Molecular marker for resistance to chlorotic yellows disease in cucurbitaceae plants and use thereof

Abstract

To provide a technique enabling precise selection of melon individuals having resistance to chlorotic yellows disease.SOLUTION: A determination method for determining presence or absence of resistance to chlorotic yellows disease in Cucurbitaceae plants, comprising a step of determining, as a molecular marker related to resistance to chlorotic yellows disease in Cucurbitaceae plants, a gene region located on chromosome 1 of the Cucurbitaceae plant and consisting of any one of the base sequences (a)-(c) below: (a) a gene region consisting of the base sequence shown in any one of SEQ ID NOs: 1-3; (b) a gene region consisting of a base sequence in which one or more and ten or fewer bases are deleted, substituted, or added in the base sequence of (a), and which exhibits the same function as the gene region of (a) with respect to resistance to chlorotic yellows disease in Cucurbitaceae plants; (c) a gene region consisting of a base sequence having 90% or more sequence identity with the base sequence of (a), and which exhibits the same function as the gene region of (a) with respect to resistance to chlorotic yellows disease in Cucurbitaceae plants.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a molecular marker for resistance to chlorotic yellows in Cucurbitaceae plants and the use thereof. [Background technology]

[0002] Melon-producing regions both in Japan and overseas are suffering from serious damage caused by chlorotic yellows disease transmitted by the tobacco whitefly. Because only a limited number of pesticides are effective against the whitefly and there are limitations to its control, there is a strong demand for the development of chlorotic yellows-resistant varieties for stable melon production. To rapidly develop chlorotic yellows-resistant varieties, it is effective to identify the gene region involved in chlorotic yellows resistance and create a selection marker by referencing that gene region.

[0003] Non-Patent Document 1 describes a gene locus involved in chlorotic yellows resistance in melon. Non-Patent Document 2 describes that the above gene locus was used as a marker for chlorotic yellows resistance, and the relationship between the genotype and chlorotic yellows disease was investigated. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yoichi Kawazu et al., Euphytica(2018)214:239. [Non-patent document 2] Mitsuhiro Sugiyama et al., NARO Research Report No. 2: 1-9 (2019) Summary of the Invention [Problem to be solved by the invention]

[0005] The gene loci involved in chlorotic yellows resistance described in Non-Patent Documents 1 and 2 are approximately 1,350 kb long, and it is not easy to create a selection marker that enables highly accurate selection of melon individuals resistant to chlorotic yellows. Therefore, there is a need for a technology that enables more accurate selection of melon individuals resistant to chlorotic yellows.

[0006] One aspect of the present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a technique for accurately selecting melon individuals resistant to chlorotic yellows disease. [Means for solving the problem]

[0007] A method for determining whether a Cucurbitaceae plant has resistance to chlorotic yellows disease according to one embodiment of the present invention comprises determining whether the plant has resistance to chlorotic yellows disease, the method comprising determining a gene region located on chromosome 1 of the Cucurbitaceae plant and consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of the base sequence of (a) in which 1 to 10 bases are deleted, substituted, or added, and which exhibits a function equivalent to that of the gene region of (a) in terms of resistance to chlorotic yellows in Cucurbitaceae plants; (c) a gene region consisting of a base sequence having 90% or more sequence identity with the base sequence of (a) and exhibiting a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; and determining this as a molecular marker for resistance to chlorotic yellows in Cucurbitaceae plants.

[0008] A production method according to one embodiment of the present invention is a method for producing a chlorotic yellows disease-resistant Cucurbitaceae plant, and includes a selection step of selecting a chlorotic yellows disease-resistant Cucurbitaceae plant from a Cucurbitaceae plant using a determination method according to one embodiment of the present invention.

[0009] A molecular marker according to one embodiment of the present invention is a molecular marker for determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease, and is located on chromosome 1 of the Cucurbitaceae plant, and is a gene region consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of the base sequence of (a) in which 1 to 10 bases are deleted, substituted, or added, and which exhibits a function equivalent to that of the gene region of (a) in terms of resistance to chlorotic yellows in Cucurbitaceae plants; (c) a gene region having a base sequence that has 90% or more sequence identity with the base sequence of (a) and that exhibits a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants;

[0010] A determination kit according to one embodiment of the present invention is a determination kit for determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease, the determination kit comprising a gene region located on chromosome 1 of the Cucurbitaceae plant and consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of the base sequence of (a) in which 1 to 10 bases are deleted, substituted, or added, and which exhibits a function equivalent to that of the gene region of (a) in terms of resistance to chlorotic yellows in Cucurbitaceae plants; (c) a gene region having a base sequence that has 90% or more sequence identity with the base sequence of (a) and that exhibits a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; and [Effects of the Invention]

[0011] According to one aspect of the present invention, a technique for accurately selecting melon individuals resistant to chlorotic yellows disease can be provided. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the relationship between the genotype of DNA markers and the average severity of disease, as investigated in the Examples. [Figure 2] FIG. 1 shows the results of the selection efficiency of DNA markers investigated in the Examples. [Figure 3] FIG. 1 shows the results of determining chlorotic yellows resistance using DNA markers in an example. [Figure 4] FIG. 1 shows the results of determining chlorotic yellows resistance using DNA markers in an example. DETAILED DESCRIPTION OF THE INVENTION

[0013] 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)."

[0014] 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.

[0015] In one embodiment of the present invention, Cucurbitaceae plants include melon (Cucumis melo), watermelon (Citrullus lanatus), and cucumber (Cucumis sativus L.). In one embodiment of the present invention, the Cucurbitaceae plant for which a molecular marker for resistance to chlorotic yellows is used may be a melon plant. Examples of melon plant varieties include "I-10," "Melon Intermediate Parent No. 5 (PL5)," and "Earl's Favorite Spring Line No. 3."

[0016] Cucurbitaceae 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 genetic engineering. Furthermore, plants obtained through a breeding process include, for example, plants obtained by intraspecific hybridization of plants belonging to the above-mentioned varieties of Cucurbitaceae, and their progeny lines. Cucurbitaceae plants may also be interspecific hybrids, such as hybrids between a Cucurbitaceae variety and another Cucurbitaceae variety, and their progeny lines. Furthermore, Cucurbitaceae plants may also be interspecific hybrids between varieties known to be resistant to chlorotic yellows disease, and their progeny lines. Furthermore, Cucurbitaceae plants may also be plants obtained by intraspecific hybridization between individuals known to be resistant to chlorotic yellows disease, and their progeny lines.

[0017] 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 seeds, fruits, leaves, flowers, tuberous roots, etc.

[0018] In this specification, chlorotic yellows disease refers to a viral disease caused by Cucurbit chlorotic yellows virus (CCYV), which belongs to the genus Crinivirus. When cucurbit plants, including melon plants, are infected with this virus, small chlorotic spots appear on the leaves, and the entire leaf may then yellow. Furthermore, when melon plants are infected with chlorotic yellows disease, fruit weight and sugar content may decrease. In watermelon, the viral disease caused by CCYV is called chlorotic necrosis disease.

[0019] As used herein, a cucurbit plant being resistant to chlorotic yellows disease means that the cucurbit plant is not infected with the pathogenic virus of chlorotic yellows disease, has a low infection rate with the pathogenic virus of chlorotic yellows disease, does not develop chlorotic yellows disease, has a low incidence of chlorotic yellows disease, does not develop symptoms of chlorotic yellows disease, or has weak symptoms of chlorotic yellows disease. Note that, in this specification, resistance to chlorotic yellows disease is sometimes referred to as chlorotic yellows disease resistance or simply as resistance.

[0020] [Molecular markers for chlorotic yellows resistance in Cucurbitaceae plants] A molecular marker according to one embodiment of the present invention is a molecular marker for chlorotic yellows resistance in a Cucurbitaceae plant, which is capable of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease and is capable of selecting a resistant Cucurbitaceae plant.

[0021] The molecular marker is a gene region consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of the base sequence of (a) in which 1 to 10 bases are deleted, substituted, or added, and which exhibits a function equivalent to that of the gene region of (a) in terms of resistance to chlorotic yellows in Cucurbitaceae plants; (c) A base sequence having 90% or more sequence identity with the base sequence of (a) and containing a gene region that exhibits a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants.

