KR101718707B1 - Method of distinguishing between the red pine and the scot pine by using PCR-RFLP marker - Google Patents
Method of distinguishing between the red pine and the scot pine by using PCR-RFLP marker Download PDFInfo
- Publication number
- KR101718707B1 KR101718707B1 KR1020150084751A KR20150084751A KR101718707B1 KR 101718707 B1 KR101718707 B1 KR 101718707B1 KR 1020150084751 A KR1020150084751 A KR 1020150084751A KR 20150084751 A KR20150084751 A KR 20150084751A KR 101718707 B1 KR101718707 B1 KR 101718707B1
- Authority
- KR
- South Korea
- Prior art keywords
- pine
- restriction enzyme
- primer
- dna
- seq
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a primer set capable of discriminating pines and pine trees and a method for identifying pine trees and pine trees using a PCR-RFLP technique using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using the primer set. In particular, one primer set and two restriction enzymes can be used to observe the restriction enzyme cleavage products specific to pine and pine trees, thereby eliminating identification errors and improving clarity in the experiment .
Description
The present invention relates to a method for identifying pine trees and pine trees using molecular markers, and more particularly, to a method of identifying pine trees and pine trees using a primer set and a primer set, -RFLP) technique to identify pines and pine trees.
Genus Pinus is distributed over 100 species and is known as the largest genus in conifer (Mirov, NT 1967. Genus Pinus . Ronald Press Company, New York, NY; Price, RA, Liston, A , and Strauss, SH 1998. Phylogeny and systematics of Pinus, In Ecology and Biogeography of Pinus . Richardson DM (Ed.), Cambridge University Press. United Kingdom.
In Korea, pine trees, pine trees, pine trees, island pine trees, snow pine trees, etc. are distributed naturally, and lignite pine trees, tegalsa trees and pine trees have been introduced and planted (Lee, National Forestry Academy).
Pine tree is a representative species of Korea widely distributed in the temperate region from Halla mountain in Jeju island to North Hamgyong province in Korea (Cho Hyun-jae and Chang-bong Cho. 2011. Vegetation type and biodiversity pattern in Korean pine forest. .
The pine tree is the most widely distributed among pine trees, and is a representative plant of Eurasia. The pine tree is distributed from Europe, Scotland and Spain to Siberia and North Asia (Mirov, NT 1967. The Genus Pinus, Ronald Press Company, New York, NY).
The pine tree is classified taxonomically as Diplozylon and Haplozylon (Price, RA, Liston, A., and Strauss, SH 1998. Phylogeny and systematics of Pinus . In Ecology and Biogeography of Pinus . Richardson DM (Ed.), Cambridge University Press, United Kingdom, pp. 49-68.), Pine trees and pine trees belong to the pine tree sub-tree.
These species are characterized by the appearance of two orifices and the bark are red or reddish brown and are very difficult to distinguish because they are very similar in appearance. Molecular phylogenetic and taxonomic studies have shown that the pine and pine trees have very similar taxonomic relationships (Gernandt, DS, LoG, G., GarciS.O., and Liston, A. 2005. Phylogeny and classification of Pinus . Taxon, 54, 29-42.).
In general, visual identification of these species is very difficult for the general public except for highly trained specialists. In particular, it is difficult to identify by visual inspection in the case of processed wood, and it is difficult to identify it by microscopic method (Park, Byeongsoo, 2006. Wood Species Identification Method, National Seminar Archive for Open Seminars, 78-89) There is a possibility that the distribution becomes problematic.
Although there is a slight difference in the shape of the leaves and cones (Fu, LG, Li, N., and Mill, RR 1999. Pinaceae. Flora of China, 4, 11-52) Because the morphological variation varies according to the development stage, it is unclear how to use it as an identification method, and there is a problem that the aspect of subjective identification error can not be excluded.
SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to search for species-specific DNA mutations that can discriminate pines and pine trees and to provide polymerase chain reaction and restriction fragment polymorphism (PCR-RFLP). ≪ / RTI >
It is another object of the present invention to provide a method and an apparatus for discriminating between an object type and a specific consistency based on a DNA variation having no influence of an external form and an environmental variation using the primer set in order to solve the problem of uncertainty of external form comparison and subjective identification error The present invention provides a method for identifying a pine tree and a pine tree.
It is still another object of the present invention to provide a kit for identification of pine and pearl pine trees, which includes the primer set and has a clear consistency with the objectivity of identification based on a DNA variation without influence of external form and environmental variation.
In order to solve the above problems, the present invention provides a primer set for identification of pine and pine tree comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2.
