KR100996968B1 - SSR primer derived from Oyster Mushroom and use of there - Google Patents

Abstract

The present invention relates to an SSR primer isolated from a mushroom and its use, and more particularly, SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 72, and performing PCR using the same. It relates to a DNA polymorphism detection method of a mushroom comprising the. SSR primer pair provided in the present invention can effectively detect the DNA polymorphism of the mushroom, it can be very useful for preparing the DNA profile of the mushroom. In addition, by efficiently evaluating the genetic resources of the mushroom through this, it is possible to effectively perform redundancy analysis and establishment of novelty with existing resources when introducing new genetic resources.

SSR Primer, Mushroom, DNA Polymorphism

Description

SSR primer derived from Oyster Mushroom and use of there}

The present invention (Oyster Mushroom; Pleurotus SSR primers isolated from Ostreatus Kummer) and their use.

The present invention (Oyster Mushroom; Pleurotus SSR primer isolated from Ostreatus Kummer and its use, and more specifically, SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 72 and performing PCR using the same It relates to a DNA polymorphism detection method of a mushroom containing.

Large quantities of genetic resources are collected annually in most major crops and cultivated crops used for breeding. However, on the other hand, the evaluation of genetic resources has not been sufficiently made. Rapid discrimination between species and subspecies varieties of collected genetic resources is very important for accurate management and protection of genetic resources. In addition, the identification of varieties, the identification and classification of trait traits of genetic resources, and the identification of soft relationships are very important for the conservation of genetic resources, the creation of new varieties, and the improvement of crops.

Recent rapid advances in molecular biology have led to the development of nucleic acid fingerprint analysis methods and various DNA markers that enable bio-diversity studies at the nucleic acid (DNA) level. This makes it possible to search useful traits, identify species of species, identify species classifications, and analyze flexible relationships of populations in a simple and quick way with little influence on the external environment. Fingerprinting methods using polymerase chain reaction (PCR) developed so far include randomly amplified polymorphic DNAs (RAPD), amplified fragment length polymorphic DNA (AFLP), and simple sequence repeat (SSR). The RAPD method has a disadvantage of poor reproducibility because the non-specific PCR product is amplified, and the AFLP method is spotlighted by high DNA polymorphism detection, but has a disadvantage of complicated appearance and analysis of a low reproducible band. In contrast, the SSR method is a method of constructing a PCR primer based on nucleotide sequence information of a microsatellite region, which is a DNA repeat sequence. Frequently used. In particular, the SSR method has the advantage that it is not affected by the external environment at all. Due to the superiority of this SSR method, studies are underway to develop SSR markers in several major crops and cultivated crops.

In rice, supersatellite markers have been developed that amplify SSR repeated in the rice genome by PCR technology to identify genotypes of rice varieties (Korean Patent Application No. 2001-34003). Tang's team in the United States developed 879 SSR markers and published a genetic map of sunflowers (Tang et al ., Theor . Appl . Genet ., 105: 1124-1136, 2002). In addition, many SSR markers have been developed in barley and soybeans. However, SSR markers have not been developed in Korea at all in mushrooms, and studies related to them have not been made in foreign countries.

Accordingly, the present inventors have completed the present invention by developing SSR primer pairs showing a large number of polymorphic variation in various mushroom strains, while continuing to develop SSR markers capable of efficiently evaluating the genetic resources of mushrooms.

It is therefore an object of the present invention to provide an SSR marker and its use capable of efficiently evaluating the genetic resources of mushrooms.

In order to achieve the above object, the present invention provides an SSR primer pair having two nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 72.

In order to achieve another object of the present invention, the present invention provides a method for detecting DNA polymorphism of mushrooms and a method for identifying a variety of mushrooms, including performing PCR using the SSR primer pair.

In order to achieve another object of the present invention, the present invention provides a kit for detecting the DNA polymorphism of the mushroom and kit for identifying the variety of the mushroom including the SSR primer pair, DNA polymerase and PCR reaction buffer.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing an SSR marker capable of specifically analyzing the genetic polymorphism of the mushroom.

In the present invention, SSR markers were developed from mushrooms by biotin-streptavidin capture (Dixit et al., Mol. Eco.Note, 5: 736-738). SSR markers isolated from mushrooms are the first to be provided by the present invention.

Specifically, the SSR marker of the present invention was developed by the following method. Genomic DNA extracted from the mushroom was treated with restriction enzymes to prepare DNA fragments and then ligated with two adapters of AP11 and AP12.

Ligation products were hybridized with biotin-labeled SSR probes and then separated using magnetic beads. PCR was performed using the AP11 primer to amplify and analyze the sequence, thereby obtaining 500 DNA fragments containing the mushroom supernatant.

By analyzing the DNA fragments obtained above, 200 pairs of bidirectional primers were designed to select only fragments containing five or more repeating units and to amplify the supersatellite based on the portion containing the repeating unit. 36 pairs of SSR primer pairs showing polymorphic variation were selected by performing DNA profiling through PCR of various cultured mushroom strains and varieties using the primer pairs.