[0022] A molecular marker according to one embodiment of the present invention may be a molecular marker located within a chlorotic yellows resistance gene region present on chromosome 1 of a Cucurbitaceae plant, or a molecular marker located within a region linked to the chlorotic yellows resistance gene region. The chlorotic yellows resistance gene region may be a region located in a region from 1,134,270 bp to 1,242,466 bp on chromosome 1 of a Cucurbitaceae plant, and consisting of the nucleotide sequence set forth in SEQ ID NO: 1. The chlorotic yellows resistance gene region may be a region located in a region from 1,130,133 bp to 1,250,268 bp on chromosome 1 of a Cucurbitaceae plant, and consisting of the nucleotide sequence set forth in SEQ ID NO: 2. The chlorotic yellows resistance gene region may be a region located in a region from 1,160,125 bp to 1,272,639 bp on chromosome 1 of a Cucurbitaceae plant, and consisting of the nucleotide sequence set forth in SEQ ID NO: 3. The nucleotide sequence shown in SEQ ID NO: 1 is derived from the melon variety "Earl's Favorite Harukei No. 3 (hereinafter referred to as Harukei No. 3)." The nucleotide sequence shown in SEQ ID NO: 2 is derived from the melon variety "I-10." The nucleotide sequence shown in SEQ ID NO: 3 is derived from the melon variety "PL5." Molecular markers are DNA markers that serve as indicators for the presence of a chlorotic yellows disease resistance gene region on chromosome 1 in Cucurbitaceae plants.

[0023] A molecular marker according to one embodiment of the present invention can be used to detect a genetic region associated with chlorotic yellows resistance. A genetic region associated with chlorotic yellows resistance refers to a quantitative trait locus or genetic region that affects chlorotic yellows resistance. The quantitative trait locus (QTL) generally refers to a chromosomal region involved in the expression of a quantitative trait. A QTL can be defined using a molecular marker that indicates a specific locus on a chromosome. Techniques for defining a QTL using such molecular markers are well known in the art.

[0024] A molecular marker refers to a genetic polymorphism in which a mutation is observed in a specific region of a DNA base sequence. In one embodiment of the molecular marker of the present invention, the genetic polymorphism is a genetic polymorphism based on the genome sequence of a melon plant. The genome sequence of the melon plant, which is the reference plant, is published in the Melonet DB (https: / / melonet-db.dna.affrc.go.jp / ap / top).

[0025] The genetic polymorphism detected by the molecular marker according to one embodiment of the present invention is a mutation in the gene region on chromosome 1 associated with chlorotic yellows resistance. The gene region on chromosome 1 associated with chlorotic yellows resistance consists of a base sequence derived from a melon plant. Other Cucurbitaceae plants may contain base sequence differences from the base sequence in addition to the above mutations. Such base sequences in other Cucurbitaceae plants are referred to as base sequences corresponding to the base sequence of the gene region on chromosome 1 associated with chlorotic yellows resistance. Because the genomic region in which this molecular marker is located is conserved among many Cucurbitaceae plants, this molecular marker can be identified by a method such as homology search.

[0026] That is, when a gene region related to chlorotic yellows resistance on chromosome 1 (i.e., a gene region that is highly conserved among plants) exists in other Cucurbitaceae plants, the nucleotide sequence of the gene region may be a "nucleotide sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 1." The "nucleotide sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 1" refers to a nucleotide sequence in the gene region related to chlorotic yellows resistance on chromosome 1 that has been determined to correspond to the "nucleotide sequence shown in SEQ ID NO: 1" by a method such as a homology search.

[0027] A molecular marker according to one embodiment of the present invention can determine whether a Cucurbitaceae plant is resistant to chlorotic yellows disease based on the genotype of the molecular marker in the Cucurbitaceae plant. The genotype of such a molecular marker can be detected, for example, using a primer pair specific to the molecular marker. Here, "a Cucurbitaceae plant is resistant to chlorotic yellows disease" means that the plant is not infected with the pathogenic virus that causes chlorotic yellows disease, does not develop chlorotic yellows disease, or does not exhibit symptoms of chlorotic yellows disease.

[0028] A molecular marker containing a gene region consisting of the base sequence (a) above can be obtained, for example, from a plant resistant to chlorotic yellowing disease (e.g., melon varieties "I-10" and "PL5") or a plant susceptible to the disease (e.g., melon variety "Harukei 3go") based on the base sequence of a gene region related to chlorotic yellowing disease resistance on chromosome 1.

[0029] A molecular marker containing a gene region consisting of the nucleotide sequence (b) above retains the function related to chlorotic yellows resistance and can have, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the nucleotide sequence (a) above. Such nucleotide sequence identity can be determined, for example, by aligning two nucleotide sequences using analysis software such as BLAST or FASTA.

[0030] A molecular marker containing a gene region consisting of the nucleotide sequence (c) above retains the function related to chlorotic yellows resistance and may be obtained by substituting, deleting, adding, or inserting, for example, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 base in the nucleotide sequence (a) above. The nucleotide sequence of such a molecular marker will be clear to those skilled in the art and can be determined by referring to the genome sequence of the melon plant described above or by decoding the nucleotide sequence of the gene region related to chlorotic yellows resistance on the genome of a chlorotic yellows-resistant Cucurbitaceae plant.

[0031] A molecular marker according to one embodiment of the present invention may be a genetic polymorphism within a chlorotic yellows resistance gene region in a Cucurbitaceae plant, and examples thereof include SNP markers, InDel markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite (SSR) markers, SCAR markers, and CAPS markers.

[0032] A molecular marker according to one embodiment of the present invention is a base itself corresponding to at least one of the following bases (i) to (iii), or a consecutive polynucleotide containing the base: (i) a base corresponding to the 251st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (ii) a base corresponding to the 251st base of the polynucleotide consisting of the base sequence set forth in SEQ ID NO: 5; (iii) a base corresponding to the 251st base of the polynucleotide consisting of the base sequence set forth in SEQ ID NO: 6; It could be.

[0033] Furthermore, a molecular marker according to one embodiment of the present invention may be an InDel marker for detecting insertions and deletions (InDels). Such a molecular marker for detecting InDels may comprise the nucleotide sequences of (iv) to (vii) below, or at least one of consecutive polynucleotides containing the nucleotide sequences: (iv) a nucleotide sequence corresponding to nucleotides 43,487 to 43,776 of the gene region consisting of the nucleotide sequence shown in SEQ ID NO: 2; (v) a nucleotide sequence corresponding to nucleotides 52,946 to 53,244 of the gene region consisting of the nucleotide sequence shown in SEQ ID NO: 2; (vi) a base sequence corresponding to bases 3,822 to 3,986 of the gene region consisting of the base sequence shown in SEQ ID NO: 2; and (vii) A nucleotide sequence corresponding to nucleotides 26,978 to 27,111 of the gene region consisting of the nucleotide sequence of SEQ ID NO: 2.

[0034] The above (iv) to (vii) correspond to the bases in the base sequence shown below. (iv) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 24 or a nucleotide sequence corresponding thereto; (v) the nucleotide sequence shown in SEQ ID NO: 25 or a nucleotide sequence equivalent thereto; (vi) the nucleotide sequence shown in SEQ ID NO: 30 or a nucleotide sequence equivalent thereto; and (vii) The nucleotide sequence shown in SEQ ID NO: 33 or a nucleotide sequence equivalent thereto.

[0035] The bases themselves corresponding to the above bases (i) to (iii), or consecutive polynucleotides containing such bases, are the SNP markers described in this Example or SNP markers that can be identified therewith. SNP markers that can be identified as the SNP markers described in this Example are SNP markers that are in linkage disequilibrium with the SNP markers described in this Example.

[0036] Here, "linkage disequilibrium" refers to the inheritance of two alleles linked to each other at a higher frequency than when each allele is inherited independently. In one embodiment of the present invention, the molecular marker is a base that is in linkage disequilibrium with a base corresponding to any of the bases (i) to (iii) above, and preferably a base that has a linkage disequilibrium coefficient r2≧0.9 with a base corresponding to any of the bases (i) to (iii) above.

[0037] Furthermore, the base in linkage disequilibrium with the base corresponding to any one of the bases (i) to (iii) above is more preferably a base that has a linkage disequilibrium coefficient r2 ≧ 0.95 with the base corresponding to any one of the bases (i) to (iii) above. Furthermore, the base in linkage disequilibrium with the base corresponding to any one of the bases (i) to (iii) above is even more preferably a base that has a linkage disequilibrium coefficient r2 ≧ 1 with the base corresponding to any one of the bases (i) to (iii) above. The base in linkage disequilibrium with any one of the bases (i) to (iii) above can be identified, for example, by sequence-analyzing DNA collected from multiple Cucurbitaceae plants using a sequencer and searching for bases in linkage disequilibrium.

[0038] An SNP marker can be (1) the base itself corresponding to the SNP, (2) a contiguous polynucleotide containing the SNP, or (3) a contiguous polynucleotide containing two or more SNPs.

[0039] (1: SNP markers and InDel markers) By using the molecular marker according to one embodiment of the present invention, it is possible to determine whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease.

[0040] Here, SNP refers to a DNA polymorphism in which a single base mutation is observed in a specific region in the DNA base sequence. In the SNP marker, SNP is a single nucleotide polymorphism based on the genomic sequence of a melon plant. InDel refers to a DNA polymorphism in which an insertion or deletion of one or more bases is observed in a specific region in the DNA base sequence. In the InDel marker, InDel is an insertion and deletion nucleotide polymorphism based on the genomic sequence of a melon plant. The genomic sequence of the melon plant, which is the reference plant, is published in the Melonet DB (https: / / melonet-db.dna.affrc.go.jp / ap / top).

