Discrimination method and discrimination kit

Abstract

To provide highly accurate methods for discriminating sweet potato variety Beniharuka or Fukumurasaki.SOLUTION: The method comprises detecting at least one first DNA marker selected from the group consisting of first DNA markers derived from a parent variety of the subject variety and at least one second DNA marker selected from the group consisting of second DNA markers derived from the other parent variety of the subject variety, where the DNA markers contained in the first and second marker groups are DNA markers derived from insertion polymorphism of retrotransposon Cl8.SELECTED DRAWING: None

Description

Technical Field

[0006] , , , , , ,

[0007]

[0001] The present invention relates to a method for discriminating sweet potato variety Beniharuka and a method for discriminating purple sweet potato variety including Beniharuka, and a discrimination kit. Specifically, it relates to these discrimination methods and discrimination kits using DNA markers.

Background Art

[0002] Sweet potatoes can be propagated by taking seedlings from the tuberous roots as products. Therefore, the overseas outflow of seeds and seedlings is likely to occur. Since varieties with the same use are similar in form among varieties and are basically imported as processed products when imported, it is difficult to distinguish a specific variety by appearance and to determine the presence or absence of use of a specific variety. Therefore, a method for discriminating varieties using DNA markers has been developed.

[0003] For example, Non-Patent Document 1 reports three markers derived from insertion polymorphisms of retrotransposon Rtsp-1 as DNA markers that can be used for discriminating Beniharuka and major sweet potato varieties.

[0004] Patent Document 1 and Non-Patent Document 2 report a method for detecting a DNA marker derived from an insertion polymorphism of retrotransposon Rtsp-1 as a method for discriminating varieties in sweet potato processed products.

[0005] Non-Patent Document 3 reports DNA markers derived from insertion polymorphisms of retrotransposon Rtsp-1 and LIb for identifying anthocyanin-containing purple sweet potato varieties.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007] [Non-Patent Document 1] Masaru Tanaka, et al., "Nissaku Kyushiho," 2015, Vol. 81, pp. 43-45, 2015. [Non-Patent Document 2] Natsuko Oe, et al., "Journal of Plant Breeding," 2004, Vol. 6, pp. 169-177. [Non-Patent Document 3] Yuki Kadota, et al., "DNA Polymorphism," 2013, Vol. 21, pp. 47-54. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] As mentioned above, while DNA markers are known that can be used to distinguish the Beni Haruka variety from other major varieties, the number of such markers is still small. Therefore, considering the existence of varieties that have recently become popular and the ongoing development of new varieties, it can be said that these markers are insufficient for highly accurate differentiation. Furthermore, there is a possibility that the differentiation between Beni Haruka and future offspring varieties of Beni Haruka may be insufficient.

[0009] On the other hand, for the Fukumurasaki variety, which has been developed in recent years, no DNA markers that can be used for variety identification have been reported.

[0010] Therefore, the present invention aims to provide an easy and accurate method for distinguishing between the varieties Beniharuka and Fukumurasaki. [Means for solving the problem]

[0011] To solve the above problems, a first aspect of the present invention provides a method for identifying the sweet potato variety Beniharuka, comprising detecting at least one first DNA marker selected from a first marker group consisting of DNA markers derived from one parent variety of Beniharuka, and at least one second DNA marker selected from a second marker group consisting of DNA markers derived from the other parent variety of Beniharuka, wherein the DNA markers included in the first and second marker groups are DNA markers derived from insertion polymorphisms of the retrotransposon Cl8.

[0012] Furthermore, in the discrimination method according to the first embodiment described above, it is preferable that either one or both of the first marker group and the second marker group further include a DNA marker derived from the insertion polymorphism of the retrotransposon Rtsp-1.

[0013] Furthermore, in the discrimination method according to the first embodiment described above, the detection of DNA markers derived from insertion polymorphisms of retrotransposon Cl8 preferably includes amplifying a nucleic acid fragment containing the terminal sequence of retrotransposon Cl8 shown in any of SEQ ID NOs: 1 to 4.

[0014] Furthermore, in the discrimination method according to the first embodiment described above, the detection of the first DNA marker preferably includes amplifying a nucleic acid fragment using the primer set of (1) or (2) below, and the detection of the second DNA marker preferably includes amplifying a nucleic acid fragment using the primer set of (3) below: (1) A set of primer 1a consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 5 or 6, and primer 1b consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 7 or 8; (2) A set of primer 2a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 9 and primer 2b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 10; (3) A set of primer 3a consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 11 or 12 and primer 3b consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 13 or 14.

[0015] Furthermore, in the discrimination method according to the first embodiment described above, it is preferable that primer 1a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 15 or 16, primer 1b is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18, primer 2a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 19, primer 2b is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 20, primer 3a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 21 or 22, and primer 3b is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18.

[0016] Furthermore, the determination method relating to the first embodiment described above may be a method for determining whether or not the raw material plant contained in the processed food is the sweet potato variety Beniharuka, which includes extracting the DNA of the raw material plant contained in the processed food.

[0017] Furthermore, the discrimination method according to the first embodiment described above may be a method that simultaneously detects the first DNA marker and the second DNA marker.

