Identification techniques for the apple variety Nishikishu

Abstract

To provide a simple and rapid method for identifying the specific apple variety Nishikishu. [Solution] According to the present invention, a method for identifying the apple variety Nishikishu is provided, comprising the following steps: a) applying a nucleic acid amplification method using at least two primer pairs, each containing a primer that binds to a retrotransposon in the genome of the apple variety Nishikishu and a primer that binds to a genomic region of Nishikishu located near the retrotransposon, with the DNA to be identified as a template, wherein the retrotransposon is CTcrm2, dem1, or TRIM; and b) identifying Nishikishu based on a combination of the presence or absence of an amplification product.

Description

[Technical Field]

[0001] The present invention relates to a method for identifying the apple cultivar Kinkei, comprising the steps of: a) applying a nucleic acid amplification method using at least two primer pairs, wherein each primer pair includes a primer that binds into a retrotransposon in the genome of the apple cultivar Kinkei and a primer that binds to a genomic region of Kinkei located near said retrotransposon, with DNA of an identification target used as a template; and b) identifying Kinkei based on the combination of the presence or absence of amplification products. The present invention also relates to a primer pair for identifying the apple cultivar Kinkei. [Background Art]

[0002] According to the status of apple cultivar registration under Japan's Seeds and Seedlings Act, there are approximately 300 cultivars including those published upon application. Many cultivars such as superior edible cultivars and rootstock cultivars have been bred and applied for registration by institutions including the Fruit Research Institute of the National Agriculture and Food Research Organization (hereinafter referred to as NARO Fruit Research Institute) (Non-Patent Document 1).

[0003] "Kinkei" is a promising apple cultivar bred by the NARO Fruit Research Institute. There are concerns both that seedlings of this cultivar may be taken out of Japan without authorization to foreign countries, and that such unauthorized seedlings may be re-imported back into Japan.

[0004] On the other hand, for cultivar identification, DNA cultivar identification technology that utilizes differences in DNA base sequences between individual cultivars is commonly used. For example, there is a demand for the introduction of DNA cultivar identification technology that enables more accurate determination in border inspections at customs and investigations related to unauthorized taking-out of seedlings and the like. Particularly, in border inspections at customs, since the time available for inspection is limited, a genetic testing method that can be carried out as simply and rapidly as possible is desirable.

[0005] A technology for identifying apple varieties using SSR markers has been published by the National Agriculture and Food Research Organization (NARO) (Non-Patent Document 1). This identification technology uses nine types of SSR markers to identify 47 apple varieties, including Nishikishu. However, the identification method using SSR markers requires expensive equipment, as it involves performing PCR followed by fragment analysis using a DNA sequencer, and the analysis of the results also requires specialized knowledge.

[0006] Furthermore, a technique for identifying apple varieties using retrotransposon insertion site polymorphism markers has been reported (Non-Patent Literature 2). This identification technique can identify 36 varieties by using at least 10 types of markers. However, since at least 10 types of markers must be analyzed to identify varieties, the experiment is complicated, or the interpretation of results based on combinations of marker positivity is complex. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] National Agriculture and Food Research Organization, "DNA Variety Identification Technology for 47 Apple Varieties and Lines Using SSR Markers," March 25, 2021, 2nd edition, https: / / www.naro.go.jp / publicity_report / publication / files / nifts_ringo_shikibetsu20210325.pdf [Non-Patent Document 2] Chikako Nishitani, Toshiya Yamamoto, Hiroshi Fujii, Kazuma Okada, Yuki Kadota, and Makoto Tahara, "Development of apple variety identification markers using retrotransposon insertion polymorphisms," 2016, Plant DNA Polymorphism 24:101-107. [Overview of the project] [Problems that the invention aims to solve]

[0008] Traditional methods for identifying apple varieties required expensive equipment, specialized knowledge, or complex preparations, which made their introduction into border inspections by customs officials, for example, difficult. Therefore, the object of the present invention is to provide a simple and rapid method for identifying the specific variety of apple, Nishikishu. [Means for solving the problem]

[0009] As a result of diligent research, the inventors of this invention have identified a retrotransposon insertion site polymorphism marker specific to the Nishikishu apple variety in 24 major apple varieties examined, thus completing the present invention.

