JP2009000071A - Primer set for discrimination of potato variety and method for discrimination of potato variety by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for specifying a DNA marker capable of discriminating a new potato variety in which the information on DNA marker usable for the variety discrimination is unknown from known potato varieties, and easily and quickly discriminating many kinds of potato varieties including these potatoes. <P>SOLUTION: The invention provides a potato variety discrimination primer set composed of a specific oligonucleotide pair selected from the oligonucleotides of potato, and a potato variety discrimination method including the PCR amplification by using the primer set and using a DNA obtained from a specimen as a template and the detection of the amplification product. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a technique for quickly and easily discriminating 16 varieties from 20 cultivars of potato.

  There are varieties of potato that have a similar appearance to the harvest. In such a case, in order to investigate the varietal contamination of the product, it is necessary to cultivate tubers suspected of being mixed and investigate the growth, which requires time, area, and labor. In addition, in order to point out and prevent infringement of the breeder's right, a technique for objectively and simply identifying the variety using not only the harvest but also the growing plant as a material is desired.

  Since the establishment of the PCR technique, various techniques for evaluating DNA polymorphism have been developed (Patent Document 1), and methods for identifying varieties using such techniques in various crops have been developed. In potatoes, attempts have been made to identify varieties using techniques such as RAPD, SSR, ISSR (Non-Patent Documents 1 to 5). In these methods, identification is made using the presence of specific DNA or length polymorphism. Do. A detectable DNA fragment that exhibits polymorphism due to such a variety is referred to as a DNA marker.

  The above objectives can be achieved by using DNA markers. However, it is necessary to newly identify and search for DNA markers for varieties that have no DNA marker information, such as newly cultivated varieties and newly introduced varieties. . At present, many potato cultivars are known, but there is no information on DNA markers for starters, sassies, crosti, rosanna and arroah introduced from abroad.

Patent No. 2818486 DNA fingerprinting service (updated April 11, 2007) http://www2.kobe-u.ac.jp/~hosaka/japanese/fingerprinting/home.html Identification of Japanese potato varieties using SSR markers Sogaku miscellaneous 75 classified 1, 327 (2006) Mapping and characterization of new EST-derived microsatellites for potato (Solanum tuberosum L.) Theor Appl Genet 111: 456 (2005) Selection of highly informative and user-friendly microsatellites (SSRs) for genotyping of cultivated potato Theor Appl Genet 108: 881 (2004) Genetic diversity in European and Argentinian cultivated potatoes (Solanum tuberosum subsp.tuberosum) detected by inter-simple sequence repeats (ISSRs) Genome 45: 481 (2002)

  Therefore, an object of the present invention is to identify DNA markers that can be distinguished from existing varieties for which potato varieties for which information on DNA markers that can be used for cultivar identification has not been obtained have been obtained so far, and various types including those potatoes. The object is to provide a means for quickly and easily identifying potato varieties.

