KR101458518B1

KR101458518B1 - SCAR marker for distingusihing cold-adapted live Pleurotus eryngii and use thereof

Info

Publication number: KR101458518B1
Application number: KR20130085952A
Authority: KR
Inventors: 조수정; 조윤진; 김수철; 류재산
Original assignee: 경남과학기술대학교 산학협력단
Priority date: 2013-07-22
Filing date: 2013-07-22
Publication date: 2014-11-07

Abstract

The present invention relates to SCAR markers and their uses for identifying low temperature adaptive shoots. It is not necessary to directly cultivate a variety of mushroom cultivars in order to identify the mushroom of low temperature adaptability using the SCAR primer set for identification of the low temperature adaptable mushroom according to the present invention, It is possible to establish a technical system for breeding.

Description

SCAR markers for identification of low temperature adaptive mushroom mushrooms and their uses {SCAR marker for distingusihing cold-adapted live Pleurotus eryngii and use thereof}

The present invention relates to SCAR markers and their uses for identifying low temperature adaptive mushroom ( Pleurotus eryngii ).

Growth conditions such as carbon dioxide, temperature, and relative humidity, together with mushroom medium, are essential for artificially cultivating the mushroom (Hashimoto and Takahashi, 1974). In Korea, a large amount of fuel cost is required to maintain the optimum temperature for growth of the mushroom mushroom in the high temperature and high humidity in summer, which is getting worse due to the warming in summer, and in the low temperature dry climate in winter. In mushroom farmers, I am looking for various ways.

From the breeding point of view, it is one of the ways to reduce the fuel cost of mushroom farmers by developing high temperature adaptive cultivars for summer climate or developing low temperature adaptable cultivars against winter climate. However, traditional cross breeding requires a lot of time and effort, and it takes much time to identify the breed species. Therefore, in case of mushroom, it is necessary to develop a genetic marker capable of discriminating the breed species without producing fruiting bodies.

RAPD method using a random primer consisting of a 10 bp nucleotide sequence has been used most often but it has a disadvantage of poor reproducibility. Recently, RAPD primers have been sequenced with SCAR (Sequence characterized amplified region) (2006), and the results of the study are as follows: 1) The number of markers is increased.

Under the above background, the present invention is based on the nucleotide sequence of a specific DNA band associated with the low-temperature adaptive Pleurotus eryngii selected through RAPD analysis, thereby developing a SCAR marker to produce a low-temperature adaptive mushroom So that they can be selected.

Korean Unexamined Patent Publication Nos. 11-0074204 and 2012-0069446 disclose methods of identifying cultivars using conventional SCAR markers or primers.

Korean Patent Publication No. 2011-0074204 Korean Patent Publication No. 2012-0069446

It is an object of the present invention to provide a SCAR (Sequence Characterized Amplified Region) primer set composition for identifying a low temperature adaptable Pleurotus eryngii , a kit using the same, and a method of identifying a low temperature adaptive shoot mushroom using the same.

According to one aspect of the present invention, a Sequence Characterized Amplified Region (SCAR) primer set composition for identifying a low temperature adaptive Pleurotus eryngii comprising a primer set of the nucleotide sequences of SEQ ID NOS: 1 and 2 can be provided.

According to another aspect of the present invention, the SCAR primer set composition; DNA polymerase; dNTPs (deoxyribonucleotides); And a buffer solution for polymerase chain reaction (PCR) can be provided.

According to another aspect of the present invention, there is provided a method for producing a mushroom, comprising: (a) extracting genomic DNA from a mushroom; (b) amplifying the genomic DNA by PCR using the extracted genomic DNA as a template DNA and using the SCAR primer set composition according to claim 1; And (c) analyzing the amplified product. The low temperature adaptive mushroom identification method may be provided.

It is not necessary to directly cultivate a variety of mushroom cultivars in order to identify the mushroom of low temperature adaptability using the SCAR primer set for identification of the low temperature adaptable mushroom according to the present invention, It is possible to establish a technical system for breeding.

