Method for determining chrysanthemum white rust resistance, production method, molecular marker, gene associated with chrysanthemum white rust resistance, and chrysanthemum plants resistant to chrysanthemum white rust

Abstract

To provide a method for determining resistance to Chrysanthemum white rust in a Chrysanthemum plant, and a method for producing a Chrysanthemum plant having resistance to Chrysanthemum white rust.SOLUTION: The present invention discloses a method for determining resistance to Chrysanthemum white rust. In a Chrysanthemum plant, the genotype of a gene determining resistance or sensitivity to Chrysanthemum white rust, included in the genome of the Chrysanthemum plant, is determined; (i) an SNP-corresponding base itself, (ii) SNP-containing contiguous polynucleotide, or (iii) contiguous polynucleotides in the region between two SNPs are amplified as the molecular marker; and the resistance to Chrysanthemum white rust in the Chrysanthemum plant is determined. There is also provided a method for producing a Chrysanthemum plant having resistance to Chrysanthemum white rust, including the step of identifying the Chrysanthemum plant having resistance to Chrysanthemum white rust.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for determining chrysanthemum white rust resistance, a production method, a molecular marker, a gene associated with chrysanthemum white rust resistance, and a chrysanthemum plant resistant to chrysanthemum white rust. [Background technology]

[0002] Chrysanthemum white rust is a disease that causes pale yellow bumps or small pinkish pustules on the underside of chrysanthemum leaves. This disease is caused by the fungus Puccinia horiana, which was first discovered in Japan in 1895 and has now spread worldwide. Puccinia horiana infects many chrysanthemum species. Chrysanthemums are one of the most important ornamental plants in the world, and are cultivated as cut flowers, potted plants, and garden plants. Chrysanthemum white rust causes significant economic losses in commercial chrysanthemum production, making control techniques essential.

[0003] Chemical control of Puccinia horiana is difficult due to the increasing number of fungicide-resistant strains and the decreasing number of registered fungicides (Non-Patent Document 1). Furthermore, environmental control, such as reducing humidity, is not always possible, especially in open-field or semi-covered cultivation.

[0004] One of the most effective methods for disease control is the use of resistant cultivars. Resistant chrysanthemum cultivars have been extensively studied. Such studies include the elucidation of the inheritance pattern of chrysanthemum white rust resistance, and many resistant cultivars with a single dominant gene have been identified (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Cook, RTA, Plant Pathol. 50: 792,2001 [Non-patent document 2] De Jong, J. and W. Rademaker, Euphytica,35: 945-952,1986 Summary of the Invention [Problem to be solved by the invention]

[0006] As described in Non-Patent Documents 1 and 2, various studies have been conducted on chrysanthemum white rust, but to date, no DNA markers associated with chrysanthemum white rust resistance that can be used for breeding chrysanthemums for white rust resistance have been known. Chrysanthemum morifolium, a chrysanthemum cultivar, has an autohexaploid genome, and in this complex genome, if monogenic inheritance at two alleles is assumed, a total of seven different allele patterns can be generated at a single locus. Due to this complex segregation pattern of markers, the development of DNA markers associated with chrysanthemum white rust resistance has been delayed.

[0007] DNA markers associated with chrysanthemum white rust resistance are useful for marker-assisted breeding (MAS) in chrysanthemum white rust resistance breeding and for determining chrysanthemum white rust resistance in Chrysanthemum plants.

[0008] An object of one aspect of the present invention is to provide a technique for determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum. [Means for solving the problem]

[0009] A method for determining chrysanthemum white rust resistance according to one embodiment of the present invention comprises detecting, in a plant of the genus Chrysanthemum, any one of the following bases (a) or (b), or (c) to (j) itself (SNP), or a consecutive polynucleotide containing such base: (a) a base corresponding to the 74th base shown in SEQ ID NO: 1; (b) a base corresponding to the 63rd base set forth in SEQ ID NO: 2; (c) a base corresponding to at least one of the 29th, 30th, 63rd, 65th, 67th, 68th, and 85th bases set forth in SEQ ID NO: 9; (d) a base corresponding to at least one of the 21st and 24th bases set forth in SEQ ID NO: 10; (e) a base corresponding to at least one of the 22nd, 23rd, 52nd, 66th, and 71st bases set forth in SEQ ID NO: 11; (f) a base corresponding to at least one of the 19th and 102nd bases set forth in SEQ ID NO: 12; (g) a base corresponding to at least one of the 29th, 48th, 50th, and 73rd bases set forth in SEQ ID NO: 13; (h) a base corresponding to at least one of the 4th and 112th bases set forth in SEQ ID NO: 14; (i) a base corresponding to at least one of the 27th and 40th bases set forth in SEQ ID NO: 15; (j) a base corresponding to at least one of the 21st and 70th bases set forth in SEQ ID NO: 16; as a molecular marker for chrysanthemum white rust resistance.

[0010] One embodiment of the present invention relates to a method for producing a Chrysanthemum plant that is resistant to chrysanthemum white rust, the method comprising: a crossbreeding step of crossbreeding a Chrysanthemum plant that is resistant to chrysanthemum white rust with another Chrysanthemum plant; and a discrimination step of discriminating a Chrysanthemum plant that is resistant to chrysanthemum white rust from the Chrysanthemum plant obtained by the crossbreeding step or a progeny line of Chrysanthemum plants using the method for determining chrysanthemum white rust resistance described above.

[0011] A molecular marker for chrysanthemum white rust resistance in a plant of the genus Chrysanthemum according to one embodiment of the present invention comprises any one of the following bases (a) or (b), or (c) to (j) themselves (SNP), or a consecutive polynucleotide containing the corresponding base: (a) a base corresponding to the 74th base shown in SEQ ID NO: 1; (b) a base corresponding to the 63rd base set forth in SEQ ID NO: 2; (c) a base corresponding to at least one of the 29th, 30th, 63rd, 65th, 67th, 68th, and 85th bases set forth in SEQ ID NO: 9; (d) a base corresponding to at least one of the 21st and 24th bases set forth in SEQ ID NO: 10; (e) a base corresponding to at least one of the 22nd, 23rd, 52nd, 66th, and 71st bases set forth in SEQ ID NO: 11; (f) a base corresponding to at least one of the 19th and 102nd bases set forth in SEQ ID NO: 12; (g) a base corresponding to at least one of the 29th, 48th, 50th, and 73rd bases set forth in SEQ ID NO: 13; (h) a base corresponding to at least one of the 4th and 112th bases set forth in SEQ ID NO: 14; (i) a base corresponding to at least one of the 27th and 40th bases set forth in SEQ ID NO: 15; (j) a base corresponding to at least one of the 21st and 70th bases set forth in SEQ ID NO: 16; It is a molecular marker.

[0012] A chrysanthemum white rust resistance-associated gene according to one embodiment of the present invention is linked to the molecular marker and is located within 3 cM of the molecular marker on the genome.

[0013] A Chrysanthemum plant according to one aspect of the present invention is a chrysanthemum white rust-resistant plant that has the chrysanthemum white rust resistance-associated gene and is obtained by the production method described above. [Effects of the Invention]

[0014] According to one aspect of the present invention, a technique for determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum can be provided. [Brief explanation of the drawings]

[0015] [Figure 1] This image compares the leaves of a white rust-resistant chrysanthemum cultivar with those infected with white rust. [Figure 2] 1 is a graph showing the results of an example. [Figure 3] FIG. 1 shows the genetic distance between the chrysanthemum white rust resistance gene and molecular markers. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. All of the documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more (including A and greater than A) to B or less (including B and less than B)."

[0017] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Here, nucleic acids can exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). As used herein, bases are represented by the single-letter symbols defined by IUPAC and IUB, as appropriate.

[0018] One aspect of the present invention is a technique for determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum. In one aspect of the present invention, the white rust resistance in a plant of the genus Chrysanthemum is determined by determining the genotype of a chrysanthemum white rust resistance gene that determines resistance or susceptibility to chrysanthemum white rust and is present in the genome of the plant of the genus Chrysanthemum.

[0019] The chrysanthemum plant may be a plant of the genus Chrysanthemum, for example, a plant belonging to the Chrysanthemum species ( Chrysanthemum morifolium ). Among the Chrysanthemum species, it may be a plant classified as a spray chrysanthemum.

[0020] Furthermore, Chrysanthemum plants may be candidate plants for breeding material or plants obtained through a breeding process. Examples of candidate plants for breeding material include parent plants used in crossbreeding and plants used in molecular breeding using recombinant DNA technology. Examples of plants obtained through a breeding process include plants obtained by intrageneric hybridization of plants of the genus Chrysanthemum, plants obtained by intraspecific hybridization of plants belonging to the species Chrysanthemum, and their progeny lines. Furthermore, Chrysanthemum plants may be interspecific hybrids, such as hybrids between plants belonging to the species Chrysanthemum and wild species of Chrysanthemum, and their progeny lines. Furthermore, Chrysanthemum plants may be the "Aries" variety, or hybrids of plants of that variety with other plants belonging to the species Chrysanthemum, or their progeny lines.

[0021] In this specification, the term "plant" may refer to a part or the whole of a plant body. Examples of a part of a plant body include propagation materials (e.g., leaves, branches, seeds, etc.).

