JP7357173B1 - sooty mold resistant tomato plants - Google Patents

Abstract

The present invention provides a tomato plant exhibiting resistance to sooty mildew. The present invention provides a tomato plant containing a polynucleotide consisting of the following polynucleotide (G1), (G2), or (G3) or a partial sequence thereof as a genomic region conferring sooty mold resistance. (G1) Polynucleotide consisting of a specific base sequence; (G2) Consisting of a base sequence in which 1 to 6011 bases are deleted, substituted, inserted and/or added to the base sequence of (G1); Polynucleotide conferring disease resistance; (G3) A polynucleotide comprising a base sequence having 90% or more identity to the base sequence of (G1) and conferring resistance to sooty mold disease. [Selection diagram] None

Description

IPOD FERM P-22459

Application of Article 30, Paragraph 2 of the Patent Act (Open to the public through meetings) Date: February 6, 2023 Meeting name and venue: Nango Tomato Producers Association General Meeting and 60th Anniversary Meeting Mikurairi Exchange Hall (Minamiaizu, Minamiaizu District, Fukushima Prefecture) Machidajima Miyamoto Higashi 22) Publisher: Hiroshi Fukunaga

Application of Article 30, Paragraph 2 of the Patent Act (Disclosure through publications) Publication date: November 23, 2022 Publication: Agriculture and Horticulture Winter 2022 Issue, Vol. 77, No. 4, pp. 11-14 Publisher: Seibundo Shinkosha Co., Ltd.

Article 30, Paragraph 2 of the Patent Act applies (Disclosure through sales 1) Sale date: April 20, 2022 to March 2, 2023 Sale location: JA Aizu Yotsuba Nango Tomato Sorting Plant (Minamiaizu Town, Minamiaizu District, Fukushima Prefecture) Miyatoko Kawakubo 22-1) Publisher: Growing Mac Co., Ltd.

Application of Article 30, Paragraph 2 of the Patent Act (Publication through sale 2) Sale date: April 11, 2022 to June 9, 2022 Publisher: Belk Fukushima Co., Ltd.

Article 30, Paragraph 2 of the Patent Act applies (Disclosure through sales 3) Sale date: March 9, 2022 to May 24, 2022 Publisher: TS Nursery Co., Ltd.

Application of Article 30, Paragraph 2 of the Patent Act (Publication through sales 4) Sales date: April 25, 2022 to June 14, 2022 Publisher: Takii Seed Co., Ltd.

The present disclosure relates to sooty mold resistant tomato plants.

Pseudocercospora fuligena , a type of filamentous fungus, is the pathogen of sooty mold on tomato plants. The sooty mold disease exhibits disease symptoms that are very similar to leaf mold diseases. In the above-mentioned sooty mold, the pathogen is mainly transmitted by wind pollination and causes the disease on tomato leaves. In the case of sooty mold, when the onset of the disease is severe, a large number of lesions appear, and the entire plant weakens and dies (Non-Patent Document 1). For this reason, when the sooty mold spreads, it becomes impossible to obtain fruit.

As a method for controlling the sooty mold, it has been known that the sooty mold can be controlled at the same time by spraying the chemical for controlling the leaf mold. However, with the spread of leaf mold-resistant varieties, the number of chemical sprays has decreased, and the occurrence of sooty mold has become more prominent.

Hartman, Glen L. and T. C. Wang. “Black leaf mold development and its effect on tomato yield.”, Plant Disease, 1992, vol. 76, pages 462-465

Therefore, there is a need for tomato plants that are resistant to sooty mildew.

Therefore, the present disclosure aims to provide tomato plants that exhibit resistance to sooty mold.

To achieve the above object, the tomato plants of the present disclosure are resistant to sooty mold.

The method for producing sooty mildew-resistant tomato plants of the present disclosure (hereinafter referred to as the "production method") includes the following steps (a) and (b):
(a) Crossing the sooty mold resistant tomato plant of the present disclosure with another tomato plant;
(b) A step of selecting sooty mildew-resistant tomato plants from the tomato plants obtained in step (a) or their progeny.

The method of imparting sooty mold resistance to tomato plants of the present disclosure (hereinafter referred to as the "imparting method") includes an introduction step of introducing a genomic region imparting sooty mold resistance into tomato plants.

The detection method of the present disclosure is a method for detecting sooty mold resistance in tomato plants (hereinafter referred to as "detection method"), which detects a genomic region that confers sooty mold resistance on a test tomato plant. Includes a detection step.

The screening method of the present disclosure is a method for screening tomato plants resistant to sooty mildew (hereinafter referred to as "screening method"), comprising:
The method includes a selection step of selecting tomato plants containing a genomic region that confers sooty mold resistance from the test tomato plants as sooty mold resistant tomato plants.

According to the present disclosure, tomato plants exhibiting resistance to sooty mold can be provided.

<Definition>
As used herein, "tomato plant" means a plant classified as a tomato species of the genus Solanum of the Solanaceae family. As a specific example, the tomato plants include, for example, S. lycopersicum , S. peruvianum , S. arcanum Peralta , S. chilense , S. corneliomulleri , S. huaylasense Peralta , S. cheesmaniae (L.Riley) Fosberg, S. chmielewskii , S. galapagense SCDarwin & Peralta, S. habrochaites , S. neorickii , S. pennelli , S. pimpinellifolium , etc., and preferred is S. lycopersicum , which can be easily crossed. In this specification, the tomato plant is, for example, a cultivated tomato plant. The tomato plant for cultivation can also be referred to as, for example, a cultivar of tomato plants.

As used herein, "sooty mold" refers in particular to an airborne disease caused by a filamentous fungus ( Pseudocercospora fuligena ). The sooty mold disease is known to cause the development of lesions and death in infected plants.

As used herein, "pathogen of sooty mold" or "pathogen" (hereinafter also referred to as "sooty mold") refers to a bacterium classified as Pseudocercospora fuligena.

As used herein, "resistance to sooty mold" refers to the ability to inhibit or suppress the occurrence of disease caused by infection with sooty mold and/or the progression of the disease. As a specific example, the above-mentioned "sooty mold resistance" means, for example, the absence of disease caused by infection with sooty mold fungi, and/or the halting, inhibition, suppression, reduction, or delay of the progression of the disease that has occurred. . The sooty mold resistance can also be referred to as sooty mold resistance or sooty mold disease resistance.

As used herein, "a genomic region that confers sooty mold resistance" refers to a quantitative trait loci (QTL) or a chromosomal region that confers sooty mold resistance. The quantitative trait locus generally refers to a chromosomal region involved in the expression of a quantitative trait.

As used herein, "plant body" or "plant" refers to an individual plant, indicating the whole plant.

As used herein, "plant body part" or "plant part" means a part of an individual plant.

Hereinafter, the present disclosure will be described using examples, but the present disclosure is not limited to the following examples and the like, and can be implemented with arbitrary changes. Further, each description in the present disclosure can be used mutually unless otherwise specified. Note that in this specification, when the expression "~" is used, it is used in a meaning that includes numerical values or physical values before and after it. Furthermore, in this specification, the expression "A and/or B" includes "A only", "B only", and "both A and B".

<Sooty mold resistant tomato plants>
In certain embodiments, the present disclosure provides tomato plants that exhibit resistance to sooty mold. The tomato plants of the present disclosure have sooty mold resistance. According to the present disclosure, tomato plants having resistance to sooty mold can be provided.

