KR20230097233A - Composition for identification of dark cap color of Pleurotus ostreatus Gonji 7ho derived strains and identification method using the same - Google Patents

Abstract

The present invention relates to a composition for detecting the dark cap color of Pleurotus ostreatus Gonji No. 7 and a method for detecting the dark cap color of Pleurotus ostreatus strains derived from Gonji No. 7 using the same. According to the configuration of the present invention, it is possible to detect a dark cap color strain with the characteristics of high-quality Pleurotus ostreatus in a mycelial state without consuming a long time or high costs, and to detect the dark cap color of Pleurotus ostreatus in a short time, thereby greatly increasing the breeding efficiency.

Description

Composition for identification of dark cap color of Pleurotus ostreatus Gonji 7ho derived strains and identification method using the same}

It relates to a composition for detecting the shade color of an oyster mushroom Gonji7-derived strain and a method for detecting the shade color of a strain derived from Gonji7 using the same. More specifically, the present invention is a composition for detecting the dark shade of oyster mushrooms that can quickly and accurately detect dark, bright and/or intermediate shades of color in a short period of time at a low cost, and a method for detecting the dark shade of oyster mushrooms using the same It is about.

Oyster mushroom is a white rotting fungus belonging to the family Pleurotusus, which grows wild throughout the world in temperate and subtropical regions. Nutritionally, it shows high protein content (Sharma and Madan 1993), excellent taste and flavor, and functional effects such as prevention of hypertension, hyperglycemia, hyperlipidemia, and anticancer effects have also been reported. It is easy to grow and has a wide range of substrates, so it is cultivated all over the world, so the production is the second largest after button mushrooms, and domestic production is also ranked first with 48,327 tons (2019 special crop production performance, Ministry of Agriculture, Food and Rural Affairs https://kass .mafra.go.kr/).

Due to increased consumer interest and increased production, new varieties of oyster mushrooms are being bred every year, and currently, 46 varieties have been established with breed protection rights (2020, National Seed Resources http://www.seed.go.kr/ ). The quality of oyster mushrooms is determined by the color of the cap, the color of the cap, and the shape of the cap. The color of the cap is highly related to storage, so the dark cap color is evaluated as excellent quality. Therefore, when breeding, the main goal is to breed dark-colored oyster mushrooms with increased yield.

However, it takes a lot of time to breed varieties with dark mustaches. In addition, since several genes regulating the color of the color exist and are regarded as quantitative traits, the color of the color cannot be predicted 100% using the molecular marker.

In addition, in oyster breeding, if the breeder wants to select a line with the desired trait, it is necessary to cultivate the fruiting body form in which the mushroom trait is expressed, which takes a long time and money. In order to improve this, a technique for detecting the high-quality oysterica trait, the dark shade line, in the mycelial state is required.

As background technology for the present invention, Korean Patent Registration No. 1835224 (August 11, 2016) describes PCR primers and their use for identifying varieties of oyster mushrooms in which DNA fragments are amplified in a unique pattern for each variety of oyster mushrooms.

In order to solve the above-mentioned conventional problems, the present invention developed a marker for detecting a major QTL marker (quantitative trait locus marker, major QTL), and applied marker assigned selection (MAS) to a breeding program.

An object of the present invention is to generate a specific band in the dark shade of oyster mushroom, and to quickly and accurately discriminate between dark, light and/or intermediate shade in a short period of time in a mycelial state at low cost. It is to provide a composition for discrimination comprising a set.

Another object of the present invention is to provide a method for detecting the dark hat color of oyster mushrooms, which can increase the breeding efficiency by detecting the dark hat color in a mycelial state in a short period of time and at low cost using the composition for detecting the dark hat color of the oyster mushroom.

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

According to one aspect, there is provided a composition for detecting the shade color of a strain derived from oyster mushroom Gonji7, including a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2.

According to one embodiment, the oyster mushroom may be a mushroom mycelium.

According to one embodiment, it may be a composition for detecting shade color of a strain derived from oyster mushroom Gonji7, capable of amplifying CAPS (cleaved amplified polymorphic sequences) marker sequences including the primers.

