Method for determining cucumber chlorosis resistance, method for producing cucumber chlorosis resistant cucumber plant, cucumber chlorosis resistant cucumber plant, molecular marker for cucumber chlorosis resistance, and gene associated with cucumber chlorosis resistance

Abstract

To provide a method for determining cucumber yellow rot resistance, a method for producing a cucumber yellow rot resistant cucumber plant, a cucumber yellow rot resistant cucumber plant, a molecular marker related to cucumber yellow rot resistance, and a cucumber yellow rot resistance-related gene.SOLUTION: A method for determining resistance to cucumber yellow mosaic disease, comprising a step of testing, in a cucumber plant, a continuous polynucleotide having at least one molecular marker selected from the group consisting of (a) to (c) or a simple sequence repeat (SSR) or singlenucleotidepolymorphism (SNP) contained in the molecular marker on chromosome 6 as a molecular marker for resistance to cucumber yellow mosaic disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for determining resistance to cucumber spotted wilt disease, a method for producing a cucumber plant resistant to cucumber spotted wilt disease, a cucumber plant resistant to cucumber spotted wilt disease, a molecular marker for resistance to cucumber spotted wilt disease, and a gene associated with resistance to cucumber spotted wilt disease. [Background technology]

[0002] Cucumber yellow spot disease is a viral disease caused by the melon yellow spot virus (MYSV) and transmitted by the southern melon thrips (Thrips palmi Karny). MYSV-infected cucumbers (Cucumis sativus L.) show mosaic, chlorotic spots, yellowing, and necrotic symptoms on the leaves. Chlorotic spots and mosaic symptoms also appear on some fruits. These symptoms can lead to reduced yields and marketable fruit rates.

[0003] Cucumber spotted wilt disease has been reported in the Kanto, Tokai, Shikoku, and Kyushu regions of Japan, causing serious damage to cucumber-producing areas, and the affected areas are showing a tendency to expand.

[0004] The most important way to control this disease is to eradicate the vector, the melon thrips. However, there are only a limited number of pesticides that are effective against melon thrips, and control is limited. For stable production of cucumbers, there is a strong need to develop resistant varieties. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sugiyama et al., Evaluation of resistance to Melon yellow spot virus in a cucumber germplasm collection. Plant Breeding. 128(6):696-700 (2009). [Non-patent document 2] Sugiyama et al., Mapping of quantitative trait loci for Melon yellow spot virus resistance in cucumber (Cucumis sativus L.). Euphytica 205:615-625 (2015). [Non-patent document 3] Sugiyama et al. (2016) Breeding process and characteristics of 'Cucumber Intermediate Parent No. 7', which has resistance to cucumber spotted wilt disease. Research Bulletin of the Vegetable and Tea Science Research Institute. 15:1-10. [Non-patent document 4] Sugiyama et al. (2019) "Midori-natsu" cucumber with resistance to cucumber spotted wilt disease. Research results information Summary of the Invention [Problem to be solved by the invention]

[0006] Although DNA selection markers are effective for rapidly developing cucumber varieties resistant to spotted wilt disease, there are currently no highly accurate DNA markers that can be used to select individuals resistant to spotted wilt disease. Therefore, despite the strong demand for cucumber varieties resistant to spotted wilt disease, varieties with sufficient resistance have not yet been developed.

[0007] An object of one aspect of the present invention is to provide a cucumber plant that is resistant to cucumber spotted wilt disease. [Means for solving the problem]

[0008] In order to achieve the above object, one embodiment of the present invention provides a method for determining resistance to cucumber spotted wilt disease, the method comprising the step of testing a cucumber plant for at least one molecular marker selected from the group consisting of the following (a) to (c), on chromosome 6, or a contiguous polynucleotide having at least an SSR (Simple Sequence Repeat) or an SNP (Single Nucleotide Polymorphism) contained in the molecular marker, as a molecular marker for resistance to cucumber spotted wilt disease: (a) Molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, (b) molecular markers consisting of the nucleotide sequences set forth in SEQ ID NOs: 11 to 15, and (c) molecular markers in linkage disequilibrium with the molecular markers (a) or (b).

[0009] A method for producing a cucumber plant resistant to cucumber spotted wilt disease according to one aspect of the present invention includes: a hybridization step of crossing a cucumber plant resistant to cucumber spotted wilt disease with another cucumber plant; and a discrimination step of identifying a cucumber plant resistant to cucumber spotted wilt disease from the cucumber plant obtained by the hybridization step or a cucumber plant of a progeny line thereof by the method for determining resistance to cucumber spotted wilt disease.

[0010] A cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention is a cucumber spotted wilt disease-resistant cucumber plant comprising a cucumber spotted wilt disease resistance locus on chromosome 6.

[0011] A molecular marker according to one embodiment of the present invention is a molecular marker for cucumber spotted wilt disease resistance in a cucumber plant, and is at least one molecular marker selected from the group consisting of the following (a) to (c), or a contiguous polynucleotide having at least an SSR or SNP contained in the molecular marker: (a) molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, (b) a molecular marker consisting of the nucleotide sequence set forth in SEQ ID NOs: 11 to 15, and (c) a molecular marker in linkage disequilibrium with the molecular marker of (a) or (b).

[0012] A cucumber spotted wilt disease resistance-associated gene according to one embodiment of the present invention is linked to the molecular marker and is located within a physical distance of 2,145 kbp from the molecular marker on the chromosome. [Effects of the Invention]

[0013] According to one aspect of the present invention, there is provided a molecular marker for cucumber spotted wilt disease resistance in a cucumber plant. Furthermore, by using the molecular marker for cucumber spotted wilt disease resistance of the present invention, it becomes possible to efficiently breed cucumber varieties that are resistant to cucumber spotted wilt disease. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the symptoms of cucumber spotted wilt disease on cucumber leaves at each disease score in an example. [Figure 2] 1 shows a linkage map of the F2 population of "Cucumber intermediate parent No. 7" and the disease-susceptible "Cucumber intermediate parent No. 4" inoculated with a common MYSV strain in an example. [Figure 3] 1 shows a linkage map of the F2 population of "Cucumber intermediate parent No. 7" and the susceptible "Cucumber intermediate parent No. 4" inoculated with a virulent MYSV strain in an example. [Figure 4] FIG. 1 shows the results of QTL analysis of the gene for resistance to spotted wilt disease caused by inoculation with a common strain of MYSV in an example. [Figure 5] FIG. 1 shows the results of QTL analysis of the gene for resistance to spotted wilt disease caused by inoculation with a virulent strain of MYSV in an example. [Figure 6] FIG. 1 shows the results of investigating the relationship between the genotype of a marker near a QTL on chromosome 6 and the degree of disease on fruit in an example. [Figure 7] This figure shows the results of an investigation into the relationship between the marker genotype and spotted wilt disease resistance in a recombinant line in which recombination has occurred within the QTL region on chromosome 6 in a line in which the resistance locus on chromosome 3 is fixed in the susceptible homozygous form, in an example. [Figure 8] FIG. 1 shows the results of investigating the relationship between the marker genotypes on chromosomes 3 and 6 and the degree of leaf disease in an example. DETAILED DESCRIPTION OF THE INVENTION

[0015] 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) and B or less (including B and less than B)."

[0016] 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.

[0017] As used herein, a "cucumber" or "cucumber plant" refers to a plant of the Cucurbitaceae family, genus Cucumis, species Cucumis sativus L.

[0018] In this specification, the term "plant" may refer to a part or the whole of a plant body. Examples of the part of a plant body include leaves, branches, stems, roots, flowers, fruits, seeds, etc., and may also be propagation materials.

