KR20170068093A - Complete sequencing of chloroplast genome and nrDNA of Ledebouriella seseloides, Peucedanum japonicum and Glehnia littoralis-derived barcoding marker, DNA primer set for discrimination of origin and species and uses thereof - Google Patents
Complete sequencing of chloroplast genome and nrDNA of Ledebouriella seseloides, Peucedanum japonicum and Glehnia littoralis-derived barcoding marker, DNA primer set for discrimination of origin and species and uses thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention relates to a complete detoxification-based genomic identification marker and primer set of chloroplast genomic and nuclear ribosomal DNA sequences of windblown, windblown and releasable winds, and its use. The molecular markers and primer sets of the present invention are useful as low-cost and high- It is possible to distinguish the windbreaking and freezing winds, and also to discriminate the raw materials in the windbreak processing products, to protect the purity of the windbreaks, to protect the breeds, and to solve the seed conflicts.
Description
The present invention relates to a complete detoxification-based species identification marker and primer set of chloroplast genomes and nuclear ribosomal DNA sequences of wind winds, edible wind winds and releasing winds, and uses thereof, and more particularly to a primer set for primers, Set, a kit for distinguishing between windshields, a windshield and a release windshield, including the primer set, and a method for distinguishing between windshields, windshields and freezes using the primer set.
The windbreak ( Ledebouriella seseloides , synonym: Saposhnikovia divaricata ) is a perennial plant of the mountain type (Umbelliferae, synonym: Apiaceae), a medicinal herb known as a medicinal herb. The windbreaks used as medicines in China and Japan are all of the same origin, and they are now commonly referred to as windbreaks in Korea.
This is one of the most frequently used medicines used in the treatment of stroke treatment. Originally, 'Roundwalk' is a plant that does not grow in Korea. In Korea, 'ginseng oil' is used as 'windshield' and 'windshield'. ' Windfight ' is defined as 'Roots of Saposhnikovia divaricata Schischkin (Umbelliferae)' in the 9th revision of the ' Korean Pharmacopoeia', 'Liberation Wind' in 'Japanese Pharmacopoeia' It is a medicinal herb that has been used as a medicinal herb and has been proven to be effective in the treatment and prevention of dryness of cigarette buttocks (勞 泻 痰 痰) (津 傷 口渴) is said to be used for, and is said to have a similar action to the four. The dry roots of the windbreak with these effects are similar to the outwardly similar species windbreaks and the windblown winds and tinnitus are the roots of Glehnia littoralis (mountain type and Umbelliferae) And rootstock and the roots of Peucedanum japonicum (mountainous and Umbelliferae).
Herbal medicines have been used for many years in Korea, China, Japan, and other countries. Herbal medicines have various criteria for their origins due to various factors such as geographical, environmental and historical differences. In addition, since the market of herbal medicines from China and other herbalist countries has been opened since the 1990s, distribution of similar herbal medicines in Korea has reached a level of concern. In the case of the one-way wind, there is no domestic cultivation, and all of the imports from China are concerned about the inclusion of similar medicines whose origins are unclear. In addition, it is difficult to distinguish the herbicide medicinal herbs from other herbaceous herbs because they are difficult to distinguish even if there is a case where the medicinal herbs of different origin are mixed with each other.
There are morphological characteristics, qualitative and quantitative analysis of components, and comparison of differences at DNA level. Morphological characteristics can be different according to the growing environment of the plant. Therefore, it is difficult to distinguish the plant species. Especially, most of the medicinal materials are dried and processed and distributed. In addition, a taste sensor, a nuclear magnetic resonance spectrometer, etc. have been used for the classification of the pharmacological composition by the component analysis. However, this method has a limitation that the ingredient of the pharmacopeptide can be affected according to the cultivation environment of the producing place. In order to solve these problems, attempts have been made to identify molecular biological plant species using polymorphisms of DNA fragments such as RAPD (Random Amplified Polymorphic DNA), SCAR (Sequence Characterized Amplified Regions) and AFLP (Amplified Fragment Length Polymorphism). Recently, the classification method using the DNA barcode proposed by CBOL Plant Working Group in the identification of herbal medicines has been recognized as useful. In particular, the nrDNA-ITS (internal transcribed spacer) region of the nuclear ribosomal DNA (nrDNA) existing in the genome not only evolves faster than the coding region of other genes, but also has advantages such as generality, simplicity and reproducibility, It is known to be a widely used base sequence for classification and interspecies genetic mutation searches.
