Novel tomato plants with tobrfv resistance

Abstract

The present invention relates to tomato plants comprising two copies of an allele associated with an increased resistance to ToBRFV infection. The present invention also relates to seeds and parts of said plants, for example fruits. The present invention further relates to methods of making, identifying, and using such seeds and plants.

Description

[0001] NOVEL TOMATO PLANTS WITH TOBRFV RESISTANCE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to tomato plants comprising two copies of an allele associated with an increased resistance to ToBRFV infection. The present invention also relates to seeds and parts of said plants, for example fruits. The present invention further relates to methods of making, identifying, and using such seeds and plants.

[0004] BACKGROUND OF THE INVENTION

[0005] Viruses, such as those belonging to the Tobamovirus genus, cause considerable damage to tomato growers, irrespective of the tomato types and cultivation methods. Infections by Tobamoviruses (e.g., by Tobacco Mosaic Virus (TMV), Tomato Mosaic Virus (ToMV) or Tomato Mild Mottle Virus (ToMMV)) typically spread rapidly across fields, either via mechanical transmission through field workers, tools and manual handling of plants and fruits, or via natural vectors such as insects, which are equally challenging to control.

[0006] In 2014 and 2015, a new Tobamovirus was first reported on tomatoes 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 caused to the crop, namely the deformation of fruits with yellow and / or brown areas. This ToBRFV outbreak is a particular concern for the tomato industry because the virus was shown to be able to overcome the tobamovirus resistance gene Tm-22. There is therefore a need for solutions for the grower and the seed industry to be able to grow ToBRFV resistant tomato crops.

[0007] Possible sources for ToBRFV resistance have been described in the patent and nonpatent literature.

[0008] For instance, WO 2018 / 219941 reports alleles located on chromosomes 6, 9 and 11 , while WO 2019 / 110821 , WO 2020 / 018783, WO 2021 / 110855, WO 2021 / 170868 and WO 2022 / 018734 also disclose alleles or genes located on chromosome 11. WO 2020 / 148021 and WO 2022 / 013452 report an allele or gene located on chromosome 8 while WO 2022 / 091104 reports a modified gene located on chromosome 9.

[0009] Finally, WO 2021 / 213892 discloses an alternative (maybe duplicated) allele of a known resistance gene, tm-1, located on chromosome 2 while WO 2022 / 018734 reports a QTL on that same chromosome 2.

[0010] Despite the number of described loci, alleles and genes, it is yet unclear what (combination of) alleles or genes will effectively provide resistance to ToBRFV in the fields because the mechanisms underlying the ToBRFV infection have yet to be determined. For instance, Zinger et al. suggest that a locus located on chromosome 2 interacts with a locus located on chromosome 11 to control ToBRFV resistance (Zinger et al., 2021 ).

[0011] Consequently, there is a need for alternative solutions to further improve ToBRFV control in plants, especially in tomato plants.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention addresses the need for an improved resistance to ToBRFV by providing novel tomato plants comprising an increased ToBRFV resistance trait. The presence of this novel allele associated with increased ToBRFV resistance in elite tomato plants greatly enhanced the ToBRFV resistance capability, which has a positive impact on overall plant performance. The ToBRFV resistance allele, located on chromosome 2, is of a semi-dominant nature, but two copies of the allele are required to provide the most improved ToBRFV resistance phenotype.

[0014] Altogether, the characteristics of the improved ToBRFV resistant tomato plant disclosed within the present invention provide a tomato grower with a novel solution to enhance economic and commercial efficiency when deploying tomato varieties in a ToBRFV pressured field.

[0015] In a first embodiment, the invention provides a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection, comprising in its genome two copies of a ToBRFV resistance allele located on chromosome 2, wherein said ToBRFV resistance allele encodes a protein having at least 99% amino acid sequence identity with SEQ ID NO: 1 and wherein said protein has an Arginine at a position corresponding to position 459 in SEQ ID NO: 1 .

[0016] In a second embodiment, the invention provides a plant according to the first embodiment, wherein said ToBRFV resistance allele encodes the protein of SEQ ID NO: 1 .

[0017] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele has at least 99% nucleotide sequence identity with SEQ ID NO: 5 and a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5.

[0018] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele further comprises at least one of the following SNP markers: a) a T genotype in the homozygous state at a position corresponding to position 114 in SEQ ID NO: 5; b) a G genotype in the homozygous state at a position corresponding to position 321 in SEQ ID NO: 5; c) a C genotype in the homozygous state at a position corresponding to position 813 in SEQ ID NO: 5; d) an A genotype in the homozygous state at a position corresponding to position 1593 in SEQ ID NO: 5; e) a T genotype in the homozygous state at a position corresponding to position 1815 in SEQ ID NO: 5; f) a G genotype in the homozygous state at a position corresponding to position 1950 in SEQ ID NO: 5; and / or, g) a T genotype in the homozygous state at a position corresponding to position 1971 in SEQ ID NO: 5.

[0019] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele has the nucleotide sequence of SEQ ID NO: 5. In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele is as comprised in tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

[0020] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said plant is obtained by crossing tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof, with a tomato plant that does not contain said ToBRFV resistance allele.

[0021] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said plant is an inbred, a dihaploid, a diploid, or a hybrid plant.

[0022] In a further embodiment, the invention provides a plant of tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132.

[0023] In a further embodiment, the invention provides a plant part of a plant according to any one of the previous embodiments.

[0024] In a further embodiment, the invention provides a seed that produces a plant or a plant part according to any one of the previous embodiments.

[0025] In a further embodiment, the invention provides a method for producing a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection comprising the steps of a) crossing a plant according to any one of the previous embodiments with a cultivated tomato plant lacking said ToBRFV resistance allele; b) selecting a progeny plant comprising said ToBRFV resistance allele located on chromosome 2, said selecting step comprising detecting a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5; thereby producing a plant with increased resistance to ToBRFV.

[0026] In a further embodiment, the invention provides a method according to the previous embodiment, wherein the method further comprises: c) selfing the selected progeny or crossing the selected progeny with another tomato plant to produce further progeny.

[0027] In a further embodiment, the invention provides a method according to the previous embodiment, wherein further progeny is selected and selfed / crossed for 2 to 10 more generations.

[0028] In a further embodiment, the invention provides a method according to any one of the previous embodiments wherein the plant of step a) is tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

[0029] In a further embodiment, the invention provides a method or producing a F1 tomato plant exhibiting increased resistance to ToBRFV, the method comprising crossing an inbred tomato plant, which is a plant according to any one of the previous embodiments, with a different inbred tomato plant to produce F1 hybrid progeny.

