KR20220074134A - KASP primer set derived on mitochondira for classifying Panax ginseng cultivar and genotype and uses thereof - Google Patents

Abstract

The present invention relates to a mitochondrial-derived KASP primer set for genotyping ginseng species and genetic resources and their use. It can be used as an efficient biomarker for maintaining seed purity and identifying species by rapidly identifying genotypes such as breeding lines, native species, and foreign ginseng. In addition, it can be used for precision breeding because it can quickly and accurately confirm the distinguishing and novelty of ginseng gene resources.

Description

KASP primer set derived on mitochondira for classifying Panax ginseng cultivar and genotype and uses thereof}

The present invention relates to a mitochondrial KASP primer set for genotyping ginseng species and genetic resources and uses thereof.

Ginseng ( Panax ginseng ) has been widely used as a medicinal plant of high value worldwide. In the case of such plants, it is important to develop excellent varieties, and molecular markers are used as an important means to increase the efficiency of breeding. However, in the case of ginseng, the development of molecular markers is very difficult due to the complex genomic structure, and there are not many high-quality markers that can be used for breeding so far, so the basis for molecular breeding is not well established.

As ginseng undergoes whole-genome duplication, there is a homozygous region in which almost all regions have more than 2 copies in the nuclear genome. have.

To solve this problem, the process of discovering SNPs by searching only a single gene region and developing molecular markers in the region is essential. In addition, the conventional agarose gel-based method is cumbersome and not suitable for large-scale analysis. However, in the case of fluorescent markers, since the experimental process and the result confirmation process consist of one step, it is possible to perform genotype analysis on a number of genetic resources very efficiently compared to the conventional method. In order to efficiently use molecular markers in breeding, a means to easily analyze a large amount of resources is required.

Recent advances in single-plex SNP (single nucleotide polymorphism) genotyping techniques such as TaqMan (Life Technologies, USA), SimpleProbe (Roche Applied Science, USA), and Kompetitive Allele Specific PCR (LGC, UK) have led to the development of markers using functional SNPs. - Enables marker-assisted selection, marker-assisted backcrossing selection and marker-assisted recurrent selection.

Currently, KASP (Kompetitive Allele Specific PCR) assay is used in various crops due to its low cost, locus specificity and efficiency. The KASP assay is based on allele-specific oligo extension and fluorescence resonance energy transfer (FRET) in homozygous SNP genotypes. In this method, sample DNA is amplified by two allele-specific forward primers, a single reverse primer and a master mix, each allele-specific primer having a unique tail at the 5' end corresponding to a FRET cassette in the master mix. contains the sequence.

On the other hand, Republic of Korea Patent No. 10-1316610 discloses 'a primer and probe set for identifying ginseng varieties by real-time PRC analysis method', and Korean Patent No. 10-1699518 discloses 'Ginseng variety Geumpoong and Hwangsoo native species are discriminated. Primer sets and uses thereof for However, the 'mitochondria-derived KASP primer set and use thereof for genotyping of ginseng species and genetic resources' of the present invention has not yet been disclosed.

An object of the present invention is to provide a primer set for KASP (kompetiive allele specific PCR) for genotype or cultivar of ginseng including 15 oligonucleotide primer sets.

In addition, another object of the present invention is Korean ginseng ( Panax ginseng ), American ginseng ( Panax quinquefolius ) and Panax notoginseng ) comprising three oligonucleotide primer sets. To provide a primer set for KASP (kompetiive allele specific PCR) for discrimination is to do

In addition, another object of the present invention is to provide a kit for KASP for determining the cultivar or genotype of ginseng including the primer set.

In addition, another object of the present invention is to provide a method for determining the cultivar or genotype of ginseng.

In order to achieve the above object, the present invention provides SEQ ID NOs: 1 to 3; SEQ ID NOs: 4 to 6; SEQ ID NOs: 7 to 9; SEQ ID NOs: 10 to 12; SEQ ID NOs: 13 to 15; SEQ ID NOs: 16 to 18; SEQ ID NOs: 19-21; SEQ ID NOs: 22-24; SEQ ID NOs: 25-27; SEQ ID NOs: 28-30; SEQ ID NOs: 31-33; SEQ ID NOs: 34 to 36; SEQ ID NOs: 37-39; SEQ ID NOs: 40-42; and oligonucleotide primer sets represented by the nucleotide sequences of SEQ ID NOs: 43 to 45; provides a primer set for kompetiive allele specific PCR (KASP) for genotype or cultivar of ginseng, including.