Accordingly, the present inventors have developed eight molecular markers capable of efficiently identifying windshields, windblown winds, and free winds by analyzing the chloroplast genome and nrDNA sequence of wind winds, windblown winds and releasing winds and comparing base sequences.
Korean Patent Laid-Open Publication No. 2015-0024581 discloses a polymorphic molecular marker for discriminating between a free wind, a free wind and a dry wind wind, and a method for distinguishing a free wind, a free wind and a wind wind using the same. Korean Patent No. 0673069, Although there has been disclosed an interspecies genetic discrimination kit for windbreaks, there has been no description of a complete decode-based genomic discrimination marker and primer set of chloroplast genomes and nuclear ribosomal DAN sequences of windshields, windshields and releasing winds of the present invention and uses thereof.
SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to provide a method for detecting chloroplast genome and nucleotide ribosomal DNA (nrDNA) sequences for Ledebouriella seseloides and Peucedanum japonicum and Glehnia littoralis , And the sequence information of the completed nucleotide sequence was compared to find seven InDel markers from the chloroplast genome sequence and one SNP marker from the nrDNA sequence. Furthermore, a primer set capable of amplifying the above-mentioned molecular marker was prepared, and DNA samples were analyzed. As a result, it was confirmed that the molecular markers of the present invention can clearly distinguish wind wind, food wind wind and free wind wind.
The oligonucleotide primer set of SEQ ID NOs: 3 and 4, the oligonucleotide primer set of SEQ ID NOs: 5 and 6, the oligo oligonucleotide primer set of SEQ ID NOs: 7 and 8, Oligonucleotide primer sets of SEQ ID Nos. 9 and 10, oligonucleotide primer sets of SEQ ID Nos. 11 and 12, oligonucleotide primer sets of SEQ ID Nos. 13 and 14, and oligonucleotide primer sets of SEQ ID Nos. 15 and 17 And at least one primer set selected from the group consisting of a primer set and a primer set.
In addition, the present invention provides a kit for distinguishing between windshields, edible winds, and releasing winds, including the primer set and the reagent for performing the amplification reaction.
Further, the present invention provides a method for distinguishing between windshields, windshields and liberated winds using the primer set.
The molecular markers of the present invention are found in a region where hundreds to thousands of copies are present in the cell, and the mutations are very rarely present in the cell. Therefore, it is possible to perform very stable verification, And the free wind can be identified. In addition, since it is possible to discriminate raw materials in windproof processing products, it can improve the value of windproof processing products, can also be used for maintaining purity of windshields, protecting varieties, and solving seed conflicts.
Figure 1 is a complete chloroplast genome map of windblown, windblown and free winds. Ledebouriella seseloides , windbreak; Peucedanum japonicum , windshield; Glehnia littoralis , freezes.
FIG. 2 shows the results of exploration of interspecies mutation regions by comparing the chloroplast genome sequences of windblown, windblown and free winds. The points indicated by the arrows are the positions of the InDel molecular markers developed in the present invention. Pj, windshield; Ls, windshield; Gl, a freeze.
FIG. 3 is a result of excavation of interspecies mutation regions through comparative analysis of nrDNA sequences of windblown winds, windblown winds and releasing winds, where the arrow indicates the position of the SNP molecular marker developed in the present invention. Pj, windshield; Ls, windshield; Gl, a freeze.
Fig. 4 shows the results of identification of wind winds, eddy wind winds and free wind winds using the molecular markers CNV01, CNV02 and CNV03 of the present invention. A, B, and C are schematic diagrams showing that the difference between the wind, wind, and drift wind genome in the other three A, B, and C target sequences appears as a difference in the number of repetition of repeating sequences, and D is a primer Is an agarose gel loading image of the PCR amplification product using sets (red arrows A, B, and C). Ls, windshield; Pj, windshield; Gl, a freeze.
5 shows the results of identification of windshields, eddy winds and free winds using the molecular markers InDel01, InDel02, InDel03 and InDel04 of the present invention. Ls, windshield; Pj, windshield; Gl, a freeze.