[0030] In a further embodiment, the invention provides a method for identifying a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection and having two copies of a ToBRFV resistance allele having at least 99% sequence identity with SEQ ID NO: 5, said method comprising the step of a) detecting a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5; thereby identifying a tomato plant exhibiting resistance to ToBRFV.

[0031] In a further embodiment, the invention provides a method according to the previous embodiment, wherein said method further comprises selecting a tomato plant comprising said genotype and crossing the selected tomato plant with a second tomato plant to produce progeny tomato plants that comprise a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5 and exhibits resistance to ToBRFV.

[0032] In a further embodiment, the invention provides a method of producing tomato seed, the method comprising growing a tomato plant from the seed of a previous embodiment and allowing the plant to produce further tomato seed. BRIEF DESCRIPTION OF THE DRAWINGS.

[0033] Figure 1 . ToBRFV pathology assay pictures representative of the disease scale used and described in Example 2C. (a) Rating Susceptible (S or 1 ); (b) Rating Intermediate Resistance (IR or 5); (c) Rating High Resistance (HR or 9).

[0034] Figure 2. Results of a ToBRFV pathology assay carried out with a susceptible check (a) and the 3T304 plant (b).

[0035] Figure 3. Sequence alignments of the Tm-1 nucleic acid coding sequences in the following plants: Solanum lycopersicum Heinz (Solyc02g062560), Solanum habrochaites PI126445 (GCR237, Ishibashi et al., 2007), tomato PAR02001 (WO 2021 / 213892), and Solanum lycopersicum 3T304. SNPs specific to the 3T304 allelic sequence are highlighted in grey and indicated with an arrow. Conserved nucleotides are indicated with a star.

[0036] Figure 4. Percent identity matrix of the nucleic acid coding sequences of Figure 3.

[0037] Figure 5. Sequence alignments of the Tm-1 protein sequences in the following plants: Solanum lycopersicum Heinz (Solyc02g062560), Solanum habrochaites PI126445 (GCR237, Ishibashi et al., 2007), tomato PAR02001 (WO 2021 / 213892), and Solanum lycopersicum 3T304. One amino acid specific to the 3T304 protein sequence is highlighted in grey and indicated with an arrow. Conserved amino acids are indicated with a star.

[0038] Figure 6. Percent identity matrix of the protein sequences of Figure 5.

[0039] BRIEF DESCRIPTION OF THE SEQUENCES.

[0040] SEQ ID NO: 1 : Amino acid sequence encoded by Solanum lycopersicum 3T304 Tm-1 allele.

[0041] SEQ ID NO: 2: Amino acid sequence encoded by Solanum lycopersicum Heinz (Solyc02g062560) Tm-1 allele.

[0042] SEQ ID NO: 3: Amino acid sequence encoded by Solanum habrochaites PI126445 Tm-1 allele.

[0043] SEQ ID NO: 4: Amino acid sequence encoded by tomato PAR02001 Tm-1 allele.

[0044] SEQ ID NO: 5: Nucleic acid sequence encoding SEQ ID NO:1. SEQ ID NO: 6: Nucleic acid sequence encoding SEQ ID NO:2.

[0045] SEQ ID NO: 7: Nucleic acid sequence encoding SEQ ID NO:3.

[0046] SEQ ID NO: 8: Nucleic acid sequence encoding SEQ ID NO:4.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] DEFINITIONS

[0049] The technical terms and expressions used within the scope of this application are generally to be given the meaning commonly applied to them in the pertinent art of plant breeding and cultivation if not otherwise indicated herein below.

[0050] 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.

[0051] A “cultivated tomato” or an “elite tomato” plant is understood within the scope of the invention to refer to a plant that is no longer in the natural state but has been developed and domesticated by human care and for agricultural use and / or human consumption, and excludes wild tomato accessions, such as Solanum habrochaites accessions. As a matter of example, in embodiments, a cultivated or elite tomato plant according to the present invention is capable of growing edible fruits. Alternatively, or additionally, the cultivated tomato plant is a hybrid plant. Alternatively, or additionally, the cultivated tomato plant is a cultivated Solanum lycopersicum plant.

[0052] An “allele” is understood within the scope of the invention to refer to alternative or variant forms of various genetic units identical or associated with different forms of a gene or of any kind of identifiable genetic determinant such as an allele, which are alternative in inheritance because they are situated at the same locus in homologous chromosomes. Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, chemical or structural modification, transcription regulation or post-translational modification / regulation. 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.

[0053] Relatively speaking, the term “improved ToBRFV resistance” or “increased ToBRFV resistance” is herein understood to mean that a plant according to the present invention, e.g., comprising an allelic sequence as defined in the claims, preferably comprising the allelic sequence of SEQ ID NO: 5, conferring resistance to ToBRFV, wherein said sequence is located on chromosome 2 and comprises at least one of the nine SNPs specific to 3T304 allelic sequence, is more resistant to ToBRFV when compared with a plant lacking said sequence.

[0054] "Improved ToBRFV resistance" is understood within the scope of the invention to mean a tomato plant which has a statistically significant improved resistance to ToBRFV compared to a control tomato plant lacking the sequence of the invention (for example as described in the Example section), for instance using standard error and / or at P < 0.05 or P < 0.01 using Student’s test and / or a Lod score > 3.

[0055] “Phenotype” is understood within the scope of the invention to refer to a distinguishable characteristic(s) of a genetically controlled trait.

[0056] A "control tomato plant" is understood within the scope of the invention to mean a tomato plant that has substantially the same genetic background as the cultivated tomato plant of the present invention wherein the control plant does not have the sequence of the present invention linked to improved ToBRFV resistance. A control tomato plant can be a tomato plant belonging to the same plant variety and does not comprise the sequence of the present invention. The control tomato plant is grown for the same length of time and under the same conditions as the cultivated tomato plant of the present invention. Plant variety is herein understood according to definition of IIPOV. Thus, an ideal control tomato plant may be a near-isogenic line, an inbred line or a hybrid provided that they have the same genetic background as the tomato plant of the present invention except the control plant does not have the sequence of the present invention linked to improved ToBRFV resistance.

[0057] The term “trait” in the context of the present invention refers to an improved ToBRFV resistance trait. A trait may be inherited in a dominant or recessive manner, or in a partial, semi-, or incomplete-dominant manner. In the context of the present invention, the ToBRFV resistance-conferring sequence located on chromosome 2 is semi-dominant and provides a stronger effect when present in two copies. A tomato plant of the invention is homozygous for the trait. Furthermore, a trait may be monogenic or polygenic, or may result from the interaction of one or more genes with the environment. In the context of the present invention, the ToBRFV resistance-conferring sequence located on chromosome 2 is sufficient to confer, alone, the improved ToBRFV resistance trait, which is therefore monogenic.

