WO2018011075A1 - Tolcndv resistant melon plants - Google Patents

Tolcndv resistant melon plants Download PDF

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Publication number
WO2018011075A1
WO2018011075A1 PCT/EP2017/067078 EP2017067078W WO2018011075A1 WO 2018011075 A1 WO2018011075 A1 WO 2018011075A1 EP 2017067078 W EP2017067078 W EP 2017067078W WO 2018011075 A1 WO2018011075 A1 WO 2018011075A1
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Prior art keywords
snp
plant
seq
tolcndv
melon
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PCT/EP2017/067078
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French (fr)
Inventor
Juan Antonio LIZARZABURU CHAVEZ
Jeffrey SKONECZKA
Daniel Bellon Doña
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Nunhems B.V.
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Publication date
Priority to EP17739942.5A priority Critical patent/EP3484276B1/en
Priority to CA3030339A priority patent/CA3030339A1/en
Priority to AU2017295610A priority patent/AU2017295610B2/en
Priority to EA201990273A priority patent/EA201990273A1/en
Priority to MX2019000534A priority patent/MX2019000534A/en
Priority to CN201780045783.7A priority patent/CN109475098B/en
Application filed by Nunhems B.V. filed Critical Nunhems B.V.
Priority to US16/317,496 priority patent/US10947557B2/en
Priority to ES17739942T priority patent/ES2974371T3/en
Priority to IL264127A priority patent/IL264127B2/en
Publication of WO2018011075A1 publication Critical patent/WO2018011075A1/en
Priority to ZA2019/00866A priority patent/ZA201900866B/en
Priority to US17/155,700 priority patent/US11591611B2/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/34—Cucurbitaceae, e.g. bitter melon, cucumber or watermelon 
    • A01H6/344—Cucumis melo [melon]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the application concerns melon plants (Cucumis melo) resistant to infection with tomato leaf curl New Dehli virus (ToLCNDV).
  • the resistant melon plants have a genomic introgression fragment on chromosome 5 which confers tolerance to ToLCNDV in a dominant manner. Also disclosed are markers for identifying those fragments, methods for identifying or producing resistant melon plants.
  • Tomato leaf curl New Dehli virus (ToLCNDV) is classified as Begomovirus belonging to the family Geminiviridae.
  • ToLCNDV has a bipartite genome consisting of two single stranded DNA molecules referred to as DNA A and DNA B (Saez et al., 2016, Annals of Applied Biology).
  • ToLCNDV was initially found to infect tomato ⁇ Solarium lycopersicum) plants in 1995 in India. Later ToLCNDV was found to infect also plants of other Solanacea species, like Solarium melongena (aubergine), chili pepper (Capsicum spp.) and Solarium tuberosum (potato).
  • Solanacea species like Solarium melongena (aubergine), chili pepper (Capsicum spp.) and Solarium tuberosum (potato).
  • curcubit species courgette, Cucurbita pepo var. giromontiina
  • Curcubitacea species such as Benincasa hispida (wax gourd), Citrullus lanatus (watermelon), Cucumis melo (melon), Cucumis melo var. flexuosus (snake melon), Cucumis sativus (cucumber), Cucurbita moschata (musky gourd), Cucurbita pepo (pumpkin), Cucurbita pepo var. giromontiina (courgette), Lagenaria siceraria (bottle gourd), Luffa cylindrica (sponge gourd), Momordica charantia (bitter gourd) have been proven.
  • Curcubitacea species such as Benincasa hispida (wax gourd), Citrullus lanatus (watermelon), Cucumis melo (melon), Cucumis melo var. flexuosus (snake melon), Cucumis
  • ToLCNDV Cucurbits upon infection of young plants with ToLCNDV show stunted growth and decreased or suppressed fruit production. Also fruits showing skin roughness and longitudinal cracking have been reported. Thus, ToLCNDV causes economic losses in various important crop species and is a major threat. Infection of plants by ToLCNDV occurs persistently by transmission of the virus by the phloem sucking whitefly (Bemisia tabaci). (European and Mediterranean Plant Protection Organization, EPPO RS 2015/114, 2016/024, 2016/040, Entry date 2015-06).
  • transgenic plants resistant to ToLCNDV have been produced by silencing virus genes (Varma & Praveen, 2006, ISB News Report).
  • An object of the present invention is to provide measurements for the control of ToLCNDV infection in melon plants.
  • the present invention discloses melon plant cells and melon plants being resistant to infections by TolCNDV.
  • ToLCNDV does infect various different plant species of the Curcubitaceae species, including melon species. It is also well known that ToLCNDV is transmitted persistently from infected plants to non-infected plants by the plant sucking pest Bemisia tabaci (whitefly). Transfer of ToLCNDV from one crop species to different crop species or even from weed species to crop species has been demonstrated. Whiteflies may pick up ToLCNDV from outside the controlled area even from different species and transfer it to melon plants grown in the controlled area. Whitefly vector control therefore is of limited effectiveness for preventing ToLCNDV infection. TolCNDV resistant melon plants have the advantage that they would withstand infection with TolCNDV without major yield losses, even if plants around the area where the melon plants are grown are infected with ToLCNDV.
  • a Quantitative Trait Locus (QTL) was identified on chromosome 5 (QTL5) and introgressed from the wild donor accession into an elite cultivated melon line. Seeds of the elite line, comprising the QTL5 introgression in homozygous form, were deposited by Nunhems B.V. under Accession number NCIMB 42585 in accordance with the Budapest Treaty. In these seeds the donor genotype for the Single Nucleotide Polymorphism (SNP) markers (SNP 01 to SNP 06) provided herein is present.
  • SNP Single Nucleotide Polymorphism
  • the donor itself is not uniform and has white fruit flesh with a low brix. It is not an accession of agronomic value. The donor is also not available anymore at the US seed bank.
  • By identifying and transferring QTL5 from the donor into cultivated melon it is now possible to make cultivated melon varieties and cultivars of high agronomic value (with uniform characteristics and marketable fruits having high brix and good shelf life) with resistance against ToLCNDV and thus it is possible to cultivate those melon varieties in ToLCNDV infested areas without yield loss.
  • Melon and the wild relatives of melon is/are diploid and has/have 12 pairs of homologous chromosomes, numbered 1 to 12.
  • the effect is measured by defining a symptom level appearing in the plant cell or plant.
  • average symptoms of several plants of a line e.g. 10 or more
  • average symptoms of several plants of a control line or variety preferably a susceptible control line or variety.
  • at least 10 or more individual plants of a line or variety are scored at one time point and the average disease score is calculated.
  • symptom level preferably young plants are infected with ToLCNDV. Young plants are preferably plants having the age of the first true leaf being expanded, preferably approximately 12-15 days after sowing. Infection is preferably carried out via feeding of the vector (Bemisia) carrying the virus. For this purpose plants are germinated and grown under optimal or close to optimal conditions.
  • the symptom level is preferably determined at least once, e.g. 30 days after infection (or later, e.g. 31, 32, 33, 34, 35 days after infection).
  • symptom level is determined twice or even three times at different time-points following infection to confirm the result, e.g.
  • a plant line is said to be resistant against TolCNDV infection if it has an average disease score of 5.0 or higher, while the susceptible control line or variety, such as variety Gandalf (or Gandalf Fl, Nunhems B.V.) or Vedantrais, has an average disease score of 2.5 or less, or 2.0 or less, when grown under the same conditions and infected in the same way.
  • the nucleotide of the introgression fragment is present in homozygous form in the deposited seeds, i.e. the donor nculeotide is present in homozygous form for SNP 01 to SNP 06 (linked to QTL5).
  • the SNPs can, therefore, be used to test the presence of the introgression fragment comprising the QTL5 in a plant cell, plant tissue, plant part, and/or in marker assisted selection (MAS) to transfer the QTLs into other elite melon lines or varieties.
  • MAS marker assisted selection
  • the SNPs can also be used to select plants comprising smaller introgressions fragments than the fragments present in the deposited seeds, whereby the smaller sub-fragments retain the QTL.
  • the SNPs can be used to identify other donors which comprise QTL5 and to introgress these QTLs into cultivated melon.
  • the present invention therefore relates to melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the donor plant in-between SNP 01 and SNP 06, preferably the sequence of the donor plant in-between SNP 01 and SNP 05, more preferably the sequence of the donor plant in- between SNP 01 and SNP 04.
  • the ToLCNDV resistance conferring QTL is present on the introgression fragment, as can be determined by a resistance assay as described herein.
  • the present invention relates to cultivated melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the donor plant in-between SNP 02 and SNP 06, preferably the sequence of the donor plant in-between SNP 02 and SNP 05, more preferably the sequence of the donor melon plant in-between SNP 02 and SNP 04.
  • the present invention further relates to melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor plant in-between SNP 03 and SNP 06, preferably the sequence of the ToLCNDV resistant donor plant in-between SNP 03 and SNP 05.
  • the present invention relates to melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP_03 and SNP_04.
  • a plant may comprise the introgression fragment comprising the sequence of the ToLCNDV resistant donor melon plant in- between SNP 03 and SNP 04, this plant in one aspect comprises a Thymine (T) at nucleotide 68 of SEQ ID NO: 3 and/or an Adenine (A) at nucleotide 227 of SEQ ID NO: 4, i.e. the donor nucleotides.
  • T Thymine
  • A Adenine
  • SNP 03 and SNP 04 are from the recipient, having e.g. a Cytosine (C) at nucleotide 68 of SEQ ID NO: 3 and e.g.
  • SNP 01 , SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06 may all have the resistant donor genotype. Or only SNP 01 and SNP 02 may have the resistant donor genotype; or only SNP 02 and SNP 03 may have the resistant donor genotype; or only SNP 03 and SNP 04 may have the resistant donor genotype, etc. Or only a single SNP, i.e. only SNP 01 , or only SNP 02 or only SNP 03, or only SNP 04, or only SNP 05 or only SNP 06 has the resistant donor genotype.
  • the SNPs that do not have the resistant donor genotype thus have another genotype, the recipient genotype.
  • the recipient genotype for a SNP may be any of the other 3 nucleotides, i.e. for SNP 01 the recipient genotype may be Adenine, Guanine or Thymine.
  • SNP 03 and SNP 04 may both have the resistant donor genotype.
  • only a single SNP, i.e. only SNP 03 or only SNP 04 may have the resistant donor genotype; or even neither SNP 03 nor SNP 04 has the resistant donor genotype, while the sequence in-between still contains QTL5.
  • the introgression fragment comprising the QTL from the donor may be smaller than the chromosome fragment introgressed e.g. in the deposited seeds, but the fragment still comprises the QTL5.
  • a plant can be recognized to contain the introgression fragment (comprising the QTL5) by the phenotype, and/or by transferring the fragment to a susceptible plant and thereby transferring the ToLCNDV resistance phenotype, or by sequencing the region between the SNP markers to identify the donor fragment, or other methods known to the skilled person, such as saturating the region with more SNP markers, allelism tests, identifying the causal gene, etc.
  • the introgression fragment from chromosome 5 of the donor plant comprising the sequence of the donor plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, in-between SNP 03 and SNP 06, in-between SNP 03 and SNP 05, preferably in-between SNP 03 and SNP 04 confers resistance to ToLCNDV to the cultivated melon plant cells according to the invention or to the cultivated melon plants according to the invention.
  • the melon plant cell according to the invention originates from a cultivated melon plant or the melon plant according to the invention, is a cultivated melon plant.
  • the present invention relates to cultivated melon plant cells or melon plants (or plant parts) comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and the introgression fragment is detectable by (comprises) the SNP genotype of the donor plant for one or more (or all) of the following SNPs: SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06, and optionally any SNP in- between SNP 01 and SNP 06.
  • the plant, plant part or plant cell comprises QTL5 and comprises the SNP donor genotype for at least SNP 03, as this SNP is most significantly associated with the ToLCNDV resistance QTL5.
  • the plant, plant part or plant cell comprises the SNP donor genotype for at least SNP 03 and SNP 04, or for at least SNP 03 and SNP 02.
  • the plant, plant part or plant cell comprises QTL5 and comprises the SNP donor genotype for SNP 01, SNP 02 and SNP 03; or for SNP_02, SNP_03 and SNP_04.
  • An introgression fragment may therefore comprise the donor SNP genotype for all SNP markers linked to QTL5 (as in the seeds deposited herein), or a smaller fragment, whereby one or more of the SNP markers is not present. As described further below, even all or all but one donor SNP markers may be absent, while QTL5 is still present on the introgression fragment.
  • the nucleotide sequences (SEQ ID NO: 1 to SEQ ID NO: 6) comprising the SNPs provided herein are the nucleotide sequences of the resistant donor, i.e. they contain the donor SNP nucleotide. Therefore, in one aspect the present invention relates to cultivated melon plant cells or melon plants (or plant parts) comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and the introgression fragment is detectable by (comprises) SEQ ID NO: 1 or an Cytosine at nucleotide 101 of SEQ ID NO: 1 or a Cytosine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO: 1 and/or by SEQ ID NO: 2 or a Thymine at nucleotide 945 of SEQ ID NO: 2 or a Thymine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID
  • Donor plant cell or “donor plant” in connection with the present invention shall mean a melon plant cell or melon plant being resistant to ToLCNDV.
  • DNA fragment or introgression fragment from the donor plant or cell shall mean a fragment of chromosome 5 of a melon plant resistant to ToLCNDV, whereby the fragment confers ToLCNDV resistance when transferred into a TolCNDV susceptible melon plant.
  • the donor plant is a wild species or wild accession of melon.
  • DNA fragments or introgression fragments from donor plant cells or plants are the donor fragments obtained from plants grown from seeds deposited under NCIMB 42585 or progeny obtained from plants grown from seeds deposited under NCIMB 42585 or plants obtained by crosses with plants grown from seeds deposited under NCIMB 42585.
  • Donor melon plants can be obtained from various sources.
  • a person skilled in the art knows how to detect other sources of ToLCNDV resistant donor plants.
  • basically melon plants can be infected with ToLCNDV, either by mechanical means, as described in Lopez et al. (2015, Euphytica 204(3), 679-691) or by transmission of the virus by whiteflies.
  • infection occurs by whitefly infection in context with the present invention.
  • Plants showing reduced symptom levels compared to susceptible controls can then be selected and used as source for genome fragments or sequences conferring ToLCNDV resistance.
  • a preferred method on how to infect melon plants with ToLCNDV and methods for determining the symptom level of infected plants are given herein under "General Methods”.
  • the donor plants preferably have an average symptom level equal to or above 5.0, more preferably equal to or above 6.0, furthermore preferred equal to or above 7.0, even more preferred equal to or above 8.0 and most preferred equal to or above 9.0.
  • the donor plant comprises the donor SNP genotype for one or more or all of SNP 01, SNP 2, SNP 3, SNP 04, SNP 05 and SNP 06, as shown in Table 2.
  • the SNP donor genotype is homozygous.
  • the donor is herein e.g. a wild melon having no agronomic value, e.g. producing white fleshed fruits, having low brix, etc.
  • Recurrent plant cell or “recurrent plant” or “recipient plant” in connection with the present invention shall be understood to be a melon plant cell or melon plant being sensitive (used herein synonymously with susceptible) or non-resistant to ToLCNDV infection. If a plant is sensitive or non-resistant to ToLCNDV can be determined by observation of the symptom levels after ToLCNDV infection.
  • a recurrent plant preferably has an average symptom level below 3.0, more preferably equal to or below 2.5 or equal to or below 2.0. Symptom levels and methods how to infect melon plants with ToLCNDV are described elsewhere herein and are applicable here accordingly.
  • the recurrent melon plant cell according to the invention originates from a cultivated melon plant or the recurrent melon plant according to the invention, is a cultivated melon plant.
  • a cultivated melon plant Preferably it is an elite line, breeding line or variety.
  • “Introgression fragment” refers to a chromosome fragment, chromosome part or region which has been introduced into another plant of the same or related species by crossing or traditional breeding techniques.
  • the introgression of the fragment from a donor plant into a recurrent plant introduces into the offspring of a cross between the donor and recurrent plant a phenotype, which was not present in the recurrent plant.
  • the phenotype transferred from the donor plant to the recurrent plant is resistance to ToLCNDV, e.g. an average disease score of 5.0.
  • the first crossing step can e.g. be followed by one or more backcrossings with the intended breeding line or variety.
  • introgression can mean a first crossing of a ToLCNDV resistant donor plant with a ToLCNDV non-resistant recurrent plant and further back-crossing one or several times ToLCNDV resistant plants obtained from the first crossing with plants of the recipient into which ToLCNDV resistance shall be introgressed.
  • the introgressed fragment is the result of breeding methods referred to by the verb "to introgress” (such as backcrossing) into a recipient variety or breeding line.
  • introgression of ToLCNDV resistance into a recurrent plant is a technical process directed by man.
  • introgression herein refers to a manmade breeding process or method.
  • One or more or all of the molecular markers (SNP markers) provided herein can be used in that process.
  • the resulting plant i.e. the cultivated line or variety comprising one introgression fragment (on chromosome 5) from a donor, i.e. comprising a recombinant chromosome 5, is also man-made and does not exist in nature.
  • the introgression fragment can be large, e.g. even half of a chromosome, but is preferably smaller, such as about 15 Mb or less, such as about 10 Mb or less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2 Mb or less, about 1 Mb (equals 1 ,000,000 base or less), or about 0.8 Mb (equals 800,000 base pairs) or less.
  • the introgression fragment can originate from a wild melon plant or wild melon accession or wild relatives of melon or landraces (donor). Wild melon plants or wild melon accessions or wild relatives of melon plants or landraces can be used to introgress fragments of the donor genome into the genome of cultivated melon, Cucumis melo, to generate breeding lines or varieties with good agronomic characteristics.
  • a cultivated melon plant thus has a "genome of cultivated C. melo ", but comprises in its genome a fragment of a donor, e.g. an introgression fragment of a related wild Cucumis genome, such as Cucumis melo ssp. agrestis, C. melo ssp.
  • telomere length a part or parts of the recurrent (recipient) melon plant and in addition parts of the donor melon plant.