Fig. 6 is a schematic diagram (A) showing a primer position for amplification of a molecular marker nrDNA01 derived from a release wind 45S nrDNA sequence, and (b) an identification result (b) of a wind wind, a wind wind and a free wind using a nrDNA01 molecular marker. Gl_nrDNA specific F, a forward primer specific for ITS1 of the dehydrated 45S nrDNA; Gl_nrDNA control F, a control forward primer based on 5.8S rRNA of three acid types and species; Gl_nrDNA control R, control reverse primer based on 5.8S rRNA of three acid types and species; Ls, windshield; Pj, windshield; Gl, a freeze.
In order to accomplish the object of the present invention, the present invention provides a primer set for distinguishing between wind winds, wind winds, and free winds.
The primer set of the present invention specifically comprises an oligonucleotide primer set of SEQ ID NOs: 1 and 2, an oligonucleotide primer set of SEQ ID NOs: 3 and 4, an oligonucleotide primer set of SEQ ID NOs: 5 and 6, an oligonucleotide primer of SEQ ID NOs: A set of oligonucleotide primers of SEQ ID NOs: 9 and 10, an oligonucleotide primer set of SEQ ID NOs: 11 and 12, an oligonucleotide primer set of SEQ ID NOs: 13 and 14, and an oligonucleotide primer set of SEQ ID NOs: 15 and 17 Lt; / RTI > primer set.
According to one embodiment of the present invention, at least one primer set selected from the group consisting of the oligonucleotide primer set of SEQ ID NOs: 1 and 2 and the oligonucleotide primer set of SEQ ID NOs: 5 and 6 is selected from the group consisting of wind, Wherein at least one primer set selected from the group consisting of an oligonucleotide primer set of SEQ ID NOs: 3 and 4, an oligonucleotide primer set of SEQ ID NOs: 7 and 8, and an oligonucleotide primer set of SEQ ID NOs: 13 and 14, And at least one primer set selected from the group consisting of an oligonucleotide primer set of SEQ ID NOs: 9 and 10 and an oligonucleotide primer set of SEQ ID NOs: 15 and 17, The oligonucleotide primer set of SEQ ID NOS: 11 and 12 is a set of primers for distinguishing the windshield from the edible winds and the releasing winds.
The primer set of the present invention is a set of oligonucleotide primers of SEQ ID NOs: 1 and 2, which can amplify seven InDel markers developed based on chloroplast genome complete sequence decoding of wind winds, edible winds and releasing winds, SEQ ID NOS: 3 and 4 An oligonucleotide primer set of SEQ ID NOS: 5 and 6, an oligonucleotide primer set of SEQ ID NOS: 7 and 8, an oligonucleotide primer set of SEQ ID NOS: 9 and 10, an oligonucleotide primer set of SEQ ID NOs: 11 and 12, A set of oligonucleotide primers of SEQ ID NOS: 13 and 14, and an oligonucleotide primer set of SEQ ID NOS: 15 and 17 capable of amplifying one SNP marker developed based on the complete decoding of the nrDNA nucleotide sequence of wind, wind, Nos. 1, 3, 5, 7, 9, 11, 13 and 15 are forward primers, SEQ ID NO: 2 , 4, 6, 8, 10, 12, 14 and 17 are reverse primers.
According to the sequence length of each primer, the primers include SEQ ID NOS: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; At least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 within SEQ ID NOs: 15 and 17, An oligonucleotide consisting of fragments of at least 26 consecutive nucleotides. For example, the primer (20 oligonucleotides) of SEQ ID NO: 1 comprises an oligonucleotide consisting of fragments of at least 15, at least 16, at least 17, at least 18, at least 19 contiguous nucleotides in the sequence of SEQ ID NO: 1 . In addition, the primers include SEQ ID NOs: 1 and 2; SEQ ID NOS: 3 and 4; SEQ ID NOS: 5 and 6; SEQ ID NOS: 7 and 8; SEQ ID NOS: 9 and 10; SEQ ID NOS: 11 and 12; SEQ ID NOS: 13 and 14; Addition, deletion or substitution of the nucleotide sequences of SEQ ID NOS: 15 and 17.
In the present invention, a "primer" refers to a single strand 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 will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.
As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.
In order to achieve still another object of the present invention,
There is provided a kit for distinguishing windshields, windshields, and freezes, comprising a primer set according to the present invention and a reagent for performing an amplification reaction.
In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual 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 manual includes instructions on the surface of the package including a brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.
In order to achieve still another object of the present invention,
Isolating the genomic DNA from a suspect sample of wind, wind, and wind;
Amplifying the polymorphic nucleotide by performing an amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set of the present invention; And
And detecting the amplification product. The present invention also provides a method for distinguishing windshields, windshields, and freezes.
