Resistance in plants of solanum lycopersicum to a tobamovirus tomato brown rugose fruit virus (TBRFV)

Abstract

To provide Solanum lycopersicum plants having improved phenotype corresponding to tolerance and / or resistance of leaf and / or fruit to tomato brown rugose fruit virus.SOLUTION: The invention relates to a Solanum lycopersicum plant having in its genome a QTL that confers the phenotype of fruit and / or foliar tolerance and / or resistance to tomato brown rugose fruit virus as compared with corresponding plants without the QTL, where the QTL is selected from QTLs present in the genome of a plant of the seeds HAZTBRFVRES1 NCIMB Accession Number 42758. Preferably the QTLs are characterized by alleles defined by different SNPs on chromosomes 6, 9 and 11. The invention is also directed to parts of these plants with improved phenotypes, as well as progeny, to the use of these plants for introgressing the phenotypes in another genetic background, as well as to different methods for obtaining tomato plants or seeds with increased fruit and / or foliar tolerance or resistance to tomato brown rugose fruit virus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to resistance or tolerance to the tobamovirus Tomato Brown Rugose Fruit virus (TBRFV) in Solanum lycopersicum plants, also known as Lycopersicum esculentum. More specifically, the present invention relates to tomato plants and fruit containing one or more genetic determinants that confer resistance or tolerance to Tomato Brown Rugose Fruit virus. The present invention further relates to markers associated with one or more genetic determinants and the use of such markers to identify or select genetic determinants and to identify or select plants having such resistance or tolerance. The present invention also relates to seeds and progeny of such plants, as well as propagation material for obtaining such plants, and 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 Solanaceae family, thought to consist of approximately 90 genera, including pepper, tobacco, and eggplant. The genus Lycopersicon is divided into two subgenera: the esculentum complex, which contains species that can easily hybridize with commercial tomatoes, and the peruvianum complex, which contains species that are significantly more difficult to hybridize (Stevens, M., and Rick, C.M. 1986). L. esculentum Miller is widespread worldwide due to its value as a crop. Although the exact origin of the cultivated tomato remains somewhat unclear, it appears to have come from the Americas, native to Ecuador, Peru, and the Galapagos Islands, where it was first cultivated by the Aztecs and Incas as early as 700 AD. Mexico is likely the site of domestication and the origin of the earliest introductions. The cherry tomato, L. esculentum var. cerasiforme, is the direct ancestor of modern cultivated forms.

[0003] Tomatoes are grown for their fruit, which is widely used as a fresh eating or processed product. As a crop, tomatoes are grown commercially wherever environmental conditions allow for the production of economically viable yields. The majority of fresh market tomatoes are harvested by hand at the ripe and mature green stages of maturity. Fresh market tomatoes are available year-round. Processed tomatoes are mostly mechanically harvested and are used in many forms, such as canned tomatoes, tomato juice, tomato sauce, puree, paste, or ketchup.

[0004] Tomatoes are typically simple diploid species with 12 differentiated chromosome pairs. However, polyploid tomatoes are also part of this invention. Cultivated tomatoes are self-fertile and almost exclusively self-pollinate. Tomato flowers are hermaphrodites. Commercial cultivars were initially open-pollinated. As hybrid vigor was identified in tomatoes, hybrids have replaced open-pollinated varieties, gaining increasing popularity among farmers due to better yields and uniformity of plant characteristics. Due to their widespread popularity and high value, tomatoes are intensively bred, which explains why such a wide variety of tomatoes is currently available. Shapes can range from small to large, and include cherry, plum, pear, block, round, and beefsteak varieties. Tomatoes can be grouped by the amount of time it takes the plant to ripen fruit for harvest; cultivars are generally considered early, mid, or late ripening. Tomatoes can also be grouped by the plant's growth habit: determinate, semi-determinate, or indeterminate. Determinate plants tend to develop leaves first, then produce flowers, which, if pollination is successful, mature into fruit. All fruits tend to ripen on the plant at roughly the same time. Indeterminate tomatoes begin with the development of some leaves and then 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-determinate tomatoes are typical of determinate types, except that they have a phenotype between determinate and indeterminate and grow larger than determinate varieties. More recent developments in tomato breeding have resulted in a variety of fruit colors. In addition to the standard red ripe color, tomatoes can be milky white, lime green, pink, yellow, gold, orange, or purple.

[0005] Commercial hybrid tomato seed can be produced by hand pollination. Pollen from the male parent is harvested and applied by hand to the stigma surface of the female inbred. Before and after hand pollination, the flowers are covered to prevent insects from introducing foreign pollen, creating a mixture or adulteration. The flowers are tagged to identify the pollinated fruit from which seeds will be harvested.

[0006] A variety of pathogens, including viruses, fungi, bacteria, nematodes, and insects, affect the productivity of tomato plants. Tomato is particularly susceptible to many viruses, so virus resistance is of great importance in agriculture.

[0007] Tobamoviruses are one of the most important plant viruses in agriculture, causing serious damage to vegetable and ornamental crops worldwide. They are easily transmitted by mechanical means and seed transmission. Tobamoviruses are generally characterized by approximately 300 nm rod-shaped particles that encapsulate a single-stranded, positive-chain RNA genome encoding four proteins. In tomatoes, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) are feared by growers worldwide because they can severely damage crop production, for example, by causing irregular ripening (fruit with yellowish spots on the surface and brownish spots below the surface). However, over the years, several genes have been identified by plant breeders, and TMV- and / or ToMV-resistant tomato varieties are now available.

[0008] In recent years, severe virus outbreaks have affected tomato-producing regions in the Middle East, including Jordan and Israel. Most of the affected tomato varieties were considered resistant to TMV and / or ToMV, yet they were still severely affected and exhibited typical TMV / ToMV-like symptoms. While leaf symptoms closely resembled those of TMV / ToMV, fruit symptoms, including fruit lesions and deformations, were significantly more frequent and severe than the usual symptoms associated with such viruses. Fruit quality was significantly poor and unmarketable. Salem et al. (Arch. Virol. 161(2)503-506. 2015) extracted RNA from the fruit and leaves of symptomatic plants and performed various tests. This identified a new tobamovirus species, which they proposed to name Tomato Brown Curly Leaf Fruit Virus (TBRFV). Resistance to TMV and / or ToMV does not confer resistance to this new virus, TBRFV. Summary of the Invention [Problem to be solved by the invention]

[0009] Tobamoviruses are not easily controlled, but through genetic improvement through the identification of resistance genes and their use in breeding, and because the resistance genes currently available to control TMV and / or ToMV are useless against the damage caused by the new Tomato Brown Curl Fruit Virus, there is an urgent need to identify resistance and / or tolerance to this new tobamovirus, failure of which could result in the failure of tomato crops to be produced in entire regions. [Means for solving the problem]

[0010] The present inventors have identified tomato plants that exhibit resistance or tolerance to Tomato Brown Leaf Fruit Virus and have been able to locate and identify the genetic determinants, hereinafter also referred to as QTLs (quantitative trait loci), that confer resistance or tolerance to Tomato Brown Leaf Fruit Virus.

[0011] The resistance or tolerance according to the present invention is conferred by a newly discovered genetic determinant that can confer resistance or tolerance to Tomato Brown Leaf Fruit Virus (TBRFV) at the leaf level of infected tomato plants, at the fruit level of infected tomato plants, or at both the leaf and fruit levels. The newly discovered genetic determinant is recessive in nature. Because fruit resistance and / or resistance is independently conferred by two QTLs and leaf resistance is conferred by one QTL, their introgression into different genetic backgrounds, i.e., into various tomatoes, can be easily performed by those skilled in the art of plant breeding, especially given the information on suitable markers associated with the QTLs provided by the present inventors.

[0012] Thus, the present invention provides these genetic determinants, also referred to herein as QTLs, which, when present in the homozygous state, confer a TBRFV resistance or tolerance phenotype at the tomato leaf and / or fruit level in TBRFV-infected tomato plants.

[0013] The present invention provides commercial S. lycopersicum plants that exhibit resistance or tolerance to TBRFV, and methods for producing or identifying S. lycopersicum plants or populations (germplasm) that exhibit resistance to TBRFV. The present invention also discloses molecular genetic markers, particularly SNPs, associated with QTLs that confer resistance or tolerance to TBRFV, which may be recessive, and which confer leaf and / or fruit resistance or tolerance. Plants obtained by the methods and use of such molecular markers are also provided.

[0014] The present invention also provides several methods and uses of information related to these SNPs associated with QTLs conferring TBRFV resistance, in particular methods for identifying TBRFV-resistant plants and identifying additional molecular markers associated with this resistance, and methods for improving tomato production yields in TBRFV-infested environments, as well as methods for protecting tomato fields from TBRFV infestation.

[0015] definition The term "resistance" is defined by the International Seed Federation (ISF) Vegetable and Ornamental Crops Section to describe plant responses to pests or pathogens and abiotic stresses in the vegetable seed industry. Specifically, resistance refers to the ability of a plant variety to limit the growth and development of a specified pest or pathogen and / or the damage it causes compared to a susceptible plant variety under similar environmental conditions and pest or pathogen pressure. Resistant varieties may exhibit some disease symptoms or damage under heavy pest or pathogen pressure.

[0016] The term "resistant" is used herein to refer to a plant phenotype in which at least some disease symptoms remain absent when the plant is exposed to an infectious dose of a virus, thereby allowing the presence of systemic or local infection, viral proliferation, the presence of viral genome sequences in at least the cells of the plant, and / or the integration of the genome to be established under at least some culture conditions. Thus, a resistant plant is resistant to the development of symptoms but is an asymptomatic virus carrier. Viral sequences may be present or even multiply in the plant without causing disease symptoms. It should be understood that a resistant plant is infected with a virus but can generally at least moderately restrict viral proliferation and development. Furthermore, some plants may be resistant under some culture conditions and resistant under different conditions. Thus, resistance and tolerance are not mutually exclusive.

[0017] In the case of TBRFV, leaf resistance or foliar resistance refers to a plant phenotype in which disease symptoms in the leaves remain absent when the plant is exposed to an infective dose of TBRFV, although disease symptoms in the fruit may be present on infected plants.

[0018] Fruit resistance, in the case of TBRFV, refers to a plant phenotype in which disease symptoms in fruit remain absent when the plant is exposed to an infectious dose of TBRFV, although disease symptoms in leaves may be present in infected plants.

[0019] Leaf symptoms of TBRFV infection generally include mosaic, twisted leaflets, and often shoelace-like symptoms. Fruit symptoms of TBRFV infection generally include typical yellow lesions and deformed fruit. Fruit often also has "chocolate spots."

[0020] Susceptibility: the inability of a plant variety to restrict the growth and development of a particular pest or pathogen; susceptible plants exhibit adverse symptoms associated with viral infection, i.e., leaf damage and fruit damage in the case of TBRFV infection.

[0021] An S. lycopersicum plant susceptible to Tomato Brown Leaf Fruit Virus infection is, for example, the commercial cultivar Candela mentioned in the 2015 Salem et al. publication. It can also be Hazera No. 2 and Hazera No. 4 mentioned in the Examples section of the present application. All commercial tomato cultivars grown in TBRFV-infected areas have, to date, i.e., before the present invention, been susceptible to TBRFV infection.

[0022] Thus, a plant according to the invention has at least improved resistance or tolerance to Tomato Brown Leaf Fruit Virus, more particularly at least improved leaf resistance or fruit resistance to TBRFV, with respect to cv. Candela, and more generally any commercial tomato cultivar, grown in an area infected by Tomato Brown Leaf Fruit Virus.

[0023] As used herein, the term "progeny" or "offspring" refers to any plant that is produced as a descendant from asexual or sexual reproduction of one or more parent plants or their offspring. For example, progeny plants can be obtained by cloning or selfing a parent plant, or by crossing two parent plants, and can include selfing and F1 or F2 or further generations. F1 is the first generation offspring produced from parents, at least one of which is used for the first time as a trait donor, and second generation (F2) offspring or subsequent generations (F3, F4, etc.) offspring are specimens produced from selfing F1, F2, etc. Thus, F1 can be (and usually is) a hybrid resulting from the cross between two true breeding parents (true breeding is homozygous for the trait), and F2 can be (and usually is) a progeny resulting from self-pollination of the above-mentioned F1 cross.

