Shine muscat discrimination method and shine muscat discrimination kit

Abstract

To provide a technique for discriminating Shine Muscat capable of reducing the labor of a worker and rapidly discriminating the food.SOLUTION: The method for discriminating Shine Muscat includes a step of amplifying retrotransposon VINE1 or Tvv1 and a region on a genome adjacent to retrotransposon using DNA of grape to be discriminated as a template by using a primer set including a primer having a base sequence specifically binding to retrotransposon VINE1 or Tvv1 contained in the genome of Shine Muscat and a primer specifically binding to a genomic region located in the vicinity of retrotransposon VINE1 or Tvv1 contained in the genome.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for distinguishing Shine Muscat grapes and a kit for distinguishing Shine Muscat grapes. [Background technology]

[0002] Shine Muscat (variety registration number 13891 under the Seed and Seedlings Act) has traditionally been known as one of the grape varieties.

[0003] For example, Patent Document 1 discloses a technique for distinguishing 24 grape varieties, including Shine Muscat, using microsatellite markers.

[0004] In the discrimination technique described in Patent Document 1, a sample obtained from grapes is first used as a template, and a primer set with a predetermined base sequence is used to amplify polynucleotides in 12 regions containing microsatellites. Based on the number of repeat sequences (sequence length) contained in the resulting amplification products, 24 grape varieties can be discriminated. Hereinafter, the grapes whose varieties are to be discriminated are also referred to as "grapes to be discriminated." [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-127637 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the discrimination technique described in Patent Document 1 requires amplification of each of the 12 regions, which poses a problem in that it takes a great deal of time and effort to discriminate between Shine Muscat grapes.

[0007] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a Shine Muscat discrimination technique that can reduce the labor required by workers and enable rapid discrimination. [Means for solving the problem]

[0008] One aspect of the present invention that solves the above problems includes the following aspects.

[0009] [1] A method for distinguishing Shine Muscat grapes, comprising the step of amplifying the retrotransposon VINE1 or Tvv1 and a region on the genome adjacent to the retrotransposon using DNA of the grape to be distinguished as a template, using a primer set including a primer having a base sequence that specifically binds to the retrotransposon VINE1 or Tvv1 contained in the genome of Shine Muscat grapes, and a primer that specifically binds to a genomic region contained in the genome and located near the retrotransposon VINE1 or Tvv1.

[0010] [2] The method for distinguishing Shine Muscat according to [1], wherein in the step, a DNA fragment is amplified by PCR using one or more primer sets selected from the following (a) to (f) with the DNA of the grape to be distinguished as a template, and when the amplified DNA fragment is obtained, the grape is distinguished as Shine Muscat; (a) a primer set comprising a first primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 1 and a second primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 2; (b) a primer set comprising the first primer and a third primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 3; (c) a primer set comprising a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 4; and a fifth primer consisting of a base sequence of 19 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 5; (d) a primer set in which one of the first primer and the second primer of the primer set (a) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography; (e) a primer set in which one of the first primer and the third primer of the primer set (b) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography; (f) a primer set in which one of the fourth primer and the fifth primer of the primer set (c) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography.

[0011] [3] The method for distinguishing Shine Muscat according to [1], wherein in the step, a DNA fragment is amplified by the LAMP method using one or more primer sets selected from the following (g) to (m) and DNA of the grape to be distinguished as a template, and when the amplified DNA fragment is obtained, the grape is distinguished as Shine Muscat;(g) a primer set comprising a sixth primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 6, a seventh primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 7, an eighth primer consisting of the base sequence shown in SEQ ID NO: 8, a ninth primer consisting of the base sequence shown in SEQ ID NO: 9, and a tenth primer consisting of the base sequence shown in SEQ ID NO: 10; (h) a primer set comprising an eleventh primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 11, a twelfth primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 12, a thirteenth primer consisting of the base sequence shown in SEQ ID NO: 13, a fourteenth primer consisting of the base sequence shown in SEQ ID NO: 14, and a fifteenth primer consisting of the base sequence shown in SEQ ID NO: 15; (i) a sixteenth primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 16, and a seventeenth primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 17. A primer set comprising a 17th primer consisting of a base sequence of 17 to 26 consecutive bases, an 18th primer consisting of the base sequence set forth in SEQ ID NO: 18, a 19th primer consisting of the base sequence set forth in SEQ ID NO: 19, and a 20th primer consisting of the base sequence set forth in SEQ ID NO: 20; (j) a primer set in which any one of the 8th, 9th, and 10th primers of the primer set (g) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography; (k) a primer set in which any one of the 13th, 14th, and 15th primers of the primer set (h) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography; (m) a primer set in which any one of the 18th, 19th, and 20th primers of the primer set (i) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography. (However, when the DNA fragment is amplified using two or more primer sets from (g) to (m), amplification by the LAMP method is performed in a different reaction field for each primer set.)

[0012] [4] The method for distinguishing Shine Muscat grapes according to [2], wherein the first primer comprises the base sequence shown in SEQ ID NO: 21, the second primer comprises the base sequence shown in SEQ ID NO: 22, the third primer comprises the base sequence shown in SEQ ID NO: 23, the fourth primer comprises the base sequence shown in SEQ ID NO: 24, and the fifth primer comprises the base sequence shown in SEQ ID NO: 25.

[0013] [5] The method for distinguishing Shine Muscat grapes according to [3], wherein the sixth primer comprises the nucleotide sequence shown in SEQ ID NO: 26, the seventh primer comprises the nucleotide sequence shown in SEQ ID NO: 27, the eleventh primer comprises the nucleotide sequence shown in SEQ ID NO: 28, the twelfth primer comprises the nucleotide sequence shown in SEQ ID NO: 29, the sixteenth primer comprises the nucleotide sequence shown in SEQ ID NO: 30, and the seventeenth primer comprises the nucleotide sequence shown in SEQ ID NO: 31.

[0014] [6] A Shine Muscat discrimination kit comprising a primer set that includes a primer having a base sequence that specifically binds to the retrotransposon VINE1 or Tvv1 contained in the Shine Muscat genome, and a primer that specifically binds to a genomic region contained in the genome and located near the retrotransposon VINE1 or Tvv1, and that is capable of amplifying the retrotransposon VINE1 or Tvv1 and the region on the genome adjacent to the retrotransposon using the Shine Muscat genome as a template.

[0015] [7] The Shine Muscat discrimination kit according to [6], wherein the primer set is one or more of the following (a) to (f): (a) a primer set comprising a first primer consisting of a 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 1 and a second primer consisting of a 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 2; (b) a primer set comprising the first primer and a third primer consisting of a 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 3; (c) a primer set comprising a fourth primer consisting of a 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 4 and a fifth primer consisting of a 19 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 5; (d) a primer set comprising the first primer and the second primer of the primer set of (a). (e) a primer set in which one of the first and third primers of the primer set (b) is assigned a tag sequence for detecting the amplification products of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography; (f) a primer set in which one of the fourth and fifth primers of the primer set (c) is assigned a tag sequence for detecting the amplification products of the retrotransposon Tvv1 and the region on the genome adjacent to the retrotransposon Tvv1 by nucleic acid chromatography.

[0016] [8] The Shine Muscat discrimination kit according to [6], wherein the primer set is one or more of the following primer sets (g) to (m): (g) a sixth primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 6, a seventh primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 7, an eighth primer consisting of the base sequence shown in SEQ ID NO: 8, a ninth primer consisting of the base sequence shown in SEQ ID NO: 9, and a tenth primer consisting of the base sequence shown in SEQ ID NO: 10. a primer set comprising (h) an 11th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 11, a 12th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 12, a 13th primer consisting of a base sequence shown in SEQ ID NO: 13, a 14th primer consisting of a base sequence shown in SEQ ID NO: 14, and a 15th primer consisting of a base sequence shown in SEQ ID NO: 15; and (i) a 1st primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 16. a primer set comprising a 6 primer, a 17 primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 17, an 18 primer consisting of the base sequence shown in SEQ ID NO: 18, a 19 primer consisting of the base sequence shown in SEQ ID NO: 19, and a 20 primer consisting of the base sequence shown in SEQ ID NO: 20; (j) a primer set in which a tag sequence is added to any of the 8 primer, the 9 primer, and the 10 primer of the primer set of (g) to detect an amplification product of the retrotransposon VINE1 and a region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography; (k) a primer set in which a tag sequence is added to any of the 13 primer, the 14 primer, and the 15 primer of the primer set of (h) to detect an amplification product of the retrotransposon VINE1 and a region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography; (m) the 18 primer of the primer set of (i);A primer set in which a tag sequence is added to either the 19th primer or the 20th primer for detecting amplification products of the retrotransposon Tvv1 and the region on the genome adjacent to the retrotransposon Tvv1 by nucleic acid chromatography.

[0017] [9] The kit for distinguishing Shine Muscat grapes according to [7], wherein the first primer comprises the nucleotide sequence shown in SEQ ID NO: 21, the second primer comprises the nucleotide sequence shown in SEQ ID NO: 22, the third primer comprises the nucleotide sequence shown in SEQ ID NO: 23, the fourth primer comprises the nucleotide sequence shown in SEQ ID NO: 24, and the fifth primer comprises the nucleotide sequence shown in SEQ ID NO: 25.

[0018]

[10] The kit for distinguishing Shine Muscat grapes according to [8], wherein the sixth primer comprises the nucleotide sequence shown in SEQ ID NO: 26, the seventh primer comprises the nucleotide sequence shown in SEQ ID NO: 27, the eleventh primer comprises the nucleotide sequence shown in SEQ ID NO: 28, the twelfth primer comprises the nucleotide sequence shown in SEQ ID NO: 29, the sixteenth primer comprises the nucleotide sequence shown in SEQ ID NO: 30, and the seventeenth primer comprises the nucleotide sequence shown in SEQ ID NO: 31. [Effects of the Invention]

[0019] According to the present invention, a technique for distinguishing Shine Muscat grapes can be provided that can reduce the labor required by workers and enable rapid discrimination. [Brief explanation of the drawings]