The SSR marker provided by the present invention refers to a pair of PCR primers, and includes a forward primer and a reverse primer. SSR primer pair provided in the present invention has two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 72. Preferably GB-PO-001 (primary pairs represented by SEQ ID NOs: 1 and 2), GB-PO-006 (primary pairs represented by SEQ ID NOs: 3 and 4), GB-PO-011 (SEQ ID NOs: 5 and 6) Primer pairs shown in the figure), GB-PO-025 (primary pairs shown in SEQ ID NOs: 7 and 8), GB-PO-026 (primary pairs shown in SEQ ID NOs: 9 and 10), GB-PO-028 (SEQ ID NO: Primer pairs represented by numbers 11 and 12), GB-PO-039 (primary pairs represented by SEQ ID NOs: 13 and 14), GB-PO-050 (primer pairs represented by SEQ ID NOs: 15 and 16), and GB-PO -051 (primary pairs represented by SEQ ID NOs: 17 and 18), GB-PO-061 (primary pairs represented by SEQ ID NOs: 19 and 20), and GB-PO-064 (primary pairs represented by SEQ ID NOs: 21 and 22) , GB-PO-076 (primary pairs represented by SEQ ID NOs: 23 and 24), GB-PO-079 (primary pairs represented by SEQ ID NOs: 25 and 26), and GB-PO-080 (SEQ ID NOs: 27 and 28) Primer pairs), GB-PO-086 (SEQ ID NOS: 29 and 30) Primer pairs), GB-PO-094 (primary pairs represented by SEQ ID NOs: 31 and 32), GB-PO-097 (primary pairs represented by SEQ ID NOs: 33 and 34), and GB-PO-102 (SEQ ID NOs 35 and Primer pair represented by 36), GB-PO-113 (primary pair represented by SEQ ID NOs: 37 and 38), GB-PO-115 (primary pair represented by SEQ ID NOs: 39 and 40), and GB-PO-117 ( Primer pairs represented by SEQ ID NOs: 41 and 42), GB-PO-124 (primer pairs represented by SEQ ID NOs: 43 and 44), GB-PO-128 (primer pairs represented by SEQ ID NOs: 45 and 46), GB- PO-131 (primary pairs represented by SEQ ID NOs: 47 and 48), GB-PO-134 (primary pairs represented by SEQ ID NOs: 49 and 50), and GB-PO-135 (primary pairs represented by SEQ ID NOs: 51 and 52) ), GB-PO-138 (primary pairs represented by SEQ ID NOs: 53 and 54), GB-PO-149 (primary pairs represented by SEQ ID NOs: 55 and 56), and GB-PO-152 (SEQ ID NOs: 57 and 58) Primer pairs shown), GB-PO-154 (SEQ ID NOs: 59 and 60) Primer pairs), GB-PO-157 (primary pairs represented by SEQ ID NOs: 61 and 62), GB-PO-171 (primary pairs represented by SEQ ID NOs: 63 and 64), and GB-PO-172 (SEQ ID NO: 65 Primer pairs represented by and 66), GB-PO-173 (primer pairs represented by SEQ ID NOs: 67 and 68), GB-PO-181 (primer pairs represented by SEQ ID NOs: 69 and 70), and GB-PO-190 (A primer pair represented by SEQ ID NOs: 71 and 72). Information on the primer pairs provided in the present invention and the repeat motifs present in the supersatellites amplified by them, the size of the supersatellites to be amplified, T _m (° C.) and the chromosomal alleles in which the supersatellites are present It is listed in Table 1 below.

Primer Name order order
number Repeating motif Range of size of supersatellite to be amplified (bp) Tm (℃) Number of alleles GB-PO-001 F: CGCAAGCTACAAACGGAC One (GCA) 3 (ATTGGC) (GCA) 1 307-313 52 2 R: AGCAGCAAGCACAAGAGC 2 GB-PO-006 F: TGTGGCAAACCCAAGTTC 3 (GGC) 4 (GAC) (GGC) 1 203-206 52 2 R: CCCAAAGGATGAGGAAGG 4 GB-PO-011 F: TCCCATACCCTGACATCG 5 (CTG) 4 (TA) (CTG) 1 166-235 52 5 R: ATCATCAAGCGCCACAAC 6 GB-PO-025 F: TGATCATGGCGAGTAGGG 7 (GGA) 10 214-217 52 2 R: GGAACTGTCAGCAGACGC 8 GB-PO-026 F: AATCGCATGGGCTCTG 9 (TTG) 3 (TTC) (TTG) 3 293-296 52 2 R: CTGTCCCTCCGTGTACCA 10 GB-PO-028 F: CTGGAGAATCGTAGCCCC 11 (GTC) 13 (CAT) 5 291-318 52 5 R: ACAAGCGCTCGGAATACA 12 GB-PO-039 F: TGTGGATGTGATGTGATGTG 13 (GGT) 2 (GGC) (GGT) 5 191-224 52 4 R: ACGTCCAGCGTCGAGTTA 14 GB-PO-050 F: CATCCGATACAGACCCGA 15 (GTT) 5 145-175 52 5 R: AGGCATCCCACAACACTG 16 GB-PO-051 F: CATAGGGACGACAGCGAG 17 (GCT) 6 272-278 52 3 R: ACTGAGCCTTCAGCACCA 18 GB-PO-061 F: TAACTTGGGCGCTTGAAA 19 (GCA) 2 (CAGTAC) (GCA) 3 258-270 52 2 R: TGGAACGCGTAGACTTGG 20 GB-PO-064 F: GTTCTGAGGGTTGAGGGG 21 (GTT) 5 (ATT) (GTT) 3 209-227 52 3 R: CCAACCACACTCTTCCCA 22 GB-PO-076 F: TCGATTGTCAGATTGTTGGA 23 (GGC) 6 233-248 52 4 R: CGGAGAAGCAGTTGGTTG 24 GB-PO-079 F: ACCCAGACGATTTGGGAG 25 (GGA) 4 (AGA) (GGA) 2 230-233 52 4 R: AGGCTGGCGTGGAATACT 26 GB-PO-080 F: CACCCATGTGCCTCAGTC 27 (GGC) 6 203-233 52 2 R: TGTCTATGGGTTACGGCG 28 GB-PO-086 F: CATCTTCGATGAACCGGA 29 (GA) 3 (GGAGAAGC) (GA) 3, (CGA) 3 378-380 52 2 R: CGAAGATGAGCCAGCAAC 30 GB-PO-094 F: CGCGAGACAATTAAACGC 31 (CCT) 2 (TCT) (CCT) 2 303-324 52 2 R: ACAGTTCCTGGAGCCCAT 32 GB-PO-097 F: CATGGAGAGAGGGCGG 33 (GGT) 5 241-247 52 3 R: CGTTTCATCGTTCGCTGT 34 GB-PO-102 F: TGTCTATGGGTTACGGCG 35 (GCC) 4 (GC) (GCC) 1 266-269 52 2 R: TGCAAAGCAAATCGGAAC 36 GB-PO-113 F: GTTCATCTGAACGCCGTC 37 (CGC) 5 270-375 52 2 R: CCTATGACGAGGGGAAGG 38 GB-PO-115 F: TGGTAGCAGGTTGTTGGG 39 (TGC) 5 (GCTGGC) (TGC) 4 213-228 52 3 R: CCGCTAAGCCACTGTTTG 40 GB-PO-117 F: TCAAACTCACGTGGTACGC 41 (TGC) 7 218-224 52 3 R: TCACATATCCGCCGGTAG 42 GB-PO-124 F: TGCGTTTGCTCGGTTAAT 43 (CG) 5 264-300 52 5 R: CGCTACTACGTCGATCCG 44 GB-PO-128 F: TGATTGGTTTGAATGGGC 45 (GTT) 10 214-238 52 5 R: GCACGATGAGGATGCAGT 46 GB-PO-131 F: CTCCCTCCTCCGTGTACC 47 (CCT) 2 (CCCC) (CCT) 2 200-212 52 3 R: CGTAACGTTCGCTTCCTG 48 GB-PO-134 F: GAGTGTGAAGAATCGGCG 49 (GA) 2 (GT) (GA) 3 115-269 52 2 R: GTGCACTCTGCCTATCGC 50