[0024] As used herein, the terms "cross," "crossing," "cross pollination," or "cross-breeding" refer to the process in which pollen from one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.

[0025] 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 may also include non-expressed DNA segments that, for example, form recognition sequences for other proteins.

[0026] 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.

[0027] As used herein, the term "grafting" refers to the process of grafting a scion onto a rootstock. The primary motivation for grafting is to avoid damage caused by soil-borne pests and pathogens when genetic or chemical approaches to disease management are unavailable. Grafting a susceptible scion onto a resistant rootstock can provide resistant varieties without the need to induce resistance in the variety. Additionally, grafting increases tolerance to abiotic stress, increases yield, and results in more efficient water and nutrient use.

[0028] 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 genetic locus.

[0029] As used herein, the term "heterozygous" refers to the presence of different alleles (forms, genetic determinants, or sequences of a given gene) at a particular genetic locus.

[0030] As used herein, "homologous chromosomes" or "homologs" (or homologues) refer to a set of one maternal and one paternal chromosome that pair with each other during meiosis. These copies have the same genes at the same loci and at the same centromeric positions.

[0031] As used herein, the term "homozygote" refers to an individual cell or plant that has the same alleles at one or more loci on all homologous chromosomes.

[0032] As used herein, the term "homozygous" refers to the presence of identical alleles at one or more loci in homologous chromosomal segments.

[0033] As used herein, the term "hybrid" refers to an individual cell, tissue, or plant that results from a cross between parents that differ in one or more genes.

[0034] As used herein, the term "locus" (plural: "loci") refers to any genetically defined site, which may be a single position (nucleotide) or a chromosomal region. A locus may be a gene, genetic determinant, or part of a gene, or a DNA sequence, and may be occupied by different sequences. A locus may also be defined by a SNP (single nucleotide polymorphism), multiple SNPs, or two adjacent SNPs.

[0035] As used herein, the term "rootstock" is the lower part of a plant that can receive a scion in the grafting process.

[0036] As used herein, the term "scion" is the top portion of a plant that can be grafted onto a rootstock in the grafting process. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present inventors have identified three QTLs that, when present homozygously in S. lycopersicum plants, alone or in combinations such as those described elsewhere in this application, provide improved tolerance and / or resistance in the fruit and / or leaves of tomato plants that are infected or capable of being infected by Tomato Brown Leaf Fruit Virus (TBRFV).

[0038] The present inventors have identified two QTLs, namely QTL1 and QTL2, that independently or in combination confer improved resistance in the fruit of tomato plants that are or may be infected with TBRFV when present homozygously in an S. lycopersicum background, particularly on chromosomes 6 and 9. Additionally, the present inventors have identified one QTL that confers improved resistance in the leaves of tomato plants that are or may be infected with TBRFV when present homozygously in an S. lycopersicum background, particularly on chromosome 11. When present homozygously on chromosomes 6, 9, and 11, the three QTLs confer improved resistance and / or resistance in both the leaves and fruit of tomato plants that are or may be infected with TBRFV.

[0039] As demonstrated in the examples, the phenotype of the plant of the present invention is best characterized as resistance, not resistance, to TBRFV in most circumstances, i.e., resistance in the form of leaves, fruits, or both. However, under certain circumstances, the plant of the present invention exhibits resistance to TBRFV. Hereinafter, reference will be made to resistance to TBRFV. However, under certain circumstances, this phenotype encompasses a resistance phenotype.

[0040] Thus, according to a first aspect, the present invention relates to S. lycopersicum plants comprising in their genome one or two QTLs, namely QTL1 and / or QTL2, particularly on chromosomes 6 and / or 9, respectively, which, when present homozygously in an S. lycopersicum background, confer improved resistance in the fruit of tomato plants infected with Tomato Brown Leaf Fruit Virus. QTL1 is located on chromosome 6, and QTL2 is located on chromosome 9. Thus, the present invention relates to S. lycopersicum plants comprising in their genome a QTL, particularly on chromosome 6, namely QTL1, which, when present homozygously in an S. lycopersicum background, confer improved fruit resistance in tomato plants to TBRFV. The present invention also relates to S. lycopersicum plants comprising in their genome a QTL, particularly on chromosome 9, namely QTL2, which, when present homozygously in an S. lycopersicum background, confer improved fruit resistance in tomato plants to TBRFV.

[0041] According to one embodiment, the present invention further relates to an S. lycopersicum plant containing in its genome one QTL, namely QTL3, in particular on chromosome 11, which, when present homozygously in the S. lycopersicum background, confers improved leaf resistance in tomato plants infected with Tomato Brown Leaf Fruit Virus.

[0042] More specifically, the present invention is directed to plants whose genomes contain homozygous QTL1 on chromosome 6 or QTL2 on chromosome 9, or both QTLs, which independently confer fruit resistance to TBRFV. The present invention also relates to plants whose genomes contain homozygous QTL3 on chromosome 11, which confers leaf resistance to TBRFV.

[0043] According to one embodiment, the present invention relates to S. lycopersicum plants containing various combinations of QTL1, QTL2, and QTL3 in their genomes that, when present in a homozygous state in the S. lycopersicum background, confer improved resistance and / or tolerance to Tomato Brown Leaf Fruit Virus-infected fruits and leaves of tomato plants. Such combinations include QTL1 and QTL3, QTL2 and QTL3, and QTL1, QTL2, and QTL3. More preferably, such combinations include QTL1, QTL2, and QTL3. It is preferred that all QTLs are present in a homozygous state.

[0044] The present invention also relates to cells of such plants of the aforementioned embodiments and seeds comprising said QTL.

[0045] QTLs that confer improved resistance to TBRFV according to the present invention are selected from QTLs present in the seed genome of HAZTBRFVRES1. This S. lycopersicum seed sample has been deposited by Hazera Seeds Ltd. Berurim, MP Shimim 79837, Israel, in compliance with and fulfillment of the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure ("Budapest Treaty"), National collection of Industrial, Food and Marine bacteria (NCIMB) (NCIMB, Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom), under accession number 42758. This tomato seed deposit is maintained by Hazera Seeds Ltd. Berurim, MP Shimim 79837, Israel.

[0046] The QTLs conferring improved resistance to TBRFV are preferably located on chromosome 6 for QTL1, on chromosome 9 for QTL2, and on chromosome 11 for QTL3. They are more preferably located within the chromosomal interval on chromosome 6 comprising SNPTO-0005197 (SEQ ID NO: 1) and SNPTO-0145581 (SEQ ID NO: 2) for QTL1, within the chromosomal interval on chromosome 9 comprising SNPTO-0180955 (SEQ ID NO: 3) and SNPTO-0196109 (SEQ ID NO: 6) for QTL2, and within the chromosomal interval on chromosome 11 comprising SNPTO-0122252 (SEQ ID NO: 7) and SNPTO-0162427 (SEQ ID NO: 18) for QTL3.

[0047] The specific polymorphisms corresponding to the SNPs (single nucleotide polymorphisms) referred to herein, and the flanking sequences of these SNPs in the S. lycopersicum genome, are shown in the Experimental Section (see, in particular, Tables 4, 5, 6, 7, 9, and 10) and the accompanying Sequence Listing. Their locations relative to the tomato genome version 2.40 on chromosomes 6, 9, and 11 are shown in Tables 4, 6, and 9, and their flanking sequences are also shown in Tables 5, 7, and 10 and in the Sequence Listing.

[0048] In this regard, it should be noted that, by definition, a SNP refers to a single nucleotide in the genome, which is variable depending on the allele present, while the flanking nucleotides are identical. To facilitate unambiguous identification of the locations of different SNPs, their locations are shown in Tables 4, 6, and 9 with reference to the tomato genome sequence, version 2.40, and with reference to the flanking sequences identified by SEQ ID NO: 1. In the sequence associated with a particular SNP in this application, e.g., SEQ ID NO: 1 for SNPTO-0005197, only one nucleotide in the sequence actually corresponds to the polymorphism, i.e., the 61st nucleotide of SEQ ID NO: 1 corresponds to the polymorphic position of SNPTO-0005197, which can be T or C, as shown in Tables 4, 5, and 6. The flanking sequences are shown to locate the SNP in the genome but are not part of such polymorphism.

[0049] The inventors have determined that the QTLs responsible for the phenotype of interest, i.e., improved resistance of leaves and / or fruits when infected with TBRFV, are located at different loci along the above region, namely 18 SNPs: TO-0005197 (SEQ ID NO: 1) and TO-0145581 (SEQ ID NO: 2) for QTL1 on chromosome 6; TO-0180955 (SEQ ID NO: 3), TO-0196724 (SEQ ID NO: 4), TO-0145125 (SEQ ID NO: 5), and TO-0196109 (SEQ ID NO: 6) for QTL2 on chromosome 9; TO-0122252 (SEQ ID NO: 7), TO-0145125 (SEQ ID NO: 8), and TO-0196109 (SEQ ID NO: 9) for QTL3 on chromosome 11; The sequences were identified to be found in the above-mentioned chromosomal regions by identifying their presence at 18 different loci defined by TO-0144317 (SEQ ID NO: 8), TO-0142270 (SEQ ID NO: 9), TO-0142294 (SEQ ID NO: 10), TO-0142303 (SEQ ID NO: 11), TO-0142306 (SEQ ID NO: 12), TO-0182276 (SEQ ID NO: 13), TO-0181040 (SEQ ID NO: 14), TO-0123057 (SEQ ID NO: 15), TO-0125528 (SEQ ID NO: 16), TO-0162432 (SEQ ID NO: 17), and TO-0162427 (SEQ ID NO: 18).

[0050] Tomato plants according to the present invention having improved fruit resistance when infected with TBRFV have a QTL conferring said phenotype at at least one locus on chromosome 6 and / or chromosome 9. Preferably, tomato plants according to the present invention having improved fruit resistance when infected with Tomato Brown Leaf Fruit Virus have a QTL at at least one locus on chromosome 6 and at least one locus on chromosome 9. Alternatively, tomato plants of the present invention have a QTL conferring fruit resistance to TBRFV at at least one of the two loci on chromosome 6 detailed above, or have a QTL conferring fruit resistance to TBRFV at at least one of the four loci on chromosome 9 detailed above.

[0051] Tomato plants according to the invention having improved resistance of leaves when infected with TBRFV have a QTL at at least one of the loci on chromosome 11.

[0052] Tomato plants according to the present invention having improved resistance at both the leaf and fruit level when infected with Tomato Brown Leaf Fruit Virus have phenotype-conferring sequences in at least one locus on chromosome 6 and / or chromosome 9, preferably at least one locus on both chromosome 6 and chromosome 9, and at least one locus on chromosome 11.

[0053] Therefore, according to another embodiment of the present invention, the QTLs present in the genome of the plant, seed, or cell of the present invention are preferably selected from the 18 loci comprising the 18 SNPs mentioned above, namely, for QTL1 on chromosome 6, a locus comprising TO-0005197 (SEQ ID NO: 1), a locus comprising TO-0145581 (SEQ ID NO: 2), for QTL2 on chromosome 9, a locus comprising TO-0180955 (SEQ ID NO: 3), a locus comprising TO-0196724 (SEQ ID NO: 4), a locus comprising TO-0145125 (SEQ ID NO: 5), a locus comprising TO-0196109 (SEQ ID NO: 6), and for QTL3 on chromosome 11, a locus comprising TO-0122252 (SEQ ID NO: 7). , the locus containing TO-0144317 (SEQ ID NO: 8), the locus containing TO-0142270 (SEQ ID NO: 9), the locus containing TO-0142294 (SEQ ID NO: 10), the locus containing TO-0142303 (SEQ ID NO: 11), the locus containing TO-0142306 (SEQ ID NO: 12), the locus containing TO-0182276 (SEQ ID NO: 13), the locus containing TO-0181040 (SEQ ID NO: 14), the locus containing TO-0123057 (SEQ ID NO: 15), the locus containing TO-0125528 (SEQ ID NO: 16), the locus containing TO-0162432 (SEQ ID NO: 17), and the locus containing TO-0162427 (SEQ ID NO: 18).

