KR102159634B1 - Marker derived from complete sequencing of chloroplast genome of three Sanguisorba species, primer set for discrimination of Sanguisorba species and uses thereof - Google Patents

Abstract

The present invention relates to a cross-species distinguishing marker and primer set based on complete sequencing of chloroplast genome of three Sanguisorba species, and a use thereof. More specifically, the present invention relates to an SNP molecular marker for discriminating three Sanguisorba species, a primer set and probe set for amplifying the SNP molecular marker, a kit comprising the primer set and probe set, a method for discriminating three Sanguisorba species using the primer set and probe set, and a microarray for discriminating three Sanguisorba species. It is possible to determine the origin species of the herbal medicine ′Sanguisorba root′ by using the single nucleotide polymorphism marker (SNP marker) according to the present invention, and through this, it can be used for distribution and quality control of herbal medicine, Sanguisorba root. In other words, it is possible to prevent mixed-use and misuse of herbal medicines through accurate identification of the origin species, and scientifically maintain the quality of raw materials for herbal medicines.

Description

Marker derived from complete sequencing of chloroplast genome of three Sanguisorba species, primer set for discrimination of Sanguisorba species and uses thereof

The present invention relates to an interspecies distinct marker and primer set based on complete translation of the genome sequence of three chloroplasts of the genus Cucumber, and a use thereof. More specifically, three kinds of SNP molecular markers for discrimination of the genus Cucumber, a primer set and probe set for amplifying the SNP molecular marker, a kit including the primer set and probe set, and three kinds of Cucumber genus using the primer set and probe set It relates to a method for discrimination of, and a microarray for discrimination of species in cucumber grass.

Cucumber plant ( Sanguisorba officinalis Linne) is a perennial plant belonging to the genus Rosaceae cucumber plant, and about five species are distributed in Korea, including cucumber plant, long cucumber plant, white cucumber plant, large cucumber plant, and mountain cucumber plant. Cucumber plant is a medicinal plant used for burn treatment, hemostatic action, vomiting control, anticancer and antibacterial, and is a plant of origin of the herbal name Jiyu ( Journal of the Korean Society for Applied Biological Chemistry , 47(1), pp.141). -145, 2004; Korean journal of medicinal crop science , 24(4), pp.303-308, 2016). Others Republic of Korea Pharmacopeia medicine (herbal medicine) in Jiyugaoka origin of plants listed in Alimentarius is Sanguisorba officinalis belongs to the rose family (Rosaceae) (Sanguisorba officinalis Linne) or jangyeop Jiyugaoka (長葉地楡;.. Sanguisorba officinalis Linne var longifolia (Bert) Yu et Li) Says the roots of

In general, because of the characteristics of medicinal plants distributed in the form of slices, it is very difficult to distinguish the plant of origin due to its external characteristics, and thus, there is a problem of mixing with a fake or other species. To solve this problem, a more systematic and scientific end-determination technology is required, and the most suitable method for this is to use a molecular marker that can identify species at the DNA level.

In the case of plant DNA barcoding using conventional molecular markers, specific genes such as rbcL, matK, and trnH-GUG-pabA, which are known to have many variations in the chloroplast genome, have been developed for only a part of the sequence of the intergenic region. However, universal barcoding primers used to amplify these regions have problems in that the PCR amplification efficiency is low or not amplified depending on the plant species, and even if amplification is performed, polymorphism of the base sequence does not exist. There is a problem that may not be. In addition, in these highly mutated regions, mutations may be found even within the same species depending on the plant, implying the possibility of classifying the same species into different species.

To date, no discrimination method has been reported for the origin species of Lichen, which is widely used as a medicinal herb at the DNA level, and therefore, the present inventors analyzed the full-length genome sequence of the chloroplast, not a part of the chloroplast genome, The purpose of this study was to develop a molecular marker that distinguishes the origin species by searching for a polymorphic and more reliable region of mutation, and to develop a more practical technique for identifying the origin species of Liu.

Journal of the Korean Society for Applied Biological Chemistry, 47(1), pp.141-145, 2004 Korean journal of medicinal crop science, 24(4), pp.303-308, 2016

An object of the present invention is to provide a set of markers and primers for distinction between species based on complete translation of the genome sequence of three species of chloroplasts of the genus Cucumber, and a use thereof.

In detail, Cucumber ( Sanguisorba officinalis ), Long Cucumber ( S. longifolia ), and White Cucumber ( S. tenuifolia f alba ) SNP molecular markers for discrimination of three kinds of Cucumber genus, primer set and probe set for amplifying the SNP molecular marker It is to provide a kit comprising the primer set and probe set, a method for discriminating three species of genus Cucumber using the primer set and probe set, and a microarray for discriminating species of genus Cucumber.

In order to solve the above problems, the present invention includes polymorphic nucleotides consisting of nucleotide sequences of SEQ ID NOs: 1 to 16 showing single nucleotide polymorphism between species of the genus Cucumber ( Sanguisorba ), Cucumber ( Sanguisorba officinalis ), Longifolia ( S. longifolia ), and white cucumber grass ( S. tenuifolia f alba ) provides a composition for distinguishing any one or more species from the species of the genus Cucumber, including.

In addition, the present invention is a set of primers represented by SEQ ID NOs: 18 and 19; A set of primers represented by SEQ ID NOs: 22 and 23; A set of primers represented by SEQ ID NOs: 26 and 27; A set of primers represented by SEQ ID NOs: 30 and 31; A set of primers represented by SEQ ID NOs: 34 and 35; A set of primers represented by SEQ ID NOs: 38 and 39; A set of primers represented by SEQ ID NOs: 42 and 43; And it provides a primer set for distinguishing any one or more species from the species of the genus Cucumber, including, cucumber grass, long cucumber grass, and white cucumber grass, including; a primer set represented by SEQ ID NOs: 46 and 47.