[0019] For each chromosome in a cucumber plant, for example, nucleotide sequence information of the cucumber genome is available, for example, from the Cucurbit Genomics Database of the International Cucurbit Genomics Initiative (http: / / www.icugi.org).

[0020] "Cucumber yellow spot disease" is a viral disease caused by the melon yellow spot virus (MYSV) and transmitted by the southern melon thrips (Thrips palmi Karny). MYSV is a tripartite single-stranded RNA virus belonging to the genus Tospovirus in the family Bunyauiridae, and several strains with different symptoms have been identified. Cucumbers infected with MYSV show symptoms such as mosaic marks, chlorotic spots, yellowing, and necrosis on the leaves. Some fruits also show symptoms such as chlorotic spots and mosaic marks.

[0021] There are several known MYSV strains that cause different symptoms and severity of disease, including a common strain isolated from cucumber (Kyushu isolate, MYSV-FuCu05P), a highly virulent strain (Shikoku isolate), and the MYSV-S strain isolated from melon. The highly virulent strain causes severe mosaic damage in the fruit.

[0022] "Cucumber spotted wilt disease resistance" refers to, for example, the ability to suppress or inhibit the onset and progression of cucumber spotted wilt disease. Therefore, a plant exhibiting cucumber spotted wilt disease resistance is one in which, even when infected with MYSV, no symptoms develop or the progression of cucumber spotted wilt disease symptoms is suppressed.

[0023] The degree of resistance to cucumber yellow spot disease can be evaluated, for example, using known criteria such as disease severity. As shown in the Examples, the disease severity on leaves is scored using a 7-point scale (0: no symptoms, 1: slight mosaic and chlorotic spots, 2: mosaic and chlorotic spots, 3: slight necrosis and yellowing (yellowing and necrosis account for up to 20% of the leaf area), 4: necrosis and yellowing (20-50%), 5: severe necrosis and yellowing (50% or more), 6: death). Furthermore, in one example, the disease severity on approximately 100g of fruit is scored using a 6-point scale (0: no symptoms, 1: slight chlorotic spots, 2: chlorotic spots, 3: mosaic with slight malformations, 4: mosaic with malformations, 5: severe mosaic with malformations). After about 8 weeks, the disease index (disease score × number of samples) / total number of samples) is calculated, and the resistance to cucumber spotted wilt virus can be evaluated.

[0024] In the present invention, resistance to cucumber spotted wilt disease is conferred by a cucumber spotted wilt disease resistance locus on chromosome 6. As described below, a cucumber spotted wilt disease-resistant cucumber plant of the present invention has a cucumber spotted wilt disease resistance locus on chromosome 6, and may also have a cucumber spotted wilt disease resistance locus on either chromosome 1 or chromosome 3.

[0025] [Molecular markers for resistance to cucumber spotted wilt disease] A molecular marker according to one embodiment of the present invention is a molecular marker for cucumber spotted wilt disease resistance in a cucumber plant, and can be used to identify a gene locus for cucumber spotted wilt disease resistance (cucumber spotted wilt disease resistance locus) on chromosome 6.

[0026] A cucumber spotted wilt disease resistance locus refers to a quantitative trait locus or gene region that confers resistance to cucumber spotted wilt disease. A quantitative trait locus (QTL) generally refers to a chromosomal region involved in the expression of a quantitative trait. A QTL can be defined as a specific locus on a chromosome using molecular markers.

[0027] A molecular marker according to one embodiment of the present invention is a marker for detecting a regional regulatory locus on chromosome 6 ranging from 4.0 Mbp to 6.2 Mbp.

[0028] A molecular marker according to one embodiment of the present invention is a molecular marker for cucumber spotted wilt disease resistance in a cucumber plant, and is at least one molecular marker selected from the group consisting of the following (a) to (c), or a contiguous polynucleotide having at least an SSR or SNP contained in the molecular marker: (a) molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, (b) a molecular marker consisting of the nucleotide sequence set forth in SEQ ID NOs: 11 to 15, and (c) a molecular marker in linkage disequilibrium with the molecular marker of (a) or (b).

[0029] A molecular marker according to one embodiment of the present invention is a molecular marker that specifies a cucumber spotted wilt disease resistance gene in a cucumber plant. Such molecular markers include SNP markers, SSR markers, STS markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, SCAR (sequence-characterized amplified region) markers, and CAPS (cleaved amplified plymorphic sequence) markers. The SNP marker may be a single SNP or a combination of two or more SNPs. It may also be a combination of SNP, SSR, and STS. The molecular marker according to one embodiment of the present invention can be used to determine cucumber spotted wilt disease resistance in a cucumber plant.

[0030] A molecular marker according to one embodiment of the present invention may be (i) the bases corresponding to the SNP itself, (ii) a contiguous polynucleotide containing the SNP, (iii) a contiguous polynucleotide in the region between two SNPs, (iv) the base sequence corresponding to the SSR itself, (v) a contiguous polynucleotide containing an SSR, or (vi) a contiguous polynucleotide containing one or more combinations of SSRs and SNPs.

[0031] (i: SSR markers and SNP markers) A molecular marker according to one embodiment of the present invention may be an SSR marker. SSRs (Simple Sequence Repeats) are DNA polymorphisms in which a repeated sequence is found within a specific region in the base sequence of DNA.

[0032] A molecular marker according to one embodiment of the present invention may be a SNP marker. SNP (single nucleotide polymorphism) refers to a DNA polymorphism in which a single nucleotide mutation is observed within a specific region in the DNA base sequence.

[0033] A molecular marker according to one embodiment of the present invention may be a marker having one or more combinations of SSRs and SNPs.

[0034] In this specification, "the Yth base (for example, when X=11, Y is 109, and when X=12, Y is 16 or 71) in the base sequence (referred to as base sequence X) shown in SEQ ID NO: X (X=1 to 15)" refers to the SNP marker described in this Example. "The base corresponding to the Yth base in the base sequence shown in SEQ ID NO: X" refers to an SNP marker that can be identified with the SNP marker described in this Example. The base sequence X is derived from a reference cucumber plant (for example, "Chinese long"), and other cucumber plants may contain a portion of the base sequence that differs in addition to the portion of the SNP marker.

[0035] That is, when a nucleotide sequence X' corresponding to the nucleotide sequence X (i.e., a nucleotide sequence highly conserved among plants) exists in another cucumber plant, "a base corresponding to the Yth base in the nucleotide sequence represented by SEQ ID NO: X" refers to a base in the nucleotide sequence X' that is determined to correspond to the Yth base by a method such as a homology search. For example, the nucleotide sequence represented by the polynucleotide described below in (ii: Polynucleotide containing SSR or SNP) is an example of the nucleotide sequence X'.

[0036] Molecular markers according to one embodiment of the present invention are, for example, the markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132 described in this Example. These are SSR markers newly identified by the present inventors, and those skilled in the art can identify the locations of these molecular markers on the genome based on their nucleotide sequences.