[0054] In tomato plants according to the present invention, the fruits of which have improved resistance when infected with TBRFV, the QTLs present in the genome of the plants, seeds or cells of such tomato plants are preferably found in at least one or more of the following loci: for QTL1 on chromosome 6, the locus comprising TO-0005197, the locus comprising TO-0145581, and / or for QTL2 on chromosome 9, the locus comprising TO-0180955, the locus comprising TO-0196724, the locus comprising TO-0145125 and the locus comprising TO-0196109.

[0055] In tomato plants according to the present invention, the leaves of which have improved resistance when infected with TBRFV, the QTL present in the genome of the plant, seed, or cell of such tomato plant are preferably found at at least one or more of the following loci: for QTL3 on chromosome 11, the locus comprising TO-0122252, the locus comprising TO-0144317, the locus comprising TO-0142270, the locus comprising TO-0142294, the locus comprising TO-0142303, the locus comprising TO-0142306, the locus comprising TO-0182276, the locus comprising TO-0181040, the locus comprising TO-0123057, the locus comprising TO-0125528, the locus comprising TO-0162432, and the locus comprising TO-0162427.

[0056] For tomato plants according to the present invention having improved resistance of both leaves and fruits when infected with TBRFV, the QTL present in the genome of the plant, seed, or cell of the present invention preferably comprises at least one or more of two loci comprising SNPs on chromosome 6, namely the locus comprising TO-0005197 and the locus comprising TO-0145581, and / or at least one or more of four loci comprising SNPs on chromosome 9, namely the locus comprising TO-0180955, the locus comprising TO-0196724, the locus comprising TO-0145125, and the locus comprising TO-0196109. The SNPs are found in one or more of the following loci on chromosome 11: the locus containing TO-0122252, the locus containing TO-0144317, the locus containing TO-0142270, the locus containing TO-0142294, the locus containing TO-0142303, the locus containing TO-0142306, the locus containing TO-0182276, the locus containing TO-0181040, the locus containing TO-0123057, the locus containing TO-0125528, the locus containing TO-0162432, and the locus containing TO-0162427.

[0057] The alleles of the 18 SNPs of the present invention corresponding to the QTLs conferring TBRFV resistance are allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, and allele T of TO-0144317. The QTLs conferring resistance to TBRFV are identified by the presence of the specific alleles listed above. Therefore, the alleles of these SNPs can reflect the presence of the QTLs of the present invention.

[0058] According to a preferred embodiment of the present invention, the QTL conferring resistance to TBRFV is located on one or more chromosomal intervals bounded by the SNPs of the present invention. According to this embodiment, QTL1 is located on the chromosomal interval of chromosome 6 bounded on one side by SNPTO-0005197 and on the other side by SNPTO-0145581.

[0059] According to another embodiment, QTL2 is located on the chromosomal interval of chromosome 9 bounded on one side by SNPTO-0180955 and on the other side by SNPTO-0196109.

[0060] According to another embodiment, QTL3 is located on the chromosomal interval of chromosome 11 bounded on one side by SNP TO-0122252 and on the other side by TO-0162427. More preferred chromosomal intervals on chromosome 11 in which QTL3 is found are the interval bounded by TO-0144317 and TO-0125528, the interval bounded by TO-0142270 and TO-0162432, the interval bounded by TO-0144317 and TO-0162432, and the interval bounded by TO-0142270 and TO-0125528. A further preferred interval is the interval bounded by TO-0142270 and TO-0125528. Another preferred interval is the interval bounded by and including TO-0142294 and TO-0125528.

[0061] In this regard, it should be noted that specific locations on chromosomes can actually be defined in terms of single nucleotide polymorphisms, as long as the flanking sequences of the SNPs are defined to unambiguously locate them on the genome. The inventors used SNPs identified by their flanking sequences, which have different alleles, to identify and track the QTLs of the present invention.

[0062] A chromosomal region delimited by two SNPs X and Y refers to the section of the chromosome that is between the locations of these two SNPs and that contains said SNPs; therefore, the nucleotide sequence of this chromosomal region begins with the nucleotide corresponding to SNP X and ends with the nucleotide corresponding to SNP Y, i.e., within the meaning of the present invention, the SNPs are contained within the region that they delimit.

[0063] In the plant, seed, or cell of the present invention, the presence of a QTL conferring a desired phenotype is preferably characterized by TO-0005197 and / or TO-0145581 for QTL1 on chromosome 6, and / or TO-0180955, TO-0196724, TO-0145125, and / or TO-0196109 for QTL2 on chromosome 9, when the resistance to TBRFV is fruit resistance, and preferably characterized by TO-0122 for QTL3 on chromosome 11, when the resistance to TBRFV is leaf resistance. 252, TO-0144317, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427, and most preferably, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, and even more preferably, TO-0182276.

[0064] When homozygously present in the genome of a tomato plant according to the present invention, QTL1 and / or QTL2 independently and collectively confer fruit resistance / tolerance to TBRFV, and QTL3 confers leaf resistance / tolerance to TBRFV. When the three QTLs are homozygously present in the genome of a tomato plant according to the present invention, or when QTL1 and QTL3 or QTL2 and QTL3 are homozygously present, such a plant will have improved fruit and leaf resistance to TBRFV.

[0065] The present invention also relates to hybrid S. lycopersicum plants having an improved phenotype, which can be obtained by crossing a plant homozygously carrying one or more QTLs of the present invention with another S. lycopersicum. A plant having an improved phenotype and homozygously carrying one or more QTLs of the present invention can be a tomato plant with fruit resistance to TBRFV, in which case it can be a tomato plant homozygously carrying QTL1 and / or QTL2 and leaf resistance to TBRFV, in which case it can be a plant homozygously carrying QTL3, or a plant with both fruit and leaf resistance to TBRFV. In the latter case, the tomato plant homozygously carries QTL1 and QTL3, QTL2 and QTL3, or QTL1, QTL2, and QTL3, preferably QTL1, QTL2, and QTL3. Because the QTL of the present invention acts in a recessive manner, the hybrid S. lycopersicum plants produced by the above crossing will have leaf and / or fruit resistance to TBRFV only if the other S. lycopersicum mating partner has the QTL of the present invention. If the other S. lycopersicum mating partner lacks the QTL of the present invention, the hybrid resulting from the crossing will have the QTL of the present invention in a heterozygous manner, and the tomato plants will not have leaf and / or fruit resistance. However, these resistant / tolerant progeny will be available to those skilled in the art of breeding.

[0066] Preferably, the S. lycopersicum plant according to the invention is a commercial plant or line. Such commercial plants or lines preferably also exhibit resistance to TMV (Tomato Mosaic Virus), for example, due to the presence of the Tm-2 (allele Tm-2 or Tm-22 (also known as Tm-2a) or Tm-1 resistance gene, which also confers resistance to ToMV (Tomato Mosaic Virus). Plants according to this aspect of the invention preferably also have the following additional characteristics: a nematode resistance trait (Mi-1 or Mi-j), and Fusarium and Verticillium resistance.

[0067] Other resistances or tolerances are also contemplated by the present invention.

[0068] According to a preferred embodiment, the plants of the present invention are not resistant to pepino mosaic virus (PepMV). According to another embodiment, the tomato plants of the present invention are also resistant to PepMV.

[0069] According to yet another embodiment, the plant of the present invention is a determinate, indeterminate, or semi-indeterminate (ie, corresponding to a determinate, indeterminate, or semi-indeterminate growth habit) plant or a seed or cell thereof.

[0070] Determinate refers to tomato plants that tend to develop leaves first, then flowers, which, if pollination is successful, mature into fruit. All fruits tend to ripen on the plant at roughly the same time. Indeterminate tomatoes begin with the development of 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-determinate tomatoes are typical of determinate varieties, except that they have a phenotype between determinate and indeterminate and grow larger than determinate varieties.

[0071] According to yet another embodiment, the plants of the present invention are used as scions or rootstocks in a grafting process. Grafting is a process that has been used for many years in crops such as cucurbits, but has recently been used only in tomatoes. Grafting can be used to provide a specific level of resistance to telluric pathogens such as Phytophthora infestans or certain nematodes. Thus, grafting is intended to prevent contact between the cultivated plant or variety and infested soil. The desired variety, optionally an F1 hybrid, used as the scion or scion is grafted onto a resistant plant used as a rootstock. The resistant rootstock remains healthy and provides a normal supply of soil to the graft, isolating it from disease.

[0072] Furthermore, commercial plants of the present invention produce fruit under suitable conditions, which fruit weighs at least 25g at full maturity, preferably at least 100g at full maturity, and even more preferably at least 200g at full maturity.

[0073] As noted above, the present invention relates to S. lycopersicum plants and seeds that produce the plants that exhibit improved phenotypes.

[0074] Plants or seeds according to the invention can be progeny or offspring of plants grown from the deposited seed HAZTBRFVRES1 deposited with NCIMB under accession number NCIMB42758. Plants grown from the deposited seeds are indeed homozygous for the QTL of the invention that confers an improved phenotype, and therefore, they carry in their genome the QTL of interest on each of the homologs of chromosomes 6, 9, and 11. They can be used to transfer these sequences into other backgrounds by crossing, selfing, and / or backcrossing.

[0075] The present invention also relates to the deposited seeds of HAZTBRFVRES1 (NCIMB42758) and plants grown from one of these seeds. These seeds homozygously contain the QTL that confers the desired phenotype. Note that these seeds do not correspond to plant varieties and are not homozygous for most genes except for the QTL of the present invention. Therefore, their phenotypes are not fixed during breeding, except for the leaf and fruit resistance / tolerance of the present invention. Most of their phenotypic characteristics segregate during breeding, except for the TBRFV leaf and fruit resistance / tolerance of the present invention.

[0076] The present invention also relates to a plant or seed as defined above, i.e., a plant or seed comprising one, two, or three QTLs of interest in a homozygous or heterozygous state, which, when present in homozygous form, confer an improved phenotype, and which can be obtained by transferring the QTLs from a S. lycopersicum plant (representative seeds of which have been deposited in NCIMB accession NCIMB-42758) into another S. lycopersicum genetic background, for example, by crossing the plant with a second tomato plant parent and selecting for a plant carrying the QTLs responsible for the desired phenotype. In such a cross, QTL1, QTL2, and / or QTL3, or any combination thereof, can be transferred. Preferably, to obtain plants with fruit resistance, only QTL1, or only QTL2, or both QTL1 and QTL2 are transferred from the deposited seed HAZTBRFVRES1 (NCIMB42758); to obtain plants with leaf resistance, QTL3 is transferred from the deposited seed HAZTBRFVRES1 (NCIMB42758); to obtain plants with both fruit and leaf resistance, QTL1 and QTL3, QTL2 and QTL3, or QTL1, QTL2, and QTL3, preferably QTL1, QTL2, and QTL3, are transferred from the deposited seed HAZTBRFVRES1 (NCIMB42758).

[0077] It should be noted that the seeds or plants of the present invention may be obtained by different processes and are not exclusively obtained by essentially biological processes.

[0078] 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 fruit and / or leaf resistance to Tomato Brown Leaf Fruit Virus, wherein the mutations are present in the genome of, for example, a plant a representative sample of which has been deposited with NCIMB under accession number NCIMB42758.

[0079] In another embodiment, the present invention relates to a method for obtaining a tomato plant or seed having one or more mutations in its genome that provide the plant with resistance to Tomato Brown Leaf Fruit Virus in its fruit and / or leaves. Such a method is illustrated in Example 7 and comprises the following steps: a) treating M0 seeds of the tomato plant to be modified with a mutagen to obtain M1 seeds; b) growing plants from the M1 seeds thus obtained to obtain M1 plants; c) self-fertilizing the M plants to produce M seeds; d) Optionally, repeating steps b) and c) n times to obtain M1+n seeds.

[0080] The M1+n seeds are grown into plants that are susceptible to Tomato Brown Leaf Fruit Virus infection. Surviving plants or plants with milder TBRFV infection symptoms are propagated for one or more additional generations while continuing to select for fruit and / or leaf resistance to Tomato Brown Leaf Fruit Virus.

[0081] 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 (des), ethyleneimine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea (enu), and sodium azide.

[0082] Alternatively, mutations are induced by irradiation, for example selected from X-rays, fast neutrons, UV radiation.

