KR101166781B1 - Specific primer for strain discrimination of Pleurotus spp. by analysis of mitochondria DNA polymorphism and uses thereof - Google Patents

Abstract

The present invention relates to a specific primer set and its use for determining the cultivars of oyster mushrooms, and more particularly, to the cultivation primer set of oyster mushrooms comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2, the primer set The present invention relates to a kit for determining a variety of oyster mushrooms, and a method for determining cultivars of oyster mushrooms using the primer set.

Pleurotus eryngii, Greater oyster mushroom, Pleurotus eryngii, Awi, Baekyounggo, Mitochondria, Primer, PCR

Description

Specific primer for cultivation of Pleurotus eryngii by mitochondrial DNA polymorphism analysis and its use {Specific primer for strain discrimination of Pleurotus spp. by analysis of mitochondria DNA polymorphism and uses approximately}

The present invention Pleurotus spp.) relates to a set of specific DNA primers that can quickly and accurately classify regions containing mitochondrial microsatellites by polymerase chain reaction (PCR). More specifically, it is a process for preparing primers for quickly and accurately diagnosing PCR specific fragment bands that are unique among varieties.

Pleurotus Pleurotus ostreatus ) , yellow locust mushroom (P. cornucopiae ) , pink locust mushroom ( P. salmoneo - stramineus ) , large locust mushroom ( P. eryngii ) Etc About 30 species are known around the world, and three species are known to be distributed in Korea, and the most are produced in Korea. Looking at the current status, excellent varieties are imported or sold by individuals or companies around the world such as Japan or China. It is confounding farmers by registering it.

In addition, large locust mushrooms do not grow in Korea, but in southern Europe and the Middle East (the Kidney Ugur region). Korea's large elk production is 46,357M / T, with 32% of the total mushroom production, the highest cultivation rate. Since its opening in 2009 under the joining of the UPOV (New Variety Protection Federation), it is urgently needed for self-development. .

The existing morphological and physicochemical methods for classifying varieties and strains are prone to variation due to environmental factors depending on the growth stage, require a lot of time and effort, and require long-term experience and expertise. However, recent advances in molecular biology have led to the development of nucleic acid fingerprint analysis technology that enables the study of biodiversity at the DNA level, making it simple and fast accurate. Nucleic acid fingerprint technology is used for various purposes such as species identification, identification of microorganisms, and analysis of soft relationships of populations.

Nucleic acid fingerprinting method developed so far is the most widely used taxonomy and genus of fungi including mushrooms, PCR amplifies a site such as internal transcribed spacer (ITS), a specific region of Ribosomal DNA (rDNA) There is a method of determining the nucleotide sequence and comparing the analysis between species phylogenetically, there is a difficulty in discriminating between genus and some species. Therefore, RAPD (random amplified polymorphic DNA) and AFLP (Amplified Fragment Length Polymorphism) have been developed and applied, but their PCR technique can generate many bands by amplifying many genes. Can be distinguished.

Korean Patent Registration No. 0682988 discloses a specific primer for identifying a species of roe deer KUMC 1008 and an identification method using the same, and Korean Patent Registration No. 0435153 discloses a PCR primer for detecting a wood mud mushroom specific DNA fragment. It is different from the primer set of the present invention.

The present invention is derived from the above requirements, the present invention is a Pleurotus ( Pleurotus) registered in GenBank Based on the mitochondrial sequences of ostreatus ), a pair of specific primers consisting of 22 bp and 20 bp, respectively, were prepared and PCR reactions were carried out on oyster mushrooms. As a result, they were able to detect specific DNA fragment bands from oyster mushrooms. The present invention could be completed.

In order to solve the above problems, the present invention is to distinguish the varieties of Pleurotus spp. Provide a specific set of PCR primers that can be used.

The present invention also provides a kit for determining a variety of oyster mushrooms comprising the primer set.

The present invention also provides a method for determining a variety of oyster mushrooms using the primer set.

The present invention relates to a cultivar specific primer of oyster mushrooms by mitochondrial DNA polymorphism analysis, and information on oyster mushrooms to be cultivated as a technique capable of quickly assaying cultivars of oyster mushrooms according to the specific band size by PCR method. It can contribute to the improvement of international competitiveness of mushrooms for export and the development of mushroom industry by preventing the confusion of varieties.