[0022] Chrysanthemum white rust refers to a disease in which lesions form on chrysanthemums due to infection with the fungus Puccinia horiana (hereinafter also referred to as chrysanthemum white rust fungus). Chrysanthemum white rust resistance refers to the ability to suppress or inhibit the formation and spread of lesions caused by infection with the chrysanthemum white rust fungus. Therefore, a plant exhibiting chrysanthemum white rust resistance is one in which, even when infected or inoculated with the chrysanthemum white rust fungus, no or only a few lesions develop, or the lesions that do develop do not spread. As shown in Figure 1 , lesions are observed on leaves infected with chrysanthemum white rust in susceptible varieties, but no lesions are observed on leaves in resistant varieties.

[0023] Plants belonging to the Chrysanthemum species are autohexaploids with 54 chromosomes. The nucleotide sequence information of the genome of each chromosome in plants belonging to the Chrysanthemum species can be determined, for example, by referring to the nucleotide sequence information of the Chrysanthemum seticus genome (CSE_r1.0). The nucleotide sequence information of the Chrysanthemum seticus genome is available, for example, from the MumGARDEN website (http: / / mum-garden.kazusa.or.jp).

[0024] [Molecular markers for white rust resistance in chrysanthemum] A molecular marker according to one embodiment of the present invention is a molecular marker for chrysanthemum white rust resistance in a plant of the genus Chrysanthemum, and is a SNP of any of the following bases (a) or (b), or (c) to (j), or a consecutive polynucleotide containing the corresponding base: (a) a base corresponding to the 74th base shown in SEQ ID NO: 1; (b) a base corresponding to the 63rd base shown in SEQ ID NO: 2; (c) a base corresponding to at least one of the 29th, 30th, 63rd, 65th, 67th, 68th, and 85th bases shown in SEQ ID NO: 9; (d) a base corresponding to at least one of the 21st and 24th bases shown in SEQ ID NO: 10; (e) a base corresponding to the 22nd base shown in SEQ ID NO: 11; (f) a base corresponding to at least one base among the 19th, 23rd, 52nd, 66th, and 71st bases shown in SEQ ID NO: 12; (g) a base corresponding to at least one base among the 29th, 48th, 50th, and 73rd bases shown in SEQ ID NO: 13; (h) a base corresponding to at least one base among the 4th and 112th bases shown in SEQ ID NO: 14; (i) a base corresponding to at least one base among the 27th and 40th bases shown in SEQ ID NO: 15; and (j) a base corresponding to at least one base among the 21st and 70th bases shown in SEQ ID NO: 16.

[0025] A molecular marker according to one embodiment of the present invention is a molecular marker that specifies a chrysanthemum white rust resistance gene in a plant of the genus Chrysanthemum. Such molecular markers include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, CAPS markers, etc. The SNP marker may be a single SNP or a combination of two or more SNPs. The molecular marker according to one embodiment of the present invention can be used to determine chrysanthemum white rust resistance in a plant of the genus Chrysanthemum.

[0026] A molecular marker according to one embodiment of the present invention may be (i) the base itself corresponding to the SNP, (ii) a contiguous polynucleotide containing the SNP, or (iii) a contiguous polynucleotide of the region between two SNPs.

[0027] (i: SNP marker) A molecular marker according to one embodiment of the present invention may be an SNP marker. SNP refers to a DNA polymorphism in which a single base mutation is observed within a specific region in the DNA base sequence.

[0028] The "base at position Y (when X=1, Y is 74; when X=2, Y is 63) of the base sequence (assumed to be base sequence X) represented by SEQ ID NO: X (X=1 to 2)" refers to the SNP marker described in this example. Also, the "base at position Y (when X=9, Y is 29, 30, 63, 65, 67, 68, or 85; when X=10, Y is 21 or 24; when X=11, Y is 22, 23, 52, 66, or 71; when X=12, Y is 19 or 102; when X=13, Y is 29, 48, 50, or 73; when X=14, Y is 4 or 112; when X=15, Y is 27 or 40; when X=16, Y is 21 or 70) of the base sequence (assumed to be base sequence X) represented by SEQ ID NO: X (X=9 to 16)" refers to the SNP marker described in this example. The "base corresponding to the Yth base in the base sequence represented by SEQ ID NO: X" is an SNP marker that can be identified with the SNP marker described in this example. The base sequence X is a base sequence derived from a reference Chrysanthemum plant ("Aries"), and other Chrysanthemum plants may contain a different base sequence in addition to the SNP marker portion.

[0029] That is, when a nucleotide sequence X' corresponding to the nucleotide sequence X (i.e., a nucleotide sequence that is highly conserved among plants) exists in another plant of the genus Chrysanthemum, the "nucleotide corresponding to the Yth nucleotide of the nucleotide sequence represented by SEQ ID NO: X" refers to the nucleotide in the nucleotide sequence X' that is determined to correspond to the Yth nucleotide by a method such as a homology search. For example, the nucleotide sequences represented by the polynucleotides described in (a2) and (a3), (b2) and (b3), and (c2) to (j2) and (c3) to (j3) described below are examples of the nucleotide sequence X'.

[0030] Molecular markers according to one embodiment of the present invention include, for example, SCSE_SC004884.1_65872 and SCSE_SC000716.1_75925 described in this Example. Molecular markers according to one embodiment of the present invention include, for example, 46.1-19, 46.5-16, 47.0-3, 47.5-2, 48.0-5, 48.5-10, 49.0-15, and 50.0-3 described in this Example. SCSE_SC004884.1_65872 and SCSE_SC000716.1_75925, as well as 46.1-19, 46.5-16, 47.0-3, 47.5-2, 48.0-5, 48.5-10, 49.0-15, and 50.0-3 are SNP markers newly identified by the present inventors, and a person skilled in the art can identify the location of these SNP markers on the genome based on the nucleotide sequences of these SNP markers. 46.1-19, 46.5-16, 47.0-3, 47.5-2, 48.0-5, 48.5-10, 49.0-15, and 50.0-3 are located between SCSE_SC004884.1_65872 and SCSE_SC000716.1_75925 on the genome of Chrysanthemum.

[0031] SCSE_SC004884.1_65872 (hereinafter also referred to as SNP(a)) indicates a polymorphism in which the base corresponding to the 74th base in SEQ ID NO: 1 is C. In other words, when SNP(a) is C, a Chrysanthemum plant can be determined to have resistance to chrysanthemum white rust.

[0032] SCSE_SC000716.1_75925 (hereinafter also referred to as SNP(b)) indicates a polymorphism in which the base corresponding to the 63rd base in SEQ ID NO: 2 is C. In other words, when SNP(b) is C, a Chrysanthemum plant can be determined to have resistance to chrysanthemum white rust.

[0033] 46.1-19 (hereinafter also referred to as SNP(c)) indicates a polymorphism in which at least one of the following is present: the base corresponding to the 29th base in SEQ ID NO: 9 is A, the base corresponding to the 30th base is T, the base corresponding to the 63rd base is C, the base corresponding to the 65th base is T, the base corresponding to the 67th base is A, the base corresponding to the 68th base is C, and the base corresponding to the 85th base is G. That is, in SNP(c), when at least one of the following is present: the base corresponding to the 29th base in SEQ ID NO: 9 is A, the base corresponding to the 30th base is T, the base corresponding to the 63rd base is C, the base corresponding to the 65th base is T, the base corresponding to the 67th base is A, the base corresponding to the 68th base is C, and the base corresponding to the 85th base is G, the Chrysanthemum plant can be determined to have resistance to chrysanthemum white rust.

[0034] 46.5-16 (hereinafter also referred to as SNP(d)) indicates a polymorphism in which the base corresponding to the 21st base in SEQ ID NO: 10 is C and the base corresponding to the 24th base is at least one of T. That is, when in SNP(d), the base corresponding to the 21st base in SEQ ID NO: 10 is C and the base corresponding to the 24th base is at least one of T, the Chrysanthemum plant can be determined to be resistant to chrysanthemum white rust.

[0035] 47.0-3 (hereinafter also referred to as SNP(e)) indicates a polymorphism in which at least one of the bases corresponding to the 22nd base in SEQ ID NO: 11 is G, the base corresponding to the 23rd base is G, the base corresponding to the 52nd base is C, the base corresponding to the 66th base is G, and the base corresponding to the 71st base is G in SEQ ID NO: 11. That is, when SNP(e) is at least one of the bases corresponding to the 22nd base in SEQ ID NO: 11 is G, the base corresponding to the 23rd base is G, the base corresponding to the 52nd base is C, the base corresponding to the 66th base is G, and the base corresponding to the 71st base is G in SEQ ID NO: 11, the Chrysanthemum plant can be determined to be resistant to chrysanthemum white rust.

[0036] 47.5-2 (hereinafter also referred to as SNP(f)) indicates a polymorphism in which at least one of the bases corresponding to the 19th base and the 102nd base in SEQ ID NO: 12 is T and the base corresponding to the 19th base and the 102nd base in SEQ ID NO: 12 is T. In other words, when at least one of the bases corresponding to the 19th base and the 102nd base in SNP(f) is T, a Chrysanthemum plant can be determined to be resistant to chrysanthemum white rust.

[0037] 48.0-5 (hereinafter also referred to as SNP(g)) indicates a polymorphism in which at least one of the bases corresponding to the 29th base in SEQ ID NO: 13 is C, the base corresponding to the 48th base is T, the base corresponding to the 50th base is C, and the base corresponding to the 73rd base is G in SEQ ID NO: 13. That is, when SNP(g) is at least one of the bases corresponding to the 29th base in SEQ ID NO: 13 is C, the base corresponding to the 48th base is T, the base corresponding to the 50th base is C, and the base corresponding to the 73rd base is G in SEQ ID NO: 13, the Chrysanthemum plant can be determined to be resistant to chrysanthemum white rust.