[0058] The terms “hybrid”, “hybrid plant”, and “hybrid progeny” refer to an individual produced from genetically different parents (e.g., a genetically heterozygous or mostly heterozygous individual).

[0059] The term "inbred line" refers to a genetically homozygous or nearly homozygous population. An inbred line, for example, can be derived through several cycles of brother / sister breeding or of selfing or in dihaploid production.

[0060] The term "dihaploid line" refers to stable inbred lines issued from anther culture. Some pollen grains (haploid) cultivated on specific medium and conditions can develop plantlets containing n chromosomes. These plantlets are then "doubled" and contain 2n chromosomes. The progeny of these plantlets is named "dihaploid" and are essentially no longer segregating (stable).

[0061] The term "genetically fixed" refers to a genetic sequence which has been stably incorporated into the genome of a plant that normally does not contain said genetic sequence. When genetically fixed, the genetic sequence can be transmitted in an easy and predictable manner to other plants by sexual crosses.

[0062] The term “rootstock” refers to a plant used as a receptacle for a scion plant. Typically, the rootstock plant and the scion plant are of different genotypes. In embodiments, plants according to the present invention are used as rootstock plants.

[0063] The term "plant" or "plant part' refers hereinafter to a plant part, organ or tissue obtainable from a tomato plant according to the invention, including but not limiting to leaves, stems, roots, flowers or flower parts, fruits, shoots, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic regions, callus tissue, seeds, cuttings, cell or tissue cultures or any other part or product of the plant which still exhibits the improved ToBRFV resistance trait according to the invention, particularly when grown into a plant that produces fruits.

[0064] A "plant" is any plant at any stage of development.

[0065] A tomato plant seed is a seed which grows into a tomato plant according to any of the embodiments.

[0066] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

[0067] "Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

[0068] A "plant organ" is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.

[0069] "Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and / or functional units.

[0070] As used herein, the term “breeding”, and grammatical variants thereof, refer to any process that generates a progeny individual. Breeding can be sexual or asexual, or any combination thereof. Exemplary non-limiting types of breeding include crossings, selfing, doubled haploid derivative generation, and combinations thereof.

[0071] As used herein, the phrase "established breeding population" refers to a collection of potential breeding partners produced by and / or used as parents in a breeding program, e.g., a commercial breeding program. The members of the established breeding population are typically well-characterized genetically and / or phenotypically. For example, several phenotypic traits of interest might have been evaluated, e.g., under different environmental conditions, at multiple locations, and / or at different times. Alternatively, or in addition, one or more genetic loci associated with expression of the phenotypic traits might have been identified and one or more of the members of the breeding population might have been genotyped with respect to the one or more genetic loci as well as with respect to one or more genetic markers that are associated with the one or more genetic loci.

[0072] As used herein, the phrase "diploid individual" refers to an individual that has two sets of chromosomes, typically one from each of its two parents. However, it is understood that in some embodiments a diploid individual can receive its “maternal” and “paternal” sets of chromosomes from the same single organism, such as when a plant is selfed to produce a subsequent generation of plants.

[0073] “Homozygous” is understood within the scope of the invention to refer to like alleles at one or more corresponding loci on homologous chromosomes. In the context of the invention, a tomato plant comprising two identical copies of a particular sequence at a particular locus, e.g., the sequence located on chromosome 2, is homozygous on the corresponding locus.

[0074] “Heterozygous” is understood within the scope of the invention to refer to unlike alleles at one or more corresponding loci on homologous chromosomes.

[0075] A “dominant” allele is understood within the scope of the invention to refer to an allele which determines the phenotype when present in the heterozygous or homozygous state. A “semi-dominant” allele is understood within the scope of the invention to refer to an allele which determines the phenotype when present in the heterozygous or homozygous state. The intensity of the phenotype is however generally higher when the allele is present in the homozygous state.

[0076] A “recessive” allele refers to an allele which determines the phenotype when present in the homozygous state only.

[0077] “Backcrossing” is understood within the scope of the invention to refer to a process in which a hybrid progeny is repeatedly crossed back to one of the parents. Different recurrent parents may be used in subsequent backcrosses.

[0078] “Locus” is understood within the scope of the invention to refer to a region on a chromosome, which comprises a gene, an allele or its corresponding genetic sequence contributing to a trait. As used herein, the phrases "sexually crossed" and "sexual reproduction" in the context of the presently disclosed subject matter refers to the fusion of gametes to produce progeny (e.g., by fertilization, such as to produce seed by pollination in plants). A "sexual cross" or "cross-fertilization" refers to, in some embodiments, fertilization of one individual by another (e.g., cross-pollination in plants). The term "selfing" refers, in some embodiments, to the production of seed by self-fertilization or self-pollination, i.e., pollen and ovule are from the same plant.

[0079] As used herein, the phrase "genetic marker" or “DNA marker” refers to a feature of an individual’s genome (e.g., a nucleotide or a polynucleotide sequence that is present in an individual’s genome) that is associated with one or more loci of interest. In some embodiments, a genetic marker is polymorphic in a population of interest, or the locus occupied by the polymorphism, depending on 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 DNAs (RAPDs), cleaved amplified polymorphic sequence (CAPS) markers, Diversity Arrays Technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs), among many other examples. Genetic markers can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits. The phrase “genetic marker” can also refer to a polynucleotide sequence complementary to a genomic sequence, such as a sequence of a nucleic acid used as probes.

[0080] As used herein, the term "genotype" refers to the genetic constitution of a cell or organism. An individual's "genotype for a set of genetic markers" includes the specific alleles, for one or more genetic marker loci, present in the individual’s haplotype.

[0081] As used herein, the term "progeny" refers to the descendant(s) of a particular cross. Typically, progeny result from breeding of two individuals, although some species (particularly some plants and hermaphroditic animals) can be selfed (i.e., the same plant acts as the donor of both male and female gametes). The descendant(s) can be, for example, of the Fi , the F2, or any subsequent generation. As used herein, the terms "quantitative trait locus" (allele) refer to an association between a genetic marker and a chromosomal region and / or gene and / or sequence that affects the phenotype of a trait of interest. Typically, this is determined statistically, e.g., based on one or more methods published in the literature. An allele can be a chromosomal region and / or a genetic locus with at least two alleles that differentially affect a phenotypic trait.

[0082] The term "recipient tomato plant" is used herein to indicate a tomato plant that is to receive DNA obtained from a donor tomato plant that comprises the improved ToBRFV resistance trait.