In addition, the present invention provides a method for identifying pine trees and pine trees using the primer set.
In addition, the present invention provides a pine tree and a pine tree identification kit comprising the primer set.
According to the present invention, since the pine tree and the pine tree can be stably and reliably distinguished by using the pine tree and the pine tree identification primer set, it can be utilized as a primer set for scientific assurance of identification of pine tree and pine tree. In particular, one primer set and two restriction enzymes can be used to observe restriction enzyme cleavage products specific to pine and pine.
According to the present invention, the identification error (uncertainty of the non-cleavage product) that may occur in the actual identification process can be efficiently excluded, and the clarity of the identification result can be improved.
In addition, when the above method of the present invention is used, the external shape comparison itself is impossible, and it can also be utilized in the case of identifying a processed wood which is difficult to identify even by a microscopic method.
FIG. 1 shows the fragmentation patterns of PCR products (
FIG. 2 shows the results of fragmentation of the PCR products using Bsa WI restriction enzymes (
FIG. 3 is a photograph showing the fragmentation pattern of the cleavage product fractions using the Sph I restriction enzyme (
The present invention provides a primer set for identification of pine and pine tree comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2.
In the present invention, the term "primer set" means a DNA marker used for distinguishing DNA base sequences by electrophoresis.
Also, in the present invention, the term "primer " refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for 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 depends on the primer usage conditions such as the complexity of the desired DNA target, temperature and ionic strength.
In the present invention, an oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, And may include intercalating angents.
The nucleotide sequences of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 constituting the primer set according to the present invention were developed based on the nucleotide sequence of chloroplast DNA of pine and guinea pine. .
In addition, the present invention provides a method for identifying pine trees and pine trees using the primer set.
The method for identifying pines and pearls according to the present invention comprises the steps of (a) extracting template DNA from a sample, (b) amplifying the template DNA extracted in the step (a) by polymerase chain reaction using the primer set, (c) treating the amplification product produced by the above step with a restriction enzyme to cleave the amplification product, and (d) fractionating the cleavage product produced by the step.
In the method for identifying the pine and the pine of the present invention, the reaction solution for the polymerase chain reaction is prepared by mixing 25 ng of pine genomic DNA, 25 ng of pearl genomic DNA, 1 x buffer, 20 μM forward primer of SEQ ID NO: 1 and reverse primer of 20μM of SEQ ID NO: 2, may comprise a DNA polymerase of MgCl 2, and 0.125 units of dNTPs, 2.5mM of 0.2mM.
In the method for identification of the pine and the pine of the present invention, the amplification of step (b) is performed at 94 ° C for 5 minutes after the initial denaturation at 94 ° C for 30 seconds, at 60 ° C for 30 seconds, at 72 ° C for 30 seconds After repeating the
In the method for identifying the pine and the pine of the present invention, the reaction product for digesting the amplification product by treating the restriction enzyme of the step (c) is 1 x reaction buffer solution and 0.5 unit restriction enzyme Enzymes.
In the identification method of the pine and Scots pine of the invention, the restriction enzyme Bsa WI, BetI, Sph I, SphI, BbuI, PaeI And SpaHI may be used, but the present invention is not limited thereto.
In the method for identification of the pine and the pine of the present invention, the treatment of the Sph I restriction enzyme may include activating at 37 캜 for 3 hours and then deactivating at 65 캜 for 20 minutes.
In the method for identifying the pine and the pine of the present invention, the treatment of the Bsa WI restriction enzyme may include activating at 60 ° C for 3 hours and then deactivating at 80 ° C for 20 minutes.
In addition, the present invention provides a pine tree and a pine tree identification kit comprising the primer set.
The constituents contained in the kit of the present invention and the method for producing the kit of the present invention are sufficient to use the components and the manufacturing method included in the conventional kit, and a detailed description thereof will be omitted.
Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited to these examples.
≪ Example 1 > Extraction and purification of template DNA
To prepare samples for template DNA extraction and purification, 200 mg of leaf samples collected from each individual were weighed and placed in 2 ml microtube with ceramic beads. 400 μl Buffer AP1 from DNeasy Plant Mini Kit (QIAGEN) and RNase A 4ul , Followed by shaking for 10 minutes at a rate of 30 times / sec using a Tissue Lyser (QIAGEN) to disrupt the leaf tissue.
DNA was extracted and purified according to the manufacturer's instructions using DNeasy Plant Mini Kit (QIAGEN). The final concentration of extracted DNA was diluted to 5 ng / ul.
Example 2: Design of primer
The nucleotide sequence of the amplification target region was determined using the pine chloroplast sequence (GenBak Accession No. JN854210) and the pine chloroplast sequence (GenBak Accession No. JN854158), and the primer was designed using Primer3 software.