[0020] [Figure 1] 1 is a diagram showing the sequences of regions on the Shine Muscat genome to which the primer set (a) and the primer set (g) bind in an example. [Figure 2] 1 is a diagram showing the sequences of regions on the Shine Muscat genome to which the primer set (b) and the primer set (h) bind in an example. [Figure 3]1 is a diagram showing the sequences of regions on the Shine Muscat genome to which the primer set (c) and the primer set (i) bind in an example. [Figure 4] (a) is an image showing the results of PCR using the primer set and DNA extracted from 24 grape varieties as a template, followed by electrophoresis. [Figure 5] (b) is an image showing the results of PCR using the primer set and DNA extracted from 24 varieties of grapes as a template, followed by electrophoresis. [Figure 6] (c) is an image showing the results of PCR using the primer set and DNA extracted from 24 varieties of grapes as a template, followed by electrophoresis. [Figure 7] This figure shows the results of PCR using DNA extracted from 24 varieties of grapes as a template, the primer sets (d) and (e) and a primer set for amplifying the grape consensus sequence, and then subjecting the result to nucleic acid chromatography. [Figure 8] This figure shows the results of PCR using DNA extracted from four varieties of grapes as a template, the primer sets (d) and (e) and a primer set for amplifying the grape consensus sequence, and then subjecting the PCR product to nucleic acid chromatography. [Figure 9] This figure shows the results of PCR using DNA extracted from four varieties of grapes as a template, primer sets (d), (e), and (f) and a primer set for amplifying a grape consensus sequence, followed by nucleic acid chromatography. [Figure 10] (h) A graph showing the results of amplifying DNA by the LAMP method for 24 grape varieties using the primer set, and measuring the changes in fluorescence intensity. [Figure 11] This is a graph showing the results of measuring the changes in fluorescence intensity after amplifying DNA by the LAMP method for 15 of 24 grape varieties using the primer set (k) and a primer set that amplifies the grape consensus sequence. [Figure 12]This graph shows the results of measuring the changes in fluorescence intensity after amplifying DNA by the LAMP method for the remaining 9 grape varieties out of the 24 varieties using the primer set (k) and a primer set that amplifies the grape common sequence. [Figure 13] This is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for 15 of 24 grape varieties using the primer set (k) and a primer set that amplifies the grape consensus sequence. [Figure 14] This is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for the remaining 9 grape varieties out of the 24 varieties using the primer set (k) and a primer set that amplifies the grape common sequence. [Figure 15] This figure shows the results of DNA amplification by the LAMP method using DNA extracted from 24 varieties of grapes as a template, the primer set (k) and a primer set for amplifying a grape consensus sequence, and nucleic acid chromatography. [Figure 16] (j) A graph showing the results of amplifying DNA by the LAMP method for 15 grape varieties out of 24 varieties using the primer set and a primer set that amplifies the grape common sequence, and measuring the changes in fluorescence intensity. [Figure 17] This graph shows the results of amplifying DNA by the LAMP method for the remaining 9 grape varieties out of the 24 varieties using the primer set (j) and a primer set that amplifies the grape common sequence, and measuring the changes in fluorescence intensity. [Figure 18] (j) A graph showing the results of association curve analysis after DNA amplification by the LAMP method for 15 of 24 grape varieties using the primer set and a primer set that amplifies the grape consensus sequence. [Figure 19] (j) A graph showing the results of association curve analysis after DNA amplification by the LAMP method for the remaining 9 varieties of grapes out of the 24 varieties using the primer set and a primer set that amplifies the grape common sequence. [Figure 20] This figure shows the results of DNA amplification by the LAMP method using DNA extracted from 24 varieties of grapes as a template, the primer set (j) and a primer set for amplifying a grape consensus sequence, and nucleic acid chromatography. [Figure 21] This is a graph showing the results of measuring the changes in fluorescence intensity after amplifying DNA by the LAMP method for 15 of 24 grape varieties using the primer set (m) and a primer set that amplifies the grape consensus sequence. [Figure 22] This graph shows the results of measuring the changes in fluorescence intensity after amplifying DNA by the LAMP method for the remaining 9 grape varieties out of the 24 varieties using the primer set (m) and a primer set that amplifies the grape common sequence. [Figure 23] This is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for 15 of 24 grape varieties using the primer set (m) and a primer set that amplifies the grape consensus sequence. [Figure 24] This is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for the remaining 9 grape varieties out of the 24 varieties using the primer set (m) and a primer set that amplifies the grape common sequence. [Figure 25] This figure shows the results of DNA amplification by the LAMP method using DNA extracted from 24 varieties of grapes as a template, the primer set (m) and a primer set for amplifying a grape consensus sequence, and nucleic acid chromatography. [Figure 26] 1 shows the results of amplified DNA fragments from a crude grape extract by the LAMP method using the primer set (k) shown in Table 5, followed by nucleic acid chromatography. [Figure 27] This figure shows the results of amplified DNA fragments from a crude grape extract by the LAMP method using the primer set (j) shown in Table 8 and subjected to nucleic acid chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0021] [How to distinguish Shine Muscat] Preferred embodiments of the present invention will now be described in detail.

[0022] A preferred embodiment of the present invention relates to a method for distinguishing Shine Muscat grapes. The method includes a step of amplifying the retrotransposon VINE1 or Tvv1 and a region on the genome adjacent to the retrotransposon using a primer set including a primer having a base sequence that specifically binds to the retrotransposon VINE1 or Tvv1 contained in the Shine Muscat genome and a primer that specifically binds to a genomic region located near the retrotransposon VINE1 or Tvv1 contained in the Shine Muscat genome, using the DNA of the grape to be distinguished as a template (amplification step). Hereinafter, for convenience of explanation, a polynucleotide having the base sequence of the retrotransposon (VINE1 or Tvv1) and the region (on the genome) adjacent to the retrotransposon amplified using the primer set is also referred to as a "DNA fragment." In other words, the DNA fragment is a DNA marker for identifying Shine Muscat, and the genomic region located near the retrotransposon VINE1 or Tvv1 is, for example, within 130 bp of the distal end base pair from the retrotransposon VINE1 or Tvv1.

[0023] In the discrimination method of this embodiment, some of the primers in the primer set are bound to a retrotransposon (VINE1 or Tvv1), and the other primers are bound to an area outside of that (an area near the retrotransposon, not the retrotransposon sequence).

[0024] Thus, by designing primers to span the retrotransposon (VINE1 or Tvv1) and the region in its vicinity and amplifying them, it becomes possible to identify varieties based on differences in the insertion positions of retrotransposons (insertion polymorphisms). That is, the discrimination method of this embodiment is a method for discriminating Shine Muscat using the insertion polymorphism of retrotransposons, and can also be regarded as a method for discriminating whether the grape to be discriminated is Shine Muscat or not.

[0025] As a method for discriminating Shine Muscat using the insertion polymorphism of retrotransposons, more specifically, after amplification by PCR (Polymerase Chain Reaction) (after the amplification operation), a method for confirming the presence or absence of amplification of DNA fragments, and after amplification by the LAMP (Loop-mediated Isothermal Amplification) method (after the amplification operation), a method for confirming the presence or absence of amplification of DNA fragments can be mentioned.

[0026] <DNA Extraction> In both the case of amplification by PCR and the case of amplification by the LAMP method, it is preferable to extract DNA from the grape to be discriminated prior to the amplification of DNA fragments.

[0027] The DNA extraction site is not particularly limited, and examples include grape stems, leaves, roots, fruits (especially the peel), flower buds, etc. Also, the method for extracting DNA from grapes is not particularly limited, and examples include using the phenol method after homogenizing any of the above-mentioned parts of grapes, or subjecting them to a commercially available DNA extraction kit.

[0028] The extracted DNA is subjected to an amplification step by PCR or an amplification step by the LAMP method. First, the amplification by PCR and the method for confirming the presence or absence of amplification of DNA fragments thereafter will be described in detail below.

[0029] <Amplification of DNA Fragments by PCR> In this discrimination method, in the amplification step, DNA fragments are amplified by PCR using one or more primer sets from the following (a) to (f) with the DNA of the grapes to be discriminated as a template. Thereafter, the presence or absence of amplification of the DNA fragments is confirmed, and if the DNA fragments are amplified (amplified DNA fragments are obtained), the grapes to be discriminated can be discriminated as Shine Muscat.

[0030] (a) a first primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 1; a second primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 2; (b) the first primer; a third primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 3; (c) a fourth primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 4; a fifth primer consisting of a base sequence of 19 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 5; (d) a primer set in which one of the first primer and the second primer of the primer set of (a) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography. (e) a primer set in which one of the first primer and the third primer of the primer set of (b) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography. (f) a primer set in which one of the fourth primer and the fifth primer of the primer set of (c) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography.

[0031] PCR using one or more primer sets from (a) to (f) above allows multiplex PCR, so two or more primer sets from (a) to (f) can be freely combined and subjected to PCR.

[0032] (Primer set (a)) Primer set (a) was able to amplify a DNA fragment containing a portion of the retrotransposon VINE1 on chromosome 9 and adjacent and contiguous regions on the genome when the genome of Shine Muscat was used as a template, whereas no DNA fragments were amplified when the genomes of 23 grape varieties (Kyoho, Pione, Delaware, Campbell Early, Koshu, Niagara, Muscat Bailey A, Steuben, Fujiminori, Akamine, Nagano Purple, Concord, Rosario Bianco, Takao, Portland, Aki Queen, Muscat of Alexandria, Queen Nina, Black Beet, Suiho, Ruby Roman, Grosse Krone, and San Verde) were used as templates.

[0033] Therefore, PCR using primer set (a) makes it possible to accurately distinguish the above 23 grape varieties from Shine Muscat. The above 23 varieties, plus Shine Muscat, make up a total of 24 varieties, which together account for approximately 95% of the total grape cultivation area in Japan. Therefore, primer set (a) makes it possible to distinguish Shine Muscat from most grape varieties cultivated in Japan.

[0034] (Primer set (b)) Primer set (b) can amplify a DNA fragment containing a portion of the retrotransposon VINE1 on chromosome 13 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas no DNA fragment is amplified when the genomes of the 23 grape varieties listed above are used as templates. Therefore, PCR using primer set (b) can accurately distinguish the 23 grape varieties listed above from Shine Muscat.

[0035] (Primer set (c)) Primer set (c) can amplify a DNA fragment containing a portion of the retrotransposon Tvv1 on chromosome 15 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas no DNA fragment is amplified when the genomes of 22 grape varieties excluding Muscat of Alexandria among the 23 grape varieties listed above are used as templates. Therefore, PCR using primer set (c) can accurately distinguish the 22 grape varieties listed above from Shine Muscat. When primer set (c) is used, a DNA fragment is amplified even when the Muscat of Alexandria genome is used as a template, making it impossible to distinguish between Muscat of Alexandria and Shine Muscat.

[0036] PCR may be performed by combining primer sets (a) to (c) (multiplex PCR). Examples of primer combinations include a combination of (a) and (b), a combination of (a) and (c), a combination of (b) and (c), and a combination of (a), (b), and (c). In these combinations, primer set (d) may be used instead of (a), primer set (e) instead of (b), and primer set (f) instead of (c).

[0037] (First primer) The first primer binds to the retrotransposon VINE1 on chromosome 9 and chromosome 13 of Shine Muscat grapes. The first primer is included in both the primer set (a) and the primer set (b). The first primer preferably consists of a base sequence of 24 to 30 consecutive bases (24 to 30 nucleotides) within the base sequence shown in SEQ ID NO: 1, more preferably a base sequence of 25 to 27 consecutive bases within the base sequence shown in SEQ ID NO: 1, and particularly preferably a base sequence of 25 or 26 consecutive bases within the base sequence shown in SEQ ID NO: 1. The first primer preferably includes the base sequence shown in SEQ ID NO: 21, and particularly preferably is 25 nucleotides consisting of the base sequence shown in SEQ ID NO: 21.

[0038] Because the sequence at the 3' end of a primer is particularly important for primer specificity, the first primer preferably contains at its 3' end (3' end side) the nucleotide sequence shown in SEQ ID NO: 21. In this case, the adenine base located at the 3' end of the nucleotide sequence shown in SEQ ID NO: 21 is located at the 3' end of the first primer.

[0039] (Second primer) The second primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon VINE1 on chromosome 9 of Shine Muscat.

[0040] The second primer preferably consists of a base sequence of 21 to 27 consecutive bases within the base sequence shown in SEQ ID NO: 2, more preferably consists of a base sequence of 22 to 24 consecutive bases within the base sequence shown in SEQ ID NO: 2, and particularly preferably consists of a base sequence of 22 or 23 consecutive bases within the base sequence shown in SEQ ID NO: 2. The second primer preferably contains the base sequence shown in SEQ ID NO: 22, more preferably contains the base sequence shown in SEQ ID NO: 22 at the 3' end (3' end side), and particularly preferably is 22 nucleotides consisting of the base sequence shown in SEQ ID NO: 22.