The tomato plant may be a pure tomato plant of each plant species, or may be a hybrid between the target tomato plant and a closely related species of the target tomato plant. Examples of the closely related species include each plant species of the tomato plant.

In the tomato plants of the present disclosure, resistance to sooty mildew may be conferred by the presence of a genomic region that confers resistance. Hereinafter, the genomic region conferring resistance to sooty mold will also be referred to as a sooty mold resistance marker.

In the tomato plant of the present disclosure, when the sooty mold resistance is conferred by the presence of a genomic region that confers sooty mold resistance, the tomato plant of the present disclosure is provided with a genomic region that confers sooty mold resistance. including. The genomic region that confers sooty mold resistance is preferably a genomic region that confers sooty mold resistance on chromosome 1.

In the present disclosure, the genomic region conferring sooty mold resistance includes, for example, the polynucleotide (G) below.
(G) Any of the following polynucleotides (G1), (G2), or (G3):
(G1) a polynucleotide consisting of the base sequence of SEQ ID NO: 1 or the base sequence of positions 457321 to 517433 of SEQ ID NO: 1;
(G2) A polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, inserted and/or added to the base sequence of (G1), and exhibits sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 80% or more identity to the base sequence of (G1) and expressing resistance to sooty mold.

The base sequence of SEQ ID NO: 1 can be obtained, for example, from the seeds of a tomato plant deposited under accession number FERM P-22459, which will be described later. More specifically, the base sequence of SEQ ID NO: 1 can be obtained from the first chromosome of the seeds. can.

In the polynucleotide of (G2), the one or several is, for example, 1 to 12022, 1 to 9016, 1 to 6011, 1 to 3005, 1 to 2402, 1 to 1803, 1 to 1202 pieces, 1-601 pieces, 1-541 pieces, 1-480 pieces, 1-420 pieces, 1-300 pieces, 1-240 pieces, 1-180 pieces, 1-120 pieces, 1-60 pieces, 1- 40 pieces, 1 to 20 pieces, 1 to 10 pieces, 1 to 5 pieces, 1 to 4 pieces, 1 to 3 pieces, 1 or 2 pieces, and 1 piece.

In the polynucleotide (G3), the identity is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99%. 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, Or 99.9% or more.

The polynucleotide (G1) may be a polynucleotide consisting of a partial sequence of the base sequence of SEQ ID NO: 1. An example of the polynucleotide consisting of the partial sequence is a polynucleotide consisting of the base sequence 457321st to 517433rd in the base sequence of SEQ ID NO: 1. In this case, in the description of the polynucleotides (G2) and (G3), the "base sequence of (G1)" is replaced with "the base sequence of SEQ ID NO: 1" in the polynucleotides of (G2) and (G3). , 457321st to 517433rd base sequence" and the explanation can be used.

The polynucleotide consisting of the partial sequence is within a range that can impart sooty mold resistance to a tomato plant containing the polynucleotide consisting of the partial sequence, for example. The lower limit, upper limit, or range of the length of the polynucleotide consisting of the partial sequence is not particularly limited, and is, for example, a range that can impart soot mold resistance to tomato plants containing the polynucleotide consisting of the partial sequence. The lower limit of the length of the polynucleotide consisting of the partial sequence is, for example, 1000 bases or more, 2500 bases or more, 5000 bases or more, 7500 bases or more, 10000 bases or more, 12500 bases or more, 15000 bases or more , 17,500 bases or more, 20,000 bases or more, 22,500 bases or more, 25,000 bases or more, 27,500 bases or more, 30,000 bases or more, 32,500 bases or more, 35,000 bases or more, 37,500 bases or more, 40,000 bases or more , 42,500 bases or more, 45,000 bases or more, 47,500 bases or more, 50,000 bases or more, 52,500 bases or more, 55,000 bases or more, or 57,500 bases or more. The upper limit of the length of the polynucleotide consisting of the partial sequence is, for example, 60,113 bases or less, 60,000 bases or less, 57,500 bases or less, 55,000 bases or less, 52,500 bases or less, 50,000 bases or less, 47,500 bases or less , 45,000 bases or less, 42,500 bases or less, 40,000 bases or less, 37,500 bases or less, 35,000 bases or less, 32,500 bases or less, 30,000 bases or less, 27,500 bases or less, 25,000 bases or less, 22,500 bases or less , 20,000 bases or less, 17,500 bases or less, 15,000 bases or less, 12,500 bases or less, 10,000 bases or less, 7,500 bases or less, or 5,000 bases or less. The length range of the polynucleotide consisting of the partial sequence is, for example, 1000 to 60113 bases, 2500 to 60000 bases, 5000 to 57500 bases, 7500 to 55000 bases, 10000 to 52500 bases, 12500 to 50000 bases. length, 15,000 to 47,500 bases in length, 17,500 to 45,000 bases in length, 20,000 to 42,500 bases in length, 22,500 to 40,000 bases in length, 25,000 to 37,500 bases in length, 27,500 to 35,000 bases in length, or 30,000 to 32,500 bases in length.

The genomic region conferring sooty mold resistance of the present disclosure may be an endogenous genomic region or an exogenous genomic region. The endogenous genomic region is, for example, a genomic region containing mutations in the tomato plant. When the genomic region containing sooty mold resistance is an endogenous genomic region, the genomic region conferring sooty mold resistance can be introduced, for example, by cross-introduction. The foreign genome region is, for example, a genome region introduced by gene recombination technology, genome editing technology, or the like. Introduction of a foreign genomic region into a tomato plant using the genetic recombination technology or the genome editing technology can be carried out by a common method in this field. For example, by constructing an artificial chromosome for plants, It can be produced by introducing into a target tomato plant and introducing it using chromosomal recombination (Reference Document 1 below).
Reference 1: Sun YD, Luo WR, Sun SY, Ni L. Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.) using the expansin 10 (CsEXP10) gene. Genet Mol Res. 2015 Dec 8;14(4): 16215-21. doi: 10.4238/2015.December.8.11. PMID: 26662414.

The sooty mold resistant tomato plants of the present disclosure exhibit resistance to sooty mold. In the present disclosure, the sooty mildew resistance of tomato plants can be expressed by the disease severity calculated from the disease severity index by evaluating the disease severity of the tomato plants according to the method of Example 1 described below. The calculation of the disease severity by this method can refer to the explanation in Example 1 described below. For example, if the disease severity is 1 or less, it can be evaluated as disease resistance, and if the disease severity is more than 1, it can be evaluated as disease susceptibility. When determining the sooty mold resistance based on the disease severity, the disease severity is the average disease severity of a plurality of tomato plants. The number of tomato plants (survey individuals or survey populations) used for the judgment of sooty mold resistance is, for example, a number that allows statistical testing with tomato plants susceptible to sooty mold, and For example, 40 to 50 stocks.