According to another aspect, (i) isolating DNA from the sample; (ii) amplifying the DNA isolated in step (i) using the composition for detecting the dark shade of oyster mushroom of the present application; and (iii) confirming the PCR product amplified in step (ii).

According to one embodiment, the step of confirming the amplified PCR product is digested with HaeIII restriction enzyme, detected in dark color when the size of the DNA fragment is 290 bp, and detected in bright color when the DNA fragment is observed at 324 bp. It may be a method for detecting the shade color of the strain derived from mushroom Gonji7.

According to one embodiment, by providing a primer set capable of generating a specific band in the dark shade line derived from Gonji7 of oyster mushroom, it is possible to quickly and accurately detect whether or not the dark shade of oyster mushroom is low in a short period of time and at low cost. can

According to one embodiment, since the oyster mushroom dark shade line can be detected at the beginning of cultivation, high-quality oyster mushroom can be produced with high yield.

According to one embodiment, since the present invention detects using the main QTL markers, the breeding efficiency can be dramatically improved because the characteristics of mushrooms can be identified in the mycelial state.

Figure 1 schematically shows the development process of CAPS markers based on SNPs related to the color of oyster mushrooms.
Figure 2 shows a schematic diagram of an analysis method for creating an association map and analyzing QTL mapping using GBS data used for detection of dark shade of oyster mushroom.
Figure 3 shows the classification according to the type of oyster mushroom Gonji No. 7 M (shade color mutation) fruit body shade color.
Figure 4 shows the pattern of DNA fragments in a light shaded line, a medium shaded line, and a dark shaded line after amplification using a primer set according to an embodiment of the present invention and treatment with DNA restriction enzymes.
Figure 5 shows the dark red color DNA sequence, bold color means a restriction enzyme recognition site, and blue color means a DNA sequence different from other red color strains (SEQ ID NO: 3).
Figure 6 shows the bright red color DNA sequence, bold color means the recognition site of the restriction enzyme, and blue means the DNA sequence that is different from other red color strains (SEQ ID NO: 4).

Since the present invention can apply various transformations and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it should be understood that this is not intended to limit the present invention to specific embodiments, and includes all transformations, equivalents, and substitutes included in the spirit and scope of the present invention.

Terms used in this application are only used to describe specific embodiments, and are not intended to limit the present invention. In this application, the terms "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

According to one aspect, there is provided a composition for detecting the shade color of a strain derived from oyster mushroom Gonji7, including primer sets represented by SEQ ID NO: 1 and SEQ ID NO: 2.

In the present application, "primer" is a short DNA manipulation used as a starting point by DNA (RNA) synthetase in synthesizing nucleic acids (DNA, RNA), and has a base sequence complementary to a nucleic acid sequence to be specifically replicated. . The base sequence and length of the primer are used in the initialization reaction that starts the synthesis of the desired nucleic acid sequence, and its specific length and base sequence are used for the complexity, reaction temperature, and ion concentration of the nucleic acid (DNA, RNA) to be synthesized and amplified. Depends on conditions.

In addition, the nucleotide sequence of the primer set according to the present invention can be modified using a label to be detected by various methods. The primer set may mainly include a label that can be detected using photochemical or spectroscopic, biochemical, immunochemical, or other chemical means.

A desired nucleic acid sequence can be amplified through polymerase chain reaction (PCR) using the primer set according to the present invention, and a method for amplifying a target sequence is to amplify a nucleic acid molecule known in the art in addition to polymerase chain reaction. Any suitable method may be applied.

Oyster mushrooms are recognized as high quality with a dark cap color, but it is not easy to breed them because the characteristics of the cap color can be confirmed only when mushrooms are generated by hybridizing mononuclear fungi of mushrooms. In addition, it takes a long time of more than 60 days for this. However, if the molecular marker for detecting the dark cap color of oyster mushroom of the present invention is used, the breeding efficiency can be greatly increased because the mushroom cap color can be detected within 3-4 days.