[0038] 48.5-10 (hereinafter also referred to as SNP(h)) indicates a polymorphism in which the base corresponding to the fourth base in SEQ ID NO: 14 is A and the base corresponding to the 112th base is at least one of G. That is, when the base corresponding to the fourth base in SEQ ID NO: 14 in SNP(h) is A and the base corresponding to the 112th base is at least one of G, the Chrysanthemum plant can be determined to be resistant to chrysanthemum white rust.

[0039] 49.0-15 (hereinafter also referred to as SNP(i)) indicates a polymorphism in which the base corresponding to the 27th base in SEQ ID NO: 15 is A and the base corresponding to the 40th base is at least one of T. That is, when in SNP(i), the base corresponding to the 27th base in SEQ ID NO: 15 is A and the base corresponding to the 40th base is at least one of T, the Chrysanthemum plant can be determined to be resistant to chrysanthemum white rust.

[0040] 50.0-3 (hereinafter also referred to as SNP(j)) indicates a polymorphism in which the base corresponding to the 21st base in SEQ ID NO: 16 is A and the base corresponding to the 70th base is at least one of T. In other words, when in SNP(j), the base corresponding to the 21st base in SEQ ID NO: 16 is A and the base corresponding to the 70th base is at least one of T, the Chrysanthemum plant can be determined to be resistant to chrysanthemum white rust.

[0041] SNP(a) to SNP(j), which are molecular markers according to one embodiment of the present invention, are in a state of linkage disequilibrium, for example, a state of linkage disequilibrium with a linkage disequilibrium coefficient of 0.9 or more. Preferably, the molecular marker according to one embodiment of the present invention is located at a short distance to a gene associated with chrysanthemum white rust resistance on the genome of a plant of the genus Chrysanthemum, thereby enabling more accurate determination of the presence or absence of resistance to chrysanthemum white rust in a test plant of the genus Chrysanthemum. Specifically, the molecular markers are preferably SNP(c) to SNP(j), more preferably SNP(c) to SNP(i), and most preferably SNP(e) to SNP(h). Details of the distances of SNP(a) to SNP(j) to genes associated with chrysanthemum white rust resistance will be described later.

[0042] SNP(a) and SNP(b) may be used in combination as a molecular marker according to one embodiment of the present invention. That is, when SNP(a) is C and SNP(b) is C, the plant may be determined to be resistant to chrysanthemum white rust. That is, SNP(a) and SNP(b) may be analyzed as a haplotype block.

[0043] Furthermore, as a molecular marker according to one embodiment of the present invention, at least two of SNPs (a) to (j) may be used in combination. That is, at least two of the following SNPs (a') and (b') and (c') to (j'): (a') the base corresponding to the 74th base in SEQ ID NO: 1 is C; (b') the base corresponding to the 63rd base in SEQ ID NO: 2 is C; (c') at least one of the following: the base corresponding to the 29th base in SEQ ID NO: 9 is A, the base corresponding to the 30th base is T, the base corresponding to the 63rd base is C, the base corresponding to the 65th base is T, the base corresponding to the 67th base is A, the base corresponding to the 68th base is C, and the base corresponding to the 85th base is G; (d') at least one of the following: the base corresponding to the 21st base in SEQ ID NO: 10 is C, and the base corresponding to the 24th base is T; (e') the base corresponding to the 22nd base in SEQ ID NO: 11 is G, the base corresponding to the 23rd base is G, the base corresponding to the 52nd base is C, the base corresponding to the 66th base is G, and the base corresponding to the 71st base is G. The plant may be determined to have resistance to chrysanthemum white rust when the base corresponding to the 19th base in SEQ ID NO: 12 is at least one of G; (f') the base corresponding to the 19th base is T and the base corresponding to the 102nd base is at least one of T; (g') the base corresponding to the 29th base in SEQ ID NO: 13 is at least one of C, T, C, and G; (h') the base corresponding to the 4th base in SEQ ID NO: 14 is at least one of A and G; (i') the base corresponding to the 27th base in SEQ ID NO: 15 is at least one of A and T; or (j') the base corresponding to the 21st base in SEQ ID NO: 16 is at least one of A and T. That is, at least two of SNPs (a) to (j) may be analyzed as a haplotype block.

[0044] (ii: Polynucleotide containing SNP) A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (a)) containing SNP (a). Polynucleotide (a) is (a1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; (a2) a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 1 except for the 74th nucleotide, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum; or (a3) ​​a polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 1 except for the 74th nucleotide, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum. When the nucleotide corresponding to SNP (a) in polynucleotide (a) is C, the plant can be determined to have chrysanthemum white rust resistance.

[0045] The polynucleotide (a1) can be obtained, for example, from "Aries", which has resistance to chrysanthemum white rust, based on the base sequence shown in SEQ ID NO: 1.

[0046] The polynucleotide (a2) may have the bases corresponding to SNP (a) conserved in the base sequence shown in SEQ ID NO: 1, but may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions) in the remaining base sequence. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0047] The polynucleotide (a3) ​​may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence of the base sequence shown in SEQ ID NO: 1, while conserving the base corresponding to SNP (a). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0048] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (b)) containing SNP (b). Polynucleotide (b) is (b1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2; (b2) a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 2 except for the 63rd nucleotide, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum; or (b3) a polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 2 except for the 63rd nucleotide, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum. When the nucleotide corresponding to SNP (b) in polynucleotide (b) is C, the plant can be determined to be resistant to chrysanthemum white rust.

[0049] The polynucleotide (b1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO:2.

[0050] The polynucleotide (b2) may have the bases corresponding to SNP (b) conserved in the base sequence shown in SEQ ID NO: 2, but may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions) in the remaining base sequence. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0051] The polynucleotide (b3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence of the base sequence shown in SEQ ID NO: 2, while conserving the base corresponding to SNP (b). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0052] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide containing SNP(c) (hereinafter also referred to as polynucleotide(c)). Polynucleotide (c) is (c1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, (c2) a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted in the nucleotide sequence of SEQ ID NO: 9 except for the nucleotides at positions 29, 30, 63, 65, 67, 68, and 85, and capable of determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum, or (c3) a polynucleotide consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence of SEQ ID NO: 9 except for the nucleotides at positions 29, 30, 63, 65, 67, 68, and 85, and capable of determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum. The plant can be determined to be resistant to chrysanthemum white rust when the base corresponding to the 29th base in SEQ ID NO: 9, which corresponds to SNP (c) in polynucleotide (c), is A, the base corresponding to the 30th base is T, the base corresponding to the 63rd base is C, the base corresponding to the 65th base is T, the base corresponding to the 67th base is A, the base corresponding to the 68th base is C, and the base corresponding to the 85th base is G, at least one of these is present.

[0053] The polynucleotide (c1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO:9.

[0054] The polynucleotide (c2) may have the bases corresponding to SNP (c) conserved in the base sequence shown in SEQ ID NO: 9, but may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions) in the remaining base sequence. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0055] The polynucleotide (c3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 9, while conserving the base corresponding to SNP (c). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0056] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (d)) containing SNP (d). Polynucleotide (d) may be (d1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10; (d2) a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 10, except for the 21st and 24th nucleotides, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum; or (d3) a polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 10, except for the 21st and 24th nucleotides, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum. When the nucleotide corresponding to the 21st nucleotide of SEQ ID NO: 10, which corresponds to SNP (d), is at least one of C and T, the nucleotide corresponding to the 24th nucleotide, the plant can be determined to be resistant to chrysanthemum white rust.

[0057] The polynucleotide (d1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO:10.

[0058] The polynucleotide (d2) may have the bases corresponding to SNP (d) conserved in the base sequence shown in SEQ ID NO: 10, but may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions) in the remaining base sequence. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0059] The polynucleotide (d3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 10, while conserving the base corresponding to SNP (d). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0060] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide containing SNP(e) (hereinafter also referred to as polynucleotide(e)). Polynucleotide (e) is (e1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11, (e2) a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted in the nucleotide sequence of SEQ ID NO: 11 except for the 22nd, 23rd, 52nd, 66th, and 71st nucleotides, and capable of determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum, or (e3) a polynucleotide consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence of SEQ ID NO: 11 except for the 22nd, 23rd, 52nd, 66th, and 71st nucleotides, and capable of determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum. When polynucleotide (e) contains at least one of the following bases, which correspond to SNP (e): the base corresponding to the 22nd base in SEQ ID NO: 11 is G, the base corresponding to the 23rd base is G, the base corresponding to the 52nd base is C, the base corresponding to the 66th base is G, and the base corresponding to the 71st base is G, the plant can be determined to be resistant to chrysanthemum white rust.

[0061] The polynucleotide (e1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO:11.

[0062] The polynucleotide (e2) may have the base sequence shown in SEQ ID NO: 11 in which the base corresponding to SNP (e) is conserved, but the remaining base sequence may contain several (for example, 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions). The base sequence of such a polynucleotide will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP on the genome of the resistant plant.

[0063] The polynucleotide (e3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 11, while conserving the base corresponding to SNP (e). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0064] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (f)) containing SNP (f). Polynucleotide (f) is (f1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12; (f2) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12 in which one or more nucleotides have been substituted, deleted, added, or inserted with respect to the nucleotide sequence other than the 19th and 102nd nucleotides, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum; or (f3) a polynucleotide consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 12 in which the nucleotide other than the 19th and 102nd nucleotides has been identified, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum. When at least one of the nucleotides corresponding to the 19th nucleotide and the 102nd nucleotide in SEQ ID NO: 12, which correspond to SNP (f), in polynucleotide (f) is T, the plant can be determined to be resistant to chrysanthemum white rust.

