Cucumber plant, method for selecting cucumber plants, and method for producing cucumber plants

Abstract

This invention provides cucumber plants resistant to anthracnose, a method for selecting them, and a method for producing them. [Solution] A cucumber plant having a first single nucleotide polymorphism marker sequence in which the eighth nucleotide in a specific nucleotide sequence is a nucleotide other than C. The eighth nucleotide may be T. When the cucumber plant is diploid, the first single nucleotide polymorphism marker sequence may be present on one or both of a pair of homologous chromosomes.

Description

Technical Field

[0001] This specification discloses cucumber plants, a method for selecting cucumber plants, and a method for producing cucumber plants.

Background Art

[0002] Cucumber anthracnose is the most important disease in open-field cucumber cultivation. Especially in Fukushima Prefecture, which is an important production area, the damage caused by anthracnose has been expanding since the end of the rainy season, resulting in a decrease in yield and excellent quality.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] Regarding cucumber anthracnose, it is believed that a functional deficiency of the stay-green gene SGR-1 confers anthracnose resistance (Non-Patent Literature 1). However, since SGR-1 is located on chromosome 5, which is the locus for existing downy mildew and powdery mildew resistance genes, its use is not suitable for developing multi-disease resistant varieties. Furthermore, it has been reported that Ban Kyuri is resistant to anthracnose, with the primary resistance locus located on chromosome 5 and a supplementary locus on chromosome 6 for race 0, and the primary resistance locus on chromosome 2 and two supplementary loci on chromosome 1 for race 1 (Non-Patent Literature 2). However, developing varieties using these multiple resistance loci requires a lot of time and effort, and commercially viable resistant varieties have not yet been developed. The objective of this disclosure is to provide a cucumber plant that is resistant to anthracnose. [Means for solving the problem]

[0005] This disclosure includes the following embodiments. Section 1. The first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A cucumber plant that has [this characteristic]. Section 2. The cucumber plant described in item 1, wherein the eighth nucleotide in the nucleotide sequence represented by sequence number 3 is T. Section 3. The cucumber plant according to item 1, wherein, when the cucumber plant is diploid, it has the first single nucleotide polymorphism marker sequence on one or both of a pair of homologous chromosomes. Section 4. The first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A second single nucleotide polymorphism marker sequence having a sequence in which the 8th nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is a nucleotide other than G, or A third single nucleotide polymorphism marker sequence having a sequence in which the 10th nucleotide in the nucleotide sequence represented by Sequence ID No. 4 is a nucleotide other than A, A cucumber plant that possesses and is resistant to anthracnose. Section 5. When the cucumber plant has the first single nucleotide polymorphism marker sequence, the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is T. When the cucumber plant has the second monosal polymorphism marker sequence, the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is A, or When the cucumber plant has the third single-nucleotide polymorphism marker sequence, the 10th nucleotide in the nucleotide sequence represented by Sequence ID No. 7 is C. The cucumber plant described in item 4. Section 6. The cucumber plant according to item 4, wherein, when the cucumber plant is diploid, it has the first single nucleotide polymorphism marker sequence, the second single nucleotide polymorphism marker sequence, or the third single nucleotide polymorphism marker sequence on one or both of a pair of homologous chromosomes. Section 7. Cucumber plants originating from seeds deposited under accession number FERM P-22504. Section 8. The first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C. A method for selecting cucumber plants, including selecting cucumber plants that possess [a certain characteristic]. Section 9. The first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A second single nucleotide polymorphism marker sequence having a sequence in which the 8th nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is a nucleotide other than G, or A third single nucleotide polymorphism marker sequence having a sequence in which the 10th nucleotide in the nucleotide sequence represented by Sequence ID No. 4 is a nucleotide other than A, A method for selecting cucumber plants, comprising selecting cucumber plants that possess and are resistant to anthracnose. Section 10. The method includes detecting at least a portion of a nucleotide sequence linked to the eighth nucleotide in the nucleotide sequence represented by SEQ ID NO: 3, within a range of 3 cM from the eighth nucleotide to the 5' and / or 3' side of the nucleotide sequence represented by SEQ ID NO: 3, wherein the eighth nucleotide is a nucleotide other than C. A method for selecting anthracnose-resistant cucumber plants. Section 11. The selection method according to item 10, wherein the linked nucleotide sequence is a nucleotide sequence found in a cucumber plant derived from seeds deposited under deposit number FERM P-22504. Section 12. A method for producing a cucumber plant having a first single nucleotide polymorphism marker sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, The method described above is The process of self-pollination or cross-pollination of cucumber plants. The process of culturing callus, The grafting process, or A process of introducing a gene locus containing the first single nucleotide polymorphism marker sequence into a cucumber plant by genetic modification. A method for producing cucumber plants, including [the specified ingredient]. Section 13. The first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A second single nucleotide polymorphism marker sequence having a sequence in which the 8th nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is a nucleotide other than G, or A third single nucleotide polymorphism marker sequence having a sequence in which the 10th nucleotide in the nucleotide sequence represented by SEQ ID NO: 4 is a nucleotide other than A, which has , and a method for producing a cucumber plant having anthracnose resistance, wherein the method comprises: a step of self-pollinating or cross-pollinating a cucumber plant; a step of culturing callus; a step of grafting, or a step of introducing into a cucumber plant a locus containing at least one of the first to third single nucleotide polymorphism marker sequences by genetic recombination A method for producing a cucumber plant, comprising. Item 14. Within a range of 3 cM on the 5' side and / or 3' side from the 8th nucleotide in the nucleotide sequence represented by SEQ ID NO: 3, detecting at least a part of a nucleotide sequence linked to the 8th nucleotide in the nucleotide sequence represented by SEQ ID NO: 3, wherein the said 8th nucleotide is a nucleotide other than C, A method for producing an anthracnose-resistant cucumber plant. Item 15. The method according to item 14, wherein the linked nucleotide sequence is a nucleotide sequence possessed by a cucumber plant derived from the seeds deposited under deposit number FERM P-22504.