Subsequently, the present invention provides SEQ ID NOs: 25 to 27; SEQ ID NOs: 28-30; And oligonucleotide primer set represented by the nucleotide sequence of SEQ ID NOs: 31 to 33; Korean ginseng ( Panax ginseng ), American ginseng ( Panax quinquefolius ) and ginseng ( Panax notoginseng ) KASP (kompetiive allele specific PCR) for discrimination, including primers provides a set.

In addition, the present invention provides a kit for KASP for determining the cultivar or genotype of ginseng including the primer set and a reagent for performing the amplification reaction.

Finally, the present invention comprises the steps of isolating genomic DNA from a ginseng sample; performing KASP using the isolated genomic DNA as a template and using the primer set of claim 1; and analyzing the amplification product of the KASP; provides a method for determining a cultivar or genotype of ginseng, including.

The mitochondria-derived KASP primer set of the present invention can discriminate Korean ginseng and foreign ginseng, as well as rapidly identify genotypes such as ginseng varieties, breeding lines, native species and foreign ginseng, and is an efficient biomarker for maintaining seed purity and identifying species. can be utilized. In addition, it can be used for precision breeding because it can quickly and accurately confirm the distinguishing and novelty of ginseng gene resources.

1 is a diagram schematically showing the mitochondrial genome map of ginseng.
2 is a graph showing the results of SNP genotype analysis using a KASP primer set.

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

The present invention provides SEQ ID NOs: 1 to 3; SEQ ID NOs: 4 to 6; SEQ ID NOs: 7 to 9; SEQ ID NOs: 10 to 12; SEQ ID NOs: 13 to 15; SEQ ID NOs: 16 to 18; SEQ ID NOs: 19-21; SEQ ID NOs: 22-24; SEQ ID NOs: 25-27; SEQ ID NOs: 28-30; SEQ ID NOs: 31-33; SEQ ID NOs: 34 to 36; SEQ ID NOs: 37-39; SEQ ID NOs: 40-42; and an oligonucleotide primer set represented by the nucleotide sequence of SEQ ID NOs: 43 to 45; provides a primer set for KASP (kompetiive allele specific PCR) for determining the cultivar, breeding line, or genotype of ginseng, including.

In addition, the primer set may consist of two forward primers and one reverse primer, and preferably, a FAM or HEX fluorescent material is attached to the 5' end of the forward primer and a single nucleotide polymorphism (SNP) is attached to the 3' end of the forward primer. is bound to, and the reverse primer may consist of the same base pair as the allele of the single nucleotide polymorphism (SNP) portion of the forward primer.

In addition, the primers of the present invention are 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, depending on the sequence length of each primer. , an oligonucleotide consisting of a fragment of 24 or more, 25 or more, 26 or more consecutive nucleotides. For example, the primers (25 oligonucleotides) of SEQ ID NO: 1 are 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, It may include an oligonucleotide consisting of a fragment of 22 or more, 23 or more, 24 or more nucleotides. In addition, the primer may also include an addition, deletion or substitution of the nucleotide sequence of SEQ ID NOs: 1 to 45. The oligonucleotide primers of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40 and 43 are forward primers to which a fluorescent material FAM is attached, and SEQ ID NO: 2, The oligonucleotide primers of 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41 and 44 are forward primers to which HEX is attached, and SEQ ID NOs: 3, 6, 9, 12, The oligonucleotide primers of 15, 18, 21, 24, 27, 30, 33, 36, 39, 42 and 45 are reverse primers.

As used herein, the term “single nucleotide polymorphism (Single Nucleotide Variation)” is also called “single nucleotide polymorphism (SNP)”, which means polymorphism in a single base. That is, it refers to a case in which some bases in the entire genome are different for each chromosome in a certain group, and it is generally known that there is about one in 300 to 1000 bases, but is not limited thereto.

As used herein, the term "polymorphism" refers to a case in which two or more alleles exist at one locus, and among polymorphic sites, only a single base differs depending on the individual. It is called a single nucleotide polymorphism. Preferred polymorphic markers have two or more alleles exhibiting an incidence of at least 1%, more preferably at least 5% or 10% in a selected population.

As used herein, the term “allele” refers to several types of one gene present at the same locus of homologous chromosomes. Alleles are also used to indicate polymorphisms.

