A novel tomato plant resistant to ToBRFV

Abstract

The present invention relates to novel tomato plants that exhibit high resistance to ToBRFV infection. The present invention also relates to seeds and parts of said plants, such as fruits. The present invention further relates to methods of making and using such seeds and plants. The present invention also relates to novel gene sequences associated with said high resistance and molecular markers associated with said novel gene sequences.

Description

[Technical field]

[0001] The present invention relates to novel tomato plants that exhibit high resistance to ToBRFV infection. The present invention also relates to seeds and parts of said plants, such as fruits. The present invention further relates to methods for the production and use of such seeds and plants. The present invention also relates to novel gene sequences associated with said high resistance, and molecular markers associated with said novel gene sequences. [Background technology]

[0002] Viruses such as those belonging to the Tobamovirus genus cause significant damage to tomato growers, regardless of the type of tomato or the cultivation method used. Infections by tobamoviruses (e.g., Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV) or Tomato mild mottle virus (ToMMV)) typically spread rapidly in the field via mechanical transmission by field workers, tools and manual handling of plants and fruit, or via natural vectors such as insects, which are also difficult to control.

[0003] In 2014 and 2015, a novel tobamovirus was first reported in tomato in Israel and Jordan, respectively (Luria et al., 2017; Salem et al., 2016). It was named Tomato Brown Rugose Fruit Virus (ToBRFV) after one of the main symptoms it caused in the crop, namely the deformation of the fruit with yellow and / or brown areas. The virus harbors the tobamovirus resistance gene Tm-2 2 This ToBRFV outbreak is of particular concern to the tomato industry, as it has been shown that ToBRFV can be overcome by tomato strains, therefore solutions are needed for growers and the seed industry to be able to grow ToBRFV resistant tomato crops.

[0004] Possible sources of ToBRFV resistance have been described in the patent and non-patent literature.

[0005] For example, WO 2018 / 219941 reports QTLs located on chromosomes 6, 9 and 11, and WO 2019 / 110821, WO 2020 / 018783, WO 2021 / 110855 and WO 2021 / 170868 also disclose QTLs or genes located on chromosome 11.

[0006] WO 2020 / 148021 and WO 2022 / 013452 report QTLs or genes located on chromosome 8.

[0007] Finally, WO 2021 / 213892 discloses alternative alleles of the known resistance gene tm-1 located on chromosome 2.

[0008] Despite the number of described loci, it is still unclear which QTLs or genes (combinations) effectively provide resistance to ToBRFV in the field, since the mechanisms underlying ToBRFV infection have yet to be determined. For example, Zinger et al. suggested that a locus located on chromosome 2 interacts with a locus located on chromosome 11 to control ToBRFV resistance (Zinger et al., 2021).

[0009] Therefore, there is a need for alternative solutions to further improve ToBRFV control in plants, especially tomato plants. Summary of the Invention [Means for solving the problem]

[0010] The present invention addresses the need for improved resistance to ToBRFV by providing a novel tomato plant that contains an increased ToBRFV resistance trait. By identifying one novel QTL associated with increased ToBRFV resistance in a breeding population and introgressing its corresponding sequence into elite tomato plants, the ToBRFV resistance ability of the tomato plant is increased, which has a positive impact on overall plant performance. The ToBRFV resistance QTL located on chromosome 1 (QTL1) and its corresponding introgressed sequence are recessive in nature, therefore, two copies of the sequence are required to provide an improved ToBRFV resistance phenotype.

[0011] Taken together, the improved ToBRFV-resistant tomato plant characteristics disclosed within the present invention provide tomato growers with a novel solution to improve the economic and commercial efficiency of deploying tomato varieties in fields affected by ToBRFV.

[0012] In a first embodiment, the present invention provides a cultured tomato plant, preferably a cultured Solanum lycopersicum plant, resistant to ToBRFV infection, comprising in its genome an introgression sequence from a Solanum galapagense plant conferring resistance to ToBRFV, said introgression sequence being located on chromosome 1 and comprising at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; b) a G genotype in the homozygous state for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) G genotype in the homozygous state for SNP marker 4 at the position corresponding to position 147 in SEQ ID NO:16.

[0013] Further, the plant of the preceding embodiment comprises: a) the A genotype of SNP marker 2 can be identified in PCR by amplification of a nucleic acid fragment using a pair of oligonucleotide primers: a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 10, and a probe of SEQ ID NO: 8; b) the G genotype of SNP marker 3 can be identified in a PCR by amplification of a nucleic acid fragment using a pair of oligonucleotide primers: forward primer SEQ ID NO: 12 and reverse primer SEQ ID NO: 15, and a probe SEQ ID NO: 13; and / or c) The G genotype of SNP marker 4 can be identified in a PCR by amplification of a nucleic acid fragment using a pair of oligonucleotide primers: forward primer SEQ ID NO: 17 and reverse primer SEQ ID NO: 20, and a probe SEQ ID NO: 18.

[0014] In a further embodiment of the invention, the ToBRFV resistance-conferring gene transfer sequence comprises at least one of SEQ ID NO:6, SEQ ID NO:11, and / or SEQ ID NO:16, or a sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identical to one or more of said sequences.

[0015] In a further embodiment of the invention, the introgression sequence is contained in tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof.

[0016] In a further embodiment, the present invention provides a plant according to any of the preceding embodiments, wherein said plant is obtained by crossing tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, with a tomato plant that does not contain the ToBRFV resistance-conferring introgression sequence.

[0017] In a further embodiment, the present invention provides a plant according to any of the preceding embodiments, wherein said plant is an inbred, dihaploid, diploid, or hybrid plant.

[0018] A further embodiment provides a plant part, organ or tissue obtained from a tomato plant according to any of the previous embodiments including, but not limited to, a leaf, stem, root, flower or inflorescence, fruit, shoot, gametophyte, spore pair, pollen, anther, microspore, egg cell, zygote, embryo, meristematic tissue part, callus tissue, seed, cutting, cell or tissue culture, or any other part or product of the plant which still exhibits the ToBRFV resistance trait according to the present invention, especially when grown into a fruit-bearing plant.

[0019] In a further embodiment, the present invention provides a seed producing a plant according to any of the preceding embodiments.

[0020] In a further embodiment, the present invention provides a method for producing a cultured tomato plant, preferably a cultivated species Solanum lycopersicum plant, that is resistant to ToBRFV infection, comprising: a) crossing a plant according to any one of the preceding embodiments with a cultivated tomato plant lacking the ToBRFV resistance-conferring introgression sequence; b) selecting progeny plants comprising said introgression sequence located on chromosome 1 that confers resistance to ToBRFV, comprising detecting at least one of the following SNP markers: i) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; ii) a G genotype that is homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or iii) a G genotype that is homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; thereby producing a plant having enhanced resistance to ToBRFV.

[0021] In a further embodiment, the present invention relates to a method of any of the preceding embodiments, wherein the method comprises: c) selfing the selected progeny or crossing the selected progeny with other tomato plants to produce additional progeny. Further includes:

[0022] In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein further progeny are selected and selfed / crossed for an additional 2-10 generations.

[0023] In a further embodiment, the present invention relates to a method according to any of the preceding embodiments, wherein the plant in step a) is tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof.