Primer Name order order
number Repeating motif Range of size of supersatellite to be amplified (bp) Tm (℃) Number of alleles GB-PO-135 F: AGGAGGGGGTGCTTGATA 51 (GGA) 2 (GGGA) (GGA) 2 244-271 52 2 R: TCCTCCGCCTTCTCTACC 52 GB-PO-138 F: TATGGAACGGTGCGAAGT 53 (CCG) 4, (TTC) 3 191-206 52 3 R: GCCGTCAAAAGGGAACTC 54 GB-PO-149 F: AGTGCATATGCCCGACAC 55 (TCC) 8 193-205 52 3 R: CGTCGTAGATGCAGGCTC 56 GB-PO-152 F: ACTGAGCCTTCAGCACCA 57 (AGC) 5 272-278 52 3 R: CATAGGGACGACAGCGAG 58 GB-PO-154 F: GTCGTAGCCAGCCATGAG 59 (CGA) 7 254-266 52 4 R: AGGGTATCTCGGGTGCAT 60 GB-PO-157 F: ATGGACGTGGTGTTCTGC 61 (GCT) 4 281-302 52 3 R: AAACCAAGCCTACCCAGC 62 GB-PO-171 F: TCTCGGGCATCATTCTTG 63 (TTG) 3, (TA) 4 295-310 52 4 R: ACGTCAGGGTGTCAAACG 64 GB-PO-172 F: GCAGAAGTTGCCCAAAGA 65 (TGC) 2 (TAC) (TGC) 5 169-229 52 2 R: ATGTCCAGCGGAAGACCT 66 GB-PO-173 F: ATGAAGTGTGAGCCGTGG 67 (GT) 8 294-309 52 4 R: TGTCCATTCATGCGTTCA 68 GB-PO-181 F: TTATTGTGAAGCCCCCG 69 (CAG) 1 (CAA) (CAG) 5 240-246 52 3 R: GACATCGGCAGAAGGTCA 70 GB-PO-190 F: TTTCCATTTCCGTTGGTG 71 (TGC) 3 (TGT) (TGC) 2 186-198 52 4 R: CAGGGGGTGATTATGCAA 72

^a F: forward primer ^b R: reverse primer

SSR primer pairs provided in the present invention can be usefully used to detect DNA polymorphism and to analyze genetic diversity in mushrooms. The SSR primers according to the present invention can be used to efficiently evaluate and preserve the genetic resources of mushrooms. Accordingly, the present invention provides a method for detecting DNA polymorphism of a mushroom comprising performing PCR using the SSR primer pair.

Specifically, the DNA polymorphism detection method of mushrooms

(a) extracting genomic DNA from the mushroom;

(b) performing PCR using the extracted genomic DNA as a template and the SSR primer pair provided by the present invention; And

(c) separating the PCR products by size.

Extraction of genomic DNA in step (a) is phenol / chloroform extraction, SDS extraction (Tai et. al ., Plant Mol . Biol . Reporter , 8: 297-303, 1990), CTAB separation method (Cetyl Trimethyl Ammonium Bromide; Murray et al . , Nuc . Res . , 4321-4325, 1980) or commercially available DNA extraction kits.

In addition, the PCR in the step (b) may be performed using a PCR reaction mixture containing a variety of components known in the art required for the PCR reaction. The PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer and water (dH ₂ O) in addition to genomic DNA extracted from the mushroom to be analyzed and the SSR primer pair provided in the present invention. The PCR buffer solution includes Tris-HCl, MgCl ₂ , KCl, and the like. At this time, MgCl ₂ Concentration greatly affects the specificity and quantity of amplification, and may be preferably used in the range of 1.5-2.5 mM. In general, when Mg ^{2 +} is insufficient if the excess will increase the non-specific PCR amplification products and, Mg ^{2 +} PCR The yield of the product is reduced. The PCR buffer may further include an appropriate amount of Triton X-100. PCR also denatured the template DNA at 94-95 ° C., followed by denaturation; Annealing; And a cycle of extension, followed by general PCR reaction conditions which are finally elongated at 72 ° C. The denaturation and amplification may be performed at 94-95 ° C. and 72 ° C., respectively, and the temperature at the time of binding may vary depending on the type of primer. Preferably it is 52-57 degreeC, More preferably, it is 55 degreeC. The time and number of cycles in each step can be determined according to the conditions generally made in the art. The optimal reaction conditions when performing PCR using the SSR primer pair according to the present invention are as follows: After denature the template DNA for 3 minutes at 95 ℃, 30 seconds at 95 ℃; 30 seconds at 55 ° C .; And repeating 36 cycles of 1 minute 30 seconds at 72 ° C., and finally reacting at 72 ° C. for 5 minutes.