[0041] "Bases (i) to (iii)" refer to the SNP markers described in this example. "Bases corresponding to bases (i) to (iii)" refer to SNP markers that can be identified with the SNP markers described in this example. "Base sequences (iv) to (vii)" refer to InDel markers described in this example. "Bases corresponding to base sequences (iv) to (vii)" refer to InDel markers that can be identified with the InDel markers described in this example. "Bases (i) to (iii)" and "base sequences (iv) to (vii)" refer to mutations in the gene region on chromosome 1 of Cucurbitaceae plants that is responsible for chlorotic yellows disease resistance. The gene region on chromosome 1 of Cucurbitaceae plants that is responsible for chlorotic yellows disease resistance consists of a base sequence derived from a reference melon plant. Other Cucurbitaceae plants may contain base sequence differences other than SNPs or InDels from the reference base sequence. The regions on the genome where these SNP markers and InDel markers are located are conserved among many Cucurbitaceae plants, and therefore these SNP markers and InDel markers can be identified by techniques such as homology search.

[0042] In other words, when a gene region on chromosome 1 associated with chlorotic yellows resistance (i.e., a region highly conserved among plants) exists in other Cucurbitaceae plants, "bases corresponding to bases (i) to (iii)" or "base sequences corresponding to the base sequences (iv) to (vii)" refer to bases or base sequences in the gene region associated with chlorotic yellows resistance on chromosome 1 that are determined to correspond to bases (i) to (iii) or the base sequence (iv) to (vii) by a method such as homology search. For example, the polynucleotides described in (i-2) to (iii-2) and the polynucleotides described in (i-3) to (iii-3) below are examples of gene regions on chromosome 1 associated with chlorotic yellows resistance.

[0043] A molecular marker according to one embodiment of the present invention is a SNP marker containing any one of the SNPs (i) to (iii) above. This SNP marker has been newly identified by the present inventors, and a person skilled in the art can identify the location of the SNP marker on the genome based on the nucleotide sequence representing each SNP of the SNP marker.

[0044] SNP (i) (hereinafter also referred to as SNP(i)) indicates a polymorphism of the 251st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 or a base corresponding thereto. SNP (ii) (hereinafter also referred to as SNP(ii)) indicates a polymorphism of the 251st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 or a base corresponding thereto. SNP (iii) (hereinafter also referred to as SNP(iii)) indicates a polymorphism of the 251st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6 or a base corresponding thereto. SNP(i), SNP(ii), and SNP(iii) correspond to marker P, marker Q, and marker R, respectively, in the examples described below.

[0045] In a molecular marker according to one embodiment of the present invention, when the base corresponding to SNP (i) is T, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease. When the base corresponding to SNP (ii) is G, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease. Furthermore, when the base corresponding to SNP (iii) is T, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease.

[0046] Here, "a Cucurbitaceae plant has resistance to chlorotic yellows disease" means that the plant has relatively strong resistance to chlorotic yellows disease compared to other Cucurbitaceae plant individuals not having the above-mentioned genotypes of SNPs (i) to (iii). Furthermore, "a Cucurbitaceae plant has resistance to chlorotic yellows disease" means that the plant has a relatively low infection rate with the pathogenic virus that causes chlorotic yellows disease, a relatively low incidence of chlorotic yellows disease, or relatively weak symptoms of chlorotic yellows disease compared to other Cucurbitaceae plant individuals not having the above-mentioned genotypes of SNPs (i) to (iii). A molecular marker according to another embodiment of the present invention is an InDel marker containing any of the above-mentioned InDels (iv) to (vii). This InDel marker has been newly identified by the present inventors, and those skilled in the art can identify the genomic location of the InDel marker based on the nucleotide sequence representing each InDel of this InDel marker.

[0047] InDel (iv) (hereinafter also referred to as InDel(iv)) is an InDel polymorphism at bases corresponding to bases 43,487 to 43,776 in the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2. InDel (v) (hereinafter also referred to as InDel(v)) is an InDel polymorphism at bases corresponding to bases 52,946 to 53,244 in the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2. InDel (vi) (corresponding to marker 2024B in the Examples, hereinafter also referred to as InDel(vi)) is an InDel polymorphism at bases corresponding to bases 3,822 to 3,986 in the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2. InDel (vii) (corresponding to marker 2024E in the examples, hereinafter also referred to as InDel(vii)) is an InDel polymorphism at bases corresponding to bases 26,978 to 27,111 of the polynucleotide consisting of the base sequence shown in SEQ ID NO:2.

[0048] For InDel(iv), if the base sequence shown in SEQ ID NO: 24 or a base sequence equivalent thereto does not exist in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 26 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 29 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant is determined to be susceptible to chlorotic yellows disease.

[0049] On the other hand, if the base sequence shown in SEQ ID NO: 24 or a base sequence equivalent thereto is present in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 26 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 29 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease.

[0050] For InDel(v), if the base sequence shown in SEQ ID NO: 25 or a base sequence equivalent thereto does not exist in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 26 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 29 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant is determined to be susceptible to chlorotic yellows disease.

[0051] On the other hand, if the base sequence shown in SEQ ID NO: 25 or a base sequence equivalent thereto is present in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 26 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 29 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease.

[0052] In one example of the present invention, a Cucurbitaceae plant is determined to be susceptible to chlorotic yellows disease when neither the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence equivalent thereto, nor the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence equivalent thereto, is present in the region of the gene region shown by the nucleotide sequence of SEQ ID NO: 2 from the nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 26 to the nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 29. On the other hand, when at least one of the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence equivalent thereto and the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence equivalent thereto is present in the region of the gene region shown by the nucleotide sequence of SEQ ID NO: 2 from the nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 26 to the nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 29, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease.

[0053] Resistant I-10 and PL5 are genotypes that have a base sequence containing an insertion sequence containing both the base sequence shown in SEQ ID NO: 24 and the base sequence shown in SEQ ID NO: 25, in a homozygous state on a homologous chromosome.

[0054] For InDel(vi), if the base sequence shown in SEQ ID NO: 30 or a base sequence equivalent thereto does not exist in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 31 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 32 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant is determined to be susceptible to chlorotic yellows disease.

[0055] On the other hand, if the base sequence shown in SEQ ID NO: 30 or a base sequence equivalent thereto is present in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 31 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 32 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease.

[0056] The resistant I-10 and PL5 strains have a genotype in which the nucleotide sequence containing the inserted sequence shown in SEQ ID NO: 30 is homozygous in the homologous chromosome.

[0057] For InDel(vii), if the base sequence shown in SEQ ID NO: 33 or a base sequence equivalent thereto does not exist in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 34 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 35 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant is determined to be susceptible to chlorotic yellows disease.

[0058] On the other hand, if the base sequence shown in SEQ ID NO: 33 or a base sequence equivalent thereto is present in the region from the base sequence corresponding to the base sequence shown in SEQ ID NO: 31 to the base sequence corresponding to the base sequence shown in SEQ ID NO: 32 in the gene region shown in the base sequence of SEQ ID NO: 2, the Cucurbitaceae plant can be determined to be resistant to chlorotic yellows disease.

[0059] The resistant I-10 and PL5 strains have a genotype in which the nucleotide sequence containing the inserted sequence having the nucleotide sequence shown in SEQ ID NO: 33 is homozygous in the homologous chromosome.

[0060] Furthermore, the molecular markers may be analyzed using at least two of SNPs (i) to (iii) and InDels (iv) to (vii) as haplotype blocks to determine whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease.

[0061] By using the molecular marker according to one embodiment of the present invention, it is possible to determine whether the alleles of the bases corresponding to SNPs (i) to (iii) and InDels (iv) to (vii) in a Cucurbitaceae plant are homozygous or heterozygous, respectively, thereby determining whether the Cucurbitaceae plant is resistant to chlorotic yellows disease.

[0062] (2: Polynucleotide containing SNP) A molecular marker according to one embodiment of the present invention may be a contiguous polynucleotide containing SNP(i) (hereinafter also referred to as polynucleotide (i)), a contiguous polynucleotide containing SNP(ii) (hereinafter also referred to as polynucleotide (ii)), or a contiguous polynucleotide containing SNP(iii) (hereinafter also referred to as polynucleotide (iii)).

[0063] Polynucleotide (i) is (i-1) a polynucleotide consisting of the base sequence of a region containing SNP(i) in the base sequence shown in SEQ ID NO: 4; (i-2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted relative to the base sequence other than SNP(i) in the base sequence of the polynucleotide (i-1), and having the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease; or (i-3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP(i) in the base sequence of the polynucleotide (i-1), and having the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease.

