Spinach plants with novel downy mildew resistance genes

Abstract

The present invention is a downy mildew resistant spinach plant having the downy mildew resistance RTM-1 gene present on chromosome 4 of the tetra spinach line CGN25466:MGK 01; the above recited downy mildew resistant spinach plant wherein the RTM-1 gene is a gene present on chromosome 4 of the tetra spinach line CGN25466:MGK 01 in the range of chr4_7962907 to chr4_8617232; the downy mildew resistant spinach plant of any one recited having resistance to at least race Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, Pfs18, Pfs19, and UA1014 race exhibited by the Pseudoperonospora farinosa f. sp. esculenta strains.

Description

TECHNICAL FIELD

[0001] The present application relates to a spinach plant having a gene showing resistance to a wide range of small races of downy mildew and a method for producing the same.

[0002] This application claims priority from Japanese Patent Application No. 2021-080911 filed on May 12, 2021, the contents of which are incorporated herein. BACKGROUND

[0003] Spinacia oleracea L. is an annual or perennial herb of the Amaranthaceae family Spinacia genus, which is native to western Asia and widely cultivated, and is believed to have been introduced to Japan from China in the Edo period. Spinach is mainly edible basal leaves (rosette), and the content of vitamins or iron, calcium components is also particularly high in vegetables, and has a very high nutritional value. In recent years, due to its nutrients and ease of use, the market for young leaves has rapidly expanded worldwide. As a result, spinach has been positioned as one of the important vegetables.

[0004] In general, plant varieties have conventional varieties and hybrid first generation (hereinafter referred to as "F1") varieties, and F1 varieties are prevalent in major crops. F1 varieties grow vigorously due to heterosis. As a result, F1 varieties have a huge advantage of growing fast, improving yield capacity, and the like, and further, can also expect an increase in the ability to resist diseases and pests, the ability to adapt to the environment such as cold tolerance / heat tolerance. In addition, since F1 varieties are heterozygous and have the same genotype, the phenotype shows extremely high uniformity. Therefore, the marketability of agricultural products is improved. Further, the parents of F1 varieties can accumulate useful traits governed by dominant genes, so rapid breeding can be performed. Due to the advantages as described above, F1 varieties have become the mainstream of the cultivated varieties in major crops. Spinach, which is considered edible, has been centered on conventional varieties until the 1960s, and F1 has been rapidly promoted since the 1970s, and most of them are currently F1 varieties.

[0005] On the other hand, one of the diseases that damages spinach is downy mildew caused by Peronospora farinosa f. sp. spinaciae (Pfs), which is a filamentous fungus. Downy mildew is the most important disease, and once it occurs, the damage will quickly spread, causing very serious damage to yield and quality. As a countermeasure against downy mildew, attempts have been made to control the pathogen by cultivation or to control the pathogen chemically by using pesticides, but from the aspects of environmental impact, cultivation labor or cost, and the like, the use of resistant varieties is the most effective method.

[0006] The differentiation of small races of downy mildew is known to be fast, and new small races that attack resistant varieties are continuously appearing, and many cases of disease occurrence in varieties that were considered to be resistant have been confirmed so far. The International Working Group on Peronospora farinosa of Spinacia (IWGP) is a joint group consisting of seed companies supported by the University of Arkansas and the University of California in the United States, and Naktuinbouw in the Netherlands, and monitors the appearance and spread of new small races of downy mildew and determines the official name. Since the initial appearance of downy mildew was reported in 1824, nineteen small races have been named so far (non-patent literature 1).

[0007] In order to cope with the continuous appearance of new small races, the search for new resistant materials is very important in the breeding of spinach. Not only in the cultivated species, but also in the wild species, the search for genetic materials resistant to downy mildew is carried out. For example, the search for genetic materials resistant to downy mildew was carried out by the Center for Genetic Resources, the Netherlands (CGN) in 2008 on Spinacia turkestanica, a wild species of spinach, and in 2011 on Spinacia tetrandra, a wild species of spinach. In addition, Correll et al. published an article in 2011 on six genes called RPF that control known resistance to downy mildew (non-patent literature 2). Further, as downy mildew resistance genes, RPF1 to RPF10, RPF11 (patent literature 1), RPF12 (patent literature 2), RPF13 (patent literature 3), RPF14 (patent literature 4), RPF15 (patent literature 5), R6 (patent literature 6), R15 (patent literature 7), and the like have been reported. By introducing a downy mildew resistance gene derived from a wild species of spinach into Spinacia oleracea L., a cultivated species of spinach, a spinach with high downy mildew resistance can be cultivated (patent literature 8).

[0008] The RPF gene is a plurality of alleles or a plurality of genes in close linkage located at a locus called the RPF locus, and in the F1 variety, a wide range of resistance is shown by having two alleles (non-patent literature 3, patent literature 5). For example, the RPF1 gene, the RPF2 gene, and the RPF3 gene are located on chromosome 3 (non-patent literature 3), and the RPF15 gene is also located on chromosome 3 (patent literature 5). In addition, although the downy mildew resistance gene described in patent literature 8 is considered to be located on linkage group 6, it is actually located on chromosome 3 according to sequence information (patent literature 5).

[0009] In the Peronospora resistance locus, depending on its structure, one or two WOLF genes are present adjacent to the alpha WOLF gene and the beta WOLF gene (Patent Literature 9). The alpha WOLF gene and the beta WOLF gene contain multiple alleles that confer each a specific resistance spectrum, and the LRR domain sequence of each of the WOLF genes and the genotype of each of the genes against the resistance pattern of Peronospora races have been disclosed. In theory, by the combination of WOLF genes having different resistance patterns, it is possible to design a desired resistance pattern. However, in the existing hybrid breeding, it is practically impossible to arbitrarily combine genes that are very tightly linked. Therefore, so far, the RPF genes have been manipulated as one gene.

[0010] In addition to this, by transforming a Peronospora resistance gene introduced from the outside (GMO) or modifying an endogenous gene (gene editing), it is also possible to produce a mutant that confers a desired resistance pattern. However, there is a problem that crops bred by GMO or gene modification have not been widely accepted by the public.

[0011] It has been reported that the Peronospora resistance gene p10 is located on chromosome 1 and shows resistance to Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, and Pfs16. However, the resistance conferred by the p10 gene is expressed only when it is homozygous, and the degree of its resistance is moderate (Patent Literature 10). Therefore, it is presumed that breeding of a resistant variety using the p10 gene is not only more difficult than breeding using a dominant resistance gene, but also the degree of resistance is not sufficient in practical terms. In addition, it has been reported that the Tetragonia tetragonoides line CGN25474:MGK 18:RNR120251 (hereinafter referred to as CGN25474:MGK 18) has a Peronospora resistance gene in chromosome 4, and shows resistance to Pfs4, Pfs7, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, and Pfs17 (Patent Literature 11).

[0012] Prior Art Documents

[0013] Patent Literature

[0014] Patent Literature 1: U.S. Patent No. 10258001 Specification

[0015] Patent Literature 2: U.S. Patent No. 10258002 Specification

[0016] Patent Literature 3: International Publication No. 2015 / 036378

[0017] Patent Literature 4: International Publication No. 2019 / 145446

[0018] Patent Literature 5: International Publication No. 2019 / 145447

[0019] Patent Literature 6: Japanese Patent No. 6457269

[0020] Patent Literature 7: U.S. Patent No. 9974276

[0021] Patent Literature 8: Japanese Patent No. 6684207

[0022] Patent Literature 9: International Publication No. 2018 / 059651

[0023] Patent Literature 10: U.S. Patent Application Publication No. 2019 / 0104700

[0024] Patent Literature 11: International Publication No. 2020 / 239215

[0025] Non-Patent Literature

[0026] Non-Patent Literature 1: Ribera et al., Euphytica, 2020, 216: 48.

[0027] Non-Patent Literature 2: Correll et al., European Journal of Plant Pathology, 2011, Vol. 129, pp. 193-205.

[0028] Non-Patent Literature 3: Feng et al., Euphytica, 2018, 214: 174.

[0029] Non-Patent Literature 4: International Seed Federation, “Differential Sets Peronospora farinosa f. sp. spinaciae (P. effusa)”, 2021, <Web page> http: / / worldseed.org / wp-content / uploads / 2021 / 11 / 20210608_DRTWG_Peronospora-effusa.pdf

[0030] Non-Patent Literature 5: Iwata and Ninomiya, Breeding Science, 2006, Vol. 56(4), pp. 371-377. SUMMARY

[0031] PROBLEMS TO BE SOLVED BY THE INVENTION

[0032] The existing downy mildew resistant lines mainly utilize the RPF gene on chromosome 3. On the other hand, in general, in the breeding of spinach, the parents of an F1 variety need to be highly fixed genetically. As a result, one parent can have only one resistance gene at the same or very close position. That is, an F1 variety obtained by crossing different parents with each other can have only two RPF genes at the same or very close position on chromosome 3. Since there is no known RPF gene that shows resistance to all of the named races, in order to breed an F1 variety that shows resistance to a wide range of races, it is necessary to cross varieties having a combination of RPF genes that complement each other's resistance with each other. This is a large limiting factor in the selection of parents.

[0033] For example, if a resistance gene present on a chromosome other than chromosome 3 can be utilized, a large effect in accumulating downy mildew resistance genes can be expected. Furthermore, if this resistance gene present on a chromosome other than chromosome 3 has polymorphism, further, diversification of downy mildew resistance genes can be promoted, and the emergence of new races of downy mildew can be suppressed.

[0034] Therefore, in view of the problems of the existing downy mildew resistant lines and F1 varieties as described above, the present application provides a novel downy mildew resistance gene that is not present on chromosome 3, shows resistance to a wider range of races than known resistance genes, and further, is different from the reported resistance gene on chromosome 4. That is, the object of the present application is to provide a spinach plant having a novel downy mildew resistance gene that shows resistance to a wide range of races, a method of manufacturing the spinach plant, a method of manufacturing an F1 plant of spinach using a line that is conferred downy mildew resistance by the gene, and the like.

