KR101481734B1 - Microsatellite marker of Korean Astragalus mongholicus and primer set for amplifying the same - Google Patents

Abstract

The present invention relates to a microsatellite marker of Hwanggi, which is cultivated in Korea, and a primer set for amplifying the same. The microsatellite markers of Hwanggi, which are cultivated in Korea according to the present invention, are polymorphic in Korea ( Astragalus mongholicus var. Dahuricus ) and A. endangered species ( A. mongholicus var. Nakaianus ) The use of light markers can be useful for molecular identification and biochemical studies of East Asian eruption, and also as a genetic tool for establishing conservation strategy of Jeju Emperor, which is a native species in Halla Mountain.

Description

Microsatellite marker of Korean astragalus mongholicus and primer set for amplifying the same.

The present invention relates to a microsatellite marker of Korean Yellow Sea era and a primer set for amplifying the same.

Population genetics is experiencing a dramatic development in recent years. In the 1960s, the main research area was to measure the frequency of heterozygosity and genetic polymorphism in natural populations using molecular markers such as allozyme. However, since the mid 1980s, sophisticated molecular biology techniques including polymerase chain reaction (PCR) have been developed to develop DNA-based molecular markers with abundant mutations such as microsatellite and AFLP (amplified fragment length polymorphism) And has been applied. In particular, microsatellite is a molecular marker that can be genotyped by simple PCR and electrophoresis, and has a very high polymorphism and is the most widely used marker in population genetics. Using this, various population genetic analyzes such as individual identification, population structure and size variation are being performed.

On the other hand, Astragalus mongholicus Bunge is a medicinal plant belonging to Leguminosae Astragalus L. and is distributed in China, Japan and Korea and includes various varieties. The scientific name of Hwanggi, which is cultivated widely in Korea, is A. mongholicus var. It was proposed by Choi et al. (2013) in lieu of the scientific name A. membranaceus Bunge, which was recently used as an unfair name for dahuricus Podlech.

Hwanggi uses dried root as a medicine, and root contains saponin such as astragaloside I to VII, isoflavonoid and amino acid v-aminobutyric acid. The pharmacological effects of Hwanggi are known to be dilation of heart contraction and cardiac action, dilation of the renal blood vessels and systemic peripheral blood vessels, lowering of blood pressure, diuretic, sedation and uterine contraction. Recently, Are reported.

These same products as the gastrointestinal (adulterant) in accordance with the commercial value of the Astragalus root Astragalus genus A. hoangchy, A. chinesis, A. adsurgens , A. complantanus and also such a distribution. In addition, it is known that Hwanggi is different in the ingredients and efficacy contained in varieties and mountain regions, and Korean hwanggi is traded at a higher price in the market than Chinese hwanggi. Therefore, their distinctions have important economic and pharmacological implications.

Most of the studies on Hwanggi are based on morphological traits (Lee and Chung, 2004), ribosome DNA sequences (Ma et al., 2000; Yip and Kwan, 2006; Gao et al., 2010), SCAR markers (Lim et al. , 2007; Yang et al., 2011) and compound analysis (Ma et al., 2002). These approaches were useful in distinguishing hwanggi from other species, but did not show sufficient judgment to identify local variants.

On the other hand, Jeju Hwanggi ( A. mongholicus var. Nakaianus Choi & Choi) is a Korean specialty plant that grows in the Alpine grassland of Mt. Halla in Cheju Island. Because of the small number of individuals remaining in the very narrow area of Mt. Halla, this species has been designated as an endangered species by the Forest Service Agency designated CR (Korea National Arboretum, 2008). Recently, the habitat fragmentation of Mt. Halla (Kang et al., 2008) and the expansion of the subalpine grassland (Kim, 2006) have been a major risk factor for the survival of this plant. However, there is no known basic genetic information about the population of this plant, so it is difficult to establish an appropriate conservation strategy.