In addition, the present invention is a set of probes represented by SEQ ID NOs: 20 and 21; A set of probes represented by SEQ ID NOs: 24 and 25; A set of probes represented by SEQ ID NOs: 28 and 29; A set of probes represented by SEQ ID NOs: 32 and 33; A set of probes represented by SEQ ID NOs: 36 and 37; A set of probes represented by SEQ ID NOs: 40 and 41; A set of probes represented by SEQ ID NOs: 44 and 45; And a probe set represented by SEQ ID NOs: 48 and 49; containing, a probe set for discriminating any one or more species from the species of the genus Cucumber, including Cucumber, Long Cucumber, and White Cucumber.

In addition, the present invention includes a kit for discriminating any one or more species from the species of the genus Cucumber plant, including the primer set, the probe set, and a reagent for carrying out an amplification reaction. to provide.

In addition, the present invention provides a method for discriminating any one or more species from species of the genus Cucumber, including cucumber grass, long cucumber grass, and white cucumber grass, using the primer set and the probe set.

In addition, the present invention in the polymorphic nucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1 to 16, one or more polymorphic nucleotides selected from polymorphic nucleotides consisting of 10 or more consecutive bases including the 150th base of each SNP position, or a complement thereof It provides a microarray for discrimination of any one or more species from species of the genus Cucumber, including Cucumber, Long Cucumber, and White Cucumber, including red polymorphic nucleotides.

It is possible to determine the origin species of the herbal medicine'lipid oil' by using the single nucleotide polymorphism marker (SNP marker) according to the present invention, and through this, it can be used for distribution and quality control of herbal medicine oil. In other words, it is possible to prevent mixed use and misuse of herbal medicines through accurate identification of the origin species, and scientifically maintain the quality of raw materials for herbal medicines.

1 is a diagram showing the complete chloroplast genome of the three types of cucumber grass, which are the origin plants of oil, cucumber grass, long cucumber grass, and white cucumber grass. Here, colored boxes represent conserved chloroplast genes classified based on the function of the gene product. Also, genes located inside the circle represent genes that are transcribed in a counterclockwise direction, and genes located outside the circle represent genes that are transcribed in a clockwise direction. Meanwhile, the dotted region of the inner circle indicates the GC content of the chloroplast genome.
2 is a diagram showing amplification curves for each of the eight molecular markers analyzed by the FenDEL-qPCR method and the results of determining +,-traits for three species of cucumber grass, cucumber grass, long cucumber grass, and white cucumber grass. Among the amplification curves for each marker, the blue line shows the amplification result of the internal control, and the red line shows the amplification result of each marker. Here, the +,-trait determination for each marker is determined by comparing the Ct value of the amplification curve of the internal control (blue) and the Ct value of the amplification curve of the SNP marker (red), that is, the Ct of the SNP marker amplification curve. If the value is less than or equal to the Ct value of the internal control amplification curve, it is determined as'+', and if it is greater or '0', it is judged as'-'
3 is a diagram showing sequence information of eight SNP markers and an internal control (IC) for three species of cucumber grass genus, cucumber grass, long cucumber grass, and white cucumber grass according to an embodiment of the present invention.

The present invention completely decodes the chloroplast genome sequence of the three genus Cucumber, Cucumber, Long Cucumber, and White Cucumber, and based on this, a SNP molecular marker for discrimination between species, a primer set and a probe set for amplifying the SNP molecular marker, the primer It relates to a kit including a set and a probe set, a method for discriminating three species of genus Cucumber using the primer set and probe set, and a microarray for discriminating species of genus Cucumber.

In one embodiment, the present invention includes polymorphic nucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1 to 16 showing single nucleotide polymorphism between species of the genus Cucumber ( Sanguisorba ), cucumber grass ( Sanguisorba officinalis ), long cucumber grass ( S. longifolia ), and white cucumber grass ( S. tenuifolia f alba ) to provide a composition for distinguishing any one or more species from the species of the genus Cucumber, including.

The polymorphic nucleotide consisting of the nucleotide sequences of SEQ ID NOs: 1 to 16 is a polymorphic nucleotide including a single nucleotide polymorphism (SNP) located at the 150th base within each nucleotide sequence, and the term "single nucleotide polymorphism: SNP )" means that one of the nucleotide sequences consisting of A, T, C, and G in the genome has been changed to another nucleotide sequence.

SNP polymorphic nucleotide information for the polymorphic nucleotide consisting of the nucleotide sequences of SEQ ID NOs: 1 to 16 according to an embodiment of the present invention is shown in FIG. 3.

In the composition for discriminating any one or more species from species of the genus Cucumber according to an embodiment of the present invention, at least one polymorphism selected from the group consisting of nucleotide sequences of SEQ ID NOs: 9, 10, 13 and 14 including SNP polymorphism Using nucleotides, it is possible to distinguish the major three species of the genus Cucumber, Cucumber, Long Cucumber or White Cucumber; Using polymorphic nucleotides consisting of nucleotide sequences of SEQ ID NOs: 1, 2, 5, 6, 7, 8, 15, and 16, it is possible to distinguish cucumber grass or white cucumber grass among the three major species of the genus Cucumber; If polymorphic nucleotides consisting of the nucleotide sequences of SEQ ID NOs: 3, 4, 11, and 12 are used, it is possible to distinguish cucumber grass or long cucumber grass among the three major species in the genus Cucumber (see FIGS. 2 and 3).

As another aspect, the present invention provides a primer set represented by SEQ ID NOs: 18 and 19; A set of primers represented by SEQ ID NOs: 22 and 23; A set of primers represented by SEQ ID NOs: 26 and 27; A set of primers represented by SEQ ID NOs: 30 and 31; A set of primers represented by SEQ ID NOs: 34 and 35; A set of primers represented by SEQ ID NOs: 38 and 39; A set of primers represented by SEQ ID NOs: 42 and 43; And it provides a primer set for distinguishing any one or more species from the species of the genus Cucumber, including, cucumber grass, long cucumber grass, and white cucumber grass, including; a primer set represented by SEQ ID NOs: 46 and 47.