[0018] To solve the above problems, a discrimination kit according to a second aspect of the present invention is a discrimination kit used for discriminating between the sweet potato variety Beniharuka, comprising a set of primers (1) and (2) below or both, and a set of primers (3) below: (1) A set of primer 1a consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 5 or 6, and primer 1b consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 7 or 8; (2) A pair of primers: primer 2a consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 9 and primer 2b consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 10; (3) A pair of primers: primer 3a consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 11 or 12 and primer 3b consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 13 or 14.

[0019] In order to solve the above problems, the discrimination method according to the third aspect of the present invention is a discrimination method for purple sweet potato including sweet potato varieties, which includes detecting at least one first DNA marker selected from a first marker group composed of DNA markers derived from one parent variety of purple sweet potato, and at least one second DNA marker selected from a second marker group composed of DNA markers derived from the other parent variety of purple sweet potato. The DNA markers included in the first marker group and the second marker group are DNA markers derived from the insertion polymorphism of retrotransposon Cl8.

[0020] Further, in the discrimination method according to the third aspect, it is preferable that either one or both of the first marker group and the second marker group further include DNA markers derived from the insertion polymorphism of retrotransposon Rtsp-1.

[0021] Further, in the discrimination method according to the third aspect, the detection of the DNA marker derived from the insertion polymorphism of retrotransposon Cl8 preferably includes amplifying a nucleic acid fragment containing the terminal sequence of retrotransposon Cl8 shown in any of SEQ ID NOs: 1 to 4.

[0022] Further, in the discrimination method according to the third aspect, the detection of the first DNA marker preferably includes amplifying a nucleic acid fragment using a primer pair of the following (4) or (5), and the detection of the second DNA marker preferably includes amplifying a nucleic acid fragment using a primer pair of the following (6) or (7): (4) A pair of primer 4a consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 6, and primer 4b consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 8; (5) A pair of primer 5a consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 23 or 24, and primer 5b consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 25 or 26; (6) A pair of primer 6a consisting of 20 or more consecutive bases in the nucleotide sequence shown in any of SEQ ID NOs: 29 to 31, and primer 6b consisting of 20 or more consecutive bases in the nucleotide sequence shown in any of SEQ ID NOs: 32 to 34; (7) A pair of primer 7a consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 28, and primer 7b consisting of 20 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 27.

[0023] In the discrimination method according to the third aspect, preferably, the primer 4a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 16, the primer 4b is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18, the primer 5a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 36 or 37, the primer 5b is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 20, the primer 6a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 38, 39 or 40, the primer 6b is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18, the primer 7a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 35, and the primer 7b is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 20.

[0024] Further, the discrimination method according to the third aspect may be a method for discriminating whether the raw material plant contained in the processed food is purple sweet potato including the Satsumaimo variety by extracting DNA of the raw material plant from the processed food.

[0025] Furthermore, the discrimination method according to the third embodiment described above may be a method that simultaneously detects the first DNA marker and the second marker.

[0026] To solve the above problems, a discrimination kit according to a fourth aspect of the present invention is a discrimination kit used for discriminating the sweet potato variety Fukumurasaki, comprising a set of one or both of the primers (4) and (5) below, and a set of one or both of the primers (6) and (7) below: (4) A set of primer 4a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 6, and primer 4b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 8; (5) A set of primer 5a consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 23 or 24 and primer 5b consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 25 or 26; (6) A set of primer 6a consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs. 29 to 31 and primer 6b consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs. 32 to 34; (7) A set of primer 7a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 28 and primer 7b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 27. [Effects of the Invention]

[0027] According to one aspect of the present invention, an easy and accurate method for distinguishing between the varieties Beni Haruka and Fukumurasaki can be realized. [Brief explanation of the drawing]

[0028] [Figure 1] This figure shows the detection of a DNA marker derived from the insertion polymorphism of the retrotransposon Cl8, used to distinguish between *Haruka Beni* and other *Haruka* species. [Figure 2] This figure shows the detection of a DNA marker derived from the insertion polymorphism of the retrotransposon Cl8, used for distinguishing between different varieties of purple. [Figure 3] This figure shows the detection of a DNA marker derived from the insertion polymorphism of the retrotransposon Rtsp-1, used to distinguish between different species of the Beni Haruka. [Figure 4] This figure shows the detection of a DNA marker derived from the insertion polymorphism of the retrotransposon Rtsp-1, used for distinguishing between purple and purplish-yellow varieties. [Figure 5] This figure shows the detection of DNA markers for distinguishing between Beni Haruka and Fukumurasaki varieties using multiplex PCR and chromatographic PAS detection. [Modes for carrying out the invention]

[0029] One embodiment of the present invention will be described in detail below.

[0030] The method for identifying sweet potato varieties according to this embodiment is a method for determining whether the sweet potato to be identified is of the Beniharuka variety or the Fukumurasaki variety. In one embodiment, it may be a method for identifying the variety of sweet potato as a plant or as a raw material plant contained in processed food. In the identification method in this embodiment, whether or not it is one of these varieties is determined with high accuracy by detecting multiple DNA markers.