Advantages of the Invention

[0006] It is possible to provide a cucumber plant having anthracnose resistance.

Brief Description of the Drawings

[0007] [Figure 1] It shows the results of cucumber anthracnose resistance QTL analysis.

Modes for Carrying Out the Invention

[0008] 1. Cucumber plant One embodiment relates to a cucumber plant.

[0009] The “cucumber plants” disclosed herein are not limited insofar as they are derived from Cucumis sativus. The cucumber plants are preferably derived from Cucumis sativus that can be cultivated in Japan. More preferably, the cucumber plants are derived from Cucumis sativus L. Examples of cucumber plant varieties include short white warts, four-leaf, British greenhouse type, slice type, and bait-alpha type. Among these, the short white wart type cucumber is preferred.

[0010] Examples of "plants" include plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plant individuals, plant callus, plant clums (ps), plant cells isolated from plants or parts of plants, leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, pistils, flowers, ovaries, ovules, seeds, fruits, stems, seedlings, etc. Examples of parts of plant individuals include organs, tissues, cells, or vegetative propagates. Examples of organs include petals, corollas, flowers, leaves, seeds, fruits, stems, roots, etc. Examples of tissues include parts of the organs. Specific examples of the plant individual parts include microspores, flowers, flower buds, pistils, anthers, pollen, ovaries, embryos, ovules, hypocotyls, embryo sacs, egg cells, cuttings, roots, root tips, trunks, stems, leaves, petioles, pith, cotyledons, cells, meristematic cells, protoplasts, seeds, etc. The pollen may be mature or immature. The plant individual parts may originate from any stage of plant development, such as pre-rooting, post-rooting, seedlings, cuttings, or mature individuals. The plant individual parts may consist of one type of organ, tissue, and / or cell, or two or more types of organs, tissues, and / or cells.

[0011] In this specification, “plant body” refers to a cucumber plant having at least roots, stems, and leaves.

[0012] The cucumber plants disclosed herein are resistant to anthracnose. Anthracnose is caused by the filamentous fungus Colletotrichum orbiculare. Anthracnose resistance can be tested by the following method.

[0013] Cucumber anthracnose fungus was cultured on potato decoction agar at 25°C for 2 weeks. Next, the conidial mass formed on the medium was suspended in sterile distilled water and filtered through double gauze, and the conidial concentration was 1 × 10⁶. 3 The inoculum is prepared by adjusting the concentration to 1 / ml. Test plants are sown at a rate of 1 seed per pot in 9cm pots filled with commercially available horticultural seedling soil, and grown at 25°C for approximately 3 weeks until two true leaves are fully developed. The second true leaves of these seedlings (individuals) are sprayed with 1 ml of the inoculum per leaf using a spray bottle, and cultivated in a greenhouse set to a maximum of 25°C and a minimum of 15°C. Disease development is checked 6 to 8 days after inoculation. Disease development is evaluated on a five-point scale: 0: no symptoms, 1: lesions less than 5 mm on the leaves, 2: lesions less than 10 mm on the leaves, 3: lesions 10 mm or larger on the leaves or wilting on part of the leaves, 4: wilting of the entire leaf. Individuals with a disease severity index of 3 or higher are judged to be susceptible to anthracnose, and individuals with a disease severity index of less than 3 are judged to be resistant to anthracnose. When evaluating multiple test plants, the average disease incidence index for each line is calculated by dividing the sum of the disease incidence indices obtained for each individual test plant by the number of individuals. An average disease incidence index of 3.5, preferably 3, is used as the baseline value. Lines with an average disease incidence index of 3.5 or higher are determined to be susceptible to anthracnose, and lines with an average disease incidence index below the baseline are determined to be resistant to anthracnose.

[0014] Here, the cucumber anthracnose fungus used in the anthracnose resistance test is, for example, Colletotrichum orbiculare isolated from cucumber, which is deposited in the National Agriculture and Food Research Organization (NARO) Agrobiological Resources Gene Bank (https: / / www.gene.affrc.go.jp / databases-micro_search.php). Examples of cucumber anthracnose fungi available from this gene bank include MAFF306867, MAFF306868, MAFF306869, and MAFF306870.