[0065] The polynucleotide (f1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO:12.

[0066] The polynucleotide (f2) may have the bases corresponding to SNP (f) conserved in the base sequence shown in SEQ ID NO: 12, but may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions) in the remaining base sequence. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0067] The polynucleotide (f3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 12, while conserving the base corresponding to SNP (f). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0068] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (g)) containing SNP (g). Polynucleotide (g) is (g1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13, (g2) a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted in the nucleotide sequence of SEQ ID NO: 13 except for the 29th, 48th, 50th, and 73rd nucleotides, and capable of determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum, or (g3) a polynucleotide consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence of SEQ ID NO: 13 except for the 29th, 48th, 50th, and 73rd nucleotides, and capable of determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum. When the base corresponding to the 29th base in SEQ ID NO: 13, which corresponds to SNP (g) in polynucleotide (g), is C, the base corresponding to the 48th base is T, the base corresponding to the 50th base is C, and the base corresponding to the 73rd base is G, the plant can be determined to be resistant to chrysanthemum white rust.

[0069] The polynucleotide (g1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO:13.

[0070] The polynucleotide (g2) may have the base sequence shown in SEQ ID NO: 13 in which the base corresponding to SNP (g) is conserved, but the remaining base sequence may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions). The base sequence of such a polynucleotide will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0071] The polynucleotide (g3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 13, while conserving the base corresponding to SNP (g). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0072] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (h)) containing SNP (h). Polynucleotide (h) is (h1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14; (h2) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14 in which one or more bases have been substituted, deleted, added, or inserted relative to the nucleotide sequence other than the 4th and 112th bases, and capable of determining chrysanthemum white rust resistance in plants of the genus Chrysanthemum; or (h3) a polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 14 in which the nucleotide other than the 4th and 112th bases has at least one of A and G, which correspond to SNP (h) in polynucleotide (h), and the plant can be determined to be resistant to chrysanthemum white rust.

[0073] The polynucleotide (h1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO:14.

[0074] The polynucleotide (h2) may have the bases corresponding to SNP (h) conserved in the base sequence shown in SEQ ID NO: 14, but may contain several (e.g., 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions) in the remaining base sequence. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0075] The polynucleotide (h3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 14, while conserving the base corresponding to SNP (h). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0076] A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (i)) containing SNP(i). Polynucleotide (i) is (i1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 15; (i2) a polynucleotide consisting of a nucleotide sequence in which one or more nucleotides have been substituted, deleted, added, or inserted relative to the nucleotide sequence of SEQ ID NO: 15, except for the 27th and 40th nucleotides, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum; or (i3) a polynucleotide consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence of SEQ ID NO: 15, except for the 27th and 40th nucleotides, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum. When the nucleotide corresponding to the 27th nucleotide of SEQ ID NO: 15, which corresponds to SNP(i), is at least one of A and T, the nucleotide corresponding to the 40th nucleotide, the plant can be determined to be resistant to chrysanthemum white rust.

[0077] The polynucleotide (i1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO: 15.

[0078] The polynucleotide (i2) may have the bases corresponding to SNP (i) conserved in the base sequence shown in SEQ ID NO: 15, but may contain modifications (substitutions, deletions, insertions, or additions) of several (for example, 1 to 10, preferably 1 to 5, and more preferably 1, 2, or 3) bases in the remaining base sequence. The base sequences of such polynucleotides will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP in the genome of the resistant plant.

[0079] The polynucleotide (i3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 15, while conserving the base corresponding to SNP (i). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0080] A molecular marker according to one embodiment of the present invention may be a contiguous polynucleotide (hereinafter also referred to as polynucleotide (j)) containing SNP(j). Polynucleotide (j) is (j1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (j2) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16 in which one or more bases have been substituted, deleted, added, or inserted relative to the nucleotide sequence other than the 21st and 70th bases, and capable of determining chrysanthemum white rust resistance in a plant of the genus Chrysanthemum; or (j3) a polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 16 in which the nucleotide other than the 21st and 70th bases has at least one of A and T, which correspond to SNP(j) in polynucleotide (j), and the plant can be determined to have chrysanthemum white rust resistance.

[0081] The polynucleotide (j1) can be obtained, for example, from the resistant plant "Aries" based on the base sequence shown in SEQ ID NO: 16.

[0082] The polynucleotide (j2) may have the base sequence shown in SEQ ID NO: 16 in which the base corresponding to SNP (j) is conserved, but the remaining base sequence may contain several (for example, 1 to 10, preferably 1 to 5, more preferably 1, 2, or 3) base modifications (substitutions, deletions, insertions, or additions). The base sequence of such a polynucleotide will be clear to those skilled in the art, and can be determined by referring to the genome sequence of the Chrysanthemum species registered in the above-mentioned database, or by decoding the base sequence of the region adjacent to the SNP on the genome of the resistant plant.

[0083] The polynucleotide (j3) may have, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the remaining base sequence in the base sequence shown in SEQ ID NO: 16, while conserving the base corresponding to SNP (j). Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0084] Polynucleotide (a) and polynucleotide (b) may be PCR amplification products amplified by a primer set that amplifies a region containing SNP (a) or SNP (b), and polynucleotides (c) to (j) may be PCR amplification products amplified by a primer set that amplifies a region containing SNP (c) to SNP (j).

[0085] Polynucleotide (a) and polynucleotide (b) may be used in combination as a molecular marker according to one embodiment of the present invention. That is, when the base corresponding to SNP (a) in polynucleotide (a) is C and the base corresponding to SNP (b) in polynucleotide (b) is C, the plant may be determined to be resistant to chrysanthemum white rust. That is, polynucleotide (a) and polynucleotide (b) may be analyzed as a haplotype block.

[0086] As a molecular marker according to one embodiment of the present invention, at least two of the polynucleotides (a) to (j) may be used in combination. That is, SNP(a) to SNP(j) in polynucleotide (a) to polynucleotide (j) are at least two of the following (a') to (j'): (a') the base corresponding to the 74th base shown in SEQ ID NO: 1 is C; (b') the base corresponding to the 63rd base shown in SEQ ID NO: 2 is C; (c') at least one of the base corresponding to the 29th base shown in SEQ ID NO: 9 is A, the base corresponding to the 30th base is T, the base corresponding to the 63rd base is C, the base corresponding to the 65th base is T, the base corresponding to the 67th base is A, the base corresponding to the 68th base is C, and the base corresponding to the 85th base is G; (d') at least one of the base corresponding to the 21st base shown in SEQ ID NO: 10 is C and the base corresponding to the 24th base is T; (e') the base corresponding to the 22nd base shown in SEQ ID NO: 11 is G, the base corresponding to the 23rd base is G, the base corresponding to the 52nd base is C, and the base corresponding to the 66th base shown in SEQ ID NO: 12 is G The plant may be determined to have resistance to chrysanthemum white rust when the polynucleotides (a) to (j) are at least one of the following: (f') the base corresponding to the 19th base in SEQ ID NO: 12 is T and the base corresponding to the 102nd base is T; (g') the base corresponding to the 29th base in SEQ ID NO: 13 is C, the base corresponding to the 48th base is T, the base corresponding to the 50th base is C, and the base corresponding to the 73rd base is G; (h') the base corresponding to the 4th base in SEQ ID NO: 14 is A and the base corresponding to the 112th base is G; (i') the base corresponding to the 27th base in SEQ ID NO: 15 is A and the base corresponding to the 40th base is T; or (j') the base corresponding to the 21st base in SEQ ID NO: 16 is A and the base corresponding to the 70th base is T. That is, at least two of the polynucleotides (a) to (j) may be analyzed as haplotype blocks.

[0087] (iii: Polynucleotide between two SNPs) A molecular marker according to one embodiment of the present invention may be a polynucleotide (hereinafter also referred to as polynucleotide (ab)) comprising a continuous region between SNP (a) and SNP (b). Polynucleotide (ab) preferably comprises both or either of SNP (a) and SNP (b). Since SNP (c) to SNP (j) are located between SNP (a) and SNP (b) on the genome of a Chrysanthemum plant, SNP (c) to SNP (j) are included in polynucleotide (ab). Polynucleotide (ab) can be obtained, for example, by referencing the region between the corresponding sites of SNP (a) and SNP (b) in a resistant plant. The nucleotide sequence of polynucleotide (ab) at least partially matches that of the resistant plant.

[0088] The method for determining chrysanthemum white rust resistance in Chrysanthemum plants using the above-mentioned molecular markers is not particularly limited, and for example, known SNP analysis methods for detecting SNPs can be used. Such known SNP analysis methods include SNP analysis methods that involve detecting SNPs in PCR-amplified fragments of a test Chrysanthemum plant. Note that the above-mentioned molecular markers only need to be found in at least one of the six genomes of a hexaploid Chrysanthemum plant, and if at least one of the six genomes is of a resistant genotype, the plant can be determined to be resistant.

