KR102401157B1 - Composition for identifying species of peyote and identification method using the same - Google Patents

Abstract

The present invention relates to a composition for identification of a species of oleifera and a method for identification of a species of quince using the same, and more particularly, a composition capable of quickly and conveniently identifying the owl species from cacti by confirming the ochre-specific SNP base in a chloroplast gene, and a composition using the same It relates to a method for identifying the species of Ou-ok.

Description

A composition for identification of a species of oyster and a method for identification of a species of oyster using the same

The present invention relates to a composition for the identification of the species of oleander and a method for identification of the species of quince of the quince using the same.

Lophophora williamsii ( Lophophora williamsii ) is a type of cactus, also called peyote or peyote. It is native to Mexico and Texas, USA, and has hairs instead of thorns, and its flowers and leaves contain mescaline, a psychoactive alkaloid. Mescaline belongs to narcotics managed internationally through the Convention on psychotropic substances of the United Nations adopted in 1971, and its possession and distribution has been prohibited since 1970 by the Controlled Substances Act in the United States. In the Republic of Korea, it falls under subparagraph 3 (a) of Article 2 of the Narcotics Control Act, so just possessing it is an illegal act. Own and distribution of Ouok as well as mescaline is prohibited internationally.

Currently, methods performed to identify the species of oyster include morphological analysis through appearance and mescaline detection using GC mass. Among them, in the case of morphological analysis through the appearance, there is a problem in that Ouok is very similar to Chwigwanok ( Lophophora diffusa ) belonging to the same genus, and thus it is difficult to distinguish between the two. In addition, there is a problem that identification based on the appearance cannot be performed when the appearance is damaged. For this reason, although the mescaline detection method is generally applied to the determination of the oyster species, there is a problem in that the detected mescaline content or the presence or absence of mescaline detection varies depending on individual differences or growth period. Therefore, there is a need for the development of a new identification method that can identify the species with higher accuracy.

On the other hand, with the recent development of molecular biology, research is being conducted on a technology to search for polymorphism in a specific DNA nucleotide sequence and use it as a molecular marker. Molecular marker refers to a DNA sequence related to a specific gene or genomic locus and a material derived therefrom, and the development of molecular markers is a very important part in genetics, and research for its development is being actively conducted. RFLP (restriction fragment length polymorphism), PCR amplification method, RAPD (Random Amplified Polymorphic DNA), SCAR (Sequence Characterized Amplified Regions), AFLP (Amplified Fragment Length Polymorphism), a variety of methods such as single base extension (SBE) The identification method is used, and the marker developed through this method is usefully used for molecular biological identification of plant species.

DNA barcode is a genetic technique that compares the nucleotide sequences of specific gene regions. The rbcL (Large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) gene is widely used because there are many points consistent with the morphological classification. The matK (maturase K) gene has a high genetic evolution rate and is widely used because it has a conserved base sequence. Unlike the above two genes, trnL -trnF IGS (trnL (UAA)-trnF (GAA) IGS) belongs to a non-coding region. It is widely used in the development of genetic markers.

KR 101804120 B1

An object of the present invention is to provide a single nucleotide polymorphism (SNP) marker capable of discriminating a species of cactus Owok ( Lophophora williamsii ) and a single nucleotide polymorphism marker composition comprising the same.

In addition, it is another object to provide a composition for identifying oook that can detect or amplify the single nucleotide polymorphism marker.

In addition, another object of the present invention is to provide a method for identification of Ohok using the single nucleotide polymorphism marker.

According to one aspect of the present invention, the 235th position in the nucleotide sequence represented by SEQ ID NO: 1, the 1497th position in the nucleotide sequence represented by SEQ ID NO: 2, the 1502th position in the nucleotide sequence represented by SEQ ID NO: 2 and A single nucleotide polymorphism present at at least one position selected from the group consisting of the 285th position in the nucleotide sequence shown in SEQ ID NO: 3, and the 304th position in the nucleotide sequence shown in SEQ ID NO: 1, shown in SEQ ID NO: 3 Owok ( A method for identifying Lophophora williamsii ) is provided.

According to another aspect of the present invention, there is provided a method of identifying oook ( Lophophora williamsii ), which includes the step of identifying the base of the single nucleotide polymorphism located at the 476th position in the nucleotide sequence represented by SEQ ID NO: 3.

In addition, when the 235th base of SEQ ID NO: 1 is C, the 1497th base of SEQ ID NO: 2 is G, the 1502th base of SEQ ID NO: 2 is A, and the 285th base of SEQ ID NO: 3 is A When it is discriminated into the genus Popophora , the 304th base of SEQ ID NO: 1 is T, the 385th base of SEQ ID NO: 3 is G, and the 476th base of SEQ ID NO: 3 is identified as G Differentiation can be performed with a species of Lophophora ) among the genus Lophophora williamsii .

In addition, when the 476th base of SEQ ID NO: 3 is identified as G, differentiation may be performed with Ouok ( Lophophora williamsii ).

According to another aspect of the present invention, the method comprising the steps of isolating a nucleic acid molecule from a subject to be identified; performing multiplex PCR using the nucleic acid molecule as a template, primer sets of SEQ ID NOs: 12 and 13, primer sets of SEQ ID NOs: 14 and 15, and primer sets of SEQ ID NOs: 16 and 17 to obtain a first amplification product; performing a single base extension (SBE) method using the primers of SEQ ID NOs: 18 to 24 using the first amplification product as a template to obtain a second amplification product; and confirming the nucleotide sequence of the second amplification product.

In addition, the nucleotide sequence of the second amplification product may include a single nucleotide polymorphism in the nucleotide sequence represented by SEQ ID NOs: 1 to 3.

According to another aspect of the present invention, position 235 in the nucleotide sequence represented by SEQ ID NO: 1, position 1497 in the nucleotide sequence represented by SEQ ID NO: 2, position 1502 in the nucleotide sequence represented by SEQ ID NO: 2 And Lophophora genus, including an agent capable of detecting or amplifying a single nucleotide polymorphism marker present at at least one position selected from the group consisting of the 285th position in the nucleotide sequence represented by SEQ ID NO: 3 A composition for identifying a cactus is provided.