  • the chromosome 5 of cultivated melon comprises an introgression fragment
  • the introgression fragment from the donor may comprise one or more or all of the donor SNP nucleotides (for SNP_01 , SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06) or one or more or all of the sequences comprising the donor SNP nucleotides (SEQ ID NO: 1 , 2, 3, 4, 5 and/or SEQ ID NO: 6). So, e.g.
  • the introgression fragment (in homozygous or heterozygous form) may comprise, and is detectable by, one or more or all of the following SNP genotypes: the CC or CT genotype for SNP 01 in SEQ ID NO: 1 , and/or the TT or TG genotype for SNP 02 in SEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/or the CC or CT genotype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6.
  • the lower part of chromosome 5 of cultivated melon, below marker CMGAAN144 on chromosome 5 on page 3 of Diaz et al 2015 (Mol. Breeding 35: 188), or between marker CMGAAN144 and marker AI 13-H12, comprises QTL5 from a donor.
  • the donor plant of the invention is not one of the five Cucumis melo subsp. agrestis accessions (subsp. agrestis var. momordica: Mom-Khalnd/ Kharbuja, Mom-PI124Ind/ PI124112, Mom- Pi 124Ind/PI414723 and subsp.
  • breeding encompasses herein crossing, backcrossing, selfing, selection, double haploid production, embryo rescue, protoplast fusion, marker assisted selection, mutation breeding etc. as known to the breeder (i.e. methods other than genetic modification/transformation/transgenic methods), by which, for example, a recombinant chromosome 5 can be obtained, identified, produced and/or transferred.
  • the introgression fragment originates form a wild Cucumis plant or a wild Cucumis accession, most preferably the introgression fragment originates from wild Cucumis melo ssp. melo, having small fruits (no more than 6 cm long), with white, acid tasting, fruit flesh.
  • This donor was used in the instant invention, but other donors can be identified by the skilled person which comprise e.g. the same SNP genotype as this donor for SNP 01 to SNP 06 and comprises a QTL in the same region of chromosome 6.
  • Plant variety is a group of plants within the same botanical taxon of the lowest grade known, which (irrespective of whether the conditions for the recognition of plant breeder's rights are fulfilled or not) can be defined on the basis of the expression of characteristics that result from a certain genotype or a combination of genotypes, can be distinguished from any other group of plants by the expression of at least one of those characteristics, and can be regarded as an entity, because it can be multiplied without any change.
  • plant variety cannot be used to denote a group of plants, even if they are of the same kind, if they are all characterized by the presence of one or two loci or genes (or phenotypic characteristics due to these specific loci or genes), but which can otherwise differ from one another enormously as regards the other loci or genes.
  • Fl, F2, F3, etc. refers to the consecutive related generations following a cross between two parent plants or parent lines. The plants grown from the seeds produced by crossing two plants or lines is called the Fl generation. Selling the Fl plants results in the F2 generation, etc.
  • Fl hybrid plant (or Fl hybrid seed) is the generation obtained from crossing two inbred parent lines.
  • Fl hybrid seeds are seeds from which Fl hybrid plants grow.
  • Fl hybrids are more vigorous and higher yielding, due to heterosis.
  • Inbred lines are essentially homozygous at most loci in the genome.
  • a "plant line” or “breeding line” refers to a plant and its progeny.
  • the term “inbred line” refers to a plant line which has been repeatedly selfed and is nearly homozygous.
  • an "inbred line” or “parent line” refers to a plant which has undergone several generations (e.g. at least 5, 6, 7 or more) of inbreeding, resulting in a plant line with a high uniformity.
  • "Uniformity" or “uniform” relates to the genetic and phenotypic characteristics of a plant line or variety. Inbred lines are genetically highly uniform as they are produced by several generations of inbreeding. Likewise, and the Fl hybrids which are produced from such inbred lines are highly uniform in their genotypic and phenotypic characteristics and performance.
  • plant cells according to the invention and plants according to the invention are characterized in that the introgression fragment conferring ToLCNDV resistance originates from the seeds deposited under NCIMB 42585 or progeny thereof.
  • the melon plant cell according to the invention originates from a cultivated melon plant or the melon plant according to the invention is a cultivated melon plant and the introgression fragment originates form a wild Cucumis plant or a wild Cucumis accession or from donor plants described herein to be preferred donor plants or obtained from seeds deposited under NCIMB 42585 or progeny thereof.
  • Chrosome 5 of a melon plant is to be understood in context of the present invention as the scaffolds, fragments, regions, markers and nucleic acid sequences assigned by the ICuGI (International Cucurbit Genomics Initiative) to belong to chromosome 5 of the melon genome.
  • Orthologous chromosome 5" refers to the chromosome 5 of wild relatives of melon, parts of which can be introgressed into cultivated melon chromosome 5.
  • a “recombinant chromosome” refers to a chromosome having a new genetic makeup arising through crossing over between homologous chromosomes, e.g. a "recombinant chromosome 5", i.e. a chromosome 5 which is not present in either of the parent plants and arose through a rare crossing-over event between homologous chromosomes of a chromosome 5 pair.
  • a recombinant melon chromosome 5 comprising a ToLCNDV-resistance conferring QTL is provided.
  • the recombinant chromosome 5 therefore is a chromosome of cultivated melon, with an introgression fragment from a wild donor, whereby the introgression fragment comprises the ToLCNDV resistance conferring QTL.
  • the current version CM 3.5.1 of the C melo genome map is of March 4th, 2012 and the map of chromosome 5 is referred to as ICuGI V (or LG V, or Linkage Group V).
  • Further information including additional markers and mapping information in addition to the ICuCI data is available from Diaz et al. (2015, Mol Breeding 35, 188) and the additional data included in the online version of the respective article.
  • Cropon plant refers to plants of Cucumis melo i.e. varieties, breeding lines or cultivars of the species C. melo, cultivated by humans and having good agronomic characteristics, especially producing edible and marketable fruits of good size and quality and uniformity; such plants are not "wild melon plants", i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and e.g. grow naturally in wild populations.
  • Wild plants include for example ecotypes, PI (Plant Introduction) lines, landraces or wild accessions or wild relatives of a species.
  • a SNP is the variation of the single nucleotide at the given position in a genome between two plants. If a wild melon plant having a ToLCNDV resistance (donor plant) shows in its corresponding sequence at a specific single position a nucleotide which is different from the corresponding nucleotide at the same position of a cultivated melon plant, the position defines a SNP between the wild melon and the cultivated melon.
  • a SNP occurs, when the cultivated plant has either of the remaining three possible nucleotides at the same corresponding sequence position.
  • a cultivated melon plant comprising an introgression fragment from a donor, it can therefore easily be determined if the single nucleotide of the SNP is from the donor or from the cultivated melon (recipient).
  • the SNP nucleotide of the ToLCNDV resistant donor is a Cytosine (C) for nucleotide 101 of SEQ ID NO: 1, while the SNP genotype of a plant or cell comprising SEQ ID NO: 1 can be CC (Cytosine on both chromosomes) or CT (Cytosine on one chromosome and Thymine on the other chromosome), whereby the ToLCNDV resistant donor SNP nucleotide (Cytosine), and thus SEQ ID NO: 1 (or a sequence substantially identical to SEQ ID NO: 1), is homozygous or heterozygous.
  • C Cytosine
  • SNP donor genotype refers to the donor SNP nucleotide being present in homozygous or in heterozygous form, i.e. for SNP 01 the SNP donor genotype is either CC or CT.
  • SNP 01 which is alternatively designated “mMEl 1320_k” is to be understood in context with the present invention to be a SNP at position 101 in SEQ ID NO 1.
  • SEQ ID NO 1 or a sequence substantially identical to SEQ ID NO 1 can be found on chromosome 5 in the ICuGI data set.
  • the relative position of SNP 01 according to markers published by ICuGI is derivable from Table 1.
  • the nucleotide sequence comprising SNP 01 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 1, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO 1 , further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 1 , even more preferred at least 97%> identity with the nucleotide sequence shown under SEQ ID NO 1, even further more preferred at least 98%> identity with the nucleotide sequence shown under SEQ ID NO 1 , in particular preferred at least 99%> identity with the nucleotide sequence shown under SEQ ID NO 1 or more particularly preferred at least 99,5%> identity with the nucleotide sequence shown under SEQ ID NO 1 under the provision that in each case the nucleotide at position 101 in SEQ ID NO 1 is different from the corresponding nucleotide at the same position of the recurrent plant.
  • the ToLCNDV resistant donor plants used in the invention have a 'C'(Cytosine) at position 101 in SEQ ID NO 1 or in a sequence comprising substantial sequence identity to SEQ ID NO: l .
  • SNP 01 is characterized in that the recurrent plant has an A, G or T at position 101 in SEQ ID NO 1.
  • the recurrent plant has a T at position 101 in SEQ ID NO 1.
  • SNP 01 is characterized in that the donor plant has a C at position 101 of SEQ ID NO 1 (or at the equivalent position of a sequence comprising substantial sequence identity to SEQ ID NO: 1) and the recurrent plant has a T at position 101 in SEQ ID NO 1.
  • SNP 02 which is alternatively designated “mME43070_k” is to be understood in context with the present invention to be a SNP at position 945 in SEQ ID NO 2.
  • SEQ ID NO 2 or a sequence substantially identical to SEQ ID NO 2 can be found on chromosome 5 in the ICuGI data set. The relative position of SNP 02 according to markers published by ICuGI is derivable from Table 1.
  • the nucleotide sequence comprising SNP 02 has a nucleotide sequence having at least 85%> identity with the nucleotide sequence shown under SEQ ID NO 2, more preferably at least 90%> identity with the nucleotide sequence shown under SEQ ID NO 2, further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 2, even more preferred at least 97% identity with the nucleotide sequence shown under SEQ ID NO 2, even further more preferred at least 98% identity with the nucleotide sequence shown under SEQ ID NO 2, in particular preferred at least 99% identity with the nucleotide sequence shown under SEQ ID NO 2 or more particularly preferred at least 99,5% identity with the nucleotide sequence shown under SEQ ID NO 2 under the provision that in each case the nucleotide at position 945 in SEQ ID NO 2 is different from the corresponding nucleotide at the same position of the recurrent plant.
  • sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 2 are referred to as having substantial sequence identity to SEQ ID NO: 2.
  • the ToLCNDV resistant donor plants used in the invention have a 'T' (Thymine) at position 945 in SEQ ID NO 2 or in a sequence comprising substantial sequence identity to SEQ ID NO: 2.
  • SNP 02 is characterized in that the recurrent plant has an A, C or G at position 945 in SEQ ID NO 2 or in a sequence comprising substantial sequence identity to SEQ ID NO: 2.
  • the recurrent plant has a 'G' (Guanine) at position 945 in SEQ ID NO 2 or in a sequence comprising substantial sequence identity to SEQ ID NO: 2.
  • SNP 02 is characterized in that the donor plant has a T at position 945 in SEQ ID NO 2 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 2) and the recurrent plant has a G at position 945 in SEQ ID NO 2 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 2).
  • SNP 03 which is alternatively designated “mME10621_k” is to be understood in context with the present invention to be a SNP at position 68 in SEQ ID NO 3.
  • SEQ ID NO 3 or a sequence substantially identical to SEQ ID NO 3 can be found on chromosome 5 in the ICuGI data set.
  • the relative position of SNP 03 according to markers published by ICuGI is derivable from Table 1.
  • the nucleotide sequence comprising SNP 03 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 3, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO 3, further more preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO 3, even more preferred at least 97% identity with the nucleotide sequence shown under SEQ ID NO 3, even further more preferred at least 98% identity with the nucleotide sequence shown under SEQ ID NO 3, in particular preferred at least 99% identity with the nucleotide sequence shown under SEQ ID NO 3 or more particularly preferred at least 99,5% identity with the nucleotide sequence shown under SEQ ID NO 3 under the provision that in each case the nucleotide at position 68 in SEQ ID NO 3 is different from the corresponding nucleotide at the same position of the recurrent plant.
  • the ToLCNDV resistant donor plants used in the invention have a ' (Thymine) at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3).
  • SNP 03 is characterized in that the recurrent plant has an A, C or G at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3).
  • the recurrent plant has a C position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3).
  • SNP 03 is characterized in that the donor plant has a 'T' at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO:
  • the recurrent plant has a 'C at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3).
  • SNP 04 which is alternatively designated “mME50729_k” is to be understood in context with the present invention to be a SNP at position 227 in SEQ ID NO 4.
  • SEQ ID NO 4 or a sequence substantially identical to SEQ ID NO 4 can be found on chromosome 5 in the ICuGI data set.
  • the relative position of SNP 04 according to markers published by ICuGI is derivable from Table 1.
  • the nucleotide sequence comprising SNP 04 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 4, more preferably at least 90%> identity with the nucleotide sequence shown under SEQ ID NO 4, further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 4, even more preferred at least 97%> identity with the nucleotide sequence shown under SEQ ID NO 4, even further more preferred at least 98%> identity with the nucleotide sequence shown under SEQ ID NO 4, in particular preferred at least 99%> identity with the nucleotide sequence shown under SEQ ID NO 4 or more particularly preferred at least 99,5%> identity with the nucleotide sequence shown under SEQ ID NO 4 under the provision that in each case the nucleotide at position 227 in SEQ ID NO 4 is different from the corresponding nucleotide at the same position of the recurrent plant.
  • the ToLCNDV resistant donor plants used in the invention have an 'A' (Adenine) at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4).
  • SNP 04 is characterized in that the recurrent plant has an C, T or G at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO:
  • the recurrent plant has a G position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4).
  • SNP 04 is characterized in that the ToLCNDV resistant donor plant has an A at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4) and the recurrent plant has a G at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4).
  • SNP 05 which is alternatively designated "mME32395_k” is to be understood in context with the present invention to be a SNP at position 839 in SEQ ID NO 5.
  • SEQ ID NO 5 or a sequence substantially identical to SEQ ID NO 5 can be found on chromosome 5 in the ICuGI data set.
  • the relative position of SNP 05 according to markers published by ICuGI is derivable from Table 1.
  • the nucleotide sequence comprising SNP 05 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 5, more preferably at least 90%> identity with the nucleotide sequence shown under SEQ ID NO 5, further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 5, even more preferred at least 97%> identity with the nucleotide sequence shown under SEQ ID NO 5, even further more preferred at least 98%> identity with the nucleotide sequence shown under SEQ ID NO 5, in particular preferred at least 99%> identity with the nucleotide sequence shown under SEQ ID NO 5 or more particularly preferred at least 99,5%> identity with the nucleotide sequence shown under SEQ ID NO 5 under the provision
  • the ToLCNDV resistant donor plants used in the invention have a 'C'(Cytosine) at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5).
  • SNP 05 is characterized in that the recurrent plant has an A, T or G at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5).
  • the recurrent plant has a T position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5).
  • SNP 05 is characterized in that the ToLCNDV resistant donor plant has a C at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5) and the recurrent plant has a T at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5).
  • SNP 06 which is alternatively designated “mME49184_k” is to be understood in context with the present invention to be a SNP at position 445 in SEQ ID NO 6.
  • SEQ ID NO 6 or a sequence substantially identical to SEQ ID NO 6 can be found on chromosome 5 in the ICuGI data set.
  • the relative position of SNP 06 according to markers published by ICuGI is derivable from Table 1.
  • the ToLCNDV resistant donor plants used in the invention have an 'A' (Adenine) at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6).
  • SNP 06 is characterized in that the recurrent plant has a C, T or G at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6).
  • the recurrent plant has a G position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6).
  • SNP 06 is characterized in that the ToLCNDV resistant donor plant has an A at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6) and the recurrent plant has a G at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6).
  • CMPSNP682 SNP 5 15.854.444 57,3 50,4 0009 4.703.882
  • CMPSNP1018 SNP 5 20.088.198 50,9 50,7 0003 808.669
  • CMPSNP741 SNP 5 23.620.132 0003 4.340.603
  • CMPSNP1115 SNP 5 26.639.938 84,6 0003 7.360.409
  • the molecular markers described herein may be detected according to standard methods.
  • SNP markers can be detected using a KASP-assay (see Www.kpbioscience.co.uk) or other assays.
  • a KASP-assay has been developed for SNPs described herein. Respective details are disclosed in the Example section. Sequences used in the respective KASP-assays are given in the Sequence Listing.
  • sequence similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity.
  • nucleic acid sequence e.g. DNA or genomic DNA
  • nucleic acid sequence having "substantial sequence identity to" a reference sequence or having a sequence identity of at least 80%, e.g. at least 85%, 90%, 95%, 98%, 99%, 99.2%, 99.5%, 99.9% nucleic acid sequence identity to a reference sequence
  • said nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using stringent hybridization conditions.
  • the nucleic acid sequence comprises one or more mutations compared to the given nucleotide sequence but still can be identified using stringent hybridisation conditions.
  • Cultivated melon plants comprising a ToLCNDV resistance conferring fragment introgressed on chromosome 5 show reduced symptoms when infected with ToLCNDV, while susceptible controls (lacking the introgression fragment) show the expected severe symptoms in the same conditions.
  • a homozygous introgression fragment comprising the donor genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06 in homozygous form
  • a plant which is resistant to ToLCNDV infection having an average disease score of at least 4.0, preferably at least 5.0, preferably at least 6.0 or preferably at least 7.0.
  • a preferred test for determining the symptom levels is given below under "General Methods”.
  • plant cells according to the invention or plants according to the invention are characterized in that they upon infection with ToLCNDV show a symptom level between 4 and 6, more preferably a symptom level of between 5 and 6, while the susceptible controls show a symptom level of 2 or less.
  • Resistance to ToLCNDV conferred by the introgression fragment is expressed in a dominant manner and thus can be observed when only one chromosome 5 comprises the introgression fragment comprising the sequence of the donor plant between the markers of chromosome 5 disclosed herein.
  • melon plant cells according to the invention or melon plants according to the invention, wherein the introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant comprising the sequence of the donor plant in- between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in- between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, in- between SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or preferably in-between SNP 03 and SNP 04 is present in heterozygous state.
  • the introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant comprising the sequence of the donor plant in- between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in- between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 03
  • At least one chromosome 5 in the plant cells according to the invention or plants according to the invention comprise the just described introgression fragment. It is however understood, that chromosome 5 in respect to the just described introgression fragment can also be present in the homozygous state without diminishing the degree of resistance, because of the dominance of the ToLCNDV resistance conferred by the just described introgression fragment.