  As a result of searching for DNA marker candidates that can be used as identification markers by performing polymorphism analysis between varieties by RAPD (Random Amplified Polymorphic DNA) method for DNA extracted from potato tissue in order to solve the above problems, Seven DNA marker candidates that were considered to be able to achieve the objective were selected. Subsequently, base sequences were determined for these DNA marker candidates, and 14 types of primers (SEQ ID NOs: 1 to 14) specific to each DNA marker candidate were prepared from the obtained base sequences. 6 varieties (including Cynthia, Dorothy, etc.) including 20 starters and sassies that had no DNA marker information so far, depending on the presence or absence of 4 DNA markers detected by PCR using the primers (SEQ ID NOs: 1 to 8). Sherry, Make Queen, Starter, Sassie), and the presence of three DNA markers detected by PCR using the remaining 6 primers, Closti, Rosanna, Arrow A total of 16 varieties, including 10 varieties (Cynthia, Dorothy, Sherry, Koror, Chelsea, May Queen, Beihai Gane, Kitaakari, Benimaru, Dejima, agriculture, forestry, item, starter, Sassy, was confirmed to be able to be identified Crosti, Rosanna, the Aroa). The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
(1) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases among the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 2 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 2.
(2) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases among the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 4.
(3) An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 5 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence set forth in SEQ ID NO: 5 and the base sequence set forth in SEQ ID NO: 6 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 6.
(4) An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 7 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence set forth in SEQ ID NO: 7 and the base sequence set forth in SEQ ID NO: 8 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 8.
(5) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide or sequence comprising the base sequence set forth in SEQ ID NO: 10 A primer set for identifying potato varieties, comprising an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence of No. 10.
(6) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 11 and the base sequence shown in SEQ ID NO: 12 A primer set for discriminating potato varieties, comprising an oligonucleotide or an oligonucleotide containing 10 consecutive bases of the base sequence set forth in SEQ ID NO: 12.
(7) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 13 and the base sequence shown in SEQ ID NO: 14 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 14.
(8) Using a DNA extracted from the sample as a template, performing PCR amplification using the four primer sets described in (1) to (4), and detecting the obtained amplification product, Identification method.
(9) The method according to (8), wherein the potato varieties identified are Cynthia, Dorothy, Shelly, Makein, Starter, Sassy.
(10) The method further comprises a step of performing PCR amplification using the DNA extracted from the sample as a template and using the three primer sets described in (5) to (7) and detecting the obtained amplification product. ) Method.
(11) The potato varieties identified are Cynthia, Dorothy, Shelly, Koror, Chelsea, Makein, Beetle, Kita Akari, Benimaru, Digima, Norin Ichigo, Starter, Sassi, Crosti, Rosanna, Arrow. The method according to 10).
(12) A kit comprising the potato variety identification primer set according to (1) to (7).

  ADVANTAGE OF THE INVENTION According to this invention, the primer set for a potato variety identification which can amplify the DNA marker which can identify a specific potato variety is provided. By using this primer set, it becomes possible to quickly and easily identify potato varieties without requiring labor, area and time without conducting cultivation or characteristic investigation in the field.

1. Potato Variety Identification Primer Set The potato variety identification primer set of the present invention comprises the following seven primer sets (a) to (g) capable of amplifying seven DNA markers that serve as indicators for potato variety identification. is there.
(a) consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 2 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 2.
(b) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 4 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 4.
(c) consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 5 and the base sequence shown in SEQ ID NO: 6 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 6.
(d) consisting of an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 7 and the base sequence shown in SEQ ID NO: 8 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 8.
(e) an oligonucleotide comprising the base sequence set forth in SEQ ID NO: 9 or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 9, and an oligonucleotide or sequence comprising the base sequence set forth in SEQ ID NO: 10 A primer set for identifying potato varieties, comprising an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence of No. 10.
(f) an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 11 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases among the base sequence shown in SEQ ID NO: 11 and the base sequence shown in SEQ ID NO: 12 A primer set for discriminating potato varieties, comprising an oligonucleotide or an oligonucleotide containing 10 consecutive bases of the base sequence set forth in SEQ ID NO: 12.
(g) An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases among the base sequence shown in SEQ ID NO: 13 and the base sequence shown in SEQ ID NO: 14 A primer set for identifying potato varieties, comprising an oligonucleotide or an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 14.

  The oligonucleotides constituting the primer set are continuous oligonucleotides having 19 to 23 bases complementary to the base sequences located at both ends (forward, reverse) of each DNA marker. Primer design may be performed using commercially available software (for example, GENETYX; Software Development Co., Ltd.).

  In addition, among the oligonucleotides constituting the primer set, “an oligonucleotide consisting of a base sequence containing 10 consecutive bases among the base sequences described in SEQ ID NOs: 1 to 14” is described in “SEQ ID NOs: 1-14”. As long as it retains the function as a primer for identifying potato varieties, the type of base other than 10 consecutive bases and the site where the 10 consecutive consecutive mutant oligonucleotides exist There is no particular limitation on (3 ′ end side, 5 ′ end side). The total length of the mutant oligonucleotide is preferably about 15 to 30 bases.