[0015] Cucumber plants resistant to anthracnose have a single nucleotide polymorphism (SNP) at a specific site in the short sequence or allele long sequence shown in Table 1. In Table 1, the short sequence or allele long sequence indicates a sequence susceptible to anthracnose. In Table 1, the underlined portion of the nucleotide sequence in the column labeled "Susceptible_allele short sequence of SNP marker" or "Susceptible_allele long sequence of SNP marker" indicates the site where the single nucleotide polymorphism exists.

[0016] Specifically, the single nucleotide polymorphism marker sequences may contain SNPs in the underlined nucleotides of the sequences represented by SEQ ID NOs: 1 to 20 shown in Table 1 (the 11th nucleotide of SEQ ID NOs: 1, the 8th nucleotide of SEQ ID NOs: 2, the 8th nucleotide of SEQ ID NOs: 3, the 10th nucleotide of SEQ ID NOs: 4, the 10th nucleotide of SEQ ID NOs: 5, the 8th nucleotide of SEQ ID NOs: 6, the 9th nucleotide of SEQ ID NOs: 8, the 101st nucleotide of SEQ ID NOs: 8, the 101st nucleotide of SEQ ID NOs: 9, the 36th nucleotide of SEQ ID NOs: 10, the 36th nucleotide of SEQ ID NOs: 11, the 36th nucleotide of SEQ ID NOs: 12, the 101st nucleotide of SEQ ID NOs: 13, the 36th nucleotide of SEQ ID NOs: 14, the 7th nucleotide of SEQ ID NOs: 15, the 8th nucleotide of SEQ ID NOs: 16, the 8th nucleotide of SEQ ID NOs: 17, the 36th nucleotide of SEQ ID NOs: 18, the 36th nucleotide of SEQ ID NOs: 19, or the 36th nucleotide of SEQ ID NOs: 20). In cucumber plants resistant to anthracnose, the underlined nucleotides described in SEQ ID NOs: 1 to 20 have SNPs in which they are substituted with other nucleotides. The preferred nucleotides present in cucumber plants resistant to anthracnose are listed in Table 1 under Resistant SNPs. Cucumber plants resistant to anthracnose do not need to possess all Resistant SNPs.

[0017] Preferably, in cucumber plants, (1) A single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence shown in Sequence ID No. 3 is a nucleotide other than C, preferably a single nucleotide polymorphism marker sequence having a sequence in which the 36th nucleotide in the nucleotide sequence shown in Sequence ID No. 10 is a nucleotide other than C; preferably, the nucleotide other than C is T;

[0018] (2) A single nucleotide polymorphism marker sequence having a sequence in which the 8th nucleotide in the nucleotide sequence shown in Sequence ID No. 2 is a nucleotide other than G, preferably a single nucleotide polymorphism marker sequence having a sequence in which the 101st nucleotide in the nucleotide sequence shown in Sequence ID No. 9 is a nucleotide other than G; preferably, the nucleotide other than G is A; or

[0019] (3) A single nucleotide polymorphism marker sequence having a sequence in which the 10th nucleotide in the nucleotide sequence shown in Sequence ID No. 4 is a nucleotide other than A, preferably a single nucleotide polymorphism marker sequence having a sequence in which the 36th nucleotide in the nucleotide sequence shown in Sequence ID No. 11 is a nucleotide other than A; preferably, the nucleotide other than A is C. It has a single nucleotide polymorphism marker sequence.

[0020] Cucumber plants only need to have at least the single nucleotide polymorphism marker sequence described in (1) above, but may also have the single nucleotide polymorphism marker sequence described in (1) above and the single nucleotide polymorphism marker sequence described in (2) above; the single nucleotide polymorphism marker sequence described in (1) above and the single nucleotide polymorphism marker sequence described in (3) above; or the single nucleotide polymorphism marker sequence described in (1) above, the single nucleotide polymorphism marker sequence described in (2) above, and the single nucleotide polymorphism marker sequence described in (3) above.

[0021] Furthermore, the single nucleotide polymorphism marker sequences shown in Table 1 may originate from SNPs and exhibit anthrax resistance, but they may also be linked to gene loci associated with anthrax resistance.

[0022] [Table 1] *In Table 1, A represents a nucleotide containing adenine as the base, G represents a nucleotide containing guanine as the base, C represents a nucleotide containing cytosine as the base, and T represents a nucleotide containing thymine as the base.

[0023] In Table 1, the SNP position indicates the physical location (location on chromosome 1) when using NCBI Reference Sequence: NC_026655.1 as the reference sequence. However, the above single nucleotide polymorphism marker sequences do not necessarily have to be located on chromosome 1. For example, they may be located on other chromosomes or on transposons.