Therefore, the primer set of the present application can quickly and accurately detect the dark and bright shade strains derived from Oyster mushroom Gonji7 in a short time.

According to one embodiment, the oyster mushroom may be a mushroom mycelium. In oyster breeding, if a breeder wants to select a line with desired traits, it is necessary to cultivate fruiting bodies in which the traits of mushrooms are expressed, which takes a long time and money. However, the oyster mushroom of the present invention does not grow to fruiting bodies, and it is possible to accurately detect whether or not the dark shade is present even at the mycelium stage.

According to one embodiment, it may be a composition for detecting shade color of a strain derived from oyster mushroom Gonji7, capable of amplifying CAPS (cleaved amplified polymorphic sequences) marker sequences including the primers.

The CAPS (cleaved amplified polymorphic sequences) marker sequence according to the present invention was derived from the most reliable red color trait QTL marker by QTL analysis, and as a result of examining the accuracy by applying it to the mononucleated hyphal population derived from Gonji7M, It showed accuracy, and it was found to have a high correlation with the color of the cap of the mushroom. The CAPS marker may include a primer designed using a Cleaved Amplified Polymorphic Sequences (CAPS) primer or a Derived Cleaved Amplified Polymorphic Sequences (dCAPS) primer using a locus to which a Cleaved Amplified Polymorphic Sequences (CAPS) primer is not applied. .

According to another aspect, (i) isolating DNA from the sample; (ii) amplifying the DNA isolated in step (i) using the composition for detecting the dark shade of oyster mushroom of the present application; and (iii) confirming the PCR product amplified in step (ii).

Step (i) of isolating the DNA may use a method known in the art, but is not limited thereto, inoculate the mycelia in a much complete medium (MCM) medium, culture the mycelia, and collect the mycelia in a microwave oven. It can be used after treatment, cooling treatment, and centrifugation. A target sequence can be amplified by performing an amplification reaction using the isolated gDNA (genomic DNA) as a template and using the primer set of the present invention as a primer.

Step (ii) of amplifying the target sequence may be performed through PCR. The primer annealing temperature of the PCR may be 50 ~ 65 ℃, 56 ~ 62 ℃ may be more suitable, the amplification temperature may be 65 ~ 80 ℃, 68 ~ 75 ℃ may be more suitable, but is limited thereto. it is not going to be

The PCR can be performed using a conventional PCR device, and the PCR device may be, for example, a real-time PCR device, a thermal block PCR (Thermal Block PCR) device, or a digital PCR device. As, it may be GeneAtlas (E02 (Astec Co Japan), but is not limited thereto.

The PCR device can simultaneously detect one or more types of oyster mushroom hat color, and can further quantify the amount of target nucleic acid.

Although not limited thereto, the oyster mushroom variety may be a gad mutant of a strain derived from Gonji7.

Furthermore, the oyster mushroom may be generated based on the analysis results of the Gonji No. 7 gad mutant and the prototype No. 1 P1.

Step (iii) of identifying the amplified product may be performed through gel electrophoresis, capillary electrophoresis, DNA chip, radioactivity measurement, fluorescence measurement, or phosphorescence measurement. Although not limited to this, the PCR amplification product after PCR is electrophoresed, stained with Safeview (iNtRON Biotechnology, Korea), and the formed DNA band is irradiated with UV to detect it, and the patterns appearing according to the color system can be confirmed at the same time. .

The step of confirming the amplified PCR product is a step of digesting the amplified PCR product with HaeIII restriction enzyme, detecting a DNA fragment with a dark red color when the size of the DNA fragment is 290 bp, and detecting a bright red color when the DNA fragment is observed at 324 bp.

In addition, although not limited to this, if the number of fragments is 5 (34bp, 50bp, 103bp, 146bp, 290bp), it can be confirmed that it is a dark red line, and the number of fragments is 4 (50bp, 103bp, 146bp, 324bp) If it is, it can be confirmed that it is a bright shade color system.

