KR101855984B1 - composition comprising SNP markers for a differentiation of Cudrania tricuspidata Bureau lines, and method for a differentiation of Cudrania tricuspidata Bureau lines and hybrid using the same - Google Patents

Abstract

The present invention relates to a marker composition for the identification of ginseng lineage using single base polymorphism, and a method for determining the lineage and crossing using the same. According to the present invention, it is possible to promptly and accurately discriminate Korean and Chinese Cudrania tricuspidata Bureau strains, and furthermore, to determine quickly whether or not hybridization, hybridization, and mixed ratios of lines mixed in herbal medicine materials can be quickly and accurately determined.

Description

The present invention relates to a marker composition for the identification of cucurbitaceous plants using single nucleotide polymorphisms, and to a method for distinguishing cucurbit lines and hybrids using the same, and a method for discriminating cucurbit lines and hybrids using the same. }

The present invention relates to a marker composition for the identification of cirrhosis using a single base polymorphism, and a method for distinguishing cirrhythmiae and hybrids using the same. More particularly, the present invention relates to PCR and HRM curves for amplification of chloroplast or nuclear gene fragments based on single nucleotide polymorphism (SNP) markers capable of discriminating the Korean and Chinese strains of Cudrania tricuspidata Bureau The present invention relates to a marker composition for analyzing high-resolution melting curves, and to a method for distinguishing cymbidium strains and hybrids using the same.

The Cudrania tricuspidata Bureau, native to the southern part of Korea, is a deciduous broad-leaved arboreous tree belonging to the Moraceae family. It has a spindle of 1.0 to 3.5 cm in the stem, It is muscular, and it is characteristic that red fruit occurs in October. It is used for edible (leaf, fruit), medicinal (stem), civil engineering work, silkworm feed (leaf). In addition, it is known that if you feed the silkworm's leaves to the silkworms, they pull the yarn and make strings such as geomungo and kayagum, which are far better than normal threads, clear and clear, and spread far away (Choi, Han-ki) No. 10, No. 2 of the Korean Agricultural Cooperative Federation, 347). In addition, Cucumisanum is used for landscaping, fiber (leaf), and fungus fungi grafting.

In addition, it is a good income tree in the field of agriculture and forestry. Although stem and roots of Cucurbitaceae are widely used for cancer treatment in the private sector, stem and roots are restricted to be used as food materials, so only fruits and leaves can be used for edible purposes. The leaves of Cucumber tree are extracted with hot water or made into powder after drying, and the fruit of Cucumber tree is used for cooking jam or soaking in alcohol for baking.

The content of protein, dietary fiber, vitamin B1, vitamin B2, calcium, potassium, GABA (γ-amino byturic acid), and rutin is higher than that of green leaf and mulberry leaf. Therefore, a large number of green tea extracts have been developed as substitutes for green tea. However, there are many thorns in the tree, so it takes a lot of labor and time to collect leaves and fruits from the farms. Therefore, it is in the process of dissemination of redeveloped species without thorns. However, the validity of the efficacy between redeveloped species and native species has not been verified, It is a reality.

In the study of Cucurbitaceae trees, substances that showed cytotoxicities to hepatocarcinoma HepG3 were identified by searching for anticancer active substances. Zou et al. (2005, Chem Biodivers. 2 (1): 131-138) Isoprenylated xanthone or isoprenylated flavanones, which inhibit cancer cell lines, were isolated. In addition, Wang et al. (2005, Planta Med. 71 (3): 273-274) isolated the isoprenylated xanthone cudrafrutixanthone A compound from roots and found Candida albicans Candida albicans) was also confirmed to have antifungal activity. An et al. (2006, Biol Pharm Bull 29: 838-840) reported efficacy in liver cancer and Tian et al. (2005, Arch Pharm Res. 28 (1): 44-48) also reported that Tacrine ) In the liver. In addition, cirrhotic trees have been shown to lower blood pressure (Kang et al., 2002, Life Sci 70: 2599-2609), antidepressant effects, Parkinson's disease prevention (Han et al., 2005, Arch Pharm Res. 28 (12): 1324-1327) And Parkinson's disease (Park et al., 2006, Bioorg Med Chem Lett 16: 5580-5583).

It is known that there are more than 1,000 species in the world and more than 10 species in the 5 genera (Moly et al., 1985, Shingo plantbtaxonomy, Hyangmunsa; 1986, Dendrology, Hyangmunsa). It is reported that three trees and two trees in Japan are indigenous to India, China and Australia (Keihin Uehara, 1959, Aristotle, Tokyo).

As the recent international trends such as the entry into the Nagoya Protocol have strengthened the discovery and preservation of genetic resources in their own countries, there has been a need to study the establishment of standards for the identification of Chinese and Korean populations. In particular, the development of cytological, biochemical, or molecular biological discrimination technologies based on the morphological research results (Kwon et al., 2014, Korean J Plant Research 27 (4): 337-343) . Recently, Sung et al. (2001, Korean J. Seric. Sci. 43 (1): 1-8) published a comparative analysis of the sequences of ITS regions. And to identify various SNPs that can be distinguished. However, there was no significant difference in the nucleotide sequence between the ginseng trees or the ginseng trees collected by region. In addition, Oh et al. (2012, J. Agri & Life Sci, 43 (2): 32-36) classified 8 families of cucurbit trees distributed in Korea into four groups, As a result of comparing the nucleotide sequences, 99.4% was more significant than 99.4%, and the single nucleotide polymorphism was confirmed in the cancer and water system in each region. However, since no additional study results have been reported, there has been a desperate need for the development of discrimination markers using advanced molecular biology techniques.

The inventors of the present invention have found that, in order to distinguish between Chinese and domestic strains which are collected and stored in Korea, the inventors of the present invention have found that fragments containing TrnL and TrnF genes, intergenic DNA, and matK gene fragments And then analyzing the base sequence and comparing the base sequence of the gene from the previously registered genbank (GenBank) to prepare a primer including a region showing SNP, and a marker And a marker composition for PCR and HRM curve analysis for amplifying a chloroplast or nucleotide gene fragment based on the marker, and a method for distinguishing a bryophyte lineage and a hybrid from the bryophyte lineage and hybrid The method was completed.

Accordingly, it is an object of the present invention to provide a marker composition for discriminating between a single nucleotide polymorphism (SNP) marker.