In addition, the ginseng may be Korean ginseng or foreign ginseng. The foreign ginseng may be American ginseng ( Panax quinquefolius ) or Jeonchilseng ( Panax notoginseng ), but is not limited thereto.

In addition, the Korean ginseng ( Panax ginseng ) is Chunpoong, Yeonpung (Yunpoong), Gopoong (Gopoong), Seonpoong (Sunpoong), Kumpoong (Kumpoong), Seonun (sunun), Seonwon (sunone), Cheongsun (cheongsun), It may be selected from the group consisting of sunhyang and cheonryang.

The breeding line of ginseng is G03001, G03002, G03069, G03127, G03141, G03187, G04001, G04002, G04007, G04014, G04120, G050004, G050007, G050095, G050097, G060001, G060002, G060003, G070001, G07000 G070057, G08001, G08002, G08006, G08009, G08065, G08066, G08075, G09001, G09043, G09127, G09185, G10004, G10005, G10016, G10033, G10064, G11001, G11002, G11011, G11047, G11049, G12001, G12002 can, but is not limited thereto.

The term "Kompetitive allele specific PCR (KASP)" used in the present invention is one of PCR-based analysis methods, and is a homogenous and fluorescence-based genotyping analysis technique. KASP is a technology based on fluorescence resonance energy transfer for allele-specific oligo extension and signal generation.

In addition, the primer set for KASP is a primer set for detecting the nucleotide type of a single nucleotide polymorphism (SNP) specifically differentiated at the locus of ginseng.

In addition, as for the KASP primer set, the 15 oligonucleotide primer sets shown in SEQ ID NOs: 1 to 45 amplify a region including the SNP present in the mitochondrial genome sequence.

In the case of the mitochondrial-derived SNP marker of the present invention, it has a significantly higher intracellular copy number compared to the existing nuclear-derived marker, so that even using a commercially available extraction product, it can be extracted at a high yield, The mitochondrial genome has the advantage of avoiding the complexity of nuclear genome because recombination does not occur. Therefore, even if the experiment is performed with a minimum PCR cycle (less than 30), it is possible to easily determine the distinguishability of the mutation. In addition, if the primer set of the present invention is used, it can be applied to large-capacity analysis equipment, and 150,000 points can be analyzed per day, so that the analysis period can be shortened to one-tenth compared to when using the existing DNA oligomer primer set. .

As used herein, the term “primer” refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and can serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required.

In the present invention, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included. In addition, the primer may incorporate additional features that do not change the basic properties of the primer to serve as the starting point of DNA synthesis. If necessary, the primer nucleic acid sequence of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means.

The suitable length of the primer is determined by the characteristics of the primer to be used, but is usually used in a length of 15 to 30 bp. The primer need not be exactly complementary to the sequence of the template, but must be complementary enough to form a hybrid complex with the template.

In addition, when the primer set of the present invention is used simultaneously, the genotypes of a total of 79 ginseng gene resources including the genotypes of 59 ginseng gene resources and 20 foreign ginseng (P. quinquefolius, P. notoginseng) including 12 Korean ginseng varieties. can be distinguished.

Subsequently, the present invention provides SEQ ID NOs: 25 to 27; SEQ ID NOs: 28-30; And oligonucleotide primer set represented by the nucleotide sequence of SEQ ID NOs: 31 to 33; Korean ginseng ( Panax ginseng ), American ginseng ( Panax quinquefolius ) and ginseng ( Panax notoginseng ) KASP (kompetiive allele specific PCR) for discrimination, including primers provides a set.

The primer set of the present invention can discriminate Korean ginseng ( Panax ginseng ), American ginseng ( Panax quinquefolius ) and Korean ginseng ( Panax notoginseng ) using three oligonucleotide primer sets represented by the nucleotide sequences of SEQ ID NOs: 25 to 33 . .

Since the primer set of the present invention includes the above-described primer set, the description of overlapping content with the primer set of the present invention is omitted in order to avoid excessive complexity of the present specification due to the description of the overlapping content.

In addition, the present invention provides a kit for KASP for determining the cultivar or genotype of ginseng including the primer set and a reagent for performing the amplification reaction.

In addition, the reagent for performing the amplification reaction may include, but is not limited to, DNA polymerase, dNTPs, and a buffer. The dNTPs include dATP, dCTP, dGTP, and dTTP, and the DNA polymerase may use a commercially available polymerase such as Taq DNA polymerase or Tth DNA polymerase as a heat-resistant DNA polymerase. In addition, the kit of the present invention may further include a user's manual describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and PCR buffer, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.