In step (c), DNAs of PCR products may be separated according to sizes according to methods well known in the art. Preferably it can be confirmed by an agarose gel (agarose gel) or polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3100 genetic analyzer-electropherogram). At this time, in order to use the fluorescence analyzer PCR is performed in the step (b) using a primer pair attached to a fluorescent die known in the art. PCR amplification results can be preferably confirmed by polyacrylamide gel electrophoresis, more preferably modified polyacrylamide gel electrophoresis. Electrophoresis results can be analyzed by silver staining after electrophoresis. General PCR performance and resulting analysis methods are well known in the art.

In addition, the present invention provides a method for identifying a variety of mushrooms, including performing PCR using the SSR primer pair.

Specifically, how to identify mushroom varieties

(a) extracting genomic DNA from mushroom and control mushroom varieties;

(b) performing PCR using each of the extracted genomic DNA as a template and the SSR primer pair provided by the present invention;

(c) separating each PCR product by size; And

(d) comparing the separation results for each size of step (c) with each other.

Genomic DNA extraction in step (a) to (c), PCR and separation of PCR products by size are as described above, and in step (d) the comparison of mushroom and control mushroom varieties is the same combination of SSR primers This is done by comparing the size of each PCR product of the mushroom and the control mushroom variety that is a sample for the pair. By comparing the size comparison results for each SSR primer pair, if the sample and the control mushroom varieties match both, the sample mushroom can be identified as the same varieties as the control mushroom varieties. The identification method of the variety can be useful when the identification of the breed, such as judging whether there is a violation of the labeling of origin through judging whether the overlapping when introducing a new genetic resource and confirm the origin.

Genomic DNA extraction, PCR, and size separation of PCR products for control mushroom varieties may be performed simultaneously with the sample mushroom, but may be performed prior to the sample mushroom and prepared as a reference for identification. Using such a reference table can be useful because it can be compared with the reference table by performing genomic DNA extraction, PCR and separation of the size of the PCR product for the mushroom to be a sample.

The present invention also provides a kit for detecting DNA polymorphism of a mushroom comprising a SSR primer pair according to the present invention. In addition, DNA polymerase and a PCR reaction buffer of the above-described composition may be additionally included in order to easily perform a PCR reaction, and components necessary for performing electrophoresis to confirm amplification of a PCR product are present. It may be further included in the kit of the invention.

The present invention also provides a kit for identifying a variety of mushrooms comprising the SSR primer pair according to the present invention. The breed identification method is as described above. In addition to the DNA polymerase and the PCR reaction buffer of the above-described composition in order to be able to easily perform the PCR reaction may be further included, the components or known to perform electrophoresis to confirm whether the PCR product amplification or not Identification criteria for selected varieties may be further included in the kits of the present invention.

Mushroom that can be applied to the present invention is Pleurotus Ostreatus Kummer and variants thereof, but are not limited thereto. Preferably Pleurotus Ostreatus It can be Kummer.

As described above, in the present invention, the first SSR marker was developed in the mushroom. SSR primer pairs provided in the present invention can be very useful for effectively preparing DNA profiles of mushrooms by effectively detecting the DNA polymorphism of mushrooms. In this way, by efficiently evaluating the genetic resources of mushrooms, it is possible to effectively perform redundancy analysis and establishment of novelty with existing resources when introducing new genetic resources, and to identify mushroom varieties.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Genome DNA  extraction

Method using SDS from cultivated mushrooms (Dept. of Genetic Resources, Agricultural Biotechnology Research Institute, Conservation No .: PO06) (Tai et al ., Plant Mol . Biol . Genomic DNA was extracted according to Reporter , 8: 297-303, 1990). This will be described in detail as follows. 0.5 g of leaf tissue was taken from the young mushroom plants at 1 month of growth and placed in a 1.5 ml tube and quenched with liquid nitrogen. The leaf was then ground finely with a plastic rod immediately. Before the ground leaves were dissolved, 750 μl of extraction buffer (200 mM Tris-HCl, pH 8.0), 200 mM NaCl, 25 mM EDTA, 0.5% SDS) was added and the mixture was stored at 65 ° C. for 30 minutes. An equivalent amount of a phenol (chloro): chloroform: isoamylalchol (25: 24: 1) solution was further added thereto, followed by stirring for about 15 minutes. It was centrifuged for 15 minutes at 13,000 rpm. The supernatant was taken and transferred to a new tube. Equal amount of isopropanol (-20 ° C.) was added to the tube and stored at −20 ° C. for 1 hour. Thereafter, the mixture was centrifuged at 13,000 rpm for 15 minutes. After discarding the supernatant, the precipitated DNA was washed with 70% ethanol. After the DNA was dried, it was sufficiently dissolved by adding TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.4) containing about 300 µl of RNase (50 µg / ml), followed by 1 hour at 37 ° C. Stored.

< Example  2> Ligation with adapters ( ligation )

500 ng of genomic DNA obtained in Example 1 was digested with Eco RV, Dra I, Sma I, Pvu I, Alu I, Hae III and Rsa I according to the instructions of each restriction enzyme manufacturer. After cleaved DNA was electrophoresed in a 1.2% agarose gel, only DNA fragments of about 300-1500 bp size were obtained by elution from the gel. The DNA fragments obtained were then lysed overnight at 12 ° C. using two adapters (AP-11 and AP-12, 50 ng each) and a T4 DNA ligase (Promega, USA) shown in Table 2. Gated. To increase ligation efficiency, the AP-12 adapter phosphorylated the 5 'end with alkaline phosphatase (Promega, USA), and the composition of the reaction solution during ligation is as follows: 1X ligation buffer (Promega). , USA), 50 ng AP11 adapter, 50 ng AP12 adapter, 150 ng DNA fragment, 3 Unit T4 ligation (Promega, USA).