[0041] (Third primer) At least a portion of the third primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) in the vicinity of the retrotransposon VINE1 on chromosome 13 of Shine Muscat.

[0042] The third primer preferably consists of a base sequence of 21 to 27 consecutive bases within the base sequence shown in SEQ ID NO: 3, more preferably consists of a base sequence of 22 to 24 consecutive bases within the base sequence shown in SEQ ID NO: 3, and particularly preferably consists of a base sequence of 22 or 23 consecutive bases within the base sequence shown in SEQ ID NO: 3. The third primer preferably contains the base sequence shown in SEQ ID NO: 23, more preferably contains the base sequence shown in SEQ ID NO: 23 at the 3' end (3' end side), and particularly preferably is 22 nucleotides consisting of the base sequence shown in SEQ ID NO: 23.

[0043] (4th primer) The fourth primer specifically binds to retrotransposon Tvv1 on chromosome 15 of Shine Muscat grapes. The fourth primer preferably consists of a 19- to 25-base contiguous base sequence from the nucleotide sequence shown in SEQ ID NO: 4, more preferably a 20- to 22-base contiguous base sequence from the nucleotide sequence shown in SEQ ID NO: 4, and particularly preferably a 20- or 21-base contiguous base sequence from the nucleotide sequence shown in SEQ ID NO: 4. The fourth primer preferably contains the nucleotide sequence shown in SEQ ID NO: 24, more preferably contains the nucleotide sequence shown in SEQ ID NO: 24 at the 3' end (3' end side), and particularly preferably is 20 nucleotides consisting of the nucleotide sequence shown in SEQ ID NO: 24.

[0044] (5th Primer) The fifth primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon Tvv1 on chromosome 15 of Shine Muscat grapes. The fifth primer preferably consists of a 20-26 contiguous base sequence within the nucleotide sequence shown in SEQ ID NO: 5, more preferably a 21-23 contiguous base sequence within the nucleotide sequence shown in SEQ ID NO: 5, and particularly preferably a 21- or 22-contiguous base sequence within the nucleotide sequence shown in SEQ ID NO: 5. The fifth primer preferably contains the nucleotide sequence shown in SEQ ID NO: 25, more preferably contains the nucleotide sequence shown in SEQ ID NO: 25 at the 3' end (3' end side), and particularly preferably is 21 nucleotides consisting of the nucleotide sequence shown in SEQ ID NO: 25.

[0045] (Primer set (d)) The primer set (d) comprises a first primer having a tag sequence (tag nucleotide) attached (bound) to its 5' end and a second primer, or a second primer having a tag sequence attached to its 5' end and a first primer. By attaching a tag sequence to the primer, each DNA fragment, which is an amplified product, also has a tag sequence.

[0046] The tag sequence is a sequence complementary to a nucleic acid printed on a test paper (solid phase carrier) made of a nitrocellulose membrane or the like, and may be, for example, 15 to 50 nucleotides in length, 15 to 25 nucleotides in length, or 17 to 25 nucleotides in length. When a DNA fragment to which a tag sequence has been added is developed on the test paper, the DNA fragment hybridizes with a nucleic acid having a sequence complementary to the tag sequence.

[0047] Here, a substance capable of specifically binding to a labeling substance for visualizing hybridized DNA fragments (hereinafter also referred to as a "binding substance") is bound to the 5' end of the other of the first and second primers, i.e., the 5' end of the primer not having a tag sequence. Therefore, by performing PCR using the primer set (d) with the Shine Muscat genome as a template, a DNA fragment having a tag sequence bound to one end and a binding substance bound to the other end is obtained.

[0048] The DNA fragments are spread on a test strip, hybridized to a nucleic acid having a sequence complementary to the tag sequence, and then a labeling substance is bound to the bindable substance, thereby visualizing the DNA fragments (in other words, detecting the DNA fragments and confirming the amplification of the DNA fragments) (nucleic acid chromatography).

[0049] The labeling substance and the binding substance may be, for example, avidin and biotin, streptavidin and biotin, or an antigen and antibody (e.g., digoxigenin and anti-digoxigenin antibody, FITC and anti-FITC antibody), but are not limited to these. The labeling substance preferably contains a substance that facilitates visualization of DNA fragments, such as a dye, pigment, chemiluminescent substance such as luminol or luciferin, fluorescent substance, or metal colloid. The labeling substance may be contained in the developing solution, or may be supplied to the test strip after hybridization with the DNA fragments.

[0050] The type of developing solution used to develop PCR products that may contain DNA fragments onto the test paper is not particularly limited, but examples include buffers such as PBS (Phosphate Buffered Saline).

[0051] The above explains nucleic acid chromatography using a tag sequence attached to a primer. However, the technology of nucleic acid chromatography using a tag sequence is a publicly known technology, as disclosed in JP 2021-078393 A, JP 2021-164429 A, JP 2016-010338 A, etc.

[0052] Primer set (d), like primer set (a) above, can amplify a DNA fragment containing a part of the retrotransposon VINE1 on chromosome 9 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas no DNA fragment is amplified when the genomes of the 23 grape varieties described above are used as templates. Therefore, PCR using primer set (d) can accurately distinguish the 23 grape varieties described above from Shine Muscat.

[0053] (Primer set (e)) The primer set (e) comprises a first primer having a tag sequence attached (bound) to its 5' end and a third primer, or a third primer having a tag sequence attached to its 5' end and the first primer.

[0054] When the first primer has a tag sequence, the third primer has the aforementioned bindable substance bound to its 5' end.When the third primer has a tag sequence, the first primer has the aforementioned bindable substance bound to its 5' end.

[0055] Primer set (e), like primer set (b) above, can amplify a DNA fragment containing a part of the retrotransposon VINE1 on chromosome 13 and the adjacent and contiguous region on the genome using the genome of Shine Muscat as a template, whereas the DNA fragment is not amplified when the genomes of the 23 grape varieties described above are used as templates. Therefore, PCR using primer set (e) can accurately distinguish the 23 grape varieties from Shine Muscat.

[0056] (Primer set of (f)) The primer set of (f) includes a primer with a tag sequence attached (linked) to the 5'-end of the fourth primer and the fifth primer, or a primer with a tag sequence attached to the 5'-end of the fifth primer and the fourth primer.

[0057] When a tag sequence is attached to the fourth primer, the above-mentioned bondable substance is bound to the 5'-end of the fifth primer. When a tag sequence is attached to the fifth primer, the above-mentioned bondable substance is bound to the 5'-end of the fourth primer.

[0058] According to the primer set of (f), similar to the primer set of (c) above, a DNA fragment containing a part of the retrotransposon Tvv1 on chromosome 15 and a region on the genome adjacent and continuous thereto can be amplified using the genome of Shine Muscat as a template, whereas when the genomes of the above 22 grape varieties are used as templates, the DNA fragment is not amplified. Therefore, by performing PCR using the primer set of (f), the above 22 grape varieties and Shine Muscat can be accurately distinguished.

[0059] Furthermore, PCR may be performed by combining (d) to (f) (multiplex PCR). Examples of the combination methods include the combination of (d) and (e), the combination of (d) and (f), the combination of (e) and (f), and the combination of (d), (e), and (f).

[0060] According to the primer sets (a) to (f) described in detail above, a DNA fragment can be specifically and efficiently amplified using the genomic DNA of Shine Muscat as a template.

[0061] <Confirmation of amplification of DNA fragment> The method for confirming whether a DNA fragment can be amplified by PCR using the above primer set is not particularly limited. For example, when PCR is performed using primer sets (a) to (c), subjecting the PCR product to electrophoresis can be mentioned. In addition, when using primer sets (d) to (f), in addition to subjecting the PCR product to electrophoresis, subjecting the PCR product to nucleic acid chromatography can be mentioned. Nucleic acid chromatography is as described above.

[0062] As a result of electrophoresis, nucleic acid chromatography, etc., if the DNA fragment has been amplified (in other words, if an amplified DNA fragment has been obtained), the grape to be discriminated can be discriminated as Shine Muscat.

[0063] On the other hand, as a result of electrophoresis, nucleic acid chromatography, etc., if the DNA fragment has not been amplified (in other words, if an amplified DNA fragment has not been obtained), it is recognized that the grape to be discriminated is a variety other than Shine Muscat.

[0064] As described above in detail, the method for confirming the presence or absence of amplification of a DNA fragment after amplification by PCR will be described below, and the amplification by the LAMP method and the method for confirming the presence or absence of amplification of the DNA fragment thereafter will be further explained.

[0065] <Amplification of DNA fragment by LAMP method> In this discrimination method, in the amplification step, using one or more primer sets of the following (g) to (m), using the DNA of the grape to be discriminated as a template, a DNA fragment is amplified by the LAMP method. Thereafter, the presence or absence of amplification of the DNA fragment is confirmed, and when the DNA fragment has been amplified (when an amplified DNA fragment has been obtained), the grape to be discriminated can be discriminated as Shine Muscat.

[0066] (g) a sixth primer consisting of a nucleotide sequence of 20 to 29 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 6, and a seventh primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 7; an eighth primer consisting of the base sequence shown in SEQ ID NO: 8; a ninth primer consisting of the base sequence shown in SEQ ID NO: 9; a primer set comprising a 10th primer consisting of the base sequence shown in SEQ ID NO: 10; (h) an 11th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 11; a 12th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 12; a 13th primer consisting of the base sequence shown in SEQ ID NO: 13; a 14th primer consisting of the base sequence shown in SEQ ID NO: 14; a primer set comprising a 15th primer consisting of the base sequence shown in SEQ ID NO: 15; (i) a 16th primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 16; a 17th primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 17; an 18th primer consisting of the base sequence shown in SEQ ID NO: 18; a 19th primer consisting of the base sequence shown in SEQ ID NO: 19; a primer set comprising a 20th primer consisting of the base sequence shown in SEQ ID NO: 20 (j) a primer set in which any one of the eighth primer, the ninth primer, and the tenth primer of the primer set (g) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography. (k) a primer set in which any one of the 13th primer, the 14th primer, and the 15th primer of the primer set (h) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography. (m) a primer set in which any one of the 18th primer, the 19th primer, and the 20th primer of the primer set (i) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography.

[0067] When amplifying the DNA fragment using one or more primer sets (g) to (m) above, only one of the primer sets (g) to (m) is used to prevent problems such as the formation of dimers between primers. When amplifying the DNA fragment using two or more primer sets (g) to (m), amplification by the LAMP method is performed in a different reaction field for each primer set. This means that a single reaction field (e.g., a solution in a tube containing primers, a master mix, template DNA, etc.) contains only one (one type) of primer sets (g) to (m). As will be described in detail in the Examples below, it is possible to perform a multiplex LAMP reaction using a primer set that amplifies a grape consensus sequence.

[0068] As described above, if the reaction field is different for each primer set, the DNA fragment may be amplified using two or more primer sets (g) to (m). That is, if the reaction field is different, the amplification of the DNA fragment using the primer set (g), the amplification of the DNA fragment using the primer set (h), the amplification of the DNA fragment using the primer set (i), the amplification of the DNA fragment using the primer set (j), the amplification of the DNA fragment using the primer set (k), and the amplification of the DNA fragment using the primer set (m) may be performed in combination.