[0089] That is, a primer set that amplifies a region containing SNP (a) or SNP (b) may be used to amplify the region in the DNA of a test Chrysanthemum plant. Alternatively, a primer set that amplifies a region containing at least one of SNPs (c) to (j) may be used to amplify the region in the DNA of a test Chrysanthemum plant. Amplification of the region in the DNA of a test Chrysanthemum plant can be performed by polymerase chain reaction (PCR) using DNA extracted from the test Chrysanthemum plant as a template and primers that amplify the region containing the SNP. The base (genotype) of the SNP in the resulting amplified fragment is then determined, and chrysanthemum white rust resistance in the Chrysanthemum plant is assessed based on data showing the relationship between the determined base (genotype) and chrysanthemum white rust resistance.

[0090] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment containing the target SNP, and the primer set may be designed to shorten the length of the amplified fragment. For example, the primer set is designed so that the length of the primer-amplified fragment is preferably 200 base pairs (bp) or less, 150 bp or less, 120 bp or less, or 100 bp or less. The primer set includes a first primer that is a forward primer and a second primer that is a reverse primer. The length of these primers may be, for example, 15 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, or 19 bp or more, or may be 50 bp or less, 40 bp or less, or 30 bp or less.

[0091] A primer set that amplifies a region containing SNP(a) is, for example, a primer set consisting of a first primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 3 and a second primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 4. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 5 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, and the base corresponding to SNP(a) can be detected.

[0092] A primer set that amplifies a region containing SNP(b) is, for example, a primer set consisting of a third primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 6 and a fourth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 7. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 8 can be obtained in Chrysanthemum plants that are resistant to chrysanthemum white rust, and the base corresponding to SNP(b) can be detected.

[0093] A primer set that amplifies a region containing SNP(c) is, for example, a primer set consisting of a fifth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 17 and a sixth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 18. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 19 can be obtained in Chrysanthemum plants that are resistant to chrysanthemum white rust, allowing the detection of bases corresponding to SNP(c).

[0094] A primer set that amplifies a region containing SNP(d) is, for example, a primer set consisting of a seventh primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 20 and an eighth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 21. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 22 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, and the base corresponding to SNP(d) can be detected.

[0095] A primer set that amplifies a region containing SNP(e) is, for example, a primer set consisting of a ninth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 23 and a tenth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 24. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 25 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, and the base corresponding to SNP(e) can be detected.

[0096] A primer set that amplifies a region containing SNP(f) is, for example, a primer set consisting of an eleventh primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a twelfth primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 27. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 28 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, and the base corresponding to SNP(f) can be detected.

[0097] A primer set that amplifies a region containing SNP(g) is, for example, a primer set consisting of a 13th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29 and a 14th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 30. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 31 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, and the base corresponding to SNP(g) can be detected.

[0098] A primer set that amplifies a region containing SNP(h) is, for example, a primer set consisting of a 15th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 32 and a 16th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 33. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 34 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, and the base corresponding to SNP(h) can be detected.

[0099] An example of a primer set that amplifies a region containing SNP(i) is a primer set consisting of a 17th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 35 and an 18th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 36. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 37 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, allowing the detection of a base corresponding to SNP(i).

[0100] A primer set that amplifies a region containing SNP(j) is, for example, a primer set consisting of a 19th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 38 and a 20th primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 39. By using this primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 40 can be obtained in a Chrysanthemum plant that is resistant to chrysanthemum white rust, and the base corresponding to SNP(j) can be detected.

[0101] Furthermore, the primer set is preferably a primer set that amplifies a region containing both SNP(a) and SNP(b). This allows SNP(a) and SNP(b) to be analyzed as a haplotype block. Furthermore, since SNP(c) to SNP(j) are located between SNP(a) and SNP(b), SNP(a) to SNP(j) can be analyzed as a haplotype block by using a primer set that amplifies a region containing both SNP(a) and SNP(b).

[0102] [Chrysanthemum white rust resistance-related gene] A chrysanthemum white rust resistance-associated gene according to one embodiment of the present invention is linked to any one of the molecular markers described above and is located within 3 cM of the molecular marker on the genome.

[0103] The chrysanthemum white rust resistance-associated gene refers to a qualitative trait gene that confers white rust resistance to chrysanthemum, and is linked to at least one of SNP (a) and SNP (b). The chrysanthemum white rust resistance-associated gene is also linked to at least one of SNPs (c) to (j). The chrysanthemum white rust resistance-associated gene is preferably located within a genetic distance of 3 cM, more preferably within 2.5 cM, from these SNPs on the genome of the Chrysanthemum plant. For example, the chrysanthemum white rust resistance-associated gene is 1.4 cM from SNP (a) and 2.1 cM from SNP (b). As another example, the chrysanthemum white rust resistance-associated gene is located 0.4 cM away from SNP (c) and SNP (d), 0 cM away from SNP (e) to SNP (h), 0.3 cM away from SNP (i), and 0.6 cM away from SNP (j). The chrysanthemum white rust resistance-associated gene is located between SNP (a) and SNP (b) on the genome of a plant of the genus Chrysanthemum.

[0104] The chrysanthemum white rust resistance-associated gene can be used to determine the presence or absence of chrysanthemum white rust resistance in a Chrysanthemum plant. By detecting the presence or absence of the chrysanthemum white rust resistance-associated gene in a test Chrysanthemum plant, the chrysanthemum white rust resistance in a Chrysanthemum plant can be determined. The method for detecting the presence or absence of the chrysanthemum white rust resistance-associated gene in a test Chrysanthemum plant is not particularly limited, and detection can be performed by a conventionally known method using the molecular marker described above.

[0105] Furthermore, by introducing a chrysanthemum white rust resistance-associated gene into a Chrysanthemum plant, chrysanthemum white rust resistance can be imparted to the plant. The method for introducing a chrysanthemum white rust resistance-associated gene into a Chrysanthemum plant is not particularly limited, and any conventionally known genetic engineering technique can be used.

[0106] [Chrysanthemum plants resistant to chrysanthemum white rust] A chrysanthemum white rust-resistant plant according to one embodiment of the present invention is a plant that has the chrysanthemum white rust resistance-associated gene described above and is obtained by the production method described below. The chrysanthemum white rust-resistant plant is a plant that has the chrysanthemum white rust resistance-associated gene region identified by the molecular marker described above and exhibits resistance to chrysanthemum white rust.

[0107] A chrysanthemum plant resistant to chrysanthemum white rust according to one embodiment of the present invention can be obtained by crossing a chrysanthemum plant resistant to chrysanthemum white rust with another plant of the genus Chrysanthemum, and identifying the chrysanthemum plant resistant to chrysanthemum white rust from the resulting plant and its progeny using the molecular markers described above, as shown in the production method described below. Note that a chrysanthemum plant resistant to chrysanthemum white rust into which a chrysanthemum white rust resistance-associated gene has been introduced by genetic engineering is also included in the scope of the present invention.

[0108] [Method for determining chrysanthemum white rust resistance] A method for determining white rust resistance of chrysanthemum according to one embodiment of the present invention comprises detecting, in a plant of the genus Chrysanthemum, the following bases (a) or (b) themselves (SNP) or a consecutive polynucleotide containing the bases: (a) a base corresponding to the 74th base of SEQ ID NO: 1; (b) a base corresponding to the 63rd base in SEQ ID NO: 2; as a molecular marker for chrysanthemum white rust resistance. Another embodiment of the present invention provides a method for determining chrysanthemum white rust resistance, comprising the steps of: detecting, in a plant of the genus Chrysanthemum, at least one of the following bases (c) to (j) itself (SNP) or a consecutive polynucleotide containing said base: (c) a base corresponding to at least one of the 29th, 30th, 63rd, 65th, 67th, 68th, and 85th bases in SEQ ID NO: 9; (d) a base corresponding to at least one of the 21st and 24th bases in SEQ ID NO: 10; (e) a base corresponding to at least one of the 22nd, 23rd, 52nd, 66th, and 71st bases in SEQ ID NO: 11; (f) a base corresponding to at least one of the 22nd, 23rd, 52nd, 66th, and 71st bases in SEQ ID NO: 12; The method includes testing the following as molecular markers for chrysanthemum white rust resistance: (a) a base corresponding to at least one of the 19th and 102nd bases shown in SEQ ID NO: 12; (g) a base corresponding to at least one of the 29th, 48th, 50th, and 73rd bases shown in SEQ ID NO: 13; (h) a base corresponding to at least one of the 4th and 112th bases shown in SEQ ID NO: 14; (i) a base corresponding to at least one of the 27th and 40th bases shown in SEQ ID NO: 15; and (j) a base corresponding to at least one of the 21st and 70th bases shown in SEQ ID NO: 16.

[0109] A method for determining chrysanthemum white rust resistance according to one embodiment of the present invention determines chrysanthemum white rust resistance in a test plant of the genus Chrysanthemum using the molecular markers SNP (a) and SNP (b) and any one of polynucleotide (a), polynucleotide (b), and polynucleotide (ab). A method for determining chrysanthemum white rust resistance according to one embodiment of the present invention determines chrysanthemum white rust resistance in a test plant of the genus Chrysanthemum using at least one molecular marker selected from SNP (c) to SNP (j) and polynucleotide (c) to polynucleotide (j). A method for determining chrysanthemum white rust resistance according to one embodiment of the present invention determines the presence or absence of chrysanthemum white rust resistance in a test plant of the genus Chrysanthemum by identifying the presence or absence of a chrysanthemum white rust resistance-associated gene in the genome of the test plant of the genus Chrysanthemum using the molecular markers described above.

