WO2022234584A1

WO2022234584A1 - Markers and methods for determining resistance to tomato brown rugose fruit virus

Info

Publication number: WO2022234584A1
Application number: PCT/IL2022/050472
Authority: WO
Inventors: Vidavski Favi SHERAGA; Assaf EYBISHITZ; Yuval JACOBSOHN; Moshe Yaakov CHOEN
Original assignee: Tomatech R&D Israel Ltd.
Priority date: 2021-05-06
Filing date: 2022-05-04
Publication date: 2022-11-10

Abstract

Markers and methods for determining and/or selecting a plant belonging to Solanaceae, e.g., S. lycopersicum, which displays resistance to the tomato brown rugose fruit virus (ToBRFV), are provided.

Description

MARKERS AND METHODS FOR DETERMINING RESISTANCE TO TOMATO

BROWN RUGOSE FRUIT VIRUS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/184,922, titled "MARKERS AND METHODS FOR DETERMINING RESISTANCE TO TOMATO BROWN RUGOSE FRUIT VIRUS", filed May 6, 2021, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[0002] The present invention is in the field of molecular markers and methods for determining viral resistance of a plant.

BACKGROUND

[0003] All cultivated and commercial forms of tomato belong to a species most frequently referred to as Lycopersicon esculentum Miller. Lycopersicon is a relatively small genus within the extremely large and diverse family Solanaceae which is considered to consist of around 90 genera, including pepper, tobacco and eggplant. The genus Lycopersicon has been divided into two subgenera, the esculentum complex which contains those species that can easily be crossed with the commercial tomato and the peruvianum complex which contains those species which are crossed with considerable difficulty. Due to its value as a crop, L. esculentum Miller has become widely disseminated all over the world. Even if the precise origin of the cultivated tomato is still somewhat unclear, it seems to come from the Americas, being native to Ecuador, Peru and the Galapagos Island and initially cultivated by Aztecs and Incas as early as 700 AD. Mexico appears to have been the site of domestication and the source of the earliest introduction. It is supposed that the cherry tomato, L. esculentum var. cerasiforme, is the direct ancestor of modern cultivated forms. [0004] Tomato is grown for its fruit, widely used as a fresh market or processed product. As a crop, tomato is grown commercially wherever environmental conditions permit the production of an economically viable yield. The majority of fresh market tomatoes are harvested by hand at vine ripe and mature green stage of ripeness. Fresh market tomatoes are available year round. Processing tomato are mostly mechanically harvested and used in many forms, as canned tomatoes, tomato juice, tomato sauce, puree, paste or even catsup. [0005] A variety of pathogens affect the productivity of tomato plants, including virus, fungi, bacteria, nematodes and insects. Tomatoes are inter alia susceptible to many viruses and virus resistance is therefore of major agricultural importance.

[0006] Tobamoviruses are among the most important plant viruses causing severe damages in agriculture, especially to vegetable and ornamental crops around the world. Tobamoviruses are easily transmitted by mechanical means, inclusive of natural vectors (e.g., Bombus bees), as well as through seed transmission. Tobamoviruses are generally characterized by a rod-shaped particle of about 300 nm, their structure consists in a single stranded, positive RNA genome encoding four proteins, encapsidated by 17KDa coat protein (CP) molecules.

[0007] In tomatoes, tobacco mosaic virus (TMV), tomato mosaic virus (ToMV) are feared by growers worldwide as they can severely damage crop production, for example through irregular ripening (fruits having yellowish patches on the surface and brownish spots beneath the surface). Several genes have however been identified by plants breeders over the years.

[0008] During 2014-2015, a severe outbreak of virus affected tomato productions areas in the middle east, such as in Jordan and in Israel. Most of the tomato varieties affected were considered TMV and/or ToMV resistant, but were still severely affected and showed typical TMV/ToMV like symptoms: while the foliar ones were quite similar to the TMV/ToMV symptoms, the fruit symptoms were much more frequent and severe than the usual symptoms from such viruses with fruits lesions and deformations. The fruit quality was very poor and rather unmarketable. A new Tobamovirus species, the tomato brown rugose fruit virus (TBRFV or ToBRFV) was identified in extracts of RNA from fruit and leaves of symptomatic plants, infected in Jordan. The comparison to other Tobamoviruses sequences showed that it is indeed a Tobamovirus, but not TMV or ToMV. The resistance to TMV and/or ToMV does not confer resistance to this new virus TBRFV.

