Resistance to ToBRFV in tomato plants

Abstract

The present invention relates to Solanum lycopersicum plants containing in their genome a QTL that confers improved resistance to Tomato Brown Rugose Fruit Virus relative to corresponding plants lacking said QTL, said QTL being introgressed from S. pimpinellifolium and found on chromosome 9 within the chromosomal region delimited by markers with SEQ ID NOS: 102 and 115, or within the chromosomal region delimited on chromosome 11 by the SNPs with SNP TO-0201220 (SEQ ID NOS: 1) and SEQ ID NOS: 101. This QTL may be selected from those present in the genome of the plant seed LVSTBRFVRES2 NCIMB Accession No. 43591. The QTL is preferably characterized by defined alleles of different SNPs on chromosomes 9 or 11. The present invention also relates to parts of these plants with improved resistance, as well as to the use of these plants for introducing improved resistance to progeny in other genetic backgrounds, and to different methods for obtaining tomato plants or seeds with increased leaf and / or fruit resistance to Tomato Brown Rugose Fruit Virus.

Description

[Technical Field]

[0001] The present invention relates to resistance to the tobamovirus Tomato Brown Rugose Fruit Virus (ToBRFV, formerly abbreviated as TBRFV) in Solanum lycopersicum (tomato) plants, also known as Lycopersicum esculentum. More specifically, the present invention relates to tomato plants and fruits containing genetic determinants conferring resistance to Tomato Brown Rugose Fruit Virus. According to the present invention, resistance is provided by a DNA sequence or QTL introgressed from S. pimpinellifolium into the genome of S. lycopersicum plants on chromosome 9 or chromosome 11. The introgressed QTL on chromosome 9 can be present homozygously or heterozygously in the genome of S. lycopersicum plants. Preferably, the introgressed QTL on chromosome 11 is present homozygously in the genome of S. lycopersicum plants. The present invention further relates to markers linked to the DNA sequences and the use of such markers to identify or select the DNA sequences or QTL and to identify or select plants having such resistance. The invention also relates to the seeds and progeny of such plants, as well as the propagation material for obtaining such plants, and the different uses of these plants. [Background technology]

[0002] All cultivated and commercial forms of tomatoes belong to the species most often referred to 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. The genus Lycopersicon has been divided into two subgenera: the Esculentum complex, which contains species that readily hybridize with commercial tomatoes, and the Peruvian complex, which contains species that hybridize more difficultly (Stevens, M., and Rick, C.M. 1986). L. esculentum Miller has become widespread worldwide due to its value as a crop. While the exact origin of the cultivated tomato remains unknown, it is believed to have originated in Ecuador, Peru, the Galapagos Islands, and, initially, the Americas, where it was cultivated by the Aztecs and Inca Islanders for as long as 700 years. Mexico was the site of domestication and likely the earliest source of introduction. The cherry tomato (L. esculentum var cerasiforme) is presumed to be the direct ancestor of modern cultivated forms.

[0003] Tomatoes are grown for their fruit, which is widely used in the fresh market or as a processed product. As an agricultural crop, tomatoes are grown commercially as long as environmental conditions allow for the production of economically viable yields. The majority of fresh market tomatoes are harvested by hand at vine-ripened and ripe-green maturity. Fresh market tomatoes are available year-round. Tomato processing is mostly mechanically harvested and used in a variety of forms, including canned tomatoes, tomato juice, tomato sauce, puree, paste, and even cutlets.

[0004] Tomatoes are usually simple diploids, possessing 12 pairs of differentiated chromosomes. However, polyploid tomatoes are also part of this invention. Cultivated tomatoes are mostly self-pollinating. Tomato flowers are hermaphrodite. Commercial cultivars were initially pollinated. Hybrid vigor has been identified in tomatoes, and hybrids are becoming increasingly popular among farmers, replacing open-pollinated varieties with better yields and uniform plant characteristics. Tomatoes have been intensively bred for their widespread popularity and high value, which explains why such a wide variety of tomatoes is currently available. Shapes range from small to large, including cherry, plum, pear, block, round, and beefsteak. Tomatoes are grouped by the time required for fruit to ripen for harvest; cultivars are generally considered to be early-, mid-, or late-maturing. Tomatoes are also classified by plant growth habit: determinate, semi-determinate, or indeterminate. If pollination is successful, the plant will tend to develop leaves first, then mature flowers into fruit. All fruits tend to ripen on the plant at roughly the same time. Indeterminate tomatoes start by developing some leaves and continue to produce leaves and flowers throughout the growing season. These plants tend to have tomato fruit at different stages of maturity at any given time. Semi-determinant tomatoes are typical determinants, with a phenotype between determinant and indeterminate, except that they grow larger than determinants. Recent advances in tomato breeding have resulted in a wider range of fruit colors. In addition to the standard ripe red, tomatoes can be creamy white, lime green, pink, yellow, golden yellow, orange, or purple.

[0005] Hybrid commercial tomato seeds can be produced by hand pollination. Pollen from the male parent is harvested and manually applied to the stigma of the female inbred. Before and after hand pollination, the flowers are coated to prevent insects from introducing foreign pollen and to prevent contamination or impurities. The flowers are labeled to identify the pollinated fruit, from which seeds are harvested.

[0006] The productivity of tomato plants is affected by a variety of pathogens, including viruses, fungi, bacteria, nematodes, and insects. Tomato is particularly susceptible to many viruses, and therefore virus resistance is of agricultural importance.

[0007] 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 seed propagation as well as mechanical means. Tobamoviruses are generally characterized by approximately 300 nm rod-shaped particles that encapsidate a single-stranded, positive-sense RNA genome encoding four proteins. In tomatoes, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) are of concern to growers worldwide because they cause significant damage to crop production through irregular ripening (fruit with yellow spots on the surface and brown spots below the surface). However, several genes have been identified by plant breeders over the years, and TMV- and / or ToMV-resistant tomato varieties are now available.

[0008] For the past few decades, modern indeterminate and many determinate tomato varieties have indeed contained the Tm-2 gene or preferably the Tm-22 allele of this gene, conferring immunity to almost all known tobamoviruses that affected commercial tomatoes (ToMV and TMV) before 2014.

[0009] From 2014 to 2015, tomato-producing regions in the Middle East, including Jordan and Israel, experienced severe virus outbreaks. 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: leaf symptoms were very similar to TMV / ToMV symptoms, while fruit symptoms, including fruit lesions and deformities, were much more frequent and severe than the usual symptoms from such viruses. Fruit quality was very poor, rendering the fruit unmarketable. Salem et al. (Arch. Virol. 161 (2), 503-506, 2015) extracted RNA from fruit and leaves of infected symptomatic plants in Jordan, performed various tests, and identified a new Tobamovirus species, the sequence of which corresponds to GenBank accession number KT383474 (SEQ ID NO: 112); Salem et al. proposed naming this Jordan virus: Tomato Brown Rugose Fruit Virus (formerly TBRFV, now ToBRFV). Comparison with other tobamovirus sequences indicated that this virus is indeed a tobamovirus, not TMV or ToMV. Resistance to TMV and / or ToMV does not confer resistance to this new virus, ToBRFV. Luria et al. (PLoS One. 2017; 12(1): e0170429) simultaneously isolated and sequenced the complete genome of an Israeli tobamovirus infecting tomatoes in Israel, corresponding to GenBank accession number Kx619418 (id no: 113 onward). Thus, very high sequence identity (>99% sequence identity) was observed between the Israeli and Jordanian viruses, leading to the conclusion that they represent two distinct isolates of Tomato Brown Rugose Fruit Virus.

[0010] Recently, the virus has been identified in Europe, particularly in Sicily, Germany, the Netherlands, France, and Mexico, and is now considered a major global threat to tomato crops. The strains identified appear to be essentially Israeli, rather than Jordanian, strains.

[0011] The identification of tomato plants exhibiting resistance to Tomato Brown Rugose Fruit Virus, and the localization and identification of genetic determinants (hereinafter referred to as QTLs (Quantitative Trait Loci)) conferring resistance to Tomato Brown Rugose Fruit Virus, referred to in this publication as tolerance, have been recently described in WO 2018 / 219941. Two QTLs, QTL1 and QTL2, are found on chromosomes 6 and 9, respectively, and when present homozygously in a S. lycopersicum background, independently or in combination, confer improved tolerance or resistance in the fruit of tomato plants infected or susceptible to ToBRFV. A third QTL, QTL3, is found on chromosome 11, and when present homozygously, confer improved tolerance or resistance in the leaves of tomato plants infected or susceptible to ToBRFV.

[0012] Although these QTLs, alone or in combination, confer tolerance or resistance to ToBRFV, the inventors have demonstrated that in most cases they fail to confer a sufficiently high level of resistance to tomato plants, resulting in a significant proportion of the fruit being affected and unmarketable. Furthermore, these QTLs have been described as conferring resistance when present in homozygosity. To the extent that QTL2 on chromosome 9 resides at the same locus as the Tm-22 gene, in regions that are transmitted "en bloc" without recombination, such QTL on chromosome 9 is therefore not suitable for combination with the Tm-22 gene.

[0013] WO2019 / 110130 and WO2019 / 110821 disclose the identification of three different QTLs on chromosomes 6, 11, and 12 that were introgressed from S. pimpinellifolium and are said to confer resistance or tolerance to ToBRFV. However, the QTL on chromosome 11 is described as being located between two markers that define a 55-Mb region corresponding to nearly the entire sequence of chromosome 11. Without clear description, this QTL on chromosome 11 cannot be used for introgression.

[0014] WO2020 / 018783 discloses a genetic region on tomato chromosome 11 that contains a Stemphylium resistance allele from S. pimpinellifolium and also contains an associated ToBRFV resistance allele. Because both alleles are linked to the same marker according to the disclosure herein, introgression of ToBRFV resistance alone is not possible, especially in plants that are already resistant to Stemphylium but susceptible to ToBRFV.

[0015] Tobamoviruses are not easily controlled, but genetic improvement through the identification and improvement of resistance genes is feasible. Furthermore, because currently available resistance genes for controlling TMV and / or ToMV are useless against damage caused by the new Tomato Brown Rugose Fruit Virus, identified tolerance or resistance QTLs are not always efficient enough, are not well characterized, and cannot be combined with Tm-22, there is an urgent need to identify resistance to this new tobamovirus. Summary of the Invention

[0016] The present inventors identified resistance to ToBRFV in wild S. pimpinellifolium plants and were able to invade this resistance into S. lycopersicum plants, thus obtaining resistant S. lycopersicum tomatoes to ToBRFV. The resistance of the present invention is conferred by a newly discovered sequence linked to an additive quantitative trait locus (QTL) that is transferable across different S. lycopersicum genetic backgrounds.

[0017] The newly discovered QTLs confer resistance to Tomato Brown Rugose Fruit Virus (ToBRFV) essentially at the level of the fruit of virus-infected tomato plants and at the level of the leaves of virus-infected tomato plants for a QTL on chromosome 9, and essentially at the level of the leaves of infected plants for a QTL on chromosome 11.

[0018] Thus, the present invention provides these transgenic sequences (also referred to herein as QTLs) that confer a ToBRFV resistance phenotype at the leaf and / or fruit level on ToBRFV-infected tomato plants.

[0019] The present invention provides S. lycopersicum plants, including commercial plants, lines, and hybrids, that exhibit resistance to ToBRFV, as well as methods for producing or identifying S. lycopersicum plants or populations (germplasm) that exhibit resistance to ToBRFV. The present invention also discloses molecular genetic markers, particularly single nucleotide polymorphisms (SNPs), linked to the QTLs of the present invention that are involved in resistance to ToBRFV. Plants obtained by the methods and use of such molecular markers are also provided.

[0020] Furthermore, the resistances described above are easily transferable to different genetic backgrounds, i.e., different tomatoes, and the invention also extends to different methods allowing the transfer or introgression of phenotype-conferring QTLs.

[0021] The present invention also provides several methods and uses of information related to these SNPs associated with QTLs conferring ToBRFV resistance, in particular methods for identifying ToBRFV-resistant plants and for identifying additional molecular markers associated with this resistance, as well as methods for improving the yield of tomato production in environments infested by ToBRFV and methods for protecting tomato fields from ToBRFV infestation.

[0022] Definition: The term "resistance" is defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Section to describe a plant's response to pests or pathogens and abiotic stresses for the vegetable seed industry. Specifically, resistance refers to the ability of a plant variety to limit the growth and development of specific pests or pathogens and / or the damage they cause compared to susceptible plant varieties under similar environmental conditions. Resistant varieties may show disease symptoms and damage under heavy pest or pathogen pressure. Two levels of resistance are defined:

[0023] Highly Resistant (HR): A plant that, when compared with susceptible plants, highly limits the growth and / or development of a particular pest and / or the damage it causes under normal pest pressure. However, these plants may exhibit certain symptoms or damage under heavy pest pressure.

[0024] Intermediately resistant (IR): A plant that is highly restrictive of, and / or causes damage to, the growth and / or occurrence of a particular pest, but may exhibit more extensive symptoms or damage than highly resistant plants. Intermediately resistant plants still exhibit less severe symptoms or damage than susceptible plants when grown under similar environmental conditions and / or pest pressure.

[0025] The term "tolerance" is commonly used to describe the ability of a plant to withstand abiotic stress without significant effects on growth, appearance and yield.

[0026] However, in literature and patents, this term is also used to refer to a plant phenotype in which, under at least some culture conditions, at least some disease symptoms remain absent when the plant is exposed to an infectious dose of a virus that can establish systemic or local infection, viral proliferation, the presence of at least viral genomic sequences in the plant's cells, and / or their genomic integration. Thus, a tolerant plant is resistant to symptom expression but is an asymptomatic virus carrier. Sometimes, viral sequences can be present or proliferate in the plant without causing disease symptoms. It should be understood that a tolerant plant can be infected with a virus, but can generally limit viral proliferation and development to at least a moderate extent.

[0027] For this reason, tolerant plants by this definition are best characterized by intermediately tolerant plants.

[0028] In the case of ToBRFV, by foliar resistance or foliar resistance is meant a plant phenotype in which disease symptoms on the leaves are absent or less significant upon exposure of the plant to an infectious dose of ToBRFV. However, infected plants may show disease symptoms on the fruit.

[0029] Fruit resistance, in the case of ToBRFV, refers to a plant phenotype in which disease symptoms on fruit are absent or insignificant upon exposure of the plant to an infectious dose of ToBRFV, although disease symptoms may appear on the leaves of infected plants.

[0030] Leaf symptoms of ToBRFV infection generally include mosaic lesions, leaflet distortion, and often sock-like symptoms. Fruit symptoms of ToBRFV infection generally include typical yellow lesions (discoloration) and fruit deformation. Often, fruit also exhibits "chocolate spots."

[0031] Susceptibility: The inability of a plant to restrict the growth and development of a particular pest or pathogen; susceptible plants show adverse symptoms associated with viral infection, i.e., leaf damage and fruit damage in the case of ToBRFV infection.

[0032] An S. licopersicum plant susceptible to Tomato Brown Rugose Fruit Virus is, for example, the commercial cultivar Candela, as described in the publication of Salem et al. in 2015. To date, i.e., prior to the present invention, all commercial cultivars of tomato grown in ToBRFV-infected areas have either been susceptible to ToBRFV or have not been sufficiently resistant to plants carrying a tolerance QTL, such as the HAZTBRFVRES1 deposit described in PCT application WO2018 / 219941.

[0033] Thus, plants of the present invention have at least improved resistance or tolerance to ToBRFV with respect to cultivar Candela, and more generally in ToBRFV-infected regions including tolerant plants, and with respect to HAZTBRFVRES1. Improved resistance to plants corresponding to HAZTBRFVRES1 is demonstrated in Example 5 of the Experimental Section.

[0034] As used herein, the term "offspring" or "progeny" refers to any plant resulting from vegetative or sexual reproduction of one or more parent plants or their offspring. For example, offspring plants can be obtained by cloning or autogamy of a parent plant or by crossing two parent plants, and include autogamy as well as F1 or F2, or other generations. F1 refers to the first generation offspring produced from the parents, at least one of which is used for the first time as a trait donor. Meanwhile, the offspring of the second (F2) or subsequent generations (F3, F4, etc.) are samples produced from autogamy, such as F1', F2', etc. Thus, F1 is a hybrid resulting from a cross between two pure-bred parents (pure-breds are homozygous for the trait), while F2 is (usually) the offspring resulting from self-pollination of an F1 hybrid.

[0035] As used herein, the terms "cross", "crossing", "cross-pollination" or "outcrossing" refer to the process by which pollen from one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.

[0036] As used herein, the terms "genetic determinant" and / or "QTL" refer to any segment of DNA 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.

[0037] 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.

[0038] As used herein, the term "grafting" refers to the process of grafting a rootstock with a cutting. The primary motivation for grafting is to avoid damage from soil-borne pests and pathogens when genetic or chemical approaches for disease management are unavailable. Grafting susceptible cuttings onto resistant rootstock allows for the creation of resistant varieties without the need to breed varieties for resistance. Furthermore, grafting may increase tolerance to abiotic stress, increase yield, and result in more efficient water and nutrient use.

[0039] As used herein, the term "heterozygote" refers to a diploid or polyploid individual cell or plant that has different alleles (forms, genetic determinants, or sequences of a given gene) present at at least one locus.

[0040] As used herein, the term "heterozygosity" refers to the presence of different alleles (forms, genetic determinants, or sequences of a given gene) at a particular genetic locus.

[0041] As used herein, "homologous chromosomes," or "homologous chromosomes" (or homologous chromosomes) refer to a set of one maternal and one paternal chromosome that pair together during meiosis. These copies have the same type of gene at the same locus and centromeric position, but may differ by their base sequence and alleles.

[0042] As used herein, the term "homozygous" refers to an individual cell or plant that has the same allele at one or more loci on all homologous chromosomes.

[0043] As used herein, the term "homozygous" refers to the presence of identical alleles at one or more loci in homologous chromosomal segments.

[0044] 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.

[0045] As used herein, the term "locus" (plural: "locuses") refers to any genetically defined site, which may be a single position (nucleotide) or a chromosomal region. A locus may be a gene, a genetic determinant, a portion 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), several SNPs, or two adjacent SNPs.

[0046] As used herein, the term "rootstock" is the lower part of a plant that can accept a cutting in the grafting process.