[0069] For example, both amplification of a DNA fragment using the primer set (g) and amplification of a DNA fragment using the primer set (h) may be performed; both amplification of a DNA fragment using the primer set (h) and amplification of a DNA fragment using the primer set (i) may be performed; both amplification of a DNA fragment using the primer set (g) and amplification of a DNA fragment using the primer set (i) may be performed; or both amplification of a DNA fragment using the primer set (g), amplification of a DNA fragment using the primer set (h), and amplification of a DNA fragment using the primer set (i) may be performed.

[0070] Furthermore, for example, both amplification of a DNA fragment using the primer set (j) and amplification of a DNA fragment using the primer set (k) may be performed; both amplification of a DNA fragment using the primer set (k) and amplification of a DNA fragment using the primer set (m) may be performed; both amplification of a DNA fragment using the primer set (j) and amplification of a DNA fragment using the primer set (m) may be performed; or all amplification of a DNA fragment using the primer set (j), amplification of a DNA fragment using the primer set (k), and amplification of a DNA fragment using the primer set (m) may be performed.

[0071] As described above, by attempting to amplify DNA fragments using multiple primer sets from (g) to (m), the accuracy of distinguishing Shine Muscat grapes can be improved compared to when attempting amplification using only one primer set.

[0072] (Primer set (g)) Primer set (g) can amplify a DNA fragment containing a portion of the retrotransposon VINE1 on chromosome 9 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas the DNA fragment (DNA marker) is not amplified when the genomes of the above 23 grape varieties are used as a template.

[0073] Therefore, by amplifying DNA fragments by the LAMP method using primer set (g), it is possible to accurately distinguish between the 23 grape varieties and Shine Muscat. Note that the region amplified using primer set (g) partially overlaps with the region amplified using primer set (a).

[0074] (Primer set (h)) Primer set (h) can amplify a DNA fragment containing a portion of the retrotransposon VINE1 on chromosome 13 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas the DNA fragment (DNA marker) is not amplified when the genomes of the 23 grape varieties listed above are used as templates. Therefore, amplifying a DNA fragment using the primer set (h) by the LAMP method allows accurate differentiation between the 23 grape varieties listed above and Shine Muscat. The region amplified using primer set (h) and the region amplified using primer set (b) partially overlap.

[0075] (Primer set (i)) Primer set (i) can amplify a DNA fragment containing a portion of the retrotransposon Tvv1 on chromosome 15 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas the DNA fragment (DNA marker) is not amplified when the genomes of the 22 grape varieties listed above are used as templates. Therefore, amplifying a DNA fragment using primer set (i) by the LAMP method allows accurate differentiation of the 22 grape varieties listed above from Shine Muscat. Furthermore, when primer set (i) is used, a DNA fragment is amplified even when the Muscat of Alexandria genome is used as a template, making it impossible to distinguish between Muscat of Alexandria and Shine Muscat. The region amplified using primer set (i) partially overlaps with the region amplified using primer set (c) above.

[0076] (6th Primer) The sixth primer specifically binds to the retrotransposon VINE1 on chromosome 9 of Shine Muscat grapes. The sixth primer preferably consists of a 21-27 contiguous base sequence (21-27 nucleotides) within the base sequence shown in SEQ ID NO: 6, more preferably a 22-24 contiguous base sequence within the base sequence shown in SEQ ID NO: 6, and particularly preferably a 22- or 23-contiguous base sequence within the base sequence shown in SEQ ID NO: 6. The sixth primer preferably contains the base sequence shown in SEQ ID NO: 26, and particularly preferably is 22 nucleotides consisting of the base sequence shown in SEQ ID NO: 26. The sixth primer preferably contains the base sequence shown in SEQ ID NO: 26 at its 3' end (3' end side).

[0077] (7th Primer) The seventh primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon VINE1 on chromosome 9 of Shine Muscat.

[0078] The seventh primer preferably consists of a base sequence of 24 to 30 consecutive bases within the base sequence shown in SEQ ID NO: 7, more preferably consists of a base sequence of 25 to 27 consecutive bases within the base sequence shown in SEQ ID NO: 7, and particularly preferably consists of a base sequence of 25 or 26 consecutive bases within the base sequence shown in SEQ ID NO: 7. The seventh primer preferably contains the base sequence shown in SEQ ID NO: 27, more preferably contains the base sequence shown in SEQ ID NO: 27 at the 3' end (3' end side), and particularly preferably is 25 nucleotides consisting of the base sequence shown in SEQ ID NO: 27.

[0079] (8th Primer) Primer 8 specifically binds to the retrotransposon VINE1 on Shine Muscat chromosome 9. Primer 8 is a sequence that joins the F1c sequence and the F2 sequence.

[0080] (9th Primer) The 9th primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon VINE1 on Shine Muscat chromosome 9. The 9th primer is a sequence that connects the B1c sequence and the B2 sequence.

[0081] (10th Primer) The tenth primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon VINE1 on chromosome 9 of Shine Muscat.

[0082] (11th Primer) The 11th primer specifically binds to the retrotransposon VINE1 on chromosome 13 of Shine Muscat grapes. The 11th primer preferably consists of a 19-25 contiguous base sequence (19-25 nucleotides) within the nucleotide sequence shown in SEQ ID NO: 11, more preferably a 20-22 contiguous base sequence within the nucleotide sequence shown in SEQ ID NO: 11, and particularly preferably a 20- or 21-contiguous base sequence within the nucleotide sequence shown in SEQ ID NO: 11. The 11th primer preferably contains the nucleotide sequence shown in SEQ ID NO: 28, and particularly preferably is 20 nucleotides consisting of the nucleotide sequence shown in SEQ ID NO: 28. The 11th primer preferably contains the nucleotide sequence shown in SEQ ID NO: 28 at its 3' end (3' end side).

[0083] (12th Primer) The 12th primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon VINE1 on chromosome 13 of Shine Muscat.

[0084] The 12th primer preferably consists of a base sequence of 19 to 25 consecutive bases within the base sequence shown in SEQ ID NO: 12, more preferably consists of a base sequence of 20 to 22 consecutive bases within the base sequence shown in SEQ ID NO: 12, and particularly preferably consists of a base sequence of 20 or 21 consecutive bases within the base sequence shown in SEQ ID NO: 12. The 12th primer preferably contains the base sequence shown in SEQ ID NO: 29, more preferably contains the base sequence shown in SEQ ID NO: 29 at the 3' end (3' end side), and particularly preferably is 20 nucleotides consisting of the base sequence shown in SEQ ID NO: 29.

[0085] (13th Primer) The 13th primer specifically binds to the retrotransposon VINE1 on chromosome 13 of Shine Muscat and a genomic region near the retrotransposon VINE1. The 13th primer is a sequence connecting the F1c sequence and the F2 sequence.

[0086] (14th Primer) The 14th primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon VINE1 on chromosome 13 of Shine Muscat. The 14th primer is a sequence that connects the B1c sequence and the B2 sequence.

[0087] (15th Primer) The 15th primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon VINE1 on chromosome 13 of Shine Muscat.

[0088] (16th Primer) The 16th primer specifically binds to the retrotransposon Tvv1 on chromosome 15 of Shine Muscat grapes. The 16th primer preferably consists of a nucleotide sequence of 18 to 24 consecutive bases (18 to 24 nucleotides) within the nucleotide sequence shown in SEQ ID NO: 16, more preferably a nucleotide sequence of 19 to 21 consecutive bases within the nucleotide sequence shown in SEQ ID NO: 16, and particularly preferably a nucleotide sequence of 19 or 20 consecutive bases within the nucleotide sequence shown in SEQ ID NO: 16. The 16th primer preferably contains the nucleotide sequence shown in SEQ ID NO: 30, and particularly preferably is 19 nucleotides consisting of the nucleotide sequence shown in SEQ ID NO: 30. The 16th primer preferably contains the nucleotide sequence shown in SEQ ID NO: 30 at its 3' end (3' end side).

[0089] (17th Primer) The 17th primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon Tvv1 on chromosome 15 of Shine Muscat.

[0090] The 17th primer preferably consists of a base sequence of 18 to 24 consecutive bases within the base sequence shown in SEQ ID NO: 17, more preferably consists of a base sequence of 19 to 21 consecutive bases within the base sequence shown in SEQ ID NO: 17, and particularly preferably consists of a base sequence of 19 or 20 consecutive bases within the base sequence shown in SEQ ID NO: 17. The 17th primer preferably contains the base sequence shown in SEQ ID NO: 31, more preferably contains the base sequence shown in SEQ ID NO: 31 at the 3' end (3' end side), and is particularly preferably 19 nucleotides consisting of the base sequence shown in SEQ ID NO: 31.

[0091] (18th Primer) Primer No. 18 specifically binds to retrotransposon Tvv1 on Shine Muscat chromosome 15. Primer No. 18 is a sequence linking the F1c sequence and the F2 sequence.

[0092] (19th Primer) Primer 19 specifically binds to the retrotransposon Tvv1 on chromosome 15 of Shine Muscat grapes and its neighboring genomic region (a non-retrotransposon sequence on the genome). Primer 19 is a sequence that joins the B1c and B2 sequences.

[0093] (20th Primer) The 20th primer specifically binds to a genomic region (a non-retrotransposon sequence on the genome) near the retrotransposon Tvv1 on chromosome 15 of Shine Muscat.

[0094] (Primer set (j)) The primer set (j) comprises a primer having a tag sequence (tag nucleotide) attached (bound) to the 5' end of the 8th primer, a 9th primer, and a 10th primer; a primer having a tag sequence attached to the 5' end of the 9th primer, the 8th primer, and the 10th primer; or a primer having a tag sequence attached to the 5' end of the 10th primer, the 8th primer, and the 9th primer.

[0095] In the primer set (j), the above-mentioned bindable substance is bound to one of the two primers among the eighth primer, the ninth primer, and the tenth primer that do not have a tag sequence. For example, if the ninth primer has a tag sequence, the above-mentioned bindable substance is bound to the 5' end of the eighth primer or the tenth primer. In this case, the primer to which the bindable substance is bound may be the eighth primer, as in the examples described below.

[0096] Primer set (j), like primer set (g) described above, can amplify a DNA fragment containing a portion of the retrotransposon VINE1 on chromosome 9 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas the DNA fragment is not amplified when the genomes of the 23 grape varieties described above are used as a template. Therefore, by amplifying the DNA fragment by the LAMP method using primer set (j), it is possible to accurately distinguish the 23 grape varieties described above from Shine Muscat.

[0097] (Primer set (k)) The primer set (k) comprises a primer having a tag sequence (tag nucleotide) attached (bound) to the 5' end of the 13th primer, a 14th primer, and a 15th primer; a primer having a tag sequence attached to the 5' end of the 14th primer, a 13th primer, and a 15th primer; or a primer having a tag sequence attached to the 5' end of the 15th primer, a 13th primer, and a 14th primer.

[0098] In the primer set (k), the 13th primer, the 14th primer, and the 15th primer have the aforementioned bindable substance bound to one of the two primers that do not have a tag sequence. For example, if the 13th primer has a tag sequence, the aforementioned bindable substance is bound to the 5' end of the 14th primer or the 15th primer. In this case, the primer to which the bindable substance is bound may be the 15th primer, as in the Examples described below.

[0099] Primer set (k), like primer set (h) described above, can amplify a DNA fragment containing a portion of the retrotransposon VINE1 on chromosome 13 and the adjacent and contiguous region on the genome using the Shine Muscat genome as a template, whereas the DNA fragment is not amplified when the genomes of the 23 grape varieties described above are used as a template. Therefore, by amplifying the DNA fragment by the LAMP method using primer set (k), it is possible to accurately distinguish between the 23 grape varieties described above and Shine Muscat.