[0064] Polynucleotide (ii) is (ii-1) a polynucleotide consisting of the base sequence of a region containing SNP (ii) in the base sequence shown in SEQ ID NO: 5; (ii-2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted relative to the base sequence other than SNP (ii) in the base sequence of the polynucleotide of (ii-1), and has the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease; or (ii-3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (ii) in the base sequence of the polynucleotide of (ii-1), and has the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease.

[0065] Polynucleotide (iii) is (iii-1) a polynucleotide consisting of the base sequence of a region containing SNP (iii) in the base sequence shown in SEQ ID NO: 6; (iii-2) a polynucleotide consisting of a base sequence in which one or more bases have been substituted, deleted, added or inserted relative to the base sequence other than SNP (iii) in the base sequence of the polynucleotide (iii-1), and has the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease; or (iii-3) a polynucleotide consisting of a base sequence that is 90% or more identical to the base sequence other than SNP (iii) in the base sequence of the polynucleotide (iii-1), and has the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease. The polynucleotides (i-1) to (iii-1) can be obtained, for example, from a resistant Cucurbitaceae plant based on the nucleotide sequence of a gene region on chromosome 1 that is involved in chlorotic yellows disease resistance.

[0066] The polynucleotides (i-2) to (iii-2) may have the base sequences of the polynucleotides (i-1) to (iii-1) conserved at the bases corresponding to any of SNPs (i) to (iii), and 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 sequences. 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 melon plant described above, or by decoding the base sequence of the region adjacent to the SNP on the genome of a resistant Cucurbitaceae plant.

[0067] The polynucleotides (i-3) to (iii-3) may have, in the base sequence of the polynucleotides (i-1) to (iii-1), bases corresponding to any of SNPs (i) to (iii), conserved, and 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. Such base sequence identity can be determined, for example, by aligning two base sequences using analysis software such as BLAST or FASTA.

[0068] When the base corresponding to SNP (i) in polynucleotide (i) is T, the cucurbit plant can be determined to be resistant to chlorotic yellows disease. When the base corresponding to SNP (ii) in polynucleotide (ii) is G, the cucurbit plant can be determined to be resistant to chlorotic yellows disease. When the base corresponding to SNP (iii) in polynucleotide (iii) is T, the cucurbit plant can be determined to be resistant to chlorotic yellows disease. Furthermore, molecular markers can be used to determine whether a cucurbit plant is resistant by analyzing at least two of polynucleotides (i) to (iii) as a haplotype block.

[0069] A molecular marker according to one embodiment of the present invention may be a consecutive polynucleotide containing InDel(iv) (hereinafter also referred to as polynucleotide (iv)), a consecutive polynucleotide containing InDel(v) (hereinafter also referred to as polynucleotide (v)), a consecutive polynucleotide containing InDel(vi) (hereinafter also referred to as polynucleotide (vi)), or a consecutive polynucleotide containing InDel(vii) (hereinafter also referred to as polynucleotide (iv)). Polynucleotides (iv) to (vii) are polynucleotides that contain modifications (substitutions, deletions, insertions, or additions) of several (e.g., 1 to 10, preferably 1 to 5, and more preferably 1, 2, or 3) bases in the base sequence of InDel(iv) to (vii) shown in SEQ ID NO: 24, 25, 30, or 33, and have the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease; or polynucleotides that have the base sequence of InDel(iv) to (vii) shown in SEQ ID NO: 24, 25, 30, or 33, and have an identity of 90% or more, and have the function of determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease.

[0070] 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 melon plant described above, or by decoding the base sequence of the region adjacent to the InDel on the genome of a resistant Cucurbitaceae plant.

[0071] (3: Polynucleotide containing three SNPs) A molecular marker according to one embodiment of the present invention may be a continuous polynucleotide (hereinafter also referred to as polynucleotide (viii)) comprising at least two of SNPs (i) to (iii). Polynucleotide (viii) comprises a region between at least two of SNPs (i) to (iii), together with at least two of SNPs (i) to (iii). Polynucleotide (viii) can be obtained, for example, by referencing a region between at least two of the corresponding SNPs (i) to (iii) in a resistant Cucurbitaceae plant. The nucleotide sequence of polynucleotide (viii) at least partially matches the nucleotide sequence of the resistant Cucurbitaceae plant.

[0072] The method for determining the presence or absence of chlorotic yellows disease resistance in a Cucurbitaceae plant using a molecular marker according to one embodiment of the present invention is not particularly limited, and for example, a known SNP analysis method 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 Cucurbitaceae plant specimen. The Cucurbitaceae plant specimen may be a leaf, stem, root, or the like of a seedling, or may also be a fruit. Another embodiment of the method for determining the presence or absence of chlorotic yellows disease resistance in a Cucurbitaceae plant using a molecular marker according to one embodiment of the present invention is the method for determining the presence or absence of chlorotic yellows disease resistance in a Cucurbitaceae plant according to one embodiment of the present invention described below.

[0073] [Chlorotic yellows disease-resistant Cucurbitaceae plants] A chlorotic yellows disease-resistant Cucurbitaceae plant according to one embodiment of the present invention is a plant obtained by the production method described below. The chlorotic yellows disease-resistant Cucurbitaceae plant is a Cucurbitaceae plant having a genotype identified by the above-mentioned molecular markers.

[0074] A chlorotic yellows disease-resistant Cucurbitaceae plant according to one embodiment of the present invention can be obtained by identifying a chlorotic yellows disease-resistant Cucurbitaceae plant from a plant obtained by crossing Cucurbitaceae plants with each other and its progeny, or from a self-pollinated progeny of a Cucurbitaceae plant, using the molecular markers described above, as shown in the production method described below. Note that a chlorotic yellows disease-resistant Cucurbitaceae plant that has been genetically engineered to have the molecular markers described above is also included in the scope of the present invention.

[0075] [Method for determining the presence or absence of chlorotic yellows disease resistance in Cucurbitaceae plants] A method for determining the presence or absence of chlorotic yellows disease resistance in a Cucurbitaceae plant according to one embodiment of the present invention comprises determining, in a Cucurbitaceae plant, a gene region consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of the base sequence of (a) in which 1 to 10 bases are deleted, substituted, or added, and which exhibits a function equivalent to that of the gene region of (a) in terms of resistance to chlorotic yellows in Cucurbitaceae plants; (c) a gene region having a nucleotide sequence with 90% or more sequence identity to the nucleotide sequence of (a) and exhibiting a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; as a molecular marker for resistance to chlorotic yellows disease in Cucurbitaceae plants.

[0076] In a determination method according to one embodiment of the present invention, the presence or absence of chlorotic yellows disease resistance is determined in a test Cucurbitaceae plant using the molecular marker according to one embodiment of the present invention described above. The determination method according to one embodiment of the present invention determines whether the test Cucurbitaceae plant has resistance by identifying the genotype of a gene region consisting of any one of the base sequences (a) to (c) above in the genome of the test Cucurbitaceae plant. The determination method according to one embodiment of the present invention may determine whether the test Cucurbitaceae plant has resistance by identifying any one of the SNPs (i) to (iii) above in the genome of the test Cucurbitaceae plant.

[0077] In one embodiment of the method of the present invention, the test cucurbit plant is a candidate plant for breeding material or a plant obtained through a breeding process. The test cucurbit plant may be, for example, a hybrid between a resistant cucurbit plant and a resistant cucurbit plant, a hybrid between a resistant cucurbit plant and a susceptible cucurbit plant, or a hybrid between a susceptible cucurbit plant and a susceptible cucurbit plant, and their progeny. The test cucurbit plant may also be a hybrid between a cucurbit plant with known chlorotic yellows disease resistance and a cucurbit plant with unknown chlorotic yellows disease resistance, or a hybrid between cucurbit plants with unknown chlorotic yellows disease resistance, and their progeny. Furthermore, the test cucurbit plant may be a self-pollinated progeny of a resistant cucurbit plant. The test cucurbit plant may be a seedling leaf, stem, root, or fruit.

[0078] In one embodiment of the present invention, the method of determination determines that a Cucurbitaceae plant is resistant to chlorotic yellows disease when (i') the 251st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is T. The method of determination also determines that a Cucurbitaceae plant is resistant to chlorotic yellows disease when (ii') the 251st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 is G. The method of determination also determines that a Cucurbitaceae plant is resistant to chlorotic yellows disease when (iii') the 251st base of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 6 is T.

[0079] A determination method according to one embodiment of the present invention determines that a Cucurbitaceae plant is resistant to chlorotic yellows disease when it (iv') has a base sequence corresponding to the base sequence shown in SEQ ID NO: 24. The determination method also determines that a Cucurbitaceae plant is resistant to chlorotic yellows disease when it (v') has a base sequence corresponding to the base sequence shown in SEQ ID NO: 25. The determination method also determines that a Cucurbitaceae plant is resistant to chlorotic yellows disease when it (vi') has a base sequence corresponding to the base sequence shown in SEQ ID NO: 30. The determination method also determines that a Cucurbitaceae plant is resistant to chlorotic yellows disease when it (vii') has a base sequence corresponding to the base sequence shown in SEQ ID NO: 33.