Accordingly, the present inventors have found 10 new microsatellite markers that exhibit polymorphism in A. mongholicus var. Dahuricus cultivated in Korea and A. mongholicus var. Nakaianus , an endangered plant, By providing a primer set, the present invention has been accomplished so as to be usefully used for molecular identification and biological systematic studies of East Asian yellow peaks using the microsatellite markers.

It is an object of the present invention to provide a microsatellite marker of Korean Acantha gigas, which is composed of any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30.

Another object of the present invention is a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; And a primer set for amplifying a microsatellite marker of Korean Yellow Emperor composed of any one of primer pairs selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.

It is still another object of the present invention to provide a PCR kit for screening Korean wild ginseng, comprising the primer set.

It is still another object of the present invention to provide a Korean proliferative stem selecting probe comprising any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30, or a complementary base sequence thereof.

It is still another object of the present invention to provide a microarray for screening Korean horseshoe, comprising a nucleotide consisting of any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30, a polypeptide encoded thereby, or cDNA thereof.

It is still another object of the present invention to provide a method for sequencing amplified DNA comprising the steps of: performing PCR using a primer set composed of the primer pairs and sequencing the amplified DNA and analyzing the amplified DNA in comparison with any microsatellite markers of SEQ ID NOS: 21 to 30 And to provide a screening method of Korean hwanggi.

The present invention provides a microsatellite markers of Korean Acantha gigas, comprising any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30.

Also, the present invention provides a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; and a primer set for amplifying a microsatellite marker of Korean Yellow Emperor composed of any one of primer pairs selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.

In addition, the present invention provides a PCR kit for screening Korean wild ginseng, comprising the above primer set.

Still another object of the present invention is to provide a probe for screening Korean wild-type eggs containing any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30, or a complementary base sequence thereof.

The present invention also provides a microarray for screening Korean wild-type mice comprising a nucleotide consisting of any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30, a polypeptide encoded thereby, or cDNA thereof.

The present invention also provides a method for amplifying DNA comprising the steps of: performing PCR using a primer set composed of the primer pairs, sequencing the amplified DNA, and analyzing the amplified DNA in comparison with any microsatellite markers of SEQ ID NOS: It provides a screening method of Korean hwanggi.

The microsatellite markers of Korean Yellow Emperor according to the present invention exhibit polymorphism in Astragalus mongholicus var. Dahuricus cultivated in Korea and A. mongholicus var. Nakaianus , an endangered plant, Can be used for molecular identification and biochemical studies of East Asian era, and it can also be used as a genetic tool for establishing conservation strategy of Jeju Emperor which is native to Halla Mountain.

The present invention provides a microsatellite marker of Korean Acantha giganteus comprising at least one nucleotide sequence selected from SEQ ID NOS: 21 to 30.

Also, the present invention provides a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; and a primer set for amplifying the microsatellite markers of Korean Yellow Emperor composed of any one of primer pairs selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.

Hereinafter, the present invention will be described in detail.

The present invention relates to ten new microsatellite markers that exhibit polymorphism in Korean horses ( Astragalus mongholicus var. Dahuricus ) and endangered plants ( A. mongholicus var. Nakaianus ) and a primer set for amplifying them , 10 DNA microsatellite loci were confirmed by cloning a DNA fragment containing the microsatellite, designing a primer for amplifying the microsatellite and optimizing the PCR conditions. The microsatellite genotype , The microsatellite markers and primers can be usefully used for molecular identification and biochemical studies on the East Asian era, and also as a genetic tool for establishing a conservation strategy of Jeju Emperor naturally grown in Mt. Lt; / RTI >

The term 'microsatellite' used in the present invention means that 1-5 short nucleotide sequences are repeated, and has a very high polymorphism in the number of repeated sequences, It is recognized as the most preferred marker for analysis. These molecular markers can be used to perform a variety of highly reliable assays by applying simple PCR techniques and utilizing a myriad of gene loci that follow Mendel's genetic rules. Therefore, it has very high discrimination power and information power compared to isozyme and protein polymorphism used in past research. It has the advantage of being able to process large amounts of data because it can semi-automate processes such as scoring and genotyping.