[0024] In a further embodiment, the present invention relates to a method of producing an F1 tomato plant exhibiting resistance to ToBRFV, the method comprising the step of crossing an inbred tomato plant, the plant being any one of the preceding embodiments, with a different inbred tomato plant to produce an F1 hybrid progeny.

[0025] In a further embodiment, the present invention provides a method for identifying cultivated tomato plants, preferably cultivated Solanum lycopersicum plants, exhibiting resistance to ToBRFV and carrying at least one copy of said ToBRFV resistance-conferring introgression sequence, said method comprising detecting at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; b) a G genotype in the homozygous state for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) a G genotype that is homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; Thereby, tomato plants exhibiting resistance to ToBRFV are identified.

[0026] In a further embodiment, the present invention relates to a method according to the aforementioned embodiment, wherein said method further comprises the steps of selecting a tomato plant comprising said at least one SNP marker, and crossing the selected tomato plant with a second tomato plant to produce a progeny tomato plant comprising at least one of said SNP markers and exhibiting enhanced resistance to ToBRFV. [Brief description of the drawings]

[0027] [Figure 1] 1 shows ToBRFV pathology assay photographs representing the disease scale used and described in Example 2C: (a) susceptibility rating (S or 1); (b) intermediate resistance rating (IR or 5); (c) high resistance rating (HR or 9). [Diagram 2] 1 shows the results of ToBRFV pathology assays performed with susceptibility check (a) and donor LA0483 plants (b). [Diagram 3] 1 shows the results of tobamovirus pathology assays performed with donor LA0483 plants inoculated with ToMV pathotype 0 (a), ToMV pathotype 2 (b) and no virus (mock test) (c). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] definition The technical terms and expressions used within the scope of this application should generally be given the meanings commonly applied to them in the relevant art of plant breeding and cultivation, unless otherwise indicated herein below.

[0029] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes one or more plants, and reference to "a cell" includes mixtures of cells, tissues, and the like.

[0030] "Cultivated tomato" or "elite tomato" plants are understood within the scope of the present invention to refer to plants that are no longer in their natural state, but have been developed and cultivated through human care and for agricultural use and / or human consumption, excluding wild tomato accessions such as Solanum galapagense accessions. By way of example, in an embodiment, a cultivated tomato or elite tomato plant according to the present invention is capable of growing edible fruit. Alternatively, or in addition, the cultivated tomato plant is a hybrid plant. Alternatively, or in addition, the cultivated tomato plant is a cultivated Solanum lycopersicum plant.

[0031] "Alleles" is understood within the scope of the present invention to refer to alternative or mutated forms of various genetic units that are identical to or related to different forms of any kind of identifiable genetic determinant, such as a gene or a QTL, which are considered alternative in inheritance because they are located at the same locus on homologous chromosomes. Such alternative forms or variants may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or may be the result of gene regulation caused, for example, by chemical or structural modifications, transcriptional or post-translational modifications / regulations. In a diploid cell or organism, the two alleles of a given gene or genetic element typically occupy corresponding loci on a pair of homologous chromosomes.

[0032] Relatively speaking, the term "improved ToBRFV resistance" or "increased ToBRFV resistance" is understood in this specification to mean a plant according to the present invention comprising an introgression sequence, e.g. from a Solanum galapagense plant, conferring resistance to ToBRFV, wherein said introgression sequence is located on chromosome 1 and comprises at least one of SNP markers 2 to 4, and which is more resistant to ToBRFV compared to a plant lacking said introgression sequence.

[0033] "Improved ToBRFV resistance" is understood within the scope of the present invention to mean a tomato plant that has a statistically significant improved resistance to ToBRFV compared to a control tomato plant lacking the introgression sequence of the present invention (e.g. as described in the Examples section) using standard error and / or with P<0.05 or P<0.01 using the Student's test and / or with a Lod score >3.

[0034] "Phenotype" is understood within the scope of the present invention to refer to the distinguishable characteristics of genetically controlled traits.

[0035] A "control tomato plant" is understood within the scope of the present invention to mean a tomato plant having substantially the same genetic background as a cultivated tomato plant of the present invention, where the control plant does not carry the introgression sequence of the present invention linked to improved ToBRFV resistance. The control tomato plant may be a tomato plant belonging to the same plant variety and not comprising the introgression sequence of the present invention. The control tomato plant is grown for the same period and under the same conditions as the cultivated tomato plant of the present invention. In the present specification, plant varieties are understood according to the definition of UPOV. Thus, the desired control tomato plant may be a near isogenic line, an inbred line or a hybrid, where the control plant has the same genetic background as the tomato plant of the present invention, but where the control plant does not carry the introgression sequence of the present invention linked to improved ToBRFV resistance.

[0036] The term "trait" refers to a characteristic or phenotype. In the context of the present invention, a ToBRFV resistance trait is an improved ToBRFV resistance trait. A trait can be inherited dominantly or recessively, or partially, semi- or incompletely dominantly. In the context of the present invention, the ToBRFV resistance-conferring introgression sequence located on chromosome 1 is recessive. The tomato plant of the present invention is therefore homozygous for the trait. Furthermore, the trait can be monogenic or polygenic, or can result from the interaction of one or more genes in the environment. In the context of the present invention, the ToBRFV resistance-conferring introgression sequence located on chromosome 1 is sufficient alone to confer an improved ToBRFV resistance trait and is therefore monogenic.

[0037] The terms "hybrid," "hybrid plant," and "hybrid progeny" refer to individuals produced from genetically distinct parents (eg, genetically heterozygous or near-heterozygous individuals).

[0038] The term "inbred" refers to a genetically homozygous or nearly homozygous population. Inbred lines are obtained, for example, through several cycles of brother / sister breeding or selfing or in dihaploid production.

[0039] The term "dizomelic haploid line" refers to a stable inbred line derived from another culture. Some pollen grains (haploids) grown in a particular medium and conditions can develop embryos containing n chromosomes. These embryos are then "doubled" and contain 2n chromosomes. The progeny of these embryos are called "dizomelic haploids" and essentially no longer segregate (are stable).

[0040] The term "genetically fixed" refers to a gene sequence that is stably integrated into the genome of a plant that does not normally contain said gene sequence. When genetically fixed, the gene sequence can be easily and predictably inherited by other plants by sexual mating.

[0041] The term "rootstock" refers to a plant used as a recipient for a cutting. Typically, the rootstock plant and the cutting are of different genotypes. In an embodiment, a plant according to the invention is used as a rootstock plant.

[0042] The term "plant" or "plant part" hereinafter refers to a plant part, organ or tissue obtainable from the tomato plant of the present invention, including, but not limited to, leaves, stems, roots, flowers or inflorescences, fruits, shoots, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic tissue sections, callus tissue, seeds, cuttings, cell or tissue cultures, or any other part or product of the plant which, especially when grown into a fruit-producing plant, still exhibits the improved ToBRFV resistance trait of the present invention.

[0043] A "plant" is any plant at any stage of development.

[0044] A "tomato plant seed" is a seed that will grow into a tomato plant as described in any of the embodiments.

[0045] A "plant cell" is the structural and physiological unit of a plant, including the protoplast and the cell wall. A plant cell can be in the form of an isolated single cell or a cultured cell, or can be part of a more highly organized unit, such as a plant tissue, a plant organ, or a whole plant.

[0046] "Plant cell culture" refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, embryo stocks, embryo sacs, zygotes and embryos at various stages of development.