It is still another object of the present invention to provide a composition for discriminating corymbunline comprising a pair of primers capable of amplifying SNP markers according to the present invention.

It is still another object of the present invention to provide a kit for discriminating a cryopresin system comprising the composition for determining cryoprecipitation according to the present invention.

It is still another object of the present invention to provide a composition for discriminating a hybrid from a primer pair capable of amplifying SNP markers according to the present invention.

It is still another object of the present invention to provide a method for distinguishing a system that can be quickly and accurately determined.

It is still another object of the present invention to provide a method for distinguishing a hybrid from a wild type.

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

According to an aspect of the present invention, there is provided a marking composition for identifying a snake root system comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 2 as an active ingredient and a single nucleotide polymorphism (SNP) marker.

According to another aspect of the present invention, there is provided a marking composition for identifying a snake root system comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 5 as an active ingredient and a single nucleotide polymorphism (SNP) marker.

According to still another aspect of the present invention, there is provided a corymbin gene identification composition comprising a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and at least one primer pair consisting of the nucleotide sequence of SEQ ID NOs: 12 and 13 do.

According to still another aspect of the present invention, there is provided a kit for discriminating gums from a composition comprising the composition.

According to still another aspect of the present invention, there is provided a marking composition for the identification of cirrhosis, comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 15 as an active ingredient and a single nucleotide polymorphism (SNP) marker.

According to still another aspect of the present invention, there is provided a corymbin gene identification composition comprising at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21 do.

According to still another aspect of the present invention, there is provided a primer pair comprising a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 12 and 13; And at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21, is provided.

According to another aspect of the present invention, there is provided a primer pair comprising a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 22 and 23, a pair of primers consisting of the nucleotide sequences of SEQ ID Nos. 24 and 25 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 26 and 27 And a primer pair is provided.

According to still another aspect of the present invention, there is provided a method for detecting genomic DNA comprising: extracting genomic DNA from a sample; Performing PCR of the DNA using the primer pair according to the present invention; And identifying the PCR product.

According to still another aspect of the present invention, there is provided a method for detecting genomic DNA comprising: (a) extracting genomic DNA from a sample; (b) performing the PCR of the DNA using at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 12 and 13 step; (c) performing a PCR of the DNA using at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21 step; And (d) identifying and comparing the PCR product of step (b) and the PCR product of step (c).

According to one embodiment of the present invention, it is possible to accurately and quickly determine the origins of the Korean native and Chinese strains using SNP markers for determining the genome.

In addition, according to one embodiment of the present invention, it is possible to further improve the promptness and accuracy of the discriminating system by providing a primer pair for ARMS-PCR and HRM curve pattern analysis.

Further, according to an embodiment of the present invention, a primer pair for PCR is provided using SNP markers existing in ITS I and II present in the nucleus, and trnL -trnF and matK gene fragments present in the primer pair and chloroplast , It is possible to discriminate whether or not the hybrid produced by mutual crossing of the two strains and the copy and reproduction of the hybridization combination of the hybrid.

Therefore, according to the present invention, it is possible to easily confirm the mixing ratio of the cucurbitan system which is mixed or mixed in various forms in the herbal medicine materials.

FIG. 1 is a diagram showing the results of comparing nucleotide sequences of the trnL-trnF gene of Korean and Chinese phloem.
FIG. 2 is a diagram showing the results of comparing nucleotide sequences of the matK gene in Korean and Chinese lines.
Fig. 3 is a schematic diagram of a trnL-trnF gene fragment including a primer site for ARMS-PCR.
FIG. 4 shows the result of ARMS-PCR using a primer prepared using a single base polymorphism of the trnL-trnF gene fragment. Sample Nos. 1, 2, 3, 4, 5, 6, 7, Sample Nos. 9, 10, 11, 12, 13, 14, 15, and 16 are Korean strains.
FIG. 5 is a diagram showing the result of discriminating the origins of giltsy lines collected in Korea using the ARMS-PCR (trnL-trnF marker) technique, and sample numbers are the collected regions described in Table 1, respectively.
FIG. 6 shows the result of comparison of nucleotide sequences of the ITS fragments in the nucleus of Korean and Chinese lines. Primers for markers for gene identification were indicated by arrows, and the sites showing two single nucleotide polymorphisms were designated as 3'- And to be placed at the distal end.
FIG. 7 is a diagram showing the result of discriminating the origins of the genetic resources collected in Korea as a primer prepared by using a single base polymorphism of an ITS gene fragment embedded in the nucleus. Respectively.
FIG. 8 is a diagram showing PCR results using a chloroplast discrimination marker ( trnL-trnF ) and an ITS discrimination marker embedded in the nucleus for a strain estimated to be a hybrid between Korean and Chinese strains (see Collection No. 2016-10, Table 1) , Lane 1, 2 and 3 are the leaves, and Lane 4, 5 and 6 are the DNAs isolated from the stem.
FIG. 9 is a graph showing the results of PCR for a combination of primers for HRM-curve analysis of trnL -trnF, matK, and ITS genes, and it is a result of confirming amplification of a single band of expected size for each gene.
FIG. 10 is a diagram showing the results of discrimination between Chinese and Korean cymidophytic strains according to HRM curve patterns using chloroplast genes ( trnL-trnF , matK) and nuclear gene (ITS) fragments.

The present invention is capable of various modifications and various embodiments, and specific embodiments are described in detail in the description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. Hereinafter, the present invention will be described in more detail.

Certain terms are hereby defined for convenience in order to facilitate a better understanding of the present invention. Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Also, unless the context clearly indicates otherwise, the singular form of the term also includes plural forms thereof, and plural forms of the term should be understood as including its singular form.

According to an aspect of the present invention, there is provided a marking composition for identifying a snake root system comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 2 as an active ingredient and a single nucleotide polymorphism (SNP) marker.

According to an embodiment of the present invention, the SNP marker is at least one of an SNP marker having a 62nd nucleotide of SEQ ID NO: 2 and a SNP marker having a 395th nucleotide of SEQ ID NO: 2.

According to one embodiment of the present invention, the nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2 is repeated at position 805 of SEQ ID NO: 2.

According to one embodiment, the gene is trnL - is derived from trnF gene.

According to another aspect of the present invention, there is provided a marking composition for identifying a snake root system comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 5 as an active ingredient and a single nucleotide polymorphism (SNP) marker.