  • the invention comprises plant cells according to the invention or plants according to the invention which comprise the just described introgression fragment in a heterozygous or homozygous state.
  • Table 2 illustrates the SNP genotype of plants or cells comprising the ToLCNDV resistant donor SNPs in homozygous form or heterozygous form, as well as the recurrent parent SNP genotype, lacking the introgression fragment.
  • a cultivated melon plant (of the species C. melo) which comprises a recombinant chromosome 5, whereby the recombinant chromosome 5 comprises an introgression fragment that confers ToLCNDV resistance onto the melon plant when present in homozygous or heterozygous form and wherein the introgression fragment is from a wild donor of the species C. melo.
  • the introgression fragment comprises the SNP donor genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and/or SNP 06.
  • the introgression fragment comprises the donor SNP genotype for at least SNP 03 and/or SNP 04.
  • the SNP genotype of the donor may be present in homozygous form (if the introgression fragment is in homozygous form) or in heterozygous form (if the introgression fragment is in heterozygous form).
  • the plant or plant cell or plant part may comprise the TT or TC genotype for SNP 03 at nucleotide 68 of SEQ ID NO: 3.
  • the plant, plant cell, or plant part may comprise SEQ ID NO: 3, or a sequence comprising at least 95%, 97%, or 98%> sequence identity to SEQ ID NO: 3, in homozygous form or in heterozygous form.
  • cultivated melon plants or cells of these plants which comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 06 (or in- between SEQ ID NO: l and SEQ ID NO:6, or a sequence comprising at least 90%, or at least 95%, or at least 97%> or 98%> sequence identity to SEQ ID NO: 1 or to SEQ ID NO 6); or whereby the introgression fragment lies in-between SNP 01 and SNP 05 (or in-between SEQ ID NO:l and SEQ ID NO:5, or a sequence comprising at least 90%>, or at least 95%>, or at least 97%> or 98%> sequence identity to SEQ ID NO: 1 or to SEQ ID NO 5).
  • the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 06 (or in-between SEQ ID NO: l and SEQ ID NO:6, or a sequence comprising at least 90%>, or at least 95%>, or at least 97%> or 98%> sequence identity to SEQ ID NO: 1 or to SEQ ID NO 6), or the fragment lies in-between SNP 01 and SNP 05 (or in-between SEQ ID NO:l and SEQ ID NO:5, or a sequence comprising at least 90%>, or at least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 1 or to SEQ ID NO 5), and whereby the introgression fragment comprises a Thymine (T) at nucleotide 68 of SEQ ID NO 3 or at the equivalent nucleotide of a sequence comprising substantial sequence identity to SEQ ID NO: 3.
  • the introgression fragment optionally further comprises an Adenine (A) at nucleotide 227 of SEQ ID NO 4 or at the equivalent nucleotide of a sequence comprising substantial sequence identity to SEQ ID NO: 4.
  • A Adenine
  • the introgression fragment optionally further comprises the donor nucleotide of SNP 02 and/or SNP 05.
  • the introgression fragment may comprise the donor genotype for SNP_03, SNP_04 and SNP_05; or for SNP_03 and SNP_02; or for SNP_03 and SNP_02 and SNP_04; or for SNP_03, SNP_02, SNP_04 and SNP_05.
  • the introgression fragment optionally further comprises the donor nucleotide of SNP 02 and/or SNP 01.
  • the introgression fragment may comprise the donor genotype for SNP 03 and SNP 02; or for SNP 03, SNP 02 and SNP 01; and optionally also for SNP 04, and further optionally also for SNP 05, and further optionally for SNP 06.
  • the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 06 (or in-between SEQ ID NO:l and SEQ ID NO:6, or a sequence comprising at least 90%, or at least 95%, or at least 97%) or 98%o sequence identity to SEQ ID NO: 1 or to SEQ ID NO 6) and whereby the introgression fragment comprises the ToLCNDV resistant SNP genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05, and SNP 06.
  • the fragment comprises the donor SNP genotype for SNP 03 and/or SNP 04.
  • the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 05 (or in-between SEQ ID NO:l and SEQ ID NO:5, or a sequence comprising at least 90%>, or at least 95%>, or at least 97% or 98% sequence identity to SEQ ID NO: 1 or to SEQ ID NO 5) and whereby the introgression fragment comprises the ToLCNDV resistant SNP genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04 and SNP 05.
  • the fragment comprises the donor SNP genotype for SNP 03 and/or SNP 04.
  • the resistant SNP genotype is also present for SNP 06.
  • the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 03 and SNP 04 (or in-between SEQ ID NO:3 and SEQ ID NO:4, or a sequence comprising at least 90%>, or at least 95%>, or at least 97%o or 98%o sequence identity to SEQ ID NO: 3 or to SEQ ID NO 4) and whereby the introgression fragment optionally comprises the ToLCNDV resistant SNP genotype for SNP 03 and/or SNP 04.
  • the donor genotype is also present for one or more or all of SNP markers selected from SNP_01, SNP_02, SNP_05 and SNP_06.
  • the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 02 and SNP 04 (or in-between SEQ ID NO:2 and SEQ ID NO:4, or a sequence comprising at least 90%, or at least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 2 or to SEQ ID NO 4) and whereby the introgression fragment optionally comprises the ToLCNDV resistant SNP genotype for SNP 02 and/or SNP 03 and/or SNP 04.
  • the donor genotype is also present for one or more or all of SNP markers selected from SNP 01, SNP 05 and SNP 06. Plants comprising plant cells according to the invention are another embodiment of the invention.
  • the melon plant according to the invention may be an inbred line, an open pollinated variety (OP) or an Fl hybrid.
  • the Fl hybrid comprises the introgression fragment in heterozygous form, i.e. produced by crossing two inbred parent lines, one of which possesses the introgression fragment (preferably in homozygous form, although not necessarily) and collecting the Fl hybrid seeds from said cross.
  • the Fl hybrid may also comprise the introgression fragment in homozygous form, i.e. produced by crossing two inbred parent lines, each comprising the introgression fragment in homozygous or heterozygous form.
  • the melon plant according to the invention may be of any type. Preferably it has good agronomic and good fruit quality characteristics, such as large average fruit size (at least 500g, 600g, 700g, 800g, 900g, lOOOg or more), high average brix of the fruits (e.g. an average refractometer % total soluble solids of at least 10%, 12%, 14%, 16%, 18% or more), many fruits being produced per plant, firm fruit flesh, etc.
  • large average fruit size at least 500g, 600g, 700g, 800g, 900g, lOOOg or more
  • high average brix of the fruits e.g. an average refractometer % total soluble solids of at least 10%, 12%, 14%, 16%, 18% or more
  • many fruits being produced per plant firm fruit flesh, etc.
  • resistances may be introduced into the melon plants of the invention, such as resistance to one or more of the following diseases: Bacterial Wilt, Root Rot, Crown Blight, Melon Rust, Powdery Mildew, Verticillum Wilt, Sulphur Bum, Scab, Watermelon Mosaic, Downy Mildew, Fusarium oxysporum fsp. melonis (Fom) race 0, Fusarium oxysporum fsp. melonis (Fom) race 1 , Fusarium oxysporum fsp. melonis (Fom) race 2, Fusarium oxysporum fsp. melonis (Fom) race 1.2, Fusarium Wilt
  • Root Knot Nematode
  • Anthracnose Cucumber Mosiac
  • Squash Mosaic and/or resistance to one or more of the following pests: Aphid resistance, Pickle Worm, Darkling Ground Beetle, Banded Cucumber Beetle, Mite, Western Spotted Cucumber Beetle, Melon Leafhopper, Melon Worm, Western Striped Cucumber Beetle or Melon Leafminer.
  • Aphid resistance Pickle Worm, Darkling Ground Beetle, Banded Cucumber Beetle, Mite, Western Spotted Cucumber Beetle, Melon Leafhopper, Melon Worm, Western Striped Cucumber Beetle or Melon Leafminer.
  • Other resistance genes, against pathogenic viruses, fungi, bacteria or pests may also be introduced.
  • a specific aspect of the invention concerns plants or plant cells comprising an introgression fragment according to the invention which introgression fragment is obtainable from seeds deposited under NCIMB 42585 or from progeny thereof.
  • the seeds deposited are cultivated melon plants of the BC4S4 generation comprising the introgression fragment in homozygous form, with the donor nucleotide being present in homozygous form for SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06.
  • the TolCNDV resistance is most likely located in-between SNP 03 and SNP 04, which means that the size of the donor introgression can be reduced, by selecting recombinants having smaller introgression fragment sizes.
  • So plants comprising sub-fragments of the introgression fragment (wherein said sub- fragments still confer TolCNDV resistance), comprising the donor SNP for SNP 03 and/or SNP 04, but having the SNP genotype of the recurrent parent for one or more or all of the other SNPs can be generated in ways known to the skilled person.
  • Whether a plant comprises the ToLCNDV resistance from the deposited seeds can be determined by various methods, such as sequencing and comparing the sequences of the introgression fragments and recombination sites.
  • Melon plants and plant parts comprising melon plant cells according to the invention are also an embodiment of the invention.
  • seeds from which such plants can be grown are encompassed herein, as well as parts of such seeds (e.g. cells or tissues of the seeds such as the seed coat, embryo, etc.).
  • a further aspect of the present invention concerns melon seeds comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, in- between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in- between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or in-between SNP 03 and SNP 04.
  • the seeds comprise an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, more preferably in-between SNP 01 and SNP 05, even more preferably in-between SNP 02 and SNP 05, furthermore preferably in-between SNP 02 and SNP 04 and most preferably in-between SNP 03 and SNP 04.
  • Another embodiment of the invention concerns melon seeds obtainable or obtained from plants according to the invention, or seeds comprising plant cells according to the invention.
  • a further aspect of the present invention concerns melon plant fruits comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or in-between SNP 03 and SNP 04.
  • the seeds comprise an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, more preferably in-between SNP 01 and SNP 05, even more preferably in-between SNP 02 and SNP 05, furthermore preferably in-between SNP 02 and SNP 04 and most preferably in-between SNP 03 and SNP 04.
  • Another embodiment of the invention concerns melon fruits obtainable or obtained from plants according to the invention, or fruits comprising plant cells according to the invention.
  • melon fruits according to the invention are characterized in that they comprise an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant comprising the sequence of the donor plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in- between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in- between SNP 02 and SNP 04, in-between SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or preferably in-between SNP 03 and SNP 04 in heterozygous or homozygous state.
  • the preferred and further embodiments described herein for melon plant cells or melon plants according to the invention are applicable to also represent preferred and further embodiments of the melon fruits of melon plants according to the invention, accordingly.
  • a further aspect of the present invention concerns melon plant propagation material comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or in-between SNP 03 and SNP 04.
  • the propagation material comprises an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, more preferably in-between SNP 01 and SNP 05, even more preferably in-between SNP 02 and SNP 05, furthermore preferably in-between SNP 02 and SNP 04 and most preferably in-between SNP 03 and SNP_04.
  • Another embodiment of the invention concerns melon plant propagation material obtainable or obtained from plants according to the invention, or melon plant propagation material comprising plant cells according to the invention.
  • propagation material comprises those components of the plant which are suitable for generating progeny via the vegetative (agamic) or generative (gamic, sexual) route.
  • Suitable for vegetative propagation are, for example, cuttings, in vitro tissue, cell, protoplast, embryo or callus cultures, micropropagation methods, rhizomes or tubers.
  • Other propagation material includes, for example, fruits, seeds, seedling, being homozygous or heterozygous for an chromosome 5 introgression fragment conferring ToLCNDV resistance etc.
  • the propagation material in one aspect takes the form of cuttings which are propagated by grafting to another rootstock or in vitro tissue culture material, in particular embryo cultures.
  • non-propagating plant cells comprising the recombinant chromosome 5 described herein are provided.
  • such non-propagating plant cells may however be part of a melon plant or melon plant part.
  • a further embodiment of the invention concerns a method for producing a ToLCNDV resistant melon plant comprising the following steps a) Selecting a ToLCNDV resistant donor plant b) Crossing the donor plant selected in step a) with a recurrent plant sensitive to ToLCNDV c) Obtaining seeds from the plants crossed in step b) and optionally d) Verifying if the plants grown from the seeds obtained in step c) are resistant to ToLCNDV and/or comprise one or more of the SNPs from the donor plant selected from the group of SNP 01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.
  • a ToLCNDV resistant donor plant in step a) in the method for producing a ToLCNDV resistant melon plant according to the invention can be selected by infection of melon plants with ToLCNDV and determining the level of symptoms of ToLCNDV infected melon plants as described elsewhere herein. The same is applicable for verification in steps b) and c) of the method for producing a ToLCNDV resistant melon plant according to the invention, if a plant is ToLCNDV sensitive or resistant, respectively.
  • the method for producing a ToLCNDV resistant melon plant according to the invention is used for producing a plant according to the invention.
  • the preferred and further embodiments as described herein for the plants according to the invention are applicable accordingly to the method for producing a ToLCNDV resistant melon plant according to the invention.
  • Plants obtainable or obtained by a method for producing a ToLCNDV resistant melon plant according to the invention are also an embodiment of the invention.
  • a further embodiment of the invention concerns methods for producing melon seeds comprising the following steps a) growing a melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, b) harvesting the fruits of the melon plants grown in step a) c) collecting the seeds from the fruits obtained in step b).
  • the melon plants of steps a) of the method for producing melon seeds according to the invention has the specific characteristics described as preferred and further embodiments of the plants according to the invention.
  • the preferred and further embodiments as described herein for the plants according to the invention are applicable accordingly to the method for producing a hybrid melon seed according to the invention.
  • Seeds obtainable the method for producing melon seeds according to the invention are also an embodiment of the invention.
  • Another embodiment of the invention concerns methods for producing hybrid melon seeds comprising the following steps a) providing a first inbred melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP_04, b) providing a second inbred melon plant with or without a chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, c) crossing the plant provided in step a) with the plant provided in step b) d) selecting seeds obtained from the cross of step c).
  • Inbred plant or “inbred line” shall mean in connection with the present invention plants which have undergone several generations of selfing and are highly uniform in respect to their genetic setup and phenotypic appearance.
  • the inbred lines of steps a) and b) of the method for producing hybrid melon seeds according to the invention has the specific characteristics described as preferred and further embodiments of the plants according to the invention.
  • the preferred and further embodiments as described herein for the plants according to the invention are applicable accordingly to the method for producing a hybrid melon seed according to the invention.
  • Hybrid seeds obtainable or obtained by the method for producing hybrid melon seeds according to the invention are also an embodiment of the invention.
  • a further embodiment of the present invention are methods for producing a melon fruit comprising the following step a) growing a plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, b) harvesting the fruits produced by the plants grown in step a).
  • the term "fruit” in its botanical meaning is commonly understood to be a seed bearing structure developed from the ovary of angiosperm flowers.
  • Melon fruits obtainable or obtained by a method for producing a melon fruit according to the invention are also an embodiment of the invention.
  • Melon donor plants being resistant to ToLCNDV can be identified with the aid of the SNP markers, in particular one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06 disclosed herein.
  • the present invention therefore for the first time enables a person skilled in the art to identify donor plants from which an introgression fragment conferring ToLCNDV resistance to melon plants can be transferred into recurrent melon plants.
  • a further embodiment of the invention therefore pertains the use of one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 or SNP 06 for identification of a ToLCNDV resistant melon plant or parts thereof (such as cells, fruits, leaves).
  • the use pertains the identification of ToLCNDV resistant donor melon plants and/or recurrent melon plants, but also to the identification of breeding lines, cultivars or varieties containing a recombinant chromosome 5, e.g. the recombinant chromosome 5 derived from the seeds deposited herein, optionally comprising a subfragment of the introgression fragment present in the seeds deposited.
  • Another embodiment is the use of one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 or SNP 06 for introgression of ToLCNDV resistance into a TolCNDV susceptible melon plant, especially a cultivated melon line or variety.
  • an embodiment of the invention is the use of one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 or SNP 06 in breeding ToLCNDV resistant melon plants.
  • the method comprises the steps of: screening the genomic DNA for the SNP genotype of one or more or all of SNP 01, SNP 02, SNP_03, SNP_04, SNP_05 and SNP_06; and optionally selecting plants or plant material which comprise the resistant donor genotype of one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.
  • a method of producing C. melo Fl hybrid plants comprising a ToLCNDV resistance phenotype comprising: crossing a first inbred melon plant comprising at least one recombinant chromosome 5, the recombinant chromosome 5 comprising an introgression fragment that confers ToLCNDV resistance onto the first inbred melon plant when present in homozygous or heterozygous form and wherein said introgression fragment is from a wild plant of the species Cucumis melo, with a second inbred melon plant with or without said at least one recombinant chromosome 5 and collecting Fl hybrid seeds from said cross.
  • a Cucumis melo plant, or part thereof, comprising a recombinant chromosome 5, the recombinant chromosome 5 comprising an introgression fragment that confers ToLCNDV resistance onto the Cucumis melo plant when present in homozygous or heterozygous form and wherein said introgression fragment comprises one or more or all of the Single Nucleotide Polymorphism (SNP) markers of the group: the CC or CT genotype for SNP 01 in SEQ ID NO: 1 , the TT or TG genotype for SNP 02 in SEQ ID NO: 2, the TT or TC genotype for the SNP 03 in SEQ ID NO: 3, the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, the CC or CT genoype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6, and wherein said introgression fragment is from a wild plant of the species Cucumis melo, said
  • the cultivated melon plant comprises at least the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4.
  • the ToLCNDV resistance QTL or the introgression fragment comprising the QTL is the obtainable from / can be obtained from / is as present in seeds of which a representative sample has been deposited under Accession Number NCIMB42585 or progeny thereof (whereby the progeny retain the ToLCNDV resistance).
  • the Applicant requests that samples of the biological material and any material derived therefrom be only released to a designated Expert in accordance with Rule 32(1) EPC or related legislation of countries or treaties having similar rules and regulation, until the mention of the grant of the patent, or for 20 years from the date of filing if the application is refused, withdrawn or deemed to be withdrawn.
