Resistance in plants of solanum lycopersicum to tobamovirus, tomato brown rugose fruit virus

Abstract

To provide a Solanum lycopersicum plant resistant to Tomato Brown Rugose Fruit virus.SOLUTION: Provided is a Solanum lycopersicum plant resistant to Tomato Brown Rugose Fruit virus (TBRFV), the genome of which comprises the combination of: a. the Tm-1 resistance gene on chromosome 2, and b. at least one quantitative trait locus (QTL) selected from QTL3 on chromosome 11, QTL1 on chromosome 6, and QTL2 on chromosome 9, which independently confer foliar and / or fruit tolerance to TBRFV, wherein the QTLs are present in the genome of a plant of the seeds HAZTBRFVRES1 (NCIMB accession number 42758).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to resistance in tomato (Solanum lycopersicum; also known as Lycopersicon esculentum) plants to the tobamovirus Tomato Brown Rugose Fruit virus (TBRFV; also known as ToBRFV). More specifically, the present invention relates to tomato plants and fruit containing one or more genetic determinants in combination with the Tm-1 resistance gene that lead to resistance to Tomato Brown Rugose Fruit virus. The present invention further relates to markers linked to these one or more genetic determinants and the Tm-1 gene, and the use of such markers to identify or select plants having such resistance. The present invention also relates to seeds and progeny of such plants, as well as propagation material for obtaining such plants, and various uses of these plants. [Background technology]

[0002] All cultivated and commercial forms of tomatoes belong to a species best known as Lycopersicon esculentum Miller. Lycopersicon is a relatively small genus within the very large and diverse family Solanaceae, thought to consist of approximately 90 genera, including pepper, tobacco, and eggplant. Lycopersicon has been divided into two subgenera: the esculentum group, which contains species that readily hybridize with commercial tomatoes, and the peruvianum group, which contains species that are highly averse to hybridization (Stevens, M. and Rick, C.M. 1986). L. esculentum Miller is a valuable crop and is widespread throughout the world. The exact origin of the cultivated tomato remains somewhat unknown, but it is thought to have originated in the Americas, native to Ecuador, Peru, and the Galapagos Islands, and first cultivated by the Aztecs and Incas as early as 700 AD. Mexico is thought to be the site of domestication and the source of first introductions. The cherry tomato, L. esculentum var. cerasiforme, is considered to be the direct ancestor of the modern cultivated form.

[0003] Tomatoes are grown for their fruit and are widely used fresh or processed. As a crop, tomatoes are grown commercially wherever environmental conditions allow for the production of economically sustainable yields. The majority of fresh tomatoes are harvested by hand at the ripened green stage on the vine. Fresh tomatoes are available year-round. Tomatoes for processing are mostly harvested by machine and are used in many forms, including canned tomatoes, tomato juice, tomato sauce, puree, paste, and even ketchup.

[0004] Tomatoes are typically simple diploid species with 12 pairs of differentiated chromosomes, but polyploid tomatoes are also part of this invention. Cultivated tomatoes are self-fertile and mostly self-pollinating. Tomato flowers are dioecious. Commercial cultivars were initially open-pollinated. As hybrid vigor has been demonstrated in tomatoes, hybrids are becoming increasingly popular among farmers for their better yields and uniformity of plant characteristics, replacing open-pollinated varieties. Due to their widespread availability and high value, tomatoes have been intensively bred, which explains the wide variety of tomatoes available today. Shapes range from small to large, including cherry, plum, pear, blocky, round, and beefsteak.

[0005] Tomatoes may be classified by the amount of time it takes the plant to mature enough to produce harvestable fruit, and cultivars are generally considered to be early, mid-season, or late. Tomatoes may also be classified by the plant's growth habit: determinate, semi-determinate, or indeterminate.

[0006] Determinate plants tend to develop leaves first, followed by flowers, which, if successfully pollinated, mature into fruit. All fruits tend to mature at roughly the same time on a single plant. Indeterminate tomatoes develop a few leaves first, then continue to produce leaves and flowers throughout the growing season. These plants tend to have tomato fruit at various stages of maturity at any given time. Semi-determinate tomatoes have a phenotype between determinate and indeterminate and are typical of determinate species, except that they grow larger than determinate varieties. Recent advances in tomato breeding have resulted in a greater variety of fruit colors. In addition to the standard red ripe color, tomatoes can be milky white, lime green, pink, yellow, golden yellow, orange, or purple.

[0007] Hybrid commercial tomato seed can be produced by hand pollination. Pollen from the male parent is harvested and applied by hand to the stigma surface of the female inbred hybrid. Before and after hand pollination, the flowers are covered to prevent insects from introducing foreign pollen or creating mixtures or impurities. The flowers are tagged and the pollinated fruit from which seeds are harvested are identified.

[0008] The productivity of tomato plants is affected by a variety of pathogens, including viruses, fungi, bacteria, nematodes, and insects. Tomatoes are particularly susceptible to many viruses, and therefore virus resistance is of great agricultural importance.

[0009] Tobamoviruses are one of the most important plant viruses in agriculture, causing severe damage to vegetable and ornamental crops worldwide. Tobamoviruses are easily transmitted by mechanical means and even seed transmission, although there is no evidence of a natural vector. Tobamoviruses are generally characterized by rod-shaped particles approximately 300 nm in size, consisting of a single-stranded positive-chain RNA genome encoding four proteins encapsidated by a 17 kDa coat protein (CP) molecule.

[0010] In tomatoes, the Tobacco Mosaic Virus (TMV) and Tomato Mosaic Virus (ToMV) are feared by growers worldwide because they can seriously impair crop yields, for example by causing irregular ripening (fruit with yellowish spots on the surface and brownish spots below the surface), although over the years plant breeders have identified several genes.

[0011] The first resistance gene identified was the Tm-1 gene, which confers resistance to TMV. Introgressed from S. habrochaites, this gene was not completely dominant; homozygosity was generally required for TMV resistance. However, the Tm-1 gene was overcome within about a year of its introduction into commercial horticulture, completely obviating any attempts to introduce it into other commercial strains (Pelham et al., 1970. "The establishment of a new strain of tobacco mosaic virus resulting from the use of resistant varieties of tomato"; Ann. Appl. Biol., 65:293-297).

[0012] This gene was also identified as conferring resistance to ToMV, but because the majority of currently circulating TMV and ToMV strains are able to infect commercial plants that possess the Tm-1 gene, this gene is no longer considered a resistance gene to TMV / ToMV infection in commercial plants. Use of the Tm-1 gene has now been almost completely abandoned in favor of alternative resistance genes.

[0013] For the past few decades, all modern indeterminate and many determinate tomato varieties have in fact lost the Tm-2 gene or, preferably, the Tm-2 gene. 2 It contains an allele that confers immunity to nearly all known races of tobamoviruses (ToMV and TMV) that affected commercial tomatoes before 2014.

[0014] Between 2014 and 2015, severe virus outbreaks affected tomato-producing regions in the Middle East, including Jordan and Israel. Although most of the affected tomato varieties were considered resistant to TMV and / or ToMV, they were still severely affected and exhibited typical TMV / ToMV-like symptoms. While leaf symptoms were very similar to those of TMV / ToMV, fruit symptoms, including fruit lesions and deformations, were much more frequent and severe than those typically associated with these viruses. Fruit quality was significantly poor, rendering the fruit unsalable. Salem et al. (Arch. Virol. 161 (2), 503-506, 2015) extracted RNA from fruit and leaves of symptomatic infected plants in Jordan and performed various tests, leading to the identification of a new tobamovirus species (the sequence corresponds to GenBank accession number KT383474 (SEQ ID NO: 25)). Salem et al. proposed naming this Jordanian virus Tomato Brown Wrinkled Fruit Virus (TBRFV or ToBRFV). Comparison with other tobamovirus sequences showed that it was indeed a tobamovirus but not TMV or ToMV. Resistance to TMV and / or ToMV did not confer resistance to this new virus, TBRFV.

[0015] Luria et al. (PLoS One. 2017; 12(1): e0170429) simultaneously isolated and sequenced the complete genome of an Israeli tobamovirus infecting tomatoes in Israel, which corresponds to GenBank accession number KX619418 (SEQ ID NO: 26). They therefore demonstrated very high sequence identity (>99% sequence identity) between the Israeli and Jordanian viruses and concluded that they represent two distinct isolates of Tomato Brown Wrinkled Fruit Virus.

[0016] The virus is now considered a significant global threat to tomato crops, having been identified in Europe in recent years, particularly in Sicily, Germany, and the Netherlands, as well as in Mexico, where the identified strain appears to be the Israeli, rather than Jordanian, strain.

[0017] In Israel, we collected isolates and sequenced seven representative isolates from all crop-producing regions (north, center, and south). Sequence comparison with the Jordanian ToBRFV sequence appears to indicate that all Israeli isolates are essentially, but not completely, identical to the Jordanian isolate, thereby confirming that they should probably be considered two different strains of the same virus in both countries.

[0018] In a previous application, the present inventors were the first to identify tomato plants that exhibit resistance to Tomato Brown wrinkled fruit virus and were able to locate and identify genetic determinants (hereinafter referred to as QTLs (quantitative trait loci)) that lead to resistance to Tomato Brown wrinkled fruit virus. Two QTLs, namely, QTL1 and QTL2, should be located on chromosomes 6 and 9, respectively, and when present homozygously in a tomato background, individually or jointly confer improved resistance to the fruit of tomato plants that are infected or susceptible to TBRFV. A third QTL, namely, QTL3, should be located on chromosome 11, and when present homozygously, confer improved resistance to the leaves of tomato plants that are infected or susceptible to TBRFV. These QTLs are referred to and described in PCT application WO2018 / 219941. In the following description, these QTLs will be referred to as resistance QTLs.

[0019] Although these QTLs individually or collectively confer resistance to TBRFV, the inventors have now demonstrated that in most cases they fail to confer resistance to tomato plants, and in particular fail to confer a level of resistance sufficient to delay, reduce, or inhibit viral replication or growth. Indeed, infected plants carrying one or more of the above resistance QTLs still propagate the virus, and the virus remains a threat to all surrounding tomato plants that do not carry these QTLs.

[0020] Because tobamoviruses are not easily controlled but rather through genetic improvement through the identification and use of resistance genes in breeding, and because currently available resistance genes for the control of TMV and / or ToMV are ineffective against the damage and proliferation caused by the new Tomato Brown Wrinkled Fruit Virus and resistance QTLs are unable to stop or sufficiently reduce viral proliferation, there is an urgent need to identify resistance to this new tobamovirus, otherwise entire regions will no longer be able to produce tomato crops. Summary of the Invention

[0021] The present inventors have been able to identify tomato plants that exhibit resistance to Tomato Brown wrinkled fruit virus and to identify the combinations of genetic determinants (i.e., combinations of QTLs (quantitative trait loci)) that lead to resistance to Tomato Brown wrinkled fruit virus, and the genes that provide this resistance or enhanced tolerance.

[0022] Resistance according to the present invention is conferred by the Tm-1 resistance gene in combination with genetic determinants, i.e., QTLs, which, when not combined with the Tm-1 resistance gene, only confer resistance to Tomato Brown wrinkled fruit virus (TBRFV) at the leaf and / or fruit level of tomato plants. According to WO 2018 / 219941, these QTLs, i.e., genetic determinants, are described as recessive. The presence of the Tm-1 resistance gene in a homozygous state is not required, unlike the primary mechanism of action of the Tm-1 gene for past resistance to TMV / ToMV (although this resistance has now been overcome by circulating TMV / ToMV strains).

[0023] Fruit resistance is independently conferred by QTL1 or QTL2, and leaf resistance by QTL3, and their introgression into different genetic backgrounds (i.e., various tomatoes) can be readily achieved by those skilled in the art of plant breeding, especially with the information on appropriate markers associated with the QTLs provided in WO 2018 / 219941. The same is true for the Tm-1 gene.

[0024] Thus, the present invention provides the following combination: - a genetic determinant (herein also referred to as QTL or resistance QTL) that, when present in the homozygous state, confers a TBRFV resistance phenotype at the tomato leaf and / or fruit level in TBRFV-infected tomato plants; -Tm-1 gene wherein the combination confers resistance to TBRFV, particularly the ability to delay, reduce, and / or inhibit viral replication, whereas neither the QTL alone or in combination nor the Tm-1 gene alone confers such a level of resistance or enhanced tolerance.