[0024] The single nucleotide polymorphism marker sequences shown in Table 1 may include nucleotide substitutions, deletions, insertions, etc., at positions other than the SNP positions listed in the table. Sequences that have 70% or more, 80% or more, 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, or 99% or more identity with the single nucleotide polymorphism marker sequence (excluding the underlined nucleotides) and have less than 100% identity may also be included.

[0025] When the cucumber plant is diploid, the above single nucleotide polymorphism marker sequence may be present on one or both of a pair of homologous chromosomes. When there are multiple types of the above single nucleotide polymorphism marker sequences (1) to (3), the multiple types of single nucleotide polymorphism marker sequences may be located on the same chromosome or on different chromosomes. Preferably, the above single nucleotide polymorphism marker sequences (1) to (3) are located on the same chromosome, preferably on chromosome 1, and more preferably between nucleotides 18384314 to 24086658 when the sequence is referenced from NCBI Reference Sequence: NC_026655.1.

[0026] The genetic distance between the SNP at the physical location indicated by SNP ID NO:2 and the SNP at the physical location indicated by SNP ID NO:7 in Table 1 is approximately 17.0 to 20 centimorgans (cM), preferably approximately 17.5 to 18.5 centimorgans (cM). The genetic distance between the SNP at the physical location indicated by SNP ID NO:2 and the SNP at the physical location indicated by SNP ID NO:6 in Table 1 is 10 to 15 centimorgans (cM), preferably 11 to 12 centimorgans (cM). The genetic distance between the SNP at the physical location indicated by SNP ID NO:2 and the SNP at the physical location indicated by SNP ID NO:5 in Table 1 is 10 to 15 centimorgans (cM), preferably 11 to 12 centimorgans (cM). The genetic distance between the SNP at the physical location indicated by SNP ID NO:2 and the SNP at the physical location indicated by SNP ID NO:4 in Table 1 is 2.8 to 4 centimorgans (cM), preferably 2 to 3 centimorgans (cM).

[0027] More preferably, the anthrax resistance gene is located between the 5' end of the 3 centimorgan (cM) upstream of SNP ID NO:3 and the 3' end of the 3 centimorgan (cM) downstream of SNP ID NO:3. However, although the locus of this anthrax resistance gene is on chromosome 1 in the above description, it does not necessarily have to be on chromosome 1.

[0028] Cucumber plants can also be defined as cucumber plants derived from seeds deposited under accession number FERM P-22504 (hereinafter also referred to as "deposited cucumber plants"). The deposit information is as follows: Type of deposit: Domestic deposit Depository name: National Institute of Technology and Evaluation (NITE), Patent Organism Depository Center Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan Accession number: FERM P-22504 Identification mark: Takii38 Date received: June 24, 2024

[0029] Deposited cucumber plants may include progeny. "Derived from seeds" means including cucumber plants grown from deposited seeds and their progeny. Progeny are offspring derived from deposited cucumber plants. Progeny are cucumber plants of which are obtained directly or indirectly by self-pollination and / or cross-pollination of deposited cucumber plants or their progeny. Progeny also include lines obtained by cross-pollinating deposited cucumber plants and their progeny. Alternatively, progeny include cucumber plants formed as plant individuals from a portion of a plant individual of a deposited cucumber plant or its progeny, or their offspring. Methods for forming plant individuals from a portion of a plant individual are publicly known. For example, methods for forming plant individuals include protoplasts and calluses.

[0030] Cucumber plants may include cucumber plants identified by having at least one single nucleotide polymorphism marker sequence from (1) to (3) above (preferably cucumber plants identified by having the single nucleotide polymorphism marker sequence from (1) above); or depositary cucumber plants (hereinafter, these cucumber plants may be collectively referred to as "parental cucumber plants"), as well as hybrid cucumber plants. Hybrid cucumber plants are cucumber plants that inherit the genome of a parental cucumber plant and possess the genome of a cucumber plant of another lineage other than the parental cucumber plant. Hybrid cucumber plants are produced by directly or indirectly crossbreeding a parental cucumber plant with a cucumber plant of another lineage. Direct crossbreeding means cross-pollinating a parental cucumber plant with a cucumber plant of another lineage. Indirect hybridization refers to methods such as self-pollination of hybrid cucumber plants, cross-pollination of hybrid cucumber plants with each other, or hybridization of a hybrid cucumber plant with a cucumber plant other than the parent plant and of a different lineage. Indirect hybridization may include backcrossing, etc.

[0031] "Hybridization" refers to the process of producing offspring from two parent plants. Hybridization includes "cross-pollination" and "self-pollination." Cross-pollination means that male and female gametes originating from different plant individuals fertilize each other. Self-pollination means that male and female gametes originating from the same individual fertilize each other.