[0083] In another embodiment of the present invention, the mutation is induced by genetic engineering. Such mutations include the insertion of sequences that confer TBRFV resistance to fruits and / or leaves, and the replacement of existing sequences with alternative sequences that confer TBRFV resistance or tolerance to fruits and / or leaves. Preferably, the mutation is the insertion of one or more of the above-mentioned QTL1, QTL2, and QTL3 in place of the homologous sequence in S. lycopersicum plants. Even more preferably, the mutation is the replacement of a sequence or a fragment thereof contained within SNPTO-0122252 (SEQ ID NO: 7) and SNPTO-0162427 (SEQ ID NO: 18) on chromosome 11 of the S. lycopersicum genome with a homologous sequence on chromosome 11 present in the genome of a plant whose representative sample has been deposited with NCIMB under accession number NCIMB42758, wherein the sequence or fragment thereof confers leaf resistance to TBRFV.

[0084] The genetic engineering tools that can be used include the use of all the techniques known as New Breeding Techniques, which are various new techniques developed and / or used to create new traits in plants through genetic variation, the purpose of which is targeted mutagenesis, i.e., targeted introduction of new genes or gene silencing (RdDM). Examples of such New Breeding Techniques are zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2, and ZFN-3, see U.S. Pat. No. 9,145,565), oligonucleotide-induced mutagenesis (ODM), cisgenesis and intragenesis, grafting (on GM rootstocks), reverse breeding, agro-infiltration (agro-infiltration "sensu stricto", agro-inoculation, 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), engineered meganucleases, engineered homing endonucleases, DNA-guided genome editing (Gao et al., Nature Targeted sequence modification is facilitated by the use of genetic engineering (Biotechnology (2016)) and synthetic genomics. Another name for novel breeding techniques, targeted genome editing is the application of induced DNA double-strand breaks (DSBs) at selected locations in the genome where modification is intended. Directed repair of DSBs allows targeted genome editing.Such applications can be used to generate mutations (e.g., targeted mutations or precise native gene editing) and precise insertions of genes (cisgene, intragene, or transgene). Mutation-causing applications are often identified as site-specific nuclease (SDN) technologies, such as SDN1, SDN2, and SDN3. In the case of SDN1, the outcome 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 the deletion, addition, or substitution of small nucleotides. In the case of SDN2, SDN is used to generate the targeted DSB, and a DNA repair template (a short DNA sequence identical to the targeted DSB DNA sequence except for one or a few nucleotide changes) is used to repair the DSB: the outcome is a targeted, predetermined point mutation in a desired gene of interest. For SDN3, SDN is used with a DNA repair template containing a new DNA sequence (such as a gene). The outcome of this technology is the insertion of that DNA sequence into the plant genome. The most likely application for the use of SDN3 would be the insertion of cisgenic, intragenic, or transgenic expression cassettes at selected genomic locations. A full description of each of these techniques is provided in a report entitled "New Plant Breeding Technologies - State of the Art and Prospects for Commercial Development," produced by the European Commission Joint Research Centre (JRC) Future Technologies Laboratory in 2011.

[0085] The present invention also relates in another aspect to any plant that may be obtained from the above-mentioned seeds or plants of the invention, and to plant parts of such plants, most preferably explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles, and any other plant parts, wherein said plants, explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems, petioles, and / or plant parts may be obtained from a seed or plant according to the first aspect of the invention, i.e. a seed or plant that has one, two or three QTLs in its genome in any combination, homozygous or heterozygous. These plant parts, particularly explants, scions, cuttings, seeds, fruits, roots, rootstocks, pollen, ovules, embryos, protoplasts, leaves, anthers, stems or petioles, contain QTL in their genomes that, when present in homozygous form, confer the desired phenotype, i.e., fruit and / or leaf resistance to TBRFV.

[0086] The QTLs referred to in this aspect of the invention are those defined above in the context of the plants of the invention. The different characteristics of the QTLs defined in relation to the first aspect of the invention apply mutatis mutandis to this aspect of the invention. Thus, the QTLs are preferably selected from QTLs present in the genome of the plant corresponding to the deposited material HAZTBRFVRES1 (NCIMB accession number 42758). Depending on the QTL of interest, they may be selected from the following: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele T the presence of allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427, preferably the presence of this allele or these alleles in homozygosity, i.e. TO-0000 Two alleles T in 5197, two alleles C in TO-0145581, two alleles G in TO-0180955, two alleles C in TO-0196724, two alleles G in TO-0145125, two alleles G in TO-0196109, two alleles T in TO-0122252, two alleles C in TO-0144317, two alleles T in TO-0142270, two alleles T in TO-0142294 The mutant is advantageously characterized by the presence of one allele G of TO-0142303, two alleles A of TO-0142306, two alleles G of TO-0182276, two alleles G of TO-0181040, two alleles G of TO-0123057, two alleles A of TO-0125528, two alleles C of TO-0162432, and / or two alleles T of TO-0162427.

[0087] The present invention also relates to cells of S. lycopersicum plants, which contain QTLs of the present invention in their genomes that confer a desired phenotype to the S. lycopersicum plants, and preferably these QTLs are present in a homozygous state. The QTLs are as previously defined within the scope of the present invention and are characterized by the same characteristics and preferred embodiments as previously disclosed for the plants and seeds according to the above-mentioned aspects of the present invention. The presence of these QTLs can be revealed by the techniques disclosed above and well known to the skilled reader. In particular, it can be determined whether the QTLs are present in the genome of such cells of the present invention in a homozygous or heterozygous state. They were identified as allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele G of TO-0142303, depending on the QTL of interest. , allele A of TO-0142306, allele A of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432 and / or allele T of TO-0162427, preferably by the presence of this or these alleles simultaneously on each chromosome, i.e. in homozygosity.

[0088] 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 the QTL of interest.

[0089] The present invention also relates to tissue cultures of non-regenerable or regenerable cells of the above-defined plants according to the 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 invention, which cells contain, in homozygous or heterozygous form, one, two, or three QTLs of interest in their genome, in any combination, which, when present in homozygous form, confer an improved phenotype, i.e., fruit resistance to TBRFV for QTL1 and / or QTL2, and leaf resistance to TBRFV for QTL3.

[0090] The tissue culture is preferably capable of regenerating plants having the physiological and morphological characteristics of the aforementioned tomato plants, and is 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.

[0091] The present invention also provides a protoplast of a plant as defined above, or a protoplast derived from a tissue culture as defined above, which protoplast comprises a QTL that confers an improved phenotype of the present invention.

[0092] In another aspect, the present invention also relates to the use of tomato plants of the present invention, preferably containing the QTLs of the present invention in a homozygous state, as breeding partners in breeding programs to obtain S. lycopersicum plants with improved phenotypes of the present invention. Indeed, such breeding partners carry the QTLs conferring the desired phenotypes in their genome in a homozygous state. Therefore, by crossing this plant with a tomato plant, particularly a line, it is possible to transfer one, two, or three QTLs of the present invention that confer the desired phenotypes to offspring. Thus, plants of the present invention can be used as breeding partners to transfer QTLs that confer the desired phenotypes to S. lycopersicum plants or germplasm, preferably QTLs responsible for leaf resistance. Plants or seeds heterozygous for the QTLs of interest can also be used as breeding partners, as detailed above, although phenotypic segregation can make breeding programs more complicated.

[0093] The improved phenotype of the present invention is resistance to TBRFV, particularly leaf resistance or fruit resistance, or a combination of fruit and leaf resistance.

[0094] The introgressed QTLs may be advantageously incorporated into varieties containing other desired genetic traits such as disease resistance, early fruit maturity, drought tolerance, fruit shape, etc.

[0095] The present invention also relates to the use of the same with plants or seeds of HAZTBRFVRES1 deposited with NCIMB under accession number NCIMB 42758. This plant is also suitable as an introgression partner in breeding programs aimed at imparting desired phenotypes to S. lycopersicum plants or germplasm.

[0096] In such breeding programs, selection of progeny exhibiting a desired phenotype or carrying a QTL associated with a desired phenotype can be advantageously carried out based on alleles of SNP markers, particularly the SNP markers of the present invention.

[0097] The progeny of the plant preferably comprises the allele T of TO-0005197 and / or the allele C of TO-0145581 for the presence of QTL1 on chromosome 6, the allele G of TO-0180955, the allele C of TO-0196724, the allele G of TO-0145125 and / or the allele G of TO-0196109 for the presence of QTL2 on chromosome 9, the allele T of TO-0122252 for the presence of QTL3 on chromosome 11, the allele T of TO-0122252 for the presence of QTL4 on chromosome 12, the allele C of TO-0145581 and / or the allele G of TO-0145581 for the presence of QTL5 on chromosome 13, the allele C of TO-0145125 and / or the allele G of TO-0196109 for the presence of QTL6 on chromosome 14, the allele T of TO-0122252 for the presence of QTL7 on chromosome 15, the allele C of TO-0145125 and / or the allele G of TO-0196109 for the presence of QTL8 on chromosome 16, the allele T of TO-0122252 for the presence of QTL9 on chromosome 17, the allele C of TO-0145125 and / or the allele G of TO-0196109 for the presence of QTL10 on chromosome 18, the allele T of TO-0122252 for the presence of QTL11 on chromosome 19, the allele C of TO-0145125 and / or the allele G of TO-0196109 for the presence of QTL12 on chromosome 20, the allele C Select for the presence of the C allele of 44317, the T allele of TO-0142270, the G allele of TO-0142294, the A allele of TO-0142303, the A allele of TO-0142306, the G allele of TO-0182276, the G allele of TO-0181040, the G allele of TO-0123057, the A allele of TO-0125528, the C allele of TO-0162432, and / or the T allele of TO-0162427. Due to the recessive nature of the QTL, plant progeny are preferably selected for the presence of the same allele on both homologs of each chromosome.

[0098] Alternatively, selection can be performed based on the presence of any one of the 18 SNP alleles of the present invention, or a combination of these alleles, associated with an improved phenotype. According to such an embodiment, selection can be performed for the presence of at least one SNP allele for QTL1, or at least one SNP allele for QTL2, or at least one SNP allele for QTL3, or the simultaneous presence of at least one SNP allele for QTL1, or at least one SNP allele for QTL2, and / or at least one SNP allele for QTL3, depending on the QTL combination achieved (QTL1 and QTL2, QTL1 and QTL3, QTL1, QTL2, and QTL3, preferably QTL1, QTL2, and QTL3). Such selection is performed for the presence of the alleles of interest in the genetic material sample of the selected plant. The presence of these alleles actually confirms the presence of the QTL of the present invention at the locus defined by the SNPs. Furthermore, in addition to point mutations or recombination events, it is conceivable that at least one or two of these alleles will be lost, with the remaining chromosomal fragment carrying the QTL of interest still conferring the phenotype of interest.

[0099] Thus, plants according to the invention or plants grown from the seeds deposited under accession number NCIMB42758 are particularly valuable in marker-assisted selection to obtain commercial tomato lines and varieties with the improved phenotypes of the invention.

[0100] The invention also relates to the use of such plants in programs aimed at identifying, sequencing, and / or cloning gene sequences that confer desired phenotypes.

[0101] 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 characterization of QTLs that confer a phenotype of interest.

[0102] The present invention also relates to a method for identifying, detecting, and / or selecting S. lycopersicum plants having a QTL of the present invention found in the genome of seeds of HAZTBRFVRES1 (NCIMB Accession No. 42758), said QTL conferring an improved phenotype of resistance and / or tolerance to Tomato Brown Leaf Fruit Virus relative to a corresponding plant lacking said sequence, the method comprising detecting in a sample of genetic material of the identified and / or selected plants the T allele of TO-0005197, the C allele of TO-0145581, the G allele of TO-0180955, the H allele of TO-0196724, the I allele of TO-0196724, the I allele of TO-0145581, the I allele of TO-0145581, the I allele of TO-0180955, the I allele of TO-0196724 ... C, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, or allele T of TO-0162427.