Examples of the tomato plants of the present disclosure include the tomato plant (S. lycopersicum) deposited under accession number FERM P-22459 or its progeny. The progeny line has, for example, the genomic region that confers the sooty mold resistance. Deposit information is shown below.
Type of deposit: Domestic deposit Name of depositary institution: National Institute of Technology and Evaluation, Patent Organism Depositary Center Address: Japan Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818 Accession number: FERM P- 22459
Display for identification: Takii31
Date of receipt: August 15, 2022

The tomato plants of the present disclosure can also be produced, for example, by introducing into a tomato plant the genomic region that confers sooty mold resistance. The method of introducing the genomic region imparting sooty mold resistance into the tomato plant is not particularly limited, and includes, for example, crossbreeding or embryo culture with the tomato plant of the present disclosure, or conventionally known genetic engineering techniques. can give. Examples of the genetic engineering methods include methods using genetic recombination technology and genome editing technology, as described above. The genomic region imparting sooty mold resistance to be introduced can be exemplified by the aforementioned genomic region conferring sooty mold resistance. When introduced by crossing with a tomato plant of the present disclosure, the tomato plant of the present disclosure used for the cross includes, for example, the genomic region conferring sooty mold resistance in a homozygous form or on both chromosomes. is preferred.

The polynucleotide (G1) is contained, for example, in the seeds of the tomato plant identified by the accession number FERM P-22459. Therefore, the polynucleotide (G1) can be subjected to cross-introgression or gene introgression, for example, by crossing with a tomato plant obtained from the seeds of the tomato plant identified by the accession number FERM P-22459 or its progeny line. may be done. Furthermore, in a tomato plant containing the polynucleotide (G2) or (G3), for example, in a tomato plant containing the polynucleotide (G1), the genomic region containing the polynucleotide (G1) is It can be produced by introducing position-specific mutations using genome editing technology.

Regarding the tomato plants of the present disclosure, characteristics other than sooty mold resistance, such as phenotypic and ecological characteristics, are not particularly limited.

Tomato plants of the present disclosure may also have other resistances.

<Part of tomato plant resistant to sooty mold>
In another aspect, the present disclosure provides parts of tomato plants that are resistant to sooty mold. The present disclosure is a portion of the sooty mildew resistant tomato plant of the present disclosure.

The plant parts may be, for example, plant cells, plant protoplasts, plant cell cultures or tissue cultures capable of regenerating plant bodies, plant callus, plant clumps, plant cells isolated from plants or plant parts. , meristematic cell, flower, petal, flower bud, pistil, corolla, pollen, leaf, petiole, leaf pith, cotyledon, ovary, embryo, ovule, hypocotyl, embryo sac, egg cell, microspore (microspore), anther, pistil, flower, ovary, cutting, scion, rootstock, root, root tip (root tip), seed, fruit, trunk, stalk, seedling, etc. It will be done. The plant parts may be any organ, tissue, cell, vegetative propagation body, etc., for example. Examples of the organs include petals, corollas, flowers, leaves, seeds, fruits, stems, roots, and the like. The tissue is, for example, a part of the organ. The pollen may be mature pollen or immature pollen.

Parts of the plant of the present disclosure can be produced, for example, by obtaining a desired part of the tomato plant of the present disclosure.

<Method for producing tomato plants resistant to sooty mildew>
In another aspect, the present disclosure provides a method for producing tomato plants that are resistant to sooty mold. The method for producing tomato plants resistant to sooty mold (hereinafter also referred to as "production method") of the present disclosure includes the following steps (a) and (b). The production method of the present disclosure may include only the step (a) below.

(a) Crossing (crossing) the sooty mold resistant tomato plant of the present disclosure with another tomato plant;
(b) A step of selecting sooty mildew-resistant tomato plants from the tomato plants obtained in step (a) or their progeny.

According to the production method of the present disclosure, by crossing the tomato plant of the present disclosure with another tomato plant, a tomato plant exhibiting resistance to sooty mold can be obtained from the resulting progeny line. According to the production method of the present disclosure, tomato plant varieties that are resistant to sooty mold can be bred using any tomato plant. Therefore, the production method of the present disclosure can also be called, for example, a breeding method or a growing method.

In the production method of the present disclosure, the description of the sooty mold resistance marker can refer to the description of the genomic region that confers sooty mold resistance in the tomato plant of the present disclosure.

In step (a), the sooty mildew-resistant tomato plant used as the first parent may be the tomato plant of the present disclosure. The tomato plant of the present disclosure is preferably, for example, the tomato plant deposited with the aforementioned accession number FERM P-22459 or its progeny. In step (a), the sooty mildew-resistant tomato plant used as the first parent can also be obtained, for example, by the screening method of the present disclosure described below. For this reason, the sooty mildew-resistant tomato plants may be prepared by selecting, for example, the following step (x) from test tomato plants, prior to the step (a).
(x) Selecting the sooty mildew-resistant tomato plants of the present disclosure from the test tomato plants.

In the step (x), the selection of the sooty mold resistant tomato plants can be carried out by, for example, directly and/or indirectly evaluating (detecting) the sooty mold resistance of the test tomato plants, and This can be carried out by selecting tomato plants resistant to rot. For the direct evaluation (detection), the description of the evaluation method using the severity of sooty mold resistance described above can be referred to.

When carried out by the indirect evaluation, in step (x), for example, the presence or absence of the sooty mold resistance marker can be used as an index to select sooty mold resistant tomato plants. Specifically, in the step (x), the selection of the sooty mold resistant tomato plants involves the selection of the sooty mold resistance marker, that is, the genomic region that confers the sooty mold resistance. This can be said to be the selection of tomato plants that have the following characteristics. Therefore, the step (x) can be performed, for example, by the steps (x1) and (x2) below.

(x1) Detection step of detecting the presence or absence of the sooty mold resistance marker on the chromosome of the test tomato plant (x2) Due to the presence of the sooty mold resistance marker, the test tomato plant is Selection process for selecting tomato plants resistant to sooty mildew

As described above, the selection in the step (x) is, for example, selection of tomato plants having the sooty mold resistance marker, and specifically, the selection of tomato plants having the sooty mold resistance marker is performed on the test tomato plants. By detecting disease resistance markers, the resistant tomato plants can be selected.

The detection of the resistance marker in the step (x1) includes, for example, the detection of the sooty mold resistance marker that confers sooty mold resistance, that is, the sooty mold resistance marker, as described in the tomato plant of the present disclosure. This can be done by detecting tomato plants that have genomic regions that confer resistance. Detection of the genomic region conferring resistance to sooty mold will be described later.

In the step (x2), for example, when the sooty mold resistance marker is present on one chromosome of a pair of chromosomes, the test tomato plant may be selected as the resistant tomato plant. If the sooty mold resistance marker is present on both chromosomes of a pair of chromosomes, the test tomato plant may be selected as the resistant tomato plant, but the latter is preferred.

Regarding the selection in the step (x), the following specific examples will be given, but the present disclosure is not limited thereto.

