Tomato plant having resistance to root-knot nematode and leaf mold

Abstract

The purpose of the present invention is to provide a tomato plant that has resistance to root-knot nematode and leaf mold. One aspect of the present invention pertains to a tomato plant that has resistance to root-knot nematode and leaf mold, said tomato plant having, in a coupled phase, Mi-1 gene that imparts resistance to root-knot nematode and Cf-5 gene that imparts resistance to leaf mold. Another aspect of the present invention pertains to a DNA fragment including the Mi-1 gene and the Cf-5 gene.

Description

Tomato plants resistant to root-knot nematodes and leaf mold

[0001] The present invention relates to tomato (Solanum lycopersicum) plants that have commercially important genes, specifically the Mi-1 gene, which confers resistance to root-knot nematodes, and the Cf-5 gene, which confers resistance to downy mildew, in the coupling phase (cis) so that they are co-inherited as if they were a single gene.

[0002] The tomato (Solanum lycopersicum) is a perennial plant of the Solanaceae family, native to South America, widely cultivated around the world, and thought to have been introduced to Japan by the Dutch during the Edo period. Tomatoes are primarily eaten for their fruit, which is rich in vitamin C, lycopene, beta-carotene, potassium, vitamin E, and other nutrients, making them extremely nutritious and considered one of the most important vegetables.

[0003] Plant varieties are generally divided into fixed varieties and first-generation hybrid (F1) varieties, with F1 varieties prevalent in major crops. F1 varieties grow vigorously due to heterosis. This provides significant advantages, such as rapid growth and increased yield. Furthermore, F1 varieties are expected to exhibit improved resistance to pests and diseases, as well as improved environmental adaptability, including cold and heat tolerance. Furthermore, because F1 varieties are heterozygous but have the same genotype, they exhibit extremely high phenotypic uniformity. This enhances the marketability of the product. Furthermore, the accumulation of useful traits controlled by dominant genes in both parents of an F1 variety allows for rapid breeding. Due to these advantages, F1 varieties have become the mainstream cultivar for major crops. While fixed varieties were the norm for edible tomatoes until the 1920s, the shift to F1 varieties rapidly progressed from the 1930s, and today, most tomatoes are F1 varieties.

[0004] On the other hand, tomatoes are susceptible to many diseases (Non-Patent Document 1). To combat these diseases, growers use chemical methods such as pesticides to control pathogens, but the most effective method is to develop resistant varieties, due to the environmental impact, cultivation labor, and costs involved.

[0005] Among these, diseases caused by nematodes and downy mildew fungi are among the most important for growers. Nematodes are widespread worldwide, with their distribution extending almost from the Arctic to the Antarctic. In addition to their wide distribution, various species of nematodes are pathogens with diverse host ranges, including plants and animals. Tomato is no exception, and the three most important species known to infect tomatoes are Meloidogyne arenaria, M. incognita, and M. javanica.

[0006] When root-knot nematodes infect plants, they cause the formation of 1- to 10-mm galls or nodules on the roots. These changes promote the transport of nutrients from the plant to the nematodes, while at the same time reducing the supply of water and nutrients to the plant, inhibiting growth. In addition, infected plants may show symptoms such as stunted growth, wilting, and yellowing (chlorosis). Because nematode-caused root galls form during the growing season, wilting becomes more pronounced and fruit formation may be affected.

[0007] Chemical control of root-knot nematodes is effective, and the nematicide methyl bromide is highly effective against nematodes in soil. However, due to concerns about its harmful effects on humans and ozone layer depletion, its use has been banned worldwide. Given this background, developing tomato varieties resistant to root-knot nematodes is a common and effective crop management strategy (Non-Patent Document 2).

[0008] The Mi-1 gene (Mi1.2 gene) is a root-knot nematode resistance gene located on chromosome 6 that was introduced into cultivated tomatoes by interspecific hybridization from the wild tomato species Solanum peruvianum. The gene was isolated in 1998, and its amino acid sequence and mRNA sequence were registered with the NCBI "National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / )" (AAC67238.1 and AF039682.1). Since then, highly reliable DNA markers, such as Mi23, have been developed near the Mi-1 gene, and the development of root-knot nematode-resistant varieties using Mi-1 is still actively underway (Non-Patent Documents 2 and 3).

[0009] Leaf mold is a disease caused by the filamentous fungus Passalora fulva (also known as Fulvia fulva or Cladosporium fulvum), and is a worldwide problem. When lesions accompanied by mold formation occupy most of the leaves, the leaves fall off, making it a serious disease that seriously damages fruit yield and quality. Chemical control of the pathogen using pesticides and other methods has been attempted as a countermeasure against leaf mold, but due to the impact on humans and the environment, the labor and cost of cultivation, and other factors, the most effective method is to use resistant varieties (Non-Patent Document 4).

[0010] Resistance genes such as Cf-2, Cf-4, Cf-5, and Cf-9 have been used to develop tomato varieties resistant to downy mildew. However, downy mildew pathogens capable of infecting tomato plants carrying these resistance genes have emerged, and a diversification of races has been observed worldwide. In particular, the emergence of downy mildew pathogen races that overcome the downy mildew resistance of the Cf-9 gene, which has been actively used in breeding for downy mildew resistance, has become a major problem in recent years (Non-Patent Document 5).

[0011] The downy mildew resistance gene Cf-5 originates from the tomato landrace Solanum lycopersicum var. cerasiforme. The gene was isolated in 1998, and its amino acid sequence and full-length coding sequence were registered with NCBI (AAC78591.1 and AF053993.1). Subsequently, a DNA marker was designed based on the genomic DNA sequence of the gene, enabling efficient breeding of tomatoes for downy mildew resistance using the Cf-5 gene (Non-Patent Documents 6 and 7).

[0012] Tomato plants carrying the Cf-5 gene in their genome exhibit resistance to races of downy mildew fungi that overcome the downy mildew resistance conferred by the Cf-9 gene. Currently, the emergence of races of downy mildew fungi that overcome the downy mildew resistance conferred by the Cf-5 gene is limited, and the Cf-5 gene remains a useful downy mildew resistance gene for cultivating tomato varieties (Non-Patent Documents 8, 9, and 10).

[0013] Because tomato is susceptible to many diseases, it is preferable to impart many disease resistance genes to tomato varieties when developing them. Disease resistance genes are located on various chromosomes, but chromosome 6 in particular is known to be home to many genes, including the root-knot nematode resistance gene Mi-1, the downy mildew resistance genes Cf-2 and Cf-5, as well as the powdery mildew resistance genes Ol-1 and Ol-4 and the yellow leaf curl resistance gene Ty-1, making it a very important chromosome for breeding (Non-Patent Document 11).

[0014] When multiple resistance genes are imparted to an F1 variety, each resistance gene is generally introduced into one of the parent lines and then crossed. When more genes are imparted to an F1 variety, the number of genes integrated into one parent line inevitably increases. However, because the loci containing the resistance genes Mi-1, Cf-2, Cf-5, OL-1, OL-4, and Ty-1 located on chromosome 6 are all closely linked genetically, it is extremely difficult to integrate multiple genes into a single fixed line. To improve breeding efficiency, it is desirable to have a fixed line that contains multiple resistance genes on chromosome 6 (Patent Document 2).

[0015] Furthermore, even if a disease resistance gene is described as being inherited dominantly, in highly susceptible environmental conditions, the incidence rate of heterozygotes is higher than that of homozygotes, often revealing that the gene is incompletely dominant (Non-Patent Document 12). Therefore, to develop highly resistant F1 varieties, it is desirable to confer resistance genes to both parent lines so that the disease resistance gene is conferred homozygously in the F1 generation. From this perspective, it is also useful to link multiple disease resistance genes present in reciprocal phases to complementary phases.

[0016] Against this background, attempts have been made to integrate genes located on chromosome 6 into fixed lines. For example, the creation of a line having the Mi-1 gene and the Ty-1 gene in a coupled phase has been reported (Patent Document 1). More specifically, from an F2 segregating population of 504 individuals obtained from a hybrid between an individual having the Mi-1 gene homozygously but not Ty-1 and an individual having the Ty-1 homozygously but not Mi-1, eight individuals having the Mi-1 gene and the Ty-1 gene in a coupled phase were successfully created.

[0017] Similarly, the creation of a line having the OL-4 gene and the Ty-1 gene in a coupling phase has been reported (Patent Document 2). More specifically, a hybrid between an individual having the Ty-1 gene in a homozygous state but not the OL-4 gene and an individual having the OL-4 gene in a homozygous state but not the Ty-1 gene was crossed with an individual having not the Ty-1 gene or the OL-4 gene, and from the resulting segregating population of 500 individuals, five individuals having the Ty-1 gene and the OL-4 gene in a coupling phase were successfully created.

[0018] Japanese Patent No. 5,312,423 U.S. Patent No. 1,535,861

[0019] “Tomato Diseases A PRACTICAL GUIDE FOR SEEDMEN, GROWERS AND AGRICULTURAL ADVISORS” Brad Gabor and Wayne Wiebe, Seminis Vegetable Seeds, inc. 1997“Development of Molecular Markers for the Mi-1 Gene in Tomato Using the KASP Genotyping Assay” Zuebeyir Devran et al.. Hortic. Environ. Biotechnol. 57(2):156-160. 2016“The Root Knot Nematode Resistance Gene Mi from Tomato Is a Member of the Leucine Zipper, Nucleotide Binding, Leucine-Rich Repeat Family of Plant Genes” Milligan et al., The Plant Cell, Vol. 10, 1307-1319, August 1998“LEAF MOLD OF GREENHOUSE TOMATOES” , University of Illinois report on PLANT DISEASE, Integrated Pest Management at the University of Illinois, August 1989Differential setsーPassalorafulva(MAY 2022), International seed federation“The Tomato Cf-5 Disease Resistance Gene and Six Homologs Show Pronounced Allelic Variation in Leucine-Rich Repeat Copy Number”, Mark S. Dixon et al., The Plant Cell, Vol. 10, 1915-1925,November 1998 “A CAPS Marker for Leaf Mold Resistance Gene Cf-5 in Tomato”, Yu Shuancang et al. , Molecular Plant Breeding, 2005, Vol. 3. No. 1, 57-60 "The first occurrence of leaf mold of tomato caused by races 4.9 and 4.9.11 of Passalora fulva (syn. Fulvia fulva) in Japan", Junichiro Enya et al., February 2009. Journal of General Plant Pathology. 75(1):76-79. Development of race-specific strains of tomato leaf mold fungus based on domestic varieties, Kubota et al., Ann. Rept. Kansai Pl. Prot. (61): 55-60 (2019) Ecology and Control of Tomato Leaf Mold, Yuichiro Iida et al., Plant Protection, Vol. 73, No. 11 (2019) "Linked, if Not the Same, Mi-1 Homology Confer Resistance to Tomato Powdery Mildew and Root-Knot Nematodes", Seifi et al., MPMI Vol. 24, No. 4, 2011, pp. 441-450. Vegetables / Resistant varieties and their use 53-54, Kunio Yamakawa (1978) "Genetic and molecular organization of the short arm and pericentromeric region of tomato chromosome 6", Van Wordragen et al., Euphytica 79: 169-174, 1994 Nematological experiments Takayuki Mizukubo and Kazuyoshi Futatsu, Kyoto University Press (2014) Fundamentals of research techniques for crop pathogens Kanichi Ohata et al. Japan Plant Protection Association (1995),

[0020] Although genetic maps of the Mi-1 and Cf-5 genes have been created based on the relative positions of each gene and surrounding molecular markers (Non-Patent Document 13), to the inventors' knowledge, no strains have actually been created that contain both genes in the coupling phase, nor have any such tomato plants been commercially sold or cultivated. It has previously been shown that homologous chromosome recombination is suppressed in the region adjacent to the Mi-1 gene. Similarly, it has been reported that homologous chromosome recombination is difficult in this region between Solanum peruvianum and Solanum lycopersicum (Patent Document 1). This supports the difficulty of linking the Mi-1 and Cf-5 genes in the coupling phase. Therefore, in order to confer resistance to root-knot nematodes and downy mildew to an F1 variety, it was previously necessary to confer both genes by conferring Mi-1 to one breeding parent and Cf-5 to the other breeding parent.