In the present invention, the term "distinguishing" refers to discriminating by judging the diversity of varieties by the primer set of the present invention from three types of Cucumber genus plant samples including Cucumber, Long Cucumber, and White Cucumber. Can be used in combination with.

In the present invention, the term "primer" refers to a single-stranded oligonucleotide sequence that is complementary to a nucleic acid strand to be copied, and serves as a starting point for the synthesis of a primer extension product. The length and sequence of the primers should allow the synthesis of the extension product to start, and the specific length and sequence of the primers depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the required DNA or RNA target. something to do.

In the present invention, the primer may further include a substance that emits DNA intercalating fluorescence, phosphorescence or radioactivity, but is not limited thereto. Preferably, the labeling substance is FAM or HEX. When performing PCR by labeling FAM or HEX at the 5'-end of an allele-specific primer when amplifying the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material.

In the present invention, the term "primer set" means a plurality of primers. In addition, the primer set may further include a container for receiving the primer. Primer refers to a short nucleotide sequence that can form a base pair with a complementary template with a short free 3'terminal hydroxyl group and serves as a starting point for template strand copying. . The primer can initiate DNA synthesis in the presence of a reagent for polymerization and four nucleosite triphosphates at an appropriate buffer and temperature. The primer may be an oligonucleotide, and the oligonucleotide may comprise a nucleotide analogue, e.g., phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or an insert ( intercalating agent). The primer may be prepared through a chemical synthesis method using a phosphoramidite method, a phosphodiester method, a diethylphosphoramidite method, or the like. In addition, the primer sequence may be modified by means known in the art.

In yet another aspect, the present invention provides a probe set represented by SEQ ID NOs: 20 and 21; A set of probes represented by SEQ ID NOs: 24 and 25; A set of probes represented by SEQ ID NOs: 28 and 29; A set of probes represented by SEQ ID NOs: 32 and 33; A set of probes represented by SEQ ID NOs: 36 and 37; A set of probes represented by SEQ ID NOs: 40 and 41; A set of probes represented by SEQ ID NOs: 44 and 45; And a probe set represented by SEQ ID NOs: 48 and 49; containing, a probe set for discriminating any one or more species from the species of the genus Cucumber, including Cucumber, Long Cucumber, and White Cucumber.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundred bases for a specific binding to a gene or mRNA, and an oligonucleotide Probes, single-stranded DNA (single stranded DNA) probes, double-stranded DNA (double stranded DNA) probes, can be produced in the form of an RNA probe, and can be labeled for easier detection.

The probe of the present invention is an allele-specific probe based on RT-PCR that uses the FEN (Flap endonuclease) specificity of DNA polymerase, and the probe has a polymorphic site in a nucleic acid fragment derived from two members of the same species. It hybridizes to a DNA fragment derived from, but does not hybridize to a fragment derived from another member.

The primers or probes of the present invention described above can be chemically synthesized using a phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g., methyl phosphonate, phosphotriester, phos Poroamidate, carbamate, etc.) or to a charged linker (eg phosphorothioate, phosphorodithioate, etc.).

As the primer or probe used in the present invention, a sequence that is completely complementary to the sequence including the SNP may be used, but a sequence that is substantially (substantially) complementary within a range that does not interfere with specific hybridization It can also be used. Specifically, the probe used in the present invention includes a sequence capable of hybridizing to a sequence containing 10 or more, preferably 10-100 consecutive nucleotide residues including the SNP of the present invention. More specifically, the 3'-end or 5'-end of the probe has a base complementary to the SNP base. In general, since the stability of the duplex formed by hybridization tends to be determined by the matching of the sequence at the end, the terminal in a probe having a base complementary to the SNP base at the 3'-end or 5'-end If the parts are not hybridized, these duplexes can disintegrate under stringent conditions.

Suitable conditions for hybridization include Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Stringent conditions used for hybridization can be determined by controlling temperature, ionic strength (buffer concentration), and the presence of compounds such as organic solvents. These stringent conditions can be determined differently depending on the sequence being hybridized.

In the present invention, the term "complementary" means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, and is substantially complementary and completely complementary ( perfectly complementary) is meant to encompass all, and specifically, it means completely complementary. In the present specification, the term "substantially complementary sequence" used in relation to a primer sequence is not only a sequence that is completely matched, but also a sequence of a comparison object within the range that can serve as a primer by annealing to a specific sequence. It means that mismatched sequences are also included.

Among the above-described primer sets and probe sets, the primer sets represented by SEQ ID NOs: 18 and 19 and the probe sets represented by SEQ ID NOs: 20 and 21 are the 150th base of SEQ ID NO: 1 or 2 (ID: SNP#1), a single nucleotide polymorphism (SNP) C and G, the primer set represented by SEQ ID NOs: 22 and 23 and the probe set represented by SEQ ID NOs: 24 and 25 is a single nucleotide polymorphism that is the 150th base of SEQ ID NO: 3 or 4 (ID: SNP#2) (SNP) A and G, the primer set represented by SEQ ID NOs: 26 and 27, and the probe set represented by SEQ ID NOs: 28 and 29, the 150th base of SEQ ID NO: 5 or 6 (ID: SNP#4) single nucleotide polymorphism ( SNP) T and C, the primer set represented by SEQ ID NOs: 30 and 31, and the probe set represented by SEQ ID NOs: 32 and 33, the 150th base of SEQ ID NO: 7 or 8 (ID: SNP#6) single nucleotide polymorphism ( SNP) T and G, the primer set represented by SEQ ID NOs: 34 and 35, and the probe set represented by SEQ ID NOs: 36 and 37, the 150th base of SEQ ID NO: 9 or 10 (ID: SNP#7) single nucleotide polymorphism ( SNP) C and T, the primer set represented by SEQ ID NOs: 38 and 39, and the probe set represented by SEQ ID NOs: 40 and 41, the 150th base of SEQ ID NO: 11 or 12 (ID: SNP#9) single nucleotide polymorphism ( SNP) G and A, the primer set represented by SEQ ID NOs: 42 and 43, and the probe set represented by SEQ ID NOs: 44 and 45, the 150th base of SEQ ID NO: 13 or 14 (ID: SNP#12) single nucleotide polymorphism ( SNP) T and C, the primer set represented by SEQ ID NOs: 46 and 47, and the probe set represented by SEQ ID NOs: 48 and 49, the 150th base of SEQ ID NO: 15 or 16 (ID: SNP#14) single nucleotide polymorphism ( SNP) characterized by distinguishing between A and G.