[0100] (Primer set (m)) The primer set (m) comprises a primer having a tag sequence (tag nucleotide) attached (bound) to the 5' end of the 18th primer, a 19th primer, and a 20th primer; a primer having a tag sequence attached to the 5' end of the 19th primer, a 18th primer, and a 20th primer; or a primer having a tag sequence attached to the 5' end of the 20th primer, a 18th primer, and a 19th primer.

[0101] In the primer set (m), the 18th primer, the 19th primer, and the 20th primer have the aforementioned bindable substance bound to one of the two primers that do not have a tag sequence. For example, if the 18th primer has a tag sequence, the aforementioned bindable substance is bound to the 5' end of the 19th primer or the 20th primer. In this case, the primer to which the bindable substance is bound may be the 20th primer, as in the Examples described below.

[0102] According to the primer set of (m), similar to the primer set of (i) described above, using the genome of Shine Muscat as a template, a DNA fragment containing a part of the retrotransposon Tvv1 on chromosome 15 and the adjacent and continuous genomic region can be amplified. In contrast, when the genomes of the above 22 grape varieties are used as templates, the DNA fragment is not amplified. Therefore, by amplifying the DNA fragment by the LAMP method using the primer set of (m), the above 22 grape varieties and Shine Muscat can be accurately distinguished. When the primer set of (m) is used, when the genome of Muscat of Alexandria is used as a template, the DNA fragment is also amplified, so Muscat of Alexandria and Shine Muscat cannot be distinguished.

[0103] As described above in detail, according to the primer sets (g) to (m), using the genomic DNA of Shine Muscat as a template, a DNA fragment can be specifically and efficiently amplified.

[0104] (Amplification by LAMP method) For amplification by the LAMP method, equipment that can maintain the temperature of the reaction solution at a substantially constant level, such as an isothermal amplification fluorescence measurement device, a heat block, a constant temperature bath, and a magic bottle, is used. The temperature during isothermal amplification is not particularly limited, but for example, it may be 58 to 67 °C or 60 to 65 °C. By amplifying within these temperature ranges, the DNA fragment can be amplified within 30 minutes.

[0105] <Confirmation of DNA fragment amplification> When attempting to amplify a DNA fragment by the LAMP method using the above primer sets (g) to (m), the method for confirming whether the DNA fragment can be amplified is not particularly limited. For example, in the case of isothermal amplification by the LAMP method using one or more of the primer sets of (g) to (i), using a master mix containing an intercalator and detecting the rise in fluorescence intensity can be mentioned.

[0106] In the case of isothermal amplification by the LAMP method using one or more primer sets (j) to (m), in addition to detecting the rise in fluorescence intensity using the master mix, the isothermal amplification reaction can be followed by nucleic acid chromatography, in which DNA fragments are hybridized to nucleic acids having a sequence complementary to the tag sequence on the test strip, as described above, and visualized with a labeling substance.

[0107] As will be described in detail later in the Examples, when multiplex amplification is performed by the LAMP method using a primer set that amplifies a grape common sequence regardless of the variety, together with any one of the primer sets (g) to (m), the use of a master mix containing an intercalator causes a rise in fluorescence for all varieties used as templates. In this case, Shine Muscat can be identified by performing association curve analysis after amplification by the LAMP method.

[0108] In association curve analysis, the temperature of the reaction mixture after LAMP amplification is raised to single-strand the DNA, which may contain the target DNA fragment, is then gradually lowered, and the temperature at which it returns to double-stranded form is measured based on the fluorescence intensity. This value is specific to the amplified DNA, so it can be used to confirm whether the amplified product is the target sequence. When the template DNA is the Shine Muscat grape genome, a peak derived from the target DNA fragment is observed in addition to the peak derived from the amplified product of the grape consensus sequence.

[0109] [Shine Muscat Grape Identification Kit] In another preferred embodiment of the present invention, the Shine Muscat discrimination kit comprises a primer having a base sequence that specifically binds to the retrotransposon VINE1 or Tvv1 contained in the Shine Muscat genome, and a primer that specifically binds to a genomic region contained in the genome and located near the retrotransposon VINE1 or Tvv1, and is equipped with a primer set that can amplify the retrotransposon VINE1 or Tvv1 and the region on the genome adjacent to the retrotransposon using the Shine Muscat genome as a template.

[0110] Examples of such a discrimination kit include a discrimination kit including one or more primer sets selected from (a) to (f) described in detail in the embodiment of the discrimination method, and a discrimination kit including one or more primer sets selected from (g) to (m). Each of the primer sets (a) to (m) is as described above.

[0111] The primer set (d) is a primer set in which one of the first primer and the second primer of the primer set (a) is provided with a tag sequence for detecting the amplification product of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography. The primer set (e) is a primer set in which one of the first primer and the third primer of the primer set (b) is provided with a tag sequence for detecting the amplification product of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography. The primer set (f) is a primer set in which one of the fourth primer and the fifth primer of the primer set (c) is provided with a tag sequence for detecting the amplification product of the retrotransposon Tvv1 and the region on the genome adjacent to the retrotransposon Tvv1 by nucleic acid chromatography. The primer set (j) is a primer set in which any one of the eighth, ninth, and tenth primers of the primer set (g) is provided with a tag sequence for detecting the amplification product of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography. The primer set (k) is a primer set in which any one of the thirteenth, fourteenth, and fifteenth primers of the primer set (h) is provided with a tag sequence for detecting the amplification product of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography. The primer set (m) is a primer set in which any one of the eighteenth, nineteenth, and twentieth primers of the primer set (i) is provided with a tag sequence for detecting the amplification product of the retrotransposon Tvv1 and the region on the genome adjacent to the retrotransposon Tvv1 by nucleic acid chromatography.The discrimination kit of this embodiment may be a discrimination kit for use in the above-mentioned discrimination method.

[0112] According to the discrimination method and discrimination kit described above in detail, Shine Muscat can be discriminated using, for example, one or more primer sets of (a) to (f) or one or more primer sets of (g) to (m), thereby reducing the labor required by the operator and enabling rapid discrimination of Shine Muscat. Note that the nucleotide sequences shown in SEQ ID NOs: 1 to 7, 10 to 12, 15 to 17, 20 to 35, and 38 to 42 are all nucleotide sequences present in the genome of Shine Muscat.

[0113] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention. [Example]

[0114] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Experimental Example 1] In this experiment, DNA was extracted from the skins of 24 varieties of grapes, and PCR was performed using the primers (a) to (c) described above, and the amplified products were then subjected to electrophoresis.

[0115] DNA was extracted using the ISOSPIN Plant DNA kit (Nippon Gene Co., Ltd.) The extracted DNA stock solution was then adjusted to a concentration of 5 ng / μL based on the absorbance at 260 nm, and 1 μL of DNA was used as a template for PCR.

[0116] As an example of the primer set (a), the "VINE1-PBS" primer (an example of the first primer) and the "VINE1-Cl155" primer (an example of the second primer) shown in Table 1 below were used. This primer set (a) is expected to amplify a 365 bp double-stranded DNA fragment. Hereinafter, the DNA fragment (DNA marker) amplified by the primer set (a) will also be referred to as "VINE1-Cl155." As an example of the primer set (b), the "VINE1-PBS" primer (an example of the first primer) and the "VINE1-Cl160" primer (an example of the third primer) shown in Table 1 were used. This primer set (b) is expected to amplify a 338-bp double-stranded DNA fragment. Hereinafter, the DNA fragment (DNA marker) amplified by the primer set (b) will also be referred to as "VINE1-Cl160." As an example of primer set (c), the "Tvv1 1st primer" (an example of the fourth primer) and the "Tvv1-Cl80" primer (an example of the fifth primer) shown in Table 1 were used. This primer set (c) is expected to amplify a 195-bp double-stranded DNA fragment. Hereinafter, the DNA fragment (DNA marker) amplified by primer set (c) will also be referred to as "Tvv1-Cl80." In addition, "rbcLa_F1" and "rbcLa_R1" were used as a primer set that can amplify the grape consensus sequence as a control, regardless of the grape variety.

[0117] [Table 1] *R is a mixed base, representing A and G.

[0118] FIG. 1 is a diagram showing the sequence of the region on the Shine Muscat genome to which the primer set (a) and the primer set (g) in the Examples bind. FIG. 2 is a diagram showing the sequence of the region on the Shine Muscat genome to which the primer set (b) and the primer set (h) in the Examples bind. FIG. 3 is a diagram showing the sequence of the region on the Shine Muscat genome to which the primer set (c) and the primer set (i) in the Examples bind. In FIGS. 1 and 2, the region surrounded by a two-dot dash line is a non-retrotransposon region near the retrotransposon VINE1, and the region not surrounded by a two-dot dash line is a region within the retrotransposon VINE1. In FIG. 3, the region surrounded by a two-dot dash line is a non-retrotransposon region near the retrotransposon Tvv1, and the region not surrounded by a two-dot dash line is a region within the retrotransposon Tvv1.

[0119] Figure 1 shows nucleotides 2303102 to 2302623 (chromosome 9) of the Shine Muscat reference genome VSMuph2.0. Figure 2 shows nucleotides 3809594 to 3810073 (chromosome 13) of the Shine Muscat reference genome VSMuph2.0. Figure 3 shows nucleotides 15465872 to 15466231 (chromosome 15) of the Shine Muscat reference genome VSMuph2.0.

[0120] The PCR reaction composition is shown in Table 2 below. The "retro primer" is a primer that specifically binds to the retrotransposon VINE1 or Tvv1, and the "insertion site Cl primer" is a primer that specifically binds to a genomic region (non-retrotransposon sequence) located near the retrotransposon VINE1 or Tvv1.

[0121] [Table 2]

[0122] The PCR conditions were as follows: Steps 2 to 4 were performed for 30 cycles. The PCR was performed as a duplex PCR using one of the primer sets (a) to (c) and a primer set that amplifies the grape consensus sequence. Step 1: 98℃ 2 minutes Step 2: 95℃ 30 seconds Step3: 55℃ 30 seconds Step4: 72℃ 30 seconds Step5: 72℃ 5 minutes

[0123] After PCR under the above conditions, 5 μL of the PCR product was subjected to electrophoresis. The electrophoresis gel used was 2% agarose (dissolved in TAE buffer), and the PCR product was electrophoresed at a voltage of 100 V for 30 minutes. Nippon Gene's Gene Ladder 100 was used as the ladder.

[0124] Figure 4 shows the results of electrophoresis of PCR using the primer set (a) and DNA extracted from 24 grape varieties as a template. The correspondence between these 24 varieties and lane numbers is shown in Table 3 below.

[0125] [Table 3]

[0126] As shown in Figure 4, a band was confirmed at position 2 for all 24 grape varieties, indicating that the grape consensus sequence had been amplified. Furthermore, a band was confirmed at position 1, indicating a DNA fragment derived from primer set (a), for only Shine Muscat out of the 24 grape varieties.

[0127] These results demonstrate that the primer set (a) can be used to distinguish between the 23 grape varieties and Shine Muscat, and to identify Shine Muscat.

[0128] Figure 5 shows the results of electrophoresis of PCR using the primer set (b) and DNA extracted from 24 grape varieties as a template. The lane numbers correspond to the varieties as shown in Table 3 above.