[0032] 2. Selection method for cucumber plants One embodiment relates to a method for selecting cucumber plants. The selection method includes detecting cucumber plants identified by having at least one single nucleotide polymorphism marker sequence from (1) to (3) above (preferably cucumber plants identified by having the single nucleotide polymorphism marker sequence in (1) above) from a group of test cucumber plants, and determining that a test cucumber plant is an anthracnose-resistant cucumber plant or a cucumber plant that may be anthracnose-resistant if it has at least one single nucleotide polymorphism marker sequence from (1) to (3) above (preferably cucumber plants identified by having the single nucleotide polymorphism marker sequence in (1) above). The step of determining a test cucumber plant as an anthracnose-resistant cucumber plant or a cucumber plant that may be anthracnose-resistant may include a step of testing whether the cucumber plant is resistant to anthracnose by the method described in 1. above.

[0033] Single nucleotide polymorphism marker sequences can be detected using a sample of part or all of the test cucumber plant, or using genomic DNA extracted from part or all of the test cucumber plant as a sample.

[0034] Methods for DNA extraction are well known. For example, cucumber plants are crushed and dissolved in Edward extraction buffer containing sodium dodecyl sulfate (SDS), and then DNA is recovered by precipitation with isopropyl alcohol or ethanol. Alternatively, cucumber plants are crushed and dissolved in a buffer containing CTAB (Cetyl trimethyl ammonium bromide), and then chloroform-isoamyl alcohol (24:1) is added to collect the aqueous layer. DNA is recovered by precipitation with isopropyl alcohol or ethanol in the aqueous layer. Examples include ISOSPIN Plant DNA (Nippon Gene), DNeasy Plant Mini Kit (Qiagen), and PureLink. TM DNA can also be extracted using spin column DNA extraction kits such as the Genomic Plant DNA Purification Kit (Thermo Fisher Scientific) and NucleoSpin® Plant II (Takara Bio). Other options include MagExtractor-Plant Genome- (Toyobo) and MagMAX. TM DNA may also be extracted using a magnetic bead DNA extraction kit such as the -96 DNA Multi-Sample Kit (Thermo Fisher Scientific).

[0035] Single nucleotide polymorphism (SNP) marker sequences can be determined, for example, by nucleotide sequencing. Nucleotide sequencing can be performed using Sanger sequencing or next-generation sequencing. Alternatively, SNP marker sequences may be detected by methods such as fingerprinting, microarrays, PCR-RFLP, or PCR-SSCP.

[0036] Another method for selecting cucumber plants includes detecting at least a portion of a nucleotide sequence linked to the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3. The linked nucleotide is within a range of 3 cM, preferably 2 cM, and more preferably 1 cM, from the eighth nucleotide to the 5' and / or 3' ends of the nucleotide sequence represented by Sequence ID No. 3. The eighth nucleotide is a nucleotide other than C, and preferably T.

[0037] Preferably, the linked nucleotide sequence is a nucleotide sequence found in a cucumber plant derived from seeds deposited under deposit number FERM P-22504.

[0038] The portion of the nucleotide sequence linked to the eighth nucleotide is approximately 1 bp to 10 kbp, 5 bp to 5,000 bp, 10 bp to 1,000 bp, preferably 15 bp to 300 bp. The portion of the nucleotide sequence linked to the eighth nucleotide may be one location or multiple locations within the above range. Multiple locations refer to 2 to 3,000 locations, preferably 2 to 1,000 locations.

[0039] Nucleotide sequences can be detected by methods such as nucleotide sequencing, fingerprinting, microarrays, PCR-RFLP, and PCR-SSCP. Specific sequencing methods are as described above.

[0040] 3. Method for producing cucumber plants This embodiment relates to a method for producing cucumber plants having the single nucleotide polymorphism marker sequence described in (1) above; the single nucleotide polymorphism marker sequence described in (1) above and the single nucleotide polymorphism marker sequence described in (2) above; the single nucleotide polymorphism marker sequence described in (1) above and the single nucleotide polymorphism marker sequence described in (3) above; or the single nucleotide polymorphism marker sequence described in (1), the single nucleotide polymorphism marker sequence described in (2), and the single nucleotide polymorphism marker sequence described in (3) above (hereinafter referred to as the "production method"). The production method includes the steps of self-pollinating or cross-pollinating cucumber plants; performing callus culture; grafting; or introducing a gene locus containing at least one of the first to third single nucleotide polymorphism marker sequences described in (1) above into cucumber plants by genetic modification.

[0041] Furthermore, the manufacturing method may involve self-pollination, cross-pollination, callus culture, or grafting of cucumber plants derived from seeds deposited under accession number FERM P-22504.

[0042] In the above, self-pollination includes the step of self-pollinating a cucumber plant having the single nucleotide polymorphism marker sequence of (1) above; the single nucleotide polymorphism marker sequence of (1) above and the single nucleotide polymorphism marker sequence of (2) above; the single nucleotide polymorphism marker sequence of (1) above and the single nucleotide polymorphism marker sequence of (3) above; or a cucumber plant having the single nucleotide polymorphism marker sequence of (1) above, the single nucleotide polymorphism marker sequence of (2) above, and the single nucleotide polymorphism marker sequence of (3) above; or a cucumber plant derived from seeds deposited under accession number FERM P-22504. Self-pollination is intended to result in cross-pollination between identical individuals. The manufacturing method including the self-pollination step may preferably include the collection of seeds obtained by self-pollination. The manufacturing method may further include sowing the collected seeds and cultivating cucumber plants.