[0031] [DNA markers] The method for identifying sweet potato varieties in this embodiment includes detecting at least one first DNA marker and at least one second DNA marker that is different from the first DNA marker.

[0032] Here, the first DNA marker is selected from a first marker group consisting of DNA markers derived from one parent variety of the target sweet potato variety. The second DNA marker is selected from a second marker group consisting of DNA markers derived from the other parent variety of the target sweet potato variety. There is no limit to the number of DNA markers that make up the first and second marker groups; they may be one or more, but preferably two or more, and more preferably three or more. The more DNA markers that are available, the more DNA markers can be actually used, and the higher the discrimination accuracy can be.

[0033] If the target sweet potato variety is "Beniharuka," one parent variety is "Kyushu 121," and the other parent variety is "Harukogane." If the target sweet potato variety is "Fukumurasaki," one parent variety is "Kyukei 255," and the other parent variety is "Purple Sweet Road."

[0034] In this specification, "DNA markers derived from parent varieties" means DNA markers detected in both the target variety and its parent varieties.

[0035] In this embodiment, the DNA marker is a marker derived from retrotransposon insertion polymorphisms present in the genomic DNA of sweet potato. The genomic sequences adjacent to retrotransposons are unique to each insertion site. Furthermore, there is inter-varietal polymorphism, where retrotransposon insertions are found in one variety's genomic location, while they are not found in another. Therefore, when the DNA marker is derived from retrotransposon insertion polymorphisms, detecting the DNA marker means detecting the presence or absence of retrotransposons at specific locations in the plant genome.

[0036] In one embodiment of this model, the DNA markers included in the first marker group and the second marker group are DNA markers derived from the insertion polymorphism of the retrotransposon Cl8. In another embodiment, either or both of the first marker group and the second marker group further include a DNA marker derived from the insertion polymorphism of the retrotransposon Rtsp-1, in addition to the DNA marker derived from the insertion polymorphism of the retrotransposon Cl8.

[0037] Although the existence of the retrotransposon Cl8 was known, its base sequence and insertion site had not been previously elucidated. In many LTR-type retrotransposons, iMet-tRNA is known to be used as a primer during transposition. Therefore, the PBS (primer binding site) adjacent to the 3' end of the 5' LTR of the retrotransposon exhibits high sequence conservation. Thus, we used this sequence to determine the sequence of Cl8. First, using the iMet-tRNA sequence, we constructed a library of PCR products extended from PBS toward the 5' LTR for several varieties (including Purple Sweet Road). Specifically, genomic DNA was digested with multiple restriction enzymes, an adapter was attached, and amplification was performed by PCR using primers corresponding to the adapter and iMet-tRNA primers to construct the library. Next, NGS analysis was performed on each fragment. Then, by comparing the results across varieties, we determined the partial sequence (including the 5' LTR) of the retrotransposon Cl8.

[0038] [Detection of DNA markers] The method for detecting DNA markers, that is, the method for detecting the presence or absence of retrotransposons at specific sites in the genome, is not particularly limited, and any known method may be appropriately selected and used, however, the use of nucleic acid amplification reactions is preferred. As an example of a method using nucleic acid amplification reactions, one can use a primer set that combines a primer designed based on the base sequence of a genomic region adjacent to the retrotransposon in the target DNA marker and a primer designed based on the base sequence of the retrotransposon, and perform a nucleic acid amplification reaction using DNA prepared from the test subject as a template to confirm the presence or absence of amplification products.

[0039] Methods for confirming the presence or absence of amplification products include electrophoresis, chromatographic PAS, and real-time PCR.

[0040] In detecting DNA markers derived from insertion polymorphisms of the retrotransposon Cl8, the method may involve amplifying a nucleic acid fragment containing the terminal sequence of the retrotransposon Cl8. Therefore, in one embodiment, a nucleic acid fragment containing the 5'LTR terminal sequence of the retrotransposon Cl8, as shown in any of SEQ ID NOs: 1 to 4, is amplified.

[0041] The nucleic acid amplification reaction can be carried out by appropriately selecting and using known nucleic acid amplification methods, such as polymerase chain reaction (PCR), ICAN, UCAN, LAMP, and primer extension methods, but is not limited to these.

[0042] Furthermore, when performing amplification by PCR, so-called multiplex PCR may be used to amplify multiple fragments in the same reaction and detect multiple DNA markers in a single reaction. That is, PCR may be performed by including both a primer set for detecting a first DNA marker and a primer set for detecting a second DNA marker in a single PCR reaction solution.

[0043] When applying multiplex PCR, it is preferable to design only one type of primer for the retrotransposon side and perform nucleic acid amplification by adding multiple insertion site-specific primers and one type of retrotransposon-side primer to a single PCR reaction solution.

[0044] Regardless of the amplification reaction method, it is sufficient to detect at least one DNA marker for both the first and second DNA markers. However, multiple (e.g., two or three) first DNA markers selected from the first DNA marker group may be detected, and similarly, multiple (e.g., two or three) second DNA markers selected from the second DNA marker group may be detected. By detecting multiple DNA markers for both the first and second DNA markers, the accuracy of discrimination can be further improved.

[0045] (1. Detection of DNA markers in the identification of the Beni Haruka variety) In the identification of the Beni Haruka variety, the detection of the first DNA marker may include amplifying the nucleic acid fragment using the primer set (1) or (2) below, and the detection of the second DNA marker may include amplifying the nucleic acid fragment using the primer set (3) below.

[0046] (1) A set of primer 1a consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 5 or 6 and primer 1b consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 7 or 8; (2) A set of primer 2a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 9 and primer 2b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 10; (3) A set of primer 3a consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 11 or 12 and primer 3b consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 13 or 14.

[0047] Here, the sequences shown in SEQ ID NOs. 5, 6, 11, and 12 are sequences of the genomic region adjacent to the 5'LTR side of retrotransposon Cl8 at different specific insertion sites. The sequences shown in SEQ ID NOs. 7, 8, 13, and 14 are complementary sequences of the 5'LTR terminal sequence of retrotransposon Cl8. Furthermore, the sequence shown in SEQ ID NO. 9 is a complementary sequence of the genomic region adjacent to the 3'LTR side of retrotransposon Rtsp-1 at a specific insertion site. The sequence shown in SEQ ID NO. 10 is the 3'LTR terminal sequence of retrotransposon Rtsp-1.

[0048] Furthermore, the first DNA marker detected using primer set (1) or (2) is a DNA marker derived from Kyushu 121, one of the parent varieties of Beniharuka. On the other hand, the second DNA marker detected using primer set (3) is a DNA marker derived from Harukogane, the other parent variety of Beniharuka.

[0049] While there is no particular upper limit to the primer length as long as it can amplify the target nucleic acid fragment, from the viewpoint of suppressing primer dimer formation, for example, a length of 50 bases or less, 45 bases or less, 40 bases or less, 35 bases or less, or 30 bases or less is preferred.

[0050] The primer sets described in (1) and (3) above are preferably the primer sets described in (1A) and (3A) below, respectively.

[0051] (1A) A set of primer 1a consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 5 and primer 1b consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 7, or a set of primer 1a consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 6 and primer 1b consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 8; (3A) A set of primer 3a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 11 and primer 3b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 13, or a set of primer 3a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 12 and primer 3b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 14.

[0052] In particular, primer 1a is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 15 or 16. Primer 1b is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18. Primer 2a is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 19. Primer 2b is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 20. Primer 3a is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 21 or 22. Primer 3b is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18.

[0053] Therefore, more preferably, the primer sets (1B) to (3B) below are the primer sets (1B) to (3B) above.

[0054] (1B) A set of primer 1a consisting of the nucleotide sequence shown in SEQ ID NO: 15 and primer 1b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18, or a set of primer 1a consisting of the nucleotide sequence shown in SEQ ID NO: 16 and primer 1b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18; (2B) A set of primer 2a consisting of the nucleotide sequence shown in SEQ ID NO: 19 and primer 2b consisting of the nucleotide sequence shown in SEQ ID NO: 20; (3B) A set of primer 3a consisting of the nucleotide sequence shown in SEQ ID NO: 21 and primer 3b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18, or a set of primer 3a consisting of the nucleotide sequence shown in SEQ ID NO: 22 and primer 3b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18.

[0055] (2. Detection of DNA markers in the identification of the Fukumurasaki variety) In the identification of the Fukumurasaki variety, the detection of the first DNA marker may include amplifying the nucleic acid fragment using the primer set of (4) or (5) below, and the detection of the second DNA marker may include amplifying the nucleic acid fragment using the primer set of (6) or (7) below.

[0056] (4) A set of primer 4a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 6 and primer 4b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 8; (5) A set of primer 5a consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 23 or 24 and primer 5b consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 25 or 26; (6) A set of primer 6a consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs. 29 to 31 and primer 6b consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs. 32 to 34; (7) A set of primer 7a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 28 and primer 7b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 27.

[0057] Here, the sequences shown in SEQ ID NOs. 6, 29-31 are the sequences of the genomic region adjacent to the 5'LTR side of retrotransposon Cl8 at different specific insertion sites. The sequences shown in SEQ ID NOs. 8, 32-34 are the complementary sequences of the 5'LTR terminal sequence of retrotransposon Cl8. Furthermore, the sequences shown in SEQ ID NOs. 23, 24, and 28 are the complementary sequences of the 3'LTR side of retrotransposon Rtsp-1 at different specific insertion sites of Rtsp-1 at the genomic region adjacent to Rtsp-1. The sequences shown in SEQ ID NOs. 25, 26, and 27 are the 3'LTR terminal sequences of retrotransposon Rtsp-1.

[0058] Furthermore, the first DNA marker detected using primer set (4) or (5) is a DNA marker derived from one of Fukumurasaki's parent varieties, Kyukei 255. On the other hand, the second DNA marker detected using primer set (6) or (7) is a DNA marker derived from the other parent variety of Fukumurasaki, Purple Sweet Road.

[0059] The length of the primer is not particularly limited as long as it can amplify the target nucleic acid fragment, but from the viewpoint of suppressing primer dimer formation, it may be, for example, 50 bases or less, 45 bases or less, 40 bases or less, 35 bases or less, or 30 bases or less.

[0060] Preferably, the primer sets described in (5) and (6) above are the primer sets described in (5A) and (6A) below, respectively.

[0061] (5A) A set of primer 5a consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 23 and primer 5b consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 25, or a set of primer 5a consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 24 and primer 5b consisting of 20 or more consecutive bases from the nucleotide sequence shown in Sequence ID No. 26; (6A) A set consisting of primer 6a made of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 29 and primer 6b made of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 32, a set consisting of primer 6a made of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 30 and primer 6b made of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 33, or a set consisting of primer 6a made of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 31 and primer 6b made of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 34.

[0062] In particular, primer 4a is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 16. Primer 4b is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18. Primer 5a is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 36 or 37. Primer 5b is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 20. Primer 6a is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 38, 39 or 40. Primer 6b is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18. Primer 7a is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 35. Primer 7b is preferably a primer consisting of the nucleotide sequence shown in SEQ ID NO: 20.

[0063] Therefore, more preferably, the primer sets (4B) to (7B) below are used as the primer sets (4B) to (7B) above.

[0064] (4B) A set of primer 4a consisting of the nucleotide sequence shown in SEQ ID NO: 16 and primer 4b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18; (5B) A set of primer 5a consisting of the nucleotide sequence shown in SEQ ID NO: 36 or 37 and primer 5b consisting of the nucleotide sequence shown in SEQ ID NO: 20; (6B) A set of primer 6a consisting of the nucleotide sequence shown in SEQ ID NO: 38 and primer 6b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18, a set of primer 6a consisting of the nucleotide sequence shown in SEQ ID NO: 39 and primer 6b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18, or a set of primer 6a consisting of the nucleotide sequence shown in SEQ ID NO: 40 and primer 6b consisting of the nucleotide sequence shown in SEQ ID NO: 17 or 18; (7B) A set of primer 7a consisting of the nucleotide sequence shown in SEQ ID NO: 35 and primer 7b consisting of the nucleotide sequence shown in SEQ ID NO: 20.

[0065] [Discrimination method] The identification method according to this embodiment may be a method for identifying the variety of a plant by extracting DNA from the plant, or a method for identifying the variety of sweet potato used in a processed product, such as a processed food, by extracting DNA from the processed product, which uses sweet potato as a raw material.

[0066] The processed foods covered by this specification are not particularly limited, as long as they contain sweet potatoes as raw material plants and allow for the extraction of DNA from the raw material plants. Furthermore, the raw material plants included in processed foods may be parts of a plant body, such as tubers, roots, leaves, stems, flowers, and seeds. A processed food is a product manufactured by subjecting a raw material plant to processing treatments such as heating, drying, or freezing. In this specification, the term is used to include not only the final product but also intermediate processed products. Moreover, processed foods are not limited to those containing a single variety of raw material plant, but may also contain multiple varieties.

[0067] DNA extraction from plants and from processed foods can be carried out using conventionally known methods.

[0068] (Identification Kit) A discrimination kit according to one embodiment of the present invention is a discrimination kit used for distinguishing between the sweet potato variety Beniharuka and a discrimination kit used for distinguishing between the sweet potato variety Fukumurasaki. Each discrimination kit comprises a set of primers used to detect a first DNA marker and a second DNA marker, respectively.

[0069] In other words, the identification kit used to distinguish the Beni Haruka variety includes one or both of the primer sets described in (1) and (2) below, and the primer set described in (3) below: (1) The set of primer 1a and primer 1b described above; (2) The set of primer 2a and primer 2b described above; (3) The set of primer 3a and primer 3b described above.

[0070] Similarly, the identification kit used to distinguish the Fukumurasaki variety includes one or both of the primer sets described in (4) and (5) below, and one or both of the primer sets described in (6) and (7) below: (4) The set of primer 4a and primer 4b described above; (5) The set of primer 5a and primer 5b described above; (6) The set of primer 6a and primer 6b described above; (7) The set of primer 7a and primer 7b described above.

[0071] Each primer can be synthesized according to conventional nucleic acid synthesis methods.

[0072] Each discrimination kit may include, as necessary, at least one of the following: various reagents used for PCR, such as polymerase, PCR buffer, and various dNTPs; various reagents for preparing a sample containing DNA for PCR, such as buffer; various reagents for analyzing PCR amplification fragments, such as electrophoresis gel material; and instructions for using the discrimination kit.

[0073] The method for identifying sweet potato varieties in this embodiment can protect seedlings and appropriate producers, and therefore can contribute to achieving the Sustainable Development Goal (SDG) of "2. Zero Hunger."

[0074] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Examples]

[0075] <1. Plant materials> Table 1 shows the varieties and lines used for the development and detection of DNA markers. Leaves for DNA extraction were collected from plants grown in a plastic greenhouse or greenhouse used for seedling cultivation. Tubers were obtained from plants cultivated at the experimental field of the Kyushu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization.

[0076] [Table 1]

[0077] <2. Obtaining insertion site sequence information for the retrotransposon Cl8 in the genome> Genomic DNA extraction and MiSeq sequencing library construction were performed according to the method described in Monden et al., DNA research 21(5), 491-498, 2014. However, the TruSeq DNA-Sampling Preparation Kit (Illumina) was not used for library construction. Instead, the MiSeq sequencing library was constructed by using primers with the necessary sequences (P5 and P7) added as an index during the second PCR of nested PCR. For the first PCR of nested PCR, AP2 and iMET_PBS primers (Table 2) were used. The iMET_PBS primers were designed based on the PBS nucleotide sequence, which is widely conserved in LTR retrotransposons in general. For the second PCR, either the P5 side sequence (D501a~D508a) or the P7 side sequence (D701a~D708a) (Table 3) was used, and the combination was adjusted to distinguish between varieties. In Table 3, the underlined portion of the D501a to D508a sequences is the index sequence, the portion 5' to the left of it is the P5 sequence, and the portion 3' to the right is the terminal sequence of the retrotransposon Cl8 (hereinafter simply referred to as "Cl8"). In addition, in the D701a to D708a sequences, the underlined portion is the index sequence, the portion 5' to the left of it is the P7 sequence, and the portion 3' to the right is the adapter sequence.

[0078] [Table 2]

[0079] [Table 3]

[0080] After sequencing using MiSeq, a cross-tabulation table summarizing the Cl8 insertion sites was created according to the method described in the aforementioned literature, and insertion sites useful for variety identification were selected.

[0081] <3. Obtaining sequence information of the insertion site of Rtsp-1 in the genome> Except for changing the primers for the first and second PCRs of nested PCR to those shown in Table 4 and Table 5, respectively, insertion sites useful for variety identification were selected in the same manner as for obtaining the Cl8 insertion site sequence information described above. In the sequences D501b to D508b shown in Table 5, the underlined portion is the index sequence, the 5' side (left) is the P5 sequence, and the 3' side (right) is the terminal sequence of Rtsp-1. In addition, in the sequences D701b to D708b, the underlined portion is the index sequence, the 5' side (left) is the P7 sequence, and the 3' side (right) is the adapter sequence.

[0082] [Table 4]

[0083] [Table 5]

[0084] <4. Detection of DNA markers> (4-1. Detection of Cl8 markers) Genomic DNA was extracted from undeveloped sweet potato leaves using QIAGEN's DNeasy Plant Mini Kit or Nippon Gene's ISOSPIN Plant DNA. Alternatively, genomic DNA was extracted from the internal tissue of tubers using ISOSPIN Plant DNA. The extraction procedure was performed according to the instructions included with the kit. DNA concentration was measured using a Thermofisher Scientific Qubit2.0 fluorometer or by measuring the absorbance at 260 nm using a spectrophotometer.

[0085] Reaction solutions were prepared with the compositions shown in Table 6, and PCR was performed under cycling conditions of denaturation at 94°C for 2 minutes, 30 cycles of 94°C for 15 seconds - 60°C for 30 seconds - 72°C for 30 seconds, and a final extension reaction at 72°C for 5 minutes. After the reaction, the mixture was cooled to 4°C. The insertion site-specific primers shown in Table 7 were used. Two types of transposon-side primers were designed, and PCR was performed for each. For example, when Cl1136 was used as the insertion site-specific primer, both PCR using the PBS_LTR and Cl1136 primer set and PCR using the PBS_LTR2 and Cl1136 primer set were performed.

[0086] PCR products were detected by electrophoresis using a Shimadzu automated electrophoresis system (MultiNA) or an agarose gel.

[0087] The results are shown in Figures 1 and 2. Figure 1 shows the results when using primers for distinguishing Beniharuka (Cl1136, Pattern 2379, Pattern 3615, or Pattern 4133) as insertion site-specific primers. On the other hand, Figure 2 shows the results when using primers for distinguishing Fukumurasaki (Pattern 1680, Pattern 2626, or Pattern 5354) as insertion site-specific primers. However, Pattern 4133 is also a primer for distinguishing Fukumurasaki. In the figures, the bands indicated by arrowheads are amplified marker fragments. Also, in the figures, the results for the varieties and lines shown in Table 1 are shown in the same lane numbers as in Table 1. That is, lanes 1 and 2 are the results for Beniharuka and Fukumurasaki, respectively. Note that in both cases, there was no difference between using PBS_LTR as the transposon-side primer and using PBS_LTR2, so only the results for one of them are shown.

[0088] Furthermore, as a control for DNA quality assurance, PCR was also performed using primers (SSII_F and SSII_R) to amplify the type II starch synthase (SSII) gene, instead of transposon-side primers and insertion site-specific primers (results are not shown).

[0089] [Table 6]

[0090] [Table 7]

[0091] (4-2. Detection of Rtsp-1 markers) Detection was performed in the same manner as for the Cl8 marker, except that the primers used for PCR were those shown in Table 8.

[0092] The results are shown in Figures 3 and 4. Figure 3 shows the results when the primer for distinguishing Beniharuka (Cl484) was used as the insertion site-specific primer. On the other hand, Figure 4 shows the results when the primer for distinguishing Fukumurasaki (Cl26, Cl561, or Pattern 1126) was used as the insertion site-specific primer. In Figures 3 and 4, the lane numbers correspond to the numbers in Table 1.

[0093] [Table 8]

[0094] (4-3.Results) In Figures 1 and 3, Pattern 3615, Pattern 4133, and Cl 484 correspond to the first marker, and Cl 1136 and Pattern 2379 correspond to the second marker. In the Beni Haruka variety, marker fragments are detected in all of these. From the results in Figures 1 and 3, Beni Haruka is the only variety in which marker fragments were detected in all of these. Therefore, it has been shown that Beni Haruka can be accurately distinguished from other varieties (including the parent varieties) by combining the detection results of the first and second markers, which are DNA markers derived from each parent variety.

[0095] Similarly, in Figures 2 and 4, Pattern 1126 and Cl 561 correspond to the first marker, and Cl 26, Pattern 1680, Pattern 2626, and Pattern 5354 correspond to the second marker. Also, Pattern 4133 in Figure 1 is the first marker in Fukumurasaki. Marker fragments are detected in all of these in the Fukumurasaki variety. From the results in Figures 1, 2, and 4, Fukumurasaki is the only variety in which marker fragments were detected in all of these. Therefore, it has been shown that Fukumurasaki can be accurately distinguished from other varieties (including the parent varieties) by combining the detection results of the first and second markers, which are DNA markers derived from each parent variety.

[0096] <5. Detection of DNA markers by multiplex PCR> Multiplex PCR was used to detect multiple DNA markers in a single PCR test.

[0097] (5-1. Multiplex PCR) As PCR reaction solutions, the reaction solutions shown in Table 9 were prepared, and PCR was performed under cycling conditions of 98°C for 10 seconds, -62°C for 1 second, and -68°C for 1 second for 30 cycles. In Table 9, "SSII Primer Mix" is a mixture of SSII_F primer and SSII_R primer in equal volumes (molar ratio). "Pattern4133 Primer Mix" is a mixture of PBS_LTR primer and Pattern4133 primer in equal volumes (molar ratio). "Pattern5354 Primer Mix" is a mixture of PBS_LTR primer and Pattern5354 primer in equal volumes (molar ratio). "Pattern2379 Primer Mix" is a mixture of PBS_LTR primer and Pattern2379 primer in equal volumes (molar ratio).

[0098] [Table 9]

[0099] (5-2. Detection by chromatographic PAS method) The amplification products of multiplex PCR were identified by chromatographic PAS according to the method described in Monden et al., Journal of Biotechnology 185, 57-62, 2014. After adding the PCR products to a dye-containing developing solution, DNA chromatographic paper was immersed in the solution and chromatographic development was performed. After standing at room temperature for about 15 minutes, the varieties were identified by the blue band patterns that appeared. The results are shown in Figure 5. In Figure 5, the lane numbers correspond to the numbers in Table 1.

[0100] In the results shown in Figure 5, the detection sites for the SSII, Pattern 4133, Pattern 5354, and Pattern 2379 markers are arranged from top to bottom on the chromatographic paper. Therefore, for Beni Haruka, blue bands will be detected in the 1st, 2nd, and 4th positions from the top of the chromatographic paper. Similarly, for Fuku Murasaki, blue bands will be detected in the 1st, 2nd, and 3rd positions from the top of the chromatographic paper. The thin lines above the detection position of SSII and between the detection positions of Pattern 5354 and Pattern 2379 are guidelines, positioned to make each detection line easily visible.

[0101] As shown in Figure 5, only Beniharuka showed bands in the first, second, and fourth positions from the top of the chromatographic paper, demonstrating that this method allows it to be distinguished from other varieties. Similarly, only Fukumurasaki showed bands in the first, second, and third positions from the top of the chromatographic paper, demonstrating that this method allows it to be distinguished from other varieties.

[0102] <6. Obtaining genome sequence information near the marker> Using whole-genome sequence information of the Beniharuka and Fukumurasaki varieties analyzed with an Illumina sequencer, read sequences containing approximately 20 bp of the insertion site sequences obtained from the analysis of the Cl8 insertion site and the Rtsp-1 insertion site were searched using seqkit software (https: / / bioinf.shenwei.me / seqkit / ). The obtained read sequences were assembled using Genetyx-ATGC software from Genetics, and from the resulting consensus sequences, those containing the retrotrolanceposon sequence were selected. Finally, by comparing these with the respective insertion site sequences obtained from the above analysis, it was confirmed that there were no sequence differences in the overlapping regions. [Industrial applicability]

[0103] This invention can be used for easy and accurate variety identification of the Beni Haruka and Fukumurasaki varieties, including in imported processed foods.

Claims

1. A method for identifying the sweet potato variety Beniharuka, At least one first DNA marker selected from a first group of markers consisting of DNA markers derived from one of the parent varieties of Beniharuka, and This includes detecting at least one second DNA marker selected from a second group of markers consisting of DNA markers derived from the other parent variety of Beniharuka, The DNA markers included in the first group of markers and the second group of markers are DNA markers derived from the insertion polymorphism of the retrotransposon Cl8. A discrimination method comprising the detection of a first DNA marker, which includes amplifying a nucleic acid fragment using the primer set (1) or (2) below, and the detection of a second DNA marker, which includes amplifying a nucleic acid fragment using the primer set (3) below: (1) A set of primer 1a consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 5 or 6, and primer 1b consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 7 or 8; (2) A set of primer 2a consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 9 and primer 2b consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 10; (3) A set of primer 3a consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 11 or 12 and primer 3b consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 13 or 14.

2. The primer 1a is a primer consisting of the base sequence shown in Sequence ID No. 15 or 16, The primer 1b is a primer consisting of the base sequence shown in SEQ ID NO: 17 or 18, The primer 2a is a primer consisting of the base sequence shown in Sequence ID No. 19, The primer 2b is a primer consisting of the base sequence shown in Sequence ID No. 20, The primer 3a is a primer consisting of the base sequence shown in SEQ ID NO: 21 or 22. The discrimination method according to claim 1, wherein the primer 3b is a primer consisting of the base sequence shown in SEQ ID NO: 17 or 18.

3. This includes extracting DNA from processed foods from the raw plant materials contained in those processed foods. A method for determining whether the raw material plant is the sweet potato variety Beni Haruka, according to claim 1 or 2.

4. The discrimination method according to any one of claims 1 to 3, wherein the detection of the first DNA marker and the detection of the second DNA marker are performed simultaneously.

5. A discrimination kit used to distinguish the sweet potato variety Beniharuka, comprising one or both of the primer sets described in (1) and (2) below, and the primer set described in (3) below: (1) A set of primer 1a consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 5 or 6, and primer 1b consisting of 20 or more consecutive bases from the nucleotide sequence shown in SEQ ID NO: 7 or 8; (2) A set of primer 2a consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 9 and primer 2b consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 10; (3) A set of primer 3a consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 11 or 12 and primer 3b consisting of 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 13 or 14.

6. A method for identifying the sweet potato variety Fukumurasaki, At least one first DNA marker selected from a first group of markers consisting of DNA markers derived from one parent variety of Fukumurasaki, and This includes detecting at least one second DNA marker selected from a second group of markers consisting of DNA markers derived from the other parent variety of Fukumurasaki, The DNA markers included in the first group of markers and the second group of markers are DNA markers derived from the insertion polymorphism of the retrotransposon Cl8. A discrimination method comprising the detection of a first DNA marker, comprising amplifying a nucleic acid fragment using the primer set of (4) or (5) below, and the detection of a second DNA marker, comprising amplifying a nucleic acid fragment using the primer set of (6) or (7) below: (4) A set of primer 4a consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 6, and primer 4b consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 8; (5) A set of primer 5a consisting of 20 or more consecutive bases in the base sequence shown in SEQ ID NO: 23 or 24 and primer 5b consisting of 20 or more consecutive bases in the base sequence shown in SEQ ID NO: 25 or 26; (6) A set of primer 6a consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs: 29 to 31 and primer 6b consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs: 32 to 34; (7) A set of primer 7a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 28 and primer 7b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No.

27.

7. The primer 4a is a primer consisting of the base sequence shown in Sequence ID No. 16, The primer 4b is a primer consisting of the base sequence shown in SEQ ID NO: 17 or 18. The primer 5a is a primer consisting of the base sequence shown in SEQ ID NO: 36 or 37, The primer 5b is a primer consisting of the base sequence shown in Sequence ID No. 20, The primer 6a is a primer consisting of the nucleotide sequence shown in SEQ ID NO: 38, 39, or 40. The primer 6b is a primer consisting of the base sequence shown in Sequence ID No. 17 or 18, The primer 7a is a primer consisting of the base sequence shown in Sequence ID No. 35, The discrimination method according to claim 6, wherein the primer 7b is a primer consisting of the base sequence shown in Sequence ID No.

20.

8. This includes extracting DNA from processed foods from the raw plant materials contained in those processed foods. The determination method according to claim 6 or 7, for determining whether the raw material plant is the sweet potato variety Fukumurasaki.

9. The discrimination method according to any one of claims 6 to 8, wherein the detection of the first DNA marker and the detection of the second DNA marker are performed simultaneously.

10. A discrimination kit used to distinguish the sweet potato variety Fukumurasaki, comprising one or both of the primer sets described in (4) and (5) below, and one or both of the primer sets described in (6) and (7) below: (4) A set of primer 4a consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 6, and primer 4b consisting of 20 or more consecutive bases in the base sequence shown in Sequence ID No. 8; (5) A set of primer 5a consisting of 20 or more consecutive bases in the base sequence shown in SEQ ID NO: 23 or 24 and primer 5b consisting of 20 or more consecutive bases in the base sequence shown in SEQ ID NO: 25 or 26; (6) A set of primer 6a consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs: 29 to 31 and primer 6b consisting of 20 or more consecutive bases from the base sequence shown in any of SEQ ID NOs: 32 to 34; (7) A set of primer 7a consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 28 and primer 7b consisting of 20 or more consecutive bases from the base sequence shown in Sequence ID No. 27.

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