[0009] Recently, the virus was identified in Europe, especially in Sicily, Germany and the Netherlands, and in Mexico, US and Canada and therefore now it is considered as a major global threat to tomato crop. The strain identified appears to be the Israeli strain, rather than the Jordanian strain.

[0010] As Tobamoviruses are not easily controlled but through genetic improvement by the identification and use in breeding of resistance genes, and as the resistance genes currently available to control TMV and/or ToMV are useless against the damages and propagation from the new ToBRFV, and the tolerance quantitative trait loci (QTLs) not able to stop or sufficiently reduce the viral propagation, there is an urgent need to identify resistance against this new Tobamovirus, failing that would result in entire regions in which tomato crop could not be produced anymore.

SUMMARY

[0011] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0012] According to a first aspect, there is provided a method for selecting a plant that displays resistance to a tomato brown rugose fruit virus (ToBRFV), the method comprising the steps of: (a) determining the presence of a plurality of SNPs selected from Table 1 in a plant or a part derived therefrom; and (b) selecting a plant determined to comprise the plurality of SNPs, thereby selecting a plant that displays resistance to ToBRFV.

[0013] According to another aspect, there is provided a progeny of a crossing obtained according to the herein disclosed method.

[0014] In some embodiments, the plurality of SNPs is selected from chromosome 1.

[0015] In some embodiments, the plurality of SNPs is selected from chromosome 2.

[0016] In some embodiments, the plurality of SNPs is selected from chromosome 3.

[0017] In some embodiments, the plurality of SNPs is selected from chromosome 4.

[0018] In some embodiments, the plurality of SNPs is selected from chromosome 6.

[0019] In some embodiments, the plurality of SNPs is selected from chromosome 9.

[0020] In some embodiments, the plurality of SNPs is selected from chromosome 11.

[0021 ] In some embodiments, the method further comprises a step of crossing the selected plant displaying resistance to ToBRFV.

[0022] In some embodiments, the crossing is intraspecies or interspecies crossing. [0023] In some embodiments, the part derived from the plant is selected from the group consisting of: a cell, a tissue, a leaf, a stem, a root, a callus, and a seed.

[0024] In some embodiments, the method further comprises a step of regenerating any one of the: cell, tissue, leaf, stem, root, callus, and seed, into a plant, thereby producing a plant displaying resistance to ToBRFV.

[0025] In some embodiments, the plant belongs to the subfamily of Solanoideae.

[0026] In some embodiments, the plant belongs to the genera Solanum or Capsicum. [0027] In some embodiments, the plant is Solanum lycopersicum.

[0028] In some embodiments, the progeny is a plant displaying resistance to ToBRFV. [0029] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0030] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION

[0031 ] The present invention, in some embodiments, is based, in part, on the findings that chromosomes 2, 4, and 9 of S. lycopersicum comprise with high probability gene(s) conferring ToBRFV resistance.

[0032] As used herein, the terms "single nucleotide polymorphism" or "SNP" refer to a substitution of a single nucleotide at a specific position in a tomato ( Solanum lycopersicum) genome, and specifically, based on the Solanum lycopersicum cv. M82 (Tomato cultivar M82) genome. Specifically, the data was downloaded from

Corresponding SRA: BioSample:

SAMN 12392445; Sample name: M82; SRA: SRS5175004.

[0033] According to some embodiments, there is provided a method for selecting a plant that displays resistance to Tomato brown rugose fmit virus (ToBRFV), the method comprising the steps of: (a) determining the presence of a plurality of SNPs selected from Table 1; and, (b) selecting a plant determined to comprise the plurality of marker nucleic acid, thereby selecting a plant that displays resistance to ToBRFV.

[0034] According to some embodiments, there is provided a method for developing a ToBRFV resistant plant comprising introgressing one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 1 and into the plant.