As used herein, the term 'primer' refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. Primers can initiate DNA synthesis in the presence of reagents (DNA polymerase or reverse transcriptase) for polymerisation and four different dNTPs (deoxynucleoside triphosphate) at appropriate buffer solutions and temperatures.

Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", substitution of one or more natural nucleotides into homologues, and modifications between nucleotides, such as uncharged linkers such as methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents.

In addition, the primer nucleic acid sequences of the present invention may, if necessary, comprise markers detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (e.g., horseradish oxidase, alkaline phosphatase), radioactive isotopes (e.g., ³² P), fluorescent molecules, chemical groups (such as biotin), and the like.

"Sorting" in the present invention means selecting a cloned colonies comprising microsatellite markers genes of Korean wild-type.

In addition to the primer set described above, the PCR kit for screening Korean kochuji can include conventional components included in the PCR kit. Typical components included in the kit may be reaction buffers, polymerases, dNTPs (dATP, dCTP, dGTP and dTTP), and joins such as Mg ²⁺ . A variety of DNA polymerases can be used in the amplification step of the present invention, including the Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) . Most of these enzymes can be isolated from the bacteria itself or commercially available. In addition, the polymerase used in the present invention can be obtained from cells expressing high levels of the cloning gene encoding the polymerase. When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component.

All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants.

In addition, the present invention provides a selection probe of Korean wild-type group consisting of any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30, or a complementary base sequence thereof.

In the present invention, 'probe' refers to a hybridization probe, and refers to an oligonucleotide capable of binding sequence-specifically to the complementary strand of a nucleic acid. Using this, the genomic DNA of the Huangji period can be extracted, and any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30 or a complementary base sequence thereof can be hybridized with the genome DNA of the Huangguan as a probe.

The nucleotide sequences of SEQ ID NOS: 21 to 30 are polymorphic sequences. A polymorphic sequence refers to a sequence comprising a polymorphic site representing a SNP in a polynucleotide sequence. The polynucleotide may be DNA or RNA.

In addition, the present invention can provide a microarray comprising any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30, or a complementary base sequence thereof.

The microarray according to the present invention can be produced by a conventional method known to those skilled in the art.

For example, the nucleotide may be immobilized on a substrate coated with an activator selected from the group consisting of aminosilane, poly-L-lysine and aldehyde. In addition, the substrate may be selected from the group consisting of a silicon wafer, glass, quartz, metal, and plastic. The method of immobilizing the polynucleotide on a substrate may be a micropipetting method using a piezoelectric method or a method using a pin-type spotter.

Also,

1) SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; Performing a PCR reaction by mixing a primer set composed of any one primer pair selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20 and a whole DNA sample extracted from the erythrocyte group;

2) DNA sequencing using the amplified DNA sample of step 1); And

3) analyzing the DNA sequencing in the step 2) and analyzing it in comparison with any one of the microsatellite markers of SEQ ID NOS: 21 to 30;

, Which provides a method for selecting Korean hulls.

Hereinafter, the method for selecting Korean horseshoe crab according to the present invention will be described in detail.

The step 1) is a step of performing a PCR reaction on the entire DNA extracted from the hull, and the "PCR reaction" means a reaction of amplifying a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT- , The method of Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822), the method of ligase chain reaction (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press LCR (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) WO 88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276) , Consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification Strand displacement amplifications 21 and 22 and loop-mediated isothermal amplification (U.S. Patent Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603) (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

The step 2) is a step of sequencing DNA using the amplified DNA. As a method for determining DNA sequence (order of arrangement of adenine, cytosine, guanine, thymine) which is widely used in DNA sequencing, there is a chemical analysis method -Gilbert method) and a chain termination method (chain termination method, Sanger method), but the method is not limited to this, and is well known in the art.

Step 3) is a step of comparing DNA sequencing analysis results. The microsatellite DNA marker amplified using the primer pair of the present invention is analyzed by a suitable method. For example, the amplification reaction products described above are subjected to gel electrophoresis, and the resultant bands are observed and analyzed to analyze characteristics of amplified microsatellite DNA markers.