  • G or A or T N G or A or T or C
  • SEQ ID NO: 1 to SEQ ID NO: 6 the SNP nucleotide of the resistant donor is shown in bold and underlined.
  • SEQ ID NO 2 Sequence of the ToLCNDV resistant donor plant comprising SNP 02.
  • SEQ ID NO 3 Sequence of the ToLCNDV resistant donor plant comprising SNP 03.
  • SEQ ID NO 4 Sequence of the ToLCNDV resistant donor plant comprising SNP 04.
  • SEQ ID NO 5 Sequence of the ToLCNDV resistant donor plant comprising SNP 05.
  • SEQ ID NO 6 Sequence of the ToLCNDV resistant donor plant comprising SNP 06.
  • SEQ ID NO 7 KASP- -assay primer for FAM allele of SNP _01.
  • SEQ ID NO 8 KASP- -assay primer for VIC allele of SNP 01.
  • SEQ ID NO 9 KASP- -assay common primer for SNP 01.
  • SEQ ID NO 10 KASP- -assay primer for FAM allele of SNP _02.
  • SEQ ID NO 12 :KASP- -assay common primer for SNP 02.
  • SEQ ID NO 14 KASP- -assay primer for VIC allele of SNP 03.
  • SEQ ID NO 15 :KASP- -assay common primer for SNP 03.
  • SEQ ID NO 16 KASP- -assay primer for FAM allele of SNP _04.
  • SEQ ID NO 17 :KASP- -assay primer for VIC allele of SNP 04.
  • SEQ ID NO 18 :KASP- -assay common primer for SNP 04.
  • SEQ ID NO 19 KASP- -assay primer for FAM allele of SNP _05.
  • SEQ ID NO 20 :KASP- -assay primer for VIC allele of SNP 05.
  • SEQ ID NO 22 :KASP- -assay primer for FAM allele of SNP _06.
  • SEQ ID NO 23 : :KASP- -assay primer for VIC allele of SNP 06.
  • Fig. 1 Shown is a ToLCNDV sensitive recurrent plant (upper picture) and a recurrent plant into which ToLCNDV resistance was integrated (lower picture) by introgression of a fragment comprising the sequence of the donor in-between SNP 01 and SNP 06. The picture was taken 25 days post infection (dpi) with ToLCNDV by whitefly transmission.
  • Fig. 2 Shown are the symptom levels 35 days post infection (dpi) with ToLCNDV by whitefly transmission of a donor plant being ToLCNDV resistant (Wild Donor), a recurrent plant (Recurrent) and a plant obtained after introgression of the ToLCNDV resistance from the donor plant into the recurrent plant (Introgression).
  • the symptom levels were determined as described herein under "General Methods”.
  • a melon plant (Cucumis melo) infecting strain of ToLCNDV is used for infection of melon plants.
  • a ToLCNDV strain isolated in Murcia, Spain was used as inoculum.
  • the ToLCNDV inoculum source is maintained on living infected melon plants. It must be ensured, that pure virus isolates are used and that neither the virus source, nor the whiteflies are contaminated with other diseases, in particular with other viruses (e.g. CGMMV, CYSDV, CYVY SqMV).
  • CGMMV CGMMV
  • CYSDV CYVY SqMV
  • whiteflies Bemisia tabaci
  • ToLCNDV sensitive susceptible
  • Infected test plants obtained as described under 1.3 were transplanted to bigger pots, transferred into a greenhouse with cooling equipment. The plants were grown at approximately 18°C night temperature and approximately 25°C day temperature in a timeframe of 14 to 16 hours daylight.
  • the infected plants for each infected genotype were grown in two replicates in two different plots, each of which comprises 6 ToLCNDV infected plants and 1 mock infected plant. The plots are randomized in respect to the growing area.
  • Scoring the symptom level of ToLCNDV infection The scoring of the symptom level may already be done approximately 15 days post infection (dpi) with ToLCNDV but is preferably done approximately 30 days post infection (dpi) with ToLCNDV, or later. In case plants are present which show recovery from the virus infection, a further scoring of the symptom is done approximately 45 days post infection (dpi) with ToLCNDV.
  • symptom levels are to be used according to the phenotypes indicated in the following:
  • symptom level 8 - 9 it is recommended to use at least one genotype highly resistant to ToLCNDV (symptom level 8 - 9) and one genotype highly sensitive to ToLCNDV (symptom level 1) in each experimental setup. It is further recommended to also include a genotype being intermediate resistant to ToLCNDV infection in each test setup. Best results are obtained when the just mentioned genotypes are included and the symptom levels of each genotype is scored relative to the results obtained for the highly resistant, highly sensitive and intermediate resistant genotypes. These genotypes also give a clear indication on the amount of infection of melon plants with ToLCNDV by the whiteflies. Furthermore, it is advisable to check infection and spreading of ToLCNDV in infected plants and control plants. This can be done by checking for the presence and amount of virus DNA in upper parts of the plants.
  • a suitable way to check for the presence and amount of ToLCNDV DNA in upper plant parts is hybridization of plant material with a probe hybridizing with the DNA of the ToLCNDV strain used.
  • Various hybridization techniques are well known in the art. A simple so called Dot Blot analysis is sufficient for obtaining valuable results. Likewise PCR or quantitative PCR techniques can be used.
  • the symptom level of wild accessions of melon plants were tested for ToLCNDV resistance according to the test described under "General Methods".
  • a wild donor plant was identified which has a high resistance to ToLCNDV infection, having a resistance level of about 7 (as seen further below the average disease score was 7.4, while the susceptible plant had an average score of 2.0).
  • Three mapping populations were developed including the use of the donor plant obtained in Example 1 to map the position of the ToLCNDV resistance conferring fragment (QTL) in the genome of donor melon plant.
  • mapping populations revealed one major QTL associated with resistance, located on chromosome 5 and showing dominant inheritance patterns.
  • a KASP-assay was developed for identifying the SNPs flanking the QTL.
  • the SNPs associated with the QTL can be determined by use of the following primers in a KASP-assay:
  • AVG average value of tested genotype
  • STDV standard deviation

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Abstract

The application concerns melon plants (Cucumis melo) resistant to infection with tomato leaf curl New Dehli virus (ToLCNDV). The resistant melon plants have a genomic introgression fragment on chromosome 5 which confers tolerance to ToLCNDV in a dominant manner. Also disclosed are markers for identifying those fragments, methods for identifying or producing resistant melon plants.

Description

ToLCNDV resistant melon plants
The application concerns melon plants (Cucumis melo) resistant to infection with tomato leaf curl New Dehli virus (ToLCNDV). The resistant melon plants have a genomic introgression fragment on chromosome 5 which confers tolerance to ToLCNDV in a dominant manner. Also disclosed are markers for identifying those fragments, methods for identifying or producing resistant melon plants.
Tomato leaf curl New Dehli virus (ToLCNDV) is classified as Begomovirus belonging to the family Geminiviridae. ToLCNDV has a bipartite genome consisting of two single stranded DNA molecules referred to as DNA A and DNA B (Saez et al., 2016, Annals of Applied Biology).
ToLCNDV was initially found to infect tomato {Solarium lycopersicum) plants in 1995 in India. Later ToLCNDV was found to infect also plants of other Solanacea species, like Solarium melongena (aubergine), chili pepper (Capsicum spp.) and Solarium tuberosum (potato). In 2012 infection of curcubit species (courgette, Cucurbita pepo var. giromontiina) by ToLCNDV was found in Spain and in 2015 the virus was identified as the disease source in melon, cucumber and courgette in Tunisia. In the meantime, infection of many Curcubitacea species such as Benincasa hispida (wax gourd), Citrullus lanatus (watermelon), Cucumis melo (melon), Cucumis melo var. flexuosus (snake melon), Cucumis sativus (cucumber), Cucurbita moschata (musky gourd), Cucurbita pepo (pumpkin), Cucurbita pepo var. giromontiina (courgette), Lagenaria siceraria (bottle gourd), Luffa cylindrica (sponge gourd), Momordica charantia (bitter gourd) have been proven. Infection of weeds (e.g. Eclipta prostrata - Asteraceae) and other crops such as Hibiscus cannabinus (kenaf - Malvaceae) and Carica papaya (papaya - Caricaceae) was also reported. In the Mediterranean region the disease does occur in various crop species in Italy (Sicilia), Spain and Tunisia. In Asian countries, infection was proven in different crops in Bangladesh, India, Indonesia, Pakistan, Philippines, Sri Lanka, Taiwan and Thailand. Further information on geographical distribution of ToLCNDV is lacking, but from the observations made today, the virus clearly seems to further spread geographically as well as to other crops. Disease symptoms in general comprise phenotypic appearance of yellow mosaic on leaves, leaf curling, vein swelling, and plant stunting. Cucurbits upon infection of young plants with ToLCNDV show stunted growth and decreased or suppressed fruit production. Also fruits showing skin roughness and longitudinal cracking have been reported. Thus, ToLCNDV causes economic losses in various important crop species and is a major threat. Infection of plants by ToLCNDV occurs persistently by transmission of the virus by the phloem sucking whitefly (Bemisia tabaci). (European and Mediterranean Plant Protection Organization, EPPO RS 2015/114, 2016/024, 2016/040, Entry date 2015-06).
In sponge gourd resistance to ToLCNDV has been shown to be controlled by a single dominant gene (Islam et al., 2010, Euphytica 174(l):83-89).
In tomato, transgenic plants resistant to ToLCNDV have been produced by silencing virus genes (Varma & Praveen, 2006, ISB News Report).
Assays for transmission of ToLCNDV by mechanically transferring the sap of an infected zucchini plant to non-infected plants from other cucurbit genera {Cucumis, Cucurbita, Citrullus, Lagenaria) have been developed. Five Cucumis melo accessions (subsp. agrestis var. momordica: Mom-Khalnd/ Kharbuja, Mom-PI124Ind/ PI 124112, Mom-PI124Ind/ PI 414723 and subsp. agrestis wild types: Ag- WM9Ind/WM9, Ag-WM7Ind/WM7) resistant to ToLCNDV were identified (Lopez et al., 2015, Euphytica 204(3), 679-691). Confirmation of resistance of these accessions by using the natural whitefly infection system was not performed.
Saez et al. 2016, pages 214-216 (Proceedings of Cucurbitaeceae 2016, the Xlth EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae July 24-28 2016, Warsaw, Poland) describe that ToLCNDV resistance of the C. melo subsp. agrestis accession WM-7 segregated in an F2 population in a 3: 1 ratio of tolerant : susceptible, suggesting a single dominant resistance gene being present in WM-7. As in Lopez et al. 2015, supra, mechanical inoculation was used, and not natural infection via whitefly vector.
Various Cucurbita species (C. pepo, C. moschata, C. maxima, C. fraternal, C. ficifolia) have been screened for resistance to ToLCNDV by the mechanical sap transmission screening assay. Four Curcubita moschata accessions (PI 604506, PI 381814, Nigerian local, Kurokawa) were found to show low symptoms upon, mechanical infection with ToLCNDV. This result however could be reproduced upon whitefly infection only for two of the accession (PI 604506, PI 381814), demonstrating that resistance to ToLCNDV should be tested not only under artificial conditions but in addition by using the naturally occurring whitefly infection system (Saez et al., 2016, Annals of Applied Biology). Therefore, it is unclear if the accessions identified to be resistant to infection by ToLCNDV are resistant under natural growing conditions when the virus is transferred by whiteflies.
Attempts to control ToLCNDV infection of crop plants comprise vector (whitefly) control by insecticides applications and adaption of cultural practices, including use of virus free crop material (transplants), establishment of crop free periods, weed management (eliminating virus infected weeds) and destruction of infected plants in the field. However, because of the complex epidemiological factors associated with the disease, these attempts are not always effective (Saez et al., 2016, Annals of Applied Biology).
Therefore, there is a need to establish further measurements for reducing ToLCNDV infections, further spreading of the virus to other geographical areas and spreading to other crop species. Breeding of varieties resistant to ToLCNDV would be essential for managing the disease.
An object of the present invention is to provide measurements for the control of ToLCNDV infection in melon plants. The present invention discloses melon plant cells and melon plants being resistant to infections by TolCNDV.
It is commonly known that ToLCNDV does infect various different plant species of the Curcubitaceae species, including melon species. It is also well known that ToLCNDV is transmitted persistently from infected plants to non-infected plants by the plant sucking pest Bemisia tabaci (whitefly). Transfer of ToLCNDV from one crop species to different crop species or even from weed species to crop species has been demonstrated. Whiteflies may pick up ToLCNDV from outside the controlled area even from different species and transfer it to melon plants grown in the controlled area. Whitefly vector control therefore is of limited effectiveness for preventing ToLCNDV infection. TolCNDV resistant melon plants have the advantage that they would withstand infection with TolCNDV without major yield losses, even if plants around the area where the melon plants are grown are infected with ToLCNDV.
The inventors found a ToLCNDV resistant donor accession of melon and have mapped the resistance in a segregating population, using whitefly infection with a Spanish ToLCNDV strain for resistance phenotyping. A Quantitative Trait Locus (QTL) was identified on chromosome 5 (QTL5) and introgressed from the wild donor accession into an elite cultivated melon line. Seeds of the elite line, comprising the QTL5 introgression in homozygous form, were deposited by Nunhems B.V. under Accession number NCIMB 42585 in accordance with the Budapest Treaty. In these seeds the donor genotype for the Single Nucleotide Polymorphism (SNP) markers (SNP 01 to SNP 06) provided herein is present. The donor itself is not uniform and has white fruit flesh with a low brix. It is not an accession of agronomic value. The donor is also not available anymore at the US seed bank. By identifying and transferring QTL5 from the donor into cultivated melon, it is now possible to make cultivated melon varieties and cultivars of high agronomic value (with uniform characteristics and marketable fruits having high brix and good shelf life) with resistance against ToLCNDV and thus it is possible to cultivate those melon varieties in ToLCNDV infested areas without yield loss. "Melon plant cells" or "melon plants" also designated as muskmelon plant cells or muskmelon plants in the art shall be understood in context with the present invention to be plant cells originating from the species Cucumis melo or to be plants belonging to the species Cucumis melo.
Cucumis melo, can be classified into: C. melo cantalupensis, C. melo inodorous and C. melo reticulatus. C. melo cantalupensis are also referred to as Cantaloupes and are primarily round in shape with prominent ribs and almost no netting. Most have orange, sweet flesh and they are usually very fragrant. In contrast to the European cantaloupe, the North American 'Cantaloupe' is not of this type, but belongs to the true muskmelons. C. melo inodorous (or winter melons) can be subdivided into different types, such as Honeydew melon, Piel de Sapo, Sugar melon, Japanese melon, etc. C. melo reticulatus is the true muskmelon, with reticulated skin (netted) and includes Galia melons, Sharlyn melons and the North American cantaloupe.
Melon and the wild relatives of melon is/are diploid and has/have 12 pairs of homologous chromosomes, numbered 1 to 12.
"Resistant" or "being resistant to" shall be understood in context of the present invention to mean a plant which is a host species of a particular pathogen and can therefore be infected by a given pathogen, but wherein the plant comprises a genetic element (e.g. an introgression fragment) resulting in reduction of pathogen growth and/or spreading in the plant after infection compared to the susceptible plant lacking the genetic element. In context of the present invention "resistant" or "being resistant to" in particular refers to plant cells or plants being resistant to ToLCNDV. Resistance is a relative term which can span a range of (different) reactions in the plant cell or plant, triggered by pathogen infection. The effect of those reactions by the plant cell or plant can be measured by various means. Typically the effect is measured by defining a symptom level appearing in the plant cell or plant. Typically average symptoms of several plants of a line (e.g. 10 or more) are compared to average symptoms of several plants of a control line or variety, preferably a susceptible control line or variety. Thus at least 10 or more individual plants of a line or variety are scored at one time point and the average disease score is calculated.
Concerning the present invention, the following commonly known symptom levels are applied according to phenotypic observations taken after ToLCNDV infection:
1 = Dead plant 2 = Severe mosaic and curling, chlorosis and growth reduction. No recovery
3 = Strong mosaic and curling, chlorosis and growth reduction. No recovery
4 = Curling and mosaic, chlorosis, no or mild growth reduction. No recovery
5 = Curling and mosaic, chlorosis, no growth reduction. Slight recovery of the upper plant zone
6 = Mild curling, mosaic and chlorosis, no growth reduction. Recovery of the upper middle plant 7 = Mild curling, mosaic and chlorosis, no growth reduction. Symptoms appear only in the lower plant zone
8 = Faint mosaic
9 = No symptoms For determining the symptom level (or disease score) preferably young plants are infected with ToLCNDV. Young plants are preferably plants having the age of the first true leaf being expanded, preferably approximately 12-15 days after sowing. Infection is preferably carried out via feeding of the vector (Bemisia) carrying the virus. For this purpose plants are germinated and grown under optimal or close to optimal conditions. The symptom level is preferably determined at least once, e.g. 30 days after infection (or later, e.g. 31, 32, 33, 34, 35 days after infection). Optionally symptom level is determined twice or even three times at different time-points following infection to confirm the result, e.g. a first scoring at approximately 15, 20 or 25 days after infection and a second scoring at approximately 30 days after infection (or later, e.g. 31, 32, 33, 34, 35 days after infection) with ToLCNDV. See also the Examples. In one aspect a plant line is said to be resistant against TolCNDV infection if it has an average disease score of 5.0 or higher, while the susceptible control line or variety, such as variety Gandalf (or Gandalf Fl, Nunhems B.V.) or Vedantrais, has an average disease score of 2.5 or less, or 2.0 or less, when grown under the same conditions and infected in the same way.