[0047] As used herein, the term "scion" is the taller part of the plant that can be grafted onto the rootstock in the grafting process.

[0048] The present invention encompasses plants of different ploidy levels, essentially diploid plants as well as triploid plants, tetraploid plants, etc.

[0049] In the context of the present invention, DNA strands and alleles are designed to be TOP according to the TOP / BOT designation method developed by Illumina: (https: / / www.illumina.com / documents / products / technotes / technote topbot.pdf). [Brief explanation of the drawings]

[0050] [Figure 1] P-value plot of QTLs associated with ToBRFV resistance for traits corresponding to AUDPC based on the F2 population (based on sources D and HMC1). The vertical axis (y-axis) represents -log10 (p-value), and the horizontal axis (x-axis) represents all SNPs as chromosomal positions on the physical map (physical distance in bp). [Figure 2A]Adjusted values ​​for fruit resistance: A Turkish trial example. [Figure 2B] Confidence intervals and adjusted values ​​for fruit resistance depending on the presence of the resistance parent allele at QTL9. [Figure 3] Adjusted values ​​of fruit resistance for different genotypes. [Figure 4] Redistribution of fruit scores by QTL9 genotype. [Figure 5] Leaf resistance adjustment value. [Figure 6] Adjusted values ​​of leaf resistance for different genotypes with confidence intervals. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present inventors have identified QTLs which, when present in S. lycopersicum plants, alone or in combination, provide improved resistance in the fruit and / or leaves of tomato plants infected or susceptible to infection with Tomato Brown Rugose Fruit Virus (ToBRFV).

[0052] The inventors identified one major QTL on chromosome 9 (hereinafter referred to as QTL9) that confers resistance to ToBRFV infection, particularly fruit resistance, when present in the S. lycopersicum background, and another QTL on chromosome 11 (hereinafter referred to as QTL11) that confers resistance to ToBRFV, particularly leaf resistance, and can be combined with QTL9 as described above.

[0053] Seeds and plants according to the invention are obtained from an initial cross between a wild plant of S. pipinellifolium, an introgression partner that is of interest but exhibits the phenotype in other species, and a plant of S. lycopersicum that is the recurrently susceptible parent of S. lycopersicum, transferring the resistance into the genetic background of S. lycopersicum.

[0054] Seeds of resistant S. lycopersicum plants obtained from this initial cross, containing homozygous QTL9 and QTL11 and designated LVSTBRFVRES2, were deposited at NCIMB on April 1, 2020, under accession number NCIMB 43591. Plants grown from these deposited seeds are S. lycopersicum tomatoes and exhibit resistance to ToBRFV, i.e., at least improved fruit resistance to this virus with respect to any known S. lycopersicum plant, particularly commercial S. lycopersicum plants.

[0055] As shown in the examples, the phenotype of the plants of the present invention is best characterized as tolerance to ToBRFV, i.e., fruit resistance, rather than both leaf and fruit resistance, and tolerance is only applicable to abiotic stress. To the extent that tolerance is also widely used to characterize resistance or intermediate resistance, the plants of the present invention can also be characterized as tolerant plants. Hereinafter, when we refer to resistance to ToBRFV, this phenotype includes the tolerant phenotype, and also includes intermediate resistance, as defined in some literature.

[0056] Furthermore, as shown in Examples 4, 5, and 8, ToBRFV resistance according to the present invention is different from the tolerance / resistance disclosed in the prior art; indeed, Example 5 shows that the level of resistance according to the present invention is higher than the level of resistance described in WO 2018 / 219941. Furthermore, Example 4 confirms that the sequences involved in ToBRFV resistance are different according to the present invention and WO 2018 / 219941. Example 8 shows that ToBRFV resistance according to the present invention is not associated with and therefore different from S. stemphilium resistance, contrary to the genetic determinants disclosed in WO 2020 / 018783.

[0057] According to a first aspect, the present invention therefore relates to S. lycopersicum plants that contain in their genome a QTL on chromosome 9 (hereinafter referred to as QTL9) and / or a QTL on chromosome 11 (hereinafter referred to as QTL11) that confer improved resistance to ToBRFV in the event of infection, particularly at the fruit level for QTL9 and at the leaf level for QTL11, as well as tomato plant seeds and cells that contain QTL9 and / or QTL11 in their genome. The QTLs conferring resistance were originally introgressed from wild S. pimpinellifolium and are therefore referred to as resistance QTLs, or QTL9 or QTL11, or introgression sequences of the invention in the following description. The present invention also relates to cells of such plants or seeds that contain these introgression sequences that confer resistance.

[0058] The tolerance / resistance phenotype can be tested and scored at the primary leaf level or at the fruit level by natural infection or artificial inoculation, as described in the experimental section, especially Example 1.

[0059] The QTL conferring improved resistance to ToBRFV is preferably located on chromosome 9 within the chromosomal interval or region bounded by SNPs TO-0201220 (SEQ ID NO: 1) and SNPs having SEQ ID NO: 101.

[0060] Indeed, we demonstrate in the Examples section that recombinant sequences within this region, corresponding to QTL9, are co-inherited with the phenotype of interest.

[0061] QTL9, conferring improved resistance to ToBRFV according to the present invention, is selected from those present in the genome of the seeds at LVSTBRFVRES2. Thus, QTL9 is present in the genome of these deposited seeds. These S. lycopersicum seed samples were deposited under the Budapest Treaty with the National Collection of Industrial, Food and Marine Bacteria (NCIMB) for patent procedures (NCIMB, Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom) on April 1, 2020, under accession number 43591, by HM Clause SA, Rue Louis Saillant, 26800 Portes-les-Valence, France. This tomato seed deposit is maintained by HM Clause SA, Rue Louis Saillant, 26800 Portes-les-Valence, France.

[0062] Other QTL according to the invention that confer improved resistance to ToBRFV are preferably located on chromosome 11, SNP TO-0201237 (SEQ ID NO: 102) and SL2.50ch11 QTL11 according to the present invention confers improved resistance to ToBRFV which can be combined with QTL9 and is selected from LVSTBRFVRES2, present in the genome of seeds of NCIMB 43591. QTL11 is indeed present in the genome of seeds of LVSTBRFVRES2 NCIMB accession number 43591.

[0063] The SNPs (single nucleotide polymorphisms) or specific polymorphisms corresponding to the markers referred to herein, as well as the flanking sequences of these SNPs or markers in the S. lycopersicum genome, are shown in the experimental section (see in particular Tables G and H for QTL9 and Table K for QTL11) and the accompanying sequence listing. For version 2.50 of the tomato genome, their locations on chromosomes 9 and 11, as well as their flanking sequences, are also shown.

[0064] In this regard, it should be noted that, by definition, an SNP refers to a single nucleotide in the genome, which varies depending on the allele present, but the flanking nucleotides are identical. To unambiguously identify the locations of different SNPs, their locations are shown in Tables G, H, and K by reference to the tomato genome sequence in version 2.50 thereof and by reference to their flanking sequences, which are identified by SEQ ID NO: 1. In a sequence related to a particular SNP herein, e.g., SEQ ID NO: 1 of SNP TO-0201220, only one nucleotide in the sequence actually corresponds to a polymorphism, i.e., only nucleotide 201 of SEQ ID NO: 1 corresponds to the polymorphic position of SNP TO-0201220, which can be A or G as shown in Table G; the flanking sequence is shown for purposes of locating the SNP in the genome but is not part of such polymorphism. Thus, detection of an SNP marker or an allele of this SNP refers to detection of the polymorphic nucleotide of this marker; not all flanking sequences need to be identical.

[0065] Similarly, other markers (not strictly SNPs) mentioned in the specification and in Table K are INDEL markers, which indicate the insertion of a single nucleotide. For example, SL2.50ch11 according to Table K Marker SL2.50ch11 at position 968449 of 9684449 (SEQ ID NO: 112) For 9684449 (SEQ ID NO: 112), position 968449 is an allele, meaning it is either C or CT corresponding to the insertion of T, and the position of "C" in SL2.50 is position 9684449 as listed in Table K. As long as the alleles relate to only one position of these INDEL markers, for simplicity, by language extension they can also be referred to as SNP markers below.

[0066] Genomic or chromosomal regions identified by flanking sequences, SNP or INDEL markers (hereafter assimilated to SNPs) are therefore clearly defined and unambiguous.

[0067] The genomic region delimited by two SNPs X and Y refers to a section of a genome, more particularly a section of a chromosome, located between the positions of these two SNPs and preferably containing said SNPs. The nucleotide sequence of this chromosomal region therefore begins with the nucleotide corresponding to SNP X and ends with the nucleotide corresponding to SNP Y, i.e. the SNPs are, according to the present invention, contained within the region they delimit.

[0068] By "introgression sequence from S. pimpinellifolium" in a given genomic region, it should be understood that the genomic sequence found in this region is the same locus as the corresponding genomic sequence found in the S. pimpinellifolium donor, i.e., introgression partner, and is the same sequence as the corresponding genomic sequence found in LVSTBRFVRES2 (NCIMB 43591) at the same locus. By having the "same sequence," it is meant that the two sequences being compared are identical with the exception of potential point mutations that may occur during transmission of the genomic region to progeny, i.e., are at least 99% identical over a length of preferably 1 kilobase.

[0069] A particular genomic region has the same sequence, within the meaning of the present invention, as the corresponding genomic region found in the S. pipinellifolium donor at the same locus if the genomic region is capable of conferring resistance to ToBRFV and is of S. pipinellifolium origin. The presence of a recombinant sequence in the genome of a S. lycopersicum plant, seed, or cell can be demonstrated, for example, by genetic in situ hybridization (GISH). GISH is a powerful technique for detecting the transfer of chromatin material from one species or subspecies to another. The advantage of GISH is that the transfer process is visualized by a "photograph of the transferred genome." This technique can also be used to establish whether a particular region of the genome is homozygous or heterozygous for the use of codominant molecular cytogenetic markers. This technique can also determine which chromosome a gene of interest is integrated into.

[0070] The present inventors identified and mapped the QTLs conferring ToBRFV resistance of the present invention primarily by identifying the presence of representative sequences of introgressed QTLs at different loci along the above-mentioned chromosome 9 and 11 regions, i.e., 101 different loci defined by 101 SNPs with SEQ ID NOS: 1 to 101 for QTL9 and 14 different loci defined by 14 markers with SEQ ID NOS: 102 to 115 for QTL11. These SNPs are hereinafter referred to as the SNPs of the present invention or the 101 SNPs of the present invention for QTL9. Preferred SNPs among them are the 14 SNPs with SEQ ID NOS: 1 to 14; in particular, the SNPs with SEQ ID NOS: 1, 2, 10, 12, and 14.

[0071] Thus, the presence of an introgression sequence or QTL conferring a resistance phenotype can be identified based on these SNP markers in the genome of the plant, seed or cell of the invention. Preferably, for QTL9, the presence of the introgression sequence or QTL is identified by one of the 101 SNPs having SEQ ID NOs: 1 to 101, preferably by one of the 14 SNPs (SNP SNPs, more preferably five SNPs, selected from the list comprising TO-0201220 (SEQ ID NO: 1), TO-0201221 (SEQ ID NO: 2), TO-0201222 (SEQ ID NO: 3), TO-0201223 (SEQ ID NO: 4), TO-0201224 (SEQ ID NO: 5), TO-0201225 (SEQ ID NO: 6), TO-0201226 (SEQ ID NO: 7), TO-0201227 (SEQ ID NO: 8), TO-0201228 (SEQ ID NO: 9), TO-0201229 (SEQ ID NO: 10), TO-0201230 (SEQ ID NO: 11), TO-0201231 (SEQ ID NO: 12), TO-0201232 (SEQ ID NO: 13) and TO-0201233 (SEQ ID NO: 14). QTL9 of the present invention may be identified or characterized in tomato plants by one of SNP TO-0201220, TO-0201221, TO-0201229, TO-0201231 and TO-0201233. QTL9 of the present invention may be identified or characterized, for example, by SNP TO-0201220 or SNP TO-0201229.

[0072] Further suitable SNPs are SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 and 101.

[0073] According to a preferred embodiment, for QTL9, the presence of the introgressed sequence in the tomato plant, cell or seed of the invention is identifiable by at least two, preferably at least three, or at least five of said 101 SNP markers, or said 14 SNP markers, preferably at least one of which is SNP TO-0201220 or SNP TO-0201229. For example, the presence of an introgressed sequence conferring ToBRFV resistance is detected by the presence of a haplotype in which at least two SNPs are present, for example, one SNP TO-0201229 and the other a different SNP selected from the SNPs having SEQ ID NOs: 1 to 101 (excluding SEQ ID NO: 10).

[0074] The alleles of these molecular markers, representing the resistance-conferring QTL or introgression sequences of the invention, are reported in the last column of Table H for the 101 SNPs of the invention. For the above 14 SNP markers, the alleles representing the introgressed QTLs are SNP TO-0201220 allele G, TO-0201221 allele G, TO-0201222 allele A, TO-0201223 allele A, TO-0201224 allele A, TO-0201225 allele A, TO-0201226 allele A, TO-0201227 allele C, TO-0201228 allele A, TO-0201229 allele C, TO-0201230 allele C, TO-0201231 allele G, TO-020132 allele G, and TO-020133 allele G.

[0075] Thus, the presence of a QTL in the genome of a tomato plant, cell or seed according to the invention can be detected or revealed by detecting a sequence representative of the QTL at said locus, more preferably by detecting one or more of the resistance alleles in tomato. For example, allele G of SNP TO-0201220, allele G of TO-0201221, allele A of TO-0201222, allele A of TO-0201223, allele A of TO-0201224, allele A of TO-0201225, allele A of TO-0201226, allele C of TO-0201227, allele C of TO-0201228, TO-0201229A, allele C of TO-0201230, allele C of TO-0201231, allele G of TO-0201232, and allele G of TO-0201233, more preferably SNP By detecting the allele G of SNP TO-0201220, the allele G of SEQ ID NO: TO-0201221, the allele A of SNP TO-0201229, the allele C of SNP TO-0201231, or the allele G of SNP TO-0201233, even more preferably the allele G of SNP TO-0201220 and / or the allele A of SNP TO-0201229.

[0076] According to a preferred embodiment, a QTL of the invention on chromosome 9, QTL9, is detected in the genome of a tomato plant, cell or seed by detecting at least two, preferably three, preferably at least five resistance alleles of SNPs having SEQ ID NOs: 1-101, preferably at least one detected resistance allele being the G allele of SNP TO-0201220 and / or the A allele of TO-0201229.

[0077] QTL9 is located on a chromosomal interval on chromosome 9 bounded on one side by or adjacent to the flanking SNP TO-0201220 and on the other side by the flanking SNP marker having SEQ ID NO: 101. A more preferred chromosomal interval on chromosome 9 in which QTL9 is found is the interval bounded by TO-0201220 and TO-0201233. Further preferred intervals are those separated by SNPs with SEQ ID NO: 20 and TO-0201233, or between SNPs with SEQ ID NO: 22 and TO-0201233, or between SNPs with SEQ ID NO: 26 and TO-0201233, or between SNPs with SEQ ID NO: 30 and TO-0201233, or between SNPs with SEQ ID NO: 34 and TO-0201233, or between SNPs with SEQ ID NO: 38 and TO-0201221 and TO-0201233, or preferably between SNPs TO-0201221 and TO-0201233.

[0078] QTL11 according to the present invention is preferably detected by one of the markers with SEQ ID NOs: 102 to 115, preferably by one of the SNP markers with SEQ ID NOs: 102 to 111, preferably by TO-0201237 (SEQ ID NO: 102), TO-0201238 (SEQ ID NO: 103), TO-0201239 (SEQ ID NO: 104), TO-0201240 (SEQ ID NO: 105) and TO-0201241 (SEQ ID NO: 106), and / or by marker SL2.50ch11. 964449 (sequence number 112), SL2.50ch11 979896 (sequence number 113), SL2.50ch11 9823405 (sequence number 114) and SL2.50ch11 9924232 (SEQ ID NO: 115). Preferably, the presence of this QTL11 can be characterized by detecting at least one resistance allele of the markers with SEQ ID NOs: 102-115, preferably one of the resistance alleles of the SNPs with SEQ ID NOs: 102-115. According to a preferred embodiment, the presence of QTL11 is characterized by detecting at least one allele CT of the markers with SEQ ID NOs: 112-115, as disclosed in the last column of Table K. SL2.50ch11 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405, SL2.50ch11 and / or SL2.50ch11, which is at least one of the allele GT of 9924232, the allele G of TO-0201237, the allele A of TO-0201238, the allele A of TO-0201239, the allele A of TO-0201240, and the allele A of TO-0201241, preferably the allele G of TO-0201237, the allele A of TO-0201238, the allele A of TO-0201239, the allele A of TO-0201240, and the allele A of TO-0201241. Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405 and SL2.50ch11 By 9924232.

[0079] Therefore, preferred markers for QTL11 are those with SEQ ID NOs: 102 to 115 (Table K), or those with SEQ ID NOs: 102 to 111, or those in the list of markers TO-0201237, TO-0201238, TO-0201239, TO-0201240, TO-0201241, SL2.50ch11 9684449, SL2.50ch11 9779896, SL2.50ch11 9823405 and SL2.50ch11 9924232, or marker list TO-0201237, TO-0201238, TO-0201239, TO-02012402 and TO-0 list marker SL2.50ch11 9684449, SL2.50ch11 9779896, SL2.50ch11 9823405, SL2.50ch11 9924232. Preferred resistance alleles correspond to these different lists and are shown in Table K, and these lists of markers and / or resistance alleles are applicable to all different aspects of the invention.

[0080] The tomato S. lycopersicum plants, cells, or seeds of the present invention can be homozygous for the QTL9, QTL11, or introgression sequence of the present invention, which confers ToBRFV resistance. However, the present invention is not limited to such homozygous plants, cells, or seeds. Indeed, the present inventors have also demonstrated that the resistance conferred by this QTL9 is additive, and that such plants heterozygously carrying the QTL9 of the present invention are also resistant to ToBRFV (see Experimental Section) at levels above those of susceptible plants that homozygously contain the QTL. Thus, the present invention also encompasses tomato S. lycopersicum plants, cells, or seeds heterozygously carrying the above-described QTL9 or introgression sequence of the present invention in their genome on chromosome 9.