[0080] For details of the molecular markers used in the determination method according to one aspect of the present invention, the above-mentioned description of the molecular markers according to one aspect of the present invention is incorporated herein by reference.

[0081] In one embodiment of the determination method of the present invention, the method of testing for the presence or absence of chlorotic yellows resistance in Cucurbitaceae plants using molecular markers is not particularly limited, and known SNP analysis methods can be used, such as a method of SNP analysis by detecting SNPs in PCR-amplified fragments of a test Cucurbitaceae plant.

[0082] In one embodiment of the present invention, the method may amplify a region in the DNA of the Cucurbitaceae plant using primers that amplify a region containing the molecular marker. One example of such primers is a primer that amplifies a region containing any of SNPs (i) to (iii) and InDels (iv) to (vii). That is, a primer that amplifies a region containing any of SNPs (i) to (iii) and InDels (iv) to (vii) can be used as a kit for determining whether a Cucurbitaceae plant is resistant to chlorotic yellows disease according to one embodiment of the present invention.

[0083] Amplification of the region in the DNA of a Cucurbitaceae plant specimen can be performed, for example, by polymerase chain reaction (PCR) using DNA extracted from the Cucurbitaceae plant specimen as a template and a primer set that amplifies a region containing an SNP or InDel. The bases (genotypes) of the SNPs in the resulting amplified fragments are then determined, and the presence or absence of chlorotic yellows disease resistance in the Cucurbitaceae plant is determined based on data showing the relationship between the determined bases (genotypes) and the presence or absence of chlorotic yellows disease resistance in the Cucurbitaceae plant. Methods for determining the genotype of the amplified fragments can be conventionally known, such as DNA sequence analysis, agarose gel electrophoresis, and TaqMan analysis using real-time PCR.

[0084] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment containing the target 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 200 bases (bases) or less, 160 b or less, 150 b or less, 140 b or less, 130 b or less, 120 b or less, or 100 b or less. The primer set includes a forward primer and 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, 19 b or more, 20 b or more, or 25 b or more, or may be 50 b or less, 40 b or less, or 30 b or less.

[0085] A primer set that amplifies a gene region consisting of any one of the base sequences (a) to (c) above is a determination kit for determining resistant Cucurbitaceae plants according to one embodiment of the present invention. Furthermore, a primer set that amplifies a region containing at least one of SNPs (i) to (iii) and at least one of InDels (iv) to (vii) as shown below can be a determination kit for determining resistant Cucurbitaceae plants according to one embodiment of the present invention. The primer set that amplifies a region containing at least one of SNPs (i) to (iii) can be a primer set used in nested PCR.

[0086] The primer set for amplifying the region containing SNP(i) may be, for example, (I) a first primer set consisting of a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 7 and a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 8, and (I') a second primer set consisting of a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 9 and a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 10. Nested PCR using this first primer set and second primer set can obtain a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 4 in chlorotic yellows-resistant Cucurbitaceae plants, allowing detection of the base corresponding to SNP(i).

[0087] The primer set for amplifying the region containing SNP(ii) may be, for example, (II) a third primer set consisting of a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 11 and a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 12, and (II') a fourth primer set consisting of a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 13 and a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 14. Nested PCR using the third and fourth primer sets can yield a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 5 in chlorotic yellows-resistant Cucurbitaceae plants, allowing detection of the base corresponding to SNP(ii).

[0088] The primer set for amplifying the region containing SNP(iii) may be, for example, (III) a fifth primer set consisting of a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 15 and a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 16, and (III') a sixth primer set consisting of a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 17 and a primer containing 15 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 18. Nested PCR using the fifth and sixth primer sets can yield a PCR amplification product consisting of the nucleotide sequence shown in SEQ ID NO: 6 in chlorotic yellows-resistant Cucurbitaceae plants, allowing detection of the base corresponding to SNP(iii).

[0089] For example, by designing a pair of primer sets that sandwich an InDel nucleotide sequence as primers capable of detecting InDels, it becomes possible to detect PCR amplification products in which an InDel nucleotide sequence has been inserted or deleted. When an InDel nucleotide sequence insertion sequence is present in the genome, a DNA fragment with a base length containing the insertion sequence is amplified. On the other hand, when the InDel nucleotide sequence is a deletion sequence, a DNA fragment with a deletion of a length corresponding to the insertion sequence is amplified.

[0090] In one example, PCR amplification products containing an InDel insertion or deletion can be detected by designing a pair of primer sets at a position within the base sequence in the region of the insertion sequence and at a position upstream or downstream in a region outside the insertion sequence (see Figure 3). If an insertion-type sequence containing an InDel base sequence is present in the genome, a DNA fragment of the base length in the region sandwiched between the primer sets designed for the regions inside and outside the insertion sequence will be amplified. On the other hand, if the InDel base sequence is a deletion sequence, no DNA fragment will be amplified.

[0091] In this way, it is possible to determine whether the sequence is an InDel insertion type or a deletion type.

[0092] In other words, in one embodiment of the determination method of the present invention, by confirming the presence or absence of an amplification product obtained by PCR using a primer set and the base length of the amplification product, it is possible to determine whether the genotype is homozygous for resistance to chlorotic yellows disease, heterozygous for resistance to chlorotic yellows disease, or homozygous for susceptibility to chlorotic yellows disease, and to determine whether the Cucurbitaceae plant is resistant to chlorotic yellows disease.

[0093] In the determination method, the primer set that amplifies the region containing InDel(v) may be, for example, (IV) a seventh primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 27, and (IV') an eighth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29.

[0094] These can also be used in PCR as (V) a ninth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26, a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 27, and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29. The ninth primer set corresponds to the first primer mix using primer p, primer q, and primer s in the Examples described below.

[0095] Another example may be (VI) a tenth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 28 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29, and (VI') an eleventh primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29.

[0096] As shown in the Examples, these can also be used in PCR as (VII) a 12th primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26, a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 28, and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29. The 12th primer set corresponds to the second primer mix using primer p, primer r, and primer s in the Examples described below.

[0097] As described in the examples below, when the 9th primer set (first primer mix) is used, resistant varieties that have the insertion sequence to be detected are amplified using the primer combination in the 7th primer set, while susceptible varieties that do not have the insertion sequence to be detected are amplified using the primer combination in the 8th primer set.

[0098] When the 12th primer set (second primer mix) is used, amplification is performed using the primer combination in the 10th primer set in resistant varieties that have the insertion sequence to be detected, and amplification is performed using the primer combination in the 11th primer set in susceptible varieties that do not have the insertion sequence to be detected.

[0099] By PCR using the above-mentioned 7th and 8th primer sets or PCR using the 9th primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 24 can be obtained in cucurbit plants resistant to chlorotic yellows disease, and the base sequence corresponding to InDel(iv) can be detected.

[0100] By PCR using the above-mentioned 9th and 10th primer sets or PCR using the 12th primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 25 can be obtained in cucurbit plants resistant to chlorotic yellows disease, and the base sequence corresponding to InDel(v) can be detected.

[0101] As described in the Examples, by using the ninth or twelfth primer set (the first or second primer mix in the Examples), a DNA fragment can be reliably amplified in a single PCR run, regardless of whether the test Cucurbitaceae plant is resistant or susceptible. Furthermore, based on the length of the amplified fragment, it is possible to easily determine whether the plant is resistant or susceptible and distinguish between homozygous and heterozygous genotypes.

[0102] An example of a primer set that amplifies a region containing InDel (vi) is (VIII) a 13th primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 31 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 32.

[0103] By PCR using the above-mentioned 13th primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 30 is obtained in cucurbit plants resistant to chlorotic yellows disease, and the base sequence corresponding to InDel(vi) can be detected.

[0104] An example of a primer set that amplifies a region containing InDel (vii) is (IX) a 14th primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 34 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 35.

[0105] By PCR using the above-mentioned 14th primer set, a PCR amplification product consisting of the base sequence shown in SEQ ID NO: 33 can be obtained in cucurbit plants resistant to chlorotic yellows, and the base sequence corresponding to InDel(vii) can be detected.

[0106] The primers of the present invention also include modified primers that have a base sequence that shares 90% or more sequence identity with the base sequences of the oligonucleotides that make up each primer in the primer set, and that are obtained by modifying some of the bases in the base sequence. The modified primers may share 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the base sequence shown in any of SEQ ID NOS: 7 to 18, 20 to 23, 26 to 29, 31, 32, 34, and 35 in the primers described above. This modified primer considered to be equivalent may have the same function as the original primer as a primer that hybridizes with the complementary DNA strand, and may be a primer consisting of an oligonucleotide having a modified base sequence in which, for example, 0 to 4, 0 to 3, 0 to 2, 1 to 3, or 1 or 2 bases have been substituted, deleted, inserted, or added in the base sequence of the oligonucleotide constituting each original primer. Here, in principle, a modified primer is considered equivalent to each primer as long as it can hybridize with the complementary DNA strand of each of the above primers.