[0037] CsSSR0039 (hereinafter referred to as SSR(a)) can be determined to be resistant to cucumber spotted wilt disease, for example, by performing PCR using DNA extracted from a test cucumber plant as a template and a combination of a primer consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 1 and a primer consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 2.If the length of the resulting amplified product is 223 bp, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0038] YK325 (hereinafter referred to as SSR(b)) can be determined to be resistant to cucumber spotted wilt disease, for example, by performing PCR using DNA extracted from a test cucumber plant as a template and a combination of a primer consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 3 and a primer consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 4.If the length of the resulting amplified product is 260 bp, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0039] CsSSR0127 (hereinafter referred to as SSR(c)) can be determined to be resistant to cucumber spotted wilt disease, for example, by performing PCR using DNA extracted from a test cucumber plant as a template and a combination of a primer consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 5 and a primer consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 6.If the length of the resulting amplified product is 364 pb, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0040] CsSSR0128 (hereinafter referred to as SSR(d)) can be determined to be resistant to cucumber spotted wilt disease, for example, by performing PCR using DNA extracted from a test cucumber plant as a template and a combination of a primer consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 7 and a primer consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 8.If the length of the resulting amplified product is 373 bp, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0041] CsRSSR0132 (hereinafter referred to as SSR(e)) can be determined to be resistant to cucumber spotted wilt disease, for example, by performing PCR using DNA extracted from a test cucumber plant as a template and a combination of a primer consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 9 and a primer consisting of an oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 10, and if the length of the resulting amplified product is 294 bp, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0042] 4,258,982-4,259,13 (hereinafter referred to as SNP(f)) indicates a polymorphism in which the base corresponding to the 109th base in the base sequence shown in SEQ ID NO: 11 is T. That is, when SNP(f) is T, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0043] 5,480,094-5,480,243 (hereinafter referred to as SNP(g)) indicates a polymorphism in which the base corresponding to the 16th base in the base sequence shown in SEQ ID NO: 12 is A and the base corresponding to the 71st base is at least one of C. In other words, when SNP(g) is at least one of A and C, the base corresponding to the 16th base in the base sequence shown in SEQ ID NO: 12, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0044] 5,571,724-5,571,873 (hereinafter referred to as SNP(h)) indicates a polymorphism in which the base corresponding to the 107th base in the base sequence shown in SEQ ID NO: 13 is T. That is, when SNP(h) is T, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0045] SNP(i) 5,888,215-5,888,361 (hereinafter referred to as SNP(i)) indicates a polymorphism in which at least one of the following is true: the base corresponding to the 11th base in the base sequence set forth in SEQ ID NO: 14 is C, the base corresponding to the 16th base is T, the base corresponding to the 20th base is G, the base corresponding to the 47th base is A, the base corresponding to the 52nd base is T, and the base corresponding to the 56th base is A in the base sequence set forth in SEQ ID NO: 14. That is, when SNP(i) is true, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease when the base corresponding to the 11th base in the base sequence set forth in SEQ ID NO: 14 is C, the base corresponding to the 16th base is T, the base corresponding to the 20th base is G, the base corresponding to the 47th base is A, the base corresponding to the 52nd base is T, and the base corresponding to the 56th base is A.

[0046] 6,194,717-6,194,865 (hereinafter referred to as SNP(j)) indicates a polymorphism in which the base corresponding to the 121st base in the base sequence shown in SEQ ID NO: 15 is G. That is, when SNP(j) is G, the cucumber plant can be determined to be resistant to cucumber spotted wilt disease.

[0047] The molecular marker according to one embodiment of the present invention enables a determination that a cucumber plant is resistant to cucumber spotted wilt disease when the alleles of the bases corresponding to SSR(a) to SNP(j) in the cucumber plant are homozygous or heterozygous, respectively.

[0048] The molecular markers according to one embodiment of the present invention, SSR(a) to SNP(j), are preferably in linkage disequilibrium with the cucumber spotted wilt disease resistance gene. For example, a molecular marker in linkage disequilibrium with any of SSR(a) to SNP(j) can be used in the same way as SSR(a) to SNP(j).

[0049] It is preferable that the molecular marker according to one embodiment of the present invention is located at a short distance from the cucumber spotted wilt disease resistance-associated gene on the genome of the cucumber plant, thereby enabling more accurate determination of the presence or absence of resistance to cucumber spotted wilt disease in a test cucumber plant.

[0050] That is, the molecular marker according to one embodiment of the present invention is preferably CsSSR0039 or YK325, more preferably CsSSR0128 or CsRSSR0132, and most preferably CsSSR0127.

[0051] Furthermore, as a molecular marker according to one embodiment of the present invention, one of SSR(a) to SNP(j) may be used, or two or more of them may be used in combination.

[0052] (ii: Polynucleotide containing SSR or SNP) A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide containing any of the above-mentioned SSR(a) to SNP(j).

[0053] For example, the polynucleotides include (1) polynucleotides consisting of the nucleotide sequences of CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, and the nucleotide sequences shown in SEQ ID NOs: 11 to 15, and (2) polynucleotides in which one or several nucleotides are substituted, deleted, or amended with respect to the nucleotide sequences other than the SNP nucleotides and SSR nucleotide sequences of CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, and the nucleotide sequences shown in SEQ ID NOs: 11 to 15. (3) a polynucleotide consisting of an added or inserted base sequence and having the function of determining the resistance of a cucumber plant to cucumber spotted wilt disease; or (4) a polynucleotide consisting of a base sequence having 90% or more identity to base sequences other than SNP base and SSR base sequences in the base sequences of CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132 and the base sequences set forth in SEQ ID NOs: 11 to 15, and having the function of determining the resistance of a cucumber plant to cucumber spotted wilt disease.

[0054] The polynucleotide (2) may be one in which the bases corresponding to the SNP bases and the SSR base sequence are conserved in the base sequences of CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, and the base sequences set forth in SEQ ID NOs: 11 to 15, and the remaining base sequences may contain modifications (substitutions, deletions, insertions, or additions) of several bases (e.g., 1 to 10, preferably 1 to 5, and more preferably 1, 2, or 3). The base sequences of such polynucleotides are clear to those skilled in the art and can be determined by referring to the cucumber genome sequence 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 (3) 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 sequences of CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, and the base sequences set forth in SEQ ID NOs: 11 to 15, while conserving the bases corresponding to the SNP bases and the SSR base sequences. Such base sequence identity can be determined by aligning two base sequences using analysis software such as BLAST or FASTA.

[0056] The polynucleotide may be a PCR amplification product amplified by a primer set that amplifies a region containing any of SSR(a) to SNP(j).

[0057] As a molecular marker according to one embodiment of the present invention, one or a combination of two or more polynucleotides containing any of SSR(a) to SNP(j) may be used.

[0058] The method for determining resistance to cucumber spotted wilt disease in a cucumber plant using the above-mentioned molecular markers is not particularly limited, and for example, known SNP analysis methods for detecting SNPs can be used, including SNP analysis methods that involve detecting SNPs in PCR-amplified fragments of test cucumber plants.

[0059] That is, a primer set that amplifies a region containing at least one of SSR(a) to SNP(j) may be used to amplify the region in the DNA of a test cucumber plant. The region in the DNA of a test cucumber plant can be amplified by polymerase chain reaction (PCR) using DNA extracted from the test cucumber plant as a template and primers that amplify a region containing the SNP and SSR. The bases (genotypes) of the SNPs in the resulting amplified fragments are then determined, and the resistance of the cucumber plant to cucumber spotted wilt disease is assessed based on data showing the relationship between the determined bases (genotypes) and resistance to cucumber spotted wilt disease. In the case of SSR markers, for example, PCR can be performed using DNA extracted from the test cucumber plant as a template and primers specific to the region containing each SSR, and the base length of the resulting amplification product can be detected to determine whether the test cucumber plant is resistant to cucumber spotted wilt disease.

[0060] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment containing the target SSR or SNP. 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 400 bases (bases) or less, 350 b or less, 300 b or less, 200 b or less, 150 b or less, 120 b or less, or 100 b 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 b or more, 16 b or more, 17 b or more, 18 b or more, or 19 b or more, or 50 b or less, 40 b or less, or 30 b or less.