[0081] QTL11 may also be present homozygously or heterozygously in the S. lycopersicum plants, cells, or seeds of the present invention, but only confers ToBRFV resistance at the homozygous stage, as the resistance allele is recessive (see Example 9 in this regard). The heterozygous or homozygous presence of QTL9 and QTL11 can be defined independently.

[0082] According to a preferred embodiment, the plant, seed or cell of the invention comprises a homozygous or heterozygous QTL9 and a homozygous or heterozygous QTL11 as defined above. Preferred combinations are QTL9 and QTL11 both present homozygously, or a homozygous QTL11 and a heterozygous QTL9, and a heterozygous QTL11.

[0083] Preferably, the S. lycopersicum plant according to the invention is a commercial plant or line. Preferably, such a commercial plant or line also exhibits resistance to ToMV (Tomato Mosaic Virus) due to the presence of the Tm-2 gene (allele Tm-2 or Tm-22 (also known as Tm-2a)), which confers resistance to TMV (Tobacco Mosaic Virus). Plants according to this aspect of the invention also preferably have the further characteristics of a nematode resistance trait (Mi-1 or Mi-j), as well as Fusarium and Verticillium resistance, and TYLCV resistance.

[0084] Other resistance or tolerability is also contemplated according to the present invention.

[0085] According to a preferred embodiment, the plants of the present invention are not resistant to Pepino Mosaic Virus (PepMV). According to yet another aspect, the tomato plants of the present invention are also resistant to PepMV.

[0086] Furthermore, commercial plants of the present invention produce fruits under appropriate conditions that are at least 10 grams, preferably 25 grams, preferably at least 100 grams, even more preferably at least 150 grams or at least 200 grams. Furthermore, the number of fruits per plant is essentially unaffected by the presence of QTL9 of the present invention. That is, the productivity of plants according to the present invention is not more than 20% less than that of plants having the same genotype but lacking QTL9. Plants of the present invention, e.g., plants grown from deposited seeds, therefore generally have at least three, preferably about four, tomatoes per cluster, with these fruits preferably weighing between 150 and 180 grams.

[0087] According to yet another embodiment, the plant of the present invention is a determinate, indeterminate or semi-indeterminate plant, or a seed or cell thereof, i.e. corresponds to a determinate, indeterminate or semi-indeterminate growth habit.

[0088] Determinant tomatoes are tomatoes that first develop leaves and then, if pollination is successful, produce flowers that will mature into fruit. All fruits tend to ripen on the plant at roughly the same time. Indeterminate tomatoes start by developing some leaves and continue to produce leaves and flowers throughout the growing season. These plants tend to have tomato fruits at different stages of maturity at any given time. Semi-determinant tomatoes are typical determinants, with a phenotype between determinant and indeterminate, except that they grow larger than determinants.

[0089] The present invention also relates to hybrid plants of S. lycopersicum obtained by crossing a plant homozygously carrying the QTL9 of the present invention with other S. lycopersicum. Because QTL9 of the present invention is additive, hybrid S. lycopersicum plants produced by the above cross are resistant to ToBRFV. Preferably, the other S. lycopersicum mating partner lacks QTL9 of the present invention but may contain one of the QTLs described in WO2018 / 219941, preferably QTL2 on chromosome 9 or QTL3 on chromosome 11. Alternatively, the other S. lycopersicum mating partner may contain a QTL on chromosome 11 described in WO2020 / 018783, WO2019 / 110130, and WO2019 / 110821 that confers ToBRFV resistance.

[0090] Thus, the present invention also relates to tomato plants, seeds or cells comprising, preferably homozygous, QTL9, as disclosed, either homozygous or heterozygous, and QTL2 on chromosome 9, as disclosed in WO2018 / 219941; heterozygous; and QTL3 on chromosome 11, as disclosed in WO2018 / 219941. One or more QTLs on chromosome 11 may be homozygous or heterozygous, as disclosed in WO2020 / 018783, WO2019 / 110130 and WO2019 / 110821.

[0091] Furthermore, insofar as the inventors of the present invention have identified a QTL on chromosome 11, hereinafter referred to as QTL11, that confers ToBRFV resistance, this QTL11 may be present in combination with QTL9 of the present invention. This QTL on chromosome 11 also corresponds to an introgression sequence from the S. pipinellifolium introgression partner that provided QTL9. The introgression sequence corresponding to QTL11 is found on chromosome 11 within the region bounded by SNP TO-0201237 (SEQ ID NO: 102) and SNP TO-0201241 (SEQ ID NO: 106). As demonstrated in the experimental section of the present application, this QTL11, when present in the genome of a tomato plant, particularly when present in combination with QTL9, provides increased resistance to ToBRFV relative to the same plant lacking said QTL11. Thus, although each QTL independently confers ToBRFV resistance, the combination of QTLs provides increased resistance, i.e., at least cumulative, relative to the resistance provided by one QTL, particularly since the resistance is essentially at the leaf level for QTL11 and at the fruit level for QTL9. Thus, the present invention is also directed to tomato plants, cells and seeds comprising this QTL11 in their genome, either homozygous or heterozygous, but preferably homozygous, to confer ToBRFV resistance, preferably leaf resistance. Preferably, this QTL11 should be found in combination with QTL9 of the present invention.

[0092] Thus, the present invention also relates to tomato plants comprising QTL9 and QTL11, thus increasing the plant's level of resistance to ToBRFV relative to corresponding plants lacking said QTL11; the present invention also encompasses cells and seeds thereof.

[0093] Furthermore, as disclosed above, the presence of a resistance gene that provides resistance to ToMV and TMV is advantageous, particularly for indeterminate commercial varieties. According to some preferred embodiments of the present invention, the Tm-22 gene (also known as Tm-22 or Tm-2(2)), which confers both ToMV and TMV resistance, is therefore combined with QTL9 of the present invention, QTL11 of the present invention, or QTL9 and QTL11. However, the inventors have noted that the Tm-22 gene and QTL9 are located on the same arm of chromosome 9, in a region that is transmitted "collectively" and is not prone to recombination events. Therefore, combining the Tm-22 gene and QTL9 on the same chromosome 9 is routinely difficult. However, because both the Tm-22 gene and QTL9 are dominant or at least cumulative, the Tm-22 gene and QTL9 are advantageously found on two different homologs of chromosome 9, i.e., they are both present in heterozygosity. In any case, the Tm-22 gene is preferably present in a heterozygous state.

[0094] According to one embodiment, the present invention is therefore directed to plants, cells, or seeds, heterozygotes, comprising the QTL9 of the present invention, as well as the Tm-22 gene or analogs thereof, which provide resistance to ToMV and TMV. The Tm-22 gene is well known to those skilled in the art, and suitable sequences for this gene are designated Solyc09g018220 for the protein sequence, or GenBank AF536201 and AAQ10736. Variants and analogs are well known in the field of the present invention. According to a preferred embodiment, the Tm-22 gene of the present invention is a gene encoding a protein having the 861 amino acid sequence reported in AAQ10736 (SEQ ID NO: 114), or a protein having at least 75%, preferably at least 80%, sequence identity to this sequence, and exhibiting Tm-22 activity, i.e., the ability to inhibit viral RNA replication of ToMV strains.

[0095] According to a preferred embodiment, the tomato plants, cells or seeds of the invention therefore comprise both heterozygous QTL9 and Tm-22 genes or variants thereof, and homozygous or heterozygous QTL11 of the invention, the combination of which provides resistance to ToBRFV, ToMV and TMV, and, if desired, further resistance.

[0096] As disclosed in application PCT / IB2019 / 00674 in the name of the same applicant, the presence of the Tm-1 gene can also improve ToBRFV resistance. Therefore, plants, cells, or seeds according to the present invention advantageously also contain the Tm-1 gene. The Tm-1 gene is defined, inter alia, as described in Ishibashi et al., 2007 (Inhibitors of viral RNA replication are encoded by plant resistance genes). PNAS August 21, 2007, 104(34)13833-13838; preferably, the term "Tm-1 gene" refers to a gene sequence encoding a protein with Tm-1 activity as reported in the literature, i.e., the ability to inhibit viral replication of wild-type ToMV strains susceptible to Tm-1, such as the strain ToMV-L disclosed herein. In a preferred embodiment, the Tm-1 gene of the present invention is a gene encoding a protein having the 754 amino acid sequence reported by Ishibashi et al. corresponding to SEQ ID NO: 115 (NCBI BA F75724), or a protein having at least 75%, preferably at least 80%, sequence identity to this sequence and exhibiting the Tm-1 activity reported in Ishibashi et al., 2007, i.e., the ability to inhibit viral RNA replication of wild-type Tm-1-susceptible ToMV strains.

[0097] Thus, the present invention also encompasses tomato plants, cells or seeds that comprise the Tm-1 gene, either homozygously or heterozygously, in addition to QTL9 of the present invention, and potentially the Tm-22 gene and QTL11, or QTL3 on chromosome 11 as defined in WO2018 / 219941. Thus, the present invention also encompasses tomato plants, cells or seeds that comprise the Tm-1 gene, preferably homozygously or heterozygously, in addition to QTL11 of the present invention.

[0098] In yet another embodiment, the plants of the present invention are used as cuttings or rootstocks in a grafting process. Grafting is a process that has been used for many years for crops such as crickets, but has only recently been used for tomatoes. Grafting can be used to provide a level of resistance to tellurium pathogens such as Phytophthora or certain nematodes. Thus, grating is intended to prevent contact between the cultivated plant or variety and infested soil. The target variety, or optionally an F1 hybrid, used as the graft or cutting is grafted onto the resistant plant, which is used as the rootstock. The resistant rootstock is healthy and serves as a normal source of disease-free grafts isolated from the soil.

[0099] As noted above, the present invention is directed to S. lycopersicum plants that exhibit improved ToBRFV resistance, as well as to those plants, and cells of these plants or seeds, or other plant parts that contain the resistance QTL9 and / or QTL11 in their genomes, introgressed from S. piminellifolium, and to progeny of the plants of the present invention that contain said QTLs.

[0100] Progeny include first, second, and further progeny from a cross with a plant of the invention, where the cross includes a cross with itself or with another plant. A plant or seed according to the invention can be a progeny or descendant of a plant grown from the deposited seed LVSTBRFVRES2, deposited with NCIMB under accession number NCIMB 43591. Plants grown from the deposited seeds are homozygous for QTL9 of the invention, which confers an improved phenotype, as well as QTL11; therefore, they have the QTL of interest in their genomes on each of the homologs on chromosomes 9 and 11. They can be used to transfer these sequences into other backgrounds by crossing, selfing, and / or backcrossing.

[0101] The present invention is also directed to the deposited seeds of LVSTBRFVRES2 (NCIMB 43591), and to plants grown from one of these seeds that homozygously contain QTL9 and QTL11 conferring a phenotype of interest. These seeds do not correspond to a plant variety and are not homozygous for most genes except for the QTL of the present invention, and therefore their phenotypes are not fixed during propagation, except for the ToBRFV resistance / tolerance of the present invention; most of their phenotypic traits segregate during propagation, except for the ToBRFV resistance of the present invention.

[0102] The present invention also provides a plant or seed as defined above containing QTL9 and / or QTL11 conferring an improved phenotype, potentially in combination with sequences conferring an improved phenotype, wherein the QTLs from a S. lycopersicum plant, representative of the plant, under NCIMB accession NCIMB-43591, have been deposited into another S. lycopersicum genetic background, e.g., by crossing the plant with a second tomato plant parent and selecting for a plant carrying QTL9 and / or QTL11 responsible for the phenotype of interest. In such a cross, QTL9 and QTL11 can be transferred, if appropriate.

[0103] It should be noted that the seeds or plants of the invention can be obtained by different methods and are not exclusively obtained by essentially biological methods.

[0104] According to this aspect, the present invention relates to a tomato plant or seed, preferably a non-naturally occurring tomato plant or seed, which may comprise one or more mutations in its genome that provide the plant with fruit and / or leaf resistance to Tomato Brown Rugose Fruit Virus, e.g., mutations such as are present in the genome of plants of which representative samples have been deposited with NCIMB under accession number NCIMB 43591.

[0105] In another embodiment, the present invention relates to a method for obtaining a plant or seed having one or more mutations in the tomato genome, the method providing the plant with fruit and / or leaf resistance to Tomato Brown Rugose Fruit Virus. Such a method is exemplified in Example 7 and may include: a) To transform M0 seeds of mutagen-modified tomato plants into M1 seeds; b) growing plants from the M1 seeds thus obtained to obtain M1 plants; c) Producing M2 seeds by self-fertilization of M1 plants; and d) Repeating steps b) and c) n times to obtain M1+n seeds.

[0106] M1+n seeds are grown into plants and subjected to ToBRFV infection. Surviving plants, or plants with milder symptoms of ToBRFV infection, are propagated for one or more generations while continuing to select for fruit and / or leaf resistance to ToBRFV.

[0107] In this method, the M1 seeds in step a) can be obtained by chemical mutagenesis, such as EMS mutagenesis. Other chemical mutagens include, but are not limited to, diethyl sulfate, ethyleneimine, propane sultone, N-methyl-N-nitrosourethane, N-nitroso-N-methylurea, N-ethyl-N-nitrosourea, and sodium azide.

[0108] Alternatively, mutations are induced by irradiation, for example selected from X-rays, fast neutrons, and UV irradiation.

[0109] In another embodiment of the present invention, the mutation is induced by genetic engineering. Such mutations include the integration of sequences that confer fruit and / or leaf resistance to ToBRFV, as well as the replacement of the resident sequence with an alternative sequence that confers fruit and / or leaf resistance or tolerance to ToBRFV. Preferably, the mutation is the integration of QTL9 and / or QTL11 in place of the homologous sequence in a S. lycopersicum plant, as described above. Even more preferably, the mutation is the replacement of the sequence contained within SNP TO-0201220 (SEQ ID NO: 1) and SNP TO-0201233 (SEQ ID NO: 14) on chromosome 9 of the S. lycopersicum genome, or a fragment thereof, with the homologous sequence on chromosome 9 present in the genome of the plant deposited with NCIMB under accession number NCIMB 43591, which sequence or fragment confers resistance to ToBRFV. According to yet another embodiment, the mutation is at SNP TO-0201237 (SEQ ID NO: 102) on chromosome 11 of the S. lycopersicum genome and at SNP SL2.50ch11 9924232 (SEQ ID NO: 115), or a fragment such as the sequence contained within SNP TO-0201237 and SNP TO-0201241, with a homologous sequence on chromosome 11 present in the genome of a plant under deposit number NCIMB 43591, wherein the sequence or fragment, when present in homozygosity, confers resistance to ToBRFV.

[0110] Available genetic engineering tools include the use of various new techniques, termed "new breeding techniques," which have been developed and / or used to generate new traits in plants through genetic variation, such as targeted mutagenesis, targeted introduction of novel genes, or gene silencing (RdDM). Examples of such new breeding techniques include zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2, and ZFN-3, see U.S. Patent No. 9,145,565), oligonucleotide-directed mutagenesis (ODM), cisgenesis and intragenesis, grafting (on GM rootstocks), reverse breeding, agroinfiltration (agroinfiltration "sensu stricto," agroinoculation, floral 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), targeted sequence alterations facilitated by the use of engineered meganucleases, re-engineered homing endonucleases, DNA-guided genome editing (Gao et al., Nature Biotechnology (2016)), and synthetic genomics. A key part of targeted genome editing, another name for a new breeding technique, is the application of inducing DNA double-strand breaks (DSBs) at selected locations in the genome where modification is intended. Direct repair of DSBs allows for targeted genome editing. Such applications can be used to generate mutations (e.g., targeted mutations or precise natural gene editing) and precise insertions of genes (e.g., cis-gene, intragenic, or transgene). Applications leading to mutations are often identified as site-specific nuclease (SDN) technologies, such as SDN1, SDN2, and SDN3.For SDN1, the result is targeted, nonspecific gene deletion mutation: 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. For SDN2, SDN is used to generate a targeted DSB and repair it using a DNA repair template (a short DNA sequence identical to the targeted DSB DNA sequence except for one or a few nucleotide changes). This results in a targeted and predetermined point mutation in the desired gene of interest. For SDN3, SDN is used with a DNA repair template containing a new DNA sequence (e.g., a gene). The outcome of this technique is the integration of that DNA sequence into the plant genome. The most likely application that illustrates the use of SDN3 is the insertion of cisgenic, intragenic, or transgenic expression cassettes at selected genomic locations. A complete description of each of these techniques can be found in the report "New Plant Breeding Technologies - State-of-the-Art and Prospects for Commercial Development," produced by the European Commission's Joint Research Center (JRC) Future Technologies Institute in 2011.

[0111] According to the present invention, resistance or moderate resistance to ToBRFV corresponds to a significant reduction in non-marketable fruit of the resistant plants of the present invention. In particular, the resistant plants disclosed herein produce tomatoes in which at least 70% of the fruit are marketable at maturity, i.e., free of discolored spots, free of browned calyxes, rough surfaces, not too small, and free of brown necrotic spots. Preferably, at least 80% of the fruit remain marketable at maturity, and preferably at least 90% of the fruit remain marketable even in the case of dual infection.

[0112] As described above, the plants of the present invention are characterized by the presence of introgressed sequences on chromosome 9 in the region of this chromosome bounded by SNP TO-0201220 and the SNP having SEQ ID NO: 101, and / or the presence of introgressed sequences on chromosome 11 in the region of this chromosome bounded by SNP TO-0201237 and SL2.50ch11 9924232, preferably SNP TO-0201237 and SNP TO-0201241. However, intragenic sequences from S. piminellifolium may be found beyond these boundaries or adjacent sequences. Similarly, although introgressed sequences are found within the above-mentioned regions, the entire region is not necessarily composed of introgressed sequences. Considering the markers that the inventors have identified and used for QTL9, the introgression sequence that confers ToBRFV resistance is preferably located in at least one or more of the 101 loci encompassing the 101 SNPs having SEQ ID NOS: 1 to 101 in the genome of the plant, seed or cell of the invention, more preferably the following 14 loci: the locus encompassing TO-0201220 on chromosome 9, the locus encompassing TO-0201221, the locus encompassing TO-0201222, the locus encompassing TO-0201223 on chromosome 9, a locus encompassing TO-0201224, a locus encompassing TO-0201225, a locus encompassing TO-0201226, a locus encompassing TO-0201227, a locus encompassing TO-0201228, a locus encompassing TO-0201229, a locus encompassing TO-0201230, a locus encompassing TO-0201231, a locus encompassing TO-0201232, and a locus encompassing TO-0201233.