In yet another aspect, the present invention comprises the primer set, the probe set, and a reagent for performing an amplification reaction, any one or more species from the species of the genus Cucumber, including Cucumber, Cucumber, and Cucumber. Provides a kit to distinguish.

Reagents for performing the amplification reaction in the kit of the present invention may include DNA polymerase, dNTPs, and a buffer. The dNTP mixture includes dATP, dCTP, dGTP, and dTTP, and the heat resistant DNA polymerase may be a commercially available heat resistant polymerase such as Taq DNA polymerase and Tth DNA polymerase. In addition, the kit of the present invention may further include a user's guide describing the optimum reaction performance conditions. The guide is a printout explaining how to use the kit, e.g., how to prepare PCR buffer, suggested reaction conditions, etc. The guide contains a brochure in the form of a pamphlet or flyer, a label affixed to the kit, and a description on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.

On the other hand, the kit according to the present invention may further include a primer set represented by SEQ ID NO: 50 and 51, and a probe represented by SEQ ID NO: 52.

Using the primer sets represented by SEQ ID NOs: 50 and 51, and the probe represented by SEQ ID NO: 52, the rbcL gene, an internal control represented by SEQ ID NO: 17, can be amplified.

The kit is a kit for discriminating any one or more species from species in the genus Cucumber, including Cucumber, Long Cucumber, and White Cucumber, characterized by using the duplex qPCR method amplifying together with the above-described internal control. It may be a kit for use.

As yet another aspect, the present invention comprises the steps of separating genomic DNA from a plant sample of the genus Cucumber; Using the isolated genomic DNA as a template, and performing an amplification reaction using the primer set of claim 3 and the probe set of claim 4 to amplify polymorphic nucleotides; And it provides a method for discriminating any one or more species from the species of the genus Cucumber, including cucumber grass, long cucumber grass, and white cucumber grass, comprising the step of detecting the product of the amplification step.

In the method of the present invention, a method of separating genomic DNA from a plant sample of the genus Cucumber may be accomplished through a conventional method known in the art, and the method includes a phenol/chloroform extraction method, an SDS extraction method (Tai et al., Plant Mol). Biol. Reporter, 8: 297-303, 1990), CTAB separation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980), or commercially available DNA extraction kits. Can be done.

Using the genomic DNA of the isolated Cucumber genus plant sample as a template, the target sequence may be amplified by performing an amplification reaction using one or more primer sets and probe sets according to an embodiment of the present invention. Methods of amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, and There are 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 pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used. PCR may be performed using a PCR reaction mixture containing various components known in the art for PCR reaction. The PCR reaction mixture contains an appropriate amount of DNA polymerase, dNTP, PCR buffer, and water in addition to the genomic DNA extracted from a plant sample of Cucumber genus to be analyzed and the primer set and probe set provided in the present invention. The PCR buffer solution includes Tris-HCl (Tris-HCl), MgCl ₂ , KCl, and the like.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling material. The labeling material may be a material that emits fluorescence, phosphorescence, or radioactivity, but is not limited thereto. The labeling material may be FAM or HEX. When performing PCR by labeling FAM or HEX at the 5'-end of the primer when amplifying the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. In addition, for labeling using a radioactive material, when a radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution when performing PCR, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product can be radiolabeled. One or more primer sets and probe sets used to amplify the target sequence are as described above.

In yet another aspect, the present invention is one or more polymorphic nucleotides consisting of 10 or more contiguous bases including the 150th base of each SNP position in the polymorphic nucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1 to 16. It provides a microarray for discriminating any one or more species from the species of the genus Cucumber, including the polymorphic nucleotide or its complementary polymorphic nucleotides, Cucumber, Long Cucumber, and White Cucumber.

The polymorphic nucleotide may be immobilized on a substrate coated with an active group of amino-silane, poly-L-lysine, or aldehyde, but is not limited thereto. The substrate may be a silicon wafer, glass, quartz, metal, or plastic, but is not limited thereto. As a method of immobilizing the polymorphic nucleotide on a substrate, a micropipetting method using a piezoelectric method, a method using a pin type spotter, or the like may be used.

Microarrays according to the present invention can be prepared by conventional methods known to those skilled in the art using the polymorphic nucleotides or their complementary nucleotides according to the present invention, the polypeptides encoded thereby, or cDNAs thereof.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1: Plant material preparation

In order to develop genetic markers for plants of'lipid oil' origin, 2 points each of Cucumber ( Sanguisorba officinalis ), Sanguisorba longifolia ( Sanguisorba longifolia ), and White Cucumber ( Sanguisorba tenuifolia f.alba ) as plant materials were collected from the Korea Institute of Oriental Medicine and the next generation base. Through sequencing (NGS; Next Generation Sequencing), the nucleotide sequence of the chloroplast genome of the three genus of Cucumber was analyzed.

Cucumber paste 68 points (commercially distributed domestic cucumber paste herbs 40 points, Chinese cucumber paste herbs 15 points, Herbal Medicine Promotion Foundation collection/cultivation 12 points, KFDA standard product 1 point), long cucumber paste 18 points (the Oriental Medicine Promotion Foundation collection/ 17 points of cultivation, 1 point of the KFDA standard product), and 11 points of white cucumber grass (11 points collected/cultivated by the Oriental Medicine Promotion Foundation) were used.