[0061] [Cucumber spotted wilt disease resistance-related gene] A cucumber spotted wilt disease resistance-associated gene in one embodiment of the present invention is linked to any of the molecular markers described above, and in one example, is located within a physical distance of 2,145 kbp from the molecular marker on the chromosome.

[0062] The cucumber spotted wilt disease resistance-associated gene refers to a qualitative trait gene that confers resistance to cucumber spotted wilt disease, and is linked to at least one of SSR (a) to SNP (j). The cucumber spotted wilt disease resistance-associated gene is located preferably within 2,145 kbp, more preferably within 1,065 kbp, of these SNPs on the genome of the cucumber plant.

[0063] Furthermore, for the gene associated with resistance to cucumber spotted wilt disease, the physical distance from the QTL peak on chromosome 6 to the start position of SNP (f) is -1,221,112 bp, the physical distance from the QTL peak on chromosome 6 to the start position of SNP (g) is 0 bp, the physical distance from the QTL peak on chromosome 6 to the start position of SNP (h) is 91,630 bp, the physical distance from the QTL peak on chromosome 6 to the start position of SNP (i) is 408,121 bp, and the physical distance from the QTL peak on chromosome 6 to the start position of SNP (j) is 714,623 bp.

[0064] The presence or absence of cucumber spotted wilt disease resistance in a cucumber plant can be determined based on the presence or absence of the cucumber spotted wilt disease resistance-associated gene. Detecting the presence or absence of the cucumber spotted wilt disease resistance-associated gene in a test cucumber plant can determine the cucumber spotted wilt disease resistance in a cucumber plant. The method for detecting the presence or absence of the cucumber spotted wilt disease resistance-associated gene in a test cucumber plant is not particularly limited, and detection can be performed by a conventionally known method using the molecular markers described above.

[0065] Furthermore, by introducing a cucumber spotted wilt disease resistance-associated gene into a cucumber plant, it is possible to confer resistance to cucumber spotted wilt disease to the cucumber plant. The method for introducing a cucumber spotted wilt disease resistance-associated gene into a cucumber plant is not particularly limited, and any conventionally known genetic engineering technique can be used.

[0066] [Cucumber plants resistant to cucumber spotted wilt disease (Cucumber spotted wilt disease resistant cucumber plants)] A cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention comprises a cucumber spotted wilt disease resistance locus on chromosome 6. In one example, the cucumber spotted wilt disease-resistant cucumber plant has the cucumber spotted wilt disease resistance locus on chromosome 6 introduced into it by hybridization or genetic engineering. In a cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention, the cucumber spotted wilt disease resistance locus on chromosome 6 is identified by the molecular markers described above, and in one example, is identified by at least one molecular marker selected from the group consisting of molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, and molecular markers in linkage disequilibrium with the molecular markers. In one embodiment of the present invention, there is provided a cucumber plant resistant to cucumber spotted wilt disease, wherein the introduced cucumber spotted wilt disease resistance locus can be identified using at least one molecular marker, and the molecular marker is a molecular marker for cucumber spotted wilt disease resistance in the cucumber plant, and the molecular marker is at least one molecular marker selected from the group consisting of the following (a) to (c), or a contiguous polynucleotide having at least an SSR or SNP contained in the molecular marker: (a) molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, (b) a molecular marker consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 11 to 15, and (c) a molecular marker in linkage disequilibrium with the molecular marker of (a) or (b).

[0067] Furthermore, a cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention further comprises a cucumber spotted wilt disease-resistant locus on at least one of chromosomes 1 and 3. In one example, in addition to the above-mentioned molecular markers, the plant may be further identified by at least one molecular marker (e.g., CSN251) on chromosomes 1 and 3.

[0068] A cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention is resistant to a highly virulent strain of MYSV. Furthermore, a cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention is resistant to the onset of fruit disease. For example, the cucumber spotted wilt disease-resistant cucumber plant exhibits reduced fruit mosaic symptoms caused by infection with a highly virulent strain, compared to a non-resistant plant.

[0069] A cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention is a plant having the cucumber spotted wilt disease-resistance-associated gene described above. A cucumber spotted wilt disease-resistant cucumber plant according to one embodiment of the present invention is a plant having the cucumber spotted wilt disease-resistance-associated gene described above and obtained by the production method described below. A cucumber spotted wilt disease-resistant cucumber plant is a plant having the cucumber spotted wilt disease-resistance-associated gene region identified by the molecular marker described above and exhibiting resistance to cucumber spotted wilt disease.

[0070] A cucumber plant resistant to cucumber spotted wilt disease according to one embodiment of the present invention is obtained by crossbreeding a cucumber plant resistant to cucumber spotted wilt disease with another cucumber plant, and by identifying and selecting cucumber plants resistant to cucumber spotted wilt disease from the resulting plants and their progeny lines using the molecular markers described above. The production method will be described in detail below. It should be noted that cucumber plants resistant to cucumber spotted wilt disease into which a gene associated with cucumber spotted wilt disease resistance has been introduced by genetic engineering are also included in the scope of the present invention.

[0071] [Method for determining resistance to cucumber spotted wilt virus] A method for determining resistance to cucumber spotted wilt disease according to one embodiment of the present invention includes a step of examining, in a cucumber plant, at least one molecular marker selected from the group consisting of the following (a) to (c) on chromosome 6, or a contiguous polynucleotide having at least an SSR or SNP contained in the molecular marker, as a molecular marker for resistance to cucumber spotted wilt disease: (a) molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, (b) molecular markers consisting of the nucleotide sequences set forth in SEQ ID NOs: 11 to 15, and (c) molecular markers in linkage disequilibrium with the molecular marker of (a) or (b).

[0072] A method for determining resistance to cucumber spotted wilt disease according to one embodiment of the present invention determines the resistance to cucumber spotted wilt disease in a test cucumber plant using at least one molecular marker selected from the above-described SSR (a) to SNP (j) and polynucleotide (a) to polynucleotide (j). A method for determining resistance to cucumber spotted wilt disease according to one embodiment of the present invention determines the presence or absence of resistance to cucumber spotted wilt disease in a test cucumber plant by identifying the presence or absence of a cucumber spotted wilt disease resistance-associated gene on a chromosome of the test cucumber plant using the above-described molecular marker.

[0073] The molecular marker used in the method for determining cucumber spotted wilt disease resistance according to one embodiment of the present invention is in linkage disequilibrium with the cucumber spotted wilt disease resistance gene, and for example, the linkage disequilibrium is such that the linkage disequilibrium coefficient is 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more.

[0074] Representative examples of molecular markers satisfying this linkage disequilibrium state are SSR(a) to SNP(j), but the present invention is not limited to these molecular markers, and for example, a molecular marker that is in linkage disequilibrium with any of SSR(a) to SNP(j) can also be used in the same way as SSR(a) to SNP(j). The molecular marker used in the method for determining resistance to cucumber spotted wilt disease according to one embodiment of the present invention preferably has a short genetic distance to the cucumber spotted wilt disease resistance-associated gene on the chromosome of the cucumber plant, thereby making it possible to more accurately determine whether or not a test cucumber plant has resistance to cucumber spotted wilt disease.

[0075] In a method for determining resistance to cucumber spotted wilt disease according to one embodiment of the present invention, the cucumber plant subject is a cucumber plant whose presence or absence of resistance to cucumber spotted wilt disease is unknown, and may be, for example, a hybrid plant between a cucumber spotted wilt disease-resistant cucumber plant and a cucumber spotted wilt disease-susceptible cucumber plant, or a progeny line thereof.

[0076] In a method for determining cucumber spotted wilt disease resistance according to one embodiment of the present invention, the testing step includes amplifying a region in DNA of the plant using a primer set that amplifies the region including the molecular marker.