Adapter name Sequence SEQ ID NO: AP-11 5'-CTCTTGCTTAGATCTGGACTA-3 ' 73 AP-12 5'-TAGTCCAGATCTAAGCAAGAGCACA-3 ' 74

< Example  3> Supersatellite  Containing DNA  Selection and Separation of Fragments

<3-1> Magnetic Bead Preparation

The solution was removed after collecting 0.6 ml of magnetic beads (SA-PMPs, Promega, USA; 1 mg / ml) with a magnetic stand (Promega, USA). 0.6 ml of 0.5x Saline-Sodium Citrate (SSC) buffer was added thereto, washed, and collected again using a magnetic separator to remove the solution. Washing with 0.5 × SSC buffer was performed three times. 100 μl of 0.5 × SSC buffer was added to the washed magnetic beads and used within 30 minutes.

<3-2> hybridization

Distilled water was added to the ligation product prepared in Example 2 to 500 μl, and then denatured at 65 ° C. for 10 minutes. Biotin-labeled SSR probe 1.5 μl (20ng) and 20 μl SSC buffer 13 μl were added thereto and left for 10 minutes at room temperature to induce binding. The eight SSR probes used for hybridization are listed in Table 3 below, and the SSR probes were labeled with biotin according to conventional methods known in the art.

order SEQ ID NO: Biotin- (GA) ₂₀ 75 Biotin- (AT) ₂₀ 76 Biotin- (CA) ₂₀ 77 Biotin- (AGC) ₁₅ 78 Biotin- (GGC) ₁₅ 79 Biotin- (AAG) ₁₅ 80 Biotin- (AAC) ₁₅ 81 Biotin- (AGG) ₁₅ 82

Through hybridization reaction, DNA fragments having the repeat base sequence of genomic DNA fragments will complementarily bind to biotin-labeled SSR probes, which are called SSR probe-template hybrids.

<3-3> Separation of Template

The magnetic bead solution prepared in <3-1> was placed in the SSR probe-template hybrid solution in <3-2> and allowed to stand at room temperature for 10 minutes. Magnetic beads in which the SSR probe-template hybrid was bound were collected by using a magnetic separator in the reaction solution, and the supernatant was removed. The magnetic beads combined with the SSR probe-template hybrid were washed four times with 300 μl of 0.1 × SSC, and then 50 μl of distilled water was added and heated at 90 ° C. for 5 minutes to separate the template.

<3-4> PCR reaction

The PCR reaction was performed using the AP-11 adapter as a primer on the template isolated in <3-3>. The composition of the PCR reaction mixture (20 μl) is as follows: 250 ng adapter-ligated DNA, 10 pmol AP11 primer, 2 mM dNTP, 2 units Taq DNA polymerase, 5 μl 10 × buffer. PCR reactions were heated at 72 ° C. for 2 minutes, 94 ° C. for 3 minutes, and then at 94 ° C. for 30 seconds; 30 seconds at 56 ° C .; And 1 minute at 72 ° C. for 24 cycles, and finally at 72 ° C. for 10 minutes.

In this manner, in the present invention, 500 DNA fragments containing supersatellites were isolated.

< Example  4> Separated DNA  Sequencing of fragments and primer  design

DNA fragments containing the supersatellite isolated in Example 3 were cloned into pGEM T-easy vector vector (Promega, USA) to analyze their sequence. Sequence analysis was performed by requesting Solgent. Only fragments containing 5 or more repeat units were selected based on the analyzed sequences. Based on the part containing the repeating unit, a primer in both directions was designed to amplify the supersatellite. A total of 200 pairs of primer pairs were designed (results not shown).

< Example  5> SSR Of marker  Selection

In order to select an SSR primer combination showing a large number of polymorphic variation in various mushroom strains among the primer pairs designed in Example 4, DNA profiling through PCR for 16-point cultivated mushrooms or mushroom varieties shown in Table 4 below ( profiling) analysis was performed.

Deposit number Depositary Institution Collection
(origin) Type PO06 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO01 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO03 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO07 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO16 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO18 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO19 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO24 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO25 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO26 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO28 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO34 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO35 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO54 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO62 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer PO73 Rural Development Administration Republic of Korea Pleurotus Ostreatus kummer

The composition of each PCR reaction mixture (20 μl) was as follows: 40 ng of mushroom template DNA isolated by the method of Example 1, 0.2 μM forward primer, 0.6 μM reverse primer, M13 primer labeled with fluorescent material (TGTAAAACGACGGCCAGT, SEQ ID NO: 83) 0.6 μM, 1 × PCR reaction solution (10 mM Tris-HCl, pH 8.8), 1.5 mM MgCl ₂ , 50 mM KCl, 0.1% Triton X-100), 1 unit DNA polymerase. PCR reaction was performed after denaturing the template DNA for 3 minutes at 94 ℃, 30 seconds at 94 ℃; 30 seconds at the temperature corresponding to the T _m of each primer (see Table 1); And 35 repetitions under conditions of 45 seconds at 72 ° C., and then 30 seconds at 94 ° C .; 45 sec at 53 ° C .; And 15 repetitions at 45 ° C. for 45 seconds, and finally at 72 ° C. for 15 minutes.

An automated sequencing device (ABI 3130 Genetic Analyzer, Applied Biosystems, USA) was used to analyze the size of PCR product fragments amplified with different fluorescent materials. For the analysis, 1.5 μl of PCR product, 9.2 μl of Hi-di formamide, and 0.3 μl of internal size standard (Genscan-500 ROX) were mixed and denatured at 94 ° C. for 3 minutes. It was.