[0103] The present invention also relates to a QTL that confers resistance to TBRFV only when present in homozygosity, and is comprised of the following alleles: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele T The present invention also relates to a method for detecting or selecting S. lycopersicum plants having at least one of the A allele of TO-0142303, the A allele of TO-0142306, the G allele of TO-0182276, the G allele of TO-0181040, the G allele of TO-0123057, the A allele of TO-0125528, the C allele of TO-0162432, or the T allele of TO-0162427, wherein the detection or selection is carried out under conditions of TBRFV infection, including inoculation of the plant being tested with TBRFV. According to a preferred embodiment, the method is for detecting or selecting S. lycopersicum plants having the G allele of TO-0182276, which has a QTL that confers leaf resistance or tolerance to TBRFV only when present in homozygosity, wherein the detection or selection is carried out under conditions of TBRFV infection, including inoculation of the leaves of the plant being tested with TBRFV.

[0104] The method is particularly adapted for breeding programs using HAZTBRFVRES1 (NCIMB Accession No. 42758) as the initial parent or its progeny, which contains a QTL of the invention that confers resistance, and detection and / or selection is carried out under conditions including infestation with TBRFV, and the transferred sequence confers resistance or tolerance to TBRFV, and is mediated by the following markers: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele G of TO-0122224, allele G of TO-0122226, allele G of TO-0122228, allele G of TO-0122229 ... more preferably, at least one of the allele T of TO-0123057, allele C of TO-0125528, allele C of TO-0162432, or allele T of TO-0162427, more preferably an allele of chromosome 11, and even more preferably an allele G of TO-0182276.

[0105] Furthermore, the present invention relates to a method for detecting and / or selecting S. lycopersicum plants having at least one of the QTLs of the present invention that confers an improved phenotype, based on detection of an allele of at least one SNP selected from 18 SNPs.

[0106] Preferably, plants having a QTL of the present invention have the following markers: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele G of TO-0144317 Plants are selected for the presence of QTL1 if at least one, and preferably at least two, three, four, five or more, or all of the following alleles are detected in a sample of genetic material of the selected plant: allele A of TO-0005197, allele C of TO-0145581, allele T of TO-0005197, allele C of TO-0145581, allele A of TO-0005197, allele C of TO-0145581, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427. Plants are selected for the presence of QTL2 if at least one, two, or three, or four of the following alleles are detected: allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109. Plants are selected for the presence of QTL3 if at least one, or two, or three, or four, or five or more of the following alleles are detected: allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and allele T of TO-0162427.

[0107] Plants are also detected if they have a combination of alleles of the invention. Plants are selected for the presence of QTL1 and QTL2 if at least one or both of the following alleles are detected: allele T of TO-0005197, allele C of TO-0145581, and at least one, two, three, or four of the following alleles are detected: allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109. Plants were identified if at least one or both of the following alleles were detected: allele T of TO-0005197, allele C of TO-0145581, and alleles T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306. Select for the presence of QTL1 and QTL3 if at least one, or two, or three, or four, or five or more of gene A, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and allele T of TO-0162427 are detected.Plants were categorized as chromosome-specific if at least one, two, three, or four of the following alleles were detected: allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, and allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele T of TO-0142295, allele C of TO-0142296, allele T of TO-0142297, allele G of TO-0142299. Select for the presence of QTL2 and QTL3 if at least one, or two, or three, or four, or five or more of allele A of 42303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and allele T of TO-0162427 are detected. The plant is characterized if at least one or both of the following alleles are detected: allele T of TO-0005197, allele C of TO-0145581, and at least one, two, three, or four of the following alleles: allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, and allele T of TO-0122252, allele C of TO-0144317, allele C of TO-0142270. Select for the presence of QTL1, QTL2, and QTL3 if at least one, or two, or three, or four, or five or more of: T, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and allele T of TO-0162427 are detected.

[0108] In all of the foregoing aspects of the invention, the preferred resistance to TBRFV is leaf resistance. Preferred SNPs for all aspects of the invention are TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, and TO-0125528, and even more preferably TO-0182276.

[0109] The present invention further relates to a method for detecting and / or selecting S. lycopersicum plants having at least one of the QTLs of the present invention that confers an improved phenotype, based on the detection of any molecular marker that reveals the presence of the above-mentioned QTL. Indeed, the QTLs of the present invention have been identified by the present inventors, and the identification and use of molecular markers in addition to the 18 SNPs of the present invention can be performed by those skilled in the art. Although the QTLs themselves are continuously characterized by the presence of at least one of the 18 SNPs of the present invention, they may also be identified by the use of different alternative markers. The method and use of any such molecular marker to identify the QTLs of the present invention in the tomato genome, wherein the QTLs confer leaf and / or fruit resistance to TBRFV relative to corresponding plants lacking the QTL, and the QTLs are identified by the following SNPs: TO-0005197, TO-0145581, TO-0180955, TO-0196724, TO-0145125, TO-01961 Included in the present invention are methods and uses characterized by the presence of at least one of TO-09, TO-0122252, TO-0144317, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427.

[0110] Methods and uses of any such molecular markers for identifying QTLs of the invention in the tomato genome, wherein the QTLs confer leaf and / or fruit resistance to TBRFV relative to corresponding plants lacking said QTLs, said QTLs being mediated by the following SNP alleles: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele G of TO-0122252. Also included are methods and uses characterized by the presence of at least one of gene T, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and allele T of TO-0162427.

[0111] According to yet another aspect, the present invention also relates to a method or process for the production of S. lycopersicum plants, particularly commercial plants and inbred parent lines, having a desired phenotype. Indeed, the present invention also aims to transfer one, two, or three QTLs of the present invention that confer a defined, improved phenotype to other tomato varieties or other species or inbred parent lines, useful for producing new types and varieties of tomatoes.

[0112] A method or process for the production of plants having these characteristics may include the following steps: a) crossing a plant grown from the deposited seed NCIMB42758 or its progeny, which carries QTL1, QTL2, and / or QTL3 conferring TBRFV resistance, with an initial S. lycopersicum plant, preferably lacking said QTL; b) selecting, from the progeny thus obtained, one plant that has one, two or three of the QTL1, QTL2 and / or QTL3 of the invention; c) Optionally, self-pollinating the plants obtained in step b) once or several times and selecting from the progeny thus obtained plants that have resistance to TBRFV, which may be fruit resistance, leaf resistance, or both, depending on the QTL present in the progeny plants.

[0113] Alternatively, the method or process may comprise the following step instead of step a): a1) crossing a plant corresponding to the deposited seed (NCIMB42758) or its progeny, carrying QTL1, QTL2, and / or QTL3 conferring TBRFV resistance with a first S. lycopersicum plant, preferably lacking said QTLs; a2) Propagating the F1 hybrids by selfing to generate an F2 population.

[0114] In the above methods or processes, SNP markers are preferably used in steps b) and / or c) to select plants having sequences that confer the desired tolerance and / or resistance phenotype.

[0115] The SNP markers are preferably one or more of the 18 SNP markers of the present invention (including all combinations thereof mentioned elsewhere in this application).

[0116] According to a preferred embodiment, the selection of plants having leaf resistance to Tomato Brown Leaf Fruit Virus is carried out based on TO-0182276 or on at least one of TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528.

[0117] It is understood that by selecting a plant based on alleles of one or more SNPs, the plant is selected as having resistance to TBRFV, which may be fruit resistance / resistance, leaf resistance / resistance, or both, for the initial plant, provided that the allele of the SNP is the allele corresponding to the allele of the HAZTBRFVRES1 parent for that SNP and is not the allele of the initial S. lycopersicum plant. For example, allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele G of TO-0142296, allele T of TO-0142298, allele G of TO-0142299, allele T of TO-014229 ... G of TO-0142299, allele T of TO-0142299, allele G of TO-0142299, allele G of TO- Plants may be selected as having the improved phenotype of the present invention if allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427 is detected.

[0118] Preferably, the S. lycopersicum plants of step a) are elite lines used to obtain plants with commercially desirable traits or desirable agronomic traits.

[0119] The method or process defined above may advantageously comprise a backcrossing step, preferably after step c), in order to obtain a plant having all the characteristic features of a S. lycopersicum plant. Consequently, the method or process for the production of a plant having these characteristics may also comprise the following additional steps: d) backcrossing the resistant plants selected in step b) or c) with S. lycopersicum plants; e) Selecting, for the initial plants, plants that have one, two or three of QTL1, QTL2 and / or QTL3 of the present invention.

[0120] 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 that carries the phenotype-conferring QTL, preferably carrying these sequences homozygously.

[0121] In step e), the SNP markers can be used to select plants with leaf resistance and / or resistance to Tomato Brown Leaf Fruit Virus from the initial plants. As explained in the previous section, the SNP markers are SNP markers of the present invention.

[0122] According to a preferred embodiment, the method or process of the present invention comprises, for at least one of the selection steps, i.e. b), c), and / or e), the selection being carried out by selecting the following alleles: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele G of TO-0144317 The detection of at least one of the following genes is based on detection of at least one of the following: allele C of TO-0142270, allele T of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427.

[0123] It should be noted that when selecting plants with an improved phenotype and homozygous for one or more of the QTLs conferring this phenotype, selection is based on the presence of one or more SNPs of the invention in combination with the absence of an allele representing the QTL, i.e., the allele HAZTBRFVRES1 parent, in combination with the absence of an allele representing the recurrent S. lycopersicum parent.

[0124] The plants selected in step e) are preferably commercial plants, in particular plants that have fruits weighing at least 25 g, at least 100 g, or at least 200 g at full maturity under normal culture conditions.

[0125] Preferably, steps d) and e) are repeated at least two times, preferably three times, not necessarily on the same S. lycopersicum plant.Said S. lycopersicum plant is preferably a breeding line.

[0126] At each selection step of the process disclosed above, one can further select for resistance to nematode traits or resistance to ToMV.

[0127] The self-pollination and backcrossing steps can be performed in any order and can be interleaved, for example, backcrossing can be performed before or after one or more self-pollinations, and self-pollination before or after one or more backcrosses can be contemplated.

[0128] Selection of progeny with the desired improved phenotype can also be based on a comparison of Tomato Brown Leaf Fruit Virus resistance from the S. lycopersicum parent, particularly according to the protocols disclosed in the Examples, where the resistance / resistance tested can be either fruit resistance / resistance, or leaf resistance / resistance, or both.

[0129] The method used for allele detection can be based on any technique that allows for the distinction between two different alleles of a SNP on a particular chromosome.

[0130] The present invention also relates to a plant obtained or obtainable by such a method, which plant is in fact a S. lycopersicum plant having an improved phenotype according to the first aspect of the invention.

[0131] The present invention also relates to a method for obtaining, from a first S. lycopersicum plant, a commercial tomato plant or an inbred parent line thereof having a desired improved phenotype corresponding to fruit and / or leaf resistance and / or resistance to Tomato Brown Leaf Fruit Virus, the method comprising the steps of: a) backcrossing a plant obtained by germinating the deposited seed HAZTBRFVRES1 NCIMB Accession No. 42758 or its progeny, which has QTL1, QTL2, and / or QTL3 conferring TBRFV resistance, with a commercial S. lycopersicum plant; b) Selecting plants that have one, two, or three of QTL1, QTL2, and / or QTL3 of the present invention.

[0132] Preferably, selection is based on one or more of the 18 SNPs of the invention, as detailed for other methods of the invention.

[0133] In all methods and processes of the present invention, the initial S. lycopersicum plant is determinate, indeterminate, or semi-determinate.

[0134] As previously disclosed, tomato plants according to the present invention are preferably resistant to tomato mosaic virus, nematodes, and also to Fusarium and Verticillium. To obtain such plants in the processes and methods of the present invention, the S. lycopersicum parents used in the breeding scheme preferably have sequences that confer resistance to tomato mosaic virus, nematodes, and Fusarium and Verticillium, and a selection step is carried out to select for plants that have these resistance sequences in addition to the QTL that confer the improved phenotype of the present invention.

[0135] The present invention also relates to S. lycopersicum plants and seeds obtainable by any of the methods and processes disclosed above. 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 stimulators, or products that stabilize the pH of the substrate and its immediate surroundings, or nutrients.

[0136] They may also be products for protecting young plants from agents unfavorable to their growth, including viruses and pathogenic microorganisms, such as fungicides, bactericides, nematicides, insecticides, or herbicides, which act by contact, ingestion, or gas diffusion. They may be, for example, any suitable essential oil, such as thyme extract. All of these products enhance the plant's resistance response and / or disinfect or condition the plant's environment. They may also be living biological materials, such as non-pathogenic microorganisms, such as at least one fungus, bacterium, or virus, if necessary, along with a medium to ensure their viability, which stimulate plant growth or protect against pathogens. For example, Pseudomonas, Bacillus, Trichoderma, Clonostachys, Fusarium, Rhizoctonia, etc.