[0083] The term "natural genetic background" is used herein to indicate the original genetic background of genetic sequence. For instance, the genetic sequence of the present invention was found at a specific location on chromosome 2 of a tomato plant. Conversely, a method that involves the transfer of DNA, via e.g., breeding, comprising this genetic sequence from chromosome 2 of tomato plant to the same position on chromosome 2 of another cultivated tomato plant, even more preferably a Solanum lycopersicum plant, will result in this genetic sequence not being in its original (initial) genetic background. When the genetic sequence of the present invention is transferred from one tomato plant background into another tomato plant background, preferably a cultivated tomato plant, even more preferably a Solanum lycopersicum plant, they are referred to as “introgressed sequence” or “introgressed genetic sequence”.

[0084] A "donor tomato plant" is understood within the scope of the invention to mean the tomato plant which provides the improved ToBRFV resistance trait.

[0085] “Marker-based selection” is understood within the scope of the invention to refer to e.g. the use of genetic markers to detect one or more nucleic acids from the plant, where the nucleic acid is associated with a desired trait to identify plants that carry alleles for desirable (or undesirable) traits, so that those plants can be used (or avoided) in a selective breeding program.

[0086] A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. A single-nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide — A, T, C, or G — in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes in an individual. For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case there are two alleles: C and T. The basic principles of SNP array are the same as the DNA microarray. These are the convergence of DNA hybridization, fluorescence microscopy, and DNA capture. The three components of the SNP arrays are the array that contains nucleic acid sequences (i.e., amplified sequence or target), one or more labelled 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 may be determined by real-time PCR using double-stranded DNA dyes or the fluorescent reporter probe method.

[0087] “PCR (Polymerase chain reaction)” is understood within the scope of the invention to refer to a method of producing relatively large amounts of specific regions of DNA or subset(s) of the genome, thereby making possible various analyses that are based on those regions. “PCR primer” is understood within the scope of the invention to refer to relatively short fragments of single-stranded DNA used in the PCR amplification of specific regions of DNA.

[0088] “Probe” as used herein refers to a group of atoms or molecules which can recognise and bind to a specific target molecule or cellular structure and thus allowing detection of the target molecule or structure. Particularly, “probe” refers to a labelled DNA or RNA sequence which can be used to detect the presence of and to quantitate a complementary sequence by molecular hybridization.

[0089] “Sequence Identity”. The terms "identical" or "identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with 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 (i.e. , % identity = # of identical positions / total # of positions x 100), considering the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described herein below. For example, sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wl 53711 ). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981 ), 482-489, to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at their pre-set ("default") values. The deviations appearing in the comparison between a given sequence and the above-described sequence of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program “fasta20u66” (version 2.0u66, September 1998 by William R. Pearson and the University of Virginia; see also W.R. Pearson (1990), Methods in Enzymology 183, 63-98, appended examples and http: / / workbench.sdsc.edu / ). For this purpose, the "default" parameter settings may be used.

[0090] EMBODIMENTS

[0091] PLANTS, SEEDS, FRUITS.

[0092] In a first embodiment, the invention provides a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection, comprising in its genome two copies of a ToBRFV resistance allele located on chromosome 2, wherein said ToBRFV resistance allele encodes a protein having at least 99% amino acid sequence identity with SEQ ID NO: 1 and wherein said protein has an Arginine at a position corresponding to position 459 in SEQ ID NO: 1 .

[0093] In a second embodiment, the invention provides a plant according to the first embodiment, wherein said ToBRFV resistance allele encodes the protein of SEQ ID NO: 1 .

[0094] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele has at least 99% nucleotide sequence identity with SEQ ID NO: 5 and a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5.

[0095] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele further comprises at least one of the following SNP markers: a) a T genotype in the homozygous state at a position corresponding to position 114 in SEQ ID NO: 5; b) a G genotype in the homozygous state at a position corresponding to position 321 in SEQ ID NO: 5; c) a C genotype in the homozygous state at a position corresponding to position 813 in SEQ ID NO: 5; d) an A genotype in the homozygous state at a position corresponding to position 1593 in SEQ ID NO: 5; e) a T genotype in the homozygous state at a position corresponding to position 1815 in SEQ ID NO: 5; f) a G genotype in the homozygous state at a position corresponding to position 1950 in SEQ ID NO: 5; and / or, g) a T genotype in the homozygous state at a position corresponding to position 1971 in SEQ ID NO: 5.

[0096] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele has the nucleotide sequence of SEQ ID NO: 5.

[0097] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said ToBRFV resistance allele is as comprised in tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

[0098] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said plant is obtained by crossing tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof, with a tomato plant that does not contain said ToBRFV resistance allele.

[0099] In a further embodiment, the invention provides a plant according to any of the previous embodiments, wherein said plant is an inbred, a dihaploid, a diploid, or a hybrid plant.

[0100] 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.

[0101] In a further embodiment, the tomato plant of the invention is a tomato plant according to any of preceding embodiments, wherein said ToBRFV resistance-conferring sequence located on chromosome 2 can be identified using any of the SNP markers 1 to 9 disclosed in Table 4 hereinbelow.

[0102] In a further embodiment, the invention provides a plant of tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132.

[0103] In a further embodiment, the invention provides a plant part of a plant according to any one of the previous embodiments.

[0104] It is a further embodiment to provide a plant part, organ or tissue obtainable from a tomato plant according to any of preceding embodiments, including but not limiting to leaves, stems, roots, flowers or flower parts, fruits, shoots, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic regions, callus tissue, seeds, cuttings, cell or tissue cultures or any other part or product of the plant which still exhibits the ToBRFV resistance trait according to the invention, particularly when grown into a plant that produces fruits.

[0105] In a further embodiment, the invention provides a seed that produces a plant or a plant part according to any one of the previous embodiments.

[0106] In a further embodiment the 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 invention relates to the use of tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof, as a tomato rootstock.

[0107] In another embodiment is considered the use of a tomato plant, plant part or seed according to any of the preceding embodiments for producing and harvesting tomato fruits.

[0108] In another embodiment the invention relates to the use of a tomato plant, plant part or seed according to any embodiments, wherein the tomato plant, plant part or seed is tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

[0109] In a further embodiment the invention relates to the use of a tomato plant, plant part or seed according to any of the preceding embodiments to sow a field, a greenhouse, or a plastic house.

[0110] In one embodiment, the invention provides tomato fruits produced by a tomato plant according to any of the preceding embodiments.

[0111] The invention further relates to the use of a tomato plant according to any of the preceding embodiments to introgress a ToBRFV resistance trait into a tomato plant lacking said ToBRFV resistance trait.

[0112] ALLELES, GENETIC SEQUENCES, MARKERS.