[0129] As shown in Figure 5, a band was confirmed at position 2 for all 24 grape varieties, indicating that the grape consensus sequence had been amplified. Furthermore, a band was confirmed at position 3, indicating a DNA fragment derived from the primer set (b), for only Shine Muscat out of the 24 grape varieties.

[0130] These results demonstrate that the primer set (b) can be used to distinguish between the 23 grape varieties and Shine Muscat, and to identify Shine Muscat.

[0131] Figure 6 shows the results of electrophoresis of PCR using primer set (c) and DNA extracted from 24 grape varieties as a template. The lane numbers correspond to the varieties as shown in Table 3 above.

[0132] As shown in Figure 6, a band was confirmed at position 2 for all 24 grape varieties, indicating that the grape consensus sequence had been amplified. In addition, a band was confirmed at position 4 for Shine Muscat and Muscat of Alexandria, two of the 24 grape varieties, indicating a DNA fragment derived from primer set (c).

[0133] These results revealed that the primer set (c) can be used to distinguish between the 22 grape varieties mentioned above and Shine Muscat, and can identify Shine Muscat, but cannot distinguish between Muscat of Alexandria and Shine Muscat.

[0134] [Experimental Example 2] In this experiment, DNA was extracted from leaves of 24 grape varieties using the ISOSPIN Plant DNA kit (Nippon Gene Co., Ltd.). Based on the absorbance at 260 nm, 1 μL of DNA adjusted to a concentration of 5 ng / μL was used as a template for PCR using the primer sets (d) and (e) described above. The amplified product was then subjected to nucleic acid chromatography (C-PAS).

[0135] The primer set (d) was the same as the primer set (a) used in Experimental Example 1, in which biotin was attached to the 5' end of "VINE1-PBS" and an F3 tag (tag sequence) was attached to the 5' end of "VINE1-Cl155."

[0136] The primer set (e) was the same as the primer set (b) used in Experimental Example 1, in which biotin was attached to the 5' end of "VINE1-PBS" and an F1 tag (tag sequence) was attached to the 5' end of "VINE1-Cl160."

[0137] The primer set used for amplifying the grape consensus sequence consisted of "rbcLa_R1" used in Experimental Example 1 with biotin attached to the 5' end and "rbcLa_F1" with an F4 tag attached to the 5' end. The ratio of the amount of primer set (d) to the primer set for amplifying the grape consensus sequence was 0.8:0.15, and the ratio of the amount of primer set (e) to the primer set for amplifying the grape consensus sequence was 0.8:0.2.

[0138] The preparation of each tag sequence or biotin-containing primer used in this example was commissioned to TBA Co., Ltd. (Miyagi Prefecture). The composition of the PCR reaction solution is shown in Table 4 below. In Table 4, "primer for VINE1-Cl160" refers to the above-mentioned primers in which biotin or a tag sequence is bound to VINE1-PBS and VINE1-Cl160 in Table 1; "primer for VINE1-Cl155" refers to the above-mentioned primers in which biotin or a tag sequence is bound to VINE1-PBS and VINE1-Cl155 in Table 1; and "primer for rbcLa" refers to the above-mentioned primers in which biotin or a tag sequence is bound to rbcLa_F1 and rbcLa_R1 in Table 1.

[0139] [Table 4]

[0140] The PCR conditions were as follows: Steps 2 to 4 were performed for 32 cycles. The PCR was a multiplex PCR using the primer sets (d) and (e) and a primer set that amplifies the grape consensus sequence. Step 1: 98℃ 10 seconds Step 2: 98℃ 10 seconds Step3: 60℃ 5 seconds Step4: 68℃ 1 second Step5: 68℃ 10 seconds Step 6: 25℃ ∞

[0141] In nucleic acid chromatography, reagents such as developing solution and latex solution were purchased from TBA.

[0142] Figure 7 shows the results of PCR performed using DNA extracted from 24 varieties of grapes as a template, primer sets (d) and (e) and a primer set for amplifying the grape consensus sequence, followed by nucleic acid chromatography. As shown in Figure 7, for Shine Muscat, the ninth test paper from the left, bands representing the DNA fragment (VINE1-Cl155) amplified using the primer set (d) and the DNA fragment (VINE1-Cl160) amplified using the primer set (e) were confirmed, whereas for the other 23 varieties, the DNA fragment (VINE1-Cl155) amplified using the primer set (d) and the DNA fragment (VINE1-Cl160) amplified using the primer set (e) were not confirmed. Furthermore, a band (rbcLa) representing the amplification product of the grape consensus sequence was confirmed for all 24 varieties, including Shine Muscat.

[0143] The above results suggest that Shine Muscat grapes can be distinguished by performing PCR using primer set (d) and primer set (e) and then subjecting the PCR product to nucleic acid chromatography.

[0144] [Experimental Example 3] In this experiment, the same procedure as in Experimental Example 2 was carried out, except that DNA extracted from four varieties, Shine Muscat, Gross Krone, Queen Nina, and Sun Verde, was used instead of the 24 varieties used in Experimental Example 2.

[0145] Figure 8 shows the results of PCR performed using DNA extracted from four grape varieties as a template, the primer sets (d) and (e) and a primer set for amplifying the grape consensus sequence, and nucleic acid chromatography. "dw" stands for distilled water, which is the negative control.

[0146] As shown in Figure 8, for Shine Muscat, the first test paper counting from the left, bands representing the DNA fragment (VINE1-Cl155) amplified using the primer set (d) and the DNA fragment (VINE1-Cl160) amplified using the primer set (e) were confirmed, whereas these two DNA fragments (VINE1-Cl155, VINE1-Cl160) were not detected in the other three test paper varieties. Furthermore, a band (rbcLa) representing the amplification product of the grape consensus sequence was confirmed for all four varieties, including Shine Muscat.

[0147] The above results suggest once again that Shine Muscat grapes can be distinguished by performing PCR using primer set (d) and primer set (e) and then subjecting the PCR product to nucleic acid chromatography.

[0148] [Experimental Example 4] In this experiment, PCR was performed in the same manner as in Experimental Example 2, except that in addition to the primer sets (d) and (e), the primer set (f) was used.

[0149] The primer set (f) was the same as the primer set (c) used in Experimental Example 1, in which biotin was attached to the 5' end of "Tvv1 1st primer" and an F2 tag was attached to the 5' end of "Tvv1-Cl80."

[0150] Figure 9 shows the results of PCR performed using DNA extracted from four varieties of grapes as a template, primer sets (d), (e), and (f) and a primer set for amplifying the grape consensus sequence, followed by nucleic acid chromatography.

[0151] As shown in Figure 9, for Shine Muscat, the first test paper from the left, bands representing the DNA fragment (VINE1-Cl155) amplified using the primer set (d), the DNA fragment (VINE1-Cl160) amplified using the primer set (e), and the DNA fragment (VINE1-Cl80) amplified using the primer set (f) were confirmed, whereas these three DNA fragments were not observed for the other three test paper varieties. Furthermore, a band (rbcLa) representing the amplification product of the grape consensus sequence was confirmed for all four varieties, including Shine Muscat.

[0152] The above results suggest that Shine Muscat grapes can be distinguished by performing PCR using primer set (d), primer set (e), and primer set (f) and then subjecting the PCR products to nucleic acid chromatography.

[0153] [Experimental Example 5] In this experiment, DNA was amplified by the LAMP method using the primer set (h), and the amplification of DNA fragments was confirmed based on the rise (increase) in fluorescence intensity.

[0154] More specifically, DNA was extracted from the 24 grape varieties mentioned above, including Shine Muscat, using the ISOSPIN Plant DNA kit (Nippon Gene Co., Ltd.). 1 μL of DNA, adjusted to a concentration of 25 ng / μL based on absorbance at 260 nm, was used as a template, and DNA fragment amplification was attempted at 60-65°C for 30 minutes using an isothermal amplification fluorometer, Genie II (Optigene). The extracted template DNA was heat-denatured at 95°C for 10 minutes and then rapidly cooled on ice.

[0155] The primer set (h) used consisted of an 11th primer consisting of the base sequence shown in SEQ ID NO: 28, a 12th primer consisting of the base sequence shown in SEQ ID NO: 29, a 13th primer consisting of the base sequence shown in SEQ ID NO: 13, a 14th primer consisting of the base sequence shown in SEQ ID NO: 14, and a 15th primer consisting of the base sequence shown in SEQ ID NO: 15.

[0156] The LAMP reaction solution was prepared by adding 12.5 μL of 2x LAMP Master Mix (Nippon Gene Co., Ltd.), 1 μL of each 10x primer mix, and 1 μL of template DNA solution to sterile distilled water to make a total of 25 μL. Details are the same as in Experimental Example 6, which will be explained later.

[0157] FIG. 10 is a graph showing the results of measuring the transition of fluorescence intensity after DNA amplification by the LAMP method for 24 grape varieties using the primer set (h). As shown in Figure 10, in the sample using DNA extracted from Shine Muscat as a template, a rise in fluorescence intensity was observed in about 15 minutes, whereas in the samples using DNA extracted from the other 23 varieties as templates, no fluorescence was detected.

[0158] These results suggest that Shine Muscat grapes can be distinguished by the LAMP method using an isothermal amplification fluorescence measurement device.

[0159] [Experimental Example 6] In this experiment, DNA was extracted from the above 24 varieties, including Shine Muscat, in the same manner as in Experimental Example 5, and then heat-denatured and rapidly cooled. The DNA fragments were amplified for 30 minutes by the LAMP method using the primer set (k), and then association curve analysis was performed using the Genie II isothermal amplification fluorescence measuring device.

[0160] Specifically, DNA was extracted from grape leaves using the ISOSPIN Plant DNA kit (Nippon Gene Co., Ltd.). The template DNA was then heated to 95°C for 10 minutes and rapidly cooled on ice. 1 μL of the 20 ng / μL DNA solution was subjected to a LAMP reaction using the Genie II isothermal amplification fluorometer. The LAMP reaction temperature was 65°C. In this experiment, duplex detection was performed using primer set (k) and a primer set amplifying the grape consensus sequence. Table 5 below lists the primer set (k) used in the LAMP method. VINE1-Cl160_PE2_FIPF1 is a primer with the F1 tag sequence attached to the 5' end of the nucleotide sequence shown in SEQ ID NO: 13, and VINE1-Cl160_PE2_LBbiotin is a primer with biotin attached to the 5' end of the nucleotide sequence shown in SEQ ID NO: 15. Furthermore, VINE1-Cl160_PE2_FIP is a sequence in which F1c and F2 in FIG. 2 are linked, and VINE1-Cl160_PE2_BIP is a sequence in which B1c and B2 in FIG. 2 are linked.

[0161] [Table 5]

[0162] The sequences of the primer set used in the LAMP method to amplify the grape consensus sequence are shown in Table 6. UFGT_PE1_FIPF4 is a primer in which the F4 tag sequence is bound to the 5' end of the nucleotide sequence shown in SEQ ID NO: 36, and UFGT_PE1_BIPbiotin is a primer in which biotin is bound to the 5' end of the nucleotide sequence shown in SEQ ID NO: 37.

[0163] [Table 6]

[0164] The LAMP reaction solution was prepared by adding 12.5 μL of 2x LAMP Master Mix (Nippon Gene), 1 μL of each 10x primer mix, 1 μL of template DNA solution, and sterile distilled water to a total volume of 25 μL. The 10x primer mix was 2.0 μL for the primer set (k) and 0.375 μL for the primer set amplifying the grape consensus sequence (0.8:0.15). The primer mix included 5 pmol of F3 and B3, 40 pmol of FIP and BIP, 20 pmol of LB (and 20 pmol of LF for the grape consensus sequence), 0.025 μL of 1 M (mol / L) Tris-HCl (pH 8.0), 0.025 μL of 0.1 M DTT, and sterile distilled water to a total volume of 2.5 μL.