[0025] The present invention also relates to methods for producing or identifying commercial tomato plants that exhibit resistance to TBRFV, as well as tomato plants or populations (germplasm) that exhibit resistance to TBRFV. The present invention also discloses molecular genetic markers (particularly single nucleotide polymorphisms (SNPs)) linked to the resistance QTL and the Tm-1 gene, which can be used in any selection method to obtain the plants of the invention. Plants obtained by the above methods and the use of such molecular markers are also provided.

[0026] The present invention also provides several methods for improving yields in tomato production in TBRFV-infested environments and methods for protecting tomato fields from TBRFV infestation.

[0027] definition The term "resistance" is defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Division to describe the plant's response to pests or pathogens and abiotic stresses for the vegetable seed industry. Specifically, resistance means that a plant variety is able to limit the growth and development of a particular pest or pathogen and / or the damage it causes compared to a susceptible plant variety under similar environmental conditions and pest or pathogen stress. A resistant variety may show some disease symptoms or damage under a high pest or pathogen stress.

[0028] The term "resistant" is used herein to refer to a plant phenotype in which, at least under some cultivation conditions, the plant is exposed to an infectious dose of the virus (an amount capable of establishing the presence of systemic or local infection, viral multiplication, the presence of viral genomic sequences in at least the cells of the plant, and / or their genomic integration) and at least some of the symptoms remain absent. Thus, a resistant plant is resistant to disease but is an asymptomatic virus reservoir. Viral sequences may be present or even multiplying in the plant without causing symptoms. Note that resistant plants are susceptible to infection by the virus, but are generally able to at least moderately restrict viral growth and development.

[0029] In the case of TBRFV, leave tolerance or foliar tolerance refers to the phenotype of a plant in which leaf symptoms remain absent when the plant is exposed to an infective dose of TBRFV, although fruit symptoms may be present on infected plants. Fruit resistance, in the case of TBRFV, refers to the phenotype of a plant in which fruit symptoms remain absent when the plant is exposed to an infectious dose of TBRFV, although leaf symptoms may be present on infected plants.

[0030] Leaf symptoms of TBRFV infection commonly include mosaic, malformed leaflets, and often shoelace-like symptoms. Fruit symptoms of TBRFV infection commonly include typical yellow lesions and deformed fruit. Fruit often also exhibits "chocolate spots."

[0031] "Susceptible" refers to the inability of a plant variety to restrict the growth and development of a particular pest or pathogen, i.e., susceptible plants exhibit the deleterious symptoms associated with viral infection, i.e., leaf damage and fruit damage in the case of TBRFV infection.

[0032] Tomato plants susceptible to Tomato Brown Creased Fruit Virus are, for example, the commercial cultivar Candela, described in Salem et al., 2015. They may also be Hazera lines 2 and 4, described in PCT Application No. WO2018 / 219941. To date, i.e., prior to the present invention, all commercial tomato cultivars grown in TBRFV-infected areas are either susceptible to TBRFV or, at best, resistant in plants carrying the resistance QTL, such as deposited seeds of HAZTBRFVRES1. This tomato seed sample has been deposited by Hazera Seeds Ltd., Berlin, MP Shikmiim 79837, Israel, with the National Collection of Industrial, Food and Marine bacteria (NCIMB) (NCIMB, Ltd., Ferguson Building, Craibstone Estate, Aberdeen AB21 9YA, United Kingdom) on 16 May 2017 under accession number 42758 in accordance with and fulfilling the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure ("Budapest Treaty").

[0033] Thus, plants according to the invention have at least improved or increased resistance to Tomato Brown Wrinkled Fruit Virus relative to the Candela cultivar, and more generally relative to any commercial tomato cultivar (including resistant plants) grown in Tomato Brown Wrinkled Fruit Virus-infected areas, and relative to HAZTBRFVRES1.

[0034] As used herein, the term "progeny" or "progeny" refers to any plant obtained as a descendant by vegetative or sexual propagation from one or more parent plants or their derivatives. For example, progeny plants may be obtained by cloning or selfing a parent plant, or by crossing two parent plants, including selfing and F1 or F2 or further generations. F1 is the first generation progeny produced from parents (at least one of which is initially used as a trait donor), while second generation (F2) or later (F3, F4, etc.) progeny are specimens produced by selfing F1, F2, etc. Thus, F1 may (and usually is) a hybrid obtained by crossing two pure-line parents (pure-line is homozygous for the trait), and F2 may (and usually is) a progeny obtained by self-pollination of the F1 hybrid. As used herein, the terms "crossing," "mating," "cross-pollination," or "cross-breeding" refer to the process by which pollen from one flower of one plant is deposited (artificially or naturally) on the ovule (stigma) of a flower of another plant.

[0035] As used herein, the terms "genetic determinant" and / or "QTL" refer to any DNA segment associated with a biological function. Thus, QTL and / or genetic determinants include, but are not limited to, genes, coding sequences, and / or regulatory sequences required for their expression. QTL and / or genetic determinants can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins.

[0036] As used herein, the term "genotype" refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., plant), or group of organisms.

[0037] As used herein, the term "grafting" refers to the process of joining a rootstock to a scion. The primary motivation for grafting is to avoid damage from soil-borne pests and pathogens when genetic or chemical disease management methods are not available. By grafting a susceptible scion onto a resistant rootstock, resistant varieties can be obtained without the need to engineer the resistance into the variety. Grafting can also enhance tolerance to abiotic stresses, potentially increasing yields and allowing for more efficient water and nutrient use.

[0038] As used herein, the term "heterozygote" refers to a diploid or polyploid individual cell or plant having different alleles (forms of a given gene, genetic determinant or sequence) present at at least one genetic locus.

[0039] As used herein, the term "heterozygous" refers to the presence of different alleles (forms of a given gene, genetic determinant or sequence) at a particular locus.

[0040] As used herein, "homologous chromosomes" or "homologs" (or homologues) refer to a set of one maternal and one paternal chromosome that pair with each other during meiosis. These copies have the same genes at the same loci and at the same centromeric positions.

[0041] As used herein, the term "homozygote" refers to an individual cell or plant that has the same allele at one or more loci on all homologous chromosomes.

[0042] As used herein, the term "homozygous" refers to the presence of identical alleles at one or more loci on homologous chromosomal intervals.

[0043] As used herein, the term "hybrid" refers to any individual cell, tissue, or plant resulting from a cross between parents that differ in one or more genes.

[0044] As used herein, the term "locus" (plural "loci") refers to any genetically defined site, which may be a single position (nucleotide) or a chromosomal region. A locus may be a gene, genetic determinant, or part of a gene, or a DNA sequence, and may be occupied by different sequences. A locus may also be defined by a SNP (single nucleotide polymorphism), multiple SNPs, or two adjacent SNPs.

[0045] As used herein, the term "rootstock" is the lower part of a plant that can receive a scion in the grafting process.

[0046] As used herein, the term "scion" is the upper part of a plant that can be joined to a rootstock by the method of grafting.

[0047] Detailed Description of the Invention The present inventors have demonstrated that the three QTLs disclosed in WO 2018 / 219941, which, when present homozygously in tomato plants, alone or in combination, improve the resistance of fruit and / or leaves of tomato plants that are infected or potentially infected with Tomato Brown Wrinkled Fruit Virus (TBRFV or ToBRFV), still fail to completely restrict viral growth. Indeed, the inventors have discovered that viral growth generally occurs within these plants, as evidenced by the detection of viral genome sequences within the cells of the plants. Thus, such plants harbor the virus and are unable to restrict the spread of the virus from plant to plant.

[0048] The three QTLs disclosed in WO2018 / 219941, i.e., QTL1, QTL2, and QTL3 located on chromosomes 6, 9, and 11, respectively, are hereinafter referred to as "resistance QTLs." More specifically, QTL1 and QTL2 are referred to as "fruit resistance QTLs," and QTL3 on chromosome 11 is referred to as "leaf resistance QTL."

[0049] Unexpectedly, the inventors have found that combining at least one of the resistance QTLs (i.e., QTL1, QTL2, and / or QTL3) with the Tm-1 resistance gene improves tolerance or resistance in tomato to TBRFV, particularly the Israeli isolate, by reducing virus titer and / or inhibiting virus growth and / or slowing the progression of the virus and thus any associated symptoms, and / or increasing the level of resistance in tomato plants, particularly with respect to at least one of the criteria including virus titer, virus progression, infected leaf symptoms, or infected fruit symptoms, relative to corresponding plants lacking the Tm-1 resistance gene.

[0050] According to a preferred embodiment, at least one of the resistance QTLs (eg QTL3, especially if there is only one QTL) is present in homozygosity.

[0051] Furthermore, it is also preferred to combine at least two resistance QTLs with the Tm-1 gene. In such cases, advantageously, at least one QTL (e.g., QTL2) is present in heterozygosity. According to one embodiment, two or three of the resistance QTLs are present in combination with the Tm-1 gene, with at least one QTL present in homozygosity and at least one other QTL present in heterozygosity.

[0052] It should be noted that the Tm-1 resistance gene was previously identified as a resistance gene to TMV and ToMV, but it no longer confers resistance to prevalent ToMV / TMV strains because prevalent ToMV and TMV strains have mutated to circumvent this resistance. Thus, the presence of the Tm-1 resistance gene in plants of the present invention does not provide ToMV and / or TMV resistance to these plants, particularly commercial plants that are particularly threatened by prevalent ToMV / TMV strains.

[0053] As demonstrated in the Examples, the phenotype of the plants of the present invention is resistance to TBRFV, i.e., resistance of the leaves and / or fruits, and the plants of the present invention can improve the restriction of viral growth at least at some stages after infection.

[0054] Improved restriction of viral growth means that the level of viral sequences or proteins detected in plants (e.g., detected by quantitative reverse transcription PCR (q-RT-PCR)) as measured by ELISA techniques at about 70-90 days post-inoculation (DPI) is at least 50% lower, preferably at least 60%, at least 70%, or at least 80% lower, than the level of viral sequences detected by the same technique simultaneously in susceptible plants or in resistant but non-resistant plants. The level of viral sequences or proteins may be measured at about 30 DPI. Levels are reduced if they are at least 20% lower than the levels measured in susceptible plants.

[0055] Thus, according to a first aspect, the present invention is directed to a tomato plant that is resistant to Tomato Brown wrinkled fruit virus (TBRFV), the plant comprising in its genome a combination of: -Homozygous or heterozygous Tm-1 resistance gene -At least one resistance quantitative trait locus (QTL) present in homozygous or heterozygous form This includes combinations with.

[0056] Preferably, there are at least two QTLs, preferably one present in homozygous form and one present in heterozygous form.

[0057] The present invention also covers cells and seeds of such plants that contain said QTL in combination with the Tm-1 gene.

[0058] The resistance QTL is selected from the group consisting of QTL3 on chromosome 11, QTL1 on chromosome 6, and QTL2 on chromosome 9. Each of these resistance QTLs independently confers leaf and / or fruit resistance to the plant and, when combined with the Tm-1 gene, confers resistance or enhanced resistance to TBRFV. The resistance QTL is present in the plant genome of seed HAZTBRFVRES1 (NCIMB accession number 42758).

[0059] The Tm-1 gene is particularly as defined in the 2007 publication by Ishibashi et al. (An inhibitor of viral RNA replication is encoded by a plant resistance gene. PNAS August 21, 2007 104 (34) 13833-13838). Preferably, the term "Tm-1 gene" refers to a gene sequence encoding a protein having the Tm-1 activity reported therein, i.e., the ability to inhibit viral replication of wild-type ToMV strains susceptible to Tm-1 (e.g., the ToMV-L strain disclosed therein). According to a preferred embodiment, the Tm-1 gene of the present invention is a gene encoding a protein having the 754 amino acid sequence reported in Ishibashi et al., corresponding to SEQ ID NO: 19 (NCBI BAF75724), or a protein having at least 75%, preferably at least 80%, more preferably at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 19 and exhibiting the Tm-1 activity reported in Ishibashi et al., 2007 (i.e., the ability to inhibit viral RNA replication of wild-type ToMV strains susceptible to Tm-1). According to a preferred embodiment, the gene has a sequence corresponding to the mRNA sequence described in Ishibashi et al., 2007, i.e., sequence NCIB AB287296 (SEQ ID NO: 20), or a sequence having at least 50%, preferably at least 60%, at least 70%, more preferably at least 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 20. Regardless of the degree of sequence identity with SEQ ID NO: 20, the Tm-1 gene according to the present invention preferably encodes a protein that exhibits the Tm-1 activity reported in Ishibashi et al., 2007, i.e., the ability to inhibit viral RNA replication of wild-type ToMV.