[0010] The present invention provides the following inventions. [1] A method for identifying the apple variety Nishikishu, comprising the following steps: a) Using the DNA to be identified as a template, A primer that binds to a retrotransposon in the genome of the apple variety Nishikishu, and A primer that binds to the genome region of Nishikishu, located near the aforementioned retrotransposon. A step of applying a nucleic acid amplification method using at least two sets of primer pairs containing, wherein the retrotransposon is CTcrm2, dem1, or TRIM. Furthermore b) A step to identify Nishikishu based on the combination of presence or absence of amplification products. [2] The identification method according to [1], wherein at least two pairs of primers in step a) are selected from the following 1) to 4): 1) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 5, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 6; 2) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 7, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 8; 3) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 9, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 10; 4) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 11, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 12. [3] The method according to [1] or [2], wherein at least two pairs of primers in step a) are selected from 1) to 4) below: 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 14; 2) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 15; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 17; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 18. [4] The method according to any one of [1] to [3], wherein the at least two primer pairs in step a) are the following combinations: a combination of the primer pair of 1) and the primer pair of 2) according to [2] or [3], a combination of the primer pair of 2) and the primer pair of 3) according to [2] or [3], or a combination of the primer pair of 2) and the primer pair of 4) according to [2] or [3]. [5] The method according to any one of [1] to [4], wherein step a) is a step of applying a nucleic acid amplification method using DNA to be identified as a template and at least two primer pairs selected from the aforementioned 1) to 4) and the following 5) to 10): 5) a pair of a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 13, and a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 19; 6) a pair of a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 13, and a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 20; 7) a pair of a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 16, and a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 21; 8) a pair of a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 22, and a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 23; 9) a pair of a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 22, and a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 24; 10) a pair of a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 13, and a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 25. [6] The method according to any one of [1] to [5], wherein the at least two primer pairs in said step a) are any one of the following combinations: a combination of the primer pair of 1) recited in [2] or [3] and the primer pair of 7) recited in [5], a combination of the primer pair of 1) recited in [2] or [3] and the primer pair of 8) recited in [5], a combination of the primer pair of 1) recited in [2] or [3] and the primer pair of 10) recited in [5], a combination of the primer pair of 3) recited in [2] or [3] and the primer pair of 7) recited in [5], a combination of the primer pair of 3) recited in [2] or [3] and the primer pair of 8) recited in [5], a combination of the primer pair of 3) recited in [2] or [3] and the primer pair of 10) recited in [5], a combination of the primer pair of 5) recited in [5] and the primer pair of 7) recited in [5], a combination of the primer pair of 5) recited in [5] and the primer pair of 8) recited in [5], a combination of the primer pair of 5) recited in [5] and the primer pair of 10) recited in [5], a combination of the primer pair of 6) recited in [5] and the primer pair of 7) recited in [5], a combination of the primer pair of 6) recited in [5] and the primer pair of 8) recited in [5], a combination of the primer pair of 6) recited in [5] and the primer pair of 10) recited in [5], a combination of the primer pair of 4) recited in [2] or [3] and the primer pair of 7) recited in [5], a combination of the primer pair of 4) recited in [2] or [3] and the primer pair of 8) recited in [5], a combination of the primer pair of 4) recited in [2] or [3] and the primer pair of 10) recited in [5], a combination of the primer pair of 7) recited in [5] and the primer pair of 9) recited in [5], or A combination of the primer pair 8) described in [5] and the primer pair 9) described in [5]. [7] The method according to one of [1] to [6], wherein the nucleic acid amplification method is the PCR method. [8] The method according to one of [1] to [7], wherein in step a), a nucleic acid amplification method using at least two sets of primer pairs is carried out in the same reaction solution. [9] A pair of primers for identifying the apple variety Nishikishu, selected from 1) to 4) and A) below: 1) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 5, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 6; 2) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 7, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 8; 3) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 9, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 10; 4) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 11, and A pair of second primers containing a polynucleotide consisting of 15 or more consecutive base sequences in the base sequence or complementary sequence described in Sequence ID No. 12; A) A pair in which tag DNA is attached to either the first primer or the second primer described in 1) to 4) above.

[10] A pair of primers for identifying the apple variety Nishikishu, selected from 1) to 10) and B) below: 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 14; 2) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 15; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 17; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 18; 5) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 19; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 7) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 21; 8) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 9) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 10) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; B) A pair in which tag DNA is attached to either the first primer or the second primer described in 1) to 10) above. [Effects of the Invention]

[0011] According to the present invention, a simple and rapid method for identifying autumn-colored flowers is provided. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the nucleotide sequences at the boundary between the retrotransposon sequence and the non-retrotransposon apple genome sequence (hereinafter referred to as the apple sequence) for four regions: CTcrm2_Cl434, CTcrm2_Cl311, dem1_pattern561, and dem1_pattern428. The bold, italicized portion is the apple genome sequence. [Figure 2] Figure 2 shows the non-retrotransposon regions of the apple genome near the retrotransposon insertion sites for CTcrm2_pattern639, CTcrm2_pattern689, dem1_pattern567, TRIM_pattern2692, TRIM_Cl924, and CTcrm2_Cl191. [Figure 3] Figure 3 shows the results of PCR performed using two primer pairs, with DNA extracted from Kinshu and Beniminori as templates, and then spread onto C-PAS test strips. There are four primer pair combination patterns: (1) CTcrm2_Cl434 and CTcrm2_Cl311, (2) dem1_pattern428 and CTcrm2_Cl311, and (3) dem1_pattern561 and CTcrm2_Cl311. Test strips numbered 1, 2, and 3 represent Kinshu, Beniminori, and sterile water, respectively. [Figure 4]Figure 4 shows the results of multiplex PCR performed using DNA extracted from 24 apple varieties as templates, employing primer pairs for amplifying dem1_pattern428, CTcrm2_Cl311, and a positive control, and then spreading the results onto C-PAS test strips. Table 6 shows the correspondence between the test strip numbers and apple varieties. [Modes for carrying out the invention]

[0013] [How to identify the apple variety Nishikishu] The present invention relates to a method for identifying the apple variety Nishikishu, comprising the following steps. a) Using the DNA to be identified as a template, A primer that binds to a retrotransposon in the genome of the apple variety Nishikishu, and A primer that binds to the genome region of Nishikishu, located near the aforementioned retrotransposon. A step of applying a nucleic acid amplification method using at least two sets of primer pairs containing the following: Furthermore b) A step to identify Nishikishu based on the combination of presence or absence of amplification products. In one embodiment, the retrotransposon is CTcrm2, dem1, or TRIM.