It has been observed that introgression of a specific fragment located on chromosome 5 in a wild melon donor plant into cultivated melon plants confers resistance to ToLCNDV infection in cultivated melon plants or cells derived therefrom. It is sufficient that the respective fragment is present only in the heterozygous state for conferring ToLCNDV resistance, demonstrating that the fragment confers resistance to TolCNDV infection in a dominant manner. Single Nucleotide Polymorphisms (SNPs) on chromosome 5 were identified which are closely linked to the fragment of chromosome 5 conferring ToLCNDV resistance. The SNP nucleotide of the resistant donor (i.e. the nucleotide of the introgression fragment) is present in homozygous form in the deposited seeds, i.e. the donor nculeotide is present in homozygous form for SNP 01 to SNP 06 (linked to QTL5). The SNPs can, therefore, be used to test the presence of the introgression fragment comprising the QTL5 in a plant cell, plant tissue, plant part, and/or in marker assisted selection (MAS) to transfer the QTLs into other elite melon lines or varieties. The SNPs can also be used to select plants comprising smaller introgressions fragments than the fragments present in the deposited seeds, whereby the smaller sub-fragments retain the QTL. Alternatively the SNPs can be used to identify other donors which comprise QTL5 and to introgress these QTLs into cultivated melon.
The present invention therefore relates to melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the donor plant in-between SNP 01 and SNP 06, preferably the sequence of the donor plant in-between SNP 01 and SNP 05, more preferably the sequence of the donor plant in- between SNP 01 and SNP 04. The ToLCNDV resistance conferring QTL is present on the introgression fragment, as can be determined by a resistance assay as described herein. In a preferred embodiment the present invention relates to cultivated melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the donor plant in-between SNP 02 and SNP 06, preferably the sequence of the donor plant in-between SNP 02 and SNP 05, more preferably the sequence of the donor melon plant in-between SNP 02 and SNP 04. The present invention further relates to melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor plant in-between SNP 03 and SNP 06, preferably the sequence of the ToLCNDV resistant donor plant in-between SNP 03 and SNP 05.
Most preferred, the present invention relates to melon plant cells or melon plants comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP_03 and SNP_04.
When referring herein to the introgression fragment comprising the sequence "in-between" two SNPs (Single Nucleotide Polymorphisms), this encompasses in one aspect that one or both of the two SNPs themselves are also from the resistant donor, i.e. have the donor nucleotide at the SNP position. In another aspect, the two SNPs are from the recipient, e.g. the susceptible melon plant, while only a region between the two SNPs is from the resistant donor and confers TolCNDV resistance, i.e. the resistance conferring donor fragment lies in-between the two SNP markers. So for example, a plant may comprise the introgression fragment comprising the sequence of the ToLCNDV resistant donor melon plant in- between SNP 03 and SNP 04, this plant in one aspect comprises a Thymine (T) at nucleotide 68 of SEQ ID NO: 3 and/or an Adenine (A) at nucleotide 227 of SEQ ID NO: 4, i.e. the donor nucleotides. In another aspect only a region (the whole region or a part thereof) between these two SNPs is from the donor, while SNP 03 and SNP 04 are from the recipient, having e.g. a Cytosine (C) at nucleotide 68 of SEQ ID NO: 3 and e.g. a Guanine (G) at nucleotide 227 of SEQ ID NO: 4. Thus, regarding the QTL on chromosome 5, SNP 01 , SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06 may all have the resistant donor genotype. Or only SNP 01 and SNP 02 may have the resistant donor genotype; or only SNP 02 and SNP 03 may have the resistant donor genotype; or only SNP 03 and SNP 04 may have the resistant donor genotype, etc. Or only a single SNP, i.e. only SNP 01 , or only SNP 02 or only SNP 03, or only SNP 04, or only SNP 05 or only SNP 06 has the resistant donor genotype. The SNPs that do not have the resistant donor genotype thus have another genotype, the recipient genotype. The recipient genotype for a SNP may be any of the other 3 nucleotides, i.e. for SNP 01 the recipient genotype may be Adenine, Guanine or Thymine. Thus, for example when stating that the introgression fragment is in-between SNP 03 and SNP 04 regarding the QTL on chromosome 5, SNP 03 and SNP 04 may both have the resistant donor genotype. Or only a single SNP, i.e. only SNP 03 or only SNP 04 may have the resistant donor genotype; or even neither SNP 03 nor SNP 04 has the resistant donor genotype, while the sequence in-between still contains QTL5.
The reason that not all of the SNPs provided herein need to have the resistant donor genotype is that the introgression fragment comprising the QTL from the donor may be smaller than the chromosome fragment introgressed e.g. in the deposited seeds, but the fragment still comprises the QTL5. Still, a plant can be recognized to contain the introgression fragment (comprising the QTL5) by the phenotype, and/or by transferring the fragment to a susceptible plant and thereby transferring the ToLCNDV resistance phenotype, or by sequencing the region between the SNP markers to identify the donor fragment, or other methods known to the skilled person, such as saturating the region with more SNP markers, allelism tests, identifying the causal gene, etc.
Thus, a combination of methods can be used to show that the QTL5 is present in a plant cell or plant, even if not for all of the linked SNPs the donor SNP genotype is present. QTL5 confers an average ToLCNDV resistance of at least 5.0 when transferred into a susceptible line or variety and is dominant.
In a preferred embodiment of the invention the introgression fragment from chromosome 5 of the donor plant comprising the sequence of the donor plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, in-between SNP 03 and SNP 06, in-between SNP 03 and SNP 05, preferably in-between SNP 03 and SNP 04 confers resistance to ToLCNDV to the cultivated melon plant cells according to the invention or to the cultivated melon plants according to the invention.
Preferably, the melon plant cell according to the invention originates from a cultivated melon plant or the melon plant according to the invention, is a cultivated melon plant.
In one aspect the present invention relates to cultivated melon plant cells or melon plants (or plant parts) comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and the introgression fragment is detectable by (comprises) the SNP genotype of the donor plant for one or more (or all) of the following SNPs: SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06, and optionally any SNP in- between SNP 01 and SNP 06.
In one aspect the plant, plant part or plant cell comprises QTL5 and comprises the SNP donor genotype for at least SNP 03, as this SNP is most significantly associated with the ToLCNDV resistance QTL5. In a further aspect the plant, plant part or plant cell comprises the SNP donor genotype for at least SNP 03 and SNP 04, or for at least SNP 03 and SNP 02. Optionally, the plant, plant part or plant cell comprises QTL5 and comprises the SNP donor genotype for SNP 01, SNP 02 and SNP 03; or for SNP_02, SNP_03 and SNP_04.
An introgression fragment may therefore comprise the donor SNP genotype for all SNP markers linked to QTL5 (as in the seeds deposited herein), or a smaller fragment, whereby one or more of the SNP markers is not present. As described further below, even all or all but one donor SNP markers may be absent, while QTL5 is still present on the introgression fragment.
The nucleotide sequences (SEQ ID NO: 1 to SEQ ID NO: 6) comprising the SNPs provided herein are the nucleotide sequences of the resistant donor, i.e. they contain the donor SNP nucleotide. Therefore, in one aspect the present invention relates to cultivated melon plant cells or melon plants (or plant parts) comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and the introgression fragment is detectable by (comprises) SEQ ID NO: 1 or an Cytosine at nucleotide 101 of SEQ ID NO: 1 or a Cytosine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO: 1 and/or by SEQ ID NO: 2 or a Thymine at nucleotide 945 of SEQ ID NO: 2 or a Thymine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO:2 and/or by SEQ ID NO: 3 or a Thymine at nucleotide 68 of SEQ ID NO: 3 or a Thymine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO: 3 and/or by SEQ ID NO: 4 or a Adenine at nucleotide 227 of SEQ ID NO: 4 or a Adenine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO: 4 and/or by SEQ ID NO: 5 or a Cytosine at nucleotide 839 of SEQ ID NO: 5 or a Cytosine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO: 5 and/or by SEQ ID NO: 6 or a Adenine at nucleotide 445 of SEQ ID NO: 6 or a Adenine at the equivalent nucleotide of a sequence having substantial sequence identity to SEQ ID NO: 6. The ToLCNDV resistance conferring QTL is present on the introgression fragment.
"Donor plant cell" or "donor plant" in connection with the present invention shall mean a melon plant cell or melon plant being resistant to ToLCNDV. Likewise, the term DNA fragment or introgression fragment from the donor plant or cell shall mean a fragment of chromosome 5 of a melon plant resistant to ToLCNDV, whereby the fragment confers ToLCNDV resistance when transferred into a TolCNDV susceptible melon plant. In a preferred embodiment of the invention, the donor plant is a wild species or wild accession of melon. In a particular preferred embodiment of the invention, DNA fragments or introgression fragments from donor plant cells or plants are the donor fragments obtained from plants grown from seeds deposited under NCIMB 42585 or progeny obtained from plants grown from seeds deposited under NCIMB 42585 or plants obtained by crosses with plants grown from seeds deposited under NCIMB 42585.
Donor melon plants can be obtained from various sources. A person skilled in the art knows how to detect other sources of ToLCNDV resistant donor plants. For detecting such sources of ToLCNDV resistant donor plants, basically melon plants can be infected with ToLCNDV, either by mechanical means, as described in Lopez et al. (2015, Euphytica 204(3), 679-691) or by transmission of the virus by whiteflies. Preferably, infection occurs by whitefly infection in context with the present invention. Plants showing reduced symptom levels compared to susceptible controls can then be selected and used as source for genome fragments or sequences conferring ToLCNDV resistance. A preferred method on how to infect melon plants with ToLCNDV and methods for determining the symptom level of infected plants are given herein under "General Methods".
In context of the present invention the donor plants preferably have an average symptom level equal to or above 5.0, more preferably equal to or above 6.0, furthermore preferred equal to or above 7.0, even more preferred equal to or above 8.0 and most preferred equal to or above 9.0. In one aspect the donor plant comprises the donor SNP genotype for one or more or all of SNP 01, SNP 2, SNP 3, SNP 04, SNP 05 and SNP 06, as shown in Table 2. Preferably the SNP donor genotype is homozygous. The donor is herein e.g. a wild melon having no agronomic value, e.g. producing white fleshed fruits, having low brix, etc.
"Recurrent plant cell" or "recurrent plant" or "recipient plant" in connection with the present invention shall be understood to be a melon plant cell or melon plant being sensitive (used herein synonymously with susceptible) or non-resistant to ToLCNDV infection. If a plant is sensitive or non-resistant to ToLCNDV can be determined by observation of the symptom levels after ToLCNDV infection. A recurrent plant preferably has an average symptom level below 3.0, more preferably equal to or below 2.5 or equal to or below 2.0. Symptom levels and methods how to infect melon plants with ToLCNDV are described elsewhere herein and are applicable here accordingly. In a preferred embodiment of the invention, the recurrent melon plant cell according to the invention originates from a cultivated melon plant or the recurrent melon plant according to the invention, is a cultivated melon plant. Preferably it is an elite line, breeding line or variety.
"Introgression fragment" refers to a chromosome fragment, chromosome part or region which has been introduced into another plant of the same or related species by crossing or traditional breeding techniques. The introgression of the fragment from a donor plant into a recurrent plant introduces into the offspring of a cross between the donor and recurrent plant a phenotype, which was not present in the recurrent plant. Concerning the present invention, the phenotype transferred from the donor plant to the recurrent plant is resistance to ToLCNDV, e.g. an average disease score of 5.0. For introgression of a fragment into a specific breeding line or variety the first crossing step can e.g. be followed by one or more backcrossings with the intended breeding line or variety. As understood herein, introgression can mean a first crossing of a ToLCNDV resistant donor plant with a ToLCNDV non-resistant recurrent plant and further back-crossing one or several times ToLCNDV resistant plants obtained from the first crossing with plants of the recipient into which ToLCNDV resistance shall be introgressed. In such a case, the introgressed fragment is the result of breeding methods referred to by the verb "to introgress" (such as backcrossing) into a recipient variety or breeding line. Thus, introgression of ToLCNDV resistance into a recurrent plant is a technical process directed by man. In particular introgression herein refers to a manmade breeding process or method. One or more or all of the molecular markers (SNP markers) provided herein can be used in that process. The resulting plant, i.e. the cultivated line or variety comprising one introgression fragment (on chromosome 5) from a donor, i.e. comprising a recombinant chromosome 5, is also man-made and does not exist in nature.
The introgression fragment can be large, e.g. even half of a chromosome, but is preferably smaller, such as about 15 Mb or less, such as about 10 Mb or less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2 Mb or less, about 1 Mb (equals 1 ,000,000 base or less), or about 0.8 Mb (equals 800,000 base pairs) or less.
The introgression fragment can originate from a wild melon plant or wild melon accession or wild relatives of melon or landraces (donor). Wild melon plants or wild melon accessions or wild relatives of melon plants or landraces can be used to introgress fragments of the donor genome into the genome of cultivated melon, Cucumis melo, to generate breeding lines or varieties with good agronomic characteristics. Such a cultivated melon plant thus has a "genome of cultivated C. melo ", but comprises in its genome a fragment of a donor, e.g. an introgression fragment of a related wild Cucumis genome, such as Cucumis melo ssp. agrestis, C. melo ssp. melo, C. melo ssp. a cidulous, C. callosus, C. trigonus, C picrocarpus, or another wild melon or wild relative of melon. It is understood that the term "introgression fragment" never includes a whole chromosome, but only a part of a chromosome. The chromosomes carrying the introgression therefore also comprise a part or parts of the recurrent (recipient) melon plant and in addition parts of the donor melon plant.
When the chromosome 5 of cultivated melon comprises an introgression fragment, this therefore means that the cultivated melon plant comprises a recombinant chromosome 5, whereby the introgressed fragment comprises the ToLCNDV resistance conferring QTL. As described elsewhere, the introgression fragment from the donor may comprise one or more or all of the donor SNP nucleotides (for SNP_01 , SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06) or one or more or all of the sequences comprising the donor SNP nucleotides (SEQ ID NO: 1 , 2, 3, 4, 5 and/or SEQ ID NO: 6). So, e.g. the introgression fragment (in homozygous or heterozygous form) may comprise, and is detectable by, one or more or all of the following SNP genotypes: the CC or CT genotype for SNP 01 in SEQ ID NO: 1 , and/or the TT or TG genotype for SNP 02 in SEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/or the CC or CT genotype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6.
In the instant invention, in one aspect the lower part of chromosome 5 of cultivated melon, below marker CMGAAN144 on chromosome 5 on page 3 of Diaz et al 2015 (Mol. Breeding 35: 188), or between marker CMGAAN144 and marker AI 13-H12, comprises QTL5 from a donor. In one aspect the donor plant of the invention is not one of the five Cucumis melo subsp. agrestis accessions (subsp. agrestis var. momordica: Mom-Khalnd/ Kharbuja, Mom-PI124Ind/ PI124112, Mom- Pi 124Ind/PI414723 and subsp. agrestis wild types: Ag-WM9Ind/WM9, Ag-WM7Ind/WM7) resistant to ToLCNDV identified by Lopez et al, 2015, Euphytica 204(3), 679-691.
The term "breeding" encompasses herein crossing, backcrossing, selfing, selection, double haploid production, embryo rescue, protoplast fusion, marker assisted selection, mutation breeding etc. as known to the breeder (i.e. methods other than genetic modification/transformation/transgenic methods), by which, for example, a recombinant chromosome 5 can be obtained, identified, produced and/or transferred.
In a preferred embodiment of the present invention, the introgression fragment originates form a wild Cucumis plant or a wild Cucumis accession, most preferably the introgression fragment originates from wild Cucumis melo ssp. melo, having small fruits (no more than 6 cm long), with white, acid tasting, fruit flesh. This donor was used in the instant invention, but other donors can be identified by the skilled person which comprise e.g. the same SNP genotype as this donor for SNP 01 to SNP 06 and comprises a QTL in the same region of chromosome 6. A suitable donor is in one aspect a wild C melo plant or accession having an average ToLCNDV disease score of at least 7.0, or at least 7.1 , 7.2, 7.3, or 7.4 or 7.5 ot 7.6, on a scale of 1 = dead plant to 9 = no symptoms and comprises one or more or all of the following SNP genotypes: the CC or CT genotype for SNP 01 in SEQ ID NO: 1, and/or the TT or TG genotype for SNP 02 in SEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/or the CC or CT genotype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6.
"Plant variety" is a group of plants within the same botanical taxon of the lowest grade known, which (irrespective of whether the conditions for the recognition of plant breeder's rights are fulfilled or not) can be defined on the basis of the expression of characteristics that result from a certain genotype or a combination of genotypes, can be distinguished from any other group of plants by the expression of at least one of those characteristics, and can be regarded as an entity, because it can be multiplied without any change. Therefore, the term "plant variety" cannot be used to denote a group of plants, even if they are of the same kind, if they are all characterized by the presence of one or two loci or genes (or phenotypic characteristics due to these specific loci or genes), but which can otherwise differ from one another enormously as regards the other loci or genes.
"Fl, F2, F3, etc." refers to the consecutive related generations following a cross between two parent plants or parent lines. The plants grown from the seeds produced by crossing two plants or lines is called the Fl generation. Selling the Fl plants results in the F2 generation, etc.
"Fl hybrid" plant (or Fl hybrid seed) is the generation obtained from crossing two inbred parent lines. Thus, Fl hybrid seeds are seeds from which Fl hybrid plants grow. Fl hybrids are more vigorous and higher yielding, due to heterosis. Inbred lines are essentially homozygous at most loci in the genome. A "plant line" or "breeding line" refers to a plant and its progeny. As used herein, the term "inbred line" refers to a plant line which has been repeatedly selfed and is nearly homozygous. Thus, an "inbred line" or "parent line" refers to a plant which has undergone several generations (e.g. at least 5, 6, 7 or more) of inbreeding, resulting in a plant line with a high uniformity.
"Uniformity" or "uniform" relates to the genetic and phenotypic characteristics of a plant line or variety. Inbred lines are genetically highly uniform as they are produced by several generations of inbreeding. Likewise, and the Fl hybrids which are produced from such inbred lines are highly uniform in their genotypic and phenotypic characteristics and performance.
In a particular preferred embodiment of the invention, plant cells according to the invention and plants according to the invention are characterized in that the introgression fragment conferring ToLCNDV resistance originates from the seeds deposited under NCIMB 42585 or progeny thereof.
In a further preferred embodiment of the invention, the melon plant cell according to the invention originates from a cultivated melon plant or the melon plant according to the invention is a cultivated melon plant and the introgression fragment originates form a wild Cucumis plant or a wild Cucumis accession or from donor plants described herein to be preferred donor plants or obtained from seeds deposited under NCIMB 42585 or progeny thereof.