Hereinafter, preferred embodiments of the present invention will be described in detail. The following examples are only for illustrating the present invention, and the scope of the present invention should not be construed as being limited by these examples. In addition, although only examples using specific compounds were exemplified in the following examples, it is obvious to those skilled in the art that equivalent or similar effects can be exerted even when equivalents thereof are used.

Example

Preparation of primer set for detection of dark shade of oyster mushroom

1. CAPS Marker Screening

Mononucleated mycelia were collected from Gonji7M, a natural shade-colored variant of Gonji7, a popular cultivar of oyster mushroom. HK-18 mononuclear mycelia, which are 100% hybridized with the collected mononuclear mycelia, were crossed with 98 individuals of Gonji No. 7 M mononuclear mycelia, and mushrooms were grown to investigate the color of the cap. The genome sequences of 98 mononucleated mycelia of Gonji7 M were analyzed and SNP (single nucleotide polymorphism) was investigated. A genetic map with 12 associated sequences was constructed using SNP data. QTL analysis was performed using the genetic map and the color data of oyster mushrooms.

A cleaved amplified polymorphic sequence (CAPS) primer was prepared by targeting a specific SNP of a highly reliable quantitative trait locus (QTL) marker selected from the QTL results. Using the reference genome sequence (MutantP2-2.fasta) and the program Primer3 (v2.3.5), SNPs satisfying the following conditions were derived (see Table 1).

In addition, we designed dCAPS (Derived Cleaved Amplified Polymorphic Sequences) primers using loci to which CAPS (Cleaved Amplified Polymorphic Sequences) primers were not applied among the significant SNPs selected from the QTL results. Up to one mutation such as SNP or In/Del within the region is allowed, and considering the characteristics of dCAPS primers, SNP loci without mutation within 8 bp to the left and right of the target SNP to which restriction enzymes can be applied were selected as marker candidates. For dCAPS Primer, dCAPS Finder 2.0 and Primer3 (v2.3.5) were used to design primers satisfying the basic values except for the following conditions. At this time, in silico PCR of the prepared primer sequence was performed on the MutantP2-2.fasta sequence (see Table 2).

The CAPS marker includes primers capable of amplifying SNP regions with mutations. Here, the DNA sequence amplified by PCR by the SNP mutation part was cut or not cut by a specific restriction enzyme, and this result showed a close correlation with the color of the mushroom cap. 1 shows the development process of the SNP-based CAPS marker, which is a molecular marker related to the color of the cap of oyster mushrooms.

2. Association map creation and QTL mapping analysis method using GBS data used to detect dark shade of oyster mushroom

A schematic diagram of the analysis method used for analyzing the QTL results is shown in FIG.

2-1. Sequence pre-processing

Demultiplexing was performed using the barcode sequence, and adapter sequence removal and sequence quality trimming were performed.

For adapter trimming, the cutadapt (version 1.8.3) program was used, and for sequence quality trimming, SolexaQA (v.1.13) packag’s Dynamic Trim and LengthSort programs were used. used Dynamic Trim performs a process of excising bad quality bases at both ends of short reads according to the press score and purifying them into good quality clean reads. In addition, LengthSort performed a process of removing reads in which too many bases were truncated in Dynamic Trim. A phred score ≧=20 of dynamic trim and a short read length ≧25 bp were used for length short process.

2-2. Alignment to reference genome

The clean reads that passed the preprocessing were mapped to the reference genome using the BWA (0.6.1-r104) program. Mapping is a preceding process for detecting raw SNPs (In/Del) between the reference genome and the sequenced sample, and a BAM format file is created, and default values are used except for the options listed in Table 3.

2-3. Raw SNP detection and consensus sequence extraction

Raw SNPs (In/Del) were detected using the SAMtools (0.1.16) program in the BAM format file generated by mapping the clean reads to the reference genome, and consensus sequences were extracted.

At this time, after SNP validation was performed using SEEDERS in-housescript before the SNP detection process, raw SNP (In/Del) detection was performed. Default values were used except for the options listed in Table 4.