Primer sizes ranged from 18 to 22 mers, Tm ranged from 57 to 63 ° C, one GC clamp, and primer amplification products ranging from 250 bp to 350 bp.
Table 1 below shows the nucleotide sequences of the primer set Pdest-cp1 of the present invention. The combination of the primer base sequences is regarded as a combination of the reverse primer base sequences and the same primer base sequence combination.
≪ Example 3 > Polymerase chain reaction (PCR)
Using the primer set Pdest-cp1 designed in Example 2, amplification products were generated by performing PCR (PCR) on pine and pearl samples.
The composition of the PCR reaction mixture is as follows.
The PCR reaction solution contained 25 ng of each of pine and pine genomic DNA, 1 × buffer, 20 μM primer, 0.2 mM dNTPs, 2.5
After the initial denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, primer binding at 60 ° C for 30 seconds, and polymerization at 72 ° C for 30 seconds were repeated 45 times, followed by final polymerization at 72 ° C for 10 minutes Respectively.
<Example 4> Production of soybean product by restriction enzyme fragment polymorphism (RFLP) technique
The amplification product generated in Example 3 was treated with a restriction enzyme to generate a cleavage product of the amplification product.
As the restriction enzyme, Bsa WI (NEB) and Sph I (NEB) were used. For the restriction enzyme cleavage of the PCR amplification product, the composition of the reaction product was determined by the restriction enzyme The mixture containing 1 × reaction buffer and 0.5 units of restriction enzyme was added. The restriction enzymes Bsa WI and Sph I were prepared as separate mixtures and were subjected to separate reactions.
The restriction enzyme Bsa WI treatment was performed at 60 ° C for 3 hours, followed by inactivation at 80 ° C for 20 minutes.
The restriction enzyme Sph I treatment was performed at 37 ° C for 3 hours and then at 60 ° C for 20 minutes.
The above Bsa WI and Sph I restriction enzyme treatments were performed separately for polymerase chain reaction products.
≪ Example 5 > Amplification products and fractions of cleavage products
5 μl of the amplification product generated in Example 3 was removed and mixed with 3 μl of loading dye. Then, 2% agarose gel electrophoresis was performed. A 100 bp ladder (Fermentas Co., 50 μg / μl) The result of checking the size of the product fragments is shown in Fig.
In addition, 5 μl of loading dye was mixed with the cleavage product produced in Example 4, followed by 2% agarose gel electrophoresis. A 100 bp ladder (Fermentas Co., 50 μg / μl) The results of confirming the size of the fragments are shown in FIG. 2 ( Bsa WI cleavage product) and FIG. 3 ( Sph I cleavage product).
As shown in FIG. 1, when the fragmentation pattern was observed by electrophoresis on agarose gel, it was found that a DNA fragment of about 300 bp was commonly observed in pine and pearl pine samples.
Further, as shown in FIG. 2, the cleavage product of the amplified product using the primer set Pidest-cp2 by the Bsa WI restriction enzyme was electrophoresed on the agarose gel and the fractionation pattern was observed. On the other hand, a fragment of DNA of about 200 bp cleavage product was observed in the pear tree.
Further, as shown in FIG. 3, the cleavage product of the amplification product using the primer set Pidest-cp2 by Sph I restriction enzyme was electrophoretographed on agarose gel, and the fragmentation pattern was observed. In the pine tree, about 200 bp cleavage product DNA While fragmentation was observed in the pine tree.
The sizes of the DNA fragments observed in the above are visually observed values based on the DNA size markers of the electrophoresis. From the nucleotide sequences used for the primer synthesis, the sizes of the base sequences amplified by the primers and the restriction enzymes The approximate size can be estimated based on the calculated length based on the recognition site.
When observing a DNA fragment through the electrophoresis, it is possible to display a slightly different value according to an observer by visual observation. However, the target DNA region can be accurately amplified and cut, have.