In the present invention, a fragment of the amplified microsatellite DNA marker is analyzed to determine its genotype. For example, by analyzing the size of the amplification product that varies depending on the number of repeats of the microsatellite DNA marker, various genotypes are classified, and Korean erythrocytes are identified based on the statistical data of the classified genotypes. In other words, it accumulates information on genotypes of microsatellite DNA markers that appear or appear at high frequency in the hibernate period, and compares the genotypes obtained from the samples to be identified based on accumulated information with existing accumulated genotyping information, It is possible to identify the cultivars of the present invention with high reliability.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

[Example 1] Sample preparation and DNA extraction

The experimental materials were 30 Jeju Emperor (30 ‰), Northeast (36 ° 23'15 "N, 128 ° 47'12" E) in Jeongseon County, Gangwon Province, The samples were collected from a total of 43 individuals.

The present inventors extracted the genomic DNA from the wild-type plants of the Halla mountain population using G-spin ^TM IIp Kit (iNtRON, Seongnam, Korea) for plants.

[Example 2] Cloning and sequencing of DNA fragments containing microsatellite

To isolate alleles of microsatellite loci, Hammond et al. (1998) enrichment protocol was partially modified and implemented. DNA fragments obtained by digesting genomic DNA with Mbo I (Promega, Madison, Wis., USA) were ligated to SAUL linker (Hammond et al. 1998) using T4 DNA ligase (Promega) ligation). The DNA fragment _{(AG) 15, (AT)} 15, (AC) 15, (ATG) 10, (AGC) 10, (CAA) 10 and (ACAT) was coupled with seven biotin-labeled probes, such as ₇ , And was attached to magnetic beads (Streptavidin MagneSphere Paramagnetic Particles) (Promega).

DNA fragment was amplified and cloned using PCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA).

Thereafter, clones with 400 to 800 bp (base pair) insertion sites were selected. To determine if the selected 196 clones were included in the microsatellite, the sequence was analyzed with ABI 3730xl sequencer (Applied Biosystems) using BigDye Terminator version 3.1.

[Example 3] Primer design and optimization of PCR conditions

The present inventors excluded primers having short repeats or short insertion sites or primers close to the vectors. Because the primers did not meet the criteria of the present invention. Primer pairs were designed from FastBCR version 5.4.51 software (Kalendar et al. 2009) from 46 base sequences with microsatellite repeats and sufficient insertions.

M13 (-21) (5'-TGTAAAACGACGGCCAGT-3 ') sequence-tag method was used to label the primers (Schuelke 2000). Allelic loci were screened at early stage using 30 individuals of Jeju Emperor. DNA was extracted according to the method described above, and PCR was performed using GeneAmp ^占 PCR System 2700 Thermal Cycler (Applied Biosystems).

Each reaction mixture contained 10 μl of DNA and a total volume of 30 μl of dNTP (GeneCraft, Ludinghausen, Germany), 1 × PCR buffer containing 1.5 mM MgCl ₂ , 1 U of Taq DNA polymerase (TaKaRa, Seoul, Korea) Lt; RTI ID = 0.0 > uL < / RTI > The mixture also contained 0.08 μM of M13 (-21) -tag forward primer, 0.3 μM of reverse primer and 0.3 μM of M13 (-21) -labeled 6-FAM fluorescent dye. PCR conditions were: initial denaturation at 94 ° C for 2 minutes; 38 times at 94 캜 for 30 seconds; 45 sec (recovery temperature along gene locus) at 54 to 61 DEG C and 1 min at 72 DEG C; Final elongation at 72 캜 for 10 minutes was performed.

