Identification technology for the Ruby Sweet apple variety

Abstract

To provide a simple and rapid method for specifically identifying the Ruby Sweet apple variety. [Solution] According to the present invention, a method for identifying the apple variety Ruby Sweet 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 CTcrm2 in the genome of the apple variety Ruby Sweet, and a primer that binds to a genomic region of Ruby Sweet located near the retrotransposon 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 Ruby Sweet, comprising the steps of: a) performing a nucleic acid amplification method using DNA of a subject to be identified as a template with a primer set comprising at least a primer that binds to retrotransposon CTcrm2 in the genome of the apple cultivar Ruby Sweet, and a primer that binds to a Ruby Sweet genomic region located in the vicinity of said retrotransposon CTcrm2; and b) identifying Ruby Sweet based on the presence or absence of an amplification product. The present invention relates to a primer pair for identifying the apple cultivar Ruby Sweet. The present invention relates to a LAMP primer set for identifying the apple cultivar Ruby Sweet. [[Background Art]]

[0002] According to the status of apple cultivar registration under the Japanese Plant Variety Protection and Seed Act, there are approximately 300 cultivars including those published upon application. Many cultivars, such as excellent edible cultivars and rootstock cultivars, have been bred and registered for application by institutions including the National Agriculture and Food Research Organization Institute of Fruit Tree and Tea Science (hereinafter referred to as NARO Institute of Fruit Tree and Tea Science) (Non-Patent Document 1).

[0003] "Ruby Sweet" is a promising apple cultivar bred by NARO Institute of Fruit Tree and Tea Science. It is a cultivar for both fresh consumption and processing with red flesh and good eating quality. There are concerns both that seedlings of this cultivar will be taken out of the country without permission, and that they will be re-imported back to Japan.