According to an embodiment of the present invention, the SNP marker is a SNP marker having the 32nd nucleotide of SEQ ID NO: 5, a SNP marker having the 55th nucleotide of SEQ ID NO: 5, a SNP marker having the 153th nucleotide of SEQ ID NO: The SNP marker having the 378th nucleotide of SEQ ID NO: 5, the SNP marker having the 409th nucleotide of SEQ ID NO: 5, the SNP marker having the 416th nucleotide of SEQ ID NO: 5, the C59th nucleotide of SEQ ID NO: And an SNP marker having G at position 692 of SEQ ID NO: 5.

According to one embodiment of the present invention, the gene is derived from the matK gene.

According to still another aspect of the present invention, there is provided a corymbin gene identification composition comprising a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and at least one primer pair consisting of the nucleotide sequence of SEQ ID NOs: 12 and 13 do.

In the present invention, a mismatch primer is prepared by changing the second or third nucleotide adjacent to the SNP after confirming the SNP, and an amplification refractory mutation system (ARMS-PCR) -PCR) technique to improve the accuracy of discrimination (Han et al., Mol Biol Rep (2016) 43: 323332).

According to still another aspect of the present invention, there is provided a kit for discriminating gums from a composition comprising the composition.

According to one embodiment of the present invention, the kit comprises an agent capable of detecting or amplifying the SNP marker.

In the present invention, the kit may be, but is not limited to, an RT-PCR kit or a microarray chip kit including a preparation capable of detecting or amplifying SNP markers.

The RT-PCR kit can comprise a respective pair of primers specific for the marker gene and can be used in combination with other test tubes or other appropriate containers, reagents necessary for PCR amplification, such as buffers, DNA polymerases (e.g., Thermus aquaticus Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermisflavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs.

The microarray chip kit may be a microarray kit for discriminating a system including the DNA markers.

In the present invention, a microarray means a group of polynucleotides immobilized on a substrate at a high density, and the polynucleotide group means a microarray immobilized in a constant region. Such microarrays are well known in the art. The microarrays are described, for example, in U.S. Patent Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The kit may be made from a number of separate packaging or compartments containing the reagent components described above.

According to still another aspect of the present invention, there is provided a marking composition for the identification of cirrhosis, comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 15 as an active ingredient and a single nucleotide polymorphism (SNP) marker.

According to an embodiment of the present invention, the SNP marker comprises a SNP marker having a 38th nucleotide of SEQ ID NO: 15, a SNP marker having a 40th nucleotide of SEQ ID NO: 15, an SNP marker having a nucleotide of 167th nucleotide of SEQ ID NO: The SNP having nucleotide No. 363 at position 36 in SEQ ID NO: 15, the nucleotide at position 363 of nucleotide G at position 15, the nucleotide sequence at nucleotide 379 at nucleotide 379 of SEQ ID NO: 15, the nucleotide sequence at nucleotide 385 of nucleotide G at position 15, And at least one of SNP markers in which the 462nd nucleotide of SEQ ID NO: 15 is G.

According to one embodiment of the present invention, the gene is derived from an intergenic transcribed spacer (ITS) gene.

According to still another aspect of the present invention, there is provided a corymbin gene identification composition comprising at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21 do.

According to still another aspect of the present invention, there is provided a kit for discriminating gums from a composition comprising the composition.

According to still another aspect of the present invention, there is provided a primer pair comprising a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 12 and 13; And at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21, is provided.

According to an embodiment of the present invention, the composition can discriminate hybrid combinations.

According to another aspect of the present invention, there is provided a primer pair comprising a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 22 and 23, a pair of primers consisting of the nucleotide sequences of SEQ ID Nos. 24 and 25 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 26 and 27 And a primer pair is provided.

According to one embodiment of the present invention, the composition is for high resolution melting analysis.

In the present invention, HRM curve analysis techniques can be used to accurately and quickly identify two strains containing only one single nucleotide polymorphism (Han et al., Mol Biol Rep (2016) 43: 323332).

According to still another aspect of the present invention, there is provided a kit for discriminating gums from a composition comprising the composition.

According to still another aspect of the present invention, there is provided a method for detecting genomic DNA comprising: extracting genomic DNA from a sample; Performing PCR of the DNA using the primer pair according to the present invention; And identifying the PCR product.

According to one embodiment of the present invention, PCR is carried out using at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 12 and 13, The nucleotide sequence of the product can be analyzed to determine that the SNP marker is a Korean SNP marker having the 62nd nucleotide of SEQ ID NO: 2 and the SNP marker having the 395th nucleotide of the SEQ ID NO: 2.

According to one embodiment of the present invention, PCR is performed using at least one primer pair consisting of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21, Analysis of the base sequence of the product revealed that the SNP marker having the 38th nucleotide of SEQ ID NO: 15 is A, the SNP marker of the 40th nucleotide of SEQ ID NO: 15 is C, the SNP marker of 167th nucleotide of SEQ ID NO: 15 is G, The SNP marker having the nucleotide No. 369 at position 36, the SNP marker having nucleotide No. 379 at position 37, the SNP marker having nucleotide No. 385 at position 38, the SNP marker having nucleotide No. 45 at position 45, And at least one of the SNP markers having the nucleotide No. 462 at position 15 of G is included, it can be discriminated as a Korean snout.

According to still another aspect of the present invention, there is provided a method for detecting genomic DNA comprising: (a) extracting genomic DNA from a sample; (b) performing a polymerase chain reaction (PCR) of the DNA using the primer pair described in (8); (c) performing a polymerase chain reaction (PCR) of the DNA using the primer pair described in claim 13; And (d) identifying and comparing the PCR product of step (b) and the PCR product of step (c).

According to one embodiment of the present invention, when the sample is judged to be cucumber in Korean as a result of checking the PCR product in step (b) and the PCR product in step (c) is confirmed as Chinese cucumbers, It is possible to distinguish it as a hybrid of the sample.

In the present invention, the ARMS-PCR technique using the single nucleotide polymorphism of the chloroplast and the nucleotide fragment in the nucleus enables discrimination between the Chinese and Korean native lines, and their hybridization rate and hybridity by crossing .