[0113] The present invention is further directed to an allele associated with ToBRFV resistance trait in the tomato plant. In a further embodiment, the allele of the present invention is located on chromosome 2. In a further embodiment of the present invention, the allele is comprised in, obtained from or obtainable from a donor plant of tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof, and comprising said allele.

[0114] In another embodiment, the allele of the present invention is located on chromosome 2 and is characterized by at least the GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5. The allele of the present invention can further be characterized by at least one of the following additional resistance genotypes at one of the SNP markers selected in the list comprising: a) a T genotype in the homozygous state at a position corresponding to position 114 in SEQ ID NO: 5; b) a G genotype in the homozygous state at a position corresponding to position 321 in SEQ ID NO: 5; c) a C genotype in the homozygous state at a position corresponding to position 813 in SEQ ID NO: 5; d) an A genotype in the homozygous state at a position corresponding to position 1593 in SEQ ID NO: 5; e) a T genotype in the homozygous state at a position corresponding to position 1815 in SEQ ID NO: 5; f) a G genotype in the homozygous state at a position corresponding to position 1950 in SEQ ID NO: 5; and / or, g) a T genotype in the homozygous state at a position corresponding to position 1971 in SEQ ID NO: 5.

[0115] The present invention also discloses the use of at least 1 , 2, 3, 4, 5, 6, 7, 8 or 9 of the SNP markers according to the invention for diagnostic selection and / or genotyping of the ToBRFV resistance trait locus in a tomato plant, particularly a cultivated tomato plant.

[0116] The present invention further discloses the use of at least 1 , 2, 3, 4, 5, 6, 7, 8 or 9 of the SNP markers according to the invention for identifying in a tomato plant, particularly a cultivated tomato plant, more particularly a tomato plant according to the invention, the presence of the ToBRFV resistance trait and / or for monitoring the introgression of the ToBRFV resistance trait in a tomato plant, particularly a cultivated tomato plant, particularly a tomato plant according to the invention and as described herein.

[0117] The present invention therefore further relates in one embodiment to derived markers, particularly to derived primers or probes, developed from an amplification product according to the invention and as described herein above by methods known in the art, which derived markers are genetically linked to the ToBRFV resistance trait locus.

[0118] METHODS OF BREEDING. In a further embodiment, the invention provides a method for producing a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection comprising the steps of a) crossing a plant according to any one of the preceding embodiments with a cultivated tomato plant lacking said ToBRFV resistance-conferring allele; b) selecting a progeny plant comprising said allele located on chromosome 2 conferring resistance to ToBRFV, said selecting step comprising detecting a GT genotype in the homozygous state at a position corresponding to positions 1376- 1377 in SEQ ID NO: 5; thereby producing a plant with enhanced resistance to ToBRFV.

[0119] In a further embodiment, the invention relates to the method of any of the preceding embodiments, wherein the method further comprises: c) selfing the selected progeny or crossing the selected progeny with another tomato plant to produce further progeny.

[0120] In a further embodiment, the invention relates to the method of the preceding embodiment, wherein further progeny is selected and selfed / crossed for 2 to 10 more generations.

[0121] In a further embodiment, the invention relates to the method of any of the preceding embodiments, wherein the plant of step a) is tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

[0122] In another embodiment the invention relates to a method of providing a ToBRFV resistant tomato plant, plant part or seed, wherein said method comprises the following steps: a) Crossing a 1stplant lacking the ToBRFV resistance-conferring allele of the invention with a 2ndtomato plant according to any embodiments, b) Obtaining a progeny tomato plant, and, c) Optionally, selecting a plant of said progeny characterized in that said plant exhibits resistance to ToBRFV.

[0123] In a further embodiment the invention relates to the method of the preceding embodiment wherein the 2ndtomato plant is tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof. In another embodiment the invention relates to a method for producing a ToBRFV resistant tomato plant comprising the following steps: a) Providing seeds of a tomato plant according to any of the preceding embodiments, b) Germinating said seed and growing a mature, fertile plant therefrom, c) Inducing self-pollination of said plant under a), growing fruits and harvesting the fertile seeds therefrom, and d) Growing plants from the seeds harvested under c) and selecting a ToBRFV resistant tomato plant.

[0124] In another embodiment the invention relates to a method for increasing the resistance to ToBRFV of a tomato plant, comprising the steps of: a) selecting a tomato, which comprises a ToBRFV resistance trait associated with one allele located on chromosome 2, wherein said trait can be identified by the presence of a resistance genotype in the homozygous state at least one of the SNP markers listed in Table 4; b) crossing said plant of step a), which comprises a ToBRFV resistance trait, with a tomato plant, particularly a cultivated tomato plant, which does not comprise a ToBRFV resistance trait and shows susceptibility to ToBRFV, as compared to the plant of step a), and c) selecting progeny from said cross which shows increased ToBRFV resistance, as compared to the plant of step b).

[0125] In a further embodiment, the invention relates to a method for producing a F1 tomato plant exhibiting resistance to ToBRFV, the method comprising crossing an inbred tomato plant, which is a plant according to any one of the preceding embodiments, with a different inbred tomato plant to produce F1 hybrid progeny.

[0126] METHODS OF SELECTION.

[0127] In a further embodiment, the invention provides a method for identifying a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, exhibiting resistance to ToBRFV and having two copies of said ToBRFV resistance-conferring allele, said method comprising the step of detecting a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5.

[0128] In a further embodiment, the invention provides a method for identifying a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant, exhibiting resistance to ToBRFV and having two copies of said ToBRFV resistance-conferring allele, said method comprising the step of detecting at least one resistance genotype for at least one of the following SNP markers: a) a T genotype in the homozygous state at a position corresponding to position 114 in SEQ ID NO: 5; b) a G genotype in the homozygous state at a position corresponding to position 321 in SEQ ID NO: 5; c) a C genotype in the homozygous state at a position corresponding to position 813 in SEQ ID NO: 5; d) an A genotype in the homozygous state at a position corresponding to position 1593 in SEQ ID NO: 5; e) a T genotype in the homozygous state at a position corresponding to position 1815 in SEQ ID NO: 5; f) a G genotype in the homozygous state at a position corresponding to position 1950 in SEQ ID NO: 5; and / or, g) a T genotype in the homozygous state at a position corresponding to position 1971 in SEQ ID NO: 5; thereby identifying a tomato plant exhibiting resistance to ToBRFV.

[0129] In a further embodiment, the invention relates to the method of the preceding embodiment, wherein said method further comprises selecting a tomato plant comprising said one or more resistance genotypes at the corresponding SNP markers, and crossing the selected tomato plant with a second tomato plant to produce progeny tomato plants that comprise at least one resistance genotype for at least one of said SNP markers and exhibits resistance to ToBRFV.