[0165] The LAMP reaction solution was basically the same for LAMP reactions using primer set (j) and primer set (m), which will be explained later, but when using primer set (j) and the primer set amplifying the grape consensus sequence, the 10x primer mix was 2.0 μL and 0.5 μL (0.8:0.2), respectively, and when using primer set (m) and the primer set amplifying the grape consensus sequence, the 10x primer mix was 2.5 μL and 0.5 μL (1.0:0.2), respectively.

[0166] In the association curve analysis, the reaction temperature was raised to 98°C to dissociate the amplified DNA from double strands to single strands, and then the temperature was gradually lowered to determine the temperature at which the amplified DNA returned to double strands. The intercalator was included in the 2x LAMP Master Mix.

[0167] Figure 11 is a graph showing the results of measuring the time course of fluorescence intensity after DNA amplification by the LAMP method for 15 of the 24 grape varieties using primer set (k) and a primer set amplifying a grape consensus sequence. Figure 12 is a graph showing the results of measuring the time course of fluorescence intensity after DNA amplification by the LAMP method for the remaining 9 of the 24 grape varieties using primer set (k) and a primer set amplifying a grape consensus sequence. The 15 grape varieties are Aki Queen, Campbell Early, Kyoho, Queen Nina, Gross Krone, Koshu, Concord, San Verde, Shine Muscat, Suiho, Steuben, Akamine, Takao, Delaware, and Niagara. The 9 grape varieties are Nagano Purple, Pione, Fujiminori, Black Beet, Portland, Muscat of Alexandria, Muscat Bailey A, Ruby Roman, and Rosario Bianco.

[0168] Figure 13 is a graph showing the results of association curve analysis after DNA amplification by LAMP for 15 of the 24 grape varieties using primer set (k) and a primer set that amplifies the grape consensus sequence. Figure 14 is a graph showing the results of association curve analysis after DNA amplification by LAMP for the remaining 9 of the 24 grape varieties using primer set (k) and a primer set that amplifies the grape consensus sequence. "NTC" is a negative control in which DNA extracted from grapes was not added during the LAMP reaction.

[0169] In this experiment, duplex detection including amplification of the grape consensus sequence was performed, and as shown in Figures 11 and 12, an increase in fluorescence intensity was confirmed for all varieties except for the negative control.

[0170] On the other hand, as shown in Figures 13 and 14, the association curve analysis showed that in addition to a peak at around 89.5°C for all 24 varieties, another peak at around 84°C was detected only in the sample using DNA extracted from Shine Muscat as a template.

[0171] From these results, it became clear that Shine Muscat can be distinguished by using the primer set (k) to amplify DNA fragments by the LAMP method and subjecting them to association curve analysis.

[0172] [Experimental Example 7] In this experiment, DNA was extracted from the 24 varieties mentioned above, including Shine Muscat, and the DNA fragments were amplified by the LAMP method using the primer set (k) shown in Table 5, and then subjected to nucleic acid chromatography (C-PAS). DNA extraction from grapes and DNA amplification by the LAMP method were performed in the same manner as in Experimental Example 6. Table 7 below shows the composition of the sample solution applied to the nucleic acid chromatography test strip. The 150 mM loading buffer in Table 7 was prepared by mixing equal amounts of 0 mM and 300 mM.

[0173] [Table 7]

[0174] The test strips were then immersed in the sample solution for about 10 minutes, and the presence or absence of bands representing DNA fragments was confirmed for each variety.

[0175] Figure 15 shows the results of DNA amplification by LAMP using DNA extracted from 24 grape varieties as a template, primer set (k) and a primer set for amplifying the grape consensus sequence, followed by nucleic acid chromatography. Figure 15 shows the following grape varieties, arranged in order from left to right: Aki Queen, Campbell Early, Kyoho, Queen Nina, Gross Krone, Koshu, Concord, San Verde, Shine Muscat, Suiho, Steuben, Akamine, Takao, Delaware, Niagara, negative control, Nagano Purple, Pione, Fujiminori, Black Beet, Portland, Muscat of Alexandria, Muscat Bailey A, Ruby Roman, Rosario Bianco, and negative control. In Figure 15, "F4" indicates the position where the amplification product of the grape consensus sequence was detected, and "F1" indicates the position where the DNA fragment was detected using primer set (k).

[0176] As shown in Figure 15, bands representing the amplification product of the grape consensus sequence were confirmed for all 24 varieties, whereas bands representing DNA fragments produced by primer set (k) were only confirmed in the amplification product produced using Shine Muscat DNA as a template.

[0177] These results demonstrate that Shine Muscat grapes can be accurately identified by performing nucleic acid chromatography after DNA amplification by the LAMP method using the primer set (k).

[0178] [Experimental Example 8] In this experiment, the same procedure as in Experimental Example 6 was carried out, except that DNA fragments were amplified by the LAMP method using primer set (j) instead of primer set (k). The primer set (j) used in the LAMP method is shown in Table 8 below. VINE1-Cl155_PE3_BIPF2 is a sequence in which an F2 tag is attached to the 5' end of the base sequence shown in SEQ ID NO: 9, which is formed by linking B1c and B2 in Figure 1, and VINE1-Cl155_PE3_FIPbiotin is a sequence in which biotin is attached to the 5' end of the base sequence shown in SEQ ID NO: 8, which is formed by linking F1c and F2 in Figure 1.

[0179] [Table 8]

[0180] Figure 16 is a graph showing the results of measuring the time course of fluorescence intensity after DNA amplification by the LAMP method for 15 of the 24 grape varieties using primer set (j) and a primer set that amplifies the grape consensus sequence. Figure 17 is a graph showing the results of measuring the time course of fluorescence intensity after DNA amplification by the LAMP method for the remaining 9 of the 24 grape varieties using primer set (j) and a primer set that amplifies the grape consensus sequence.

[0181] Figure 18 is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for 15 of the 24 grape varieties using the primer set (j) and a primer set that amplifies the grape consensus sequence. Figure 19 is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for the remaining 9 of the 24 grape varieties using the primer set (j) and a primer set that amplifies the grape consensus sequence. The 15 varieties in Figures 16 and 18 are the same as the 15 varieties in Figure 11.

[0182] In this experiment, as in Experimental Example 6, duplex detection including amplification of the grape consensus sequence was performed, and as shown in Figures 16 and 17, an increase in fluorescence intensity was confirmed for all varieties except for the negative control.

[0183] On the other hand, as shown in Figures 18 and 19, the results of the association curve analysis showed that in addition to a peak at around 89.5°C for all 24 varieties, a peak was also detected at around 82 to 84°C only for the sample using DNA extracted from Shine Muscat as a template.

[0184] From these results, it became clear that Shine Muscat can be distinguished by using the primer set (j) to amplify DNA fragments by the LAMP method and subjecting them to association curve analysis.

[0185] [Experimental Example 9] In this experiment, DNA extraction, amplification by the LAMP method, and nucleic acid chromatography (C-PAS) were performed in the same manner as in Experimental Example 7, except that primer set (j) shown in Table 8 was used instead of primer set (k).

[0186] Figure 20 shows the results of DNA amplification by the LAMP method using DNA extracted from 24 grape varieties as a template, the primer set (j) and the primer set for amplifying the grape consensus sequence, and nucleic acid chromatography. In Figure 20, the order of the varieties from left to right is the same as in Figure 15, "F4" is the detection position of the amplification product of the grape consensus sequence, and "F2" is the detection position of the DNA fragment amplified by the primer set (j).

[0187] As shown in Figure 20, bands representing the amplification product of the grape consensus sequence were confirmed for all 24 varieties, whereas bands representing the DNA fragment amplified by primer set (j) were only confirmed in the amplification product using Shine Muscat DNA as a template.

[0188] These results demonstrate that Shine Muscat grapes can be accurately identified by performing nucleic acid chromatography after DNA amplification by the LAMP method using the primer set (j).

[0189] [Experimental Example 10] In this experiment, the same procedure as in Experimental Example 8 was carried out, except that the DNA fragment was amplified by the LAMP method using primer set (m) instead of primer set (j), and the amplification by the LAMP method was carried out at 60°C. Table 9 below shows the primer set (m) used in the LAMP reaction. Tvv1-Cl-80_PE2_FIPF3 is a primer in which the tag sequence of F3 is linked to the 5' end of the nucleotide sequence shown in SEQ ID NO: 18, which is formed by linking F1c and F2 in Figure 3; Tvv1-Cl80_PE2_BIP is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 19, which is formed by linking B1c and B2 in Figure 3; and Tvv1-Cl80_PE2_LBbiotin is a primer in which biotin is linked to the 5' end of the nucleotide sequence shown in SEQ ID NO: 20.

[0190] [Table 9]

[0191] Figure 21 is a graph showing the results of measuring the time course of fluorescence intensity after DNA amplification by the LAMP method for 15 of the 24 grape varieties using primer set (m) and a primer set that amplifies the grape consensus sequence. Figure 22 is a graph showing the results of measuring the time course of fluorescence intensity after DNA amplification by the LAMP method for the remaining 9 of the 24 grape varieties using primer set (m) and a primer set that amplifies the grape consensus sequence.

[0192] Figure 23 is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for 15 of the 24 grape varieties using primer set (m) and a primer set that amplifies the grape consensus sequence. Figure 24 is a graph showing the results of association curve analysis after DNA amplification by the LAMP method for the remaining 9 of the 24 grape varieties using primer set (m) and a primer set that amplifies the grape consensus sequence. The 15 varieties in Figures 21 and 23 are the same as the 15 varieties in Figure 11.

[0193] In this experiment, as in Experimental Example 8, duplex detection including amplification of the grape consensus sequence was performed, and as shown in Figures 21 and 22, an increase in fluorescence intensity was confirmed for all varieties except for the negative control.

[0194] On the other hand, as shown in Figures 23 and 24, the association curve analysis showed a peak at around 89.5°C for all 24 varieties, and also a peak at around 80.5°C for only the samples prepared using DNA extracted from Shine Muscat and Muscat of Alexandria as templates.

[0195] These results demonstrate that when primer set (m) is used, Shine Muscat can be distinguished from the 22 cultivars listed above, excluding Muscat of Alexandria, by amplifying DNA fragments using the LAMP method.

[0196] [Experimental Example 11] In this experiment, DNA extraction, amplification by the LAMP method, and nucleic acid chromatography (C-PAS) were performed in the same manner as in Experimental Example 9, except that DNA fragments were amplified by the LAMP method using primer set (m) instead of primer set (j), and amplification by the LAMP method was carried out at 60°C for 30 minutes.

[0197] Figure 25 shows the results of DNA amplification by the LAMP method using DNA extracted from 24 grape varieties as a template, primer set (m) and a primer set for amplifying the grape consensus sequence, and nucleic acid chromatography. In Figure 25, the order of the varieties from left to right is the same as in Figure 15, "F4" indicates the detection position of the amplification product of the grape consensus sequence, and "F3" indicates the detection position of the DNA fragment amplified with primer set (m).

[0198] As shown in Figure 25, bands representing the amplification products of the grape consensus sequence were confirmed for all 24 varieties, whereas bands representing DNA fragments amplified with primer set (m) were confirmed only in the amplification products using DNA from Shine Muscat and Muscat of Alexandria as templates.