In addition, the agent can detect or amplify 10 to 500 consecutive nucleotide sequences including the single nucleotide polymorphism or a complementary nucleotide sequence thereof.

In addition, the preparation comprises at least one primer set selected from the group consisting of primer sets of SEQ ID NOs: 12 and 13, primer sets of SEQ ID NOs: 14 and 15, and primer sets of SEQ ID NOs: 16 and 17 or SEQ ID NOs: 18, 20, It may include at least one primer selected from the group consisting of primers 21 and 22.

According to another aspect of the present invention, the 304th position in the nucleotide sequence shown in SEQ ID NO: 1, the 385th position in the nucleotide sequence shown in SEQ ID NO: 3, and the 476th position in the nucleotide sequence shown in SEQ ID NO: 3 Including an agent capable of detecting or amplifying a single nucleotide polymorphism marker present in at least one position selected from the group consisting of A polymorphic marker composition for distinguishing between

In addition, the agent can detect or amplify 10 to 500 consecutive nucleotide sequences including the single nucleotide polymorphism or a complementary nucleotide sequence thereof.

In addition, the preparation comprises at least one primer set selected from the group consisting of a primer set of SEQ ID NOs: 12 and 13, a primer set of SEQ ID NOs: 14 and 15, and a primer set of SEQ ID NOs: It may include at least one primer selected from the group consisting of 24 primers.

The single nucleotide polymorphism marker for identification of Ogne according to an aspect of the present invention can be used as a molecular marker that appears specifically only in the species of Og by constructing a SNP profile of the species of Og, and information for more simple and high-accuracy identification of the species of Og. It can be applied to the delivery method.

According to another aspect of the present invention, the composition for identification of oleifera can easily identify owl species from other cacti by detecting or amplifying the single nucleotide polymorphism.

The method for identifying the species of Ouok according to another aspect of the present invention is the mescaline component detection method performed for the identification of the species of Ouok in the prior art by using a single nucleotide polymorphism (SNP) and an agent capable of detecting the same. Compared to the sequencing method, it is possible to identify the oook species more quickly and conveniently.

In addition, since the analysis is performed on a short DNA fragment within 300 bp, it is possible to analyze even an old or decomposed sample, multiple samples can be analyzed at once, and the process for identification is quick and the experiment and its interpretation are quick as the process is simplified. Since it can be done in time, it is possible to reduce the time and effort required for the identification of the quince species.

1 is a view showing the identification of the SNP site for the specific Lopopora genus at the rbcL gene position and for distinguishing between Ouok and Chwigwanok;
Figure 2 shows by confirming the specific SNP site of the genus Lopopora at the matK gene position,
Figure 3 shows by confirming the SNP site for the distinction between woook and chwigwanok at the trnL -trnF gene position,
4 is a result of confirming the multiplex PCR (multiplex PCR) result by electrophoresis,
Figure 5 shows the binding position of the SBE primer in each region;
6 is a result of confirming the SNP profile through multiplex mini-sequencing,
7 is an electropherogram result of multiplex mini sequencing.

The present invention relates to a marker for identifying the species of oyster, a composition for identification using the same, and a method for providing information.

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

Technical and scientific terms used in this specification may be interpreted as meanings commonly understood by those of ordinary skill in the art to which this invention belongs.

The term polymorphism used in the present invention means a case in which two or more alleles exist at one locus, and single nucleotide polymorphism (single nucleotide) that differs only in a single base from among polymorphic sites polymorphism (SNP).

Confirmation of the presence of the polymorphism may be performed through identification of nucleotides. In the present invention, identifying the nucleotide means identifying the type of the base of the nucleotide. Identification of a base or a base sequence may be used interchangeably with determination of a base or a base sequence.

According to one aspect of the present invention, there is provided an identification method for identifying a species of Lophophora genus Lophophora in a cactus. The identification method according to one aspect of the present invention may be performed using a genetic marker of the chloroplast of Ouok or a single nucleotide polymorphism (SNP) marker in the gene sequence.

The genetic marker may include at least one selected from the group consisting of rbcL , matK , and trnL-trnF IGS. The single nucleotide polymorphism marker may be associated with the gene and form a species-specific single nucleotide polymorphism profile. Specifically, position 235 in the rbcL sequence, position 304 in the rbcL sequence, position 1497 in the matK sequence, position 1502 in the matK sequence, position 285 in the trnL-trnF IGS sequence, the trnL- It may be at least one single nucleotide polymorphism selected from the group consisting of position 385 in the trnF IGS sequence and position 476 in the trnL-trnF IGS sequence. Here, the gene serving as a reference for the position of the single nucleotide polymorphism may be a gene derived from Ohok. Specifically, the rbcL sequence may be represented by SEQ ID NO: 1, the matK sequence may be represented by SEQ ID NO: 2, and the trnL-trnF IGS sequence may be represented by SEQ ID NO: 3.

The identification method according to an aspect of the present invention may be performed using a single nucleotide polymorphism for identification at a genus level and a single nucleotide polymorphism for identification at a species level. Specifically, a single nucleotide polymorphism that can identify the genus Lophophora among cacti, and a single nucleotide polymorphism that can identify the genus Lophophora and Lophophora williamsii and Lophophora diffusa within the genus. Polymorphisms can be used to discriminate eucalyptus species from cacti.

In an example of a method using a single nucleotide polymorphic marker, a polymorphic sequence including a polymorphic site may be utilized. The polymorphic sequence refers to a sequence including a polymorphic site in which a single nucleotide polymorphism is located among polynucleotide sequences. The polynucleotide sequence may be DNA or RNA.

The base of the polymorphic site can be identified by known means. The known means include, for example, sequencing, direct sequencing, mini-sequencing, dynamic allele-specific hybridization (DASH), PCR amplification. , allele specific PCR, Single Base Extension (SBE), restriction fragment length polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Sequence Characterized Amplified Regions (SCAR), AFLP ( Amplified Fragment Length Polymorphism), ligase reaction (LCR), hybridization by microarray, SNPlex platform, mass spectrometry, Molecular Inversion, Probe array, Bead Array and SNP chip from the group consisting of At least one selected may be used, but is not limited thereto. Identification of the base sequence may be performed using a gene analyzer. The gene analyzer may be based on capillary electrophoresis.