[0021] The present invention aims to provide a tomato plant that has the Mi-1 gene and the Cf-5 gene in a coupling phase, which has not yet been produced due to the particular difficulty of chromosomal recombination for disease resistance genes located on chromosome 6, thereby facilitating breeding for integrating disease resistance genes located on chromosome 6 into F1 varieties. Another object of the present invention is to provide a tomato plant, seeds, seedlings, or products containing parts of a tomato plant that are easily resistant to root-knot nematodes and downy mildew by utilizing a tomato plant that has the Mi-1 gene and the Cf-5 gene in a coupling phase.

[0022] As a result of research aimed at solving the above-mentioned problems, the present inventors have succeeded in producing a tomato plant containing both genes in a coupled phase by repeatedly crossing a tomato plant having Mi-1 with a tomato plant having Cf-5, self-pollinating the plants, and selecting the plants using DNA markers, inoculation tests, etc.

[0023] That is, the present invention encompasses one or more of the following aspects: [1] A tomato plant resistant to root-knot nematodes and downy mildew, the tomato plant having, in coupled phase, the Mi-1 gene that confers resistance to root-knot nematodes and the Cf-5 gene that confers resistance to downy mildew. [2] The tomato plant according to [1], which contains a DNA fragment comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. [3] The plant according to [1] or [2], in which the Mi-1 gene and the Cf-5 gene are located on chromosome 6. [4] The tomato plant according to any of [1] to [3], which contains, on the same genomic DNA of chromosome 6, a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. [5] The plant according to any of [1] to [4], in which the Mi-1 gene and the Cf-5 gene are each homozygous or heterozygous. [6] The plant according to any of [1] to [5], wherein the Mi-1 gene is identified by a genotyping method using a primer set comprising a primer having the nucleotide sequence of SEQ ID NO: 3 and a primer having the nucleotide sequence of SEQ ID NO: 4, and / or a genotyping method using a primer set comprising a primer having the nucleotide sequence of SEQ ID NO: 5, a primer having the nucleotide sequence of SEQ ID NO: 6, and a primer having the nucleotide sequence of SEQ ID NO: 7, and the Cf-5 gene is identified by a genotyping method using a primer set comprising a primer having the nucleotide sequence of SEQ ID NO: 8 and a primer having the nucleotide sequence of SEQ ID NO: 9. [7] The plant according to any of [1] to [6], wherein the plant is resistant to at least one species of root-knot nematode of the genus Meloidogyne and to at least one species of a race of Passalora fulva that cannot infect tomato plants expressing the Cf-5 gene.[8] The tomato plant according to any one of [1] to [7], which has resistance to root-knot nematodes and downy mildew and is derived from the tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477. [9] The tomato plant according to any one of [1] to [8], which is a hybrid plant obtained using the tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477 as a parent line, or a progeny of the hybrid plant.

[10] The tomato plant according to any of [1] to [9], comprising genomic DNA of chromosome 6, the nucleotide sequence being: (1) a nucleotide sequence of a portion of genomic DNA of chromosome 6 of a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477, the portion containing the Mi-1 gene and the Cf-5 gene; or (2) a nucleotide sequence having 50% or more identity with the nucleotide sequence described in (1), the nucleotide sequence including a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[11] The tomato plant according to

[10] , wherein the portion of the genomic DNA of chromosome 6 of the tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477 containing the Mi-1 gene and the Cf-5 gene is included in the range from the base specified by ch06_2151188 to the base specified by ch06_2742590 in the genomic DNA of chromosome 6 of the tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477.

[12] A part of the tomato plant according to any of [1] to

[11] .

[13] The part of the tomato plant according to

[12] , wherein the part is a tomato plant cell incapable of regeneration, and comprises a DNA fragment comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[14] A fruit of the tomato plant according to any one of [1] to

[11] .

[15] A seed of the tomato plant according to any one of [1] to

[11] .

[16] A DNA fragment comprising the Mi-1 gene and the Cf-5 gene.

[17] The DNA fragment according to

[16] , comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[18] The DNA fragment according to

[16] or

[17] , comprising: (1) a nucleotide sequence of a portion of the genomic DNA of chromosome 6 of a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477, the portion containing the Mi-1 gene and the Cf-5 gene; or (2) a nucleotide sequence having 50% or more identity to the nucleotide sequence according to (1), and comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[19] The DNA fragment according to

[18] , wherein the portion of the genomic DNA of chromosome 6 of the tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477 containing the Mi-1 gene and the Cf-5 gene is included in the range from the base specified by ch06_2151188 to the base specified by ch06_2742590 in the genomic DNA of chromosome 6 of the tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477.

[20] A method for producing F1 seeds from the tomato plant according to any of [1] to

[11] , comprising the steps of: crossing the tomato plant according to any of [1] to

[11] with the tomato plant according to any of [1] to

[11] or another plant that is crossable with the tomato plant according to any of [1] to

[11] ; and collecting F1 seeds from individuals obtained by the cross.

[21] A method for producing a tomato plant that has the Mi-1 gene and the Cf-5 gene in a coupled phase and is resistant to root-knot nematodes and downy mildew, the method comprising: self-pollinating the tomato plant according to any one of [1] to

[11] ; and repeatedly self-pollinating the individual plant obtained by the self-pollination for one or more generations.

[22] A method for producing a tomato plant that is resistant to root-knot nematodes and downy mildew, and that has the Mi-1 gene and the Cf-5 gene in a coupling phase, comprising: a first crossbreeding step of crossbreeding the tomato plant according to any one of [1] to

[11] with a first tomato plant; a second crossbreeding step of subjecting the F1 individual obtained by the first crossbreeding step to selfing, backcrossing, or interspecific or intraspecific crossbreeding with a second tomato plant different from the first tomato plant used in the first crossbreeding step to obtain a segregating population; and a selection step of selecting, from the segregating population, a tomato plant that has the Mi-1 gene and the Cf-5 gene in a coupling phase.

[23] The method according to

[22] , wherein the selection step comprises at least one of: detecting the Mi-1 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 3 and a primer comprising the nucleotide sequence of SEQ ID NO: 4; detecting the Mi-1 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 5, a primer comprising the nucleotide sequence of SEQ ID NO: 6 and a primer comprising the nucleotide sequence of SEQ ID NO: 7; detecting the Cf-5 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 8 and a primer comprising the nucleotide sequence of SEQ ID NO: 9; confirming resistance to root-knot nematodes; and confirming resistance to downy mildew.

[24] A method for producing a product comprising a tomato plant, a seed, a seedling, or a part of a tomato plant, the method comprising: confirming, in the tomato plant according to any one of [1] to

[11] , one trait selected from root-knot nematode resistance, downy mildew resistance, the presence of the Mi-1 gene, and the presence of the Cf-5 gene; and preparing a product comprising the tomato plant, a seed, a seedling, or a part of a tomato plant for which the trait has been confirmed.

[25] The method according to

[24] , wherein the product is a product comprising a seed of the tomato plant and packaging material containing the seed, or a product comprising a seedling of the tomato plant and a tray containing the seed.

[26] A product comprising a tomato plant, a seed, a seedling, or a part of a tomato plant, produced by the method according to

[24] or

[25] .

[27] The tomato plant according to any one of [1] to

[11] , which is not obtained essentially solely by biological methods.

[28] The method according to

[21] , further comprising a selection step of selecting tomato plants having the Mi-1 gene and the Cf-5 gene in a coupling phase from the individuals obtained by self-pollination and / or the individuals obtained by repeated self-pollination for one or more generations.

[29] The method according to

[28] , wherein the selection step comprises at least one of: detecting the Mi-1 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 3 and a primer comprising the nucleotide sequence of SEQ ID NO: 4; detecting the Mi-1 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 5, a primer comprising the nucleotide sequence of SEQ ID NO: 6 and a primer comprising the nucleotide sequence of SEQ ID NO: 7; detecting the Cf-5 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 8 and a primer comprising the nucleotide sequence of SEQ ID NO: 9; confirming resistance to root-knot nematodes; and confirming resistance to downy mildew.

[30] A method for selecting a tomato plant that has resistance to root-knot nematodes and downy mildew and that has the Mi-1 gene and the Cf-5 gene in a coupled phase, the method comprising: (1) analyzing the presence of the Mi-1 gene and the Cf-5 gene in the genomic DNA of one chromosome of a test tomato plant using one or more assays, and (2) selecting a test tomato plant in which the presence of the Mi-1 gene and the Cf-5 gene in the genomic DNA of one chromosome is confirmed.

[31] The method according to

[30] , wherein the one or more assays in step (1) comprise: determining the presence or absence of the Mi-1 gene, and, if present, whether the Mi-1 gene is heterozygous or homozygous, based on a genotyping method that detects a DNA marker of the Mi-1 gene, and determining the presence or absence of the Cf-5 gene, and, if present, whether the Cf-5 gene is heterozygous or homozygous, based on a genotyping method that detects a DNA marker of the Cf-5 gene.

[32] The DNA marker for the Mi-1 gene and the DNA marker for the Cf-5 gene are each independently an SNP marker, an SSR marker, an RFLP marker, a SCAR marker, a CAPS marker, and a KASP marker. ＴＭ

[33] The method according to

[31] , wherein the DNA marker for the Mi-1 gene is one or more selected from the group consisting of Mi23 marker and Mi-1K marker, and the DNA marker for the Cf-5 gene is the Cf-5 marker.

[34] The method according to

[33] , wherein the Mi23 marker comprises a sequence identified by a genotyping method using a primer set of SEQ ID NOs: 3 and 4, the Mi-1K marker comprises a sequence identified by a genotyping method using a primer set of SEQ ID NOs: 5, 6 and 7, and the Cf-5 marker comprises a sequence identified by a genotyping method using a primer set of SEQ ID NOs: 8 and 9.

[35] The method according to any of

[30] to

[34] , wherein the one or more assays in step (1) comprise: determining the presence or absence of the Mi-1 gene, and, if present, whether the Mi-1 gene is heterozygous or homozygous, based on a genotyping method using a primer set of SEQ ID NO:3 and SEQ ID NO:4, and / or a primer set of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; and determining the presence or absence of the Cf-5 gene, and, if present, whether the Cf-5 gene is heterozygous or homozygous, based on a genotyping method using a primer set of SEQ ID NO:8 and SEQ ID NO:9.