[0035] According to some embodiments, there is provided a method for developing a ToBRFV resistant plant comprising hybridizing or crossing a non resistant plant with a resistant plant comprising one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0036] According to some embodiments, there is provided a method for developing a ToBRFV resistant plant comprisng germinating seed comprising in its genome one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0037] According to some embodiments, there is provided a method for selecting a plant that displays resistance to Tomato brown rugose fmit vims (ToBRFV), the method comprising the steps of: (a) determining the presence of a plurality of SNPs selected from Table 2; and, (b) selecting a plant determined to comprise the plurality of marker nucleic acid, thereby selecting a plant that displays resistance to ToBRFV.

[0038] According to some embodiments, there is provided a ToBRFV resistant plant or plant material, the plant or plant material comprising in its genome one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0039] According to some embodiments, there is provided a plant, a plant part, a tissue, a cells, a seed, or any combination thereof, being derived from a ToBRFV resistant plant as disclosed herein.

[0040] In some embodiments, a tissue comprises a foliar tissue (e.g., related or derived from a plant foliage or canopy). [0041] In some embodiments, a foliar tissue comprises: seed, fmit, flower, leaf, or any combination thereof.

[0042] According to some embodiments, there is provided a method for developing a ToBRFV resistant plant comprising introgressing one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 2 and into the plant.

[0043] According to some embodiments, there is provided a ToBRFV resistant plant or plant material, the plant or plant material comprising in its genome one or more SNPs of the plurality of SNPs of Table 2.

[0044] In some embodiments, the plant or plant material comprises any combination SNPs of the plurality of SNPs of Table 2.

[0045] According to some embodiments, there is provided a method for obtaining or producing a ToBRFV resistant plant comprising one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0046] Methods for sexually producing plants are common and would be apparent to one of ordinary skill in the art. Non-limiting example for sexual reproduction of plants, includes, but is not limited to, crossing or breeding.

[0047] In some embodiments, the method comprises sexually producing a ToBRFV resistant plant comprising one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0048] In some embodiments, the sexually produing comprises crossing or back-crossing a ToBRFV resistant plant as disclosed herein, with other germplasm so as to produce a progeny plant.

[0049] In some embodiments, at least one cell of a progeny plant, as disclosed herein, comprises one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0050] In some embodiments, the method comprises asexually producing a ToBRFV resistant plant comprising one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0051] In some embodiments, a ToBRFV resistant plant as disclosed herein is asexually produced from a plant cell, plant tissue, or both, comprising one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both. [0052] Methods for asexual reproduction of plants are common and would be apparent to one of ordinary skill in the art. Non-limiting example for such methodology includes, but is not limited to vegetative propagation.

[0053] In some embodiments, the method comprises introgressing one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both into the plant.

[0054] In some embodiments, the method comprises transforming the plant with one or more polyucleotide squences comprising one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0055] Methods for introgressing and/or transforming plant or plant cells are common and would be apparent to one of ordinary skill in the art. Non-limiting example for such a method inucldes, but is not limited to, use of agrobacterium vector so as to introduce a nucleic acid sequence to a plant cell, a plant cell genome, or both.

[0056] In some embodiments, the one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both, comprises at least 1, at least 2, at least 3, at least 5, at least 7, at least 9, at least 10, at least 12, at least 15, at least 17, or at least 20 SNPs of the plurality of SNPs of Table 1, Table 2, or both, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both, comprises 1-8, 2-20, 3-15, 1- 15, 2-17, or 1-20 SNPs of the plurality of SNPs of Table 1, Table 2, or both. Each possibility represents a separate embodiment of the invention.

[0057] According to some embodiments, the plurality of SNPs is selected from chromosome 2.

[0058] According to some embodiments, plurality of SNPs is selected from chromosome 4.

[0059] According to some embodiments, the plurality of SNPs is selected from chromosome 9.

[0060] In some embodiments, the plant is seleced from Solanales.

[0061] In some embodiments, the plant is a Solanales fruit.

[0062] In some embodiments, the fruit comprises a tomato fruit.

[0063] In some embodiments, the plant belongs to the genera of Solanoideae or Capsicum. [0064] In some embodiments, the plant comprises a tomato plant, e.g., Solanum lycopersicum, including any line, species, subspecies, variant, or derivative thereof, or any combination thereof.

[0065] In some embodiments, the plant comprises a pepper. In some embodiments, the plant comprises a potato. In some embodiments, the plant comprises an eggplant.

[0066] As used herein, the term "plant" encompasses a plant, a plant material, a plant part, or any combination thereof.