[0060] Preferably, in the genome of a plant, seed, or cell of the invention, the Tm-1 gene is present on chromosome 2. However, the invention also encompasses plants, seeds, or cells that contain the Tm-1 gene at a locus that does not correspond to the locus described in Ishibashi et al., 2007.

[0061] Thus, the present invention encompasses tomato plants, cells, or seeds containing various combinations of QTL1, QTL2, and QTL3 in their genomes (preferably with at least one QTL in a homozygous state and / or at least one in a heterozygous state) along with the Tm-1 gene. Preferably, at least two QTLs are present, at least one in a homozygous state and at least one in a heterozygous state. Thus, the present invention encompasses plants containing combinations of QTL3 and Tm-1, QTL1 and Tm-1, QTL2 and Tm-1, QTL3, QTL2 and Tm-1, QTL1, QTL2 and Tm-1, and QTL1, QTL2, QTL3 and Tm-1. Particularly preferred combinations are QTL3 and Tm-1, and QTL2, QTL3 and Tm-1. Various alternative combinations are disclosed in Table 1 below. It is particularly preferred that QTL3 is present in a homozygous state and QTL2 in a heterozygous state. The Tm-1 resistance gene may be present in either a heterozygous or homozygous state. A preferred combination, for example, is that QTL3 is homozygous or heterozygous, QTL2 is heterozygous, and Tm-1 is homozygous.

[0062] In the present invention, it is preferred that at least one of the resistance QTLs is present in the genome of the plant in a homozygous state, but the Tm-1 resistance gene may be present in either a heterozygous or homozygous state.

[0063] According to one embodiment, QTL2 on chromosome 9 is present in a heterozygous state in a plant according to the present invention.

[0064] In another preferred embodiment, the plant contains QTL3 homozygously in combination with Tm-1 homozygously or heterozygously. Such plants may advantageously contain QTL2 (preferably heterozygously). According to a preferred embodiment, the Tm-1 resistance gene should also be present in a homozygous state, for example, in a preferred embodiment, the plant contains QTL3 and Tm-1 homozygously and QTL2 heterozygously.

[0065] The resistance QTLs according to the present invention, i.e., QTL1, QTL2, and QTL3, which confer resistance to TBRFV when combined with the Tm-1 gene or in the absence of such combination, are selected from those present in the genome of the seeds of HAZTBRFVRES1. A sample of this tomato seed has been deposited by Hazera Seeds Ltd. (Berlin, MP Shikmiim 79837, Israel) with the National Collection of Industrial, Food, and Marine bacteria (NCIMB, Ltd., Ferguson Building, Craibstone Estate, Aberdeen AB21 9YA, United Kingdom) on May 16, 2017, in accordance with and fulfilling the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (the "Budapest Treaty"), under accession number 42758. This tomato seed deposit is maintained by Hazera Seeds Ltd., Berlin, MP Shikmiim 79837, Israel.

[0066] Resistance QTLs that confer resistance to TBRFV and confer resistance when combined with the Tm-1 gene are located: QTL1 on chromosome 6, QTL2 on chromosome 9, and QTL3 on chromosome 11. More preferably, QTL1 is located within the chromosomal interval containing SNPs TO-0005197 (SEQ ID NO: 1) and TO-0145581 (SEQ ID NO: 2) on chromosome 6, QTL2 is located within the chromosomal interval containing SNPs TO-0180955 (SEQ ID NO: 3) and SNPs TO-0196109 (SEQ ID NO: 6) on chromosome 9, and QTL3 is located within the chromosomal interval containing SNPs TO-0122252 (SEQ ID NO: 7) and SNPs TO-0162427 (SEQ ID NO: 18) on chromosome 11.

[0067] The specific polymorphisms present in the tomato genome that correspond to the SNPs (single nucleotide polymorphisms) referred to herein and the flanking sequences of these SNPs are shown in the experimental section (see Tables 3 and 4) and in the accompanying sequence listing. Their locations on chromosomes 6, 9 and 11, relative to version 2.40 of the tomato genome, are shown in Table 3, and their flanking sequences are also shown in Table 4 and the sequence listing.

[0068] In this regard, it should be noted that, by definition, a SNP refers to a single nucleotide that may vary depending on the allele present in the genome, while the adjacent nucleotides are identical. To facilitate clear identification of the locations of different SNPs, the tomato genome sequence, version 2.40, is provided, along with the adjacent sequences identified by SEQ ID NOs., and their locations are shown in Tables 3 and 4. In sequences associated with particular SNPs herein (e.g., SEQ ID NO: 1 for SNP TO-0005197), only one nucleotide in the sequence actually corresponds to the polymorphism, i.e., nucleotide 61 of SEQ ID NO: 1 corresponds to the polymorphic position of SNP TO-0005197, which can be either T or C, as shown in Table 4. The adjacent sequences are provided to identify the location of the SNP within the genome, but are not themselves part of the polymorphism.

[0069] The present inventors have confirmed that resistance QTLs responsible for resistance or enhanced resistance when combined with the Tm-1 gene should be present in the above chromosomal regions by using different loci on the regions (i.e., the following 18 SNPs: TO-0005197 (SEQ ID NO: 1) and TO-0145581 (SEQ ID NO: 2) for QTL1 on chromosome 6, TO-0180955 (SEQ ID NO: 3), TO-0196724 (SEQ ID NO: 4), TO-0145125 (SEQ ID NO: 5), and TO-0196109 (SEQ ID NO: 6) for QTL2 on chromosome 9, and TO-0196724 (SEQ ID NO: 6) for QTL3 on chromosome 11. The identity of the sequences was confirmed by confirming the presence of sequences at 18 different loci defined by TO-0122252 (SEQ ID NO:7), TO-0144317 (SEQ ID NO:8), TO-0142270 (SEQ ID NO:9), TO-0142294 (SEQ ID NO:10), TO-0142303 (SEQ ID NO:11), TO-0142306 (SEQ ID NO:12), TO-0182276 (SEQ ID NO:13), TO-0181040 (SEQ ID NO:14), TO-0123057 (SEQ ID NO:15), TO-0125528 (SEQ ID NO:16), TO-0162432 (SEQ ID NO:17), and TO-0162427 (SEQ ID NO:18).

[0070] These 18 SNPs are associated or genetically linked to at least one of the resistance QTLs. Association or genetic association, or more specifically genetic linkage, is understood as the co-occurrence of a genetic polymorphism of a marker (i.e., a particular allele of a SNP marker) and a phenotype of interest as a result of their proximity on the same chromosome, i.e., they are inherited together more often than would be expected by chance, i.e., a non-random association due to the alleles and gene sequences responsible for the phenotype.

[0071] The molecular markers of the present invention, i.e., any one of the 18 markers or surrogate markers disclosed above, are preferably inherited with the phenotype of interest in greater than 90% of meiotic divisions, preferably greater than 95%, 96%, 98%, or 99% of meiotic divisions.

[0072] According to another embodiment of the present invention, the resistance QTLs present in the genome of the plant, seed, or cell of the present invention are preferably selected from 18 loci containing the above-mentioned 18 SNPs (i.e., for QTL1 on chromosome 6, a locus containing TO-0005197 (SEQ ID NO: 1) and a locus containing TO-0145581 (SEQ ID NO: 2); for QTL2 on chromosome 9, a locus containing TO-0180955 (SEQ ID NO: 3), a locus containing TO-0196724 (SEQ ID NO: 4), a locus containing TO-0145125 (SEQ ID NO: 5), and a locus containing TO-0196109 (SEQ ID NO: 6); and for QTL3 on chromosome 11, a locus containing TO-0122252 (SEQ ID NO: 7). locus containing TO-0144317 (SEQ ID NO: 8), locus containing TO-0142270 (SEQ ID NO: 9), locus containing TO-0142294 (SEQ ID NO: 10), locus containing TO-0142303 (SEQ ID NO: 11), locus containing TO-0142306 (SEQ ID NO: 12), locus containing TO-0182276 (SEQ ID NO: 13), locus containing TO-0181040 (SEQ ID NO: 14), locus containing TO-0123057 (SEQ ID NO: 15), locus containing TO-0125528 (SEQ ID NO: 16), locus containing TO-0162432 (SEQ ID NO: 17), and locus containing TO-0162427 (SEQ ID NO: 18).

[0073] In one embodiment, in a tomato plant according to the invention, the QTLs that are present in the genome of the plant, a seed or a cell of such a tomato plant and that are combined with the Tm-1 gene are preferably located at at least one or more of the following loci: on chromosome 6 for QTL1 locus comprising TO-0005197 and locus comprising TO-0145581, and / or on chromosome 9 for QTL2 locus comprising TO-0180955, locus comprising TO-0196724, locus comprising TO-0145125, and locus comprising TO-0196109.

[0074] In another embodiment of the invention, the QTL present in the genome of the plant, seed or cell of the tomato plant and to be combined with the Tm-1 gene is preferably located at at least one or more of the following loci: for QTL3 on chromosome 11, the locus comprising TO-0122252, the locus comprising TO-0144317, the locus comprising TO-0142270, the locus comprising TO-0142294, the locus comprising TO-0142303, the locus comprising TO-0142306, the locus comprising TO-0182276, the locus comprising TO-0181040, the locus comprising TO-0123057, the locus comprising TO-0125528, the locus comprising TO-0162432 and the locus comprising TO-0162427.

[0075] The alleles of 18 SNPs linked to the resistance QTL conferring TBRFV resistance are allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, and allele G of TO-0196109. The specific alleles are the T allele of TO-0142270, the G allele of TO-0142294, the A allele of TO-0142303, the A allele of TO-0142306, the G allele of TO-0182276, the G allele of TO-0181040, the G allele of TO-0123057, the A allele of TO-0125528, the C allele of TO-0162432, and the T allele of TO-0162427. The presence of these specific alleles can reveal the presence of resistance QTLs. Therefore, the alleles of these SNPs can reflect the presence of resistance QTLs according to the present invention that should be combined with the Tm-1 gene.

[0076] According to a preferred embodiment of the present invention, the QTL conferring resistance to TBRFV to be combined with the Tm-1 gene is located on one or more chromosomal intervals bounded by SNPs as disclosed. According to this embodiment, QTL1 is located on the chromosomal interval bounded on one end by SNP TO-0005197 and on the other end by SNP TO-0145581 on chromosome 6.

[0077] In another embodiment, QTL2 is located on the chromosomal interval of chromosome 9 bounded on one end by SNP TO-0180955 and on the other end by SNP TO-0196109.

[0078] In another embodiment, QTL3 is located on chromosome 11 in a chromosomal interval bounded at one end by SNP TO-0122252 and at the other end by TO-0162427. More preferred chromosomal intervals on chromosome 11 in which QTL3 should be located are the interval bounded by TO-0144317 and TO-0125528, the interval bounded by TO-0142270 and TO-0162432, the interval bounded by TO-0144317 and TO-0162432, and the interval bounded by TO-0142270 and TO-0125528. A further preferred interval is the interval bounded by TO-0142270 and TO-0125528. Another preferred interval is the interval bounded by and including TO-0142294 and TO-0125528.

[0079] In this regard, it should be noted that specific locations within a chromosome may in fact be defined for single nucleotide polymorphisms only if the flanking sequences of said SNPs are defined to unambiguously locate them on the genome. The inventors have used SNPs identified in their flanking sequences with different alleles to identify and track the QTLs of the present invention.

[0080] A chromosomal region delimited by two SNPs X and Y refers to the chromosomal section that is located between the positions of these two SNPs and that contains said SNPs; therefore, the nucleotide sequence of this chromosomal region begins with the nucleotide corresponding to SNP X and ends with the nucleotide corresponding to SNP Y. That is, within the meaning of the present invention, the SNPs are contained within the region that they delimit.