[36] The method according to

[31] or

[35] , wherein in the one or more assays, the test tomato plant having the Mi-1 gene in a homozygous form and the Cf-5 gene in a heterozygous form, the test tomato plant having the Mi-1 gene in a heterozygous form and the Cf-5 gene in a homozygous form, or the test tomato plant having the Mi-1 gene in a homozygous form and the Cf-5 gene in a homozygous form is a tomato plant in which the Mi-1 gene and the Cf-5 gene are present in the genomic DNA of a single chromosome.

[37] The method according to

[30] , wherein the one or more assays in step (1) comprise decoding the genomic DNA sequence of a homologous chromosome of chromosome 6 of the test tomato plant, and confirming the presence of a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and the presence of a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[38] The method according to any one of

[30] to

[37] , wherein the test tomato plant is a progeny of a tomato plant having resistance to root-knot nematodes and downy mildew and having the Mi-1 gene and the Cf-5 gene in a coupling phase.

[0024] This specification includes the disclosure of Japanese Patent Application No. 2023-132701, from which the present application claims priority. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

[0025] The present invention provides a tomato plant containing the Mi-1 gene and the Cf-5 gene in a coupled phase. Furthermore, by using the tomato plant of the present invention as a parent line, it becomes easy to integrate multiple resistance genes located on chromosome 6 into an F1 variety, enabling the development of products with high industrial value.

[0026] Figure 1 is a schematic diagram showing the relative positions of the Mi-1 gene, the Cf-5 gene, and each DNA marker on chromosome 6 of a tomato plant (Example 2). Figure 2 is a photograph of a tomato plant seedling of the deposited line (accession number FERM BP-22476) 18 days after inoculation with late mold race 2.4.9 in Example 5. Figure 3 is a photograph of a tomato plant seedling of the tomato line SKTom-C, which has the Mi-1 gene in a homozygous state but does not have the Cf-5 gene, 18 days after inoculation with late mold race 2.4.9 in Example 5. The top row of Figure 4 is a photograph of a leaf of a tomato plant seedling of the deposited line (accession number FERM BP-22476) 18 days after inoculation with late mold race 2.4.9 in Example 5. The lower panel of Figure 4 is a photograph of leaves of tomato seedlings of the tomato line SKTom-C, which has the Mi-1 gene homozygously but does not have the Cf-5 gene, 18 days after inoculation with late mold race 2.4.9 in Example 5.

[0027] Seed Deposit In connection with the present disclosure, seeds of two tomato plant lines have been deposited at the Patent Organism Depositary of the National Institute of Technology and Evaluation (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code 292-0818) and have been assigned the following accession numbers: (Deposited line 1) Accession number: FERM BP-22476 Identification mark: SSC-TOM-23-001 Deposit date: May 12, 2023 (Deposited line 2) Accession number: FERM BP-22477 Identification mark: SSC-TOM-23-002 Deposit date: May 12, 2023

[0028] In the present disclosure, tomato plants are plants classified in the tomato clade of the genus Solanum (Bedinger et al., 2011). Cultivated tomatoes include Solanum lycopersicum, and wild tomatoes include Solanum peruvianum and the like. Landrace tomatoes include Solanum lycopersicum var. cerasiforme and the like. In the present disclosure, "cultivated species" refers to plants of a lineage used for cultivation, and includes not only fixed species but also F1 varieties.

[0029] In the present disclosure, root-knot nematodes are nematodes of the genus Meloidogyne.

[0030] In the present disclosure, leaf mold is a disease caused by the filamentous fungus Passalora fulva, and several races have been identified.

[0031] In the present disclosure, "chX_Y" (X and Y are integers) or "a base specified by chX_Y" of a tomato plant means "a base corresponding to the Yth base of chromosome number X (also referred to as chromosome X) in the reference genome (SL4.0) published on Sol Genomics Network (https: / / solgenomics.net / )" in the base sequence of the genomic DNA of the tomato plant. Note that "a base corresponding to the Yth base of chromosome X on the reference genome of Sol Genomics Network" for a certain tomato plant can be determined by aligning the base sequence of the genomic DNA of the tomato plant with the base sequence of the reference genome of Sol Genomics Network so as to achieve the highest homology (sequence identity). Therefore, the X chromosome of a certain tomato plant can be represented as the X chromosome of the tomato plant determined by aligning the base sequence of the genomic DNA of the tomato plant with the base sequence of the reference genome of the Sol Genomics Network so as to have the highest homology (sequence identity).

[0032] In the present disclosure, a "chromosome" includes not only the entire chromosome but also a part thereof. That is, a "part of a chromosome" may also be simply referred to as a "chromosome."

[0033] An "X locus" is a "site in a chromosome occupied by an X gene." Therefore, in the present disclosure, a "tomato plant having an X locus" is a tomato plant having a site in a chromosome occupied by an X gene, and is synonymous with a "tomato plant having an X gene."

[0034] Furthermore, the term "vicinity" as used in the present disclosure refers to a distance that can be easily understood by a person skilled in the art from the relationship between the position of the marker and the root-knot nematode resistance gene Mi-1, or the relationship between the position of the marker and the downy mildew resistance gene Cf-5, and further the relationship between the root-knot nematode resistance gene Mi-1 and the downy mildew resistance gene Cf-5, as well as from common knowledge of a person skilled in the art. For example, depending on the analysis conditions, the distance can be about 10 cM or less (e.g., 7 cM).

[0035] Therefore, for example, by using the Mi23 marker identified by the primer set of SEQ ID NOs: 3 and 4 and / or the Mi-1K marker identified by the primer set of SEQ ID NOs: 5, 6 and 7 shown in Table 1 in the Examples, the presence of the Mi-1 gene located nearby the locus indicated by these markers can be estimated or confirmed.

[0036] In the present disclosure, "coupling phase" and "cis phase" refer to a genetic state in which alleles at two different loci occur linked to each other on a single (homologous) chromosome. For example, when both the Mi-1 gene and the Cf-5 gene are located on one of the homologous chromosomes, these alleles are in "coupling phase" or "cis phase." In contrast, when the Mi-1 gene and the Cf-5 gene are located on different homologous chromosomes of a homologous pair, they are said to be in "reciprocal phase" or "trans phase." Furthermore, this "coupling phase" or "cis phase" refers to a state in which genes or base sequences originally present at the same locus in "reciprocal phase" or "trans phase" are linked not only on the same chromosome or chromosomal fragment, but also on the same DNA fragment.

[0037] In the present disclosure, the term "production method" can also be referred to as a "generation method," "creation method," "growing method," or "production method." That is, the terms "production," "generation," "creation," "growing," and "production" are used interchangeably herein.

[0038] In the present disclosure, the term "part of a plant" refers to a cell, tissue, or organ of the plant, specifically a fruit, seed, flower, pollen, anther, leaf, stem, root, embryo, hypocotyl, meristematic cell, callus, etc. It also includes protoplasts obtained from cells of the plant. Furthermore, in tomato plants, regeneration from the hypocotyl, explants from young expanded leaves, seeds, shoot apical tissue, and stem and leaf primordia has been reported (Kita 1987, Toyoda 1987, Sakata 1991). In particular, tomato fruit consists of the exocarp (including epidermal cells), mesocarp, endocarp, seeds, placenta, ovule, vascular bundles, pedicel, sepals, etc. (Matsui 1981). However, to the inventors' knowledge, there have been no reports of totipotent regeneration from fully differentiated plant cells, tissues, or organs of fruit other than seeds.

[0039] <Root-knot nematode-resistant and downy mildew-resistant tomato plants> The root-knot nematode-resistant and downy mildew-resistant tomato plants according to the present disclosure have the root-knot nematode resistance gene Mi-1 and the downy mildew resistance gene Cf-5 linked in a coupling phase. The root-knot nematode-resistant and downy mildew-resistant tomato plants according to the present disclosure can contain the Mi-1 gene and the Cf-5 gene in an expressible manner in the coupling phase.

[0040] The Mi-1 gene is a gene that confers resistance to a wide range of root-knot nematode species, such as Meloidogyne arenaria (arenarian root-knot nematode), Meloidogyne incognita (sweet potato root-knot nematode), and Meloidogyne javanica (Javanese root-knot nematode). It has been shown that the Mi-1 gene loses its function in environments with soil temperatures of 28°C or higher. However, because the Mi-1 gene is still effective against a wide range of root-knot nematode species, it is still widely used in cultivar development and is important in breeding.

[0041] The amino acid sequence of the Mi-1 gene (SEQ ID NO: 1) has been registered with NCBI as AAC67238.1, and the nucleotide sequence of its mRNA has been registered as AF039682.1 (SEQ ID NO: 10). It is well known in the art that the root-knot nematode resistance of the Mi-1 gene is due to this amino acid sequence. Of the mRNA sequence of the Mi-1 gene shown in SEQ ID NO: 10 and the DNA sequence in which all U's are replaced with T's, the partial nucleotide sequence from base 87 to base 3860 is a coding sequence that encodes the amino acid sequence of SEQ ID NO: 1.

[0042] The Mi-1 gene is a dominantly inherited gene. Therefore, the tomato plant according to the present disclosure may be a tomato plant that is homozygous for the Mi-1 gene, i.e., a tomato plant that has the Mi-1 gene in a homozygous form, or a tomato plant that has the Mi-1 gene in a heterozygous form.

[0043] The Cf-5 gene is a gene that confers resistance to Passalora fulva race 0 (Pf:0), race 2 (Pf:A), race 4 (Pf:B), race 2.4 (Pf:C), race 2.9 (Pf:F), race 9 (Pf:G), race 4.9 (Pf:H), and race 2.6.9 (Pf:J) as defined by the ISF (International Seed Federation), as well as race 2.4.9, which has been reported to occur in Japan.

[0044] The amino acid sequence of the Cf-5 gene (SEQ ID NO: 2) has been registered with NCBI as AAC78591.1, and the nucleotide sequence of its full-length coding sequence has been registered as AF053993.1 (SEQ ID NO: 11). It is well known in the art that the downy mildew resistance of the Cf-5 gene is due to this amino acid sequence. Of the nucleotide sequence of the Cf-5 gene shown in SEQ ID NO: 11, the partial nucleotide sequence from base 654 to base 3560 is the coding sequence that encodes the amino acid sequence of SEQ ID NO: 2.

[0045] The Cf-5 gene is a dominantly inherited gene. Therefore, the tomato plant according to the present disclosure may be a tomato plant that is homozygous for the Cf-5 gene, i.e., has the Cf-5 gene in a homozygous form, or a tomato plant that has the Cf-5 gene in a heterozygous form.

[0046] The root-knot nematode-resistant and downy mildew-resistant tomato plants according to the present disclosure can be produced by introducing a DNA fragment containing the Mi-1 gene and the Cf-5 gene linked in coupling phase into the genomic DNA of a tomato plant. The site in a cell into which the DNA fragment containing the Mi-1 gene and the Cf-5 gene linked in coupling phase is introduced is not particularly limited, and may be intrachromosomal or extrachromosomal. For example, the root-knot nematode-resistant and downy mildew-resistant tomato plants according to the present disclosure include tomato plants in which a region corresponding to the DNA fragment on chromosome 6 has been replaced with a DNA fragment containing the Mi-1 gene and the Cf-5 gene linked in coupling phase, and tomato plants in which a DNA fragment containing the Mi-1 gene and the Cf-5 gene linked in coupling phase has been introduced, by translocation or the like, into a region on chromosome 6 of the tomato plant other than the region corresponding to the Mi-1 gene locus and the Cf-5 gene locus.