[0014] The Kinshu apple variety is a new mid-season apple variety known for its high sweetness and excellent flavor. It features a deep red skin that colors easily, and is characterized by its juiciness and good flesh texture. The variety's registration number is 27428 (April 23, 2019). Because the Kinshu apple variety is a superior variety, there are concerns about infringement such as the unauthorized removal of fruit and seedlings.

[0015] This invention provides a simple and rapid genetic testing method for identifying autumn-colored flowers. Because the test can be performed in a short time, this invention can be used, for example, in border inspections at customs.

[0016] Step a) of the present invention involves using the DNA to be identified as a template and amplifying the retrotransposon present in the genome of Nishikishu and the genomic region near its insertion site by nucleic acid amplification using at least two sets of primer pairs.

[0017] In the identification method of the present invention, the object to be identified may be any plant or its processed product, but for example, the object to be identified may be a plant of the genus Malus Mill. of the Rosaceae family or its processed product, based on morphological characteristics, external characteristics, etc.

[0018] In the identification method of the present invention, the DNA to be identified can be extracted from any part of a plant, for example. Specifically, it can be extracted from leaves, stems, trunks, branches, bark, buds, flowers, fruit pulp, fruit peel, etc., and due to the ease of extraction, it is preferable to extract from leaves, fruit peel, etc. The plant sample for DNA extraction may be fresh, refrigerated, frozen, or dried (silica gel dried). The object to be identified may be a processed product, such as dried fruit pulp (dried fruit, dried apples), jam, juice, confectionery, etc. Since DNA may not be extractable from processed products, it is particularly important to confirm the amplification of a positive control to confirm that DNA extraction has been successful.

[0019] Samples for DNA extraction can be prepared by shredding, grinding (liquid nitrogen grinding, bead grinding, etc.), or pulverizing. DNA extraction can be performed by methods such as CTAB (cetyltrimethylammonium bromide), phenol-chloroform extraction, SDS (sodium dodecyl sulfate) extraction, ethanol precipitation, silica membrane technology (spin column method), and magnetic bead technology, but is not limited to these; any method capable of DNA extraction can be used. DNA extraction can be performed using commercially available kits, such as ISOSPIN Plant DNA (Nippon Gene Co., Ltd.) and DNeasy Plant Mini Kit (Qiagen).

[0020] Nucleic acid amplification methods include, but are not limited to, PCR, LAMP (loop-mediated isothermal amplification), HDA (helicase-dependent amplification), RPA (recombinase polymerase amplification), SDA (strand displacement amplification), and RCA (rolling cycle amplification). Methods for confirming the presence or absence of amplification products include electrophoresis and chromatographic PAS.

[0021] Retrotransposon sequences are scattered throughout the genome, and once inserted, they are stably inherited. Therefore, copy sequences inserted in different regions between varieties can be used as DNA markers. When retrotransposons transpose, they insert their own replicated sequences at random locations in the genome, so it is statistically highly unlikely that insertions would occur independently in the same location across different varieties within the entire genome.

[0022] The identification method of the present invention utilizes inter-varietal insertion polymorphisms of the retrotransposon families "CTcrm2," "dem1," and "TRIM." "CTcrm2," "dem1," and "TRIM" are present in multiple regions of the Nishikishu genome. The inventors have found that Nishikishu can be identified by combining two regions of retrotransposon insertion sites as DNA markers.

[0023] Step a) of the present invention is a step of amplifying "a retrotransposon and the genomic region near its insertion site" by nucleic acid amplification using a primer that binds to a retrotransposon within the genome of the apple variety Nishikishu, and a primer that binds to a genomic region of Nishikishu located near the retrotransposon.

[0024] Examples of "retrotransposons and the genomic regions near their insertion sites" are the regions named CTcrm2_Cl434 (sequence number 1), CTcrm2_Cl311 (sequence number 2), dem1_pattern561 (sequence number 3), and dem1_pattern428 (sequence number 4), shown in Figure 1. In Figure 1, the retrotransposon is inserted before an apple genome sequence that is not a retrotransposon sequence (hereinafter referred to as the apple sequence) (the apple sequence is the sequence shown in bold italics in Figure 1).

[0025] Retrotransposon sequences of the same type ("CTcrm2" type, "dem1" type, and "TRIM" type) are considered to have very high sequence identity. On the other hand, the apple sequence near the insertion site is completely different. The above-mentioned "retrotransposon and the genomic region near its insertion site" can be specifically amplified by designing and using primers that specifically bind to the apple sequence near the insertion site. On the other hand, "primers that bind to retrotransposons in the genome of the apple variety Nishikishu" can be used as primers that can be commonly used for amplification of retrotransposon insertion sites of the same type. That is, the base sequences of retrotransposons of the same type can be aligned, and primers can be set in the region without mutations. In another embodiment, primers can be set including the region with mutations, and individual primers can be designed for each region to be amplified.