[0004] On the other hand, for cultivar identification, DNA-based cultivar identification technology that utilizes differences in DNA nucleotide sequences between individual cultivars is common. For example, the introduction of DNA-based cultivar identification technology that enables more accurate determination is required for border inspections at customs and investigations related to the unauthorized taking out of seedlings and other materials. In particular, for border inspections at customs, since the time available for inspection is limited, a genetic testing method that can be performed 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 Ruby Sweet. 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 Initiative] [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 Ruby Sweet. [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 Ruby Sweet apples 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 Ruby Sweet, comprising the following steps: a) Using the DNA to be identified as a template, A primer that binds to the retrotransposon CTcrm2 in the genome of the apple variety Ruby Sweet, and A primer that binds to the ruby ​​sweet genomic region located near the retrotransposon 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 ruby ​​sweet 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 3) 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. 4, 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. 5; 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. 6, 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. 7; 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. 8, 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. 9. [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 3) below: 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 11; 2) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 12; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 13. [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. 17, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases as described in Sequence ID No. 18, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 19 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 20 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: 21 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 22 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 27. [5] 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 the base sequence described in Sequence ID No. 23, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 25, and A set of BIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 26; 2) The F3, B3, FIP, and BIP primers described in 1) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 27. [6] A pair of primers selected from 1) to 7) below to identify the apple variety Ruby Sweet: 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. 4, 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. 5; 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. 6, 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. 7; 3) a first primer comprising a polynucleotide consisting of a contiguous nucleotide sequence of 15 or more nucleotides in the nucleotide sequence set forth in SEQ ID NO: 8 or a complementary sequence thereof, and a pair of a second primer comprising a polynucleotide consisting of a contiguous nucleotide sequence of 15 or more nucleotides in the nucleotide sequence set forth in SEQ ID NO: 9 or a complementary sequence thereof; 4) a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 10 or a complementary sequence thereof, and a pair of a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 11 or a complementary sequence thereof; 5) a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 10 or a complementary sequence thereof, and a pair of a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 12 or a complementary sequence thereof; 6) a first primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 10 or a complementary sequence thereof, and a pair of a second primer comprising a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 13 or a complementary sequence thereof; 7) a pair of a first primer and a second primer, wherein a tag DNA is added to any one of the first primer and the second primer according to 1) to 6) above. [7] A primer set for LAMP method for identifying the apple cultivar Ruby Sweet, selected from the following 1) to 5): 1) an F3 primer comprising a polynucleotide consisting of a contiguous nucleotide sequence of 18 to 30 nucleotides in the nucleotide sequence set forth in SEQ ID NO: 17, a B3 primer comprising a polynucleotide consisting of a contiguous nucleotide sequence of 18 to 30 nucleotides in the nucleotide sequence set forth in SEQ ID NO: 18, an FIP primer comprising a polynucleotide having a contiguous nucleotide sequence of 20 to 30 nucleotides in the nucleotide sequence set forth in SEQ ID NO: 19 on the 3'-terminal side and a contiguous nucleotide sequence of 20 to 30 nucleotides in the nucleotide sequence set forth in SEQ ID NO: 20 on the 5'-terminal side, and A set of BIP primers containing polynucleotides having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 21 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 22 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 27; 3) F3 primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 23, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 25, and A set of BIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 26; 4) The F3, B3, FIP, and BIP primers described in 3) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 27; 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 Ruby Sweet, 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. 29, 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. 30, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 31 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 32 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: 33 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 34 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 SEQ ID NO: 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 40; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 35, A B3 primer containing a polynucleotide consisting of the base sequence described in SEQ ID NO: 36, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 37, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 38; 4) The F3, B3, FIP, and BIP primers described in 3) above, and An LF primer containing a polynucleotide consisting of the base sequence described in SEQ ID NO: 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 40; 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 ruby ​​sweet potatoes specifically 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 three retrotransposon insertion sites named CTcrm2_pattern559, CTcrm2_pattern648, and CTcrm2_pattern738. 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 CTcrm2_pattern559 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 CTcrm2_pattern648 amplification (A) and for positive control Mdtl1 gene amplification (B). [Figure 4] Figure 4 shows the results of performing a LAMP reaction using a primer set for CTcrm2_pattern648 amplification, 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. NTC is the negative control (sterile water). [Figure 5] Figure 5 shows the results of LAMP reactions performed using DNA extracted from six apple varieties as templates, employing a LAMP primer set for CTcrm2_pattern648 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 Ruby Sweet apple variety] The present invention relates to a method for identifying the apple variety Ruby Sweet, comprising the following steps. a) Using the DNA to be identified as a template, A primer that binds to the retrotransposon CTcrm2 in the genome of the apple variety Ruby Sweet, and A primer that binds to the ruby ​​sweet genomic region located near the retrotransposon 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 ruby ​​sweet based on the presence or absence of amplification products.

[0015] The Ruby Sweet apple variety has red skin, is a mid-season variety that can be harvested at the same time as the 'Kogyoku' apple, and is a large fruit weighing about 450g. Its flesh is red, sweeter and tastier than existing red-fleshed varieties. It can be used to produce distinctive processed products by utilizing the color of its flesh and juice, and is also suitable for eating fresh, so it is expected to expand the demand for apples. The variety's registration number is 24268 (March 26, 2015). The Ruby Sweet 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 Ruby Sweet grapes. 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 present invention involves using the DNA to be identified as a template and a primer set to amplify the retrotransposon CTcrm2 present in the ruby ​​sweet genome 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 or its processed product belonging to the genus Malus Mill. of the family Rosaceae, 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 "CTcrm2," a member of the retrotransposon family. "CTcrm2" is present in multiple locations in the Ruby Sweet genome, and a specific insertion site is found only in Ruby Sweet among the 24 varieties being identified. Therefore, the insertion site of "CTcrm2" can serve as a highly effective DNA marker for identifying Ruby Sweet.

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

[0025] Examples of "retrotransposon CTcrm2 and the genomic region near its insertion site" are the regions named CTcrm2_pattern559 (sequence number 1), CTcrm2_pattern648 (sequence number 2), and CTcrm2_pattern738 (sequence number 3) shown in Figure 1. In Figure 1, the retrotransposon 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 sequence identity is very high in at least the three regions mentioned above, but the apple sequence near the insertion site is completely different, as it does not have homology in at least the three regions mentioned above. The above "retrotransposon CTcrm2 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, a primer that binds to the retrotransposon CTcrm2 within the genome of the apple variety Ruby Sweet can be used to design primers that can be commonly used for amplification of the three regions and other insertion sites mentioned above. That is, the base sequences of the retrotransposon CTcrm2 in the three regions and other insertion sites can be aligned, and primers can be set in the parts without mutations. In another embodiment, primers can be set including the parts with mutations, and individual primers can be designed for each region to be amplified.