[0043] In the above, cross-pollination includes a cross-pollination step of crossing a first parent line plant and a second parent line plant selected from each of the following lines of cucumber plants: a line having the single nucleotide polymorphism marker sequence of (1) above; a line having the single nucleotide polymorphism marker sequence of (1) above and the single nucleotide polymorphism marker sequence of (2) above; a line having the single nucleotide polymorphism marker sequence of (1) above and the single nucleotide polymorphism marker sequence of (3) above; or a line having the single nucleotide polymorphism marker sequence of (1), the single nucleotide polymorphism marker sequence of (2), and the single nucleotide polymorphism marker sequence of (3) above; or a line of cucumber plants derived from seeds deposited under accession number FERM P-22504. The first and second parent line plants may be from the same line or different lines. Alternatively, the first or second parent line plant may be cross-pollinated with a cucumber plant that does not have anthracnose resistance. The manufacturing method including the cross-pollination step may preferably include recovering the seeds obtained by the cross-pollination. The manufacturing method may further include sowing the recovered seeds and cultivating cucumber plants.

[0044] Callus culture is well known (References: *Breeding Journal*, 29(1):33-38, 1979; *Plant Tissue Culture*, 7(3), 181-186, 1990). Briefly, the callus culture process may include, for example, collecting the hypocotyl of a cucumber plant, culturing the collected hypocotyl on MS medium containing plant growth regulators (auxins such as naphthaleneacetic acid and cytokines such as benzyladenine), and transferring the cultured callus to a redifferentiation medium with plant growth regulators (auxins such as naphthaleneacetic acid and cytokines such as benzyladenine) to redifferentiate the callus and obtain a cucumber plant. This manufacturing method may also include growing the cucumber plant redifferentiated from the callus.

[0045] Grafting methods are publicly known (References: https: / / www.takii.co.jp / tsk / y_garden / spring / point03 / index.html; https: / / www.naro.affrc.go.jp / org / tarc / seika / jyouhou / H05 / tnaes93134.html). The method includes the steps of grafting a scion onto a rootstock and acclimatizing the grafted seedling. Approach grafting is preferred. The manufacturing method may also include cultivating cucumber plants in which the grafted portion has taken root.

[0046] A method for genetically modifying a cucumber plant to produce a locus containing at least one of the first to third single nucleotide polymorphism marker sequences described above is not limited as long as the locus containing at least one of the first to third single nucleotide polymorphism marker sequences described above is introduced into the cucumber plant. The cucumber plant undergoing genetic modification (the recipient cucumber plant into which the locus containing at least one of the first to third single nucleotide polymorphism marker sequences described above is introduced) may be a cucumber plant that does not exhibit anthracnose resistance or a cucumber plant that exhibits anthracnose resistance. For a cucumber plant that does not exhibit anthracnose resistance undergoing genetic modification, it is preferable that the disease incidence index in the anthracnose resistance evaluation described in 1. above is 3.5 or higher, preferably 3 or higher. For a cucumber plant that exhibits anthracnose resistance undergoing genetic modification, the disease incidence index in the anthracnose resistance evaluation described in 1. above may be less than 3 or less than 3.5. At this time, it is preferable to introduce genes from the gene locus from the 5' side 3 centimorgan (cM) upstream of the first single nucleotide polymorphism marker shown in Table 1 to the 3' side 3 centimorgan (cM) downstream of SNP ID NO:3. More preferably, gene loci containing the first to third single nucleotide polymorphism marker sequences are introduced into the cucumber plant. Gene recombination methods are well known. Examples include the Agrobacterium method using Ti plasmids or Ri plasmids, particle gun method, electroporation method, and genome editing methods utilizing homologous recombination repair (HDR: Homology-Directed Repair) (Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system, CompoZr Zinc Finger Nuclease (ZFN) system, or TAL effector nuclease (TALEN) system). The method for producing cucumber plants through genetic modification further involves regenerating the plant body from the genetically modified cells.

[0047] A method for producing cucumber plants, in another form, includes detecting at least a portion of a nucleotide sequence linked to the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3. The linked nucleotide is within a range of 3 cM, preferably within a range of 2 cM, more preferably within a range of 1 cM, from the 5' and / or 3' side of the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3. The eighth nucleotide is a nucleotide other than C, and preferably the eighth nucleotide is T.

[0048] Preferably, the linked nucleotide sequence is a nucleotide sequence found in a cucumber plant derived from seeds deposited under deposit number FERM P-22504.

[0049] For a description of the nucleotide sequence linked to the eighth nucleotide, and the method for detecting the nucleotide sequence, refer to the description of the selection method for cucumber plants in section 3 above.

[0050] The above-described method for producing cucumber plants may further include selecting cucumber plants having at least one single nucleotide polymorphism marker sequence (preferably a cucumber plant identified by having the single nucleotide polymorphism marker sequence of (1)) according to the method described in 2.