[0047] A "plant organ" is a distinct, visually structured differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0048] As used herein, "plant tissue" refers to a group of plant cells organized into a structural and functional unit. Any plant tissue in a plant or in culture is included. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into a structural and / or functional unit.

[0049] As used herein, the term "breeding" and grammatical variations thereof refer to any process of producing offspring individuals. Breeding can be sexual or asexual or any combination thereof. Exemplary non-limiting types of breeding include crossing, selfing, derivative production of doubled haploids, and combinations thereof.

[0050] As used herein, the phrase "established breeding population" refers to a collection of potential breeding partners that are generated by and / or used as parents in a breeding program, e.g., a commercial breeding program. Members of an established breeding population are typically well characterized genetically and / or phenotypically. For example, some phenotypic traits of interest may be evaluated, for example, under different environmental conditions, in multiple locations and / or at different times. Alternatively or in addition, one or more loci associated with the expression of the phenotypic trait may be identified, and one or more members of the breeding population may be genotyped for one or more loci and for one or more genetic markers associated with the one or more loci.

[0051] As used herein, the phrase "diploid individual" refers to an individual having two sets of chromosomes, typically one from each of its two parents. However, it is understood that in some embodiments, a diploid individual may inherit its "maternal" and "paternal" sets of chromosomes from the same single organism, for example, when the plant self-pollinates to generate the next generation of the plant.

[0052] "Homozygous" is understood within the scope of the present invention to refer to similar alleles at one or more corresponding loci in homologous chromosomes. In the context of the present invention, a tomato plant containing two identical copies of a particular introgression sequence at a particular locus, such as an introgression sequence located on chromosome 1, is homozygous at the corresponding locus.

[0053] "Heterozygous" is understood within the scope of the present invention to refer to different alleles at one or more corresponding loci of homologous chromosomes.

[0054] A "dominant" allele is understood within the scope of the present invention to refer to an allele which determines the phenotype when present in the heterozygous or homozygous state.

[0055] A "semi-dominant" allele is understood within the scope of the present invention to refer to an allele that determines a phenotype when present in a heterozygous or homozygous state, however the strength of the phenotype is generally higher when the allele is present in a homozygous state.

[0056] A "recessive" allele refers to an allele that determines a phenotype when only present in the homozygous state.

[0057] "Backcrossing" is understood within the scope of the present invention to refer to a process in which the progeny of a hybrid are repeatedly crossed with one of the original parents. Different recurrent parents can be used in subsequent backcrosses.

[0058] A "locus" is understood within the scope of the present invention to refer to a region on a chromosome that contains a gene, a QTL or its corresponding genetic sequence that contributes to a trait.

[0059] "Genetic linkage" is understood within the scope of the present invention to refer to the association of inherited traits due to the location of adjacent genes on the same chromosome, measured by the recombination rate between loci (centimorgans, cM).

[0060] As used herein, the phrases "sexual mating" and "sexual reproduction" in relation to the subject matter of this disclosure refer to the production of offspring by the fusion of gametes (e.g., by fertilization, such as the production of seeds by pollination in a plant). "Sexual mating" or "cross-fertilization" refers, in some embodiments, to the fertilization of one individual by another individual (e.g., cross-pollination in a plant). The term "selfing" refers, in some embodiments, to the production of seeds by self-fertilization or self-pollination, i.e., the pollen and ovules are from the same plant.

[0061] As used herein, the phrase "genetic marker" or "DNA marker" refers to a feature of an individual's genome (e.g., a nucleotide or polynucleotide sequence present in an individual's genome) that is associated with one or more loci of interest. In certain embodiments, a genetic marker is a locus that is polymorphic or occupied by a polymorphism in a population of interest, depending on the context. Genetic markers include, for example, single nucleotide polymorphisms (SNPs), indels (i.e., insertions / deletions), simple sequence repeats (SSRs), restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), truncated amplified polymorphic sequences (CAPS) markers, diversity array technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs), among many other examples. Genetic markers can be used, for example, to identify the location of loci containing alleles on chromosomes that contribute to the variability of a phenotypic trait. The phrase "genetic marker" can also refer to a polynucleotide sequence that is complementary to a genomic sequence, such as the sequence of a nucleic acid used as a probe.

[0062] As used herein, the term "genotype" refers to the genetic makeup of a cell or organism. An individual's "genotype for a set of genetic markers" includes the particular alleles for one or more genetic marker loci that are present in the individual's haplotype.

[0063] As used herein, the term "progeny" refers to the offspring of a particular cross. Typically, progeny result from the breeding of two individuals, although some species (particularly some plants and hermaphroditic animals) can self-fertilize (i.e., the same plant serves as the donor of both male and female gametes). Progeny can be, for example, F 1 , F 2 Or it could be of any future generation.

[0064] As used herein, the term "quantitative trait locus" (QTL) refers to an association between a genetic marker and a chromosomal region and / or a gene and / or an introgression sequence that influences the phenotype of a trait of interest. Typically, this is determined statistically; for example, based on one or more methods published in the literature. A QTL can be a chromosomal region and / or a locus with at least two alleles that differentially affect a phenotypic trait.

[0065] The term "recipient tomato plant" is used herein to refer to a tomato plant that receives DNA obtained from a donor tomato plant that contains an improved ToBRFV resistance trait.

[0066] The term "natural genetic background" is used herein to indicate the original genetic background of a gene sequence. For example, the gene sequence of the present invention was found at a specific location on chromosome 1 of a Solanum galapagense plant. Conversely, a method including the transfer of DNA comprising this gene sequence from chromosome 1 of a Solanum galapagense plant to the same location on chromosome 1 of another tomato species, preferably a cultivated tomato plant, even more preferably a Solanum lycopersicum plant, such as the transfer of DNA via breeding, would result in this gene sequence not being in its natural genetic background. When the gene sequences of the present invention are transferred from a Solanum galapagense background to another tomato species, preferably a cultivated tomato plant, even more preferably a Solanum lycopersicum plant, they are referred to as "introgression sequences" or "introgression gene sequences".

[0067] "Donor tomato plant" is understood within the scope of the present invention to mean a tomato plant which provides an improved ToBRFV resistance trait.

[0068] "Marker-based selection" is understood within the scope of the present invention to refer to the use of genetic markers to detect, for example, one or more nucleic acids from a plant, which nucleic acids are associated with a desired trait to identify plants carrying an allele for the desired (or undesirable) trait so that those plants can be used (or avoided) in selective breeding programs.

[0069] Single nucleotide polymorphisms (SNPs), which are mutations at a single site in DNA, are the most common type of genomic variation. Single nucleotide polymorphisms (SNPs) are variations in DNA sequence that occur when a single base (A, T, C, or G) in a genome (or other shared sequence) differs between members of a biological species or between paired chromosomes of an individual. For example, two sequenced DNA fragments, AAGCCTA and AAGCTTA, from different individuals contain a single base difference. In this case, there are two alleles: C and T. The basic principles of SNP arrays are the same as DNA microarrays. These are the confluence of DNA hybridization, fluorescence microscopy, and DNA capture. The three components of an SNP array are an array containing nucleic acid sequences (i.e., amplified sequences or targets), one or more labeled allele-specific oligonucleotide probes, and a detection system that records and interprets the hybridization signal. The presence or absence of the desired SNP marker allele can be determined by real-time PCR using double-stranded DNA dyes or fluorescent reporter probe methods.