[0047] The DNA fragment having the Mi-1 gene and the Cf-5 gene linked in coupling phase may be, for example, a fragment that exhibits the Mi-1 genotype by a genotyping method using a primer set of SEQ ID NO: 3 and SEQ ID NO: 4 and / or a primer set of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and that exhibits the Cf-5 genotype by a genotyping method using a primer set of SEQ ID NO: 8 and SEQ ID NO: 9, but is not particularly limited as long as it contains, on the same DNA fragment, a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 in an expressible manner. In other words, the root-knot nematode-resistant and downy mildew-resistant tomato plant according to the present disclosure is preferably a tomato plant that contains, in its genomic DNA, a DNA fragment that expressibly contains both a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Furthermore, the DNA fragment having the Mi-1 gene and the Cf-5 gene linked in complementary phase may be a DNA fragment that exhibits the Mi-1 genotype by a genotyping method using a primer set of SEQ ID NO: 3 and SEQ ID NO: 4 and / or a primer set of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and that exhibits the Cf-5 genotype by a genotyping method using a primer set of SEQ ID NO: 8 and SEQ ID NO: 9.

[0048] That is, in one or more embodiments of the present disclosure, the Mi-1 gene is identified by a genotyping method using a primer set including a primer comprising the nucleotide sequence of SEQ ID NO: 3 and a primer comprising the nucleotide sequence of SEQ ID NO: 4, and / or a genotyping method using a primer set including a primer comprising the nucleotide sequence of SEQ ID NO: 5, a primer comprising the nucleotide sequence of SEQ ID NO: 6, and a primer comprising the nucleotide sequence of SEQ ID NO: 7. Furthermore, in one or more embodiments of the present disclosure, the Cf-5 gene is identified by a genotyping method using a primer set including a primer comprising the nucleotide sequence of SEQ ID NO: 8 and a primer comprising the nucleotide sequence of SEQ ID NO: 9. Because the markers used in these genotyping methods are co-dominant markers, it is possible to determine whether the Mi-1 gene and the Cf-5 gene are each heterozygous or homozygous in a tomato plant.

[0049] The above-mentioned genotyping method will now be described. In the present disclosure, a "primer containing the nucleotide sequence of SEQ ID NO: N" refers to a primer containing the nucleotide sequence of SEQ ID NO: N at its 3' end, and may also be a primer to which another nucleotide sequence is further added on the 5' end of the nucleotide sequence of SEQ ID NO: N. Furthermore, each primer may contain a labeling substance as needed.

[0050] A primer set including a primer containing the nucleotide sequence of SEQ ID NO: 3 and a primer containing the nucleotide sequence of SEQ ID NO: 4 can amplify, through a nucleic acid amplification reaction using DNA extracted from a tomato plant as a template, the nucleotide sequence of Mi23 that is specifically linked to the Mi-1 gene and the nucleotide sequence of another allele of Mi23 that is linked to another allele of the Mi-1 gene, as amplification products that are different in size and can be distinguishably detected. Therefore, a genotyping method using this primer set can determine the presence or absence of the Mi-1 gene, and, if the Mi-1 gene is present, whether the strain is heterozygous or homozygous.

[0051] A primer set including a primer (referred to as Mi1K_1F) containing the nucleotide sequence of SEQ ID NO: 5, a primer (referred to as Mi1K_2F) containing the nucleotide sequence of SEQ ID NO: 6, and a primer (referred to as Mi1K_R) containing the nucleotide sequence of SEQ ID NO: 7 can amplify the nucleotide sequence of Mi-1K that is specifically linked to the Mi-1 gene as an amplification product by Mi1K_1F and Mi1K_R, and the nucleotide sequence of another allele of Mi-1K that is linked to another allele of the Mi-1 gene as an amplification product by Mi1K_2F and Mi1K_R, by a nucleic acid amplification reaction using DNA extracted from a tomato plant as a template. Mi1K_1F and Mi1K_2F contain nucleotide sequences (tail sequences) that are distinctly detectable and added to the 5'-end of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively. By using probes in which a labeling substance for distinguishing each Tail sequence has been added to the nucleotide sequence that anneals to each Tail sequence, it is possible to distinguish between the amplification products of MilK_1F and MilK_R and the amplification products of MilK_2F and MilK_R in the nucleic acid amplification reaction. Therefore, a genotyping method using this primer set can determine the presence or absence of the Mi-1 gene and, if the Mi-1 gene is present, whether the gene is heterozygous or homozygous.

[0052] A primer set including a primer containing the nucleotide sequence of SEQ ID NO: 8 and a primer containing the nucleotide sequence of SEQ ID NO: 9 can amplify a partial nucleotide sequence contained specifically in the Cf-5 gene and a partial nucleotide sequence contained in another allele of the Cf-5 gene through a nucleic acid amplification reaction using DNA extracted from a tomato plant as a template, as amplification products that differ in cleavage pattern with the restriction enzyme TaqI (length and / or number of fragments after TaqI treatment) and can be distinguishably detected. Therefore, by using this primer set and, if necessary, a genotyping method using TaqI, it is possible to determine the presence or absence of the Cf-5 gene and, if the Cf-5 gene is present, whether the gene is heterozygous or homozygous.

[0053] The Mi-1 gene and the Cf-5 gene can be linked onto the same DNA fragment by a method utilizing homologous chromosomal recombination, or by a method well known in the art in which DNA fragments containing the respective gene sequences are linked in vitro using a restriction enzyme and DNA ligase. Alternatively, by using an artificial gene synthesis service provided by Eurofin genomics or the like, a DNA sequence linking both genes can be artificially synthesized.

[0054] Here, the DNA fragments containing the respective gene sequences may be any DNA fragments that ensure gene expression, including DNA sequences derived from genomic DNA, and may also be DNA fragments that contain cDNA DNA sequences.

[0055] The length of the DNA fragment containing the Mi-1 gene and the Cf-5 gene linked in coupling phase is not particularly limited as long as it contains the nucleotide sequences of the genes linked together. The DNA fragment may be, for example, 700 kb or more or less than 700 kb. Furthermore, the DNA fragment may be less than 650 kb, less than 400 kb, less than 200 kb, less than 100 kb, less than 50 kb, or less than 10 kb.

[0056] The amplification of a DNA fragment containing the Mi-1 gene and the Cf-5 gene artificially linked in coactivation phase as described above can be carried out by cloning using a vector such as a BIBAC vector (U.S. Pat. No. 5,733,744) or a large-capacity binary shuttle vector (Japanese Patent No. 3,350,753) and E. coli, or by LA PCR. ＴＭ The PCR can be carried out by methods well known in the art, such as those described above. Kits containing PCR enzymes used in LA PCR are commercially available from companies that deal in products in the field of molecular biology, such as Takara Bio, and by using these kits, it is possible to amplify long DNA fragments of tens to hundreds of Kb.

[0057] Furthermore, by using a BIBAC vector or a large-capacity binary shuttle vector (Japanese Patent No. 3350753) or the like and combining a DNA fragment containing the Mi-1 gene and the Cf-5 gene artificially linked in coupling phase as described above in an expressible manner with a method well known in the art such as the Agrobacterium method, it is possible to introduce the Mi-1 gene and the Cf-5 gene artificially linked in coupling phase into the cells of a tomato plant and produce a tomato plant that is resistant to root-knot nematodes and downy mildew.

[0058] As mentioned above, methods of linkage and introduction include not only cross-breeding using chromosomal recombination, but also in vitro recombinant DNA technology to link and introduce the two genes (genetic recombination), genetic engineering (GE), etc. However, there is a problem in that crops produced by genetic recombination or genetic engineering, etc., have not yet been widely accepted by the general public.

[0059] An example of a root-knot nematode-resistant and downy mildew-resistant tomato plant according to the present disclosure is a tomato plant produced using as parent lines a tomato plant having root-knot nematode resistance derived from the Mi-1 gene and a tomato plant having downy mildew resistance derived from the Cf-5 gene, wherein the Mi-1 gene and the Cf-5 gene are linked in a coupling phase. The term "tomato plant" includes not only plants resulting from intraspecific or interspecific hybridization within species belonging to the Solanum-tomato clade, but also somatic hybrid plants resulting from intraspecific or interspecific cell fusion of plants belonging to the Solanum-tomato clade, and grafted hybrid plants obtained by intraspecific or interspecific grafting of plants belonging to the Solanum-tomato clade.

[0060] In the tomato production method according to the present disclosure, for example, when chromosomal recombination is utilized in crossbreeding, the tomato plants used as parent lines are not particularly limited as long as they are tomato plants having root-knot nematode resistance derived from the Mi-1 gene as one parent and having downy mold resistance derived from the Cf-5 gene as the other parent, but are preferably varieties cultivated as agricultural crops. Tomato plants currently distributed in normal commercial transactions in Japan mainly include large-sized tomato varieties, medium-sized tomato varieties, cherry tomato varieties, and rootstock tomato varieties, and any of these may be used as long as they satisfy the above conditions. For example, F1 varieties such as "Reiyo" and "Gohoubi" sold in Japan by Sakata Seed Corporation are tomato plants in which one parent has the Mi-1 gene in a homozygous state and the other parent has the Cf-5 gene in a homozygous state, and therefore are varieties that have the Mi-1 gene and the Cf-5 gene in a heterozygous and reciprocal phase (trans phase). Such tomato varieties can be used as parental lines for producing tomatoes according to the present disclosure.

[0061] Furthermore, as described above, the tomato plants used as parent lines are not particularly limited, as long as they are a combination of a tomato plant having root-knot nematode resistance derived from the Mi-1 gene as one parent and a tomato plant having downy mold resistance derived from the Cf-5 gene as the other parent. Such tomato plants used as parent lines can be selected from a variety of commonly available tomato plants by confirming the presence of root-knot nematode resistance derived from the Mi-1 gene or downy mold resistance derived from the Cf-5 gene using the genotyping methods, inoculation tests, or nucleotide sequence analysis described in this disclosure. Furthermore, the tomato plants used as parent lines do not necessarily have to be varieties owned by Sakata Seed Corporation.

[0062] In this disclosure, "progeny of a tomato plant" includes not only progeny obtained by intraspecific crossing of the tomato plant, but also individuals and their progeny obtained using the tomato plant as a parent line, somatic hybrid plants and their progeny obtained by cell fusion between cells of the tomato plant and cells of other plants, and individuals and their progeny obtained by grafting using the tomato plant as a rootstock or scion. "Progeny" includes both individuals obtained as intraspecific hybrids and individuals obtained as interspecific hybrids. Furthermore, "individuals obtained using (a plant) as a parent line" refers to individuals obtained by intraspecific crossing, interspecific crossing, cell fusion, tissue culture, or grafting using the plant as a parent line.

[0063] The root-knot nematode-resistant and downy mildew-resistant tomato plant according to the present disclosure is not particularly limited as long as it has the Mi-1 gene and the Cf-5 gene linked in a coupling phase, but is preferably a tomato plant that has the traits of a cultivated tomato.