The detection of the genomic region conferring sooty mold resistance in step (x1) can be carried out, for example, by detecting the polynucleotide of (G) or a polynucleotide consisting of a partial sequence thereof. In the following description, unless otherwise specified, in the present disclosure, the description of the polynucleotide in (G) above can be referred to in the description of the polynucleotide consisting of a partial sequence of the polynucleotide in (G). Furthermore, in the following description, unless otherwise specified, in the present disclosure, a polynucleotide consisting of a partial sequence of the polynucleotide (G) may be used instead of the polynucleotide (G). Specifically, in the step (x1), the base sequence of the genome (genomic DNA) of the test tomato plant is decoded. Then, in step (x1), the obtained base sequence is compared with the base sequence of the polynucleotide (G) to detect whether or not the base sequences match. The base sequence can be decoded using, for example, a sample containing the genome (genomic DNA) of the tomato plant to be tested, a sequencing reagent, and a sequencer. As a specific example, the decoding in step (x1) can be determined by performing scaffolding using a single molecule sequencer, for example, with reference to Reference 2 below. Further, the comparison of the base sequences can be performed using, for example, base sequence analysis software (eg, the above-mentioned BLAST, etc.). In step (x1), a tomato plant having a base sequence in its genomic DNA that matches the polynucleotide (G) is identified (identified or detected) as a test tomato plant having the polynucleotide (G). can. In the step (x1), the detection may be performed on the entire genome of the test tomato plant, or may be performed on a part of the genome. Furthermore, the detection may be performed on all chromosomes or on some chromosomes. In step (x2), for example, the test tomato plant containing the polynucleotide (G) is selected as the tomato plant of the present disclosure or its progeny line, that is, the tomato plant exhibiting sooty mold resistance. .
Reference 2: Kenta Shirasawa et.al., “Chromosome-scale genome assembly of Eustoma grandiflorum, the first complete genome sequence in family Gentianaceae, Ryohei Arimoto”, bioRxiv 2021.09.09.459690; doi: https://doi.org/10.1101 /2021.09.09.459690

The detection of the genomic region conferring sooty mold resistance in step (x1) can be carried out, for example, by specifically detecting the polynucleotide of (G). In this case, in the step (x1), the genome imparting the sooty mildew resistance is detected in the test tomato plant by detecting a molecular marker that can specifically detect the polynucleotide (G). Detect areas. The polynucleotide (G) may be detected, for example, by using a molecular marker that can specifically detect the polynucleotide (G) designed in advance, or by detecting the polynucleotide (G) prior to the step (x1). , the molecular marker may be designed. The types of molecular markers include, for example, SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP (restriction fragment length polymorphism) markers, microsatellite markers, and SCAR (sequence-characterized amplified region) markers. , or a CAPS (cleaved amplified polymorphic sequence) marker. In the present disclosure, one type of molecular marker may be used, or two or more types may be used in combination. When using a SNP marker as the molecular marker, the SNP marker may be, for example, one SNP as the SNP marker, or a combination of two or more SNPs as the SNP marker, that is, a SNP marker set. . The molecular marker is preferably a SNP marker.

The molecular marker can be designed, for example, by a common design method in this technical field. The molecular marker is, for example, a base or base sequence specific to the base sequence of the polynucleotide (G), using the base sequence of the polynucleotide (G) and a reference sequence with which the base sequence is compared. This can be done by specifying the As a specific example, the molecular marker may be designed using, for example, the polynucleotide (G) and, as the reference sequence, the nucleotide sequence information (SL3.0) of the Heinz 1706 genome corresponding to the polynucleotide (G). A molecular marker can be designed by comparing the two and identifying different bases or base sequences between the two. The corresponding Heinz 1706 genome nucleotide sequence information is, for example, the nucleotide sequence 678023 to 1337393 of chromosome 1 in SL3.0.

The molecular marker can be designed, for example, with reference to References 3 to 5 below. As a specific example, first, using the base sequence of SEQ ID NO: 1 (BLM_Chr) as a reference sequence, base sequence information obtained by short read sequencing or the like is mapped to detect polymorphisms such as SNPs. The molecular marker can be set, for example, by creating a marker based on the obtained polymorphism.
Reference 3: Hamilton JP et al., “Single Nucleotide Polymorphism Discovery in Cultivated Tomato via Sequencing by Synthesis.”, 2012, The Plant Genome 5.
Reference 4: Sim SC et al., “Development of a Large SNP Genotyping Array and Generation of High-Density.”, 2012, Genetic Maps in Tomato. PLoS ONE 7 (7)
Reference 5: Blanca J et al., “Variation Revealed by SNP Genotyping and Morphology Provides Insight into the Origin of the Tomato.”, 2012, PLoS ONE 7 (10)

The chromosome for detecting the presence or absence of the genomic region conferring sooty mold resistance is preferably chromosome 1.

The tomato plants of the present disclosure can be produced, for example, by the application method described below. Therefore, the production method of the present disclosure may include the following step (y). For the following step (y), for example, the explanation of the application method of the present disclosure described below can be referred to.
(y) Step of producing the sooty mold resistant tomato plant of the present invention from the target tomato plant (creation step)

Further, in the above step (a), the tomato plant used as the other parent is not particularly limited, and may be, for example, a tomato plant that has known sooty mold resistance or does not have sooty mold resistance. , with or without other resistances, or the sooty mildew-resistant tomato plants of the present disclosure.

In step (a), the method of crossing the sooty mildew-resistant tomato plant with the other tomato plant is not particularly limited, and any known method can be employed.

In step (b), the target for selecting sooty mildew-resistant tomato plants may be, for example, the tomato plant obtained in step (a), or the progeny line obtained from the tomato plant. . Specifically, the target may be, for example, the F1 tomato plant obtained by the cross in step (a), or its progeny line. The progeny line may be, for example, a self-cross progeny or a backcross progeny of the F1 tomato plant obtained by the cross in step (a), or may be a cross between the F1 tomato plant and another tomato plant. It may be a tomato plant obtained by.

In the step (b), selection of tomato plants resistant to sooty mold can be carried out, for example, by directly or indirectly confirming sooty mold resistance.

In the step (b), the direct confirmation can be performed by, for example, evaluating the sooty mildew resistance of the F1 tomato plants obtained or their progeny lines based on the disease severity described above. Specifically, for example, the F1 tomato plant or its progeny line is inoculated with the sooty mold fungus, and the sooty mold resistance is evaluated based on the aforementioned disease severity. In the following cases, if the disease resistance or disease severity exceeds 1, it can be evaluated as susceptibility. In this case, for example, the F1 tomato plant or its progeny that meets the predetermined criteria can be selected as a sooty mildew-resistant tomato plant.

Moreover, in the step (b), the selection by indirect confirmation can be performed, for example, by the steps (b1) and (b2) below.
(b1) Detection step of detecting the presence or absence of a sooty mold resistance marker on the chromosome of the tomato plant obtained in step (a) or its progeny (b2) Presence of the sooty mold resistance marker A selection step of selecting the tomato plant obtained in step (a) or its progeny line as a sooty mildew-resistant tomato plant by

Selection by indirect confirmation of sooty mold resistant tomato plants in step (b) is, for example, the same as the method described in step (x), and includes detection of the presence or absence of the sooty mold resistant marker. This can be done by

In the production method of the present disclosure, it is preferable to further grow the sooty mildew-resistant tomato plant selected in the step (b).

In this way, the tomato plant or its progeny line whose resistance to sooty mold has been confirmed can be selected as a tomato plant resistant to sooty mold.

The production method of the present disclosure may further include a seed collecting step of collecting seeds from the progeny line obtained by crossing.

Although the production method of the present disclosure includes the step (a) and the step (b), the present disclosure is not limited thereto and may include only the step (a). In this case, the tomato plant of the present disclosure used in step (a) preferably contains the genome region containing the resistance in a homozygous form or in both chromosomes.