The method of the present invention includes isolating genomic DNA from suspect samples of windblown, edible and wind blows. A method known in the art may be used for separating the genomic DNA from the sample. For example, a CTAB method may be used, or a Wizard prep kit (Promega, USA) may be used. The target sequence can be amplified by performing amplification reaction using the separated genomic DNA as a template and using a primer set according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a 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 can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as 32 P or 35 S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The primer set used to amplify the target sequence is as described above.
The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As a method of detecting the amplification product, gel electrophoresis can be performed, and gel electrophoresis can be performed using acrylamide gel electrophoresis or agarose gel electrophoresis according to the size of the amplification product. In addition, capillary electrophoresis can be performed. Capillary electrophoresis can be performed, for example, using the ABI Genetic Analyzer. In the fluorescence measurement method, when a fluorescent dye is labeled at the 5'-end of the primer and PCR is performed, the target sequence is labeled with a fluorescent label capable of detecting the fluorescence. The fluorescence thus labeled can be measured using a fluorescence analyzer. In addition, in the case of performing the PCR, the radioactive isotope such as 32 P or 35 S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
Materials and methods
1. Experimental material
Windbreaks and windbreaks were collected at the National Institute of Horticultural Science, Rural Development Administration of Chungbuk Province and collected at the Yeongdeok County Office, Yeongdeok County, Gyeongbuk Province. The leaves of the collected plants were stored at -70 ° C until use.
2. Sequence analysis method
Genomic DNA of windblown, windblown and offspring plants was extracted using CTAB (cetyltrimethylammonium bromide) method. Approximately 2 의 of the genomic DNA extracted was prepared with a genome library of 300 bp insert size according to a paired-end standard protocol provided by the manufacturer at the National Institute of Agronomic Science (NICEM), Seoul National University, And tagged separately. Sequencing was performed 101 times on both ends of a lane using a pooled library on an Illumina HiSeq-2000 instrument.
3. Assembly of the chloroplast genome and nucleus 45S nrDNA sequence
Large quantities of nucleotide sequences generated by next-generation sequencing (NGS) can be used to generate small amounts of DNA covering the dnaLCW ( de) sequence using a small amount of data, covering a low coverage of 0.5-1X of the plant genome size (300-400X chloroplast size) novo assembly of low-coverage whole genome shotgun sequencing method (Application No. 10-2013-0167982). After assembly, gene regions of the chloroplast genome were determined through DOGMA (http://dogma.ccbb.utexas.edu/) program and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search .
4. Polymerase chain reaction (PCR)
The PCR reaction was performed in a total volume of 25 μl, and 20 ng template DNA, 10 mM dNTPs, 10 pmol of each primer, and 1 U of Taq polymerase (Inclone, Korea) were used. PCR was carried out for 5 min at 94 ° C for 5 min and DNA amplification was performed 35 times for 45 sec at 94 ° C, 45 sec at 54 ° C and 45 sec at 72 ° C for DNA amplification. The final extension process was carried out at 72 ° C for 7 minutes. PCR amplification products were confirmed by 1.3% agarose gel electrophoresis.
Example 1. Chloroplast genome and nrDNA sequencing of wind, wind,
The chloroplast genome and 45S nrDNA sequences of wind, windblown and dehydrated winds were assembled and analyzed using NGS method. Initial conditions were created using 1.06 ~ 2.08 Gbp of primordial data, and the chloroplast genome was assembled from 4 to 6 contigs. The size of the completely decoded chloroplast genome was 147,880 bp for wind, 164,653 bp for dry wind, and 147,467 bp for free wind (Fig. 1). 45S nrDNA in the nucleus could be completed by using 1 contig, and the size of 45S nrDNA in the nucleus was 5,815 bp for wind and wind, 5,812 bp for free wind.
Example 2. Identification of interspecies mutation regions by comparison of base sequences
The interspecies mutation regions were compared and sequenced by mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) program and visualized by comparing the nucleotide sequence of the chloroplast genome. Exon region was blue, gene region was red, tRNA and rRNA were blue, and interspecies were white. Interspecific regions of the chloroplast genome sequence contain 1,672 single nucleotide polymorphisms (SNPs), four insert deletions (InDel), and 41 copy repeat (TR) tandem repeats (CNV, copy number variation ), And barcoding markers were developed for three CNV regions and four InDel regions (Fig. 2). In the interspecific mutation region of the 45S nrDNA sequence in the nucleus, 66 SNPs were identified, and identification markers were developed based on the SNPs of ITS1 (internal transcribed spacer 1) region (FIG. 3).