[0064] The root-knot nematode-resistant and downy mildew-resistant tomato plants according to the present disclosure are produced, for example, from the progeny of F1 individuals obtained by crossing a tomato plant having the Mi-1 gene but not the Cf-5 gene with a tomato plant having the Cf-5 gene but not the Mi-1 gene. For example, seeds of tomato plants having the Mi-1 gene and the Cf-5 gene in heterozygote and reciprocal phase are obtained by crossing a tomato plant having the Mi-1 gene in homozygote and not the Cf-5 gene with a tomato plant having the Cf-5 gene in homozygote and not the Mi-1 gene. Subsequently, plants grown from the obtained seeds are selfed to obtain seeds of a segregating population. Furthermore, individuals in which chromosomal recombination has occurred between the Mi-1 gene and the Cf-5 gene and the two genes are linked in the coupling phase can be produced by selecting from the segregating population using DNA markers, inoculation tests, or the like.

[0065] Selection of individuals in which the Mi-1 gene and the Cf-5 gene are linked in coupling phase from the progeny obtained by crossing can be carried out by a selection method using the sequences of the genomic regions containing both genes as DNA markers. Such markers include markers that are amplified by primers having the nucleotide sequences set forth in SEQ ID NOs: 3 to 9. More specifically, the presence of the Mi-1 gene can be detected by a genotyping method using a primer set of SEQ ID NOs: 3 and 4 and / or a primer set of SEQ ID NOs: 5, 6 and 7. Furthermore, the presence of the Cf-5 gene can be detected by a genotyping method using a primer set of SEQ ID NOs: 8 and 9. Alternatively, detection can be carried out by confirming that the genome contains a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, for example, by decoding a portion of the genome sequence.

[0066] More specifically, by using the above method, it can be determined that an individual having the Mi-1 gene in a homozygous state and the Cf-5 gene in a heterozygous state, or an individual having the Mi-1 gene in a heterozygous state and the Cf-5 gene in a homozygous state, contains a DNA fragment having the Mi-1 gene and the Cf-5 gene linked in coupling phase in one of its homologous chromosomes. Furthermore, an individual having both the Mi-1 gene and the Cf-5 gene in a homozygous state can be determined that both homologous chromosomes contain a DNA fragment having the Mi-1 gene and the Cf-5 gene linked in coupling phase. These confirmation methods can also be used in the process of fixing both genes by backcrossing or selfing, allowing the selection of progeny with resistance to root-knot nematodes and downy mildew.

[0067] The root-knot nematode-resistant and downy mildew-resistant tomato plants according to the present disclosure may be the progeny of an F1 individual obtained by crossing a tomato plant having the Mi-1 gene and the Cf-5 gene in the coupling phase with another tomato plant. For example, a tomato plant having the Mi-1 gene and the Cf-5 gene in the coupling phase is crossed with another tomato plant to produce F1 seeds, and seeds are obtained by backcrossing the other tomato plant with an individual plant grown from the F1 seeds, or by self-pollinating the individual plant grown from the F1 seeds. Subsequently, individuals having the Mi-1 gene and the Cf-5 gene in the coupling phase are selected from the individual plants grown from the resulting seeds. By repeatedly selfing the selected plant individuals or crossing them with tomato plants and selecting individuals having the Mi-1 gene and the Cf-5 gene in a coupling phase from the progeny obtained by the crossing or selfing, it is possible to confer resistance to root-knot nematodes and downy mildew to any tomato plant. Selfing, crossing with tomato plants, cultivation, and seed collection can be carried out using standard methods for tomato cultivation.

[0068] Selection of individuals having the Mi-1 gene and the Cf-5 gene in a coupled phase from progeny plants can be carried out, for example, by examining root-knot nematode resistance and downy mildew resistance. Specifically, for root-knot nematode resistance, the roots of a test plant individual to be examined for root-knot nematode resistance are inoculated with the root-knot nematode species, and the presence or absence of root galls is examined. If the degree of root galls is slight, the test plant individual is evaluated as being resistant to the inoculated root-knot nematode species. For downy mildew resistance, the leaves of a test plant individual to be examined for downy mildew resistance are inoculated with Passalora fulva, and the presence or absence of downy mildew lesions on the leaves is examined. Furthermore, races of Passalora fulva that are invulnerable to tomato plants expressing the Cf-5 gene include race 2, race 4, race 2.4, race 9, race 2.9, race 4.9, and race 2.4.9, and it is possible to examine downy mildew resistance using at least one of these races. For example, downy mildew race 2.4.9 infects tomato plants expressing the resistance genes Cf-2, Cf-4, and Cf-9 but does not infect tomato plants expressing Cf-5, making it one of the races suitable for confirming resistance via Cf-5.

[0069] Therefore, according to one preferred aspect of the present disclosure, the cultivation method of the present disclosure includes determining the genotype of the Mi-1 gene based on a genotyping method using a primer set of SEQ ID NO: 3 and SEQ ID NO: 4 and / or a primer set of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, and determining the genotype of the Cf-5 gene based on a genotyping method using a primer set of SEQ ID NO: 8 and SEQ ID NO: 9, and can also be performed by, for example, decoding a portion of the genome sequence, confirming that the genome contains a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0070] In the present disclosure, a DNA marker includes a DNA sequence on a chromosome that serves as an index for distinguishing between chromosomal fragments containing alleles with different sequences. Examples of DNA markers include SNP markers, SSR markers, RFLP markers, SCAR markers, CAPS markers, and KASP markers. ＴＭ Markers and the like.

[0071] Detection of DNA markers can be performed by conventional methods. For example, DNA extracted from each plant individual is used as a template, and a nucleic acid amplification reaction is performed using primers capable of specifically hybridizing with a specific SNP or SSR and polymerase. The presence or absence of an amplified product can be detected using electrophoresis or the like, thereby identifying each polymorphism. Alternatively, DNA extracted from each plant individual can be treated with a restriction enzyme, and then the pattern of DNA fragments can be detected using electrophoresis or the like, thereby identifying each polymorphism. Primers capable of specifically hybridizing with a specific SNP or SSR can be designed by conventional methods using commonly used primer design tools or the like, depending on the base sequence of tomato genomic DNA or the base sequence of the SNP or SSR to be detected, and synthesized by methods well known in the art.

[0072] Specific examples of tomato plants according to the present disclosure include the aforementioned seeds deposited under accession numbers FERM BP-22476 (SSC-TOM-23-001) and FERM BP-22477 (SSC-TOM-23-002). The tomato plants according to the present disclosure include the following aspects based on the deposited specific plants:

[0073] In other words, the present disclosure also includes tomato plants containing "a gene in which the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene are linked in coupling phase" or "a gene having the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene linked in coupling phase" derived from the deposited plants.

[0074] In the present disclosure, "including" can also be replaced with "having." That is, the terms "including" and "having" are used herein with the same meaning.

[0075] The present disclosure also includes tomato plants containing "a gene in which the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene are linked in coupling phase" or "a gene having the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene linked in coupling phase" identified or represented by the deposited plants.

[0076] In the present disclosure, a gene, genetic trait, base sequence, chromosomal fragment, or DNA fragment that is "identified" or "represented" by the deposited plant includes a gene that has a function equivalent to that of the gene, genetic trait, base sequence, chromosomal fragment, or DNA fragment present in the deposited plant, even if it is not exactly the same.

[0077] The present disclosure also includes tomato plants containing "a gene in which the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene are linked in coupling phase" or "a gene having the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene linked in coupling phase" that can be found in the deposited plants.

[0078] Here, in the present disclosure, a gene, genetic trait, base sequence, chromosomal fragment, or DNA fragment that is "able to be found" in the deposited plant is meant to include genes with equivalent functions, even if they are not exactly identical to the gene, genetic trait, base sequence, chromosomal fragment, or DNA fragment that is "found" in the deposited plant.

[0079] The present disclosure also includes a hybrid plant obtained using a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477, which has a root-knot nematode resistance Mi-1 gene and a downy mildew resistance Cf-5 gene in a coupling phase as a parent line, or a tomato plant which has a root-knot nematode resistance Mi-1 gene and a downy mildew resistance Cf-5 gene in a coupling phase and is a progeny of the hybrid plant.

[0080] The tomato plant containing "a gene in which the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene are linked in coupling phase" or "a gene having the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene linked in coupling phase" derived from, found in, or identified or represented by the tomato plant identified under Accession No. FERM BP-22476 or Accession No. FERM BP-22477 includes: (1) the nucleotide sequence of a portion of the genomic DNA of chromosome 6 of the tomato plant identified under Accession No. FERM BP-22476 or Accession No. FERM BP-22477 that contains the Mi-1 gene and the Cf-5 gene; or (2) A plant containing genomic DNA on chromosome 6, which contains a nucleotide sequence that has 50% or more identity with the nucleotide sequence described in (1) and contains a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. The nucleotide sequence described in (1) or (2) can expressibly contain a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Here, in (1), the portion of the genomic DNA of chromosome 6 of a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477, which contains the Mi-1 gene and the Cf-5 gene, is preferably a portion of the genomic DNA ranging from the base identified by ch06_2151188 to the base identified by ch06_2742590, and more preferably a portion of the genomic DNA ranging from the base identified by ch06_2151188 to the base identified by ch06_2742590. Furthermore, the nucleotide sequence described in (2) preferably has an identity of 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and most preferably 99% or more to the nucleotide sequence described in (1) (however, the identity is less than 100%).In (2), the nucleotide sequence encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 has an exon portion that is preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and most preferably 100% identical to the partial nucleotide sequence from base 87 to base 3860 of the DNA sequence in which all U's in the mRNA sequence of SEQ ID NO: 10 have been replaced with T. Furthermore, in (2), the nucleotide sequence encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 has an exon portion that is preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and most preferably 100% identical to the partial nucleotide sequence from base 654 to base 3560 of SEQ ID NO: 11.

[0081] Another preferred aspect of the present disclosure relates to a part of a tomato plant having the Mi-1 gene and the Cf-5 gene in coupling phase. Here, preferred aspects of the tomato plant having the Mi-1 gene and the Cf-5 gene in coupling phase are as described above. Furthermore, the term "part of a plant" is as defined above. A preferred example of a part of a tomato plant having the Mi-1 gene and the Cf-5 gene in coupling phase according to this aspect includes a tomato plant cell that does not have the ability to regenerate, and that contains a DNA fragment comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Here, examples of the DNA fragment include those described above as DNA fragments comprising the Mi-1 gene and the Cf-5 gene linked in coupling phase. Specific aspects of the nucleotide sequence encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and the nucleotide sequence encoding the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 are as described above. In the tomato plant cell according to this aspect, the location where the DNA fragment is present is not particularly limited and may be intrachromosomal or extrachromosomal, but preferably is present as part of intrachromosomal chromosomal DNA. Examples of tomato plant cells that do not have the ability to regenerate include cells of tomato fruit other than seeds, such as cells of the exocarp (including epidermal cells), mesocarp, endocarp, placenta, locus, vascular bundles, stalk, or sepals of tomato fruit. The tomato plant cell according to this aspect is preferably a cell isolated from a plant.

[0082] In the present disclosure, a "healthy product" refers to a high-purity tomato product, which is a tomato plant having the Mi-1 gene and the Cf-5 gene in a complementary phase, and which contains at least a plant body, a seed, a seedling, or a part of a plant body of a tomato plant that is free from infection with at least a root-knot nematode to which the Mi-1 gene provides resistance and a downy mildew race to which the Cf-5 gene provides resistance, and is produced by the production method described below.