[0130] In another embodiment the invention relates to a method of identifying a tomato plant comprising the ToBRFV resistance-conferring introgressed sequence of the invention, wherein said method comprises the steps of: a) providing a population segregating for the ToBRFV resistance trait, b) screening the segregating population for a member exhibiting resistance to ToBRFV, wherein said trait can be identified by the presence of ToBRFV resistance-conferring allele of the invention, c) selecting one member of the segregating population, wherein said member comprises the ToBRFV resistance trait.

[0131] In a further embodiment, the invention provides a method for identifying a cultivated tomato plant comprising an allele on chromosome 2, wherein said allele confers resistance to ToBRFV, comprising: a) providing a population segregating for ToBRFV resistance, b) screening said population using a kit which detects at least one resistance genotype for at least one of the SNP markers listed in Table 4, and, c) identifying a plant comprising at least one resistance genotype for at least one SNP marker selected in the list of Table 4.

[0132] In a further embodiment, the invention provides a method for identifying a tomato source of ToBRFV resistance trait on chromosome 2, comprising: a) providing a tomato accession or a plurality of tomato accessions, b) screening said tomato accession or plurality of tomato accessions using a kit which detects at least one resistance genotype for at least one of the SNP markers listed in Table 4, and, c) identifying a wild tomato accession comprising said one resistance genotype for at least one SNP marker selected in the list of Table 4.

[0133] In yet another embodiment, the 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 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof, 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 the ToBRFV resistance trait in a tomato plant, to identify a tomato plant that comprises and exhibits the ToBRFV resistance trait. In a further embodiment, the invention relates to a method for assessing the genotype of a cultivated tomato plant, preferably a Solanum lycopersicum plant, exhibiting resistance to ToBRFV, said method comprising the steps of: a) providing a sample from said plant, and, b) detecting in said sample an allele locus located on chromosome 2 and associated with said ToBRFV resistance, said allele locus comprising a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5.

[0134] In a further embodiment, the invention relates to a method for assessing the genotype of a cultivated tomato plant, preferably a Solanum lycopersicum plant, exhibiting resistance to ToBRFV, said method comprising the steps of: a) providing a sample from said plant, and, b) detecting in said sample an allele locus located on chromosome 2 and associated with said ToBRFV resistance, said allele locus comprising at least one of the following SNP markers: i) a T genotype in the homozygous state at a position corresponding to position 114 in SEQ ID NO: 5; ii) a G genotype in the homozygous state at a position corresponding to position 321 in SEQ ID NO: 5; iii) a C genotype in the homozygous state at a position corresponding to position 813 in SEQ ID NO: 5; iv) an A genotype in the homozygous state at a position corresponding to position 1593 in SEQ ID NO: 5; v) a T genotype in the homozygous state at a position corresponding to position 1815 in SEQ ID NO: 5; vi) a G genotype in the homozygous state at a position corresponding to position 1950 in SEQ ID NO: 5; vii) a T genotype in the homozygous state at a position corresponding to position 1971 in SEQ ID NO: 5; and / or viii) any other DNA marker associated with said allele locus. USES.

[0135] The present invention also relates to the use of ToBRFV resistance-propagating material obtainable from a tomato plant according to any of the preceding embodiments for growing a tomato plant to produce ToBRFV resistant tomato plants wherein said ToBRFV resistance may be assessed in a standard assay, particularly an assay as described in Example 2 below.

[0136] The present invention also relates to the use of ToBRFV resistance propagating material obtainable from a tomato plant according to any of the preceding embodiments for producing tomato fruits.

[0137] In another embodiment the invention relates to the use a cultivated tomato plant, plant part or seed, more preferably a cultivated Solanum lycopersicum plant, plant part or seed according to any of the preceding embodiments for growing a plant and producing and harvesting crops and / or fruits.

[0138] In another embodiment the invention relates to the use of a cultivated tomato plant, more preferably a cultivated Solanum lycopersicum plant, according to any of the preceding embodiments for producing fruits for the fresh market or for food processing.

[0139] In another embodiment the 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 preceding embodiments, wherein said cultivated tomato plant, plant part or seed, preferably the cultivated Solanum lycopersicum plant, plant part or seed is of tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

[0140] In a further embodiment the 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 preceding embodiments to sow a field, a greenhouse, or a plastic house.

[0141] In a further embodiment the invention relates to the use of a tomato plant according to any of the preceding embodiments to confer the 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 to introgress an increased ToBRFV resistance trait into a tomato plant lacking said trait.

[0142] In a further embodiment the invention relates to the use of any of SEQ ID NOs 5-8 for screening a population of tomato plants for the presence or absence of an allele locus located on chromosome 2 and associated with an increased ToBRFV resistance.

[0143] In a further embodiment the invention relates to the use of SEQ ID NO 5 for screening a population of tomato plants for the presence of an allele locus located on chromosome 2 and associated with an increased ToBRFV resistance.

[0144] Based on the description of the present invention, the skilled person who is in possession of tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny thereof, comprising said introgressed genetic sequence, as described herein, has no difficulty to transfer the said introgressed genetic sequence of the present invention to other tomato plants of various types using breeding techniques well-known in the art with the support of sequence information herein disclosed.

[0145] SEED DEPOSIT DETAILS

[0146] Applicant has made a deposit of 625 seeds of tomato plant 22TES320003 with NCIMB (National Collection of Industrial, Food and Marine Bacteria, NCIMB Limited, Wellheads Place, Dyce, Aberdeen, AB21 7GB, Scotland) on 29 March 2023 under NCIMB Accession No. 44132.

[0147] Applicant elects for the expert solution and requests that the deposited material be released only to an Expert according to Rule 32(1 ) EPC or corresponding laws and rules of other countries or treaties (Expert Witness clause), until the mention of the grant of the patent publishes, or from 20 years from the date of filing if the application is refused, withdrawn, or deemed to be withdrawn.

[0148] Tomato hybrid plant 22TES320003 is homozygous for the ToBRFV resistance allele on chromosome 2, i.e., tomato plant 22TES320003 comprises two copies of the allele on chromosome 2 that is associated with increased ToBRFV resistance. EXAMPLES

[0149] Example 1 : GERMPLASM AND POPULATION DEVELOPMENT

[0150] An internal Syngenta line 3T304 was identified as resistant to several Tobamoviruses, including ToBRFV, in internal disease screenings.

[0151] An F1 hybrid resulting from a cross between 3T304 and a susceptible elite line PI4B09TES160854 was self-pollinated to get a F2 population.