[0035] Technical means for solving the technical problem

[0036] The present inventors conducted research in order to solve the above problems, and as a result, found that a dominant downy mildew resistance gene that shows resistance to a wide range of races is present on chromosome 4 of the tetraploid spinach line CGN25466:MGK 01:RNR120234 (hereinafter referred to as CGN25466:MGK 01). Further, it was found that by performing interspecific crossing of wild species spinach having this downy mildew resistance gene with cultivated species spinach, and further repeatedly crossing the F1 individual having downy mildew resistance with cultivated species spinach, a spinach plant having downy mildew resistance due to this downy mildew resistance gene and traits close to cultivated species can be bred, thereby completing the present application.

[0037] That is, the present application is as described below.

[0038] [1] A downy mildew resistant spinach plant, which is a spinach plant having a downy mildew resistance RTM-1 gene located on chromosome 4 of Spinacia oleracea line CGN25466:MGK 01, wherein Spinacia oleracea is excluded.

[0039] [2] The downy mildew resistant spinach plant of [1], wherein the RTM-1 gene is a gene present in a range of chr4_7962907 to chr4_8617232 on chromosome 4 of Spinacia oleracea line CGN25466:MGK 01.

[0040] [3] The downy mildew resistant spinach plant of [1] or [2], wherein, in at least one allele of chromosome 4, a fragment comprising a range of chr4_8488603 to chr4_8510715 on chromosome 4 of Spinacia oleracea line CGN25466:MGK 01 is contained.

[0041] [4] The downy mildew resistant spinach plant of any one of [1] to [3], wherein the RTM-1 gene is homozygous or heterozygous.

[0042] [5] The downy mildew resistant spinach plant of any one of [1] to [4], which is resistant to at least races Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, Pfs18, Pfs19, and UA1014 races exhibited by the downy mildew.

[0043] [6] The downy mildew resistant spinach plant of any one of [1] to [5], wherein, in at least one allele of chromosome 4,

[0044] the SNP identified on chr4_8488603 is cytosine,

[0045] the SNP identified on chr4_8494600 is thymine, or

[0046] the SNP identified on chr4_8510715 is guanine.

[0047] [7] The downy mildew resistant spinach plant of any one of [1] to [5], wherein, in at least one allele of chromosome 4,

[0048] the SNP identified on chr4_7962907 is guanine,

[0049] The SNP identified at chr4_8152986 is cytosine,

[0050] The SNP identified at chr4_8190990 is guanine,

[0051] The SNP identified at chr4_8488603 is cytosine,

[0052] The SNP identified at chr4_8494600 is thymine,

[0053] The SNP identified at chr4_8510715 is guanine, or

[0054] The SNP identified at chr4_8617232 is guanine.

[0055] [8] The downy mildew resistant spinach plant according to any one of [1] to [7], wherein the downy mildew resistant spinach plant is derived from an interspecific hybrid plant of Tetragonia tetragonoides and Spinacia oleracea.

[0056] [9] The downy mildew resistant spinach plant according to any one of [1] to [8], which has the downy mildew resistance derived from a plant defined by Accession No. FERM BP-22404 (TNKH-1 line).

[0057]

[10] The downy mildew resistant spinach plant according to any one of [1] to [8], which has the downy mildew resistance derived from a plant defined by Accession No. FERM BP-22405 (TNKH-2 line).

[0058]

[11] The downy mildew resistant spinach plant according to any one of [1] to

[10] , which has at least one or more downy mildew resistance genes other than the RTM-1 gene.

[0059]

[12] A downy mildew resistant spinach plant, which is a downy mildew resistant spinach plant defined by Accession No. FERM BP-22404 (TNKH-1 line), a hybrid plant obtained using the downy mildew resistant spinach plant as a parent, or a progeny thereof.

[0060]

[13] A downy mildew resistant spinach plant, which is a downy mildew resistant spinach plant defined by Accession No. FERM BP-22405 (TNKH-2 line), a hybrid plant obtained using the downy mildew resistant spinach plant as a parent, or a progeny thereof.

[0061]

[14] A method for predicting the downy mildew resistance of a spinach plant, wherein the genotype of the SNP of chr4_8488603 to chr4_8510715 of chromosome 4 is investigated in a test spinach plant, and in at least one allele,

[0062] the SNP identified at chr4_8488603 is cytosine,

[0063] the SNP identified at chr4_8494600 is thymine, or

[0064] in the case where the SNP identified at chr4_8510715 is guanine,

[0065] the possibility that the test spinach plant has downy mildew resistance is predicted to be high.

[0066]

[15] A method for screening a downy mildew resistant spinach plant, wherein the genotype of the SNP of chr4_8488603 to chr4_8510715 of chromosome 4 of a test spinach plant is investigated, and in at least one allele,

[0067] the SNP identified at chr4_8488603 is cytosine,

[0068] the SNP identified at chr4_8494600 is thymine, or

[0069] in the case where the SNP identified at chr4_8510715 is guanine,

[0070] the test spinach plant is selected as a downy mildew resistant spinach plant.

[0071]

[16] A method for producing a downy mildew resistant spinach plant, comprising the steps of:

[0072] a first hybridization step of hybridizing a spinach plant having an RTM-1 gene and an arbitrary spinach plant;

[0073] a second hybridization step of selfing, backcrossing, or intercrossing or intracrossing with a spinach plant different from the parent used in the first hybridization step, with respect to an Fl individual obtained by the first hybridization step, to obtain a segregating population; and

[0074] a selection step of selecting a spinach plant having an RTM-1 gene from the segregating population.

[0075]

[17] The method for producing a downy mildew resistant spinach plant according to the

[16] , wherein the spinach plant having an RTM-1 gene is Spinacia oleracea or the downy mildew resistant spinach plant according to any one of the [1] to

[13] .

[0076]

[18] A method for producing the downy mildew resistant spinach plant according to

[16] or

[17] , wherein the arbitrary spinach plant, or the parents used in the inter-species crossing or the intra-species crossing in the second hybridization step is a spinach plant having at least one downy mildew resistance gene other than the RTM-1 gene.

[0077]

[19] A part of a plant body of the downy mildew resistant spinach plant according to any one of [1] to

[13] .

[0078]

[20] A leaf of the downy mildew resistant spinach plant according to any one of [1] to

[13] .

[0079]

[21] A seed of the downy mildew resistant spinach plant according to any one of [1] to

[13] .

[0080]

[22] A DNA marker for selecting a spinach plant having the RTM-1 gene, which is composed of one or more selected from the group consisting of a SNP identified on chr4_8488603, a SNP identified on chr4_8494600, a SNP identified on chr4_8510715, and a SNP in strong linkage with these SNPs in genetics.

[0081]

[23] A kit for selecting a spinach plant having the RTM-1 gene, which comprises one or more primer sets selected from the group consisting of:

[0082] a primer set for identifying the genotype of the SNP identified on chr4_8488603,

[0083] a primer set for identifying the genotype of the SNP identified on chr4_8494600, and

[0084] a primer set for identifying the genotype of the SNP identified on chr4_8510715.

[0085]

[24] The kit according to

[23] , wherein,

[0086] the primer set for identifying the genotype of the SNP identified on chr4_8488603 is composed of a primer consisting of the base sequence represented by SEQ ID NO: 13, a primer consisting of the base sequence represented by SEQ ID NO: 14, and a primer consisting of the base sequence represented by SEQ ID NO: 15;

[0087] The primer set for identifying the genotype of the SNP identified at chr4_8494600 consists of a primer composed of the base sequence represented by SEQ ID NO: 16, a primer composed of the base sequence represented by SEQ ID NO: 17, and a primer composed of the base sequence represented by SEQ ID NO: 18.

[0088] The primer set for identifying the genotype of the SNP identified at chr4_8510715 consists of a primer composed of the base sequence represented by SEQ ID NO: 19, a primer composed of the base sequence represented by SEQ ID NO: 20, and a primer composed of the base sequence represented by SEQ ID NO: 21.

[0089] Effects of the Invention

[0090] According to the present application, a downy mildew resistant spinach plant showing resistance to a wide range of races is provided.

[0091] In addition, by using the spinach plant according to the present application as a parent, a new spinach line showing resistance to a wide range of races can be bred. BRIEF DESCRIPTION OF DRAWINGS

[0092] [ Figure 1 ]In Example 6, a linkage map in the vicinity of the RTM-1 locus of chromosome 4 of spinach. DETAILED DESCRIPTION

[0093] In the present application and the present specification, a spinach plant is a plant classified as Spinacia. As wild species of spinach, Spinacia tetrandra, Spinacia turkestanica, and the like can be given. In addition, as cultivated species of spinach, Spinacia oleracea L. can be given. Furthermore, in the present application and the present specification, the so-called "cultivated species" is a plant of a line for cultivation, and includes not only a conventional species but also an Fl variety.

[0094] In the present application and the present specification, the so-called "downy mildew" refers to a disease caused by a bacterium belonging to the family Peronosporaceae. The main pathogenic bacterium of spinach downy mildew is Pfs (Spinacia Peronospora differentiation type pathogen), and currently numbered as Pfs 1 to Pfs 19. In addition to these, there are many races that are not numbered, such as the race of the pathogenicity of the UA1014 strain.

[0095] In the present application and the present specification, "chrX_Y" (X and Y are integers) of a spinach plant means "a base corresponding to the Yth base of chromosome number X (also referred to as chromosome X) in the reference genome (Spinach Genome Sequence (V1)) disclosed in SpinachBase (http: / / spinachbase.org / ) of the spinach plant". In addition, the "base corresponding to the Yth base of chromosome X in the reference genome of SpinachBase" in a certain spinach plant can be determined by aligning the base sequence of the genomic DNA of the spinach plant with the base sequence of the reference genome of SpinachBase in such a way that the homology (sequence identity) becomes the highest.

[0096] Thus, chromosome X of a certain spinach plant can be represented as the chromosome X of the spinach plant determined by aligning the base sequence of the genomic DNA of the spinach plant with the base sequence of the reference genome of SpinachBase in such a way that the homology (sequence identity) becomes the highest.

[0097] In the present application and the present specification, "chromosome" includes not only the entire chromosome but also a part thereof. That is, "a part of the chromosome" can also be simply referred to as "chromosome".