2-4. Generate SNP matrix

In order to perform SNP comparative analysis between analysis subjects, an integrated SNP matrix between samples was created. A list of unions is constructed by comparing the raw SNP positions obtained by comparing each sample with the standard genome sequence as candidates, and at this time, the non-SNP loci are filled in from the consensus sequence of the sample through a filling process was written. Thereafter, a final SNP matrix was prepared by filtering mis-calling SNP loci through SNP comparison between samples. Based on the corresponding loci, SNPs were classified according to the type classification criteria (see Table 5).

2-5. SNP filtering and genotyping

In order to create a genome map, selection of SNP markers at a level usable for analysis was required, and SNP filters were performed based on the following criteria. Imputation was performed using the LinkImpute 1.1.4 program to compensate for deletions in the SNP matrix.

2-6. Construction of genetic map

The SNP markers finally selected through the filtering process were used with the JoinMap 4.0 program. A genetic linkage map was created.

2-7. QTL mapping

QTL mapping was performed using the QTL Cartographer V2.5 program with linkage map information and trait data. Table 7 shows the results accordingly.

3. Results

3-1. genetic mapping

Using the filtered SNP1,866 locus through the above analysis method. As a result of linkage grouping, 1,321 SNPs were used to form 12 linkage groups.

Table 8 shows the association mapping statistics of 96 samples of oyster mushroom progeny.

3-2. QTL analysis

QTL analysis using linkage map positions (SNP marker 1,321) of 96 samples of oyster mushroom offspring and 12 phenotypic traits of Gonji No. 7 m * HK18 (tester) and Gonji No. 7 m * prototype. No. 1 P1 (tester) was performed. Table 9 shows the character information of Gonji No. 7 m * HK18 (tester), and Table 10 shows the character information of Gonji No. 7 m * Prototype No. 1 P1 (tester).

3-3. Selection of markers within QTL candidate regions

for each trait. As a result of performing 1,000 permutations (p < 0.05), the LOD threshold value for each trait was calculated. If there is no marker that satisfies the standard LOD threshold or higher, a LOD threshold value was arbitrarily set and significant markers higher than the standard were selected.

Tables 11 and 12 show examples of marker lists located within the QTL candidate regions of Gonji No. 7m * HK18 (tester) and Gonji No. 7m * Prototype.No. 1P1 (tester).

SNPs of the most reliable shaded trait QTL markers were developed as cleaved amplified polymorphic sequences (CAPS) markers and applied to mononucleated mycelial populations derived from Gonji7M to examine their accuracy. The CAPS marker contained a primer capable of amplification including the SNP region with a mutation, where the DNA sequence amplified by PCR by the mutation portion of the SNP was cut or not cut by a specific restriction enzyme, which resulted in the color of the mushroom cap. showed a close correlation with

4. Production of specific primers for detection of dark cap color of oyster mushrooms

In order to develop the SNP of the most reliable red trait QTL marker derived by the analysis method of 2-1 to 2-7 as a CAPS marker, a primer for DNA amplification of a specific site that can be amplified including the mutated SNP region was produced. Table 13 shows the forward sequence (SEQ ID NO: 1) and reverse sequence (SEQ ID NO: 2) of the primer.

Primer name Sequence (5'-3') Size (bp) Forward (SEQ ID NO: 1) GTG GGT AAT TTC GTC TCG GC 20 Reverse (SEQ ID NO: 2) CGA TCG CAT AGT GGT GCA AA 20

5. Extraction of gDNA (genomic DNA) from hyphae of oyster mushrooms

Mushroom complete medium (MCM) was inoculated with mycelia of the dark shade of oyster mushrooms, and after culturing the mycelia, the mycelia were collected using a toothpick. The mycelium was put in 100 μl of TE buffer (pH 7.4) and treated twice at 1 minute intervals in a microwave oven (700 w). The culture of the mycelia was cooled at -20 ° C for at least 10 minutes, and centrifugation was performed at 10,000 rpm for 5 minutes. Then, the concentration was measured, and PCR (polymerase chain reaction) was performed.