[0070] "PCR (polymerase chain reaction)" is understood within the scope of the present invention to refer to a method for generating relatively large amounts of specific regions or subsets of genomic DNA, thereby allowing a variety of analyses based on those regions. "PCR primers" is understood within the scope of the present invention to refer to relatively short pieces of single-stranded DNA used in PCR amplification of specific regions of DNA.

[0071] As used herein, "probe" refers to a group of atoms or molecules that can recognize and bind to a specific target molecule or cellular structure, thus allowing the detection of the target molecule or structure. In particular, "probe" refers to a labeled DNA or RNA sequence that can be used to detect the presence of and quantify a complementary sequence by molecular hybridization.

[0072] "Sequence identity". The term "identical" or "identity" in reference to two or more nucleic acid or protein sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection. When the two sequences compared to each other are of different lengths, the sequence identity preferably relates to the percentage of nucleotide residues of the shorter sequence that are identical to the nucleotide residues of the longer sequence. As used herein, the percent identity / homology between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % identity = number of identical positions / total number of positions x 100). Comparison of sequences and determination of the percent identity between two sequences can be performed using a mathematical algorithm as described later in this specification. For example, sequence identity can be conventionally determined using a computer program such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711). Bestfit utilizes the locus homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, to find the segment with the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has, for example, 95% identity with the reference sequence of the present invention, the parameters are preferably adjusted so that the percentage of identity is calculated over the entire length of the reference sequence and so that homology gaps of up to 5% of the total number of nucleotides in the reference sequence are allowed. When using Bestfit, the so-called optional parameters are preferably left at their preset ("initial") values.Deviations found in the comparison between a given sequence and the above sequences of the invention may be caused, for example, by additions, deletions, substitutions, insertions or recombinations. Such sequence comparisons may also be preferably carried out using the program "fasta20u66" (Version 2.0u66 by William R. Pearson and the University of Virginia, September 1998; W.R. Pearson (1990), Methods in Enzymology 183, 63-98, see also the appended examples and http: / / workbench.sdsc.edu / ). For this purpose, the "default" parameter settings may be used.

[0073] Embodiment Plants, seeds, fruits. In a first embodiment, the present invention provides a cultured tomato plant, preferably a cultured Solanum lycopersicum plant, resistant to ToBRFV infection, comprising in its genome an introgression sequence from Solanum galapagense conferring resistance to ToBRFV, said introgression sequence being located on chromosome 1 and comprising at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; b) a G genotype in the homozygous state for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) G genotype in the homozygous state for SNP marker 4 at the position corresponding to position 147 in SEQ ID NO:16.

[0074] Further, the plant of the preceding embodiment comprises: a) the genotype of SNP marker 2 can be identified in PCR by amplification of a nucleic acid fragment using a pair of oligonucleotide primers: a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 10, and a probe of SEQ ID NO: 8; b) the G genotype of SNP marker 3 can be identified in a PCR by amplification of a nucleic acid fragment using a pair of oligonucleotide primers: forward primer SEQ ID NO: 12 and reverse primer SEQ ID NO: 15, and a probe SEQ ID NO: 13; and / or c) The G genotype of SNP marker 4 can be identified in a PCR by amplification of a nucleic acid fragment using a pair of oligonucleotide primers: forward primer SEQ ID NO: 17 and reverse primer SEQ ID NO: 20, and a probe SEQ ID NO: 18.

[0075] In a further embodiment of the invention, the ToBRFV resistance-conferring gene transfer sequence comprises at least one of SEQ ID NO:6, SEQ ID NO:11, and / or SEQ ID NO:16, or a sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identical to one or more of said sequences.

[0076] In a further embodiment of the invention, the plant comprises SEQ ID NO:11.

[0077] In a further embodiment of the invention, the introgression sequence is comprised in tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof.

[0078] In a further embodiment, the present invention provides a plant according to any of the preceding embodiments, wherein said plant is obtained by crossing tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, with a tomato plant that does not comprise the ToBRFV resistance-conferring introgression sequence.

[0079] In a further embodiment, the present invention provides a plant according to any of the preceding embodiments, wherein said plant is an inbred, haploid, diploid, or hybrid plant.

[0080] In another embodiment, the plant according to the invention is male sterile. In another embodiment, the plant according to the invention is cytoplasmic male sterile.

[0081] In a further embodiment, the tomato plant of the invention is a tomato plant according to any of the preceding embodiments, wherein said ToBRFV resistance-conferring introgression sequence located on chromosome 1 can be identified using any of SNP markers 2 to 4 disclosed in Table 4 below.

[0082] In a further embodiment, the present invention provides a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, resistant to ToBRFV infection, comprising in its genome an introgression sequence from Solanum galapagense that confers resistance to ToBRFV located on chromosome 1, said plant genome comprising: a) the A genotype in the homozygous state of SNP marker 1 at a position corresponding to position 47 in SEQ ID NO:1; and b) a C genotype in the heterozygous or homozygous state at SNP marker 5 at a position corresponding to position 94 in SEQ ID NO: 21; and c) at least one of the resistance genotypes in any one of SNP markers 2 to 4 disclosed in Table 4 Includes.

[0083] In a further embodiment, the cultivated tomato plant of the previous embodiment further comprises at least one second resistance genotype at any of SNP markers 2-4 disclosed in Table 4.

[0084] In a further embodiment, the present invention provides a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, resistant to ToBRFV infection, comprising in its genome an introgression sequence from Solanum galapagense that confers resistance to ToBRFV located on chromosome 1, said plant genome comprising: a) G genotype in the homozygous state for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO:11 Includes.

[0085] In a further embodiment, the tomato plant of the invention is a tomato plant according to any of the preceding embodiments, wherein tomato plant 19TEP080670 (representative seeds of which have been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, or Solanum galapagense accession LA0483, is the source of said ToBRFV resistance-conferring introgression sequence.

[0086] A further embodiment provides a plant part, organ or tissue obtained from a tomato plant according to any of the previous embodiments including, but not limited to, a leaf, stem, root, flower or inflorescence, fruit, shoot, gametophyte, spore pair, pollen, anther, microspore, egg cell, zygote, embryo, meristematic tissue part, callus tissue, seed, cutting, cell or tissue culture, or any other part or product of the plant which still exhibits the ToBRFV resistance trait according to the present invention, especially when grown into a fruit-bearing plant.

[0087] In a further embodiment, the present invention provides a seed producing a plant according to any of the previous embodiments.

[0088] In a further embodiment, the present invention relates to the use of a tomato plant according to any of the preceding embodiments as a rootstock, preferably a tomato rootstock. In a further embodiment, the present invention relates to the use of tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, as a tomato rootstock.

[0089] In other embodiments, the use of a tomato plant, plant part, or seed according to any of the previous embodiments to produce and harvest tomato fruit is contemplated.

[0090] In other embodiments, the present invention relates to the use of a tomato plant, plant part, or seed according to any of the embodiments, wherein the tomato plant, plant part, or seed is tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof.

[0091] In a further embodiment, the present invention relates to the use of a tomato plant, plant part or seed according to any of the previous embodiments for sowing in a field, a greenhouse or a plastic greenhouse.

[0092] In one embodiment, the present invention provides a tomato fruit produced by a tomato plant according to any of the preceding embodiments.