[0098] The "X locus" is "a site occupied by an X gene in a chromosome". Therefore, in the present application and the present specification, the "spinach plant having an X locus" means a spinach plant having a site occupied by an X gene in a chromosome, and has the same meaning as "spinach plant having an X gene".

[0099] In addition, in the present application and the present specification, "manufacturing method" can be replaced with "breeding method", "cultivation method", or "production method". That is, the terms "manufacture", "breed", "cultivate", and "produce" are used with the same meaning.

[0100] In the present application and the present specification, the "part of the plant body" includes a cell or a tissue of the plant body, and specifically, leaf, seed, flower, stem, root, fruit, and the like can be mentioned. In addition, a protoplast obtained from a cell of the plant body is also included.

[0101] <Spinach plant resistant to downy mildew>

[0102] The downy mildew resistant spinach plant according to the present application is a spinach plant other than Spinacia oleracea L. var. tetrandra Tsigar-Sokolska having an RTM-1 gene which is a downy mildew resistance gene located on chromosome 4 of Spinacia oleracea L. var. tetrandra Tsigar-Sokolska strain CGN25466:MGK 01 which is a wild species of spinach. The RTM-1 gene is a gene present in the range of chr4_7962907 to chr4_8617232 of chromosome 4 of Spinacia oleracea L. var. tetrandra Tsigar-Sokolska strain CGN25466:MGK 01.

[0103] The RTM-1 gene confers resistance to at least races Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, Pfs18, Pfs19 and UA1014 races of downy mildew. That is, the so-called "downy mildew resistance derived from the RTM-1 gene" means resistance to at least races Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, Pfs18, Pfs19 and UA1014 races of downy mildew. Since it is possible to confer resistance to such a wide range of races with a single gene, the RTM-1 gene is an unprecedentedly very excellent downy mildew resistance gene.

[0104] The RTM-1 gene is a gene that is dominantly inherited to offspring. Therefore, the spinach plant according to the present application can be either a spinach plant in which the RTM-1 gene is homozygous (that is, a spinach plant having a homozygous RTM-1 gene) or a spinach plant having a heterozygous RTM-1 gene. The spinach plant according to the present application is very excellent as a downy mildew resistant spinach due to having the RTM-1 gene which is a dominant downy mildew resistance gene that confers resistance to a very wide range of downy mildew races alone.

[0105] The downy mildew resistant spinach plant according to the present application can be cultivated by introducing the RTM-1 gene into the genomic DNA of a spinach plant other than Spinacia oleracea. The introduction of the RTM-1 gene into the genomic DNA can be performed, for example, by introducing a chromosomal fragment containing the site of the RTM-1 gene (RTM-1 locus) on chromosome 4 of the Spinacia oleracea line CGN25466:MGK 01. The site in the chromosome into which the chromosomal fragment containing the RTM-1 locus is introduced is not particularly limited and can be extrachromosomal. For example, in the downy mildew resistant spinach plant according to the present application, there are included spinach plants in which a region corresponding to the chromosomal fragment of chromosome 4 of Spinacia oleracea line CGN25466:MGK 01 is replaced by a chromosomal fragment containing the RTM-1 locus, or spinach plants in which a chromosomal fragment containing the RTM-1 locus is introduced outside a region corresponding to the RTM-1 locus of chromosome 4 of Spinacia oleracea by translocation or the like.

[0106] As the chromosomal fragment containing the RTM-1 locus, there can be used a fragment containing the entire region of chr4_7962907 to chr4_8617232 on chromosome 4 of Spinacia oleracea line CGN25466:MGK 01, or a fragment containing a portion of the region of chr4_7962907 to chr4_8617232 including the RTM-1 locus. As the fragment containing a portion of the RTM-1 locus, there is preferably used a fragment containing the region of chr4_8488603 to chr4_8510715 on chromosome 4 of Spinacia oleracea line CGN25466:MGK 01. As such a fragment, there is preferably used a chromosomal fragment containing the region of chr4_8488603 to chr4_8510715 in the region of chr4_7962907 to chr4_8617232. That is, as the downy mildew resistant spinach plant according to the present application, there is preferably used a spinach plant in which at least one allele of chromosome 4 contains a fragment containing the range of chr4_8488603 to chr4_8510715 on chromosome 4 of Spinacia oleracea line CGN25466:MGK 01. In addition, as the chromosomal fragment containing the RTM-1 locus, there can be used a chromosomal fragment containing the region of chr4_7962907 to chr4_8617232 on chromosome 4 of Spinacia oleracea line CGN25466:MGK 01, or a chromosomal fragment containing the region of chr4_7421480 to chr4_10710358.

[0107] The introduction of the RTM-1 gene into the genomic DNA can be performed by a hybridization breeding method using Spinacia oleracea as a parent, a genome editing method, or the like.

[0108] For example, the downy mildew resistant spinach plant according to the present application is an interspecific hybrid plant of Spinacia oleracea and a spinach plant other than Spinacia oleracea, which is derived from a plant having downy mildew resistance derived from the RTM-1 gene. Furthermore, in the "interspecific hybrid plant", in addition to the plant obtained by hybridization between different species within the genus Spinacia, a somatic hybrid plant obtained by cell fusion between different species of the genus Spinacia, or a grafted hybrid plant obtained by grafting between different species of the genus Spinacia is also included. In addition, in the "interspecific hybrid plant of Spinacia oleracea and a spinach plant other than Spinacia oleracea", in addition to the interspecific hybrid plant, progeny of the interspecific hybrid plant is also included.

[0109] In the present application and the present specification, the "progeny of a spinach plant" includes, in addition to the progeny obtained by intraspecific hybridization of the spinach plant, an individual obtained using the spinach plant as a parent and its progeny, a somatic hybrid plant obtained by cell fusion between a cell of the spinach plant and a plant cell of another variety and its progeny, and an individual obtained by grafting using the spinach plant as a rootstock or a scion and its progeny. The "progeny" includes both an individual obtained by intraspecific hybridization and an individual obtained by interspecific hybridization. In addition, the "individual obtained using the (plant) as a parent" means an individual obtained by intraspecific hybridization, interspecific hybridization, or cell fusion or grafting using the plant as a parent.

[0110] For example, the downy mildew resistant spinach plant according to the present application can be cultivated by crossing Spinacia oleracea and a spinach plant other than Spinacia oleracea. In the F1 individual obtained by interspecific hybridization using Spinacia oleracea as a parent, one allele of chromosome 4 has the RTM-1 gene derived from Spinacia oleracea. Since the RTM-1 gene is a dominant gene, the F1 individual having chromosome 4 derived from Spinacia oleracea has downy mildew resistance derived from the RTM-1 gene.

[0111] As the spinach plant used as a cultivation material of the downy mildew resistant spinach plant according to the present application, there is no particular limitation as long as it has the RTM-1 gene possessed by CGN25466:MGK 01. For example, as a strain of Spinacia oleracea, in addition to CGN25466:MGK 01, a progeny strain that inherits the RTM-1 gene from CGN25466:MGK 01, and the like can be mentioned.

[0112] In order to breed the downy mildew resistant spinach plant according to the present application, as a parent to be crossed with Spinacia oleracea, there is no particular limitation as long as it is a spinach plant other than Spinacia oleracea, and a cultivated species spinach plant that is cultivated as a crop is preferred. Spinacia oleracea is a wild species, and the traits of germination, pollen quality, leaf color, and leaf shape are inferior to those of a cultivated species spinach plant as a crop. By using a cultivated species spinach plant as a parent to be crossed with Spinacia oleracea, the traits other than the downy mildew resistance of the downy mildew resistant spinach plant obtained can be made close to the traits preferred as a crop. As a cultivated species spinach plant, a Persian spinach or an interspecific hybrid spinach plant bred using a Persian spinach as a parent can be mentioned.

[0113] The downy mildew resistant spinach plant according to the present application can also be a descendant of an Fl individual obtained by interspecific crossing of Spinacia oleracea with a spinach plant other than Spinacia oleracea. For example, Fl seeds are obtained by crossing Spinacia oleracea and a cultivated species spinach plant, and seeds are obtained by backcrossing or self-crossing a plant individual of the cultivated species spinach plant with a plant individual grown from the obtained Fl seeds. Then, a plant individual having the RTM-1 gene is selected from among plant individuals grown from the obtained seeds. With respect to the selected plant individual, self-crossing or crossing with a cultivated species spinach plant is repeated, and a plant individual having the RTM-1 gene is selected from among the descendants obtained by the crossing, whereby a spinach plant variety having stable downy mildew resistance can be obtained. The self-crossing, the crossing with a cultivated species spinach plant, the cultivation, and the seed harvesting can be performed by a conventional method of spinach cultivation.

[0114] The plant individual having the RTM-1 gene is selected from among the descendants obtained by the crossing, for example, by investigating the downy mildew resistance. Specifically, with respect to the leaves of a plant individual to be investigated for the downy mildew resistance, Pfs of each race is inoculated, and the occurrence of downy mildew is investigated. In the case where downy mildew does not occur, it is evaluated that the plant individual has resistance to the inoculated race. From among the descendants obtained by the crossing, a plant individual having resistance to at least the races exhibited by the Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, Pfs18, Pfs19, and UA1014 strains of downy mildew is selected as a plant individual having the RTM-1 gene.

[0115] From the offspring obtained by the cross, an individual having the RTM-1 gene is selected, for example, by a method of investigating whether or not the genomic DNA contains a chromosomal fragment containing the RTM-1 gene. Specifically, using the DNA markers in the region of chr4_7962907 to chr4_8617232 in chromosome 4 of the spinach plant, or in the vicinity thereof, it is identified whether these regions are chromosomal fragments derived from the tetra spinach line CGN25466:MGK 01 or chromosomal fragments derived from a spinach plant other than tetra spinach.

[0116] In the present application and the present specification, the so-called DNA marker is capable of detecting a difference in the DNA sequence on the chromosome, which enables the distinction of the chromosomal fragments derived from different varieties. As the DNA marker, for example, a SNP (Single Nucleotide Polymorphism) marker, or a SSR (Simple Sequence Repeats) marker, a RFLP (Restriction Fragment Length Polymorphism) marker, and the like can be cited.