As a result, we identified 36 pairs of SSR primer pairs showing polymorphisms in mushroom cultivars and mushroom varieties collected domestically and internationally. GB-PO-001 (primary pairs represented by SEQ ID NOs: 1 and 2), GB-PO- 006 (primary pairs represented by SEQ ID NOs: 3 and 4), GB-PO-011 (primary pairs represented by SEQ ID NOs: 5 and 6), GB-PO-025 (primary pairs represented by SEQ ID NOs: 7 and 8), GB-PO-026 (primary pairs represented by SEQ ID NOs: 9 and 10), GB-PO-028 (primary pairs represented by SEQ ID NOs: 11 and 12), and GB-PO-039 (SEQ ID NOs: 13 and 14) Primer pairs), GB-PO-050 (primary pairs represented by SEQ ID NOs: 15 and 16), GB-PO-051 (primary pairs represented by SEQ ID NOs: 17 and 18), and GB-PO-061 (SEQ ID NOs: 19 and Primer pair represented by 20), GB-PO-064 (primary pair represented by SEQ ID NOs: 21 and 22), GB-PO-076 (primary pair represented by SEQ ID NOs: 23 and 24), and GB-PO-079 ( Shown in SEQ ID NOs: 25 and 26 Is a primer pair), GB-PO-080 (primary pairs represented by SEQ ID NOs: 27 and 28), GB-PO-086 (primary pairs represented by SEQ ID NOs: 29 and 30), and GB-PO-094 (SEQ ID NO: 31 And primer pairs represented by 32), GB-PO-097 (primary pairs represented by SEQ ID NOs: 33 and 34), GB-PO-102 (primary pairs represented by SEQ ID NOs: 35 and 36), GB-PO-113 (Primary pairs represented by SEQ ID NOs: 37 and 38), GB-PO-115 (primary pairs represented by SEQ ID NOs: 39 and 40), GB-PO-117 (primary pairs represented by SEQ ID NOs: 41 and 42), GB -PO-124 (primary pairs represented by SEQ ID NOs: 43 and 44), GB-PO-128 (primary pairs represented by SEQ ID NOs: 45 and 46), and GB-PO-131 (primers represented by SEQ ID NOs: 47 and 48) Pairs), GB-PO-134 (primary pairs represented by SEQ ID NOs: 49 and 50), GB-PO-135 (primary pairs represented by SEQ ID NOs: 51 and 52), GB-PO-138 (SEQ ID NOs 53 and 54) Primer pair), GB-PO-149 (SEQ ID NOs. 55 and 56) Primer pairs), GB-PO-152 (primary pairs represented by SEQ ID NOs: 57 and 58), GB-PO-154 (primary pairs represented by SEQ ID NOs: 59 and 60), and GB-PO-157 (SEQ ID NO: Primer pairs represented by 61 and 62), GB-PO-171 (primary pairs represented by SEQ ID NOs: 63 and 64), GB-PO-172 (primary pairs represented by SEQ ID NOs: 65 and 66), and GB-PO- 173 (primary pairs represented by SEQ ID NOs: 67 and 68), GB-PO-181 (primary pairs represented by SEQ ID NOs: 69 and 70), and GB-PO-190 (primary pairs represented by SEQ ID NOs: 71 and 72) Screened (see Table 1). The size of each PCR fragment amplified by the 36 pairs of primers is shown in Table 5 below.

Deposit number
primer PO06 PO01 PO03 PO07 PO16 PO18 PO19 PO24 GB-PO-001 307 307 307 313 307 307 307 307 GB-PO-006 203 203 206 203 206 203 203 203 GB-PO-011 175 235 190 235 181 190 235 190 GB-PO-025 217 217 214 217 214 217 217 217 GB-PO-026 296 296 296 296 296 296 296 296 GB-PO-028 300 303 291 300 300 318 300 300 GB-PO-039 206 206 206 197 206 224 197 206 GB-PO-050 145 169 175 145 175 145 169 163 GB-PO-051 272 272 278 278 275 272 272 272 GB-PO-061 258 258 258 258 270 258 258 258 GB-PO-064 227 227 227 227 227 227 227 227 GB-PO-076 248 236 233 236 248 248 236 236 GB-PO-079 323 320 320 296 323 230 320 320 GB-PO-080 230 230 230 230 230 230 230 230 GB-PO-086 378 378 380 378 378 378 378 378 GB-PO-094 324 324 324 324 303 324 324 324 GB-PO-097 244 241 244 244 247 244 241 241 GB-PO-102 266 269 266 266 266 266 269 266 GB-PO-113 270 270 270 270 375 270 270 270 GB-PO-115 228 225 213 225 228 228 213 213 GB-PO-117 224 218 224 218 221 221 218 221 GB-PO-124 294 294 294 278 264 294 272 278 GB-PO-128 214 232 223 232 214 214 232 229 GB-PO-131 212 212 200 200 200 200 200 209 GB-PO-134 269 115 115 115 115 115 115 115 GB-PO-135 244 244 244 244 244 244 244 244/271 GB-PO-138 194 194 206 194 206 191 206 194 GB-PO-149 193 205 196 205 193 193 205 196 GB-PO-152 272 272 278 278 275 275 272 272 GB-PO-154 254 266 266 266 254 254 254 257 GB-PO-157 281 281 293 302 293 302 302 281 GB-PO-171 301 301 295 301 298 310 301 301 GB-PO-172 169 169 169 169 229 169 169 169 GB-PO-173 306 300 306 294 300 309 300 300 GB-PO-181 243 243 246 243 240 246 243 243 GB-PO-190 186 189 186 186 195 186 186 198