[0113] By "locus encompassing a SNP marker" is meant the sequence surrounding the SNP polymorphism, preferably extending from about 2 megabases upstream to about 2 megabases downstream of the SNP, preferably 1 megabase, preferably about 0.5 megabases upstream and downstream of the SNP.

[0114] The interrogres sequences at these loci are the sequences found at the corresponding loci of the seed LVSTBRFVRES2 corresponding to NICMB43591.

[0115] For QTL11, the intorogens sequence that confers ToBRFV resistance when present in homozygosity is preferably found in the genome of the plant, seed or cell of the present invention at at least one of 14 loci encompassing 14 markers having SEQ ID NOs: 102 to 115 listed in Table K, and the intorogens sequences at these loci are those found in the corresponding loci of seed LVSTBRFVRES2 corresponding to NICMB43591.

[0116] In another aspect, the present invention also relates to any plant, and plant parts of such plants, which may be obtained from the seeds or plants of the invention described above, most preferably explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles, cotyledons, flowers, root tips, hypocotyls and other plant parts, wherein said plants, explants, cuttings, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles, cotyledons, flowers, root tips, hypocotyls and / or plant parts are seeds or plants according to the first aspect of the invention, i.e. which are homozygous or heterozygous in the genome for the QTL9 and / or QTL11 of interest. These plant parts, in particular explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles, cotyledons, flowers, root tips or hypocotyls, contain in their genome the phenotype of interest, i.e. QTL9 and / or QTL11, which confer resistance to ToBRFV, in particular fruit resistance for QTL9 and foliar resistance for QTL11.

[0117] According to a preferred embodiment, the present invention is directed to seeds as described above, grown into plants according to the first aspect of the invention, and thus exhibiting resistance to ToBRFV infection due to the presence of resistance QTL9, or QTL11, or an introgressed sequence as described above.

[0118] The QTL9 and QTL11 referred to in this aspect of the invention are those described above in the context of the plants of the invention.

[0119] The plant part may advantageously comprise QTL11 as defined above in addition to or instead of QTL9.

[0120] The different characteristics of the QTL defined in relation to the first aspect of the invention apply mutatis mutandis to this aspect of the invention. Thus, QTL9 is preferably selected from those present in the genome of the plant corresponding to the deposited material LVSTBRFVRES2 (NCIMB accession number 43591). It is advantageously characterized by the presence of at least one resistance allele of the SNPs of Table H, preferably allele G of SNP TO-0201220, allele G of TO-0201221, allele A of TO-0201222, allele A of TO-0201223, allele A of TO-0201224, allele A of TO-0201225, allele A of TO-0201226, allele C of TO-0201227, allele C of TO-0201228, allele A of TO-0201229, allele C of TO-0201230, allele C of TO-0201231, allele G of TO-0201232 and / or allele G of TO-020133, more preferably SNP allele G of SEQ ID TO-0201220, allele G of SEQ ID TO-0201221, allele A of TO-0201229, allele C of TO-0201231 or allele G of TO-0201233, even more preferably by SNP allele G of TO-0201220 and / or allele A of TO-0201229.

[0121] QTL11 is preferably selected from those present in the genome of the plant corresponding to the deposited material LVSTBRFVRES2 (NCIMB accession number 43591). It is advantageously characterized by the presence of at least one resistance allele of the markers of Table K, preferably allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201241, allele A of SL2.50ch11 Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405 and SL2.50ch11 by the allele GT of 9924232, for example, by the presence of at least one of the allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, and allele A of TO-0201241.

[0122] The present invention also relates to cells of S. lycopersicum plants, such that the cells of the plants contain in their genome a QTL9 or QTL11 of the present invention, which independently confers a phenotype of interest to the S. lycopersicum plant, potentially QTL9 and QTL11. QTL9, like QTL11, has been previously defined in the context of the present invention and is characterized by the same features and preferred embodiments as those previously disclosed for the plants and seeds according to the above aspects of the present invention. The presence of this QTL, i.e., QTL9 or QTL11, can be revealed by the techniques disclosed above and well known to those skilled in the art. In particular, it can be determined whether the QTL is present homozygously or heterozygously in the genome of such cells of the present invention. QTL9 is advantageously characterized by the presence of at least one resistance allele of the SNPs of Table H, preferably allele G of SNP TO-0201220, allele G of TO-0201221, allele A of TO-0201222, allele A of TO-0201223, allele A of TO-0201224, allele A of TO-0201225, allele A of TO-0201226 due to the presence of allele A, allele C of TO-0201227, allele C of TO-0201228, allele A of TO-0201229, allele C of TO-0201230, allele C of TO-0201231, allele G of TO-0201232 and / or allele G of TO-020133, more preferably due to SNP allele G of SEQ ID TO-0201220, allele G of SEQ ID TO-0201221, allele A of TO-0201229, allele C of TO-0201231 or allele G of TO-0201233, even more preferably by SNP allele G of TO-0201220 and / or allele A of TO-0201229.QTL11 is advantageously determined by the presence of at least one resistance allele of a marker of Table K, preferably allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201241, SL2.50ch11. Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405, SL2.50ch11 By the presence of the allele GT of 9924232, for example, by the presence of at least one of the allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, and allele A of TO-0201241.

[0123] The cells according to the invention can be any type of S. lycopersicum cell, in particular isolated cells and / or cells capable of regenerating whole S. lycopersicum plants, carrying QTL9 and / or QTL11 of interest.

[0124] The present invention is also directed to tissue cultures of non-regenerable or regenerable cells of the plants defined above in accordance with the present invention. Preferably, the regenerable cells are derived from embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, stems, petioles, roots, root tips, fruits, seeds, flowers, cotyledons, and / or hypocotyls of the present invention, and the cells contain QTL9 and / or QTL11 in their genome, independently conferring the improved phenotypes of fruit resistance to ToBRFV of QTL9 and foliar resistance to ToBRFV of QTL11.

[0125] Preferably, such cells contain QTL9 and also QTL11, either homozygous or heterozygous, as defined in the context of the present invention.

[0126] Such cells also advantageously comprise any additional resistance or tolerance genes, as disclosed in the context of the first aspect of the invention, also applicable herein.

[0127] The tissue culture is preferably capable of regenerating plants having the physiological and morphological characteristics of the aforementioned tomato plants and 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.

[0128] The present invention also provides protoplasts of the plants defined above, or said protoplasts from tissue cultures defined above, containing QTL9 and / or QTL11, which confer the improved phenotype of the present invention.

[0129] The present invention also relates to tissues of the plants of the present invention, which may be undifferentiated or differentiated tissues, such tissues comprising one or more cells containing a QTL of the present invention.

[0130] The present invention also relates to propagation material capable of generating resistant tomato plants according to the invention, comprising a transferred sequence or a QTL as defined above.

[0131] In another aspect, the present invention also relates to the use of tomato plants of the present invention, preferably containing a QTL9 of the present invention in a homozygous form, as breeding partners in breeding programs to obtain S. lycopersicum plants with improved phenotypes of the present invention. Indeed, such breeding partners homozygously carry the QTL9 of the present invention conferring the desired phenotype. By crossing this plant with a tomato plant, particularly a line, it is possible to transfer the QTL9 of the present invention conferring the desired phenotype to offspring. Thus, plants of the present invention can be used as breeding partners to introgress the QTL9 of the present invention conferring the desired phenotype to S. lycopersicum plants or germplasm, i.e., ToBRFV resistance. Plants or seeds heterozygous for the QTL9 of interest can also be used as breeding partners as described above, although phenotypic segregation is likely to make breeding programs more complicated.

[0132] The improved phenotype of the present invention is resistance to ToBRFV, in particular fruit resistance or leaf resistance, or fruit and leaf resistance.

[0133] The breeding partner may also contain QTL11 as defined in the present invention, preferably homozygous.

[0134] The introduced QTL9 is advantageously introduced into a variety that contains other desirable genetic traits such as disease resistance, early fruit maturity, drought tolerance, fruit shape, etc. Preferably, the introduced QTL9 is advantageously introduced into a plant or variety that contains the Tm-22 gene.

[0135] According to yet another aspect, the present invention also relates to the same use of a tomato plant of the present invention, which comprises, in homozygote form, QTL11 of the present invention as a breeding partner.

[0136] The present invention also relates to the use of plants or seeds of LVSTBRFVRES2 deposited at NCIMB under accession number NCIMB 43591, and / or plants derived therefrom comprising homozygous QTL9 and / or QTL11, which plants are also suitable as introgression partners in breeding programs aimed at imparting desired phenotypes to S. lycopersicum plants or germplasm.

[0137] In such breeding programs, the selection of offspring exhibiting a desired phenotype or having a QTL9 linked to a desired phenotype can advantageously be carried out based on alleles of SNP markers, in particular the SNP markers of the present invention having SEQ ID NOs: 1 to 101.

[0138] In the case of QTL9, plant progeny are preferably selected based on the presence of SNPs TO-0201220 allele G, TO-0201221 allele G, TO-0201222 allele A, TO-0201223 allele A, TO-0201224 allele A, TO-0201225 allele A, TO-0201226 allele C, TO-0201227 allele C, TO-0201228 allele A, TO-0201229 allele C, TO-0201230 allele C, TO-0201231 allele G, TO-0201232 allele G and / or TO-020133, more preferably based on the presence of SNPs by the allele G of SNP TO-0201220, the allele G of SEQ ID NO: TO-0201221, the allele A of TO-0201229, the allele C of TO-0201231, or the allele G of TO-0201233, even more preferably by the allele G of SNP TO-0201220 and / or the allele A of TO-0201229.

[0139] Alternatively, selection can be based on the presence of any one of the resistance alleles of the 101 SNPs of the present invention that are associated with an improved phenotype or combinations of these alleles.

[0140] Such a selection occurs when the allele of interest is present in the sample of genetic material of the selected plant. The presence of this or these alleles indeed confirms the presence of the QTL9 or introgression sequence of the present invention at the locus defined by said SNPs. However, following a point mutation or recombination event, it is conceivable that at least one or two of these alleles will be lost and the remaining chromosomal fragment carrying the QTL9 of interest will still confer the phenotype of interest.

[0141] The selection of offspring carrying QTL11 is advantageously carried out by selecting alleles of markers with SEQ ID NOs: 102 to 115, preferably alleles of SNP markers with SEQ ID NOs: 102 to 111, preferably allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201241, allele A of SL2.50ch11 Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405 and SL2.50ch11 by the presence of at least one of the allele GT of 9924232, for example, the allele G of TO-0201237, the allele A of TO-0201238, the allele A of TO-0201239, the allele A of TO-0201240, and the allele A of TO-0201241.

[0142] Thus, plants according to the invention, or plants grown from seeds deposited under accession number NCIMB 43591, are particularly useful in marker-assisted selection to obtain commercial tomato lines and varieties with the improved phenotypes of the invention.

[0143] The invention also relates to the use of said plants in programs aimed at identifying, sequencing and / or cloning gene sequences that confer a desired phenotype.

[0144] Any particular embodiment described for the previous aspect of the invention is also applicable to this aspect of the invention, particularly with respect to the traits of QTL9 and QTL11 that confer phenotypes of interest.

[0145] In yet another aspect, the present invention also relates to methods or processes for the production or propagation of S. lycopersicum plants with desired phenotypes, particularly commercial plants and inbred lines. Indeed, the present invention is also directed to the introgression of the QTL or transgenic sequences of the present invention conferring ToBRFV resistance to other tomato plants, particularly other tomato varieties, or other species or inbred parent lines, useful for producing new types and varieties of tomato.

[0146] In this regard, the present invention also includes a method for breeding S. lycopersicum plants with ToBRFV resistance, comprising the step of crossing a plant grown from the deposited species LVSTBRFVRES2 NCIMB 4591 or a progeny thereof, which carries QTL9 of the present invention conferring ToBRFV resistance, with an initial S. lycopersicum plant, preferably lacking the QTL. The QTL is defined as above, i.e., is introgressed from S. piminellifolium and is preferably present in the genome of seeds of LVSTBRFVRES2, NCIMB accession number 43591. The QTL can be identified by at least one resistance allele of the SNP markers having SEQ ID NOS: 1-101.

[0147] The present invention also relates to a method for conferring ToBRFV resistance to S. lycopersicum plants, comprising genetically modifying said plants to introduce the resistance QTL9 of the present invention, said QTL9 being defined as above and preferably present in the genome of seed LVSTBRFVRES2, NCIMB accession number 43591. The genetic modification can be carried out by any method or means known to those skilled in the art.

[0148] The present invention also relates to the same methods for breeding S. lycopersicum plants with ToBRFV resistance and for conferring ToBRFV resistance to S. lycopersicum plants in conjunction with QTL11 instead of QTL9. In such cases, the QTL is identifiable by at least one of the resistance alleles of the markers having SEQ ID NOs: 102-115.

[0149] The present invention therefore relates to a method for breeding S. lycopersicum plants resistant to ToBRFV, the method comprising the step of crossing a plant grown from deposited seed NCIMB 43591 or a progeny thereof, carrying QTL9, introgressed from S. pimpinellifolium and conferring ToBRFV resistance, with an initial S. lycopersicum plant lacking said QTL9, wherein said QTL9 on chromosome 9 is present in the genome of seed of plant LVSTBRFVRES2, NCIMB accession number 4591, and is identified by the G allele of SNP TO-0201220, the G allele of SEQ ID NO: TO-0201221, the A allele of TO-0201229, the C allele of TO-0201231, or the G allele of TO-0201233.

[0150] The present invention also relates to a method for breeding S. lycopersicum plants with resistance to ToBRFV, comprising the step of crossing a plant grown from deposited seed NCIMB 43591 or a progeny thereof, which has been introgressed from S. pimpinellifolium and carries QTL11 on chromosome 11 conferring ToBRFV resistance, with an initial S. lycopersicum plant lacking said QTL11, wherein said QTL11 on chromosome 11 is present in the genome of the seed of plant LVSTBRFVRES2, NCIMB accession number 43591, and contains allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201249, allele CT of SL2.50ch11, allele B of TO-0201249, allele C of TO-0201249, allele D of TO-0201249, allele E of TO-0201249, allele F of TO-0201249, allele G of TO-0201249, allele F ... They can be distinguished by the allele AT of 9779896, the allele C of SL2.50ch11 9823405 or the allele GT of SL2.50ch11 9924232.

[0151] Specifically, the present invention also provides the following: a) crossing a plant grown from the deposited seed NCIMB 43591 or a progeny thereof, which contains QTL9 conferring ToBRFV resistance, with an initial S. lycopersicum plant, preferably lacking said QTL; b) selecting one plant among the resulting progeny that contains a QTL9 of the present invention; c) optionally self-pollinating one to several times the number of plants obtained in step b), and selecting from the progeny thus obtained plants that are resistant to ToBRFV, and selecting plants that are resistant to fruit, leaves, or both. The present invention relates to a method or process for producing a plant having ToBRFV resistance, comprising:

[0152] Alternatively, the method or process may comprise, instead of step a), the following: a1) crossing a plant corresponding to the deposited seed (NCIMB 43591), or a progeny thereof, containing QTL9 conferring ToBRFV resistance with an initial S. lycopersicum plant preferably lacking said QTL; a2) Propagating F1 hybrids on their own to create an F2 population may include:

[0153] In the above method or process, SNP markers are preferably used in steps b) and / or c) to select plants carrying sequences that confer the desired resistance phenotype.

[0154] The SNP markers are preferably one or more of the 101 SNP markers of the present invention having SEQ ID NOs: 1 to 101, including all combinations thereof as mentioned elsewhere in this application, preferably SNPs having SEQ ID NOs: 1 to 14.

[0155] It should be understood that by selecting a plant based on the alleles of one or more SNPs, the plant is selected as having ToBRFV resistance, whether fruit tolerant / resistant, leaf tolerant / resistant, or both, with respect to the initial plant, provided that the alleles of the SNPs are the alleles corresponding to the alleles of the LVSTBRFVRES2 parent for that SNP and are not the alleles of the initial S. lycopersicum plant. For example, when the allele G of SNP TO-0201220, the allele G of TO-0201221, the allele A of TO-0201222, the allele A of TO-0201223, the allele A of TO-0201224, the allele A of TO-0201225, the allele A of TO-0201226, the allele C of TO-0201227, the allele C of TO-0201228, the allele A of TO-0201229, the allele C of TO-0201230, the allele C of TO-0201231, the allele G of TO-0201232 and / or the allele G of TO-020133 are detected, more preferably the allele G of SNP TO-0201220, the allele A of SEQ ID NO: 1 Plants can be selected as having an improved phenotype of the invention if allele G of TO-0201221, allele A of TO-0201229, allele G of TO-0201231, allele G of TO-0201232, or allele G of TO-0201233 are detected, and even more preferably if allele G of SNP TO-0201220 and / or allele A of TO-0201229 are detected. Other resistance alleles of the SNPs listed in Table G can also be used.

[0156] Preferably, the S. lycopersicum plants of step a) are elite lines used to obtain plants with commercially or horticulturally desirable traits. Advantageously, such plants are resistant to TMV due to the presence of the homozygous or heterozygous Tm-22 gene.

[0157] The method or process defined above can advantageously comprise a backcrossing step, preferably after step c), to obtain a plant having all the characteristics of a S. lycopersicum plant. Thus, the method or process for the production of a plant having these characteristics can comprise the following additional steps: d) crossing the resistant plant selected in b) or c) with a S. lycopersicum plant; e) Selecting plants carrying the QTL9 or introgression sequence of the present invention may include:

[0158] The plant used in step a), i.e. the plant corresponding to the deposited seed, may be a plant grown from the deposited seed; or it may be any plant according to the first aspect of the invention, which carries QTL9 or an introgressed sequence, preferably conferring the phenotype of carrying these sequences homozygously.

[0159] Preferably, such plants also contain QTL11, which is preferably defined as homozygous.

[0160] In step e), SNP markers can be used to select plants with ToBRFV resistance relative to the initial plants. The SNP markers are those of the present invention, as described in the previous section. According to a preferred embodiment, the method or process of the present invention is carried out such that for at least one of the selection steps, i.e., b), c), and / or e), the selection is based on the detection of at least one resistance allele of the SNPs having SEQ ID NOs: 1-101. Preferred alleles and combinations have already been disclosed and are applicable to this embodiment of the present invention.