6. Amplification of oyster mushroom hat color specific sequence

The total amount of PCR reaction was 20 μl, 30 ng of genomic DNA isolated from the mycelia of each strain, 0.4 μl of 10 mM dNTP, 1.0 unit of heat-resistant DNA polymerase (E-taq polymerase, Solgent, Korea), 10 pmol primer and buffer Mix thoroughly. Table 14 and Table 15 below show the PCR reaction solution and PCR conditions, respectively.

Reagent name 1x (μl) E-Taq 0.1 10mM-dNTPs 0.4 Primer (10 pmol) F+R 2 10x buffer 2 H ₂ O 14.5 DNA (30ng) One Total 20

reaction Tm (℃) Time Initial denaturation 95 3min Denaturation 35 cycles 95 30sec Annealing 60 40sec Extension 72 1min Final Extension 72 5min

7. Oyster mushroom mustard color specific sequence restriction enzyme and its cleavage

For the detection of dark red color of oyster mushroom, the DNA amplified with SEQ ID NOs: 1 and 2 of the present invention was digested with "HaeIII" restriction enzyme (see Table 16), and the reaction conditions for the restriction enzyme digestion reaction are shown in Table 17.

restriction enzyme Recognition seat amputation Reaction temperature (℃) reaction time HaeIII CCGG CG 37 5h

8. DNA Fragmentation Pattern Results

In order to confirm the DNA cleavage pattern of the dark red color restriction enzyme, the amplified DNA was treated with the restriction enzyme using the primers of the present invention for the strains classified as light red color, medium red color, and dark red color, and then the DNA fragment pattern was confirmed. .

Table 18 shows the shade brightness and color type of strains for which DNA fragment patterns were confirmed.

No. Species( Pleurotus ostreatus ) Freshness (L) shade type One Gonji No. 7 m98 93.1 bright shade 2 Gonji No. 7 m75 92.7 bright shade 3 Gonji 7 m92 92.0 bright shade 4 Gonji No. 7 m83 91.7 bright shade 5 Gonji No. 7 m85 91.6 bright shade 6 Gonji No. 7 m48 85.2 medium shade 7 Gonji 7 m17 85.1 medium shade 8 Gonji No. 7 m88 84.3 medium shade 9 Gonji 7 m36 81.3 medium shade 10 Gonji 7 m60 81.1 medium shade 11 Gonji 7 m34 71.6 dark shade 12 Gonji No. 7 m82 71.6 dark shade 13 Gonji No. 7 m50 70.2 dark shade 14 Gonji 7 m49 69.9 dark shade 15 Gonji 7 m90 64.1 dark shade

Figures 5 and 6 show the DNA sequences of the dark shade and bright shade lines of oyster mushrooms. Referring to this, in the case of DNA sequences of dark color lineages, when HaeIII restriction enzymes are used, 5 DNA fragments are generated by the recognition sites of 4 restriction enzymes, but in the case of light lineage DNA sequences, the base is at G. It is substituted with A, so it has 3 restriction enzyme recognition sites, resulting in 4 DNA fragments.

As a result of PCR reaction after amplification using the primers of the present invention, DNA of 623 bp was amplified in both the light and dark shade lines. However, as a result of digestion with HaeIII, which recognizes CCGG, the polymorphism was confirmed according to the shade color. In order to confirm the DNA cleavage pattern, electrophoresis was performed on a 3% agarose gel, and a loading volume of 10 μl was used.

Table 19 shows the size of the fragment appearing according to each strain as a result of electrophoresis. Referring to this, the most notable difference is that a 290 bp DNA fragment was observed in the dark shade, and a 324 bp DNA fragment was observed in the light shade.

item bright shade dark shade Amplification product size 623 623 Different cutting site (Cutting_site) 137 137 Cutting_site 103, 427, 573 103, 137, 427, 573 Cutted size 50, 103, 146, 324 34, 50, 103, 146, 290

Although the present invention has been described in detail through specific examples, this is for explaining the present invention in detail, the present invention is not limited thereto, and within the technical spirit of the present invention, by those skilled in the art It is clear that it can be modified or improved. All simple modifications or changes of the present invention fall within the scope of the present invention, and the specific protection scope of the present invention will be clarified by the appended claims.