Here, the single base extension (SBE) method, also called the SNaPshot method, is a method used for genotyping a plurality of single base polymorphisms at a time. There is an advantage in that the cost required for the experiment is low and the experiment can be performed at once by multiplexing a large number of single nucleotide polymorphisms. In a preferred embodiment of the present invention, the base of the single nucleotide polymorphism can be identified from the result of SBE PCR.

The single nucleotide polymorphism capable of identifying the genus Lophophora is the 235th position in the 5' to 3' direction in the nucleotide sequence shown in SEQ ID NO: 1, and the 1497th position in the nucleotide sequence shown in SEQ ID NO: 2 It may be present at at least one position selected from the group consisting of the position, the 1502th position in the nucleotide sequence shown in SEQ ID NO: 2, and the 285th position in the nucleotide sequence shown in SEQ ID NO:3. When the 235th nucleotide of SEQ ID NO: 1 is C, the 1497th nucleotide of SEQ ID NO: 2 is G, the 1502th nucleotide of SEQ ID NO: 2 is A, and the 285th nucleotide of SEQ ID NO: 3 is A Differentiation can be performed into the genus.

The single nucleotide polymorphism capable of discriminating Owok and Chwigwanok in the genus Lophophora is the 304th position in the nucleotide sequence shown in SEQ ID NO: 1, the 385th position in the nucleotide sequence shown in SEQ ID NO:3 And it may be present at at least one position selected from the group consisting of the 476th position in the nucleotide sequence represented by SEQ ID NO: 3. When it is confirmed that the 304th base of SEQ ID NO: 1 is T, the 385th base of SEQ ID NO: 3 is G, and the 476th base of SEQ ID NO: 3 is G, the differentiation is not as a woook species in the genus Lopopora, not chwigwanok. can be performed.

Among the above-mentioned single nucleotide polymorphisms, the single nucleotide polymorphism at position 476 in the nucleotide sequence represented by SEQ ID NO: 3 is a owl-specific single nucleotide polymorphism that is distinguished from other cacti. There are advantages to being implemented.

According to another aspect of the present invention, there is provided a composition for identification in a cactus Lophophora genus Lophophora williamsii species for identifying. The composition may include a single nucleotide polymorphism marker for identification of the species Lopopora genus Ouok. In addition, the composition may include an agent capable of detecting or amplifying a single nucleotide polymorphism marker for the identification of a species of Lopopora genus Ouch. The above-described identification method may be performed using the composition.

In one embodiment of the present invention, the formulation is the 235th position in the nucleotide sequence represented by SEQ ID NO: 1, the 1497th position in the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: An agent capable of detecting or amplifying a single nucleotide polymorphism marker present at at least one position selected from the group consisting of the 1502th position in the nucleotide sequence represented by 2 and the 285th position in the nucleotide sequence represented by SEQ ID NO: 3 can be selected. Alternatively, the 235th position in the nucleotide sequence represented by SEQ ID NO: 1, the 1497th position in the nucleotide sequence represented by SEQ ID NO: 2, the 1502th position in the nucleotide sequence represented by SEQ ID NO: 2 and SEQ ID NO: 3 An agent capable of detecting or amplifying 10 to 500 consecutive nucleotide sequences containing a single nucleotide polymorphism present at at least one position selected from the group consisting of the 285th position in the nucleotide sequence or a complementary nucleotide sequence thereof is selected can be Here, the continuous nucleotide sequence may be derived from the sequence of SEQ ID NO: including the corresponding single nucleotide polymorphism, respectively.

In another example of the present invention, the formulation is the 304th position in the nucleotide sequence represented by SEQ ID NO: 1, within the nucleotide sequence represented by SEQ ID NO: 3, in order to distinguish between Ou-ok and Chwi-gwan-ok in the genus Lopopora. An agent capable of detecting or amplifying a single nucleotide polymorphism marker present at at least one position selected from the group consisting of position 385 and position 476 in the nucleotide sequence represented by SEQ ID NO: 3 may be selected. Or, at least selected from the group consisting of the 304th position in the nucleotide sequence represented by SEQ ID NO: 1, the 385th position in the nucleotide sequence represented by SEQ ID NO: 3, and the 476th position in the nucleotide sequence represented by SEQ ID NO: 3 An agent capable of detecting or amplifying 10 to 500 consecutive nucleotide sequences including a single nucleotide polymorphism present at one position or a complementary nucleotide sequence thereof may be selected. Here, the continuous nucleotide sequence may be derived from the sequence of SEQ ID NO: including the corresponding single nucleotide polymorphism, respectively.

The composition according to another aspect of the present invention may be applied to a kit or a microarray for identifying eucalyptus species from cacti, but is not limited thereto.

Hereinafter, preferred embodiments are presented to help the understanding of the present invention, but these examples are merely illustrative of the present invention and do not limit the appended claims, and are within the scope and spirit of the present invention. It is obvious to those skilled in the art that various changes and modifications are possible, and it is natural that such variations and modifications fall within the scope of the appended claims.

Example

<Sample collection>

The plant sample information used is shown in Table 1 below. In the mescaline item of Table 1 below, D means that mescaline was detected by performing the mescalin detection method on the sample, ND means that mescaline was detected but mescaline was not detected, and NT is Mescaline detection method was not performed.

division scientific name source mescaline L.w-1 Lophophora williamsii National Arboretum D L.w-2 Lophophora williamsii National forensic service evidence D L.w-3 Lophophora williamsii National Forensic Service evidence D L.w-4 Lophophora williamsii National Forensic Service evidence ND L.d-1 Lophophora diffusa Internet ND L.d-2 Lophophora diffusa Internet ND L.d-3 Lophophora diffusa Internet ND C1 Astrophytum myriostigma National Arboretum NT C2 Astrophytum ornatum National Arboretum NT C3 Copiapoa bridgesii National Arboretum NT C4 Copiapoa haseltoniana National Arboretum NT C5 Coryphantha difficilis National Arboretum NT C6 Echinocereus arizonicus National Arboretum NT C7 Echinopsis multiplex National Arboretum NT C8 Ferocactus flavovirens National Arboretum NT C9 Ferocactus latispinus National Arboretum NT C10 Gymnocalycium comarapeuse National Arboretum NT C11 Gymnocalycium mihanovichii National Arboretum NT C12 Gymnocalycium tilcarense National Arboretum NT C13 Gymnocalycium zegarrae National Arboretum NT C14 Gymnocalycium horsesei National Arboretum NT C15 Mammillaria decipiens National Arboretum NT C16 Mammillaria polythele National Arboretum NT C17 Matucana madisoniorum National Arboretum NT C18 Thelocactus ehrenbergii National Arboretum NT C19 Thelocactus rinconensis National Arboretum NT

Some tissues of the Korea National Arboretum, which were donated from the Munich Institute in Germany, were received and used (L.w-1). Three samples of Ohok (L.w-2,3,4) submitted for analysis to the National Forensic Service were used. For the use of Ohok, it has been approved by the Ministry of Food and Drug Safety in accordance with Articles 5 and 6 of the 「Enforcement Regulations of the Narcotics Control Act」.