[0107] By using such a primer set, a region containing a molecular marker can be specifically amplified, and the genotype can be determined by agarose electrophoresis, thereby determining the presence or absence of chlorotic yellows resistance in a Cucurbitaceae plant. A determination kit for determining the presence or absence of chlorotic yellows resistance in a Cucurbitaceae plant, which includes such a primer set, is also included in the scope of the present invention.

[0108] PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR device 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: a denaturation step, an annealing step, and an extension step; and a two-step PCR method, in which one cycle consists of two steps: a denaturation step, an annealing step, and an extension step. An example of reaction conditions for three-step PCR is 10 to 40 cycles (e.g., 38 cycles, 35 cycles, etc.) of a denaturation step at 90 to 100°C for 10 to 60 seconds (e.g., 95°C for 15 seconds, 95°C for 30 seconds, etc.), an annealing step at 55 to 65°C for 10 to 90 seconds (e.g., 64°C for 15 seconds, 55°C for 90 seconds, etc.), and an extension step at 65 to 80°C for 10 to 90 seconds (e.g., 72°C for 15 seconds, 72°C for 60 seconds, etc.). For example, the annealing temperature can be reduced stepwise (e.g., by 1°C per cycle) from an initial annealing temperature of 55–70°C (e.g., 64°C) to a final annealing temperature of 40–60°C (e.g., 60°C) for each cycle. PCR reaction conditions can be adjusted to ensure consistent SNP detection depending on the state of the template DNA. Such PCR reactions can be performed using, for example, a highly parallel real-time PCR device, the BioMark HD System (manufactured by Standard Biotools).

[0109] 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 having different wavelengths (e.g., NED, 6-FAM, VIC, PET, HEX) may be mixed.

[0110] As the 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 discrimination 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.

[0111] The method for extracting DNA to be amplified by PCR from a Cucurbitaceae 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 step. 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 one or two restriction enzymes, and the resulting restriction enzyme fragments may be amplified by PCR.

[0112] In addition, in the determination method according to one aspect of the present invention, genetic polymorphisms that are in linkage disequilibrium with SNP(i) to SNP(iii) may be analyzed to identify SNP(i) to SNP(iii). The linkage disequilibrium state is, for example, a linkage disequilibrium state with a linkage disequilibrium coefficient of 0.9 or more.

[0113] According to one aspect of the present invention, the method for determining whether a test Cucurbitaceae plant is resistant to chlorotic yellows disease can be determined using molecular markers, and thus chlorotic yellows-resistant Cucurbitaceae plants and their progeny can be selected based on the determination results.

[0114] According to the method of determining resistance to chlorotic yellows disease in a Cucurbitaceae family plant, it is possible to select chlorotic yellows disease-resistant plants at the seedling stage, thereby significantly shortening the time required for breeding selection. Furthermore, by planting and growing only the chlorotic yellows disease-resistant seedlings selected at the seedling stage, the efficiency of field utilization can be improved.

[0115] [Method for producing a chlorotic yellows disease-resistant Cucurbitaceae plant] A production method according to one embodiment of the present invention is a method for producing a chlorotic yellows disease-resistant Cucurbitaceae plant, and includes a selection step of selecting a chlorotic yellows disease-resistant Cucurbitaceae plant from a Cucurbitaceae plant using a determination method according to one embodiment of the present invention.

[0116] In addition, the production method may include a hybridization step in which Cucurbitaceae plants are hybridized with each other before the selection step, and in the selection step, a Cucurbitaceae plant resistant to chlorotic yellows disease may be selected from the Cucurbitaceae plants obtained by the hybridization step or other propagated Cucurbitaceae plants by a determination method according to one embodiment of the present invention.

[0117] Furthermore, the production method may include a step of obtaining a self-pollinated progeny of a chlorotic yellows disease-resistant Cucurbitaceae plant before the selection step, and in the selection step, a chlorotic yellows disease-resistant Cucurbitaceae plant may be selected from the self-pollinated progeny of the chlorotic yellows disease-resistant Cucurbitaceae plant obtained by the determination method according to one embodiment of the present invention.

[0118] Therefore, the explanations regarding the molecular marker, the chlorotic yellows disease-resistant cucurbit plant, and the determination method according to one embodiment of the present invention are incorporated herein by reference to explain the method for producing a chlorotic yellows disease-resistant cucurbit plant.

[0119] In the crossbreeding step, the cucurbit plants used as parent plants may be, for example, chlorotic yellows-resistant cucurbit plants, susceptible cucurbit plants, cucurbit plants whose resistance to chlorotic yellows is unknown, and their progeny lines.

[0120] Furthermore, the Cucurbitaceae plant used as a parent plant in the hybridization step may be a chlorotic yellows disease-resistant Cucurbitaceae plant according to one embodiment of the present invention. Furthermore, the Cucurbitaceae plant used in the hybridization step may be a chlorotic yellows disease-resistant Cucurbitaceae plant selected by the determination method according to one embodiment of the present invention. That is, the production method according to one embodiment of the present invention may further include, prior to the hybridization step, a discrimination step of discriminating a chlorotic yellows disease-resistant Cucurbitaceae plant from test Cucurbitaceae plants by the determination method according to one embodiment of the present invention. In the discrimination step, a chlorotic yellows disease-resistant Cucurbitaceae plant is discriminated from the Cucurbitaceae plant obtained in the hybridization step or a progeny Cucurbitaceae plant by the determination method according to one embodiment of the present invention.

[0121] According to a production method of one aspect of the present invention, it is possible to determine whether a Cucurbitaceae plant has resistance to chlorotic yellows disease using a molecular marker, and to produce a Cucurbitaceae plant resistant to chlorotic yellows disease that is selected based on the determination results.

[0122] 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]

[0123] [1. Gene region involved in resistance to chlorotic yellows disease] We developed recombinant lines in which recombination had occurred within a gene locus (approximately 1350 kbp) involved in resistance to chlorotic yellows disease, as described in Non-Patent Document 1. The genotypes of the polymorphic markers set at the locus in the developed recombinant lines were examined, and resistance tests were conducted on the recombinant lines. The results are shown in Figure 1.

[0124] The genomic DNA sequences of Earl's Favorite Spring Line No. 3 (hereinafter referred to as Spring Line No. 3), which is susceptible to chlorotic yellows (causative virus: CCYV), and I-10, which is resistant to the disease, were compared, and polymorphic DNA markers (SSR markers, SNP markers) were created between the two lines (Markers No. 1 to 23 in Figure 1).

[0125] The lines used in the CCYV resistance test (HA01 to HA12 in Figure 1) are the progeny (F6 generation) of a cross between Harukei 3 and the resistant I-10. In the F2 generation, individuals heterozygous for markers 1 to 23 were selected, and self-fertilized seeds (F3 generation) were obtained. In the F3 generation, individuals showing recombination within the QTL region were selected, and self-fertilized seeds (F4 generation) were obtained. In the F4 generation, individuals showing fixation (homozygous) within the QTL region were selected, and self-fertilized seeds (F5 generation) were obtained.

[0126] The 12 F6 generation lines (HA01-HA12) were further selfed and tested for chlorotic yellows resistance at the Kyushu Okinawa Agricultural Research Center (Kurume).

[0127] Melon seeds were sown in 9 cm pots filled with a 1:2 mixture of Super Mix A (Sakata Seed Co., Ltd.) and Kennae (Yae Nogei Co., Ltd.) on August 5 and 11, 2021 (re-seeding of strains with poor germination). The seedlings were grown in a greenhouse. Bemisia tabaci, previously infected with CCYV, was propagated in a screened greenhouse, feeding on cucumber plants. The potted seedlings were then transferred to the screened greenhouse and inoculated with CCYV insects between August 13 and 23 (August 20 and 30 for re-seeding). On August 23 (August 30 for re-seeding), the seeds were planted in a greenhouse in a two-row staggered arrangement with 30 cm spacing between plants and 40 cm spacing between rows. The training method was to train the plant to one main branch, and the primary side branches at nodes 11 to 13 were targeted to bear fruit (one fruit per plant).

[0128] After planting in the greenhouse, insecticides and fungicides were sprayed as needed to suppress the infestation and proliferation of Bemisia tabaci and the occurrence of other diseases within the greenhouse. On October 15, the 16th to 20th true leaves were scored for disease severity (0: no symptoms, 1: chlorotic spots visible, 2: yellowing visible (1-20% of the total), 3: yellowing spreading and becoming patchy (20-50% of the total), 4: yellowing widespread (more than 50% of the total), 5: nearly 100% yellowing), and the average disease severity score for the 16th to 20th true leaves was calculated. The average of four to five plants of each line was then calculated to represent the average disease severity for each line.