Figure 1 shows the results of RAPD analysis of low temperature adaptive mushroom and control using OP-S primer.
Fig. 2 shows the results of RAPD analysis of low-temperature adaptive mushroom and control using OP-S3 primer.
FIG. 3 shows the results of cloning confirmation of bands specifically detected in the low-temperature adaptive mushroom by RAPD analysis.
Fig. 4 shows the nucleotide sequences of the bands specifically detected in the low-temperature adaptive mushroom by RAPD analysis.
FIG. 5 shows PCR results of a low-temperature adaptive mushroom and a control using a SCAR primer set according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

The term " low temperature "or" low temperature adaptability " as used in the present invention means the characteristic of the mushroom cultivated or capable of being cultivated in the temperature range of 10 to 15 캜.

The term " SCAR (Sequence Characterized Amplified Region) "or" SCAR marker "used in the present invention is produced by analyzing the nucleotide sequence of RAPD (Marked Amplification Fragment Length Polymorphism) or AFLP Allele-Specific Associated Primer) method. Because SCAR markers are relatively insensitive to amplification environments and can read results easily compared to other molecular markers, they are recognized as an efficient molecular markers for breed identification with reproducibility, universality and simplicity. That is, it can be used as a more accurate and detailed primer.

As used herein, the term "primer " means a single stranded oligonucleotide sequence complementary to a nucleic acid strand to be amplified and can serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer may depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

[SEQ ID NO: 1]

5'-CAG AGG TCC CGG TCA GCA CT-3 '

[SEQ ID NO: 2]

5'-CAG CGC CCA TTC GTC CCA CTC-3 '

The nucleotide sequence of SEQ ID NO: 1 is a forward primer and the nucleotide sequence of SEQ ID NO: 2 is a reverse primer.

In one embodiment, the nucleotide or oligonucleotide used as the primer also includes a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid. Or it may comprise an intercalating agent.

In one embodiment, the SCAR primer set is a primer set for amplification of a SCAR marker for identifying a low-temperature adaptive mushroom, and the reagent for performing the amplification reaction may include a heat-resistant DNA polymerase, dNTPs, and a buffer solution. The buffer solution has a composition that is commonly used in the art to which the present invention belongs, and may be, for example, Tris-HCl, MgCl ₂ or KCl.

Further, in one embodiment, the kit may further include a user guide describing optimal reaction performing conditions. The brochure is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. 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 disclosed or provided through an electronic medium such as the Internet.

In one embodiment, the method for extracting the genomic DNA in step (a) may be performed by a conventional method known in the art. For example, a CRAB method, a phenol / chloroform extraction method, or an SDS extraction method may be used, or a DNA extraction kit may be used.

In one embodiment, the SCAR primer set composition used in step (b) is a composition comprising the nucleotide sequence of SEQ ID NOS: 1 and 2. In addition, amplification of genomic DNA can be performed by PCR (Polymerase Chain Reaction). The PCR may be carried out using a PCR mixture containing components known to be necessary for PCR in the art to which the present invention belongs or using a commercially available kit. The PCR mixture may contain the genomic DNA extracted from Shiitake mushroom and the SCAR primer set composition according to the present invention, an appropriate amount of DNA polymerase, dNTP, a buffer solution and water.

In one embodiment, step (c) can be performed by DNA chip, electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement. Preferably, the amplified DNA product is analyzed using gel electrophoresis, more specifically, the amplification product is electrophoresed on an agarose gel or an acrylamide gel, and the product is mixed with ethidium bromide (EtBr), silver staining You can see the band by.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. RAPD analysis for polymorphism search

The template DNA for RAPD (Random Amplified Polymorphic DNA) was prepared by sequencing the genomic DNA of 8 mushroom strains of eight selected mushroom strains and 8 mushroom strains of control (not forming fruiting bodies at low temperature) OP-A (20), OP-B (20), OP-L (20) and OP-P (20) were used for bulking OP-R (20), and OP-S (20) were used.