[0161] It should be noted that when plants are selected that have an improved phenotype and that are homozygous for the QTL conferring this phenotype, selection should be based on one or more SNPs of the invention, based on the presence of the allele representing the QTL, i.e., the parent of LVSTBRFVRES2, in combination with the absence of the allele representing the parent of the recurrently susceptible S. lycopersicum.

[0162] Selection can also be based on any other markers linked to the introgressed sequences and indicating the presence of these introgressed sequences by contrasting them with the resident sequences of the susceptible parent. Methods for defining additional markers are within the scope of this invention and are disclosed in separate sections.

[0163] The plants selected in step e) are preferably commercial plants, in particular plants bearing fruits having a total maturity of at least 10 g, preferably 25 g, at least 100 g, at least 150 g, or at least 200 g under normal culture conditions.

[0164] Preferably, steps d) and e) are repeated at least two times, preferably three times, not necessarily with the same S. lycopersicum plant, which is preferably a breeding line.

[0165] Resistance to nematode traits or resistance to ToMV may be further selected for at each selection step in the process disclosed above.

[0166] Self-pollination and backcrossing can be performed in any order and can be intercalated, for example, backcrossing can be performed before or after one or more self-pollinations, or self-pollination can be performed before or after one or more backcrosses.

[0167] Selection of progeny with the desired improved phenotype can also be performed based on a comparison of ToBRFV resistance from S. lycopersicum parents, particularly through the protocols disclosed in the Examples; the resistance / tolerance tested can be fruit resistance / tolerance, or leaf resistance / tolerance, or both.

[0168] The method used for allele detection can be based on any technique that allows for differentiation between two different alleles of a SNP on a particular chromosome.

[0169] The present invention also relates to the same method, wherein in step a) a plant grown from deposited seed NCIMB 43591, or a progeny thereof, containing QTL11 conferring ToBRFV resistance is used.All detection / selection steps are then carried out using markers with SEQ ID NOs: 102 to 115 for QTL11, in particular allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201241, allele A of TO-0201242, allele G of TO-0201243, allele A of TO-0201244, allele A of TO-0201245, allele G of TO-0201246, allele A of TO-0201247, allele G of TO-0201248, allele A of TO-0201249, allele A of TO-0201250, allele A of TO-0201251, allele G of TO-0201252, allele A of TO-0201253, allele A of TO-0201254, allele A of TO-0201255, allele G of TO-0201256, allele A of TO-0201257, allele A of TO-0201258, allele A of TO-0201259, allele A of TO-0201260, allele A of TO-0201261, allele B of TO-0201262, allele B of TO-0201263, allele B of TO-0201264, allele C of TO-0201265, allele C of TO-0201266 Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405 and / or SL2.50ch11 based on the presence of the 9924232 allele GT; for example, by the presence of at least one of the following: allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, and allele A of TO-0201241. Another preferred list of markers and resistance alleles has already been disclosed above.

[0170] Therefore, the present invention also relates to a method for conferring resistance to ToBRFV in S. lycopersicum plants, a) crossing a plant grown from the deposited seed NCIMB 43591, or a progeny thereof, and carrying QTL9 on chromosome 9 and / or QTL11 on chromosome 11 introgressed from S. pimpinellifolium, which independently confer ToBRFV resistance in NCIMB 43591, with an initial S. lycopersicum plant, preferably lacking said QTL(s); b) selecting plants in the progeny thus obtained that carry QTL9 and / or QTL11; c) A step of optionally self-pollinating one to several times the number of plants obtained in b) and selecting plants that are resistant to ToBRFV from the progeny thus obtained. Includes:

[0171] In another aspect, the present invention relates to a method for conferring resistance to ToBRFV to a S. lycopersicum plant, the method comprising the steps of: a) crossing a plant grown from the deposited seed NCIMB 43591, or its progeny, carrying QTL9 on chromosome 9 and / or QTL11 on chromosome 11 introgressed from S. pimpinellifolium, which independently confer ToBRFV resistance in NCIMB 43591, with an initial S. lycopersicum plant, preferably lacking said QTL(s), thus generating an F1 population; a2) self-directing the F1 hybrids to produce an F2 population; b) A step of selecting offspring individuals that have thus acquired resistance to ToBRFV Includes:

[0172] SNP markers are advantageously used to select plants carrying QTL9 and / or QTL11 conferring ToBRFV resistance independently in steps b) and / or c).

[0173] The present invention also relates to a method for obtaining a commercial tomato plant or an inbred line thereof, having a desired improved phenotype corresponding to tolerance and / or resistance of the fruit and / or foliage to Tomato Brown Rugose Fruit Virus, with respect to the initial commercial S. lycopersicum plant, a) germinating the deposited seed LVSTBRFVRES2 NCIMB Accession No. 43591, or a progeny thereof, which has QTL9 conferring ToBRFV resistance, with a commercially available S. lycopersicum plant and backcrossing the resulting plant; b) Selecting plants that have the QTL9 of the present invention Includes:

[0174] Preferably, the selection is based on one or more of the 101 or more SNPs of the invention, as detailed for other methods of the invention.

[0175] Alternatively, the progeny of step a) are progeny carrying QTL11 and the selection of step b) is based on QTL11, preferably on one or more of the 14 markers of the invention having SEQ ID NOs: 102 to 115, as detailed for other methods of the invention.

[0176] In all methods and processes of the present invention, the initial S. lycopersicum plant is definitive, indeterminate, or semi-determinative.

[0177] As previously disclosed, the tomato plants according to the invention are preferably also resistant to tomato mosaic virus, nematodes, TYLCV, and Fusarium and Verticillium. To obtain such plants in the methods and processes of the invention, the S. lycopersicum parents used in the breeding scheme preferably carry sequences that confer resistance to tomato mosaic virus, nematodes, TYLCV, and Fusarium and Verticillium, and a selection process is carried out to select for plants that carry these resistance sequences in addition to the QTL conferring the improved phenotype of the invention.

[0178] The present invention also relates to S. lycopersicum plants and seeds obtained or obtainable by any of the methods and processes disclosed above. Such plants are S. lycopersicum plants with improved phenotypes according to the first aspect of the present invention. Such S. lycopersicum seeds are preferably coated or pelleted with individual or combined active ingredients, such as plant nutrients, enhancing microorganisms, or products for disinfecting the seed and plant environment. Such seeds and chemicals may be products that promote plant growth, such as hormones, or products that increase resistance to environmental stress, such as defense stimulants, or products that stabilize the pH of the substrate and its immediate environment, or nutrients.

[0179] They may also be products for protecting young plants from agents detrimental to their growth, including viruses and pathogenic microorganisms, such as fungicidal, fungicidal, cidal, insecticidal or herbicidal products that act by contact, ingestion or gas diffusion, and any suitable essential oil, such as thyme extract. All of these products strengthen the plant's resistance response and / or disinfect or regulate the plant's environment. They may also be live biological materials, such as non-pathogenic microorganisms, such as at least one fungus, bacterium, or virus, optionally accompanied by a medium that ensures their viability; these microorganisms, such as those of the genera Pseudomonas, Bacillus, Trichoderma, Clonostachys, Fusarium, Rhizoctonia, etc., stimulate plant growth or protect plants from pathogens.

[0180] In all conventional methods and methods, identification of plants carrying a QTL or introgression sequence involved in ToBRFV resistance can be achieved by detecting at least one allele of a SNP associated with resistance QTL9, potentially in combination with the absence of other allelic SNPs of the invention, to confirm the homozygous status of the QTL if necessary. Thus, identification of plants carrying a homozygous QTL or introgression sequence of the invention will be based on the identification of at least one resistance allele of the SNPs having SEQ ID NOs: 1-101 for QTL9, as well as the absence of susceptibility alleles of said SNPs. For example, identification of plants homozygous for QTL9 of the present invention is based on the identification of the G allele of SNP TO-0201220, the G allele of TO-0201221, the A allele of TO-0201222, the A allele of TO-0201223, the A allele of TO-0201224, the A allele of TO-0201225, the A allele of TO-0201226, the C allele of TO-0201227, the C allele of TO-0201228, the A allele of TO-0201229, the C allele of TO-0201230, the C allele of TO-0201231, the G allele of TO-0201232 and / or the G allele of TO-020133, and the absence of the corresponding susceptibility allele, i.e., SNP based on the absence of allele A of TO-0201220, allele A of TO-0201221, allele G of TO-0201222, allele G of TO-0201223, allele C of TO-0201224, allele G of TO-0201225, allele G of TO-0201226, allele A of TO-0201227, allele G of TO-0201228, allele G of TO-0201229, allele A of TO-0201230, allele A of TO-0201231, allele A of TO-0201232 and / or allele A of TO-0201233.

[0181] Similarly, identification of a plant carrying a homozygous QTL or introgression sequence of the invention will be based on the identification of at least one of the resistance alleles of the markers having SEQ ID NOS: 102-115 for QTL11, and the absence of the susceptibility allele of said SNP. For example, identification of a plant carrying QTL11 of the invention homozygously will be based on the identification of allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201241, allele A of SL2.50ch11. Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405 and / or SL2.50ch11 Based on the identification of the allele GT of 9924232, as well as the absence of the corresponding susceptibility alleles, namely, allele A of TO-0201237, allele T of TO-0201238, allele C of TO-0201239, allele C of TO-0201240, allele C of TO-0201241, SL2.50ch11 Allele C of 9684449, SL2.50ch11 Allele A of 9779896, SL2.50ch11 Allele T of 9823405 and SL2.50ch11 This may be due to the absence of the G allele of 9924232.

[0182] The present invention also relates to the use of information provided by the present inventors, i.e., the presence of QTL9 and QTL11 in the deposited seed of LVSTBRFVRES2 and their ability to confer improved phenotypes to S. lycopersicum plants, as well as the disclosure of molecular markers associated with these QTLs or invaded sequences. This knowledge can be used, inter alia, to precisely map QTLs, define their sequences, identify tomato plants containing QTLs conferring improved phenotypes, and identify additional or alternative markers associated with these QTLs. Such additional markers are characterized by their location, i.e., positions close to the 101 markers disclosed herein, preferably from the 14 SNPs with SEQ ID NOS: 1-14 for QTL9, and their association with ToBRFV resistance revealed by the present invention. For QTL11, applicable markers are those with SEQ ID NOS: 102-115.

[0183] In this regard, the present invention also relates to a method for identifying, detecting and / or selecting S. lycopersicum plants carrying the QTL9 of the present invention found in the genome of seeds of LVSTBRFVRES2 (NCIMB Accession No. 43591), said QTL conferring improved resistance to ToBRFV with respect to corresponding plants lacking said sequence, the method comprising detecting in a sample of genetic material of the identified and / or selected plants at least one resistance allele of the SNP markers of Table H, in particular SNP one of the allele G of TO-0201220, allele G of TO-0201221, allele A of TO-0201222, allele A of TO-0201223, allele A of TO-0201224, allele A of TO-0201225, allele A of TO-0201226, allele C of TO-0201227, allele C of TO-0201227, allele C of TO-0201228, allele A of TO-0201229, allele C of TO-0201230, allele C of TO-0201231, allele G of TO-0201232 and allele G of TO-020133, more preferably allele G of SNP TO-0201220, SEQ ID detecting one of the following: allele G of TO-0201221, allele A of TO-0201229, allele C of TO-0201231, allele G of TO-0201233, for example, one of the following SNPs: allele G of SNP TO-0201220 and allele A of SNP TO-0201229. Preferably, detecting at least two or three or five resistance alleles of SNPs having SEQ ID NOs: 1 to 101.

[0184] The present invention also relates to methods for detecting or selecting S. lycopersicum plants carrying QTL9 conferring resistance to ToBRFV, and S. lycopersicum plants carrying at least one resistance allele of a SNP having SEQ ID NOS: 1-101, particularly a SNP having SEQ ID NOS: 1-14, wherein the detection or selection is carried out under conditions of ToBRFV infection, either artificial or natural, including inoculating the plant to be tested with ToBRFV. The presence of a phenotype of interest beneficially indicates the presence of a QTL9 or introgression sequence of the present invention, particularly in breeding schemes including a parent carrying QTL9 of the present invention.

[0185] The present invention also relates to a method for identifying, detecting and / or selecting S. lycopersicum plants carrying QTL11 of the invention found in the genome of seeds of LVSTBRFVRES2, said QTL conferring improved resistance to ToBRFV with respect to plants lacking said sequence, the method comprising the step of detecting in a sample of genetic material of the identified and / or selected plants at least one of the resistance alleles of the markers of Table K, in particular allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201241, allele A of TO-0201242, allele B of TO-0201243, allele C of TO-0201244, allele D of TO-0201245, allele E of TO-0201246, allele F of TO-0201247, allele G of TO-0201248, allele F of TO-0201249, allele G of TO-0201250, allele F of TO-0201251, allele G of TO-0201252, allele F of TO-0201253, allele F of TO-0201254, allele F of TO-0201255, allele F of TO-0201256, allele F of TO-0201257, allele F of TO-0201258, allele F of TO-0201259, allele F of TO-0201260, allele F of TO-0201261, allele F of TO-0201262, allele F of TO-0201263, allele F of TO-0201264, allele F of TO-02 Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405, SL2.50ch11 Preferably, at least two, three or five resistance alleles of the SNPs with SEQ ID NOs: 102 to 115 are detected, or one of the nine markers TO-0201237, TO-0201241 and SL2.50ch11 9684449, SL2.50ch11 979896, SL2.50ch11 9823405 and SL2.50ch11 According to another embodiment, two, three or four of the markers SL2.50ch11 should be detected. 968449, SL2.50ch11 979896, SL2.50ch11 9823405 and SL2.50ch11 At least one, two, or three of the resistance alleles of 9924232 should be detected. Another preferred list of markers and resistance alleles has already been disclosed above. Detection or selection can be performed under conditions of ToBRFV infection. The presence of the desired phenotype is informative, homozygously indicating the presence of QTL11 of the present invention or the introgressed sequence.

[0186] The present invention also relates to a method for detecting and / or selecting S. lycopersicum plants, in particular commercial tomato plants, which have a QTL of the invention, by detecting in a sample of genetic material of the selected plants the resistance alleles described above, i.e. for a QTL on chromosome 9, the allele G of SNP TO-0201220, the allele G of TO-0201221, the allele A of TO-0201222, the allele A of TO-0201223, the allele A of TO-0201224, the allele A of TO-0201225, the allele A of TO-0201226, the allele C of TO-0201227, the allele C of TO-0201228, the allele A of TO-0201229, the allele C of TO-0201230, the allele C of TO-0201231, the allele G of TO-0201232, the allele G of TO-0201233, preferably the allele G of SNP TO-0201220, SEQ ID at least one of allele G of TO-0201221, allele A of TO-0201229, allele C of TO-0201231, allele G of TO-0201232, or allele G of TO-0201233; or TO-0201237 allele G, TO-0201238 allele A, TO-0201239 allele A, TO-0201240 allele A, TO-0201241 allele A, SL2.50ch11 Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405 and SL2.50ch11 allele GT of 9924232, e.g., allele G of TO-0201237, allele A of TO-0201238, alleles of TO-0201239, allele A of TO-0201240 or allele A of TO-0201241 The method includes detecting at least one of the following:

[0187] Another preferred list of resistance alleles has already been disclosed above.

[0188] The method is particularly adapted to breeding programs in which LVSTBRFVRES2 (NCIMB Accession No. 43591), which contains the QTL of the present invention that confers ToBRFV resistance, is the initial parent, or its progeny.

[0189] The present invention further relates to methods for detecting and selecting S. lycopersicum plants carrying QTL9 and / or QTL11 of the present invention, which confer ToBRFV resistance, based on the detection of any molecular marker that reveals the presence of said QTL. Indeed, since QTL9 and QTL11 of the present invention have been identified by the present inventors, the identification and use of molecular markers in addition to the 101 SNPs (SEQ ID NOS: 1-101) or 14 markers (SEQ ID NOS: 102-115) of the present invention can be readily achieved by those skilled in the art. QTL9 can be characterized by the presence of at least one of the 101 SNPs of the present invention, but can also be identified through the use of different surrogate markers. The same applies to QTL11. The present invention therefore also includes methods and uses of any such molecular markers for identifying QTLs of the present invention in the tomato genome, where the QTL confers resistance to ToBRFV relative to a corresponding plant lacking the QTL, and the QTL is characterized by the presence of at least one resistance allele of a SNP having SEQ ID NOS: 1-101, preferably 1-14.

[0190] Also included are methods and uses of any such surrogate molecular markers for identifying QTL9 of the present invention in the tomato genome, said QTL conferring ToBRFV resistance, said QTL being characterized by the presence of at least one resistance allele of a SNP having SEQ ID NOs: 1 to 101, preferably SNP and more preferably by SNPs TO-0201220 allele G, TO-0201221 allele G, TO-0201222 allele A, TO-0201223 allele A, TO-0201224 allele A, TO-0201225 allele A, TO-0201226 allele A, TO-0201227 allele C, TO-0201228 allele C, TO-0201229 allele C, TO-0201230 allele C, TO-0201231 allele C, TO-0201232 allele G and TO-020133. It is characterized by one of the following: allele G of SNP TO-0201220, allele G of SEQ ID NO: TO-0201221, allele A of SNP TO-0201229, allele C of SNP TO-0201231 and allele G of SNP TO-0201233, and even more preferably by one of SNPs allele G of SNP TO-0201220 and allele A of SNP TO-0201229.

[0191] Also included are methods and uses of any such surrogate molecular markers for identifying QTL11 of the invention in the tomato genome, said QTL conferring ToBRFV resistance when present in homozygous form, said QTL being identified by the presence of at least one resistance allele of markers having SEQ ID NOs: 102 to 115, preferably at least one resistance allele of SNPs having SEQ ID NOs: 102 to 111, more preferably allele G of TO-0201237, allele A of TO-0201238, allele A of TO-0201239, allele A of TO-0201240, allele A of TO-0201241, allele A of TO-0201242, allele B of TO-0201243, allele C of TO-0201244, allele D of TO-0201245, allele E of TO-0201246, allele F of TO-0201247, allele G of TO-0201248, allele F of TO-0201249, allele G of TO-0201250, allele F of TO-0201251, allele G of TO-0201252, allele F of TO-0201253, allele F of TO-0201254, allele F of TO-0201255, allele F of TO-0201256, allele F of TO-0201257, allele F of TO-0201258, allele F of TO-0201259, allele F of TO-0201260, allele F of TO-0201261, allele F of TO-0201262, allele F of TO-0201263, allele F of TO-0201264, all Allele CT of 9684449, SL2.50ch11 Allele AT of 9779896, SL2.50ch11 Allele C of 9823405 and SL2.50ch11 for example, the presence of any one of the allele G of TO-0201237, the allele A of TO-0201238, the allele A of TO-0201239, the allele A of TO-0201240, and the allele A of TO-0201241. Another preferred list of resistance alleles has already been disclosed above.