Finally, the present invention comprises the steps of isolating genomic DNA from a ginseng sample; performing KASP using the isolated genomic DNA as a template and using the primer set of claim 1; and analyzing the amplification product of the KASP; provides a method for determining a cultivar or genotype of ginseng, including.

Since the discrimination method of the present invention includes the primer set described above, the description of overlapping content with the primer set of the present invention is omitted in order to avoid excessive complexity of the present specification due to the description of the overlapped content.

The method of the present invention includes isolating genomic DNA from a ginseng sample. A method for isolating the genomic DNA may use a method known in the art, for example, the CTAB method may be used, or a Wizard prep kit (Promega) may be used. The target sequence may be amplified by performing an amplification reaction using the isolated genomic DNA as a template and using the 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, and transcription-based amplification systems. amplification system), strand displacement amplification or amplification via Qβ 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 primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be a material emitting fluorescence, phosphorescence, or radioactivity, but is not limited thereto. Preferably, the labeling material may be FAM, HEX, VIC, JOE, ROX, TAMRA, Cy3 or Cy5. When PCR is performed by labeling the labeling material at the 5'-end of the primer during amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material.

In addition, the step of analyzing the amplification product of KASP may be performed through genotyping. Specifically, ginseng varieties can be distinguished by genotype discrimination through the fluorescent material FAM or HEX attached to the 5' end of the forward primer and the SNP bound to the 3' end. If the genotype is homozygous, only one of the two fluorescence (FAM or HEX) fluorescence is detected. If HEX is detected, the genotype is 'AA', and if FAM is detected, the genotype is 'BB'. If it is heterozygous, both fluorescence (FAM or HEX) are detected and it can be determined as genotype 'AB'.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only intended to materialize the contents of the present invention, and the present invention will not be limited thereby.

<Example 1> Mitochondrial genome analysis of ginseng

The leaves of the 5-year-old 'Geumpung' variety were collected from the ginseng field of the Ginseng Special Production Department of the National Institute of Horticultural and Herbal Science. The obtained leaf sample was washed with tap water to remove soil and foreign matter from the root surface, and then the sample was ground using liquid nitrogen and a mortar and DNA was extracted at 100 mg. DNA was extracted using the modified CTAB method, and the method is as follows.

8.6% CTAB, 5M NaCl, 5% sarcosyl stock, PVP (final 2%), 2-mercaptoethanol (final 1%) was added to 100 mg of the ground plant, and incubated in a water bath at 65 degrees for 15 minutes. Then, 400 μl of PCI was added to the lysate and centrifuged at 20°C for 10 minutes. 60 μl of 3M NaOAC (PH5.2) was placed in a new tube, and 400 μl of the layer separated through PCI was carefully picked and transferred to a new tube. After 1ml of ice cold lsopropanol was added, incubated at -80°C for 30 minutes and centrifuged at 4°C for 12 minutes. The supernatant was all discarded, 1 ml of ice cold 70% EtOH was added, and centrifuged at 4°C for 5 minutes. After that, 50 μl of DW was added, the pellet was dissolved, and whole genome NGS analysis was performed using genomic DNA. The base sequence of ginseng was deciphered using Illumina's MiSeq and Oxford Nanopore Technologies (ONT)'s GridION equipment, respectively. The amount of Illumina raw data is about 5.58 Gbp, and the CLC quality trim (ver. 4.21) program in the CLC Assembly Cell package (version 4.2.1, https://www.qiagenbioinformatics.com/products/clc-assembly-cell/) was used. Through the trimming process, only high-quality sequences (Phred score of > 20) were used for analysis. The amount of ONT raw data is about 6.78 Gbp, and it was used for analysis after removing the adapter trimming process using Porechop sw and sequencing control lambda DNA sequence using NanoLyse sw. De novo assembly was performed through the SMARTdenovo (https://github.com/ruanjue/smartdenovo) assembler using ONT trimmed reads, and only contigs derived from the mitochondrial genome were then selected. Afterwards, the Illumina trimmed reads were polished using the clc read mappiong program.

As a result, the length of the completed mitochondrial genome sequence was found to be 464,705 bp, aligned reads were confirmed to be Illumina 293,142, ONT 29,150, and the organelle genome average coverage was Illumina 165.41 X, ONT 110.81 X. To determine the gene site of the completed mitochondrial genome sequence, the primary gene site was identified through the GeSeq program, and the final gene site was confirmed by performing manual curation based on BLAST search. The identified genomic regions of the mitochondrial genome were displayed on the circlar genome map through the OGDraw program (FIG. 1). The mitochondrial genes of ginseng are shown in Table 1.