[0026] Regarding step a) of the present invention, the primers that bind to the retrotransposon within the genome of the apple variety Nishikishu, and the primers that bind to the genomic region of Nishikishu located near the retrotransposon, can be designed to amplify all or part of the "retrotransposon and the genomic region near its insertion site" described in SEQ ID NOs: 1, 2, 3, or 4. When amplifying a part of the above region, the length of the target sequence (number of bases between primers) can be appropriately set for the primer pair used in the PCR method, and they may be set to be in complete proximity or to be several bases or more apart, for example, 10 bases or more. Similarly, primers can be set and amplified for the genomic region near the retrotransposon insertion site and the region containing the inserted retrotransposon described in SEQ ID NOs: 28, 29, 30, 31, or 32. In the sequences described in SEQ ID NOs: 28, 29, 30, 31, or 32, the "primer that binds to the genomic region of Nishikishu located near the retrotransposon" can be set, and the "primer that binds to the retrotransposon within the genome of the apple variety Nishikishu" can be the one set for the same type of retrotransposon.

[0027] Step b) of the present invention is a step of identifying Kinshu based on the combination of the presence or absence of amplification products. In one embodiment, the reaction solution of the nucleic acid amplification method in step a) is analyzed, and if amplification products of the two target regions are confirmed, it is indicated that the subject to be identified is Kinshu. If only one of the amplification products of the two target regions is confirmed, or if neither is confirmed, it is indicated that the subject to be identified is not Kinshu.

[0028] The following examples demonstrate that Kinshu can be identified from 23 representative apple varieties: Fuji, Tsugaru, Ohrin, Jonagold, Shinano Sweet, Shinano Gold, Hokuto, Mutsu, Akie, Kogyoku, Kiou, Kinsei, Yoko, Senshu, Sekaiichi, Sansa, Delicious varieties, Rose Pearl, Ruby Sweet, Beni Minori, Mori no Kagayaki, Toki, and Gunma Meigetsu. According to the identification method of the present invention, it is possible that Kinshu can also be identified from other varieties.

[0029] In this specification, "proximity of a retrotransposon" refers to the genomic region located outside the retrotransposon, specifically the apple-side sequence into which the retrotransposon is inserted. For example, this includes the apple-side sequence extending approximately 1000 base pairs from the boundary between the retrotransposon and the apple-side sequence towards the outside of the retrotransposon.

[0030] One embodiment of the present invention relates to a method for identifying the apple variety Nishikishu, wherein at least two sets of primer pairs in step a) are selected from the following 1) to 4). 1) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 5, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 6; 2) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 7, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 8; 3) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 9, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 10; 4) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 11, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 12.

[0031] In this specification, one of the primer pair is referred to as the first primer, and the other as the second primer. The first and second primers are a combination in which one binds to the upstream portion (5' end) of the target DNA and synthesizes in the 5'→3' direction, and the other binds to the opposite strand in the 3'→5' direction at the downstream portion (3' end) of the target DNA and synthesizes in the 5'→3' direction.

[0032] The primer pairs described in 1) to 4) above are primer pairs that target the retrotransposon and the genomic regions CTcrm2_Cl434, CTcrm2_Cl311, dem1_pattern561, and dem1_pattern428 near its insertion site for amplification, respectively. The first primer in each of the primer pairs described in 1) to 4) above corresponds to the "primer that binds to the retrotransposon within the genome of the apple variety Nishikishu." The nucleotide sequences shown in SEQ ID NOs. 5, 7, 9, or 11 are retrotransposon sequences. The second primer in the primer pairs described in 1) to 4) above corresponds to "a primer that binds to the Kinshu genomic region located near the retrotransposon." Sequence IDs 6, 8, 10, or 12 are the Kinshu genomic regions located near the retrotransposon. Sequence IDs 6, 8, 10, or 12 are the reverse complementary sequences of the apple-side sequence (bold italicized portion) shown in Figure 1.

[0033] The first primer described above contains a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in SEQ ID NOs. 5, 7, 9, or 11. The second primer described above contains a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in SEQ ID NOs. 6, 8, 10, or 12.

[0034] The primer length may be 15 bases or more, and can be 16 bases or more, 17 bases or more, 18 bases or more, 19 bases or more, or 20 bases or more. The primer length may be, for example, 50 bases or less, and can be 45 bases or less, 40 bases or less, 35 bases or less, or 30 bases or less. The position and length of the primer can be determined by considering the melting temperature (Tm), GC content, sequence uniformity, sequence specificity, and efficiency. In PCR, it is preferable to set the primer Tm to be in the range of approximately 50-65°C and the GC content to be in the range of approximately 40-60%, but it is not limited to these ranges, and it is possible to set it outside of the above range depending on the sequence composition and specificity. For example, it is possible to set the Tm to be in the range of approximately 50-80°C and the GC content to be in the range of approximately 35-65%.

[0035] In a particular embodiment, the present invention relates to a method for identifying the apple variety Nishikishu, wherein at least two sets of primer pairs in step a) are selected from the following 1) to 4). 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 14; 2) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 15; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 17; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 18.

[0036] The primer pairs described in 1) to 4) above are, in order, primer pairs that amplify the retrotransposon and the genomic regions CTcrm2_Cl434, CTcrm2_Cl311, dem1_pattern561, and dem1_pattern428 near the insertion site, as shown in Table 1 of the examples described below.