[0083] Tomato F1 products are usually produced by artificial hand-crossing. The cross-pollinator performs each step of the process, such as identifying flowers suitable for F1 production, emasculating, pollinating, and isolating, following protocols and taking into account past experience, while constantly washing and disinfecting their hands and tools to avoid contamination with unwanted pollen. However, mistakes during cross-pollination or unexpected accidents during logistics can sometimes result in a decrease in the purity of the F1 product.

[0084] On the other hand, customers who purchase tomato products, such as seedling growers, farmers, and general consumers, always expect 100% pure F1 products. In order to fulfill this responsibility, seed companies generally conduct purity testing directly or indirectly before selling the products, and then process and sell the products. In the case of tomatoes, it takes about four months to harvest the fruit, and the harvestable period is long, about five months, so any defects in the fruit due to imperfections ...

[0085] It is essential that purity testing be accurate and rapid, but it is also important that it be inexpensive, as it directly affects the manufacturing cost of the product. Therefore, for product distributors, it is important to significantly reduce the number of testing steps, reduce work time and labor, and supply healthy products with 100% purity in order to meet customer expectations.

[0086] The forms of healthy products include seeds, seedlings (sprouts), whole plants, parts of plants, etc., and also include various forms such as packaged products in which these healthy products are appropriately packaged in packaging materials such as containers, bags, trays, etc.

[0087] That is, as one aspect of a healthy product in the present disclosure, for example, a tomato plant or a part thereof having the Mi-1 gene and the Cf-5 gene in a complementary phase can be packaged in a container, bag, tray, or other packaging material and sold after confirming that the plant is not infected with at least the root-knot nematode to which the Mi-1 gene is resistant and the downy mildew race to which the Cf-5 gene is resistant.

[0088] <DNA Fragment Comprising Mi-1 Gene and Cf-5 Gene> Another embodiment of the present disclosure relates to a DNA fragment comprising the Mi-1 gene and the Cf-5 gene.

[0089] The DNA fragment of this embodiment can be introduced into a tomato plant, if necessary, in a state where it has been incorporated into a nucleic acid vector. In the DNA fragment of this embodiment, the Mi-1 gene and the Cf-5 gene can be present in a state where each gene can be expressed in the cells of the tomato plant.

[0090] The Mi-1 gene can comprise a nucleotide sequence that encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. The Cf-5 gene can comprise a nucleotide sequence that encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0091] The DNA fragment according to this embodiment particularly preferably comprises: (1) a nucleotide sequence of a portion of the genomic DNA of chromosome 6 of a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477, the portion containing the Mi-1 gene and the Cf-5 gene; or (2) a nucleotide sequence having 50% or more identity to the nucleotide sequence described in (1), and containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Specific aspects of the nucleotide sequences described in (1) and (2) are as described above.

[0092] The DNA fragment according to this embodiment is preferably isolated. The DNA fragment according to this embodiment is also preferably artificially constructed.

[0093] <Method for producing root-knot nematode- and downy mildew-resistant tomato plants> The method for cultivating root-knot nematode- and downy mildew-resistant tomato plants having the Mi-1 gene and the Cf-5 gene in a coupling phase according to the present disclosure comprises, as described above, introducing resistance to root-knot nematodes and downy mildew into any tomato plant by crossbreeding using as a starting material a tomato plant having the Mi-1 gene and the Cf-5 gene in a coupling phase, thereby producing a novel resistant tomato plant.

[0094] Once a plant in which the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene are linked in coupling phase is cultivated, the probability of chromosomal recombination occurring between the two genes is extremely low. Therefore, after the plant has been cultivated, it is possible to reliably determine whether the progeny plants have both the traits of root-knot nematode resistance and downy mildew resistance by testing the progeny plants for only one trait selected from root-knot nematode resistance, downy mildew resistance, the presence of the Mi-1 gene, and the presence of the Cf-5 gene using a conventional DNA test or inoculation test.

[0095] The method according to the present disclosure has the advantage that by testing only one of four traits, namely, root-knot nematode resistance, downy mildew resistance, the presence of the Mi-1 gene, and the presence of the Cf-5 gene, to confirm the presence or absence of the Mi-1 gene or the Cf-5 gene, the number of testing steps can be significantly reduced, thereby reducing work time and labor. One example of this advantage is the following. When inoculating and selecting for root-knot nematodes and downy mildew on the same individual during the cultivation process, as described below, after inoculation and selection for root-knot nematodes, it is necessary to excise a growing point from the root-knot nematode-inoculated tomato plant, and then, after approximately one month of aging (depending on the conditions), inoculate with downy mildew. In conventional tomato breeding, it is necessary to cultivate tomato plants with traits superior to those of conventional plants while also selecting for other traits. If a plant is developed that has the root-knot nematode resistance gene Mi-1 and the downy mildew resistance gene Cf-5 in a complementary phase, the progeny plants obtained from that parent line will not need to be tested for both root-knot nematode resistance and downy mildew resistance. This will make it possible to avoid the approximately one-month time lag mentioned above for each individual plant, thereby contributing to the acceleration of breeding.

[0096] Furthermore, because both genes linked in the coupling phase are inherited to progeny as if they were a single gene, they can be easily introduced into any line, further accelerating the development of tomato varieties. Furthermore, by using the tomato plant of the present disclosure, which has the root-knot nematode resistance gene Mi-1 and the downy mildew resistance gene Cf-5 in the coupling phase, as a hybrid parent, it becomes easy to accumulate useful genes, such as the powdery mildew resistance gene and yellow leaf curl resistance gene located on chromosome 6, in F1 varieties.

[0097] In the method for producing a tomato plant having resistance to root-knot nematodes and downy mildew according to the present disclosure, which has the Mi-1 gene and the Cf-5 gene in a coupling phase, hybridization can be performed using a tomato plant having the Mi-1 gene and the Cf-5 gene in a coupling phase as a parent line. One specific example of the method includes a first crossing step of crossing a tomato plant having the Mi-1 gene and the Cf-5 gene in a coupling phase with an arbitrary first tomato plant, a second crossing step of selfing, backcrossing, or interspecific or intraspecific crossing the F1 individual obtained by the first crossing step with a second tomato plant different from the first tomato plant used as the parent line in the first crossing step to obtain a segregating population, and a selection step of selecting from the segregating population a tomato plant having the Mi-1 gene and the Cf-5 gene in a coupling phase. Self-pollination, backcrossing, interspecific crossing, intraspecific crossing, cultivation of tomato plants, and seed collection can be carried out by conventional methods for tomato cultivation.

[0098] The tomato plant having the Mi-1 gene and the Cf-5 gene in the coupling phase used as the parent line in the first hybridization step can be the root-knot nematode-resistant and downy mildew-resistant tomato plant according to the present disclosure. In the present disclosure, the tomato plant having the Mi-1 gene and the Cf-5 gene in the coupling phase used as the parent line is preferably a root-knot nematode-resistant and downy mildew-resistant tomato plant that has the Mi-1 gene and the Cf-5 gene linked in the coupling phase and also has the traits of a cultivated species. Examples of such root-knot nematode-resistant and downy mildew-resistant tomato plants include the SSC-TOM-23-001 line, individuals obtained using the SSC-TOM-23-001 line as a parent line, the SSC-TOM-23-002 line, and individuals obtained using the SSC-TOM-23-002 line as a parent line, as well as progeny thereof, which tomato plants have resistance to root-knot nematodes and downy mildew derived from the Mi-1 gene and the Cf-5 gene.

[0099] The first tomato plant used as a parent line in the first hybridization step and the second tomato plant used as a parent line for interspecific or intraspecific hybridization in the second hybridization step are not particularly limited, but are more preferably cultivated tomato plants having at least one disease resistance gene other than the Mi-1 gene and the Cf-5 gene. Even more preferably, by using a tomato plant having at least one disease resistance gene on chromosome 6 other than the Mi-1 gene and the Cf-5 gene as a parent line, it is possible to relatively easily produce a novel tomato line having an additional disease resistance gene in addition to the Mi-1 gene and the Cf-5 gene on chromosome 6.

[0100] In the selection step, selection of individuals having the Mi-1 gene and the Cf-5 gene in the coupling phase can be carried out using DNA markers near the Mi-1 gene and the Cf-5 gene on chromosome 6 of the tomato plant. The DNA markers and specific methods used can be the same as those used in the above-mentioned case of producing individuals having the Mi-1 gene and the Cf-5 gene in the coupling phase using DNA markers. Note that when the first tomato plant used as a parent line in the first hybridization step or the second tomato plant used as a parent line for interspecific or intraspecific hybridization in the second hybridization step does not have the Mi-1 gene or the Cf-5 gene, individuals having both genes in a heterozygous or homozygous state in the segregating population can be selected as individuals having the Mi-1 gene and the Cf-5 gene in the coupling phase. Furthermore, when the first tomato plant used as a parent line in the first hybridization step or the second tomato plant used as a parent line for interspecific hybridization or intraspecific hybridization in the second hybridization step has the Mi-1 gene or the Cf-5 gene, individuals having a heterozygous Mi-1 gene and a homozygous Cf-5 gene, individuals having a homozygous Mi-1 gene and a heterozygous Cf-5 gene, or individuals having both the Mi-1 gene and the Cf-5 gene in a homozygous form will appear in the segregating population, and these can be selected as individuals having the Mi-1 gene and the Cf-5 gene in a coupling phase.

[0101] When selecting tomato plant individuals having additional disease resistance in addition to resistance to root-knot nematodes and downy mildew, in addition to selecting individuals having the Mi-1 gene and Cf-5 gene in the coupling phase, selection can be performed based on a DNA marker or inoculation test that can identify the presence or absence of other disease resistance genes. As a DNA marker that can identify the presence or absence of other disease resistance genes, SNPs located at the locus of other disease resistance genes and in their vicinity can be used.

[0102] Thereafter, by repeatedly crossing seeds collected from the selected progeny individuals, it is possible to obtain root-knot nematode-resistant and downy mildew-resistant tomato plants with more stable traits and with germination rates, yields, seed yields, etc. that are sufficient for stable cultivation as agricultural crops. The number of times crossing is repeated is not particularly limited as long as it is one or more, and may be two to three times, or may be three or more times.

[0103] <Method for producing a healthy product comprising a tomato plant according to the present disclosure> According to the present disclosure, there is provided a method for producing a highly pure healthy product comprising a tomato plant, a seed, a seedling, or a part of the plant, the method comprising: confirming, in a tomato plant according to the present disclosure, one trait selected from root-knot nematode resistance, downy mildew resistance, the presence of the Mi-1 gene, and the presence of the Cf-5 gene; and preparing a product comprising a plant, a seed, a seedling, or a part of the plant of the tomato plant in which the trait has been confirmed.

[0104] As described above, the tomato plant according to the present disclosure contains the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene in a coupling phase. According to a preferred embodiment, once a plant in which the root-knot nematode resistance Mi-1 gene and the downy mildew resistance Cf-5 gene are linked in coupling phase has been cultivated, products using the plant or its progeny can be tested for the presence or absence of other traits by testing for only one trait selected from root-knot nematode resistance, downy mildew resistance, the presence of the Mi-1 gene, and the presence of the Cf-5 gene using a conventional DNA test or inoculation test.