Function Genes Complex I
(NADH dehydrogenase) nad1*,T, nad2*, nad3, nad4*, nad4L, nad5*,T, nad6, nad7*, nad9 Complex II
(succinate dehydrogenase) sdh3, sdh4 Complex III
(ubiquinol-
cytochrome c reductase) cob Complex IV
(cytochrome c oxidase) cox1, cox2*, T, cox3 Complex V
(ATP synthase) atp1, atp4, atp6, atp8, atp9, atpE Cytochrome c biogenesis ccmB, ccmC, ccmFc*, ccmFn Large subunit
ribosomal proteins rpl10, rpl14, rpl16, rpl22 Small subunit
ribosomal proteins rps1, rps10, rps11, rps12, rps3, rps4, rps7, rps8 Maturase matK, matR Ribosomal RNAs rrn18, rrn26, rrn5 Transfer RNAs trnA-UGC*, trnC-GCA, trnD-GUC, trnE-UUC, trnF-AAA, trnF-GAA, trnG-GCC, trnH-GUG, trnI-GAU, trnK-CUU, trnK-UUU, trnM-CAU -GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnS-GCU, trnS-UGA, trnStop-UUA*, trnT-GGU, trnV-GAC, trnV-UAC*, trnW-CCA, trnY-GUA other genes infA, petD, psbA, psbD, rpoA, tatC

<Example 2> Mitochondrial genome mutation confirmation

Sequence alignment using MAFFT sequence alignment (version7, https://mafft.cbrc.jp/alignment/server/index.html) for Panax ginseng mitochondrion sequence (GenBank accession KF735063) treated with UNVERIFIED in NCBI for mutation detection was performed.

After that, 213 SNPs were identified as a result of checking the SNP variation using MsaToVcf (http://lindenb.github.io/jvarkit/MsaToVcf.html) and in-house perl script.

<Example 3> Development and application of KASP markers

A total of 15 sets of KASP primers were synthesized by selecting a site that can be designed as a primer based on the secured SNP. The primer set consisted of three consecutive oligonucleotides of SEQ ID NO: 3 forming one primer set, and two forward primers and one reverse primer. Specifically, a FAM or HEX fluorescent material is attached to the 5' end of the forward primer and a base representing SNP is bound to the 3' end of the forward primer, and the reverse primer was configured with the same base pair to the allele of the SNP portion of the forward primer. The primer sets constructed in the present invention are shown in Table 2.

Then, the genotypes of 59 ginseng gene resources and 20 foreign ginsengs (P. quinquefolius, P. notoginseng) including 12 Korean ginseng varieties were analyzed using 15 sets of KASP primers in Table 2 below (Table 3).

For analysis, make a final volume of 5 μl by mixing 0.068 μl of primer with 1 ng DNA, FAM or HEX fluorescent material attached, 2.5 μl of KASP reaction mix (ROX) and 2.43 μl of sterile water, and then dispensing in large quantities. Using equipment (Nexar, Douglas Scientific LLC, USA), it was aliquoted into a silicon PCR plate. The aliquoted PCR plates were sealed using an automatic sealing device (ABgene ALPS 3000, Thermo Scientific, USA). The sealed plate was subjected to gene amplification using a large-capacity PCR equipment (Soellex, Douglas Scientific LLC, USA). For initial denaturation, the amplification conditions were repeated at 94 °C for 10 min, denaturing at 94 °C for 1 min, synthesizing at 57 °C for 30 sec, and elongating at 72 °C for 30 sec 30 times. After that, the final renal reaction was analyzed at 72 °C for 5 minutes. Thereafter, genotyping was performed by measuring the fluorescence intensity of FAM or HEX of each sample using a high-capacity/high-speed fluorescence analyzer (Araya, Douglas Scientific LLC, USA). If the genotype is homozygous, only one of the two fluorescence (FAM or HEX) fluorescence is detected. If HEX is detected, the genotype is 'AA', and if FAM is detected, the genotype is 'BB'. In the case of a heterozygous (heterozygous), both fluorescence (FAM or HEX) were detected and it was determined as genotype 'AB'.

The results are shown in Table 4 and FIG. 2 .