In order to achieve the object of the present invention, the present invention provides at least 15 oligonucleotides selected from the group consisting of oligonucleotides consisting of fragments of at least 15 contiguous nucleotides in the sequence of SEQ ID NO. Provided is a set of cultivars for discriminating oyster mushrooms, comprising one or more oligonucleotides selected from the group consisting of oligonucleotides consisting of segments of contiguous nucleotides.

According to the present invention, primers of the present invention, MtPO1F and MtPO1R, were prepared using a nucleotide sequence specifically present in the mitochondria of Pleurotus spp. Registered in GenBank when PCR nucleic acid fingerprinting was performed with MtPO1F and MtPO1R. A gene fragment is a primer comprising SEQ ID NO: 1 and SEQ ID NO: 2.

The oligonucleotide may preferably be an oligonucleotide consisting of segments of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides in the sequence of SEQ ID NO: 1. In addition, the oligonucleotide may preferably be an oligonucleotide consisting of segments of at least 16, at least 17, at least 18, at least 19 consecutive nucleotides in the sequence of SEQ ID NO: 2.

The primer set of the present invention may preferably comprise an oligonucleotide primer set of SEQ ID NOs: 1 and 2. In addition, the primer may include an addition, deletion or substituted sequence of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primers will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

As used herein, oligonucleotides used as primers may also include nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

In order to achieve another object of the present invention, the present invention

Oligonucleotide primer sets according to the present invention; And it provides a kit for determining the variety of oyster mushrooms, including a reagent for performing the amplification reaction.

In the kit of the present invention, the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, buffers and the like. In addition, the kits of the present invention may further comprise a user guide describing the optimal reaction performance conditions. The guide is a printed document that explains how to use the kit, such as how to prepare a PCR buffer, and the reaction conditions presented. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

In order to achieve another object of the present invention, the present invention

Separating genomic DNA from Pleurotus eryngii;

Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using a primer set according to the present invention; And

It provides a method for determining the variety of oyster mushrooms, comprising the step of detecting the amplification products.

The method of the present invention comprises the step of isolating genomic DNA from Oyster mushroom samples. As a method for separating genomic DNA from the sample, a method known in the art may be used. For example, the CTAB method may be used, or a wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, an amplification reaction may be performed using an oligonucleotide primer set according to an embodiment of the present invention as a primer to amplify a target sequence. Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification systems, There are any suitable methods for amplifying via strand displacement amplification or Qβ replication or for amplifying nucleic acid molecules known in the art. Among these, PCR is a method of amplifying a target nucleic acid from a pair of primers specifically binding to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. The oligonucleotide primer set used to amplify the target sequence is as described above.

The method of the present invention includes detecting the amplification product. Detection of the amplification product may be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, capillary electrophoresis can be performed. Capillary electrophoresis can use, for example, an ABi Sequencer. In addition, gel electrophoresis may be performed, and gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, radioactivity measuring method is to add a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.

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  One. primer  making

Genetic information of oyster mushrooms registered in Genbank to access the preservation of mitochondrial genes that are highly reproducible and responsible for maternal inheritance rather than RAPD polymorphism of known genomes (Accession no. EF204913) As shown in FIG. 1, a primer including a microsatellite region located between atp9 and nad1 genes was designed. Considerations in primer design include GC content and the formation of complexes or melting points between primers, and the base sequences and product sizes of primers prepared using the "primer3" software program (NCBI) considering these are shown in Table 1.

Table 1.