Deposit number
primer PO25 PO26 PO28 PO34 PO35 PO54 PO62 PO73 GB-PO-001 307 307 307 307 307 307 307 307 GB-PO-006 203 206 203 206 203 203 206 203 GB-PO-011 190 181 190 175 190 166 175 166 GB-PO-025 217 214 217 214 217 217 214 217 GB-PO-026 293 296 296 296 296 296 296 296 GB-PO-028 297 300 300 300 318 303 300 303 GB-PO-039 224 206 206 206 224 206 206 191 GB-PO-050 145 169 175 163 145 157 163 163 GB-PO-051 272 275 272 272 272 272 272 272 GB-PO-061 258 270 258 258 258 258 258 258 GB-PO-064 227 227 227 227 227 227 212 209 GB-PO-076 248 248 236 236 248 242 236 236 GB-PO-079 230 323 320 320 230 323 320 320 GB-PO-080 230 230 230 233 230 230 233 230 GB-PO-086 378 378 378 378 378 378 378 378 GB-PO-094 324 303 324 303 324 324 303 324 GB-PO-097 244 247 241 241 244 241 241 241 GB-PO-102 266 266 266 269 266 266 269 269 GB-PO-113 270 270 270 270 270 270 270 270 GB-PO-115 228 228 213 225 228 213 225 228 GB-PO-117 221 221 221 224 221 221 224 221 GB-PO-124 294 264 300 294 294 294 294 294 GB-PO-128 214 214 229 214 238 229 229 229 GB-PO-131 200 200 209 212 200 209 212 212 GB-PO-134 115 115 115 115 115 115 115 115 GB-PO-135 244 244 244 244 244 244 244 244/271 GB-PO-138 191 206 194 191 191 194 191 206 GB-PO-149 193 193 196 196 193 193 196 193 GB-PO-152 275 275 272 272 275 272 272 272 GB-PO-154 254 254 257 266 254 263 254 263 GB-PO-157 302 293 281 293 302 281 293 281 GB-PO-171 310 298 301 298 310 301 298 298 GB-PO-172 169 229 169 169 169 169 169 169 GB-PO-173 309 294 300 309 309 294 300 294 GB-PO-181 246 240 243 246 246 243 246 243 GB-PO-190 195 195 198 195 195 195 195 195

<110> Rural Development Administration <120> SSR primer derived from Oyster Mushroom and use of there <160> 83 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-001 <400> 1 cgcaagctac aaacggac 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> revese primer of GB-PO-001 <400> 2 agcagcaagc acaagagc 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-006 <400> 3 tgtggcaaac ccaagttc 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-006 <400> 4 cccaaaggat gaggaagg 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-011 <400> 5 tcccataccc tgacatcg 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-011 <400> 6 atcatcaagc gccacaac 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-025 <400> 7 tgatcatggc gagtaggg 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-025 <400> 8 ggaactgtca gcagacgc 18 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-026 <400> 9 aatcgcatgg gctctg 16 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-026 <400> 10 ctgtccctcc gtgtacca 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-028 <400> 11 ctggagaatc gtagcccc 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-028 <400> 12 acaagcgctc ggaataca 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-039 <400> 13 tgtggatgtg atgtgatgtg 20 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-039 <400> 14 acgtccagcg tcgagtta 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-050 <400> 15 catccgatac agacccga 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-050 <400> 16 aggcatccca caacactg 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-051 <400> 17 catagggacg acagcgag 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-051 <400> 18 actgagcctt cagcacca 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-061 <400> 19 taacttgggc gcttgaaa 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-061 <400> 20 tggaacgcgt agacttgg 18 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-064 <400> 21 gttctgaggg ttgagggg 18 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-064 <400> 22 ccaaccacac tcttccca 18 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-076 <400> 23 tcgattgtca gattgttgga 20 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-076 <400> 24 cggagaagca gttggttg 18 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-079 <400> 25 acccagacga tttgggag 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-079 <400> 26 aggctggcgt ggaatact 18 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-080 <400> 27 cacccatgtg cctcagtc 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-080 <400> 28 tgtctatggg ttacggcg 18 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-086 <400> 29 catcttcgat gaaccgga 18 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-086 <400> 30 cgaagatgag ccagcaac 18 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-094 <400> 31 cgcgagacaa ttaaacgc 18 <210> 32 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-094 <400> 32 acagttcctg gagcccat 18 <210> 33 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-097 <400> 33 catggagaga gggcgg 16 <210> 34 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-097 <400> 34 cgtttcatcg ttcgctgt 18 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-102 <400> 35 tgtctatggg ttacggcg 18 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-102 <400> 36 tgcaaagcaa atcggaac 18 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-113 <400> 37 gttcatctga acgccgtc 18 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-113 <400> 38 cctatgacga ggggaagg 18 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-115 <400> 39 tggtagcagg ttgttggg 18 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-115 <400> 40 ccgctaagcc actgtttg 18 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-117 <400> 41 tcaaactcac gtggtacgc 19 <210> 42 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-117 <400> 42 tcacatatcc gccggtag 18 <210> 43 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-124 <400> 43 tgcgtttgct cggttaat 18 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-124 <400> 44 cgctactacg tcgatccg 18 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-128 <400> 45 tgattggttt gaatgggc 18 <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-128 <400> 46 gcacgatgag gatgcagt 18 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-131 <400> 47 ctccctcctc cgtgtacc 18 <210> 48 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-131 <400> 48 cgtaacgttc gcttcctg 18 <210> 49 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer GB-PO-134 <400> 49 gagtgtgaag aatcggcg 18 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-134 <400> 50 gtgcactctg cctatcgc 18 <210> 51 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-135 <400> 51 aggagggggt gcttgata 18 <210> 52 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-135 <400> 52 tcctccgcct tctctacc 18 <210> 53 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-138 <400> 53 tatggaacgg tgcgaagt 18 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-138 <400> 54 gccgtcaaaa gggaactc 18 <210> 55 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-149 <400> 55 agtgcatatg cccgacac 18 <210> 56 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-149 <400> 56 cgtcgtagat gcaggctc 18 <210> 57 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-152 <400> 57 actgagcctt cagcacca 18 <210> 58 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse pimer of GB-PO-152 <400> 58 catagggacg acagcgag 18 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-154 <400> 59 gtcgtagcca gccatgag 18 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-154 <400> 60 agggtatctc gggtgcat 18 <210> 61 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-157 <400> 61 atggacgtgg tgttctgc 18 <210> 62 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-157 <400> 62 aaaccaagcc tacccagc 18 <210> 63 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-171 <400> 63 tctcgggcat cattcttg 18 <210> 64 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-171 <400> 64 acgtcagggt gtcaaacg 18 <210> 65 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-172 <400> 65 gcagaagttg cccaaaga 18 <210> 66 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-172 <400> 66 atgtccagcg gaagacct 18 <210> 67 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-173 <400> 67 atgaagtgtg agccgtgg 18 <210> 68 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-173 <400> 68 tgtccattca tgcgttca 18 <210> 69 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-181 <400> 69 ttattgtgaa gcccccg 17 <210> 70 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-181 <400> 70 gacatcggca gaaggtca 18 <210> 71 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer of GB-PO-190 <400> 71 tttccatttc cgttggtg 18 <210> 72 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of GB-PO-190 <400> 72 cagggggtga ttatgcaa 18 <210> 73 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> adapter, AP-11 <400> 73 ctcttgctta gatctggact a 21 <210> 74 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> adapter, AP-12 <400> 74 tagtccagat ctaagcaaga gcaca 25 <210> 75 <211> 40 <212> DNA <213> Artificial Sequence <220> Biotin- (GA) 20 <400> 75 gagagagaga gagagagaga gagagagaga gagagagaga 40 <210> 76 <211> 40 <212> DNA <213> Artificial Sequence <220> Biotin- (AT) 20 <400> 76 atatatatat atatatatat atatatatat atatatatat 40 <210> 77 <211> 40 <212> DNA <213> Artificial Sequence <220> Biotin- (CA) 20 <400> 77 cacacacaca cacacacaca cacacacaca cacacacaca 40 <210> 78 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AGC) 15 <400> 78 agcagcagca gcagcagcag cagcagcagc agcagcagca gcagc 45 <210> 79 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (GGC) 15 <400> 79 ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcggc 45 <210> 80 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AAG) 15 <400> 80 aagaagaaga agaagaagaa gaagaagaag aagaagaaga agaag 45 <210> 81 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AAC) 15 <400> 81 aacaacaaca acaacaacaa caacaacaac aacaacaaca acaac 45 <210> 82 <211> 45 <212> DNA <213> Artificial Sequence <220> Biotin- (AGG) 15 <400> 82 aggaggagga ggaggaggag gaggaggagg aggaggagga ggagg 45 <210> 83 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> M13 primer <400> 83 tgtaaaacga cggccagt 18  