2 is a graph showing the results of SNP genotype analysis using a KASP primer set.

As shown in Table 4 and FIG. 2, genotype analysis using 15 sets of KASP primers confirmed that the genotype was divided into 11 genogroups. Specifically, the SNP combination genotypes of the 11 groups are 'TCTAGTACTTACAAT', 'GAGCTGCATTAACAT', 'GAGTTGCATTAACAT', 'TAGCTGCATTAAAAT', 'TAGCTTAATTAAAAT', 'TATAAGTAAATTAAAAT', 'TATATTAAGTGAACC', 'TATAATTAGTAT' , was identified as 'TNTATTAAGCGANCC'. In addition, it was confirmed that 3 breeding lines (G03001, G03002, G04002) out of 79 genetic resources had a unique genotype that was distinguished from other resources. The SNP combination genotype of G03001 was identified as ‘TCTAGTACTTACAAT’, G03002 as GAGTTGCATTAACAT’, and G04002 as TCTAGTACTTAAAAT, demonstrating genetic differentiation and novelty from varieties and other breeding lines. In addition, it was confirmed that the three types (P. ginseng, P. quinquefolius, and P. notoginseng) were clearly distinguished through the three markers of Pgmt139, Pgmt157, and Pgmt169.

These results mean that the 15 types of KASP markers of the present invention can be applied to identify Korean ginseng and two types of foreign ginseng, and to prove the genetic novelty of ginseng genetic resources and differentiation from existing varieties.

No. Name Origin Classification Remarks One G03001 ENG Breeding line purple stem 2 G03002 ENG Breeding line purple stem 3 G03069 ENG Breeding line purple stem 4 G03127 ENG Breeding line yellow seed coat 5 G03141 ENG Breeding line purple stem 6 G03187 ENG Breeding line purple stem 7 G04001 ENG Breeding line purple stem 8 G04002 ENG Breeding line purple stem 9 G04007 ENG Breeding line purple stem 10 G04014 ENG Breeding line yellow seed coat 11 G04120 ENG Breeding line Orange seed coat 12 G050004 ENG Breeding line purple stem 13 G050007 ENG Breeding line purple stem 14 G050095 ENG Breeding line yellow seed coat 15 G050097 ENG Breeding line purple stem 16 G06001 ENG Breeding line purple stem 17 G060002 ENG Breeding line purple stem 18 G060003 ENG Breeding line purple stem 19 G070001 ENG Breeding line purple stem 20 G070003 ENG Breeding line purple stem 21 G070007 ENG Breeding line purple stem 22 G070031 ENG Breeding line Orange seed coat 23 G070057 ENG Breeding line Orange seed coat 24 G08001 ENG Breeding line purple stem 25 G08002 ENG Breeding line purple stem 26 G08006 ENG Breeding line yellow seed coat 27 G08009 ENG Breeding line yellow seed coat 28 G08065 ENG Breeding line green stem 29 G08066 ENG Breeding line green stem 30 G08075 ENG Breeding line Orange seed coat 31 G09001 ENG Breeding line purple stem 32 G09043 ENG Breeding line purple stem 33 G09127 ENG Breeding line green stem 34 G09185 ENG Breeding line Orange seed coat 35 G10004 ENG Breeding line purple stem 36 G10005 ENG Breeding line purple stem 37 G10016 ENG Breeding line Orange seed coat 38 G10033 ENG Breeding line yellow seed coat 39 G10064 ENG Breeding line green stem 40 G11001 ENG Breeding line purple stem 41 G11002 ENG Breeding line purple stem 42 G11011 ENG Breeding line purple stem 43 G11047 ENG Breeding line Orange seed coat 44 G11049 ENG Breeding line Orange seed coat 45 G12001 ENG Breeding line purple stem 46 G12002 ENG Breeding line purple stem 47 G12014 ENG Breeding line purple stem 48 Chunpoong ENG Cultivar 49 Yunpoong ENG Cultivar 50 Gopoong ENG Cultivar 51 Sunpoong ENG Cultivar 52 Gumpoong ENG Cultivar 53 Sunun ENG Cultivar 54 Sunone ENG Cultivar 55 Cheongsun ENG Cultivar 56 Sunhyang ENG Cultivar 57 Cheonryang ENG Cultivar 58 Kowon ENG Cultivar 59 cheonmyeong ENG Cultivar 60 P. quinquefolius_ 1 USA American access 61 P. quinquefolius_ 2 USA American access 62 P. quinquefolius_ 3 USA American access 63 P. quinquefolius_ 4 USA American access 64 P. quinquefolius_ 5 USA American access 65 P. quinquefolius_ 6 USA American access 66 P. quinquefolius_ 7 USA American access 67 P. quinquefolius_ 8 USA American access 68 P. quinquefolius_ 9 USA American access 69 P. quinquefolius_ 10 USA American access 70 P. notoginseng_ 1 CHN Chinese access 71 P. notoginseng_ 2 CHN Chinese access 72 P. notoginseng_ 3 CHN Chinese access 73 P. notoginseng_ 4 CHN Chinese access 74 P. notoginseng_ 5 CHN Chinese access 75 P. notoginseng_ 6 CHN Chinese access 76 P. notoginseng_ 7 CHN Chinese access 77 P. notoginseng_ 8 CHN Chinese access 78 P. notoginseng_ 9 CHN Chinese access 79 P. notoginseng_ 10 CHN Chinese access