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

<Examples>

sample

The Gyujipong system used in the following experiment has a well-known system of Chinese or Korean origin in the farmhouse (Kim Kwang-yeon) in Sangcheong-gun, Gyeongnam Province. In addition to this, the Gyungju daegok-myeon, Gyeongnam, Daeyeon-myeon, Gyeongnam- (See Table 1). The results of this study are summarized as follows.

Table 1 shows the current status of the system being collected and kept.

&Lt; Example 1 >

Genomic DNA isolation by system

Genomic DNA was isolated according to the method provided by the company using GeneAll's Exgene ^™ Plant SV kit using a powder obtained by rapidly freezing a predetermined amount of roots, seeds, leaves, etc. in liquid nitrogen in each collected system, and finely ground powder . The purified DNA was electrophoresed on 1% agarose gel, and the degree of degradation in the DNA isolation was determined. The DNA was purified using a Micro-spectrometer (BioPrince, SD-2000, Gangwon, South Korea) at 234 nm, 260 nm, and 280 nm Absorbance was measured to confirm whether the A ₂₆₀ / A ₂₈₀ value ranged from 1.8-2.2 and the A ₂₃₄ / A ₂₆₀ value ranged from 0.5-0.8 to confirm the degree of contamination of RNA and protein to confirm pure DNA.

&Lt; Example 2 >

Chloroplast genes trnL-trnF  Wow matK ) And nucleotide sequence analysis

To confirm the single nucleotide polymorphism in the chloroplast gene, primers were prepared using the nucleotide sequence information of known genes as summarized in Table 2 for the amplification of the trnL -trnF gene fragment and the matK gene fragment. That is trnL-trnF gene fragment was prepared for each of the primers from the nucleotide sequence of Accession number (JN006418.1), and matK gene fragment (JF317421.1) in GenBank.

Table 2 shows the primer information used for the SNP development of the chloroplast genome.

The amplification of the gene was minimized by using a buffer solution for PCR reaction and i-pfu DNA polymerase provided by the iNtRON company (Gyeonggi Province, South Korea), which could be caused by polymerization reaction. 50 ng of pure DNA and 10 pmole of primer were mixed according to the manufacturer's instructions and denatured at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, 60 ° C for 10 seconds, and 72 ° C at 30 ° C After repeated 35 cycles of 1 cycle, the reaction was terminated at 4 ° C for 5 minutes at 72 ° C. The amplified DNA band was electrophoresed using 2.0% agarose gel. Since mutagenization may occur during the cloning of each amplified gene fragment, the DNA fragment amplified without cloning was subjected to Direct Sequence Analysis after restriction of net fertilization using Expin ^™ PCR SV (GeneAll, Seoul, South Korea) Kit . In addition, the final confirmed single nucleotide polymorphism (SNP) was verified by performing at least 5 repetitions.

&Lt; Example 3 >

Chloroplast gene fragment ( trnL-trnF  Wow matK ) Of single nucleotide polymorphisms

Kkujippong the target system, known as the species introduced from Korea and China, known as the national indigenous trnL - trnF and matK Gene fragments were amplified and subjected to sequencing analysis, and the results were compared. As a result, two trnL-trnF gene fragments (SEQ ID NOS: 1 and 2) ), matK In the case of the gene fragments (SEQ ID NOS: 4 and 5), substitution of the base sequence at 10 positions (see FIG. 2) occurred and different single base single nucleotide polymorphism patterns could be confirmed. If enough of Korean specificity has trnL - it was confirmed that about 805bp trnF gene region (TTTTTAATTGAC) 12 bp of the fragment is repeated (see FIG. 1).

<Example 4>

Chloroplast gene fragment ( trnL-trnF ) Using the primers for ARMS-PCR for marker identification in Korea and China

In general, attempts have been made to identify specific strains using mismatch primers made using single nucleotide polymorphisms, but primers for one or two mismatch sequences are not working properly And often do not get the desired results. Therefore, it is possible to increase the specificity of the trnL-trnF gene by using a primer in which the base representing the single nucleotide polymorphism of the trnL-trnF gene is positioned at the 3'-terminal and the first or second base is artificially modified toward the 5'- And to introduce ARMS-PCR technology.

FIG. 3 is a diagram illustrating the results of preparing primers for ARMS-PCR by substituting different bases for the second base at the 5'-terminal side based on the SNP of the trnL-trnF gene. The nucleotide sequence information for each of these primers is summarized in Table 3 and the other mismatches of bases are indicated in red. That is, primers for ARMS-PCR were prepared for both bidirectional primers, and PCR was performed to establish temperature-dependent conditions using the respective primers. As shown in FIG. 4, the trnL- All the Chinese specific primers gave good results at 55 ° C.

Table 3 relates to primer information for ARMS-PCR centered on single base polymorphism of trnL-trnF gene.

Based on these results, genomic DNA was isolated from the resources described in Table 1 in order to carry out an experiment for discriminating the origins of gypsophila strains collected in each region, and the result of performing PCR using the primers described above was also shown 5. Using ARMS-PCR primers constructed by artificially altering bases in both the forward and reverse primers, a line of well-known origin of the Chinese or Korean lineage collected at the Kim Kyeon-yeon farm in Sancheong- 36, 36-1), Gyeongsangnam-do, Dae-myeon (37), Sancheong-gun livestock (38), Gyeongsangnam-do (43) (39) of Micheon-myeon in Jinju and 45 (30, 31) of Song-hori Mountain in Songnam-myeon, Chonnam Province, all of which were found to be of Korean origin (Fig. 5).

&Lt; Example 5 >

Development of a DNA Marker for Identification of Nuclei in Korean and Chinese Cyprinidae Hybrids

It is assumed that the Korean and Chinese cypress lines can be cross - linked with each other by paper. Especially, recently, it has been grown in China as a large quantity of Chinese paper,

Such hybrids may not be distinguishable from the discrimination markers present in the chloroplast. Therefore, genes present in the chloroplast are inherited by the mother, and the genes present in the nucleus are inherited by crossing. Therefore, an intergenic transcribed spacer (ITS) in the transcription unit of the gene encoding the ribosomal DNA present in the nucleus, We have identified single nucleotide polymorphisms in the gene fragments of the region and developed discriminant markers that can distinguish each on the basis of it.