Primer Name Sequence (5'-3 ') Product size (bp) MtPO1F (SEQ ID NO: 1) 5'-CCGGCCCTACCTTAATTATTTT-3 ' 120-400
MtPO1R (SEQ ID NO: 2) 5'-GTTTACCCCGCCTTTAGGTC-3 '

Example  2. Specific Detection for Differentiation of Pleurotus eryngii Primer  Applicability Check

Standard strains used in this example were disclosed by introducing strains used in domestic and overseas research institutes, universities, and commercial or farming, including CBS (Cetraalburea voor Schimmercultures), the Dutch microbial strain preservation center. In Pleurotus cornucopiae (Yellow), 15 strains of P leurot us ostreatus were reported in Pleurotus ostreatus . Pleurotus One strain of salmoneo- stramineus (pink elk ) and one hybrid were disclosed. In Pleurotus eryngii ( Pleurotus eryngii ) 5 strains, Pleurotus A total of 50 strains including 17 strains of ferulae (Awi) and 10 strains of Pleurotus nebrodensis (Baekyounggo) were collected from various regions at home and abroad and disclosed as shown in Table 2.

Table 2.

To separate DNA Pleurotus The varieties were cultured in PDA (Difco) solid medium for 10 days and the bacterial flora formed on the medium was scraped, lyophilized in liquid nitrogen, and dried using a lyophilizer. The freeze-dried mycelium was finely ground with a toothpick, and then 100 μg was transferred to a 1.5 ml tube and 400 μl of extraction buffer (200 mM Tris-HCl, pH 8.0; 200 mM NaCl; 25 mM EDTA; 0.5% SDS) and Proteinase K ( 20 mg / ml) was added and mixed well on the buffer with a glass rod and then incubated at 37 ° C. for 1 hour.

2X CTAB buffer was added to the mixture in the same amount and left at 65 ° C. for 15 minutes, chloroform: isoamyl alcohol (24: 1) was added thereto, thoroughly mixed, and centrifuged at 12,000 rpm. The supernatant was transferred to a new tube, 0.7 volume of isopropanol was added, left for 10 minutes at room temperature, and centrifuged at 12,000 rpm for 5 minutes to precipitate DNA. DNA precipitate was washed with 70% ethanol, dried in vacuo and dissolved in 100 μl of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). 2 μl of 10 mg / ml RNase was added and the RNA was removed by treatment at 37 ° C. for 30 minutes. The amount of separated DNA was determined by electrophoresis on agarose gel (1.0%) and compared with the quantified DNA.

The reaction solution for the PCR reaction was 20 μl in total, 50ng of DNA isolated from each mushroom sample, 4 dNTPs (2.5 mM each), Taq polymerase (2.5 unit, manufactured by Promega), 20 pmol primer and buffer ( 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl ₂ , 0.01% gelatin).

The PCR device of GeneAmp PCR system 9700 (Applied Biosystem) was used for this PCR chain reaction. PCR conditions using the MtPO1F and MtPO1R primers were first performed by heat treatment at 94 ° C. for 4 minutes to degenerate genomic DNA into single strand, followed by denaturing DNA at 94 ° C. for 1 minute in the cycle; Contacting with a URP1F primer having a concentration of 20 pmol at 59 ° C. for 1 minute; DNA synthesis was performed at 72 ° C. for 1 minute (DNA polymerization). After the reaction, the PCR amplification product was subjected to electrophoresis on 2% agarose gel, and then stained with ethidium bromide to detect DNA diversity bands by UV irradiation. Polaroid photographs were taken. It is shown in Figure 2, the results for the large locust mushroom is shown in FIG. As shown in Figures 2 and 3, the primer pair of the present invention only detected a specific band in the published oyster mushrooms and showed various band sizes.

Therefore, it was confirmed that the primer pair of MtPO1F and MtPO1R of the present invention can be used to specifically detect oyster mushrooms and to determine the authenticity of cultivar of oyster mushrooms.

Figure 1 is a schematic diagram of the development of cultivar specific primers (MtPO1F / MtPO1R) of oyster mushrooms by the mitochondrial DNA polymorphism analysis according to the present invention.

Figure 2 is an electrophoresis picture after PCR amplification of Pleurotus eryngii using MtPO1F / MtPO1R primer according to the present invention.