Three samples (L.d-1,2,3) of Chwikwanok were purchased from the website (http://www.xplant.co.kr). In addition, for verification of the oook species identification system according to one aspect of the present invention, 19 species of cactus (Cactaceae) tissues (C1-19) belonging to 10 genera were used after being sold from the National Arboretum. The tissue was stored at -20°C without drying, and genomic DNA (gDNA) was extracted therefrom. Mescaline detection was analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (SCIEX, Concord, Canada).

<Extraction of DNA from plant tissue>

About 100 mg of plant-derived material, each collected from plant tissues, was pulverized with a bead beater for 3 minutes. DNA was extracted using the DNeasy Plant Mini Kit (QIAgen, Hilden, Germany) according to the manufacturer's manual, and confirmed by agarose gel electrophoresis.

<Distinguishing species through chloroplast DNA barcodes>

First, PCR was performed to directly sequencing the rbcL , matK and trnL-trnF ( trnL -F ) genes using gDNA derived from two samples (Lw-2,3) of Ohok submitted to the National Forensic Research Institute. did Primer information prepared for this is shown in Table 2 below. The trnL-trnF IGS gene was amplified by dividing it into two regions (cFdR and eFfR) to secure the correct nucleotide sequence. In Table 2, the length means the size of the expected PCR product.

gene Primer sequence (5'-3') Length (bp) Tm (℃) GenBank Accession No. rbcL F: SEQ ID NO: 4 584 57 KC777011 R: SEQ ID NO: 5 matK 390F: SEQ ID NO:6 841 48 HM041711 1326R: SEQ ID NO:7 trnL-F cF: SEQ ID NO: 8 524-530 57 AB362488
AB362489
AB362490 HM041292 dR: SEQ ID NO: 9 eF: SEQ ID NO: 10 378 fR: SEQ ID NO: 11

PCR was performed with 10 pmol of each primer, 2.5 units of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 2.5 μL of Gold ST*R 10X buffer (Promega, Madison, WI, USA) and 6- 10ng was mixed and the volume was adjusted to a final 25 μL with distilled water, and performed on an ABI 9700 system (Thermo Fisher Scientific). PCR conditions are shown in Table 3 below. As a positive control, a sample of Ouok (L.w-1) received from the National Arboretum was analyzed together, and distilled water was used as a negative control.

rbcL matK390F-1326R trnLF_cFeF-dRfR cycle Pre-
denaturation 95℃ 10 minutes 95℃ 10 minutes 95℃ 10 minutes One Denaturation 94℃ 30 seconds 95℃ 1 minute 95℃ 1 minute 35 Annealing 57℃ 30 seconds 48℃ 1 minute 57℃ 1 minute Extension 72℃ 1 minute 72℃ 1 minute 72℃ 1 minute Final Extension 72℃ 5 minutes 72℃ 7 minutes 72℃ 7 minutes One

The PCR amplification product was purified after confirming whether it was amplified by 2% agarose electrophoresis, and the purified amplification product was directly sequenced using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Residual fluorescent material was removed with DyeEX ^TM 2.0 Spin Kit (QIAgen, Germany). The final purified amplification product was sequenced using Applied Biosystems 3500xL Genetic Analyzer (Applied BioSystems, USA).

Gene sequences obtained as a result of sequencing were aligned and compared with MEGA software (Molecular Evolution Genetics Analysis 5.0 software, USA). Thereafter, the most similar species was searched for by comparing homology with the registered sequence using BLAST (Basic Local Alignment Search Tool) of NCBI (National Center for Biological Information, USA) GenBank.

In the two samples of Ohok submitted to the National Forensic Service, the sequences analyzed for the rbcL gene showed the same nucleotide sequence, and 100% homology to the 96-533bp sequence of the Ohok rbcL sequence (KC777011) registered with the NCBI. showed However, it was confirmed that the sequence information of Chwigwanok was not registered in the NCBI, so it could not be used directly for comparison between the two species.

The nucleotide sequence of the matK gene was also found to be identical in the two samples, and showed 100% homology to the 1021-1907 bp sequence of the NCBI registered Ohok matK sequence (HM041711). However, in some species of the genera Mammillaria and Coryphantha , it was confirmed that it was difficult to distinguish the species from other genera with the matK gene alone, because of the high homology of 99.25% due to the difference of two bases as a result of the NCBI search.

Although the nucleotide sequence of the trnL-trnF IGS gene was different in the two samples, it showed 100% homology with a part of the sequence (HM041292 or AB362488) of Ohok registered with the NCBI, respectively. This difference is a TA repeat that appears from 171 bp based on one sequence of Ohok (AB362488), and was confirmed to be due to a simple sequence repeat (SSR) region. When comparing the sort by percent identity with other species through BLAST search of NCBI, the value was 97.55% or less, confirming that the discriminating power was higher than that of the rbcL (99.5%) and matK (99.25%) genes.

A phylogenetic tree analysis was performed on the above three genes, and as a result, it was found that at least one gene among the above three genes could be used to identify the Ouko species. In order to increase the accuracy of identification, it was confirmed that it is preferable to analyze at least two of the three genes in combination.

<The genus Lopopora and oysteroid bell Identification of specific SNPs>

In the case of direct sequencing, there is a problem in that the experimental process is complicated and the number of steps to be performed is large, so it is difficult to perform it at once using a large sample. Accordingly, it was attempted to derive a new analysis technique that could more easily analyze the species of Ou-ok from other cacti.