[0026] With respect to step a) of the present invention, a primer that binds to the retrotransposon CTcrm2 in the genome of the apple variety Ruby Sweet, and a primer that binds to a genomic region of Ruby Sweet located near the retrotransposon CTcrm2, can be designed to amplify all or part of the "retrotransposon CTcrm2 and the genomic region near its insertion site" as described in Sequence ID No. 1, 2, or 3. 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 ruby ​​sweet 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 target to be identified is ruby ​​sweet. If the target amplification product is not confirmed, it is indicated that the target to be identified is not ruby ​​sweet. The presence or absence of an amplification product can be confirmed by electrophoresis (agarose gel, etc.), nucleic acid chromatography PAS method, real-time PCR, DNA sequencing, etc.

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

[0029] In this specification, "near the retrotransposon CTcrm2" refers to the genomic region located outside the retrotransposon CTcrm2, specifically the apple-side sequence into which the retrotransposon CTcrm2 is inserted. For example, this includes the apple-side sequence extending approximately 1000 base pairs from the boundary between the retrotransposon 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 Ruby Sweet, wherein the nucleic acid amplification method is PCR, and the primer set is a primer pair selected from the following 1) to 3). 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. 4, 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. 5; 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. 6, 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. 7; 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. 8, 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. 9. The primer pairs described in 1) to 3) above allow for the amplification of the retrotransposon CTcrm2 and the genomic region near its insertion site, as shown in Sequence IDs 1 to 3, using ruby ​​sweet 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 in the primer pairs described in 1) to 3) above corresponds to "a primer that binds to the retrotransposon CTcrm2 within the genome of the apple variety Ruby Sweet." The nucleotide sequences shown in SEQ ID NOs. 4, 6, or 8 are the CTcrm2 retrotransposon sequences. The second primer in the primer pairs described in 1) to 3) above corresponds to "a primer that binds to the ruby ​​sweet genomic region located near the retrotransposon CTcrm2." Sequence IDs 5, 7, or 9 are the ruby ​​sweet genomic regions located near the retrotransposon CTcrm2. Sequence IDs 5, 7, or 9 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 NO: 4, 6, or 8. 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 NO: 5, 7, or 9.

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

[0035] In a particular embodiment, the present invention relates to a method for identifying the apple variety Ruby Sweet, wherein the nucleic acid amplification method is PCR, and the primer set is a primer pair selected from 1) to 3) below. 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 11; 2) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 12; 3) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 13. The primer pairs described in 1) to 3) above allow for the amplification of the retrotransposon CTcrm2 and the genomic region near its insertion site, as shown in SEQ ID NOs: 1 to 3, using ruby ​​sweet DNA as a template. The present invention is not limited to the method using the primer pairs described in 1) to 3) above, and it is possible to design and use primer pairs capable of amplifying the retrotransposon CTcrm2 and the genomic region near its insertion site, as shown in SEQ ID NOs: 1 to 3.

[0036] (LAMP method) The present invention provides a method for identifying the apple variety Ruby Sweet. Using the DNA of the target to be identified as a template, the LAMP method amplifies "the retrotransposon CTcrm2 and the genomic region near its insertion site," and identifies Ruby Sweet based on the presence or absence of the amplified product. The LAMP method amplifies nucleic acids under isothermal conditions (60-65°C), eliminating the need for a thermal cycler. The reaction can be carried out using a constant temperature device (e.g., a heat block), and the amplification reaction is completed in about 30 minutes to 1 hour. Therefore, the LAMP method is considered suitable for border inspections, for example, at customs, due to its simplicity and speed.

[0037] 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 examples described below, primers are designed to amplify CTcrm2_pattern648 and the genomic region near its insertion site. However, LAMP primers can also be designed for CTcrm2_pattern559 and the genomic region near its insertion site, and for CTcrm2_pattern738 and the genomic region near its insertion site.