[0137] In all of the above methods and processes, identification of plants homozygously carrying the QTL responsible for fruit and / or leaf resistance to TBRFV can be achieved not only by detecting at least one of the alleles associated with each QTL, but also by combining it with the absence of other allelic forms of the SNPs of the present invention. Thus, identification of plants homozygously carrying QTL1 of the present invention is based on the identification of the T allele of TO-0005197 and / or the C allele of TO-0145581, and the absence of the C allele of TO-0005197 and the T allele of TO-0145581. Similarly, identification of plants homozygous for QTL2 of the present invention is based on the identification of allele G of TO-0180955 and / or allele C of TO-0196724 and / or allele G of TO-0145125 and / or allele G of TO-0196109, and the absence of allele A of TO-0180955, allele T of TO-0196724, allele A of TO-0145125, and allele T of TO-0196109.Similarly, identification of plants homozygous for a QTL3 of the present invention can be performed by identifying the T allele of TO-0122252, and / or the C allele of TO-0144317, and / or the T allele of TO-0142270, and / or the G allele of TO-0142294, and / or the A allele of TO-0142303, and / or the A allele of TO-0142306, and / or the G allele of TO-0182276, and / or the G allele of TO-0181040, and / or the G allele of TO-0123057, and / or the A allele of TO-0125528, and and / or allele C of TO-0162432, and / or allele T of TO-0162427, and the absence of allele A of TO-0122252, allele T of TO-0144317, allele C of TO-0142270, allele A of TO-0142294, allele C of TO-0142303, allele G of TO-0142306, allele A of TO-0182276, allele A of TO-0181040, allele T of TO-0123057, allele G of TO-0125528, allele T of TO-0162432, and allele C of TO-0162427.

[0138] The present invention also relates to the use of the information provided herein by the inventors, i.e., the disclosure of the existence of three QTLs present in the deposited seeds of HAZTBRFVRES1 that confer improved phenotypes to S. lycopersicum plants, and the molecular markers associated with these QTLs. This knowledge can be used, inter alia, to precisely map the QTLs, define their sequences, identify tomato plants containing QTLs that confer improved phenotypes, and identify additional or alternative markers associated with these QTLs. Such additional markers are characterized by their location, i.e., their location near the 18 markers disclosed in the present invention, preferably derived from the 12 SNPs on chromosome 11, and by their association with the phenotype of interest revealed by the present invention, i.e., leaf resistance, fruit resistance, or both, to TBRFV.

[0139] By association, or genetic association, and more specifically genetic linkage, it is understood that a genetic polymorphism of a marker (i.e., a particular allele of a SNP marker) and a phenotype of interest co-occur, i.e., are inherited together more frequently than would be expected by chance occurrence, i.e., there is a non-random association of the genetic sequences responsible for the alleles and phenotype as a result of their proximity on the same chromosome.

[0140] The molecular markers of the present invention, any one of the 18 markers disclosed above or the alternative markers, are preferably inherited with the desired phenotype in more than 90% of meiotic divisions, preferably more than 95%, 96%, 98%, or 99% of meiotic divisions.

[0141] Thus, the present invention relates to the use of one or more molecular markers for fine mapping or identifying QTL in the tomato genome, said QTL which, when present in homozygous form, confers the improved phenotype of the invention to S. lycopersicum plants, said one or more markers being located in the following chromosomal regions: the chromosomal region bounded on chromosome 6 by TO-0005197 and TO-015581, the chromosomal region bounded on chromosome 9 by TO-0180955 and TO-0196109, the chromosomal region bounded on chromosome 11 by TO-0122252 and TO-0162427 or TO-0142270 and TO-0125528. The SNP markers are located in one of the truncated chromosomal regions or are located less than 2 megabase units from the locus of one of the 18 SNP markers of the present invention, namely, TO-0005197, TO-015581, TO-0180955, TO-0196724, TO-0145125, TO-0196109, TO-0122252, TO-0144317, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427.

[0142] The improved phenotype is resistance to TBRFV, either fruit or foliar resistance.

[0143] More specifically, for fine mapping or identification of QTL conferring fruit resistance, the one or more markers are located in the chromosomal region of chromosomes 6 and 9 defined above, or less than 2 megabases from the TO-0005197, TO-015581, TO-0180955, TO-0196724, TO-0145125, or TO-0196109 locus. More specifically, for fine mapping or identification of QTL conferring leaf resistance, the one or more markers described above are located in the chromosomal region of chromosome 11 defined above or are located less than 2 megabases from the locus of TO-0122252, TO-0144317, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427.

[0144] According to a preferred embodiment, the one or more markers are located in the chromosomal region bounded by TO-0122252 and TO-0162427, or TO-0144317 and TO-0125528, or TO-0142270 and TO-0162432, or TO-0144317 and TO-0162432, or TO-0142270 and TO-0125528.

[0145] The one or more molecular markers further preferably have a p-value of 0.05 or less and are selected from the following SNP alleles: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, allele G of TO-0196109, allele T of TO-0122252, allele C of TO-0144317, allele G of TO-01 associated with at least one of the following: allele T of TO-014270, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427.

[0146] The molecular marker is preferably a SNP marker, which is more preferably less than 1 megabase from the locus of at least one of the 18 SNPs of the present invention.

[0147] The QTL is found in the deposited seed NCIMB42758.

[0148] The p-value is preferably less than 0.01.

[0149] The present invention also relates to QTLs TO-0005197, TO-015581, TO-0180955, TO-0196724, TO-0145125, TO-0196109, TO-0122252 ... The present invention also relates to the use of at least one of the following SNP markers for identifying a surrogate molecular marker associated with said QTL: TO-0005197, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427, wherein said surrogate molecular marker is selected from the group consisting of TO-0005197, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427. and TO-015581 on chromosome 6, the chromosomal region bounded by TO-0180955 and TO-0196109 on chromosome 9, the chromosomal region bounded by TO-0122252 and TO-0162427 or TO-0142270 and TO-0125528 on chromosome 11, or the chromosomal region bounded by the 18 SNP markers of the present invention, namely, TO-0005197, TO-015581, T Located less than 2 megabases from the loci O-0180955, TO-0196724, TO-0145125, TO-0196109, TO-0122252, TO-0144317, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427.

[0150] According to a preferred embodiment, said surrogate markers are in the chromosomal region bounded by TO-0122252 and TO-0162427, or TO-0144317 and TO-0125528, or TO-0142270 and TO-0162432, or TO-0144317 and TO-0162432, or TO-0142270 and TO-0125528.

[0151] Preferably, the surrogate molecular marker is associated with the above QTL with a p-value of 0.05 or less, preferably less than 0.01. The QTL is found in deposited seed NCIMB42758.

[0152] The present invention also relates to a method for identifying a molecular marker associated with a QTL that, when present in a homozygous state, confers fruit resistance to TBRFV, the method comprising identifying a molecular marker that is located in a chromosomal region bounded on chromosome 6 by SNP markers TO-0005197 and TO-015581, or in a chromosomal region bounded on chromosome 9 by SNP markers TO-0180955 and TO-0196109, or that is less than 2 megabase units from at least one of the loci SNP markers TO-0005197, TO-015581, TO-0180955, TO-0196724, TO-0145125, and TO-0196109, and determining whether the molecular marker is associated with or linked to fruit resistance to TBRFV in a segregating population derived from plants exhibiting the improved phenotype. The population preferably originates from plants grown from the fruit resistant deposited seed of the present invention, NCIMB42758, or its progeny.

[0153] The above-mentioned QTL on chromosomes 6 and 9 that confers fruit resistance according to the present invention is a QTL present in HAZTBRFVRES1 (NCIMB42758).

[0154] Genetic association or genetic linkage is as defined above. Preferably, the association or linkage has a p-value of less than 0.05, most preferably a p-value of less than 0.01 or even less.

[0155] The molecular marker and the resistance phenotype are preferably inherited together in more than 90% of meiotic divisions, preferably more than 95%.

[0156] The present invention also provides a method for identifying molecular markers associated with QTLs that, when present in homozygosity, confer leaf resistance to TBRFV, the QTLs being located in the chromosomal region bounded on chromosome 11 by SNP markers TO-0122252 and TO-0162427 or by TO-0142270 and TO-0125528, or in the region bounded on chromosome 11 by SNP markers TO-0122252, TO-0144317, TO-0142270, TO-0142294, TO-0142295, TO-0142296, TO-0142297, TO-0142298, TO-014229 ... The present invention also relates to a method for identifying a molecular marker that is less than 2 megabases from at least one of the loci selected from the group consisting of TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427, and determining whether the molecular marker is associated with or linked to leaf resistance to TBRFV in a segregating population derived from plants exhibiting the improved phenotype. The population is preferably derived from plants grown from the deposited seed NCIMB42758 or its progeny that exhibit the leaf resistance of the present invention.

[0157] The above-mentioned QTL on chromosome 11 that confers leaf resistance according to the present invention is a QTL present in HAZTBRFVRES1 (NCIMB42758).

[0158] Molecular markers according to this aspect of the invention are most preferably SNP markers. They are more preferably less than 1 megabase from the locus of at least one of the 18 SNPs of the invention.

[0159] The present invention also relates to the use of molecular markers for identifying or selecting tomato plants that contain in their genome a QTL that, when present in homozygous form, confers fruit resistance to TBRFV in S. lycopersicum plants, wherein the markers are located in the chromosomal region delimited on chromosome 6 by SNP markers TO-0005197 and TO-015581, or in the chromosomal region delimited on chromosome 9 by SNP markers TO-0180955 and TO-0196109, or in the chromosomal region delimited on chromosome 9 by SNP markers TO-0005197, TO-015581, TO-0180955 and TO-0196109, and the molecular marker is associated with at least one of the following SNP alleles with a p-value of 0.05 or less, preferably 0.01 or less, of the following SNP alleles: allele T of TO-0005197, allele C of TO-0145581, allele G of TO-0180955, allele C of TO-0196724, allele G of TO-0145125, and allele G of TO-0196109. When the QTL is homozygous, it confers fruit resistance to the TBRFV phenotype.

[0160] The present invention also relates to the use of molecular markers for identifying or selecting tomato plants that contain a QTL in their genome that, when present in homozygous form, confers leaf resistance to TBRFV to S. lycopersicum plants, wherein the marker is located in a chromosomal region bounded on chromosome 11 by SNP markers TO-0122252 and TO-0162427, or is located in a chromosomal region bounded on chromosome 11 by at least one of SNP markers TO-0122252, TO-0144317, TO-0142270, TO-0142294, TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, and TO-0162427. and the molecular markers are associated with at least one of the following SNP alleles with a p-value of 0.05 or less, preferably 0.01 or less, of the following SNP alleles: allele T of TO-0122252, allele C of TO-0144317, allele T of TO-0142270, allele G of TO-0142294, allele A of TO-0142303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and / or allele T of TO-0162427. When present in a homozygous state, the QTL confers leaf resistance to the TBRFV phenotype.

[0161] The molecular markers used in this embodiment can be obtained by the methods for identifying additional or alternative molecular markers disclosed above. The molecular markers are preferably SNP markers. They are more preferably located less than 1 megabase from the locus of at least one of the 18 SNPs of the present invention.