[0199] These results demonstrate that nucleic acid chromatography after DNA amplification by the LAMP method using primer set (m) can accurately distinguish between the 22 grape varieties listed above, excluding Muscat of Alexandria, and Shine Muscat.

[0200] [Experimental Example 12] In this experiment, a crude extract was prepared from grape skins without extracting DNA. DNA fragments were amplified by the LAMP method using primer set (j) shown in Table 8 and primer set (k) shown in Table 5, and then subjected to nucleic acid chromatography (C-PAS).

[0201] Specifically, one or two 5 mm square pieces (10 mg) of skin were first cut from grape berries using a scalpel, and 200 μL of GenCheck (registered trademark) DNA Extraction Reagent (Fasmac Co., Ltd.) was added to the resulting skin, which was then crushed and shaken in a vortex mixer.

[0202] The mixture was then heated to 100°C (above 95°C) for 10 minutes and then placed on ice for 1 minute. The mixture was then centrifuged at 15,000 x g for 1 minute at room temperature, and the supernatant was collected. The crude extract thus obtained was used as a template for LAMP. The crude extract also contained proteins, sugars, and other components in addition to DNA.

[0203] The DNA fragments were amplified at 65°C by the LAMP method using a heat block (aluminum block thermostatic bath, TAITEC Dry Thermo Unit DTU-1BN), and then subjected to nucleic acid chromatography (C-PAS).

[0204] Figure 26 shows the results of nucleic acid chromatography of DNA fragments amplified from a crude grape extract by the LAMP method using primer set (k) listed in Table 5. Figure 27 shows the results of nucleic acid chromatography of DNA fragments amplified from a crude grape extract by the LAMP method using primer set (j) listed in Table 8. Figures 26 and 27 show, from left to right, Shine Muscat, Queen Nina, San Verde, Pione, Delaware, Gros Krone, Koshu, Kyoho, Muscat of Alexandria, Rosario Bianco, Shine Muscat (DNA extracted from leaves, PC), and a negative control sample in which no grape template DNA was added to the LAMP reaction. "F4" indicates the detection position of the amplification product of the grape consensus sequence, "F1" indicates the detection position of the DNA fragment amplified with primer set (k), and "F2" indicates the detection position of the DNA fragment amplified with primer set (j).

[0205] As shown in Figures 26 and 27, bands representing amplification products of the grape consensus sequence were confirmed for all 10 varieties, whereas bands representing DNA fragments amplified by primer set (j) and primer set (k) were only confirmed in the amplification products using Shine Muscat DNA as a template.

[0206] These results demonstrate that Shine Muscat grapes can be accurately identified by performing nucleic acid chromatography after DNA amplification with the primer sets (j) and (k), even when a crude extract obtained from the skin is used instead of a grape DNA extract. [Industrial Applicability]

[0207] According to the present invention, Shine Muscat grapes can be identified by amplifying the retrotransposon VINE1 or Tvv1 and the region on the genome adjacent to the retrotransposon, and therefore, the present invention is industrially applicable. [Explanation of symbols]

[0208] 1...position showing the DNA fragment derived from primer set (a), 2...position showing that the grape consensus sequence was amplified, 3...position showing the DNA fragment derived from primer set (b), 4...position showing the DNA fragment derived from primer set (c).

Claims

1. a primer having a base sequence that specifically binds to retrotransposon VINE1 or Tvv1 contained in the genome of Shine Muscat; a primer set including a primer that specifically binds to a genomic region located near the retrotransposon VINE1 or Tvv1 contained in the genome, A method for distinguishing Shine Muscat grapes, comprising a step of amplifying the retrotransposon VINE1 or Tvv1 and a region on the genome adjacent to the retrotransposon, using DNA of grapes to be distinguished as a template.

2. In the above step, a DNA fragment is amplified by PCR using one or more primer sets selected from the following (a) to (f) and using the DNA of the grapevine to be identified as a template; The method for distinguishing Shine Muscat according to claim 1, wherein when the amplified DNA fragment is obtained, the grape is distinguished as Shine Muscat; (a) a first primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 1; a second primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 2; (b) the first primer; and a third primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 3; (c) a fourth primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 4; a fifth primer consisting of a base sequence of 19 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 5; (d) a primer set in which one of the first primer and the second primer of the primer set of (a) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography; (e) a primer set in which one of the first primer and the third primer of the primer set of (b) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography; (f) A primer set in which one of the fourth primer and the fifth primer of the primer set of (c) has a tag sequence added thereto for detecting the DNA fragment by nucleic acid chromatography.

3. In the above step, a DNA fragment is amplified by the LAMP method using one or more primer sets selected from the following (g) to (m) and using the DNA of the grapevine to be identified as a template: The method for distinguishing Shine Muscat according to claim 1, wherein when the amplified DNA fragment is obtained, the grape is distinguished as Shine Muscat; (g) a sixth primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 6; a seventh primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 7; an eighth primer consisting of the base sequence shown in SEQ ID NO: 8; a ninth primer consisting of the base sequence shown in SEQ ID NO: 9; a primer set comprising a 10th primer consisting of the base sequence shown in SEQ ID NO: 10; (h) an 11th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 11; a 12th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 12; a 13th primer consisting of the base sequence shown in SEQ ID NO: 13; a 14th primer consisting of the base sequence shown in SEQ ID NO: 14; a primer set comprising a 15th primer consisting of the base sequence shown in SEQ ID NO: 15; (i) a 16th primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 16; a 17th primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 17; an 18th primer consisting of the base sequence shown in SEQ ID NO: 18; A 19th primer consisting of the base sequence shown in SEQ ID NO: 19; a primer set comprising a 20th primer consisting of the base sequence shown in SEQ ID NO: 20; (j) a primer set in which a tag sequence for detecting the DNA fragment by nucleic acid chromatography is added to any one of the eighth primer, the ninth primer, and the tenth primer of the primer set of (g); (k) a primer set in which a tag sequence for detecting the DNA fragment by nucleic acid chromatography is added to any one of the 13th primer, the 14th primer, and the 15th primer of the primer set of (h); (m) A primer set in which any one of the 18th primer, the 19th primer, and the 20th primer of the primer set (i) is provided with a tag sequence for detecting the DNA fragment by nucleic acid chromatography. (However, when the DNA fragment is amplified using two or more primer sets from (g) to (m), amplification by the LAMP method is performed in a different reaction field for each primer set.)

4. the first primer comprises the base sequence shown in SEQ ID NO: 21, the second primer comprises the base sequence shown in SEQ ID NO: 22, the third primer comprises the nucleotide sequence shown in SEQ ID NO: 23, the fourth primer comprises the nucleotide sequence shown in SEQ ID NO: 24, The method for distinguishing Shine Muscat grapes according to claim 2 , wherein the fifth primer comprises the base sequence shown in SEQ ID NO:

25.

5. the sixth primer comprises the base sequence shown in SEQ ID NO: 26, the seventh primer comprises the base sequence shown in SEQ ID NO: 27, the 11th primer comprises the base sequence shown in SEQ ID NO: 28, the 12th primer comprises the base sequence shown in SEQ ID NO: 29, the 16th primer comprises the base sequence shown in SEQ ID NO: 30; The method for distinguishing Shine Muscat grapes according to claim 3 , wherein the 17th primer comprises the base sequence shown in SEQ ID NO:

31.

6. a primer having a base sequence that specifically binds to retrotransposon VINE1 or Tvv1 contained in the genome of Shine Muscat; a primer that specifically binds to a genomic region located near the retrotransposon VINE1 or Tvv1 contained in the genome; A kit for distinguishing Shine Muscat, comprising a primer set capable of amplifying the retrotransposon VINE1 or Tvv1 and a region on the genome adjacent to the retrotransposon, using the Shine Muscat genome as a template.

7. The Shine Muscat discrimination kit according to claim 6, wherein the primer set is one or more of the following primer sets (a) to (f): (a) a first primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 1; a second primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 2; (b) the first primer; and a third primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 3; (c) a fourth primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 4; a fifth primer consisting of a base sequence of 19 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 5; (d) a primer set in which one of the first primer and the second primer of the primer set of (a) is provided with a tag sequence for detecting amplification products of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography; (e) a primer set in which one of the first primer and the third primer of the primer set of (b) is provided with a tag sequence for detecting amplification products of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography; (f) A primer set in which one of the fourth primer and the fifth primer of the primer set of (c) is provided with a tag sequence for detecting amplification products of the retrotransposon Tvv1 and the region on the genome adjacent to the retrotransposon Tvv1 by nucleic acid chromatography.

8. The Shine Muscat discrimination kit according to claim 6, wherein the primer set is one or more of the following primer sets (g) to (m): (g) a sixth primer consisting of a base sequence of 20 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 6; a seventh primer consisting of a base sequence of 23 to 32 consecutive bases in the base sequence shown in SEQ ID NO: 7; an eighth primer consisting of the base sequence shown in SEQ ID NO: 8; a ninth primer consisting of the base sequence shown in SEQ ID NO: 9; a primer set comprising a 10th primer consisting of the base sequence shown in SEQ ID NO: 10; (h) an 11th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 11; a 12th primer consisting of a base sequence of 18 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 12; a 13th primer consisting of the base sequence shown in SEQ ID NO: 13; a 14th primer consisting of the base sequence shown in SEQ ID NO: 14; a primer set comprising a 15th primer consisting of the base sequence shown in SEQ ID NO: 15; (i) a 16th primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 16; a 17th primer consisting of a base sequence of 17 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 17; an 18th primer consisting of the base sequence shown in SEQ ID NO: 18; A 19th primer consisting of the base sequence shown in SEQ ID NO: 19; a primer set comprising a 20th primer consisting of the base sequence shown in SEQ ID NO: 20; (j) a primer set in which a tag sequence is added to any one of the eighth primer, the ninth primer, and the tenth primer of the primer set of (g) for detecting amplification products of the retrotransposon VINE1 and the region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography; (k) a primer set in which any one of the 13th primer, the 14th primer, and the 15th primer of the primer set of (h) is provided with a tag sequence for detecting an amplification product of the retrotransposon VINE1 and a region on the genome adjacent to the retrotransposon VINE1 by nucleic acid chromatography; (m) A primer set in which any one of the 18th primer, the 19th primer, and the 20th primer of the primer set of (i) is provided with a tag sequence for detecting the amplification products of the retrotransposon Tvv1 and the region on the genome adjacent to the retrotransposon Tvv1 by nucleic acid chromatography.

9. the first primer comprises the base sequence shown in SEQ ID NO: 21, the second primer comprises the base sequence shown in SEQ ID NO: 22, the third primer comprises the nucleotide sequence shown in SEQ ID NO: 23, the fourth primer comprises the nucleotide sequence shown in SEQ ID NO: 24, The Shine Muscat discrimination kit according to claim 7 , wherein the fifth primer comprises the base sequence shown in SEQ ID NO:

25.

10. the sixth primer comprises the base sequence shown in SEQ ID NO: 26, the seventh primer comprises the base sequence shown in SEQ ID NO: 27, the 11th primer comprises the base sequence shown in SEQ ID NO: 28, the 12th primer comprises the base sequence shown in SEQ ID NO: 29, the 16th primer comprises the base sequence shown in SEQ ID NO: 30; The Shine Muscat discrimination kit according to claim 8, wherein the 17th primer comprises the base sequence shown in SEQ ID NO: 31.

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