"Chromosome 5 of a melon plant" is to be understood in context of the present invention as the scaffolds, fragments, regions, markers and nucleic acid sequences assigned by the ICuGI (International Cucurbit Genomics Initiative) to belong to chromosome 5 of the melon genome.
"Orthologous chromosome 5" refers to the chromosome 5 of wild relatives of melon, parts of which can be introgressed into cultivated melon chromosome 5.
A "recombinant chromosome" refers to a chromosome having a new genetic makeup arising through crossing over between homologous chromosomes, e.g. a "recombinant chromosome 5", i.e. a chromosome 5 which is not present in either of the parent plants and arose through a rare crossing-over event between homologous chromosomes of a chromosome 5 pair. Herein, for example, a recombinant melon chromosome 5 comprising a ToLCNDV-resistance conferring QTL is provided. The recombinant chromosome 5 therefore is a chromosome of cultivated melon, with an introgression fragment from a wild donor, whereby the introgression fragment comprises the ToLCNDV resistance conferring QTL.
"ICuGI" refers herein to the Cucumis melo data published by the International Cucurbit Genomics Initiative, which publishes genetic maps of e.g. Cucumis melo (http:// www.icugi.org/cgi-bin/cmap/map set_info?species acc=CM). The current version CM 3.5.1 of the C melo genome map is of March 4th, 2012 and the map of chromosome 5 is referred to as ICuGI V (or LG V, or Linkage Group V). Further information including additional markers and mapping information in addition to the ICuCI data is available from Diaz et al. (2015, Mol Breeding 35, 188) and the additional data included in the online version of the respective article.
"Cultivated melon plant" refers to plants of Cucumis melo i.e. varieties, breeding lines or cultivars of the species C. melo, cultivated by humans and having good agronomic characteristics, especially producing edible and marketable fruits of good size and quality and uniformity; such plants are not "wild melon plants", i.e. plants which generally have much poorer yields and poorer agronomic characteristics than cultivated plants and e.g. grow naturally in wild populations. "Wild plants" include for example ecotypes, PI (Plant Introduction) lines, landraces or wild accessions or wild relatives of a species. A "SNP (= Single Nucleotide Polymorphism)" in context with the present invention is to be understood as a variation in a single nucleotide that occurs at a specific position in the genome. A SNP is the variation of the single nucleotide at the given position in a genome between two plants. If a wild melon plant having a ToLCNDV resistance (donor plant) shows in its corresponding sequence at a specific single position a nucleotide which is different from the corresponding nucleotide at the same position of a cultivated melon plant, the position defines a SNP between the wild melon and the cultivated melon. If the donor plant has one of the four possible nucleotides (A, C, T or G) at a specific position, a SNP occurs, when the cultivated plant has either of the remaining three possible nucleotides at the same corresponding sequence position. In a cultivated melon plant comprising an introgression fragment from a donor, it can therefore easily be determined if the single nucleotide of the SNP is from the donor or from the cultivated melon (recipient).
'SNP nucleotide' refers to the single nucleotide, while 'SNP genotype' refers to the pair of nucleotides in a diploid plant cell. So, for SNP 01, the SNP nucleotide of the ToLCNDV resistant donor is a Cytosine (C) for nucleotide 101 of SEQ ID NO: 1, while the SNP genotype of a plant or cell comprising SEQ ID NO: 1 can be CC (Cytosine on both chromosomes) or CT (Cytosine on one chromosome and Thymine on the other chromosome), whereby the ToLCNDV resistant donor SNP nucleotide (Cytosine), and thus SEQ ID NO: 1 (or a sequence substantially identical to SEQ ID NO: 1), is homozygous or heterozygous. The term "SNP donor genotype" refers to the donor SNP nucleotide being present in homozygous or in heterozygous form, i.e. for SNP 01 the SNP donor genotype is either CC or CT. "SNP 01" which is alternatively designated "mMEl 1320_k" is to be understood in context with the present invention to be a SNP at position 101 in SEQ ID NO 1. SEQ ID NO 1 or a sequence substantially identical to SEQ ID NO 1 can be found on chromosome 5 in the ICuGI data set. The relative position of SNP 01 according to markers published by ICuGI is derivable from Table 1. Preferably the nucleotide sequence comprising SNP 01 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 1, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO 1 , further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 1 , even more preferred at least 97%> identity with the nucleotide sequence shown under SEQ ID NO 1, even further more preferred at least 98%> identity with the nucleotide sequence shown under SEQ ID NO 1 , in particular preferred at least 99%> identity with the nucleotide sequence shown under SEQ ID NO 1 or more particularly preferred at least 99,5%> identity with the nucleotide sequence shown under SEQ ID NO 1 under the provision that in each case the nucleotide at position 101 in SEQ ID NO 1 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%>, 90%>, 95%>, 97%>, 98%>, 99%> or more sequence identity to SEQ ID NO: 1 are referred to as having substantial sequence identity to SEQ ID NO: l .
The ToLCNDV resistant donor plants used in the invention have a 'C'(Cytosine) at position 101 in SEQ ID NO 1 or in a sequence comprising substantial sequence identity to SEQ ID NO: l . In a preferred embodiment of the invention, SNP 01 is characterized in that the recurrent plant has an A, G or T at position 101 in SEQ ID NO 1. In one aspect, the recurrent plant has a T at position 101 in SEQ ID NO 1. In one embodiment of the invention, SNP 01 is characterized in that the donor plant has a C at position 101 of SEQ ID NO 1 (or at the equivalent position of a sequence comprising substantial sequence identity to SEQ ID NO: 1) and the recurrent plant has a T at position 101 in SEQ ID NO 1.
"SNP 02" which is alternatively designated "mME43070_k" is to be understood in context with the present invention to be a SNP at position 945 in SEQ ID NO 2. SEQ ID NO 2 or a sequence substantially identical to SEQ ID NO 2 can be found on chromosome 5 in the ICuGI data set. The relative position of SNP 02 according to markers published by ICuGI is derivable from Table 1. Preferably the nucleotide sequence comprising SNP 02 has a nucleotide sequence having at least 85%> identity with the nucleotide sequence shown under SEQ ID NO 2, more preferably at least 90%> identity with the nucleotide sequence shown under SEQ ID NO 2, further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 2, even more preferred at least 97% identity with the nucleotide sequence shown under SEQ ID NO 2, even further more preferred at least 98% identity with the nucleotide sequence shown under SEQ ID NO 2, in particular preferred at least 99% identity with the nucleotide sequence shown under SEQ ID NO 2 or more particularly preferred at least 99,5% identity with the nucleotide sequence shown under SEQ ID NO 2 under the provision that in each case the nucleotide at position 945 in SEQ ID NO 2 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 2 are referred to as having substantial sequence identity to SEQ ID NO: 2. The ToLCNDV resistant donor plants used in the invention have a 'T' (Thymine) at position 945 in SEQ ID NO 2 or in a sequence comprising substantial sequence identity to SEQ ID NO: 2.
In one embodiment of the invention, SNP 02 is characterized in that the recurrent plant has an A, C or G at position 945 in SEQ ID NO 2 or in a sequence comprising substantial sequence identity to SEQ ID NO: 2. In one aspect the recurrent plant has a 'G' (Guanine) at position 945 in SEQ ID NO 2 or in a sequence comprising substantial sequence identity to SEQ ID NO: 2.
In a particular embodiment of the invention, SNP 02 is characterized in that the donor plant has a T at position 945 in SEQ ID NO 2 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 2) and the recurrent plant has a G at position 945 in SEQ ID NO 2 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 2).
"SNP 03" which is alternatively designated "mME10621_k" is to be understood in context with the present invention to be a SNP at position 68 in SEQ ID NO 3. SEQ ID NO 3 or a sequence substantially identical to SEQ ID NO 3 can be found on chromosome 5 in the ICuGI data set. The relative position of SNP 03 according to markers published by ICuGI is derivable from Table 1. Preferably the nucleotide sequence comprising SNP 03 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 3, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO 3, further more preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO 3, even more preferred at least 97% identity with the nucleotide sequence shown under SEQ ID NO 3, even further more preferred at least 98% identity with the nucleotide sequence shown under SEQ ID NO 3, in particular preferred at least 99% identity with the nucleotide sequence shown under SEQ ID NO 3 or more particularly preferred at least 99,5% identity with the nucleotide sequence shown under SEQ ID NO 3 under the provision that in each case the nucleotide at position 68 in SEQ ID NO 3 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 3 are referred to as having substantial sequence identity to SEQ ID NO: 3.
The ToLCNDV resistant donor plants used in the invention have a ' (Thymine) at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3). In one embodiment of the invention, SNP 03 is characterized in that the recurrent plant has an A, C or G at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3). In one aspect the recurrent plant has a C position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3).
In one embodiment of the invention, SNP 03 is characterized in that the donor plant has a 'T' at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO:
3) and the recurrent plant has a 'C at position 68 in SEQ ID NO 3 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 3).
"SNP 04" which is alternatively designated "mME50729_k" is to be understood in context with the present invention to be a SNP at position 227 in SEQ ID NO 4. SEQ ID NO 4 or a sequence substantially identical to SEQ ID NO 4 can be found on chromosome 5 in the ICuGI data set. The relative position of SNP 04 according to markers published by ICuGI is derivable from Table 1. Preferably the nucleotide sequence comprising SNP 04 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 4, more preferably at least 90%> identity with the nucleotide sequence shown under SEQ ID NO 4, further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 4, even more preferred at least 97%> identity with the nucleotide sequence shown under SEQ ID NO 4, even further more preferred at least 98%> identity with the nucleotide sequence shown under SEQ ID NO 4, in particular preferred at least 99%> identity with the nucleotide sequence shown under SEQ ID NO 4 or more particularly preferred at least 99,5%> identity with the nucleotide sequence shown under SEQ ID NO 4 under the provision that in each case the nucleotide at position 227 in SEQ ID NO 4 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%>, 90%>, 95%>, 97%>, 98%>, 99%> or more sequence identity to SEQ ID NO 4 are referred to as having substantial sequence identity to SEQ ID NO: 4.
The ToLCNDV resistant donor plants used in the invention have an 'A' (Adenine) at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4). In one embodiment of the invention, SNP 04 is characterized in that the recurrent plant has an C, T or G at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO:
4) . In one aspect the recurrent plant has a G position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4). In one embodiment of the invention, SNP 04 is characterized in that the ToLCNDV resistant donor plant has an A at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4) and the recurrent plant has a G at position 227 in SEQ ID NO 4 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 4). "SNP 05" which is alternatively designated "mME32395_k" is to be understood in context with the present invention to be a SNP at position 839 in SEQ ID NO 5. SEQ ID NO 5 or a sequence substantially identical to SEQ ID NO 5 can be found on chromosome 5 in the ICuGI data set. The relative position of SNP 05 according to markers published by ICuGI is derivable from Table 1. Preferably the nucleotide sequence comprising SNP 05 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 5, more preferably at least 90%> identity with the nucleotide sequence shown under SEQ ID NO 5, further more preferably at least 95%> identity with the nucleotide sequence shown under SEQ ID NO 5, even more preferred at least 97%> identity with the nucleotide sequence shown under SEQ ID NO 5, even further more preferred at least 98%> identity with the nucleotide sequence shown under SEQ ID NO 5, in particular preferred at least 99%> identity with the nucleotide sequence shown under SEQ ID NO 5 or more particularly preferred at least 99,5%> identity with the nucleotide sequence shown under SEQ ID NO 5 under the provision that in each case the nucleotide at position 839 in SEQ ID NO 5 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%>, 90%>, 95%>, 97%>, 98%>, 99%> or more sequence identity to SEQ ID NO 5 are referred to as having substantial sequence identity to SEQ ID NO: 5.
The ToLCNDV resistant donor plants used in the invention have a 'C'(Cytosine) at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5). In one embodiment of the invention, SNP 05 is characterized in that the recurrent plant has an A, T or G at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5). In one aspect the recurrent plant has a T position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5).
In one embodiment of the invention, SNP 05 is characterized in that the ToLCNDV resistant donor plant has a C at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5) and the recurrent plant has a T at position 839 in SEQ ID NO 5 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 5).
"SNP 06" which is alternatively designated "mME49184_k" is to be understood in context with the present invention to be a SNP at position 445 in SEQ ID NO 6. SEQ ID NO 6 or a sequence substantially identical to SEQ ID NO 6 can be found on chromosome 5 in the ICuGI data set. The relative position of SNP 06 according to markers published by ICuGI is derivable from Table 1. Preferably the nucleotide sequence comprising SNP 06 has a nucleotide sequence having at least 85% identity with the nucleotide sequence shown under SEQ ID NO 6, more preferably at least 90% identity with the nucleotide sequence shown under SEQ ID NO 6, further more preferably at least 95% identity with the nucleotide sequence shown under SEQ ID NO 6, even more preferred at least 97% identity with the nucleotide sequence shown under SEQ ID NO 6, even further more preferred at least 98% identity with the nucleotide sequence shown under SEQ ID NO 6, in particular preferred at least 99% identity with the nucleotide sequence shown under SEQ ID NO 6 or more particularly preferred at least 99,5% identity with the nucleotide sequence shown under SEQ ID NO 6 under the provision that in each case the nucleotide at position 445 in SEQ ID NO 6 is different from the corresponding nucleotide at the same position of the recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 6 are referred to as having substantial sequence identity to SEQ ID NO: 6.
The ToLCNDV resistant donor plants used in the invention have an 'A' (Adenine) at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6). In one embodiment of the invention, SNP 06 is characterized in that the recurrent plant has a C, T or G at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6). In one aspect the recurrent plant has a G position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6).
In a particular embodiment of the invention, SNP 06 is characterized in that the ToLCNDV resistant donor plant has an A at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6) and the recurrent plant has a G at position 445 in SEQ ID NO 6 (or in a sequence comprising substantial sequence identity to SEQ ID NO: 6).
MarPseudo- cM Garcia- Scaffold v3.5.1 ker molecule Mas et al. cM Argyris et (world wide web at Scaffold
Marker name type Chrom coordinates (2012) al. (2015) LG ICuGI cM ICuGI melonomics.net) coordinates
CM3.5.1_scaffoldO
CMPSNP460 SNP 5 12.966.671 57,3 0009 1.816.109
CM3.5.1_scaffoldO
SNP 06 SNP 5 0009 2.607.437
CM3.5.1_scaffoldO
CMPSNP682 SNP 5 15.854.444 57,3 50,4 0009 4.703.882
CM3.5.1_scaffoldO
CMN62 05 SSR 5 16.570.623 0009 5.420.061
CM3.5.1_scaffoldO
CMPSNP1005 SNP 5 17.239.621 57,3 50,4 0009 6.089.059
CM3.5.1_scaffoldO
CMN61 15 SSR 5 17.453.772 0009 6.303.210
CM3.5.1_scaffoldO
CMPSNP39 SNP 5 17.651.074 57,3 0009 6.500.512
CM3.5.1_scaffoldO
PS 03-B08 SNP 5 18.119.560 57,3 50,4 0009 6.968.998
CM3.5.1_scaffoldO
ECM203 SSR 5 18.459.732 0009 7.309.170
CMPSNP2005 SNP 5 50,4 CM3.5.1 scaffoldO 7.682.475
18.833.037 0009
CM3.5.1_scaffoldO
CMN01 49 SSR 5 19.305.580 0003 26.051
CM3.5.1_scaffoldO
ECM142 SSR 5 19.743.282 5 54 0003 463.753
CM3.5.1_scaffoldO
CMGAN3 SSR 5 19.871.767 5 56 0003 592.238
CM3.5.1_scaffoldO
CMPSNP1018 SNP 5 20.088.198 50,9 50,7 0003 808.669
CM3.5.1_scaffoldO
CMPSNP613 SNP 5 20.406.864 0003 1.127.335
CM3.5.1_scaffoldO
CMN05 89 SSR 5 21.514.552 0003 2.235.023
CM3.5.1_scaffoldO
CMN23 06 SSR 5 21.949.502 0003 2.669.973
CM3.5.1_scaffoldO
GCM295 SSR 5 23.318.454 0003 4.038.925
CM3.5.1_scaffoldO
CMPSNP741 SNP 5 23.620.132 0003 4.340.603
CM3.5.1_scaffoldO
ECM115 SSR 5 23.786.719 0003 4.507.190
CMPSNP1136 SNP 5 67,0 67,7 CM3.5.1 scaffoldO 4.820.475
24.100.004 0003
CM3.5.1_scaffoldO
ECM206 SSR 5 24.175.232 0003 4.895.703
CM3.5.1_scaffoldO
CMCTN2 SSR 5 24.313.118 5 73 0003 5.033.589
CM3.5.1_scaffoldO
CMGAAN144 SSR 5 24.403.405 5 73 0003 5.123.876
CM3.5.1_scaffoldO
SNP 05 SNP 5 0003 5.202.092
CM3.5.1_scaffoldO
3J84-496 SNP 5 24.884.331 0003 5.604.802
CM3.5.1_scaffoldO
3J84-19 SNP 5 24.884.834 0003 5.605.305
CM3.5.1_scaffoldO
60k53-404 SNP 5 25.036.462 0003 5.756.933
CM3.5.1_scaffoldO
60k53-147 SNP 5 25.036.692 0003 5.757.163
CM3.5.1_scaffoldO
60k53-80 SNP 5 25.036.780 0003 5.757.251
CM3.5.1_scaffoldO
60k49-351 SNP 5 25.040.592 0003 5.761.063
60k49-307 SNP 5 CM3.5.1 scaffoldO 5.761.160
25.040.689 0003
CM3.5.1_scaffoldO
60k49-182 SNP 5 25.040.770 0003 5.761.241
CM3.5.1_scaffoldO
60k45.389 SNP 5 25.043.452 73,4 0003 5.763.923
CM3.5.1_scaffoldO
60k45.288 SNP 5 25.043.521 0003 5.763.992
CM3.5.1_scaffoldO
60k45.213 SNP 5 25.043.624 0003 5.764.095
CM3.5.1_scaffoldO
60k45.14 SNP 5 25.043.807 73,4 0003 5.764.278
CM3.5.1_scaffoldO
60k42-490 SNP 5 25.045.798 0003 5.766.269
CM3.5.1_scaffoldO
60k42-411 SNP 5 25.045.907 0003 5.766.378
CM3.5.1_scaffoldO
60k42-126 SNP 5 25.046.183 76,6 0003 5.766.654
CM3.5.1_scaffoldO
60k42-29 SNP 5 25.046.269 0003 5.766.740
CM3.5.1_scaffoldO
60k41.333 SNP 5 25.047.374 0003 5.767.845
60k41.243 SNP 5 73,4 CM3.5.1 scaffoldO 5.767.920
25.047.449 0003
CM3.5.1_scaffoldO
60k41.49 SNP 5 25.047.654 0003 5.768.125
CM3.5.1_scaffoldO
SNP 04 SNP 5 0003 5.785.550
CM3.5.1_scaffoldO
CMTAAN128 SSR 5 25.081.134 5 72 0003 5.801.605
CM3.5.1_scaffoldO
CMTAN138 SSR 5 25.081.134 5 70 0003 5.801.605
CM3.5.1_scaffoldO
CMTAN139 SSR 5 25.081.134 5 70 0003 5.801.605
CM3.5.1_scaffoldO
CMPSNP588 SNP 5 25.271.831 75,0 78,3 0003 5.992.302
CM3.5.1_scaffoldO
CMPSNP464 SNP 5 25.639.164 75,8 0003 6.359.635
CM3.5.1_scaffoldO
SNP 03 SNP 5 0003 6.553.350
CMPSNP CM3.5.1_scaffoldO
1155 SNP 5 26.124.693 79,8 0003 6.845.164
CM3.5.1_scaffoldO
SNP 02 SNP 5 0003 6.950.286
CMPSNP690 SNP 5 79,8 90,0 CM3.5.1 scaffoldO 7.061.100
26.340.629 0003
CM3.5.1_scaffoldO
CMTCN227 SSR 5 26.453.003 5 61 0003 7.173.474
CM3.5.1_scaffoldO
CMPSNP1115 SNP 5 26.639.938 84,6 0003 7.360.409
CM3.5.1_scaffoldO
ECM213 SSR 5 26.907.003 0003 7.627.474
CM3.5.1_scaffoldO
SNP 01 SNP 5 0003 7.824.960
CM3.5.1_scaffoldO
AI 13-H12 SNP 5 27.276.249 89,4 0003 7.996.720
Table 1
The molecular markers described herein may be detected according to standard methods. For example SNP markers can be detected using a KASP-assay (see Www.kpbioscience.co.uk) or other assays. A KASP-assay has been developed for SNPs described herein. Respective details are disclosed in the Example section. Sequences used in the respective KASP-assays are given in the Sequence Listing. For developing KASP-assays for the SNPs two allele specific forward primers and one allele specific reverse primer were designed according to common general knowledge (see e.g. Allen et al. 2011, Plant Biotechnology J. 9, 1 0 86-1 099, especially p097-098 for KASP assay method).
"Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as "substantially identical" or "essentially similar" when they are optimally aligned by for example the programs GAP or BESTFIT or the Emboss program "Needle" (using default parameters, see below) share at least a certain minimal percentage of sequence identity defined further below). These programs use the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 10 and gap extension penalty = 0.5 (both for nucleotide and protein alignments). For nucleotides the default scoring matrix used is DNAFULL and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1 992, PNAS 89, 1 09 1 5-1 09 1 9). Sequence alignments and scores for percentage sequence identity may for example be determined using computer programs, such as EMBOSS as available on the world wide web under ebi.ac.uk/Tools/psa/emboss_needle/). Alternatively sequence similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences have "substantial sequence identity" if the percentage sequence identity is at least 85%, 90%, 95%, 98%, 99% or more (e.g. at least 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or more (as determined by Emboss "needle" using default parameters, i.e. gap creation penalty = 10, gap extension penalty = 0.5, using scoring matrix DNAFULL for nucleic acids an Blosum62 for proteins).
When reference is made to a nucleic acid sequence (e.g. DNA or genomic DNA) having "substantial sequence identity to" a reference sequence or having a sequence identity of at least 80%, e.g. at least 85%, 90%, 95%, 98%, 99%, 99.2%, 99.5%, 99.9% nucleic acid sequence identity to a reference sequence, in one embodiment said nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using stringent hybridization conditions. In another embodiment, the nucleic acid sequence comprises one or more mutations compared to the given nucleotide sequence but still can be identified using stringent hybridisation conditions.
Cultivated melon plants comprising a ToLCNDV resistance conferring fragment introgressed on chromosome 5 show reduced symptoms when infected with ToLCNDV, while susceptible controls (lacking the introgression fragment) show the expected severe symptoms in the same conditions.
In a preferred embodiment of the invention, plant cells according to the invention or plants according to the invention are characterized in that they upon infection with ToLCNDV show an average symptom level of at least 4.0, more preferably at least 5.0. As mentioned previously, QTL5 confers resistance when the QTL is in homozygous or heterozygous form. A ToLCNDV susceptible plant into which the introgression fragment comprising QTL5 is introduced by e.g. backcrossing and which is optionally selfed to generate a homozygous introgression fragment (comprising the donor genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06 in homozygous form), will result in a plant which is resistant to ToLCNDV infection, having an average disease score of at least 4.0, preferably at least 5.0, preferably at least 6.0 or preferably at least 7.0. Symptom levels occurring after infection with ToLCNDV between 1 and 9, wherein 1 is defined to be the level with the most severe symptoms and 9 is defined as the highest resistance level, have been described herein above and are applicable here accordingly. A preferred test for determining the symptom levels is given below under "General Methods". In another embodiment of the invention plant cells according to the invention or plants according to the invention are characterized in that they upon infection with ToLCNDV show a symptom level between 4 and 6, more preferably a symptom level of between 5 and 6, while the susceptible controls show a symptom level of 2 or less.
Resistance to ToLCNDV conferred by the introgression fragment is expressed in a dominant manner and thus can be observed when only one chromosome 5 comprises the introgression fragment comprising the sequence of the donor plant between the markers of chromosome 5 disclosed herein.
Other specific embodiments of the present invention therefore relate to melon plant cells according to the invention, or melon plants according to the invention, wherein the introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant comprising the sequence of the donor plant in- between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in- between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, in- between SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or preferably in-between SNP 03 and SNP 04 is present in heterozygous state. Accordingly, it is sufficient that at least one chromosome 5 in the plant cells according to the invention or plants according to the invention comprise the just described introgression fragment. It is however understood, that chromosome 5 in respect to the just described introgression fragment can also be present in the homozygous state without diminishing the degree of resistance, because of the dominance of the ToLCNDV resistance conferred by the just described introgression fragment. Thus, the invention comprises plant cells according to the invention or plants according to the invention which comprise the just described introgression fragment in a heterozygous or homozygous state.
Table 2 illustrates the SNP genotype of plants or cells comprising the ToLCNDV resistant donor SNPs in homozygous form or heterozygous form, as well as the recurrent parent SNP genotype, lacking the introgression fragment.
Figure imgf000028_0001
In one embodiment a cultivated melon plant (of the species C. melo) is provided which comprises a recombinant chromosome 5, whereby the recombinant chromosome 5 comprises an introgression fragment that confers ToLCNDV resistance onto the melon plant when present in homozygous or heterozygous form and wherein the introgression fragment is from a wild donor of the species C. melo. In one aspect the introgression fragment comprises the SNP donor genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and/or SNP 06. In one embodiment the introgression fragment comprises the donor SNP genotype for at least SNP 03 and/or SNP 04. The SNP genotype of the donor may be present in homozygous form (if the introgression fragment is in homozygous form) or in heterozygous form (if the introgression fragment is in heterozygous form). So, for example, the plant or plant cell or plant part may comprise the TT or TC genotype for SNP 03 at nucleotide 68 of SEQ ID NO: 3. Thus, the plant, plant cell, or plant part may comprise SEQ ID NO: 3, or a sequence comprising at least 95%, 97%, or 98%> sequence identity to SEQ ID NO: 3, in homozygous form or in heterozygous form. In one embodiment cultivated melon plants or cells of these plants are provided which comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 06 (or in- between SEQ ID NO: l and SEQ ID NO:6, or a sequence comprising at least 90%, or at least 95%, or at least 97%> or 98%> sequence identity to SEQ ID NO: 1 or to SEQ ID NO 6); or whereby the introgression fragment lies in-between SNP 01 and SNP 05 (or in-between SEQ ID NO:l and SEQ ID NO:5, or a sequence comprising at least 90%>, or at least 95%>, or at least 97%> or 98%> sequence identity to SEQ ID NO: 1 or to SEQ ID NO 5).
In one aspect the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 06 (or in-between SEQ ID NO: l and SEQ ID NO:6, or a sequence comprising at least 90%>, or at least 95%>, or at least 97%> or 98%> sequence identity to SEQ ID NO: 1 or to SEQ ID NO 6), or the fragment lies in-between SNP 01 and SNP 05 (or in-between SEQ ID NO:l and SEQ ID NO:5, or a sequence comprising at least 90%>, or at least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 1 or to SEQ ID NO 5), and whereby the introgression fragment comprises a Thymine (T) at nucleotide 68 of SEQ ID NO 3 or at the equivalent nucleotide of a sequence comprising substantial sequence identity to SEQ ID NO: 3.
In a further aspect the introgression fragment optionally further comprises an Adenine (A) at nucleotide 227 of SEQ ID NO 4 or at the equivalent nucleotide of a sequence comprising substantial sequence identity to SEQ ID NO: 4.
In a further aspect the introgression fragment optionally further comprises the donor nucleotide of SNP 02 and/or SNP 05. Thus, the introgression fragment may comprise the donor genotype for SNP_03, SNP_04 and SNP_05; or for SNP_03 and SNP_02; or for SNP_03 and SNP_02 and SNP_04; or for SNP_03, SNP_02, SNP_04 and SNP_05. In another aspect the introgression fragment optionally further comprises the donor nucleotide of SNP 02 and/or SNP 01. Thus, the introgression fragment may comprise the donor genotype for SNP 03 and SNP 02; or for SNP 03, SNP 02 and SNP 01; and optionally also for SNP 04, and further optionally also for SNP 05, and further optionally for SNP 06.
In yet another aspect the introgression fragment optionally further comprises the donor nucleotide of SNP_05 and/or SNP_06.
Thus in one aspect, the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 06 (or in-between SEQ ID NO:l and SEQ ID NO:6, or a sequence comprising at least 90%, or at least 95%, or at least 97%) or 98%o sequence identity to SEQ ID NO: 1 or to SEQ ID NO 6) and whereby the introgression fragment comprises the ToLCNDV resistant SNP genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05, and SNP 06. In one aspect, the fragment comprises the donor SNP genotype for SNP 03 and/or SNP 04.
Thus in one aspect, the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 01 and SNP 05 (or in-between SEQ ID NO:l and SEQ ID NO:5, or a sequence comprising at least 90%>, or at least 95%>, or at least 97% or 98% sequence identity to SEQ ID NO: 1 or to SEQ ID NO 5) and whereby the introgression fragment comprises the ToLCNDV resistant SNP genotype for one or more or all of SNP 01, SNP 02, SNP 03, SNP 04 and SNP 05. In one aspect, the fragment comprises the donor SNP genotype for SNP 03 and/or SNP 04. Optionally the resistant SNP genotype is also present for SNP 06.
Thus another aspect, the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 03 and SNP 04 (or in-between SEQ ID NO:3 and SEQ ID NO:4, or a sequence comprising at least 90%>, or at least 95%>, or at least 97%o or 98%o sequence identity to SEQ ID NO: 3 or to SEQ ID NO 4) and whereby the introgression fragment optionally comprises the ToLCNDV resistant SNP genotype for SNP 03 and/or SNP 04. Optionally the donor genotype is also present for one or more or all of SNP markers selected from SNP_01, SNP_02, SNP_05 and SNP_06.
In a further aspect, the cultivated melon plants or cells of these plants comprise an introgression fragment from a wild donor on chromosome 5, which introgression fragment confers the ToLCNDV resistance, whereby the introgression fragment lies in-between SNP 02 and SNP 04 (or in-between SEQ ID NO:2 and SEQ ID NO:4, or a sequence comprising at least 90%, or at least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 2 or to SEQ ID NO 4) and whereby the introgression fragment optionally comprises the ToLCNDV resistant SNP genotype for SNP 02 and/or SNP 03 and/or SNP 04. Optionally the donor genotype is also present for one or more or all of SNP markers selected from SNP 01, SNP 05 and SNP 06. Plants comprising plant cells according to the invention are another embodiment of the invention.
The melon plant according to the invention may be an inbred line, an open pollinated variety (OP) or an Fl hybrid. In one aspect the Fl hybrid comprises the introgression fragment in heterozygous form, i.e. produced by crossing two inbred parent lines, one of which possesses the introgression fragment (preferably in homozygous form, although not necessarily) and collecting the Fl hybrid seeds from said cross. The Fl hybrid may also comprise the introgression fragment in homozygous form, i.e. produced by crossing two inbred parent lines, each comprising the introgression fragment in homozygous or heterozygous form.
The melon plant according to the invention may be of any type. Preferably it has good agronomic and good fruit quality characteristics, such as large average fruit size (at least 500g, 600g, 700g, 800g, 900g, lOOOg or more), high average brix of the fruits (e.g. an average refractometer % total soluble solids of at least 10%, 12%, 14%, 16%, 18% or more), many fruits being produced per plant, firm fruit flesh, etc.
Also other resistances may be introduced into the melon plants of the invention, such as resistance to one or more of the following diseases: Bacterial Wilt, Root Rot, Crown Blight, Melon Rust, Powdery Mildew, Verticillum Wilt, Sulphur Bum, Scab, Watermelon Mosaic, Downy Mildew, Fusarium oxysporum fsp. melonis (Fom) race 0, Fusarium oxysporum fsp. melonis (Fom) race 1 , Fusarium oxysporum fsp. melonis (Fom) race 2, Fusarium oxysporum fsp. melonis (Fom) race 1.2, Fusarium Wilt
R2, Root Knot (Nematode), Anthracnose, Cucumber Mosiac, and Squash Mosaic, and/or resistance to one or more of the following pests: Aphid resistance, Pickle Worm, Darkling Ground Beetle, Banded Cucumber Beetle, Mite, Western Spotted Cucumber Beetle, Melon Leafhopper, Melon Worm, Western Striped Cucumber Beetle or Melon Leafminer. Other resistance genes, against pathogenic viruses, fungi, bacteria or pests may also be introduced.
A specific aspect of the invention concerns plants or plant cells comprising an introgression fragment according to the invention which introgression fragment is obtainable from seeds deposited under NCIMB 42585 or from progeny thereof. The seeds deposited are cultivated melon plants of the BC4S4 generation comprising the introgression fragment in homozygous form, with the donor nucleotide being present in homozygous form for SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06. The TolCNDV resistance is most likely located in-between SNP 03 and SNP 04, which means that the size of the donor introgression can be reduced, by selecting recombinants having smaller introgression fragment sizes. So plants comprising sub-fragments of the introgression fragment (wherein said sub- fragments still confer TolCNDV resistance), comprising the donor SNP for SNP 03 and/or SNP 04, but having the SNP genotype of the recurrent parent for one or more or all of the other SNPs can be generated in ways known to the skilled person.
Whether a plant comprises the ToLCNDV resistance from the deposited seeds (either an introgression fragment of the same size or a sub-fragment thereof) can be determined by various methods, such as sequencing and comparing the sequences of the introgression fragments and recombination sites.
Melon plants and plant parts (such as leaves, stems, roots, fruits, pollen, flowers, etc.) comprising melon plant cells according to the invention are also an embodiment of the invention. Likewise seeds from which such plants can be grown are encompassed herein, as well as parts of such seeds (e.g. cells or tissues of the seeds such as the seed coat, embryo, etc.).
A further aspect of the present invention concerns melon seeds comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, in- between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in- between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or in-between SNP 03 and SNP 04. In a preferred embodiment of the invention the seeds comprise an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, more preferably in-between SNP 01 and SNP 05, even more preferably in-between SNP 02 and SNP 05, furthermore preferably in-between SNP 02 and SNP 04 and most preferably in-between SNP 03 and SNP 04.
Another embodiment of the invention concerns melon seeds obtainable or obtained from plants according to the invention, or seeds comprising plant cells according to the invention.
A further aspect of the present invention concerns melon plant fruits comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or in-between SNP 03 and SNP 04. In a preferred embodiment of the invention the seeds comprise an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, more preferably in-between SNP 01 and SNP 05, even more preferably in-between SNP 02 and SNP 05, furthermore preferably in-between SNP 02 and SNP 04 and most preferably in-between SNP 03 and SNP 04.
Another embodiment of the invention concerns melon fruits obtainable or obtained from plants according to the invention, or fruits comprising plant cells according to the invention.
Preferably melon fruits according to the invention are characterized in that they comprise an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant comprising the sequence of the donor plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in- between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in- between SNP 02 and SNP 04, in-between SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or preferably in-between SNP 03 and SNP 04 in heterozygous or homozygous state. The preferred and further embodiments described herein for melon plant cells or melon plants according to the invention are applicable to also represent preferred and further embodiments of the melon fruits of melon plants according to the invention, accordingly.
A further aspect of the present invention concerns melon plant propagation material comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or in-between SNP 03 and SNP 04. In a preferred embodiment of the invention the propagation material comprises an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 and SNP 06, more preferably in-between SNP 01 and SNP 05, even more preferably in-between SNP 02 and SNP 05, furthermore preferably in-between SNP 02 and SNP 04 and most preferably in-between SNP 03 and SNP_04.
Another embodiment of the invention concerns melon plant propagation material obtainable or obtained from plants according to the invention, or melon plant propagation material comprising plant cells according to the invention.
The preferred and further embodiments described herein for plant cells or plants according to the invention are applicable to also represent preferred and further embodiments of the propagation material of melon plants according to the invention, accordingly.