[0067] As used herein, the terms "plant material" or "plat part" are interchangeable, and refer to any material or part being derived or obtained from a plant, such as, but not limited to, cell, root, stem, stalk, leaf, seed, flower, fruit, extract, lysate, homogenate, any fraction thereof, or any combination thereof.

[0068] In some embodiments, the plant material comprises a seed, a fruit, a flower, or any combination thereof.

[0069] In some embodiments, the plant material is a seed.

[0070] In some embodiments, there is provided a seed of a ToBRFV resistant plant comprising in its genome one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0071] In some embodiments, there is provided a seed characterized by being capable of germinating and/or developing into a ToBRFV resistant plant comprising in its genome one or more SNPs of the plurality of SNPs of Table 1, Table 2, or both.

[0072] Classification and identification of plants, including methods for determining same, are common and would be apparent to one of ordinary skill in the art of botanies. Non-limiting examples for guidance on plant classifaction and determination can be found in any geographic’s flora.

[0073] The following definitions are provided as an aid to understand the invention. [0074] The term “plant” includes whole plants, plant cells, plant protoplast, plant cell or tissue cultures from which the plants can be regenerated, plant cells that are intact in plants or parts of plants, such as seeds, heads, flowers, cotyledons, leaves, stems, buds, roots, root tips, and the like.

[0075] As used herein, the term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus. [0076] An allele is “associated with” a trait when it is linked to it and when the presence of the allele is an indicator that the desired trait or trait form will occur in or be manifested by a plant comprising the allele.

[0077] As used herein, the term “amplicon” refers to an in vitro amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).

[0078] As used herein, the term “amplifying” in the context of nucleic acid amplification refers to any process whereby additional copies of a selected nucleic acid for a transcribed form thereof are produced. Typical amplification methods include various polymerase- based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase-based amplification (e.g., by transcription) methods.

[0079] As used herein, the term “backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al., (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross gives rise to the FI generation: the term “BC1” then refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.

[0080] As used herein, the term "centimorgan" (“cM”) refers to the unit of measure of recombination frequency. One (1) cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.

[0081] As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA that resides in plants on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%, respectively. [0082] As used herein, the term “chromosomal interval” designates any and all intervals defined by any of the markers set forth in this invention.

[0083] As used herein, the term “complement” refers to a nucleotide sequence that is complementary to a given nucleotide sequence, i.e., the sequences are related by the base- pairing rules.

[0084] As used herein, the term “contiguous DNA” refers to overlapping contiguous genetic fragments.

[0085] As used herein, the term “crossed” or “cross” means the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant). The term “crossing” refers to the act of fusing gametes via pollination to produce progeny.

[0086] As used herein, the term “elite line” refers to any line that has resulted from breeding and selection for superior agronomic performance. An elite plant is any plant from an elite line.

[0087] As used herein, the term “favorable allele” is the allele at a particular locus that confers, or contributes to, a desirable phenotype, e.g., increased resistance, or alternatively, is an allele that allows the identification of plants with decreased resistance that can be removed from a breeding program or planting (“counter-selection”). A favorable allele of a marker is a marker allele that segregates with the favorable phenotype, or alternatively, segregates with the unfavorable plant phenotype, therefore providing the benefit of identifying plants.

[0088] As used herein, the term “fragment” is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using methods disclosed herein.

[0089] As used herein, the term “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or chromosomes) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by the recombination frequencies between them, and recombinations between loci can be detected using a variety of molecular genetic markers (also called molecular markers). A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci can differ from one genetic map to another. However, information such as marker position and order can be correlated between maps by determining the physical location of the markers on one of the sunflower linkage groups, Ha412HO bronze assembly reference genome, which is publicly available on the internet. One of ordinary skill in the art can use the publicly available genome browser to determine the physical location of markers on a chromosome.