[0093] The present invention further relates to the use of a tomato plant according to any of the previous embodiments for introgressing a ToBRFV resistance trait into a tomato plant not comprising said ToBRFV resistance trait.

[0094] In a further embodiment, the tomato plant of any of the preceding embodiments is resistant to tobamovirus. In a further embodiment, the tomato plant is also resistant to TMV and ToMV viruses. In a further embodiment, the tomato plant is also resistant to ToMV pathotypes 0 and 2.

[0095] Gene sequences, markers. The present invention further relates to an introgressed genetic sequence linked to a ToBRFV resistance trait in a tomato plant. In a further embodiment, the genetic sequence of the present invention is located on chromosome 1. In a further embodiment of the present invention, the genetic sequence is contained in, obtained from, or can be obtained from tomato plant 19TEP080670 (a representative of which has been deposited under NCIMB accession number 43938), or a donor plant that is a descendant or ancestor thereof and comprises said genetic sequence, or Solanum galapagense accession LA0483.

[0096] In another embodiment, the introgression gene sequence of the present invention is characterized by at least one of the following resistance genotypes at one of the SNP markers located on chromosome 1 and selected in the list comprising: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; b) a G genotype in the homozygous state for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) G genotype in the homozygous state for SNP marker 4 at the position corresponding to position 147 in SEQ ID NO:16.

[0097] The present invention also discloses the use of at least one, at least two or at least three of the SNP markers of the present invention for diagnostic selection and / or genotyping of ToBRFV resistance trait loci in tomato plants, in particular cultivated tomato plants.

[0098] The present invention further discloses the use of at least one, at least two or at least three of the SNP markers according to the present invention for identifying the presence of a ToBRFV resistance trait in a tomato plant, in particular a cultivated tomato plant, more particularly a tomato plant according to the present invention, and / or for monitoring the introgression of a ToBRFV resistance trait in a tomato plant, in particular a cultivated tomato plant, in particular a tomato plant as described herein according to the present invention.

[0099] It is further disclosed that the gene transfer sequence of the present invention provides broad spectrum resistance to tobamoviruses, preferably TMV and ToMV viruses, more preferably ToMV pathotypes 0 and 2.

[0100] The present invention therefore in one embodiment also relates to a derived marker, in particular a derived primer or probe, developed from the amplification products described herein above by methods known in the art, which is genetically linked to a ToBRFV resistance trait locus.

[0101] Breeding methods. In a further embodiment, the present invention provides a method for producing a cultured tomato plant, preferably a cultivated species Solanum lycopersicum plant, that is resistant to ToBRFV infection, comprising: a) crossing a plant according to any one of the preceding embodiments with a cultivated tomato plant lacking the ToBRFV resistance-conferring introgression sequence; b) selecting progeny plants comprising said introgression sequence located on chromosome 1 conferring resistance to ToBRFV, comprising detecting a resistance genotype for at least one of the following SNP markers: i) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; ii) a G genotype that is homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or iii) a G genotype that is homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; thereby producing a plant having enhanced resistance to ToBRFV.

[0102] In a further embodiment, the present invention relates to a method of any of the preceding embodiments, wherein the method comprises: c) selfing the selected progeny or crossing the selected progeny with other tomato plants to produce additional progeny. Further includes:

[0103] In a further embodiment, the present invention relates to a method according to the preceding embodiment, where further progeny are selected and selfed / crossed for an additional 2-10 generations.

[0104] In a further embodiment, the present invention relates to the method of any of the previous embodiments, wherein the plant of step a) is tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, or Solanum galapagense accession LA0483.

[0105] In another embodiment, the present invention relates to a method for providing a ToBRFV resistant tomato plant, plant part or seed, comprising the steps of: a) crossing a first plant lacking a ToBRFV resistance-conferring introgression sequence with a second tomato plant according to any embodiment; b) obtaining progeny tomato plants; and c) optionally selecting said progeny plants, characterized in that they exhibit resistance to ToBRFV.

[0106] In a further embodiment, the method relates to the method of the previous embodiment, wherein the second tomato plant is tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, or Solanum galapagense accession LA0483.

[0107] In another embodiment, the present invention relates to a method for producing a ToBRFV resistant tomato plant, comprising the steps of: a) providing a seed of a tomato plant according to any of the preceding embodiments. b) germinating the seeds and growing mature fertile plants therefrom; c) inducing self-pollination in the plant of a), causing fruit to grow, and harvesting fertile seeds therefrom; and d) growing plants from the seeds harvested in c) and selecting ToBRFV-resistant tomato plants.

[0108] In another embodiment, the present invention relates to a method for increasing resistance of a tomato plant to ToBRFV, the method comprising the steps of: a) selecting tomatoes containing a ToBRFV resistance trait associated with an introgression sequence located on chromosome 1, said trait being identifiable by the presence of a resistance genotype in at least one of the SNP markers listed in Table 4; b) crossing the plant of step a) comprising the ToBRFV resistance trait with a tomato plant, in particular a cultivated tomato plant, that does not comprise the ToBRFV resistance trait and that exhibits a susceptibility to ToBRFV compared to the plant of step a); and c) selecting progeny resulting from said cross that exhibit increased ToBRFV resistance compared to the plants of step b).

[0109] In a further embodiment, the present invention relates to a method of producing an F1 tomato plant exhibiting resistance to ToBRFV, the method comprising the step of crossing an inbred tomato plant, the plant being any one of the preceding embodiments, with a different inbred tomato plant to produce an F1 hybrid progeny.

[0110] How to select. In a further embodiment, the present invention provides a method for identifying cultivated tomato plants, preferably cultivated Solanum lycopersicum plants, exhibiting resistance to ToBRFV and carrying at least one copy of said ToBRFV resistance-conferring introgression sequence, said method comprising the steps of detecting at least one resistance genotype for at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; b) a G genotype in the homozygous state for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) a G genotype that is homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; Thereby, tomato plants exhibiting resistance to ToBRFV are identified.

[0111] In a further embodiment, the present invention relates to the method of the preceding embodiment, further comprising selecting a tomato plant comprising said one or more resistance genotypes at corresponding SNP markers; and crossing the selected tomato plant with a second tomato plant to produce a progeny tomato plant comprising at least one resistance genotype for at least one of said SNP markers and exhibiting resistance to ToBRFV.

[0112] In another embodiment, the present invention relates to a method for identifying a tomato plant comprising a ToBRFV resistance conferring introgression sequence of the present invention, said method comprising the steps of: a) providing a population segregating for a ToBRFV resistance trait; b) screening the segregating population for elements exhibiting resistance to ToBRFV, said trait being identifiable by the presence of a ToBRFV resistance-conferring introgression sequence of the invention; c) selecting an element of the segregating population, said element comprising a ToBRFV resistance trait.

[0113] In a further embodiment, the present invention provides a method for identifying a cultured tomato plant comprising an introgression sequence on chromosome 1 that confers resistance to ToBRFV, the method comprising: a) providing a population segregating for ToBRFV resistance; b) screening said population with a kit that detects at least one resistance genotype for at least one of the SNP markers listed in Table 4; and c) identifying plants that contain at least one resistance genotype for at least one SNP marker selected from the list in Table 4; Includes.