[0081] In the plant, seed, or cell of the present invention, the presence of resistance QTLs to be combined with the Tm-1 resistance gene is preferably determined by TO-0005197 and / or TO-0145581 for QTL1 on chromosome 6, and / or TO-0180955, TO-0196724, TO-0145125, and / or TO-0196109 for QTL2 on chromosome 9, and / or TO-0122252, TO-0144317, TO-0144318 for QTL3 on chromosome 11. 142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427, most preferably TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, and even more preferably TO-0182276.

[0082] When present homozygously in the genome of a tomato plant, QTL1 and / or QTL2 independently or collectively confer TBRFV resistance in fruit and QTL3 confer TBRFV resistance in leaves, unless combined with the Tm-1 resistance gene to confer resistance or enhanced resistance to TBRFV in accordance with the present invention.

[0083] The resistance QTL defined above is present in combination with the Tm-1 gene in the genome of the plant, seed or cell of the present invention.

[0084] The Tm-1 gene may be present in the genome of the plant, seed, or cell of the present invention in a heterozygous or homozygous state, although it is preferable that the gene be present in a homozygous state.

[0085] The present inventors have also found suitable markers for detecting the presence of the Tm-1 gene in the genome of a plant, seed, or cell of the present invention. The presence of the Tm-1 resistance gene, which should be combined with one or more resistance QTLs, is preferably characterized by SNP TO-0200838 (SEQ ID NO: 21).

[0086] The allele of SNP TO-0200838 corresponding to the Tm-1 gene is TO-0200838 allele A. The presence of this particular allele can reveal the presence of the Tm-1 gene, which confers resistance to TBRFV when combined with at least one resistance QTL.

[0087] According to one embodiment, the tomato plant, cell or seed according to the invention contains in its genome a Tm-2 resistance gene, in particular Tm-2 or Tm-2 2 Also includes the Tm-2 and Tm-2a alleles. 2 Alleles are well known to those skilled in the art and are well described in the literature. 2 The alleles may be present in the genome of the plant, cell or seed according to the invention in either a homozygous or heterozygous state, but are preferably heterozygous.

[0088] In a preferred embodiment, the plants of the present invention contain a Tm-2 gene, preferably Tm-2 2 The plant contains an allele on one homolog of chromosome 9 and QTL2 on the other homolog. Such plants further contain at least one of QTL1 and QTL3, more preferably QTL3, in a homozygous state. The plant also contains the Tm-1 gene in a homozygous or heterozygous state.

[0089] Alternatively, but less preferably, the plants, seeds, or cells of the present invention do not exhibit TMV or ToMV resistance unless the Tm-1 resistance gene confers TMV or ToMV resistance to most of the widespread TMV and ToMV strains, and in particular unless the Tm-2 resistance gene is included.

[0090] The present invention encompasses tomato plants comprising, for example, the genotype combinations in Table 1. In the table, "Hom" means that the resistance QTL or resistance gene is homozygous, "Het" means that the resistance QTL or resistance gene is heterozygous, and "φ" means that the resistance QTL or resistance gene is absent.

[0091] [Table 1]

[0092] The presence of the resistance QTL and the Tm-1 gene in a homozygous or heterozygous state can be detected using the various SNP markers disclosed herein.

[0093] Preferably, the tomato plants according to the invention are commercial plants or lines, which preferably also exhibit additional resistances, such as nematode resistance traits (Mi-1 or Mi-j) and resistance to Fusarium and Verticillium.

[0094] Other resistances or tolerances are also contemplated according to the present invention. According to a preferred embodiment, the plants of the present invention are not resistant to pepino mosaic virus (PepMV). According to another embodiment, the tomato plants of the present invention are also resistant to PepMV.

[0095] In yet another embodiment, the plants of the present invention are determinate, indeterminate or semi-determinate plants, or seeds or cells thereof, i.e., correspond to a determinate, indeterminate or semi-determinate growth habit.

[0096] Determinate refers to tomato plants that tend to develop leaves first and then produce flowers, which, if successfully pollinated, mature into fruit. The fruits tend to all mature at roughly the same time on a single plant. Indeterminate tomatoes develop a few leaves first and then continue to produce leaves and flowers throughout the growing season. These plants tend to have tomato fruit at various stages of maturity at any given time. Semi-determinate tomatoes have a phenotype between determinate and indeterminate and are typical of determinate species, except that they grow larger than determinate varieties.

[0097] In yet another embodiment, the plants of the present invention are used as scions or rootstocks in grafting methods. Grafting is a method that has been used for many years in crops such as cucurbits, but has only recently been used in tomatoes. Grafting may be used to provide a specific level of resistance to soil-borne pathogens or specific nematodes. Thus, the purpose of grafting is to prevent contact between the plant or variety being cultivated and infected soil. The desired variety (optionally an F1 hybrid) used as the graft or scion is joined to a resistant plant used as the rootstock. The resistant rootstock remains healthy and provides a normal supply of soil to the graft, which it isolates from the disease.

[0098] Additionally, commercial plants of the present invention, under appropriate conditions, produce fruit that is at least 25 grams when ripe, preferably at least 100 grams when ripe, and / or more preferably at least 200 grams when ripe.

[0099] As detailed above, the present invention is directed to tomato plants that exhibit a TBRFV resistance phenotype and the seeds that give rise to those plants.

[0100] The plants or seeds according to the present invention may be the progeny or descendants of a hybrid between a plant cultivated from the deposited seed HAZTBRFVRES1, deposited with NCIMB under accession number NCIMB 42758, and a tomato plant carrying the Tm-1 gene. The plants cultivated from the deposited seeds are indeed homozygous for the resistance QTL and therefore carry the QTL of interest in their genomes on the respective homologues of chromosomes 6, 9, and 11. These can be used to combine these QTLs with the Tm-1 gene by crossing, selfing, and / or backcrossing processes, as shown in the examples of the present application.

[0101] It should be noted that the deposited seeds of HAZTBRFVRES1 (NCIMB 42758) do not correspond to plant varieties and are not homozygous for most genes other than the resistance QTL. Therefore, their phenotypes other than the leaf and fruit resistance of the present invention are not fixed during breeding. Therefore, with the exception of TBRFV leaf and fruit resistance, their phenotypic traits segregate during breeding.

[0102] According to one embodiment of the present invention, the plant, seed, or cell is more specifically resistant to the Israeli strain of TBRFV. The Israeli strain of TBRFV refers to the strain of TBRFV first identified and sequenced by Luria et al., i.e., a strain that infects tomato and has a sequence with a very high degree of sequence identity to KX619418 (SEQ ID NO: 26) (i.e., a higher degree of sequence identity with SEQ ID NO: 26 than with SEQ ID NO: 25), for example, greater than 99%, preferably greater than 99.5%, or even greater. Thus, according to one embodiment, the plant, seed, or cell of the present invention is more resistant to the Israeli strain than the Jordanian strain, for example, is resistant only to the Israeli strain.

[0103] The present invention also covers plants or seeds obtained by introgressing the resistance QTL from a tomato plant (representative seeds of which have been deposited under NCIMB Accession No. NCIMB-42758) into another tomato genetic background containing the Tm-1 gene (e.g., by crossing the plant with a parent tomato plant containing the Tm-1 gene and selecting for plants carrying the resistance QTL(s) or at least one of them and the Tm-1 gene). Such crosses can transfer QTL1, QTL2, and / or QTL3, or any combination thereof. In one embodiment, only QTL1, or only QTL2, or both QTL1 and 2, are introgressed. In another embodiment, QTL3 is introgressed. Alternatively, QTL1 and QTL3, QTL2 and QTL3, or QTL1, QTL2 and QTL3, preferably QTL2 and QTL3, are introgressed from the seed deposit of HAZTBRFVRES1 (NCIMB 42758) into a tomato genetic background containing the Tm-1 gene. The resulting progeny are preferably selfed, so that at least one resistance QTL is present in the resulting genome in a homozygous state.

[0104] According to a preferred embodiment, the plant comprises at least two QTLs selected from QTL1, QTL2, and QTL3, at least one of which is heterozygous, and preferably at least one of which is homozygous. It should be noted that the seeds or plants of the present invention may be obtained by other methods and are not necessarily obtained exclusively by biological methods.

[0105] According to this aspect, the invention relates to a tomato plant or seed, preferably a non-naturally occurring tomato plant or seed, which optionally comprises two or more mutations in its genome, thereby conferring resistance to Tomato Brown Wrinkled Fruit Virus on the plant, wherein at least one mutation is present in the genome of, for example, a plant a representative sample of which has been deposited with NCIMB under accession number NCIMB 42758, and at least one additional mutation is present on chromosome 2 and corresponds to the sequence of the Tm-1 gene.

[0106] In another embodiment, the present invention relates to a method for obtaining a tomato plant or seed that has two or more mutations in its genome that confer resistance to Tomato Brown Wrinkled Fruit Virus. Such a method is exemplified in Example 4, comprising: a) treating M0 seeds of a tomato plant to be improved with a mutagen to obtain M1 seeds; b) cultivating plants from the M1 seeds thus obtained to obtain M1 plants; c) Producing M2 seeds by selfing M1 plants; and d) Optionally, steps b) and c) are repeated n times to obtain M1+n seeds. may also include:

[0107] The M1+n seeds are grown into plants and subjected to Tomato Brown wrinkled fruit virus infection. Surviving plants or plants with milder TBRFV infection symptoms are selected for resistance to Tomato Brown wrinkled fruit virus and propagated for one or more additional generations. M0 seeds are derived from tomato plants that contain, for example, the Tm-1 gene.

[0108] In this method, the M1 seeds in step a) can be obtained by chemical mutagenesis, such as ethyl methanesulfonate (EMS) mutagenesis. Other chemical mutagens include, but are not limited to, diethyl sulfate (des), ethyleneimine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (enu), and sodium azide. Alternatively, mutations are induced by irradiation selected from, for example, X-rays, fast neutrons, and UV irradiation.

[0109] In another embodiment of the present invention, mutations are induced by genetic engineering. Such mutations include not only replacing existing sequences with alternative sequences that confer TBRFV resistance, but also incorporating sequences corresponding to resistance QTLs and the Tm-1 gene. Preferably, the mutations involve the integration of one or more of the above-mentioned QTL1, QTL2, and QTL3 in place of the homologous sequences in the tomato plant, as well as the integration of the Tm-1 gene (preferably on chromosome 2). More preferably, the mutations involve the replacement of sequences or fragments thereof contained in SNP TO-0122252 (SEQ ID NO: 7) and SNP TO-0162427 (SEQ ID NO: 18) on chromosome 11 of the tomato genome with the homologous sequences on chromosome 11 present in the genome of a plant, a representative sample of which has been deposited with NCIMB under accession number NCIMB 42758, and also include the integration of the Tm-1 resistance gene. Here, the combination of the integrated sequences confers resistance to TBRFV. The substitution on chromosome 11 corresponding to QTL3 is preferably homozygous, and the integration of the Tm-1 gene may be homozygous or heterozygous.