<110> Korea National College of Agriculture and Fisheries <120> Composition for identification of dark cap color of Pleurotus ostreatus Gonji 7ho derived strains and identification method using the same <130> NPF35126 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 1 gtgggtaatt tcgtctcggc 20 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 2 cgatcgcata gtggtgcaaa 20 <210> 3 <211> 623 <212> DNA <213> unknown <220> <223> Pleurotus ostreatus Gonji 7ho <400> 3 gtgggtaatt tcgtctcggc gatctcccgc gatcagtctt cccactagaa ctgctattac 60 ccctcgacga ctgggcagct ccgacgctca acgtagagtc caccggtgaa gggaaggtcg 120 ggagttgcgt cggcgcccgg ctgctgctgt ggaatatatc acgagcgtct attcgttttg 180 gtaagcgttg gttgataagc ggcggtgcac tatgtgcagc atgctctgat gatatacgtg 240 aaagaagagg aagcgtcagg ccgtcgttat catcatctga atagaggtcg gcgttcgacg 300 ctgctgcagc ggccgaggtg gagtcggtat tgattcccag cggctgcggt gacggaggat 360 agatgttacc cagcggcgaa tgcggtgctt cacggtgttg atgtatgacg gaattatctc 420 cagtccccgg tcttgaatac tgatgtacaa catcctggct atcgaacttg atggtggggc 480 gtttgccgat ggctgaaggg ctaccaggcg gagacaaagc ggatgaggtg ggtcttggga 540 cgggtgcgtc atagagttgg tgtctggtca tcccggctag cgcttcgcgc tcagcggtcg 600 gactttgcac cactatgcga tcg 623 <210> 4 <211> 623 <212> DNA <213> unknown <220> <223> Pleurotus ostreatus Gonji 7ho <400> 4 gtgggtaatt tcgtctcggc gatctcccgc gatcagtctt cccactagaa ctgctattac 60 ccctcgacga ctgggcagct ccgacgctca acgtagagtc caccggtgaa gggaaggtcg 120 ggagttgcgt cggcgcccga ctgctgctgt ggaatatatc acgagcgtct attcgttttg 180 gtaagcgttg gttgataagc ggcggtgcac tatgtgcagc atgctctgat gatatacgtg 240 aaagaagagg aagcgtcagg ccgtcgttat catcatctga atagaggtcg gcgttcgacg 300 ctgctgcagc ggccgaggtg gagtcggtat tgattcccag cggctgcggt gacggaggat 360 agatgttacc cagcggcgaa tgcggtgctt cacggtgttg atgtatgacg gaattatctc 420 cagtccccgg tcttgaatac tgatgtacaa catcctggct atcgaacttg atggtggggc 480 gtttgccgat ggctgaaggg ctaccaggcg gagacaaagc ggatgaggtg ggtcttggga 540 cgggtgcgtc atagagttgg tgtctggtca tcccggctag cgcttcgcgc tcagcggtcg 600 gactttgcac cactatgcga tcg 623

Claims (5)

A composition for detecting shade color of a strain derived from oyster mushroom Gonji7, comprising primer sets represented by SEQ ID NO: 1 and SEQ ID NO: 2.
According to claim 1,
The oyster mushroom is a mushroom mycelium, a composition for detecting the shade of a strain derived from oyster mushroom Gonji No. 7.
A composition for detecting the shade of a strain derived from oyster mushroom Gonji7, capable of amplifying CAPS (cleaved amplified polymorphic sequences) marker sequences, including the primer according to claim 1.
(i) isolating DNA from the sample;
(ii) amplifying the DNA isolated in step (i) using the composition for detecting the dark shade of oyster mushroom of claim 1; and
(iii) confirming the PCR product amplified in step (ii);
According to claim 4,
The step of confirming the amplified PCR product is digested with HaeIII restriction enzyme, detected in dark color when the size of the DNA fragment is 290 bp, and detected in bright color when the DNA fragment is observed at 324 bp. A method for detecting the red color of

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