Example 2: Genomic DNA extraction

Genomic DNA of each specimen prepared in Example 1 was extracted using NucleoSpin® 96 Plant II (MACHEREY-NAGEL GmbH & Co. KG) according to the manufacturer's instructions. The amount of extracted genomic DNA was measured using Qubit dsDNA BR Assay Kit (Invitrogen). The DNA concentration of the sample for next-generation sequencing (NGS) was 200 ng or more in total, and 0.5 ng/µl of the sample for marker verification was prepared.

Example 3: Next Generation Sequencing (NGS)

An NGS analysis library was prepared for the genomic DNA of each specimen obtained in Example 2 using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEBNext-NGS Kit). Index adapters classified for each sample to be analyzed for NGS were used, and the detailed library production process was performed according to the product manual. Each of the prepared libraries was analyzed using TapStation HSD5000 (Agilent), and the suitability of NGS analysis was determined.

For the production of large amounts of genome sequence data using NGS, the Illumina® platform was used, and the shape and length of the analysis lead were analyzed by the pair-end, 101 method. A data amount of 1 Gb per sample was generated.

Example 4: Assembly of chloroplast genome sequences of three species of plant specimens of the genus Cucumber

Analysis of the genomic DNA of each specimen obtained in Example 2, and the entire chloroplast genome sequence of each specimen was completed using Genotech's'cellular organelle genome sequence assembly and verification system' (Fig. 1 Reference). The length of the chloroplast genome sequence of two assembled and verified cucumber grass, two long cucumber grass, and two white cucumber grass ranges from 155,393 bp to 155,415 bp (see Table 1), and the sequence and orientation of genes are very well preserved between species. Was confirmed. It was confirmed that the chloroplast genome of plants in the genus Cucumber is composed of 82 protein-generating genes, 52 tRNA genes, and 4 rRNA genes in common.Structurally, it was confirmed that one LSC (large single copy region) and one SSC ( small single copy region), and two IR (inverted repeat regions).

Evaluation of chloroplast genome sequence assembly results for each specimen division Plant name Total lead
(Total reads) Aligned leads
(Aligned reads) Organelle genome%
(Organelle genome %) Cov.
(x) Genome length (bp) Genomic morphology 1-1 Cucumber grass 6,222,080 239,662 8.99 364 155,393 circle 1-2 Cucumber grass 6,585,316 237,590 8.29 355 155,401 circle 2-1 Long cucumber grass 7,404,878 166,728 3.94 189 155,415 circle 2-2 Long cucumber grass 6,010,712 171,652 5.06 198 155,412 circle 3-1 White cucumber 8,410,896 188,944 4.10 224 155,385 circle 3-2 White cucumber 6,506,648 218,028 5.01 212 155,393 circle

Example 5: Analysis of chloroplast genome sequence variation between species and selection of molecular marker candidates

The genus of three kinds of cucumber plants prepared as plant materials in Example 1, specifically, a total of six chloroplast genome sequences of two points each of cucumber grass, long cucumber grass, and white cucumber grass, using Genotech's MPPC (Modified Phred Phrap Consed) program. Genetic diversity was confirmed through mutual comparison. Genetic diversity and mutation were found as single nucleotide polymorphism (SNP) and indel (InDel), and as a result, the region where the mutation was confirmed is a molecule that can identify each individual and distinguish it from its relatives. Used for marker development.

The location of mutations between species was confirmed through MPPC analysis, and individual mutations within the same species were also confirmed at the same time. Among the mutations, a total of 8 cases were identified between individuals within the same species, and as a result of excluding them and analyzing inter-species mutations, a total of 179 mutations that could be used as genetic markers, of which SNP-type mutations were 101, InDel-type mutations. There were 78 mutations in, and InDel mutations were in the form of insertions and deletions of 1-7 bases (see Table 2).

Chloroplast genome sequence variation of cucumber grass, long cucumber grass, and white cucumber grass division Genetic variant form system SNP InDel Count 101 pcs 78 pieces 179 pcs

Example 6: Marker verification method for end determination and selection of molecular markers to be verified

In consideration of the type and number of molecular markers for distinguishing the genus Cucumber and its fauna and flora, and the species of origin discovered through the NGS-based comparative genome performed in Example 3, and the number of samples that can be used for verification, Geno Co., Ltd. Tech's FenDEL-qPCR technique was applied. FenDEL-qPCR is a genetic diversity and mutation analysis technique that is easy to commercialize, and it is easy to discriminate single nucleotide polymorphism and indel mutations.

In order to apply the FenDEL-qPCR method, Genotech's FenDEL primer/probe design standard was applied, and finally, 8 SNPs suitable for the standard were selected as FenDEL-qPCR experiment verification targets (see Table 3).

Example 7: SNP marker verification using FenDEL-qPCR technique

As described in Example 6, the genotyping of SNP markers for each sample was performed using Genotech's FenDEL-qPCR method, and primers and probe sets as shown in Table 4 below were used. The primers and probe sets in Table 4 below were prepared using Genotech's oligonucleotide synthesis system.

In addition, for the FenDEL-qPCR reaction, a reaction product was prepared under the conditions shown in Table 5 below for each SNP marker, and a real-time polymerase chain reaction was performed. At this time, PCR was performed using the ABI7500 Real-Time PCR System.

In order to verify the marker for the identification of the'lipid' origin, a total of 97 samples (68 points of cucumber grass, 18 points of long cucumber grass, 11 points of white cucumber grass) prepared in Example 1 were selected as the final target in Example 6 above. The genotypes for one of the eight SNPs were analyzed as described above, and based on the results (see Table 6), eight SNP markers (see FIG. 3) were determined as markers capable of discriminating the origin species of the herbal medicine oil.