[0110] The genetic engineering tools that can be used include the use of all such techniques, referred to as new breeding techniques. New breeding techniques are a variety of new techniques that have been developed and / or are being used to generate new traits in plants through genetic diversity, for the purpose of targeted mutagenesis, targeted introduction of new genes, or gene silencing (RNA-directed DNA methylation (RdDM)). Examples of such new breeding techniques include zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2, and ZFN-3, see U.S. Pat. No. 9,145,565), oligonucleotide-directed mutagenesis (ODM), cisgenesis and intragenesis, grafting (onto genetically modified (GM) rootstocks), reverse breeding, agro-infiltration (also known as "agro-infiltration" in the "narrow sense," agro-inoculation, and floral dip). dip), transcription activator-like effector nucleases (TALENs, see U.S. Patent Nos. 8,586,363 and 9,181,535), CRISPR / Cas systems (see U.S. Patent Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233, and 8,999,641), engineered meganucleases, re-engineered homing endonucleases, DNA-guided genome editing (Gao et al., Nature Biotechnology (2016)), and targeted sequence alterations facilitated by the use of synthetic genomics. A key component of targeted genome editing, another name for new breeding techniques, is the use of inducing DNA double-strand breaks (DSBs) at selected locations within the genome where the alteration is intended. Targeted genome editing can be achieved by directed repair of the DSBs.Such applications can be used to generate mutations (e.g., targeted mutations or precise natural gene editing) and precise insertions of genes (e.g., cisgenes, intragenes, or transgenes). Mutation-generating applications are often identified as site-specific nuclease (SDN) technologies, such as SDN1, SDN2, and SDN3. In the case of SDN1, the result is targeted, nonspecific gene deletion mutations. That is, the location of the DNA DSB is precisely selected, but DNA repair by the host cell is random, resulting in small nucleotide deletions, additions, or substitutions. In the case of SDN2, SDN is used to generate a targeted DSB, and a DNA repair template (a short DNA sequence identical to the DNA sequence of the targeted DSB except for one or a few nucleotide changes) is used to repair the DSB. This results in a targeted, specific point mutation in the desired gene of interest. In the case of SDN3, SDN is used with a DNA repair template containing a new DNA sequence (e.g., a gene). The result of this technology is the integration of that DNA sequence into the plant genome. The most likely application describing the use of SDN3 is the insertion of expression cassettes into selected genomic locations by cisgenesis, intragenesis, or transgenesis. A full description of each of these techniques is given in a report prepared in 2011 by the Joint Research Center (JRC) of the European Commission's Institute for Prospective Technological Studies entitled "New plant breeding techniques - State-of-the-art and prospects for commercial development."

[0111] In another aspect, the present invention also relates to any plant that may be obtained from the above-described seeds or plants of the invention, as well as plant parts of such plants, most preferably explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles, and any other plant parts, wherein said plants, explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles, and / or plant parts are obtained from seeds or plants according to the first aspect of the invention (i.e., having one, two, or three of the resistance QTLs of interest in combination with the Tm-1 gene). These plant parts, particularly explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasm, leaves, anthers, stems, or petioles, contain resistance QTLs in their genomes that, when present homozygously without the Tm-1 gene, confer a phenotype of TBRFV resistance in the fruit and / or leaves, or that, when present in combination with the Tm-1 gene, confer TBRFV resistance.

[0112] Resistance QTLs in this aspect of the invention are as defined above in relation to the plants of the invention. The various features of resistance QTLs defined in relation to the first aspect of the invention apply, mutatis mutandis, to this aspect of the invention. Thus, resistance QTLs are preferably selected from those present in the genome of the plant corresponding to the deposited material HAZTBRFVRES1 (NCIMB accession number 42758). They are advantageously selected from the following QTLs depending on the QTL of interest: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele T It is characterized by the presence of allele A of O-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427, preferably by the presence of this or these alleles in a homozygous state.

[0113] The presence of the Tm-1 resistance gene to be combined with one or more resistance QTLs is preferably characterized by the SNP TO-0200838 (SEQ ID NO: 21), more particularly the allele A of TO-0200838. The TBRFV resistance is advantageously resistance to the Israeli strain of TBRFV.

[0114] The present invention is also directed to cells of tomato plants that therefore comprise in their genome a homozygous or heterozygous Tm-1 gene in combination with at least one resistance QTL that, when present homozygously in the absence of the Tm-1 gene, confers a phenotype of TBRFV resistance in fruit and / or leaves, or that, when present in combination with the Tm-1 gene, confers TBRFV resistance. The resistance QTLs are as previously defined herein and are characterized by the same features and preferred embodiments as previously disclosed for the plants and seeds according to the foregoing aspects of the invention.

[0115] The presence of these resistance QTLs can be revealed by the techniques disclosed above and known to those skilled in the art. In particular, it can be determined whether the QTLs are present in the genome of such cells of the invention in homozygous or heterozygous form. These can advantageously be selected from the following QTLs depending on the resistance QTL of interest: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele G of TO-014230 3, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427, and preferably characterized by the simultaneous (i.e. homozygous) presence of this or these alleles on each chromosome. Preferably, at least one QTL is present in homozygous form and at least one QTL is present in heterozygous form.

[0116] In one embodiment, QTL2 on chromosome 9 is present in heterozygous form in the cells according to the invention. In a particular embodiment, the other homologue on chromosome 9 is the Tm-2 gene or Tm-22 These cells contain the allele and are therefore resistant to TMV / ToMV. Preferred genotypes for the cells of the present invention are disclosed in Table 1.

[0117] The presence of the Tm-1 resistance gene to be combined with one or more resistance QTLs is preferably characterized by the SNP TO-0200838, more particularly the allele A of TO-0200838.

[0118] The cells according to the invention can be any type of tomato cell carrying one or more (preferably two) resistance QTLs of interest and the Tm-1 gene, in particular cells that can be regenerated into isolated cells and / or whole tomato plants.

[0119] The present invention also relates to tissue cultures of non-regenerable or regenerable cells of plants according to the invention as defined above. Preferably, the regenerable cells are derived from embryos, protoplasts, meristem cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, flowers, cotyledons, and / or hypocotyls of the invention, wherein the cells comprise a combination of the Tm-1 gene with one, two, or three of the resistance QTLs of interest, always in combination, whether homozygous or heterozygous within their genomes, which QTLs, when present homozygously, confer TBRFV resistance in fruits for QTL1 and / or QTL2, or TBRFV resistance in leaves for QTL3, and, when present in combination with Tm-1, confer resistance or enhanced resistance to TBRFV.

[0120] The tissue cultures are preferably capable of regenerating plants having physiological and morphological characteristics of the aforementioned tomato plants, and are capable of regenerating plants having substantially the same genotype as the aforementioned tomato plants. The present invention also provides tomato plants regenerated from the tissue cultures of the present invention.

[0121] The present invention also provides a plant protoplast as defined above (or obtained from a tissue culture as defined above), said protoplast comprising a combination of a Tm-1 gene and a resistance QTL that confers TBRFV resistance.

[0122] In another aspect, the present invention also relates to the use of tomato plants of the present invention, which contain at least one of the QTLs of the present invention, preferably homozygously, and also preferably homozygously, the Tm-1 gene, as breeding partners in breeding programs to obtain tomato plants with TBRFV resistance. Indeed, such breeding partners contain at least one of the resistance QTLs homozygously in their genome. Therefore, by crossing these plants with tomato plants, particularly lines, it is possible to transfer one, two, or three resistance QTLs and the Tm-1 gene to progeny. Thus, plants of the present invention can be used as breeding partners for introgressing the resistance QTLs and the Tm-1 gene into tomato plants or germplasm. Plants or seeds heterozygously carrying the resistance QTL or the Tm-1 gene can also be used as breeding partners, as detailed above, although phenotypic segregation can make breeding programs more complicated.

[0123] The introgressed resistance QTL and Tm-1 gene are advantageously introduced into varieties containing other desirable genetic traits, such as disease resistance (especially resistance to TMV / ToMV), early fruit maturity, drought tolerance, fruit shape, etc.

[0124] The present invention also contemplates the use of plants or seeds that contain, preferably homozygously, at least one of the resistance QTLs (e.g., tomato plants or seeds homozygously carrying a QTL that confers resistance to TBRFV infection, deposited at NCIMB under accession number NCIMB 42758) or their progeny as breeding partners in breeding programs with tomato plants that contain the Tm-1 gene. Such breeding programs can result in tomato plants or seeds that are resistant to TBRFV.

[0125] In such breeding programs, selection of progeny exhibiting the desired phenotype of TBRFV resistance, i.e., carrying at least one of the resistance QTLs and the Tm-1 gene, can be advantageously carried out based on alleles of SNP markers, particularly the SNP markers disclosed above.

[0126] Preferably, for the presence of QTL1 on chromosome 6, the presence of the allele T of TO-0005197 and / or the allele C of TO-0145581; for the presence of QTL2 on chromosome 9, the presence of the allele G of TO-0180955, the allele C of TO-0196724, the allele G of TO-0145125, and / or the allele G of TO-0196109; for the presence of QTL3 on chromosome 11, the allele T of TO-0122252, TO-0144 The progeny of the plants are selected for the presence of the C allele of TO-013333, the T allele of TO-0142270, the G allele of TO-0142294, the A allele of TO-0142303, the A allele of TO-0142306, the G allele of TO-0182276, the G allele of TO-0181040, the G allele of TO-0123057, the A allele of TO-0125528, the C allele of TO-0162432, and / or the T allele of TO-0162427. Preferably, the progeny of the plants are selected for the presence of the same allele on both homologs of each chromosome. For the presence of the Tm-1 gene, the progeny are preferably selected for allele A of TO-0200838.

[0127] Alternatively, in addition to selecting for the presence of the Tm-1 gene, selection can be based on the presence of any one of the 18 SNP alleles linked to the resistance QTL, or a combination of these alleles. Such selection is based on the presence of the alleles of interest in the sample of genetic material of the plant being selected. The presence of these alleles indeed confirms the presence of the resistance QTL at the locus defined by the SNPs. Furthermore, in addition to point mutations or recombination events, it is possible that at least one or two of these alleles are deleted, with the remaining chromosomal segment carrying the resistance QTL.

[0128] Thus, plants according to the invention are particularly valuable for marker-assisted selection to obtain commercial tomato lines and varieties with the improved phenotypes of the invention.

[0129] The present invention is also directed to methods for identifying, detecting, and / or selecting tomato plants resistant to TBRFV, which can inhibit, reduce, or delay viral replication and / or reduce viral titers in the plants. Such methods include detecting a combination of the Tm-1 gene and at least one resistance QTL in the plants to be tested or selected, where the QTL is preferably present in homozygosity. Thus, in addition to detecting the Tm-1 gene, the method can also include detecting at least one of the following markers in a sample of genetic material from the identified and / or selected plants: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252 ... G of TO-0122252, allele C of TO-0196724, allele G of TO-019610 allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, or allele T of TO-0162427 (preferably in the homozygous state). Preferably, the resistance QTL is QTL3, which is detected by the presence of T at TO-0122252, allele C at TO-0144317, allele T at TO-0142270, allele G at TO-0142294, allele A at TO-0142303, allele A at TO-0142306, allele G at TO-0182276, allele G at TO-0181040, allele G at TO-0123057, allele A at TO-0125528, allele C at TO-0162432, and allele T at TO-0162427 (preferably in the homozygous state).

[0130] Advantageously, the method comprises the detection of two resistance QTLs, at least one of which is heterozygous and preferably at least one of which is homozygous. Preferably, QTL2 is present in heterozygous form and QTL3 in homozygous form.

[0131] The present invention is also directed to methods for detecting or selecting tomato plants that have at least one of the resistance QTLs in combination with the Tm-1 gene, i.e., the marker alleles disclosed herein, wherein the detection or selection is performed under conditions of TBRFV infection, including inoculating a test plant with TBRFV and detecting an inhibition, reduction, or delay in viral replication and / or a reduction in viral titer in the plant.

[0132] The present invention is further directed to a method for detecting and / or selecting tomato plants having at least one of the Tm-1 gene and a resistance QTL, wherein the detection of the resistance QTL is based on the detection of any molecular marker that reveals the presence of said QTL. Indeed, the identification and subsequent use of molecular markers other than the 18 SNPs disclosed above can be readily performed by those skilled in the art. Thus, various alternative markers can be used to identify resistance QTLs.

[0133] Therefore, the present invention is also directed to a method for detecting and / or selecting tomato plants that are resistant to TBRFV and inhibit, reduce or delay viral replication, the method comprising: a) Tomato plants are analyzed using the genome The presence of the Tm-1 resistance gene on chromosome 2 The presence of at least one genetic marker genetically linked to a resistance QTL selected from QTL3 on chromosome 11, QTL1 on chromosome 6, and QTL2 on chromosome 9. and testing for a combination of b) selecting a plant containing the Tm-1 gene, genetic markers, and resistance QTLs linked to the genetic markers in its genome, where the selected QTLs and genetic markers are located within the chromosomal region bounded by TO-0005197 (SEQ ID NO: 1) and TO-015581 (SEQ ID NO: 2) for QTL1 on chromosome 6, within the chromosomal region bounded by TO-0180955 (SEQ ID NO: 3) and TO-0196109 (SEQ ID NO: 6) for QTL2 on chromosome 9, and within the chromosomal region bounded by TO-0122252 (SEQ ID NO: 7) and TO-0162427 (SEQ ID NO: 18) for QTL3 on chromosome 11. The genetic markers under consideration are preferably SNP markers. Resistance QTLs are as defined herein and are present in the genome of the seed HAZTBRFVRES1 plant (NCIMB accession number 42758).