As described above, although specific embodiments of the present invention have been described in detail, those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, and other degenerate inventions However, other embodiments included within the scope of the present invention may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the claims described later rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> KASP primer set derived on mitochondira for classifying Panax ginseng cultivar and genotype and uses thereof <130> 2020-0448-10-C <160> 45 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pgmt17 Allele-specific primer - FAM <400> 1 gagtcgcccc actcacttga g 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pgmt17 Allele-specific primer - HEX <400> 2 agagtcgccc cactcacttg at 22 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pgmt17 Common primer <400> 3 taggcaagtg ggaaacaagg aattgaatt 29 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> pgmt20 Allele-specific primer - FAM <400> 4 cgttgatatg caaaacagag gaaaagat 28 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pgmt20 Allele-specific primer - HEX <400> 5 gttgatatgc aaaacagagg aaaagag 27 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt20 Common primer <400> 6 gagtgcgcga aggtacaatc ctaaa 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt38 Allele-specific primer - FAM <400> 7 tgaggctttc tttcccttat tagtc 25 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pgmt38 Allele-specific primer - HEX <400> 8 gttgaggctt tctttccctt attagta 27 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> pgmt38 Common primer <400> 9 gaacgtcccg cgccccctt 19 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt48 Allele-specific primer - FAM <400> 10 agtcgataat aaggtcagct acctg 25 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pgmt48 Allele-specific primer - HEX <400> 11 aagtcgataa taaggtcagc tacctt 26 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt48 Common primer <400> 12 atgcgaaggc tcacctcatt ggaaa 25 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pgmt57 Allele-specific primer - FAM <400> 13 gcccgttact tcatcaagat agactt 26 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt57 Allele-specific primer - HEX <400> 14 cccgttactt catcaagata gactg 25 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt57 Common primer <400> 15 ttgcacttgg tggtatcccg acttt 25 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt93 Allele-specific primer - FAM <400> 16 atctcgtcta ataagaagag cgctc 25 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pgmt93 Allele-specific primer - HEX <400> 17 aaatctcgtc taataagaag agcgcta 27 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pgmt93 Common primer <400> 18 cgagactagc gtgatgtaag acagaa 26 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pgmt130 Allele-specific primer - FAM <400> 19 gagataagga gctagccttt tagaag 26 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pgmt130 Allele-specific primer - HEX <400> 20 agagataagg agctagcctt ttagaat 27 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> pgmt130 Common primer <400> 21 aggtactagt gacttcttgc atctcaaa 28 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pgmt134 Allele-specific primer - FAM <400> 22 aggctttttt tatagattaa gaggtgagta 30 <210> 23 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pgmt134 Allele-specific primer - HEX <400> 23 ggcttttttt atagattaag aggtgagtc 29 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt134 Common primer <400> 24 agttggggct ctttggtctt ccatt 25 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pgmt139 Allele-specific primer - FAM <400> 25 aaaaccgggt ccagaaggag ag 22 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pgmt139 Allele-specific primer - HEX <400> 26 caaaaccggg tccagaagga gat 23 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt139 Common primer <400> 27 ccccgataaa atccctttcc agctt 25 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pgmt157 Allele-specific primer - FAM <400> 28 gaaagagcaa taaaccggac cag 23 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt157 Allele-specific primer - HEX <400> 29 aggaaagagc aataaaccgg accaa 25 <210> 30 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt157 Common primer <400> 30 acggagggac cgtttcgttt ttgta 25 <210> 31 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> pgmt169 Allele-specific primer - FAM <400> 31 tttttcttag tttagtcaat ttctcatgaa ag 32 <210> 32 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> pgmt169 Allele-specific primer - HEX <400> 32 ttttcttagt ttagtcaatt tctcatgaaa a 31 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt169 Common primer <400> 33 cacacggttc ctttaccccc tttta 25 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pgmt198 Allele-specific primer - FAM <400> 34 atagaactcc tagctctgga gct 23 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pgmt198 Allele-specific primer - HEX <400> 35 agaactccta gctctggagc g 21 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pgmt198 Common primer <400> 36 gcgaagcctt tcggtagctg ttaaa 25 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pgmt199 Allele-specific primer - FAM <400> 37 tggtttttct ctttctacgt tcttcc 26 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pgmt199 Allele-specific primer - HEX <400> 38 gtttggtttt tctctttcta cgttcttca 29 <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pgmt199 Common primer <400> 39 cttctagcaa ctaagcactc ggacaa 26 <210> 40 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pgmt200 Allele-specific primer - FAM <400> 40 atattcgtaa agcagaaaca tagacgc 27 <210> 41 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> pgmt200 Allele-specific primer - HEX <400> 41 catattcgta aagcagaaac atagacga 28 <210> 42 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pgmt200 Common primer <400> 42 gactaatccg gtatattttc cacgttcat 29 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> pgmt213 Allele-specific primer - FAM <400> 43 aaattgactt attatactcc tgactatgac 30 <210> 44 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> pgmt213 Allele-specific primer - HEX <400> 44 caaattgact tattatactc ctgactatga t 31 <210> 45 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> pgmt213 Common primer <400> 45 ggaatgctgc caagatatca gtatcttt 28