The rbcL , matK and trnL-trnF IGS sequences obtained through the analysis of the preceding examples and the nucleotide sequences of genera belonging to the cactus family registered in NCBI GenBank were analyzed. Sequences of rbcL (~500bp), matK (~850bp), and trnL -trnF IGS (~1,000bp) of Ohok were searched through NCBI GenBank, and nucleotide sequences with high similarity were extracted for each region from 100 cacti. . These sequences were divided into four oysters and three chimneys. The DNA sequence was aligned using MEGA software (Molecular Evolution Genetics Analysis 5.0 software, USA). Based on the aligned sequence, At the genus level between Lopopora and other genera, oysters and chrysanthemums within the genus Lopopora Comparisons were made at the species level between the two. Some of the comparison results for each gene are shown in FIGS. 1 to 3 , respectively. Referring to FIGS. 1 to 3 , as a result of the analysis, SNP sites that are distinguished from other species in Ouok for each gene region were identified.

Based on the rbcL , matK and trnL-trnF IGS sequences of 7 cacti samples of the genus Lopopora and NCBI GenBank data, rbcL235, matK1497, matK1502R and trnLF285R were identified as four SNPs specific to the genus Lopopora. In addition, Ou-ok and Chwi-gwan-ok Three distinct SNPs were identified between rbcL304, trnLF385R and trnLF476. Here, SNP means a base difference existing at a corresponding position in the NCBI GeneBank sequence of the corresponding gene. The NCBI GeneBank sequence may be represented by the sequence of SEQ ID NO: 1 for rbcL , SEQ ID NO: 2 for matK , and SEQ ID NO: 3 for trnL-trnF IGS. For example, Lopopora The genus-specific SNP, rbcL235, is interpreted to mean a base difference at position 235 of SEQ ID NO: 1. 4 SNPs specific to the genus Lopopora and Ou-ok and Chwi-gwan-ok The three SNPs distinguished between them are shown in Table 4 below.

gene rbcL matK trnL-trnF IGS SNP site 235 304 1497 1502 285 385 476 oh ok C T G A A G G chwigwanok C C G A A T A other group T C, T A C G G, A A

The identified SNPs of rbcL235, rbcL304 and matK1497 were present in the protein coding region, but were not shown to alter transcriptional proteins as synonymous-SNPs. In contrast, matK1502R is a non-synonymous SNP. In other genera, cytosine (C) exists and threonine (Thr) is transcribed, but in the genus Lopopora, adenine (A) is present by conversion mutation. It has been shown to transcribe lysine (K). In addition to the matK1502R SNP, trnLF285R and trnLF385R were designed as reverse primers for easy SBE multiplex PCR amplification. trnL-trnF IGS is a non-coding region, but morphologically or related to mescaline content It can be used importantly in identification.

<SNP specificity test>

Lopophora genus and eucalyptus After each species-specific SNP was identified, PCR and mini-sequencing were performed using all cactus samples to confirm their specificity. The detailed execution method was the same as described above except for the parts described separately, and the 7 SNP sites identified above were amplified using PCR, and the results were confirmed using electrophoresis and mini-sequencing.

First, multiplex PCR was performed. Specifically, SNaPshot Multiplex PCR Assay was performed. Primers for this operation were prepared with three primer pairs: rbcL_FR, matK_FR and trnLF_FR to amplify chloroplast DNA containing the identified SNPs. Prepared primer information is shown in Table 5 below.

primer Primer sequence (5'-3') Primer length Primer Concentration
(μM) Amplification product size
(bp) rbcL-F SEQ ID NO: 12 22 0.05 243 rbcL-R SEQ ID NO: 13 21 0.05 matK_F SEQ ID NO: 14 22 0.05 258 matK_R SEQ ID NO: 15 20 0.05 trnLF_F SEQ ID NO: 16 21 1.00 339 trnLF_R SEQ ID NO: 17 24 1.00

PCR amplification products were 243, 258 and 339 bp, and Tm values were prepared according to amplification through multiplex PCR. PCR is performed by mixing 3 units of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 1 μL of Gold ST*R 10X buffer (Promega, Madison, WI, USA) and 2-5 ng of template DNA, The volume was adjusted to a final 10 μL with distilled water and performed on an ABI 9700 system (Thermo Fisher Scientific). PCR treatment conditions were 35 cycles of pre-denaturation at 95°C for 11 minutes, denaturation at 94°C for 20 seconds, annealing at 57°C for 1 minute, and extension at 72°C for 30 seconds, followed by final extension at 72°C for 7 minutes. made to do

Purification of the PCR amplification product was performed by adding 2 μL of ExoSAP-IT ^® (Thermo Fisher Scientific) to 5 μL of the PCR amplification product, reacting at 37° C. for 45 minutes, and then reacting at 80° C. for 15 minutes.

The results of amplification in 7 Lopopora using the primer set of Table 5 are shown in FIG. 4 . Referring to FIG. 4 , it can be confirmed that multiple amplification has been successfully performed. For verification purposes, some bands were deleted or bands different from the expected size were confirmed in C1 to C19 other cactus species analyzed together.

For 1 μL of the purified PCR amplification product, mini-sequencing was performed using the ABI PRISM ^® SNaPshot™ Multiplex Kit (Thermo Fisher Scientific). Single-base extension (SBE) multiplex primer information obtained through mini-sequencing for SNP site identification is shown in Table 6 below. The binding positions of the SBE primers in Table 6 below are shown in FIG. 5 . In Table 6, the identification level was described as a genus when the genus Lopopora can be distinguished, and a species when the genus Lopopora can be distinguished.

primer Primer sequence (5'-3') Primer length Primer Concentration
(μM) Identification level rbcL235 SEQ ID NO: 18 25 0.1 inside rbcL304 SEQ ID NO: 19 61 0.6 bell matK1497 SEQ ID NO: 20 35 0.3 inside matK1502R SEQ ID NO:21 30 0.3 inside trnLF285R SEQ ID NO:22 41 1.0 inside trnLF385R SEQ ID NO:23 55 1.0 bell trnLF476 SEQ ID NO:24 66 1.5 bell

Purification of the mini-sequencing reaction product was performed by adding 1 μL of shrimp alkaline phosphatase (Thermo Fisher Scientific) to 10 μL of the reaction product, reacting at 37° C. for 45 minutes, and then reacting at 80° C. for 15 minutes. Capillary electrolysis was performed by adding 0.2 μL of GeneScan™ 120 LIZ ^® Size Standard (Thermo Fisher Scientific) and 10 μL of Hi-Di™ Formamide (Thermo Fisher Scientific) to 1 μL of purified amplification product and adding an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific) Scientific). The results were confirmed through GeneMapper™ ID-X software Version 1.4 (Thermo Fisher Scientific).