[0134] In yet another aspect, the present invention also relates to methods for producing tomato plants, particularly commercial plants and inbred parent lines, that are resistant to TBRFV. Indeed, the present invention also covers the introgression of one or more of the resistance QTLs and / or the Tm-1 gene into other tomato varieties, or other species or inbred parent lines, to confer TBRFV resistance, particularly resistance to the Israeli strain of TBRFV, and is useful for producing new tomato varieties and cultivars. These methods encompass the introgression of at least one resistance QTL and the Tm-1 gene into another plant, in addition to the introgression of at least one QTL into another plant that has Tm-1.

[0135] A method or process for producing a plant having these characteristics may, for example, comprise the following steps: a) crossing a plant cultivated from the deposited seed NCIMB 42758 or its progeny, which has QTL1, QTL2, and / or QTL3 conferring TBRFV resistance, with a tomato plant preferably lacking said QTLs and carrying the Tm-1 gene; b) selecting from the progeny thus obtained plants that have one, two, or three of the resistance QTL1, QTL2, and / or QTL3 in combination with the Tm-1 gene; and c) Optionally, the method may include a step of self-pollinating the plants obtained in step b) one or more times and selecting plants that are resistant to TBRFV from the progeny thus obtained. TBRFV resistance slows, reduces, or inhibits the replication or proliferation of TBRF virus in plants and / or reduces viral titer.

[0136] Alternatively, the method may further comprise the following step instead of step a): a1) crossing a plant cultivated from the deposited seed NCIMB 42758 or its progeny, which has QTL1, QTL2, and / or QTL3 conferring TBRFV resistance, with a tomato plant preferably lacking said QTL and having the Tm-1 gene, to produce an F1 hybrid; and a2) The method may include a step of self-breeding the F1 hybrids to generate an F2 population.

[0137] In the above method, preferably, in steps b) and / or c), SNP markers are used to select plants having the resistance QTL and / or Tm-1 gene. The SNP markers for the resistance QTL are preferably one or more of the 18 SNP markers previously disclosed herein (including all combinations thereof, as described elsewhere herein).

[0138] In a preferred embodiment, the selection of plants with resistance QTL is carried out using TO-0182276 as a reference or at least one of TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528 as a reference.

[0139] Selection of a plant on the basis of one or more SNP alleles means that the plant is selected as carrying a resistance QTL if the (those) SNP alleles are the alleles corresponding to the alleles of the HAZTBRFVRES1 parent for this SNP and are not the alleles of the original tomato plant that does not carry said QTL, e.g., allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele G of TO- If allele A of 0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432 and / or allele T of TO-0162427 is detected, the plant can be selected as having the resistance QTL of the present invention.

[0140] Preferably, the tomato plant of step a) or a1) is an elite line used to obtain a plant with commercially desirable or horticultural traits. The plant has preferably been previously improved to incorporate the Tm-1 gene. According to one embodiment, the plant is susceptible to TBRFV. The plant preferably comprises the Tm-1 gene and preferably the Tm-2 gene or its Tm-2 variant. 2 This also includes alleles.

[0141] Selection of plants carrying the Tm-1 gene is preferably carried out by detecting allele A of SNP TO-0200838.

[0142] The method defined above may advantageously comprise a step of backcrossing, preferably after step c), in order to obtain a plant having all the characteristics that characterize the tomato plant. The method for producing a plant having these characteristics therefore comprises the following further steps: d) backcrossing the resistant plants selected in step b) or c) with tomato plants; e) selecting plants that have one, two, or three of the resistance QTL1, QTL2, and / or QTL3 in combination with the Tm-1 gene.

[0143] The plant used in step a), i.e., the plant corresponding to the deposited seed, may be a plant cultivated from the deposited seed. Alternatively, it may be any plant according to the first aspect of the invention that carries the phenotype-conferring QTL, preferably carrying at least one of these sequences in homozygosity. In such a case, in step a), the plant is crossed with a tomato plant that preferably does not carry the QTL, but not necessarily carries the Tm-1 gene.

[0144] In step e), SNP markers can be used to select plants carrying the resistance QTL and the Tm-1 gene. SNP markers that can be used are, for example, those described in the previous section of this specification.

[0145] Note that when selecting plants that are homozygous for at least one resistance QTL, selection should be based on the presence of an allele representing the QTL, i.e., the allele of the HAZTBRFVRES1 parent, while simultaneously not representing the allele of the re-emerged susceptible tomato parent, using one or more SNPs linked to the resistance QTL as the criterion. When selecting plants that are heterozygous for at least one resistance QTL, selection should be based on the presence of both alleles of the SNP, i.e., the allele of the HAZTBRFVRES1 parent and the allele of the re-emerged susceptible tomato parent, using one or more SNPs linked to the resistance QTL as the criterion.

[0146] The plants selected in steps b), c) or e) are preferably commercial plants, in particular plants that bear fruit weighing at least 25 grams, at least 100 g, or at least 200 g when fully ripe under normal growing conditions. Preferably, steps d) and e) are repeated at least twice, preferably three times, on (not necessarily the same) tomato plants, which are preferably breeding lines. At each selection step in the methods disclosed above, one may additionally select for a nematode resistance trait or ToMV resistance.

[0147] The self-pollination and backcrossing steps may be performed in any order or interspersed, for example, a backcross may be performed before or after one or more self-pollinations, or a self-pollination may be envisaged before or after one or more backcrosses.

[0148] Selection of progeny with the desired TBRFV resistance that delays, reduces, and / or inhibits viral replication and / or reduces viral titer in plants can also be based on comparison of Tomato Brown Wrinkled Fruit Virus resistance from tomato parents, particularly according to the experimental design disclosed in the Examples. The method used for allele detection may be based on any technique that is able to distinguish between two different alleles of a SNP on a particular chromosome.

[0149] The present invention also relates to plants obtained or obtainable by such a method, such plants being in fact tomato plants having TBRFV resistance according to the first aspect of the invention. In all methods according to the present invention, the initial TBRFV-susceptible tomato plants may be determinate, indeterminate or semi-determinate.

[0150] As previously disclosed, tomato plants according to the invention are preferably resistant to Tomato Mosaic Virus, nematodes, and Fusarium and Verticillium. To obtain such plants in the methods of the invention, the tomato parents used in the breeding program preferably carry sequences that confer resistance to Tomato Mosaic Virus, nematodes, and Fusarium and Verticillium, and a selection process is carried out to select for plants that carry these resistance sequences in addition to the resistance QTL and the Tm-1 gene.

[0151] The present invention is also directed to a method for breeding a tomato plant that is resistant to TBRFV, comprising the step of crossing a plant cultivated from the deposited seed NCIMB 42758 or its progeny, and having QTL1, QTL2, and / or QTL3 that confer TBRFV resistance, with a tomato plant that has the Tm-1 gene. The present invention is also directed to tomato plants and seeds obtainable by any of the methods disclosed above.

[0152] Preferably, any tomato seed of the present invention is coated or embedded with, alone or in combination, an active species, such as a plant nutrient, a fortifying microorganism, or a product that disinfects the seed or plant environment. Such species or chemicals may be products that promote plant growth (e.g., hormones), products that increase resistance to environmental stress (e.g., defense stimulants), or products that stabilize the pH of the substrate and its immediate surroundings, or nutrients.

[0153] They may be products for protecting young plants from substances unfavorable to their growth (such as viruses and pathogenic microorganisms in this context), such as fungicidal, bactericidal, nematicidal, insecticidal, or herbicidal products that act by contact, uptake, or gaseous diffusion. They may be, for example, any suitable essential oil, such as an extract of thyme. All of these products enhance the plant's defense response and / or disinfect or regulate the plant's environment. They may also be live biological substances, such as non-pathogenic microorganisms, such as at least one fungus, bacterium, or virus (if necessary, accompanied by a culture medium to ensure that they remain viable). These microorganisms, such as species of the genera Pseudomonas, Bacillus, Trichoderma, Clonostachys, Fusarium, and Rhizoctonia, stimulate plant growth or protect plants from pathogens.

[0154] In all of the above methods, plants homozygously carrying the resistance QTL can be identified by detecting at least one of the alleles linked to each QTL (but in combination with the absence of the other allelic form of the SNP of the invention). Thus, plants homozygously carrying QTL3 of the invention can be identified by detecting the T allele of TO-0122252, and / or the C allele of TO-0144317, and / or the T allele of TO-0142270, and / or the G allele of TO-0142294, and / or the A allele of TO-0142303, and / or the A allele of TO-0142306, and / or the G allele of TO-0182276, and / or the G allele of TO-0181040, and / or the G allele of TO-0123057, and / or the A allele of TO-0125528, and / or the T allele of TO-0142294, and / or the A allele of TO-0142303, and / or the A allele of TO-0142306, and / or the G allele of TO-0182276, and / or the G allele of TO-0181040, and / or the G allele of TO-0123057, and / or the A allele of TO-0125528, and / or the A allele of TO-0125528. Based on the confirmation of allele C of TO-0162432 and / or allele T of TO-0162427, and the absence of allele A of TO-0122252, allele T of TO-0144317, allele C of TO-0142270, allele A of TO-0142294, allele C of TO-0142303, allele G of TO-0142306, allele A of TO-0182276, allele A of TO-0181040, allele T of TO-0123057, allele G of TO-0125528, allele T of TO-0162432, and allele C of TO-0162427.

[0155] When selecting plants heterozygous for one of the resistance QTLs, preferably heterozygous for resistance QTL2, this identification means that the G allele of TO-0180955 and / or the C allele of TO-0196724 and / or the G allele of TO-0145125 and / or the G allele of TO-0196109 are detected, and simultaneously the A allele of TO-0180955, the T allele of TO-0196724, the A allele of TO-0145125 and the T allele of TO-0196109 are detected.

[0156] In all of the above methods, preferred combinations of QTL and Tm-1 genes to be associated or detected are as disclosed in relation to the first aspect of the invention, i.e. homozygous Tm-1, heterozygous QTL2 and homozygous or heterozygous QTL3, and heterozygous Tm-1, heterozygous QTL2 and homozygous or heterozygous QTL3.

[0157] Given the ability of the resistant plants of the present invention to limit damage caused by TBRFV infection, reduce virus titer, and delay, reduce, and / or inhibit virus replication and therefore its growth, they are advantageously grown in environments where TBRFV (especially the Israeli strain or isolate) is prevalent, likely to be prevalent, or infected. Under these conditions, the resistant plants of the present invention produce more saleable tomatoes than susceptible plants. Furthermore, they protect less resistant plants by limiting the spread of the virus to other fields, thus indirectly improving their yield.

[0158] Thus, the present invention also relates to a method for improving the yield of tomato plants in an environment where TBRFV, particularly the Israeli strain or isolate, is prevalent or likely to infect, the method comprising cultivating tomato plants resistant to TBRFV according to the present invention (i.e., containing in their genome at least one resistance QTL in combination with the Tm-1 gene, i.e., QTL1, QTL2, and / or QTL3 as defined in WO 2018 / 219941, in a homozygous or heterozygous state on chromosomes 6, 9, and 11, respectively) in accordance with the present invention. Preferably, at least one of the resistance QTLs is present in a homozygous state. According to another embodiment, at least one is present in a heterozygous state, and preferably another is present in a homozygous state. Preferably, the method comprises a first step of selecting or screening tomato plants having at least one of the resistance QTLs and the Tm-1 gene. This method may also be defined as a method for increasing the productivity of tomato plants in a field, tunnel, greenhouse, or glasshouse.

[0159] As disclosed in the previous aspect, the tomato plants cultivated are preferably Tm-2 or Tm-2 2 The alleles are also preferably contained in heterozygotes. Preferred genotypes of tomato plants or seeds to be cultivated are shown in Table 1.

[0160] According to a preferred embodiment, the method comprises cultivating a tomato plant which comprises the above-defined, preferably homozygous, QTL3 and Tm-1 genes on chromosome 11. The present invention is also directed to a method of reducing tomato yield losses in the context of TBRFV infestation or infection, comprising cultivating a tomato plant resistant to TBRFV as defined above. These methods are particularly valuable for populations of tomato plants, whether grown in the field, tunnels, greenhouses or glasshouses.