[0117] The detection of the DNA marker can be performed by a conventional method. For example, the DNA extracted from each plant individual can be used as a template, a nucleic acid amplification reaction can be performed using primers capable of specifically hybridizing to a specific SNP or SSR and a polymerase, and the presence or absence of the amplification product can be detected using an electrophoresis method or the like, whereby each polymorphism can be identified. In addition, the DNA extracted from each plant individual can be subjected to restriction enzyme treatment, and the pattern of the DNA fragments can be detected using an electrophoresis method or the like, whereby each polymorphism can be identified. Furthermore, the primers capable of specifically hybridizing to a specific SNP or SSR can be designed using a general primer design tool or the like according to the base sequence of the genomic DNA of the spinach, or the base sequence of the SNP or SSR of the detection object, and synthesized by a conventional method.

[0118] As the DNA marker for selecting an individual having the RTM-1 gene, for example, a SNP in the region of chr4_7962907 to chr4_8617232 or its vicinity can be mentioned. As the SNP, specifically, chr4_7421480, chr4_7962907, chr4_8152986, chr4_8190990, chr4_8488603, chr4_8494600, chr4_8500200, chr4_8502319, chr4_8502334, chr4_8502394, chr4_8510715, chr4_8617232, chr4_10710358 can be mentioned. The base recognizing these SNPs can be performed, for example, using KASP (Kompetitive Allele Specific PCR) genotyping assay (manufactured by LGC Genomics). As to these SNPs, the genotype of tetra-cultivated Spinach line CGN25466:MGK01 and the genotype of the reference genome disclosed in SpinachBase are shown in Table 1. In Table 1, G means guanine, C means cytosine, A means adenine, and T means thymine.

[0119] [Table 1]

[0120] SNP Genotype of Tetraena mongolica Genotype of reference genome chr4_7421480 C G chr4_7962907 G A chr4_8152986 C T chr4_8190990 G A chr4_8488603 C T chr4_8494600 T C chr4_8500200 T C chr4_8502319 C A chr4_8502334 G A chr4_8502394 G A chr4_8510715 G C chr4_8617232 G A chr4_10710358 G C

[0121] For example, in the downy mildew resistant spinach plant according to the present application, a spinach plant having homozygous or heterozygous for the fragment comprising the region ranging from chr4_8488603 to chr4_8510715 of chromosome 4 of Spinacia oleracea strain CGN25466:MGK 01, in at least one allele of chromosome 4, the SNP identified on chr4_8488603 is cytosine, the SNP identified on chr4_8494600 is thymine, the SNP identified on chr4_8510715 is guanine. Further, as a spinach plant having homozygous or heterozygous for the fragment comprising the region ranging from chr4_7962907 to chr4_8617232 of chromosome 4 of Spinacia oleracea strain CGN25466:MGK 01, a spinach plant in which, in at least one allele of chromosome 4, the SNP identified on chr4_7962907 is guanine, the SNP identified on chr4_8152986 is cytosine, the SNP identified on chr4_8190990 is guanine, the SNP identified on chr4_8488603 is cytosine, the SNP identified on chr4_8494600 is thymine, the SNP identified on chr4_8510715 is guanine, the SNP identified on chr4_8617232 is guanine is preferred. As a spinach plant having homozygous or heterozygous for the fragment comprising the region ranging from chr4_7962907 to chr4_8617232 of chromosome 4 of Spinacia oleracea strain CGN25466:MGK 01, a spinach plant in which, in at least one allele of chromosome 4, the SNP identified on chr4_7962907 is guanine, the SNP identified on chr4_8152986 is cytosine, the SNP identified on chr4_8190990 is guanine, the SNP identified on chr4_8488603 is cytosine, the SNP identified on chr4_8494600 is thymine, the SNP identified on chr4_8500200 is thymine, the SNP identified on chr4_8502319 is cytosine, the SNP identified on chr4_8502334 is guanine, the SNP identified on chr4_8502394 is guanine, the SNP identified on chr4_8510715 is guanine, the SNP identified on chr4_8617232 is guanine is also preferred.

[0122] The Peronospora infestans resistant spinach plant according to the present application preferably has the same or similar traits to cultivated spinach other than the Peronospora infestans resistance, particularly traits as a crop plant. As traits as a crop plant, there can be mentioned taste, shape of leaves, yield, easiness of cultivation, and the like. Further, the "traits as a cultivated plant" means traits as a crop plant that are suitable as a crop plant.

[0123] As the Peronospora infestans resistant spinach plant according to the present application, there can be mentioned, for example, the TNKH-1 line (deposited under Accession No. FERM BP-22404) and the TNKH-2 line (deposited under Accession No. FERM BP-22405). These two lines have broken the linkage of the undesirable traits possessed by Tetragonia tetrandra and the RTM-1 gene, and have maintained the Peronospora infestans resistance derived from the RTM-1 gene, while taking into account the traits as a cultivated plant. Further, a spinach plant having the Peronospora infestans resistance derived from a plant defined by these lines is also included in the Peronospora infestans resistant spinach plant according to the present application.

[0124] As the spinach plant having the Peronospora infestans resistance derived from a plant defined by the TNKH-1 line, there can be mentioned, in addition to the TNKH-1 line, hybrid plants obtained using the TNKH-1 line as a parent, and their progeny (spinach plants having the Peronospora infestans resistance derived from the RTM-1 gene). The same applies to the spinach plant having the Peronospora infestans resistance derived from a plant defined by the TNKH-2 line.

[0125] As the Peronospora infestans resistant spinach plant according to the present application, it is preferable that the spinach plant have at least one or more Peronospora infestans resistance genes other than the RTM-1 gene. Although the RTM-1 gene is capable of conferring resistance to a wider range of races alone as compared to known resistance genes, by also having other Peronospora infestans resistance genes, it is expected that the emergence of new races that break the resistance will be suppressed. As the Peronospora infestans resistance genes other than the RTM-1 gene, there is no particular limitation, and there can be mentioned, for example, the RPF1 gene to the RPF15 gene, the R6 gene, the R15 gene, and the like.

[0126] The Peronospora infestans resistant spinach plant according to the present application can be any of the whole and a part of the plant body. That is, the Peronospora infestans resistant spinach plant according to the present application also includes any of the whole plant body of a spinach plant having the RTM-1 gene, the aboveground portion of the spinach plant, and the tissue of the spinach plant. Further, the Peronospora infestans resistant spinach plant according to the present application also includes a cell obtained from one tissue of a spinach plant having the RTM-1 gene. As the tissue, there can be mentioned an embryo, a meristematic cell, a callus, pollen, a leaf, an anther, a stem, a petiole, a root, a root tip, a fruit, a seed, a flower, a cotyledon, and a hypocotyl.

[0127] The downy mildew resistance of the downy mildew resistant spinach plant according to the present application is expressed dominantly. Therefore, by performing interspecific crossing or intraspecific crossing using the downy mildew resistant spinach plant according to the present application as a parent, a new strain of spinach plant having downy mildew resistance derived from the RTM-1 gene can be bred.

[0128] <Method for screening downy mildew resistant spinach plant>

[0129] In the method for screening the downy mildew resistant spinach plant according to the present application, whether or not the test spinach plant has the RTM-1 gene is investigated, and in the case where it is judged that the test spinach plant has the RTM-1 gene, the test spinach plant is selected as the downy mildew resistant spinach plant. In the method for screening the downy mildew resistant spinach plant according to the present application, whether or not the test spinach plant has the RTM-1 gene is judged based on the genotype of the DNA marker at the RTM-1 locus or in the vicinity thereof on chromosome 4, or the DNA marker strongly linked to these DNA markers. In at least one allele of chromosome 4, in the case where the genotype of the DNA marker at the RTM-1 locus or in the vicinity thereof, or the DNA marker strongly linked to these DNA markers is the same as the genotype of Spinacia oleracea having the RTM-1 gene such as CGN25466:MGK01 strain, and exhibiting downy mildew resistance, the test spinach plant is selected as the downy mildew resistant spinach plant.

[0130] As the DNA marker for judging the presence or absence of the RTM-1 gene, for example, the SNP described in Table 1 can be cited. Among these, one or more selected from the group consisting of the SNP identified at chr4_8488603, the SNP identified at chr4_8494600, the SNP identified at chr4_8510715, and the SNP strongly linked genetically to these SNPs are suitable as the DNA marker for judging the presence or absence of the RTM-1 gene. For example, in the case where, in at least one allele, the SNP identified at chr4_8488603 is cytosine, the SNP identified at chr4_8494600 is thymine, or the SNP identified at chr4_8510715 is guanine, the test spinach plant is selected as the downy mildew resistant spinach plant.

[0131] By kitizing a primer set for identifying a genotype of a DNA marker for judging the presence or absence of the RTM-1 gene in advance and by using the kit, it is possible to more easily judge the presence or absence of the RTM-1 gene, thereby selecting a downy mildew resistant spinach plant. For example, a kit that aggregates one or more primer sets selected from the group consisting of a primer set for identifying a genotype of a SNP identified at chr4_8488603 of a spinach plant, a primer set for identifying a genotype of a SNP identified at chr4_8494600, and a primer set for identifying a genotype of a SNP identified at chr4_8510715 is useful for identifying a genotype of these SNPs for judging the presence or absence of the RTM-1 gene in a test spinach plant.

[0132] <Method for predicting downy mildew resistance of a spinach plant>

[0133] The method for predicting downy mildew resistance of a spinach plant according to the present application is a method for investigating whether a test spinach plant has the RTM-1 gene, and predicting that the test spinach plant has a high possibility of having downy mildew resistance when judged to have the RTM-1 gene. In the method for predicting downy mildew resistance of a spinach plant according to the present application, whether a test spinach plant has the RTM-1 gene is judged based on a genotype of a DNA marker at or near the RTM-1 locus of chromosome 4, or a DNA marker in strong linkage with these DNA markers. In at least one allele of chromosome 4, when the genotype of the DNA marker at or near the RTM-1 locus, or the DNA marker in strong linkage with these DNA markers is the same genotype as a tetraploid spinach having the RTM-1 gene such as CGN25466:MGK 01 and showing downy mildew resistance, it is judged that the test spinach plant has the RTM-1 gene, and when the genotype is different from that of the tetraploid spinach having the RTM-1 gene such as CGN25466:MGK 01 and showing downy mildew resistance, it is judged that the test spinach plant does not have the RTM-1 gene.