  Among the 20 potato cultivars, potato varieties that can be identified by PCR using the above primer set include Cynthia, Dorothy, Shelly, Koror, Chelsea, Makein, Shoakaganet, Kita Akari, Benimaru, Digima, Norinichi No. 1, Includes 16 varieties of starter, sash, crosti, rosanna, and arrow. Of the 20 potato cultivars, four varieties of Nishiyutaka, Toyoshiro, Danshak and Sayaka other than the 16 varieties cannot be discriminated between Nishiyutaka and Toyoshiro, Danshak and Sayaka, but differ from the 16 varieties described above. Can be judged.

  It is not necessary to use all of the above primer sets depending on the type to be identified, the number of types to be identified, and the purpose of identification. For example, only 4 sets of primer sets (a) to (d) may be used. Specifically, PCR amplification is performed using the primer sets (a) to (d), and when it is confirmed that all four DNA markers shown in Table 3 below are detected, the test subject is Cynthia. I can say that.

  The oligonucleotide used as a primer can be synthesized by a method known in the art as an oligonucleotide synthesis method, for example, a phosphotriethyl method, a phosphodiester method, etc., using a commonly used automatic DNA synthesizer. It is.

  The above primer set can also be made into a kit. The kit of the present invention only needs to contain the above-mentioned seven primer sets (a) to (g), and depending on the variety to be identified, the number of types to be identified, and the purpose of identification, these primers An appropriate set may be appropriately selected from the sets and used. In addition, the kit includes DNA extraction reagents, PCR reagents such as PCR buffers and DNA polymerase (excluding primers), detection reagents such as gels for staining electrophoresis, instructions, etc., as necessary. May be included.

2. Method for identifying potato varieties The method for identifying potato varieties of the present invention includes performing PCR amplification using DNA extracted from a sample as a template and the above primer set, and detecting the obtained amplification product. Potato varieties identifiable by this method are as described above.

  The potato variety identification method of the present invention comprises the following two steps: a primary test and a secondary test. A secondary test should be performed only when necessary.

(Primary test)
Primer sets (a) to (d) are mixed to perform PCR amplification, and the reaction products are separated by electrophoresis and visualized to test the presence of each of the four DNA markers. By this test, 20 varieties of Cynthia, Dorothy, Shelly, Koror, Chelsea, Makein, Beetle, Kita Akari, Nishiyutaka, Toyoshiro, Danshak, Sayaka, Benimaru, Digima, Norinichi, Starter, Sassy, Crosti, Rosanna, Arrow It is possible to identify six varieties from Cynthia, Dorothy, Shelly, Makeney, Starter, and Sassi. In this primary test, (i) Crosti and Koror, (ii) Nishitaka and Toyoshiro, (iii) Rosanna and Scarlet, (iv) Danshak, Sayaka, Benimaru, Kitaakari, (v) Chelsea and Arrow, (vi) Digima and agriculture and forestry Six varieties of No. 1 show the same result for the presence or absence of four DNA markers.

(Secondary test)
If it is necessary to identify varieties that cannot be identified by the above-mentioned primary test, it is performed as follows. To distinguish (i) Closti and Koror, and (v) Chelsea and Arrow, PCR amplification is performed using the primer set (e), and the reaction products are separated and visualized by electrophoresis. Just test for existence. (iii) When discriminating between Rosanna and the fungus, PCR amplification is performed using the primer set (f), and the presence of this DNA marker may be assayed by separating and visualizing the reaction product by electrophoresis. (iv) When discriminating between Digima and Norin Ichigo, the presence of this DNA marker can be tested by performing PCR amplification using the primer set (g) and separating and visualizing the reaction products by electrophoresis. . (iv) When discriminating Danshak, Sayaka, Benimaru, Kitaakari, perform PCR amplification using the primer set (f), separate and visualize the reaction products by electrophoresis, and use the primers from the primer set (g) The presence of these two DNA markers may be assayed by performing PCR amplification and separating and visualizing the reaction products by electrophoresis. However, Danshak and Sayaka cannot be distinguished from each other because both DNA markers are detected.