[0038] One embodiment of the present invention relates to a method for identifying the apple variety Ruby Sweet, 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. 17, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases as described in Sequence ID No. 18, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 19 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 20 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: 21 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 22 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 27.

[0039] The above F3 primer corresponds to "a primer that binds to the retrotransposon CTcrm2 within the genome of the apple variety Ruby Sweet." The nucleotide sequence shown in Sequence ID No. 17 is the sequence within the retrotransposon CTcrm2. The B3 primer described above corresponds to a "primer that binds to the ruby ​​sweet genomic region located near the retrotransposon CTcrm2." Sequence ID No. 18 is the sequence of the ruby ​​sweet genomic region located near the retrotransposon CTcrm2.

[0040] 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.

[0041] In a particular embodiment, the present invention relates to a method for identifying the apple variety Ruby Sweet, 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. 23, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 25, and A set of BIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 26; 2) The F3, B3, FIP, and BIP primers described in 1) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 27. The primer set described in 1) or 2) above allows for the amplification of the retrotransposon CTcrm2 and the genomic region near its insertion site, as shown in Sequence ID No. 2, using ruby ​​sweet 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 also be carried out by designing and using a LAMP primer set capable of amplifying the retrotransposon CTcrm2 and the genomic region near its insertion site, as shown in Sequence ID Nos. 1 to 3.

[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] The present invention provides a method for identifying the above-mentioned apple variety Ruby Sweet, 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. 29, 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. 30, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 31 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 32 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: 33 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 34 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 SEQ ID NO: 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 40; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 35, A B3 primer containing a polynucleotide consisting of the base sequence described in SEQ ID NO: 36, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 37, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 38; 4) The F3, B3, FIP, and BIP primers described in 3) above, and An LF primer containing a polynucleotide consisting of the base sequence described in SEQ ID NO: 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 40.

[0044] (Nucleic acid chromatography PAS) Step b) of the present invention is a step of identifying ruby ​​sweet 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 target to be identified is ruby ​​sweet; if the target amplification product is not confirmed, it is indicated that the target to be identified is not ruby ​​sweet. In certain embodiments of the present invention, step b) is preferably performed by nucleic acid chromatography-PAS (C-PAS) method.

[0045] 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.

[0046] 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.

[0047] The C-PAS method using tagged DNA is a known technique, as disclosed in Japanese Patent Publication No. 2016-010338.

[0048] (Primer and Kit) The present invention relates to a pair of primers for identifying the apple variety Ruby Sweet, selected from the following 1) to 7). 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. 4, 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. 5; 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. 6, 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. 7; 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. 8, 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. 9; 4) A first primer comprising a polynucleotide consisting of the base sequence or complementary sequence described in SEQ ID NO: 10, and A pair of second primers containing a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 11; 5) A first primer comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 12; 6) A first primer comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 13; 7) 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 6) above. Furthermore, the present invention relates to a kit for identifying the apple variety Ruby Sweet, comprising a primer pair selected from 1) to 7) above.

[0049] The present invention relates to a set of primers for the LAMP method for identifying the apple variety Ruby Sweet, 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. 17, A B3 primer containing a polynucleotide consisting of a sequence of 18 to 30 consecutive bases as described in Sequence ID No. 18, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 19 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 20 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: 21 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 22 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 27; 3) F3 primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 23, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 25, and A set of BIP primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 26; 4) The F3, B3, FIP, and BIP primers described in 3) above, and A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 27; 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 a kit for identifying the apple variety Ruby Sweet, comprising a set of primers selected from 1) to 5) above.

[0050] 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. 29, 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. 30, An FIP primer containing a polynucleotide having a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 31 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 32 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: 33 at its 3' end, and a sequence of 20-30 consecutive bases in the sequence described in SEQ ID NO: 34 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 SEQ ID NO: 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 40; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 35, A B3 primer containing a polynucleotide consisting of the base sequence described in SEQ ID NO: 36, FIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 37, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 38; 4) The F3, B3, FIP, and BIP primers described in 3) above, and An LF primer containing a polynucleotide consisting of the base sequence described in SEQ ID NO: 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in SEQ ID NO: 40; 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 Ruby Sweet. Furthermore, the present invention relates to a kit for identifying the apple variety Ruby Sweet, comprising a set of primers selected from 1) to 5) above for amplifying the Mdtl1 gene as a positive control.