[0161] Alternatively, the method for improving yield or reducing tomato production losses may comprise a first step of identifying tomato plants that are resistant to TBRFV and that contain in their genomes homozygous or heterozygous resistance QTLs on chromosomes 6, 9, and / or 11 in combination with the Tm-1 gene, and then cultivating the resistant plants in an environment where the virus has spread or is likely to spread. Preferably, the plants contain the Tm-1 gene, a heterozygous QTL on chromosome 9, and a heterozygous Tm-2 or Tm-2 2 In combination with the allele, it contains the resistance QTL on chromosome 11 in homozygosity. According to a preferred embodiment, the plants identified in the first step contain the G allele of TO-0182276.

[0162] The resistant plants of the present invention can limit or even inhibit the growth of TBRFV, particularly Israeli isolates or strains of TBRFV, thereby limiting infection of additional plants and viral proliferation. Accordingly, the present invention also relates to methods for protecting fields, tunnels, greenhouses, or glasshouses, or any other type of cultivated land, from the spread of TBRFV, or at least limiting the level of TBRFV infestation in said fields, tunnels, greenhouses, or glasshouses, or limiting the spread of TBRFV in fields, tunnels, greenhouses, or glasshouses, particularly tomato fields. Such methods preferably include cultivating resistant plants of the present invention, i.e., plants containing, in their genomes, preferably homozygous resistance QTLs on chromosomes 6, 9, and / or 11 and the Tm-1 gene. The plants used of the present invention preferably contain QTL3 on chromosome 11, and more preferably, the plants exhibit the G allele of TO-0182276. Other preferred resistant plants have one of the genome combinations disclosed in Table 1.

[0163] Preferably, the method comprises a first step of selecting or selecting tomato plants which carry a resistance QTL, in particular QTL3 on chromosome 11 and the Tm-1 resistance gene. These methods may also include the subsequent step of harvesting the tomatoes.

[0164] The present invention also relates to the use of TBRFV-resistant plants for controlling TBRFV infection or spread in fields, tunnels, greenhouses or glasshouses, or other cultivated areas. Such plants are plants of the present invention that contain in their genome at least one of the resistance QTLs on chromosomes 6, 9, and / or 11, as defined above, preferably homozygous, and the Tm-1 gene. In a preferred embodiment, the plant contains two resistance QTLs in its genome, at least one of which is heterozygous, e.g., one heterozygous and one homozygous.

[0165] Therefore, according to this use, the plants of the present invention are used to protect fields, tunnels, greenhouses or glasshouses from the spread of TBRFV. The plants of the present invention used preferably contain QTL3 on chromosome 11, and more preferably, they exhibit the G allele of TO-0182276. Other preferred resistant plants have one of the genome combinations disclosed in Table 1. According to a preferred embodiment, the TBRFV is an Israeli strain or isolate of TBRFV. The resistance QTL is preferably present in the genome of the seed HAZTBRFVRES1 (NCIMB 42758) plant.

[0166] In all of these uses, preferred combinations of associated or detected QTL and Tm-1 genes are as disclosed in relation to the first aspect of the invention, i.e. homozygous Tm-1, heterozygous QTL2 and homozygous or heterozygous QTL3, and heterozygous Tm-1, heterozygous QTL2 and homozygous or heterozygous QTL3. [Brief explanation of the drawings]

[0167] [Figure 1]Results of the first ELISA test performed 45 DPI (first "Microlab" score 45 DPI) showing the presence or absence of TBRFV coat protein in leaves of test plants. The figure reports the optical density measured at 405 nm in the ELISA test for four different plants. [Figure 2] Results of an ELISA test performed 75 DPI (second "Microlab" scoring 75 DPI) showing the presence or absence of TBRFV coat protein in leaves of test plants. The figure reports the optical density measured at 405 nm in the ELISA test for four different plants. [Figure 3] ELISA results performed approximately 110 DPI showing the presence or absence of TBRFV coat protein in leaves of test plants. The figure reports the optical density measured at 405 nm in the ELISA test for four different plants. [Figure 4] ELISA results performed 70 DPI for various QTL combinations showing the presence or absence of TBRFV coat protein in leaves of test plants. [Figure 5] ELISA results performed 91 DPI for various QTL combinations showing the presence or absence of TBRFV coat protein in leaves of test plants. [Figure 6] The results show the results of evaluation of leaf symptoms 31 days after ToBRFV inoculation. Ch11-S, Ch11-H, and Ch11-R indicate the absence of QTL3 on chromosome 11 (S), the presence of QTL3 in heterozygote (H), or the presence of QTL3 in homozygote (R), respectively. Ch9-S, Ch9-H, and Ch9-R indicate the absence of QTL2 on chromosome 9 (S), the presence of QTL2 in heterozygote (H), or the presence of QTL2 in homozygote (R), respectively. Tm1-S, Tm1-H, and Tm1-R indicate the absence of the Tm-1 gene on chromosome 2 (S), the presence of the Tm-1 gene in heterozygote (H), or the presence of the Tm-1 gene in homozygote (R), respectively. [Figure 7]ELISA results for various QTL combinations performed 35 DPI showing the presence or absence of TBRFV coat protein in leaves of test plants. The figure reports the optical density measured at 405 nm in the ELISA assay. The QTL and Tm1 gene combinations are as described in Figure 6. [Figure 8] Fruit symptom assessment results 112 days after ToBRFV inoculation. Genotypes tested are detailed in Figure 6. [Example]

[0168] Example 1: Materials and Methods Lineage Description: Haz-Tm1 line: This line is a commercial, indeterminate, loose-growing tomato with normal, round, red fruit weighing approximately 120 g. The plants have light green leaves and are resistant to TMV race 0.

[0169] Resistance testing: Haz-Tm1 lines were tested for TBRFV resistance in two replicates of 10 plants (total of 20 plants). The susceptible controls used were as follows (Table 2):

[0170] [Table 2]

[0171] NB2 strain: used to form the population This line is an indeterminate, single-yielding tomato producing round, deep red fruits weighing approximately 160 g. The plants have dark green leaves and are resistant to Stemphylium, Verticillium, nematodes, Folliculorum wilt (Fol) races 1 and 2, and TMV race 2.

[0172] Symptoms: Symptoms of TBRFV infection include: Mild leaf symptoms: Usually a non-severe mosaic with no gross malformations in leaflet shape. Severe leaf symptoms: Leaflets are deformed, often with "shoestring" symptoms, and in most cases the mosaic is severe. Mild fruit symptoms: A few yellow lesions (which may look like "blotchy" symptoms) but no malformed or misshapen fruit. Severe fruit symptoms: Typical misshapen fruit, sometimes with "chocolate spots". Scoring of TBRFV symptoms: 4-point scale as described in WO2018 / 219941, where 4 corresponds to no symptoms and 1 corresponds to severe symptoms.

[0173] ELISA experimental design: Each sample containing one to two tomato leaves is crushed in a crusher. 3 ml of buffer SEB (sample extraction buffer) is added and the sample is disrupted in a bag mixer for 30 seconds. The PrimeDiagnostics ToMV primed ELISA protocol was then performed. This diagnostic test was chosen because it is designed for ToMV infection but allows for the detection of ToBRFV infection.

[0174] Student's t-test A t-test is used to determine whether the means of two data sets are significantly different from each other. In the comparative pie chart (see figure), the positions of the circles correspond to the means of the various groups. The distance between the centers of the circles represents the actual difference. The outer angle of the intersection of the comparison circles provides a guide as to whether the group means are significantly different. When the means are significantly different, the circles do not intersect or only intersect slightly, so that the outer angle of the intersection is less than 90 degrees.

[0175] marker: Suitable SNP markers for detecting resistance QTL are disclosed below. Table 3 lists the SNPs, their location in susceptible plants, and the alleles found (first nucleotide of the list: S allele) and the allele of the marker linked to resistance (second nucleotide of the list: T allele). Table 4 lists the sequences of the SNPs.

[0176] [Table 3]

[0177] [Table 4-1] [Table 4-2]

[0178] For Tm-1, a marker was developed based on information from the 2007 publication by Ishibashi et al. Four intragenic SNPs were defined, and the KASPar test was developed, with only one found to be favorable. Marker number: TO-0200838 SNP sequence: The allele associated with viral resistance is listed first in brackets (i.e., A): [ka] [ka] [ka] [ka]

[0179] Example 2: Origins of resistance The first source of resistance The inventors first identified a cultivated tomato (Solanum lycopersicum) line, Haz-Tm1, as having a high level of leaf TBRFV resistance, which was also known to contain the gene Tm-1. According to the literature and as is well known to experienced breeders, Tm-1 was originally introgressed from the wild tomato species Solanum habrochaites PI126445 into the cultivated tomato species Solanum lycopersicum to confer ToMV / TMV resistance. However, the ToMV resistance conferred by this gene was overcome within a year of its introduction into commercial tomato varieties in the 1960s. Therefore, this gene is rarely, if ever, found in current commercial varieties and can no longer be considered a resistance gene to ToMV or TMV.

[0180] Based on the published gene sequence, markers were developed for the Tm-1 gene (chromosome 2). Four SNPs were defined and a KASPar test was developed, with only one found to be favorable. The inventors first found that the Haz-Tm1 line was highly resistant to TBRFV in two artificial laboratory tests. Subsequently, the inventors also screened the Haz-Tm1 line for fruit resistance under field conditions in greenhouse trials (natural infection). 2 The plants were then transplanted into a greenhouse at a temperature equivalent to 100°C (equivalent to 100°F). The results showed that the Haz-Tm1 line showed mild TBRFV symptoms mainly in the fruit at the final stage of plant development. It was concluded that the Haz-Tm1 line probably has high resistance to leaf symptoms and mild and insufficient resistance to fruit symptoms. The Haz-Tm1 line was subsequently retested in tests such as ELISA tests, including: (1) Sow in a tray labeled "54" (2) mechanical inoculation of young seedlings; (3) Scoring - Observe the symptoms of tobamovirus, (4) Confirm the presence or absence of virus at three time points using Immunostrip kits (AGDIA) and ELISA tests; (5) From seedlings planted in a greenhouse to a complete growth cycle.

[0181] Sowing seeds in seedling trays: October 9th Machine vaccination: October 31st Transplanting part of the trial to Brurim (greenhouses GH3 and 4): November 5th

[0182] Transplanting part of the trial to the Mivtahim greenhouse: November 13th First scoring and ELISA test sample collection: December 16th and 17th Second scoring and ELISA test sample collection: January 14th Third scoring and ELISA test sample collection: February 19th. The results of the first scoring, approximately 45 days post inoculation (DPI), are detailed in Table 5. At this stage, there is no fruit, so only leaf resistance is assessed.

[0183] [Table 5]

[0184] The results of the ELISA test are shown in FIG.

[0185] Second scoring Phenotypic scoring of the second round showed similar results to those obtained with the first round. The results of the ELISA tests are shown in Figure 2.

[0186] Third scoring The results of the third scoring, approximately 110 DPI, are detailed in Table 6. At this stage, the fruit is present, so the leaves and fruit are scored for resistance.

[0187] [Table 6]

[0188] The results of the ELISA test are shown in FIG.

[0189] The ELISA results suggest that the Haz. Tm-1 line possesses a defense mechanism that delays viral replication in plants.

[0190] A second source of resistance WO2018219941 discloses resistance QTLs to TBRFV, essentially a leaf resistance QTL, QTL3 on chromosome 11, and two fruit resistance QTLs, QTL1 and 2 on chromosomes 6 and 9, respectively.

[0191] Example 3: Combination by crossing two sources Group formation A cross was made between the Haz-Tm1 line and the NB2 line to produce F1 seeds, which were then self-pollinated to produce F2 seeds. The F2 seeds were sown in trays and selected for homozygosity of resistance QTL3 (i.e., a QTL on chromosome 11) using one representative marker, such as TO-0142306. These plants were grown to produce F3 seeds, which are referred to as population 1 in the examples (see Table 7).