[0192] The present invention also relates to a method for detecting and / or selecting tomato plants carrying a resistance QTL as described above that confers ToBRFV resistance, said method comprising: a) analyzing tomato plants for the presence of at least one genetic marker genetically linked to or associated with QTL9 or QTL11 involved in ToBRFV resistance, which confers said resistance, in particular in tomato plants; b) selecting plants containing genetic markers involved in ToBRFV resistance and the associated QTL9 or QTL11 wherein the QTL and genetic markers should be found in the genomic region bounded by TO-0201220 and the SNP with SEQ ID NO: 101 for QTL9, preferably in the region bounded by TO-0201220 and TO-0201233 in the S. lycopersicum genome, and in the genomic region bounded by markers with SEQ ID NOs: 102 and 115 for QTL11, preferably in the region bounded by markers with SEQ ID NOs: 102 and 111.

[0193] By association, or genetic association, or more specifically genetic linkage, it should be understood that a polymorphism in a genetic marker (e.g., a particular allele of a SNP marker) and a phenotype of interest co-occur, i.e., are inherited together more often than would be expected by chance occurrence, i.e., there is a non-random association of the allele with the genetic sequence responsible for the phenotype as a result of their genomic proximity.

[0194] The genetic marker is one of the 101 markers disclosed above for QTL9 or any of the alternative markers, and is preferably inherited in the phenotype of interest in more than 90% of meiosis, preferably in more than 95%, 96%, 98% or 99% of meiosis. The same is true for QTL11.

[0195] The definitions and preferred characteristics of the QTL or introgressed sequence of the present invention are as defined in other sections of this specification. A QTL conferring ToBRFV resistance is advantageous as found in the genome of seed LVSTBRFVRES2.

[0196] Thus, the present invention relates to the use of one or more molecular or genetic markers for fine mapping or identifying QTLs in the tomato genome that confer ToBRFV resistance according to the present invention, wherein said one or more markers are located in the following chromosomal regions: - in the chromosomal region bounded on chromosome 9 by SNPs TO-0201220 (SEQ ID NO: 1) and SNPs with SEQ ID NO: 101, - less than 2 megabase units from one locus of the 101 SNP markers of the present invention, preferably from one locus of the SNPs having SEQ ID NOs: 1 to 14, more preferably from one locus of TO-0201220, TO-0201221, TO-0201229, TO-0201231 or TO-0201233 Located in one of the

[0197] According to a preferred embodiment, the one or more markers are within the chromosomal region bounded by TO-0201210 and the SNP with SEQ ID NO: 101, or by TO-0201210 and TO-0201233, or by TO-0201221 and TO-0201233.

[0198] More preferably, the one or more molecular or genetic markers are associated with at least one of the following resistance alleles of SNPs having SEQ ID NOs: 1 to 101 with a p-value of 0.05 or less: for example, allele G of SNP TO-0201220, allele G of TO-0201221, allele A of TO-0201222, allele A of TO-0201223, allele A of TO-0201224, allele A of TO-0201225, allele A of TO-0201226, allele C of TO-0201227, allele C of TO-0201228, allele A of TO-0201229, allele C of TO-0201230, allele C of TO-0201231, allele G of TO-0202132 and / or allele G of TO-020133.

[0199] The molecular or genetic marker is preferably a SNP marker, which is preferably less than 0.5 megabases and less than 1 megabase from the locus of at least one of the 101 SNPs of the present invention.

[0200] The p-value is preferably less than 0.01.

[0201] Furthermore, the present invention relates to the use of at least one of the 101 SNP markers of the present invention associated with a QTL on chromosome 9 that confers ToBRFV resistance, and to the use of at least one of the 101 SNP markers of the present invention for identifying one or more alternative molecular or genetic markers associated with said QTL, wherein said one or more alternative molecular or genetic markers are: - in the chromosomal region bounded on chromosome 9 by SNPs TO-0201220 (SEQ ID NO: 1) and SNPs with SEQ ID NO: 101, - less than 2 megabase units from one locus of the 101 SNP markers of the present invention, preferably from one locus of the SNPs having SEQ ID NOs: 1 to 14, more preferably from one locus of TO-0201220, TO-0201221, TO-0201229, TO-0201231 or TO-0201233 is located.

[0202] According to a preferred embodiment, the surrogate marker is within the preferred chromosomal region described above. Genetic association or linkage can advantageously be detected by tracing different markers and detecting the presence of the QTL in progeny derived from plants containing the QTL of interest.

[0203] The further molecular marker is preferably associated with said QTL with a p-value of less than or equal to 0.05, preferably less than 0.01. The QTL is preferably one found in the genome of the deposited seed NCIMB 43591.

[0204] The molecular or genetic marker and the resistance phenotype are preferably inherited together in more than 90% of meiotic divisions, preferably more than 95%.

[0205] The molecular or genetic markers according to this aspect of the invention are preferably SNPs. They are preferably less than 0.5 megabases and less than 1 megabase from the locus of at least one of the 101 SNPs of the invention.

[0206] The present invention also provides a method for detecting a marker comprising: - in the chromosomal region delimited on chromosome 11 by the marker with SEQ ID NO: 102 and the marker with SEQ ID NO: 115, from one locus of the 14 markers of Table K, preferably TO-0201237, TO-0201238, TO-0201239, TO-0201240, TO-0201240, TO-0201241, SL2.50ch11 968449, SL2.50ch11 979896, SL2.50ch11 9823405 or SL2.50ch11 Less than 2 megabases from one of the 9,924,232 loci and localized / localized in the same methods and uses.

[0207] Similarly, the present invention also encompasses a method for identifying molecular or genetic markers associated with QTLs conferring ToBRFV resistance in tomato plants, as described in the present application, comprising the following steps: - identifying molecular or genetic markers in the genomic interval bounded by TO-0201220 and TO-0201233 or bounded by less than 2 megabases, preferably less than 0.5 megabases, from the locus of one of the 101 SNPs of the invention; - determining whether the allele or state of said molecular or genetic marker is associated or related to the ToBRFV resistance phenotype in a segregant population derived from a plant that exhibits ToBRFV resistance, for example a segregant population derived from a plant that corresponds to the deposited seed. Includes:

[0208] According to yet another aspect, the present invention is also directed to a method for genotyping plants, preferably S. lycopersicum plants or tomato germplasm, for the presence of at least one genetic marker associated with resistance or tolerance to ToBRFV infection, comprising determining or detecting in the genome of a test plant a nucleic acid comprising at least one of the 101 markers of the present invention or comprising at least one of the surrogate molecular markers disclosed above. Preferably, the method comprises the step of identifying in a sample of the plant to be tested a specific sequence associated with resistance to ToBRFV in a nucleic acid comprising at least one of the resistance alleles of the SNPs of the present invention.

[0209] According to a most preferred embodiment of the method, the method comprises the detection in the tested plants of the presence of a nucleic acid comprising the allele G of SNP TO-0201220 or the allele A of SNP TO-0201229.

[0210] The invention also relates to the same method with respect to QTL11 and with respect to the chromosomal region delimited on chromosome 11 by the marker with SEQ ID NO: 102 and the marker with SEQ ID NO: 115. Associated markers or SNPs in this region have already been disclosed in the present invention, as well as a preferred list.

[0211] In view of the ability of the resistant plants of the invention to limit damage caused by ToBRFV infection, they are advantageously grown in environments where they may be infested or infected by ToBRFV; under such conditions, the resistant or tolerant plants of the invention produce tomatoes that are more marketable than susceptible plants. Thus, the present invention also relates to a method for improving the yield of tomato plants in an environment infected by ToBRFV, comprising growing tomato plants that contain in their genome QTL9 on chromosome 9 and / or QTL11 on chromosome 11, as defined according to the previous aspect of the invention, and conferring resistance of said plants to ToBRFV.

[0212] Preferably, the method comprises a first step of selecting or selecting tomato plants which contain a QTL or introgression sequence of interest. The method may also be defined as a method for improving tomato field, tunnel or greenhouse productivity or for reducing the intensity or number of chemical or fungicide applications in tomato production.

[0213] The present invention also relates to a method for reducing losses in tomato production in conditions of infestation or infection with ToBRFV, comprising growing a tomato plant as defined above.

[0214] These methods are particularly useful for tomato plant populations, whether in the field, tunnels, or greenhouses.

[0215] Alternatively, the method for improving yield or reducing losses in tomato production may comprise a first step of identifying tomato plants that are resistant / tolerant to ToBRFV and that contain QTL9 and / or QTL11 of the present invention in their genomes, conferring ToBRFV resistance to the plants, and then growing the resistant plants in an environment that is or may be infested by the virus. According to a preferred embodiment, the plants identified in the first step comprise allele A of TO-0201229 or at least one resistance allele of the SNPs having SEQ ID NOs: 1-101.

[0216] The resistant plants of the present invention can also limit the proliferation of ToBRFV, thereby limiting further plant infection and viral propagation. Therefore, the present invention also relates to a method for protecting a field, tunnel, greenhouse, or other type of plantation from ToBRFV infection, or at least limiting the level of ToBRFV infection in said field, tunnel, or greenhouse, or for limiting the spread of ToBRFV in a field, tunnel, or greenhouse, particularly for tomatoes. Such a method preferably comprises growing a resistant or tolerant plant of the present invention, i.e., a plant containing in its genome QTL9 on chromosome 9, conferring ToBRFV resistance to the plant. The plants of the present invention used preferably contain allele A of TO-0201229 or at least one resistance allele of the SNPs having SEQ ID NOS: 1-101. According to another embodiment, the plants used contain at least one resistance allele of the markers having SEQ ID NOS: 102-115.

[0217] The present invention also relates to the use of ToBRFV-resistant plants for controlling ToBRFV infection or infestation in fields, tunnels, greenhouses, or other plantations. Such plants are plants of the present invention that contain in their genome QTL9 and / or QTL11, or recombinant genetic sequences from S. piminellifolium on chromosome 9 or 11 as defined above. This use or method is also a method for disinfecting fields, tunnels, or greenhouses by reducing the virus population therein.

[0218] All preferred characteristics of the QTLs are as defined in relation to other aspects of the invention, i.e. preferably present in seeds of LVSTBRFVRES2 (NCIMB accession number 43591) and by SNP markers with SEQ ID NOs: 1 to 101 in the case of QTL9; preferably by SNP markers with SEQ ID NOs: 1 to 14 in the case of QTL9, preferably SNPs: allele G of TO-0201220, allele G of TO-0201221, allele A of TO-0201222, allele A of TO-0201223, allele A of TO-0201224, allele A of TO-0201225, allele A of TO-0201226, allele C of TO-0201227, allele C of TO-0201228, allele A of TO-0201229, allele C of TO-0201230, allele C of TO-020123 and for QTL11, the markers have SEQ ID NOs: 102 to 115, preferably for QTL11, the allele G of TO-0201237, the allele A of TO-0201238, the allele A of TO-0201239, the allele A of TO-0201240, the allele A of TO-0201241, the allele A of TO-0201242, the allele A of TO-0201243, the allele C of TO-0201234, the allele G of TO-0201235, the allele C of TO-0201236, the allele G of TO-0201237, the allele A of TO-0201238, the allele A of TO-0201239, the allele A of TO-0201240, the allele A of TO-0201241, the allele A of TO-0201242, the allele A of TO-0201243, the allele C of TO-0201238, the allele A of TO-0201239, the allele A of TO-0201244, the allele A of TO-0201245, the allele A of TO-0201246, the allele A of TO-0201247, the allele A of TO-0201248, the allele A of TO-0201249, the allele A of TO-0201250, the allele C of TO-0201251, the allele C of TO-0201252, the allele G of TO-0201253, the allele C of TO-0201254, the allele G of TO-0201255, the allele C of TO-0201 Allele CT of 9684449, SL2.50ch Allele AT of 977989611, SL2.50ch11 Allele C of 9823405 and / or SL2.50ch11 It can be distinguished by the GT allele of 9924232.

[0219] In yet another aspect, the present invention also relates to a method of producing a tomato comprising: a) growing a S. lycopersicum plant of the present invention comprising QTL9 and / or QTL11; b) bearing fruit; and c) Harvesting the fruits of the plant, preferably at and / or before maturity.

[0220] All preferred embodiments relating to QTL9 and QTL11 have already been disclosed in the context of the previous aspects of the invention. The method may advantageously comprise the further step of processing the tomatoes into a processed tomato product. [Example]

[0221] Example 1 : Materials and methods. 1.A. Source Validation Testing The study was carried out with three replicates of approximately 15 plants per line or genetic background tested. Plants were inoculated and infected at the two-leaf stage and then scored by visual evaluation of the leaves at 7, 14 and 28 days post-infection. In this test, the scale was as follows: 9: Asymptomatic 5: Moderate mosaic and / or necrotic symptoms 1: Strong mosaic and / or necrotic symptoms ELISA tests were performed in plants on the four lines and in bulk on the two susceptible controls after 32 days.

[0222] 1.B. Phenotyping of the F2 population Plants were inoculated and infected at the two-leaf stage and then scored by visual evaluation of the leaves at 7, 14 and 28 days post-infection. In this test, the scale was as follows: 9: Asymptomatic 7: Mild mosaic symptoms 5: Moderate mosaicism 3: Strong mosaicism and / or bubbling 1: Very strong mosaicism and / or bubbling and / or deformation For QTL analysis, different phenotypic variables / traits were used by the inventors, in particular annotation at 14 dpi, annotation at 21 dpi, annotation at 28 dpi, AUDPC were used. AUDPC (area under the disease progression curve) was calculated using the following formula:

[0223]

number

[0224] Here, "n" is the number of symptom assessments, "y" is the symptom intensity (1 to 9 times), and "t" is the time dpi (days post-inoculation).

[0225] 1.3.DNA Extraction: DNA was extracted from crushed leaves using the NucleoMag® Plant Kit (Macherey-Nagel) according to the manufacturer's protocol. DNA purification was based on magnetic bead technology for isolating genomic DNA from plant tissue. DNA concentration was quantified with the Quant-iT™ PicoGreen® dsDNA Assay Kit.

[0226] 1.4. Protocol for assessing ToBRFV resistance under field conditions Inoculation stage 10 and 17 days after planting. The plants were thus infected twice. At each time point, the two youngest leaves were inoculated. Finally, four different leaves were inoculated.

[0227] Inoculum preparation : Isolate: ToBRFV Jordan local strain 2017. ToBRFV-infected young leaves were collected from naturally infected plants resistant to TYLCV (Ty) and TMV to ensure the absence of several viruses in the inoculum. One gram of young leaves with ToBRFV symptoms is needed to prepare 4 mL of crushed inoculum.

[0228] Verification of vaccination and vaccination : Before inoculation, the presence of ToBRFV in the inoculum is checked using a TMV immunostrip (this immunostrip is not specific but also recognizes ToBRFV), and the absence of PepMV (Pepino) is checked using a PepMV immunostrip. As long as the infected plants are TMV-resistant, the presence of ToBRFV in the inoculum can be detected by the TMV immunostrip.

[0229] The inoculum is positive on TMV immunostrips and negative on PepMV immunostrips.

[0230] The inoculum is applied to two young leaves of the plants to be tested by gently rubbing the leaves with a coarse sponge soaked in the inoculum.

[0231] Symptom assessment The first evaluation is performed when the first set of fruits is red and the second set of fruits is red. A second evaluation is performed if at least the third cluster is red.

[0232] Leaf Symptom Scale 9: No symptoms / 7: Weak symptoms on a few leaves / 5: Moderate symptoms on some leaves / 3: Moderate symptoms on all leaves / 1: Severe symptoms on all leaves.

[0233] Fruit symptom scale : 9: Symptomless fruit 7: Mild symptoms (discoloration) in one or several fruits 5: Mild / moderate fruit discoloration on 2-3 or more fruits 3: Moderate / severe fruit discoloration and / or minor fruit deformation in more than 30% of the fruit 1: Very severe discoloration and / or moderate / severe deformation and / or necrotic spots on more than 50% of the fruit

[0234] 1.5.Stemphylium spp. Resistance Assessment Protocol Stemphylium spp. are plant pathogens and cause gray leaf spot of tomato. The Sm gene from Lycopersicum pipinellifolium confers genetically dominant resistance to Stemphylium.

[0235] Inoculum preparation : Stemphylium, Sicilian strain, is stored at -80°C and inocula are prepared directly from frozen tubes after cultivation in V8 medium. Conidia are obtained by scratching the surface of the medium, suspending in water with 1% glucose, and then filtering over muslin. 4 ~10 5 Obtain a solution containing 100 conidia / mL.

[0236] inoculation : The plantlets to be tested are at the three unfolded leaf stage, which corresponds to 17-24 days after sowing. The inoculum is applied by spraying on all leaf surfaces until droplets form.

[0237] Symptom evaluation : Scoring was then carried out by visual assessment of the leaves 7-8 days after infection. In this test, the scale was as follows: 9: Asymptomatic 7: Brown necrotic lesions, fewer in number than on susceptible plants 1: Small or large brown necrotic lesions on both sides of the leaves

[0238] Example 2 Identification of donors for ToBRFV resistance and preliminary mapping of QTLs Identification of suitable wild donors for ToBRFV resistance More than 500 different wild accessions were screened by the inventors to identify potential sources of resistance to ToBRFV, with the aim of potentially injecting resistance-conferring sequences in these wild accessions into the S. lycopersicum background, particularly in commercial plants.

[0239] The type of resistance we expected was fruit tolerance / resistance, but such tests are not applicable to wild accessions with different fruit sizes and morphologies. Therefore, we decided to rank sources of leaf symptom tests as a proxy for fruit resistance since we cannot screen for this parameter.

[0240] Among the wild strains screened, only four potential sources of infection (0.9%) were identified in the species S. habrochaites, S. chilense, and S. pipinellifolium, indicating that, unlike other viruses, it is difficult to find resistant sources of infection for ToBRFV.