[0037] As shown in Table 5 of the examples described below, Nishikishu can be distinguished from 23 other major varieties by using two sets of primer pairs selected from primer pairs for amplifying retrotransposons and the genomic regions CTcrm2_Cl434, CTcrm2_Cl311, dem1_pattern561, and dem1_pattern428, respectively. In a particular embodiment, there are three patterns of primer pair combinations that can distinguish Nishikishu: (1) CTcrm2_Cl434 and CTcrm2_Cl311, (2) dem1_pattern428 and CTcrm2_Cl311, and (3) dem1_pattern561 and CTcrm2_Cl311. These showed good amplification in multiplex PCR (a method of simultaneously amplifying multiple target genes in a single PCR reaction) including a primer pair for positive control amplification.

[0038] In a particular embodiment, the present invention relates to a method for identifying the apple variety Nishikishu, wherein step a) is a step of applying a nucleic acid amplification method using DNA to be identified as a template and at least two sets of primer pairs selected from 1) to 4) and 5) to 10) below. 5) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 19; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 7) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 21; 8) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 9) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 10) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25.

[0039] The primer pairs described in 5) to 10) above are, in order, primer pairs that amplify the retrotransposon and the genomic regions near its insertion site, CTcrm2_pattern639, CTcrm2_pattern689, dem1_pattern567, TRIM_pattern2692, TRIM_Cl924, and CTcrm2_Cl191, as shown in Table 1 of the Examples described below.

[0040] As shown in Table 5 of the examples described below, by using two sets of primer pairs selected from primer pairs for amplifying retrotransposons and the genomic regions CTcrm2_Cl434, CTcrm2_Cl311, dem1_pattern561 and dem1_pattern428, and CTcrm2_pattern639, CTcrm2_pattern689, dem1_pattern567, TRIM_pattern2692, TRIM_Cl924 and CTcrm2_Cl191 near their insertion sites, Nishikishu can be distinguished from the other 23 major varieties.

[0041] In a particular embodiment, the primer pair combinations that can identify Nishikishu include the following 17 patterns in addition to the above three patterns: (1) CTcrm2_Cl434 and CTcrm2_Cl311, (2) dem1_pattern428 and CTcrm2_Cl311, and (3) dem1_pattern561 and CTcrm2_Cl311: CTcrm2_Cl434 and dem1_pattern567, CTcrm2_Cl434 and TRIM_pattern2692, CTcrm2_Cl434 and CTcrm2_C l191, dem1_pattern561 and dem1_pattern567, dem1_pattern561 and TRIM_pattern2692, dem1_pattern561 and CTcrm2_Cl191, CTcrm2_pattern639 and dem1_pattern567, CTcrm2_pattern639 and TRIM_pattern2692, CTcrm2_pattern639 and CTcrm2_Cl191, CTcrm2_pattern689 and dem1_pattern567, CTcrm2_pattern689 and TRIM_pattern2692, CTcrm2_pattern689 and CTcrm2_Cl191, dem1_pattern428 and dem1_pattern567, dem1_pattern428 and TRIM_pattern2692, dem1_pattern428 and CTcrm2_Cl191, dem1_pattern567 and TRIM_Cl924, or TRIM_pattern2692 and TRIM_Cl924. According to the identification method of the present invention, Nishikishu can be distinguished from other varieties by using only two sets of primer pairs.

[0042] (Positive control) In one embodiment, the nucleic acid amplification method preferably includes a positive control to confirm that the reaction has proceeded normally. Amplification of the positive control can be performed using a positive control amplification primer set. So-called housekeeping genes can be used as positive controls. Examples include the ACC synthase isogene (MdACS1) gene, the thaumatin-like protein Mdtl1 gene, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, the actin gene, the elongation factor 1α gene, the ubiquitin gene, and the rbcL gene (chloroplast genome).

[0043] (Nucleic acid chromatography PAS) Step b) of the present invention is a step of identifying Nishiki-shu based on the combination of the presence or absence of amplification products. In certain embodiments of the present invention, step b) is preferably performed by nucleic acid chromatography PAS (C-PAS) method.

[0044] The C-PAS method is a lateral flow assay that utilizes a single-strand tag hybridization reaction to detect the desired amplification product on a test strip. In a specific embodiment, nucleic acid amplification is performed using a biotin-coated primer and a primer coated with tag DNA, and the reaction mixture is mixed with avidin-coated latex (blue) and spread onto the test strip. Complementary tag DNA is immobilized on the test strip in a line, and the amplification product is trapped by the hybridization reaction between the tag DNA and the complementary tag DNA. The trapped amplification product appears as a blue line, allowing for visual confirmation of amplification.

[0045] Labeling and capture systems usable in the C-PAS method include, but are not limited to, those using avidin and biotin, streptavidin and biotin, or antigens and antibodies (e.g., digoxigenin and anti-digoxigenin antibody, FITC and anti-FITC antibody). The labeling substance preferably includes a dye, pigment, chemiluminescent substance such as luminol or luciferin, fluorescent substance, or metal colloid, which facilitates the visualization of DNA fragments.

[0046] The C-PAS method using tagged DNA is a known technique, as disclosed in Japanese Patent Publication No. 2016-010338.

[0047] (Primer and Kit) The present invention relates to a primer pair for identifying the apple variety Nishikishu, selected from the following 1) to 4) and A). 1) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 5, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 6, 2) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 7, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 8, 3) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 9, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 10, 4) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 11, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 12, A) A pair in which tag DNA is attached to either the first primer or the second primer described in 1) to 4) above. Furthermore, the present invention relates to a kit for carrying out the method for identifying the apple variety Nishikishu, comprising a primer pair selected from 1) to 4) and A) above.

[0048] The present invention relates to a pair of primers for identifying the apple variety Nishikishu, selected from the following 1) to 10) and B). 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 14; 2) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 15; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 17; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 18; 5) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 19; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 7) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 21; 8) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 9) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 10) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; B) A pair in which tag DNA is attached to either the first primer or the second primer described in 1) to 10) above.

[0049] In certain embodiments, the identification kit for the apple variety Nishikishu of the present invention may include, in addition to a primer pair, a C-PAS test strip, a developing solution, a latex solution, reagents for nucleic acid amplification reactions, such as polymerase, dNTPs (deoxynucleoside triphosphates), DTT (dithiothreitol), BSA (bovine serum albumin), reaction buffer, MgCl2, reagents for DNA extraction (e.g., a spin column, a surfactant), containers (such as tubes) for nucleic acid amplification reactions or nucleic acid extraction, and instructions for using the kit. [Examples]

[0050] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0051] Example 1: Design of primers for amplifying the insertion site of Nishiki-Aki retrotransposons. In this example, a primer for amplifying the insertion site of a retrotransposon specific to Nishikishu was designed. The genomes of 24 major apple varieties were comprehensively analyzed using next-generation sequencing to identify insertion sites of the CTcrm2 retrotransposon family, the dem1 retrotransposon family, and the TRIM retrotransposon family. As a result, 10 regions were identified in the Kinshu apple variety that contained retrotransposons and genomic regions near their insertion sites. These 10 regions were named CTcrm2_Cl434, CTcrm2_Cl311, dem1_pattern561, dem1_pattern428, CTcrm2_pattern639, CTcrm2_pattern689, dem1_pattern567, TRIM_pattern2692, TRIM_Cl924, and CTcrm2_Cl191. Figure 1 shows the nucleotide sequences at the boundary between the retrotransposon sequence and the non-retrotransposon apple genome sequence (hereinafter referred to as the apple sequence) for four regions: CTcrm2_Cl434, CTcrm2_Cl311, dem1_pattern561, and dem1_pattern428. The complete genome sequence of the apple variety Golden Delicious has been sequenced and made public (Velasco, R. et al. (2010) The genome of the domesticated apple (Malus × domestica Borkh.). Nature Genetics. 42: 833-839.). The location of the above retrotransposon was identified using the Golden Delicious apple genome sequence as a reference genome. For the retrotransposon CTcrm2_Cl434, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 10 of the reference genome, from 40446254 to 40446153. The insertion site of CTcrm2_Cl434 corresponds to the 3' side of chromosome 10, 40446254, in the reference genome. For the retrotransposon CTcrm2_Cl311, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 13 of the reference genome, 8069677-8069584. The insertion site of CTcrm2_Cl311 corresponds to the 3' side of chromosome 13, 8069677, in the reference genome. For the retrotransposon dem1_pattern561, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 10 of the reference genome, from 40555291 to 40555260. The insertion site of dem1_pattern561 corresponds to the 3' side of chromosome 10, 40555291, in the reference genome. Regarding the retrotransposon CTcrm2_pattern639, the apple-side sequence near the insertion site corresponds to chromosome 2 of the reference genome, specifically sequences 13526002 to 13525887. The insertion site of CTcrm2_pattern639 corresponds to the 3' side of chromosome 2, specifically sequence 13526002, in the reference genome. For the retrotransposon dem1_pattern567, the apple-side sequence near the insertion site corresponds to chromosome 10 of the reference genome, from 33424199 to 33424329. The insertion site of dem1_pattern567 corresponds to the 5' side of chromosome 10, 33424199, in the reference genome. For the retrotransposon TRIM_pattern2692, the apple-side sequence near the insertion site corresponds to chromosome 9 of the reference genome, 15014638~15014505. The insertion site of TRIM_pattern2692 corresponds to the 3' side of chromosome 9, 15014638, in the reference genome. For the retrotransposon CTcrm2_Cl191, the apple-side sequence near the insertion site corresponds to chromosome 7 of the reference genome, from 20328030 to 20328145. The insertion site of CTcrm2_Cl191 corresponds to the 5' side of chromosome 7, 20328030, in the reference genome.

[0052] We designed primer pairs for PCR amplification of 10 regions, each corresponding to a retrotransposon and the genomic region near its insertion site. An example of a primer pair is shown in Table 1. [Table 1] JPEG2026144212000003.jpg158170

[0053] In the following experiment, DNA was extracted from the peel of the Nishikishu orange and used for PCR amplification. The ISOSPIN Plant DNA kit (Nippon Gene Co., Ltd.) was used for DNA extraction. The extracted DNA stock solution was diluted to a concentration of 5-10 ng / μL based on the absorbance at 260 nm.

[0054] Table 2 shows the composition of the PCR reaction solution and the amplification cycle. [Table 2]

[0055] The primer mix in Table 2 includes the primer pairs listed in Table 1 and a positive control amplification primer pair to confirm that the PCR reaction was performed correctly. The positive control amplification primer pair used amplified the ACC synthase isogene (MdACS1) gene (gene sequence, international nucleotide sequence database accession number U89156.1, Malus domestica (apple)). [Table 3] For the 10 primer pairs shown in Table 1, we confirmed that target retrotransposon DNA fragments could be amplified even when mixed with a positive control amplification primer pair using Nishikishu DNA as a template.