A primer set for identifying the identification markers developed through comparative analysis of nucleotide sequences of the chloroplast genome and nucleus 45S nrDNA was prepared using primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) Design. Primer sets were all developed in the intergenic region.
Ls / Pj / Gl (bp)
(Ls, windshield; Pj, windshield; Gl, free wind)
Example 3 Classification of windshield, windshield, and blowhole using a molecular marker
The above-mentioned primer set was used to verify the molecular markers of the present invention. As a result of PCR analysis using a DNA sample of wind, wind, and freezing wind, the CNV01 marker showed that the number of TRs of 18 bp in the region between trn I-CAU and trn L-CAA gene was 6 windshields, 4, and 3, respectively, so that all three species could be distinguished (FIGS. 4A and 4D). The CNV02 markers were able to distinguish between the trn N- GUU and ycf 1 genes by a difference of 22 bp TR, one for each of the windshield and the free wind, and four for the windshield (Figs. 4B and 4D). CNV03 markers were found to be 18 bp TR in the region between psa C and ndh E, two windshields, one windshield and one free wind, and 18 bp InDel were present in windshields and windshields. (Figs. 4C and 4D). The InDel01 marker was distinguishable from the 23 bp InDel gene located in the trn S-GCU to trn R-UCU gene region. The InDel02 marker was identified as a 37 bp InDel in the region between ndh F and rpl 32 genes, was possible to distinguish, InDel03 marker was the wind guard is possible separated by a 159bp InDel of existing in the area between the rps 12 ~ trn V-GAC gene, InDel04 marker is present in the region between trn E-UUC ~ trn T- GGU gene A 454 bp InDel was able to distinguish the diaphragm (Fig. 5). Finally, the nrDNA01 marker was constructed so as to amplify all three types of windshields, windshields, and blown breezes, and a primer was made in the ITS1 region so that only the freezing wind was amplified (Fig. 6).
As a result, it was confirmed that CNV01, CNV03 markers of the present invention are markers capable of simultaneously distinguishing three types of wind, wind, and blowing winds. CNV02, InDel01, and InDel04 markers were classified as food type wind markers. InDel02 and nrDNA01 markers And the InDel03 marker could be used as a windbreak markers.
<110> SNU R & DB FOUNDATION National Institute of Biological Resources <120> Complete sequencing of chloroplast genome and nrDNA of Ledebouriella seseloides, Peucedanum japonicum and Glehnia littoralis-derived barcoding marker, DNA primer set for discrimination of origin and species and uses thereof <130> PN15406 <160> 17 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggtcaaatac ctagcgacaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ttatgcaagg agacattgct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 catccatatc ccaattccat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gctcggagaa ggaagagata 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cattgagtgc accctataca 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tcgtaacaga aaatcaactc g 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aacgaatcct acggtttctc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgtcgaacag ggataatttg 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tctcgctttt tagtcagttt g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcctaatgaa aagcctaatg a 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 caggaggata gcaagttaca a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 caacgccact attcttgaac 20 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ctatatgtat atacaataac gaatca 26 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gttcaagaat agtggcgttg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gttaacaatt agggcgagca 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gcatcgatga agaacgtagc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gcgttcaaag actcgatggt 20
Claims (7)
Amplifying the polymorphic nucleotide by performing an amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set of any one of claims 1 to 5; And
And detecting said amplification product. ≪ Desc / Clms Page number 19 >
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| KR102380784B1 (en) * | 2020-10-16 | 2022-03-29 | 서울대학교산학협력단 | Molecular marker for discriminating genetic resources of Peucedanum japonicum and uses thereof |
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| KR100673069B1 (en) | 2005-08-31 | 2007-01-22 | 대구한의대학교산학협력단 | A kit for discriminating genetical identification between Saposhnikovia divaricata Turcz. Schiskin and Peucedanum japonicum Thunberg |
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| KR102393484B1 (en) * | 2020-10-30 | 2022-05-04 | 서울대학교산학협력단 | Molecular marker based on nuclear genome sequence for discriminating genotype of Peucedanum japonicum resources and uses thereof |
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