Method for identifying tea varieties, polynucleotide, microsatellite marker and primer set

Abstract

To provide methods for identifying tea cultivars that facilitates the determination of genotypes in identification of tea cultivars.SOLUTION: Provided is a method for identifying tea cultivars, comprising identifying tea cultivars using at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any base sequence represented by a sequence consisting of a specific sequence. The tea cultivars can include Ryofu, Harumidori, Sofu, Sun Rouge, Shuntaro, Saeakari, Nanmei, Seimei, Sayamakaori, Yabukita, Asatsuyu, Okumidori, Kanayamidori, Saemidori, Yutakamidori, Kiyoka, Kanaemaru, Danshin 37, Okuharuka, Kirari31, Sainomidori, Sakimidori, Nagomiyutaka, Haruto34, Harunonagori, Harumoegi, Miyamakaori, Musashikaori, Yumekaori, Yumewakaba, Benifuki, and Sayamaakari and the like.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for identifying tea varieties, polynucleotides, microsatellite markers and primer sets.

Background Art

[0002] Tea (Camellia sinensis (L.) O. Kuntze) is an evergreen tree belonging to the genus Camellia of the family Theaceae. It is native to southern China and is cultivated in about 50 countries around the world. Tea is regarded as one of the three major non-alcoholic beverages, and it is said that 2 billion cups are consumed worldwide every day. In addition, due to the attention paid to its health functionality, etc., its popularity is increasing around the world, and the global production volume has increased from about 1.9 million tons in 1980 to about 6.3 million tons in 2018 (FAO STAT).

[0003] In recent years, the total production volume of rough tea in the main tea-producing prefectures in Japan has been around 75,000 to 80,000 tons. Although the consumption volume of green tea in the domestic market is stagnant, the export of green tea has increased with the improvement of the popularity of Japanese food overseas, and the export value has increased more than fourfold in 10 years from 3.4 billion yen in 2010 to 14.6 billion yen in 2019 (Ministry of Finance trade statistics).

[0004] The total tea cultivation area in the country in 2019 was 40,600 ha (Ministry of Agriculture, Forestry and Fisheries production statistics). Looking at the share by variety, "Yabukita" accounts for 71.5%, "Yutakamidori" accounts for 6.3%, "Saemidori" accounts for 4.0%, and "Okumidori" accounts for 3.3% (Ministry of Agriculture, Forestry and Fisheries). Looking at the country as a whole, the cultivation area of "Yabukita" has occupied most of it for many years, but in some production areas, the picking period has been dispersed by introducing early and late varieties.

[0005] Thus, approximately 70% of the tea cultivation area in Japan is occupied by "Yabukita," a variety selected during the Meiji period. However, this over-reliance on a single variety is problematic because it leads to a concentration of optimal harvesting periods and frequent outbreaks of pests and diseases. Furthermore, over-reliance on a single variety is undesirable in the face of changing market needs due to the diversification of consumer preferences. Therefore, the development of new tea varieties is actively underway. For example, "Seimei," a variety registered by the National Agriculture and Food Research Organization on March 30, 2020, is suitable not only for sencha (green tea) but also for matcha (powdered green tea) and powdered tea. It is expected to contribute to improving the productivity and added value of matcha and powdered tea, for which demand has been increasing both domestically and internationally in recent years.

[0006] The mislabeling of tea variety names, including these superior varieties, and the outflow of registered varieties overseas not only have the potential to infringe on intellectual property rights (breeder's rights), but also have a significant impact on tea producers and distributors who handle varieties fairly, and pose a major problem from the perspective of ensuring food safety and security for consumers.

[0007] While the Plant Variety Control Law distinguishes varieties based on differences in traits, accurately identifying varieties from the appearance of the plant or parts thereof is not easy. In this respect, variety identification using DNA markers has an advantage because it can provide objective distinctions based on genotype data.

[0008] Non-Patent Document 1 publishes a manual for identifying 47 varieties of tea using seven types of CAPS markers. Also in Patent Document 1, a technique for identifying 16 varieties of tea using nine types of SSR (Simple Sequence Repeat) markers is disclosed. Non-Patent Document 2 discloses a technique for identifying 41 varieties of tea using six types of SSR markers. Furthermore, several reports indicate that polymorphisms between tea varieties can be shown using SSR markers (Non-Patent Documents 3-6). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2010-11764 [Non-patent literature]

[0010] [Non-Patent Document 1] "Green Tea Variety Identification Manual," National Agriculture and Biotechnology Research Organization, Kanaya Tea Research Center, Vegetable and Tea Research Institute, February 1, 2005, URL <http: / / www.naro.affrc.go.jp / publicity_report / publication / pamphlet / tech-pamph / 004264.html> [Non-Patent Document 2] Kato et al. (2008) "Tea variety identification using simple repeating sequence (SSR) markers," Journal of the Japanese Society for Food Science and Technology, Vol. 55, No. 2, pp. 49-55. [Non-Patent Document 3] Ma et al (2010) Am J Bot, 97, e153-e156. [Non-Patent Document 4] Yao et al (2012) Tree Genet Genomes, 8, 205-220 [Non-Patent Document 5] Tan et al (2013) PLOS One, 8, e81611 [Non-Patent Document 6] Ma et al (2014) PLOS One, 9, e93131 [Overview of the project] [Problems that the invention aims to solve]

[0011] However, the CAPS markers described in Non-Patent Document 1 sometimes yield inconsistent results depending on the type of reagent used and the operator. Furthermore, SSR markers with small repeating motifs of 2 to 3 bases often produce stutter bands due to replication errors during PCR, hindering the interpretation of analytical results (Patent Document 1, Non-Patent Document 2). While Non-Patent Documents 3-6 mention SSR markers with repeating units of 4 to 5 bases, it is unclear whether they are widely applicable to the identification of varieties and strains developed in Japan.

[0012] This invention was made to solve the above-mentioned problems, and aims to provide a method for identifying tea varieties that facilitates the determination of genotypes. Furthermore, the present invention aims to provide a marker that can be used in a method for identifying tea varieties, and a primer set capable of amplifying said marker. [Means for solving the problem]

[0013] As a result of diligent research to solve the above problems, the inventors have discovered a new excellent marker that can be used to identify tea varieties and that facilitates genotype determination, thus completing the present invention. That is, the present invention has the following aspects.

[0014] [1] A method for identifying a tea variety, comprising identifying a tea variety using at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any of the base sequences represented by SEQ ID NOs: 1 to 2. [2] The method for identifying a tea variety according to [1], further comprising identifying the tea variety using at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any of the base sequences represented by sequence numbers 3 to 15. [3] The method for identifying the tea cultivar according to [1] or [2] above, which comprises identifying the tea cultivar using at least one combination of the following combinations 1 to 4 of microsatellite markers. (Combination 1) A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 1, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 2, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 13, and A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 15 (Combination 2) A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 1, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 3, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 4, and A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 15 (Combination 3) A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 1, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 4, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 8, and A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 13 (Combination 4) A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 1, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO:4, A microsatellite marker containing a region corresponding to the nucleotide sequence represented by SEQ ID NO: 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 [4] A method for identifying a tea variety according to [1] or [2], comprising identifying a tea variety using microsatellite markers of at least one combination of combinations 5 to 8 described below. (Combination 5) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 2, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 3, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 6) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 2, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 8, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 7) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 2, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 12, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 8) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 3, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 6, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 [5] A method for identifying a tea variety according to [1] or [2], comprising identifying a tea variety using all microsatellite markers selected from the group consisting of microsatellite markers containing a region corresponding to any of the base sequences represented by sequence numbers 1 to 15. [6] The tea varieties mentioned above include Ryofu, Tea Intermediate Mother No. 3, Harumidori, Sofu, Tea Intermediate Mother No. 4, Tea Intermediate Mother No. 5, Tea Intermediate Mother No. 6, Sunrouge, Shuntaro, Saeakari, Nanmei, Seimei, Sayamakaori, Yabukita, Asatsuyu, Okumidori, Kanayamidori, Saemidori, Yutakamidori, Kiyoka (Nochaken No. 4), MK5601 (Makurakei 56-01), Kanaemaru (Kanaya No. 33), Nochaken No. 1, Nochaken No. 2, A method for identifying a tea variety described in any one of the above [1] to [5], wherein the tea variety is selected from the group consisting of Nochaken No. 09, Nochaken No. 10, Kokuken No. 01, Danshin 37 (Miyazaki No. 37), Miyazaki No. 39, Miyazaki No. 40, Okuharuka, Kirari 31, Sai no Midori, Saki Midori, Nagomi Yutaka, Haruto 34, Haruno Nagori, Harumoegi, Miyama Kaori, Musashi Kaori, Yume Kaori, Yume Wakaba, Benifuki, and Sayama Akari, wherein at least one is selected from the above group. [7] A polynucleotide consisting of the base sequence represented by Sequence ID No. 1 or Sequence ID No. 2. [8] A microsatellite marker for identifying tea varieties, consisting of a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. [9] A primer set for tea variety identification that can amplify at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any of the base sequences represented by Sequence ID No. 1 to 2.

[10] The primer set described in [9] above, which is either primer set 1 or primer set 2 below. Primer Set 1: (1F-1) A polynucleotide containing the base sequence described in Sequence ID No. 16, or (1F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 16, and (1R-1) A polynucleotide containing the base sequence described in Sequence ID No. 17, or (1R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 17. Primer Set 2: (2F-1) A polynucleotide containing the base sequence described in Sequence ID No. 18, or (2F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 18, and (2R-1) A polynucleotide containing the base sequence described in Sequence ID No. 19, or (2R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 19. [Effects of the Invention]

[0015] According to the present invention, a method for identifying tea varieties that facilitates the determination of genotypes can be provided. Furthermore, according to the present invention, it is possible to provide a marker that can be used in a method for identifying tea varieties, and a primer set that can amplify the marker. [Brief explanation of the drawing]

[0016] [Figure 1] These are waveform diagrams of the PCR products of the standard set prepared in the examples and referenced when determining the genotype of each marker. [Figure 2] These are waveform diagrams of the PCR products of the standard set prepared in the examples and referenced when determining the genotype of each marker. [Figure 3] These are waveform diagrams of the PCR products of the standard set prepared in the examples and referenced when determining the genotype of each marker. [Modes for carrying out the invention]

[0017] The following describes embodiments of the tea variety identification method, polynucleotides, microsatellite markers, and primer sets of the present invention.

[0018] ≪Methods for Identifying Tea Varieties≫ The method for identifying a tea variety according to the embodiment (hereinafter sometimes simply referred to as the "method for identifying the embodiment" or "the method for identifying the variety") includes identifying a tea variety using at least one microsatellite marker selected from the group consisting of microsatellite markers that include a region corresponding to any of the base sequences represented by SEQ ID NOs: 1 to 2.

[0019] Microsatellites are repetitive sequences found in the genome that consist of short sequences of a few bases (e.g., 1 to 6 bases) as repeating units (motifs). They are also known as SSRs (Simple Sequence Repeats) or STRs (short tandem repeats).

[0020] The inventors found that each microsatellite marker, which has each repeat sequence in the region corresponding to any of the base sequences represented by Sequence IDs 1-2, exhibits polymorphism among major tea varieties and is extremely useful as a polymorphic marker for identifying tea varieties.

[0021] Microsatellite markers are known to exhibit polymorphism due to differences in the number of repeats of the aforementioned repeat sequence. Since the length of the region changes depending on the number of repeats of the aforementioned repeat sequence, polymorphism can be easily analyzed by obtaining information on the length of the region, for example, by obtaining the amplification product of PCR.

[0022] In this specification, the term "tea variety" includes Camellia taliensis and its descendants.

[0023] The following 44 varieties can be given as examples of major tea varieties. Ryofu, Tea Intermediate Mother No. 3, Harumidori, Sofu, Tea Intermediate Mother No. 4, Tea Intermediate Mother No. 5, Tea Intermediate Mother No. 6, Sunrouge, Shuntaro, Saeakari, Nanmei, Seimei, Sayamakaori, Yabukita, Asatsuyu, Okumidori, Kanayamidori, Saemidori, Yutakamidori, Kiyoka (Nochaken No. 4), MK5601 (Makurakei 56-01), Kanaemaru (Kanaya No. 33), Nochaken No. 1, Examples include at least one tea variety selected from the group consisting of Nochaken No. 02, Nochaken No. 09, Nochaken No. 10, Kokuken No. 01, Danshin 37 (Miyazaki No. 37), Miyazaki No. 39, Miyazaki No. 40, Okuharuka, Kirari 31, Sai no Midori, Saki Midori, Nagomi Yutaka, Haruto 34, Haruno Nagori, Harumoegi, Miyama Kaori, Musashi Kaori, Yume Kaori, Yume Wakaba, Benifuki, and Sayama Akari. Hereafter, these 44 tea varieties may simply be referred to as "44 varieties".

[0024] Tea Intermediate Parent No. 6 is an interspecific hybrid of Camellia taliensis and Camellia sinensis, both closely related to tea. Sunrouge and MK5601 are progeny of Tea Intermediate Parent No. 6.

[0025] These 44 varieties include the top 7 varieties in terms of cultivated area in Japan, and are expected to cover almost all (over 99%) of the varieties currently considered for identification in Japan.

[0026] Polymerases are prone to errors during replication reactions, particularly with repetitive sequences. The microsatellite markers according to this embodiment have a long number of repeating bases (4-5 bases), which makes them less susceptible to replication errors and thus reduces the possibility of misidentification.

[0027] The inventors analyzed a total of 213 SSR markers, including 190 known markers with 4-base or 5-base repeat motifs that are considered usable for identifying tea varieties, and 23 newly designed markers, and obtained inter-varietal polymorphism data for the 44 varieties mentioned above. Based on the obtained data, the inventors narrowed down the selection to markers that facilitate genotype determination based on all of the following criteria 1) to 6). In other words, markers that meet each of these criteria enable highly accurate variety identification.

[0028] 1) Varieties that exhibit polymorphism among 44 varieties and have three or more alleles (genotypes). Markers that meet these criteria can be widely used for variety identification across many varieties.

[0029] 2) The amplification product peaks observed when the genome of each of the 44 varieties is amplified are two or fewer. Since Cha is typically diploid, the amplification product peaks observed when the target region is amplified usually consist of two or fewer peaks. Markers that meet this criterion are less likely to produce nonspecific amplification products and can accurately determine the genotype.

[0030] 3) Stutter is at a negligible level, or the signal intensity is less than 1 / 10 of the main peak. Markers that meet these criteria are less likely to produce stutter bands and do not show significant amplification differences between the peaks to be determined, making accurate genotype determination easier.

[0031] 4) The difference in peak height of the amplification product between alleles is small, with a signal intensity ratio of approximately 1:2 or less. Markers that meet this standard contribute to improving the accuracy of variety identification because they have similar amplification efficiencies for each variety.

[0032] 5) There are no alleles in the amplification product whose size differs by one or two bases. Markers that meet these criteria allow for easy peak identification, thus facilitating accurate genotyping.

[0033] 6) The amplification size is 300 bp or less, and no broad peak is observed. Markers that meet these criteria allow for easy peak identification, thus facilitating accurate genotyping.

[0034] Furthermore, the peak and signal intensities of the amplification products in each criterion can be determined using the results obtained under the fragment analysis conditions shown in the examples described later.

[0035] As a result of the analysis, we found 15 microsatellite markers that satisfy all of the above criteria 1) to 6), including microsatellite markers containing a region corresponding to any of the nucleotide sequences represented by SEQ ID NOs: 1 to 15. In other words, these 15 markers are excellent markers that allow for easy determination of genotypes and highly accurate identification of tea varieties.

[0036] The microsatellite markers containing regions corresponding to any of the base sequences represented by Sequence ID No. 1-2 are novel markers designed based on the base sequences of the newly acquired tea genome by the inventors, and are excellent markers that contribute significantly to the identification of tea varieties, as will be shown later.

[0037] Furthermore, by appropriately combining a microsatellite marker containing a region corresponding to any of the base sequences represented by SEQ ID NOs: 1 to 2, and at least one microsatellite marker selected from the group consisting of a microsatellite marker containing a region corresponding to any of the base sequences represented by SEQ ID NOs: 3 to 15, it is possible to identify a larger number of tea varieties with high accuracy.

[0038] Below, the following marker names are listed for the 15 microsatellite markers mentioned above, and their details are explained below.

[0039] Microsatellite markers related to Sequence IDs 1-2: MSE0348, MSE0354

[0040] Microsatellite markers related to Sequence IDs 3-15: ·TM043, TM107 (see non-patent document 3) ·TM336, TM348, TM350, TM464, TM485, TM553, TM626 (see non-patent document 6) • CsFM1097, CsFM1206, CsFM1566, CsFM1595 (See Non-Patent Document 5)

[0041] The nucleotide sequence represented by Sequence ID No. 1 was obtained from Camellia sinensis (variety: Sayama Kaori) and contains a microsatellite with acca as a repeating unit at nucleotides 154-175, which can be used as a microsatellite marker (marker name: MSE0348) for identifying tea varieties. In Sequence ID No. 1, the number of acca repeats is 5, and the repeat sequence is (acca)5ac.

[0042] The nucleotide sequence represented by Sequence ID No. 2 was obtained from Camellia sinensis (variety: Sayama Kaori) and contains a microsatellite with agaaa as a repeating unit at nucleotides 39 to 71, which can be used as a microsatellite marker (marker name: MSE0354) for identifying tea varieties. In Sequence ID No. 2, the number of agaaa repeats is 6, and the repeat sequence is (agaaa)6ag.

[0043] The nucleotide sequence represented by Sequence ID No. 3 was obtained from Camellia sinensis (variety: Longjing43) and contains a microsatellite with tttc as a repeating unit at bases 135-156, which can be used as a microsatellite marker (marker name: TM043) for identifying tea varieties. In Sequence ID No. 3, the number of tttc repeats is 5, and the repeat sequence is (tttc)5tt.

[0044] The nucleotide sequence represented by Sequence ID No. 4 was obtained from Camellia sinensis (variety: P1258) and contains a microsatellite with tttgt as a repeating unit at nucleotides 142 to 165, which can be used as a microsatellite marker (marker name: TM107) for identifying tea varieties. In Sequence ID No. 4, the number of tttgt repeats is 4, and the repeat sequence is (tttgt)4tttg.

[0045] The base sequence represented by Sequence ID No. 5 was obtained from Camellia sinensis (variety: Asatsuyu) and contains a microsatellite with ataaa as a repeating unit at bases 57 to 74, which can be used as a microsatellite marker (marker name: TM336) for identifying tea varieties. In Sequence ID No. 5, the number of ataaa repeats is 3, and the repeat sequence is aaa(ataaa)3.

[0046] The nucleotide sequence represented by Sequence ID No. 6 was obtained from Camellia sinensis (variety: Musashikaori) and contains a microsatellite with tatc as a repeating unit at nucleotides 132 to 162, which can be used as a microsatellite marker (marker name: TM348) for identifying tea varieties. In Sequence ID No. 6, the number of tatc repeats is 7, and the repeat sequence is (tatc)7tat.

[0047] The nucleotide sequence represented by Sequence ID No. 7 was obtained from Camellia sinensis (variety: Asatsuyu) and contains a microsatellite with tggtc as a repeating unit at bases 29 to 47, which can be used as a microsatellite marker (marker name: TM350) for identifying tea varieties. In Sequence ID No. 7, the number of tggtc repeats is 3, and the repeat sequence is (tggtc)3tggt.

[0048] The base sequence represented by Sequence ID No. 8 was obtained from Camellia sinensis (variety: Asatsuyu) and contains a microsatellite with acca as a repeating unit at bases 47-66, which can be used as a microsatellite marker (marker name: TM464) for identifying tea varieties. In Sequence ID No. 8, the number of acca repeats is 5, and the repeat sequence is (acca)5.

[0049] The nucleotide sequence represented by Sequence ID No. 9 was obtained from Camellia sinensis (variety: Asatsuyu) and contains a microsatellite with tttc as a repeating unit at bases 142-159, which can be used as a microsatellite marker (marker name: TM485) for identifying tea varieties. In Sequence ID No. 9, the number of tttc repeats is 4, and the repeat sequence is (tttc)4tt.

[0050] The nucleotide sequence represented by Sequence ID No. 10 was obtained from Camellia sinensis (variety: Nanmei) and contains a microsatellite with agaag as a repeating unit at nucleotides 94 to 117, which can be used as a microsatellite marker (marker name: TM553) for identifying tea varieties. In Sequence ID No. 10, the number of agaag repeats is 4, and the repeat sequence is (agaag)4agaa.

[0051] The nucleotide sequence represented by Sequence ID No. 11 was obtained from Camellia sinensis (variety: Asatsuyu) and contains a microsatellite with caaac as a repeating unit at nucleotides 39 to 65, which can be used as a microsatellite marker (marker name: TM626) for identifying tea varieties. In Sequence ID No. 11, the number of caaac repeats is 5, and the repeat sequence is (caaac)5ca.

[0052] The nucleotide sequence represented by Sequence ID No. 12 was obtained from Camellia sinensis (variety: Fudingdabaicha) and contains a microsatellite with ctttt as a repeating unit at nucleotides 91 to 115, which can be used as a microsatellite marker (marker name: CsFM1097) for identifying tea varieties. In Sequence ID No. 12, the number of ctttt repeats is 5, and the repeat sequence is (ctttt)5.

[0053] The nucleotide sequence represented by Sequence ID No. 13 was obtained from Camellia sinensis (variety: Fudingdabaicha) and contains a microsatellite with gagtt as a repeating unit at bases 78 to 103, which can be used as a microsatellite marker (marker name: CsFM1206) for identifying tea varieties. In Sequence ID No. 13, the number of gagtt repeats is 5, and the repeat sequence is (gagtt)5g.

[0054] The nucleotide sequence represented by Sequence ID No. 14 was obtained from Camellia sinensis (variety: Fudingdabaicha) and contains a microsatellite with tcttt as a repeating unit at bases 87 to 113, which can be used as a microsatellite marker (marker name: CsFM1566) for identifying tea varieties. In Sequence ID No. 14, the number of tcttt repeats is 5, and the repeat sequence is (tcttt)5tc.

[0055] The nucleotide sequence represented by Sequence ID No. 15 was obtained from Camellia sinensis (variety: Fudingdabaicha) and contains a microsatellite with aagg as a repeating unit at nucleotides 156 to 181, which can be used as a microsatellite marker (marker name: CsFM1595) for identifying tea varieties. In Sequence ID No. 15, the number of aagg repeats is 6, and the repeat sequence is (aagg)6aa.

[0056] Furthermore, the regions corresponding to any of the nucleotide sequences represented by SEQ ID NOs: 1 to 15 do not necessarily have to be perfectly identical, even in the regions other than the repetitive sequences, due to variations between varieties. A person skilled in the art can easily identify the regions corresponding to any of the nucleotide sequences represented by SEQ ID NOs: 1 to 15 based on each nucleotide sequence. Each region corresponding to any of the nucleotide sequences represented by SEQ ID NOs: 1 to 15 may correspond to all or part of the regions of any of the nucleotide sequences represented by SEQ ID NOs: 1 to 15. For example, each region corresponding to the nucleotide sequences represented by Sequence IDs 1 to 15 is a region that can be amplified by the primer set exemplified later, so a person skilled in the art can easily obtain the corresponding region. The region sandwiched between the binding sites of the primer set may be defined as the region corresponding to the nucleotide sequences represented by Sequence IDs 1 to 15.

[0057] In each region corresponding to any of the base sequences represented by sequence numbers 1 to 15 above, the length of the repeating sequence may differ for each variety, and by treating this difference in sequence length as a polymorphism, tea varieties can be identified.

[0058] The 15 microsatellite markers listed above can be used to classify polymorphisms based on differences in their sequence lengths. For example, the relevant genomic region can be amplified using PCR, and the resulting fragments (e.g., PCR products) can be classified. Based on this information, tea varieties can be identified.

[0059] For example, the types of polymorphisms detected by each microsatellite marker can be pre-associated with the polymorphism patterns of known tea varieties. Then, by comparing the information on the polymorphism patterns identified from the test sample with the information on the polymorphism patterns of known tea varieties and analyzing the patterns for agreement and disagreement, the tea variety of the test sample can be identified.

[0060] Figures 1-3 illustrate the fragments obtained by amplifying the region of each marker using the primer set described later in the embodiments described below. For example, in the marker MSE0348 shown in Figure 1, the fragments A, B, C, D, and E shown are identified based on their sequence length.

[0061] While fragments A and B can be identified by their sequence length (bp), to improve the accuracy of analysis using general-purpose capillary DNA sequencers, a pre-prepared set of size standards for polymorphic patterns is available for each marker, allowing for genotype determination by comparison. Therefore, the polymorphisms obtained through analysis are identified and represented here using symbols such as A and B.

[0062] Table 1 shows the genotypes (polymorphism patterns) obtained from the analysis of each microsatellite marker for each tea variety.

[0063] [Table 1]

[0064] In Table 1, for example, the notation AA indicates that the organism is homozygous for polymorphism A, which is obtained as a fragment of A, and the notation AB indicates that the organism is heterozygous for polymorphism A, which is obtained as a fragment of A, and polymorphism B, which is obtained as a fragment of B.

[0065] As shown in Table 1, by obtaining information about the fragment patterns that the target tea plant exhibits through microsatellite marker analysis, it is possible to identify the tea variety.

[0066] Of the 15 types of microsatellite markers listed above, the microsatellite marker used for identifying tea varieties should be determined appropriately according to the tea variety being identified.

[0067] By appropriately using the 15 types of microsatellite markers shown here, it is possible to identify the 44 types of tea varieties listed above, as shown in Table 1.

[0068] While using only one type may not always allow for the identification of a single variety, if the varieties contained in the test sample can be predicted to some extent, then only that polymorphism should be used.

[0069] Referring to Table 1, for example, when the polymorphism pattern of the microsatellite marker MSE0348 identified from the test sample is BB and the polymorphism pattern of MSE0354 is CD, the tea variety of the test sample can be identified as one of the following varieties from the 44 tea varieties listed above: Shuntaro, Seimei, Yutakamidori, Sainomidori, Sakimidori, Haruto 34, or Yumewakaba. Furthermore, when the polymorphism pattern of the microsatellite marker TM107 identified from the test sample is DD, the tea variety of the test sample can be identified as Seimei.

[0070] From the contents of Table 2, it can be seen that the microsatellite marker (MSE0348) associated with Sequence ID No. 1 and the microsatellite marker (MSE0354) associated with Sequence ID No. 2 have a wide variety of polymorphisms among varieties, making them markers that enable highly accurate identification of tea varieties.

[0071] As illustrated above, Table 2 shows examples of markers or combinations thereof that can be used to identify varieties.

[0072] [Table 2A]

[0073] [Table 2B]

[0074] [Table 2C]

[0075] [Table 2D]

[0076] As an example of an embodiment identification method, at least one of the marker combinations listed in Table 2 is used to identify the corresponding varieties: Ryofu, Tea Intermediate Parent No. 3, Harumidori, Sofu, Tea Intermediate Parent No. 4, Tea Intermediate Parent No. 5, Tea Intermediate Parent No. 6, Sunrouge, Shuntaro, Saeakari, Nanmei, Seimei, Sayamakaori, Yabukita, Asatsuyu, Okumidori, Kanayamidori, Saemidori, Yutakamidori, Kiyoka (Nochaken No. 04), MK5601 (Makurakei 56-01), Kana An example of a method for identifying tea varieties can be given for identifying at least one tea variety selected from the group consisting of Emaru (Kanaya No. 33), Nochaken No. 01, Nochaken No. 02, Nochaken No. 09, Nochaken No. 10, Kokuken No. 01, Danshin 37 (Miyazaki No. 37), Miyazaki No. 39, Miyazaki No. 40, Okuharuka, Kirari 31, Sai no Midori, Saki Midori, Nagomi Yutaka, Haruto 34, Haruno Nagori, Harumoegi, Miyama Kaori, Musashi Kaori, Yume Kaori, Yume Wakaba, Benifuki, and Sayama Akari.

[0077] Of these 44 varieties, MSE0348 was necessary to identify the intermediate tea parent variety No. 4. MSE0354 was necessary to identify Kanayamidori. This indicates that both MSE0348 and MSE0354 are useful markers that contribute to the identification of a wider range of tea varieties.

[0078] The identification method of the embodiment may use all of the microsatellite markers related to SEQ ID NOs: 1 to 15, but it is also possible to distinguish between the above 44 varieties by using a specific combination of markers, including the microsatellite marker MSE0348 related to SEQ ID NOs: 1 to 2, or MSE0348 and MSE0354. Such combinations are shown in Tables 3 and 4.

[0079] [Table 3]

[0080] [Table 4]

[0081] By using one or more of the four marker combinations listed in Table 3 from the 15 markers mentioned above, it is possible to distinguish between the 44 varieties and lines based on differences in genotype using at least one marker. By using one or more of the four marker combinations listed in Table 4 from the 15 markers mentioned above, it is possible to distinguish between the 44 varieties and lines based on differences in genotypes using at least two markers, thus enabling clearer identification.

[0082] Regarding the combinations listed in Table 3 above, an example of a method for identifying tea varieties can be given, which includes identifying tea varieties using microsatellite markers from at least one of the following combinations 1 to 4. (Combination 1) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 2, A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 2) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 3, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 3) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 8, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by SEQ ID NO: 13 (Combination 4) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15

[0083] Regarding the combinations listed in Table 4 above, an example of a method for identifying tea varieties can be given, which includes identifying tea varieties using microsatellite markers from at least one of the following combinations 5 to 8. (Combination 5) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 2, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 3, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 6) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 2, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 8, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 7) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 2, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 12, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15 (Combination 8) A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 1, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 3, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 4, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 6, A microsatellite marker containing a region corresponding to the base sequence represented by Sequence ID No. 13, and Microsatellite marker containing the region corresponding to the nucleotide sequence represented by Sequence ID No. 15

[0084] On the other hand, the more markers used, the wider the range of identifiable objects can be, and the higher the accuracy can be.

[0085] From this perspective, a method for identifying tea varieties can be illustrated, which includes identifying tea varieties using all microsatellite markers selected from the group consisting of microsatellite markers containing regions corresponding to any of the base sequences represented by sequence numbers 1 to 15.

[0086] When all 15 of the above markers are used, it is possible to distinguish between 44 varieties and strains based on differences in genotype using at least three markers, and even clearer identification is possible.

[0087] Table 5 shows the number of markers with different genotypes for each of the 44 varieties / lines when all 15 markers listed above are used. In Table 5, the Yabukita vs. Saemidori pair has 3 markers with different genotypes, and for the other pairs, they can be distinguished by having 4 or more markers with different genotypes.

[0088] [Table 5]

[0089] In this specification, "identification of tea variety" may refer to information that can be estimated regarding the identification of a variety from the genotype information obtained by a marker for the subject being identified, and it is not required to definitively determine the variety.

[0090] Examples of variety identification include: identifying the tea varieties contained in the test sample; identifying tea varieties not contained in the sample; identifying tea varieties likely to be contained in the test sample; identifying tea varieties unlikely to be contained in the sample; confirming whether the sample contains one or more tea varieties; confirming whether the tea varieties contained in the sample belong to a specific group of varieties; confirming whether a sample supposedly containing a specific variety actually contains that variety (e.g., authenticity of product labeling); and confirming whether a sample supposedly containing a specific variety contains other varieties.

[0091] In the identification method of the embodiment, when identifying tea varieties using microsatellite markers, it is possible to identify polymorphisms in any of the above-mentioned microsatellite markers in the genome of the tea contained in the test sample. Furthermore, even without clearly determining the base sequence of the polymorphism, the polymorphism of the microsatellite marker can be identified by comparing the sequence lengths of each corresponding region in the genome of the tea contained in the sample.

[0092] In the identification method of the embodiment, for example, polymorphisms can be identified by amplifying a region containing a sequence corresponding to the repeat sequence of the region in question for at least one microsatellite marker selected from the group consisting of the 12 types of microsatellite markers. For this amplification, an amplification reaction using a primer set consisting of two or more primers capable of amplifying a region containing a sequence corresponding to the repeat sequence can be used.

[0093] The amplification reaction is not particularly limited as long as it allows for DNA amplification using two or more primers, but examples include amplification reactions under isothermal conditions and amplification reactions under non-isothermal conditions. Examples of amplification reactions under isothermal conditions include the LAMP (Loop-mediated Isothermal Amplification) method, the ICAN (Isothermal and Chimeric Primer-initiated Amplification of Nucleic Acids) method, the HDA (Helicase-dependent Amplification) method, and the RPA (Recombinase Polymerase Amplification) method. An example of amplification reaction under non-isothermal conditions is the PCR method. In this embodiment, the LAMP method and the PCR method are preferred as amplification reactions.

[0094] The amplification size of the amplification product can be determined as appropriate, but for example, it may be 50-500 bp, 70-400 bp, or 90-350 bp.

[0095] Methods for analyzing the length of amplification products obtained from the above amplification reactions are well known and can be carried out appropriately using various methods. For example, this can be done by capillary electrophoresis using a sequencer or by polyacrylamide electrophoresis.

[0096] In the identification method of the embodiment, the sample subjected to the amplification reaction may be a sample containing, or potentially containing, genomic DNA derived from a tea variety. Here, "variety" may be a concept that includes lines, hybrids, and crosses that are considered to be stages in the breeding of a variety. Examples of tea varieties include at least one selected from the group consisting of the above 44 tea varieties. The tea may be the plant body, a part of the plant body, or processed products thereof. Examples of the part of the plant body include leaves, stems, fruits, flowers, roots, etc., and leaves are preferred. Examples of the processed products include, for example, sencha, powdered tea, matcha, aracha, stem tea, powdered green tea, bud tea, pan-fired tea, gyokuro, etc. According to the inventors, it has been confirmed that the identification method of the embodiment is also applicable to sencha (processed product).

[0097] In addition, processed products of tea may be commercially available with two or more varieties blended. In that case, it is recommended to extract DNA using, as the test sample, the separated product for each tissue piece derived from tea (for example, tea leaf pieces) to obtain the genotype results. As another method, it is also possible to estimate the blended varieties from the genotype results obtained from the test sample in which two or more varieties are blended.

[0098] Regarding general precautions in DNA variety identification analysis and experiments, and the method for DNA extraction from samples, it can also be carried out in accordance with the content disclosed in <Basic Considerations on DNA Variety Identification of Plants - Guidelines for Technology Development and Use - (http: / / www.hinshu2.maff.go.jp / pvr / dna_manual / guideline.pdf)> and <Guidelines for Confirming the Validity of DNA Variety Identification Technology (http: / / www.hinshu2.maff.go.jp / pvr / dna_meeting / H20_2nd / guideline.pdf)>.

[0099] The identification method of the embodiment is an excellent method with few misjudgments and capable of obtaining stable results in the identification of tea varieties.

[0100] According to the identification method of the embodiment, it can be used for the identification of at least 44 tea varieties including widely popular varieties and the latest varieties, and it is expected to greatly contribute to the resolution of variety troubles including infringement of breeding rights.

[0101] ≪Polynucleotide, Microsatellite Marker≫ Polynucleotides consisting of the base sequences represented by Sequence ID No. 1 or Sequence ID No. 2 above can be used as microsatellite markers for identifying tea varieties. A polynucleotide consisting of any one of the above sequence numbers 1-15 can be used as a microsatellite marker for identifying tea varieties.

[0102] The polynucleotide or microsatellite marker of the embodiment may be a polynucleotide that includes a base sequence in which one or more bases are deleted, substituted, or added in each base sequence represented by any of the above sequence numbers 1 to 15, and which can be used as a microsatellite marker for identifying the above tea varieties. Here, "one or more" could mean, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0103] The polynucleotide or microsatellite marker of the embodiment may be a polynucleotide that contains a base sequence with 80% or more sequence identity with each base sequence represented by any of the above SEQ ID NOs: 1 to 15, and that can be used as a microsatellite marker for identifying the above tea varieties. Here, sequence identity may be 85% or higher, 90% or higher, or 95% or higher. Sequence identity can be determined, for example, using BLAST (Basic Local Alignment Search Tool), which is made available for use by DDBJ.

[0104] Primer Set Furthermore, as one embodiment of the present invention, a primer set for tea variety identification is provided that can amplify at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any of the base sequences represented by SEQ ID NOs: 1 to 2.

[0105] Furthermore, as a primer set usable for the method of identifying tea varieties in the embodiment, we provide a primer set for identifying tea varieties that can amplify at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any of the base sequences represented by SEQ ID NOs: 3 to 15.

[0106] Examples of such primer sets include a primer set consisting of two or more primers capable of detecting polymorphisms in at least one microsatellite marker selected from the group of the 15 microsatellite markers mentioned above.

[0107] Such primer sets include those capable of amplifying the sequences (microsatellites) corresponding to each repeat sequence contained in the 15 microsatellite markers mentioned above.

[0108] The number of primers included in each primer set varies depending on the amplification reaction used, but is typically between 2 and 6.

[0109] The number of nucleotide residues constituting the primer (complementary nucleotide residues that anneal to the target site) is not particularly limited as long as the primer anneals to the target site and amplifies the target product, and may be, for example, 15 or more, 16 or more, 17 or more, 18 or more, or 20 or more. Alternatively, the number of nucleotide residues constituting the primer may be, for example, 50 or fewer, 40 or fewer, or 30 or fewer.

[0110] Primers may be labeled with a fluorescent substance or other material to facilitate detection of the amplified product. Examples of such fluorescent substances include 6-FAM, VIC, NED, and PET. Of course, primers do not necessarily need to be labeled with such a substance. For example, differences in the size of the amplified products can be confirmed by gel electrophoresis. Alternatively, in LAMP detection, the presence or absence of amplification can be confirmed by visually observing the turbidity of magnesium pyrophosphate, a byproduct of the amplification reaction. In such cases, labeling of the primers with fluorescent substances or other materials is unnecessary.

[0111] The base sequence of the primer set can be appropriately designed based on the base sequence information of the microsatellite marker so that its annealing position is, for example, located on either side of the microsatellite region. In this case, when amplifying the microsatellite, the surrounding regions upstream and downstream of the microsatellite may also be amplified along with the microsatellite.

[0112] Table 6 shows the primer set sequences for each microsatellite marker used in the examples described later. Some primers have fluorescent labeling and tail sequences (gtttctt) added to stabilize the waveform.

[0113] As an example of a primer set for the embodiment, the following primer sets are exemplified based on the sequence of the primer set used in the embodiment.

[0114] Primer set 1 capable of amplifying the microsatellite marker MSE0348: (1F-1) A polynucleotide containing the base sequence described in Sequence ID No. 16, or (1F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 16, and (1R-1) A polynucleotide containing the base sequence described in Sequence ID No. 17, or A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in (1R-2) Sequence ID No. 17.

[0115] Primer set 2 capable of amplifying the microsatellite marker MSE0354: (2F-1) A polynucleotide containing the base sequence described in Sequence ID No. 18, or (2F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 18, and (2R-1) A polynucleotide containing the base sequence described in Sequence ID No. 19, or (2R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 19.

[0116] Primer set 3 capable of amplifying the microsatellite marker TM043: (3F-1) A polynucleotide containing the base sequence described in Sequence ID No. 20, or (3F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 20, and (3R-1) A polynucleotide containing the base sequence described in Sequence ID No. 21, or (3R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 21.

[0117] Primer set 4 capable of amplifying the microsatellite marker TM107: (4F-1) A polynucleotide containing the base sequence described in Sequence ID No. 22, or (4F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 22, and (4R-1) A polynucleotide containing the base sequence described in Sequence ID No. 23, or (4R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 23.

[0118] Primer set 5 capable of amplifying the microsatellite marker TM336: (5F-1) A polynucleotide containing the base sequence described in Sequence ID No. 24, or (5F-2) A polynucleotide containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence described in Sequence ID No. 24, and (5R-1) A polynucleotide containing the base sequence described in Sequence ID No. 25, or (5R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 25.

[0119] Primer set 6 capable of amplifying the microsatellite marker TM348: (6F-1) A polynucleotide containing the base sequence described in Sequence ID No. 26, or (6F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 26, and (6R-1) A polynucleotide containing the base sequence described in Sequence ID No. 27, or (6R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 27.

[0120] Primer set 7 for amplifying the microsatellite marker TM350: (7F-1) A polynucleotide containing the base sequence described in Sequence ID No. 28, or (7F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 28, and (7R-1) A polynucleotide containing the base sequence described in Sequence ID No. 29, or (7R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 29.

[0121] Primer set 8 capable of amplifying the microsatellite marker TM464: (8F-1) A polynucleotide containing the base sequence described in Sequence ID No. 30, or (8F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 30, and (8R-1) A polynucleotide containing the base sequence described in Sequence ID No. 31, or (8R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 31.

[0122] Primer set 9 capable of amplifying the microsatellite marker TM485: (9F-1) A polynucleotide containing the base sequence described in Sequence ID No. 32, or (9F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 32, and (9R-1) A polynucleotide containing the base sequence described in Sequence ID No. 33, or (9R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 33.

[0123] Primer set 10 capable of amplifying the microsatellite marker TM553: (10F-1) A polynucleotide containing the base sequence described in Sequence ID No. 34, or (10F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 34, and (10R-1) A polynucleotide containing the base sequence described in Sequence ID No. 35, or (10R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 35.

[0124] Primer set 11 capable of amplifying the microsatellite marker TM626: (11F-1) A polynucleotide containing the base sequence described in Sequence ID No. 36, or (11F-2) A polynucleotide containing a base sequence in which one or more bases are deleted, substituted, or added in the base sequence described in Sequence ID No. 36, and (11R-1) A polynucleotide containing the base sequence described in Sequence ID No. 37, or (11R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 37.

[0125] Primer set 12 capable of amplifying the microsatellite marker CsFM1097: (12F-1) A polynucleotide containing the base sequence described in Sequence ID No. 38, or (12F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 38, and (12R-1) A polynucleotide containing the base sequence described in Sequence ID No. 39, or (12R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 39.

[0126] Primer set 13 capable of amplifying the microsatellite marker CsFM1206: (13F-1) A polynucleotide containing the base sequence described in Sequence ID No. 40, or (13F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 40, and (13R-1) A polynucleotide containing the base sequence described in Sequence ID No. 41, or (13R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 41.

[0127] Primer set 14 capable of amplifying the microsatellite marker CsFM1566: (14F-1) A polynucleotide containing the base sequence described in Sequence ID No. 42, or (14F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 42, and (14R-1) A polynucleotide containing the base sequence described in Sequence ID No. 43, or (14R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 43.

[0128] Primer set 15 capable of amplifying the microsatellite marker CsFM1595: (15F-1) A polynucleotide containing the base sequence described in Sequence ID No. 44, or (15F-2) A polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 44, and (15R-1) A polynucleotide containing the base sequence described in Sequence ID No. 45, or (15R-2) A primer set comprising a polynucleotide containing a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence described in Sequence ID No. 45.

[0129] In the polynucleotides of the primer set described above, the form of deletion, substitution, or addition of one or more bases is not particularly limited, as long as it is possible to amplify the sequences (microsatellites) corresponding to each repeat sequence contained in the 15 types of microsatellite markers described above. One or more bases may be, for example, 1 to 7, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0130] It is preferable that a tail sequence (e.g., gtttctt) is added to the 5' end of the above-mentioned reverse primers (1R-1) to (15R-1) and (1R-2) to (15R-2). [Examples]

[0131] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0132] 1. DNA extraction from leaves Tea leaves (fully unfolded or hardened mature leaves) of each of the 44 tea varieties shown in Table 1 were collected and used for DNA extraction. If DNA extraction was not performed immediately after collection, the leaves were stored by one of the following methods: sealed and dried with silica gel, freeze-dried and stored in a desiccator, or frozen at -20°C or below without drying. DNA extraction was performed using the DNeasy Plant Mini Kit (QIAGEN) according to the kit protocol provided by QIAGEN, yielding sample DNA, which is DNA extract from the 44 varieties listed above.

[0133] 2. PCR reaction using SSR markers Using 15 types of SSR markers (Table 6) selected based on the criteria of ease of waveform checking and genotype determination, PCR reactions were performed on 44 varieties.

[0134] <Equipment and reagents used> • 1.5ml or 2.0ml tube (sterilized) • 96-well PCR plate or 8-tube strip (manufactured by Thermo Fisher Scientific / Applied Biosystems, hereafter referred to as ABI) • Cap or full plate cover (manufactured by ABI) • Sample DNA solution (prepared to 5 ng / μl) • Go Taq® Colorless Master Mix or Go Taq® Green Master Mix (manufactured by Promega) • 1 / 10 TE buffer ·Sterile ultrapure water • SSR primer solution (a solution containing 10 pmol / μl each of fluorescently labeled forward and reverse primers, diluted with 1 / 10 TE buffer) • PCR System ProFlex® (registered trademark) PCR System (manufactured by ABI Corporation)

[0135] <SSRマーカー> Table 6 shows the sequence number (No.), marker name, motif, nucleotide sequences of the forward and reverse primers and their sequence numbers (No.) (5' - 3'), target size (size of the amplified product), number of alleles, and Accession No. for each SSR marker. All reverse primers have a tail (gtttctt) added to the 5' end to stabilize the waveform. Sequence numbers 5-11 are newly determined sequences for which no publicly available information exists.

[0136] [Table 6]

[0137] <Basic operations> (1) The PCR reaction solution was prepared as follows: ·Go Taq Colorless Master Mix or Go Taq Green Master Mix 5.0μl • SSR primer solution 1.0 μl ·Sterile ultrapure water 3.0μl • Sample DNA solution 1.0 μl Total 10.0 μl (2) PCR reactions were performed using the ProFlex PCR System with the following program. • Heat denaturation at 94°C for 5 minutes. The reaction is repeated 35 times, with intervals of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. After reacting at 72°C for 7 minutes, the temperature rises to infinity at 10°C.

[0138] 3. Creating the Standard Set To determine genotypes, a standard set (a mixture of 2-5 PCR products) unique to each marker was created to cover the fragment patterns for each marker. The reference varieties for the standard sets are shown in Table 7, and the waveform data for each are shown in Figures 1-3.

[0139] [Table 7]

[0140] <Basic operations> (1) PCR reactions were performed under the conditions shown in "2. PCR reaction using SSR markers" above, and amplified products were obtained from each reference variety. (2) The PCR products were mixed and diluted 20 to 100 times with 1 / 10 TE buffer or the like, and dispensed into 50 μl portions. The date of preparation was written on the dispensed tubes, and they were stored in a light-shielded place in a cryogenic freezer or the like, and could be used within approximately two years.

[0141] 4. Fragment analysis using DNA sequencers The SSR-PCR products obtained in "2. PCR reaction using SSR markers" and the standard sets obtained in "3. Preparation of standard sets" were analyzed using a DNA sequencer, and fragment analysis was performed. The basic analytical procedures followed ABI's fragment analysis protocol.

[0142] <Equipment and reagents used> • 96-well PCR plate 1 / 10 TE Buffer • Applied Biosystems SeqStudio Genetic Analyzer (manufactured by ABI) • SeqStudio Cartridge (manufactured by ABI) • SeqStudio Cathode Buffer Container (manufactured by ABI) • Hi-Di Formamide (manufactured by ABI) • GeneScan® 400HD ROX Standard Size (manufactured by ABI) • GeneMapper® software (manufactured by ABI Inc.) • PCR System ProFlex® (registered trademark) PCR System (manufactured by ABI Corporation)

[0143] <Basic operations> (1) The PCR product obtained in "2. PCR reaction using SSR markers" above was diluted with 1 / 10 TE buffer at a dilution suitable for fragment analysis, taking into consideration PCR amplification. (2) 1.5 μl of diluted PCR product or standard set, 0.09 μl of 400HD ROX size standard, and 8.41 μl of Hi-Di formamide were placed in a 96-well PCR plate and mixed. (3) After heat denaturation at 95°C for 5 minutes using the ProFlex™ PCR System, the samples were immediately left to stand on ice for at least 5 minutes. (4) Analysis was performed using the SeqStudio Cartridge and SeqStudio Cathode Buffer Container, following the protocol for the Applied Biosystems SeqStudio Genetic Analyzer. (5) The results were analyzed using GeneMapper software.

[0144] 5. Genotyping and results by fragment analysis The SSR genotype was determined by comparison with the standard set prepared in "3. Preparation of Standard Set" above. In determining the genotype, factors such as the difference in electrophoretic movement between capillaries were considered, and if the difference compared to the standard peak size was less than ±0.5 bp, the genotype was determined to be the corresponding genotype. The genotypes revealed by the 15 markers are shown in Table 1.

[0145] When all 15 markers were used, it was possible to distinguish 44 varieties / lines based on genotype differences using at least three markers (Table 5).

[0146] Furthermore, by using the four combinations of markers listed in Table 3 from the 15 markers mentioned above, it was possible to distinguish between 44 varieties and lines based on differences in genotype using at least one marker.

[0147] Furthermore, by using the four combinations of markers listed in Table 4 from the 15 markers mentioned above, it was possible to distinguish between 44 varieties and lines based on differences in genotypes using at least two markers.

[0148] Each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited by each embodiment, but is limited only by the scope of the claims.

Claims

1. A method for identifying tea varieties, comprising identifying tea varieties using at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any one of the base sequences represented by SEQ ID NOs: 1 and 2.

2. The method for identifying tea varieties according to claim 1, further comprising identifying tea varieties using at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any one of the base sequences represented by SEQ ID NOs: 3 to 15.

3. The method for identifying tea varieties according to claim 1 or 2, comprising identifying tea varieties using microsatellite markers in at least one combination of the following combinations 1 to 4: (Combination 1) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 2; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 13; and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 15 (Combination 2) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 3; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 4; and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 15 (Combination 3) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 4; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 8; and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 13 (Combination 4) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 4; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 13; and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 15

4. The method for identifying tea varieties according to claim 1 or 2, comprising identifying tea varieties using microsatellite markers in at least one combination of the following combinations 5 to 8. (Combination 5) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 2; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 3; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 4; A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 13, and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 15 (Combination 6) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 2; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 4; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 8; A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 13, and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 15 (Combination 7) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 2; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 4; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 12; A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 13, and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 15 (Combination 8) a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 1; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 3; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 4; a microsatellite marker comprising a region corresponding to the base sequence represented by SEQ ID NO: 6; A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 13, and A microsatellite marker containing a region corresponding to the base sequence represented by SEQ ID NO: 15

5. The method for identifying tea varieties according to claim 1 or 2, comprising identifying tea varieties using all microsatellite markers selected from the group consisting of microsatellite markers containing regions corresponding to any of the base sequences represented by SEQ ID NOs: 1 to 15.

6. The tea varieties are Ryofu, Chachukan Mother No. 3, Harumidori, Sofu, Chachukan Mother No. 4, Chachukan Mother No. 5, Chachukan Mother No. 6, Sanrouge, Shuntaro, Saeakari, Nanmei, Seimei, Sayamakaori, Yabukita, Asatsuyu, Okumidori, Kanayamidori, Saemidori, Yutakamidori, Kiyoka (Nochaken 04), MK5601 (Makurakei 56-01), Kanaemaru (Kanaya 33), Nochaken 01, and Nochaken 02.

6. The method for identifying tea varieties according to any one of claims 1 to 5, wherein the tea variety is at least one selected from the group consisting of: Nochaken 09, Nochaken 10, Kokuken 01, Danshin 37 (Miyazaki 37), Miyazaki 39, Miyazaki 40, Okuharuka, Kirari 31, Saimidori, Sakimidori, Nagomiyutaka, Haruto 34, Harunonagori, Harumoegi, Miyamakaori, Musashikaori, Yumekaori, Yumewakaba, Benifuuki, and Sayamaakari.

7. A polynucleotide consisting of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO:

2.

8. A microsatellite marker for identifying tea varieties, consisting of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO:

2.

9. A primer set for identifying tea varieties, capable of amplifying at least one microsatellite marker selected from the group consisting of microsatellite markers containing a region corresponding to any one of the base sequences represented by SEQ ID NOs: 1 and 2.

10. The primer set according to claim 9 , which is the following primer set 1 or primer set 2: Primer set 1: (1F-1) A polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 16, or (1F-2) A polynucleotide comprising a base sequence in which one or more bases are deleted, substituted, or added in the base sequence set forth in SEQ ID NO: 16; (1R-1) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 17, or (1R-2) a polynucleotide comprising a base sequence in which one or more bases are deleted, substituted, or added in the base sequence set forth in SEQ ID NO: 17; and Primer set 2: (2F-1) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 18, or (2F-2) A polynucleotide comprising a base sequence in which one or more bases are deleted, substituted, or added in the base sequence set forth in SEQ ID NO: 18; (2R-1) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 19, or (2R-2) a polynucleotide comprising a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of SEQ ID NO: 19; and

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