In order to secure the nucleotide sequence of the ITS gene fragment, GenBank searches for a gene coding for 18S rRNA and 28S rRNA present in the transcription unit of ribosomal DNA of Cucumisponin (GenBank), and obtains a primer from known nucleotide sequence information ) Were prepared and the fragments obtained by PCR amplification using the genomic DNA isolated from Chinese and Korean cyprinol system (Sample Nos. 41 and 42, see Table 1) were subjected to direct sequence analysis (SEQ ID NOS: 4 and 5) The results were very similar as expected, with only about 8 bases showing substitution (Figure 6). A pair of bi-directional primers containing two single base polymorphisms was constructed based on the given base sequence to discriminate between these two systems (see Table 4). The positions of the primers are indicated by arrows in FIG. Since both bidirectional primers contain two SNPs, ARMS-PCR technology, which artificially induces mutations, has not been applied.

Table 4 provides information on primers containing SNPs in the nuclear ITS DNA to discriminate Korean and Chinese Cyprinidae crossbreds.

PCR was carried out on all of the cloned resources collected in the regions identified as Korean by trnL-trnF gene markers using the primers described above, and the result was also found to be Korean in all the resources (see FIG. 7). This result shows that the single nucleotide polymorphism of the ITS gene fragment existing in the nucleus for the strains which could be clearly distinguished using the Chinese and Korean specific primers prepared from the nucleotide sequence of the trnL-trnF gene present in the chloroplast of the mother lineage The same results were obtained with the primers prepared on the basis of these primers. Therefore, it can be said that all of these genetic resources originated in Korea.

In addition, Korea and China are using the same scientific name, so they are different from each other in the same species or country. Therefore, in the case of cross-hybridization, discriminative primers were prepared so as to utilize the ITS gene existing in the nucleus, since it is impossible to distinguish them as chloroplast genetic markers. By using the primers ITS determine a strains collected from farmers in Gyeongsangnam Milyang (see collection number 2016-10, Table 1) on a result of the analysis of, trnL-trnF genetic marker is found to be both, but in Korea ITS marker PCR products of the same size were generated when not only Korean but also Chinese specific primers were used (see FIG. 8). These results suggest that the pollen of the Chinese system is pollinated by the Korean system as a model, resulting in the fertilization. Similar findings such as Zeng (2015, PLoS One, 13 (8): e0135411), Moraceae (Moraceae), suggested that the comparative analysis of eight results group of ITS gene sequence of mulberry in (Morus) to separate the species , And interspecific hybrids were also identified. In addition, Burgess et al. (2007, New Phytologist 177: 276-284) also reported that the mulberry ( Morus ruba ) bearing red berries belonging to the same genus Mulberry, the white mulberry ( Morus alba ) , And the frequency is high enough to allow easy discrimination.

In the future, further studies using more molecular biologic markers should be performed in order to find a cross-breeding combination in which the Chinese system becomes a model and the pollen of the Korean system becomes pollinated, contrary to the 2016-10 hybrid (see Table 1) In addition to the morphological differences, studies on the index components and pharmacological effects are not only academically useful, but also can be easily distinguished from their distribution processes.

&Lt; Example 6 >

High Resolution Melting (HRM)) Development of discrimination technology of origin of Chinese and Chinese cymbals by analyzing curve pattern

A primer for HRM curve analysis was designed based on the single nucleotide polymorphism pattern found by comparing nucleotide sequences of trnL -trnF and matK genes present in the chloroplast genome and ITS gene fragments present in the nucleus. A primer was designed to produce a PCR product of about 200 bp in size with one or more single nucleotide polymorphisms located at the center. The sequence information is summarized in Table 5.

Table 5 shows the nucleotide sequence information of primers for discriminating origin of Chinese and Korean strains using the HRM curve pattern.

Using primer combinations designed to include a single nucleotide polymorphism inherent in the three genes described in Table 5, first, in order to investigate whether or not the target gene fragments were correctly amplified by these primers, the method described in Example 2 PCR was performed on the Chinese and Korean strains using the primer combination, and the result was confirmed by electrophoresis on 2.0% agarose gel. As a result, a single-band DNA fragment was amplified for each gene at the predicted position as shown in FIG. I could confirm.

In addition, these bands were purified from agarose gels and confirmed to be 100% identical to the nucleotide sequences of trnL -trnF or matK genes and ITS gene fragments identified in each species. The other bands were amplified Respectively.

To analyze the HRM curve pattern for each gene, China and South Korea lines into each 10 ng of genomic DNA isolated from kkujippong trees, respectively, the primers described in Table 6 into each by 5 pmol SsoFast of 10 ul ™ EvaGreen ^® Supermix BIO-RAD, 172-5200 premixture was added and the total reaction volume was adjusted to 20 μl HRM curve analysis was performed using Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, USA). The conditions were as follows: first, the enzyme was activated at 98 ° C for 2 minutes, denatured the DNA at 98 ° C for 5 seconds, maintained at 58 ° C for 20 seconds, annealing and progressing the extension of the complementary strand, Thereafter, the temperature was lowered to 65 ° C., and the melting curve pattern was analyzed by the input program while increasing the temperature to 95 ° C. The results are shown in FIG. It was found that the discrimination of matK , trnL -trnF and ITS gene fragments was possible by showing different patterns of HRM curves in Chinese and Korean cypresses. In particular, the trnL-trnF gene, which contains a 12 -bp repeat sequence, has a pattern that can be more clearly differentiated between the two strains, and it can be applied to commercialization in the future distribution market.

As described above, the present invention relates to a method for isolating strains of TrnL and TrnF present in chloroplasts, fragments containing intergenic DNA (DNA) and matK genes The fragments were amplified by PCR, and the nucleotide sequences were analyzed. The nucleotide sequences of the genes were compared from the previously registered genbank (GenBank) to prepare primers containing the regions showing SNPs. Markers were developed. In addition, in order to increase the specificity of the PCR reaction, a mismatch primer (ARMS-PCR marker) prepared by artificially altering the nucleotide sequence of the third base adjacent to the 3'-terminal of the base showing the existing single base polymorphism . In order to discriminate these strains by applying HRM technology, primers designed to center SNPs were developed and compared with HRM curve patterns using these primers, the conditions suitable for discrimination were established. Furthermore, recently, in order to solve the problem of mixing and misuse of the two lines in the process of distribution or processing of the product, we have developed a technology capable of measuring not only qualities but also mixing ratios. In addition, in view of the fact that they are introduced and cultivated in large quantities in recent years, in order to identify the hybrids produced by mutual crossing with the same native species, the transcription units of ribosomal DNA present in the nucleus are separately amplified After identifying the SNP, we developed a discriminative marker that can distinguish the strains in the internal transcribed sequence (ITS) I and ITS II regions. These strains were identified and, in combination with the chloroplast discrimination markers described above, We have established a technology that can be distinguished.