[0162] According to yet another aspect, the present invention also relates to a method for analyzing the genotype of a plant, preferably a S. lycopersicum plant or tomato germplasm, for the presence of at least one genetic marker associated with resistance or tolerance to TBRFV infection, the method comprising determining or detecting in the genome of the plant to be tested a nucleic acid comprising at least one of the markers of the present invention or comprising at least one of the surrogate molecular markers disclosed above. Preferably, the method comprises determining or detecting in the genome of the plant to be tested a nucleic acid comprising at least one of the markers of the present invention or comprising at least one of the surrogate molecular markers disclosed above. Preferably, the method comprises determining in the genome of the plant of interest to be tested a nucleic acid comprising at least one of the markers of the present invention or comprising at least one of the surrogate molecular markers disclosed above. The method includes identifying specific sequences associated with resistance / tolerance to TBRFV in a nucleic acid comprising at least one of TO-0142303, TO-0142306, TO-0182276, TO-0181040, TO-0123057, TO-0125528, TO-0162432, TO-0162427, and / or TO-0162427. More preferably, the method comprises detecting, in a sample of the plant being tested, a specific sequence associated with resistance to TBRFV in a nucleic acid comprising the T allele of TO-0122252, the C allele of TO-0144317, the T allele of TO-0142270, the G allele of TO-0142294, the A allele of TO-0142303, the A allele of TO-0142306, the G allele of TO-0182276, the G allele of TO-0181040, the G allele of TO-0123057, the A allele of TO-0125528, the C allele of TO-0162432, and / or the T allele of TO-0162427.

[0163] According to a most preferred embodiment of the method, the method comprises detecting the presence of a nucleic acid comprising the G allele of TO-0182276 in a test plant.

[0164] Detection of the specific allele of a SNP can be performed by any method known to the skilled reader.

[0165] Given the ability of the resistant plants of the present invention to limit damage caused by TBRFV infection, the plants are advantageously grown in environments infested with TBRFV or environments potentially infested or infected with TBRFV. Under these conditions, the resistant or tolerant plants of the present invention produce tomatoes that are more marketable than susceptible plants. Therefore, the present invention also relates to a method for improving the yield of tomato plants in a TBRFV-infected environment, comprising growing a tomato plant defined by the above-described embodiment of the present invention, the tomato plant having homozygous QTLs on chromosome 6, chromosome 9, and / or chromosome 11 in its genome that confer resistance or tolerance to TBRFV to the plant. Preferably, the method includes a first step of selecting or sorting tomato plants that are homozygous for one or more QTLs of interest. The method can also be defined as a method for improving the productivity of a tomato field, alley, or greenhouse.

[0166] According to one embodiment, the method comprises growing a tomato plant comprising a QTL3 on chromosome 11 as defined above that confers leaf resistance to TBRFV.

[0167] The present invention also relates to a method for reducing tomato production losses in conditions of TBRFV infestation or infection, comprising growing a tomato plant as defined above.

[0168] These methods are particularly valuable for populations of tomato plants in the field, alley, or greenhouse.

[0169] Alternatively, the method for improving yield or reducing losses in tomato production may comprise a first step of identifying a tomato plant that is resistant / tolerant to TBRFV and contains in its genome a QTL on chromosome 6, 9, and / or 11 that confers resistance or tolerance to TBRFV on the plant, and then growing the tolerant or resistant plant in an environment that is infested or likely to be infested with the virus. Preferably, the plant contains a QTL on chromosome 11 defined according to the present invention that, when present in homozygous form, confers leaf resistance to TBRFV. According to a preferred embodiment, the plant identified in the first step comprises the G allele of TO-0182276.

[0170] Resistant plants of the present invention can also limit the proliferation of TBRFV, thus limiting further plant infection and viral proliferation. Accordingly, the present invention also relates to methods for protecting fields, alleys, or greenhouses, or any other type of cultivated land, from TBRFV infestation, or at least limiting the level of TBRFV infestation in said fields, alleys, or greenhouses, or limiting the spread of TBRFV in fields, alleys, greenhouses, particularly tomato fields. Such methods preferably include growing resistant or tolerant plants of the present invention, i.e., plants whose genomes contain homozygous QTLs on chromosomes 6, 9, and / or 11 that confer resistance or tolerance to TBRFV to the plants. The plants of the present invention used preferably contain QTL3 on chromosome 11, and more preferably, the plants exhibit the G allele of TO-0182276.

[0171] Preferably, the method comprises a first step of selecting or sorting tomato plants that are homozygous for a QTL of interest, in particular QTL3 on chromosome 11.

[0172] The present invention also relates to the use of a plant resistant or tolerant to TBRFV to control TBRFV infestation or infection in fields, walkways, greenhouses, or other cultivated areas. Such plants are plants of the present invention that contain at least one of QTL1, QTL2, and / or QTL3 on chromosomes 6, 9, and 11, respectively, as defined above, in their genome in a homozygous state. Therefore, according to this use, the plants of the present invention are used to protect fields, walkways, or greenhouses from TBRFV infestation. The plants of the present invention used preferably contain QTL3 on chromosome 11, and more preferably, they exhibit the G allele of TO-0182276. [Brief explanation of the drawings]

[0173] [Figure 1] Figure 1 shows a p-value plot of QTLs associated with TBRFV resistance in fruit based on the HAZ1 x HAZ2 F2 population. This figure is a Manhattan plot showing the mapping results of a biparental mapping population (HAZ1 x HAZ2, see Example 4) for fruit resistance and / or tolerance to Tomato Brown Leaf Fruit Virus. The vertical axis (y-axis) shows -loglO(p-value), and the horizontal axis (x-axis) represents all SNPs by their position per chromosome along the physical map (physical distance in bp). [Figure 2] Figure 2 shows a p-value plot of QTLs associated with leaf TBRFV resistance based on the HAZ1 x HAZ2 F2 population. This figure is a Manhattan plot showing the mapping results of a biparental mapping population (HAZ1 x HAZ2, see Example 4) for leaf tolerance and / or resistance to Tomato Brown Leaf Fruit Virus. The vertical axis (y-axis) shows -loglO(p-value), and the horizontal axis (x-axis) represents all SNPs by their position per chromosome along the physical map (physical distance in bp). [Figure 3]Figure 3 shows a p-value plot of QTLs associated with leaf TBRFV resistance based on the HAZ3 x HAZ4 F2 population. This figure is a Manhattan plot showing mapping results for a biparental mapping population (HAZ3 x HAZ4, see Example 6) for leaf tolerance and / or resistance to Tomato Brown Leaf Fruit Virus. The vertical axis (y-axis) shows -loglO(p-value), and the horizontal axis (x-axis) represents all SNPs by their position per chromosome along the physical map (physical distance in bp). [Example]

[0174] Example 1: Collection and identification of Tomato Brown Leaf Fruit Virus: We created a collection of different isolates infected with Tomato Brown Curl Fruit Virus from different production regions in Israel (North, Central, and South Israel): seven different isolates were collected and analyzed according to the protocol described by Salem et al. Sequence comparison with the Jordanian Tomato Brown Curl Fruit Virus showed that all Israeli isolates were identical to the Jordanian isolates, confirming that the same virus is present in both countries.

[0175] Example 2: Identification of resistance The present inventors screened tomato breeding genetic material in a naturally infected greenhouse in the Bsor region of southern Israel, a major tomato crop-producing region in Israel. Approximately 443 different tomatoes were screened. Each tomato was planted in two replicates, with 10 plants per replicate, in different locations in the greenhouse.

[0176] Each row in the greenhouse contained 120 plants. Each row was planted with 10 plants as susceptible line controls. To spread the controls across different locations in the greenhouse, the controls were placed diagonally along different rows in the greenhouse.

[0177] In this screening, a small number of tomatoes showed no TBRFV symptoms on leaves and few TBRFV symptoms on fruit. Of these, two symptom-free tomatoes and two susceptible tomatoes were selected for the next stage.

[0178] The results of these experiments are shown in Table 1. The two susceptible tomatoes selected were representative of the 441 susceptible tomatoes in the sense that they were considered susceptible to Tomato Brown Leaf Fruit Virus.

[0179] Hazera No. 1 (or HAZ1) is a loose, indeterminate tomato with regular, round, dark red fruits of approximately 170g. The plants have dark green leaves and are resistant to Verticillium dahlia, Meloidogyne incognita, Tomato yellow leaf curl virus, and Stemphylium solani.

[0180] Hazera No. 2 (or HAZ2) is a beef-type indeterminate tomato with regular, medium-sized, flattened, deep dark red fruits weighing approximately 280g. The plant is resistant to Verticillium dahliae, Fusarium oxysporum f.sp. lycopersici 1,2, tomato mosaic virus, Fulvia fulva, sweet potato root-knot nematode, and tomato spotted wilt virus.

[0181] Hazera No. 3 (or HAZ3) is a beefy, indeterminate tomato with medium-sized, flat, red fruits weighing approximately 270g. The plant is resistant to Tomato spotted wilt virus, Verticillium dahliae, Fusarium oxysporum f. sp. lycopersici 1, 2, and Stemphylium solani.

[0182] Hazera No. 4 (or HAZ4) is a minibeef-type indeterminate tomato with round, red fruits weighing approximately 180g. The plant is resistant to Tomato mosaic virus, Tomato yellow leaf curl virus, Cladosporium fulvum (CF9), Verticillium dahliae, and Fusarium oxysporum f. sp. lycopersici 1, 2.

[0183] [Table 1]

[0184] Example 3: Confirmation of resistance To better understand the genetics underlying the tolerance / resistance phenotype and to validate the leads identified in the first screening, we performed a second screening under similar conditions to those of the first screening: each row in a greenhouse under natural infection contained 120 plants, and in each row, susceptible controls (10 plants) were planted. To spread the controls in different locations in the greenhouse, the controls were placed diagonally along different rows in the greenhouse.

[0185] In addition to the resistant tomatoes identified in the first screening, F1 and F2 plants obtained from crosses between resistant plants and susceptible lines were included in the tests.

[0186] Table 2 shows the results of the second screening for leaf evaluation. Plants were considered susceptible as soon as there was some mosaic and twisting at the shoot apex. Tolerant / resistant plants had no symptoms at the shoot apex.

[0187] [Table 2]

[0188] Phenotyping data from F1 and F2 plants tend to indicate that leaf tolerance and / or resistance to Tomato Brown Leaf Fruit Virus is controlled in a recessive manner by a single gene or QTL.

[0189] Table 3 shows the results of the second screening for fruit evaluation. Plants were scored on a scale of 1 to 4, whereby plants with a score of 1 to 3 were considered susceptible, with grade 1 plants having severe symptoms of typical fruit lesions and some fruit deformation, grade 2 plants having moderate lesions on only some fruits, and grade 3 having mild symptoms. Only plants with grades 3, 5, and 4, i.e., no symptoms on the fruit, were considered resistant.

[0190] [Table 3]

[0191] Phenotyping data from F2 plants tend to indicate that fruit tolerance and / or resistance to Tomato Brown Leaf Fruit Virus is controlled in a recessive manner by a small number of QTLs, one or two.

[0192] Example 4: Genetic mapping association analysis Tomato plants Hazera 1 and Hazera 2 were used to construct an F2 biparental mapping population. Tomato plant Hazera 1, which exhibited a resistance phenotype (fruit and leaves) to Tomato Brown Leaf Fruit Virus, was crossed with a susceptible plant to generate an F1, which was then used to generate an F2 segregating population. Additional biparental populations used for validation (leaf QTL) based on Hazera 3 and Hazera 4 were generated in the same way (see Example 6).

[0193] 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 to isolate genomic DNA from plant tissue. DNA concentration was quantified using a NanoDrop spectrophotometer.

[0194] Genotyping of the F2 population (based on Hazera 1 and Hazera 2) was performed using a custom-made Affymetrix Axium chip array (multiplexed genotyping technology) containing approximately 9500 SNPs for tomato.

[0195] Tomato SNP markers were selected and discovered from different sources, including the public domain, LVS projects, and collaborations. All SNPs were validated in pre-screening (previous experience with other techniques) and selected according to: Polymorphism / allele frequency Representing world wide variation SNP cluster removal SNPs evenly spaced according to physical map distance Low expression in heterochromatic (pericentromeric) regions – high LD. Genotyping on Affymetrix Axiom chip arrays was performed using the standard protocol recommended by the manufacturer. The procedure included the following steps: DNA amplification, fragmentation, precipitation, resuspension and hybridization preparation, hybridization to the chip, washing, ligation, staining, and scanning. The last two steps were performed by the Affymetrix GeneTitan instrument. Analysis was performed by an automated clustering algorithm developed by Affymetrix.

[0196] A mixed linear model association was used for both fruit and leaf symptoms independently.

[0197] Mapping results revealed one candidate QTL associated with leaf resistance and / or resistance to Tomato Brown Leaf Fruit Virus located on chromosome 11, and two candidate QTL associated with fruit resistance and / or resistance to Tomato Brown Leaf Fruit Virus located on tomato chromosome 6 and chromosome 9.