[0152] This F2 population, 15TES320144, was used to perform a genetic mapping based on genotyping and phenotyping data of individual F2 plants. A total of 445 F2 plants were sampled and genotyped to establish a genetic map of the population. After sampling, those 445 plants were mechanically inoculated with a ToBRFV strain on seedlings and assessed according to a qualitative phenotyping score described in Example 2A-C below. An allele associated with increased ToBRFV resistance located on chromosome 2 was identified in this F2 population (see Examples 3 to 5).

[0153] 22TES320003 was retained and deposited at NCIMB on 29 March 2023 under NCIMB Accession No. 44132. Tomato plant 22TES320003 is homozygous for the ToBRFV resistance trait, i.e., it comprises two copies of the allele on chromosome 2 that is associated with increased ToBRFV resistance.

[0154] Example 2: PROTOCOLS.

[0155] Example 2A. ToBRFV isolate.

[0156] A ToBRFV strain was collected in Israel in 2015 and thereafter stored on dried tomato leaves. From 1 gram of these tomato leaves, an inoculum production was performed by crushing the leaves and adding 5 ml of buffer, 0,1 gram of charcoal and 0.1 gr of carborundum (abrasive dust). The obtained solution was used to mechanically inoculate cotyledons of susceptible materials. T rays containing seedlings were maintained in growth chamber at 18°C during night / 24°C during day for 10-14 days to increase inoculum. Day length was fixed at for 15 hours under full light (around 10 000 lux). Symptomatic leaves were then harvested from the seedlings and stored in a - 80°C freezer. This storage is considered as the source of inoculum. Following this production, a first step of calibration was performed to establish the lower concentration of inoculum allowing to infect 100% of susceptible controls, without escape. The best dilution fitting with this requirement was fixed at % (1 g viral leaves in 10ml buffer) and was used in the phenotyping experiment.

[0157] Example 2B. Preparation and inoculation of plants.

[0158] An artificial method was used to inoculate plants with the ToBRFV strain. Seeds of the F2 lines were sown in specific trays and placed in a growth room in Sarrians, France. In addition, twenty-four seeds of parental lines PI4B09TES160854 and 3T304, resulting F1 plant 13TES160105 and susceptible control 16TEP071665 were sown to use as checks. Four days before inoculation, depending on germination rate, 445 plants of F2 and 16 check plants were selected for inoculation. For each material tested, 1 plant was transplanted in another support. Plants in trays were inoculated 16 days (2 true leaves) after sowing using ToBRFV inoculum prepared as described above. The inoculation was done by rubbing gently twice the leaves. Transplanting plants were inoculated without virus (mock) to evaluate the inoculation effect.

[0159] Example 2C. Scoring of ToBRFV resistance.

[0160] The first symptoms such as distortion and mosaic on the leaves appeared 13 days postinoculation (dpi). Plants were monitored and symptoms were assessed when susceptible checks are fully symptomatic, in this case at 13, 27 and 37 dpi. Plants were scored in a qualitative scale as described below and illustrated in Figure 1.

[0161] Rating Symptoms

[0162] HR (9) Healthy plants with no symptoms.

[0163] IR (5) Low symptoms expression compared to the susceptible

[0164] S (1 ) Susceptible plants with strong leaves distortion.

[0165] Example 2D. Method of identifying the QTL and allele underlying the ToBRFV increased resistance trait.

[0166] For genetic mapping, 445 plants of the “3T304 x PI4B09TES 160854” F2 population were genotyped with 155 genetic markers spanning the genome. These plants were grown and evaluated for ToBRFV as described in Example 2A-C above. The allele detection was performed using the R / qtl package in the R statistical framework. First, the function ‘calc.genoprob’ was used to calculate the genotype probabilities (step 1 cM). Haley-Knott regression was performed to provide an approximation of the results of standard interval mapping. Then, the function ‘stepwiseqtl’ was invoked, which provides a fully automated model selection forward / backward algorithm. LOD threshold for main effect was determine by 1 ,000 permutations. The function ‘fitqtl’ was used to fit the final allele model and the function ‘qtlStats’ to obtain estimates of allele effects.

[0167] EXAMPLE 3: IDENTIFICATION OF ONE QTL ASSOCIATED WITH INCREASED TOBRFV RESISTANCE

[0168] One QTL was identified based on the ToBRFV resistance phenotypes from the F2 population. Table 1 shows the chromosomal location, the effect of the QTL measured as LOD score, and the percentage of variation explained by the QTL on chromosome 2 for ToBRFV resistance. The QTL showed a semi-dominant effect in the F2 population. Table 2 shows the position of the allele on the genetic map.

[0169] Table 1 : Significant QTL associated with ToBRFV resistance.

[0170] "LOD" = log likelihood score, "%var" = percent phenotypic variation explained by the allele, "Rvalue (F)" = the probability of the allele detected due to random chance by F test.

[0171] Table 2. Genetic map of the QTL on chromosome 2 EXAMPLE 4: FINE MAPPING OF THE INCREASED TOBRFV RESISTANCE ALLELE

[0172] The QTL identified on chromosome 2 initially spanned a 30 Mbp region, it was therefore decided to carry out a fine mapping experiment to reduce the size of the region. Nine thousand two hundred fifty-six (9,256) F2 plants and 149 F3 families (20 plants each) were subsequently analysed with markers spanning the QTL interval. Thereafter, a subset of 89 F4 families (10 plants each) have been inoculated with ToBRFV strain and 26 F4 recombinants were analysed in a 25K fingerprinting Axiom experiment. The size of the QTL region was reduced to a 3 Mbp interval containing 227 genes.

[0173] Table 3. Genetic map of the fine-mapped QTL on chromosome 2

[0174] This 3 Mbp interval comprises the Tm-1 gene, Solyc02g062560, an allele of which was previously described for its involvement in controlling resistance to ToMV / TMV pathotype 0 (Ishibashi and Ishikawa, 2013). However, the Tm-1 allele described in this publication (SEQ ID NO: 7) has been tested against ToBRFV and did not show resistance.

[0175] EXAMPLE 5: CHARACTERIZATION OF THE INCREASED TOBRFV RESISTANCE ALLELE ON CHROMOSOME 2 AS A NEW TM-1 ALLELIC VARIANT IN SYNGENTA LINE 3T304

[0176] Zinger et al. identified a QTL conferring resistance to ToBRFV on chromosome 2 in the vicinity of Tm-1 , in a F2 population obtained between a resistant plant, VC554, and a susceptible plant, Moneymaker. WO 2021 / 213892 and WO 2022 / 018734 are also reporting QTL / genes on chromosome 2, allegedly in proximity with Tm-1 .

[0177] The 3T304 donor and a set of 27 sensitive accessions from various genetic origins were re-sequenced using Illumina short read technology at a sequencing depth of 10x. A total of 70,944 SNPs were identified on chromosome 2 when mapping the generated reads against Heinz 1706 SL4.0. Among these SNPs, a total of 3,595 were specific to 3T304 plant (alternative alleles present only in 3T304) and 2,462 of them were comprised in the QTL interval. 1 ,032 SNPs out of the 3,595 SNPs were located + / - 100 kb from the Tm-1 gene (Solyc02g062560).

[0178] To determine the specificity of the 3T304 allele of Tm-1 versus the reported ones, sequence comparisons were performed using the 3T304 consensus coding sequence (CDS). Comparisons indicated that the haplotype present in 3T304 is different from the haplotype described in WO 2021 / 213892 (Figure 3 and Figure 4). Among the three SNPs described in WO 2021 / 213892 as being specific to the so-called Rug-1 allele, only one was identified in the 3T304 Tm-1 consensus CDS and no evidence for gene duplication was spotted in the read alignments of 3T304.

[0179] On the other end, 9 SNPs were identified as being specific to the 3T304 Tm-1 allele (Figure 3, arrows). Out of these 9 SNPs, a pair of SNPs located at positions 1376-1377 of SEQ ID NO: 5 translate into one specific amino acid change at position 459 of SEQ ID NO: 1 , such amino acid Arginine being specific to the 3T304 protein sequence when compared with the other Tm-1 alleles (Figure 5, arrow). It is hypothesized that this amino acid change might account for the specificity of the 3T304 allele and its improved ToBRFV resistance effect.

[0180] EXAMPLE 6: SEQUENCE AND SNP MARKER INFORMATION FOR THE 3T304 ALLELE

[0181] The sequence information of SNP markers 1 to 9 (by sequence order in the 3T304 Tm-1 allelic variant) is summarized in Table 4 below.

[0182] Table 4.

[0183] As a matter of example, SNP marker 1 is characterized by a particular sequence polymorphism (3T304 allele vs. other alleles) at position 114 of the sequence of SEQ ID NO: 5.

[0184] BIBLIOGRAPHY

[0185] • Ishabashi et al., 2007, An inhibitor of viral RNA replication is encoded by a plant resistance gene, Proc Natl Acad Sci USA 104(34): 13833-13838.

[0186] • Ishibashi and Ishikawa, 2013, The resistance protein Tm-1 inhibits formation of a Tomato Mosaic Virus replication protein-host membrane protein complex, J Virol 87(14): 7933-7939.

[0187] • Luria etal., 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

[0188] • Salem etal., 2016, A new tobamovirus infecting tomato crops in Jordan, Arch. Virol. 161 :503-506.

[0189] • 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

CLAIMS1. A cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection, comprising in its genome two copies of a ToBRFV resistance allele located on chromosome 2, wherein said ToBRFV resistance allele encodes a protein having at least 99% amino acid sequence identity with SEQ ID NO: 1 and wherein said protein has an Arginine at a position corresponding to position 459 in SEQ ID NO: 1.

2. The plant according to claim 1 , wherein said ToBRFV resistance allele encodes the protein of SEQ ID NO: 1 .

3. The plant according to claims 1 -2, wherein said ToBRFV resistance allele has at least 99% nucleotide sequence identity with SEQ ID NO: 5 and comprises a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5.

4. The plant according to claim 3, wherein said ToBRFV resistance allele further comprises at least one of the following SNP markers: a) a T genotype in the homozygous state at a position corresponding to position 114 in SEQ ID NO: 5; b) a G genotype in the homozygous state at a position corresponding to position 321 in SEQ ID NO: 5; c) a C genotype in the homozygous state at a position corresponding to position 813 in SEQ ID NO: 5; d) an A genotype in the homozygous state at a position corresponding to position 1593 in SEQ ID NO: 5; e) a T genotype in the homozygous state at a position corresponding to position 1815 in SEQ ID NO: 5; f) a G genotype in the homozygous state at a position corresponding to position 1950 in SEQ ID NO: 5; and / or,g) a T genotype in the homozygous state at a position corresponding to position 1971 in SEQ ID NO: 5.

5. The plant according to claims 1 -4, wherein said ToBRFV resistance allele has the nucleotide sequence of SEQ ID NO: 5.

6. The plant of any one of claims 1 -5, wherein said ToBRFV resistance allele is as comprised in tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

7. The plant of any one of claims 1 -6 wherein said plant is obtained by crossing tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof, with a tomato plant that does not contain said ToBRFV resistance allele.

8. The plant of any one of claims 1 -7 wherein said plant is an inbred, a dihaploid, a diploid, or a hybrid plant.

9. A plant of tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132.

10. A plant part of a plant according to any one of claims 1 -9.

11. A seed that produces a plant or a plant part according to any one of claims 1 -10.

12. A method for producing a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection comprising the steps of a) crossing a plant according to any one of claims 1 -9 with a cultivated tomato plant lacking said ToBRFV resistance allele; b) selecting a progeny plant comprising said ToBRFV resistance allele located on chromosome 2, said selecting step comprising detecting a GT genotype in thehomozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5; thereby producing a plant with increased resistance to ToBRFV.

13. The method according to claim 12, wherein the method further comprises: c) selfing the selected progeny or crossing the selected progeny with another tomato plant to produce further progeny.

14. The method according to claim 13, wherein further progeny is selected and selfed / crossed for 2 to 10 more generations.

15. The method according to any one of claims 12 to 14 wherein the plant of step a) is tomato plant 22TES320003, representative seed of which is deposited under NCIMB Accession No. 44132, or a progeny or an ancestor thereof.

16. A method for producing a F1 tomato plant exhibiting increased resistance to ToBRFV, the method comprising crossing an inbred tomato plant, which is a plant according to any one of claims 1 to 9, with a different inbred tomato plant to produce F1 hybrid progeny.

17. A method for identifying a cultivated tomato plant, preferably a cultivated Solanum lycopersicum plant resistant to ToBRFV infection and having two copies of a ToBRFV resistance allele having at least 99% sequence identity with SEQ ID NO: 5, said method comprising the step of a) detecting a GT genotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5; thereby identifying a tomato plant exhibiting resistance to ToBRFV.

18. The method according to claim 17, wherein said method further comprises selecting a tomato plant comprising said genotype and crossing the selected tomato plant with a second tomato plant to produce progeny tomato plants that comprise a GTgenotype in the homozygous state at a position corresponding to positions 1376-1377 in SEQ ID NO: 5 and exhibits resistance to ToBRFV.

19. A method of producing tomato seed, the method comprising growing a tomato plant from the seed of claim 11 and allowing the plant to produce further tomato seed.

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