<110> GYEONGNAM NATIONAL UNIVERSITY OF SCIENCE AND TECHNOLOGY <120> composition comprising SNP markers for a differentiation of          Cudrania tricuspidata Bureau lines, and method for a          Differentiation of Cudrania tricuspidata Bureau lines and hybrid          using the same <130> NPF29923 <160> 27 <170> PatentIn version 3.2 <210> 1 <211> 993 <212> DNA <213> Cudrania tricuspidata Bureau <400> 1 nnncgtagac gctacggact taattgaatt gagccttggt atggaaacct accaagtgac 60 aactttcaaa ctcagagaaa ccctggaatt aaaaatgggc aatcntgagc caaatccggt 120 tttctgaaaa caaacaaggg ttcagaaggc aataataaaa agggataggt gcagagactc 180 aatggaagct gttctaacaa atggagttgg ctgcggtgcg ttagtaaagg aatcactcca 240 gaaagaatga agaataaacg tatatatgta tacgtactga aatactatct tcaaatgatt 300 aatgacaacc caaatccgta tttcttttca ttttcatgaa aaatgaaaga attgttgtga 360 atcaattata aggtgaaaaa agaatcaaat atttattgat caaatcattt actccatcaa 420 aatctgatag atcttttgaa gaattgatta atcggacgag aataaagata gagtcctatt 480 ctacatgtca atattggcaa caatgaaatt tatagtaaga ggaaaatccg tcgactttaa 540 aaatcgtgag ggttcaagtc cctctatccc caaaaaggcc catttaatcc cctaagttaa 600 ttatttatcc taccttctca tttcgttagc ggttccaaat tcgttatctt tctcgttcat 660 tctaattcta caaacgtatc tgagcaaaaa gttttttctt atcacaagcc ttgtgatcta 720 tatgaaacac gtacaaatga agatctttga gaaaggaatc ccaatgttaa atttgaataa 780 ttaataattc attttattac tcgtactgta ctgaaactta caaagtcctt ttttttgaag 840 atccaagaaa tttcaccaag gcccggataa gactttgcaa ttcccctttc atctttttaa 900 ttgacataga cccaagtcct ctattaaaat gaggatgatg cgtaaggaat ggtcgggata 960 gctcagctgg tagagcagag acgaatnctn nnn 993 <210> 2 <211> 1007 <212> DNA <400> 2 nnntcgtaga cgctacggac ttaattgaat tgagccttgg tatggaaacc taccaagtga 60 cacttcaaac tcagagaaac cctggaatta aaaatgggca atcntgagcc aaatccggtt 120 ttctgaaaac aaacaagggt tcagaaggca ataataaaaa tggataggtg cagagactca 180 atggaagctg ttctaacaaa tggagttggc tgcggtgcgt tagtaaagga atcactccag 240 aaagaatgaa gaataaacgt atatatgtat acgtactgaa atactatctt caaatgatta 300 atgacaaccc aaatccgtat ttcttttcat tttcatgaaa aatgaaagaa ttgttgtgaa 360 tcaattataa ggtgaaaaaa gaatcaaata tttattgatc aaatcattta ctccatcaaa 420 atctgataga tcttttgaag aattgattaa tcggacgaga ataaagatag agtcctattc 480 tacatgtcaa tatcggcaac aatgaaattt atagtaagag gaaaatccgt cgactttaaa 540 aatcgtgagg gttcaagtcc ctctatcccc aaaaaggccc atttaatccc ctaagttaat 600 tatttatcct accttctcat ttcgttagcg gttccaaatt cgttatcttt ctcgttcatt 660 ctaattctac aaacgtatct gagcaaaaag ttttttctta tcacaagcct tgtgatctat 720 atgaaacacg tacaaatgaa gatctttgag aaaggaatcc caatgttaaa tttgaataat 780 taataattca ttttattact cgtactgtac tgaaacttac aaagtccttt tttttgaaga 840 tccaagaaat ttcaccaagg cccggataag actttgcaat tcccctttca tctttttaat 900 tgacttttta attgacatag acccaagtcc tctattaaaa tgaggatgat gcgtaaggaa 960 tggtcgggat agctcagctg gtagagcaga gactaattnt tnncnnn 1007 <210> 3 <211> 12 <212> DNA <400> 3 tttttaattg ac 12 <210> 4 <211> 968 <212> DNA <400> 4 ttttnttnat tgcggggggt tttntctcac gactattata catgggaata gtcttattat 60 tcaaataaat atatttcttt tttttcaaaa agtaatacaa gattattctt gttcctatat 120 aattctcatg tgtgtgaata cgaatccatc ttactttttc tacgtaagca atcttctcat 180 ttacgattaa catcttctgg gggctttttt gagcgaatat atttctatgg aaaaataaaa 240 catcccgtag aaaaagtctt tgctaatgat tttccgacta gcttatggtt cttcgaggat 300 ctcttcatgc attatgttag atatcaagga aaatcaattc tggcttcaaa ggatacgcct 360 cttttcatga ataaatggaa atattacctt gtccttttat ggcaatgtca tttttatgtg 420 tggtctcaac caggaagtat gtatataaac caattatgca aacattccct cagctttctg 480 ggttatcttt caagtatgca aataaatcgt tcagtggtac ggagtcaaat gctagaaaat 540 tcatttctaa tggataatgc tatgaagaag attgatacat tagttccaat tagtcctctg 600 attggatcgt tggctaaaat gagattttgt aacgtattag ggcatcccat tagtaagtcg 660 acctgggccg attcatcaga ttttgatatt atcgaccgat ttgcgtgtat atgcagaaat 720 ctttttcatt attacagtgg atcctcaaaa aaaaagagtt tgtatcgagt aaaatatata 780 cttcgacttt cttgtgttaa aactttagct cgtaaacaca aaagtactgt acgcgctttt 840 ttgaaaagat taggttctaa attattggaa gatttcttta cggaggaaga agaggttctt 900 tctttgattt ttccaagaac ttattccgct tttcgaagtt catataaggt gcgaattgtc 960 nnnnnnnn 968 <210> 5 <211> 946 <212> DNA <400> 5 ttnnctcacg actattataa ttggaatagt cttattattt caaataaata