[0198] Markers significantly associated with various QTLs for leaf and / or fruit resistance and / or resistance to Tomato Brown Leaf Fruit Virus and their locations on the tomato genome are summarized in Table 4. The sequences of the SNPs, including flanking sequences, are reported in Table 5 and in the accompanying sequence listing section of this application.

[0199] The results show that one QTL responsible for fruit resistance and / or resistance to Tomato Brown Leaf Fruit Virus (QTL1 of the present invention) is located on chromosome 6 between positions 33932438 and 33933905, and a second QTL responsible for fruit resistance and / or resistance to Tomato Brown Leaf Fruit Virus (QTL2 of the present invention) is located on chromosome 9 between positions 4800680 and 59014540, these physical locations on the genome being based on version 2.40 of the tomato genome (Bombarely 2011). The region of chromosome 9 is a region of low recombination rate.

[0200] The region of chromosome 6 is prone to introgression, and several genes of interest have already been mapped to this region, notably the introgression of genes involved in salt tolerance from S. lycopersicoides, S. pennellii, and S. pimpinellifolium (Li et al., Euphytica (2011) 178: 403), the introgression of genes involved in powdery mildew resistance from S. habrochaites and S. neorickii (Seifi et al., Eur J Plant Pathol (2014) 138: 641), and the introgression of genes involved in pepino mosaic virus (WO2013 / 064641).

[0201] Results showed that the QTL responsible for leaf tolerance and / or resistance to Tomato Brown Leaf Fruit Virus was located on chromosome 11 between positions 9548029 and 10015478, and such physical location on the genome was based on version 2.40 of the tomato genome (Bombarely 2011).

[0202] Further analyses were performed using additional markers to better characterize the QTL on chromosome 11 responsible for leaf resistance. The results are shown in Table 6 and the sequences of the SNPs are reported in Tables 5 and 7.

[0203] These additional results indicate that the p-value and R 2Based on the values ​​and the variation in these values, it can be defined that the QTL responsible for leaf resistance to Tomato Brown Leaf Fruit Virus was broadly located on chromosome 11 between SNPs TO-0122252 and TO-0162427, i.e., between positions 8090264 and 10018811, with such physical location on the genome being based on version 2.40 of the tomato genome. SNPs TO-0122252 and TO-0162427, which are adjacent to the broader-defined QTL locus, are indicated by an asterisk (*) in Table 6. The location of the narrower-defined QTL on chromosome 11 is the region defined by SNPs TO-0142270 and TO-0162432. These flanking markers of the narrower-defined locus are indicated by an (**) in Table 6. SNPs with more significant association with QTLs conferring leaf resistance / tolerance are indicated by (+) in Table 6, namely TO-0181040, TO-0123057, and TO60125528.

[0204] [Table 4]

[0205] [Table 5]

[0206] [Table 6]

[0207] [Table 7]

[0208] Example 5: Further validation of markers One marker most associated with leaf resistance to TBRF virus was defined at the edge of the QTL3 region, a candidate marker close to the resistance gene. This SNP was designed for SNP monoplex KASPar technology: the KASPar assay used for validation was based on the KASP method from KBioscience (LGC Group, Teddington, Middlesex, UK).

[0209] Primers for the KASP SNP assay were designed using LGC's Primer Picker software. Two allele-specific forward primers and one general reverse primer were designed for each SNP assay. The KASP genotyping assay is based on competitive allele-specific PCR, allowing biallelic scoring of SNPs at specific loci. Briefly, a SNP-specific KASP assay mix and a universal KASP Master mix were added to a DNA sample, followed by a thermal cycling reaction and an endpoint fluorescence readout. Biallelic discrimination was achieved by competitive binding of two allele-specific forward primers, each with a unique tail sequence corresponding to two universal FRET (fluorescence resonance energy transfer) cassettes, one labeled with FAM™ dye and the other with VIC™ dye (LGC, www.lgcgroup.com).

[0210] A volume of 3 μl of DNA was pipetted into a black 384-well hard shell PCR plate and allowed to dry at room temperature. For genotyping, the DNA was suspended by adding 3 μl of PCR mix according to the manufacturer's protocol (KBioscience). Genotyping PCR results were analyzed using the software KlusterCaller (KBioscience). The marker used in this study was TO-0182276 (SEQ ID NO: 13).

[0211] The HAZ3 x HAZ4 F2 population (Table 2) was used for this marker validation. The F2 plants were genotyped using this marker and also phenotyped for leaf symptoms as described in Example 3. Association was 100% based on data from 251 plants.

[0212] Summary data of leaf symptom phenotyping and candidate marker genotyping are shown in Table 8. R markers mean homozygous for the resistance / tolerance allele, S markers mean homozygous for the susceptibility allele, and H markers mean heterozygous for two alleles.

[0213] [Table 8]

[0214] Example 6: Association analysis for gene mapping An F2 biparental mapping population was constructed using tomato plants Hazera 3 and Hazera 4. Tomato plant Hazera 3, which exhibits a leaf resistance phenotype to Tomato Brown Leaf Fruit Virus, was crossed with the susceptible plant Hazera 4 to create an F1, which was then used to generate an F2 segregating population.

[0215] Using HAZ1 and HAZ2, crosses, phenotyping, and associations were performed as described in Example 4.

[0216] QTLs and markers with the most significant associations for leaf resistance were identified on chromosome 11, as detailed in Table 9 and shown in FIG.

[0217] As in Example 4, the broader locus containing the QTL is defined by the flanking markers marked with asterisks in Table 9, namely SNPs TO-012252 and TO0162427. These SNPs are the same as those flanking the broader QTL location inferred from results obtained with another source of resistance, namely HAZ1. This strongly supports the conclusion that the QTL for leaf resistance is the same in HAZ1 and HAZ3.

[0218] HAZ1 corresponds to the seed HAZTBRFVRES1 deposited at the NICMB under accession number 42758.

[0219] The locus of the more narrowly defined QTL inferred from the results for the HAZ3 population is defined by adjacent markers TO-0144317 and TO-0125528 (markers ** in Table 9) on chromosome 11. The markers with the most significant association with TBRFV leaf tolerance / resistance are those indicated with a (+), namely TO-0142303, TO-0142306, and TO60142294.

[0220] [Table 9]

[0221] [Table 10]

[0222] Taken together, these results confirm the presence of a QTL conferring leaf resistance broadly located within the chromosomal region bounded by TO-012252 and TO0162427, and more precisely, TO-0144317 and TO-0125528.

[0223] Therefore, taking into account the results of Example 4, these results indicate that the location of this QTL can be advantageously defined as between TO-0142270 and TO-0125528.

[0224] Example 7: Genetic modification of tomato seeds with ethyl methanesulfonate (EMS) Treat seeds of tomato cultivars with EMS by soaking approximately 2,000 seeds per cultivar in an aerated solution of either 0.5% (w / v) EMS or 0.7% EMS for 24 h at room temperature.

[0225] Approximately 1500 treated seeds per variety and per EMS dose are germinated and the resulting plants are grown, preferably in a greenhouse, for example from May to September, to produce seed.

[0226] After maturity, M2 seeds are harvested and pooled together per treatment and per cultivar. The resulting M2 seed pools are used as starting material to identify individual M2 seeds and plants with fruit and / or leaf resistance to Tomato Brown Leaf Curl Fruit Virus.

Claims

1. A method for detecting and / or selecting S. lycopersicum plants having QTLs that confer fruit and / or leaf resistance to tomato brown leaf fruit virus, In the selected plant genetic material sample, the following markers are present: Allele T of TO-0005197 (Sequence ID 1), Allele C of TO-0145581 (Sequence ID 2), Allele G of TO-0180955 (Sequence ID 3), Allele C of TO-0196724 (Sequence ID 4), Allele G of TO-0145125 (Sequence ID 5), Allele G of TO-0196109 (Sequence ID 6), Allele T of TO-0122252 (Sequence ID 7), Allele C of TO-0144317 (Sequence ID 8), Allele T of TO-0142270 (Sequence ID 9), Allele G of TO-0142294 (Sequence ID 10), Allele A of TO-0142303 (Sequence ID 11), Allele A of TO-0142306 (Sequence ID 12), Allele G of TO-0182276 (Sequence ID 13), Allele G of TO-0181040 (Sequence ID 14), Allele G of TO-0123057 (Sequence ID 15), Allele A of TO-0125528 (Sequence ID 16), Allele C of TO-0162432 (SEQ ID NO: 17), and Allele T of TO-0162427 (Sequence ID 18) A method comprising the detection of at least one of the following.

2. Allele T of TO-0005197, Allele C of TO-0145581, Allele G of TO-0180955, Allele C of TO-0196724, Allele G of TO-0145125, Allele G of TO-0196109, Allele T of TO-0122252, Allele C of TO-0144317, Allele T of TO-0142270, Allele G of TO-0142294, TO-01 The method according to claim 1, wherein at least two, three, four, five or more alleles are detected from among allele A of 42303, allele A of TO-0142306, allele G of TO-0182276, allele G of TO-0181040, allele G of TO-0123057, allele A of TO-0125528, allele C of TO-0162432, and allele T of TO-0162427.

3. The following alleles: Allele T of TO-0122252 (Sequence ID 7), Allele C of TO-0144317 (Sequence ID 8), Allele T of TO-0142270 (Sequence ID 9), Allele G of TO-0142294 (Sequence ID 10), Allele A of TO-0142303 (Sequence ID 11), Allele A of TO-0142306 (Sequence ID 12), Allele G of TO-0182276 (Sequence ID 13), Allele G of TO-0181040 (Sequence ID 14), Allele G of TO-0123057 (Sequence ID 15), Allele A of TO-0125528 (Sequence ID 16), Allele C of TO-0162432 (SEQ ID NO: 17), and Allele T of TO-0162427 (Sequence ID 18) The method according to claim 1, wherein if at least three, four, five or more of the above are detected, the plant is selected for the presence of QTLs that confer leaf tolerance.

4. The method according to claim 3, wherein if at least four of the alleles are detected, the plant is selected for the presence of QTLs that confer leaf tolerance.

5. The method according to claim 3, wherein if more than five alleles are detected among the aforementioned alleles, the plant is selected for the presence of QTLs that confer leaf tolerance.

6. The following alleles: Allele G of TO-0180955 (Sequence ID 3), Allele C of TO-0196724 (Sequence ID 4), Allele G of TO-0145125 (SEQ ID NO: 5), and Allele G of TO-0196109 (SEQ ID NO: 6) The method according to claim 1, wherein if at least one, two, three, or four of the following are detected, the plant is selected for the presence of QTLs that confer fruit tolerance.

7. The method according to any one of claims 1 to 6, wherein the allele of the marker is detected in a homozygous state.

8. The method according to any one of claims 1 to 6, wherein the allele of the marker is detected in the absence of other alleles of the marker.

9. A method for detecting the presence of at least one genetic marker related to resistance to TBRFV infection in the germplasm of a S. lycopersicum plant or tomato, In the genome of the plant or germplasm being examined, (i) Allele G of TO-0180955 (Sequence ID 3), Allele C of TO-0196724 (Sequence ID 4), Allele G of TO-0145125 (SEQ ID NO: 5), and Allele G of TO-0196109 (SEQ ID NO: 6) At least two of them, or (ii) Allele T of TO-0122252 (Sequence ID 7), Allele C of TO-0144317 (Sequence ID 8), Allele T of TO-0142270 (Sequence ID 9), Allele G of TO-0142294 (Sequence ID 10), Allele A of TO-0142303 (Sequence ID 11), Allele A of TO-0142306 (Sequence ID 12), Allele G of TO-0182276 (Sequence ID 13), Allele G of TO-0181040 (Sequence ID 14), Allele G of TO-0123057 (Sequence ID 15), Allele A of TO-0125528 (Sequence ID 16), Allele C of TO-0162432 (SEQ ID NO: 17), and Allele T of TO-0162427 (Sequence ID 18) More than five of the alleles A method comprising the step of detecting nucleic acids containing the following:

10. The method according to claim 9, wherein the allele of the marker is detected in a homozygous state.

11. The method according to claim 9, wherein the allele of the marker is detected in the absence of other alleles of the marker.

Images