[0051] In certain embodiments, the identification kit for the Ruby Sweet apple variety 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]

[0052] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0053] Example 1: Design of a primer for amplifying the insertion site of ruby ​​sweet specific retrotransposons. In this example, a primer for amplifying the insertion site of ruby ​​sweet-specific retrotransposons was designed. Next-generation sequencing analysis was used to comprehensively analyze the insertion sites of the CTcrm2 retrotransposon family in the genomes of 24 major apple varieties, and three genomic regions with retrotransposon insertions were identified that exist only in Ruby Sweet apples. These three regions were named CTcrm2_pattern559, CTcrm2_pattern648, and CTcrm2_pattern738. 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 three 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 CTcrm2_pattern559, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 3 of the reference genome, from 30602514 to 30602480. The insertion site of CTcrm2_pattern559 is the 3' side corresponding to chromosome 30602514 of the reference genome. For the retrotransposon CTcrm2_pattern648, the apple-side sequence near the insertion site (the region shown in bold italics in Figure 1) corresponds to chromosome 13, 2161918-2161974, in the reference genome. The insertion site of CTcrm2_pattern648 corresponds to the 5' side of chromosome 13, 2161918, in the reference genome.

[0054] We designed primer pairs for PCR amplification of each of these three regions. An example of a primer pair is shown in Table 1. [Table 1]

[0055] In the following experiment, DNA was extracted from the peel of Ruby Sweet sweet potatoes 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.

[0056] Table 2 shows the composition of the PCR reaction solution and the amplification cycle. [Table 2]

[0057] 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 for the three primer pairs shown in Table 1, even when mixed with a positive control amplification primer pair using ruby ​​sweet DNA as a template.

[0058] 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. For the CTcrm2_pattern559, CTcrm2_pattern648, and CTcrm2_pattern738 primer pairs, the 5' end of the first primer in Table 1 was labeled with biotin, and the 5' end of the second primer was conjugated with an F1 tag (tag 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.

[0059] 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 CTcrm2_pattern559 amplification and a primer pair for positive control amplification, 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 Ruby Sweet test strip #19, 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 Ruby Sweet-specific retrotransposon insertion site was amplified by the primer pair for CTcrm2_pattern559 amplification, confirming that the primer pair for CTcrm2_pattern559 amplification can be used to distinguish Ruby Sweet from other apple varieties. We also confirmed that primer pairs for amplifying CTcrm2_pattern648 and CTcrm2_pattern738 can similarly be used to identify Ruby Sweet from other apple varieties. By using the C-PAS method in combination with a primer pair for amplifying Ruby Sweet-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 Ruby Sweet from other apple varieties.

[0060] Example 3: LAMP-C-PAS In this example, a primer set was designed for LAMP amplification of the retrotransposon CTcrm2_pattern648, 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 amplification of the retrotransposon CTcrm2_pattern648. An example of the primer set is shown in Table 5. [Table 5]

[0061] The 648_PE1_BIP primer in Table 5 had biotin conjugated to its 5' end, and the 648_PE1_LoopF ​​primer had an F1 tag (tagged DNA) conjugated to its 5' end. Figure 3 shows the positions of the primers.

[0062] 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]

[0063] 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 65°C, and incubated for 20 minutes to perform the LAMP amplification reaction.

[0064] 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 CTcrm2_pattern648. In the association curve analysis, the temperature of the reaction solution after LAMP amplification was increased to single-strand the DNA, denaturing various DNAs potentially containing 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.

[0065] 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.

[0066] Figure 4 shows the results of performing a LAMP reaction using a primer set for CTcrm2_pattern648 amplification, with DNA extracted from 24 apple varieties as a template, and then spreading the results onto test strips. The correspondence between the test strip numbers and apple varieties is the same as in Table 4 of Example 2. In the Ruby Sweet apple test strip #19, a blue line was observed at the F1 position, but no line was observed at the F1 position in the other 23 varieties. The blue line at F1 indicates that the DNA fragment of a Ruby Sweet-specific retrotransposon was amplified by the primer set for CTcrm2_pattern648 amplification, confirming that the primer pair for CTcrm2_pattern648 amplification can be used to distinguish Ruby Sweet from other apple varieties.