Astna1-F, Astna1-R, Astna2-F, Astna2-R, Astna3-F, Astna3- R (SEQ ID NO: 6), Astna4-F (SEQ ID NO: 7), Astna4-R (SEQ ID NO: 8), Astna5-F Astna6-R (SEQ ID NO: 12), Astna7-R (SEQ ID NO: 13), Astna7-F (SEQ ID NO: 14), Astna8- (SEQ ID NO: 17), Astna9-R (SEQ ID NO: 18), Astna10-F (SEQ ID NO: 19) and Astna10-R (SEQ ID NO: 20)

As shown in Table 1, M13 (5'-TGTAAAACGACGGCCAGT-3 ') was tagged at the 5' end of all the forward primers. After repeated adjustments, significant deviations from the Hardy-Weinberg equilibrium (HWE) were shown ( P <0.005). The P value for HWE is shown for each marker. [ T _a : PCR annealing temperature; N _a: the number of alleles in the population group; H _o : observed heterozygosity; H _e: expected heterozygosity].

Of the 46 pairs of primer pairs, 36 pairs of primers were excluded because of the difficulty of PCR amplification failure or data interpretation. The present inventors have found that A. mongholicus var. Nakaianus , native to Mt. Halla, Microsatellite representing 10 polymorphisms was generated from the plants, with clear and strong bands for each allele. Genotypic data of A. mongholicus var. Dahuricus and A. mongholicus var. Nakaianus were obtained. Parameters for genetic diversity in each population are shown in Table 1 above. Of these, Astna3 was not amplified in the Hwanggi cultivated in Korea. Overall, the number of alleles was 10 to 22 (mean 16.4), and the observed heterozygosity ( H _o ) and expected heterozygosity ( H _e ) of the allele were 0.385 to 0.900 And 0.432 to 0.942 ( P < 0.005).

[Example 4] Genotyping analysis of microsatellite

Fluorescently labeled PCR products were analyzed with GeneScan ^TM -500LIZ ^TM Size Standard (Applied Biosystems) and ABI 3730xl sequencer. The length of the allele was determined using GENEMAPPER version 3.7 (Applied Biosystems). Diversity statistics, deviations from Hardy-Weinberg equilibrium (HWE), and linkage disequilibrium (LD) were assessed using GENEPOP version 4.0 software (Rousset 2008). The frequency of null alleles was calculated by MICRO-CHECKER version 2.2.3 (Van Oosterhout et al. 2004). Astna1 (SEQ ID NO: 21), Astna2 (SEQ ID NO: 22), Astna2 (SEQ ID NO: 23), Astna4 (SEQ ID NO: 24), and the like were used as repetitive motifs of the microsatellite locus Astna5 (SEQ ID NO: 25), Astna6 (SEQ ID NO: 26), Astna7 (SEQ ID NO: 27), Astna8 (SEQ ID NO: 28), Astna9 (SEQ ID NO: 29) and Astna10 (SEQ ID NO: 30)); And allele exhibited a dielectric can be (N _a) of Table 1 above.

Three of the 10 allele loci (Astna2, Astna5, and Astna8) showed significant deviations in the modified Hardy-Weinberg equilibrium (HWE) after Bonferroni correction in the Hallasan population P < 0.005; Table 1). Based on Hardy-Weinberg equilibrium (Hardy-Weinberg equilibrium; HWE), significant differences were observed within the Hallasan population. All deviations from the Hardy-Weinberg equilibrium are due to defects in heterozygosity. This is interpreted as a result of induced high inbreeding rates due to the limited and fragmented population structure. Severe linkage disequilibrium (LD) was not observed in the gene locus pairs at all. Detailed allele locus characterization analysis and GenBank Accession Number are shown in Table 1 above.