[0110] The molecular markers SNP(a) to SNP(j) used in a method for determining chrysanthemum white rust resistance according to one embodiment of the present invention are in linkage disequilibrium, for example, a state of linkage disequilibrium with a linkage disequilibrium coefficient of 0.9 or more. The molecular markers used in a method for determining chrysanthemum white rust resistance according to one embodiment of the present invention are preferably located close to the chrysanthemum white rust resistance-associated gene on the genome of a plant of the genus Chrysanthemum, thereby enabling more accurate determination of the presence or absence of chrysanthemum white rust resistance in a test plant of the genus Chrysanthemum. That is, the molecular markers are preferably SNP(c) to SNP(j), more preferably SNP(c) to SNP(i), and most preferably SNP(e) to SNP(h). Furthermore, when at least two of SNP(a) to SNP(j) are analyzed as a haplotype block, it is preferable that the haplotype block be at least two of SNP(c) to SNP(j), more preferably at least two of SNP(c) to SNP(i), and even more preferably at least two of SNP(e) to SNP(h).

[0111] In a method for determining chrysanthemum white rust resistance according to one embodiment of the present invention, the subject Chrysanthemum plant is a Chrysanthemum plant whose presence or absence of chrysanthemum white rust resistance is unknown, and may be, for example, a hybrid plant between a chrysanthemum white rust-resistant plant and a chrysanthemum white rust-susceptible plant, and its progeny line.

[0112] A method for determining chrysanthemum white rust resistance according to one embodiment of the present invention determines chrysanthemum white rust resistance using any of the molecular markers described above. That is, a method for determining chrysanthemum white rust resistance according to one embodiment of the present invention can determine that a plant has chrysanthemum white rust resistance when (a') the base corresponding to the 74th base in SEQ ID NO: 1 is C, or (b') the base corresponding to the 63rd base in SEQ ID NO: 2 is C. Furthermore, a method for determining chrysanthemum white rust resistance according to one embodiment of the present invention may determine that a plant has chrysanthemum white rust resistance when the base corresponding to the 74th base in SEQ ID NO: 1 is C and the base corresponding to the 63rd base in SEQ ID NO: 2 is C.

[0113] Furthermore, a method for determining chrysanthemum white rust resistance according to one embodiment of the present invention is any of the following (c') to (j'): (c') at least one of the base corresponding to the 29th base in SEQ ID NO: 9 being A, the base corresponding to the 30th base being T, the base corresponding to the 63rd base being C, the base corresponding to the 65th base being T, the base corresponding to the 67th base being A, the base corresponding to the 68th base being C, and the base corresponding to the 85th base being G; (d') at least one of the base corresponding to the 21st base in SEQ ID NO: 10 being C and the base corresponding to the 24th base being T; (e') at least one of the base corresponding to the 22nd base in SEQ ID NO: 11 being G, the base corresponding to the 23rd base being G, the base corresponding to the 52nd base being C, the base corresponding to the 66th base being G, and the base corresponding to the 71st base being G; (f') at least one of the following is true: (f') the base corresponding to the 19th base in SEQ ID NO: 12 is T and the base corresponding to the 102nd base is T; (g') at least one of the following is true: (29th base in SEQ ID NO: 13 is C, the base corresponding to the 48th base is T, the base corresponding to the 50th base is C, and the base corresponding to the 73rd base is G; (h') at least one of the following is true: (4th base in SEQ ID NO: 14 is A and the base corresponding to the 112th base is G; (i') at least one of the following is true: (27th base in SEQ ID NO: 15 is A and the base corresponding to the 40th base is T; or (j') at least one of the following is true: (21st base in SEQ ID NO: 16 is A and the base corresponding to the 70th base is T).

[0114] For details of the molecular markers used in the method for determining chrysanthemum white rust resistance according to one embodiment of the present invention, the above-mentioned description of the molecular markers according to one embodiment of the present invention is incorporated herein by reference.

[0115] In a method for determining white rust resistance in chrysanthemum according to one embodiment of the present invention, the method for testing the white rust resistance of a Chrysanthemum plant using a molecular marker is not particularly limited, and known SNP analysis methods can be used, such as a method for SNP analysis by detecting SNPs in PCR-amplified fragments of a test Chrysanthemum plant. That is, in a method for determining white rust resistance in chrysanthemum according to one embodiment of the present invention, the testing step may involve amplifying a region in the DNA of the plant using a primer set that amplifies the region containing the molecular marker. The details of such primer sets are incorporated herein by reference.

[0116] PCR in SNP analysis may be either singleplex PCR, which amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR, which amplifies genes in a reaction system containing multiple primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances with different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

[0117] PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR instrument used, the length of the amplified fragment, and other factors. Cycling conditions include a three-step PCR method, in which one cycle consists of three steps: denaturation, annealing, and extension; and a two-step PCR method, in which one cycle consists of two steps: denaturation, annealing, and extension. Examples of PCR reaction conditions include 90-100°C for 40-60 seconds (e.g., 95°C for 50 seconds), 30-60 cycles (e.g., 40 cycles) of 90-100°C (e.g., 95°C) for 5 seconds, annealing for 10-20 seconds (e.g., 15 seconds), and 65-80°C for 10-30 seconds (e.g., 72°C for 20 seconds). The annealing temperature can be gradually decreased every specified cycle from an initial annealing temperature of 60-70°C (e.g., 66°C) to a final annealing temperature of 50-60°C (e.g., 56°C). Depending on the state of the template DNA, PCR reaction conditions may be adjusted to stably detect SNPs.

[0118] As PCR in SNP analysis, real-time PCR such as TaqMan®-PCR, which performs PCR amplification and identifies SNP markers, may be used. That is, a TaqMan® probe may be further used to detect SNPs contained in the amplified fragments amplified using a primer set. The use of real-time PCR can provide a high-throughput identification method. Furthermore, SNPs in the amplified fragments amplified by PCR may be identified by analyzing the nucleotide sequence of the amplified fragments using an automated DNA sequencer or the like.

[0119] The method for extracting DNA to be amplified by PCR from a test specimen of a Chrysanthemum plant is not particularly limited, and known DNA extraction methods can be used. Alternatively, DNA may be extracted using a commercially available DNA extraction kit. Depending on the type of test specimen and the amount of contaminants, appropriate pretreatment may be performed before the DNA extraction step. Furthermore, the DNA extracted from the test specimen may be washed or purified as necessary for use as a template in a PCR reaction. Furthermore, the DNA extracted from the test specimen may be digested with two restriction enzymes, and the resulting restriction enzyme fragments may be amplified by PCR.

[0120] According to a method for determining chrysanthemum white rust resistance according to one embodiment of the present invention, the presence or absence of chrysanthemum white rust resistance in a Chrysanthemum plant can be determined using a molecular marker, and therefore, chrysanthemum plants resistant to chrysanthemum white rust and their progeny lines can be selected based on the determination results.

[0121] [Manufacturing method] A production method according to one embodiment of the present invention is a method for producing a Chrysanthemum plant that is resistant to chrysanthemum white rust, and includes a hybridization step of crossing a Chrysanthemum plant that is resistant to chrysanthemum white rust with another Chrysanthemum plant, and a discrimination step of identifying a Chrysanthemum plant that is resistant to chrysanthemum white rust from the Chrysanthemum plant obtained by the hybridization step or a Chrysanthemum plant of a progeny lineage by the method for determining chrysanthemum white rust resistance described above.

[0122] Therefore, the above-mentioned descriptions of the molecular markers, the genes associated with chrysanthemum white rust resistance, the Chrysanthemum plants resistant to chrysanthemum white rust, and the methods for determining chrysanthemum white rust resistance are incorporated herein by reference in the description of the methods for producing Chrysanthemum plants resistant to chrysanthemum white rust.

[0123] In the crossing step, the Chrysanthemum plant having resistance to chrysanthemum white rust used as a parent plant may be a chrysanthemum white rust-resistant plant of the present invention. Furthermore, the Chrysanthemum plant having resistance to chrysanthemum white rust used in the crossing step may be a chrysanthemum white rust-resistant plant selected by the method of determining chrysanthemum white rust resistance of the present invention. That is, a production method according to one embodiment of the present invention may further include, prior to the crossing step, a discrimination step of discriminating a Chrysanthemum plant having resistance to chrysanthemum white rust from test Chrysanthemum plants by any of the above-described methods for determining chrysanthemum white rust resistance.

[0124] In the discrimination step, the above-mentioned chrysanthemum white rust resistance is determined from the Chrysanthemum plant obtained by the crossbreeding step or a progeny line of Chrysanthemum plants by any of the above-mentioned methods for determining chrysanthemum white rust resistance.

[0125] According to one embodiment of the production method of the present invention, the presence or absence of chrysanthemum white rust resistance in a Chrysanthemum plant can be determined using a molecular marker, and a chrysanthemum plant resistant to chrysanthemum white rust selected based on the determination results can be produced.

[0126] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0127] [Example 1] Materials and Methods (Pathogen) The chrysanthemum white rust pathogen (Puccinia horiana) was collected from within Japan. A single teliospore mass was used to inoculate disease-free "Shuho no Chikara" plants, which were then maintained for subsequent generations. The growth chamber was maintained at 20°C and 70% relative humidity under fluorescent white light (50 μmol m -2 ·s -1 The plants were maintained under a 16-hour photoperiod using a 1000-millimeter-long (1000-millimeter) irradiated ...