[0129] As shown in Figure 1, the genotype of each marker and the average disease severity were examined for each line. In Figure 1, the genotype of each marker for each line is shown in gray (resistant line type) or white (susceptible line type). As shown in Figure 1, the line with gray markers 16 and 17 had a lower average disease severity than the white line, suggesting that the chlorotic yellows resistance gene region is approximately 100 kbp between markers 15 and 18 (position surrounded by a bold frame).

[0130] (2. Selection efficiency using polymorphic markers) Polymorphic markers were designed in the chlorotic yellows resistance gene region narrowed down as described above, and the selection efficiency was compared with that of polymorphic markers outside the region. The results are shown in Figure 2.

[0131] CCYV resistance was evaluated in the same manner as described above in [1. Genetic regions involved in resistance to chlorotic yellows disease]. CCYV resistance was evaluated for four populations, populations 1 to 4. CCYV resistance tests were conducted for populations 1 to 4 in 2010, 2014, 2015, and 2022, respectively.

[0132] In the test for group 1, seeds were sown on September 1, 2010, inoculated with CCYV from September 8 to 13, planted in a greenhouse on September 16, and the disease score was checked on November 5, with the average disease score for the 16th to 20th true leaves being calculated. In the test for group 2, seeds were sown on August 18, 2014, inoculated with CCYV from August 26 to September 3, planted in a greenhouse on September 4, and the disease score was checked on November 5, with the average disease score for the 5th to 14th true leaves being calculated.

[0133] In the test for group 3, seeds were sown on September 3, 2015, inoculated with CCYV insects from September 11 to September 24, planted in a greenhouse on September 24, and the disease scores were checked on December 4, with the average disease score for the 5th to 14th true leaves being calculated. In the test for group 4, seeds were sown on July 25, 2022, inoculated with CCYV insects from August 3 to August 11, planted in a greenhouse on August 11, and the disease scores were checked on September 14, with the average disease score for the 5th to 14th true leaves being calculated.

[0134] To examine the genotype of polymorphic markers in each individual plant, genomic DNA was extracted from the true leaves of each plant using the DNeasy 96 Plant Kit (QIAGEN). Genotyping using single nucleotide polymorphism (SNP) markers was then performed using the BioMark HD System (Standard Biotools), a highly parallel real-time PCR system. SNP genotyping was performed using the BioMark HD (Standard Biotools) polymorphism analysis system.

[0135] Markers P and R shown in Figure 2 are located at both ends of the narrowed-down genetic region in [1. Genetic region involved in resistance to chlorotic yellows]. Marker Q is located within the genetic region, and marker Z is a marker near the end of the QTL described in Non-Patent Document 1 (approximately 680 kbp away from the narrowed-down genetic region).

[0136] Marker Z is an SNP marker that distinguishes 454,686 bp of bases on chromosome 1 of Harukei No. 3 (T is Harukei No. 3 type, C is I-10 type). The genomic DNA sequence surrounding Marker Z is shown in SEQ ID NO: 19 (the sequence of Harukei No. 3). The genomic DNA sequence surrounding Marker Z was amplified by nested PCR using a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 20 and 21 and a primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 22 and 23, and the genotype was identified.

[0137] Marker P is identical to Marker No. 15 in Figure 1 and is an SNP marker that distinguishes 1,134,270 bp of bases on chromosome 1 of Harukei No. 3 (A is Harukei No. 3 type, T is I-10 type). The genomic DNA sequence surrounding Marker P is shown in SEQ ID NO: 4 (the sequence of Harukei No. 3). The genomic DNA sequence surrounding Marker P was amplified by nested PCR using a first primer set consisting of the base sequences shown in SEQ ID NOs: 7 and 8 and a second primer set consisting of the base sequences shown in SEQ ID NOs: 9 and 10, and the genotype was identified.

[0138] Marker Q is an SNP marker that distinguishes 1,194,791 bp of bases on chromosome 1 of Harukei No. 3 (A is Harukei No. 3 type, G is I-10 type). The genomic DNA sequence surrounding Marker Q is shown in SEQ ID NO: 5 (the sequence of Harukei No. 3). The genomic DNA sequence surrounding Marker Q was amplified by nested PCR using a third primer set consisting of the base sequences shown in SEQ ID NOs: 11 and 12 and a fourth primer set consisting of the base sequences shown in SEQ ID NOs: 13 and 14, and the genotype was identified.

[0139] Marker R is identical to Marker No. 18 in Figure 1 and is an SNP marker that distinguishes 1,242,466 bp of bases on chromosome 1 of Harukei No. 3 (A is Harukei No. 3 type, T is I-10 type). The genomic DNA sequence surrounding Marker R is shown in SEQ ID NO: 6 (the sequence of Harukei No. 3). The genomic DNA sequence surrounding Marker R was amplified by nested PCR using a fifth primer set consisting of the base sequences shown in SEQ ID NOs: 15 and 16 and a sixth primer set consisting of the base sequences shown in SEQ ID NOs: 17 and 18, and the genotype was identified.

[0140] As shown in Figure 2, for markers P, Q, and R, the disease score of the YY type population is lower than that of the other populations (XY type, XX type). For example, if population 1 is divided into YY type, XY type, and XX type by marker P, the average disease score of YY type (1.0) is lower than the average disease score of XY type (2.5) and the average disease score of XX type (2.7). For marker Z, which is far from the narrowed gene region, the average disease scores of YY type, XY type, and XX type are 1.5, 2.3, and 2.6, respectively, narrowing the difference in disease score and resulting in low selection efficiency.

[0141] Thus, when markers were designed in the gene region narrowed down in the above-mentioned [1. Gene region involved in resistance to chlorotic yellows disease], the selection efficiency was higher than that of markers designed in the QTL described in Non-Patent Document 1, suggesting the presence of a chlorotic yellows disease resistance gene in that region. [2. Identifying chlorotic yellows resistance using DNA markers 1] The gene region consisting of the base sequence shown in SEQ ID NO: 2 on chromosome 1 of chlorotic yellows-resistant varieties (e.g., I-10) contains a long insertion sequence that is not present in susceptible varieties. As shown in Figure 3, primers were designed for the inner and outer regions of the insertion sequence and used in the following experiments.

[0142] Genomic DNA was extracted from the true leaves of each individual using the DNeasey Plant Mini Kit (QIAGEN).

[0143] Genomic DNA fragments surrounding the insertion sequence were amplified by PCR using a first primer mix consisting of primers p, q, and s, each consisting of the nucleotide sequences shown in SEQ ID NOs: 26, 27, and 29. Similarly, they were amplified by PCR using a second primer mix consisting of primers p, r, and s, each consisting of the nucleotide sequences shown in SEQ ID NOs: 26, 28, and 29. This allowed the genotype to be identified.

[0144] When the first primer mix "p, q, s" is used, in resistant varieties (with long insertion sequences), a DNA fragment is amplified by primer p, which is located in the outer region of the long insertion sequence, and primer q, which is located in the inner region of the long insertion sequence, but primers p and s, which are located at both ends of the long insertion sequence, do not amplify a DNA fragment because the amplified region is very long.

[0145] On the other hand, in susceptible varieties (without a long insertion sequence), no DNA fragment is amplified with primers p and q, but a short DNA fragment is amplified with primers p and s.

[0146] When the second primer mix "p, r, s" is used, in resistant varieties (with long insertion sequences), a DNA fragment is amplified by primer r, which is located in the inner region of the long insertion sequence, and primer s, which is located in the inner region of the long insertion sequence, but primers p and s, which are located at both ends of the long insertion sequence, do not amplify a DNA fragment because the amplified region is very long.

[0147] On the other hand, in susceptible varieties (without a long insertion sequence), no DNA fragment is amplified with primers r and s, but a short DNA fragment is amplified with primers p and s.

[0148] In resistant varieties (with long insertion sequences), the lengths of the DNA fragments amplified with primers p and q and those amplified with primers r and q are both designed to be longer than the lengths of the DNA fragments amplified with primers p and s in susceptible varieties (without long insertion sequences).

[0149] Therefore, by using the above-mentioned first or second primer mix, a DNA fragment can be reliably amplified in a single PCR run, regardless of whether the Cucurbitaceae plant sample is a resistant or susceptible variety. Furthermore, based on the length of the amplified fragment, it is possible to easily determine resistance or susceptibility and distinguish between homozygous and heterozygous genotypes.

[0150] The PCR composition was 2.5 μL of 2X Master Mix from the QIAGEN Multiplex PCR Kit (QIAGEN), 1.5 pmol of each primer (final concentration 0.3 μM), and approximately 10 ng of genomic DNA, for a total reaction volume of 5 μL.

[0151] The PCR reaction conditions were as follows: a 5-minute reaction at 95°C, followed by 35 cycles of a 30-second denaturation step at 95°C, a 90-second annealing step at 55°C, and a 60-second extension step at 72°C, followed by a 10-minute reaction at 68°C.