[0114] In a further embodiment, the present invention provides a method for identifying a tomato source of a ToBRFV resistance trait on chromosome 1, the method comprising: a) providing a tomato accession or a number of tomato accessions; b) screening the tomato accession or tomato accessions with a kit that detects at least one resistance genotype for at least one of the SNP markers listed in Table 4; and c) identifying wild tomato accessions that contain said one resistance genotype for at least one SNP marker selected from the list in Table 4; Includes.

[0115] In yet another embodiment, the present invention relates to the use of at least one SNP marker amplified from the genome of a tomato plant according to any of the preceding embodiments, preferably from the genome of tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or its descendants or ancestors, or Solanum galapagense accession LA0483, for identifying a tomato plant comprising and exhibiting a ToBRFV resistance trait, wherein said SNP marker is identified using one of the SNP markers listed in Table 4, and wherein said SNP marker is indicative of the presence of a ToBRFV resistance trait in the tomato plant.

[0116] In a further embodiment, the present invention relates to a method for assessing the genotype of a cultivated tomato plant, preferably a Solanum lycopersicum plant, exhibiting resistance to ToBRFV, comprising: a) providing a sample from said plant; and b) detecting in said sample a QTL locus located on chromosome 1 and associated with said ToBRFV resistance, said QTL locus being flanked by SNP markers 1 and 5 and at least one of the following SNP markers: i) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; ii) G genotype, homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO:11; iii) a G genotype that is homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; and / or iv) any other DNA markers associated with the QTL locus flanking SNP markers 1 and 5. Includes.

[0117] In a further embodiment, the present invention relates to a method for identifying an introgression sequence associated with increased resistance to ToBRFV in a cultivated tomato plant, preferably in a cultivated Solanum lycopersicum plant, said method comprising detecting in said plant an allele of at least one DNA marker genetically linked to a QTL locus associated with increased resistance to ToBRFV, wherein said allele is located within 10 cM, preferably within 5 cM, of said QTL locus located on chromosome 1 in a genomic region flanked by SNP markers 1 and 5.

[0118] In a further embodiment, the present invention relates to the method of the previous embodiment, wherein said QTL locus can be identified by at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO:6; b) a G genotype in the homozygous state for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) G genotype in the homozygous state for SNP marker 4 at the position corresponding to position 147 in SEQ ID NO:16.

[0119] In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein said method further comprises the step of selecting a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, comprising said introgression sequence.

[0120] In a further embodiment, the present invention relates to a method for identifying a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, exhibiting high resistance to ToBRFV by identifying a QTL associated with high resistance to ToBRFV, comprising: a) detecting at least one DNA marker from a tomato plant linked to a chromosomal interval associated with high resistance to ToBRFV, said chromosomal interval being flanked on both sides by SNP markers having at least 80% sequence identity to SEQ ID NOs: 1 and 21; and b) identifying said tomato plants containing said at least one DNA marker. Includes.

[0121] use. The present invention also relates to the use of ToBRFV-resistant propagation material obtained from a tomato plant according to any of the preceding embodiments for growing a tomato plant to produce a ToBRFV-resistant tomato plant, wherein said ToBRFV resistance can be assessed in a standard assay, in particular the assay described in Example 2 below.

[0122] The present invention also relates to the use of ToBRFV-resistant propagation material obtained from a tomato plant according to any of the preceding embodiments for producing tomato fruits.

[0123] In another embodiment, the present invention relates to the use of a cultivated tomato plant, plant part or seed, more preferably a cultivated Solanum lycopersicum plant, plant part or seed, according to any of the previous embodiments, for growing the plant and for producing and harvesting a crop and / or fruit.

[0124] In another embodiment, the present invention relates to the use of a cultivated tomato plant, more preferably a cultivated Solanum lycopersicum plant, as described in any of the previous embodiments, for producing fruits for the fresh market or for food processing.

[0125] In another embodiment, the present invention relates to the use of a cultivated tomato plant, plant part or seed, preferably a cultivated Solanum lycopersicum plant, plant part or seed according to any of the previous embodiments, wherein said cultivated tomato plant, plant part or seed, preferably a cultivated Solanum lycopersicum plant, plant part or seed, is of tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof.

[0126] In a further embodiment, the present invention relates to the use of a cultivated tomato plant, plant part or seed, more preferably a cultivated Solanum lycopersicum plant, plant part or seed, according to any of the previous embodiments, for sowing in a field, a greenhouse or a plastic greenhouse.

[0127] In a further embodiment, the invention relates to the use of a tomato plant according to any of the preceding embodiments for conferring an increased ToBRFV resistance trait to a tomato plant lacking said trait. The invention further relates to the use of a tomato plant according to any of the preceding embodiments for introgressing an increased ToBRFV resistance trait into a tomato plant lacking said trait. The invention further relates to the use of a tomato plant according to any of the preceding embodiments for introgressing an increased tobamovirus resistance trait into a tomato plant lacking said trait. The invention further relates to the use of a tomato plant according to any of the preceding embodiments for introgressing an increased ToMV pathotype 0 and 2 resistance trait into a tomato plant lacking said trait.

[0128] In a further embodiment, the present invention relates to the use of any of SEQ ID NOs: 1-25 for screening a population of tomato plants for the presence of a QTL locus located on chromosome 1 and associated with increased ToBRFV resistance.

[0129] In a further embodiment, the present invention relates to the use of SEQ ID NOs: 6, 11 and / or 16 for screening a population of tomato plants for the presence of a QTL locus located on chromosome 1 and associated with increased ToBRFV resistance.

[0130] Based on the present description, one of skill in the art who possesses tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB Accession No. 43938) containing the introgression gene sequence described herein, or a progeny thereof, will have no difficulty in introducing the introgression gene sequence of the present invention into other tomato plants of various types by using breeding techniques well known in the art with the aid of the SNP markers disclosed herein.

[0131] Seed deposit details The applicant has made a deposit of 625 seeds of tomato plant 19TEP080670 under NCIMB accession number 43938 on 11 February 2022 with NCIMB (National Collection of Industrial, Food and Marine Bacteria, NCIMB Limited, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Scotland).

[0132] The Applicant requests that, having chosen an expert solution, the deposited material shall be made available only to experts in accordance with Rule 32(1) EPC or corresponding regulations of other countries or treaties (expert witness clause) until notice of grant of the patent has been published or, if the application is refused, withdrawn or deemed withdrawn, for a period of 20 years from the filing date.

[0133] Tomato hybrid plant 19TEP080670 is heterozygous for the ToBRFV resistance QTL on chromosome 1, i.e., tomato plant 19TEP080670 contains one copy of the introgression sequence on chromosome 1 associated with increased ToBRFV resistance. EXAMPLES

[0134] Example 1: Germplasm and population generation Solanum galapagense accession number LA0483 was identified in a ToBRFV screen as exhibiting increased ToBRFV resistance and was therefore used as the source for the creation of the discovery population.

[0135] The F1 population resulting from a cross between LA0483 and the susceptible elite line 16TEP070330 was self-pollinated to obtain 20 F2 lines.

[0136] One of these F2 lines, 20TEP070778, was used for QTL mapping based on genotyping and phenotyping data of individual F2 plants. A total of 443 F2 plants were sampled and genotyped to establish a genetic map of the population. In parallel, a subset of 349 plants was mechanically inoculated on seedlings with a ToBRFV strain and evaluated according to the qualitative phenotypic scores described in Examples 2A-C below. A QTL associated with increased ToBRFV resistance located on chromosome 1 was identified in this F2 population (see Examples 3-5).