[0105] According to this method, purity can be tested by confirming only one trait selected from root-knot nematode resistance, downy mildew resistance, the presence of the Mi-1 gene, and the presence of the Cf-5 gene, which has the advantage of significantly reducing the number of testing steps compared to conventional methods, thereby reducing work time and labor. This method makes it possible to provide products containing tomato plants with such high commercial value. Meanwhile, customers such as nursery breeders and farmers who purchase products containing tomato plants according to the present disclosure can prevent defects in fruits and vegetables due to accidental contamination or cross-pollination.

[0106] The sample used in the step of confirming the trait can be a tomato plant seed, a sprout, or a part thereof. For example, a part of prepared tomato plant seeds can be sampled and used to carry out the step; if the trait is confirmed in the step, the remaining part of the tomato plant seeds can be used in a product as seeds in which the trait has been indirectly confirmed. Alternatively, a sprout or a part thereof can be sampled from prepared tomato plant seedlings and used to carry out the step; if the trait is confirmed in the step, the tomato plant seedling can be used in a product as a seedling in which the trait has been indirectly or directly confirmed.

[0107] In this method, the product containing a tomato plant, seed, seedling, or plant part typically includes a tomato plant, seed, seedling, or plant part for which the trait has been confirmed, and packaging material such as a container, bag, or tray for containing the plant. For example, the product is a packaged product such as a product including a tomato seed and packaging material for containing the tomato seedling, or a product including a tomato seedling and a tray for containing the tomato seedling. Therefore, in this method, the step of preparing a product containing a tomato plant, seed, seedling, or plant part for which the trait has been confirmed includes, for example, packaging the tomato plant, seed, seedling, or plant part for which the trait has been confirmed in packaging material to form a packaged product. The product may be refrigerated as needed, or may be subjected to further processing.

[0108] Example 1 Inoculation Test to Determine Root-Knot Nematode Resistance The resistance of tomato plants to root-knot nematodes was determined by the following method, with reference to Non-Patent Document 14.

[0109] Susceptible tomato plants were cultivated for two months in soil containing a single-coccal strain of Meloidogyne incognita collected in Japan. Second-stage larvae were collected from root galls formed on tomato roots and from the soil, and a suspension of 100 larvae / ml was prepared with sterilized water and used as an inoculum.

[0110] Tomato seedlings sown in commercially available horticultural soil and left for 14 days were transplanted as single plants into 9 cm pots filled with sterilized soil, and cultivated in a greenhouse at a room temperature of 22 to 28°C for 14 days to prepare test tomato plants.

[0111] 50 ml of the nematode suspension was irrigated into the base of each test tomato plant, and the plants were cultivated in the greenhouse for a further 35 days. After cultivation, the roots of the test tomatoes were washed and examined for disease occurrence.

[0112] The disease score was determined according to the five-level scale for the degree of root gall formation for tomatoes described in Non-Patent Document 14. Scores of 0 and 1 were judged to be resistant, and scores of 2 to 4 were judged to be susceptible. Score 0: No root galls Score 1: Few root galls are observed, but damage is not noticeable Score 2: Root galls are visible at a glance. Few large or connected root galls Score 3: Numerous large and small root galls are observed. Some roots are covered with root galls and have become thicker, but the formation accounts for less than 50% of the entire root zone Score 4: Many roots are covered with root galls and have become thicker.

[0113] <Inoculation Test to Examine Resistance to Leaf Mold> Resistance of tomato plants to leaf mold was determined using the following method with reference to Non-Patent Document 15.

[0114] Race 2.4.9 of the leaf mold fungus collected in Japan was transferred to a PDA plate medium and cultured at 23°C for 2 weeks. After the culture, conidia were collected from the mycelium and 1 x 10 ５ A spore suspension adjusted to spores / mL with sterile water was used as the inoculum source.

[0115] Tomato seedlings sown in commercially available horticultural soil and left for 14 days were transplanted as single plants into 9 cm pots filled with sterilized soil, and cultivated for 14 days in a greenhouse at a temperature adjusted to 22-28°C to prepare test tomato plants.

[0116] When the same tomato plant was inoculated with root-knot nematodes and late mold, the growing point was cut from a tomato plant inoculated with root-knot nematodes, inserted into sterilized soil, and grown in a greenhouse at 22 to 28°C for 28 days. The rooted cuttings were used as test tomato plants.

[0117] 3 mL of the spore suspension per test tomato plant was sprayed onto the underside of the tomato leaves, and the plants were cultivated in an acclimation room at 23°C and 80-100% humidity for 18 days. After cultivation, the test tomato leaves were observed for disease symptoms to investigate the occurrence of the disease.

[0118] The disease survey was conducted according to the criteria for determining the race of tomato leaf mold described in Non-Patent Documents 5 and 8, and the plants were judged as either susceptible or resistant. Susceptible: Leaf spots present Resistant: Leaf spots absent

[0119] Example 2 To produce tomato plants in which the Mi-1 gene and the Cf-5 gene are linked in coupling phase, the inventors first crossed large-fruited tomato lines SKTom-A and SKTom-C, which have the Mi-1 gene in a homozygous state and are susceptible to Downy mildew race 2.4.9, with large-fruited tomato lines SKTom-B and SKTom-D, which have the Cf-5 gene in a homozygous state and are susceptible to root-knot nematodes (both owned by Sakata Seed Corporation), in combinations of SKTom-A and SKTom-B, and SKTom-C and SKTom-D, to obtain seeds of large-fruited F1 tomato plants that have both genes in reciprocal phase and heterozygous. These F1 tomato plants were then grown and self-pollinated to obtain F2 seeds. The characteristics of the parental lines SKTom-A, SKTom-B, SKTom-C and SKTom-D are shown in Table 5.

[0120] Genotyping using DNA markers was initiated to produce individuals in which the Mi-1 and Cf-5 genes were linked in coupling phase from the tomato plant population obtained by sowing the F2 seeds. The DNA markers used to determine the genotypes of the Mi-1 and Cf-5 genes are listed below (Table 1). Figure 1 also shows a schematic representation of the relative positions of the Mi-1 and Cf-5 genes and the DNA markers. According to Non-Patent Document 6, the Cf-5 gene is located closer to the centromere than the Cf-2.2 gene. Furthermore, according to Sol Genomics Network, the most centromeric base of the Cf-2.2 gene is the base identified as Ch06_2151188 on the reference genome, which means that the Cf-5 gene is located closer to the centromere than this base (Figure 1).

[0121]

[0122] Among the markers shown in Table 1, Mi23 is a marker designed near the Mi-1 gene described in Non-Patent Document 2, and therefore, genotyping was performed according to the method described in Non-Patent Document 2.

[0123] Among the markers shown in Table 1, Mi-1K is a KASP marker that detects a SNP at Ch06_2742590 on the opposite side (centromere side) of the Mi-1 gene from Mi23 in the present disclosure. ＴＭ It is a marker, and in the sequences of Mi1K_1F and Mi1K_2F, one base at the 3' end is designed to hybridize with the SNP to be identified. Note that the Mi-1K marker is designed based on the sequence of the complementary strand of the sequence shown in Reference Genome SL4.0, and therefore, in the sequence shown in Reference Genome SL4.0, the SNP sequence to be identified by the one base at the 3' end of Mi1K_1F is G (guanine), and the SNP sequence to be identified by the one base at the 3' end of Mi1K_2F is T (thymine). Furthermore, like Mi23, Mi-1K was designed to be able to specifically detect the Mi-1 gene in various strains.

[0124] When the genomic DNA of a plant individual having the Mi-1 gene is used as a template, an amplified product is obtained by PCR using Mi1K_1F and Mi1K_R as the forward and reverse primers, respectively. When the genomic DNA of a plant individual not having the Mi-1 gene is used as a template, an amplified product is obtained by PCR using Mi1K_2F and Mi1K_R as the forward and reverse primers, respectively. The genotype of the SNP is identified based on the presence or absence of this amplified product. In this way, these three types of oligo DNA are used as a primer set to detect KASP ＴＭ The assay can be performed to distinguish the genotype of each SNP.

[0125] For example, a tomato plant that has the Mi-1 gene in a homozygous state has a homozygous allele whose genotype at the SNP identified by ch06_2742590 is G (guanine), and a susceptible line that does not have the Mi-1 gene has a homozygous allele whose genotype at the SNP identified by ch06_2742590 is T (thymine).Furthermore, in an F1 that has the Mi-1 gene in a heterozygous state, the genotype at the SNP identified by ch06_2742590 is G / T, and the SNP has a heterozygous Mi-1 allele whose G is the SNP and a heterozygous susceptible allele whose T is the SNP.

[0126] As described above, both the Mi23 and Mi-1K markers are located near the Mi-1 gene, flanking the Mi-1 gene. Therefore, by selecting individuals in the segregating population in which both of these markers indicate the Mi-1 genotype, it is possible to reliably obtain individuals that have the Mi-1 gene.

[0127] Of the markers shown in Table 1, the Cf-5 marker is a marker designed based on the specific sequence of the Cf-5 gene itself in the aforementioned Non-Patent Document 7. For this reason, by performing genotyping according to the method described in Non-Patent Document 7 and selecting individuals exhibiting the Cf-5 genotype, it is possible to reliably obtain individuals having the Cf-5 gene.

[0128] DNA testing of the F2 population derived from the above multiple lines using the Mi23 marker and the Cf-5 marker showed that the marker genotypes of two of the cumulative 1,899 individuals were those with the Mi-1 gene and the Cf-5 gene in the coupling phase. These two individuals were subsequently tested using the Mi-1K marker, and the Mi-1K marker also showed a root-knot nematode resistance genotype. Furthermore, as shown in Table 5, these two individuals were combinations of breeding lines SKTom-A and SKTom-B, and SKTom-C and SKTom-D, each with different genetic backgrounds and characteristics, suggesting that it is possible to link the Mi-1 gene and the Cf-5 gene in the coupling phase regardless of genetic background and characteristics. Subsequently, individuals homozygous for both genes were produced from each self-fertilized progeny, and these were designated lines SKTom-E and SKTom-F. SKTom-E is suitable for winter cultivation and has drooping leaves. In contrast, SKTom-F is suitable for summer / autumn cultivation and has an upright habit with small leaves, resulting in a compact plant appearance. These two lines were inoculated with sweet potato root-knot nematode and sweet potato leaf mold race 2.4.9, and were confirmed to be resistant to both diseases. The results are shown in Table 2.

[0129]

[0130] Example 3 Next, the SKTom-E line was crossed with the SKTom-G line (owned by Sakata Seed Corporation) susceptible to root-knot nematodes and downy mildew to produce F1 tomato plants. These F1 plants were then self-pollinated to obtain seeds of an F2 population. These seeds were then sown and cultivated, and the tomato plants of the F2 population were assayed using DNA markers and inoculation in the same manner as above. It was found that the Mi-1 gene and the Cf-5 gene were inherited linked in the coupling phase. The results are shown in Table 3.

[0131]

[0132] From the tomato plants obtained by sowing seeds from the F2 population, individuals homozygous for both the Mi-1 and Cf-5 genes were selected using DNA markers. These individuals were then inoculated with Meloidogyne incognita and Downy mildew race 2.4.9 in the same manner as in Example 2, and were confirmed to be resistant to both pathogens. This line was designated SKTom-H (Table 3).