[0077] One embodiment of the primer set is at least one of the following (a) to (j):

[0078] (a) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 1 with an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (b) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 3 with an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (c) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 5 with an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 6; (d) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 7 with an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 8; (e) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 9 with an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 10; (f) a primer set that amplifies a region including a base corresponding to the 109th base of the base sequence shown in SEQ ID NO: 11; (g) a primer set that amplifies a region including at least one of a base corresponding to the 16th base and a base corresponding to the 71st base of the base sequence shown in SEQ ID NO: 12; (h) a primer set that amplifies a region including a base corresponding to the 107th base of the base sequence shown in SEQ ID NO: 13; (i) a primer set that amplifies a region including at least one of a base corresponding to the 11th base, a base corresponding to the 16th base, a base corresponding to the 20th base, a base corresponding to the 47th base, a base corresponding to the 52nd base, and a base corresponding to the 56th base of the base sequence shown in SEQ ID NO: 14; and (j) a primer set that amplifies a region including a base corresponding to the 121st base of the base sequence shown in SEQ ID NO: 15.

[0079] A method for determining resistance to cucumber yellow spot disease according to one embodiment of the present invention uses any of the molecular markers described above to determine resistance to cucumber yellow spot disease. That is, the method for determining resistance to cucumber yellow spot disease in one example of the present invention includes the steps of: (a') determining the base length of the amplification product amplified using the primer set (a) above is 223 bp; (b') determining the base length of the amplification product amplified using the primer set (b) above is 260 bp; (c') determining the base length of the amplification product amplified using the primer set (c) above is 364 bp; (d') determining the base length of the amplification product amplified using the primer set (d) above is 373 bp; (e') determining the base length of the amplification product amplified using the primer set (e) above is 294 bp; (f') determining the base corresponding to the 109th base of the base sequence shown in SEQ ID NO: 11 in the amplification product amplified using the primer set (f) above is T; or (g') determining the base corresponding to the 16th base of the base sequence shown in SEQ ID NO: 12 in the amplification product amplified using the primer set (g) above is T. A plant can be determined to be resistant to cucumber spotted wilt when the base corresponding to the 11th base in the base sequence shown in SEQ ID NO: 14 is C, the base corresponding to the 16th base is T, the base corresponding to the 20th base is G, the base corresponding to the 47th base is A, the base corresponding to the 52nd base is T, or the base corresponding to the 56th base is A in the base sequence shown in SEQ ID NO: 14; or (j') the base corresponding to the 121st base in the base sequence shown in SEQ ID NO: 15 is G in the amplification product amplified using the primer set shown in (j).

[0080] In addition, in one embodiment of the method for determining resistance to cucumber spotted wilt disease of the present invention, the plant can be determined to be resistant to cucumber spotted wilt disease when the plant exhibits a combination of one, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten of the above (a') to (j').

[0081] For details of the molecular markers used in the method for determining cucumber spotted wilt disease 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.

[0082] In the method for determining resistance to cucumber spotted wilt disease according to one embodiment of the present invention, the method for testing the resistance of a cucumber plant to cucumber spotted wilt disease using a molecular marker is not particularly limited, and any known SNP analysis method can be used, such as a method of SNP analysis by detecting SNPs in PCR-amplified fragments of a test cucumber plant.

[0083] 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.

[0084] 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 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) every specified cycle. Depending on the state of the template DNA, PCR reaction conditions may be adjusted to stably detect SNPs, SSRs, and STSs.

[0085] As PCR for 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. SNPs in the amplified fragments amplified by PCR may be identified by analyzing the amplified fragments by determining their base sequences using an automated DNA sequencer or the like.

[0086] The method for extracting DNA to be amplified by PCR from a cucumber plant specimen 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 specimen and the amount of contaminants, appropriate pretreatment may be performed before the DNA extraction process. Furthermore, the DNA extracted from the specimen may be washed or purified as necessary for use as a template in a PCR reaction. Furthermore, the DNA extracted from the specimen may be digested with two restriction enzymes, and the resulting restriction enzyme fragments may be amplified by PCR.

[0087] According to the method for determining resistance to cucumber spotted wilt disease according to one embodiment of the present invention, the presence or absence of resistance to cucumber spotted wilt disease in a cucumber plant can be determined using molecular markers, and therefore, cucumber plants resistant to cucumber spotted wilt disease and their progeny lines can be selected based on the determination results.

[0088] In the determination method according to one aspect of the present invention, the test cucumber plant is a candidate plant for breeding material, a plant obtained through a breeding process, etc. Candidate plants for breeding material include, for example, parent plants used for crossbreeding and plants used in molecular breeding using genetic engineering. Furthermore, the test cucumber plant includes a cucumber plant used in molecular breeding using genetic engineering and a cucumber plant obtained by molecular breeding.

[0089] [Method for producing cucumber plants resistant to cucumber spotted wilt disease] A production method according to one embodiment of the present invention is a method for producing a cucumber plant resistant to cucumber spotted wilt disease, and includes the steps of: a hybridization step of crossing a cucumber plant resistant to cucumber spotted wilt disease with another cucumber plant; and a discrimination step of identifying a cucumber plant resistant to cucumber spotted wilt disease from the cucumber plant obtained in the hybridization step or a cucumber plant of its progeny by the method for determining resistance to cucumber spotted wilt disease described above. The production method can also be referred to as a creation method.

[0090] Therefore, the above-mentioned descriptions of the molecular marker, the gene associated with resistance to cucumber spotted wilt disease, the cucumber spotted wilt disease-resistant cucumber plant, and the method for determining resistance to cucumber spotted wilt disease are incorporated herein by reference in the description of the method for producing a cucumber plant having resistance to cucumber spotted wilt disease.

[0091] The cucumber plant having resistance to cucumber spotted wilt disease used as a parent plant in the crossing step may be a cucumber spotted wilt disease-resistant cucumber plant according to the present invention. Furthermore, the cucumber plant having resistance to cucumber spotted wilt disease used in the crossing step may be a cucumber plant having resistance to cucumber spotted wilt disease selected by the method of the present invention for determining resistance to cucumber spotted wilt disease. That is, the production method according to one aspect of the present invention may further comprise, prior to the crossing step, a discrimination step of discriminating a cucumber plant having resistance to cucumber spotted wilt disease from test cucumber plants by any of the above-described methods for determining resistance to cucumber spotted wilt disease.

[0092] In the discrimination step, the above-mentioned resistance to cucumber spotted wilt disease is determined from the cucumber plant obtained in the crossbreeding step or a cucumber plant of its progeny line by any of the above-mentioned methods for determining resistance to cucumber spotted wilt disease.

[0093] According to a production method according to one embodiment of the present invention, the presence or absence of resistance to cucumber spotted wilt disease in a cucumber plant can be determined using a molecular marker, and a cucumber plant resistant to cucumber spotted wilt disease selected based on the determination result can be produced.

[0094] 〔summary〕 The present invention includes any of the following aspects. <1> A method for determining resistance to cucumber spotted wilt disease, comprising the steps of: A method for detecting resistance to cucumber spotted wilt disease, comprising a step of testing a cucumber plant for at least one molecular marker selected from the group consisting of (a) to (c) below, on chromosome 6, or a contiguous polynucleotide having at least an SSR (Simple Sequence Repeat) or an SNP (Single Nucleotide Polymorphism) contained in the molecular marker: (a) Molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132; (b) a molecular marker consisting of the nucleotide sequence shown in SEQ ID NOs: 11 to 15, and (c) A molecular marker that is in linkage disequilibrium with the molecular marker in (a) or (b) above. <2> The cucumber plant is a candidate plant for breeding material or a plant obtained through a breeding process. <1> The method described below. <3> In the testing step, the region in the DNA of the plant is amplified using a primer set that amplifies the region containing the molecular marker. <1> or <2> The method described below. <4> The primer set is at least one of the following (a) to (j): <3> The method described below.