[0134] As the DNA marker for judging the presence or absence of the RTM-1 gene, the SNPs described in Table 1 can be given. Among these, one or more selected from the group consisting of the SNP identified at chr4_8488603, the SNP identified at chr4_8494600, the SNP identified at chr4_8510715, and SNPs in strong linkage disequilibrium with these SNPs are suitable as the DNA marker for predicting downy mildew resistance. For example, in a case where the SNP identified at chr4_8488603 is cytosine, the SNP identified at chr4_8494600 is thymine, or the SNP identified at chr4_8510715 is guanine in at least one allele, it is predicted that the test spinach plant has the RTM-1 gene and has a high possibility of having downy mildew resistance. The primer set for identifying the genotype of the DNA marker for judging the presence or absence of the RTM-1 gene and the kit thereof can use the same primer set and the kit as described above.

[0135] <Method for producing a downy mildew resistant spinach plant>

[0136] By crossing a spinach plant having the RTM-1 gene as a material, a new downy mildew resistant spinach plant can be produced by introducing downy mildew resistance derived from the RTM-1 gene into a spinach plant not having the RTM-1 gene. The RTM-1 gene, which is a monogenic resistance gene, is easily introduced into any strain, and can accelerate the breeding of a spinach variety. Further, the RTM-1 gene can be present in the same haplotype as other monogenic resistance alleles. Therefore, by using a spinach plant having the RTM-1 gene as a parent, a variety having resistance to a wider range of races can be easily bred.

[0137] The method for producing a downy mildew resistant spinach plant according to the present application is a method of using hybrid breeding using a spinach plant having the RTM-1 gene as a parent. Specifically, it includes a first crossing step of crossing a spinach plant having the RTM-1 gene and an arbitrary spinach plant, a second crossing step of selfing, backcrossing, or inter-species crossing or intra-species crossing with a spinach plant different from the parent used in the first crossing step with respect to an Fl individual obtained by the first crossing step to obtain a segregating population, and a selection step of selecting a spinach plant having the RTM-1 gene from the segregating population. Selfing, backcrossing, inter-species crossing, intra-species crossing, cultivation of a spinach plant, and seed production can be performed by a conventional method of spinach cultivation.

[0138] As the spinach plant having the RTM-1 gene used as the parent in the first hybridization step, tetra spinach can be used, and the above-mentioned spinach plant of the present application having resistance to downy mildew can also be used. In the present application, as the spinach plant having the RTM-1 gene used as the parent, a spinach plant having the RTM-1 gene and also having traits as a cultivated species is preferred. As such a spinach plant having resistance to downy mildew, TNKH-1 line, hybrid plants obtained using the TNKH-1 line as the parent, TNKH-2 line, and hybrid plants obtained using the TNKH-2 line as the parent, and progeny of these plants, i.e., spinach plants having resistance to downy mildew derived from the RTM-1 gene can be mentioned.

[0139] As the arbitrary spinach plant used as the parent in the first hybridization step, or the arbitrary spinach plant used as the parent for inter-species hybridization or intra-species hybridization in the second hybridization step, various spinach plants not having the RTM-1 gene can be appropriately used without particular limitation. In the present application, a cultivated species spinach plant is preferably used as the parent. For example, by using a spinach plant having the RTM-1 gene and also having traits as a cultivated species as the parent, and hybridizing the same with a cultivated species spinach plant not having the RTM-1 gene, selfing, backcrossing, or inter-species hybridization or intra-species hybridization with a cultivated species spinach plant different from the parent used in the first hybridization step is performed on the obtained Fl individual, a new spinach line having resistance to downy mildew derived from the RTM-1 gene and also having traits suitable as a cultivated species can be comparatively easily bred.

[0140] As the arbitrary spinach plant used as the parent in the first hybridization step, or the arbitrary spinach plant used as the parent for inter-species hybridization or intra-species hybridization in the second hybridization step, a spinach plant having at least one or more downy mildew resistance genes other than the RTM-1 gene is preferred, and a cultivated species spinach plant having at least one or more downy mildew resistance genes other than the RTM-1 gene is more preferred. By using a spinach plant having at least one or more downy mildew resistance genes other than the RTM-1 gene as the parent, a new spinach line containing both the RTM-1 gene and other downy mildew resistance genes can be comparatively easily bred.

[0141] In the selection step, a spinach plant having the RTM-1 gene is selected from the isolated groups, for example, the investigation of downy mildew resistance can be performed by inoculating leaves of the individual plants under test with each race of Pfs. From the isolated groups, an individual plant showing resistance to at least the race of downy mildew exhibited by the Pfs 1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs 10, Pfs 11, Pfs 12, Pfs 13, Pfs 14, Pfs 15, Pfs 16, Pfs 17, Pfs 18, Pfs 19, and UA1014 strains is selected as an individual having the RTM-1 gene.

[0142] The selection of the individual having the RTM-1 gene in the selection step can be performed using the DNA markers in the vicinity of the RTM-1 locus on chromosome 4 of the spinach plant. As for the DNA markers used or the specific method, the same as described in the screening method relating to the present application can be performed.

[0143] In the case where the individual plant containing both the RTM-1 gene and the other downy mildew resistance gene is selected, a DNA marker capable of identifying the presence or absence of the other downy mildew resistance gene is used together with the DNA marker described above for the selection of the individual having the RTM-1 gene. As the DNA marker capable of identifying the presence or absence of the other downy mildew resistance gene, a SNP or the like present in the vicinity of the locus of the other downy mildew resistance gene can be used.

[0144] Then, the seeds collected from the selected offspring individuals are selfed repeatedly, whereby a spinach plant having downy mildew resistance, which is more stable in traits, and the germination rate, the harvest amount, the seed collection amount, and the like reach a level capable of being stably cultivated as a crop plant, can be obtained. The number of times of repetition of selfing is not particularly limited as long as it is one or more times, and can be 2 to 3 times, or 3 or more times.

[0145] Example

[0146] Hereinafter, the present application will be described in more detail with reference to Examples. However, the present application is not limited to the following Examples.

[0147] <Investigation of the race of downy mildew resistance>

[0148] The inoculation test of Pfs performed on the spinach plant is performed by the following method.

[0149] The spinach seeds were sown in trays filled with culture soil "Super Mixture A" (manufactured by Sakata Seed Co., Ltd.), and covered with culture soil "Metro Mix 350" (manufactured by Hyponex Japan Co., Ltd.). At the time of sowing, seeds of a strain showing susceptibility to the inoculated race were sown on each tray, and set as a diseased control area. The trays were grown in a glass greenhouse for 2 weeks, and at the second true leaf stage, the spore suspension (5 x 10 4 / mL) of Pfs was sprayed. Immediately after inoculation, the trays were covered with plastic covers, and managed under conditions of temperature 15°C, humidity 100%, and light for 12 hours.

[0150] Seven to ten days after inoculation, the disease of the control area was confirmed sufficiently, and then the individuals were judged for resistance, susceptibility, respectively. The plants in which the disease was confirmed by the appearance of hyphae and spores on the cotyledon or true leaf were regarded as having susceptibility, and the plants in which the disease was not confirmed on both the cotyledon and true leaf were regarded as having resistance.

[0151] Each race of downy mildew was obtained from within Japan, The University of Arkansas (Dr. Jim Correll), and Naktuinbouw. In addition, in the discrimination of the races of downy mildew, a known spinach variety (differential set) showing different resistance patterns to each race can be used as a discrimination variety. The discrimination variety for the discrimination of the downy mildew resistance of each race can be obtained from the USDA (United States Department of Agriculture) and Naktuinbouw (Non-patent literature 4).

[0152] The resistance reported for each discrimination variety is shown in Table 2 and Table 3. In the tables, "-" means resistance, "(-)" means moderate resistance, "+" means susceptibility, "(+)" means that the symptoms of downy mildew and spore formation were observed only on the cotyledon, and not on the true leaf. In addition, "*" means that the results of resistance were different in each test.

[0153] [Table 2]

[0154]

[0155] [Table 3]

[0156]

[0157] [Example 1]

[0158] A new downy mildew resistant strain was bred using the wild species Spinacia oleracea strain CGN25466:MGK 01, which was obtained from CGN in the Netherlands in 2013, as a parent.

[0159] First, wild species Tetragonia tetragonoides line CGN25466:MGK 01 and CGN25474:MGK 18 obtained from CGN in the Netherlands were sowed respectively, and the germinated individuals were inoculated with Pfs6. As a result, Tetragonia tetragonoides line CGN25466:MGK 01 showed resistance, and Tetragonia tetragonoides line CGN25474:MGK 18 showed susceptibility. Therefore, Tetragonia tetragonoides line CGN25466:MGK 01 was used as a parent.

[0160] First, 60 seeds of Tetragonia tetragonoides line CGN25466:MGK 01 were sowed, and 25 individuals that germinated were inoculated with Pfs10. As a result, all of the 25 individuals showed resistance.

[0161] Then, 4 individuals among the 25 individuals were crossed with RPF3 line (elite parent having RPF3 gene), and F1 seeds were obtained. In addition, the so-called elite parent means a parent line suitable for being used as a parent line for breeding a line expressing suitable traits as a cultivated species.

[0162] The obtained 40 F1 seeds were sowed, and as a result, plants of 10 individuals grew. 2 individuals among the 10 individuals that grew were backcrossed with the RPF3 line, and BC1 seeds were obtained.

[0163] The obtained 24 BC1 seeds were sowed, and as a result, plants of 10 individuals grew. The 10 individuals that grew were inoculated with Pfs10 that RPF3 did not show resistance to, and as a result, 7 individuals showed resistance. 1 individual among them was crossed with RPF4 line (elite parent having RPF4 gene), and BC1F1 seeds were obtained.

[0164] The obtained 50 BC1F1 seeds were sowed, and as a result, plants of 43 individuals grew. The 43 individuals that grew were inoculated with Pfs10 that neither RPF3 nor RPF4 showed resistance to, and as a result, 15 individuals showed resistance. 11 individuals among them were self-crossed, and BC1F1S1 seeds were obtained.