[0133] Seeds of the line SKTom-F (accession number FERM BP-22477, identification code: SSC-TOM-23-002) and the line SKTom-H (accession number FERM BP-22476, identification code: SSC-TOM-23-001), in which the Mi-1 gene and the Cf-5 gene were linked in coupling phase, obtained through the above tests, have been deposited.

[0134] Example 4 In Examples 2 and 3, we successfully produced tomato plants resistant to root-knot nematodes and downy mildew by linking the Mi-1 gene and the Cf-5 gene in a coupling phase from two lines with different genetic backgrounds and characteristics. However, since both were large-fruited tomato lines, we attempted to produce cherry tomato plants that were similarly resistant to root-knot nematodes and downy mildew.

[0135] A cherry tomato line SKTom-I, which has the Mi-1 gene homozygously and does not have the Cf-5 gene, was crossed with a cherry tomato line SKTom-J, which has the Cf-5 gene homozygously and does not have the Mi-1 gene (both owned by Sakata Seed Corporation), to obtain an F2 cherry tomato plant population. Similarly, a cherry tomato line SKTom-K, which has the Mi-1 gene homozygously and does not have the Cf-5 gene, was crossed with a cherry tomato line SKTom-L, which has the Cf-5 gene homozygously and does not have the Mi-1 gene (both owned by Sakata Seed Corporation), to obtain an F2 cherry tomato plant population. The characteristics of each parent line are shown in Table 5.

[0136] DNA testing was performed on 4,262 plant individuals from the F2 population obtained by crossing line SKTom-I with line SKTom-J using the Mi23 marker and the Cf-5 marker. The results showed that two of the 4,262 individuals had the Mi-1 gene in a heterozygous state and the Cf-5 gene in a homozygous state. In addition, one of the 4,262 individuals had the Mi-1 gene in a homozygous state and the Cf-5 gene in a heterozygous state. This indicated that the Mi-1 gene and the Cf-5 gene had been successfully linked in coupling phase on either of the homologous chromosomes of chromosome 6 of these three individuals. Subsequently, testing of these three individuals with the Mi-1K marker showed the same genotype of the Mi-1 gene as that of the Mi23 marker.

[0137] Next, DNA testing was performed on 1,719 plants from the F2 population obtained by crossing line SKTom-K with line SKTom-L using the Mi23 marker and the Cf-5 marker. The results showed that one of the 1,719 plants had the Mi-1 gene in a heterozygous state and the Cf-5 gene in a homozygous state. In addition, one of the 1,719 plants had the Mi-1 gene in a homozygous state and the Cf-5 gene in a heterozygous state. In other words, these two plants demonstrated successful linkage of the Mi-1 gene and the Cf-5 gene in coupling phase on either of the homologous chromosomes of chromosome 6. These two plants were then tested using the Mi-1K marker, and showed the same genotype of the Mi-1 gene as the Mi23 marker.

[0138] From the two cherry tomato F2 populations, a total of five individuals were shown to have the marker genotype of the Mi-1 gene and the Cf-5 gene in the coupling phase. Individuals that were homozygous for both genes were then selected from each selfed progeny to obtain the lines SKTom-M, SKTom-N, SKTom-O, SKTom-P, and SKTom-Q. These five lines were inoculated with nematode and late mold race 2.4.9 in the same manner as in Examples 2 and 3, and were confirmed to be resistant to both diseases. The results are shown in Table 4.

[0139]

[0140] From the above, it has become clear that linking the root-knot nematode resistance gene Mi-1 and the downy mildew resistance gene Cf-5 in the coupling phase is possible not only in large-berry tomato lines but also in a variety of tomato plants with different genetic backgrounds. Similarly, it has become clear that once linked in the coupling phase, the Mi-1 gene and the Cf-5 gene are inherited to progeny as if they were a single gene not only in large-berry tomato lines but also in a variety of tomato plants with different genetic backgrounds.

[0141]

[0142] Example 5 Next, seeds of the deposited line (accession number FERM BP-22476) produced in Example 3 were germinated in a 72-well tray to prepare tomato plant seedlings. As a control, seeds of SKTom-C, a line that has Mi-1 homozygously but does not have Cf-5, were also germinated in a 72-well tray to prepare tomato plant seedlings.

[0143] These prepared tomato seedlings were inoculated with late mold race 2.4.9 and observed 18 days later. No disease was observed in the trays of the deposited line (accession number FERM BP-22476), indicating that the tomato maintained its value as a plant product (Figs. 2 and 4). On the other hand, severe late mold disease was observed in the trays of SKTom-C, indicating that infection with late mold would cause the tomato to lose its value as a plant product (Figs. 3 and 4).

[0144] Furthermore, when the Mi-1 gene was tested for these prepared tomato plants using the Mi23 marker, all individuals of both lines were found to have the Mi-1 gene in a homozygous state. This indicates that in lines in which the Mi-1 gene and the Cf-5 gene are linked in coupling phase, such as the deposited lines, the Cf-5 gene can be detected by confirming only the genotype of the Mi-1 gene, as is clear from Examples 3, 4, and 5 above.

Claims

1. A tomato plant having resistance to root-knot nematodes and downy mildew, the tomato plant having, in complementary phase, the Mi-1 gene that confers resistance to root-knot nematodes and the Cf-5 gene that confers resistance to downy mildew.

2. The tomato plant according to claim 1, comprising a DNA fragment comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:

2.

3. The plant according to claim 1 or 2, wherein the Mi-1 gene and the Cf-5 gene are located on chromosome 6.

4. The tomato plant according to any one of claims 1 to 3, which contains, on the same genomic DNA of chromosome 6, a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:

2.

5. A plant described in any one of claims 1 to 4, wherein the Mi-1 gene and the Cf-5 gene are each homozygous or heterozygous.

6. The plant according to any one of claims 1 to 5, wherein the Mi-1 gene is identified by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO:3 and a primer comprising the nucleotide sequence of SEQ ID NO:4, and / or a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO:5, a primer comprising the nucleotide sequence of SEQ ID NO:6 and a primer comprising the nucleotide sequence of SEQ ID NO:7, and the Cf-5 gene is identified by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO:8 and a primer comprising the nucleotide sequence of SEQ ID NO:

9.

7. The plant according to any one of claims 1 to 6, which is resistant to at least one species of root-knot nematode of the genus Meloidogyne and is resistant to at least one race of Passalora fulva that cannot infect tomato plants expressing the Cf-5 gene.

8. The tomato plant according to any one of claims 1 to 7, which has resistance to root-knot nematodes and resistance to downy mildew and is derived from a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477.

9. The tomato plant according to any one of claims 1 to 8, which is a hybrid plant obtained by using the tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477 as a parent line, or a progeny of said hybrid plant.

10. The tomato plant according to any one of claims 1 to 9, comprising genomic DNA of chromosome 6, the nucleotide sequence being a portion of the genomic DNA of chromosome 6 of a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477, the portion comprising the Mi-1 gene and the Cf-5 gene; or (2) a nucleotide sequence having 50% or more identity with the nucleotide sequence described in (1) and comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:

2.

11. The tomato plant according to claim 10, wherein the portion of the genomic DNA of chromosome 6 of the tomato plant identified under accession number FERM BP-22476 or accession number FERM BP-22477, which contains the Mi-1 gene and the Cf-5 gene, is included in the range from the base identified at ch06_2151188 to the base identified at ch06_2742590 of the genomic DNA of chromosome 6 of the tomato plant identified under accession number FERM BP-22476 or accession number FERM BP-22477.

12. A part of the tomato plant according to any one of claims 1 to 11.

13. A part of the tomato plant body according to claim 12, which is a tomato plant cell incapable of regeneration, and which comprises a DNA fragment comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:

2.

14. A fruit of a tomato plant according to any one of claims 1 to 11.

15. A seed of the tomato plant according to any one of claims 1 to 11.

16. A DNA fragment containing the Mi-1 gene and the Cf-5 gene.

17. A DNA fragment according to claim 16, comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:

2.

18. The DNA fragment according to claim 16 or 17, comprising: (1) a nucleotide sequence of a portion of the genomic DNA of chromosome 6 of a tomato plant identified by accession number FERM BP-22476 or accession number FERM BP-22477, the portion containing the Mi-1 gene and the Cf-5 gene; or (2) a nucleotide sequence having 50% or more identity with the nucleotide sequence described in (1) and comprising a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:

2.

19. The DNA fragment according to claim 18, wherein the portion of the genomic DNA of chromosome 6 of a tomato plant identified under accession number FERM BP-22476 or accession number FERM BP-22477, which contains the Mi-1 gene and the Cf-5 gene, is included in the range from the base specified by ch06_2151188 to the base specified by ch06_2742590 of the genomic DNA of chromosome 6 of a tomato plant identified under accession number FERM BP-22476 or accession number FERM BP-22477.

20. A method for producing F1 seeds from the tomato plant according to any one of claims 1 to 11, comprising the steps of: crossing the tomato plant according to any one of claims 1 to 11 with the tomato plant according to any one of claims 1 to 11 or with another plant that is crossable with the tomato plant according to any one of claims 1 to 11; and harvesting F1 seeds from individuals obtained by the cross.

21. A method for producing a tomato plant having resistance to root-knot nematodes and downy mildew, comprising the steps of: self-pollinating a tomato plant according to any one of claims 1 to 11; and repeatedly self-pollinating an individual obtained by the self-pollination for one or more generations.

22. A method for producing a tomato plant which is resistant to root-knot nematodes and downy mildew, comprising: a first crossing step of crossing the tomato plant according to any one of claims 1 to 11 with a first tomato plant; a second crossing step of performing selfing, backcrossing, or interspecific or intraspecific crossing of an F1 individual obtained in the first crossing step with a second tomato plant different from the first tomato plant used in the first crossing step to obtain a segregating population; and a selection step of selecting, from the segregating population, a tomato plant which has the Mi-1 gene and the Cf-5 gene in a coupling phase.

23. The method of claim 22, wherein the selection step comprises at least one of: detecting the Mi-1 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 3 and a primer comprising the nucleotide sequence of SEQ ID NO: 4; detecting the Mi-1 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 5, a primer comprising the nucleotide sequence of SEQ ID NO: 6 and a primer comprising the nucleotide sequence of SEQ ID NO: 7; detecting the Cf-5 gene by a genotyping method using a primer set comprising a primer comprising the nucleotide sequence of SEQ ID NO: 8 and a primer comprising the nucleotide sequence of SEQ ID NO: 9; confirming resistance to root-knot nematodes; and confirming resistance to downy mildew.

24. A method for producing a product containing a tomato plant, a seed, a seedling, or a part of a tomato plant, the method comprising: a step of confirming, in the tomato plant according to any one of claims 1 to 11, one trait selected from root-knot nematode resistance, downy mildew resistance, the presence of the Mi-1 gene, and the presence of the Cf-5 gene; and a step of preparing a product containing the tomato plant, a seed, a seedling, or a part of a tomato plant in which the trait has been confirmed.

25. The method of claim 24, wherein the product is a product including a seed of the tomato plant and a packaging material containing the seedling, or a product including a seedling of the tomato plant and a tray containing the seedling.

26. A product comprising a plant, seed, seedling or part of a plant of a tomato plant produced by the method of claim 24.

Images