Example 8. End determination using SNP marker genotype combination

The genotype of each of the 8 SNP markers analyzed by the FenDEL-qPCR method in Example 7 was determined as'-' or'+' based on the Ct value of the internal control (IC), and then, the'Lolipid origin species discrimination table (Table 7)' and the species were identified. The + and-traits of each SNP marker were determined by comparing the Ct value of the'FAM' fluorescence (red amplification curve) confirmed by FenDEL-qPCR and the Ct value of the'JOE' fluorescence (blue amplification curve) (Fig. 3). Reference). That is, if the Ct value of the SNP marker amplification curve is less than or equal to the Ct value of the internal control amplification curve, it is determined as'+', and if it is greater or '0', it is determined as'-'. On the other hand, the FenDEL-qPCR method is a polymerization for analyzing the eight SNP markers (SNP#1, SNP#2, SNP#4, SNP#6, SNP#7, SNP#9, SNP#12, SNP#14). It uses the principle of inhibiting the FEN (flap endonuclease) activity of the enzyme.

Specifically, according to the analysis by the FenDEL-qPCR method, 8 SNP markers (SNP#1, SNP#2, SNP#4, SNP#6, SNP#7, SNP#9, SNP#12, SNP#14) If all are amplified, it was determined as cucumber grass, and if SNP#2, SNP#6, SNP#7, SNP#9, and SNP#12 are amplified, it is determined as long cucumber grass, and SNP#1, SNP#4, SNP#7 , SNP#12, SNP#14 was determined to be white cucumber grass when amplified.