[0165] The obtained 817 BC1F1S1 seeds were sowed, and as a result, plants of 676 individuals grew. When all of the plants that grew were inoculated with Pfs10, as a result, plants of 500 individuals showed resistance. Among the individuals that showed resistance, 22 individuals were self-crossed, and BC1F1S2 seeds were obtained.

[0166] The obtained 7696 BC1F1S2 seeds were sowed, and as a result, plants of 3551 individuals grew. 76 individuals among the plants that grew were self-crossed, and BC1F1S3 seeds were obtained.

[0167] The obtained 12971 seeds of BC1F1S3 were sown, and as a result, 9815 individual plants grew. 115 individuals of the grown plants were selfed, and seeds of BC1F1S4 were obtained.

[0168] 7377 seeds of BC1F1S4 obtained from the selfed 115 individuals were sown, and as a result, 4717 individual plants grew. All of the grown plants were inoculated with RPF3 and RPF4, and none of the plants showed resistance to the race expressed by the Pfs10 or UA1014 strain. As a result, all of the plants of the five lines showed resistance to the race expressed by the Pfs10 and UA1014 strains. This gave the following implications: the five lines can have a homozygous unknown downy mildew resistance gene different from RPF3 and RPF4, and the unknown downy mildew resistance gene is not an allele of the RPF3 gene and the RPF4 gene, and can exist in a different locus. 34 individuals of the five lines showing resistance were selfed, and seeds of BC1F1S5 were obtained.

[0169] For the five lines showing resistance to all of the individuals, the RPF3 marker and the RPF4 marker were used to investigate the presence or absence of the RPF3 gene and the RPF4 gene. According to the results, an RPF3 homologous line was selected and named as the TNKH-1 line. In addition, an RPF4 homologous line was selected and named as the TNKH-2 line. Further, a line in which RPF3 and RPF4 were segregated was selected and named as the TNKH-3 line. The unknown downy mildew resistance gene and the locus thereof possessed by these lines, which originated from Spinacia tetrandra, were named as RTM-1.

[0170] In addition, in Spinacia tetrandra and early generations in which the Spinacia tetrandra strongly expressed genetic influence, many seeds were clustered, and germination was also poor. Therefore, seed harvesting, selection, and attention to the germination environment after sowing were required in order to improve the germination rate. Also, pollen was heavy and not easily dispersed, so crossing was difficult. Also, in each step, not only selection for disease resistance was performed, but also individuals having traits as close as possible to the cultivation type in terms of leaf color, leaf shape, leaf size, and the like were selected.

[0171] As to the TNKH-1 strain and the TNKH-2 strain, the applicant deposited seeds thereof in the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (2-5-8 Kazusakamimichi, Kisarazu-shi, Chiba-ken, Room No. 120). The depositor attached to the TNKH-1 strain a certificate of identification: SSC-SPI-20-001, and the accession number: FERM BP-22404 (date of deposit: December 25, 2020), and the depositor attached to the TNKH-2 strain a certificate of identification: SSC-SPI-20-002, and the accession number: FERM BP-22405 (date of deposit: December 25, 2020).

[0172] [Example 2]

[0173] The Viroflay strain, which has no resistance to any of the sub-species, was used as a seed parent, and the TNKH-3 strain, which was bred in Example 1, was used as a pollen parent to perform hybridization. As to the F1 individuals obtained, inoculation tests using the Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, and UA1014 strains were performed, and the resistance to these sub-species was investigated. The results are shown in Table 4. In Table 4, as to the “resistant individuals” and the “number of individuals in the resistant individuals and susceptible individuals” columns of the “Viroflay x TNKH-3 F1” column, “P (Q)” (both P and Q are integers) is the number of F1 individuals in the resistant individuals or the susceptible individuals, and Q is the number of individuals (self) obtained by selfing of Viroflay in the resistant individuals or the susceptible individuals. In addition, “nt” means that no experiment was performed, and no data is available. Further, in the column of each sub-species in the “RPF3 strain”, the “RPF4 strain”, and the “Viroflay” strain, “-” means resistance, and “+” means susceptibility. Furthermore, in the RTM-1 genotype described in Table 4, “RTM-1” means a resistance genotype derived from Tetragonia tetragonoides, and “rtm-1” means a susceptibility genotype derived from Viroflay. Thus, “RTM-1 / RTM-1” means a homozygote for resistance, “rtm-1 / rtm-1” means a homozygote for susceptibility, and “RTM-1 / rtm-1” means a heterozygote for resistance and susceptibility. Likewise, in the RPF genotype, “rpfV” means a susceptibility genotype derived from Viroflay.

[0174]

Table 4

[0175]

[0176] Spinach is a crop of dioecious plants, but there are also lines that show intersexuality (produce both pistils and stamens in the same individual). In F1 seed production with Viroflay as the seed parent, there are sometimes self-seeds of Viroflay included. Whether it is a self-seed of Viroflay or an F1 seed can be distinguished by investigating the RPF genotype using the RPF3 marker and the RPF4 marker.

[0177] In the F1 population obtained by crossing the Viroflay line and the TNKH-3 line, the RPF genotype of susceptible individuals was investigated, and it was confirmed that none of the RPF3 gene and the RPF4 gene (rpfV / rpfV) were possessed by all of the susceptible individuals obtained by selfing of Viroflay. That is, as shown in Table 4, the F1 individuals obtained by crossing the Viroflay line and the TNKH-3 line showed resistance to all of the races except for Pfs9, and there were no susceptible individuals. From the downy mildew resistance of the RPF3 gene and the RPF4 gene, it was found that the RTM-1 gene can confer resistance to at least the races exhibited by Pfs6, Pfs7, Pfs10, Pfs13, Pfs17, and the UA1014 strain.

[0178] In the F1 population, RPF3 / rpfV and RPF4 / rpfV segregated approximately at a ratio of 1:1. RPF3 is susceptible to Pfs2, Pfs4, and Pfs15, and RPF4 is susceptible to Pfs5, Pfs8, Pfs11, Pfs12, Pfs14, and Pfs16, and it is assumed that the RTM-1 gene does not have resistance to these races. However, in fact, all of the F1 individuals had resistance to these races. From this, it was found that the RTM-1 gene can also confer dominant resistance to Pfs2, Pfs4, Pfs5, Pfs8, Pfs11, Pfs12, Pfs14, Pfs15, and Pfs16.

[0179] [Example 3]

[0180] The Viroflay line was used as a seed parent, and the TNKH-1 line raised in Example 1 was used as a pollen parent to perform a cross, and the F1 individuals obtained were subjected to inoculation tests using the strains of Pfs2, Pfs4, Pfs10, Pfs15, Pfs17, Pfs18, and UA1014 to investigate the resistance to these strains. The results are shown in Table 5. In the table, "P(Q)" (P and Q are integers) in the column of the number of individuals of "resistant individuals" and "susceptible individuals" in the column of "Viroflay x TNKH-1 F1", P is the number of individuals of the F1 individuals that are resistant or susceptible, and Q is the number of individuals that are obtained by selfing of Viroflay (self). In addition, "nt" indicates that no experiment was performed, and no data is available. Further, in the column of each strain in "RPF3" and "Viroflay", "-" means resistance, and "+" means susceptibility. Furthermore, in the RTM-1 genotype described in Table 5, "RTM-1" means the resistance genotype derived from Tetragonia tetrandra, and "rtm-1" means the susceptibility genotype derived from Viroflay. Thus, "RTM-1 / RTM-1" means a homozygote for resistance, "rtm-1 / rtm-1" means a homozygote for susceptibility, and "RTM-1 / rtm-1" means a heterozygote for resistance and susceptibility. Similarly, in the RPF genotype, "rpfV" means the susceptibility genotype derived from Viroflay.

[0181] [Table 5]

[0182]

[0183] As shown in Table 5, the F1 individuals obtained by crossing the Viroflay line with the TNKH-1 line showed resistance to the strains of Pfs2, Pfs4, Pfs10, Pfs15, Pfs17, Pfs18, and UA1014, and no susceptible individuals were present. As a result of investigating the RPF genotype, it was confirmed that all of the susceptible individuals were obtained by selfing of Viroflay. From the downy mildew resistance of RPF3 gene, it was confirmed that the RTM-1 gene can confer at least the dominant resistance to the strains of Pfs2, Pfs4, Pfs10, Pfs15, Pfs17, Pfs18, and UA1014.

[0184] [Example 4]

[0185] The Viroflay line was used as the seed parent, and the TNKH-2 line raised in Example 1 was used as the pollen parent to perform a cross. The F1 individuals obtained were inoculated with the strains of Pfs5, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs14, Pfs16, Pfs17, Pfs18, Pfs19, and UA1014, and the resistance to these strains was investigated. The results are shown in Table 6. In Table 6, "P(Q)" (P and Q are both integers) in the column of the number of individuals of "resistant individuals" and "susceptible individuals" in the column of "Viroflay x TNKH-2 F1", P is the number of individuals of the F1 individuals that are resistant or susceptible, and Q is the number of individuals that are obtained by selfing of Viroflay (self). In addition, "nt" indicates that no experiment was performed, and no data is available. Further, in the column of each strain in the lines of "RPF4" and "Viroflay", "-" means resistance, and "+" means susceptibility. Furthermore, in the RTM-1 genotype described in Table 6, "RTM-1" means the resistance genotype derived from Tetragonia tetragonoides, and "rtm-1" means the susceptibility genotype derived from Viroflay. Thus, "RTM-1 / RTM-1" means a homozygote for resistance, "rtm-1 / rtm-1" means a homozygote for susceptibility, and "RTM-1 / rtm-1" means a heterozygote for resistance and susceptibility. Similarly, in the RPF genotype, "rpfV" means the susceptibility genotype derived from Viroflay.

[0186] [Table 6]

[0187]

[0188] As shown in Table 6, the F1 individuals obtained by crossing the Viroflay line with the TNKH-2 line showed resistance to the strains of Pfs5, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs14, Pfs16, Pfs17, Pfs18, Pfs19, and UA1014, and no susceptible individuals were present. As a result of investigating the RPF genotype, it was confirmed that all of the susceptible individuals were obtained by selfing of Viroflay. From the downy mildew resistance of the RPF4 gene, it was found that the RTM-1 gene can confer resistance that is at least dominant to the strains of Pfs5, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs14, Pfs16, Pfs17, Pfs18, Pfs19, and UA1014.