[0128] (Inoculation method) The inoculation test of chrysanthemum white rust fungus was carried out in a growth chamber using a polystyrene foam box (50.8 cm long x 36.0 cm wide x 34.9 cm deep). Individuals of the F1 population and unrooted scions of both parents were inserted into a 200-hole cell tray containing horticultural medium (Metro Mix 360; Scotts) and placed at the bottom of the polystyrene foam box. The top opening of the box was covered with a plastic net (5 mm mesh). The diseased leaves of "Shuho no Chikara" were inoculated approximately 1 cm apart. 2 The inoculum was cut into small pieces and placed on a net at a density of 3 cm x 3 cm, with the teliospore pile facing downward. To ensure high humidity, the pieces, the inside of the box, and the net holding the inoculum were sprayed with demineralized water using a sprayer. The box was sealed and placed in a growth chamber at 19°C in the dark. 16 hours after the start of inoculation, the 200-well cell tray was placed in a transparent plastic container (37.5 cm long x 24.7 cm wide x 12.9 cm high) and placed in the growth chamber, where it was maintained at 22°C under a 16-hour photoperiod. Disease symptoms were evaluated 28 days after inoculation.

[0129] The above inoculation test was performed three times. If at least one teliospore pile was observed on a plant in any of the analyses, it was rated as "susceptible (S)." If no teliospore pile was observed in any of the three analyses, the plant was rated as "resistant (R)."

[0130] (Varieties and F1 populations) An F1 population was obtained from a reciprocal cross between "Aries" and "Yellow Queen." 64 seedlings were obtained from the cross between the ovary parent "Aries" and the pollen parent "Yellow Queen," and 219 seedlings were obtained from the cross between the ovary parent "Yellow Queen" and the pollen parent "Aries." The mother plants of the F1 individuals were planted in plastic pots (inner diameter 12 cm, one seedling per pot) containing commercially available horticultural soil (Kureha horticultural soil, manufactured by Kureha Chemical Co., Ltd.) and maintained as cuttings in a greenhouse at 18-25°C with a natural day length plus a 6-hour dark break.

[0131] Genomic DNA of the parental varieties and F1 individuals was extracted from the shoot tips (30 mg fresh weight) using the DNeasy Plant Mini Kit (Qiagen).

[0132] (dd-RAD-Seq analysis) To generate a dd-RAD-Seq library, the extracted DNA was digested with two restriction enzymes, PstI and MspI, and the nucleotide sequence of the library was determined on a HiSeq2000 (Illumina) platform.

[0133] (Data processing and simplex SNP data mining) Data processing for simplex SNP determination was performed as summarized below. Reads obtained from dd-RAD-Seq analysis were mapped to the C. seticuspe genome sequence (CSE_r1.0), which was used as a reference. For each SNP locus, the read data from all F1 individuals was pooled, and the allele frequency (AAF) at each position was calculated by dividing the number of reads containing the variant base at each SNP locus by the total number of reads. Simplex and double-simplex SNPs were selected according to the allele frequency and the frequency of heterozygous individuals in the population.

[0134] (Genome-wide association study in F1 individuals) The association between genotype and phenotype was analyzed by general linear model using the TASSEL program.

[0135] (linkage analysis) Linkage analysis was performed using JoinMap 4.1 software (Kyazma BV). The dd-RAD-seq genotype data of SNP markers significantly associated with chrysanthemum white rust resistance and the phenotype data of chrysanthemum white rust resistance were imported into JoinMap 4.1 software, and a map was constructed using the initial regression mapping parameters.

[0136] (SNP marker-resistance association analysis) In the F1 population derived from a reciprocal cross between "Yellow Queen" and "Aries," we identified the genotypes of two SNP markers by PCR. Two SNP-specific primers were designed that were highly associated with chrysanthemum white rust resistance in "Aries" (Table 1).

[0137] [Table 1] SNP-discriminating touchdown PCR was performed under the following conditions: 95°C for 50 seconds, 40 cycles of 95°C for 5 seconds, annealing for 15 seconds, and 72°C for 20 seconds. The annealing temperature was decreased stepwise by 2°C every three cycles, from an initial annealing temperature of 66°C to a final annealing temperature of 56°C. PCR was performed in a 15-μL volume containing 8 ng of genomic DNA, 0.3 μM of each primer, and a 2× TB Green Premix Ex Taq II Tli RNase H plus kit (TaKaRa Bio) using a Thermal Cycler Dice Real-Time system (TaKaRa Bio).

[0138] 〔result〕 (Inheritance of chrysanthemum white rust resistance derived from "Aries") A total of 283 F1 individuals obtained from the intercross between "Aries" and the susceptible "Yellow Queen" segregated with a resistant:susceptible ratio of 145:138. This result follows the expected segregation pattern of a single mutation x no mutation (Aaaaaa x aaaaaa) in an autohexaploid inheritance pattern, with a 1:1 ratio (χ 2= 0.17, P = 0.68), indicating that Aries has a single dominant gene for resistance to chrysanthemum white rust.

[0139] (SNP marker for chrysanthemum white rust resistance in Aries) Using individuals (n=283) obtained from a cross between "Aries" and the susceptible "Yellow Queen," DNA markers closely linked to resistance genes in "Aries" were examined. A total of 10,779 SNP candidates were identified from sequence reads. GLM analysis of the 10,779 SNP markers identified 82 SNP markers significantly associated with chrysanthemum white rust resistance.

[0140] Among the SNP markers, SCSE_SC004884.1_65872 and SCSE_SC000716.1_75925 showed a high association with chrysanthemum white rust resistance. The genotypes of these two SNPs were determined by PCR analysis using specific primers (Figure 2). dd-RAD-seq results indicated that the genotype of SCSE_SC004884.1_65872 was heterozygous (TTTTTC) in the resistant "Aries" variety, while it was homozygous (TTTTTT) in the susceptible "Yellow Queen" variety. The genotype of SCSE_SC000716.1_75925 was TTTTTC in the resistant "Aries" variety, and TTTTTT in the susceptible "Yellow Queen" variety.

[0141] The locus for the chrysanthemum white rust resistance gene (Phr2) in Aries was located between SCSE_SC004884.1_65872 and SCSE_SC000716.1_75925, 1.4 cM from the former and 2.1 cM from the latter. The allele pattern of Aries at the nearest SNP marker SCSE_SC004884.1_65872 was TTTTTC, indicating that the C allele of the SNP and the Phr2 locus are interconnected in the Aries genome. Similarly, the C allele of TTTTTC for SCSE_SC000716.1_75925 also showed interconnection with Phr2. The segregation of the C allele in SCSE_SC004884.1_65872 was 143:140 present:absent, and the segregation of the C allele in SCSE_SC000716.1_75925 was 151:132.

[0142] Regarding the relationship between the genotypes of both markers and chrysanthemum white rust resistance, 142 plants with the "Aries" allele were resistant, while 131 plants lacked either allele and were all susceptible.

[0143] [Example 2] Materials and Methods (Pathogen and inoculation method) The pathogen and inoculation method in Example 2 were the same as those in Example 1 described above.

[0144] (Varieties and F1 populations) An F1 population was obtained from a reciprocal cross between "Aries" and "Yellow Queen." 64 seedlings were obtained from the cross between the ovary parent "Aries" and the pollen parent "Yellow Queen," and 592 seedlings were obtained from the cross between the ovary parent "Yellow Queen" and the pollen parent "Aries." A total of 656 F1 mother plants were planted in plastic pots (inner diameter 12 cm, one seedling per pot) containing commercially available horticultural soil (Kureha horticultural soil, manufactured by Kureha Chemical Co., Ltd.) and maintained as cuttings in a greenhouse at 18°C ​​to 25°C with a natural day length plus a 6-hour dark break.

[0145] Genomic DNA of "Aries" and "Yellow Queen" was extracted from shoot tips (30 mg fresh weight) using the DNeasy Plant Mini Kit (Qiagen).

[0146] (dd-RAD-Seq analysis) After preparing a paired-end library with an insert length of 500 bases, the nucleotide sequence of the library was determined on a HiSeq X Ten (Illumina) platform.

[0147] (Data Processing) The resulting read data were assembled using SOAPdenovo2 software with k-mer = 101. Assembly sequences with a base length of 500 or more were extracted and mapped onto the genome sequence of C. seticuspe, which was used as a reference, using minimap2 software.

[0148] (linkage analysis) Linkage analysis was performed using JoinMap 4.1 software (Kyazma BV). For 656 F1 individuals, genotype data for SNPs linked to molecular marker (a) (SCSE_SC004884.1_65872) and molecular marker (b) (SCSE_SC000716.1_75925) and phenotype data for chrysanthemum white rust resistance were imported into JoinMap 4.1 software, and a map was constructed using the initial regression mapping parameters.

[0149] (SNP marker-resistance association analysis) The base sequence information of "Aries" and "Yellow Queen" was compared for the region between the two SNPs, molecular marker (a) and molecular marker (b), to identify the SNP. From the identified SNPs, those linked to molecular marker (a) and molecular marker (b) in "Aries" were selected by PCR. The PCR conditions were the same as in Example 1.

[0150] Eight SNPs were selected between molecular markers (a) and (b) in "Aries": 46.1-19 (molecular marker (c)), 46.5-16 (molecular marker (d)), 47.0-3 (molecular marker (e)), 47.5-2 (molecular marker (f)), 48.0-5 (molecular marker (g)), 48.5-10 (molecular marker (h)), 49.0-15 (molecular marker (i)), and 50.0-3 (molecular marker (j)). SNP-specific primers were designed for each SNP (Table 2).