[0137] One progeny of the initial tomato cross, 19TEP080670, was retained and deposited at NCIMB under NCIMB accession number 43938 on February 11, 2022. Tomato plant 19TEP080670 is heterozygous for the ToBRFV resistance trait, i.e., it contains one copy of the ToBRFV resistance-conferring introgression sequence from LA0483.

[0138] Example 2: Protocol. Example 2A. ToBRFV Isolates. ToBRFV strains were collected in Israel in 2015 and then preserved on dried tomato leaves. From 1 g of these tomato leaves, the preparation of the inoculum was carried out by crushing the leaves and adding 5 ml of buffer, 0.1 g of charcoal, and 0.1 gr of carborundum (abrasive powder). The resulting solution was used to mechanically inoculate the cotyledons of the susceptible material. The trays with the seedlings were kept in a growth chamber at 18 °C night / 24 °C day for 10-14 days to increase the inoculum dose. The day length was fixed at 15 h under full light (approximately 10000 lux). Symptomatic leaves were then collected from the seedlings and stored in a freezer at -80 °C. This storage is considered as the source of the inoculum. Following this preparation, a first step of calibration was carried out to establish lower concentrations of the inoculum, which infected 100% of the susceptible controls without escape. The best dilution meeting this requirement was fixed at 1 / 2 (1 g of virus leaves in 10 ml buffer) and used for the phenotyping experiments.

[0139] Example 2B. Plant preparation and inoculation. The plants were inoculated with the ToBRFV strain using an artificial method. Seeds of the F2 line were sown in specific trays and placed in a growth room in Saliens, France. In addition, 24 seeds of the parental lines 16TEP070330 and LA0483, the resulting F1 plant 19TEP080670 and the susceptible control 16TEP071665 were sown to be used as checks. Four days before inoculation, 349 plants of the F2 and 16 plants of the check were selected for inoculation according to the germination rate. For each material tested, one plant was transplanted to another support. The plants in the trays were inoculated 14 days after sowing (at two true leaves) using the ToBRFV inoculum prepared as described above. Inoculation was performed by gently rubbing the leaves twice. To evaluate the inoculation effect, the transplanted plants were inoculated without the virus (mock).

[0140] Example 2C. Scoring of ToBRFV resistance. The first symptoms, such as leaf distortion and mosaic, appeared 16-19 days post inoculation (dpi). If the susceptibility check was fully symptomatic, in this case at 20 and 27 dpi, the plants were monitored and symptoms were assessed. The plants were scored on a qualitative scale as described below and illustrated in Figure 1. Evaluation Symptoms HR(9) Healthy plant with no symptoms. Lower symptom expression compared to IR(5) sensitivity S(1) Susceptible plants with severe leaf distortion.

[0141] Example 2D. Methods to identify QTLs and corresponding introgression sequences underlying ToBRFV increased resistance traits.

[0142] For QTL discovery, 349 plants of the "LA0483x16TEP070330" population were genotyped with 191 genetic markers spanning the genome. These plants were grown and evaluated for ToBRFV as described above in Examples 2A-C.

[0143] QTL detection was performed using the R / qtl package in the R statistical framework. First, genotype probabilities were calculated using the function "calc.genoprob" (step 1cM). Haley-Knott regression was performed to approximate the standard interval mapping results. Then, the function "stepwiseqtl" was called, which results in a fully automated model selection forward / backward algorithm. The LOD threshold for the main effects was determined by 10,000 permutations. The final QTL model was fitted using the function "fitqtl" and the function "qtlStats" to obtain estimates of QTL effects.

[0144] Example 3: Identification of one QTL associated with increased ToBRFV resistance One QTL was identified based on the ToBRFV resistance phenotype from the F2 population. Table 1 shows the chromosomal location, the effect of the QTL measured as the LOD score, and the percentage of variation explained by the QTL on chromosome 1 for ToBRFV resistance. The QTL showed a recessive effect in the F2 population.

[0145] [Table 1]

[0146] Example 4: Evaluation of ToBRFV resistance in a panel of tomato plants Solanum galapagense accession LA0483 is a wild tomato plant with odorous leaves, reproductive habit, and small orange hairy fruits. Susceptible material 16TEP070330 is an elite accession from an indeterminate cluster tomato type for passive greenhouse cultivation.

[0147] The LA0483 source showed strong resistance to ToBRFV, whereas the 16TEP070330 susceptible parent and the F1 resulting from the "LA0483x16TEP070330" cross show a severe susceptibility phenotype. F2 plants carrying a gene sequence associated with increased resistance to ToBRFV present on chromosome 1 of the Solanum galapagense accession LA0483 in homozygous state showed a strong resistance phenotype. On the other hand, F2 plants that are heterozygous in this same genomic region or that carry two copies of the susceptible 16TEP070330 allele show a susceptibility phenotype.

[0148] The phenotyping results, together with the results of testing for the presence or absence of representative markers at QTL1, are summarized in Table 2 below.

[0149] [Table 2]

[0150] The existing susceptible check plant (no. 2) and the F1 plant (no. 3) and F2 lines containing 0 (no. 4 and 5) or 1 copy (no. 6 and 7) of the introgression sequence at QTL1 showed a susceptible phenotype. On the other hand, the original donor plant (no. 1) and F2 lines (no. 8 and 9) containing 2 copies of the introgression sequence at QTL1 with resistance genotypes at SNP markers spanning the introgression sequence showed increased ToBRFV resistance and achieved high resistance scores.

[0151] Within the QTL1 region, three SNP markers within the QTL interval, ST4450, ST1840 and ST4079 (SNP markers 2, 3 and 4, respectively), showed specificity for the selection of donor resistance alleles from resistant donors, and from them, SNP marker ST1840 (SNP marker 3) was most closely associated with resistance.

[0152] Table 3 shows both the genetic and physical location of the QTL on chromosome 1, as well as the locations of three SNP markers closely associated with the QTL. The physical locations are provided relative to the public genome assembly of Heinz 1706 SL4.0 (released September 2019, https: / / solgenomics.net / ).

[0153] [Table 3]

[0154] Example 5: Sequence and SNP marker information of QLT1 The sequence information of SNP markers 1 to 5 (ST2752, ST4450, ST1840, ST4079 and ST0435A) is summarized in Table 4 below.

[0155] [Table 4]

[0156] As an example, SNP marker 1 (ST2752) at position 82,311,506 bp on chromosome 1 (based on the Heinz 1706 SL4.0 assembly) is characterized by a specific sequence polymorphism (resistant donor allele vs. susceptible allele) at position 47 of the target sequence of SEQ ID NO:1.

[0157] Example 6: Evaluation of resistance to other tobamoviruses In addition to being screened for ToBRFV resistance, Solanum galapagense accession LA0483 was also challenged against other tobamoviruses, such as TMV and ToMV pathotypes 0 and 2. This accession was found to provide strong resistance, for example, to ToMV pathotypes 0 (aucuba) and 2. It is hypothesized that QTL1 of the present invention is not only involved in ToBRFV resistance, but also in broad-spectrum resistance to tobamoviruses. The phenotyping results are summarized in Table 5 below and FIG. 3.

[0158]

Table 5

[0159] References ·Luria et al., 2017, A new Israeli Tobamovirus isolate infects tomato plants harboring Tm-22 resistance genes, PLoS ONE 12(1): e0170429. doi:10.1371 / journal.pone.0170429 ·Salem et al., 2016, A new tobamovirus infecting tomato crops in Jordan, Arch. Virol. 161: 503 - 506. ·Zinger et al., 2021, Identification and mapping of tomato genome loci controlling tolerance and resistance to Tomato Brown Rugose Fruit Virus, Plants 10(1): 179. doi:10.3390 / plants10010179.

Claims

1. 1. A cultivated tomato plant, preferably a Solanum lycopersicum plant, that is resistant to ToBRFV infection, comprising in its genome an introgression sequence from Solanum galapagense that confers resistance to ToBRFV, said introgression sequence being located on chromosome 1 and comprising at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO: 6; b) a G genotype that is homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) G genotype, homozygous for SNP marker 4 at the position corresponding to position 147 in SEQ ID NO:

16.

2. a) the A genotype for SNP marker 2 is identifiable in PCR by amplification of a nucleic acid fragment with a pair of oligonucleotide primers: a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 10, and a probe of SEQ ID NO: 8; b) the G genotype for SNP marker 3 is identifiable in PCR by amplification of a nucleic acid fragment with a pair of oligonucleotide primers: a forward primer of SEQ ID NO: 12 and a reverse primer of SEQ ID NO: 15, and a probe of SEQ ID NO: 13; and / or c) the G genotype for SNP marker 4 is identifiable in PCR by amplification of a nucleic acid fragment with a pair of oligonucleotide primers: a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 20, and a probe of SEQ ID NO: 18; The plant of claim 1.

3. 2. The plant of claim 1, wherein the introgression sequence comprises at least one of SEQ ID NO: 6, SEQ ID NO: 11, and / or SEQ ID NO: 16, or a sequence that is at least 80% identical to one or more of the foregoing sequences.

4. 2. The plant of claim 1, wherein the introgression sequence is contained in tomato plant 19TEP080670 (a representative seed of which has been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof.

5. 2. The plant of claim 1, wherein the plant is obtained by crossing tomato plant 19TEP080670 (representative seeds of which have been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, or Solanum galapagence accession LA0483, with a tomato plant that does not contain the ToBRFV resistance-conferring introgression sequence.

6. 10. The plant of claim 1, wherein the plant is an inbred, dihaploid, diploid, or hybrid plant.

7. Tomato plant 19TEP080670, a representative seed of which has been deposited under NCIMB accession number 43938.

8. A plant part of the plant according to claim 1.

9. A seed that produces a plant part of the plant of claim 1 or the plant of claim 8.

10. 1. A method for producing a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, that is resistant to ToBRFV infection, comprising the steps of: a) crossing the plant of any one of claims 1 to 7 with a cultivated tomato plant lacking the ToBRFV resistance-conferring introgression sequence; b) selecting progeny plants comprising said introgression sequence located on chromosome 1 that confers resistance to ToBRFV, comprising detecting at least one of the following SNP markers: i) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO: 6; ii) a G genotype that is homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or iii) G genotype, homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; Including, thereby producing plants with enhanced resistance to ToBRFV.

11. c) selfing the selected progeny or crossing the selected progeny with other tomato plants to produce additional progeny. The method of claim 10 further comprising:

12. The method of claim 11, wherein further progeny are selected and selfed / crossed for 2 to 10 generations.

13. 11. The method of claim 10, wherein the plant of step a) is tomato plant 19TEP080670 (representative seeds of which have been deposited under NCIMB accession number 43938), or a descendant or ancestor thereof, or Solanum galapagense accession LA0483.

14. 10. A method for producing an F1 tomato plant that exhibits resistance to ToBRFV, the method comprising the step of crossing an inbred tomato plant, the plant being the plant of any one of claims 1 to 7, with a different inbred tomato plant to produce F1 hybrid progeny.

15. 1. A method for identifying cultivated tomato plants, preferably cultivated Solanum lycopersicum plants, that are resistant to ToBRFV infection and that carry at least one copy of a ToBRFV resistance-conferring introgression sequence, comprising detecting at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO: 6; b) a G genotype that is homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) a G genotype that is homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; This method identifies tomato plants that exhibit resistance to ToBRFV.

16. 16. The method of claim 15, further comprising the steps of selecting a tomato plant that contains the one or more SNP markers, and crossing the selected tomato plant with a second tomato plant to produce a progeny tomato plant that contains at least one of the SNP markers and exhibits resistance to ToBRFV.

17. 10. A method of producing tomato seeds, comprising growing a tomato plant from the seed of claim 9 and causing the plant to produce additional tomato seeds.

18. 1. A method for assessing the genotype of cultivated tomato plants, preferably cultivated Solanum lycopersicum plants, that exhibit resistance to ToBRFV, comprising the steps of: a) providing a sample from said plant; and b) detecting in said sample a QTL locus located on chromosome 1 and associated with said ToBRFV resistance, said QTL locus being flanked by SNP markers 1 and 5 and at least one of the following SNP markers: i) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO: 6; ii) G genotype, homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; iii) a G genotype that is homozygous for SNP marker 4 at a position corresponding to position 147 in SEQ ID NO: 16; and / or iv) Any other DNA markers associated with the QTL locus that are adjacent to SNP markers 1 and 5.

19. 1. A method for identifying an introgression sequence associated with increased resistance to ToBRFV in cultivated tomato plants, preferably cultivated Solanum lycopersicum plants, comprising detecting in said plant an allele of at least one DNA marker genetically linked to a QTL locus associated with said increased resistance to ToBRFV, wherein said allele is located within 10 cM, preferably within 5 cM, of said QTL locus located on chromosome 1 in a genomic region flanked by SNP markers 1 and 5.

20. 20. The method of claim 19, wherein the QTL locus can be identified by at least one of the following SNP markers: a) A genotype that is homozygous for SNP marker 2 at a position corresponding to position 79 in SEQ ID NO: 6; b) a G genotype that is homozygous for SNP marker 3 at a position corresponding to position 51 in SEQ ID NO: 11; and / or c) G genotype, homozygous for SNP marker 4 at the position corresponding to position 147 in SEQ ID NO:

16.

21. 21. The method of claim 20, further comprising the step of selecting a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, comprising the introgression sequence.

22. 1. A method for identifying a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, exhibiting high resistance to ToBRFV by identifying a QTL associated with said high resistance to ToBRFV, comprising the steps of: a) detecting at least one DNA marker from a tomato plant linked to a chromosomal interval associated with high resistance to ToBRFV, wherein the chromosomal interval is flanked on both sides by SNP markers having at least 80% sequence identity to SEQ ID NOs: 1 and 21; and b) identifying said tomato plants containing said at least one DNA marker A method comprising:

23. 1. A method for identifying a wild tomato source for a ToBRFV resistance trait on chromosome 1, comprising: a) providing a wild tomato accession or a plurality of wild tomato accessions; b) screening the wild tomato accession or accessions with a kit that detects at least one of the SNP markers listed in Table 4 , and c) identifying wild tomato accessions containing said at least one SNP marker selected from the list in Table 4; A method comprising:

24. Use of any of SEQ ID NOs: 6, 11, or 16 to screen a population of tomato plants for the presence of a QTL locus located on chromosome 1 and associated with increased ToBRFV resistance.

Images