[0090] As used herein, the term “genetic marker” shall refer to any type of nucleic acid based marker, and can be determined by any method known to one of ordinary skill in the art of molecular biology, including but not limited to, Restriction Fragment Length Polymorphism (RFLP) (Botstein et al, 1998), Simple Sequence Repeat (SSR) (Jacob et al., 1991), Random Amplified Polymorphic DNA (RAPD) (Welsh et al., 1990), Cleaved Amplified Polymorphic Sequences (CAPS) (Rafalski and Tingey, 1993, Trends in Genetics 9:275-280), Amplified Fragment Length Polymorphism (AFLP) (Vos et al, 1995, Nucleic Acids Res. 23:4407-4414), Single Nucleotide Polymorphism (SNP) (Brookes, 1999, Gene 234:177-186), Sequence Characterized Amplified Region (SCAR) (Pecan and Michelmore, 1993, Theor. Appl. Genet, 85:985-993), Sequence Tagged Site (STS) (Onozaki et al. 2004, Euphytica 138:255-262), Single Stranded Conformation Polymorphism (SSCP) (Orita et al., 1989, Proc Natl Aced Sci USA 86:2766-2770). Inter-Simple Sequence Repeat (ISR) (Blair et al. 1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon Amplified Polymorphism (IRAP), Retrotransposon-Microsatellite Amplified Polymorphism (REMAP) (Kalendar et al., 1999, Theor. Appl. Genet 98:704-711), an RNA cleavage product (such as a Lynx tag), and the like.

[0091] As used herein, the term “genetic recombination frequency” is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and/or traits following meiosis. [0092] As used herein, the term “genome” refers to the total DNA, or the entire set of genes, DNA interspace regions, non-coding DNA sequences, or others, all of which carried and/or organized in a chromosome or chromosome set.

[0093] As used herein, the term “genotype” refers to the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple led, or, more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome.

[0094] As used herein, the term “germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leafs, stems, pollen, or cells that can be cultured into a whole plant. [0095] As used herein, the term “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term “haplotype” can refer to sequence, polymorphisms at a particular locus, such as a single marker locus, or sequence polymorphisms at multiple loci along a chromosomal segment in a given genome. The former can also be referred to as “marker haplotypes” or “marker alleles”, while the latter can be referred to as “long-range haplotypes”.

[0096] As used herein, the term “heterozygous” means a genetic condition wherein different alleles reside at corresponding loci on homologous chromosomes.

[0097] As used herein, the term “homozygous” means a genetic condition wherein identical alleles reside at corresponding loci on homologous chromosomes. [0098] As used herein, the terms “hybridization” or “nucleic acid hybridization” refer to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.

[0099] As used herein, the term “hybridize” means the formation of base pairs, e.g., via hydrogene bonds, between complementary regions of nucleic acid strands.

[0100] The term “introgression” or “introgressing” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species or of different species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a selected allele of a marker, a QTL, a transgene, or the like. In any case, offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background. For example, the linkage group 4 locus described herein may be introgressed into a recurrent parent that is susceptible to Orobanche resistance. The recurrent parent line with the introgressed gene or locus then has increased Orobanche resistance.

[0101] As used herein, the term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus or some other locus (for example, a Orobanche resistance locus). The linkage relationship between a molecular marker and a phenotype is given as a “probability” or “adjusted probability”. Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units for cM). In some aspects, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes. Thus, “closely linked loci” such as a marker locus and a second locus display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10 (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” each other. Since one cM is the distance between two markers that show a 1% recombination frequency, any marker is closely linked (genetically and physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distant. Two closely linked markers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each other.

[0102] As used herein, the term “linkage disequilibrium” refers to a non-random segregation of genetic loci or traits for both loci. In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency (in the case of co-segregating traits, the loci that underlie the traits are in sufficient proximity to each other). Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51% to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same chromosome.) As used herein, linkage can be between two markers, or alternatively between a marker and a phenotype. A marker locus can be “associated with” (linked to) a trait, e.g., increased Orobanche resistance. The degree of linkage of a molecular marker to a phenotypic trait is measured, e.g. as a statistical probability of co-segregation of that molecular marker with the phenotype.

[0103] As used herein, the term “locus” refers to a position on a chromosome where a gene or marker is located.

[0104] SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus having the potential to provide the highest genetic map resolution (Bhattramakki et al. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called ‘ultra- high-throughput’ fashion, as they do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp, 475-492: Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100: Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R, J Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode™. (Qiagen), Invader® (Third Wave Technologies), Snapshot® (Applied Biosystems), Taqman® (Applied Biosystems) and Beadarrays™ (Illumina).

[0105] A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than, single SNPs and can be more descriptive of any particular genotype.