[0151] [Table 2]

[0152] 〔result〕 Using individuals (n = 656) obtained from a cross between "Aries" and the susceptible "Yellow Queen," we investigated the linkage relationship between the chrysanthemum white rust resistance gene (Phr2) in "Aries" and molecular markers (a) to (j). The genetic distances from Phr2 were 0.9 cM for molecular marker (a), 2.2 cM for molecular marker (b), 0.4 cM for molecular markers (c) and (d), 0 cM for molecular markers (e) and (h), 0.3 cM for molecular marker (i), and 0.6 cM for molecular marker (j) (Figure 3). Figure 3 shows the genetic distances between the chrysanthemum white rust resistance gene and the molecular markers.

[0153] For molecular markers (e) to (h) with a genetic distance of 0 cM from Phr2, all F1 individuals with the "Aries" allele were resistant, indicating that these molecular markers are completely linked to the chrysanthemum white rust resistance gene. [Industrial Applicability]

[0154] The present invention can be used in the fields of agriculture, plant breeding, etc.

Claims

1. A method for determining chrysanthemum white rust resistance, comprising: In a plant of the genus Chrysanthemum, the following base (a) or (b), or any one of (c) to (j) itself (SNP), or a consecutive polynucleotide containing the base: (a) a base corresponding to the 74th base shown in SEQ ID NO: 1; (b) a base corresponding to the 63rd base set forth in SEQ ID NO: 2; (c) a base corresponding to at least one of the 29th, 30th, 63rd, 65th, 67th, 68th, and 85th bases set forth in SEQ ID NO: 9; (d) a base corresponding to at least one of the 21st and 24th bases set forth in SEQ ID NO: 10; (e) a base corresponding to at least one of the 22nd, 23rd, 52nd, 66th, and 71st bases set forth in SEQ ID NO: 11; (f) a base corresponding to at least one of the 19th and 102nd bases set forth in SEQ ID NO: 12; (g) a base corresponding to at least one of the 29th, 48th, 50th, and 73rd bases set forth in SEQ ID NO: 13; (h) a base corresponding to at least one of the 4th and 112th bases set forth in SEQ ID NO: 14; (i) a base corresponding to at least one of the 27th and 40th bases set forth in SEQ ID NO: 15; (j) a base corresponding to at least one of the 21st and 70th bases set forth in SEQ ID NO: 16; as a molecular marker for chrysanthemum white rust resistance, Any of the following (a') or (b'), or (c') to (j'): (a') the base corresponding to the 74th base in SEQ ID NO: 1 is C; (b') the base corresponding to the 63rd base in SEQ ID NO: 2 is C; (c') at least one of the following: A for the base corresponding to the 29th base, T for the base corresponding to the 30th base, C for the base corresponding to the 63rd base, T for the base corresponding to the 65th base, A for the base corresponding to the 67th base, C for the base corresponding to the 68th base, and G for the base corresponding to the 85th base as set forth in SEQ ID NO: 9; (d') at least one of the following: the base corresponding to the 21st base of SEQ ID NO: 10 is C and the base corresponding to the 24th base is T; (e') at least one of the bases corresponding to the 22nd base, 23rd base, 52nd base, 66th base, and 71st base shown in SEQ ID NO: 11 is G, C, G, and G, respectively; (f') at least one of the bases corresponding to the 19th base and the 102nd base shown in SEQ ID NO: 12 being T; (g') at least one of the bases selected from the group consisting of C for the 29th base, T for the 48th base, C for the 50th base, and G for the 73rd base as set forth in SEQ ID NO: 13; (h') at least one of A corresponding to the 4th base and G corresponding to the 112th base as set forth in SEQ ID NO: 14; (i') at least one of A corresponding to the 27th base and T corresponding to the 40th base as set forth in SEQ ID NO: 15; (j') at least one of A corresponding to the 21st base and T corresponding to the 70th base as set forth in SEQ ID NO: 16; and determining that the plant is resistant to chrysanthemum white rust when

2. 2. The method of claim 1, wherein the Chrysanthemum plant is a candidate plant for breeding material or is a plant obtained by a breeding process.

3. At least two of the following (a') and (b'), and (c') to (j'): (a') the base corresponding to the 74th base in SEQ ID NO: 1 is C; (b') the base corresponding to the 63rd base of SEQ ID NO: 2 is C; (c') at least one of the following: A for the base corresponding to the 29th base, T for the base corresponding to the 30th base, C for the base corresponding to the 63rd base, T for the base corresponding to the 65th base, A for the base corresponding to the 67th base, C for the base corresponding to the 68th base, and G for the base corresponding to the 85th base as set forth in SEQ ID NO: 9; (d') at least one of the bases corresponding to the 21st base and the 24th base shown in SEQ ID NO: 10 being C and T; (e') at least one of the bases corresponding to the 22nd base, 23rd base, 52nd base, 66th base, and 71st base shown in SEQ ID NO: 11 is G, C, G, and G, respectively; (f') at least one of the bases corresponding to the 19th base and the 102nd base shown in SEQ ID NO: 12 being T; (g') at least one of the bases selected from the group consisting of C for the 29th base, T for the 48th base, C for the 50th base, and G for the 73rd base as set forth in SEQ ID NO: 13; (h') at least one of A corresponding to the 4th base and G corresponding to the 112th base as set forth in SEQ ID NO: 14; (i') at least one of A corresponding to the 27th base and T corresponding to the 40th base as set forth in SEQ ID NO: 15; (j') at least one of A corresponding to the 21st base and T corresponding to the 70th base as set forth in SEQ ID NO: 16; The method according to claim 1 or 2, wherein the plant is determined to be resistant to chrysanthemum white rust when

4. The method according to claim 1 , wherein in the step of examining, the region in the DNA of the plant is amplified using a primer set that amplifies the region containing the molecular marker.

5. The primer set includes at least one of the following (a) and (b), and (c) to (j): (a) a base corresponding to the 74th base shown in SEQ ID NO: 1; (b) a base corresponding to the 63rd base set forth in SEQ ID NO: 2; (c) a base corresponding to at least one of the 29th, 30th, 63rd, 65th, 67th, 68th, and 85th bases set forth in SEQ ID NO: 9; (d) a base corresponding to at least one of the 21st and 24th bases set forth in SEQ ID NO: 10; (e) a base corresponding to at least one of the 22nd, 23rd, 52nd, 66th, and 71st bases set forth in SEQ ID NO: 11; (f) a base corresponding to at least one of the 19th and 102nd bases set forth in SEQ ID NO: 12; (g) a base corresponding to at least one of the 29th, 48th, 50th, and 73rd bases set forth in SEQ ID NO: 13; (h) a base corresponding to at least one of the 4th and 112th bases set forth in SEQ ID NO: 14; (i) a base corresponding to at least one of the 27th and 40th bases set forth in SEQ ID NO: 15; (j) a base corresponding to at least one of the 21st and 70th bases set forth in SEQ ID NO: 16; The method according to claim 4, wherein the primer set amplifies a region comprising:

6. 6. The method according to claim 1, wherein the plant of the genus Chrysanthemum is a plant belonging to the species Chrysanthemum morifolium.

7. A method for producing a plant of the genus Chrysanthemum that is resistant to chrysanthemum white rust, comprising: A hybridization step of hybridizing a plant of the genus Chrysanthemum having resistance to chrysanthemum white rust with another plant of the genus Chrysanthemum; a discrimination step of discriminating a Chrysanthemum plant having resistance to chrysanthemum white rust from the Chrysanthemum plants obtained by the hybridization step or from Chrysanthemum plants of progeny lineages by the method according to any one of claims 1 to 6; A manufacturing method comprising:

8. The production method according to claim 7, further comprising a discrimination step of discriminating a Chrysanthemum plant having resistance to chrysanthemum white rust from test Chrysanthemum plants by the method according to any one of claims 1 to 6, prior to the hybridization step.

9. A primer set for amplifying a region containing a molecular marker for chrysanthemum white rust resistance in a plant of the genus Chrysanthemum, comprising: The molecular marker is any one of the following bases (a) or (b), or (c) to (j) themselves (SNP), or a consecutive polynucleotide containing the corresponding base: (a) a base corresponding to the 74th base shown in SEQ ID NO: 1; (b) a base corresponding to the 63rd base set forth in SEQ ID NO: 2; (c) a base corresponding to at least one of the 29th, 30th, 63rd, 65th, 67th, 68th, and 85th bases set forth in SEQ ID NO: 9; (d) a base corresponding to at least one of the 21st and 24th bases set forth in SEQ ID NO: 10; (e) a base corresponding to at least one of the 22nd, 23rd, 52nd, 66th, and 71st bases set forth in SEQ ID NO: 11; (f) a base corresponding to at least one of the 19th and 102nd bases set forth in SEQ ID NO: 12; (g) a base corresponding to at least one of the 29th, 48th, 50th, and 73rd bases set forth in SEQ ID NO: 13; (h) a base corresponding to at least one of the 4th and 112th bases set forth in SEQ ID NO: 14; (i) a base corresponding to at least one of the 27th and 40th bases set forth in SEQ ID NO: 15; (j) a base corresponding to at least one of the 21st and 70th bases set forth in SEQ ID NO: 16; This is the primer set.

10. A chrysanthemum plant resistant to chrysanthemum white rust obtained by the production method according to claim 7 or 8, The chrysanthemum plant resistant to chrysanthemum white rust is A chrysanthemum plant which is a descendant line of a chrysanthemum plant resistant to chrysanthemum white rust obtained by crossing a chrysanthemum plant resistant to chrysanthemum white rust with another chrysanthemum plant.

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