<Screening method for tomato plants resistant to sooty mold>
In another aspect, the disclosure provides a method of screening tomato plants for resistance to sooty mold. The screening method of the present disclosure is a method for screening tomato plants resistant to sooty mold, in which tomato plants containing a genomic region that confers resistance to sooty mold are selected from test tomato plants to produce sooty mold resistant tomato plants. Includes a selection process to select candidates. According to the screening method of the present disclosure, tomato plants that may exhibit resistance to sooty mold can be screened. Furthermore, according to the screening method of the present disclosure, parents for producing tomato plants resistant to sooty mold by crossing can be screened.

When selecting a tomato plant having a genomic region conferring sooty mold resistance as a sooty mold resistant tomato plant, for example, in the selection step, the genomic region conferring sooty mold resistance is selected as a sooty mold resistant tomato plant. A test tomato plant containing the polynucleotide (G) is selected as a sooty mildew-resistant tomato plant. For the selection, for example, the explanation of step (x) in the method for producing a sooty mildew-resistant tomato plant of the present disclosure can be referred to.

In the screening method of the present disclosure, the selection step can refer to the description of the indirect selection (selection using indirect evaluation) in the step (x) or the step (x1).

<Method for detecting sooty mold resistance in tomato plants>
In another aspect, the disclosure provides a method of detecting resistance to sooty mold in tomato plants. The detection method of the present disclosure is a method for detecting sooty mold resistance in tomato plants, and includes a detection step of detecting a genomic region that confers sooty mold resistance on a test tomato plant. According to the detection method of the present disclosure, it is possible to detect whether a target tomato plant exhibits resistance to sooty mold. The method for detecting sooty mold resistance in tomato plants of the present disclosure can also be referred to as, for example, a method for screening for sooty mold resistance in tomato plants.

Detection of the sooty mold resistant tomato plant is, for example, indirect selection in the step (x) in the method for producing a sooty mold resistant tomato plant of the present disclosure, that is, the explanation of the step (x1). can be used.

<Method for imparting sooty mold resistance to tomato plants>
In another aspect, the present disclosure provides a method of conferring sooty mold resistance to tomato plants. The method of imparting sooty mold resistance to a tomato plant of the present disclosure is characterized by including an introduction step of introducing a genomic region imparting sooty mold resistance into a tomato plant. According to the method for imparting sooty mold of the present disclosure, resistance to sooty mold can be imparted to tomato plants by introducing a genomic region imparting sooty mildew resistance, that is, the sooty mildew resistance marker of the present disclosure.

In the introduction step, the method for introducing the genomic region conferring resistance to sooty mold is not particularly limited. Examples of the introduction method include crossing with the resistant tomato plant, embryo culture, and conventionally known genetic engineering techniques. The genomic region that confers sooty mold resistance to be introduced can be exemplified by the genomic region that confers sooty mold resistance described above in the description of the tomato plants of the present disclosure. When introduced by crossing with the resistant tomato plant, the resistant tomato plant preferably contains the genome region containing the resistance in a homozygous form or on both chromosomes, for example.

When introducing the genomic region imparting sooty mold resistance, the introduction step may be carried out by introducing an expression vector and/or an artificial chromosome containing the genomic region. The expression vector and artificial chromosome can be introduced into a target tomato plant by, for example, a polyethylene glycol method, an electroporation method, an Agrobacterium-mediated method, a particle gun method, or the like. The target tomato plant may be, for example, a plant cell, a callus, a plant tissue, or an individual plant.

When introducing the genomic region that confers sooty mold resistance, the introduction step may be carried out by introducing a nucleic acid containing the genomic region (donor nucleic acid) and a genome editing unit. In this case, at the 5' end of the nucleic acid, a 5' homology arm having homology with the base sequence on the 5' end side of the introduction position in the chromosome into which the nucleic acid is introduced is arranged, and at the 3' end of the nucleic acid, a 5' homology arm is arranged. , a 3' homology arm having homology with the base sequence on the 3' end side of the introduction position in the chromosome into which the nucleic acid is introduced is arranged. Further, the genome editing unit system is configured to be capable of cutting the introduction site. As a result, after the introduction step, in the introduced tomato plant, the genomic DNA is cleaved at the introduction position, and the nucleic acid can be integrated into the chromosome by homologous recombination. The introduction positions may be two. In this case, the introduction position can be designed at the upstream end where the base sequence is exchanged and the downstream end of the chromosome into which the nucleic acid is introduced. Further, the 5' homology arm has homology with the base sequence on the 5' end side of the introduction position at the upstream end, and the 3' homology arm has homology with the base sequence on the 3' end side of the introduction position at the downstream end. It can be designed to have homology. By providing two introduction positions, the region between the two introduction positions in the chromosome is excised, and the nucleic acid can be introduced instead of this region.

Examples of the genome editing unit include ZFN (Zinc Finger Nuclease), TALEN (Transcription Activator-Like Effector Nuclease), CRISPR-CAS system, and the like. The CRISPR-CAS system includes, for example, a guide strand and a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme. The CRISPR enzymes include, for example, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Cs m3 , Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, etc. can be given . Examples of the nucleic acids include crRNA and tracrRNA, or single-stranded nucleic acids in which these are linked via a linker. In this case, in the nucleic acid, for example, a base sequence that anneals with a target sequence in crRNA is designed to be complementary to a base sequence encoding the target sequence.

The nucleic acid (donor nucleic acid) and genome editing unit can be introduced into a target tomato plant by, for example, a polyethylene glycol method, an electroporation method, an Agrobacterium-mediated method, a particle gun method, or the like. The target tomato plant may be, for example, a plant cell, a callus, a plant tissue, or an individual plant.

In the introduction step, when the expression vector, the artificial chromosome, or the nucleic acid is introduced, the imparting method of the present disclosure further includes selecting tomato plants into which the expression vector, the artificial chromosome, or the nucleic acid has been introduced. It may also include a selection process. In this case, the selection can be carried out, for example, by detecting the introduced expression vector, artificial chromosome, or nucleic acid, and selecting tomato plants in which the expression vector, artificial chromosome, or nucleic acid has been detected.

<Sooty mold resistance marker for tomato plants>
In certain embodiments, the present disclosure provides markers that can be used as indicators of sooty mold resistance in tomato plants. The markers of the present disclosure are sooty mold resistance markers for tomato plants and include genomic regions that confer sooty mold resistance. According to the marker of the present disclosure, the presence or absence of sooty mold resistance can be detected.

Hereinafter, the present disclosure will be explained in detail using Examples, but the present invention is not limited to the embodiments described in the Examples.

[Example 1]
A new tomato plant resistant to sooty mold was confirmed to exhibit resistance to sooty mold fungi. Furthermore, a genomic region conferring novel sooty mold resistance in the sooty mold resistant tomato plants was identified.

In order to select tomato plants that are resistant to Pseudocercospora fuligena, we conducted an inoculation test using Pseudocercospora fuligena. Specifically, the inoculation test for sooty mold was conducted as follows.

(1) Test plants In order to develop new tomato plants that are resistant to sooty mold, a large number of seeds of tomato lines collected through succession breeding at the Takii Seed Co., Ltd. farm were bred and tested for sooty mold. A resistance test was conducted. Specifically, tomato plants to be used for inoculation were sown in 200-hole cell trays filled with TM-1 unfertilized potting soil (Takii Seed Co., Ltd.), and the seedlings were raised for about 2 weeks. After raising the seedlings, the tomato plants were transplanted into 55-hole cell trays filled with vegetable soil.