tatttctttt 60 ttttcaaaaa gtaatccaag attattcttg ttcctatata attctcatgt ttgtgaatac 120 gaatccatct tacttttttc tacgtaagca atcttctcat ttacgattaa catcttctgg 180 ggactttttt gagcgaatat atttctatgg aaaaataaaa catcccgtag aagaagtctt 240 tgctaatgat tttccgacta gcttatggtt cttcgaggat ctcttcatgc attatgttag 300 atatcaagga aaatcaattc tggcttcaaa ggatacgcct cttttcatga ataaatggaa 360 atattacctt gtccttttat ggcaatgtca tttttatgtg tggtctcaac caggaaggat 420 gtatataaac caattatgca aacattccct cagctttttg ggttatcttt caagtatgca 480 aataaatctt tcagttgtac ggagtcaaat gctagaaaat tcatttctaa tggataatgc 540 tatgaagaag attgatacat tagttccaat tagtcctctg attggatcgt tggctaaaat 600 gagattttgt aacgtattag ggcatcccat tagtaagtcg acctgggccg attcatcaga 660 ttttgatatt atcgaccgct ttgcgtgtat atgcagaaat ctttttcatt attacagtgg 720 atcctcaaaa aaaaagagtt tgtatcgagt aaaatatata cttcgacttt cgtgtgttaa 780 aactttagct cgtaaacaca aaagtactgt acgcgctttt ttgaaaagat taggttctaa 840 attattggaa gatttcttta cggaggaaga agaggttctt tctttgattt ttccaagaac 900 ttattccgct tttcgaagtt catataaggt gcgaattgtc nnnnnt 946 <210> 6 <211> 23 <212> DNA <400> 6 attgcggttt tttcttcacg act 23 <210> 7 <211> 23 <212> DNA <400> 7 atgattgacc agatcgttga tgc 23 <210> 8 <211> 22 <212> DNA <400> 8 gatatggcga aatcggtaga cg 22 <210> 9 <211> 22 <212> DNA <400> 9 tgccaggaac cagatttgaa ct 22 <210> 10 <211> 25 <212> DNA <400> 10 gggttcagaa ggcaataata aacat 25 <210> 11 <211> 28 <212> DNA <400> 11 cctcttacta taaatttcat tgttgtcg 28 <210> 12 <211> 25 <212> DNA <400> 12 gggttcagaa ggcaataata aatag 25 <210> 13 <211> 25 <212> DNA <400> 13 cttactataa atttcattgt tggca 25 <210> 14 <211> 711 <212> DNA <400> 14 ncctgcgtaa ggatcattgt cgaaagctgc ccagcagaaa gacccgtgaa cctgttgtac 60 ctcgtgatgg ggtggggggt gcagcatgca cccttcgctc atcatgccaa gtgcgtgccg 120 ctctgtgtca cgccctcggc cctgaacgaa ccccggcgcg gaaagcgtca aggaaagaca 180 atgaaacgag ggctaccaag tcactagaat tggtgtatat cttggtggtc gcatcgtggt 240 tggttgaagt ctataacgac tctcggcaac ggatatctcg gctctcgcat cgatgaagaa 300 cgtagcgaaa tgcgatactt ggtgtgaatt gcagaatccc gtgaaccatc gagtctttga 360 acgcaagttg cgcccgaagc catccggtcg agggcacgtc tgcctgggcg tcacacactg 420 ttgcccccct aatcccttgt cactcgttct gtagtgcatg tggagtaaaa agggcgtata 480 ttggcctccc gtgcgatatg gacttcatgg ttggcccaaa ctcaagtccc ttgtcacgtg 540 catcgtgaca aaaggtggtt gtcgatcagt cggtgccccg tcacttgatg ccggacacgt 600 aaagggactc ccagcgaccc caatgcatcg atagtgtagg tgctttgaaa gcgaccccag 660 gtcaggcggg gctacccgct gagttaagca tatcaatagc gngannnnnn n 711 <210> 15 <211> 723 <212> DNA <400> 15 cctccccgng aggggacctg cggaatgatc attgtcgaaa gctgcccagc agaaagaccc 60 gcgaacctgt tgtacctcgt gatgggtggg gggtgcagca tgcacccttc gctcatcatg 120 ccaagtgcgt gccgctcagc gtcacgccct cggccctgaa cgaaccccgg cgcggaaagc 180 gtcaaggaaa gacaatgaaa cgagggctac caagtcacta gaattggtgt atatcttggt 240 ggtcgcatcg tggttggttg aagtctgtaa cgactctcgg caacggatat ctcggctctc 300 gcatcgatga agaacgtagc gaaatgcgat acttggtgtg aattgcagaa tcccgtgaac 360 catcgagtct ttgaacgcaa gttgcgcccg aagccatccg gtcgagggca cgtctgcctg 420 ggcgtcacac actgttgccc ccctaatccc ttgtcactcg ttgtgtagtg catgtggact 480 aaaaggggcg tatattggcc tcccgtgcga tatggacttc atggttggcc caaactcaag 540 tcccttgtca cgtgcattgt ggcaaaaggt ggttgtcgat cagtcggtgc cccgtcactt 600 gatgccggac acgtaaaggg actcccagcg accccaatgc atcgatagtg taggtgcttt 660 gaaagcgacc ccaggtcagg cggggctacc cgctgagtta agcatatcaa taagncgaan 720 nna 723 <210> 16 <211> 19 <212> DNA <400> 16 tccgtaggtg aacctgcgg 19 <210> 17 <211> 22 <212> DNA <400> 17 gccgttacta ggggaatcct tg 22 <210> 18 <211> 21 <212> DNA <400> 18 gccaagtgcg tgccgctcag c 21 <210> 19 <211> 21 <212> DNA <400> 19 cgacaaccac cttttgccac a 21 <210> 20 <211> 21 <212> DNA <400> 20 gccaagtgcg tgccgctctg t 21 <210> 21 <211> 21 <212> DNA <400> 21 cgacaaccac cttttgtcac g 21 <210> 22 <211> 24 <212> DNA <400> 22 gaagatcttt gagaaaggaa tccc 24 <210> 23 <211> 22 <212> DNA <400> 23 tgccaggaac cagatttgaa ct 22 <210> 24 <211> 20 <212> DNA <400> 24 gtgtggtctc aaccaggaag 20 <210> 25 <211> 20 <212> DNA <400> 25 gccaacgatc caatcagagg 20 <210> 26 <211> 19 <212> DNA <400> 26 tcccgtgaac catcgagtc 19 <210> 27 <211> 19 <212> DNA <400> 27 gcacgtgaca agggacttg 19