[0192] Plant genotyping and selection: The F3 seeds (population 1) were sown in trays to obtain approximately 500 seedlings. From each F3 seedling, leaf discs were collected for DNA extraction, and the DNA was used for molecular marker analysis. Two molecular markers were used for selection: one for the Tm-1 gene on chromosome 2, and the second for a QTL (QTL2) on chromosome 9. A QTL (resistance QTL3) on chromosome 11 was already fixed in the F2 as a resistance homozygote (see population formation).

[0193] result: A cross was made between the Haz. Tm-1 line and one breeding line, NB2, which contains a QTL on chromosome 11 and a QTL on chromosome 9. F3 seeds were obtained as disclosed above. Molecular markers linked to the resistance QTL and the Tm-1 gene were used to preselect F3 plants in trays, and the selected plants were mechanically inoculated at the young seedling level. The seedlings were planted in a greenhouse at Bsor and grown in the greenhouse. Molecular marker analysis included one marker per QTL. Tables 7 and 9 show the different F3 plants from population 1 containing different genotypes at three loci (QTL2, QTL3, and Tm-1) and their resistance based on the phenotypic scoring and ELISA results for each plant. Controls are also shown. Healthy controls are uninfected controls. Table 7 shows the results at 70 DPI and Table 9 shows the results at 91 DPI. Some of the leaf symptoms reported in the table may have increased due to the presence of pepinoviruses or the harsh temperature conditions in the greenhouse. Indeed, it is well known that tobamovirus infection symptoms increase with increasing temperature. Thus, the moderate to severe symptoms observed in this assay may be considered only mild symptoms under more moderate conditions. In fact, this assay was designed to distinguish between resistant plants on the one hand and resistant or susceptible plants on the other hand, but not between resistant / tolerant plants and susceptible plants.

[0194] [Table 7-1] [Table 7-2]

[0195] [Table 8]

[0196] Figure 4 shows the results of the ELISA test at 70 DPI for various combinations of QTLs and controls. It can be inferred that combining the Tm-1 gene with at least one of the resistance QTLs significantly reduces the level of ToBRFV virus coat protein detection in plants, and that combining the Tm-1 gene with two resistance QTLs results in ToBRFV detection levels as low as those found in uninfected, healthy plants (Chr11-R, Tm-1-R, Chr9-R).

[0197] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0198] [Table 10]

[0199] Figure 5 shows the results of the ELISA tests for various combinations of QTLs and controls at 91 DPI.

[0200] The results shown in Tables 9 and 10 confirm the resistance of plants containing Tm-1 and at least one resistance QTL and demonstrate that this resistance is still present three months after infection, protecting the plants from leaf and fruit damage.

[0201] Example 4: Genetic modification of tomato seeds with ethyl methanesulfonate (EMS) Seeds of tomato cultivars are EMS-treated by soaking approximately 2000 seeds per cultivar in an aerated solution of 0.5% (w / v) or 0.7% EMS for 24 hours at room temperature. Approximately 1500 treated seeds per EMS dose and per variety are germinated and the resulting plants are grown, preferably in a greenhouse, for example from May to September, to produce seed. After maturity, M2 seeds are harvested and pooled together, one for each variety and treatment. The resulting M2 seed pools are used as the starting material to identify individual M2 seeds and plants with fruit and / or leaf resistance to Tomato Brown Wrinkled Fruit Virus.

[0202] Example 5: ToBRFV Segregation Tracking - Testing Various Combinations of QTL and Tm-1 In this study, the inventors tested various combinations of a QTL on chromosome 11 (QTL3, or Ch11 as in Example 3), a QTL on chromosome 9 (QTL2, or Ch9 as in Example 3) with Tm-1 on chromosome 2, and a susceptible control (Haz Tm-R) carrying the Tm2 gene. Healthy controls correspond to plants not exposed to the virus. Materials and methods were as disclosed for Example 3, particularly with regard to leaf and fruit labeling and ELISA testing.

[0203] Calendar Sowing: T0 Sample collection for DNA extraction: T0+14 days Mechanical inoculation: T0+28 days Transplantation into quarantine: T0+29 days = 1 DPI First scoring (leaf symptoms): 31 DPI Sample collection for ELISA: 35 DPI Second scoring (fruit symptoms): 112 DPI

[0204] result: First scoring (leaf symptoms) and ELISA (31 DPI) General observations: All plants of susceptible genotypes, which had not yet set fruit, showed significant leaf symptoms. Leaf symptom index: 1 is severe leaf symptom, 9 is no visible symptom. The average leaf symptom is shown in Figure 6. It is observed that the addition of the Tm-1 gene improves the score, and this improvement is even greater when the Tm-1 gene is present in homozygous form. Furthermore, it is observed that three genotypes, namely [Ch11-R Tm1-R Ch9-H], [Ch11-R Tm1-R Ch9-R], and [Ch11-R Tm1-R Ch9-S], do not show symptoms.At this early stage (31 DPI), QTL2 (fruit resistance QTL) on chromosome 9 does not contribute to leaf resistance. Three other genotypes, namely [Ch11-R Tm1-H Ch9-H], [Ch11-R Tm1-H Ch9-R], and [Ch11-R Tm1-H Ch9-S], also exhibit significant leaf resistance, although it is slightly less significant than the above three genotypes. ELISA tests were performed 4 days later, 35 DPI. The results are reported in Table 11 and shown in FIG.

[0205] [Table 11]

[0206] It is observed that plants homozygous for both QTL3 and Tm1 (Ch11-R Tm1-R) exhibit lower levels of virus than all other genotypes. From these leaf symptom scoring and ELISA testing at 31 and 35 DPI, the following can be concluded: 1. All plants with the Ch11-R and Tm-1-R combinations (three combinations with Ch9 (QTL2) in all three states) are symptomless. These combinations are ELISA positive, but show much lower virus loads than all other combinations. 2. Three combinations of Chr-11-R and Tm-1-H yield almost completely symptomless plants, but their ELISAs do not appear statistically different from susceptible genotypes at this particular stage of 30 DPI.

[0207] Second scoring - Fruit symptoms (112 DPI) General observations: Most plants have a few clusters with red berries. Fruit symptom index: 1 is severe leaf symptoms, 9 is no visible symptoms. The symptoms on the leaves are consistent with the first findings. Fruit symptoms are reported in Figure 8 and detailed in Table 12.

[0208] [Table 12]

[0209] It can be assumed that the homozygous or heterozygous presence of QTL2 on chromosome 9 (Ch9) significantly improves fruit resistance (see, for example, the first three genotypes in Figure 8). In Figure 8, fruit symptoms are absent when QTL2 is present in the homozygous state (Ch9-R) and mild when QTL2 is present in the heterozygous state (Ch9-H), whereas these symptoms are greater when QTL2 is absent (Ch9-S).

[0210] This test cannot distinguish between the genotypes [Ch11-R, Tm1-R / H, Ch9-H] and [Ch11-R, Tm1-S, Ch9-H] because both genotypes are scored as 9 in this experimental design. However, the results of Example 3 suggest that the presence of homozygous or heterozygous Tm1 genes confers an enhanced level of resistance to plants corresponding to the genotype [Ch11-R, Ch9-H] under various conditions of ToBRFV infection or at later stages of infection.

[0211] conclusion The preferred homozygous presence of QTL3 on chromosome 11 in combination with the Tm1 gene results in the best leaf resistance with reduced virus titers when both are present homozygously (Ch11-R; Tm1-R). Therefore, this combination provides the best resistance to the initial stage of ToBRFV infection. Furthermore, leaf resistance ensures proper plant development, which in turn ensures healthy plants with better photosynthesis and the expected higher fruit yield. Furthermore, a reduced virus titer means that plants are less likely to contaminate other plants around them and propagate the virus, and the slower virus progression may prevent more serious infection stages, especially in the case of late-onset infections. This combination should preferably be combined with QTL2 on chromosome 9 (Ch9-H or Ch9-R) to ensure good fruit resistance. In conclusion, genotypes corresponding to Ch11-R, Tm1-R, Ch9-H or R provide the best overall results for the combined criteria of leaf resistance (advantageous for photosynthesis and yield), virus titer (less contamination and slower progression), and fruit resistance (increased yield of saleable fruit).

Claims

1. 1. A method for conferring resistance to TBRFV to a tomato plant, the method comprising: a) crossing a plant having at least one QTL conferring TBRFV resistance, selected from QTL2 on chromosome 9 and QTL3 on chromosome 11, with a tomato plant having the Tm-1 gene; b) selecting plants that have one or two of the QTL2 and / or QTL3 in combination with the Tm-1 gene from the progeny thus obtained; wherein the resistance delays, reduces, or inhibits replication or proliferation of the virus, and wherein the QTL2 and QTL3 are present in the genome of a plant, representative seeds of which have been deposited under NCIMB Accession No. 42758.

2. 1. A method for conferring resistance to TBRFV to a tomato plant, the method comprising: a1) crossing a plant having at least one QTL conferring TBRFV resistance selected from QTL2 on chromosome 9 and QTL3 on chromosome 11 with a tomato plant having the Tm-1 gene to produce an F1 hybrid; a2) selfing the F1 hybrids to generate an F2 population; b) A step of selecting individuals having resistance to TBRFV from the progeny thus obtained. wherein the resistance delays, reduces, or inhibits replication of the virus, and wherein the QTL2 and QTL3 are present in the genome of a plant, representative seeds of which have been deposited under NCIMB Accession No. 42758.

3. c) self-pollinating the plants obtained in step b) one or more times and selecting plants that are resistant to TBRFV from the progeny thus obtained.

3. The method of claim 1 or 2, further comprising:

4. The following additional steps: d) backcrossing the resistant plants selected in step b) or c) with tomato plants; e) Selecting plants that have one or two of the resistance QTL2 and / or QTL3 in combination with the Tm-1 gene The method of any one of claims 1 to 3, further comprising:

5. The method according to any one of claims 1 to 4, wherein in steps b) and / or c), SNP markers are used to select plants having QTL2 and / or QTL3 that confer TBRFV resistance and / or to select plants having the Tm-1 gene.

6. Selection of plants carrying QTL2 and / or QTL3 was carried out using the G allele of TO-0180955 (SEQ ID NO: 3), the C allele of TO-0196724 (SEQ ID NO: 4), the G allele of TO-0145125 (SEQ ID NO: 5) and / or the G allele of TO-0196109 (SEQ ID NO: 6) for the presence of QTL2 on chromosome 9, and the T allele of TO-0122252 (SEQ ID NO: 7), the C allele of TO-0144317 (SEQ ID NO: 8), the T allele of TO-0142270 (SEQ ID NO: 9), the T allele of TO-014229 6. The method of claim 5, wherein the method is performed based on the presence of the G allele of TO-0142304 (SEQ ID NO: 10), the A allele of TO-0142303 (SEQ ID NO: 11), the A allele of TO-0142306 (SEQ ID NO: 12), the G allele of TO-0182276 (SEQ ID NO: 13), the G allele of TO-0181040 (SEQ ID NO: 14), the G allele of TO-0123057 (SEQ ID NO: 15), the A allele of TO-0125528 (SEQ ID NO: 16), the C allele of TO-0162432 (SEQ ID NO: 17) and / or the T allele of TO-0162427 (SEQ ID NO: 18).

7. 7. The method of claim 6, wherein the selection of plants carrying QTL2 and / or QTL3 is based on the presence of said alleles in homozygosity.

8. The method of claim 5, wherein the selection of plants containing the Tm-1 gene is based on the presence of allele A of TO-0200838 (SEQ ID NO: 21).

9. The method according to any one of claims 1 to 7, wherein the tomato plant in step a) or step a1) is an elite line.

10. 1. A method for breeding a tomato plant having resistance to TBRFV, comprising the step of crossing a plant having at least one QTL selected from QTL2 on chromosome 9 and QTL3 on chromosome 11 that confers resistance to TBRFV with a tomato plant having the Tm-1 gene, wherein the QTL2 and QTL3 are present in the genome of the plant, and representative seeds thereof have been deposited under NCIMB Accession No. 42758.

11. The method according to claim 10, further comprising the step of self-pollinating the obtained plant body at least once and selecting a plant body having resistance to TBRFV from the progeny thus obtained.

12. The method according to claim 10 or 11, wherein a plant having at least one QTL selected from QTL2 on chromosome 9 and QTL3 on chromosome 11 contains the QTL in a homozygous state.

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