (gDNA).

In the method of the present invention, the genomic DNA may be a medium of shiitake, a shiitake mushroom or a shiitake mycelium. Specifically, it is obtained from mycelium of shiitake mushrooms. Plants as a source of nucleic acid molecules can be used as sources, whatever the stage of development.

When the starting material in the method of the present invention is gDNA, the separation of gDNA can be carried out according to conventional methods known in the art (Rogers & Bendich (1994)).

According to another aspect of the present invention, the present invention provides a method for differentiating shiitake mushroom variety 1, comprising the steps of:

(a) separating nucleic acid molecules from shiitake;

(b) performing a polymerase chain reaction using the nucleic acid molecule as a template and using a primer set consisting of a nucleotide sequence of the second and third sequences of the sequence listing;

(c) treating the amplification product of the polymerase chain reaction with a restriction enzyme recognizing the 4-20 consecutive nucleotide sequences including the 120 < th > nucleotide of the first sequence of the sequence listing; And

(d) analyzing the result of step (c). If the result is not cleaved by a restriction enzyme, it is judged to be a first mushroom variety.

Since the method of the present invention utilizes the same method, primer and / or restriction enzyme as the above kit, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a discriminating kit and a discriminating method, which can quickly and easily discriminate a mushroom cultivar Rimaru 1.

(b) The present invention provides a kit for discriminating the mushroom cultivar, which is low in cost and time for discriminating the cultivars and easy to interpret the result, and a method for distinguishing it.

Fig. 1 shows the result of PCR performed using a primer set for discriminating the mushroom cultivar 1 and sequencing the PCR product to confirm the SNP.
Fig. 2 shows the result of PCR using the primer set for discriminating the mushroom cultivar Rimoru 1 and confirming the CAPS marker using the restriction enzyme TspR I.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / Liquid / liquid is (volume / volume)%

Example

Production of primer

Table 1 shows the marker sequence (RL-LE-116) and the primer sequence amplified in the mushroom cultivar Rimaru 1 by the PCR method using the primer sequence. A nucleotide sequence with specific mutations in the Ridge 1 was obtained from the genomic sequence of the mushroom, and a primer was prepared using the sequence.

- order Sequence List RL-LE-116 5'-CTGGAAAAGGGCCTCATTCTaaagagtcactaagtacatgcaaatgatcatgaatttctcaagtcaaaagtctctgaattatttctcagggtctacttacatagtactattcatcatca T tgatttgtttgtacatgtgtacaaaagacaccaggaactgcttcaattgtcggtggcagatagaccttgatgccggtttgccgacacccataggcaaaccatAGTCTTCGAATGGGCATGTC-3 ' (242 bp) First sequence Forward primer 5'-CTGGAAAAGGGCCTCATTCT-3 '(20 bp) Second sequence Reverse primer 5'-GACATGCCCATTCGAAGACT-3 '(20 bp) Third sequence

PCR methods and results

PCR was performed using the primer sequences shown in Table 1 and sequencing was performed to confirm the mutation (Fig. 1). Genomic DNA was extracted from B17, a standard mushroom strain, and the mushroom cultivar Rimaru 1 to obtain 20 ng PCR was performed using DNA. DNA extraction and PCR are performed as follows.

The mushroom mycelium cultivated in PDB (Potato Dextrose Broth) medium was filtered through miracloth and washed with PBS buffer (NaCl 135 mM, KCl 2.7 mM, Na ₂ HPO ₄ 4.3 mM and KH ₂ PO ₄ 1.4 mM) And the water was removed with a kitchen towel. The dried hyphae were frozen in liquid nitrogen and finely ground in a mortar and then genomic DNA was extracted using the GeneAll® GenEx ™ Plant kit. 500 μl of PL buffer was added to the mycelium transferred to the tube, and the mixture was heated at 65 ° C for 10 minutes and mixed well using a pipette. After centrifugation at 13000 rpm for 1 min, the supernatant was separated and transferred to a new tube. One third of the supernatant was added to the supernatant, and the supernatant was mixed well using a Vortex. After standing for 5 minutes on ice, centrifugation was carried out at 13000 rpm for 5 minutes. The supernatant was removed and transferred to a new tube. The same volume of PCI (phenol: Chloroform: isoamylalcohol = 25: 24: 1) was added and the mixture was inverted. After centrifugation at 13000 rpm for 10 minutes, the supernatant was separated, transferred to a new tube, equilibrated with isopropanol, and mixed in turn. The ice was left for 10 minutes and then centrifuged at 13000 rpm for 1 minute. After removal of the supernatant, 1 ml of 70% ethanol was added to shake the DNA pellet and centrifuged at 13000 rpm for 1 minute. The supernatant was removed and centrifuged once more at 13000 rpm for 1 min. After the remaining ethanol was completely removed, the DNA pellet was dried at room temperature for 5 minutes, and 100 μl of RE buffer was added to dissolve the DNA pellet. PCR was carried out using 20 ng of the extracted genomic DNA as a template using the primer prepared in the present invention.