Although the present invention has been described in connection with the specific embodiments of the present invention, it is to be understood that the present invention is not limited thereto. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
<110> Republic of Korea
<120> Method of distinguishing between the red pine and the scot pine
by using PCR-RFLP marker
<130> 10143
<160> 2
<170> KoPatentin 3.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> forward primer of Pdest-cp2
<400> 1
Claims (10)
(b). Amplifying the template DNA extracted in the above step by a polymerase chain reaction using a primer set for identifying a pine and a pine tree comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2;
(c). Treating the amplification product produced in step (b) with SphI and Bsa WI as a restriction enzyme to cleave the amplification product; And
(d). And fractionating the cleavage product produced in step (c).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150084751A KR101718707B1 (en) | 2015-06-16 | 2015-06-16 | Method of distinguishing between the red pine and the scot pine by using PCR-RFLP marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150084751A KR101718707B1 (en) | 2015-06-16 | 2015-06-16 | Method of distinguishing between the red pine and the scot pine by using PCR-RFLP marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20160148151A KR20160148151A (en) | 2016-12-26 |
| KR101718707B1 true KR101718707B1 (en) | 2017-03-24 |
Family
ID=57733663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150084751A KR101718707B1 (en) | 2015-06-16 | 2015-06-16 | Method of distinguishing between the red pine and the scot pine by using PCR-RFLP marker |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101718707B1 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101409012B1 (en) * | 2012-06-04 | 2014-06-18 | 충북대학교 산학협력단 | Specific primers for Pines distinction, analysis kit using its primers, and Pines distinciton method using thereof |
-
2015
- 2015-06-16 KR KR1020150084751A patent/KR101718707B1/en active IP Right Grant
Non-Patent Citations (3)
| Title |
|---|
| Plant Syst. Evol. (2000), Vol.220, pp.21-36* |
| Theor Appl Genet (1993) Vol.86, pp.159-165 |
| Theor Appl Genet (1995) Vol.91, pp.1222-1236 |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20160148151A (en) | 2016-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108660246B (en) | InDel molecular markers of Ma (Citrus paradisi) pomelos and application of InDel molecular markers in early-stage differentiation of rough-peel Ma pomelos of citrus varieties | |
| CN107287300B (en) | A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application | |
| CN106367496B (en) | kiwi species associated specific single nucleotide molecular marker, detection primer group and application thereof | |
| CN114672588B (en) | A SNP molecular marker Cpa03g017250:8, amplification primer, detection kit and application thereof | |
| KR101409012B1 (en) | Specific primers for Pines distinction, analysis kit using its primers, and Pines distinciton method using thereof | |
| CN113637787B (en) | DNA fragment related to quality of single oil tea fruit and application thereof | |
| CN113637786B (en) | DNA fragment and SNP molecular marker related to linoleic acid content in oil tea seed oil and application thereof | |
| CN105112557B (en) | The method of Rapid identification epidendrum | |
| JP5799600B2 (en) | Species identification method of Eucalyptus hybrids | |
| KR101718707B1 (en) | Method of distinguishing between the red pine and the scot pine by using PCR-RFLP marker | |
| CN108517373B (en) | InDel labeled primer pair for distinguishing five pepper cultivars and application thereof | |
| KR101828774B1 (en) | Method of distinguishing between the red pine and the scot pine using molecular marker | |
| KR101940286B1 (en) | Primer Set for Identifying Maternal origin of Ilex x wandoensis and Uses Thereof | |
| CN113584203B (en) | DNA fragment related to single fruit quality of camellia oleifera, SNP molecular marker closely linked with DNA fragment and application of DNA fragment | |
| KR101634346B1 (en) | Method of distinguishing between the pitch pine and the loblolly pine using molecular marker | |
| Güçlü et al. | Genetic characterization of Turkish commercial opium poppy (Papaver somniferum L.) cultivars using ISSR and SSR markers | |
| KR101718703B1 (en) | Method of distinguishing the Korean pine from the dwarf stone pine using molecular marker | |
| CN113430298A (en) | DNA fragment related to content of linolenic acid in camellia seed oil, SNP molecular marker closely linked with DNA fragment and application of SNP molecular marker | |
| CN107345252B (en) | Primer combination and kit for identifying 96 of flue-cured tobacco Qin tobacco, application and identification method | |
| KR101788785B1 (en) | A method, composition, and kit for the identification of Goodyera x maximo-velutina | |
| KR102511995B1 (en) | Primer set for discriminating Adenophora triphylla var. japonica Hara, Adenophora remotiflorus Miquel and Codonopsis lanceolata (Siebold Zucc.) Trautv DNA Barcode and uses thereof | |
| CN111518938B (en) | CAPs molecular marker for identifying single seed of Amaranthus praecox, Amaranthus praecox and Amaranthus sanderi based on SNP locus and application thereof | |
| KR101793911B1 (en) | Primers for distinguishing between Hinoki Cypress and Sawara Cypress and method using the same | |
| KR100452087B1 (en) | Method for identifying wild ginseng by DNA Finger-printing method | |
| Ghaemmaghami et al. | Characterization of genetic diversity in three Chara L. species (Characeae) in Iran using RAPD-PCR technique |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |