Identification technology for the apple variety Rose Pearl

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to a method for identifying the apple cultivar Rose Pearl, comprising the steps of: a) applying a nucleic acid amplification method using a primer set comprising at least a primer that binds to the retrotransposon dem1 or CTcrm2 in the genome of the apple cultivar Rose Pearl, and a primer that binds to a genomic region of Rose Pearl located in the vicinity of said retrotransposon dem1 or CTcrm2, with DNA from a subject to be identified used as a template; and b) identifying Rose Pearl based on the presence or absence of an amplification product. The present invention relates to a primer pair for identifying the apple cultivar Rose Pearl. The present invention relates to a primer set for the LAMP method for identifying the apple cultivar Rose Pearl. [Background Art]

[0002] Approximately 300 apple cultivars have been registered for publication of application under Japan's Seed and Seedling Act, and many cultivars including excellent edible cultivars and rootstock cultivars have been bred and applied for registration by organizations such as the Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization (referred to hereinafter as NARO Institute of Fruit Tree Science) (Non-Patent Document 1).

[0003] "Rose Pearl" is a promising apple cultivar bred by NARO Institute of Fruit Tree Science. It is a cultivar for both raw consumption and processing that has pink-colored flesh and moderate acidity. There are concerns both that seedlings of this cultivar will be taken out of foreign countries without permission and that they will be imported back into Japan illegally.

[0004] On the other hand, for cultivar identification, DNA cultivar identification technology that utilizes differences in DNA base sequences between cultivars is generally used. For example, introduction of DNA cultivar identification technology capable of more accurate determination is required for border inspections at customs and investigations related to unauthorized taking-out of seedlings and the like. In particular, for border inspections at customs, there are restrictions on the time available for inspection, so 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 Rose Pearl. However, the identification method using SSR markers requires expensive equipment, as it involves 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 specifically identifying the apple variety Rose Pearl. [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 rose pearl in 24 major apple varieties that were examined, thus completing the present invention.

[0010] The present invention provides the following inventions. [1] A method for identifying the apple variety Rose Pearl, comprising the following steps: a) Using the DNA to be identified as a template, Primers that bind to the retrotransposon dem1 or CTcrm2 within the genome of the apple variety Rose Pearl, and Primers that bind to the genomic region of rose pearl, located near the retrotransposon dem1 or CTcrm2. A step of applying a nucleic acid amplification method using a primer set containing at least the following, and b) A step of identifying rose pearls based on the presence or absence of amplification products. [2] The identification method according to [1], wherein the nucleic acid amplification method is PCR and the primer set is a pair of primers selected from 1) to 6) 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. 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; 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. 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; 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. 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; 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. 13, 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. 14; 5) 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. 15, 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. 16; 6) 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. 17, 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. 18. [3] The identification method according to [1], wherein the nucleic acid amplification method is PCR and the primer set is a pair of primers selected from 1) to 6) below: 1) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 19, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 2) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 22; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 5) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 26. [4] The identification method according to [1], wherein the nucleic acid amplification method is the LAMP method and the primer set is one of the following sets: 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 30, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 31, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 32 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 33 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 34 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 35 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and An LB primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 40 and / or A set of LF primers comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 41. [5] The identification method according to [1], wherein the nucleic acid amplification method is the LAMP method, and the primer set is any one of the following sets: 1) An F3 primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 36, a B3 primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 37, an FIP primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 38, and a set of BIP primers comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 39; 2) The F3, B3, FIP, and BIP primers according to 1) above, and an LB primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 40 and / or a set of LF primers comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 41. [6] A primer pair for identifying the apple cultivar Rose Pearl, selected from the following 1) to 13): 1) A first primer comprising a polynucleotide consisting of a continuous nucleotide sequence of 15 or more bases in the nucleotide sequence set forth in SEQ ID NO: 7 or a complementary sequence thereof, and a pair of a second primer comprising a polynucleotide consisting of a continuous nucleotide sequence of 15 or more bases in the nucleotide sequence set forth in SEQ ID NO: 8 or a complementary sequence thereof; 2) A first primer comprising a polynucleotide consisting of a continuous nucleotide sequence of 15 or more bases in the nucleotide sequence set forth in SEQ ID NO: 9 or a complementary sequence thereof, and a pair of a second primer comprising a polynucleotide consisting of a continuous nucleotide sequence of 15 or more bases in the nucleotide sequence set forth in SEQ ID NO: 10 or a complementary sequence thereof; 3) A first primer comprising a polynucleotide consisting of a continuous nucleotide sequence of 15 or more bases in the nucleotide sequence set forth in SEQ ID NO: 11 or a complementary sequence thereof, 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; 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. 13, 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. 14; 5) 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. 15, 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. 16; 6) 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. 17, 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. 18; 7) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 19, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 8) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 22; 9) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23, 10) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 11) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; 12) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 26; 13) A pair of first primers and second primers in which a tag DNA is attached to either the first primer or the second primer described in 1) to 12) above. [7] A set of LAMP primers for identifying the apple variety Rose Pearl, selected from the following 1) to 5): 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 30, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 31, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 32 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 33 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 34 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 35 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and LB primers and / or containing polynucleotides comprising the base sequence described in SEQ ID NO: 40 A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 41; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 36, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 37, FIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 38, and A set of BIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 39; 4) The F3, B3, FIP, and BIP primers described in 3) above, and LB primers and / or containing polynucleotides comprising the base sequence described in SEQ ID NO: 40 A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 41; 5) A set in which tag DNA is attached to one of the FIP primers, BIP primers, or LF primers, if present, as described in 1) to 4) above. [8] A LAMP primer set for amplifying the Mdtl1 gene as a positive control in the identification of the apple variety Rose Pearl, selected from 1) to 5) below: 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 43, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 44, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 45 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 46 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 47 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 48 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and An LF primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 53, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 49, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 50, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 51, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 52; 4) The F3, B3, FIP, and BIP primers described in 3) above, and Sequence ID 53 LF primers comprising polynucleotides consisting of the base sequence described above, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54; 5) A set in which tag DNA is attached to either the FIP primer, BIP primer, or, if present, the LF primer or LB primer described in 1) to 4) above. [Effects of the Invention]

[0011] According to the present invention, a simple and rapid method for identifying rose pearls 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 for six retrotransposon insertion sites named dem1_pattern666, CTcrm2_pattern600, CTcrm2_pattern606, CTcrm2_pattern643, CTcrm2_pattern649, and CTcrm2_pattern669. The bold, italicized portion is the non-retrotransposon apple genome sequence. [Figure 2]Figure 2 shows the results of PCR performed using DNA extracted from 24 apple varieties as templates, employing a primer pair for dem1_pattern666 amplification and a primer pair for positive control amplification, and then spreading the results onto C-PAS test strips. Table 4 shows the correspondence between the test strip numbers and apple varieties. Test strip 28 is the negative control (sterile water). [Figure 3] Figure 3 shows the positions of the LAMP primers for amplifying dem1_pattern666 (A) and for amplifying the positive control Mdtl1 gene (B). [Figure 4] Figure 4 shows the results of performing a LAMP reaction using a primer set for amplifying dem1_pattern666, with DNA extracted from 24 apple varieties as templates, and spreading the results onto C-PAS test strips. Table 4 shows the correspondence between the test strip numbers and apple varieties. The NTC is the negative control (sterile water). [Figure 5] Figure 5 shows the results of performing a LAMP reaction using DNA extracted from six apple varieties as templates, employing a LAMP primer set for dem1_pattern666 amplification and a primer set for positive control amplification, and spreading the results onto test strips. Table 8 shows the correspondence between the test strip numbers and apple varieties. NTC is the negative control (sterile water). [Modes for carrying out the invention]

[0013] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, numerical ranges represented by "~" mean a range that includes the numbers before and after "~" as the lower and upper limits. In this specification, when a numerical value is accompanied by the term "approximately", it is intended to include a range of ±10% of that value.

[0014] [How to identify the apple variety Rose Pearl] The present invention relates to a method for identifying the apple variety Rose Pearl, comprising the following steps. a) Using the DNA to be identified as a template, Primers that bind to the retrotransposon dem1 or CTcrm2 within the genome of the apple variety Rose Pearl, and Primers that bind to the genomic region of rose pearl, located near the retrotransposon dem1 or CTcrm2. A step of applying a nucleic acid amplification method using a primer set containing at least the following, and b) A step of identifying rose pearls based on the presence or absence of amplification products.

[0015] The Rose Pearl apple variety is a versatile apple variety suitable for both fresh consumption and processing, with pink flesh and a pleasant acidity. Its distinctive flesh and juice color can be utilized in the production of unique processed products. Compared to existing varieties with red flesh, it has a milder acidity and a better texture, making it suitable for both cooking and fresh consumption, and is expected to expand the demand for apples. The variety's registration number is 24267 (March 26, 2015). The Rose Pearl apple variety is a superior variety, and there are concerns about infringement such as the unauthorized removal of fruit and seedlings.

[0016] This invention provides a simple and rapid genetic testing method for identifying rose pearls. Because the test can be performed in a short time, this invention can be used, for example, in border inspections at customs.

[0017] Step a) of the method of the present invention involves using the DNA to be identified as a template and a primer set to amplify the retrotransposon dem1 or CTcrm2 present in the genome of rose pearl and the genomic region near its insertion site by nucleic acid amplification.

[0018] 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.

[0019] 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.

[0020] 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).

[0021] 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.

[0022] 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.

[0023] The identification method of the present invention utilizes insertion polymorphisms of the retrotransposon family "dem1" or "CTcrm2". "dem1" and "CTcrm2" are present at multiple locations in the rose pearl genome, and certain insertion sites are found only in rose pearl among the 24 varieties being identified. Therefore, the insertion sites of "dem1" and "CTcrm2" can serve as highly effective DNA markers for identifying rose pearl.

[0024] Step a) of the present invention is a step of amplifying "the retrotransposon dem1 or CTcrm2 and the genomic region near its insertion site" by nucleic acid amplification using a primer that binds to the retrotransposon dem1 or CTcrm2 within the genome of the apple variety Rose Pearl, and a primer that binds to a genomic region of Rose Pearl located near the retrotransposon dem1 or CTcrm2.

[0025] An example of "retrotransposon dem1 and the genomic region near its insertion site" is the region named dem1_pattern666 (sequence number 1) shown in Figure 1. Examples of "retrotransposon CTcrm2 and the genomic region near its insertion site" are the regions named CTcrm2_pattern600 (sequence number 2), CTcrm2_pattern606 (sequence number 3), CTcrm2_pattern643 (sequence number 4), CTcrm2_pattern649 (sequence number 5), and CTcrm2_pattern669 (sequence number 6) shown in Figure 1. In Figure 1, the retrotransposon dem1 or CTcrm2 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). The retrotransposon CTcrm2 sequence is inserted at multiple locations within the apple genome, and exhibits very high sequence identity in at least the five regions mentioned above. However, the apple sequences near the insertion sites are completely different, as they lack homology in at least the five regions mentioned above. The "retrotransposon CTcrm2 and the genomic regions near its insertion sites" can be specifically amplified by designing and using primers that specifically bind to the apple sequences near the insertion sites. The retrotransposon dem1 sequence is inserted at multiple locations within the apple genome, but the apple sequences near the insertion sites are completely different for each insertion site. The "retrotransposon dem1 and the genomic regions near its insertion sites" can be specifically amplified by designing and using primers that specifically bind to the apple sequences near the insertion sites. On the other hand, the "primer that binds to the retrotransposon CTcrm2 in the genome of the apple variety Rose Pearl" allows for the creation of a primer that can be commonly used for amplification of the five regions and other insertion sites mentioned above. That is, the nucleotide sequences of the retrotransposon CTcrm2 in the five regions and other insertion sites can be aligned, and a primer can be set in the region without mutation. Similarly, for the "primer that binds to the retrotransposon dem1 in the genome of the apple variety Rose Pearl," the nucleotide sequences of multiple retrotransposons dem1 with different insertion sites can be aligned, and a primer can be set in the region without mutation. In another embodiment, primers can be set including the mutated region, and individual primers can be designed for each region to be amplified.

[0026] With respect to step a) of the present invention, primers that bind to the retrotransposon dem1 or CTcrm2 within the genome of the apple variety Rose Pearl, and primers that bind to a genomic region of Rose Pearl located near the retrotransposon dem1 or CTcrm2, can be designed to amplify all or part of the "retrotransposon dem1 or CTcrm2 and the genomic region near its insertion site" as described in Sequence ID No. 1, 2, 3, 4, 5, or 6. 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 may be set to be in complete proximity or to be several bases or more apart, for example, 10 bases or more.

[0027] Step b) of the present invention is a step of identifying rose pearl based on the presence or absence of amplification products. The reaction solution of the nucleic acid amplification method in step a) is analyzed, and if the target amplification product is confirmed, it is indicated that the object to be identified is rose pearl. If the target amplification product is not confirmed, it is indicated that the object to be identified is not rose pearl. The presence or absence of amplification products can be confirmed by electrophoresis (agarose gel, etc.), nucleic acid chromatography PAS method, real-time PCR, DNA sequencing, etc.

[0028] The following examples demonstrate that Rose Pearl 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, Ruby Sweet, Beni Minori, Kinshu, Mori no Kagayaki, Toki, and Gunma Meigetsu. According to the identification method of the present invention, it is possible that Rose Pearl can also be identified from other varieties.

[0029] In this specification, "near retrotransposon dem1 or CTcrm2" refers to the genomic region located outside the retrotransposon dem1 or CTcrm2, specifically the apple-side sequence into which the retrotransposon dem1 or CTcrm2 is inserted. For example, this includes the apple-side sequence extending approximately 1000 base pairs from the boundary between the retrotransposon dem1 or CTcrm2 and the apple-side sequence towards the outside of the retrotransposon.

[0030] (PCR method) One embodiment of the present invention relates to a method for identifying the apple variety Rose Pearl, wherein the nucleic acid amplification method is PCR, and the primer set is a primer pair selected from the following 1) to 6). 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. 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; 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. 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; 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. 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; 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. 13, 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. 14; 5) 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. 15, 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. 16; 6) 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. 17, 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. 18. The primer pair described in 1) above allows for the amplification of "retrotransposon dem1 and the genomic region near its insertion site" shown in SEQ ID NO: 1, using rose pearl DNA as a template. The primer pairs described in 2) to 6) above allow for the amplification of "retrotransposon CTcrm2 and the genomic region near its insertion site" shown in SEQ ID NOs: 2 to 6, respectively, using rose pearl DNA as a template.

[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 first primer of the primer pair described in 1) above corresponds to "a primer that binds to the retrotransposon dem1 within the genome of the apple variety Rose Pearl." The base sequence shown in Sequence ID No. 7 is the retrotransposon dem1 sequence. The second primer of the primer pair described in 1) above corresponds to "a primer that binds to a genomic region of Rose Pearl located near the retrotransposon dem1." Sequence ID No. 8 is the genomic region of Rose Pearl located near the retrotransposon dem1. Sequence ID No. 8 is the reverse complementary sequence of the apple side sequence (bold italicized portion) shown in Sequence ID No. 1 in Figure 1.

[0033] The first primer in the primer pairs described in 2) to 6) above corresponds to a "primer that binds to the retrotransposon CTcrm2 within the genome of the apple variety Rose Pearl." The nucleotide sequences shown in SEQ ID NOs. 9, 11, 13, 15, or 17 are the retrotransposon CTcrm2 sequences. The second primer in the primer pairs described in 2) to 6) above corresponds to a "primer that binds to a genomic region of Rose Pearl located near the retrotransposon CTcrm2." SEQ ID NOs. 10, 12, 14, 16, or 18 are genomic regions of Rose Pearl located near the retrotransposon CTcrm2. SEQ ID NOs. 10, 12, 14, 16, or 18 are the reverse complementary sequences of the apple-side sequences (bold italicized portion) shown in SEQ ID NOs. 2 to 6 in Figure 1.

[0034] 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: 7, 9, 11, 13, 15, or 17. 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: 8, 10, 12, 14, 16, or 18.

[0035] 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 60-70°C and the GC content to be in the range of approximately 30-60%.

[0036] In a particular embodiment, the present invention relates to a method for identifying the apple variety Rose Pearl, wherein the nucleic acid amplification method is PCR, and the primer set is a primer pair selected from the following 1) to 6). 1) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 19, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 2) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 22; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 5) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 26. The primer pairs described in 1) to 6) above allow for the amplification of the retrotransposon dem1 or CTcrm2 and the genomic region near its insertion site, as shown in SEQ ID NOs: 1 to 6, using rose pearl DNA as a template. The present invention is not limited to the method using the primer pairs described in 1) to 6) above, and it is possible to design and use primer pairs capable of amplifying the retrotransposon dem1 or CTcrm2 and the genomic region near its insertion site, as shown in SEQ ID NOs: 1 to 6.

[0037] (LAMP method) The present invention provides a method for identifying the apple variety Rose Pearl. Using the DNA of the target to be identified as a template, the LAMP method amplifies "the retrotransposon dem1 or CTcrm2 and the genomic region near its insertion site," and identifies Rose Pearl based on the presence or absence of the amplified product. Since the LAMP method amplifies nucleic acids under isothermal conditions (60-65°C), a thermal cycler is not required, and the reaction can be carried out in a constant temperature device (e.g., a heat block), with the amplification reaction completed in about 30 minutes to 1 hour. Therefore, the LAMP method is considered suitable for border inspections at customs, for example, due to its simplicity and speed.

[0038] In the LAMP method, three regions are defined on the target DNA from the 3' end: F3c, F2c, and F1c, and three regions are defined from the 5' end: B3, B2, and B1. Four types of primers, F3, B3, FIP, and BIP, are designed using these six regions. FIP is designed to have an F2 region at the 3' end, which is a sequence complementary to the F2c region, and the same sequence as the F1c region at the 5' end. BIP is designed to have a B2 region at the 3' end, which is a sequence complementary to the B2c region, and the same sequence as the B1c region at the 5' end. LAMP can be performed with the four types of primers F3, B3, FIP, and BIP, and loop primers called LoopF ​​(hereinafter referred to as LF) and / or LoopB (hereinafter referred to as LB) can be optionally used. In the embodiments described below, primers are designed to amplify dem1_pattern666 and the genomic region near its insertion site. However, LAMP primers can also be designed for CTcrm2_pattern600 and the genomic region near its insertion site, CTcrm2_pattern606 and the genomic region near its insertion site, CTcrm2_pattern643 and the genomic region near its insertion site, CTcrm2_pattern649 and the genomic region near its insertion site, and CTcrm2_pattern669 and the genomic region near its insertion site.

[0039] One embodiment of the present invention relates to a method for identifying the apple variety Rose Pearl, wherein the nucleic acid amplification method is the LAMP method, and the primer set is a primer set selected from 1) or 2) below. 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 30, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases as described in Sequence ID No. 31; An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 32 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 33 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 34 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 35 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and LB primers and / or containing polynucleotides comprising the base sequence described in SEQ ID NO: 40 A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 41.

[0040] The above F3 primer corresponds to "a primer that binds to the retrotransposon dem1 in the genome of the apple variety Rose Pearl." The nucleotide sequence shown in Sequence ID No. 30 is the sequence within the retrotransposon dem1. The B3 primer described above corresponds to a "primer that binds to the genomic region of rose pearl located near the retrotransposon dem1." Sequence ID 37 is the sequence of the genomic region of rose pearl located near the retrotransposon dem1.

[0041] The length of the primers can be approximately 18-30 bases for the outer primers (F3 / B3), approximately 40-60 bases for the inner primers (FIP / BIP), and approximately 18-30 bases for the loop primers (LF / LB). In another embodiment, the length of the primers can be approximately 18-24 bases for the outer primers (F3 / B3), approximately 40-45 bases for the inner primers (FIP / BIP), and approximately 18-24 bases for the loop primers (LF / LB). The melting temperature (Tm) can be approximately 55-70°C for the outer primers (F3 / B3) and approximately 60-70°C for the loop primers (LF / LB). The GC content of the primers can be 40-60%. Furthermore, the inner primers (FIP / BIP) are designed to prevent hairpin structures and dimer formation.

[0042] In a particular embodiment, the present invention relates to a method for identifying the apple variety Rose Pearl, wherein the nucleic acid amplification method is the LAMP method, and the primer set is a primer set selected from 1) or 2) below. 1) An F3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 36, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 37, FIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 38, and A set of BIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 39; 2) The F3, B3, FIP, and BIP primers described in 1) above, and LB primers and / or containing polynucleotides comprising the base sequence described in SEQ ID NO: 40 A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 41. The primer set described in 1) or 2) above allows for the amplification of "retrotransposon dem1 and the genomic region near its insertion site" shown in SEQ ID NO: 1, using rose pearl DNA as a template. The present invention is not limited to the method using the primer set described in 1) or 2) above, and can be carried out by designing a primer set for the LAMP method that can amplify "retrotransposon dem1 and the genomic region near its insertion site" shown in SEQ ID NO: 1. The present invention can be carried out by designing a primer set for the LAMP method that can amplify "retrotransposon CTcrm2 and the genomic region near its insertion site" shown in SEQ ID NOs: 2 to 6.

[0043] (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).

[0044] The present invention provides a method for identifying the apple variety Rose Pearl, comprising using a set of primers selected from the following 1) to 4) to amplify the Mdtl1 gene as a positive control. 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 43, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 44, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 45 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 46 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 47 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 48 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and An LF primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 53, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 49, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 50, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 51, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 52; 4) The F3, B3, FIP, and BIP primers described in 3) above, and Sequence ID 53 LF primers comprising polynucleotides consisting of the base sequence described above, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54.

[0045] (Nucleic acid chromatography PAS) Step b) of the present invention is a step of identifying rose pearl based on the presence or absence of an amplification product. The reaction solution of the nucleic acid amplification method in step a) is analyzed, and if the target amplification product is confirmed, it is indicated that the object to be identified is rose pearl; if the target amplification product is not confirmed, it is indicated that the object to be identified is not rose pearl. In certain embodiments of the present invention, step b) is preferably performed by nucleic acid chromatography PAS (C-PAS).

[0046] 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.

[0047] 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.

[0048] The C-PAS method using tagged DNA is a known technique, as disclosed in Japanese Patent Publication No. 2016-010338.

[0049] (Primer and Kit) The present invention relates to a pair of primers for identifying the apple variety Rose Pearl, selected from the following 1) to 13). 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. 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; 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. 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; 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. 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; 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. 13, 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. 14; 5) 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. 15, 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. 16; 6) 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. 17, 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. 18; 7) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 19, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 8) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 22; 9) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23, 10) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 11) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; 12) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 26; 13) A pair of first primers and second primers in which a tag DNA is attached to either the first primer or the second primer described in 1) to 12) above. Furthermore, the present invention relates to an identification kit for the apple variety Rose Pearl, comprising a primer pair selected from 1) to 13) above.

[0050] The present invention relates to a LAMP method primer set for identifying the apple variety Rose Pearl, selected from the following 1) to 5). 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 30, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 31, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 32 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 33 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 34 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 35 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and LB primers and / or containing polynucleotides comprising the base sequence described in SEQ ID NO: 40 A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 41; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 36, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 37, FIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 38, and A set of BIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 39; 4) The F3, B3, FIP, and BIP primers described in 3) above, and LB primers and / or containing polynucleotides comprising the base sequence described in SEQ ID NO: 40 A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 41; 5) A set in which tag DNA is attached to one of the FIP primers, BIP primers, or LF primers, if present, as described in 1) to 4) above. Furthermore, the present invention relates to an identification kit for the apple variety Rose Pearl, comprising a set of primers selected from 1) to 5) above.

[0051] The present invention relates to a primer set for the LAMP method for amplifying the Mdtl1 gene as a positive control, selected from the following 1) to 5). 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 43, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 44, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 45 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 46 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 47 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 48 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and An LF primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 53, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 49, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 50, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 51, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 52; 4) The F3, B3, FIP, and BIP primers described in 3) above, and Sequence ID 53 LF primers comprising polynucleotides consisting of the base sequence described above, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54; 5) A set in which tag DNA is attached to either the FIP primer, BIP primer, or, if present, the LF primer or LB primer described in 1) to 4) above. In one embodiment, a primer set selected from 1) to 5) above can be used in a method for identifying the apple variety Rose Pearl. Furthermore, the present invention relates to a kit for identifying the apple variety Rose Pearl, comprising a set of primers selected from 1) to 5) above for amplifying the Mdtl1 gene as a positive control.

[0052] In certain embodiments, the identification kit for the apple variety Rose Pearl of the present invention may include, in addition to a primer pair or primer set, 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]

[0053] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0054] Example 1: Design of a primer for amplifying the insertion site of rose pearl-specific retrotransposons. In this example, a primer for amplifying the insertion site of a rose pearl-specific retrotransposon was designed. Next-generation sequencing analysis was used to comprehensively analyze the insertion sites of the dem1 and CTcrm2 retrotransposon families in the genomes of 24 major apple varieties, and six genomic regions with retrotransposon insertions were identified only in the rose pearl variety. These six regions were named dem1_pattern666, CTcrm2_pattern600, CTcrm2_pattern606, CTcrm2_pattern643, CTcrm2_pattern649, and CTcrm2_pattern669. 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 these six regions. 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 dem1_pattern666, 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 34948028 to 34947909. The insertion site of dem1_pattern666 corresponds to the 3' side of chromosome 10, 34948028, in the reference genome. For the retrotransposon CTcrm2_pattern606, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 7 of the reference genome, 863360-863246. The insertion site of CTcrm2_pattern606 corresponds to the 3' side of chromosome 7, 863360, in the reference genome. For the retrotransposon CTcrm2_pattern643, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 7 of the reference genome, 6974319~6974423. The insertion site of CTcrm2_pattern643 corresponds to the 5' side of chromosome 7, 6974319, in the reference genome. For the retrotransposon CTcrm2_pattern649, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 16 of the reference genome, from 13748044 to 13748148. The insertion site of CTcrm2_pattern649 corresponds to the 5' side of chromosome 16, 13748044, in the reference genome. For the retrotransposon CTcrm2_pattern669, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 7 of the reference genome, 30194053-30193945. The insertion site of CTcrm2_pattern669 corresponds to the 3' side of chromosome 7, 30194053, in the reference genome.

[0055] We designed primer pairs for PCR amplification of each of these six regions. An example of a primer pair is shown in Table 1. [Table 1] CTcrm2_pattern606 (SEQ ID NO: 3) had a single-nucleotide substitution mutation in the region where the first primer (SEQ ID NO: 21) was applied, compared to other CTcrm2 sequences, but it was still possible to amplify it with the above primer pair. This is thought to be because the mutation was not located at either end of the primer sequence.

[0056] In the following experiment, DNA was extracted from the pericarp of rose pearl and used for PCR amplification. An 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.

[0057] Table 2 shows the composition of the PCR reaction solution and the amplification cycle. [Table 2]

[0058] 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 was one that amplified the ACC synthase isogene (MdACS1) gene (gene sequence, international nucleotide sequence database accession number U89156.1, Malus domestica (apple)) as shown in Table 3. [Table 3] Agarose gel electrophoresis confirmed that the DNA fragments of target retrotransposons could be amplified using the three primer pairs shown in Table 1, even when mixed with a positive control amplification primer pair using rose pearl DNA as a template.

[0059] Example 2: PCR-C-PAS In the following experiment, DNA was extracted from the peels of 24 apple varieties, DNA fragments of target retrotransposons were amplified by PCR, and the presence or absence of amplification was confirmed by 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. The first primer in Table 1 had its 5' end labeled with biotin, and the second primer had its 5' end conjugated to an F1 tag (tagged DNA). 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.

[0060] 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 2 shows the results of PCR performed using DNA extracted from 24 apple varieties as templates, with a primer pair for amplifying dem1_pattern666 and a primer pair for amplifying a positive control, and then spreading the results onto test strips. Table 4 shows the correspondence between the test strip numbers and apple varieties. [Table 4] As shown in Figure 2, 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 Rose Pearl test strip #18, a blue line was observed at the F1 position, but no line was observed at the F1 position in the test strips of the other 23 varieties. The blue line at F1 indicates that the DNA fragment at the Rose Pearl-specific retrotransposon insertion site was amplified by the primer pair for dem1_pattern666 amplification, confirming that the primer pair for dem1_pattern666 amplification can be used to distinguish Rose Pearl from other apple varieties. We confirmed that the primer pairs for amplifying CTcrm2_pattern600, CTcrm2_pattern606, CTcrm2_pattern643, CTcrm2_pattern649, and CTcrm2_pattern669 can also be used to identify Rose Pearl from other apple varieties. By using the C-PAS method in combination with a primer pair for amplifying Rose Pearl-specific retrotransposon insertion sites, it was demonstrated that the presence or absence of PCR amplification can be quickly and easily determined visually, allowing for rapid identification of Rose Pearl from other apple varieties.

[0061] Example 3: LAMP-C-PAS In this example, a primer set was designed for LAMP amplification of the retrotransposon dem1_pattern666, and the presence or absence of amplification was confirmed by the C-PAS method after the LAMP reaction. We designed a LAMP primer set for amplifying the retrotransposon dem1_pattern666. An example of the primer set is shown in Table 5. [Table 5]

[0062] We used primers with biotin attached to the 5' end of the 666_PE1_BIP primer shown in Table 5, and primers with an F2 tag (tagged DNA) attached to the 5' end of the 666_PE1_FIP primer. Figure 3 shows the positions of the primers.

[0063] DNA was extracted from the leaves of the same 24 apple varieties as in Example 2 and used for LAMP amplification. The DNA extraction method was the same as described in Example 1. The preparation of each tagged DNA or biotin-tagged primer used in this example was commissioned to TBA Corporation (Miyagi Prefecture). The composition of the LAMP reaction solution is shown in Table 6. [Table 6]

[0064] A tube containing the LAMP reaction solution was placed in a heat block (aluminum block constant temperature bath, TAITEC's Dry Thermo Unit DTU-1BN) set to 63°C, and incubated for 20 minutes to perform the LAMP amplification reaction.

[0065] LAMP amplification was performed using a reaction solution containing a double-stranded DNA-binding fluorescent dye, separate from the C-PAS analysis. The amplified product was then single-stranded at high temperature, and association curve analysis was performed to confirm that the amplified product originated from dem1_pattern666. In the association curve analysis, the temperature of the reaction solution after LAMP amplification was increased to single-strand the DNA, denaturing various DNAs that may contain the target DNA fragment. The temperature was then gradually lowered, and the temperature at which the DNA returned to double-stranded state was measured based on fluorescence intensity. Since this value is specific to the amplified DNA, it can be used to confirm whether the amplified product is the target sequence.

[0066] After the LAMP amplification reaction, the amplified product was developed onto test paper. Specifically, latex solution and developing agent were added to the amplified product and then developed onto the test paper. The test paper, latex solution, developing agent, and other reagents for the C-PAS method were purchased from TBA Corporation.

[0067] Figure 4 shows the results of performing a LAMP reaction using a primer set for amplifying dem1_pattern666, with DNA extracted from 24 apple varieties as a template, and then developing the results on test paper. The correspondence between the test strip numbers and apple varieties is the same as in Table 4 of Example 2. In the Rose Pearl test strip #18, a blue line was observed at the F2 position, but no line was observed at the F2 position in the other 23 varieties. The blue line at F2 indicates that the DNA fragment of a Rose Pearl-specific retrotransposon was amplified by the primer set for dem1_pattern666 amplification, confirming that the primer pair for dem1_pattern666 amplification can be used to distinguish Rose Pearl from other apple varieties.

[0068] Next, a primer set targeting the Mdtl1 gene was designed as a positive control for LAMP. The Mdtl1 gene sequence was referenced from Malus domestica thaumatin-like protein precursor Mdtl1 (MDTL1) mRNA, complete cds, Accession: AF090143. An example of the primer set is shown in Table 7. Figure 3 shows the positions of the primers. [Table 7]

[0069] We used the Mdtl1_2_BIP2 primer shown in Table 7, with biotin conjugated to its 5' end, and the Mdtl1_2_FIP primer, with an F4 tag (tag DNA) conjugated to its 5' end. As a 10x primer mix, a mixture of the LAMP primer set for dem1_pattern666 amplification (Table 5) and the positive control amplification primer set (Table 7) was used, and LAMP amplification and C-PAS were performed under the same conditions as above. The template DNA used was a crude extract from the perilla peel. The results are shown in Figure 5.

[0070] Table 8 shows the correspondence between the test strips and apple varieties. [Table 8]

[0071] In Figure 5, a blue line was observed at position F4 in all seven test strips. The blue line at F4 indicates that the target DNA fragment was amplified by the positive control amplification primer pair, thus indicating that the PCR reaction was performed successfully in all seven samples. In the Rose Pearl test strips b and g, a blue line was observed at the F2 position, but no line was observed at the F2 position in the test strips of other varieties. The blue line at F2 indicates that the DNA fragment of the Rose Pearl-specific retrotransposon was amplified by the primer set for dem1_pattern666 amplification, confirming that the primer pair for dem1_pattern666 amplification can be used to distinguish Rose Pearl from other apple varieties. Furthermore, DNA fragments were amplified in crude DNA extracted from the peel, similar to the DNA extracted from the leaves (g in Table 8). Combining the LAMP method and the C-PAS method demonstrated that Rose Pearl apples can be rapidly identified from other apple varieties.

Claims

1. A method for identifying the apple variety Rose Pearl, comprising the following steps: a) Using the DNA to be identified as a template, Primers that bind to the retrotransposon dem1 or CTcrm2 within the genome of the apple variety Rose Pearl, and Primers that bind to the genomic region of rose pearl, located near the retrotransposon dem1 or CTcrm2. A step of applying a nucleic acid amplification method using a primer set containing at least the following, and b) A step of identifying rose pearls based on the presence or absence of amplification products.

2. The identification method according to claim 1, wherein the nucleic acid amplification method is a PCR method and the primer set is a pair of primers selected from 1) to 6) 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. 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; 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. 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; 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. 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; 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. 13, 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. 14; 5) 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. 15, 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. 16; 6) 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. 17, 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.

18.

3. The identification method according to claim 1, wherein the nucleic acid amplification method is a PCR method and the primer set is a pair of primers selected from 1) to 6) below: 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 19, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 2) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 22; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23; 4) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 5) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; 6) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No.

26.

4. The identification method according to claim 1, wherein the nucleic acid amplification method is the LAMP method and the primer set is one of the following sets: 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 30, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 31, An FIP primer comprising a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 32 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 33 at its 5' end, and A set of BIP primers containing a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 34 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 35 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, LB primers and / or containing a polynucleotide comprising the base sequence described in Sequence ID No. 40 A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No.

41.

5. The identification method according to claim 1, wherein the nucleic acid amplification method is the LAMP method and the primer set is one of the following sets: 1) An F3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 36, A B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 37, FIP primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 38, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 39; 2) The F3, B3, FIP, and BIP primers described in 1) above, LB primers and / or containing a polynucleotide comprising the base sequence described in Sequence ID No. 40 A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No.

41.

6. A pair of primers for identifying the apple variety Rose Pearl, selected from the following 1) to 13): 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. 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; 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. 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; 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. 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; 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. 13, 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. 14; 5) 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. 15, 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. 16; 6) 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. 17, 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. 18; 7) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 19, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 20; 8) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 22; 9) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23, 10) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24; 11) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25; 12) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 21, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 26; 13) A pair of first primers and second primers in which a tag DNA is attached to either the first primer or the second primer described in 1) to 12) above.

7. A LAMP primer set for identifying the apple variety Rose Pearl, selected from the following 1) to 5): 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 30, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 31, An FIP primer comprising a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 32 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 33 at its 5' end, and A set of BIP primers containing a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 34 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 35 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, LB primers and / or containing a polynucleotide comprising the base sequence described in Sequence ID No. 40 A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 41; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 36, A B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 37, FIP primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 38, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 39; 4) The F3, B3, FIP, and BIP primers described in 3) above, LB primers and / or containing a polynucleotide comprising the base sequence described in Sequence ID No. 40 A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 41; 5) A set in which tag DNA is attached to one of the FIP primers, BIP primers, and, if present, an LF primer, as described in 1) to 4) above.

8. A LAMP primer set for amplifying the Mdtl1 gene as a positive control in the identification of the apple variety Rose Pearl, selected from the following 1) to 5): 1) An F3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 43, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases in the base sequence described in Sequence ID No. 44, An FIP primer comprising a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 45 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 46 at its 5' end, and A set of BIP primers containing a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 47 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 48 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, An LF primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 53, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 49, A B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 50, An FIP primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 51, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 52; 4) The F3, B3, FIP, and BIP primers described in 3) above, Sequence ID 53 An LF primer comprising a polynucleotide consisting of the base sequence described above, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 54; 5) A set comprising the FIP primer, BIP primer, and, if present, either the LF primer or LB primer, to which tag DNA has been attached.

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