  According to the RAPD analysis, for example, when trying to identify the arrow or corrole from the above 20 varieties, it may be possible to identify with the minimum PCR reaction, but it is still necessary to perform the PCR reaction twice (see Example 1 below). . On the other hand, according to the method of the present invention, it is possible to identify 6 varieties of Cynthia, Dorothy, Shelly, Make Queen, Starter and Sassie from the above 20 varieties by one PCR reaction (Example 2 described later). See) 3 times of PCR reaction, Cynthia, Dorothy, Sherry, Koror, Chelsea, May Queen, Scarlet, Kita Akari, Benimaru, Digima, Norinichi, Starter, Sassi, Crosti, Rosanna, Arrow 16 types can be clearly identified (see Example 3 below).

  Specific procedures of the method of the present invention will be described below. First, DNA is extracted from a potato tissue to be a test sample. As the potato tissue, for example, leaves (young leaves, adult leaves), tubers, roots and the like can be used.

  For extraction of DNA from potato, any method known to those skilled in the art as a nucleic acid extraction method may be used, and examples thereof include a phenol extraction method, a cetyltrimethylammonium bromide (CTAB) method, and an alkaline SDS method. In addition, these methods may be appropriately modified, and various DNA extraction kits sold by reagent manufacturers may be used. Depending on the type of sample, filtration with a membrane filter or homogenization is performed.

  After DNA extraction, purification treatment such as chloroform / isoamyl alcohol treatment, isopropanol precipitation, deproteinization with phenol / chloroform, and ethanol precipitation may be performed.

  Next, using the DNA sample obtained by the above operation as a template, PCR amplification is performed using the primer set, and the presence or absence of the obtained amplification product (DNA marker) is detected. PCR amplification may be performed in accordance with a conventional method, and there is no particular limitation except that the above primer set is used. The composition of the PCR reaction solution and the PCR conditions (temperature cycle, number of cycles, etc.) are determined in PCR using each primer set. Those skilled in the art can appropriately select and set conditions for obtaining a PCR amplification product with high sensitivity by a preliminary experiment or the like. For example, mix 0.1 to 100 ng of template DNA, 10 × PCR buffer, 0.25 to 1 μM of primer, 0.25 to 2.5 U of DNA polymerase (Taq polymerase, TthDNA polymerase, etc.), 250 μM of dNTP, 94-96 ° C. 5 minutes × 1 cycle, (94-96 ° C. 30 seconds, 52-58 ° C. 30 seconds, 70-74 ° C. 1 minute) × 30 cycles for those diluted to a volume of 10-100 μl The reaction is performed at 70 to 74 ° C. for 5 minutes × 1 cycle. This is merely an example, and the composition, reaction temperature, and time of the PCR reaction solution can be appropriately set according to the length of the oligonucleotide sequence that serves as a primer, the base composition, and the like. A series of these PCR operations can be performed using a commercially available PCR kit or PCR apparatus according to the operating instructions.

  Each DNA marker is detected as a band at this position by separating the PCR amplification product of each primer set by electrophoresis. It becomes possible to identify potato varieties from the presence or absence of these markers. As the electrophoresis method, any method using agarose gel, denaturing and non-denaturing acrylamide gel may be used. In general, band detection is performed by ethidium bromide staining. However, when a primer to which a fluorescent dye (FAM, NED, HEX, etc.) is added is used, fluorescent color development may be used as an index.

  In carrying out the method of the present invention, a correspondence table in which the potato varieties are associated with the DNA markers possessed by the varieties is prepared, and the DNA extracted from the test potato varieties is amplified using the primer set described above. What is necessary is just to determine a kind by collating with the obtained detection result.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
(Example 1) Potato variety identification by RAPD method
(1) Test potato variety Twenty varieties of Crosti, Rosanna and Arroa were used.

(2) Preparation of DNA sample
Preparation of DNA samples is described in the literature (Walbot, V. and Warren, C. (1988) Mol., Described in the literature, Cell Engineering, Separate Plant Cell Engineering Series 2 “Plant PCR Experiment Protocol”, Shujunsha (1995) pages 47-48. Gen. Genet. 211, 27) A method partially modified with reference to the method described in 27) was used as follows.

  The above 20 varieties of potato leaves or tubers were used as samples for DNA extraction. The tubers of the varieties used in this example are available from Japan Potato Co., Ltd. (Larle Building 4F, 2-7-7 Shinkawa, Chuo-ku, Tokyo 104-0033, TEL: 03-5541-5335) . In the case of leaves, about 1 cm in diameter was collected, and in the case of tubers, the skin was peeled and a 3 to 5 mm square (about 50 mg) of a healthy part inside was cut off with a scalpel and used. Place the sample in a 1.5 ml tube, grind with a micro pestle, and add 500 μl of DNA extraction buffer (15% sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 500 mM NaCl) when the tissue is disrupted. Grind until the tissue is gone. Milling was performed without using liquid nitrogen. Centrifuge the sample after grinding for 5 min at 4 ° C and 4000 rpm, remove the supernatant, add 300 µl of 2T1E (20 mM Tris-HCl (pH 8.0), 10 mM EDTA), 40 µl of 10% SDS, and vortex well. Mixed and treated at 70 ° C. for 20 minutes. Thereafter, 150 μl of 7.5M ammonium acetate was added, and the mixture was further vortexed and placed on ice for 30 minutes. Thereafter, the mixture was centrifuged at 4 ° C. and 15000 rpm for 10 minutes, and the supernatant was transferred to a new 1.5 ml tube, and 700 μl of isopropanol was added and mixed by inversion. Further, the mixture was centrifuged at 20 ° C. and 8000 rpm for 5 minutes to remove the supernatant, and 500 μl of 70% ethanol was added and mixed by inversion. Centrifugation was carried out at 20 ° C. and 15000 rpm for 5 minutes, and the supernatant was removed and dried until the ethanol odor disappeared. 200 μl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) was added to dissolve the DNA, and the DNA was completely dissolved by treatment at 70 ° C. for 15 minutes.

(3) RAPD analysis RAPD analysis was performed using a commercially available reagent kit (Ready-To-Go RAPD Analysis Beads, GE Healthcare Biosciences) according to the attached protocol. For the primers 1, 3, 4, 5, 6, and 7 shown in Table 1 below, 1 μl of the DNA sample prepared in (1), 1.25 μl of the primer (25 pmol), milliliter in the tube (with Analysis Beads) attached to the kit Q. 22.75 μl of water was added and mixed, and then transferred to a 0.2 ml tube for PCR amplification. For primer 2 only, 2.5 µl primer (25 pmol), 2.5 µl buffer attached to TaKaRa Ex Taq, 0.25 µl TaKaRa Ex Taq (Takara Biosciences) After adding 42.75 μl of Q water and mixing, half the volume (24 μl) was transferred to a 0.2 ml tube and 1 μl of the DNA sample prepared in (1) was added to perform PCR amplification. PCR amplification conditions were set at 95 ° C. for 5 minutes × 1 cycle (95 ° C. for 1 minute, 36 ° C. for 1 minute, 72 ° C. for 2 minutes) × 45 cycles. After the reaction, 10 μl of the RAPD reaction product was run on a 2% agarose gel for 30 minutes (100 V). Agarose is prepared by dissolving in 1 × TAE buffer (Tris-base 4.84 g / l, acetic acid 1.14 ml / l, 0.5M EDTA 2 ml / l), and 1 × TAE buffer is used as the electrophoresis buffer. It was. The agarose gel after electrophoresis was stained with an ethidium bromide (0.5 μg / ml) solution for 20 minutes, and the polymorphism was confirmed under UV.

  FIG. 1 shows the results of RAPD analysis, and Table 2 summarizes the results.

  As shown in Table 2, potato varieties Cynthia, Dorothy, Shelly, Koror, Chelsea, Mayine, Hokoyokogane, Kita Akari, Nishiyutaka, Toyoshiro, Danshak, Sayaka, Benimaru, Digima, No. 1 From a total of 20 varieties of starter, sassi, rosti, rosanna, and arrow, it was possible to distinguish a total of 16 varieties excluding 4 species of Nishiyutaka, Toyoshiro, Kitakari and Sayaka, which share the same markers. However, there were cases where the results were not clear in the RAPD analysis, as in the (+) results. It was also expected that the analysis of the results would be misleading because of amplification products other than DNA markers.

(Example 2) Primary verification for potato variety identification
The DNA sample extracted in Example 1 was used. Primers were used by mixing the following 8 oligonucleotides.

Sequence number 1: 5'-GACCGTTAAACTCCGCGCT-3 '
Sequence number 2: 5'-CTGAAATGAGGCATAGTTGATT-3 '
Sequence number 3: 5'-CACACTTTTTCTTTAGACTTCACT-3 '
Sequence number 4: 5'-ATCAAGGATCACATGGACAGC-3 '
Sequence number 5: 5'-ATATTTGGAGAGATGACAACC-3 '
Sequence number 6: 5'-CATACGATCAGTTAAATCAGTAC-3 '
Sequence number 7: 5'-TAAGAGCCCTGAGTGTTTTAC-3 '
Sequence number 8: 5'-AACATGACAAGGGTGGATAAC-3 '

  DNA sample 1 μl, buffer 2 μl attached to TaKaRa Ex Taq, dNTPs (2.5 mM) 2 μl, primers (20 μM) 8 species 3.74 μl (SEQ ID NO: 1, 0.75 μl each, SEQ ID NO: 3, 4 0.5 μl each, sequence Nos. 5 and 6, each 0.37 μl, SEQ ID Nos. 7 and 8 each 0.25 μl), TaKaRa Ex Taq (Takara Bioscience Co., Ltd.) 0.1 μl, and Milli-Q water 11.16 μl were mixed for PCR amplification. The conditions for PCR amplification were 95 ° C. for 5 minutes × 1 cycle, (95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute) × 30 cycles, 72 ° C. for 5 minutes × 1 cycle. After the reaction, 10 μl of the PCR product was run on a 2% agarose gel for 30 minutes (100 V). In addition, agarose gel electrophoresis and visualization of amplification products were performed in the same manner as in Example 1.

  As a result, polymorphism as shown in FIG. 2 was observed. The presence or absence of each DNA marker is summarized in Table 3.

  As can be seen from the above results, 20 varieties, namely Cynthia, Dorothy, Shelly, Koror, Chelsea, Mayine, Beetle, Kita Akari, Nishiyaka, Toyoshiro, Danshak, Sayaka, Benimaru, Digima, Norinichi, Starter, Sassy, Cross It was possible to identify six varieties, namely Cynthia, Dorothy, Sherry, Makeyne, Starter and Sassie, from Tee, Rosanna and Arrow. However, in this primary test, (i) Crosti and Koror, (ii) Nishitaka and Toyoshiro, (iii) Rosanna and Scarlet, (iv) Danshak, Sayaka, Benimaru, Kitaakari, (v) Chelsea and Arrow, (vi) Digima The six varieties of No. 1 and No. 1 also showed the same results for the presence or absence of four DNA markers.

(Example 3) Secondary test for potato variety identification
The DNA sample extracted in Example 1 was used. The primer is a set of two oligonucleotides selected from the following six oligonucleotides (set of oligonucleotides SEQ ID NO: 9 and 10; set of oligonucleotides SEQ ID NO: 11 and 12; SEQ ID NO: A set of 13 and 14 oligonucleotides) was used.

Sequence number 9: 5'-TCTATATGGAGCAACCACCTA-3 '
SEQ ID NO: 10: 5′-TGACTTCACAAATGTTGTAGAGT-3 ′
Sequence number 11: 5'-GTCCACACGGGATGAGGCT-3 '
Sequence number 12: 5'-GTCCACACGGTCCAAGATATA-3 '
SEQ ID NO: 13: 5'-TGGGGGACTCCAAATCACTAT-3 '
SEQ ID NO: 14: 5′-TGGGGGACTCAGGGCCGA-3 ′

  DNA sample 1μl, buffer 2μl attached to TaKaRa Ex Taq, dNTPs (2.5mM) 2μl, primer (20μM) 0.5μl for each DNA marker, TaKaRa Ex Taq (Takara Biosciences) 0.1μl, milli PCR amplification was performed by mixing 13.9 μl of Q water. PCR amplification conditions, agarose gel electrophoresis, and visualization of amplification products were performed as in Example 1.

  As a result, polymorphism as shown in FIG. 3 was observed. (I) Crosti and Koror, (ii) Nishitaka and Toyoshiro, (iii) Rosanna and Scarlet, (iv) Danshak, Sayaka, Benimaru, Kitaakari, (v) Chelsea and Arrow, (vi) Among Digima and Norin Ichigo, (i) Closti and Koror and (v) Chelsea and Arrow were able to be distinguished by secondary test using oligonucleotides of SEQ ID NOs: 9 and 10 (FIG. 3A). (iii) Rosanna and Scarlet radish were distinguishable by a secondary test using the oligonucleotides of SEQ ID NOs: 11 and 12 (FIG. 3B). (vi) Digima and Norinichi No. 1 could be distinguished by a secondary test using the oligonucleotides of SEQ ID NOs: 13 and 14 (FIG. 3C). (iv) Benimal and Kitaakali could be distinguished from each other by a secondary test using the oligonucleotides of SEQ ID NOs: 11 and 12 and SEQ ID NOs: 13 and 14 among Danshak, Sayaka, Benimaru, and Kitaakari (FIG. 3D, E).

The result of RAPD analysis is shown. A is an agarose gel electrophoresis photograph of the PCR amplification product using primer 1, B is an agarose gel electrophoresis photograph of the PCR amplification product using primer 2, and C is an agarose gel electrophoresis photograph of the PCR amplification product using primer 3. , D is an agarose gel electrophoresis photograph of a PCR amplification product using primer 4 (in A to D of FIG. 1-1, M: molecular weight marker (φX174 HinCII digest), 1: Cynthia, 2: Shelley, 3 : Chelsea, 4: Koror, 5: Rosanna, 6: Arrow, 7: Starter, 8: Crosti, 9: Sassie, 10: Nishitaka, 11: Digima, 12: Scarlet, 13: Kitaakari, 14: Dunshak, 15: Mayeen, 16: Dorothy, 17: Sayaka, 18: Benimaru, 19: Toyoshiro, 20: No. 1) The result of RAPD analysis is shown. E is an agarose gel electrophoresis photograph of a PCR amplification product using primer 5, F is an agarose gel electrophoresis photograph of a PCR amplification product using primer 6, and G is an agarose gel electrophoresis photograph of a PCR amplification product using primer 7. (E in FIG. 1-2, M: molecular weight marker (φX174 HinCII digest), 1: Cynthia, 2: Shelley, 3: Chelsea, 4: Koror, 5: Rosanna, 6: Arrow, 7: Starter, 8: Crosti, 9: Sassy, 10: Nishitaka, 11: Digima, 12: Beetle, 13: Kita Akari, 14: Danshak, 15: Makein, 16: Dorothy, 17: Sayaka, 18: Benimaru, 19: Toyoshiro, 20: No. 1 The results of the primary test are shown (M: molecular weight marker (φX174 HinCII digest), 1: Cynthia, 2: Starter, 3: Crosty, 4: Koror, 5: Nishiyutaka, 6: Toyoshiro, 7: Rosanna, 8: Beetle , 9: Danshak, 10: Sayaka, 11: Benimaru, 12: Kita Akari, 13: Dorothy, 14: Shelley, 15: Chelsea, 16: Arroa, 17: Digima, 18: Ichigo Norin, 19: Sassy, 20: Mei (Queen). The result of the secondary test is shown. A: Agarose gel electrophoresis photograph of PCR amplification product using oligonucleotides of SEQ ID NOs: 9 and 10 (M: molecular weight marker (φX174 HinCII digest), 1: Crosty, 2: Koror, 3: Chelsea, 4: Arrow B) Agarose gel electrophoresis photograph of PCR amplification product using oligonucleotides of SEQ ID NOs: 11 and 12 (M: molecular weight marker (φX174 HinCII digested product), 5: Rosanna, 6: scallop) C: SEQ ID NOs: 13, 14 Agarose gel electrophoresis photograph of PCR amplification product using oligonucleotides of (M: molecular weight marker (φX174 HinCII digested product), 7: Digima, 8: Noichi Nobayashi) D: oligonucleotides of SEQ ID NOs: 11 and 12 were used Agarose gel electrophoresis photograph of PCR amplification product (M: molecular weight marker (φX174 HinCII digest), 9: Danshak, 10: Sayaka, 11: Benimaru, 12: Kitakari) E: Sequence Agarose gel electrophoresis photograph of PCR amplification products using the issue 13, 14 oligonucleotides (M: molecular weight marker (? X174 HincII digest), 9: Baron, 10: Sayaka, 11: Benimaru, 12: Kitaakari)

Claims (12)

  An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 1 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases among the base sequence set forth in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 2; A primer set for identifying potato varieties comprising an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 2.   An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 3 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence set forth in SEQ ID NO: 3, and an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 4 or A primer set for identifying potato varieties comprising an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 4.   An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 5 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6 A primer set for identifying potato varieties comprising an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 6.   An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence shown in SEQ ID NO: 7 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 8; A primer set for identifying potato varieties comprising an oligonucleotide having a base sequence comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 8.   An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 9 or an oligonucleotide containing 10 consecutive bases among the base sequence shown in SEQ ID NO: 9 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 10 A primer set for identifying potato varieties comprising an oligonucleotide having a base sequence containing 10 consecutive bases among the base sequences described.   An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 11 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases among the base sequence set forth in SEQ ID NO: 11, and an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 12 or A primer set for identifying varieties of potato, comprising an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 12.   An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 13 or an oligonucleotide consisting of a base sequence containing 10 consecutive bases of the base sequence set forth in SEQ ID NO: 13, and an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 14 or A primer set for identifying potato varieties, comprising an oligonucleotide comprising 10 consecutive bases of the base sequence set forth in SEQ ID NO: 14.   A method for identifying potato varieties, comprising a step of performing PCR amplification using DNA extracted from a sample as a template and using the four primer sets according to claim 1 to 4 and detecting the obtained amplification product.   9. The method of claim 8, wherein the potato varieties identified are Cynthia, Dorothy, Shelly, Makein, Starter, Sassy.   The method according to claim 8, further comprising a step of performing PCR amplification using the DNA extracted from the sample as a template and using the three primer sets according to claims 5 to 7, and detecting the obtained amplification product. .   Claim 10 wherein the potato varieties identified are Cynthia, Dorothy, Shelly, Koror, Chelsea, Mayeen, Beetle, Kita Akari, Benimaru, Digima, Norin No. 1, Starter, Sassy, Crosti, Rosanna, Arrow The method described.   A kit comprising the potato variety identification primer set according to claim 1.

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