[0067] 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]

[0068] 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 CTcrm2_pattern648 amplification (Table 5) and the positive control amplification primer set (Table 7) was used. LAMP amplification and C-PAS were performed under the same conditions as described above. The template DNA used was a crude extract from the perilla peel. The results are shown in Figure 5.

[0069] Table 8 shows the correspondence between the test strips and apple varieties. [Table 8]

[0070] 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 Ruby Sweet test strips b and g, a blue line was observed at the F1 position, but no line was observed at the F1 position in the test strips of other varieties. The blue line at F1 indicates that the DNA fragment of a Ruby Sweet-specific retrotransposon was amplified by the primer set for CTcrm2_pattern648 amplification, confirming that the primer pair for CTcrm2_pattern648 amplification can be used to distinguish Ruby Sweet 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 Ruby Sweet apples can be rapidly identified from other apple varieties.

Claims

1. A method for identifying the apple variety Ruby Sweet, comprising the following steps: a) Using the DNA to be identified as a template, A primer that binds to the retrotransposon CTcrm2 in the genome of the apple variety Ruby Sweet, and A primer that binds to the ruby ​​sweet genomic region located near the retrotransposon CTcrm2. A step of applying a nucleic acid amplification method using a primer set containing at least the following, and b) A step to identify ruby ​​sweet 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 3) 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. 4, 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. 5; 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. 6, 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. 7; 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. 8, 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.

9.

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 3) below: 1) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 11; 2) A first primer comprising a polynucleotide consisting of the base sequence described in SEQ ID NO: 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 12; 3) A first primer comprising a polynucleotide consisting of the base sequence described in Sequence ID No. 10, and A pair of second primers containing a polynucleotide consisting of the base sequence described in Sequence ID No.

13.

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. 17, 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. 18, An FIP primer comprising a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 19 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 20 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 21 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 22 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No.

27.

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. 23, A B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24, An FIP primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 26; 2) The F3, B3, FIP, and BIP primers described in 1) above, A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No.

27.

6. A pair of primers for identifying the apple variety Ruby Sweet, selected from the following 1) to 7): 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. 4, 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. 5; 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. 6, 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. 7; 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. 8, 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. 9; 4) A first primer comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 10, and A pair of second primers comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 11; 5) A first primer comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 10, and A pair of second primers comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 12; 6) A first primer comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 10, and A pair of second primers comprising a polynucleotide consisting of the base sequence or complementary sequence described in Sequence ID No. 13; 7) 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 6) above.

7. A LAMP primer set for identifying the apple variety Ruby Sweet, 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. 17, 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. 18, An FIP primer comprising a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 19 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 20 at its 5' end, and A set of BIP primers containing polynucleotides having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 21 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 22 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 27; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 23, A B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 24, An FIP primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 25, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 26; 4) The F3, B3, FIP, and BIP primers described in 3) above, A set of LF primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 27; 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 Ruby Sweet, 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. 29, 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. 30, An FIP primer comprising a polynucleotide having a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 31 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 32 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: 33 at its 3' end, and a sequence of 20 to 30 consecutive bases in the sequence described in SEQ ID NO: 34 at its 5' end; 2) The F3, B3, FIP, and BIP primers described in 1) above, An LF primer comprising a polynucleotide having the base sequence described in Sequence ID No. 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 40; 3) F3 primers containing a polynucleotide consisting of the base sequence described in Sequence ID No. 35, B3 primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 36, An FIP primer containing a polynucleotide consisting of the base sequence described in Sequence ID No. 37, and A set of BIP primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 38; 4) The F3, B3, FIP, and BIP primers described in 3) above, An LF primer comprising a polynucleotide having the base sequence described in Sequence ID No. 39, and / or A set of LB primers containing polynucleotides consisting of the base sequence described in Sequence ID No. 40; 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.

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