[0051] Furthermore, the cucumber plants produced may be tested for anthracnose resistance using the method described in 1. above. [Examples]

[0052] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[0053] The following examples were performed by creating F2, F3, and F4 generation isolated populations and NILs (near isogenic lines) using the method of the present invention with a typical Japanese cucumber parent line of Takii.

[0054] (1) Methods for testing for disease resistance First, a novel cucumber strain (hereinafter referred to as "PR") exhibiting anthracnose resistance was selected from among the various cucumber strains owned by Takii Seed Co., Ltd. through inoculation testing. The selected PR was crossed with the susceptible variety 'TCU-093' (hereinafter referred to as "PS"), and self-pollinated seeds were collected from the resulting F1 plants. F3 generation seeds were obtained from 92 F2 generation plants. Next, 7 individuals from each of the 87 strains of the F3 generation that germinated in sufficient numbers for testing were used for inoculation. The inoculation test was conducted using the following method, referring to Non-Patent Literature 1. Specifically, the cucumber anthracnose fungus (strain number Tf1172) maintained by Takii Seed Co., Ltd. was cultured on potato decoction agar medium at 25°C for 2 weeks. Next, the conidial mass formed on the medium was suspended in sterile distilled water, filtered through double gauze, and the conidial concentration was 1 × 10⁶. 3 The inoculum was prepared to achieve a concentration of 1 seed / ml. The test plants were sown at a rate of 1 seed per pot in 9cm pots filled with commercially available horticultural seedling soil, and grown at 25°C for approximately 3 weeks until two true leaves had fully developed. The second true leaves of these seedlings were inoculated with 1 ml of the inoculum per leaf using a spray bottle, and the disease was allowed to progress in a greenhouse set at a maximum temperature of 25°C and a minimum temperature of 15°C. Disease development was checked 6 to 8 days after inoculation. Disease severity was evaluated using a disease severity index of 5 levels (0: no symptoms, 1: lesions less than 5 mm on the leaf, 2: lesions less than 10 mm on the leaf, 3: lesions 10 mm or larger on the leaf or wilting on part of the leaf, 4: wilting of the entire leaf). The average disease severity index for each line was calculated by summing the disease severity indices obtained for each individual plant and dividing by the number of individuals per line.

[0055] While susceptible varieties had an average disease severity index of around 3, the F3 generation lines believed to possess this disease resistance gene locus had an average disease severity index of around 1-2, demonstrating significant resistance. Furthermore, compared to existing varieties, harvested fruits showed a reduction in lesions and suppression of mycelial formation.

[0056] (2) Identification of resistance loci DNA was extracted from each of the 92 F2 generation plants mentioned above, and polymorphism analysis was performed on 48 SNPs covering all chromosomes where polymorphism was observed between PR and PS. QTL analysis was performed using QTL cartographer based on the genotypes obtained here and the average disease incidence index obtained in (1). As a result, in the data from each of the surveys at 6 days (hereinafter, "p1"), 7 days (hereinafter, [p2]), and 8 days (hereinafter, "p3") after inoculation, a single QTL with an LOD value of approximately 6-9 was detected around 14.4Mb to 19.8Mb on chromosome 1 (physical location when the reference sequence is NCBI Reference Sequence: NC_026655.1) (Figure 1).

[0057] Next, the physical and genetic distances were calculated for the detected resistance loci. First, individuals with heterozygous genotypes (i.e., heterozygous for both the susceptible and resistive SNPs listed in Table 1) of the four SNPs on chromosome 1—Cs1_18384414 (SNP1), Cs1_19836089 (SNP4), Cs1_22078282 (SNP5), and Cs1_24086623 (SNP7)—were selected from the F3 generation, and F4 generation seeds were obtained by self-pollination. The obtained F4 generation seeds were sown, and DNA was extracted from 268 germinated individuals to determine the genotype of the four SNPs. From the obtained genotype data, the genetic distance between each SNP was calculated using MSTmap online (http: / / www.mstmap.org / ).

[0058] The results are shown in Table 2. In Table 2, genotypes are denoted as follows: PS homozygous (i.e., homozygous for the susceptible SNPs listed in Table 1) is A, PR homozygous (i.e., homozygous for the resistive SNPs listed in Table 1) is B, and heterozygous for susceptible and resistive SNPs is H.

[0059] The genetic distance between Cs1_18384414 (SNP1) and Cs1_24086623 (SNP7) was approximately 20 cM, and it was revealed that 1 cM in this region corresponds to approximately 284 kb. Furthermore, when anthracnose resistance tests were performed on each of the aforementioned individuals, the average disease incidence index for each SNP in the PR homozygous state was lowest for Cs1_18384414 (SNP1), followed by Cs1_19836089 (SNP4), Cs1_22078282 (SNP5), and Cs1_24086623 (SNP7). For all SNPs, the average disease incidence index was lowest in the PR homozygous state, followed by heterozygous and then PS homozygous. These results reveal that the PR resistance locus is located upstream of Cs1_19836089 (SNP4) on chromosome 1 and is controlled by a single gene exhibiting incomplete dominance.