Claims (24)

An agent capable of detecting or amplifying a single nucleotide polymorphism (SNP) marker,
Wherein the SNP marker is an SNP marker having a 62nd nucleotide of SEQ ID NO: 2 and a SNP marker having a 395th nucleotide of SEQ ID NO: 2,
The nucleotide sequence of SEQ ID NO: 2 repeats the nucleotide sequence of SEQ ID NO: 3 at position 805,
The SNP marker is a SNP marker having the 32nd nucleotide of SEQ ID NO: 5, the SNP marker having the 55th nucleotide of SEQ ID NO: 5, the SNP marker having the 153th nucleotide of SEQ ID NO: 5, the 378th nucleotide of SEQ ID NO: The SNP marker having the nucleotide No. 409 of SEQ ID NO: 5, the SNP marker having the nucleotide number 409 of SEQ ID NO: 5, the SNP marker having the nucleotide number 416 of SEQ ID NO: 5, the SNP marker having the nucleotide number 599 of SEQ ID NO: The SNP marker in which the 692nd nucleotide is G,
The SNP marker is a SNP marker having the 38th nucleotide of SEQ ID NO: 15, the SNP marker having the 40th nucleotide of SEQ ID NO: 15, the SNP having the 167th nucleotide of SEQ ID NO: 15, the 363rd nucleotide of SEQ ID NO: 15 The SNP having nucleotide number 379 in SEQ ID NO: 15, the SNP having nucleotide number 385 in SEQ ID NO: 15, the SNP having nucleotide number 458 in SEQ ID NO: 15, and the 462nd nucleotide in SEQ ID NO: SNP marker &lt; / RTI &gt;
A composition for the identification of a bryophyte lineage which is capable of discriminating one or more of hybrids produced by interbreeding between Korean, Chinese and Korean native lines and Chinese lines.
delete delete The method according to claim 1,
The nucleotide sequence of SEQ ID No. 2 is trnL - trnF the system determines kkujippong composition derived from a gene.
delete delete The method according to claim 1,
Nucleotide sequence of the SEQ ID NO: 5 is the composition for the system comes from kkujippong matK gene determination.
The method according to claim 1,
The agent capable of amplifying the single nucleotide polymorphism (SNP) marker comprises a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 12 and 13, / RTI &gt;
8. A kit for discriminating a cirrhotic plant comprising the composition of claim 8.
delete delete The method according to claim 1,
The nucleotide sequence of SEQ ID NO: 15 is derived from ITS (intergenic transcribed spacer) gene.
The method according to claim 1,
The agent capable of amplifying the single nucleotide polymorphism (SNP) marker comprises a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21, / RTI &gt;
13. A kit for discriminating a cirrhotic plant comprising the composition according to claim 13.
The method according to claim 1,
The agent capable of amplifying a single nucleotide polymorphism (SNP) marker comprises at least one primer pair of a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and a pair of primers consisting of nucleotide sequences of SEQ ID NOs: 12 and 13; And
A primer pair consisting of the nucleotide sequences of SEQ ID NOS: 18 and 19, and a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 20 and 21, respectively.
16. The method of claim 15,
The composition of claim 1, wherein the composition is capable of distinguishing hybridization.
The method according to claim 1,
The agent capable of amplifying the single nucleotide polymorphism (SNP) marker comprises a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 22 and 23, a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 24 and 25, and a nucleotide sequence of SEQ ID NOs: 26 and 27 Wherein the primer pair comprises at least one primer pair.
18. The method of claim 17,
Wherein the composition is for high resolution melting analysis.
17. A kit for discriminating between at least one hybrid produced by mutual crossing of Korean, Chinese and Korean native and Chinese strains, comprising the composition of claim 17.
delete Extracting the genomic DNA from the sample;
A pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 10 and 11, a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 12 and 13, a pair of primers consisting of nucleotides of SEQ ID NOS: 18 and 19, Performing PCR of the DNA using a primer pair; And
Identifying the PCR product,
PCR was carried out to analyze the base sequence of the amplified PCR product to determine the SNP marker having the 62nd nucleotide as T in SEQ ID NO: 2, the SNP marker having the 395th nucleotide as C in SEQ ID NO: 2, the nucleotide having the 38th nucleotide as SEQ ID NO: 15 as A The SNP marker having the 40th nucleotide of SEQ ID NO: 15, the SNP marker having the 167th nucleotide of SEQ ID NO: 15, the SNP marker having the 363th nucleotide of SEQ ID NO: 15, the 379th nucleotide of SEQ ID NO: 15, The SNP marker of SEQ ID NO: 15, the SNP marker of SEQ ID NO: 15, the SNP marker of SEQ ID NO: 15, the SNP marker of SEQ ID NO: 15, the SNP marker of SEQ ID NO: 15, and the SNP marker of SEQ ID NO: Identification of cymidophyllum lineage from Korean cymidophyllum line.
delete (a) extracting genomic DNA from a sample;
(b) performing a PCR of the DNA using a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 10 and 11 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOS: 12 and 13;
(c) performing a PCR of the DNA using a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19 and a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 20 and 21; And
(d) Identifying and comparing the PCR product of step (b) and the PCR product of step (c) and one of the hybrid products generated by interbreeding between Korean, Chinese, and Korean native lines and Chinese lines Identification method of crossbred hybrids that can discriminate species.
24. The method of claim 23,
When the sample was identified as a Korean cymbidium in step (b), and a PCR product in step (c) was identified as a Chinese cymbidium in step (b), the hybrid sample was a Korean hybrid, Identification method of crossbred hybrids that can discriminate one or more crossbreds produced by interbreeding between Korean native line and Chinese line.

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