The results of sequencing and mini-sequencing using the purified PCR product as a template are shown in FIGS. 6 and 7 . In FIG. 6 , bases with a background color ( ^a ) are the results of mini-sequencing, and bases without a background color ( ^b ) and deleted bases ( ^c ) are results of sequencing. In FIG. 7 , ag denotes SNP, a denotes rbcL235, b denotes matK1502R, c denotes matk1497, d denotes trnLF285R, e denotes trnLF385R, f denotes rbcL304 and g denotes trnLF476. The peaks of ad are for identifying oysters at the genus level, and the peaks eg at the species level. Fig. 7(C) does not show the SNP profile of Ou-ok as a result of one of the cacti of the genus Lopopora analyzed together for verification. Another non-Ropophora not shown in FIG. 7 The cactus of the genus also did not show the SNP profile of Oukok.

6 and 7, it was confirmed that the SNP profile was different when comparing the results with other cactus species. In the Lopopora genus identification SNP, four oysters containing the positive control of the present invention and only in the three chimney houses. In addition, it was confirmed that the four chimneys and the three chimneys each exhibited the same SNP profile for each species.

Guanine (G) displayed in blue was confirmed in g (trnLF476 site), but was not confirmed in Chwikwanok. Mono-PCR was additionally performed on the trnLF476 site, but no peak was identified through additional experiments. It was predicted that PCR amplification was not performed in this sample due to a problem caused by a difference in the primer binding sequence. It was confirmed that one base on the sequence of the position at which the corresponding primer binds was different between the two. Specifically, in Ohok, 5'-… In TTAA-3′, in the chimney building, 5′-… As for TGAA-3', thymine (T) and guanine (G) were found to have different bases at the corresponding positions. Due to the difference in bases, it was confirmed that PCR in the chwigwanok sample did not work well in g of FIG. 6 .

Here, in the SBE PCR result using the reverse primer, the bases complementary to the bases identified in the result should be interpreted as the bases located in this sequence in consideration of the characteristics of the reverse primers. For example, through the base T identified in the matK1502R SBE PCR result, the base at the corresponding position in SEQ ID NO: 2, which is the present sequence, is identified as the base A complementary to T. As another example, through the base T identified in the trnLF285R SBE PCR result, the base at the corresponding position in SEQ ID NO: 3, which is the present sequence, is identified as the base A complementary to T. As another example, through the base C identified in the trnLF385R SBE PCR result, the base at the corresponding position in SEQ ID NO: 3, which is the present sequence, is identified as the base G complementary to C.

Only the samples in which the 7 SNP profiles identified previously were confirmed were considered to be oook, and as a result of the experiment, Lopopora It was found that none of the 19 non-genus cactus species exhibited the SNP profile of the identified eucalyptus. The SNP profile including 7 SNPs identified according to an embodiment of the present invention is a unique profile of Ohok, and can be utilized to identify Ohok.