[0241] Although each of these sources had a high proportion of symptomless plants, all of these plants were ELISA positive and no total resistance or immunity was observed.

[0242] Control plants S1 and S2 were used, which are known to be susceptible to ToBRFV, but the control plant S2 was resistant to TMV due to the presence of the Tm-22 gene.

[0243] The potential for resistance / tolerance was assessed on plant leaves; indeed, given the different shape, color, and size of the fruits of these wild accessions, it was not possible to rank resistance at the fruit level.

[0244] Table A reports the results obtained.

[0245] [Table 1]

[0246] However, after 32 dpi, all plants tested positive by ELISA, indicating that the resistance carried by these plants was not total (Table A).

[0247] Considering the results obtained, we decided to focus on source D, which showed better leaf resistance levels at 28 dpi, although the aim of this study was to identify the source fruit resistance, not the cause of leaf resistance.

[0248] F2 Mapping - HMC1* Source D - Artificial Test Four F2 populations of 240 individuals each were developed by crossing the susceptible parent HMC1 with the resistant wild type line D. HMC1 is an indeterminately propagated line with red round fruits weighing approximately 100 g and contains the Tm-22 gene.

[0249] Visual scoring was performed on different dates at 7, 14, and 28 dpi (see Table B above).

[0250] The results of the F2 screening under artificial conditions are shown in Table B. That is, on a scale of 1 to 9, plants with a score of "1" or "3" are considered susceptible, plants with a score of "5" are considered moderately resistant, and plants with a score of "7" or "9" are considered highly resistant in terms of leaf resistance.

[0251] Columns 4-8 report the number of plants with scores of 1, 3, 5, 7, and 9. Column 9 (1=S) indicates the number of plants with a score of 1, 3, 5, or 7. Column 10 (9=R) indicates the number of plants with a score of 9. Columns 11 and 12 report the percentage of plants rated "S" (score 1, 3, 5, or 7) and "R" (score 9).

[0252] [Table 2]

[0253] QTL analysis DNA was extracted as detailed in Example 1. For QTL analysis, we used different phenotypic variables / traits: 14 dpi, 21 dpi, 28 dpi, AUDPC. AUDPC (area under the disease progression curve) was calculated using the following formula:

[0254]

number

[0255] Here, "n" is the number of symptom assessments, "y" is the symptom intensity (1 to 9 times), and "t" is the time dpi (days post-inoculation).

[0256] Genotyping of the F2 population (based on sources D and HMC1) was performed using a set of 169 SNPs. These SNPs were selected according to: Polymorphism / allele frequency SNPs evenly spaced according to physical map distance

[0257] QTL analysis was performed using a QTL detection (ANOVA) model for biparental populations in the MAST-A marker-assisted selection tool (proprietary software).

[0258] The mapping results (see Figure 1, which shows the P-value plots for traits corresponding to AUDPC) revealed two candidate QTLs associated with ToBRFV resistance, located on chromosomes 9 (QTL9) and 11 (QTL11), and were positive alleles derived from Source D.

[0259] Markers significantly associated with QTL9 and QTL11 related to ToBRFV resistance and their locations on the tomato genome were identified. Table C To summarize:

[0260] [Table 3]

[0261] The results showed that QTL9, which is involved in ToBRFV resistance, is located on chromosome 9 between positions 3987296 and 40039587 on the tomato genome version SL2.50. This region of chromosome 9 is known to have a low recombination rate.

[0262] QTL11, responsible for ToBRFV resistance, is located on chromosome 11 at position 4524671 based on the tomato genome version SL2.50.

[0263] Example 3 : QTL mapping and validation. Field test. BC1F2 QTL mapping and validation A BC1F2 population between source D and the susceptible parent HMC1 was developed using the SNPs listed in Table C, and 158 individual plants were phenotyped under field inoculation conditions in Jordan as described in Example 1.

[0264] Fruit symptoms were rated on a scale of 9 to 1: 9: No symptoms - 7: Mild symptoms on a few fruits - 5: Moderate symptoms on some fruits - 3: Moderate symptoms on all fruits - 1: Severe symptoms.

[0265] Table D shows the results of BC1F2 screening under field inoculation conditions. Plants were rated on a scale of 1 to 9, with plants with a score of 1 or 3 considered susceptible, plants with a score of 5 considered intermediately resistant, and plants with a score of 7 or 9 considered highly resistant. Fruit evaluation of Source D was not performed due to the small size of the fruit from S. pimpinellifolium.

[0266] [Table 4]

[0267] DNA was extracted from the leaves as described in Example 1.

[0268] The BC1F2 population was genotyped using a subset of SNPs significantly associated with ToBRFV resistance in the F2 mapping population. Marker-trait associations were performed by cross-ANOVA in the MAST-A marker-assisted selection tool (proprietary software).

[0269] Mapping results revealed that QTL9 is associated with fruit resistance to ToBRFV, the same region already associated with leaf resistance.

[0270] BC3F2 QTL mapping - field test Approximately 140 BC3 3F2 populations each between Source D and HMC1 were evaluated under inoculated field conditions. The inoculation and symptom evaluation protocols were identical to those used for BC1F2 and described in Example 1. The scores for these plants are detailed in Table E, and the score scale of 1 to 9 is the same as for BC1F2.

[0271] [Table 5]

[0272] DNA was extracted from the leaves as described in Example 1.

[0273] BC3F2 individuals were genotyped at a subset of polymorphic SNPs on chromosomes 9 and 11 that were identified as associated with ToBRFV resistance in the F2 mapping population.

[0274] Genetic pap of chromosomes 9 and 11 was generated using JoinMap software to confirm the position and order of the markers.

[0275] QTL detection was performed using MapQTL software and genetic maps were made in these pedigrees.

[0276] The QTL mapping results confirm the presence of a major QTL for fruit resistance to ToBRFV on chromosome 9. Peak associated markers are listed in Table F.

[0277] [Table 6]

[0278] Example 4 : Resequencing of strains and identification of unique SNPs Six tomato accessions were resequenced, including three tolerant / resistant sources of ToBRFV, including source D and accessions used in WO2018 / 219941 (HAZTBRFVRES1), and three susceptible accessions used in the recurrent mapping population.

[0279] Seeds were sown, DNA was extracted from fresh leaves, and whole genome sequencing was performed.

[0280] The ordered sequencing depth was a minimum of 20. Sequencing was performed using Illumina NovaSeq 2x150nt technology.

[0281] Reads were mapped to the SL2.40 tomato reference genome, and variant calling analysis was performed using samtools. To identify SNPs from Source D as uniquely as possible, SNPs were filtered and alleles found in Source D were compared with alleles of SNPs identified in other lines in the project and with 360 tomato genomes ("Genomic analyses provide insights into the history of tomato breeding"; Lin et al. Nature Genetics, 2014).

[0282] Based on this, 310 unique SNPs from source D were identified in the interval containing QTL9 (SL2.40ch09: 10 Mb to 55 Mb). To select the best ones, several quality filters were applied (% AT in unique Blast and flanking sequences on the tomato genome). A list of 101 SNPs was selected (see Table H). Fourteen SNPs (see Table G) were tested in a large background panel to confirm their ability to track the presence of QTL9. Of these, five SNPs (see Table G) performed very well in terms of specificity for source D.

[0283] Table G: List of 14 specific SNPs from source D resequencing in the QTL9 interval. The table shows the names of the 14 SNPs, their location in the SL2.50 genome, the sequence with the polymorphism in parentheses, and the susceptible and resistant alleles. Five SNPs showing very good results are marked with an asterisk.

[0284] Polymorphisms are indicated in parentheses. The "S" column reports the susceptible allele, i.e., present in the recurrent parental HMC, while the "R" column indicated the resistant allele, as found in source D.

[0285] [Table 7-1]

[0286] [Table 7-2]

[0287] [Table 7-3]

[0288] Table H : A list of 101 specific SNPs from source D resequencing in the QTL9 interval. Table H shows the location in the SL2.50 genome, the SEQ ID NO of the sequence in the sequence listing, and the susceptibility and resistance alleles of the 101 SNPs identified by the inventors. 14 SNPs and 5 SNPs mentioned above showed very good results, and are indicated by one or two asterisks, respectively.

[0289] [Table 8-1]

[0290] [Table 8-2]

[0291] [Table 8-3]

[0292] [Table 8-4]

[0293] These SNPs therefore allow for discrimination between plants carrying QTL9 as described in the present invention and plants derived from HAZTBRFVRES1 as described in WO2018 / 219941 which carry a different QTL on chromosome 9 that confers tolerance to ToBRFV. The QTL disclosed in the present invention therefore clearly differs from the QTL disclosed in WO2018 / 219941 in terms of sequence.

[0294] Example 5 : Characterization of a new QTL9 in a field trial Two different trials were conducted in Jordan and confirmed that in addition to the sequence differences between QTL9 of the present invention and the QTL described in WO2018 / 219941, these different sequences confer different types of resistance to ToBRFV.

[0295] The first trial, T1 (372 plants), was carried out in one tunnel in summer using different elite lines (check), the controls S1 and S2 described in Examples 2-4, and plants containing QTLs on chromosomes 9 and 11 described in WO2018 / 219941.

[0296] The second trial T2 (1165 plants) was carried out the following winter in two tunnels consisting of the same elite line (check) and control as in the previous trial T1, and BC3F2 issued from source D described in Example 4.

[0297] In this example, the type / origin of the sequences found at the QTL9 and QTL11 loci on chromosomes 6 and 9 of the plants tested is defined as follows: → The sequences found in elite lines at the loci of QTL9 and QTL11, and the “alleles” due to the extension of these loci in elite lines, are coded as “Re”; → The sequences found in susceptible strains S1 and S2 at the QTL9 and QTL11 loci (the “alleles” of these loci in susceptible strains) are coded as “S”; → The sequences found at the QTL9 and QTL11 loci in tolerant / resistant plants derived from HAZTBRFVRES1 described in WO2018 / 219941 (the "alleles" of these loci in HAZTBRFVRES1) are coded as "Rh"; →The sequences found in the resistant plant BC3F2 issued from source D at the loci QTL9 and QTL11 (the "alleles" of these loci in the plants of the invention) are coded as "Rd".

[0298] As described in Example 1, plants were inoculated twice, once at 1 week and twice at 2 weeks after planting.

[0299] Plants were then scored for fruit and leaf symptoms according to the scale described in Example 1.4. However, as leaf symptoms were assessed at the mature leaf stage (plants with red fruit), leaf symptoms were difficult to assess given the scarce young leaves.

[0300] Statistical analysis of results The data were analyzed using a mixed model to determine the level of resistance conferred by the "allele" found at the QTL9 locus in plants, taking into account other potential influences, namely the "allele" found at the QTL11 locus, the plant genotype, and the tunnel effect. The mixed model was as follows: Score = μ + (genotype) random + tunnel + QTL9 + QTL11 + ε where score (fruit resistance or leaf resistance) is the observed variable, μ is the trait mean, ε is the residual error, and (genotype) Random, Tunnel, QTL9 and QTL11 are the effects.

[0301] The tunnel effect was estimated as a result of checking that all tunnels exist.

[0302] Genotype effects were treated as random effects to capture variation in the populations in which the tested varieties occur.

[0303] For each genotype at the QTL9 locus and for each trait (fruit symptoms and leaf symptoms), we extracted adjusted values ​​to better estimate the likelihood of each genotype, independent of other effects.

[0304] For example, for the genotype corresponding to Rd / Rd, the adjustment value is: μ + QTL9[RdRd] + average(genotype) + average(tunnel) + average(QTL11) where μ is the estimated mean value of the trait (fruit symptom or leaf symptom), QTL9[Rd / Rd] is the estimated effect of the genotype RdRd at QTL9, and mean(genotype), mean(tunnel), and mean(QTL11) are the means of the corresponding estimated effects. Multiple comparisons between adjusted values ​​were performed using Tukey's test.

[0305] result : Effect of QTL9 on fruit symptoms: For traits corresponding to fruit symptoms, the adjusted values ​​of the different test genotypes for the QTL9 locus were calculated using the mixed model detailed above. The results are reported in Table I and shown in Figures 2A and 2B.

[0306] [Table 9]

[0307] From these results, it can be deduced that the presence of QTL9 defined in the present invention (which in this example corresponds to the Rd genotype) confers a significantly higher level of resistance than the level of resistance provided by QTL2 defined in WO2018 / 219941 (which in this example corresponds to the Rh genotype).

[0308] Furthermore, analysis of the allelic effect of QTL9 on fruit resistance, as shown below (extracts in Table I), indicates a significant additive effect of the Rd allele, as shown in Figure 3 .

[0309] [Table 10]

[0310] The redistribution of fruit scores was also determined by the QTL9 genotype. The results are shown in Figure 4.

[0311] These results show that QTL9 of the present invention, when present in the homozygous state (genotype RdRd in this example), confers over 80% of plants with fruit scores of 5, 7 or 9 (48, 61 and 59 plants out of 208, respectively). Approximately 60% of the plants (120 out of 208) show a score of 7 or 9 after two infections, i.e., the fruit is largely symptom-free.

[0312] These results also confirm that QTL9 according to the invention, when present in the heterozygous state (genotype RdS in this example), gives about 50% of the plants a fruit score of 5, 7 or 9, respectively (63, 36 and 13 plants out of 225, respectively). About 22% of the plants (49 out of 225) show a score of 7 or 9 after two infections, i.e. the fruit is almost symptomless.

[0313] In contrast, for the RhRh genotype, approximately 80% of the plants had fruit scores of 5, 7, and 9, but less than 35% of the plants had scores of 7 and 9, respectively (30 and 1 out of 89, respectively), and the fruit was largely symptomless after two infections.

[0314] QTL9 effect on leaf symptoms : For traits corresponding to leaf symptoms, adjusted values ​​for the different test genotypes for the QTL9 locus were calculated using the mixed model detailed above. The results are reported in Table J and shown in FIG.

[0315] [Table 11]

[0316] From these results, it can be deduced that the presence of QTL9 defined in the present invention (corresponding to the Rd genotype in this example) provides a significantly higher level of leaf resistance that is significantly different from the level of leaf resistance provided by QTL2 defined in WO2018 / 219941 (corresponding to the Rh genotype in this example). Furthermore, the results also show that the heterozygous presence of QTL9 is sufficient to provide a high level of leaf resistance (adjusted values ​​for the RdRd and RdS genotypes are statistically identical).

[0317] Example 6 : Line resequencing and identification of unique SNPs on chromosome 11 The same experiment as disclosed in Example 4 for the QTL on chromosome 9 was applied to the QTL identified by the inventors on chromosome 11. SNP analysis was performed on the same plant population.

[0318] Informative SNPs for the presence of QTL11 according to the present invention are reported in Table K below (SEQ ID NOs: 102 to 111), as well as their location in the SL2.50 version of the genome and the susceptible and resistant alleles.

[0319] We then conducted further investigations to identify additional markers that are informative for the presence of recombinant sequences conferring ToBRFV resistance. These markers are highly specific for recombinant sequences from Source D that confer ToBRFV resistance.

[0320] [Table 12-1]

[0321] [Table 12-2]

[0322] [Table 12-3]

[0323] Example 7 Genetic modification of tomato seeds with ethyl methanesulfonate (EMS) Seeds of tomato cultivars are treated with EMS by soaking approximately 2000 seeds per cultivar in an aerated solution of either 0.5% (w / v) or 0.7% EMS for 24 hours at room temperature.

[0324] Approximately 1,500 treated seeds per variety per EMS dose are germinated, and the resulting plants are preferably grown in a greenhouse, for example, from May to September, to produce seeds. After maturation, M2 seeds are harvested and bulked in pools, one per variety per treatment. The resulting M2 seed pools are used as starting materials to identify individual M2 seeds and plants resistant to tomato brown rugose fruit virus.

[0325] Example 8 : Resistance to Stemphylium WO 2020 / 018783 discloses a genetic region on tomato chromosome 11 containing a Stemphylium resistance allele from S. pimpinellifolium, and also a TBRFV resistance allele, which are allegedly so closely linked that they are characterized by the same markers and therefore co-introgressed. To confirm that the QTL on chromosome 11 identified by the present inventors is different from the resistance allele disclosed in WO 2020 / 018783, the present inventors tested plants according to the present invention for resistance to Stemphylium. The protocol for Stemphylium resistance is as disclosed in Example 1.5.

[0326] result: Over 157 different plants from five different genotypes were tested, with at least 18 replicates per genotype. The genotypes / varieties tested were as follows: -Stempphylium-resistant control (R) 1 case -One case of intermediate Stemphylium resistance (IR) -1 Stemphylium-susceptible control (S) -Source D -BC5F3 HMC1*Source D

[0327] The Stemphylium resistance results are shown in Table L below.

[0328] [Table 13]

[0329] Table L: Stemphylium resistance. "Mean" indicates the average score for all plants of the same family according to the symptom assessment detailed in 1.5. The interpretation indicates whether the variety is resistant (R) or susceptible (S). QTL11 is a QTL according to the present invention, the presence of which is tested with the markers disclosed in the preceding examples. NT means not tested.

[0330] Conclusion: QTL11 from source D according to the invention, which confers ToBRFV resistance, contains QTL11 according to the invention but is not associated with Stemphylium resistance, in contrast to ToBRFV genetic resistance according to WO2020 / 018783, since the test plants are not resistant to Stemphylium.

[0331] Therefore, it can be concluded that QTL11 of the present invention is different from the resistance disclosed in WO2020 / 018783 on chromosome 11.

[0332] Example 9 Analysis of ToBRFV resistance provided by QTL11 An F2 population was obtained between source D and the susceptible parent HMC2. HMC2 is a line that is highly susceptible to ToBRFV at the leaf level. 134 individual plants were phenotyped after ToBRFV inoculation for leaf symptoms and genotyped based on SNP markers at QTL11, as described in Example 1.B.

[0333] For traits corresponding to leaf symptoms, adjusted values ​​for the different test genotypes for the QTL11 locus were calculated using a mixed model similar to the model used for QTL9. The results are reported in Table M and shown in Figure 6.

[0334] [Table 14]

[0335] Analysis of the effect of alleles at QTL11 on leaf resistance shows a significant recessive effect of the resistance allele, as shown in Table M and in Figure 6 .