PCR was carried out using Bioneer premix kit, 2 μl of genomic DNA (50 ng), 3 μl of primer (20 pmoles) and 15 μl of distilled water were added to Bioneer premix kit under the following conditions. PCR was carried out by predenaturation at 94 ° C for 4 minutes, followed by denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extensin for 2 minutes at 72 ° C. Lt; / RTI > for 10 minutes. The amplified PCR product was electrophoresed with 1.5% agarose gel to confirm the DNA band. The PCR products of OP-S3 (480 bp) and OP-S10 (320) primers were used as primers in the control and low temperature adaptive lines. (See Fig. 1).

In order to identify the specific bands of the low-temperature adaptive strain, which were differentiated from the control, RAPD was performed on the control and low temperature adaptive lines using OP-S3 and OP-S11 primers. As a result, primers showing specific bands in the low temperature adaptive system OP-S3 primer (see Fig. 2).

Example 2. Cloning and Identification of Low Temperature Adaptive System-Specific PCR Products

PCR was performed using OP-S3 primer. The DNA of the low-temperature adaptive specific band (480 bp) was extracted with a gel extraction kit (Bioneer, Korea) and the extracted DNA was purified using a T-blunt PCR cloning kit (SolGent, Korea) . To confirm that the cloning was correctly performed, the selected clones were cultured in a liquid medium, and the plasmids were isolated using a plasmid isolation kit (Intron, Korea). The plasmids isolated were digested with EcoR I (Promega, USA) (See FIG. 3), and the nucleotide sequence of the low temperature adaptive specific band is shown in FIG.

Example 3. Development of a SCAR primer set

(5'-CAG AGG TCC CGG TCA GCA CT-3 ') to be used as a SCAR marker by adding a base sequence of 10 to 12 bp to the base containing the OP-S3 primer based on the nucleotide sequence of the low temperature adaptive specific band And a reverse primer (5'-CAG CGC CCA TTC GTC CCA CTC-3 ').

PCR using the primer set to be used as the SCAR marker was performed by predioneating at 94 ° C for 4 minutes using a Bioneer premix kit, followed by denaturation at 94 ° C for 1 minute, annealing at 62 ° C for 1 minute, and 2 minutes at 72 ° C The extensin process was repeated 30 cycles, followed by a last extensin process at 72 ° C for 10 minutes.

The amplified PCR product was electrophoresed with 1.5% agarose gel to confirm the DNA band. The low frequency adaptive strain and the control group were analyzed using a SCAR primer set according to SEQ ID NOS: 1 and 2, and a single band discriminated from the control was confirmed in the low temperature adaptive mushroom. The results showed that the low - temperature adaptive mushroom was easily distinguishable from other varieties and showed higher reproducibility than the random primers used.

Therefore, it is not necessary to directly cultivate a variety of mushroom cultivars in order to identify the mushroom having low temperature adaptability by using the SCAR primer set for identification of the low-temperature adaptive mushroom according to the present invention. Accordingly, It is possible to establish a technical system for identification and breeding.

<110> ACADEMY AND INDUSTRY COLLABORATION OF GYEONGNAM NATIONAL UNIVERSITY OF SCIENCE AND TECHNOLOGY <120> SCAR marker for distingusihing cold-adapted live Pleurotus eryngii and use thereof <130> NPF-24056 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of SCAR marker for distingusihing cold-adapted live Pleurotus eryngii <400> 1 cagaggtccc ggtcagcact 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of SCAR marker for distingusihing cold-adapted live Pleurotus eryngii <400> 2 cagcgcccat tcgtcccact c 21

Claims

A Sequence Characterized Amplified Region (SCAR) primer set composition for identifying a low temperature adaptive Pleurotus eryngii comprising a primer set of the nucleotide sequences of SEQ ID NOs: 1 and 2.

A SCAR primer set composition according to claim 1;
DNA polymerase;
dNTPs (deoxyribonucleotides); And
A kit for identifying a low temperature adaptive mushroom comprising a buffer solution for PCR.

(a) extracting genomic DNA from the mushroom;
(b) amplifying the genomic DNA by PCR using the extracted genomic DNA as a template DNA and using the SCAR primer set composition according to claim 1; And
(c) analyzing the amplified product;
Lt; RTI ID = 0.0 > mushroom. &Lt; / RTI >

The method of claim 3,
Wherein the step (c) is performed by DNA chip, electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement.