Identification method and identification kit

Abstract

To provide a technique for identifying Diospyros kaki having heavy fruits.SOLUTION: A method for identifying a large-fruited persimmon includes a step of determining a type of a fruit weight suggestive base, which is a base at a position corresponding to a base located at position 95497234 from the 5 ' end of the 15 * chromosome on the reference genome of the persimmon to be identified, among the bases of the DNA of the persimmon to be identified, and identifies the persimmon to be identified as a large-fruited persimmon when one or more alleles in which the fruit weight suggestive base is adenine are present as a result of the determination.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method and kit for identifying persimmon trees with heavy fruit weight. [Background technology]

[0002] Conventionally, methods for identifying or estimating plant traits using DNA markers based on genotypes have been known.

[0003] For example, Patent Document 1 discloses a technique for distinguishing between completely sweet and non-sweet persimmons.

[0004] This technology amplifies the base sequences of the region linked to the non-completely sweet persimmon sex (the non-completely sweet persimmon sex linked region base sequence) and the region linked to the completely sweet persimmon sex (the completely sweet persimmon sex linked region base sequence). Then, based on the presence or absence of amplification and the results of restriction enzyme treatment, it is possible to distinguish between completely sweet persimmons and non-completely sweet persimmons.

[0005] Persimmon trees take a long time to flower, so by using genotypes to understand the characteristics of completely sweet persimmons and non-completely sweet persimmons, especially in the early stages of growth such as seedlings, it is possible to select strains with desirable characteristics and improve the efficiency of breeding. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2022-076414 Summary of the Invention [Problem to be solved by the invention]

[0007] In addition to the trait of being completely sweet, there are many other traits that are widely desired in persimmons, such as yield, ease of management, resistance to pests, taste, and texture.

[0008] The present invention has been made in consideration of these circumstances, and aims to provide a novel technique for identifying or estimating persimmon trees with heavy fruit weight. [Means for solving the problem]

[0009] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0010] [1] A method for identifying persimmons with heavy fruit weight, comprising a step of determining the type of fruit weight-indicating base, which is a base at a position corresponding to the base located at position 9497243 from the 5' end of chromosome 15X on the reference genome of the persimmon, among the bases in the DNA of the persimmon to be identified; if the determination shows that there is one or more alleles in which the fruit weight-indicating base is adenine, the persimmon to be identified is identified as a persimmon with heavy fruit weight.

[0011] [2] In the step of identifying the type of fruit weight-indicating base, a competitive allele-specific PCR is performed using DNA of the persimmon to be identified as a template, with a first primer, a second primer, and a third primer, wherein the first primer consists of one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and a third base sequence following the 3' end of the one base sequence, and the third base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 16, and is a base sequence of 6 or more bases from the 5' end of the base sequence shown in SEQ ID NO: 16. the second primer comprises the other of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14 and a fourth base sequence following the 3' end of the other base sequence, the fourth base sequence being a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 17 and comprising a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 17; and the third primer is a base sequence of 18 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 18.

[0012] [3] The identification method described in [1] includes a step of determining the type of floury-indicative base, which is a base at a position corresponding to the base located at position 22156180 from the 5' end of chromosome 3 on the reference genome of the persimmon, among the bases in the DNA of the persimmon to be identified; if the result of the determination shows that there is one or more alleles in which the floury-indicative base is thymine, the persimmon is identified as having low floury quality inside the fruit.

[0013] [4] In the step of identifying the type of the powdery-indicative base, a competitive allele-specific PCR is performed using a fourth primer, a fifth primer, and a sixth primer with DNA of the persimmon to be identified as a template, and the fourth primer is composed of one base sequence selected from the group consisting of the first base sequence shown in SEQ ID NO: 13 and the fifth base sequence shown in SEQ ID NO: 15 and the second base sequence shown in SEQ ID NO: 14, and a sixth base sequence following the 3' end of either of the base sequences, and the sixth base sequence is a base sequence of 25 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 19, and a base sequence consisting of the fifth or subsequent base from the 5' end of the base sequence shown in SEQ ID NO: 19. and when the fourth primer has one base sequence selected from the group, the fifth primer consists of the second base sequence and a seventh base sequence following the 3' end of the second base sequence; when the fourth primer has the second base sequence, the fifth primer consists of the first base sequence and a seventh base sequence following the 3' end of the first base sequence, the seventh base sequence is a base sequence of 24 to 29 contiguous bases in the base sequence shown in SEQ ID NO: 20 and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 20; and the sixth primer is a base sequence of 18 to 23 contiguous bases in the base sequence shown in SEQ ID NO: 21.

[0014] [5] The identification method described in [1] includes a step of identifying a main trunk diameter suggestive base sequence, which is the base sequence of a region corresponding to the base sequence located at positions 12281277 to 12281283 from the 5' end of chromosome 5 on the reference genome of the persimmon, from the base sequence of the DNA of the persimmon to be identified, and as a result of the identification, the fewer alleles with the main trunk diameter suggestive base sequence CTTTTTA, the larger the main trunk diameter of the persimmon is identified.

[0015] [6] A method for identifying one or more bases selected from the group consisting of a base at a position corresponding to the base located at position 2032946, a base at a position corresponding to the base located at position 35347759, a base at a position corresponding to the base located at position 2032922, a base at a position corresponding to the base located at position 11610850, a base at a position corresponding to the base located at position 11610854, a base at a position corresponding to the base located at position 26462429, a base at a position corresponding to the base located at position 27467699, a base at a position corresponding to the base located at position 27467690, and a base at a position corresponding to the base located at position 997141, from the 5' end of chromosome 9 on the reference genome of the persimmon to be identified, and wherein the identification result indicates that there is one or more alleles in which one or more bases selected from the group are bases indicative of a persimmon with less branch and trunk damage. The method of identifying a persimmon tree with less damage to its trunks and branches by insects when the base is at the 2032946th position from the 5' end, the 35347759th position from the 5' end, the 11610854th position from the 5' end, and the 997141st position from the 5' end are adenine, thymine, guanine, and thymine are, respectively, when the base is at the 11610850th position from the 5' end, the 26462429th position from the 5' end, the 27467699th position from the 5' end, and the 27467690th position from the 5' end are guanine.

[0016] [7] When, in the step of discriminating the type of one or more bases selected from the group, the bases on chromosome 9 whose type is to be discriminated are one or more bases selected from the group consisting of the base at a position corresponding to the base located at position 35347759 from the 5' end on the reference genome of persimmon, the base at a position corresponding to the base located at position 11610850, and the base at a position corresponding to the base located at position 11610854 from the 5' end, among the bases of the DNA of the persimmon to be discriminated, and the type of the base at the position corresponding to the base located at position 35347759 from the 5' end is discriminated and performing competitive allele-specific PCR using the DNA of the persimmon to be identified as a template and a seventh primer, an eighth primer, and a ninth primer, wherein the seventh primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and an eighth base sequence following the 3' end of the one base sequence, and the eighth base sequence is a base sequence of 26 to 31 consecutive bases in the base sequence shown in SEQ ID NO: 22, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 22. the eighth primer comprises the other of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a ninth base sequence following the 3' end of the other base sequence, the ninth base sequence being a base sequence of 23 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 23, and including a base sequence consisting of the 6th and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 23; the ninth primer being a base sequence of 20 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 24, and including a base sequence consisting of the 11610850th and subsequent bases from the 5' end and the base at the position corresponding to the 11610854th base, competitive allele-specific PCR is performed using DNA of a persimmon to be identified as a template, and a tenth primer, an eleventh primer, and a twelfth primer, wherein the tenth primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and a tenth base sequence following the 3' end of the one base sequence, and the tenth base sequence isThe discrimination method according to [6], wherein the 12th primer is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 25 and includes a base sequence consisting of the sixth base and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 25, the 11th primer consists of the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and an 11th base sequence following the 3' end of the other base sequence, the 11th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 26 and includes a base sequence consisting of the sixth base and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 26, and the 12th primer is a base sequence of 19 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 27.

[0017] [8] An identification kit for identifying persimmons with heavy fruit weight, comprising a primer set having a first primer, a second primer, and a third primer, wherein the first primer consists of one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and a third base sequence following the 3' end of the one base sequence, and the third base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 16, and consists of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 16. a base sequence, wherein the second primer comprises the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a fourth base sequence following the 3' end of the other base sequence, wherein the fourth base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 17 and comprises a base sequence consisting of the sixth base and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 17; and the third primer is a base sequence of 18 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 18.

[0018] [9] The present invention further includes a primer set including a fourth primer, a fifth primer, and a sixth primer, wherein the fourth primer comprises a base sequence selected from the group consisting of a first base sequence shown in SEQ ID NO: 13 and a fifth base sequence shown in SEQ ID NO: 15, and a second base sequence shown in SEQ ID NO: 14, and a sixth base sequence following the 3' end of either of the base sequences, wherein the sixth base sequence is a base sequence of 25 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 19, and is a base sequence of 5 or more bases from the 5' end of the base sequence shown in SEQ ID NO: 19. the fifth primer comprises the other of a base sequence selected from the group and the second base sequence shown in SEQ ID NO: 14, and a seventh base sequence following the 3' end of the other base sequence, the seventh base sequence being a base sequence of 24 to 29 contiguous bases in the base sequence shown in SEQ ID NO: 20 and comprising a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 20; and the sixth primer being a base sequence of 18 to 23 contiguous bases in the base sequence shown in SEQ ID NO: 21.

[0019]

[10] The present invention further includes either or both of a primer set including a seventh primer, an eighth primer, and a ninth primer, and a primer set including a tenth primer, an eleventh primer, and a twelfth primer, wherein the seventh primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and an eighth base sequence following the 3' end of the one base sequence, and the eighth base sequence comprises 26 to 31 consecutive bases in the base sequence shown in SEQ ID NO: 22. the eighth primer comprises the other of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a ninth base sequence following the 3' end of the other base sequence, the ninth base sequence being a base sequence of 23 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 23, and comprising a base sequence consisting of the sixth base and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 23; the primer is a base sequence of 20 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 24, the 10th primer consists of one of the base sequences of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a 10th base sequence following the 3' end of the one base sequence, the 10th base sequence being a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 25 and including a base sequence consisting of the 6th and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 25, and the 11th primer is The identification kit according to [8] or [9], comprising the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and an 11th base sequence following the 3' end of the other base sequence, wherein the 11th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 26 and includes a base sequence consisting of the sixth base and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 26, and the 12th primer is a base sequence of 19 to 27 consecutive bases in the base sequence shown in SEQ ID NO: 27. [Effects of the Invention]

[0020] According to the present invention, a novel technique for identifying or estimating persimmon trees with heavy fruit weight can be provided. [Brief explanation of the drawings]

[0021] [Figure 1] This is an explanatory diagram of analysis using the additive model, 1-dom model, and dip-add model. [Figure 2] Graph showing the −log10P value of the correlation between the locus and the trait for each trait screened. [Figure 3] 1 is a graph showing the relationship between the number of normal and mutant alleles and actual fruit weight in the five populations shown in Table 1 for bases suggesting fruit weight. [Figure 4] 1 is a graph showing the relationship between the number of normal and mutant alleles and actual powderiness in the five populations shown in Table 1 for powderiness-indicating bases. [Figure 5] 1 is a graph showing the relationship between the number of normal and mutant alleles and the actual main trunk diameter in the four populations excluding E shown in Table 1 for the base sequences suggesting the main trunk diameter. [Figure 6] This is a graph showing the relationship between the number of normal and mutant alleles and actual damage caused by herbivory in the five populations shown in Table 1 for bases that indicate damage caused by herbivory (bases at positions corresponding to the 35,347,759th base from the 5' end of the reference genome). [Figure 7] This is a graph showing the relationship between the number of normal and mutant alleles and actual damage caused by feeding damage in the five populations shown in Table 1 for bases that indicate feeding damage (the base at the position corresponding to the 11610850th base from the 5' end of the reference genome, and the base at the position corresponding to the 11610854th base from the 5' end). [Figure 8] 1 is a graph plotting the fluorescence intensity as a result of competitive allele-specific PCR performed using the first primer set. [Figure 9] 10 is a graph plotting the fluorescence intensity of the results of competitive allele-specific PCR performed using the second primer set. [Figure 10] 10 is a graph plotting the fluorescence intensity of the results of competitive allele-specific PCR performed using the third primer set. [Figure 11] 10 is a graph plotting the fluorescence intensity of the results of competitive allele-specific PCR performed using the fourth primer set. DETAILED DESCRIPTION OF THE INVENTION

[0022] Preferred embodiments of the present invention will now be described in detail. [How to identify persimmons with heavy fruit weight] A preferred embodiment of the present invention provides a method for identifying persimmons with heavy fruit weight, which comprises determining the type of fruit-weight-indicating base, which is a base at a position corresponding to the base located at position 9497243 from the 5' end of chromosome 15X on the persimmon reference genome, among the bases in the DNA of the persimmon to be identified as being a plant with heavy fruit weight; if the determination reveals that the persimmon has one or more alleles in which the fruit-weight-indicating base is adenine (A), the persimmon is identified as having heavy fruit weight.

[0023] <Fruit weight suggestion base discrimination process> The base located at position 9497243 from the 5' end of chromosome 15X in the persimmon reference genome is a single nucleotide polymorphism (SNP). As will be described in detail in the Examples below, persimmons that have one or more mutant alleles in which the base is adenine are statistically more likely to have heavy fruit than persimmons that do not.

[0024] Therefore, by determining the type of fruit weight-indicating base (adenine, thymine, guanine, or thymine) of a persimmon tree of interest that one wishes to identify (estimate) as a plant with heavy fruit weight, one can identify (estimate) whether that persimmon tree (scientific name: Diospyros kaki) is a plant with heavy fruit weight. In this specification, a persimmon tree of interest that one wishes to identify as a plant with desirable traits such as heavy fruit weight is also referred to as a "persimmon tree to be identified" or a "persimmon tree to be estimated."

[0025] Among the bases in the DNA of the persimmon to be identified, the fruit weight-indicating base, which is the base at a position corresponding to the base located at position 9497243 from the 5' end of chromosome 15X on the reference genome, can be used as a base to indicate whether the plant has heavy fruit weight.

[0026] In this specification, the step of identifying the type of fruit weight-indicating base is also referred to as the "fruit weight-indicating base identification step." If the fruit weight-indicating base is not a mutant type (adenine), it is usually guanine (G). If the fruit weight-indicating base of the persimmon to be identified is guanine as a result of the identification, it is estimated that the plant does not have relatively heavy fruit weight.

[0027] Heavy fruit weight refers to the weight of a persimmon tree's fruit (commonly known as a persimmon) being relatively heavy. There is no specific weight standard for identifying a fruit as heavy, but for example, a fruit weighing 280g or more is generally considered to be relatively heavy. A fruit can also be said to be relatively heavy if it is heavier than the average fruit weight for that variety. In general, persimmon trees (trees) with heavy fruit weights tend to be more popular with producers, and heavier persimmons (fruits) tend to be more popular with producers and consumers.

[0028] Persimmons are hexaploid, meaning they have six alleles, and if the base indicating fruit weight in one or more of these alleles is adenine, they can be identified (estimated) as a plant with heavy fruit weight. Persimmons with one or more alleles that have the fruit weight-indicating base adenine can be expected to bear relatively heavy (large) fruit.

[0029] The persimmon genome contains chromosome 15X and chromosome 15Y as chromosome 15. If the persimmon to be identified has one or more 15X chromosomes among the six alleles of chromosome 15, it will be able to produce flower buds (and thus fruit), and the identification method of this embodiment can be used to identify (estimate) whether the plant has heavy fruit weight.

[0030] In this specification, the reference genome of persimmon is the reference genome (reference genome name: DLO_r1.0.pseudomolecule, file name: "DLO_r1.0.pseudomolecule.fasta.gz", variety: Taishu) published on the website "http: / / persimmon.kazusa.or.jp / index.html", and is widely used as a reference genome for persimmon, as described in, for example, the following literature: Horiuchi A. et al., Ongoing Rapid Evolution of a Post-Y Region Revealed by Chromosome-Scale Genome Assembly of a Hexaploid Monoecious Persimmon (Diospyros kaki). Molecular Biology and Evolution, 40, 7, 2023. (https: / / doi.org / 10.1093 / molbev / msad151)

[0031] (Oysters to be identified) In this specification, the persimmon to be identified is not particularly limited as long as it is Diospyros kaki. The persimmon to be identified may be, for example, a seed, a seedling, a young tree, or an old tree.

[0032] The DNA of the persimmon to be identified that is subjected to the fruit weight suggestive base discrimination step is preferably DNA extracted from the persimmon. Therefore, the identification method of this embodiment may include a step of extracting DNA from the persimmon to be identified prior to the fruit weight suggestive base discrimination step.

[0033] The method for extracting DNA from persimmon is not particularly limited, and examples include methods using commercially available DNA extraction kits. Note that it is not always necessary to perform a step of extracting DNA from persimmon; for example, a crude extract of persimmon or already extracted DNA may be subjected to the fruit weight-indicating base discrimination step.

[0034] The part of the persimmon tree to be identified and used in the process of extracting persimmon DNA or preparing a crude extract is not particularly limited, and may be, for example, a leaf, stem, root, seed, flower, fruit, or calyx.

[0035] In the fruit weight-indicating base discrimination step, the method for discriminating the fruit weight-indicating base is not particularly limited, but examples include subjecting the DNA of the persimmon to be identified to a next-generation sequencer to read a wide range of base sequences, designing primers in a region highly conserved among persimmons near the fruit weight-indicating base and amplifying it by PCR, and then subjecting the PCR product to sequencing or electrophoresis, or amplifying it by PCR and measuring its fluorescence intensity. When subjecting the PCR product to electrophoresis, restriction enzyme treatment may be performed after amplification prior to electrophoresis.

[0036] A person skilled in the art can easily identify which base in the DNA sequence of the persimmon to be identified, obtained by PCR amplification or next-generation sequencing, corresponds to the base at position 9,497,243 from the 5' end of chromosome 15X in the persimmon reference genome (the fruit weight-indicating base), for example, by comparing the persimmon reference genome with the base sequence read by a sequencer such as a next-generation sequencer. An example of a method for reading the base sequence using a next-generation sequencer is ddRAD-seq, which will be described in detail later in the Examples.

[0037] One way to determine which bases in the DNA sequence of the persimmon tree being identified, as read by a sequencer, are indicative of fruit weight is to use an alignment program such as bwa mem to align the sequence information read by sequencing to the reference genome. It is also possible to determine which bases (or base sequences) in the DNA sequence of the persimmon tree being identified, as read by a sequencer, are indicative of flouriness, trunk diameter, or damage, as described in detail below.

[0038] On the other hand, when amplifying the region near the fruit weight-indicating base by PCR and measuring the fluorescence intensity, for example, competitive allele-specific PCR can be performed using the DNA of the persimmon to be identified as a template. Competitive allele-specific PCR is known as Kompetitive allele-specific PCR (KASP, registered trademark), and as disclosed in, for example, JP 2023-505138 A, this amplification technique using fluorescence is a known genotyping technique.

[0039] For competitive allele-specific PCR to distinguish the type of fruit weight-indicating base, for example, the first, second and third primers described in detail below can be suitably used.

[0040] The first primer consists of one of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a third base sequence following the 3' end of the one base sequence.

[0041] The third base sequence is a base sequence of 20 to 25 consecutive bases from the base sequence shown in SEQ ID NO: 16, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 16. The base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 16 is the base sequence from the sixth thymine to the adenine at the 3' end. The third base sequence may include the base sequence shown in SEQ ID NO: 28.

[0042] The third base sequence preferably consists of a base sequence of 21 to 24 consecutive bases (21 to 24 nucleotides) from the base sequence shown in SEQ ID NO: 16, more preferably a base sequence of 22 to 23 consecutive bases from the base sequence shown in SEQ ID NO: 16, and particularly preferably consists of the base sequence shown in SEQ ID NO: 28. The third base sequence may be 24 bases or less.

[0043] The third base sequence contained in the first primer complementarily and specifically binds to an allele in which the fruit weight-indicating base is a mutant (adenine). The base sequence shown in SEQ ID NO: 16 is the sequence of the genomic region near the fruit weight-indicating base on the allele (see the underlined portion in Table 8 below).

[0044] When the first primer has the above-mentioned nucleotide sequence from the sixth thymine to the 3'-terminal adenine at its 3'-end, it can amplify an allele in which the fruit weight-indicating nucleotide is mutant, as in the first primer having the nucleotide sequence shown in SEQ ID NO: 1 used in the Examples described later in detail. If the third nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO: 28, the first primer binds to (a region corresponding to) the region of nucleotides 9497222 to 9497243 from the 5'-end of chromosome 9 of the reference genome.

[0045] The nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, which may be contained in the first primer, are each a universal tail sequence (tag sequence). In the nucleotide sequence of the first primer, the base located one 3' end of the base located at the 3' end of the universal tail sequence is the 5' end base of the third nucleotide sequence. In other words, the first primer has a nucleotide sequence in which the universal tail sequence is attached to the 5' end of the third nucleotide sequence.

[0046] The second primer consists of the other of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a fourth base sequence following the 3' end of the other base sequence. If the first primer contains the first base sequence at its 5' end, the second primer contains the second base sequence at its 5' end, and if the first primer contains the second base sequence at its 5' end, the second primer contains the first base sequence at its 5' end.

[0047] In the base sequence of the second primer, the base located one 3' away from the 3'-end of the universal tail sequence is the 5'-end base of the fourth base sequence. That is, the second primer has a base sequence in which the universal tail sequence is added to the 5'-end of the fourth base sequence.

[0048] The fourth base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 17, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 17. The base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 17 is the base sequence from the sixth thymine to the 3' end guanine. The fourth base sequence may include the base sequence shown in SEQ ID NO: 29.

[0049] The fourth base sequence preferably consists of a base sequence of 21 to 24 consecutive bases (21 to 24 nucleotides) from the base sequence shown in SEQ ID NO: 17, more preferably a base sequence of 22 to 23 consecutive bases from the base sequence shown in SEQ ID NO: 17, and particularly preferably consists of the base sequence shown in SEQ ID NO: 29. The fourth base sequence may be 24 bases or less.

[0050] The fourth base sequence contained in the second primer complementarily and specifically binds to an allele in which the fruit weight-indicating base is normal (guanine). The base sequence shown in SEQ ID NO: 17 is the sequence of the genomic region near the fruit weight-indicating base on the allele (see the underlined portion in Table 8 below).

[0051] By having the nucleotide sequence from the sixth thymine to the 3'-terminal guanine described above at its 3' end, the second primer can amplify alleles in which the fruit weight-indicating nucleotide is of the normal type, as in the second primer having the nucleotide sequence shown in SEQ ID NO: 2 used in the Examples described later. The normal type refers to the so-called reference type, meaning that the nucleotide is the same as that in the reference genome (Taishu). If the fourth nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO: 29, the second primer will bind to the region of nucleotides 9497222 to 9497243 from the 5' end of chromosome 9 of the reference genome (the region corresponding to this region).

[0052] The third primer is a nucleotide sequence of 18 to 26 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO: 18. The third primer may contain the nucleotide sequence shown in SEQ ID NO: 3. When the third primer consists of the nucleotide sequence shown in SEQ ID NO: 3, the third primer binds to the region of nucleotides 9497222 to 9497243 from the 5' end of chromosome 9 of the reference genome.

[0053] The third primer preferably consists of a base sequence of 19 to 24 consecutive bases (19 to 24 nucleotides) within the base sequence shown in SEQ ID NO: 18, more preferably consists of a base sequence of 20 to 23 consecutive bases within the base sequence shown in SEQ ID NO: 18, even more preferably consists of a base sequence of 20 to 22 consecutive bases within the base sequence shown in SEQ ID NO: 18, particularly preferably consists of a base sequence of 20 to 21 consecutive bases within the base sequence shown in SEQ ID NO: 18, and most preferably consists of the base sequence shown in SEQ ID NO: 3. The third primer may be 24 nucleotides or less, 23 nucleotides or less, or 22 nucleotides or less.

[0054] By performing competitive allele-specific PCR using the first primer, the second primer, the third primer, and a Master MIX containing a FRET cassette with a fluorescent dye for competitive allele-specific PCR, different fluorescent dyes are attached to the PCR products amplified using an allele with a mutant fruit weight-indicating base as a template and the PCR products amplified using an allele with a normal fruit weight-indicating base as a template.

[0055] As a result, the fluorescence wavelength differs between PCR products amplified using an allele in which the fruit weight-indicating base is mutant as a template and PCR products amplified using an allele in which the fruit weight-indicating base is normal as a template.

[0056] Therefore, after competitive allele-specific PCR, by detecting the presence or absence and intensity of fluorescence at wavelengths corresponding to the type of fluorescent dye contained in the amplified product derived from the first primer and the type of fluorescent dye contained in the amplified product derived from the second primer, it is possible to confirm whether the genomic DNA of the persimmon to be identified contains mutant alleles and normal alleles, as well as the number of mutant (or normal) alleles (the ratio of the amount of mutant to normal alleles among the six alleles).

[0057] Furthermore, by comparing the results (fluorescence intensity at each wavelength) of competitive allele-specific PCR of the persimmon tree to be identified with the results of competitive allele-specific PCR using DNA from a persimmon tree as a template, for which the quantitative ratio between mutant and normal forms of the six alleles of fruit weight-indicating bases has already been determined, it is possible to more accurately determine whether or not the persimmon tree to be identified has alleles containing mutant fruit weight-indicating bases, as well as the quantitative ratio between mutant and normal forms of the six alleles.

[0058] For example, in Fuyu and Amaaki, all alleles have normal bases that indicate fruit weight. In Taigetsu, there is one allele in which the base that indicates fruit weight is mutant (adenine) (see Figure 3, described in detail below). Therefore, if the persimmon to be identified is subjected to competitive allele-specific PCR and the detected fluorescence intensity of the fluorescent dye contained in the amplification product derived from the first primer is equal to or greater than that of Taigetsu, the persimmon to be identified can be identified (or estimated) as a persimmon with a relatively heavy fruit weight.

[0059] Examples of fluorescent dyes include, but are not limited to, FAM (fluorescein) and HEX (hexachlorofluorescein). A commercially available Master Mix containing a FRET cassette with a fluorescent dye can be used as the Master Mix. The above describes competitive allele-specific PCR, but the same applies to competitive allele-specific PCR that can be performed in the flouriness-indicative base discrimination step, main stem diameter-indicative base sequence discrimination step, and herbivore damage-indicative base discrimination step, which will be described in detail later.

[0060] According to the identification method of this embodiment, it is possible to identify (or estimate) whether the persimmon being identified will bear heavy fruit in the future at an early growth stage, such as the seedling stage, thereby improving the efficiency of strain selection and variety improvement.

[0061] [How to identify persimmons with low powdery contents inside the fruit] According to another preferred embodiment of the present invention, a method for identifying persimmons with low flouriness inside the fruit includes a step of determining the type of flouriness-indicating base, which is a base at a position in the DNA of the persimmon to be identified that corresponds to the base located at position 22156180 from the 5' end of chromosome 3 on the reference genome of the persimmon. If the determination shows that there is one or more alleles in which the flouriness-indicating base is thymine, the persimmon is identified as having low flouriness inside the fruit.

[0062] <Powder quality suggestive base discrimination process> The base located at position 22156180 from the 5' end of chromosome 3 in the persimmon reference genome is an SNP. As will be described in detail in the Examples below, persimmons that have one or more normal alleles in which the floury base is thymine are statistically more likely to have low flouriness in the fruit interior than persimmons that do not have one or more normal alleles. Furthermore, it has been revealed that the more alleles in which the floury base is thymine that are present, the lower the degree of flouriness.

[0063] Therefore, by determining the type of base that indicates powdery quality of the persimmon to be identified, it is possible to identify (estimate) whether the persimmon (scientific name: Diospyros kaki) is a plant with low powdery quality inside the fruit and to what extent the powdery quality is.

[0064] Among the bases in the DNA of the persimmon to be identified, the floury-indicating base, which is the base at a position corresponding to the 22156180th base from the 5' end of chromosome 3 on the reference genome, can be used as a base to indicate whether the persimmon to be identified is a strain with low floury quality inside the fruit.

[0065] If the flour-indicating base of one or more of the six alleles is thymine, the persimmon can be identified (presumed) as having low flouriness, and it can be expected that such a persimmon will bear fruit with low flouriness.

[0066] In this specification, the process of determining the type of powdery-indicative base is also referred to as the "powdery-indicative base determination process." When the powdery-indicative base is not the normal type (thymine) but a mutant type, it is often adenine. Therefore, if the determination of the powdery-indicative base reveals that the fruit weight-indicative base of the persimmon to be identified is adenine, it is estimated that the plant does not have a relatively low powdery texture inside the fruit (it has a high powdery texture).

[0067] Low powdery texture inside the fruit refers to a state in which, when eating persimmon fruit, the interior does not have a gritty, rough texture (commonly referred to as "stale") like that of an old apple with a high powdery texture (or a low texture). For example, Fuyu is known to have no powdery texture, while Nishimura Wase is known to have a powdery texture. Also, for example, when an experienced evaluator performs a sensory evaluation of persimmons, if no powdery texture is detected, the persimmon can be rated as low powdery, and if the powdery texture is clearly detected, the persimmon can be rated as high powdery. Generally, persimmons with a low powdery texture inside the fruit tend to be more popular with consumers.

[0068] The floury quality suggestive base discrimination step and the persimmon DNA extraction step that may precede it can be carried out in the same manner as the fruit weight suggestive base discrimination step described above, except that the primer sequences are different.

[0069] In the powdery-indicative base discrimination step, the method for discriminating the powdery-indicative base is not particularly limited, but examples include, for example, subjecting the DNA of the persimmon to be identified to a next-generation sequencer to read a wide range of base sequences, designing primers in a region highly conserved among persimmons near the powdery-indicative base and amplifying it by PCR, and subjecting the PCR product to sequencing or electrophoresis, or amplifying it by PCR and measuring fluorescence intensity. When subjecting the PCR product to electrophoresis, restriction enzyme treatment may be performed after amplification prior to electrophoresis. Examples of methods for reading base sequences using a next-generation sequencer include ddRAD-seq, which will be described in detail in the Examples below.

[0070] One example of amplifying the region near the protein-indicating base by PCR and measuring the fluorescence intensity is to perform competitive allele-specific PCR using DNA from the persimmon tree to be identified as a template.

[0071] For competitive allele-specific PCR to distinguish between the nucleotides that indicate maltiness, the fourth, fifth, and sixth primers described in detail below can be suitably used, for example.

[0072] The fourth primer consists of one base sequence selected from the group consisting of the first base sequence shown in SEQ ID NO: 13 and the fifth base sequence shown in SEQ ID NO: 15, and the second base sequence shown in SEQ ID NO: 14, and a sixth base sequence following the 3' end of that base sequence.

[0073] As with the first and second primers described above, the fourth primer can use either the first or second base sequence as the universal tail sequence. However, a fifth base sequence, which adds an adenine to the 3' end of the first base sequence, can be used in place of the first base sequence. In some cases, the sequence upstream of the genomic region to which the fourth primer binds is "CT." Adding an adenine to the 3' end of the first base sequence can make the Tm value consistent with that of the fifth primer. Furthermore, because the universal tail sequence does not anneal to the genome, adding an adenine to the 3' end still allows amplification of regions containing nucleotide-indicating bases.

[0074] In the base sequence of the fourth primer, the base located 3' to one of the bases located at the 3' end of any of the three types of universal tail sequences is the 5'-terminal base of the sixth base sequence. That is, the fourth primer has a base sequence in which the universal tail sequence is added to the 5' end of the sixth base sequence.

[0075] The sixth base sequence is a base sequence of 25 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 19, and includes a base sequence consisting of the fifth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 19. The base sequence consisting of the fifth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 19 is the base sequence from the fifth guanine to the adenine at the 3' end. The sixth base sequence may include the base sequence shown in SEQ ID NO: 30.

[0076] The sixth base sequence preferably consists of a base sequence of 26 to 28 consecutive bases (26 to 28 nucleotides) from the base sequence shown in SEQ ID NO: 19, more preferably a base sequence of 27 to 28 consecutive bases from the base sequence shown in SEQ ID NO: 19, and particularly preferably consists of the base sequence shown in SEQ ID NO: 30. The sixth base sequence may be 29 bases or less.

[0077] The sixth base sequence contained in the fourth primer complementarily and specifically binds to an allele in which the chromosomal base is normal (thymine). The base sequence shown in SEQ ID NO: 19 is the sequence of the genomic region near the chromosomal base on the allele (see the underlined portion in Table 8 below).

[0078] The fourth primer has the base sequence from the fifth guanine to the 3'-terminal adenine at its 3' end, allowing amplification of alleles in which the nucleotide-indicating base is normal, as in the fourth primer having the base sequence shown in SEQ ID NO: 4 used in the Examples described later in detail. If the sixth base sequence consists of the base sequence shown in SEQ ID NO: 30, the fourth primer binds to (a region corresponding to) the region from bases 22156180 to 22156206 from the 5' end of chromosome 3 of the reference genome.

[0079] When the fourth primer has a base sequence selected from the group consisting of the first base sequence and the fifth base sequence, the fifth primer consists of the second base sequence and a seventh base sequence following the 3' end of the second base sequence. When the fourth primer has the second base sequence, the fifth primer consists of the first base sequence and a seventh base sequence following the 3' end of the first base sequence.

[0080] In the base sequence of the fifth primer, the base located on the 3' side of the base located at the 3' end of the first or second base sequence, which is the universal tail sequence, is the 5'-end base of the seventh base sequence. That is, the fifth primer has a base sequence with a universal tail sequence attached to the 5' end of the seventh base sequence.

[0081] The seventh base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 20, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 20. The base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 20 is the base sequence from the sixth guanine to the thymine at the 3' end. The seventh base sequence may include the base sequence shown in SEQ ID NO: 31.

[0082] The seventh base sequence preferably consists of a base sequence of 25 to 28 consecutive bases (25 to 28 nucleotides) from the base sequence shown in SEQ ID NO: 20, more preferably a base sequence of 26 to 27 consecutive bases from the base sequence shown in SEQ ID NO: 20, and particularly preferably consists of the base sequence shown in SEQ ID NO: 31. The seventh base sequence may be 28 bases or less.

[0083] The seventh base sequence contained in the fifth primer complementarily and specifically binds to an allele in which the maltogenic base is a mutant (adenine). The base sequence shown in SEQ ID NO: 20 is the sequence of the genomic region near the maltogenic base on the allele (see the underlined portion in Table 8 below).

[0084] The fifth primer has the nucleotide sequence from the sixth guanine to the thymine at the 3' end, as described above, and can amplify alleles in which the mutation-indicating base is a mutant, as in the fifth primer having the nucleotide sequence shown in SEQ ID NO: 5 used in the Examples described later. If the seventh nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO: 31, the fifth primer binds to (a region corresponding to) the region of bases 22156180 to 22156205 from the 5' end of chromosome 3 of the reference genome.

[0085] The sixth primer is a base sequence of 18 to 23 consecutive bases within the base sequence shown in SEQ ID NO: 21. The sixth primer may contain the base sequence shown in SEQ ID NO: 6. When the sixth primer consists of the base sequence shown in SEQ ID NO: 6, the sixth primer binds to a region of bases 22156160 to 22156179 from the 5' end of chromosome 3 of the reference genome.

[0086] The sixth primer preferably consists of a base sequence of 19 to 22 consecutive bases (19 to 22 nucleotides) from the base sequence shown in SEQ ID NO: 21, more preferably a base sequence of 20 to 21 consecutive bases from the base sequence shown in SEQ ID NO: 21, and even more preferably consists of the base sequence shown in SEQ ID NO: 6. The sixth primer may be 22 nucleotides or less.

[0087] By performing competitive allele-specific PCR using the fourth, fifth, and sixth primers and a Master MIX containing a FRET cassette with a fluorescent dye for competitive allele-specific PCR, different fluorescent dyes are attached to the PCR products amplified using an allele with a mutant nucleotide-indicating base as a template and the PCR products amplified using an allele with a normal nucleotide-indicating base as a template.

[0088] After competitive allele-specific PCR, by detecting the presence or absence and intensity of fluorescence at wavelengths corresponding to the type of fluorescent dye contained in the amplification product derived from the fourth primer and the type of fluorescent dye contained in the amplification product derived from the fifth primer, it is possible to confirm whether the genomic DNA of the persimmon to be identified contains mutant alleles and normal alleles, as well as the number of mutant (or normal) alleles (the ratio of the amount of mutant to normal alleles among the six alleles).

[0089] Furthermore, by comparing the results (fluorescence intensity at each wavelength) of competitive allele-specific PCR of the persimmon tree to be identified with the results of competitive allele-specific PCR using DNA from a persimmon tree as a template, for which the quantitative ratios of mutant and normal forms of the six alleles of the floury-indicating bases have already been determined, it is possible to more accurately determine whether or not the persimmon tree to be identified has alleles containing mutant floury-indicating bases, as well as the quantitative ratios of mutant and normal forms of the six alleles.

[0090] For example, in the Taigetsu, Fuyu, and Kanshu varieties, the floury base indicative of all alleles is normal (thymine) (see Figure 4, described in detail below). Therefore, if the target persimmon is subjected to competitive allele-specific PCR and the fluorescence intensity of the fluorescent dye contained in the amplified products derived from primers 4 and 5 is equivalent to that of, for example, Taigetsu, Fuyu, or Kanshu, the target persimmon can be identified (or estimated) as a persimmon with low floury internal fruit. Furthermore, in the Seido persimmon, the floury base indicative of some alleles is mutant (adenine), and this can be obtained by applying for distribution to, for example, the National Agriculture and Food Research Organization Genebank.

[0091] According to the identification method of this embodiment, it is possible to identify (or estimate) whether the persimmon being identified will bear fruit with low internal flouriness in the future at an early growth stage, such as the seedling stage, thereby improving the efficiency of strain selection and variety improvement.

[0092] [How to identify persimmon trees with large trunk diameters] According to yet another preferred embodiment of the present invention, a method for identifying persimmons with large main trunk diameters includes a step of determining a main trunk diameter-indicative base sequence, which is a base sequence of a region (position) of the DNA base sequence of the persimmon to be identified that corresponds to the base sequence located at positions 12281277 to 12281283 from the 5' end of chromosome 5 on the persimmon reference genome. As a result of the determination, the fewer alleles in the main trunk diameter-indicative base sequence where CTTTTTA is present in order from the 5' end, the larger the main trunk diameter of the persimmon is identified.

[0093] <Step of determining the base sequence that suggests the main stem diameter> In the persimmon reference genome, the base sequence from 12281277 to 12281283 from the 5' end of chromosome 5 is the normal base sequence CTTTTTT (with A at the 3' end), whereas in mutants it is often CTTTTTA. As will be described in detail later in the Examples, the fewer mutant alleles with the main trunk diameter suggestive base sequence CTTTTTA, the higher the statistical probability that the persimmon to be identified will be a plant with a large main trunk diameter, especially in a population where the seed parent and pollen parent are the same.

[0094] Therefore, by determining the trunk diameter-suggestive base sequence of the persimmon to be identified, it is possible to determine (estimate) whether the persimmon (scientific name: Diospyros kaki) is a plant that will grow into a persimmon with a large trunk diameter. In this specification, the process of determining the trunk diameter-suggestive base sequence (specifically, confirming whether it is CTTTTTA) is also referred to as the "main trunk diameter-suggestive base sequence determination process."

[0095] In this specification, the term "main trunk" refers to the thickest trunk (meaning not the branches) of a persimmon. A thick main trunk is a necessary condition for a persimmon to produce a high yield; if the diameter of the main trunk is not large, a high fruit yield cannot be expected. There are no particular limitations on the specific dimensions that constitute a large main trunk diameter, but for example, if the diameter of the main trunk at a height of 1 m from the ground is larger than the average diameter at the same position of the same variety, the plant can be said to have a large main trunk diameter.

[0096] If the discrimination result shows that there are three or more alleles with the main trunk diameter suggestive base sequence CTTTTTA, the persimmon may be identified as having a larger main trunk diameter as the number of alleles decreases. In this case, if there are 0 to 2 alleles, the main trunk diameter is not identified. In particular, if there are three or four (particularly three) alleles with the main trunk diameter suggestive base sequence CTTTTTA among the six alleles, the persimmon to be identified may be identified as having a larger main trunk diameter.

[0097] The process of identifying the base sequence suggesting the main trunk diameter and the process of extracting the DNA of the persimmon tree to be identified that may precede this can be carried out in the same manner as the above-mentioned process of identifying the base sequence suggesting the fruit weight, except that the primer sequence design is different.

[0098] In the main trunk diameter suggestive base sequence discrimination step, the method for discriminating the main trunk diameter suggestive base sequence is not particularly limited, but examples include, for example, subjecting the DNA of the persimmon to be identified to a next-generation sequencer to read a wide range of base sequences, designing primers in a region highly conserved among persimmons near the main trunk diameter suggestive base sequence and amplifying it by PCR, and then subjecting the PCR product to sequencing or electrophoresis, or PCR to measure fluorescence intensity. When subjecting the PCR product to electrophoresis, restriction enzyme treatment may be performed after amplification prior to electrophoresis. An example of a method for reading base sequences using a next-generation sequencer is ddRAD-seq, which will be described in detail in the Examples below.

[0099] According to the identification method of this embodiment, it is possible to identify (or estimate) whether the persimmon being identified will grow into a plant with a large trunk diameter in the future at an early growth stage, such as the seedling stage, thereby improving the efficiency of strain selection and variety improvement.

[0100] [How to identify persimmon trees with less damage to trunks and branches by insects] According to yet another preferred embodiment of the present invention, a method for identifying persimmon trees with less damage to branches and trunks by insects includes a step of determining the type of one or more bases selected from the group consisting of the base at a position corresponding to the base located at position 2032946, the base at a position corresponding to the base located at position 35347759, the base at a position corresponding to the base located at position 2032922, the base at a position corresponding to the base located at position 11610850, the base at a position corresponding to the base located at position 11610854, the base at a position corresponding to the base located at position 26462429, the base at a position corresponding to the base located at position 27467699, the base at a position corresponding to the base located at position 27467690, and the base at a position corresponding to the base located at position 997141 from the 5' end of chromosome 9 on the reference genome of the persimmon to be identified. If the discrimination results in one or more alleles in which one or more bases selected from the group indicate that the persimmon has little damage to its branches and trunks, the persimmon is identified as having little damage to its branches and trunks by insects.

[0101] Among the bases in the DNA of the persimmon to be identified, in the persimmon reference genome, the base at the position corresponding to the base at position 2032946 from the 5' end of chromosome 9, the base at the position corresponding to the base at position 35347759, the base at the position corresponding to the base at position 11610854, and the base at the position corresponding to the base at position 997141, the base that indicates that the persimmon has little damage to its branches and trunks is adenine.

[0102] Among the bases in the DNA of the persimmon to be identified, in the persimmon reference genome, the base at the position corresponding to the base at position 11610850 from the 5' end of chromosome 9, the base at the position corresponding to the base at position 26462429, the base at the position corresponding to the base at position 27467699, and the base at the position corresponding to the base at position 27467690 are the bases that indicate that the persimmon has little damage to its branches and trunks.

[0103] Among the bases in the DNA of the persimmon tree being identified, if the base at a position corresponding to the base located at 2,032,922nd position from the 5' end of chromosome 9 on the persimmon reference genome is guanine, the base that indicates that the persimmon has suffered little damage to its branches and trunks.

[0104] <Base discrimination process indicating pest damage> As will be described in detail later in the Examples, if the type of each base in the above group is a mutant base that indicates a persimmon tree with less damage to branches and trunks, there is a statistically higher probability that the tree will suffer less damage to branches and trunks by insects than if the base type is a normal type (base type in the reference genome).

[0105] Therefore, by determining the type of one or more bases selected from the above group of the persimmon to be identified, it is possible to identify (estimate) whether the persimmon (scientific name: Diospyros kaki) is a plant with little damage to its branches and trunks caused by pests. Each base in the above group can be used as a base that indicates whether a plant is heavily damaged by pests, and is therefore also referred to as a pest damage indicative base. The process of determining the pest damage indicative base is also referred to as a pest damage indicative base determination process.

[0106] Among the bases in the DNA of the persimmon to be identified, the base at the position corresponding to the base at position 2032946 from the 5' end of chromosome 9 on the persimmon reference genome is thymine in the case of the normal type, the base at the position corresponding to the base at position 35347759 is guanine in the case of the normal type, the base at the position corresponding to the base at position 2032922 is cytosine in the case of the normal type, and the base at the position corresponding to the base at position 11610850 is adenine in the case of the normal type. In the normal form, the base at the position corresponding to the base at position 11610854 is thymine, in the normal form the base at the position corresponding to the base at position 26462429 is cytosine, in the normal form the base at the position corresponding to the base at position 27467699 is cytosine, in the normal form the base at the position corresponding to the base at position 27467690 is adenine, and in the normal form the base at the position corresponding to the base at position 997141 is guanine.

[0107] Insects that feed on the branches and trunks of persimmon trees include, but are not limited to, Lepidoptera such as the persimmon moth and the Asian persimmon moth.

[0108] Without being bound by any particular theory, one possibility is that the degree of damage caused by insects is related to the smoothness of the bark surface. A smooth bark surface makes it difficult for insects to penetrate the bark cavities, making it easier to exterminate them with insecticides. Therefore, it is thought that trees with smooth bark surfaces are less susceptible to damage caused by insects. However, it is also possible that other factors, such as bark hardness, are involved.

[0109] Regarding the bases indicative of damage caused by pests at each of the above positions, the more alleles in the genome of the persimmon being identified that contain the above-mentioned ``bases indicating a persimmon with less damage to branches and trunks,'' the more likely it is that the plant will have less damage to branches and trunks.

[0110] The process of identifying bases indicative of pest damage, and the process that may precede it of extracting DNA from the persimmon tree to be identified, can be carried out in the same manner as the above-mentioned process of identifying bases indicative of fruit weight, except that the primer sequences are different.

[0111] In the step of identifying bases indicative of herbivory damage, the method for identifying the bases indicative of herbivory damage is not particularly limited. Examples include, for example, subjecting the DNA of the persimmon to be identified to a next-generation sequencer to read a wide range of base sequences, designing primers in a region highly conserved among persimmons near the bases indicative of herbivory damage and amplifying them by PCR, and then sequencing or electrophoresing the PCR product, or amplifying the PCR product and measuring its fluorescence intensity. When subjecting the PCR product to electrophoresis, restriction enzyme treatment may be performed after amplification prior to electrophoresis. Examples of methods for reading base sequences using a next-generation sequencer include ddRAD-seq, which will be described in detail in the Examples below.

[0112] One example of amplifying the region near the bases indicative of herbivory damage by PCR and measuring the fluorescence intensity is to perform competitive allele-specific PCR using the DNA of the persimmon tree to be identified as a template.

[0113] As primers that can be used in competitive allele-specific PCR to distinguish the type of base that indicates damage caused by pests, for example, from the above group, primers 7, 8, and 9 can be suitably used when distinguishing the type of base at a position corresponding to the base located at position 35347759 from the 5' end of chromosome 9 on the persimmon reference genome.

[0114] The seventh primer consists of one of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and an eighth base sequence following the 3' end of the one of the base sequences.

[0115] In the base sequence of the 7th primer, the base located on the 3' side of the base located at the 3' end of the 1st or 2nd base sequence, which is the universal tail sequence, is the 5'-end base of the 8th base sequence. That is, the 7th primer has a base sequence with a universal tail sequence attached to the 5' end of the 8th base sequence.

[0116] The eighth base sequence is a base sequence of 26 to 31 consecutive bases in the base sequence shown in SEQ ID NO: 22, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 22. The sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 22 are the base sequence from the sixth thymine to the thymine at the 3' end. The eighth base sequence may include the base sequence shown in SEQ ID NO: 32.

[0117] The eighth base sequence preferably consists of a base sequence of 27 to 30 consecutive bases (27 to 30 nucleotides) from the base sequence shown in SEQ ID NO: 22, more preferably a base sequence of 28 to 29 consecutive bases from the base sequence shown in SEQ ID NO: 22, and particularly preferably consists of the base sequence shown in SEQ ID NO: 32. The eighth base sequence may be 30 bases or less.

[0118] The eighth base sequence contained in the seventh primer complementarily and specifically binds to an allele in which the base at the position corresponding to the 35347759th base from the 5' end of chromosome 9 in the persimmon reference genome is a mutant (adenine). The base sequence shown in SEQ ID NO: 22 is the sequence of the genomic region near the base indicative of damage caused by pests on the allele (see the underlined portion in Table 8 below).

[0119] By having the 7th primer have the above-mentioned nucleotide sequence from the 6th thymine to the 3'-terminal thymine at their 3'-end, it is possible to amplify an allele in which the nucleotide indicative of pest damage at 35347759 is mutant, as in the 7th primer having the nucleotide sequence shown in SEQ ID NO: 7 used in the Examples described in detail below. If the 8th nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO: 32, the 7th primer will bind to (a region corresponding to) the region from 35347759 to 35347786 bases from the 5'-end of chromosome 9 of the reference genome.

[0120] The eighth primer consists of the other of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a ninth base sequence following the 3' end of the other base sequence. If the seventh primer contains the first base sequence at its 5' end, the eighth primer contains the second base sequence at its 5' end, and if the seventh primer contains the second base sequence at its 5' end, the eighth primer contains the first base sequence at its 5' end.

[0121] In the base sequence of the 8th primer, the base located one 3' away from the 3'-end of the universal tail sequence is the 5'-end base of the 9th base sequence. That is, the 8th primer has a base sequence in which the universal tail sequence is added to the 5'-end of the 9th base sequence.

[0122] The ninth base sequence is a base sequence of 23 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 23, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 23. The base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 23 is the base sequence from the sixth thymine to the 3' end cytosine. The ninth base sequence may include the base sequence shown in SEQ ID NO: 33.

[0123] The ninth base sequence preferably consists of a base sequence of 24 to 27 consecutive bases (24 to 27 nucleotides) from the base sequence shown in SEQ ID NO: 23, more preferably a base sequence of 25 to 26 consecutive bases from the base sequence shown in SEQ ID NO: 23, and particularly preferably consists of the base sequence shown in SEQ ID NO: 33. The ninth base sequence may be 27 bases or less.

[0124] The ninth base sequence contained in the eighth primer complementarily and specifically binds to an allele in which the base at the position corresponding to the 35347759th base from the 5' end of chromosome 9 in the persimmon reference genome is normal (guanine). The base sequence shown in SEQ ID NO: 23 is the sequence of the genomic region near the base indicative of damage caused by herbivory on the allele (see the underlined portion in Table 8 below).

[0125] By having the 8th primer have the above-mentioned nucleotide sequence from the 6th thymine to the 3'-terminal cytosine at their 3'-end, it is possible to amplify an allele in which the nucleotide at position 35347759 indicating damage caused by pests is normal, as in the 8th primer having the nucleotide sequence shown in SEQ ID NO: 8 used in the Examples described later in detail. If the 9th nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO: 33, the 8th primer binds to (a region corresponding to) the region from 35347759 to 35347783 bases from the 5'-end of chromosome 9 of the reference genome.

[0126] The ninth primer is a nucleotide sequence of 20 to 28 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO: 24. The ninth primer may contain the nucleotide sequence shown in SEQ ID NO: 9. When the ninth primer consists of the nucleotide sequence shown in SEQ ID NO: 9, the ninth primer binds to the region of nucleotides 35347683 to 35347704 from the 5' end of chromosome 9 of the reference genome.

[0127] The ninth primer preferably consists of a base sequence of 21 to 26 consecutive bases (21 to 26 nucleotides) in the base sequence shown in SEQ ID NO: 24, more preferably consists of a base sequence of 22 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 24, even more preferably consists of a base sequence of 22 to 24 consecutive bases in the base sequence shown in SEQ ID NO: 24, particularly preferably consists of a base sequence of 22 to 23 consecutive bases in the base sequence shown in SEQ ID NO: 18, and most preferably consists of the base sequence shown in SEQ ID NO: 9. The ninth primer may be 26 nucleotides or less, 25 nucleotides or less, or 24 nucleotides or less.

[0128] By performing competitive allele-specific PCR using the seventh, eighth, and ninth primers and a Master MIX containing a FRET cassette with a fluorescent dye for competitive allele-specific PCR, different fluorescent dyes are attached to the PCR products amplified using an allele in which the base indicative of plant damage is mutant as a template and the PCR products amplified using an allele in which the base indicative of plant damage is normal as a template.

[0129] After competitive allele-specific PCR, by detecting the presence or absence and intensity of fluorescence at wavelengths corresponding to the type of fluorescent dye contained in the amplification product derived from the seventh primer and the type of fluorescent dye contained in the amplification product derived from the eighth primer, it is possible to determine whether the genomic DNA of the persimmon being identified contains mutant alleles and normal alleles, as well as the number of mutant (or normal) alleles (the ratio of the amount of mutant to normal alleles among the six alleles).

[0130] Furthermore, by comparing the results (fluorescence intensity at each wavelength) of competitive allele-specific PCR performed on the persimmon tree to be identified with the results of competitive allele-specific PCR performed using persimmon DNA as a template, for which the quantitative ratio between mutant and normal types of six alleles has already been determined, it is possible to more accurately determine whether mutant and normal alleles are present, and the number of mutant (or normal) alleles (in other words, the number of alleles containing mutant bases indicative of damage from feeding (or normal bases indicative of damage from feeding)).

[0131] For example, in Taigetsu, Amasuki, and Fuyu, the base indicative of damage caused by pests (the base at the position corresponding to the base at position 35347759 above) in all alleles is normal (guanine), while in Saijo, the base indicative of damage caused by pests (the base at the position corresponding to the base at position 35347759 above) in some alleles is mutant (adenine).

[0132] Furthermore, as primers that can be used in competitive allele-specific PCR to read bases indicative of damage caused by pests, from the above group, primers 10, 11, and 12 can be suitably used to determine the type of base at one or both of the bases at the position corresponding to the base located at position 11610850 from the 5' end of chromosome 9 on the persimmon reference genome and the base located at position 11610854.

[0133] The tenth primer consists of one of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a tenth base sequence following the 3' end of the one base sequence.

[0134] In the base sequence of the 10th primer, the base located on the 3' side of the base located at the 3' end of the first or second base sequence, which is the universal tail sequence, is the 5'-end base of the 10th base sequence. In other words, the 10th primer has a base sequence with a universal tail sequence attached to the 5' end of the 10th base sequence.

[0135] The 10th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 25, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 25. The sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 25 are the base sequence from the sixth guanine to the adenine at the 3' end. The 10th base sequence may include the base sequence shown in SEQ ID NO: 34.

[0136] The 10th base sequence preferably consists of a base sequence of 25 to 28 consecutive bases (25 to 28 nucleotides) from the base sequence shown in SEQ ID NO: 25, more preferably consists of a base sequence of 26 to 27 consecutive bases from the base sequence shown in SEQ ID NO: 25, and particularly preferably consists of the base sequence shown in SEQ ID NO: 34. The 10th base sequence may be 28 bases or less.

[0137] The tenth base sequence contained in the tenth primer complementarily and specifically binds to an allele in which the base at the position corresponding to the 11610850th base from the 5' end of chromosome 9 in the persimmon reference genome is normal (adenine) and the base at the position corresponding to the 11610854th base is normal (thymine). The base sequence shown in SEQ ID NO: 25 is the sequence of the genomic region near the base indicative of damage caused by feeding on the allele (see the underlined portion in Table 8 below).

[0138] The tenth primer has the above-mentioned nucleotide sequence from the sixth guanine to the 3'-terminal adenine at its 3' end, so that it can amplify alleles in which the bases indicative of pest damage located at positions 11610850 and 11610854 are normal, as in the tenth primer having the nucleotide sequence shown in SEQ ID NO: 10 used in the Examples described later. If the tenth nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO: 34, the tenth primer binds to (a region corresponding to) the region from the 5' end of chromosome 9 of the reference genome to bases 11610829 to 11610854.

[0139] The 11th primer consists of the other of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and the 11th base sequence following the 3' end of the other base sequence. If the 10th primer contains the first base sequence at its 5' end, the 11th primer contains the second base sequence at its 5' end, and if the 10th primer contains the second base sequence at its 5' end, the 11th primer contains the first base sequence at its 5' end.

[0140] In the base sequence of primer 11, the base located one 3' away from the base located at the 3' end of the universal tail sequence is the 5'-end base of base sequence 11. In other words, primer 11 has a base sequence in which the universal tail sequence is added to the 5' end of base sequence 11.

[0141] The 11th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 26, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 26. The base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 26 is the base sequence from the sixth guanine to the thymine at the 3' end. The 11th base sequence may include the base sequence shown in SEQ ID NO: 35.

[0142] The 11th base sequence preferably consists of a base sequence of 25 to 28 consecutive bases (25 to 28 nucleotides) from the base sequence shown in SEQ ID NO: 26, more preferably consists of a base sequence of 26 to 27 consecutive bases from the base sequence shown in SEQ ID NO: 26, and particularly preferably consists of the base sequence shown in SEQ ID NO: 35. The 11th base sequence may be 28 bases or less.

[0143] The 11th base sequence contained in the 11th primer complementarily and specifically binds to an allele in which the base at the position corresponding to the 11610850th base from the 5' end of chromosome 9 on the persimmon reference genome is a mutant (thymine) and the base at the position corresponding to the 11610854th base is a mutant (adenine). The base sequence shown in SEQ ID NO: 26 is the sequence of the genomic region near the base indicative of damage caused by feeding on the allele (see the underlined portion in Table 8 below).

[0144] By having the 11th primer have the above-mentioned nucleotide sequence from the 6th guanine to the 3'-terminal thymine at their 3'-end, it is possible to amplify alleles in which the nucleotides indicative of pest damage located at positions 11610850 and 11610854 are mutant, as in the 11th primer having the nucleotide sequence shown in SEQ ID NO: 11 used in the Examples described later in detail. If the 11th nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO: 35, the 11th primer binds to (a region corresponding to) the region from the 5'-end of chromosome 9 of the reference genome to positions 11610829 to 11610854.

[0145] In addition, if the base at the position corresponding to the base at position 11610850 is mutant, the base at the position corresponding to the base at position 11610854 is also mutant, and if the base at the position corresponding to the base at position 11610850 is normal, the base at the position corresponding to the base at position 11610854 is also normal in most cases.

[0146] The 12th primer is a nucleotide sequence of 19 to 27 consecutive nucleotides from the nucleotide sequence shown in SEQ ID NO: 27. The 12th primer may contain the nucleotide sequence shown in SEQ ID NO: 12. When the 12th primer consists of the nucleotide sequence shown in SEQ ID NO: 12, the 12th primer binds to (a region corresponding to) the region from 11610899 to 11610919 nucleotides from the 5' end of chromosome 9 of the reference genome.

[0147] The 12th primer preferably consists of a base sequence of 20 to 25 consecutive bases (20 to 25 nucleotides) in the base sequence shown in SEQ ID NO: 27, more preferably consists of a base sequence of 21 to 24 consecutive bases in the base sequence shown in SEQ ID NO: 27, even more preferably consists of a base sequence of 21 to 23 consecutive bases in the base sequence shown in SEQ ID NO: 27, particularly preferably consists of a base sequence of 21 to 22 consecutive bases in the base sequence shown in SEQ ID NO: 18, and most preferably consists of the base sequence shown in SEQ ID NO: 12. The 12th primer may be 25 nucleotides or less, 24 nucleotides or less, or 23 nucleotides or less.

[0148] By performing competitive allele-specific PCR using the 10th, 11th, and 12th primers and a Master MIX containing a FRET cassette with a fluorescent dye for competitive allele-specific PCR, different fluorescent dyes are attached to the PCR products amplified using an allele in which the base indicative of feeding damage is mutant as a template and the PCR products amplified using an allele in which the base indicative of feeding damage is normal as a template.

[0149] After competitive allele-specific PCR, by detecting the presence or absence and intensity of fluorescence at wavelengths corresponding to the type of fluorescent dye contained in the amplification product derived from primer 10 and the type of fluorescent dye contained in the amplification product derived from primer 11, it is possible to determine whether the genomic DNA of the persimmon being identified contains mutant alleles and normal alleles, as well as the number of mutant (or normal) alleles (the ratio of mutant to normal amounts of the six alleles).

[0150] Furthermore, by comparing the results (fluorescence intensity at each wavelength) of competitive allele-specific PCR performed on the persimmon tree to be identified with the results of competitive allele-specific PCR performed using persimmon DNA as a template, for which the quantitative ratio between mutant and normal types of six alleles has already been determined, it is possible to more accurately determine whether mutant and normal alleles are present, and the number of mutant (or normal) alleles (in other words, the number of alleles containing mutant bases indicative of damage from feeding (or normal bases indicative of damage from feeding)).

[0151] For example, Taigetsu and Amaaki have one allele in which the base indicating damage caused by pests is mutant (the base at the position corresponding to the base at position 11610850 above is thymine, and the base at the position corresponding to the base at position 11610854 above is adenine) (see Figure 7).In addition, all alleles of Fuyu have normal bases indicating damage caused by pests (the base at position 11610850 above is adenine, and the base at position 11610854 above is thymine).

[0152] According to the identification method of this embodiment, it is possible to identify (or estimate) whether the persimmon to be identified is a plant that is less susceptible to insect damage at an early growth stage, such as the seedling stage, thereby improving the efficiency of strain selection and variety improvement.

[0153] The above describes in detail methods for identifying persimmons with various desirable traits. For example, the method for identifying persimmons with heavy fruit weight described above may include, in addition to the step of identifying bases indicative of fruit weight, a step of identifying bases indicative of flouriness, a step of identifying base sequences indicative of main trunk diameter, and / or a step of identifying bases indicative of pest damage.

[0154] In addition, the above-mentioned method for identifying persimmons with low flouriness inside the fruit may include, in addition to the flouriness-indicative base discrimination step, a fruit weight-indicative base sequence discrimination step, a main trunk diameter-indicative base sequence discrimination step, and / or a pest damage-indicative base discrimination step.

[0155] In addition, the above-mentioned method for identifying persimmons with large main trunk diameters may include, in addition to the main trunk diameter suggestive base sequence discrimination step, a fruit weight suggestive base discrimination step, a floury quality suggestive base discrimination step, and / or a pest damage suggestive base discrimination step.

[0156] In addition, the method for identifying persimmon trees with less damage to the branches and trunks by insects described above may include, in addition to the step of determining bases indicative of damage caused by insects, a step of determining bases indicative of fruit weight, a step of determining bases indicative of flouriness, and / or a step of determining base sequences indicative of main trunk diameter.

[0157] In this way, by including other steps, it is possible to identify (predict) persimmons that have multiple desirable traits. Note that any of the above-mentioned identification methods can also be considered as prediction methods.

[0158] [Identification kit for identifying heavy-fruited persimmons] According to another preferred embodiment of this embodiment, the identification kit for identifying persimmons with heavy fruit weight includes a primer set (first primer set) having a first primer, a second primer, and a third primer. The first to third primers are the same as the first to third primers described in detail in the above-mentioned embodiment of the identification method.

[0159] The identification kit of this embodiment may be used in the method for identifying persimmons with heavy fruit weight described above. The first primer set can be suitably used for competitive allele-specific PCR. Competitive allele-specific PCR using the first primer set makes it easy to identify (estimate) whether the persimmon to be identified is a persimmon with heavy fruit weight.

[0160] The identification kit of this embodiment may further include the following second primer set, third primer set, and / or fourth primer set, in addition to the first primer set.

[0161] The second primer set includes a fourth primer, a fifth primer, and a sixth primer, which are the same as the fourth primer, the fifth primer, and the sixth primer described in detail in the above-mentioned embodiment of the identification method, respectively.

[0162] The third primer set includes a seventh primer, an eighth primer, and a ninth primer, which are the same as the seventh primer, the eighth primer, and the ninth primer described in detail in the above-described embodiment of the identification method, respectively.

[0163] The fourth primer set includes a tenth primer, an eleventh primer, and a twelfth primer, which are the same as the tenth primer, the eleventh primer, and the twelfth primer, respectively, described in detail in the embodiment of the identification method described above.

[0164] The second to fourth primer sets can each be suitably used in competitive allele-specific PCR. Competitive allele-specific PCR using the second primer set makes it easy to identify (estimate) whether the persimmon to be identified is a strain with low flouriness inside the fruit. Competitive allele-specific PCR using the third primer set and competitive allele-specific PCR using the fourth primer set make it easy to identify (estimate) whether the persimmon to be identified is a strain with little damage to the branches and trunks by insects.

[0165] [Identification kit for identifying persimmons with low powdery interior fruit] In yet another preferred embodiment of this embodiment, an identification kit for identifying persimmons with low flouriness inside the fruit includes the second primer set.

[0166] The identification kit of this embodiment may be used in the method for identifying persimmons with low fruit flouriness. The second primer set can be suitably used in competitive allele-specific PCR. Competitive allele-specific PCR using the second primer set can easily identify (estimate) whether the persimmon to be identified is a strain with low fruit flouriness.

[0167] The identification kit of this embodiment may further include the above-mentioned first primer set, third primer set, and / or fourth primer set in addition to the second primer set.

[0168] [Identification kit for identifying persimmon trees with less damage to trunks and branches by insects] In yet another preferred embodiment of this embodiment, an identification kit for identifying persimmon trees with less damage to branches and trunks by insects comprises the third primer set and / or the fourth primer set.

[0169] The identification kit of this embodiment may be used in the method for identifying persimmon trees with less damage to the trunks and branches by insects described above. The third primer set and the fourth primer set can each be suitably used for competitive allele-specific PCR. Competitive allele-specific PCR using the third primer set and competitive allele-specific PCR using the fourth primer set can easily identify (estimate) whether the persimmon tree to be identified is a strain with less damage to the trunks and branches by insects.

[0170] The identification kit of this embodiment may further include the first primer set and / or the second primer set, in addition to the third primer set and / or the fourth primer set.

[0171] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention. [Example]

[0172] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0173] [Experimental Example 1] In this experiment, five seedling populations A to E (n = 269) obtained by crossing the seed parents and pollen parents shown in Table 1 below were subjected to ddRAD-seq to obtain genome sequences.

[0174] For ddRAD-seq, the genomes of five seedling populations A to E were first digested with EcoRI and BglII, and EcoRI and BglII adapters were ligated to the respective ends of each fragment.

[0175] The fragments were then purified using AMPureXP (Beckman Coulter) and PCR amplified using a pair of indexed TruSeq universal primers. The pooled PCR products were purified using AMPureXP and size-selected (320 bp) using E-Gel SizeSelect agarose gels (Life Technologies). Paired-end 100-bp or 150-bp sequencing was performed.

[0176] The genome sequences of the five seedling populations A to E obtained in this manner were aligned to the persimmon reference genome described above, and a genome-wide search for mutations (polymorphisms) that differed from the normal type was conducted (approximately 30,000 locations). The bwa mem program was used for alignment to the reference genome. The GATK program was used to search for mutation sites (locuses). Subsequently, quantitative genotypes were estimated based on the read ratio between the normal (Ref) allele and the mutant (Alt) allele using the GATK and updog programs, and the quantitative genotypes were corrected. Finally, a genome-wide association analysis between traits and loci was conducted in the five seedling populations using the GWASpoly program.

[0177] [Table 1] *In Table 1, the underlined parents are completely sweet persimmons, and the others are not completely sweet persimmons.

[0178] For the analysis using the GWASpoly program, data on a total of 28 traits, including fruit weight, powdery texture inside the fruit, damage to branches and trunks by insect pests, and trunk diameter, were entered as traits of the five seedling populations.

[0179] For example, actual fruit weight data was entered as fruit weight data, data on the degree of mealiness of the actual fruit, sensory evaluation data by an experienced breeder in 0.5 increments from 0 to 2, data on trunk diameter, data on the results of actual diameter measurements, and data on trunk and branch damage, data on whether or not there were actual traces of feeding damage (0 or 1), were entered for analysis. Note that when data on the degree of mealiness was available over multiple years, the average value over multiple years was entered instead of in 0.5 increments. Furthermore, each seedling was grafted onto a persimmon rootstock as a scion, and data on the diameter of the scion side (upper side) of the grafting surface onto the rootstock was entered as trunk diameter data.

[0180] In the association analysis between traits and gene loci using the GWASpoly program, analyses were performed using the additive model, 1-dom model, and dip-add model. In addition, for the four populations (5pops-popFY) excluding population E shown in Table 1, the additive model was used to find highly correlated traits and gene loci (-log above the threshold). 10 P value) were screened.

[0181] Figure 1 is an explanatory diagram of the analysis using the additive model, 1-dom model, and dip-add model.

[0182] As shown in Figure 1, the additive (add) model is an analytical model that screens loci in which the phenotype changes as the number of mutant types (Alt) increases among the six alleles.

[0183] The 1-dom-ref model is an analytical model that screens for loci that change traits only when the wild type is dominant and all six alleles are mutant.

[0184] The 1-dom-alt model is an analytical model that screens for loci that change traits when the mutant type is dominant and there is one or more mutant alleles among six alleles.

[0185] The dip-add model is an analytical model that screens for loci that have different traits when there are 0, 1 to 5, or 6 mutations among six alleles.

[0186] Figure 2 shows the -log correlation between locus and trait for each trait screened. 10 Graph showing P values.

[0187] As a result of the association analysis using the above model, for four traits, fruit weight, fruit mealiness, damage to branches and trunks by insect pests, and trunk diameter, the -log 10 We were able to screen for loci with P values ​​(logarithmic values ​​of statistical significance). These loci are significant at the 5% level (p<0.05).

[0188] Specifically, for the trait of fruit weight, the above-mentioned fruit weight suggestive bases located on chromosome 15X were screened for significant correlation between the trait and the locus (mutation) in the 1-dom-alt model.

[0189] In addition, with regard to the trait of fruit powdery texture, the above-mentioned powdery-suggestive bases located on chromosome 3 were screened for a statistically significant correlation between the trait and the gene locus (mutation) in the additive model, 1-dom-alt model, dip-add model, and 5pops-popFY, additive model.

[0190] In addition, with regard to the trait of trunk diameter, the above-mentioned trunk diameter suggestive base sequence located on chromosome 5 was screened, as it was found that there was a statistically significant correlation between the trait and the gene locus (mutation) in the additive model for the above four populations.

[0191] Furthermore, for the trait of stem and branch damage, the above-mentioned bases indicative of damage by herbivory located on chromosome 9 were screened for statistically significant correlations between the trait and the gene locus (mutation) in the additive, 1-dom-alt, and dip-add models. In addition to these three models, a statistically significant correlation was also observed in the 5pops-popFY and additive models for the base indicative of damage by herbivory located at the position corresponding to the 2,032,946th base from the 5' end of chromosome 9 in the reference genome.

[0192] For each locus screened, -log 10 There are no loci with high P values ​​and the log 10 Loci with high P values ​​were removed and, together with the surrounding loci, the -log 10 Loci where a gently sloping peak in the P value was confirmed were selected, which allows for more accurate selection of DNA markers that are suspected to be linked to causative genes that directly determine traits.

[0193] FIG. 3 is a graph showing the relationship between the number of normal and mutant alleles and actual fruit weight in the five populations shown in Table 1 for the bases indicative of fruit weight.

[0194] In Figures 3 to 7, the numbers in parentheses next to each of populations A to E on the right side of the figures indicate the number of mutant alleles in the seed parent and pollen parent. For example, for the fruit weight-indicating base in Figure 3, population A is (1,0), so in the genome of the seed parent (Taigetsu), there is only one allele that has a mutant base (adenine) as the fruit weight-indicating base, and in the genome of the pollen parent (109-27), there are zero alleles (all normal) that have a mutant base as the fruit weight-indicating base.

[0195] Table 2 below shows the relationship between the types of fruit weight suggestive bases and traits, and the -log 10 P-values ​​(scores) are shown. -log 10 The higher the P value, the stronger the correlation between the base type and the trait.

[0196] [Table 2]

[0197] As shown in Figure 3, when there is one allele in which the fruit weight-indicating base is mutant (Alt, adenine) (AAAAAB), the fruit weight is heavier on average than when all six alleles are normal (Ref, guanine) (AAAAAA).

[0198] Figure 4 is a graph showing the relationship between the number of normal and mutant alleles and actual flouriness in the five populations shown in Table 1 for flouriness-indicating bases. Table 3 below also shows the relationship between the type of flouriness-indicating base and the trait, as well as the -log 10 P values ​​are indicated.

[0199] [Table 3]

[0200] As shown in Figure 4, it can be seen that the average chalkiness is higher when there is at least one allele in which the chalkiness-indicating base is a mutant type (Alt, adenine) (AAAAAB) than when there are only alleles in which the chalkiness-indicating base is the normal type (Ref, thymine) (AAAAAA).

[0201] Figure 5 is a graph showing the relationship between the number of normal and mutant alleles and the actual main trunk diameter for the four populations excluding E shown in Table 1 for the main trunk diameter suggestive base sequences. Table 4 below also shows the relationship between the type of main trunk diameter suggestive base sequence and traits, and the -log 10P values ​​are shown. Because screening was performed using an additive model, there is a tendency for the number of variants in each population to be larger.

[0202] [Table 4]

[0203] The fewer alleles with the variant (Alt, CTTTTTA) main trunk diameter suggestive base sequence, the larger the main trunk diameter.

[0204] Figure 6 is a graph showing the relationship between the number of normal and mutant alleles and actual damage caused by herbivory in the five populations shown in Table 1 for the bases indicative of herbivory damage (the bases at the positions corresponding to the 35347759th base from the 5' end of the reference genome). Table 5 below also shows the relationship between the type of base indicative of herbivory damage and traits, as well as the -log 10 P values ​​are shown. -log 10 The higher the P value of a locus (referred to as "position" in the table), the stronger the correlation between the base type and the trait.

[0205] [Table 5]

[0206] As shown in Figure 6, when there is one allele in which the base indicative of damage from insect pests (the base at the position corresponding to the 35347759th base from the 5' end of the reference genome) is a mutant (Alt, adenine) (AAAAAB), there is less damage to the branches and trunks from insect pests than when there is only one allele in which the base indicative of damage from insect pests (the base at the position corresponding to the 35347759th base from the 5' end of the reference genome) is a normal (guanine) base (AAAAAA).

[0207] Figure 7 is a graph showing the relationship between the number of normal and mutant alleles and actual damage caused by feeding damage in the five populations shown in Table 1 for bases that indicate feeding damage (the base at the position corresponding to the 11610850th base from the 5' end of the reference genome and the base at the position corresponding to the 11610854th base from the 5' end).

[0208] As shown in Figure 7, when there is one or more alleles in which the bases indicative of damage from pests are mutant (Alt, the base at the position corresponding to the 11610850th base is thymine, and the base at the position corresponding to the 11610854th base is adenine), damage from pests from branches and trunks is less than when there are only alleles in which the bases indicative of damage from pests are normal.

[0209] In addition, it can be seen that the more alleles there are in which the two bases indicative of damage from insect damage are mutant (the further to the right in Figure 7), the less damage there is from insect damage to the branches and trunks.

[0210] [Experimental Example 2] In this experiment, competitive allele-specific PCR was carried out using the DNA of the persimmon to be identified as a template and the first to fourth primer sets described above.

[0211] <Method and materials for competitive allele-specific PCR> (1) Competitive allele-specific PCR using the first, third, and fourth primer sets A reaction solution having the composition shown in Table 6 below was prepared and subjected to competitive allele-specific PCR. The KASP® Master MIX (x2) in Table 6 is specifically KASP® V4.0 2X Master mix 96 / 384 Std Rox (LGC genomics / KBS-1016-003 250 mL).

[0212] [Table 6]

[0213] Information about the primer mix in Table 6 is shown in Table 7, and information about each primer in the first to fourth primer sets is shown in Table 8. Primers ASP1, ASP2, and LSP, shown at the end of the primer names in Table 8, were used in the amounts shown in Table 7 for competitive allele-specific PCR using the first primer set, competitive allele-specific PCR using the second primer set, competitive allele-specific PCR using the third primer set, and competitive allele-specific PCR using the fourth primer set.

[0214] [Table 7]

[0215] [Table 8] *In Table 8, the underlined bases are the bases corresponding to the target SNPs on the genome (each suggested base or its complementary base). *In Table 8, the fluorescent dye column indicates the type of fluorescent dye that is added to the amplified product during amplification by competitive allele-specific PCR, and a FRET cassette labeled with one of these fluorescent dyes is contained in the KASP (registered trademark) Master MIX (x2) shown in Table 6.

[0216] The reaction mixture shown in Table 10 was placed in a MicroAmp Fast 96-well Reaction Plate (Thermo Fisher / 4346907) for parental polymorphism investigation of the five populations, and in a MicroAmp Optical 384-well Reaction Plate (Thermo Fisher / 4309849) for population analysis of the five populations. The plates were covered with MicroAmp Optical Adhesive Film (Thermo Fisher / 4311971) and subjected to competitive allele-specific PCR. Thermo's Quantstudio 3 was used for competitive allele-specific PCR to investigate parental polymorphisms of the five populations. Thermo's Quantstudio 5 was used for population analysis of the five populations.

[0217] The conditions for competitive allele-specific PCR are shown in Table 9. Competitive allele-specific PCR was analyzed using QuantStudio Design & Analysis Software v1.5.3, with the data analysis setting set to "Analyze Real-Time Rn - Medan (Rna to Rnb)."

[0218] [Table 9] *Scan refers to the measurement of fluorescence intensity. Pre-scan and post-scan were performed to plot the amplification trajectory, but are not necessarily required for competitive allele-specific PCR.

[0219] (2) Competitive allele-specific PCR using a second primer set A reaction solution having the composition shown in Table 10 below was prepared and subjected to competitive allele-specific PCR. The KASP® Master MIX (x2) in Table 10 is specifically KASP® V4.0 2X Master mix 96 / 384 Std Rox (LGC genomics / KBS-1016-003 250 mL).

[0220] [Table 10]

[0221] The primer mix information in Table 10 is as shown in Table 7 above. The information on each primer in the second primer set is as shown in Table 8 above.

[0222] The reaction mixture shown in Table 6 was placed in a 384-well Hard Shell PCR Plate CLR / WHT (BIORAD / HSP3805), covered with an Optically Clear Heat Seal (BIORAD / 1814030), and subjected to competitive allele-specific PCR. Thermo's ProFlex PCR System-2x384 well was used for competitive allele-specific PCR. Table 11 below shows the conditions for competitive allele-specific PCR using the second primer set. A homemade tool was used for competitive allele-specific PCR analysis.

[0223] [Table 11]

[0224] <Results of competitive allele-specific PCR> First, we will explain the results of competitive allele-specific PCR performed on the five seedling populations described above using the first primer set described above.

[0225] Figure 8 is a graph plotting the fluorescence intensity results of competitive allele-specific PCR performed using primer set 1. The vertical and horizontal axes of Figures 8 to 11 plot the relative intensity of fluorescence derived from FAM or HEX.

[0226] As shown in Figure 8, the results of competitive allele-specific PCR showed that the fluorescence intensity at two wavelengths differed between the population with the mutant fruit weight-indicating base and the population containing only the allele with the normal fruit weight-indicating base.

[0227] This suggests that by performing competitive allele-specific PCR using the first primer set, it is possible to confirm the number of alleles containing mutant fruit weight-indicating bases and identify persimmon trees with heavy fruit weight.

[0228] Next, we will explain the results of competitive allele-specific PCR performed on population A of the five seedling populations mentioned above using the second primer set mentioned above.

[0229] Figure 9 is a graph plotting the fluorescence intensity of the results of competitive allele-specific PCR performed using primer set 2. In Figure 9, ND01 to ND08 are negative controls in which template DNA was not included in the reaction composition of the competitive allele-specific PCR. As shown in Figure 9, the results of competitive allele-specific PCR showed that the fluorescence intensity at two wavelengths differed between population B, which contained one allele with a mutant base indicative of floury texture, and population A, which contained only an allele with the normal base indicative of fruit weight.

[0230] This suggests that by performing competitive allele-specific PCR using the second primer set, it is possible to confirm the number of alleles containing mutant fruit weight-indicating bases and identify persimmons with low flouriness.

[0231] Next, we will explain the results of competitive allele-specific PCR performed on the five seedling populations mentioned above using the third primer set mentioned above.

[0232] FIG. 10 is a graph plotting the fluorescence intensity of the results of competitive allele-specific PCR performed using the third primer set. As shown in Figure 10, the results of competitive allele-specific PCR showed that the fluorescence intensities at two wavelengths differed between population C, which contains one allele with a mutant base that indicates damage from insect pests (the base at the position corresponding to the base at position 35347759 mentioned above), and population B, which contains only alleles with the normal base that indicates damage from insect pests.

[0233] This suggests that by performing competitive allele-specific PCR using the third primer set, it is possible to confirm the number of alleles containing mutant bases that indicate damage from insect pests and identify persimmon trees that have less damage to their branches and trunks.

[0234] Next, we will explain the results of competitive allele-specific PCR performed on the five seedling populations mentioned above using the fourth primer set mentioned above.

[0235] FIG. 11 is a graph plotting the fluorescence intensity of the results of competitive allele-specific PCR performed using the fourth primer set. As shown in Figure 11, the results of competitive allele-specific PCR showed that the fluorescence intensity at two wavelengths differed between population C, which contained one allele with a mutant base that indicates damage from plant damage (the base at the position corresponding to the base at position 11610850 and the base at the position corresponding to the base at position 11610854 mentioned above), and population B, which contained only an allele with a normal base that indicates damage from plant damage.

[0236] This suggests that by performing competitive allele-specific PCR using the fourth primer set, it is possible to confirm the number of alleles containing mutant bases that indicate damage caused by insects, and to identify persimmon trees that are less susceptible to damage to their trunks and branches by insects. [Industrial Applicability]

[0237] According to the present invention, by determining the fruit weight-indicating bases of the persimmon to be identified, it is possible to identify whether the persimmon to be identified is a plant with heavy fruit weight, and therefore the present invention is applicable industrially.

Claims

1. A method for identifying persimmon trees with heavy fruit weight, comprising: The method includes a step of determining the type of a fruit weight-indicating base, which is a base at a position corresponding to the base located at position 9,497,243 from the 5' end of chromosome 15X on the reference genome of the persimmon, among bases in the DNA of the persimmon to be identified; This is an identification method in which, if the result of the discrimination shows that there is one or more alleles in which the fruit weight-indicating base is adenine, the persimmon to be identified is identified as a persimmon with a heavy fruit weight.

2. In the step of identifying the type of the fruit weight-indicating base, a competitive allele-specific PCR is performed using DNA of the persimmon to be identified as a template and a first primer, a second primer, and a third primer; the first primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and a third base sequence following the 3' end of the one base sequence; the third base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 16, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 16, the second primer comprises the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a fourth base sequence following the 3' end of the other base sequence; the fourth base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 17, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 17, 2. The method according to claim 1, wherein the third primer is a base sequence of 18 to 26 consecutive bases in the base sequence shown in SEQ ID NO:

18.

3. The method includes a step of determining the type of a base that indicates malodor, which is a base at a position corresponding to the base located at position 22156180 from the 5' end of chromosome 3 on the reference genome of the persimmon, among bases in the DNA of the persimmon to be identified; 2. The method of claim 1, wherein if the discrimination result shows that there is one or more alleles in which the floury-indicating base is thymine, the persimmon is identified as having low floury quality inside the fruit.

4. In the step of identifying the type of the powdery-indicative base, a competitive allele-specific PCR is carried out using DNA of the persimmon to be identified as a template and a fourth primer, a fifth primer, and a sixth primer; the fourth primer comprises a base sequence selected from the group consisting of a first base sequence shown in SEQ ID NO: 13 and a fifth base sequence shown in SEQ ID NO: 15 and a second base sequence shown in SEQ ID NO: 14, and a sixth base sequence following the 3' end of any of the base sequences; the sixth base sequence is a base sequence of 25 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 19, and includes a base sequence consisting of the fifth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 19, when the fourth primer has one base sequence selected from the group, the fifth primer consists of the second base sequence and a seventh base sequence following the 3'-end of the second base sequence, and when the fourth primer has the second base sequence, the fifth primer consists of the first base sequence and a seventh base sequence following the 3'-end of the first base sequence, the seventh base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 20, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 20, 4. The method according to claim 3, wherein the sixth primer is a base sequence of 18 to 23 consecutive bases in the base sequence shown in SEQ ID NO:

21.

5. The method includes a step of determining a trunk diameter suggestive base sequence, which is a base sequence of a region corresponding to the base sequence located at positions 12281277 to 12281283 from the 5' end of chromosome 5 on the reference genome of the persimmon, from the base sequence of the DNA of the persimmon to be identified; The method of claim 1, wherein the fewer alleles in which the main trunk diameter suggestive base sequence is CTTTTTA, the larger the diameter of the main trunk of the persimmon tree is identified as the result of discrimination.

6. the method comprises a step of determining the type of one or more bases selected from the group consisting of a base at a position corresponding to the base located at position 2032946, a base at a position 35347759, a base at a position corresponding to the base located at position 2032922, a base at a position corresponding to the base located at position 11610850, a base at a position corresponding to the base located at position 11610854, a base at a position corresponding to the base located at position 26462429, a base at a position corresponding to the base located at position 27467699, a base at a position corresponding to the base located at position 27467690, and a base at a position corresponding to the base located at position 997141 from the 5' end of chromosome 9 on a reference genome of persimmon to be identified, If the discrimination result shows that there is one or more alleles in which one or more bases selected from the group are bases indicative of a persimmon tree with less damage to its branches and trunks, the persimmon is identified as one with less damage to its branches and trunks by insects; The bases that indicate that the persimmon has little damage to the trunk and branches are: The base at the position corresponding to the base located at position 2,032,946 from the 5' end, the base at the position corresponding to the base located at position 35,347,759, the base at the position corresponding to the base located at position 1,161,0854, and the base at the position corresponding to the base located at position 997,141 are adenine, The base at the position corresponding to the base located at position 11610850 from the 5' end, the base at the position corresponding to the base located at position 26462429, the base at the position corresponding to the base located at position 27467699, and the base at the position corresponding to the base located at position 27467690 are thymine, The method of claim 1 , wherein the base at the position corresponding to the 2,032,922nd base from the 5′ end is guanine.

7. in the step of discriminating the type of one or more bases selected from the group, the bases of chromosome 9 whose type is to be discriminated are one or more bases selected from the group consisting of the base at a position corresponding to the base located at position 35347759 from the 5' end on the reference genome of the persimmon to be identified, the base at a position corresponding to the base located at position 11610850, and the base at a position corresponding to the base located at position 11610854, among the bases of the DNA of the persimmon to be discriminated, When determining the type of base at a position corresponding to the 35,347,759th base from the 5'-end, performing competitive allele-specific PCR using DNA of the persimmon to be identified as a template and the seventh, eighth, and ninth primers; the seventh primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and an eighth base sequence following the 3' end of the one base sequence; the eighth base sequence is a base sequence of 26 to 31 consecutive bases in the base sequence shown in SEQ ID NO: 22, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 22, the eighth primer comprises the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a ninth base sequence following the 3' end of the other base sequence; the ninth base sequence is a base sequence of 23 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 23, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 23; the ninth primer is a base sequence of 20 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 24, When determining the type of one or both of the base at the position corresponding to the base located at the 11610850th position from the 5'-end and the base at the position corresponding to the base located at the 11610854th position from the 5'-end, performing competitive allele-specific PCR using DNA of a persimmon to be identified as a template and using the tenth primer, the eleventh primer, and the twelfth primer; the tenth primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and a tenth base sequence following the 3' end of the one base sequence; the 10th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 25, and includes a base sequence consisting of the 6th and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 25; the 11th primer comprises the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and an 11th base sequence following the 3' end of the other base sequence; the 11th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 26, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 26; 7. The method according to claim 6, wherein the twelfth primer is a base sequence of 19 to 27 consecutive bases in the base sequence shown in SEQ ID NO:

27.

8. An identification kit for identifying persimmons with heavy fruit weight, a primer set including a first primer, a second primer, and a third primer; the first primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and a third base sequence following the 3' end of the one base sequence; the third base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 16, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 16, the second primer comprises the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a fourth base sequence following the 3' end of the other base sequence; the fourth base sequence is a base sequence of 20 to 25 consecutive bases in the base sequence shown in SEQ ID NO: 17, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 17, an identification kit, wherein the third primer is a base sequence of 18 to 26 consecutive bases in the base sequence shown in SEQ ID NO: 18;

9. further comprising a primer set comprising a fourth primer, a fifth primer, and a sixth primer; the fourth primer comprises a base sequence selected from the group consisting of a first base sequence shown in SEQ ID NO: 13 and a fifth base sequence shown in SEQ ID NO: 15 and a second base sequence shown in SEQ ID NO: 14, and a sixth base sequence following the 3' end of any of the base sequences; the sixth base sequence is a base sequence of 25 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 19, and includes a base sequence consisting of the fifth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 19, when the fourth primer has one base sequence selected from the group, the fifth primer consists of the second base sequence and a seventh base sequence following the 3'-end of the second base sequence, and when the fourth primer has the second base sequence, the fifth primer consists of the first base sequence and a seventh base sequence following the 3'-end of the first base sequence, the seventh base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 20, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 20, 9. The identification kit according to claim 8, wherein the sixth primer is a base sequence of 18 to 23 consecutive bases in the base sequence shown in SEQ ID NO:

21.

10. The nucleic acid sequence further includes either or both of a primer set including a seventh primer, an eighth primer, and a ninth primer, and a primer set including a tenth primer, an eleventh primer, and a twelfth primer; the seventh primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and an eighth base sequence following the 3' end of the one base sequence; the eighth base sequence is a base sequence of 26 to 31 consecutive bases in the base sequence shown in SEQ ID NO: 22, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 22, the eighth primer comprises the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and a ninth base sequence following the 3' end of the other base sequence; the ninth base sequence is a base sequence of 23 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 23, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 23; the ninth primer is a base sequence of 20 to 28 consecutive bases in the base sequence shown in SEQ ID NO: 24, the tenth primer comprises one of a first base sequence shown in SEQ ID NO: 13 and a second base sequence shown in SEQ ID NO: 14, and a tenth base sequence following the 3' end of the one base sequence; the 10th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 25, and includes a base sequence consisting of the 6th and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 25; the 11th primer comprises the other base sequence of the first base sequence shown in SEQ ID NO: 13 and the second base sequence shown in SEQ ID NO: 14, and an 11th base sequence following the 3' end of the other base sequence; the 11th base sequence is a base sequence of 24 to 29 consecutive bases in the base sequence shown in SEQ ID NO: 26, and includes a base sequence consisting of the sixth and subsequent bases from the 5' end of the base sequence shown in SEQ ID NO: 26; 9. The identification kit according to claim 8, wherein the twelfth primer is a base sequence of 19 to 27 consecutive bases in the base sequence shown in SEQ ID NO:27.

Images