[0189] [Example 5]

[0190] The Viroflay line was used as the female parent and the TNKH-1 line, developed in Example 1, was used as the male parent to produce F1 seeds. The F1 seeds were selfed to produce an F2 segregating population. The F2 population was inoculated with the races expressed by the Pfs1, Pfs3, Pfs4, Pfs8, Pfs10, Pfs11, Pfs12, Pfs14, Pfs15 and UA1014 strains and the resistance to these races was investigated. The results are shown in Table 7. In the table, "nt" means that no experiment was performed and no data are available. Further, in the column for each race in the lines "RPF3" and "Viroflay", "-" means resistance and "+" means susceptibility. In addition, in the genotypes of the RTM-1 line described in Table 7, "RTM-1" means the resistance genotype derived from the Tetragonia tetrandra and "rtm-1" means the susceptibility genotype derived from Viroflay. Thus, "RTM-1 / RTM-1" means a homozygote for resistance, "rtm-1 / rtm-1" means a homozygote for susceptibility and "RTM-1 / rtm-1" means a heterozygote for resistance and susceptibility. Similarly, in the RPF genotype, "rpfV" means the susceptibility genotype derived from Viroflay.

[0191] [Table 7]

[0192]

[0193] As shown in Table 7, with respect to the downy mildew resistance of the TNKH-1 line, the resistant individuals: susceptible individuals segregated approximately 3:1 for the races expressed by the Pfs4, Pfs10, Pfs15 and UA1014 strains, which are susceptible to the RPF3 gene. Thus, it was assumed that the RTM-1 gene is monogenic. This assumption was also supported by the results of the chi-square test (p>0.05).

[0194] With respect to the downy mildew resistance of the TNKH-1 line, the resistant individuals: susceptible individuals segregated approximately 15:1 for the Pfs1, Pfs3, Pfs8, Pfs11, Pfs12 and Pfs14 strains, which are resistant to the RPF3 gene. Thus, it was assumed that the RTM-1 gene and the RPF3 gene are digenic. This assumption was also supported by the results of the chi-square test (p>0.05).

[0195] These results indicate that the RTM-1 gene is not an allele of the RPF gene, but is located on a different chromosome and is able to confer monogenic resistance to the races expressed by the Pfs1, Pfs3, Pfs4, Pfs8, Pfs10, Pfs11, Pfs12, Pfs14, Pfs15 and UA1014 strains.

[0196] [Example 6]

[0197] In order to identify the chromosomal location of the region of the RTM-1 gene, genetic analysis and genetic marker making were performed.

[0198] First, the F1 obtained by crossing the Viroflay line with the TNKH-1 line raised in Example 1 was selfed to make a F2 segregating population.

[0199] 330 individuals of the F2 segregating population obtained were subjected to inoculation test using the race expressed by the UA1014 strain to investigate the resistance thereto. As a result, 234 individuals were resistant individuals and 96 individuals were susceptible individuals.

[0200] The individuals were subjected to genetic analysis to search for a region that becomes a resistant homozygote or heterozygote in the resistant individuals or a susceptible homozygote in the susceptible individuals from the genome.

[0201] The individuals subjected to the trait investigation were used as a population for linkage analysis, and genomic DNA was extracted from each individual. Genotyping was performed using the DNA of the population for linkage analysis and SNP markers designed on the reference genome of SpinachBase. The genotyping was performed using KASP genotyping assay.

[0202] The base sequences of the primers for KASP genotyping assay for identifying each SNP are shown in Table 8 and Table 9. In Table 8, Table 9, respectively, “allele A” indicates the allele of the TNKH-1 type (Spinacia oleracea type), and “allele B” indicates the allele of the Viroflay type. Each SNP is identified by genotype by three primers of allele-specific primer_1, allele-specific primer_2, and universal primer. In the sequences of allele-specific primer_1 and allele-specific primer_2, one base at the 3’-terminal end is designed to hybridize to the SNP to be identified.

[0203] In the case of using the genomic DNA of the plant individual having the allele of the TNKH-1 type as a template, the allele-specific primer_1 and the universal primer were used as the forward primer and the reverse primer, respectively, and an amplification product was obtained by PCR. In the case of using the genomic DNA of the plant individual having the allele of the Viroflay type as a template, the allele-specific primer_2 and the universal primer were used as the forward primer and the reverse primer, respectively, and an amplification product was obtained by PCR. The genotype of the SNP was identified depending on the presence or absence of the amplification product. In this way, by using the three kinds of oligo DNAs as one set of primer sets, KASP assay was performed, and the genotype of each SNP could be identified.

[0204]

Table 8

[0205]

[0206]

Table 9

[0207]

[0208] For example, the genotype of the SNP identified on chr4_8510715 in the TNKH-1 line is the homologous allele G (guanine), and the genotype of the SNP identified on chr4_8510715 in the Viroflay line is the homologous allele C (cytosine). In addition, in the Fl of the TNKH-1 line and the Viroflay line, the genotype of the SNP identified on chr4_8510715 is G / C, and the SNP has the TNKH-1 type allele G and the Viroflay type allele C.

[0209] Furthermore, the KASP markers used for linkage analysis are used for identifying the position of the locus of the RTM-1 gene, and it is not necessarily required to use these KASP markers as DNA markers when selecting a spinach having downy mildew resistance. For example, a DNA polymorphism other than the SNPs described in Tables 8 and 9 within the range of the spinach genome identified on chr4_7962907 to chr4_8617232, a DNA polymorphism showing a high correlation with the downy mildew resistance trait derived from the locus on chr4_7962907 to chr4_8617232, and the like can be used as a DNA marker when selecting a spinach having downy mildew resistance.

[0210] By using the genomic DNA extracted from the individual of the test plant and the KASP markers described in Tables 8 and 9, the genotype of each SNP can be easily investigated in the linkage analysis population. After obtaining the genotyping data by the KASP markers, linkage analysis was continued using the ultra-high density genetic linkage map generation software AntMAP Ver. 1.1 (http: / / lbm.ab.a.u-tokyo.ac.jp / ~iwata / antmap / (Non-Patent Literature 5). In the use of this software, the Criterion of Grouping was changed to Distance (cM) / Haldane / Threshold 5, and the default settings were set except for this.

[0211] In Table 10, the genotypes of 7 individuals (sample No. 112 / 143 / 322 / 294 / 53 / 232 / 6) in which recombination occurred in the vicinity of the RTM-1 gene were excerpted according to the results of genotyping using the KASP markers shown in Tables 8 and 9, and were recorded together with the phenotypes. In the table, "A" indicates the TNKH-1 type allele, and "B" indicates the Viroflay type allele. For example, in the case of "A / A" being recorded, it indicates that the homologous TNKH-1 type allele is possessed, in the case of "B / B" being recorded, it indicates that the homologous Viroflay type allele is possessed, and in the case of "A / B" being recorded, it indicates that the heterologous TNKH-1 type allele and Viroflay type allele are possessed.

[0212] [Table 10]

[0213]

[0214] As shown in Table 10, in the SNPs of chr4_8488603, chr4_8494600, and chr4_8510715, 2 individuals of sample 232 and sample 6 having the genotype B / B had the phenotype S (susceptibility), and sample 112, sample 143, sample 322, sample 294, and sample 53 having the genotype A / B had the phenotype R (resistance), and thus it was found that these SNPs had a high correlation with the phenotype. In addition, the results of linkage analysis indicated that, on the reference genome of SpinachBase, in the range of chr4_7962907 to chr4_8617232, the RTM-1 gene that causes resistance to downy mildew was present. In addition, the SNPs of chr4_8488603, chr4_8494600, and chr4_8510715 were linked to the RTM-1 gene at a genetic distance of 0 cM. Figure 1

[0215] ​SNPs were searched from the sequencing analysis data of the total base sequence in the range of chr4_7962907 to chr4_8617232 where the RTM-1 gene is located, with respect to the four-radiate spinach line CGN25466:MGK 01, the TNKH-1 line, and the TNKH-2 line. As a result, in addition to the SNPs described in Tables 8 and 9, a plurality of SNPs were confirmed. Among them, for example, four SNPs of chr4_8500200, chr4_8502319, chr4_8502334, and chr4_8502394 were different from the reference genome. Specifically, in the four-radiate spinach line CGN25466:MGK 01, the TNKH-1 line, and the TNKH-2 line, the SNP identified at chr4_8500200 was thymine, the SNP identified at chr4_8502319 was cytosine, the SNP identified at chr4_8502334 was guanine, and the SNP identified at chr4_8502394 was guanine (Table 1). Like the SNPs described in Tables 8 and 9, these four SNPs can also be used as markers for identifying plant individuals having the TNKH-1 type of allele of downy mildew resistance.

[0216] Accession No.:

[0217] FERM BP-22404

[0218] FERM BP-22405. SEQUENCE LISTING <110> Sakata Seeds Corporation <120> Spinach plant having a novel downy mildew resistance gene <130> PC-34414 <160> 27 <210> 1 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic allele-specific primer_1 for chr4_7421480. <400> 1 gaaggtgacc aagttcatgc tatccccggc tttcttatcc tg 42 <210> 2 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Description of Artificial Sequence: Allele specific primer_2 for synthetic chr4_7421480. <400> 2 gaaggtcgga gtcaacggat tatccccggc tttcttatcc tc 42 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Universal primer for synthetic chr4_7421480. <400> 3 catgtgaagg aagggacacc a 21 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_1 for synthetic chr4_7962907. <400> 4 gaaggtgacc aagttcatgc tatgctcgta tacgcaatgg c 41 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_2 for synthetic chr4_7962907. <400> 5 gaaggtcgga gtcaacggat tcatgctcgt atacgcaatg gt 42 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Universal primer for synthetic chr4_7962907. <400> 6 ccaattgacc agaattgtca cc 22 <210> 7 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic allele specific primer_1 for chr4_8152986. <400> 7 gaaggtgacc aagttcatgc tgggagcaac atttatgcaa gac 43 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic allele specific primer_2 for chr4_8152986. <400> 8 gaaggtcgga gtcaacggat tgggagcaac atttatgcaa gat 43 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic universal primer for chr4_8152986. <400> 9 atgtatggtc tgacaacggc a 21 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic allele specific primer_1 for chr4_8190990. <400> 10 gaaggtgacc aagttcatgc tatcgggagg aaagctctaa cc 42 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> DESCRIPTION OF ARTIFACT: Allele specific primer_2 for synthetic chr4_8190990. <400> 11 gaaggtcgga gtcaacggat tatcgggagg aaagctctaa ct 42 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DESCRIPTION OF ARTIFACT: Universal primer for synthetic chr4_8190990. <400> 12 tgtgtaccca tattggccag a 21 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> DESCRIPTION OF ARTIFACT: Allele specific primer_1 for synthetic chr4_8488603. <400> 13 gaaggtcgga gtcaacggat ttaatgggat ctgctgcctc g 41 <210> 14 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> DESCRIPTION OF ARTIFACT: Allele specific primer_2 for synthetic chr4_8488603. <400> 14 gaaggtgacc aagttcatgc tctaatggga tctgctgcct ca 42 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Universal primer for synthetic chr4_8488603. <400> 15 caaaggcttg acggtccagt a 21 <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_1 for synthetic chr4_8494600. <400> 16 gaaggtgacc aagttcatgc ttctcgcttg tcattgtcag ca 42 <210> 17 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_2 for synthetic chr4_8494600. <400> 17 gaaggtcgga gtcaacggat tttcgcttgt cattgtcagc g 41 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Universal primer for synthetic chr4_8494600. <400> 18 ctgtggagtc ggaagaacac a 21 <210> 19 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_1 for synthetic chr4_8510715. <400> 19 gaaggtgacc aagttcatgc tctggtccat actcatactc aggg 44 <210> 20 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_2 for synthetic chr4_8510715. <400> 20 gaaggtcgga gtcaacggat tctggtccat actcatactc aggc 44 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Universal primer for synthetic chr4_8510715. <400> 21 agctaattct gagccaaggc c 21 <210> 22 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_1 for synthetic chr4_8617232. <400> 22 gaaggtcgga gtcaacggat ttgagacagc gggataaaga gtaag 45 <210> 23 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_2 for synthetic chr4_8617232. <400> 23 GAAGGTGACC AAGTTCATGC TTGAGACAGC GGGATAAAG AGTAA 45 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Universal primer for synthetic chr4_8617232. <400> 24 TCAGTTCGAC AAGCTGCCTA G 21 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_1 for synthetic chr4_10710358. <400> 25 GAAGGTCGGA GTCAACGGAT TCGAGTTGAT CACCTCGTCG 40 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Allele specific primer_2 for synthetic chr4_10710358. <400> 26 GAAGGTGACC AAGTTCATGC TCGAGTTGAT CACCTCGTCC 40 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Universal primer for synthetic chr4_10710358. <400> 27 TGTA CCCAGT TGTCGAGCC 19

Claims

1. A cell of a downy mildew-resistant spinach plant that cannot develop into a whole plant, and which is a spinach plant possessing the downy mildew-resistant RTM-1 gene on chromosome 4 of the four-stamen spinach cultivar CGN25466:MGK 01, wherein, Except for four-stemmed spinach; The RTM-1 gene is a gene located on chromosome 4 of the four-stamen spinach cultivar CGN25466:MGK 01, ranging from chr4_7962907 to chr4_8617232. At least one allele on chromosome 4 contains a segment ranging from chr4_8488603 to chr4_8510715 on chromosome 4, which includes the four-stamen spinach cultivar CGN25466:MGK 01.

2. The cells of the downy mildew-resistant spinach plant according to claim 1, wherein, At least one allele on chromosome 4 contains a segment ranging from chr4_7962907 to chr4_8617232 on chromosome 4, which includes the four-stamen spinach cultivar CGN25466:MGK 01.

3. The cells of the downy mildew-resistant spinach plant according to claim 1, wherein, The RTM-1 gene is either homozygous or heterozygous.

4. Cells of downy mildew-resistant spinach plants according to claim 1, wherein the cells are resistant to at least the races of downy mildew Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15, Pfs16, Pfs17, Pfs18, Pfs19 and UA1014 strains.

5. The cells of the downy mildew-resistant spinach plant according to claim 1, wherein, In at least one allele on chromosome 4, The SNP identified on chr4_8488603 is cytosine. The SNP identified on chr4_8494600 is thymine, or The SNP identified on chr4_8510715 is guanine.

6. The cells of the downy mildew-resistant spinach plant according to claim 1, wherein, In at least one allele on chromosome 4, The SNP identified on chr4_7962907 is guanine. The SNP identified on chr4_8152986 is cytosine. The SNP identified on chr4_8190990 is guanine. The SNP identified on chr4_8488603 is cytosine. The SNP identified on chr4_8494600 is thymine. The SNP identified on chr4_8510715 is guanine, or The SNP identified on chr4_8617232 is guanine.

7. The cells of the downy mildew-resistant spinach plant according to claim 1, wherein, The downy mildew-resistant spinach plants are derived from interspecific hybrids of spinach and cultivated spinach.

8. Cells of the downy mildew-resistant spinach plant according to claim 1, which have downy mildew resistance derived from the plant specified by accession number FERM BP-22404 (TNKH-1 strain).

9. Cells of the downy mildew-resistant spinach plant according to claim 1, having downy mildew resistance derived from the plant specified by accession number FERM BP-22405 (TNKH-2 strain).

10. The cells of the downy mildew-resistant spinach plant according to claim 1, wherein the spinach has at least one downy mildew resistance gene other than the RTM-1 gene.

11. A cell of a downy mildew-resistant spinach plant that cannot develop into a whole plant, which is a downy mildew-resistant spinach plant defined by accession number FERM BP-22404 (TNKH-1 strain), a hybrid plant obtained using the downy mildew-resistant spinach plant as a parent, or a progeny thereof.

12. A cell of a downy mildew-resistant spinach plant that cannot develop into a whole plant, which is a downy mildew-resistant spinach plant defined by accession number FERM BP-22405 (TNKH-2 strain), a hybrid plant obtained from said downy mildew-resistant spinach plant as a parent, or a progeny thereof.

13. A method for predicting downy mildew resistance in spinach plants, wherein, The genotypes of SNPs on chromosome 4 from chr4_8488603 to chr4_8510715 in the tested spinach plants were investigated, and in at least one allele, The SNP identified on chr4_8488603 is cytosine. The SNP identified on chr4_8494600 is thymine, or In the case where the SNP identified on chr4_8510715 is guanine. The tested spinach plants are predicted to have a high probability of being resistant to downy mildew.

14. A method for screening downy mildew-resistant spinach plants, wherein, The genotypes of SNPs on chromosome 4 from chr4_8488603 to chr4_8510715 in the tested spinach plants were investigated, and in at least one allele, The SNP identified on chr4_8488603 is cytosine. The SNP identified on chr4_8494600 is thymine, or In the case where the SNP identified on chr4_8510715 is guanine. The tested spinach plant was selected as a downy mildew-resistant spinach plant.

15. A method for manufacturing a downy mildew-resistant spinach plant, comprising the following steps: The first hybridization step involves crossing spinach plants carrying the RTM-1 gene with any other spinach plant. The RTM-1 gene is a gene located on chromosome 4 of the four-stamen spinach cultivar CGN25466:MGK 01, ranging from chr4_7962907 to chr4_8617232. Among them, at least one allele on chromosome 4 contains a segment in the range of chr4_8488603 to chr4_8510715 on chromosome 4 containing the four-stamen spinach strain CGN25466:MGK 01. The second hybridization step involves self-crossing, backcrossing, or interspecific or intraspecific hybridization with spinach plants that are different from the parents used in the first hybridization step, using the F1 individuals obtained through the first hybridization step to obtain segregating populations. as well as In the selection step, spinach plants with the RTM-1 gene are selected from the isolated population.

16. The method for manufacturing downy mildew-resistant spinach plants according to claim 15, wherein, The spinach plant with the RTM-1 gene is either spinach with four stamens or downy mildew resistant spinach plant according to any one of claims 1 to 13.

17. The method for manufacturing downy mildew-resistant spinach plants according to claim 15, wherein, The parent plant used in any of the spinach plants, or in the interspecific or intraspecific hybridization in the second hybridization step, is a spinach plant with at least one downy mildew resistance gene other than the RTM-1 gene.

18. A DNA marker for selecting spinach plants possessing the RTM-1 gene, comprising one or more of the following groups: SNPs identified on chr4_8488603, chr4_8494600, and chr4_8510715 of spinach plants, and SNPs genetically strongly linked to these SNPs, wherein... The RTM-1 gene is a gene located on chromosome 4 of the four-stamen spinach variety CGN25466:MGK 01, ranging from chr4_7962907 to chr4_8617232.

19. A kit for selecting spinach plants with the RTM-1 gene, comprising one or more primer sets selected from the group consisting of: Primer set used to identify the genotype of the SNPs identified on chr4_8488603 Primer set used to identify the genotype of SNPs identified on chr4_8494600, and The primer set used to identify the genotype of the SNP identified on chr4_8510715, wherein The RTM-1 gene is a gene located on chromosome 4 of the four-stamen spinach variety CGN25466:MGK 01, ranging from chr4_7962907 to chr4_8617232.

20. The kit according to claim 19, wherein, The primer set used to identify the genotype of the SNP identified on chr4_8488603 consists of the following primers: primers consisting of the base sequence represented by sequence number 13, primers consisting of the base sequence represented by sequence number 14, and primers consisting of the base sequence represented by sequence number 15. The primer set used to identify the genotype of the SNP identified on chr4_8494600 consists of the following primers: primers consisting of the base sequence represented by sequence number 16, primers consisting of the base sequence represented by sequence number 17, and primers consisting of the base sequence represented by sequence number 18. The primer set used to identify the genotype of the SNP identified on chr4_8510715 consists of the following primers: primers consisting of the base sequence represented by sequence number 19, primers consisting of the base sequence represented by sequence number 20, and primers consisting of the base sequence represented by sequence number 21.

Images