Claims (7)

Primer pairs represented by SEQ ID NOs: 5 and 6; Primer pairs represented by SEQ ID NOs: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; Primer pairs represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 23 and 24; Primer pairs represented by SEQ ID NOs: 31 and 32; Primer pairs represented by SEQ ID NOs: 35 and 36; Primer pairs represented by SEQ ID NOs: 39 and 40; Primer pairs represented by SEQ ID NOs: 43 and 44; Primer pairs represented by SEQ ID NOs: 45 and 46; Primer pairs represented by SEQ ID NOs: 47 and 48; Primer pairs represented by SEQ ID NOs: 55 and 56; Primer pairs represented by SEQ ID NOs: 59 and 60; Primer pairs represented by SEQ ID NOs: 61 and 62; Primer pairs represented by SEQ ID NOs: 65 and 66; Primer pairs represented by SEQ ID NOs: 67 and 68; And a pair of primers represented by SEQ ID NOs: 69 and 70. The method of claim 1, wherein the primer pair represented by SEQ ID NO: 1 and 2; Primer pairs represented by SEQ ID NOs: 3 and 4; Primer pairs represented by SEQ ID NOs: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; Primer pairs represented by SEQ ID NOs: 11 and 12; Primer pairs represented by SEQ ID NOs: 19 and 20; Primer pairs represented by SEQ ID NOs: 21 and 22; Primer pairs represented by SEQ ID NOs: 25 and 26; Primer pairs represented by SEQ ID NOs: 27 and 28; Primer pairs represented by SEQ ID NOs: 29 and 30; Primer pairs represented by SEQ ID NOs: 33 and 34; Primer pairs represented by SEQ ID NOs: 37 and 38; Primer pairs represented by SEQ ID NOs: 41 and 42; Primer pairs represented by SEQ ID NOs: 49 and 50; Primer pairs represented by SEQ ID NOs: 51 and 52; Primer pairs represented by SEQ ID NOs: 53 and 54; Primer pairs represented by SEQ ID NOs: 57 and 58; Composition for identifying a variety of mushrooms, characterized in that it further comprises at least one primer pair selected from the group consisting of a primer pair represented by SEQ ID NO: 63 and 64 and a primer pair represented by SEQ ID NO: 71 and 72. The composition of claim 1, wherein the primer pair is derived from Oyster Mushroom ( Pleurotus Ostreatus Kummer). (a) extracting genomic DNA from the mushroom; (b) PCR using the extracted genomic DNA as a template and each primer pair included in the composition of claim 1 or 2; And (C) method for detecting DNA polymorphism of mushroom comprising the step of separating the PCR product by size. (a) extracting genomic DNA from mushroom and control mushroom varieties; (b) subjecting each extracted genomic DNA to a template and performing PCR using each primer pair contained in the composition of claim 1 or 2; (c) separating each PCR product by size; And (d) a method of identifying a variety of mushrooms comprising the step of comparing the results of the separation of the mushroom and the size of the control mushroom varieties. A kit for detecting DNA polymorphism of a mushroom comprising the composition of claim 1. A kit for identifying a variety of mushrooms comprising the composition of claim 1.