[0106] For a non-limiting example, a single SNP may be allele ‘T’ for a specific line or variety with increased Orobanche resistance, but the allele ‘T’ might also occur in the sunflower breeding population being utilized for recurrent parents. In this case, a haplotype, e.g. a combination of alleles at linked SNP markers, may be more informative. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. See, for example, W02003054229. Using automated high throughput marker detection platforms known to those of ordinary skill in the art makes this process highly efficient and effective.

[0107] The sequences listed in Table 1 can be readily used to obtain additional polymorphic SNPs (and other markers) within the QTF interval listed in this disclosure. [0108] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

[0109] Table 1. SNPs identified in ToBRFV resistant tomato residing in Chromosomes 1, 2, 3, 4, 6, 9, and 11.

EXAMPLES

[0110] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0111] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8^th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Materials and Methods

[0112] A wide variety range of Tomatech's genetic sources were sown in a nursery, a 28- day-old seedlings were planted in a quarantine greenhouse, the plants were mechanically controlled inoculated with a solution containing virus and grown under a commercial growth protocol. Throughout all the growing period an assessment of the symptoms level was made, the plants were sampled and tested for the presence of a virus by three different methods: (1) a biological test (inoculation of tester plants); (2) a serological test (enzyme- linked immunosorbent assay (ELISA) test); and (3) a PCR test. The plants were grown up to the fruit ripening stage. Fruits from a plant that was found to be relevant to the experiment, were harvested and the seeds were extract. Those seeds were used for growing and testing the next generation in order to establish and examine the inheritance of the trait.

EXAMPLE 1

Molecular analysis revelas SNPs linked to ToBRFV resistance in a Tomato plant

[0113] The inventors have perforfm a wide genomice comparative analysis comparing the genomes of ToBRFV resistant tomamto parent plants with ToBRFV sensitive parent plants. [0114] The results show that chromosomes 1, 2, 3, 4, 6, 9, and 11 with high probability contain gene(s) comprising numerous SNPs which may convey resistance to the ToBRFV (Table 2). These SNPs were identified in the genome of resistant parent plants and were absent in sensitivie parent plants.

[0115] Other SNPs identified by the inventors as suitable for determining resistance, as disclosed herein, include for example positions provided in Table 2, and any combination thereof.

Table 2.

Claims

CLAIMS What is claimed is:

1. A method for selecting a plant that displays resistance to a tomato brown rugose fruit virus (ToBRFV), the method comprising the steps of: a. determining the presence of a plurality of SNPs selected from Table 1 in a plant or a part derived therefrom; and b. selecting a plant determined to comprise the plurality of SNPs, thereby selecting a plant that displays resistance to ToBRFV.

2. The method of claim 1, wherein the plurality of SNPs is selected from chromosome

1.

3. The method of claim 1, wherein the plurality of SNPs is selected from chromosome

2.

4. The method of claim 1, wherein the plurality of SNPs is selected from chromosome

3.

5. The method of claim 1, wherein the plurality of SNPs is selected from chromosome

4.

6. The method of claim 1, wherein the plurality of SNPs is selected from chromosome

6.

7. The method of claim 1, wherein the plurality of SNPs is selected from chromosome

9.

8. The method of claim 1, wherein the plurality of SNPs is selected from chromosome

11.

9. The method of any one of claims 1 to 8, further comprising a step of crossing said selected plant displaying resistance to ToBRFV.

10. The method of claim 9, wherein said crossing is intraspecies or interspecies crossing.

11. The method of any one of claims 1 to 10, wherein said part derived from said plant is selected from the group consisting of: a cell, a tissue, a leaf, a stem, a root, a callus, and a seed.

12. The method of claim 11, further comprising a step of regenerating any one of said: cell, tissue, leaf, stem, root, callus, and seed, into a plant, thereby producing a plant displaying resistance to ToBRFV.

13. The method of any one of claims 1 to 12, wherein said plant belongs to the subfamily of Solanoideae.

14. The method of any one of claims 1 to 13, wherein said plant belongs to the genera Solanum or Capsicum.

15. The method of any one of claims 1 to 14, wherein said plant is Solanum lycopersicum.

16. A progeny of a crossing obtained according to the method of any one of claim 9 to 15.

17. The progeny of claim 16, being a plant displaying resistance to ToBRFV.