Claims

1. A Solanum lycopersicum plant resistant to Tomato Brown Rugos Fruit Virus (ToBRFV), wherein the plant's genome contains a quantitative trait locus (QTL) (QTL9) on chromosome 9 and / or a QTL (QTL11) on chromosome 11, each of which is derived from S. pimpinellifolium and confers resistance to ToBRFV to the plant. - The QTL on chromosome 9 is located within a chromosomal region delimited by SNPs identified by SNP TO-0201220 (SEQ ID NO: 1) and SEQ ID NO:

101. - The QTL on chromosome 11 is located within a chromosomal region delimited by the SNP identified by sequence number 102 and the marker identified by sequence number 115. Herein, the QTL(s) (single or multiple) are present in the genome of the seed of LVSTBRFVRES2, accession number NCIMB 43591, of the Solanum lycopersicum plant.

2. The plant according to claim 1, wherein the QTL on chromosome 9 can be identified by one of the SNP markers selected from a list including SNP TO-0201220 (SEQ ID NO: 1), TO-0201221 (SEQ ID NO: 2), TO-0201222 (SEQ ID NO: 3), TO-0201223 (SEQ ID NO: 4), TO-0201224 (SEQ ID NO: 5), TO-0201225 (SEQ ID NO: 6), TO-0201226 (SEQ ID NO: 7), TO-0201227 (SEQ ID NO: 8), TO-0201228 (SEQ ID NO: 9), TO-0201229 (SEQ ID NO: 10), TO-0201230 (SEQ ID NO: 11), TO-0201231 (SEQ ID NO: 12), TO-0201232 (SEQ ID NO: 13), and TO-0201233 (SEQ ID NO: 14).

3. The plant according to claim 2, wherein the QTL on chromosome 9 can be identified by SNP TO-0201220 (SEQ ID NO: 1) and / or SNP TO-0201229 (SEQ ID NO: 10).

4. The QTL on chromosome 9 is composed of the following alleles: Allele G of SNP TO-0201220 (SEQ ID NO: 1), Allele G of TO-0201221 (SEQ ID NO: 2), Allele A of TO-0201222 (SEQ ID NO: 3), Allele A of TO-0201223 (SEQ ID NO: 4), Allele A of TO-0201224 (SEQ ID NO: 5), Allele A of TO-0201225 (SEQ ID NO: 6), Allele A of TO-0201226 (SEQ ID NO: 7), and Allele A of TO-0201227 (SEQ ID NO: 8) A plant according to any one of claims 1 to 3, which can be identified by at least one of allele C of TO-0201228 (SEQ ID NO: 9), allele A of TO-0201229 (SEQ ID NO: 10), allele C of TO-0201230 (SEQ ID NO: 11), allele C of TO-0201231 (SEQ ID NO: 12), allele G of TO-0201232 (SEQ ID NO: 13), and allele G of TO-0201233 (SEQ ID NO: 14).

5. The plant according to claim 1, wherein the QTL on chromosome 9 is found within the region delimited by TO-0201221 (SEQ ID NO: 2) and TO-0201233 (SEQ ID NO: 14).

6. The plant according to any one of claims 1 to 5, wherein the QTL on chromosome 9 is present in the plant genome in a homozygous or heterozygous manner.

7. The plant according to claim 1, wherein the QTL on chromosome 11 can be identified by one of the SNPs selected from a list including TO-0201237 (SEQ ID NO: 102), TO-0201238 (SEQ ID NO: 103), TO-0201239 (SEQ ID NO: 104), TO-0201240 (SEQ ID NO: 105), and TO-0201241 (SEQ ID NO: 106), or by one of the markers selected from a list including SL2.50ch11 9684449 (SEQ ID NO: 112), SL2.50ch11 9779896 (SEQ ID NO: 113), SL2.50ch11 9823405 (SEQ ID NO: 114), and SL2.50ch11 9924232 (SEQ ID NO: 115).

8. The QTL on chromosome 11 is the following alleles: allele G of TO-0201237 (sequence number 102), allele A of TO-0201238 (sequence number 103), allele A of TO-0201239 (sequence number 104), allele A of TO-0201240 (sequence number 105), allele A of TO-0201241 (sequence number 106), allele CT of SL2.50ch11 9684449 (sequence number 112), allele AT of SL2.50ch11 9779896 (sequence number 113), allele C of SL2.50ch11 9823405 (sequence number 114), and SL2.50ch11 The plant according to any one of claims 1 and 7, which can be identified by at least one of the alleles GT of 9924232 (SEQ ID NO: 115).

9. The plant according to any one of claims 1 and 7 to 8, wherein the QTL on chromosome 11 is found in a region delimited by TO-0201237 (SEQ ID NO: 102) and TO-0201241 (SEQ ID NO: 106).

10. The plant according to any one of claims 1 and 7 to 9, wherein the QTL on chromosome 11 is homozygous in the plant genome.

11. The plant according to any one of claims 1 to 10, wherein the plant is a descendant of the seeds of LVSTBRFVRES2 deposited with NCIMB under accession number NCIMB 43591.

12. The plant also has Tm-2 in its genome that confers resistance to TMV. ２ A plant containing a gene, according to any one of claims 1 to 11.

13. The Tm-2 ２ The plant according to claim 12, wherein the genes are heterozygous.

14. A plant according to any one of claims 1 to 13, further comprising the Tm-1 gene.

15. A cell of an S. lycopersicum plant according to any one of claims 1 to 14, wherein the genome of the cell of an S. lycopersicum plant contains QTL9 on chromosome 9 and / or QTL11 on chromosome 11, and the QTL9 on chromosome 9 and / or QTL11 on chromosome 11 independently confer resistance to ToBRFV.

16. A tissue culture of plant cells as defined in any one of claims 1 to 14, wherein the cells are derived from an embryo, protoplast, meristem cell, callus, pollen, leaf, anther, stem, petiole, root, root tip, seed, flower, cotyledon, and / or hypocotyl, and the genome of the cells contains QTL9 on chromosome 9 and / or QTL11 on chromosome 11, the QTLs independently conferring resistance to ToBRFV.

17. A plant part, seed, explant, reproductive material, scion, cutting, seed, fruit, root, root, pollen, ovule, embryo, protoplast, leaf, anther, stem, petiole, or flower of a S. lycopersicum plant as described in any one of claims 1 to 14, wherein the plant part, seed, explant, reproductive material, scion, cutting, seed, fruit, root, root, pollen, ovule, embryo, protoplast, leaf, anther, stem, petiole, or flower contains the cells described in claim 15.

18. Seeds of a S. lycopersicum plant, which occur on a plant as defined in any one of claims 1 to 14.

19. A plant derived from the seeds of S. lycopersicum deposited with NCIMB as accession number NCIMB 43591, or a part thereof or its offspring, which homozygously possesses QTL9 and / or QTL11 that independently confer resistance to ToBRFV infection. Use as a breeding partner in a breeding program to confer ToBRFV resistance to lycopersicum plants, wherein the QTL on chromosome 9 is located within a chromosomal region delimited by SNPs identified by SNP TO-0201220 (SEQ ID NO: 1) and SEQ ID NO: 101, the QTL on chromosome 11 is located within a chromosomal region delimited by SNPs identified by SEQ ID NO: 102 and markers identified by SEQ ID NO: 115, and the QTL(s) are present in the genome of seeds of LVSTBRFVRES2 with accession number NCIMB 43591.

20. A method for detecting and / or selecting S. lycopersicum plants containing a QTL (Quadruple Threshold Leptolum) transfected from S. pimpinellifolium, wherein the QTL confers ToBRFV resistance, and the method comprises detecting at least one gene marker linked to the QTL, wherein the gene marker is - Selected from the group including SNP TO-0201220 (SEQ ID NO: 1), TO-0201221 (SEQ ID NO: 2), TO-0201222 (SEQ ID NO: 3), TO-0201223 (SEQ ID NO: 4), TO-0201224 (SEQ ID NO: 5), TO-0201225 (SEQ ID NO: 6), TO-0201226 (SEQ ID NO: 7), TO-0201227 (SEQ ID NO: 8), TO-0201228 (SEQ ID NO: 9), TO-0201229 (SEQ ID NO: 10), TO-0201230 (SEQ ID NO: 11), TO-0201231 (SEQ ID NO: 12), TO-0201232 (SEQ ID NO: 13), and TO-0201233 (SEQ ID NO: 14), or The method described above, selected from the group including markers TO-0201237 (SEQ ID NO: 102), TO-0201238 (SEQ ID NO: 103), TO-0201239 (SEQ ID NO: 104), TO-0201240 (SEQ ID NO: 105), TO-0201241 (SEQ ID NO: 106), SL2.50ch11 9684449 (SEQ ID NO: 112), SL2.50ch11 9779896 (SEQ ID NO: 113), SL2.50ch11 9823405 (SEQ ID NO: 114), and SL2.50ch11 9924232 (SEQ ID NO: 115).

21. A method for detecting and / or selecting S. lycopersicum plants containing a QTL on chromosome 9 and / or a QTL on chromosome 11, wherein the QTL is translocated from S. pimpinellifolium and independently confers ToBRFV resistance, and the method involves detecting the following alleles in a genetic material sample of the plant to be selected: - Regarding QTLs on chromosome 9, allele G of SNP TO-0201220 (SEQ ID NO: 1), allele G of TO-0201221 (SEQ ID NO: 2), allele A of TO-0201222 (SEQ ID NO: 3), allele A of TO-0201223 (SEQ ID NO: 4), allele A of TO-0201224 (SEQ ID NO: 5), allele A of TO-0201225 (SEQ ID NO: 6), allele A of TO-0201226 (SEQ ID NO: 7), TO-0 Allele C of 201227 (SEQ ID NO: 8), Allele C of TO-0201228 (SEQ ID NO: 9), Allele A of TO-0201229 (SEQ ID NO: 10), Allele C of TO-0201230 (SEQ ID NO: 11), Allele C of TO-0201231 (SEQ ID NO: 12), Allele G of TO-0201232 (SEQ ID NO: 13) and Allele G of TO-0201233 (SEQ ID NO: 14), or - Regarding QTLs on chromosome 11, allele G of TO-0201237 (sequence number 102), allele A of TO-0201238 (sequence number 103), allele A of TO-0201239 (sequence number 104), allele A of TO-0201240 (sequence number 105), allele A of TO-0201241 (sequence number 106), allele CT of SL2.50ch11 9684449 (sequence number 112), allele AT of SL2.50ch11 9779896 (sequence number 113), allele C of SL2.50ch11 9823405 (sequence number 114), or allele GT of SL2.50ch11 9924232 (sequence number 115) The above method, which includes detecting at least one of the following.

22. The use of a genetic marker to detect the presence of a sequence translocated from S. pimpinellifolium, wherein the sequence confers ToBRFV resistance, and the use involves detecting the genetic marker, wherein the genetic marker is - Selected from the group including SNP TO-0201220 (SEQ ID NO: 1), TO-0201221 (SEQ ID NO: 2), TO-0201222 (SEQ ID NO: 3), TO-0201223 (SEQ ID NO: 4), TO-0201224 (SEQ ID NO: 5), TO-0201225 (SEQ ID NO: 6), TO-0201226 (SEQ ID NO: 7), TO-0201227 (SEQ ID NO: 8), TO-0201228 (SEQ ID NO: 9), TO-0201229 (SEQ ID NO: 10), TO-0201230 (SEQ ID NO: 11), TO-0201231 (SEQ ID NO: 12), TO-0201232 (SEQ ID NO: 13), and TO-0201233 (SEQ ID NO: 14), or - The above use is selected from the group including markers TO-0201237 (SEQ ID NO: 102), TO-0201238 (SEQ ID NO: 103), TO-0201239 (SEQ ID NO: 104), TO-0201240 (SEQ ID NO: 105), TO-0201241 (SEQ ID NO: 106), SL2.50ch11 968449 (SEQ ID NO: 112), SL2.50ch11 9779896 (SEQ ID NO: 113), SL2.50ch11 9823405 (SEQ ID NO: 114), and SL2.50ch11 9924232 (SEQ ID NO: 115).

23. The use of a marker for identifying a QTL in the tomato genome, wherein the QTL is translocated from S. pimpinellifolium and confers fruit resistance to ToBRFV, and the use comprises detecting the gene marker, wherein the marker is localized to a chromosomal region delimited on chromosome 9 by SNPs identified by SNP TO-0201220 (SEQ ID NO: 1) and SEQ ID NO: 101, wherein the marker is the following SNP allele: SNP The above use is characterized by association with one of the following alleles: G in TO-0201220 (SEQ ID NO: 1), allele G in TO-0201221 (SEQ ID NO: 2), allele A in TO-0201229 (SEQ ID NO: 10), allele C in TO-0201231 (SEQ ID NO: 12), or allele G in TO-0201233 (SEQ ID NO: 14), with a p-value of 0.05 or less, wherein the gene marker is disclosed in Table H and selected from gene markers identified by SEQ ID NOs: 1 to 101.

24. The use of a marker for identifying QTLs in the tomato genome, wherein the QTL is S. The gene is transferred from pimpinellifolium and confers resistance to ToBRFV in the fruit, wherein the use includes detecting the gene marker, where the marker is localized to a chromosomal region delimited on chromosome 11 by the marker identified by SEQ ID NOs: 102 and 115, where the marker is the following resistance allele: allele G of TO-0201237 (SEQ ID NO: 102), allele A of TO-0201238 (SEQ ID NO: 103), allele A of TO-0201239 (SEQ ID NO: 104), allele A of TO-0201240 (SEQ ID NO: 105), allele A of TO-0201241 (SEQ ID NO: 106), allele CT of SL2.50ch11 9684449 (SEQ ID NO: 112), SL2.50ch11 The above use is selected from gene markers that are associated with one of the alleles AT of 9779896 (SEQ ID NO: 113), allele C of SL2.50ch11 9823405 (SEQ ID NO: 114), or allele GT of SL2.50ch11 9924232 (SEQ ID NO: 115) with a p-value of 0.05 or less, and the gene marker is disclosed in Table K and identified by SEQ ID NOs: 102 to 115.

25. A method for conferring resistance to ToBRFV to the plant S. lycopersicum, a) A step of crossing a plant grown from seeds deposited in NCIMB 43591 or its offspring, which possesses QTL9 on chromosome 9 and / or QTL11 on chromosome 11, with an early S. lycopersicum plant, wherein the QTLs are gene-transferred from S. pimpinellifolium and independently confer ToBRFV resistance in NCIMB 43591, and the QTL on chromosome 9 is located within a chromosomal region delimited by SNPs identified by SNP TO-0201220 (SEQ ID NO: 1) and SEQ ID NO: 101, and the QTL on chromosome 11 is located within a chromosomal region delimited by SNPs identified by SEQ ID NO: 102 and markers identified by SEQ ID NO:

115. b) A step of selecting plants that possess QTL9 and / or QTL11 based on SNP marker detection among the offspring obtained in this manner; c) The plants obtained in step b) are to be self-pollinated one or more times as desired, and from the offspring obtained in this way, plants that have resistance to ToBRFV are selected. The above method, including.

26. A method for improving the yield of tomato plants in an environment where ToBRFV is prevalent, comprising growing tomato plants in which the genome of the tomato plant contains QTL9 on chromosome 9 and / or QTL11 on chromosome 11, wherein the QTLs are translocated from S. pimpinellifolium and independently confer resistance to ToBRFV to the plants, wherein the QTLs are present in the genome of plants derived from seeds of LVSTBRFVRES2 with accession number NCIMB 43591, and QTL9 is an SNP It is identifiable by at least one of TO-0201220 (SEQ ID NO: 1), TO-0201221 (SEQ ID NO: 2), TO-0201229 (SEQ ID NO: 10), TO-0201231 (SEQ ID NO: 12), and TO-0201233 (SEQ ID NO: 14), and for QTL11 it is identifiable by at least one of the markers TO-0201237 (SEQ ID NO: 102), TO-0201238 (SEQ ID NO: 103), TO-0201239 (SEQ ID NO: 104), TO-0201240 (SEQ ID NO: 105), TO-0201241 (SEQ ID NO: 106), SL2.50ch11 9684449 (SEQ ID NO: 112), SL2.50ch11 9779896 (SEQ ID NO: 113), SL2.50ch11 The above method, which is identifiable by at least one of 9823405 (sequence number 114) and SL2.50ch11 9924232 (sequence number 115).

27. A method for reducing tomato production losses in a ToBRFV-infested state, comprising cultivating tomato plants containing QTL9 on chromosome 9 and / or QTL11 on chromosome 11 in the genome of the tomato plants, wherein the QTLs are genetically transferred from S. pimpinellifolium and independently confer to the plants resistance to ToBRFV, wherein the QTLs are present in the genome of plants derived from seeds of LVSTBRFVRES2 with accession number NCIMB 43591, and QTL9 is an SNP. It is identifiable by at least one of TO-0201220 (SEQ ID NO: 1), TO-0201221 (SEQ ID NO: 2), TO-0201229 (SEQ ID NO: 10), TO-0201231 (SEQ ID NO: 12), and TO-0201233 (SEQ ID NO: 14), and for QTL11 it is identifiable by at least one of the markers TO-0201237 (SEQ ID NO: 102), TO-0201238 (SEQ ID NO: 103), TO-0201239 (SEQ ID NO: 104), TO-0201240 (SEQ ID NO: 105), TO-0201241 (SEQ ID NO: 106), SL2.50ch11 9684449 (SEQ ID NO: 112), SL2.50ch11 9779896 (SEQ ID NO: 113), SL2.50ch11 The above method, which is identifiable by at least one of 9823405 (sequence number 114) and SL2.50ch11 9924232 (sequence number 115).

28. - A step to identify a tomato plant exhibiting resistance to ToBRFV, wherein the tomato plant genome contains QTL9 on chromosome 9, the QTL9 being introduced from S. pimpinellifolium and conferring fruit resistance to ToBRFV to the plant; or a step to identify a tomato plant exhibiting resistance to ToBRFV, wherein the tomato plant genome contains QTL11 on chromosome 11, the QTL11 being introduced from S. pimpinellifolium and conferring leaf resistance to ToBRFV to the plant, wherein the QTL is present in the genome of a plant derived from seeds of LVSTFVRES2 with accession number NCIMB 43591; - The process of growing the plant in an environment where ToBRFV is prevalent or in a state where ToBRFV is prevalent. The method according to any one of claims 26 to 27, including the method described in any one of claims 26 to 27.

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