<110> National Institute for Korean Medicine Development <120> Marker derived from complete sequencing of chloroplast genome of three Sanguisorba species, primer set for discrimination of Sanguisorba species and uses thereof <130> PA-19-0159 <160> 52 <170> KoPatentIn 3.0 <210> 1 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#1_ndhA_C <400> 1 ataaattcgc agtaaaaata ttgagtctca ttttccatgt atacgaatta aagagttaag 60 cggaaataaa cataagaaac cgtttacccc aagattggtt gattagtcat cctgacttga 120 agcgggctca aaagatcacc atattgtatc atttatatgt attaacatac atgtattatc 180 ataatggcgg gttaaacaaa aacgagtgga tggttagaaa caccaagata cgtaacggat 240 ttgtaatgga aatgaaaatt atgtaaatta tccaaacaga catttttaca taatataca 299 <210> 2 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#1_ndhA_G <400> 2 ataaattcgc agtaaaaata ttgagtctca ttttccatgt atacgaatta aagagttaag 60 cggaaataaa cataagaaac cgtttacccc aagattggtt gattagtcat cctgacttga 120 agcgggctca aaagatcacc atattgtatg atttatatgt attaacatac atgtattatc 180 ataatggcgg gttaaacaaa aacgagtgga tggttagaaa caccaagata cgtaacggat 240 ttgtaatgga aatgaaaatt atgtaaatta tccaaacaga catttttaca taatataca 299 <210> 3 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#2_ndhD_A <400> 3 tagtaggcac aattcaaatt atctatgcag ctttaacatc tcctggtcag cgtaatttaa 60 aaaagagaat agcctattcc tctgtatcgc atatgggttt cataattata ggaattggtt 120 ctataagtga tacagggctc aatggagcca ttttacaaat aatttcgcac ggatttattg 180 gcgctgcgct ttttttcttg gctggaacga gttatgatag aatacgactt gtttatctcg 240 acgaaatggg cggaatggct atcgcaattc caaaaatatt cacgactttc agtatctta 299 <210> 4 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#2_ndhD_G <400> 4 tagtaggcac aattcaaatt atctatgcag ctttaacatc tcctggtcag cgtaatttaa 60 aaaagagaat agcctattcc tctgtatcgc atatgggttt cataattata ggaattggtt 120 ctataagtga tacagggctc aatggagccg ttttacaaat aatttcgcac ggatttattg 180 gcgctgcgct ttttttcttg gctggaacga gttatgatag aatacgactt gtttatctcg 240 acgaaatggg cggaatggct atcgcaattc caaaaatatt cacgactttc agtatctta 299 <210> 5 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#4_trnL_T <400> 5 attccttatc cattttgtct ttgtccagta ttttcatacc acagaaatta ggattaggaa 60 taaagaatcc atatcaaaga aaagccacag gctcgctgtt ggagtatcca ttcttatttg 120 atgcattgtg gtcaggaaca ttgatgaatt aggtgaagaa tacggaaaga gagggattcg 180 aaccctcggt aaacagaagt ctacatagca gttccaatgc tacgccttca accactcggc 240 catctctcct acctaatgat tatggctcaa aaaccgagtg aatagtgagt cattcatat 299 <210> 6 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#4_trnL_C <400> 6 attccttatc cattttgtct ttgtccagta ttttcatacc acagaaatta ggattaggaa 60 taaagaatcc atatcaaaga aaagccacag gctcgctgtt ggagtatcca ttcttatttg 120 atgcattgtg gtcaggaaca ttgatgaatc aggtgaagaa tacggaaaga gagggattcg 180 aaccctcggt aaacagaagt ctacatagca gttccaatgc tacgccttca accactcggc 240 catctctcct acctaatgat tatggctcaa aaaccgagtg aatagtgagt cattcatat 299 <210> 7 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#6_trnStop_T <400> 7 gaagaacata ttgtttcttt gctctttcca aaaacttctt ctactttgac gaggttttat 60 agagggcgta tttggtattt ggatattttg tgtatcaatg atctaattaa tcatgaatga 120 ttggttatgc gaccgtggaa atggaaattt tggttaaata ataaccagat agcaaatttt 180 ttctattatg tattatgaaa tgttcatgca ataagggttg atcaactgag tattcacctt 240 tcttttctta gagtcgtgta taagtaagga actacatttt agatgtatac atagagaaa 299 <210> 8 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#6_trnStop_G <400> 8 gaagaacata ttgtttcttt gctctttcca aaaacttctt ctactttgac gaggttttat 60 agagggcgta tttggtattt ggatattttg tgtatcaatg atctaattaa tcatgaatga 120 ttggttatgc gaccgtggaa atggaaattg tggttaaata ataaccagat agcaaatttt 180 ttctattatg tattatgaaa tgttcatgca ataagggttg atcaactgag tattcacctt 240 tcttttctta gagtcgtgta taagtaagga actacatttt agatgtatac atagagaaa 299 <210> 9 <211> 296 <212> DNA <213> Artificial Sequence <220> <223> SNP#7_ndhD_C <400> 9 atggagttat taccccacgc ccattcgata ttttctccat ggttgatgat agtaggcaca 60 attcaaatta tctatgcagc tttaacatct cctggtcagc gtaatttaaa aaagagaata 120 gcctattcct ctgtatcgca tatgggtttc ataattatag gaattggttc tataagtgat 180 acagggctca atggagccag ttttacaaat aatttcgcac ggatttattg gcgctgcgct 240 ttttttcttg gctggaacga gttatgatag aatacgactt gtttatctcg acgaaa 296 <210> 10 <211> 296 <212> DNA <213> Artificial Sequence <220> <223> SNP#7_ndhD_T <400> 10 atggagttat taccccacgc ccattcgata ttttctccat ggttgatgat agtaggcaca 60 attcaaatta tctatgcagc tttaacatct cctggtcagc gtaatttaaa aaagagaata 120 gcctattcct ctgtatcgca tatgggtttt ataattatag gaattggttc tataagtgat 180 acagggctca atggagccag ttttacaaat aatttcgcac ggatttattg gcgctgcgct 240 ttttttcttg gctggaacga gttatgatag aatacgactt gtttatctcg acgaaa 296 <210> 11 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#9_petB-petD_G <400> 11 cctctgtatg gggcttacgc gatggttgga acaaagacac attttttttt tgtaaattca 60 aatgtggcag ataaagctat atatctgcac ggcccgatca atagttcaag tcacacactc 120 ccataatcca ttttagcttc ggagaattcg cttcaactat tcaactacta agtgtcaaaa 180 atatatattt ttgtagagtt gaagagaaat atccaaatac atgtcttatt tttttcaata 240 atccatattt gtagcaataa aatactattg ttcctacccc aagtgataaa tgattgatt 299 <210> 12 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#9_petB-petD_A <400> 12 cctctgtatg gggcttacgc gatggttgga acaaagacac attttttttt tgtaaattca 60 aatgtggcag ataaagctat atatctgcac ggcccgatca atagttcaag tcacacactc 120 ccataatcca ttttagcttc ggagaattca cttcaactat tcaactacta agtgtcaaaa 180 atatatattt ttgtagagtt gaagagaaat atccaaatac atgtcttatt tttttcaata 240 atccatattt gtagcaataa aatactattg ttcctacccc aagtgataaa tgattgatt 299 <210> 13 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#12_ndhJ-trnF_T <400> 13 aaatatgtat gaagtagtaa tatttttttt actggcggag acacgaacaa ataaataaag 60 taagaggaaa cgaaatggaa ttcgtttctt ttgtatactt tgaatataca atacccatcg 120 tttcttttgc ctgttttata tacataaaat agaattacta agctaaggga tatagctccg 180 acacttagat ggataagata agtatgtatc atgggctaga actagaatac tgaatatgta 240 tgtcgaccca tatcaattaa tttgtagaat atatactcat acaaattact catgtgcca 299 <210> 14 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#12_ndhJ-trnF_C <400> 14 aaatatgtat gaagtagtaa tatttttttt actggcggag acacgaacaa ataaataaag 60 taagaggaaa cgaaatggaa ttcgtttctt ttgtatactt tgaatataca atacccatcg 120 tttcttttgc ctgttttata tacataaaac agaattacta agctaaggga tatagctccg 180 acacttagat ggataagata agtatgtatc atgggctaga actagaatac tgaatatgta 240 tgtcgaccca tatcaattaa tttgtagaat atatactcat acaaattact catgtgcca 299 <210> 15 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#14_psaA_A <400> 15 ggagacttct tggttcatca tgctattgcg ctaggtttac atacaactac attaatctta 60 gtaaaaggtg ctttagatgc acgcggttcc aagttaatgc cagataaaaa agattttggt 120 tatagttttc cctgtgatgg tccgggacga ggcggtactt gtgatatttc ggcttgggac 180 gcattttact tggcagtttt ttggatgtta aataccattg gatgggttac tttttattgg 240 cattggaagc acatcacatt atggcagggt aacgtttcac agtttaatga gtcttccac 299 <210> 16 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP#14_psaA_G <400> 16 ggagacttct tggttcatca tgctattgcg ctaggtttac atacaactac attaatctta 60 gtaaaaggtg ctttagatgc acgcggttcc aagttaatgc cagataaaaa agattttggt 120 tatagttttc cctgtgatgg tccgggacgg ggcggtactt gtgatatttc ggcttgggac 180 gcattttact tggcagtttt ttggatgtta aataccattg gatgggttac tttttattgg 240 cattggaagc acatcacatt atggcagggt aacgtttcac agtttaatga gtcttccac 299 <210> 17 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> Internal Control <400> 17 gcggaatctt ctactggtac atggacaact gtatggactg acgggcttac cagtcttgat 60 cgttacaaag ggcgctgcta ccatattgaa cctg 94 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SNP#1_ndhA_F <400> 18 tcaaaagatc accatattgt gtc 23 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SNP#1_ndhA_R <400> 19 tccgttacgt atcttggtgt 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SNP#1_ndhA_FenDEL-P <400> 20 gacacaatat ggtgatcttt tg 22 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SNP#1_ndhA_Combo-P <400> 21 ttaaacaaaa acgagtggat ggtta 25 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SNP#2_ndhD_F <400> 22 tgatacaggg ctcaatgga 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SNP#2_ndhD_R <400> 23 ccatttcgtc gagataaaca 20 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SNP#2_ndhD_FenDEL-P <400> 24 tgactccatt gagccctgt 19 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SNP#2_ndhD_Combo-P <400> 25 ttgcccttac agaaagtgaa ga 22 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SNP#4_trnL_F <400> 26 tggtcaggaa cattgatgat tt 22 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SNP#4_trnL_R <400> 27 gtaggagaga tggccgag 18 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SNP#4_trnL_FenDEL-P <400> 28 aactcatcaa tgttcctgac c 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SNP#4_trnL_Combo-P <400> 29 tccaatgcta cgccttcaac c 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SNP#6_trnStop_F <400> 30 cgaccgtgga aatggaaaat t 21 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SNP#6_trnStop_R <400> 31 ggtgaatact cagttgatca ac 22 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SNP#6_trnStop_FenDEL-P <400> 32 aagtttccat ttccacggtc 20 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SNP#6_trnStop_Combo-P <400> 33 tgttcatgca ataagggttg atcaa 25 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SNP#7_ndhD_F <400> 34 cctctgtatc gcatatgggt 20 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SNP#7_ndhD_R <400> 35 ttctatcata actcgttcca gc 22 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SNP#7_ndhD_FenDEL-P <400> 36 gagacccata tgcgatacag a 21 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SNP#7_ndhD_Combo-P <400> 37 taatttcgca cggatttatt ggcg 24 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SNP#9_petB-petD_F <400> 38 tccattttag cttcggagaa t 21 <210> 39 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SNP#9_petB-petD_R <400> 39 ttgctacaaa tatggattat tgaa 24 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SNP#9_petB-petD_FenDEL-P <400> 40 cgtattctcc gaagctaaaa t 21 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> SNP#9_petB-petD_Combo-P <400> 41 tagagttgaa gagaaatatc caaatac 27 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SNP#12_ndhJ-trnF_F <400> 42 gtttcttttg cctgttttat atac 24 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SNP#12_ndhJ-trnF_R <400> 43 aattgatatg ggtcgacata ca 22 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SNP#12_ndhJ-trnF_FenDEL-P <400> 44 atcttatgta tataaaacag gcaaa 25 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SNP#12_ndhJ-trnF_Combo-P <400> 45 tagctccgac acttagatgg ataa 24 <210> 46 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> SNP#14_psaA_F <400> 46 cctgtgatgg tccggga 17 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SNP#14_psaA_R <400> 47 cataatgtga tgtgcttcca at 22 <210> 48 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SNP#14_psaA_FenDEL-P <400> 48 tcatcccgga ccatcaca 18 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SNP#14_psaA_Combo-P <400> 49 ttgggacgca ttttacttgg cagt 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Internal Control_rbcL_IC-F <400> 50 gcggaatctt ctactggtac atgg 24 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Internal Control_rbcL_IC-R <400> 51 caggttcaat atggtagcag cg 22 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Internal Control_rbcL_IC-P <400> 52 gactgacggg cttaccagtc ttga 24