[0056] Example 2: Analysis of 24 varieties Using DNA extracted from the 24 varieties listed below as templates, PCR was performed using the primer pairs shown in Table 1, and the presence or absence of amplified products and their size were confirmed by agarose gel electrophoresis. The results are shown in Table 4. [Table 4] A "+" indicates that PCR amplification occurred. A blank space indicates that PCR amplification did not occur.

[0057] The results in Table 4 show that at least two primer pair combinations are sufficient to identify *Nishikishu*. The combinations that can identify *Nishikishu* are indicated by ● in Table 5. [Table 5]

[0058] Example 3: PCR-C-PAS Of the combinations in Table 5, three combinations yielded good amplification even when multiplexed, particularly when two primer pairs were used in the same reaction solution: CTcrm2_Cl434 and CTcrm2_Cl311, dem1_pattern428 and CTcrm2_Cl311, and dem1_pattern561 and CTcrm2_Cl311. Using Kinshu and Kinshu's DNA as templates, DNA fragments of target retrotransposons were amplified by PCR using these three sets, and the presence or absence of amplification was confirmed by the C-PAS method. The DNA extraction method is as described in Example 1.

[0059] The preparation of each tagged DNA or biotin-tagged primer used in this example was outsourced to TBA Corporation (Miyagi Prefecture). Reagents such as C-PAS test strips, latex solution, and developing agent were purchased from TBA Corporation. In the experiment described below, a pair of primers for positive control amplification was used in which biotin was bound to the 5' end of one primer and an F4 tag (tag DNA) was bound to the 5' end of the other primer. The PCR reagents and PCR conditions were the same as those described in Example 1. (1) CTcrm2_Cl434 and CTcrm2_Cl311 For CTcrm2_Cl434 amplification, the primer pair used consisted of the first primer (Table 1) with biotin attached to its 5' end and the second primer with an F2 tag (tagged DNA) attached to its 5' end. For CTcrm2_Cl311 amplification, the primer pair used consisted of the first primer (Table 1) with biotin attached to its 5' end and the second primer with an F1 tag (tag DNA) attached to its 5' end. (2) dem1_pattern428 and CTcrm2_Cl311 For the dem1_pattern428 amplification primer pair, we used the first primer shown in Table 1 with biotin attached to its 5' end and the second primer with an F2 tag (tagged DNA) attached to its 5' end. For CTcrm2_Cl311 amplification, the primer pair used consisted of the first primer (Table 1) with biotin attached to its 5' end and the second primer with an F1 tag (tag DNA) attached to its 5' end. (3) dem1_pattern561 and CTcrm2_Cl311 For the dem1_pattern561 amplification primer pair, we used the first primer shown in Table 1 with biotin attached to its 5' end and the second primer with an F2 tag (tagged DNA) attached to its 5' end. For CTcrm2_Cl311 amplification, the primer pair used consisted of the first primer (Table 1) with biotin attached to its 5' end and the second primer with an F1 tag (tag DNA) attached to its 5' end. After the PCR amplification reaction, the amplified product was subjected to the C-PAS method. Specifically, latex solution and developing solution were added to the amplified product, and the product was developed on test paper. The results are shown in Figure 3. The blue line for F4 (MdACS1) indicates that the target DNA fragment was amplified by the positive control amplification primer pair, meaning that the PCR reaction was performed successfully. In the Nishikishu test strip, blue lines were observed at the F1 and F2 positions, but no lines were observed at the F1 and F2 positions in Beniminori. It was confirmed that Nishikishu can be distinguished from other apple varieties by the primer pair combinations described in (1) to (3) above. Furthermore, it was shown that the presence or absence of PCR amplification can be quickly and easily determined visually using the C-PAS method, allowing for rapid identification of Nishikishu from other apple varieties.

[0060] Example 4: PCR-C-PAS In the following experiment, DNA was extracted from the peels of 24 apple varieties, and DNA fragments of target retrotransposons were amplified by PCR. The presence or absence of amplification was then confirmed using the C-PAS method. The DNA extraction method is as described in Example 1. The preparation of each tagged DNA or biotin-tagged primer used in this example was outsourced to TBA Corporation (Miyagi Prefecture). Reagents such as C-PAS test strips, latex solution, and developing agent were purchased from TBA Corporation. For the dem1_pattern428 amplification primer pair, we used the first primer shown in Table 1 with biotin attached to its 5' end and the second primer with an F2 tag (tagged DNA) attached to its 5' end. For CTcrm2_Cl311 amplification, the primer pair used consisted of the first primer (Table 1) with biotin attached to its 5' end and the second primer with an F1 tag (tag DNA) attached to its 5' end. A pair of primers for positive control amplification was used, in which biotin was bound to the 5' end of one primer and an F4 tag (tagged DNA) was bound to the 5' end of the other primer. The PCR reagents and PCR conditions are the same as those described in Example 1.