(2) Inoculation test As the sooty mold fungus ( Pseudocercospora fuligena ), a strain transferred from the Gifu Prefecture Agricultural Research Institute was used.

The cells were cultured on V8 agar medium at 25°C for 14 days. After the culture, the cultured mycelium was suspended in water and filtered through double gauze, and the spore concentration was adjusted to 1×10 ⁴ spores/ml. Thereafter, the prepared spores were used as an inoculation source and subjected to an inoculation test. Furthermore, the tomato plants obtained in Example 1 (1) were used as the tomato plants to be tested in the above-mentioned test. In the inoculation test, the inoculum was sprayed on the day the seedlings were transplanted in Example 1 (1), and after the spraying, the seedlings were grown for 3 weeks at 25° C. and under a 12-hour photoperiod. After the above-mentioned growth, a disease onset investigation was conducted.

The disease onset investigation was carried out by evaluating the disease index according to the following criteria for sporulation per field of view when the diseased site of the inoculated leaf was observed using an optical microscope with a magnification of 100 times.

Disease index 0: No sporulation is observed.
Disease index 1: Several sporulations (9 or less) are observed.
Disease index 2: 10-20 spores are observed.
Disease index 3: 21 or more spores are observed.

Then, the disease index of each individual was investigated according to the above-mentioned criteria, and the disease severity of each strain was determined from the following formula.
Disease severity (N) = [(0 x n ₀ ) + (1 x n ₁ ) + (2 x n ₂ ) + (3 x n ₃ )] / Number of surveyed individuals In the above formula, "0, 1, 2, 3 and 4" respectively indicate the disease index, and "n ₀ , n ₁ , n ₂ , n ₃ " indicate the number of individuals with disease index 0, disease index 1, disease index 2, and disease index 3, respectively. .

(3) Establishment of deposited line As a result of the above inoculation test, a new sooty mold resistant tomato line showing a high degree of sooty mold resistance was obtained. Hereinafter, this sooty mildew-resistant tomato plant will be referred to as the parent line. The parent line was backcrossed three times with a sooty mildew-susceptible tomato plant "Momotaro Hope" (Takii Seed Co., Ltd.) as a female parent (BC3). The above-mentioned inoculation test was performed on the obtained BC3, and a tomato line exhibiting a high degree of resistance to sooty mold was obtained. The obtained tomato line was selfed three times (BC3F3). The resulting sooty mildew-resistant tomato line (BC3F3) was deposited under accession number FERM P-22459. Hereinafter, the parent strain will also be referred to as the deposited strain.

(4) Identification of the genomic region conferring resistance The deposited line having sooty mold resistance was crossed with the sooty mold susceptible tomato plant "Momotaro Hope" (Takii Seed Co., Ltd.) as the female parent, and F1 Obtained generation. Next, seeds of the F2 generation were obtained by self-pollinating the F1 generation. Selection was made from the 3000 F2 generation individuals using SNP markers. Furthermore, an inoculation test was conducted on the selected F2 generation individuals. Specifically, an inoculation test was conducted in the same manner as in Example 1 (2), except that the F2 generation individuals were used as test plants for the inoculation test. In the inoculation test, individuals exhibiting a phenotype resistant to Pseudocercospora fuligena were obtained. As a result of the above acquisition, the genomic region conferring resistance to Pseudocercospora fuligena is a region between 678,023bp and 1,337,393bp on chromosome 1 when the known reference sequence Heinz1706 (SL3.0) is used. (SEQ ID NO: 1) was strongly suggested.

Next, the F1 generation individuals were backcrossed four times with "Momotaro Hope" as a female parent. After the crossbreeding, plants in which all chromosomes other than the sooty mold resistant genomic region were replaced with "Momotaro Hope" were selected using SNP markers set genome-wide. By self-pollinating the selected individuals twice, a plant line B in which the sooty mildew resistant genome region was fixed was created.

For the plant line B, the whole genome was sequenced using a single molecule sequencer (Pacbio sequencer). Based on the results of the whole genome sequencing, a pseudochromosome was constructed by de novo assembly. Based on the sequence information obtained from the similar chromosomes, nine SNP markers were designed based on the above-mentioned references 6 to 8, and using the nine SNP markers obtained, the genetic Individuals in which recombination had occurred were selected. After the selection, 5 lines of self-fertilized seeds (F3) were obtained by self-pollinating each strain. Furthermore, an inoculation test was conducted in the same manner as described above for the F3 line (lines 3 to 7) of the selected individuals of the five lines, the deposited line (line 1), and the tomato plant susceptible to sooty mildew (line 2). Ta. Specifically, an inoculation test was conducted in the same manner as in Example 1 (2), except that individuals of the five lines were used as test plants for the inoculation test. Then, based on the obtained inoculation results, it was evaluated whether each strain was a resistant homogeneous population, a susceptible (susceptible) homogeneous population, or a population in which resistant/susceptible individuals were separated.

As a result, for tomato plants having the nucleotide sequence from 457321st to 517433rd (1,133,392bp and 1,121,649bp on chromosome 1 in reference sequence Heinz1706 (SL3.0)) of the nucleotide sequence of SEQ ID NO: 1, F3 line The phenotype was found to be resistant. From this result, it was found that the 457321st to 517433rd nucleotide sequences in the nucleotide sequence of SEQ ID NO: 1 contribute to imparting sooty mildew resistance to tomato plants.

Next, regarding strain A, the base sequence between 1,133,392bp and 1,121,649bp on chromosome 1 in the reference sequence Heinz1706 (SL3.0) (strain A (deposited strain), positions 457321 to 517433 in the base sequence of SEQ ID NO: 1) nucleotide sequence), Heinz1706 (strain B), LA1589 (strain C, S. pimpinellifolium), LA2093 (strain D, S. pimpinellifolium), LA0716 (strain E, S. penellii), AJ002235.1 (strain F, leaf It was compared with AJ002236.1 (line G, which has a leaf mold resistance gene (Cf-9) and is derived from S. pimpinellifolium).

In strains A to G, the nucleotide sequences corresponding to the 457321st to 517433rd nucleotide sequences in the nucleotide sequence of SEQ ID NO. The nucleotide sequence (SEQ ID NO: 1) was confirmed to be a genomic region conferring novel sooty mold resistance. Furthermore, when the nucleotide sequence of SEQ ID NO: 1 of strain A was compared with the corresponding nucleotide sequences of other strains, strain G had the highest nucleotide sequence homology. Furthermore, when the nucleotide sequence containing the sooty mold resistance gene region derived from the deposited strain (SEQ ID NO: 1) is compared with the nucleotide sequence of strain G (AJ002236.1), the identity is 46.87%. , the base sequence between 1,133,392bp and 1,121,649bp on chromosome 1 in the reference sequence Heinz1706 (SL3.0) was significantly different between the deposited strain and other strains.

(5) Analysis of SNPs in commercially available cultivars It was confirmed that no other tomato plants possess the genomic region that confers resistance, which the new soot mold resistant tomato plant possesses.