[0095] (a) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (b) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 3 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (c) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 5 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 6; (d) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 7 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 8; (e) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 9 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 10; (f) a primer set that amplifies a region containing a base corresponding to 109 bases of the base sequence shown in SEQ ID NO: 11; (g) a primer set that amplifies a region containing at least one of the bases corresponding to the 16th and 71st bases in the base sequence shown in SEQ ID NO: 12; (h) a primer set for amplifying a region containing a base corresponding to the 107th base in the base sequence shown in SEQ ID NO: 13; (i) a primer set that amplifies a region containing at least one of the bases corresponding to the 11th base, the 16th base, the 20th base, the 47th base, the 52nd base, and the 56th base in the base sequence shown in SEQ ID NO: 14; and (j) A primer set that amplifies a region containing the base corresponding to the 121st base in the base sequence shown in SEQ ID NO: 15. <5> A plant is determined to be resistant to cucumber spotted wilt disease when any of the following conditions (a') to (j') is met: <4> The method described below.

[0096] (a') The length of the amplification product amplified using the primer set (a) is 223 bp; (b') The length of the amplification product amplified using the primer set (b) is 260 bp; (c') The length of the amplification product amplified using the primer set (c) above is 364 bp; (d') The length of the amplification product amplified using the primer set (d) above is 373 bp; (e') The length of the amplification product amplified using the primer set (e) is 294 bp; (f') in the amplification product amplified using the primer set (f), the base corresponding to the 109th base in the base sequence shown in SEQ ID NO: 11 is T; (g') in the amplification product amplified using the primer set (g), at least one of the base corresponding to the 16th base of the base sequence shown in SEQ ID NO: 12 being A and the base corresponding to the 71st base being C; (h') in the amplification product amplified using the primer set (h), the base corresponding to the 107th base in the base sequence shown in SEQ ID NO: 13 is T; (i') in the amplification product amplified using the primer set of (i) above, at least one of the following bases in the base sequence shown in SEQ ID NO: 14: C for the 11th base, T for the 16th base, G for the 20th base, A for the 47th base, T for the 52nd base, and A for the 56th base; and (j') In the amplification product amplified using the primer set of (j) above, the base corresponding to the 121st base in the base sequence shown in SEQ ID NO: 15 is G. <6> A method for producing a cucumber plant having resistance to cucumber spotted wilt disease, comprising: A hybridization step of hybridizing a cucumber plant having resistance to cucumber spotted wilt disease with another cucumber plant; From the cucumber plant obtained by the hybridization step or a cucumber plant of its progeny, <1> ~ <5> a step of identifying a cucumber plant having resistance to cucumber spotted wilt disease by the method described in A manufacturing method comprising: <7> A cucumber plant resistant to cucumber spotted wilt disease, comprising a cucumber spotted wilt disease resistance locus on chromosome 6. <8> the cucumber spotted wilt disease resistance locus on chromosome 6 is identified by at least one molecular marker selected from the group consisting of molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, and molecular markers in linkage disequilibrium with the molecular markers; <7> 1. A cucumber plant resistant to cucumber spotted wilt disease according to claim 1. <9> the cucumber spotted wilt disease resistance locus on chromosome 6 has been introduced by mating or genetic engineering techniques; <7> or <8> 1. A cucumber plant resistant to cucumber spotted wilt disease according to claim 1. <10> and further comprising a cucumber spotted wilt disease resistance locus on at least one of chromosomes 1 and 3; <7> ~ <9> 1. A cucumber plant resistant to cucumber spotted wilt disease according to claim 1. <11> Resistant to virulent strains of melon spotted wilt virus. <7> ~ <10> 1. A cucumber plant resistant to cucumber spotted wilt disease according to claim 1. <12> Resistant to fruit disease, <7> ~ <11> 1. A cucumber plant resistant to cucumber spotted wilt disease according to claim 1. <13> A molecular marker for cucumber spotted wilt disease resistance in a cucumber plant, the molecular marker being at least one molecular marker selected from the group consisting of (a) to (c) below, or a contiguous polynucleotide having at least an SSR or SNP contained in the molecular marker: (a) Molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132; (b) a molecular marker consisting of the nucleotide sequence shown in SEQ ID NOs: 11 to 15, and (c) A molecular marker that is in linkage disequilibrium with the molecular marker in (a) or (b) above. <14> <13> and is located within a physical distance of 2,145 kbp from the molecular marker on the chromosome.

[0097] 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]

[0098] Example 1. Genetic analysis of spotted wilt disease resistance (1. Investigation of the disease incidence by inoculation with a virulent strain of Melon yellowing fusarium virus (MYSV)) <Investigation of the inoculation method of a virulent strain of Melon yellowing fusarium virus (MYSV) and the method for evaluating disease incidence> As materials, 109 individuals of the F2 population obtained from the cross between the yellowing fusarium disease-resistant 'Cucumber intermediate female parent Nong 7' and the susceptible 'Cucumber intermediate female parent Nong 4' were used. On September 1, 2021, seeds were sown in 7.5 cm pots filled with a culture soil mixture of Super Mix A (manufactured by Sakata Seed Corporation) and Nippi Horticultural Culture Soil No. 1 (manufactured by Nippon Fertilizer Co., Ltd.) at a ratio of 1:1, and seedlings were grown in a glass greenhouse. On September 6, 2021, MYSV (a virulent strain that causes severe mosaic on fruits) was inoculated onto the cotyledons. The inoculation method was to grind the frozen leaves infected with the MYSV virulent strain stored at -80°C with 10 times the volume of 0.1 M phosphate buffer pH 7.0 (containing 0.1% 2-mercaptoethanol), and rub the sap onto the cotyledons sprinkled with carborundum (#600). On September 16, 2021, they were transplanted in a single-row planting pattern with a 45 cm spacing between plants and a 120 cm spacing between rows in a PO film greenhouse. The training method was to train with a single main branch and pinch the main branch at the 20th node.

[0099] On October 15, 2021, the disease incidence scores (0: no symptoms, 1: slight mosaic, chlorotic spots, 2: mosaic, chlorotic spots, 3: mild fusarium, yellowing (area of yellowing and fusarium parts on the leaf ~20%), 4: fusarium, yellowing (20 - 50%), 5: severe fusarium, yellowing (50% or more), 6: death) were investigated for the 6th to 15th true leaves on the main branch. In addition, the disease incidence scores (0: no symptoms, 1: slightly chlorotic spot symptoms observed, 2: chlorotic spot symptoms observed, 3: mosaic symptoms with slight deformity observed, 4: mosaic symptoms with deformity observed, 5: severe mosaic symptoms with deformity observed) for fruits weighing about 100 g were investigated for 3 to 10 fruits per plant.