These microsatellite markers grown in Korea will be a valuable tool for improving understanding of the process for identifying groups within the Hwanggi species complex that are cultivated in Korea in East Asia. Furthermore, the present inventor has completed the present invention by obtaining genetic information that will be very important for the conservation of Jeju Emperor, which is native to Mt.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Microsatellite marker of Korean Astragalus mongholicus and primer          set for amplifying the same <130> INHA-P92 <160> 30 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Astna1-Forword primer <400> 1 ggaatctgaa tagacgggaa a 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna1-Reverse primer <400> 2 ccagcactcg tacgcttttt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna2-Forword primer <400> 3 cgaaggtagg ggaaacatga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astn2-Reverse primer <400> 4 aaaaacgaag ctcccacttg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna3-Forword primer <400> 5 gcgaactaga gagggggttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna3-Reverse primer <400> 6 atgtaggcac actgttcatc 20 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Astna4-Forword primer <400> 7 ctacaccatc cttagaca 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Astna4-Reverse primer <400> 8 aagaacaaga cacagtgc 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Astna5-Forword primer <400> 9 cgccagtgtg agcaaaag 18 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna5-Reverse primer <400> 10 cacgcattcc atcatgtttc 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Astna6-Forword primer <400> 11 cgagcagtct gaccaggtg 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna6-Reverse primer <400> 12 tcacactccc tttcgctctc 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Astna7-Forword primer <400> 13 acatgtgccg tttggtctc 19 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Astna7-Reverse primer <400> 14 cctctccatc tccgaaaact t 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna8-Forword primer <400> 15 gacagttctg accgcttgac 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna8-Reverse primer <400> 16 ctggtattcc cgttgcacac 20 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Astna9-Forword primer <400> 17 tggtggatac tcacgtatcc tg 22 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Astna9-Reverse primer <400> 18 tgcaggcagt tgactttggg t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna10-Forword primer <400> 19 tttgatggac gcgaaaggtt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Astna10-Reverse primer <400> 20 tgccacaaag agcaactcaa 20 <210> 21 <211> 39 <212> DNA <213> Astragalus mongholicus <400> 21 atgatgatga tgatgatgat gatgatgatg atgatgatg 39 <210> 22 <211> 46 <212> DNA <213> Astragalus mongholicus <400> 22 agagagagag agagagagag agagagagag agagagagag agagag 46 <210> 23 <211> 56 <212> DNA <213> Astragalus mongholicus <400> 23 gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagaga 56 <210> 24 <211> 44 <212> DNA <213> Astragalus mongholicus <400> 24 tctctctctc tctctctctc tctctctctc tctctctctc tctc 44 <210> 25 <211> 60 <212> DNA <213> Astragalus mongholicus <400> 25 gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga 60                                                                           60 <210> 26 <211> 54 <212> DNA <213> Astragalus mongholicus <400> 26 gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gaga 54 <210> 27 <211> 56 <212> DNA <213> Astragalus mongholicus <400> 27 gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagaga 56 <210> 28 <211> 52 <212> DNA <213> Astragalus mongholicus <400> 28 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tg 52 <210> 29 <211> 50 <212> DNA <213> Astragalus mongholicus <400> 29 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 50 <210> 30 <211> 46 <212> DNA <213> Astragalus mongholicus <400> 30 gagagagaga gagagagaga gagagagaga gagagagaga gagaga 46

Claims (7)

A microsatellite marker of Erysipelas grown in Korea consisting of at least one nucleotide sequence selected from SEQ ID NOS: 21 to 30.
SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; A primer set for amplifying microsatellite markers of erythroblasts grown in Korea consisting of a pair of primers selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.
A PCR kit for screening Huanggi growing in Korea containing the primer set of claim 2.
31. A method for screening a stem cell of any one of SEQ ID NOS: 21 to 30, or a complementary base sequence thereof, which is cultured in Korea,
Wherein any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30 is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; And SEQ ID NO: 19 and SEQ ID NO: 20, wherein the primer pair is amplified by a primer pair selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.
delete A nucleotide consisting of any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30; A polypeptide encoded thereby; Or a cDNA thereof, which is cultivated in Korea,
Wherein any one of the nucleotide sequences selected from SEQ ID NOS: 21 to 30 is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; And SEQ ID NO: 19 and SEQ ID NO: 20, wherein the primer pair is selected from the group consisting of primers selected from the group consisting of:

1) SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; Performing PCR by mixing a primer set composed of any one primer pair selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20, and a whole DNA sample extracted from the erythrocyte group;
2) DNA sequencing using the amplified DNA sample of step 1); And
3) analyzing the DNA sequencing in the step 2) and analyzing it in comparison with any one of the microsatellite markers of SEQ ID NOS: 21 to 30;
, Which is cultivated in Korea.