M: DNA size marker (100 bp), 1: Pleurotus cornucopiae ASI 2295 (Yellow), 2: P. ostreatus ASI 2001 (Farm No. 2-1), 3: P. ostreatus ASI 2736 (Bupyeong Ebony No. 1), 4: P. ostreatus ASI 2730 (Qingdao 22), 5: P. ostreatus ASI 2706 (Black Pyeong), 6: P. ostreatus ASI 2596 (3), 7: P. ostreatus ASI 2180 (1), 8: P. ostreatus ASI 2018 (Agricultural 201), 9: P. ostreatus ASI 2726 (Shin Nong No. 11), 10: P. ostreatus ASI 2228 (Spring 1), 11: P. ostreatus ASI 2504 (Suhan 1), 12: P. ostreatus ASI 2792 (Halla 1) ), 13: P. ostreatus ASI 2487 (Cheongpung), 14: P. ostreatus ASI 2344 (Spring 2), 15: P. ostreatus ASI 2194 (Attari), 16: Hybrid ASI 0308 (Round Black), 17: P. ostreatus ASI 2181 ( September 2), 18: P. salmoneo - stramineus ASI 2104 (pink elk ).

Figure 3 is an electrophoresis picture after PCR amplification of the oyster (mushroom mushroom) using the MtPO1F / MtPO1R primer according to the present invention.

M: DNA size marker (100 bp), 19: Pleurotus fuscus var. ferulae ASI 2623 (20th), 20: P. fuscus var. ferulae ASI 2776 (21), 21: P. fuscus there is . ferulae ASI 2780 ( Augo ), 22: P. nebrodensis ASI 2685 (large suba ), 23: P. nebrodensis ASI 2720 ( baeksong ), 24: P. nebrodensis ASI 2852 (Baekyounggo), 25: P. nebrodensis ASI 2882 (Baekyounggo), 26: P. nebrodensis ASI 2883 (Baekyounggo), 27: P. nebrodensis ASI 2884 (Baekyounggo), 28: P. nebrodensis ASI 2885 (Baekyounggo), 29: P. nebrodensis ASI 2886 (Baekyounggo), 30: P. var. nebrodensis ASI 2802 (Baek Younggo), 31: P. var. nebrodensis ASI 2804 (Baek Younggo), 32: P. eryngii ASI 2824 (Great 3), 33: P. eryngii ASI 2887 (Children 3), 34: P. eryngii ASI 2302 (Great No. 1), 35: P. eryngii ASI 2865 (big horn), 36: P. eryngii ASI 2839 (A large herd ), 37: P. ferulae ASI 2831 ( Agogo ), 38: P. ferulae ASI 2847 (39), P. ferulae ASI 2848 (Awi), 40: P. ferulae ASI 2849 (Awi), 41: P. ferulae ASI 2850 (Awi), 42: P. ferulae ASI 2880 (Awi), 43: P. ferulae ASI 2881 (Awi), 44: P. var. ferulae ASI 2798 (Ab: CBS282.32), 45: P. var. ferulae ASI 2801 (Awi), 46: P. var. ferulae ASI 2803 (Awi), 47: P. var. ferulae ASI 2806 (Awi), 48: P. var. ferulae ASI 2807 (Awi), 49: P. var. ferulae ASI 2808 (Awi), 50: P. var. ferulae ASI 2809 (Awi).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Specific primer for strain discrimination of Pleurotus spp. by          analysis of mitochondria DNA polymorphism and uses <130> PN09275 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MtPO1F primer <400> 1 ccggccctac cttaattatt tt 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MtPO1R primer <400> 2 gtttaccccg cctttaggtc 20  

Claims (6)

At least one oligonucleotide selected from the group consisting of fragments of at least 15 contiguous nucleotides in the sequence of SEQ ID NO: 1 and oligonucleotides consisting of fragments of at least 15 contiguous nucleotides in the sequence of SEQ ID NO: 2 Pleurotus , comprising one or more oligonucleotides selected from the group spp.) variety discriminating primer set. The variety discriminating primer set of oyster mushroom according to claim 1, comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2. A primer set according to claim 1; And a kit for determining a variety of oyster mushrooms, including a reagent for performing an amplification reaction. The kit of claim 3, wherein the reagent for carrying out the amplification reaction comprises DNA polymerase, dNTPs, and a buffer. Separating genomic DNA from Pleurotus eryngii; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to claim 1; And Method for determining a variety of oyster mushrooms, comprising the step of detecting the amplified product. The method of claim 5, wherein the detection of the amplification product is carried out through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement.

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