[0061] After the PCR amplification reaction, the amplified product was subjected to the C-PAS method. Specifically, latex solution and developing solution were added to the amplified product, and the product was developed on test paper. Figure 4 shows the results of PCR performed using DNA extracted from 24 apple varieties as templates, with amplification primer pairs for dem1_pattern428 and CTcrm2_Cl311, as well as a positive control amplification primer pair, and then spreading the results onto test strips. Table 6 shows the correspondence between the test strip numbers and apple varieties. [Table 6] As shown in Figure 4, a blue line was visually confirmed at position F4 in all 24 test strip varieties. The blue line at F4 indicates that the target DNA fragment was amplified by the positive control amplification primer pair, meaning that the PCR reaction was performed successfully. In the Nishikishu test strip #21, blue lines were observed at the F1 and F2 positions, but in the test strips of the other 23 varieties, no lines appeared at both the F1 and F2 positions. It was confirmed that Nishikishu can be distinguished from other apple varieties by the combination of the dem1_pattern428 amplification primer pair and the CTcrm2_Cl311 amplification primer pair. Furthermore, it was demonstrated that the C-PAS method allows for rapid and simple visual determination of whether or not PCR amplification has occurred, enabling rapid identification of Nishikishu apples from other varieties.

Claims

1. A method for identifying the apple variety Nishikishu, comprising the following steps: a) Using the DNA to be identified as a template, A primer that binds to a retrotransposon in the genome of the apple variety Nishikishu, and A primer that binds to the genome region of Nishikishu, located near the aforementioned retrotransposon. A step of applying a nucleic acid amplification method using at least two sets of primer pairs containing, wherein the retrotransposon is CTcrm2, dem1, or TRIM. Furthermore b) A step of identifying Nishikishu based on the combination of presence or absence of amplification products.

2. The identification method according to claim 1, wherein at least two pairs of primers in step a) are selected from the following 1) to 4): 1) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 5, and A pair of second primers comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 6; 2) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 7, and A pair of second primers comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 8; 3) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 9, and A pair of second primers comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 10; 4) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 11, and A pair of second primers containing a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No.

12.

3. The method according to claim 1, wherein at least two pairs of primers in step a) are selected from the following 1) to 4): 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 14; 2) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 15; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 17; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No.

18.

4. The method according to claim 2 or 3, wherein at least two pairs of primers in step a) are in the following combinations: A combination of the primer pair 1) and the primer pair 2) according to claim 2 or 3, A combination of the primer pair of 2) and the primer pair of 3) as described in claim 2 or 3, or A combination of the primer pair of 2) and the primer pair of 4) as described in claim 2 or 3.

5. The method according to claim 1, wherein step a) is a step of applying a nucleic acid amplification method using DNA to be identified as a template and at least two sets of primer pairs selected from 1) to 4) and 5) to 10) below: 5) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 19; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 7) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 21; 8) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 9) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 10) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No.

25.

6. The method according to claim 5, wherein at least two pairs of primers in step a) are in the following combinations: A combination of the primer pair 1) described in claim 2 or 3 and the primer pair 7) described in claim 5, A combination of the primer pair 1) described in claim 2 or 3 and the primer pair 8) described in claim 5, A combination of the primer pair 1) described in claim 2 or 3 and the primer pair 10) described in claim 5, A combination of the primer pair 3) described in claim 2 or 3 and the primer pair 7) described in claim 5, A combination of the primer pair 3) described in claim 2 or 3 and the primer pair 8) described in claim 5, A combination of the primer pair 3) described in claim 2 or 3 and the primer pair 10) described in claim 5, A combination of the primer pair 5) described in claim 5 and the primer pair 7) described in claim 5, A combination of the primer pair 5) described in claim 5 and the primer pair 8) described in claim 5, A combination of the primer pair 5) described in claim 5 and the primer pair 10) described in claim 5, A combination of the primer pair 6) described in claim 5 and the primer pair 7) described in claim 5, A combination of the primer pair 6) described in claim 5 and the primer pair 8) described in claim 5, A combination of the primer pair 6) described in claim 5 and the primer pair 10) described in claim 5, A combination of the primer pair 4) described in claim 2 or 3 and the primer pair 7) described in claim 5, A combination of the primer pair 4) described in claim 2 or 3 and the primer pair 8) described in claim 5, A combination of the primer pair 4) described in claim 2 or 3 and the primer pair 10) described in claim 5, A combination of the primer pair 7) described in claim 5 and the primer pair 9) described in claim 5, or A combination of the primer pair 8) described in claim 5 and the primer pair 9) described in claim 5.

7. The method according to claim 1, wherein the nucleic acid amplification method is PCR.

8. The method according to claim 1, wherein in step a), a nucleic acid amplification method using at least two sets of primer pairs is carried out in the same reaction solution.

9. A pair of primers for identifying the apple variety Nishikishu, selected from 1) to 4) and A) below: 1) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 5, and A pair of second primers comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 6; 2) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 7, and A pair of second primers comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 8; 3) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 9, and A pair of second primers comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 10; 4) A first primer comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 11, and A pair of second primers comprising a polynucleotide consisting of a sequence of 15 or more consecutive bases in the base sequence or complementary sequence described in Sequence ID No. 12; A) A pair in which tag DNA is attached to either the first primer or the second primer described in 1) to 4) above.

10. A pair of primers for identifying the apple variety Nishikishu, selected from 1) to 10) and B) below: 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 14; 2) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 15; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 17; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 18; 5) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 19; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 7) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 16, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 21; 8) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 9) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 22, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 10) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 13, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; B) A pair in which tag DNA is attached to either the first primer or the second primer described in 1) to 10) above.

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