[0152] After the PCR reaction was completed, the product was electrophoresed on a 4% agarose gel, stained with EtBr, and then exposed to ultraviolet light to detect bands. The results are shown in Figure 3.

[0153] As shown in Figure 3, when the first primer mix "p, q, s" and the second primer mix "p, r, s" were used, small amplification product bands (201 bp) were obtained in the susceptible Harukei-3 and AnMP-5. Furthermore, both small and large amplification product bands were obtained in the F1 hybrids of susceptible Harukei-3 and I-10 and the F1 hybrid of AnMP5 and Chubono-5 (PL5). Large amplification product bands (290 bp with the first primer mix and 299 bp with the second primer mix) were obtained in the resistant I-10 and PL5.

[0154] From the above, it was demonstrated that the genotypes of chlorotic yellows disease-susceptible homozygotes, chlorotic yellows disease-resistant heterozygotes, and chlorotic yellows disease-resistant homozygotes can be distinguished using the InDel marker of the above insertion sequence.

[0155] Thus, it was demonstrated that the presence or absence of resistance to chlorotic yellows disease in Cucurbitaceae plants can be determined by detecting amplification products by PCR using the first primer mix and the second primer mix.

[0156] [3. Identifying chlorotic yellows resistance using DNA markers 2] Marker 2024B is an InDel marker that distinguishes the nucleotide sequence from positions 3,822 to 3,986 in the gene region consisting of the nucleotide sequence shown in SEQ ID NO: 2 on chromosome 1 of the resistant I-10 variety. Compared with the nucleotide sequence corresponding to the marker region in the susceptible variety, the nucleotide sequence of the resistant variety contains an inserted sequence that is not present in the nucleotide sequence corresponding to the marker region in the susceptible variety. The genomic DNA sequence of the marker 2024B region was amplified by PCR using a primer set consisting of a primer (2024B-Fw) consisting of the nucleotide sequence shown in SEQ ID NO: 31 and a primer (2024B-Rv) consisting of the nucleotide sequence shown in SEQ ID NO: 32, and the genotype was identified.

[0157] Marker 2024E is an InDel marker that distinguishes the nucleotide sequence from positions 26,978 to 27,111 in the gene region consisting of the nucleotide sequence shown in SEQ ID NO: 2 on chromosome 1 of I-10. Compared with the nucleotide sequence corresponding to the above marker region in the susceptible variety, the nucleotide sequence corresponding to the above marker region in the resistant variety is missing a portion of the sequence present in the nucleotide sequence in the susceptible variety.

[0158] The genomic DNA sequence of the marker 2024B region was amplified by PCR using a primer set consisting of a primer (2024E-Fw) consisting of the base sequence shown in SEQ ID NO: 34 and a primer (2024E-Rv) consisting of the base sequence shown in SEQ ID NO: 35, and the genotype was identified.

[0159] The PCR composition and reaction conditions were the same as those described above in [2. Identification of chlorotic yellows resistance using DNA markers 1].

[0160] After the PCR reaction was completed, the product was electrophoresed on a 4% agarose gel, stained with EtBr, and then exposed to ultraviolet light to detect bands. The results are shown in Figure 4. As shown in Figure 4, when marker 2024B was used, a small amplification product (134 bp) band was obtained in the susceptible Harukei-3 and AnMP-5. Furthermore, both small and large amplification product bands were obtained in the F1 cross between susceptible Harukei-3 and I-10 and the F1 cross between AnMP5 and Chumomo Nou 5 (PL5). A large amplification product (165 bp) band was obtained in the resistant I-10 and PL5.

[0161] On the other hand, when marker 2024E was used, a large amplification product band (219 bp) was obtained in the susceptible Harukei-3 and AnMP-5. Furthermore, both small and large amplification product bands were obtained in the F1 hybrid of the susceptible Harukei-3 and I-10 and the F1 hybrid of AnMP5 and PL5. A small amplification product band (134 bp) was obtained in the resistant I-10 and PL5.

[0162] From the above, it was demonstrated that the genotypes of chlorotic yellows disease-susceptible homozygotes, chlorotic yellows disease-resistant heterozygotes, and chlorotic yellows disease-resistant homozygotes can be distinguished using the above InDel marker 2024B or 2024E.

[0163] Thus, it was demonstrated that the presence or absence of chlorotic yellows disease resistance in Cucurbitaceae plants can be determined by detecting amplification products by PCR using the 2024B primer set or the 2024E primer set. [Industrial Applicability]

[0164] The present invention can be used in the fields of agriculture, plant breeding, etc.

Claims

1. A method for determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease, comprising: A gene region located on chromosome 1 of a Cucurbitaceae plant, and consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of a base sequence in which 1 to 10 bases are deleted, substituted, or added in the base sequence of (a), and which exhibits a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; (c) a gene region consisting of a base sequence having a sequence identity of 90% or more with the base sequence of (a) and exhibiting a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; as a molecular marker for resistance to chlorotic yellows disease in Cucurbitaceae plants.

2. In the determining step, In a Cucurbitaceae plant, at least one of the following bases (i) to (iii) themselves or a continuous polynucleotide containing said bases, and the following base sequences (iv) to (vii) themselves or a continuous polynucleotide containing said base sequences: (i) a base corresponding to the 251st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4; (ii) a base corresponding to the 251st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5; (iii) a base corresponding to the 251st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6; (iv) a nucleotide sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 24; (v) a nucleotide sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 25; (vi) a nucleotide sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 30; and (vii) a nucleotide sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 33 The method of claim 1, wherein the genotype of the gene region is detected by:

3. In the determining step, In a Cucurbitaceae plant, when at least one of the following (i') to (vii') is satisfied: (i') the base corresponding to the 251st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is T; (ii') the base corresponding to the 251st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 is G; (iii') the base corresponding to the 251st base of the polynucleotide consisting of the base sequence shown in SEQ ID NO: 6 is T; (iv') has a nucleotide sequence corresponding to the nucleotide sequence set forth in SEQ ID NO: 24; (v') has a nucleotide sequence corresponding to the nucleotide sequence set forth in SEQ ID NO: 25; (vi') having a nucleotide sequence corresponding to the nucleotide sequence set forth in SEQ ID NO: 30; and (vii') has a nucleotide sequence corresponding to the nucleotide sequence set forth in SEQ ID NO: 33; The method according to claim 2, wherein the Cucurbitaceae plant is determined to have resistance to chlorotic yellows disease.

4. 4. The method according to claim 2 or 3, wherein in the determining step, the region in the DNA of the Cucurbitaceae plant is amplified using primers that amplify a region including at least one of the bases corresponding to the bases (i) to (iii) themselves, or a consecutive polynucleotide containing the bases, and the base sequence of the bases (iv) to (vii) themselves, or a consecutive polynucleotide containing the base sequence.

5. A method for producing a chlorotic yellows disease-resistant Cucurbitaceae plant, comprising: A step of selecting a cucurbitaceous plant resistant to chlorotic yellows disease by the method of claim 1 or 2. A manufacturing method comprising:

6. A molecular marker for determining whether or not a plant in the Cucurbitaceae family has resistance to chlorotic yellows disease, A gene region located on chromosome 1 of a Cucurbitaceae plant, and consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of a base sequence in which 1 to 10 bases are deleted, substituted, or added in the base sequence of (a), and which exhibits a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; (c) a gene region consisting of a base sequence having a sequence identity of 90% or more with the base sequence of (a) and exhibiting a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; Molecular markers, including:

7. A kit for determining whether or not a Cucurbitaceae plant has resistance to chlorotic yellows disease, comprising: A gene region located on chromosome 1 of a Cucurbitaceae plant, and consisting of any one of the following base sequences (a) to (c): (a) a gene region consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 3; (b) a gene region consisting of a base sequence in which 1 to 10 bases are deleted, substituted, or added in the base sequence of (a), and which exhibits a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; (c) a gene region consisting of a base sequence having a sequence identity of 90% or more with the base sequence of (a) and exhibiting a function equivalent to that of the gene region of (a) with respect to resistance to chlorotic yellows in Cucurbitaceae plants; A determination kit comprising a primer for amplifying the above.

8. A primer set used in the determination method according to claim 4, comprising at least one primer set shown below: (I) a first primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 7 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 8, and (I') a second primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 9 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 10; (II) a third primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 11 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 12, and (II') a fourth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 13 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 14; (III) a fifth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 15 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 16, and (III') a sixth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 17 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 18; (IV) a seventh primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 27, and (IV') an eighth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29; (V) A ninth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26, a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 27, and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO:

29. (VI) a tenth primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 28 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29, and (VI') an eleventh primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29; (VII) a 12th primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 26, a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 28, and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 29; (VIII) a 13th primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 31 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 32; and (IX) A 14th primer set consisting of a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 34 and a primer containing 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 35.

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