<110> Republic of Korea (National Forensic Service Director Ministry of Interior and Safety) <120> COMPOSITION FOR IDENTIFYING SPECIES OF PEYOTE AND IDENTIFICATION METHOD USING THE SAME <130> NP-2020-0417 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 533 <212> DNA <213> Unknown <220> <223> Lophophora williamsii <400> 1 ttacaaattg acttattata ctcctgaata tcaaccccag gataccgaca tcttggcagc 60 attccgagta actcctcaac ccggagttcc gtcagaagaa gcaggagccg cagtagctgc 120 cgaatcttct actggtacat ggacaactgt atggaccgac ggacttacca gtcttgatcg 180 ttacaaagga cgatgctacc acatcgatgc cgttcctgga aaagacaatc aatacatttg 240 ttatgtagct taccccttag acctttttga agaaggttct gttactaata tgtttacttc 300 cattgtgggt aatgtatttg gattcaaagc cctgcgtgct ttacggttgg aggatttgcg 360 aatccctgtt gcttatataa aaactttcca aggcccgcct cacggtatcc aagttgagag 420 ggataaattg aacaagtatg gccgtcctct actgggatgc actattaagc cgaaattggg 480 gttatctgct aaaaactatg gtcgagcagt ttatgaatgt cttcgcggtg gac 533 <210> 2 <211> 2340 <212> DNA <213> Unknown <220> <223> Lophophora williamsii <400> 2 cgactgatcc tganaggaaa tgaatggaaa aaagagcatg tcgtatcaat agagaattcg 60 gagaatattt catgggttcc aaatcggtac caaatattat ttgaattgaa cgaaatgaat 120 tcgcaagctt ggtcgattga ataaatggat cgagccctat ggttcanatt ctagggaaag 180 aaagagcaac gagcttatct tcgtaatttg aatgattacc cgatctaatt aaacgttaaa 240 aaaaaattag tgcctggtac gggaaaggct tctcacacga gtgaattttt tcgtttttag 300 tgaatcctaa ctattatatt atccattttc tatatggaga tgaatgtgta gaagaaacag 360 tatattgata aagatacttt tccaaaatca aaagagcgat tgggttgaaa aaataaagga 420 tttctancca tcttgctttg ttatcctata aaaaaaccaa ttagatggaa aaaatngaga 480 atccgttgat gaatttacct atctccgagg tacttantta tttcaaaatg gaataccttg 540 ttttgactgt atcgcactat gtatcatttg ataatccaag aaaccccctc tgcttttttt 600 tagttttcgg tctaatttga aatggaagaa ttccaaagat atatagaact agataggtct 660 tggcaacaca actttttcta tccacttatc tttcaggaat atatttatgg atttgcatat 720 gatcatggtt taaataaatc gattttgttg gaaaatgcag gcgacaagaa atatagttta 780 ctgattgtaa aacgtttaat taaccgaatg tatcaacaga ctcatttgat tctttctgct 840 aatcattcta gtcaaaatga cttttttggg cacaagcaca agaagaattt gtattatcaa 900 ataatatcag aaggatttgc agtcattgtg gaaattccat tttccctact gttaatatct 960 tccctggagg caaaagaaaa taaaatagta aaatcccata atttgcgatc aattcattca 1020 atatttcctt ttttcgagga caaattctta catttaaatt atgtgttaga aatattaata 1080 ccttacccca ttcatctgga aatcttggtt caaactcttc gttactgggt gaaagatgct 1140 tcttctttgc atttattacg attctttctt tatgagtatc gtaattggaa tagtcttatt 1200 actccccaaa aatccaattc gattttttca aaaaggaatc aacgattatt cttgttcctc 1260 tataatttct atgtatgtga atacgaatcc attttctttt ttctctgtaa ccaatcctct 1320 catttacgat caacatcttt tggagccctt cttgaacgaa attattttta tggaaagcta 1380 gaatatctag taaaagtcaa aacttttact aaggattttt gccttatctt atggcctttc 1440 aaagaccctt tcctgcatta tgttaggtat cgaggaaaat caattctggc ttcaaagggg 1500 aaatctcttc tgatgcataa atggaaatat tatcttctca atttttggca atgtcatttt 1560 tccctgtggt ctcaaccaag aagaatctat atcaatcggt tatcaaagca ttctctcgac 1620 tttatgggtt ttttttcaag tgttcgactc aattcttcag tggtacggag tcaaatggta 1680 gcaaattctt ttctaataga taatcgtatt aagaaattcg ataccatagt tcgaattatt 1740 cctctggttg gatcgttggc taaagcgaaa ttttgtaacg tattaggaca tcccattagt 1800 aagtcggtct ggaccgattt attagattct gatattattg atcggtttgg gcgcatatgc 1860 agaaatcttt ctcattatta tggtggctct tcaagaaaaa agagtttgta tcgaataaag 1920 tatatacttc tactttcttg tgctagaact ttggctcgta aacacaaaag tactgtacgc 1980 gcttttttga aaagattagg ttcagcattt ttggaagaat tctttactac agaggaagaa 2040 aaagttcttt ctttgatctt accaagagat tcttctatct caggaggatt atataggggt 2100 cctttttggt atttggatat tatttgtatc cacgatttgg ccaatgatga atgattggtt 2160 atgagactgt ctaaatgaaa tggaaattat acctaaatga atgacggaat aagtaatata 2220 aaaaaaatgc attcatttct atactgaaat gtatatcttt atgcttatgc agtaagggtt 2280 gaatcatttg cttgagtatt caactttgtt agagtctctt ctaggaaagg aactgaattt 2340 2340 <210> 3 <211> 1166 <212> DNA <213> Unknown <220> <223> Lophophora williamsii <400> 3 tccncaaann ngggggggcgc tacggactta attggcgtgc gcccttgtna gggaacctac 60 taagtgaaaa ctttcaaatt cagagaaacc ctggaattaa ttaaaatggg caatcctgag 120 ccaactcctt ttttcaaaaa aaaaaggcaa aaaataaggg ttcagaaagc aagaataaaa 180 aaaaggaaag gtgcagagac tcaaaggaag ttgttctaac aaatggggtt gactgtgctg 240 tgttcttata agaattcttc catcaaaact ccaataaaaa agggagtaaa gcatatacgt 300 gtagtgaact atatcaaatg attaaagaca acccgaaccc atacgtaatc cttatatata 360 tatatata tatatata atcagataat ctgaattcta ctttatataa taagaatatg 420 aaatatatat tctaaaatgg ataattctat ctaaatagaa attttagaat atgaagtgaa 480 aagatttcta tgaaaaaatg gaagaatttt tgggttatga atcgattcca agttgaaaaa 540 agaataaaat atgcatttac tccatagtct gatagatctt ttgaagaact gattaatcgg 600 acgagaatga agatagagtc ccgttttaca tgtcaatact gacaacaaag aaatttctag 660 taagaagaaa atccgttgac tttcaaaatc gtgagggttc aagtccctct atccccaaaa 720 gcttatttca ctcccgaact agttatcctt tttttataaa cagtttgaag gtggttatgt 780 tcctcatttt ttttgttttc aaattcaaaa aggctaaagg ctccggacga aaatgctttt 840 cccttatcaa aagtcttgtg atatacgtaa aaatgaatat ctttgagcaa ggaataatct 900 tttgagtgat tcacaatcaa tatcattaca cgtactaaaa cttaaataaa cttagagaca 960 agggagacaa agtccttctt tttgaagacc aaagaaattg cggtacctag ataagacctt 1020 gttagacttt tcgtcctttt aattgacata gacccgagtt ctctattaaa atgagtagat 1080 gatgcgccag aagggggaat ggccgggata gctcagctgg tagagcagag gactgaaaat 1140 cctcgtgtcc aggntttcca aataaa 1166 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> rbcL F primer <400> 4 agttccgtca gaagaagcag gag 23 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> rbcL R primer <400> 5 cattcaagta atgccctttg attt 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> matK 390F primer <400> 6 cgatctattc attcaatatt tc 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> matK 1326R primer <400> 7 tctagcacac gaaagtcgaa gt 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> trnL-F cF primer <400> 8 cgaaatcggt agacgctacg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> trnL-F dR primer <400> 9 ggggatagag ggacttgaac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> trnL-F eF primer <400> 10 ggttcaagtc cctctatccc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> trnL-F fR primer <400> 11 atttgaactg gtgacacgag 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rbcL_F primer <400> 12 tcgttacaaa ggacgatgct ac 22 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rbcL_R primer <400> 13 ctctcaactt ggataccgtg a 21 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> matK_F primer <400> 14 tgccttatct tatggccttt ca 22 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> matK_R primer <400> 15 catttgactc cgtaccactg 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> trnLF_F primer <400> 16 gtgcagagac tcaaaggaag t 21 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> trnLF_R primer <400> 17 caacttggaa tcgattcata accc 24 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> rbcL235 SBE primer <400> 18 tcgttcctgg aaaagacaat caata 25 <210> 19 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> rbcL304 SBE primer <400> 19 ttttttttttt tttttttttt tttttttttt tttttctgtt actaatatgt ttacttccat 60 60 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> matK1497 SBE primer <400> 20 tttttttttt tttgaaaatc aattctggct tcaaa 35 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> matK1502R SBE primer <400> 21 ttttttttcc atttatgcat cagaagagat 30 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> trnLF285R SBE primer <400> 22 tttttttttt tttttttttc actacacgta tatgctttac 40 <210> 23 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> trnLF385R SBE primer <400> 23 tttttttttt tttttttttt ttttttctta ttatataaag tagaattcag attat 55 <210> 24 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> trnLF476 SBE primer <400> 24 tttttttttt tttttttttt tttttttttt tttttttttt ctaaatagaa attttagaat 60 atgaa 65