[0060] Next, based on polymorphism information of all chromosomes, NIL lines were created in which the genotype of the relevant region was as shown in Table 3, and the genotypes of all remaining chromosomal regions were identical. Inoculation tests were performed on 15-20 individuals per line according to standard procedures. The results showed that the genotype of Cs1_19025793 (SNP3) had the strongest correlation with the phenotype, and that the resistance gene was located within approximately 3 cM between SNP2 and SNP3 or between SNP3 and SNP4 (Table 3).

[0061] Table 2. Physical and genetic distances of QTL regions Genotypes were denoted as follows: PS homozygous (A), PR homozygous (B), and heterozygous (H). Genetic distance was calculated using MSTmap online. [Table 2]

[0062] Table 3. Genotypes of NIL lines Genotypes were denoted as A for PS homozygous and B for PR homozygous. Estimated genetic distance was calculated based on the ratio of known physical distance to genetic distance, using the measured values ​​in Table 2. [Table 3]

Claims

1. A first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A cucumber plant that has [this characteristic].

2. The cucumber plant according to claim 1, wherein the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is T.

3. The cucumber plant according to claim 1, wherein, when the cucumber plant is diploid, it has a first single nucleotide polymorphism marker sequence on one or both of a pair of homologous chromosomes.

4. A first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A second single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is a nucleotide other than G, or A third single nucleotide polymorphism marker sequence having a sequence in which the 10th nucleotide in the nucleotide sequence represented by Sequence ID No. 4 is a nucleotide other than A, A cucumber plant that possesses and is resistant to anthracnose.

5. When the cucumber plant has the first single-nucleotide polymorphism marker sequence, the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is T. When the cucumber plant has the second monosal polymorphism marker sequence, the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is A, or When the cucumber plant has the third single-nucleotide polymorphism marker sequence, the tenth nucleotide in the nucleotide sequence represented by Sequence ID No. 7 is C. The cucumber plant according to claim 4.

6. The cucumber plant according to claim 4, wherein, when the cucumber plant is diploid, it has a first single nucleotide polymorphism marker sequence, a second single nucleotide polymorphism marker sequence, or the third single nucleotide polymorphism marker sequence on one or both of a pair of homologous chromosomes.

7. Cucumber plants originating from seeds deposited under deposit number FERM P-22504.

8. A first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C. A method for selecting cucumber plants, including selecting cucumber plants that possess [a certain characteristic].

9. A first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A second single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is a nucleotide other than G, or A third single nucleotide polymorphism marker sequence having a sequence in which the 10th nucleotide in the nucleotide sequence represented by Sequence ID No. 4 is a nucleotide other than A, A method for selecting cucumber plants, comprising selecting cucumber plants that possess and are resistant to anthracnose.

10. The method includes detecting a portion of a nucleotide sequence linked to the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3, within a range of 3 cM from the eighth nucleotide to the 5' and / or 3' side of the nucleotide sequence represented by Sequence ID No. 3, wherein the eighth nucleotide is a nucleotide other than C. A method for selecting anthracnose-resistant cucumber plants.

11. The selection method according to claim 10, wherein the linked nucleotide sequence is a nucleotide sequence possessed by a cucumber plant derived from seeds deposited under deposit number FERM P-22504.

12. A method for producing a cucumber plant having a first single nucleotide polymorphism marker sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, The method described above is The process of self-pollination or cross-pollination of cucumber plants. The process of culturing callus, The grafting process, or A process of introducing a gene locus containing the first single nucleotide polymorphism marker sequence into a cucumber plant by genetic modification. A method for producing cucumber plants, including [the specified ingredient].

13. A first single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3 is a nucleotide other than C, A second single nucleotide polymorphism marker sequence having a sequence in which the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 2 is a nucleotide other than G, or A third single nucleotide polymorphism marker sequence having a sequence in which the 10th nucleotide in the nucleotide sequence represented by Sequence ID No. 4 is a nucleotide other than A, A method for producing cucumber plants that have anthracnose resistance, The method described above is The process of self-pollination or cross-pollination of cucumber plants. The process of culturing callus, The grafting process, or A process of introducing a gene locus containing at least one of the first to third single nucleotide polymorphism marker sequences into a cucumber plant by genetic modification. A method for producing cucumber plants, including [the specified ingredient].

14. The method includes detecting a portion of a nucleotide sequence linked to the eighth nucleotide in the nucleotide sequence represented by Sequence ID No. 3, within a range of 3 cM from the eighth nucleotide to the 5' and / or 3' side of the nucleotide sequence represented by Sequence ID No. 3, wherein the eighth nucleotide is a nucleotide other than C. A method for producing anthracnose-resistant cucumber plants.

15. The method according to claim 14, wherein the linked nucleotide sequence is a nucleotide sequence found in a cucumber plant derived from seeds deposited under deposit number FERM P-22504.

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