[0100] (2. Investigation of the disease incidence of yellowing fusarium disease by inoculation with a common strain of MYSV) <Investigation of the inoculation method of a common strain of MYSV and the method for evaluating disease incidence> The seedlings were an F2 population of 200 individuals from a cross between the yellow spot disease-resistant "Cucumber Intermediate Parent No. 7" and the disease-susceptible "Cucumber Intermediate Parent No. 4." On September 15, 2021, seeds were sown in 7.5 cm pots filled with a 1:1 mixture of Super Mix A (Sakata Seed Co., Ltd.) and Nippi Engei Baido No. 1 (Nihon Hiryo Co., Ltd.) and grown in a greenhouse. On September 21, 2021, cotyledons were inoculated with MYSV (a virulent strain that causes severe fruit mosaic). Frozen leaves infected with the MYSV common strain stored at -80°C were ground in 10 volumes of 0.1 M phosphate buffer, pH 7.0 (containing 0.1% 2-mercaptoethanol), and the resulting juice was rubbed onto cotyledons dusted with carborundum (#600). The plants were planted in a PO film greenhouse on September 30, 2021, in single rows with 45cm spacing between plants and 120cm furrow spacing. The plants were trained to a single main branch, with the top pinched off at the 20th node. The disease scores for the 6th to 15th true leaves of the main branch were assessed (0: no symptoms, 1: slight mosaic, chlorotic spots, 2: mosaic, chlorotic spots, 3: slight necrosis, yellowing (yellowing / necrotic area of ​​up to 20% of the leaf), 4: necrosis, yellowing (20-50%), 5: severe necrosis, yellowing (over 50%), 6: death).

[0101] <QTL analysis of spotted wilt disease resistance> (Double-digest restriction-site-associated DNA sequencing (ddRAD-Seq)) Genomic DNA was extracted from each individual of the F2 population using the DNeasy Plant Kit 96 (Qiagen, Hilden, Germany), and the final DNA concentration of each individual was adjusted to 20 ng / μL. Library preparation and sequencing were performed using a modified method of Shirasawa et al. (2016). For library preparation, two restriction enzymes, PstI and MspI (Thermo Fisher Scientific, Waltham, MA, USA), were used. The digested DNA was ligated with two types of adapters using T4 DNA ligase (Takara Bio Inc., Shiga, Japan), and then purified with Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to remove short DNA fragments (<300 bp). The purified DNA fragments were amplified by PCR using primers containing Illumina index sequences. The amplified fragments were concentrated using the QIAquick® PCR Purification Kit (Qiagen). The concentrated solution was electrophoresed on a 2.0% agarose gel (Kanto Chemical Co., Inc., Tokyo, Japan), and DNA fragments of 300 - 1000 bp were excised by physical means and then recovered using the MiniElute® Gel Extraction Kit (Qiagen). The library was sequenced on a HiSeq X sequencer (Illumina Inc., San Diego, USA) with 150 bp paired - end reads.

[0102] <NGS Data Acquisition and SNP Calling> The resulting sequencing data were filtered to remove low-quality sequences. The sequences were mapped to the cucumber reference genome sequence 'Chinese Long' v3 (Li et al. 2019) published in the Cucurbit Genomics Database of the International Cucurbit Genomics Initiative (http: / / www.icugi.org) using BWA-MEM2 v. 2.2.1 (Vasimuddin et al. 2019). The resulting SAM (sequence alignment / map) files were converted to BAM (binary sequence alignment / map) files and subjected to SNP calling. SNP calling and genotyping were performed using Stacks v. 2.64 (Catchen et al. 2011, Catchen et al. 2013).

[0103] <Linkage map construction and QTL analysis> Using the ddRAD-seq SNP data obtained for each individual F2 cross, a linkage map was constructed using Onemap v. 3.0.0 (Margarido et al. 2007) in R (R Core Team 2023). QTL analysis was performed using Windows® QTL Cartographer v. 2.5 software (Wang et al. 2007) with composite interval mapping (CIM). Permutation tests were performed 1000 times at a significance level of 0.05, and the threshold value for the logarithm of the odds (LOD) of the QTL was calculated.

[0104] 〔result〕 The symptoms of cucumber yellow spot disease on cucumber leaves at each disease score are shown in Figure 1.

[0105] Figure 2 shows the linkage map of the F2 population of "Cucumber intermediate parent No. 7" inoculated with the MYSV common strain and the susceptible "Cucumber intermediate parent No. 4."

[0106] Figure 3 shows the linkage map of the F2 population of the cucumber intermediate parent No. 7 and the susceptible No. 4, both inoculated with a virulent MYSV strain. The underlined areas on each chromosome in Figures 2 and 3 indicate the position where the QTL was detected (leaf disease score).

[0107] Figure 4 shows the results of QTL analysis of the gene for resistance to spotted wilt disease induced by inoculation with a common MYSV strain. Figure 5 shows the results of QTL analysis of the gene for resistance to spotted wilt disease induced by inoculation with a virulent MYSV strain.

[0108] As a result of QTL analysis, in addition to the existing QTL on chromosome 3, a new QTL on chromosome 6 was detected.

[0109] Example 2: Narrowing down the gene region on chromosome 6 involved in resistance to spotted wilt disease To narrow the region containing the resistance gene for a new QTL (approximately 25 cM) on chromosome 6, we developed lines that had undergone recombination within this region. From the F2 generation of a cross between "Cucumber Intermediate Parent No. 7" and the susceptible "Cucumber Intermediate Parent No. 4," heterozygous individuals within the QTL region were selected, and selfed seeds (F3 generation) were obtained. In the F3 generation, individuals with a fixed QTL (homozygous) were selected, and selfed seeds (F4 generation) were obtained. This F4 generation was used for spotted wilt disease resistance testing. For genotyping, PCR products were fluorescently labeled using the one-tube multiplex postlabeling method developed by Shimizu et al. (BMC Research Notes, 2011). The PCR products were analyzed using a DNA sequencer (Applied Biosystems 3730xl DNA Analyzer) and genotyped using GeneMapper software. The test for resistance to spotted wilt disease and the investigation of disease scores were carried out in the same manner as in (2. Investigation of the degree of spotted wilt disease caused by inoculation with a common MYSV strain).

[0110] Using DNA extracted from each line as a template, PCR reactions were carried out to specifically amplify each genotype using DNA markers A to G within an approximately 25 cM region of the QTL on cucumber chromosome 6.

[0111] 〔result〕 FIG. 6 shows the results of examining the relationship between the genotype of the marker near the QTL on chromosome 6 and the degree of disease on fruit.

[0112] This QTL on chromosome 6 was found to have the effect of reducing the mosaic symptoms in fruit caused by infection with virulent strains.

[0113] Figure 7 shows the results of an investigation into the relationship between marker genotype and spotted wilt disease resistance in a recombinant line in which recombination has occurred within the QTL region on chromosome 6 in a line in which the resistance locus on chromosome 3 is fixed in the susceptible homozygous form.

[0114] The marker genotypes of each line (F4 line of resistant "Cucumber intermediate parent No. 7" x susceptible "Cucumber intermediate parent No. 4") are shown in gray (resistant type), light gray (heterozygous type), and white (susceptible type).

[0115] As shown in Figure 7, the resistance gene was thought to be located between markers D (CsSSR0039) and E (YK325) (inside the bold frame). The region between markers D and E was 2,145 kb.

[0116] Further investigation of the relationship between marker genotypes within the 2,145 kb region between markers D and E obtained above and spotted wilt disease resistance narrowed down the resistance genes to a 1,065 kb region between markers CsSSR0132 and CsSSR0128.

[0117] Example 3: Creation of a selection marker near a gene on chromosome 6 The region where the resistance gene for the new QTL on chromosome 6 is located was narrowed by approximately 2,145 kb, and the amplification size of the SSR marker was examined in "Cucumber Intermediate Parent Nou 7," the susceptible cultivars "Cucumber Intermediate Parent Nou 4," and "Tokiwa" with the aim of developing an SSR marker that can distinguish between the resistant "Cucumber Intermediate Parent Nou 7" and susceptible cultivars within this region. Genotyping was performed using the same method as in Example 2, "Narrowing Down the Gene Region on Chromosome 6 Involved in Spotted Wilt Resistance."