Claims (12)

The 235th position in the nucleotide sequence shown in SEQ ID NO: 1, the 1497th position in the nucleotide sequence shown in SEQ ID NO: 2, the 1502 position in the nucleotide sequence shown in SEQ ID NO: 2, and the nucleotide sequence shown in SEQ ID NO: 3 A single nucleotide polymorphism present at at least one position selected from the group consisting of the 285th position in the And In the nucleotide sequence represented by SEQ ID NO: 3, comprising the step of identifying the base of the single nucleotide polymorphism present at at least one position selected from the group consisting of the 476th position, Ohok ( Lophophora williamsii ) A method of identifying.
A method of identifying Ohok ( Lophophora williamsii ), comprising the step of identifying the base of the single nucleotide polymorphism located at the 476th position in the nucleotide sequence represented by SEQ ID NO: 3.
The method of claim 1,
When the 235th base of SEQ ID NO: 1 is C, the 1497th base of SEQ ID NO: 2 is G, the 1502th base of SEQ ID NO: 2 is A, and the 285th base of SEQ ID NO: 3 is A, Lopophora ( Lophophora ) to differentiate into the genus,
When the 304th base of SEQ ID NO: 1 is T, the 385th base of SEQ ID NO: 3 is G, and the 476th base of SEQ ID NO: 3 is G, Lophophora among the genus Ohok ( Lophophora williamsii ) species How to identify the oyster ( Lophophora williamsii ).
3. The method of claim 2,
When the 476th base of SEQ ID NO: 3 is identified as G, oook ( Lophophora williamsii ) to discriminate, ohok ( Lophophora williamsii ) A method of identifying.
isolating the nucleic acid molecule from the subject of identification;
performing multiplex PCR using the nucleic acid molecule as a template, primer sets of SEQ ID NOs: 12 and 13, primer sets of SEQ ID NOs: 14 and 15, and primer sets of SEQ ID NOs: 16 and 17 to obtain an amplification product; and
Using the amplification product as a template and the primers of SEQ ID NOs: 18 to 24, performing a single base extension (SBE) method; comprising, a method of identifying Ohok ( Lophophora williamsii ).
delete The 235th position in the nucleotide sequence shown in SEQ ID NO: 1, the 1497th position in the nucleotide sequence shown in SEQ ID NO: 2, the 1502 position in the nucleotide sequence shown in SEQ ID NO: 2, and the nucleotide sequence shown in SEQ ID NO: 3 A composition for identifying a cactus in the genus Lophophora , comprising an agent capable of detecting or amplifying a single nucleotide polymorphism marker present at at least one position selected from the group consisting of the 285th position within.
8. The method of claim 7,
The agent is capable of detecting or amplifying 10-500 consecutive nucleotide sequences or their complementary nucleotide sequences including the single nucleotide polymorphism, Lophophora genus Cactus identification composition.
8. The method of claim 7,
The agent is a primer of SEQ ID NO: 18 capable of detecting a single nucleotide polymorphism marker present at position 235 in the nucleotide sequence shown in SEQ ID NO: 1, and at position 1497 within the nucleotide sequence shown in SEQ ID NO: 2 A primer of SEQ ID NO: 20 capable of detecting an existing single nucleotide polymorphism marker, a primer of SEQ ID NO: 21 capable of detecting a single nucleotide polymorphism marker present at position 1502 within the nucleotide sequence represented by SEQ ID NO: 2, and Lophophora comprising at least one primer selected from the group consisting of primers of SEQ ID NO: 22 capable of detecting a single nucleotide polymorphism marker present at position 285 in the nucleotide sequence represented by SEQ ID NO: 3 ) composition for identifying cacti of the genus.
At least one selected from the group consisting of the 304th position in the nucleotide sequence shown in SEQ ID NO: 1, the 385th position in the nucleotide sequence shown in SEQ ID NO: 3, and the 476th position in the nucleotide sequence shown in SEQ ID NO: 3 A polymorphic marker composition for distinguishing between Lophophora williamsii and Lophophora diffusa within the genus Lophophora , comprising an agent capable of detecting or amplifying a single nucleotide polymorphic marker present at a position.
11. The method of claim 10,
The agent is capable of detecting or amplifying 10 to 500 consecutive nucleotide sequences including the single nucleotide polymorphism or a complementary nucleotide sequence thereof, Lophophora within the genus Lophophora williamsii and odor A polymorphic marker composition for distinguishing the crown jewel ( Lophophora diffusa ).
11. The method of claim 10,
The agent is a primer of SEQ ID NO: 19 capable of detecting a single nucleotide polymorphism marker present at position 304 in the nucleotide sequence shown in SEQ ID NO: 1, and at position 385 in the nucleotide sequence shown in SEQ ID NO: 3 A primer of SEQ ID NO: 23 capable of detecting an existing single nucleotide polymorphism marker, and a primer of SEQ ID NO: 24 capable of detecting a single nucleotide polymorphism marker present at position 476 in the nucleotide sequence represented by SEQ ID NO: 3 A polymorphic marker composition for discriminating between Lophophora ( Lophophora ) and Owok ( Lophophora williamsii ) and Bwigwanok ( Lophophora diffusa ), comprising at least one primer selected from the group consisting of.

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