Claims (9)

Cucumbers ( Sanguisorba ) containing polymorphic nucleotides consisting of nucleotide sequences of SEQ ID NOs: 1 to 16 showing single nucleotide polymorphism between species of the genus Sanguisorba ( Sanguisorba officinalis ), long cucumber grass ( S. longifolia ), and white Cucumber ( S. tenuifolia f alba ) A composition for discriminating any one or more species from species of the genus Cucumber, including.
The composition of claim 1, wherein the polymorphic nucleotide consisting of the nucleotide sequences of SEQ ID NOs: 1 to 16 comprises a single nucleotide polymorphism (SNP) located at the 150th base within each nucleotide sequence.
A set of primers represented by SEQ ID NOs: 18 and 19; A set of primers represented by SEQ ID NOs: 22 and 23; A set of primers represented by SEQ ID NOs: 26 and 27; A set of primers represented by SEQ ID NOs: 30 and 31; A set of primers represented by SEQ ID NOs: 34 and 35; A set of primers represented by SEQ ID NOs: 38 and 39; A set of primers represented by SEQ ID NOs: 42 and 43; And a primer set represented by SEQ ID NOs: 46 and 47; containing, a primer set for discriminating any one or more species from the species of the genus Cucumber, including Cucumber, Long Cucumber, and White Cucumber.
A set of probes represented by SEQ ID NOs: 20 and 21; A set of probes represented by SEQ ID NOs: 24 and 25; A set of probes represented by SEQ ID NOs: 28 and 29; A set of probes represented by SEQ ID NOs: 32 and 33; A set of probes represented by SEQ ID NOs: 36 and 37; A set of probes represented by SEQ ID NOs: 40 and 41; A set of probes represented by SEQ ID NOs: 44 and 45; And a probe set represented by SEQ ID NOs: 48 and 49; containing, a probe set for distinguishing any one or more species from the species of the genus Cucumber, including Cucumber grass, Long cucumber grass, and White cucumber grass.
A kit for discriminating any one or more species from the species of the genus Cucumber, including the Cucumber plant, Long Cucumber plant, and Cucumber plant, comprising the primer set of claim 3, the probe set of claim 4, and a reagent for performing an amplification reaction.
The kit according to claim 5, wherein the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs, and buffer.
The kit according to claim 5, wherein the kit further comprises a primer set represented by SEQ ID NO: 50 and 51, and a probe represented by SEQ ID NO: 52.
Separating genomic DNA from a plant sample of the genus Cucumber;
Using the isolated genomic DNA as a template, and performing an amplification reaction using the primer set of claim 3 and the probe set of claim 4 to amplify polymorphic nucleotides; And
A method for discriminating any one or more species from the species in the genus Cucumber, including cucumber grass, long cucumber grass, and white cucumber grass, comprising the step of detecting the product of the amplification step.
In the polymorphic nucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 to 16,
One or more polymorphic nucleotides selected from polymorphic nucleotides consisting of 10 or more contiguous nucleotides including the 150th base of each SNP position, or a complementary polymorphic nucleotide thereof, in Cucumber, Cucumber, and Cucumber genus Microarray for discriminating one or more species from species.

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