Claims (8)

SEQ ID NOs: 1 to 3; SEQ ID NOs: 4 to 6; SEQ ID NOs: 7 to 9; SEQ ID NOs: 10 to 12; SEQ ID NOs: 13 to 15; SEQ ID NOs: 16 to 18; SEQ ID NOs: 19-21; SEQ ID NOs: 22-24; SEQ ID NOs: 25-27; SEQ ID NOs: 28-30; SEQ ID NOs: 31-33; SEQ ID NOs: 34 to 36; SEQ ID NOs: 37-39; SEQ ID NOs: 40 to 42; and oligonucleotide primer sets represented by the nucleotide sequences of SEQ ID NOs: 43 to 45; KASP (kompetiive allele specific PCR) primer sets for ginseng varieties, breeding lines or genotype determination, including.
According to claim 1,
The primer set is a primer set for KASP (kompetiive allele specific PCR) for determining the variety, breeding line or genotype of ginseng, characterized in that it consists of two forward primers and one reverse primer.
According to claim 1,
The ginseng is Korean ginseng ( Panax ginseng ), American ginseng ( Panax quinquefolius ) and ginseng ( Panax notoginseng ), characterized in that, KASP (kompetiive allele specific PCR) primer set for ginseng breed, breeding line or genotype determination.
4. The method of claim 3,
The varieties of Korean ginseng are Chunpoong, Yunpoong, Gopoong, Sunpoong, Kumpoong, Sunun, Sunone, Cheongsun, and Sunhyang. And cheonryang (cheonryang), characterized in that it comprises at least one selected from the group consisting of, KASP (kompetiive allele specific PCR) primer set for ginseng breed, breeding line or genotype discrimination.
SEQ ID NOs: 25-27; SEQ ID NOs: 28-30; and oligonucleotide primer sets represented by the nucleotide sequences of SEQ ID NOs: 31 to 33; including, Korean ginseng ( Panax ginseng ), American ginseng ( Panax quinquefolius ) and ginseng ( Panax notoginseng ) for discrimination KASP (kompetiive allele specific PCR) primer set.
A kit for KASP for genotype or cultivar of ginseng, comprising the primer set of claim 1 and a reagent for performing an amplification reaction.
7. The method of claim 6,
The reagent for performing the amplification reaction is a kit for KASP for cultivar, breeding line or genotype determination of ginseng, characterized in that it comprises a DNA polymerase, dNTPs and a buffer.
isolating genomic DNA from the ginseng sample;
performing KASP using the isolated genomic DNA as a template and using the primer set of claim 1; and
Analyzing the amplification product of the KASP; Containing, a method for determining a cultivar, breeding line or genotype of ginseng.

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