PCR was performed by amplifying 35 cycles at 95 ° C for 3 minutes, again at 95 ° C for 30 seconds, at 58 ° C for 30 seconds, at 72 ° C for 20 seconds, and then at 72 ° C for 5 minutes.

Sequencing results were aligned and compared after PCR, confirming the presence of specific mutations in Ridge 1.

Fig. 2 shows the result of PCR using the primer sequences shown in Table 1 and the restriction enzyme TspR I treatment. Genomic DNA was extracted from shiitake mushroom standard genomic strain B17 and mushroom cultivar Baekhwa incense, Rimaru 1, Rimaru 2, acid lavender, Chalam, Sanjo 701, Sanjo 707, Sanjo 708, and 20 ng of DNA Were used for PCR. The restriction enzyme TspR Ⅰ confirmed that Ridge 1 was differentiated from other cultivars. The division of Ridge No. 1 can be confirmed by a black band pattern.

As can be seen in FIG. 2, the product of the mushroom cultivar Rimaru 1 was not cleaved by restriction enzymes and showed a band size of 242 bp. The products of other mushroom cultivars truncated by Tsp RI were 118 bp and 124 bp Lt; / RTI >

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> CAPS Marker for Differentiation of Shiitake Mushroom Cultivar          Sanmaru-1 and use thereof <130> PN170039 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 242 <212> DNA <213> Unknown <220> <223> Shiitake Mushroom Cultivar Sanmaru-1 <400> 1 ctggaaaagg gcctcattct aaagagtcac taagtacatg caaatgatca tgaatttctc 60 aagtcaaaag tctctgaatt atttctcagg gtctacttac atagtactat tcatcatcat 120 tgatttgttt gtacatgtgt acaaaagaca ccaggaactg cttcaattgt cggtggcaga 180 tagaccttga tgccggtttg ccgacaccca taggcaaacc atagtcttcg aatgggcatg 240 tc 242 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 ctggaaaagg gcctcattct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 gacatgccca ttcgaagact 20

Claims (6)

Comprising a primer or a probe specifically binding to 10-100 consecutive nucleotide sequences comprising a single nucleotide polymorphism site having a thymine at the 120th nucleotide position of the first sequence of the sequence listing, Varieties Ridge No. 1 kit for discrimination.
(a) a primer set consisting of a nucleotide sequence of a second sequence and a third sequence of the sequence listing; And (b) a restriction enzyme that recognizes the 4-20 consecutive nucleotide sequences comprising the 120th nucleotide of Sequence Listing 1. Sequence identification kit.
3. The kit according to claim 2, wherein the restriction enzyme is TspR I, TscA I or Bts I Mut I.
Differentiating method of Ridge Mushroom Varieties 1 including the following steps:
(a) separating nucleic acid molecules from shiitake;
(b) detecting a base type of single nucleotide polymorphism (SNP) of the nucleic acid molecule, wherein the SNP is a SNP located at a 120 th nucleotide position of the first sequence of the sequence listing; And
(c) The step of discriminating the mushroom cultivar ridge number 1 from the base type of the detected SNP position. When the base at the SNP position is thymine, it is judged to be the mushroom ridge ridge number 1.
Differentiating method of Ridge Mushroom Varieties 1 including the following steps:
(a) separating nucleic acid molecules from shiitake;
(b) performing a polymerase chain reaction using the nucleic acid molecule as a template and using a primer set consisting of a nucleotide sequence of the second and third sequences of the sequence listing;
(c) treating the amplification product of the polymerase chain reaction with a restriction enzyme recognizing the 4-20 consecutive nucleotide sequences including the 120th nucleotide of the first sequence of the sequence listing; And
(d) analyzing the result of step (c). If the result is not cleaved by a restriction enzyme, it is judged to be a first mushroom variety.
6. The method according to claim 5, wherein the restriction enzyme of step (c) is TspR I, TscA I or Bts I Mut I.

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