[0118] 〔result〕 The amplification sizes of three SSR markers that are promising as selection markers in the new QTL region on chromosome 6 are summarized in Table 1 below.

[0119] [Table 1]

[0120] Example 4. Marker-based selection for spotted wilt disease resistance The source material was an F2 population derived from a cross between the yellow spot disease-resistant "Cucumber Intermediate Parent No. 7" and the disease-susceptible "Cucumber Intermediate Parent No. 5." On September 12, 2022, seeds were sown in 7.5 cm pots filled with a 1:1 mixture of Super Mix A (Sakata Seed Co., Ltd.) and Nippi Engei Baido No. 1 (Nihon Hiryo Co., Ltd.), and grown in a greenhouse. On September 16, 2022, the cotyledons were inoculated with MYSV (a virulent or normal strain). On September 26, 2022, the seeds were planted in a single row with 45 cm spacing and 120 cm furrow spacing in a PO film greenhouse. The plants were trained to a single main shoot, with the main shoot pinched at the 20th node. On November 11, 2022, disease scores were assessed for the 6th to 15th true leaves of the main shoot (0: no symptoms, 1: slight mosaic and chlorotic spots, 2: mosaic and chlorotic spots, 3: mild necrosis and yellowing (yellowing and necrosis account for up to 20% of the leaf area), 4: necrosis and yellowing (20-50%), 5: severe necrosis and yellowing (over 50%), 6: death). Disease scores were compared for each genotype for CSN251, located near the QTL on chromosome 3, and SSR marker CsSSR0127, located near a newly detected QTL on chromosome 6. Genotyping was performed in the same manner as in Example 2, "Narrowing down the gene region on chromosome 6 involved in resistance to spotted wilt disease."

[0121] 〔result〕 FIG. 8 shows the results of examining the relationship between the marker genotypes on chromosomes 3 and 6 and the degree of disease on leaves.

[0122] It was shown that by utilizing DNA markers near the QTL on chromosome 3 and the newly detected QTL on chromosome 6, it is possible to efficiently select individuals resistant to spotted wilt disease. [Industrial Applicability]

[0123] The present invention can be used in the fields of agriculture, plant breeding, etc.

Claims

1. A method for determining resistance to cucumber spotted wilt disease, comprising the steps of: A method for detecting resistance to cucumber spotted wilt disease, comprising a step of testing a cucumber plant for at least one molecular marker selected from the group consisting of (a) to (c) below, on chromosome 6, or a contiguous polynucleotide having at least an SSR (Simple Sequence Repeat) or an SNP (Single Nucleotide Polymorphism) contained in the molecular marker: (a) Molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128 and CsRSSR0132; (b) a molecular marker consisting of the base sequence shown in SEQ ID NOs: 11 to 15, and (c) A molecular marker that is in linkage disequilibrium with the molecular marker (a) or (b) above.

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

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

4. The method according to claim 3, wherein the primer set is at least one of the following (a) to (j): (a) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 1 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 2; (b) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 3 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 4; (c) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 5 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 6; (d) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 7 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 8; (e) a combination of an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 9 and an oligonucleotide containing the nucleotide sequence shown in SEQ ID NO: 10; (f) a primer set that amplifies a region containing a base corresponding to 109 bases of the base sequence shown in SEQ ID NO: 11; (g) a primer set that amplifies a region including at least one of a base corresponding to the 16th base and a base corresponding to the 71st base in the base sequence shown in SEQ ID NO: 12; (h) a primer set that amplifies a region containing a base corresponding to the 107th base in the base sequence shown in SEQ ID NO: 13; (i) a primer set that amplifies a region containing at least one of a base corresponding to the 11th base, a base corresponding to the 16th base, a base corresponding to the 20th base, a base corresponding to the 47th base, a base corresponding to the 52nd base, and a base corresponding to the 56th base in the base sequence shown in SEQ ID NO: 14; and (j) A primer set that amplifies a region containing a base corresponding to the 121st base of the base sequence shown in SEQ ID NO:

15.

5. The method according to claim 4, wherein the plant is determined to be resistant to cucumber spotted wilt disease when any of the following (a') to (j') is met: (a') the base length of the amplification product amplified using the primer set (a) is 223 bp; (b') the base length of the amplification product amplified using the primer set (b) is 260 bp; (c') the base length of the amplification product amplified using the primer set (c) is 364 bp; (d') the base length of the amplification product amplified using the primer set (d) is 373 bp; (e') the base length of the amplification product amplified using the primer set (e) is 294 bp; (f') in the amplification product amplified using the primer set (f), the base corresponding to the 109th base in the base sequence shown in SEQ ID NO: 11 is T; (g') in the amplification product amplified using the primer set of (g), at least one of the base corresponding to the 16th base of the base sequence shown in SEQ ID NO: 12 being A and the base corresponding to the 71st base being C; (h') in the amplification product amplified using the primer set of (h), the base corresponding to the 107th base of the base sequence shown in SEQ ID NO: 13 is T; (i') in the amplification product amplified using the primer set (i), at least one of the base corresponding to the 11th base of the base sequence shown in SEQ ID NO: 14 is C, the base corresponding to the 16th base is T, the base corresponding to the 20th base is G, the base corresponding to the 47th base is A, the base corresponding to the 52nd base is T, and the base corresponding to the 56th base is A; and (j') In the amplification product amplified using the primer set (j) above, the base corresponding to the 121st base in the base sequence shown in SEQ ID NO: 15 is G.

6. A method for producing a cucumber plant having resistance to cucumber spotted wilt disease, comprising: A hybridization step of hybridizing a cucumber plant having resistance to cucumber spotted wilt disease with another cucumber plant; a step of identifying a cucumber plant having resistance to cucumber spotted wilt disease from the cucumber plants obtained by the hybridization step or from the cucumber plants of the progeny line by the method of claim 1; A manufacturing method comprising:

7. A cucumber plant resistant to cucumber spotted wilt disease, comprising a cucumber spotted wilt disease resistance locus on chromosome 6.

8. 8. The cucumber spotted wilt virus-resistant cucumber plant according to claim 7, wherein the cucumber spotted wilt virus resistance locus on chromosome 6 is identified by at least one molecular marker selected from the group consisting of molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128, and CsRSSR0132, and molecular markers in linkage disequilibrium with the molecular markers.

9. 8. The cucumber spotted wilt disease-resistant cucumber plant according to claim 7, into which the cucumber spotted wilt disease-resistant gene locus on chromosome 6 has been introduced by crossbreeding or genetic engineering techniques.

10. 8. The cucumber spotted wilt disease-resistant cucumber plant of claim 7, further comprising a cucumber spotted wilt disease resistance locus on at least one of chromosomes 1 and 3.

11. The cucumber spotted wilt disease-resistant cucumber plant according to claim 7, which has resistance to a virulent strain of melon spotted wilt virus.

12. 8. The cucumber spotted wilt disease-resistant cucumber plant of claim 7, which is resistant to the disease on its fruit.

13. A molecular marker for cucumber spotted wilt disease resistance in cucumber plants, comprising: A molecular marker which is at least one molecular marker selected from the group consisting of (a) to (c) below, or a contiguous polynucleotide having at least an SSR or SNP contained in the molecular marker: (a) Molecular markers CsSSR0039, YK325, CsSSR0127, CsSSR0128 and CsRSSR0132; (b) a molecular marker consisting of the base sequence shown in SEQ ID NOs: 11 to 15, and (c) A molecular marker that is in linkage disequilibrium with the molecular marker (a) or (b) above.

14. A cucumber spotted wilt disease resistance-associated gene linked to the molecular marker according to claim 13 and located within a physical distance of 2,145 kbp from the molecular marker on the chromosome.

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