For each tomato plant including the commercial varieties in Table 1 below, using the SNP corresponding to 1,133,392 bp on chromosome 1 in the reference sequence Heinz1706 (SL3.0), which is located in the sooty mold resistant genome region, We investigated whether it was possible to detect genomic regions that confer resistance to sooty mold. It has been confirmed that none of the tomato plants exhibits resistance to sooty mold. These results are shown in Table 1 below. In Table 1 below, S indicates that the strain has a different SNP from the deposited strain, that is, a susceptibility SNP, and R indicates that the strain has the same SNP as the deposited strain, that is, a resistance SNP. As shown in Table 1 below, each tomato plant has a genotype different from that of the deposited line, and it is possible to design a genomic region-specific SNP that confers sooty mildew resistance, and using this SNP. It was found that resistance to sooty mold can be evaluated.

Based on the above, by crossing the deposited line as the first parent and a tomato plant without sooty mold resistance as the second parent, we can obtain tomato plants resistant to sooty mold (Pseudocercospora fuligena). I found out that it is possible. Furthermore, using a primer set or probe that detects a SNP specific to a genomic region that confers sooty mold resistance, and/or detecting and selecting a sequence that includes a genomic region that confers sooty mold resistance. It was found that the tomato plants resistant to sooty mildew of the present disclosure could be selected.

Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various changes can be made to the structure and details of the present disclosure that can be understood by those skilled in the art within the scope of the present disclosure.

<Additional notes>
<Sooty mold resistant tomato plants>
(Additional note 1)
A tomato plant that is resistant to sooty mold.
(Additional note 2)
The tomato plant according to Supplementary Note 1, which contains the following polynucleotide (G) or a polynucleotide consisting of a partial sequence thereof as a genomic region conferring sooty mildew resistance:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Additional note 3)
A polynucleotide comprising the polynucleotide of (G) or a partial sequence thereof,
The tomato plant according to supplementary note 2, wherein the tomato plant is imparted with the sooty mildew resistance by the polynucleotide or a polynucleotide consisting of a partial sequence thereof.
(Additional note 4)
The tomato plant according to any one of appendices 1 to 3, wherein the sooty mildew-resistant tomato plant is a tomato plant obtained from the seeds of a tomato plant identified by accession number FERM P-22459 or a progeny line thereof.
<Part of tomato plant resistant to sooty mold>
(Appendix 5)
A part of a tomato plant resistant to sooty mildew according to any of Supplementary Notes 1 to 4.
(Appendix 6)
The plant part according to appendix 5, wherein the plant part is a seed.
<Method for producing tomato plants resistant to sooty mildew>
(Appendix 7)
A method for producing tomato plants resistant to sooty mold, characterized by comprising the following steps (a) and (b):
(a) a step of crossing the sooty mildew-resistant tomato plant according to any one of Supplementary Notes 1 to 4 with another tomato plant;
(b) A step of selecting sooty mildew-resistant tomato plants from the tomato plants obtained in step (a) or their progeny.
(Appendix 8)
The production method according to appendix 7, which includes the following step (x) prior to the step (a).
(x) Selecting a sooty mildew-resistant tomato plant described in any of Appendixes 1 to 4 from the test tomato plants (Appendix 9)
In the step (x),
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
The production method according to appendix 8, which selects a sooty mold resistant tomato plant containing the genomic region conferring sooty mold resistance:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Appendix 10)
In the step (x),
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
Selecting a candidate tomato plant resistant to sooty mold that includes the genomic region conferring resistance to sooty mold,
The candidate tomato plant or its progeny line is subjected to an inoculation test with a sooty mold fungus, and tomato plants exhibiting sooty mold resistance are selected as sooty mold resistant tomato plants, according to appendix 8 or 9. Production method:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Appendix 11)
Prior to step (x), the base sequence of the polynucleotide (G) or a partial sequence thereof and the reference sequence of the corresponding region in a tomato plant are used to determine the polynucleotide of (G) or a partial sequence thereof. The production method according to appendix 9 or 10, which includes the step of designing a molecular marker that can specifically detect the base sequence.
(Appendix 12)
The production according to any one of Supplementary Notes 8 to 11, wherein the selection in the step (x) is selection of a sooty mildew-resistant tomato plant containing the polynucleotide of the following (G) or a polynucleotide comprising a partial sequence thereof Method:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) A polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted, and/or added to the base sequence of (G1), and exhibits sooty mold resistance;
(G3) A polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of (G1) and expressing resistance to sooty mold.
(Appendix 13)
13. The production method according to any one of Supplementary Notes 7 to 12, wherein the selection in the step (b) is selection of a sooty mold resistant tomato plant containing a genomic region that confers sooty mold resistance.
(Appendix 14)
In the step (b),
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
The production method according to any one of Supplementary Notes 7 to 13, wherein a sooty mold resistant tomato plant containing the genomic region imparting sooty mold resistance is selected:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Appendix 15)
In the step (b),
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
Selecting a candidate tomato plant resistant to sooty mold that includes the genomic region conferring resistance to sooty mold,
Any one of appendices 7 to 14, wherein the candidate tomato plants or their progeny lines are subjected to an inoculation test with sooty mold fungi, and tomato plants exhibiting sooty mold resistance are selected as sooty mold resistant tomato plants. Production method described in:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Appendix 16)
Prior to step (b), the base sequence of the polynucleotide (G) or a partial sequence thereof and the reference sequence of the corresponding region in a tomato plant are used to determine the polynucleotide of (G) or a partial sequence thereof. 16. The production method according to appendix 14 or 15, which includes the step of designing a molecular marker that can specifically detect the base sequence.
(Appendix 17)
The production according to any one of Supplementary Notes 7 to 16, wherein the selection in the step (b) is selection of a sooty mildew-resistant tomato plant containing a polynucleotide comprising the polynucleotide of (G) below or a partial sequence thereof Method:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) A polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted, and/or added to the base sequence of (G1), and exhibits sooty mold resistance;
(G3) A polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of (G1) and expressing resistance to sooty mold.
<Method for imparting sooty mold resistance to tomato plants>
(Appendix 18)
A method for imparting sooty mold resistance to tomato plants, the method comprising the step of introducing into the tomato plant a genomic region imparting sooty mildew resistance.
(Appendix 19)
Introducing the polynucleotide according to Appendix 2 or 3 as a genomic region conferring resistance to sooty mold by crossing with a tomato plant resistant to sooty mold according to any one of Appendixes 1 to 4. The granting method described in Appendix 18.
<Method for detecting sooty mold resistance>
(Additional note 20)
A method for detecting sooty mold resistance in tomato plants, the method comprising:
A method comprising a detection step of detecting a genomic region conferring resistance to sooty mold in a test tomato plant.
(Additional note 21)
In the detection step, the sooty mildew resistance is detected in the test tomato plants by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. The method according to appendix 20 for detecting the genomic region to be assigned:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Additional note 22)
Prior to the detection step, the base sequence of the polynucleotide (G) or a partial sequence thereof is determined using the base sequence of the polynucleotide (G) or a partial sequence thereof and a reference sequence of the corresponding region in a tomato plant. including the step of designing a molecular marker that can specifically detect the
22. The method according to appendix 21, wherein in the detection step, the genomic region conferring the sooty mildew resistance is detected by detecting the molecular marker in the test tomato plant.
(Additional note 23)
Supplementary Notes 20 to 22, in the detection step, detecting, in the test tomato plant, a genomic region conferring resistance to sooty mildew, which includes a polynucleotide comprising the polynucleotide shown below (G) or a partial sequence thereof; Methods described in any of:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Additional note 24)
24. The method according to any one of appendices 20 to 23, wherein in the detection step, a genomic region conferring the sooty mildew resistance is detected on chromosome 1 of the test tomato plant.
<Screening method for tomato plants resistant to sooty mold>
(Additional note 25)
A method for screening tomato plants resistant to sooty mildew, the method comprising:
A screening method comprising a selection step of selecting tomato plants containing a genomic region that confers sooty mold resistance from test tomato plants as sooty mold resistant tomato plants.
(Additional note 26)
In the selection process,
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
The screening method according to appendix 25, which selects a sooty mold resistant tomato plant containing the genomic region conferring sooty mold resistance:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Additional note 27)
In the selection process,
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
Selecting a candidate tomato plant resistant to sooty mold that includes the genomic region conferring resistance to sooty mold,
According to appendix 25 or 26, the candidate tomato plants or their progeny lines are subjected to an inoculation test with sooty mold fungi, and candidate tomato plants exhibiting sooty mold resistance are selected as sooty mold resistant tomato plants. Screening method:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Additional note 28)
Prior to the selection step, the base sequence of the polynucleotide (G) or a partial sequence thereof is determined using the base sequence of the polynucleotide (G) or a partial sequence thereof and the reference sequence of the corresponding region in a tomato plant. 28. The screening method according to appendix 26 or 27, comprising the step of designing a molecular marker that can specifically detect.
(Additional note 29)
In the selection step, for the test tomato plants, the test tomato plants having the genomic region imparting sooty mold resistance, which includes the polynucleotide (G) below, are selected as sooty mold resistant tomato plants. The screening method according to any one of appendices 25 to 28 for selecting:
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.
(Additional note 30)
From appendix 25, in the selection step, a test tomato plant containing the genomic region conferring sooty mold resistance on chromosome 1 of the test tomato plant is selected as a sooty mold resistant tomato plant. 30. The screening method according to any one of 29.

As described above, according to the present disclosure, it is possible to provide tomato plants that exhibit resistance to sooty mold. Therefore, the present disclosure is extremely useful in, for example, the agricultural field, the breeding field, and the like.

Claims (17)

A sooty mold resistant tomato plant containing a polynucleotide consisting of the following polynucleotide (G) as a genomic region conferring sooty mold resistance :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold. The genomic region conferring sooty mold resistance includes a polynucleotide consisting of a partial sequence of the polynucleotide shown below (G) ,
A tomato plant resistant to sooty mold, wherein the partial sequence is 5000 bases or more in length :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold. 3. The tomato plant according to claim 2, wherein the tomato plant is imparted with the sooty mildew resistance by the polynucleotide consisting of the partial sequence. The tomato plant according to claim 1 or 2, wherein the sooty mildew-resistant tomato plant is a tomato plant obtained from the seeds of a tomato plant identified by accession number FERM P-22459 or a progeny line thereof. Parts of a sooty mildew resistant tomato plant according to claim 1 or 2. 6. A plant part according to claim 5, wherein the plant part is a seed. A method for producing tomato plants resistant to sooty mold, characterized by comprising the following steps (a) and (b):
(a) a step of crossing the sooty mold resistant tomato plant according to claim 1 or 2 with another tomato plant;
(b) A step of selecting sooty mildew-resistant tomato plants from the tomato plants obtained in step (a) or their progeny. The production method according to claim 7, comprising the following step (x) prior to the step (a).
(x) Selecting tomato plants resistant to sooty mold from test tomato plants In the step (x),
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
Selecting a sooty mold resistant tomato plant containing the genomic region conferring sooty mold resistance,
The production method according to claim 8, wherein the length of the partial sequence is 5000 bases or more :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold. In the step (x),
For the test tomato plants, a genomic region that confers sooty mold resistance is detected by detecting a molecular marker that can specifically detect the polynucleotide shown below (G) or a polynucleotide containing a partial sequence thereof. ,
Selecting a candidate tomato plant resistant to sooty mold that includes the genomic region conferring resistance to sooty mold,
Conducting an inoculation test with sooty mold fungi on the candidate tomato plants or their progeny lines, and selecting tomato plants exhibiting sooty mold resistance as sooty mold resistant tomato plants ;
The production method according to claim 9 , wherein the length of the partial sequence is 5000 bases or more :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold. Prior to step (x), the base sequence of the polynucleotide (G) or a partial sequence thereof and the reference sequence of the corresponding region in a tomato plant are used to determine the polynucleotide of (G) or a partial sequence thereof. The production method according to claim 9, comprising the step of designing a molecular marker that can specifically detect the base sequence. The selection in step (x) is selection of sooty mildew-resistant tomato plants containing a polynucleotide comprising the following polynucleotide (G) or a partial sequence thereof,
The production method according to claim 8, wherein the length of the partial sequence is 5000 bases or more :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) A polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted, and/or added to the base sequence of (G1), and exhibits sooty mold resistance;
(G3) A polynucleotide consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence of (G1) and expressing resistance to sooty mold. An introduction step of introducing a polynucleotide of the following (G) or a polynucleotide consisting of a partial sequence thereof into a tomato plant as a genomic region that confers sooty mold resistance ,
A method for imparting soot mold resistance to tomato plants, characterized in that the length of the partial sequence is 5000 bases or more :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold. The sooty mold resistance can be achieved by crossing with a sooty mold resistant tomato plant containing the polynucleotide shown below (G) or a polynucleotide consisting of a partial sequence thereof as a genomic region conferring sooty mold resistance. Introducing the polynucleotide shown below (G) or a polynucleotide consisting of a partial sequence thereof as the genomic region to be added,
The imparting method according to claim 13 , wherein the length of the partial sequence is 5000 bases or more :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold. A method for detecting sooty mold resistance in tomato plants, the method comprising:
By detecting a molecular marker that can specifically detect a polynucleotide containing the polynucleotide shown below (G) or a partial sequence thereof in a test tomato plant, a polynucleotide containing the polynucleotide shown below (G) or a partial sequence thereof is detected. a detection step of detecting a genomic region conferring sooty mold resistance containing a nucleotide ;
The length of the partial sequence is 5000 bases or more , a method :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold. Prior to the detection step, the base sequence of the polynucleotide (G) or a partial sequence thereof is determined using the base sequence of the polynucleotide (G) or a partial sequence thereof and a reference sequence of the corresponding region in a tomato plant. including the step of designing a molecular marker that can specifically detect the
16. The method according to claim 15 , wherein in the detection step, the genomic region conferring the sooty mildew resistance is detected by detecting the molecular marker in the test tomato plant. A method for screening tomato plants resistant to sooty mildew, the method comprising:
By detecting a molecular marker that can specifically detect a polynucleotide containing the polynucleotide (G) below or a partial sequence thereof from a test tomato plant , the following ( G) a selection step of selecting a tomato plant containing a polynucleotide consisting of the polynucleotide as a candidate tomato plant resistant to sooty mildew ;
A screening method in which the length of the partial sequence is 5000 bases or more :
(G) Polynucleotide of the following (G1), (G2), or (G3):
(G1) Polynucleotide consisting of the base sequence 457321st to 517433rd of SEQ ID NO: 1;
(G2) a polynucleotide consisting of a base sequence in which 1 to 6011 bases have been deleted, substituted, inserted and/or added to the base sequence of (G1), and confers sooty mold resistance;
(G3) A polynucleotide consisting of a base sequence having 90% or more identity to the base sequence of (G1) and imparting resistance to sooty mold.