The term "propagation material" comprises those components of the plant which are suitable for generating progeny via the vegetative (agamic) or generative (gamic, sexual) route. Suitable for vegetative propagation are, for example, cuttings, in vitro tissue, cell, protoplast, embryo or callus cultures, micropropagation methods, rhizomes or tubers. Other propagation material includes, for example, fruits, seeds, seedling, being homozygous or heterozygous for an chromosome 5 introgression fragment conferring ToLCNDV resistance etc. The propagation material in one aspect takes the form of cuttings which are propagated by grafting to another rootstock or in vitro tissue culture material, in particular embryo cultures. In particular preferred is propagation material in the form of in vitro tissue culture material, particularly in vitro embryo cultures.
In one aspect non-propagating plant cells comprising the recombinant chromosome 5 described herein are provided. In one aspect such non-propagating plant cells may however be part of a melon plant or melon plant part. A further embodiment of the invention concerns a method for producing a ToLCNDV resistant melon plant comprising the following steps a) Selecting a ToLCNDV resistant donor plant b) Crossing the donor plant selected in step a) with a recurrent plant sensitive to ToLCNDV c) Obtaining seeds from the plants crossed in step b) and optionally d) Verifying if the plants grown from the seeds obtained in step c) are resistant to ToLCNDV and/or comprise one or more of the SNPs from the donor plant selected from the group of SNP 01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.
A ToLCNDV resistant donor plant in step a) in the method for producing a ToLCNDV resistant melon plant according to the invention can be selected by infection of melon plants with ToLCNDV and determining the level of symptoms of ToLCNDV infected melon plants as described elsewhere herein. The same is applicable for verification in steps b) and c) of the method for producing a ToLCNDV resistant melon plant according to the invention, if a plant is ToLCNDV sensitive or resistant, respectively. In a preferred embodiment of the method for producing a ToLCNDV resistant melon plant according to the invention the ToLCNDV resistant donor plant in step a) comprises a fragment on chromosome 5 conferring ToLCNDV resistance, the fragment comprising the sequence of the donor plant in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, in-between SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or in-between SNP 03 and SNP 04. Most preferably the fragment on chromosome 5 conferring ToLCNDV resistance comprises the sequence in- between SNP 03 and SNP 04. In a specifically preferred embodiment of the invention, the method for producing a ToLCNDV resistant melon plant according to the invention the ToLCNDV resistant donor plant in step a) comprises the fragment of chromosome 5 conferring ToLCNDV resistance comprising the sequence in-between SNP 01 and SNP 06, in-between SNP 01 and SNP 05, in-between SNP 01 and SNP 04, in-between SNP 02 and SNP 06, in-between SNP 02 and SNP 05, in-between SNP 02 and SNP 04, in-between SNP 03 and SNP 06, in-between SNP 03 and SNP 05 or preferably in- between SNP 03 and SNP 04 as found in the seeds deposited under NCIMB 42585.
In a preferred embodiment of the invention the method for producing a ToLCNDV resistant melon plant according to the invention is used for producing a plant according to the invention. The preferred and further embodiments as described herein for the plants according to the invention are applicable accordingly to the method for producing a ToLCNDV resistant melon plant according to the invention. Plants obtainable or obtained by a method for producing a ToLCNDV resistant melon plant according to the invention are also an embodiment of the invention.
A further embodiment of the invention concerns methods for producing melon seeds comprising the following steps a) growing a melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, b) harvesting the fruits of the melon plants grown in step a) c) collecting the seeds from the fruits obtained in step b).
In a preferred embodiment of the invention the melon plants of steps a) of the method for producing melon seeds according to the invention has the specific characteristics described as preferred and further embodiments of the plants according to the invention. The preferred and further embodiments as described herein for the plants according to the invention are applicable accordingly to the method for producing a hybrid melon seed according to the invention.
Seeds obtainable the method for producing melon seeds according to the invention are also an embodiment of the invention.
Another embodiment of the invention concerns methods for producing hybrid melon seeds comprising the following steps a) providing a first inbred melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP_04, b) providing a second inbred melon plant with or without a chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, c) crossing the plant provided in step a) with the plant provided in step b) d) selecting seeds obtained from the cross of step c).
"Inbred plant" or "inbred line" shall mean in connection with the present invention plants which have undergone several generations of selfing and are highly uniform in respect to their genetic setup and phenotypic appearance.
In a preferred embodiment of the invention the inbred lines of steps a) and b) of the method for producing hybrid melon seeds according to the invention has the specific characteristics described as preferred and further embodiments of the plants according to the invention. The preferred and further embodiments as described herein for the plants according to the invention are applicable accordingly to the method for producing a hybrid melon seed according to the invention.
Hybrid seeds obtainable or obtained by the method for producing hybrid melon seeds according to the invention are also an embodiment of the invention.
A further embodiment of the present invention are methods for producing a melon fruit comprising the following step a) growing a plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, b) harvesting the fruits produced by the plants grown in step a). The term "fruit" in its botanical meaning is commonly understood to be a seed bearing structure developed from the ovary of angiosperm flowers.
Melon fruits obtainable or obtained by a method for producing a melon fruit according to the invention are also an embodiment of the invention.
Melon donor plants being resistant to ToLCNDV can be identified with the aid of the SNP markers, in particular one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06 disclosed herein. The present invention therefore for the first time enables a person skilled in the art to identify donor plants from which an introgression fragment conferring ToLCNDV resistance to melon plants can be transferred into recurrent melon plants.
A further embodiment of the invention therefore pertains the use of one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 or SNP 06 for identification of a ToLCNDV resistant melon plant or parts thereof (such as cells, fruits, leaves). Preferably the use pertains the identification of ToLCNDV resistant donor melon plants and/or recurrent melon plants, but also to the identification of breeding lines, cultivars or varieties containing a recombinant chromosome 5, e.g. the recombinant chromosome 5 derived from the seeds deposited herein, optionally comprising a subfragment of the introgression fragment present in the seeds deposited. Another embodiment is the use of one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 or SNP 06 for introgression of ToLCNDV resistance into a TolCNDV susceptible melon plant, especially a cultivated melon line or variety.
Also an embodiment of the invention is the use of one or more or all of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 or SNP 06 in breeding ToLCNDV resistant melon plants.
Also provided is a method of screening plants or plant material or DNA derived therefrom for the presence of a fragment on chromosome 5 conferring TolCNDV resistance. The method comprises the steps of: screening the genomic DNA for the SNP genotype of one or more or all of SNP 01, SNP 02, SNP_03, SNP_04, SNP_05 and SNP_06; and optionally selecting plants or plant material which comprise the resistant donor genotype of one or more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.
Also provided is a method for producing a cultivated C. melo plant comprising an introgression fragment on chromosome 5, wherein said introgression fragment comprises a ToLCNDV QTL, comprising: crossing a first cultivated melon plant being susceptible to ToLCNDV with a second wild melon plant being resistant to ToLCNDV, wherein said second melon plant comprises the CC or CT genotype for SNP 01 in SEQ ID NO: 1, and/or the TT or TG genotype for SNP 02 in SEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/or the CC or CT genotype for SNP 05 in SEQ
ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6; collecting Fl seeds from said cross and backcrossing an Fl plant to the first melon plant to produce a backcross (BC1) population, or selling said Fl plants one or more times to produce an F2 or F3 population, and optionally selling the backcross population to produce a BC1 S1 population, wherein said F2, F3, BC1 or BC1 S1 plant comprises the CC or CT genotype for SNP 01 in SEQ ID NO: 1, and/or the TT or TG genotype for SNP 02 in SEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/or the CC or CT genotype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6.
Also provided is a method for identifying or detecting a cultivated C. melo plant comprising an introgression fragment on chromosome 5, wherein said introgression fragment comprises a ToLCNDV- resistance allele, comprising: screening a Cucumis melo plant using a molecular marker assay which detects at least one of SNP marker selected from the group consisting of: SNP 01 in SEQ ID NO: 1, SNP 02 in SEQ ID NO: 2, SNP 03 in SEQ ID NO: 3, SNP 04 in SEQ ID NO: 4, SNP 05 in SEQ ID NO: 5 and/or SNP 06 in SEQ ID NO: 6; and identifying and/or selecting a plant comprising the CC or CT genotype for SNP 01 in SEQ ID NO: 1, and/or the TT or TG genotype for SNP 02 in SEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/or the CC or CT genoype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6.
A method of producing C. melo Fl hybrid plants comprising a ToLCNDV resistance phenotype is provided comprising: crossing a first inbred melon plant comprising at least one recombinant chromosome 5, the recombinant chromosome 5 comprising an introgression fragment that confers ToLCNDV resistance onto the first inbred melon plant when present in homozygous or heterozygous form and wherein said introgression fragment is from a wild plant of the species Cucumis melo, with a second inbred melon plant with or without said at least one recombinant chromosome 5 and collecting Fl hybrid seeds from said cross.
Further encompassed is a method for producing a melon plant comprising ToLCNDV resistance on chromosome 5, said method comprising: a) screening a wild melon accession or several wild melon accessions using a molecular marker assay which detects at least one of SNP marker selected from the group consisting of: SNP 01 in SEQ ID NO: 1, SNP 02 in SEQ ID NO: 2, SNP 03 in SEQ ID NO: 3, SNP 04 in SEQ ID NO: 4, SNP 05 in SEQ ID NO: 5 and/or SNP 06 in SEQ ID NO: 6 b) identifying and/or selecting a wild melon plant comprising the CC or CT genotype for
SNP 01 in SEQ ID NO: 1, and/or the TT or TG genotype for SNP 02 in SEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/or the CC or CT genotype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6. c) optionally confirming ToLCNDV resistance in a resistance assay; d) and optionally introgressing said ToLCNDV resistance from said wild accession into a cultivated melon plant.
A Cucumis melo plant, or part thereof, is provided comprising a recombinant chromosome 5, the recombinant chromosome 5 comprising an introgression fragment that confers ToLCNDV resistance onto the Cucumis melo plant when present in homozygous or heterozygous form and wherein said introgression fragment comprises one or more or all of the Single Nucleotide Polymorphism (SNP) markers of the group: the CC or CT genotype for SNP 01 in SEQ ID NO: 1 , the TT or TG genotype for SNP 02 in SEQ ID NO: 2, the TT or TC genotype for the SNP 03 in SEQ ID NO: 3, the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, the CC or CT genoype for SNP 05 in SEQ ID NO: 5, and/or the AA or AG genotype for SNP 06 in SEQ ID NO: 6, and wherein said introgression fragment is from a wild plant of the species Cucumis melo, said wild plant having an average ToLCNDV disease score of at least 7.0 on a scale of 1 = dead plant to 9 = no symptoms.
In one aspect the cultivated melon plant comprises at least the TT or TC genotype for the SNP 03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4.
In one aspect the ToLCNDV resistance QTL or the introgression fragment comprising the QTL is the obtainable from / can be obtained from / is as present in seeds of which a representative sample has been deposited under Accession Number NCIMB42585 or progeny thereof (whereby the progeny retain the ToLCNDV resistance).
Seed deposit information
A representative sample of seeds of a cultivated melon, designated Cucumis melo TOLCHR5, comprising an introgression fragment comprising TolCNDV resistance introgressed on chromosome 5 (backcross 4, selling 4 generation, BC4S4), was deposited by Nunhems B.V. on 6 June 2016 at the NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn Aberdeen, Scotland AB21 9YA, UK) according to the Budapest Treaty, under the Expert Solution (EPC 2000, Rule 32(1)). Seeds were given the following deposit numbers NCIMB 42585.
The Applicant requests that samples of the biological material and any material derived therefrom be only released to a designated Expert in accordance with Rule 32(1) EPC or related legislation of countries or treaties having similar rules and regulation, until the mention of the grant of the patent, or for 20 years from the date of filing if the application is refused, withdrawn or deemed to be withdrawn.
Access to the deposit will be available during the pendency of this application to persons determined by the Director of the U.S. Patent Office to be entitled thereto upon request. Subject to 37 C.F.R. § 1.808(b), all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent. The deposit will be maintained for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent whichever is longer, and will be replaced if it ever becomes nonviable during that period. Applicant does not waive any rights granted under this patent on this application or under the Plant Variety Protection Act (7 USC 2321 et seq.). Description of sequences
Characters other than G (guanine), A (adenine), T (thymine) and C (cytosine) have the following meaning in the SEQ ID NOs shown in the sequence listing:
R: G or A
Y: T or C
M: A or C
K: G or T
S: G or C
W: A or T H: A or C or T
B: G or T or C V: G or C or A
D: G or A or T N: G or A or T or C
In SEQ ID NO: 1 to SEQ ID NO: 6 the SNP nucleotide of the resistant donor is shown in bold and underlined.
SEQ ID NO 1 : Sequence of the ToLCNDV resistant donor plant comprising SNP 01.
SEQ ID NO: 1 :
agctggtgca aagctggcat tcaaatcgaa tgaagaaata gcagtacaag tgaagtcaat 60 tccactagat gaagtaatcc cggattcaga acgagtgctt ctaataaaaa tcgatgttca 120 aggctgggaa tatcatgtgc taaaaggggc aaagagaatt ttgtcaagga agggcactga 180 agctccatat ctcatctatg 200
SEQ ID NO 2: Sequence of the ToLCNDV resistant donor plant comprising SNP 02.
SEQ ID NO: 2
ttgtaatcat ggccattgcc tgatytgcag aatgtggcgt cttgattggg cacggtctgg 60 atgaagttac tccaaagctt gtggaagtta atgggttctt ctcttcaccr tctgrtctcc 120 ttcccatcga caatgtggtt tcttcttcaa acacaaaaga aacccctgtt gaagctgtag 180 aagaaagctg tgagttcttt tctccactca gttctaggtt tagtttttta tttgtttttt 240 ttttcttttt tcctttttac ttgctttgag ctgtaggaca caacacaaaa tgataagaac 300 aaaaatcaat ctatgttatt gttgtcattg tctcgcctgc ctcttcaagg taactaggct 360 ataacactga tccaaaatgt ataatcttta ttgctaagta acttatattg caaagattgg 420 gatggaatag gagcataggt tgcatctaat agttttgtcc aactttggtt atagtttcag 480 ccagtttgtt cagatgaaat tcttagtgat tatcactaat ggaattgttt ttataaatgt 540 tgtaggtgtt gaaagaagtg aagaatatga aaaggaaagc aggggaaccg agaaggctga 600 aatcttaccg acaaaagcaa catctgaagc aggttctgaa gtccaacctg tttccagtga 660 ttctgctcag atggtaccca atatgttgga gctcggtgat gcttataagc tagctgtagg 720 tgctagagga ggaagacaat tgtctggcaa gcttttggaa caatggatyg ggaaggaatc 780 ttcaaaagtt agtgaagatc tgaagcttct cttgacacaa ctctcattta atcgtttgaa 840 tgaccaatca cgggagatga gtccaaggct gtccgtaaat ggagacgagg tgaggaactt 900 tgattacttg agygctgttg ggatgcaaat gctacaaaaa aggwtttcgc ttgaaagaaa 960 tgagtccggt gtagaatctt tagatggaag cataattagt gaaatcgatg gggaaaacat 1020 ggctgatagg ttgaaacgac agattgagta tgataagaag gttmtgartt ctttatacaa 1080 ggaattggag gaagaaagaa atgcatccgc aattgctgca aatcaggcaa tggccatgat 1140 tacaa 1145
SEQ ID NO 3: Sequence of the ToLCNDV resistant donor plant comprising SNP 03.
SEQ ID NO 3
agacggcgta catgcagcat tctgttaccc cgcgatgccc agcgatgaaa caactccttt 60 gcatccttat ggtccagagg ttccagatca tacgactcac caaatgcagg aaacttaaat 120 ctagaggtga ccaaaatttt gcagttgggt aatcgggaga acttttca 168
SEQ ID NO 4: Sequence of the ToLCNDV resistant donor plant comprising SNP 04.
SEQ ID NO 4
ggagkcttca tccgtcttta ccttcctcac ttttttacct aaccaagtta cgtcttgtag 60 gttgcaagat aacaaatttg gatttcttag aaacaattgt ttatgttgcc ccttcrttga 120 aagagttgga cttgtccgaa aacaactttt gtagamtacc ctcrtgtatt attaatttta 180 aatccctgaa atatctttat acaatggatt gtgagttgct ygaagaaatt tcaaaggttc 240 cagaaggtgt aatttgtacg agtgccgcag gatgcaaatc attggctaga tttcccgaca 300 acttagctga tttcatatct tgtggtaatt ctgcggtgcg taccatatct ctttctcatg 360 acttcaccat tatctctagc tcatgtatat ttaatttcat tcatataata tatattactt 420 ataactattt actgatctca tggtgcagga atgttgtaaa ggtggatg 468
SEQ ID NO 5: Sequence of the ToLCNDV resistant donor plant comprising SNP 05.
SEQ ID NO: 5 tgcaggtagt caaaccaatt gataatattt gctcaccatc aaaattggta accatagaca 60 tagaytgacc aagcccaggc aaaacggcat catctttttg ttttctatca ttactattct 120 gattacccaa aagaaatctt ctttttgtac ttggagagaa ataatccccg tctgcacatt 180 gcatggaagg tgtgctgtca tgagcatcag cattacacac accagaatta gatttcttgc 240 tagccaagat ttcacaacca ccaagagaag caggacctac gcctggggtc tctgcctyag 300 aaagccaags atcaaagcac acttgtgaag gacctgaaac tgtctcagat ccagggattt 360 ctcctacatt gggtaagcta gcctgttcaa agccatcaca cacatctaca agcggcagtc 420 taccatacat ggaactatta gaataagcag gcttattttc ataaaacaca ttaacgcttg 480 catttggatt agttaaacat ggcatcaaat gggaagaatc ccgaaatctc aagctgttct 540 cattggaccg cagcactcca gaattcaagt ctcttgacca tacttttttt tgcctgctat 600 caacagtaaa attttgagct ttaccatatc cacagtcaag tggctcattt cgagagttac 660 gcttcctcct cattaaatca cktacagaac ggccttccct ggagttatgt gtgcctgtac 720 ctgcatttcg taatgtaaca tccacctgcc ctccatcttc acttgaaaag caactaatga 780 actcagaatc tttggtcgaa cttgtccata tgtcaggcct gcaagaattg acacacgtcg 840 aatctgtatt cactttctca gcctcatgaa aaggtaaaga tccccaaaat gatttctctt 900 tcttgtgact gcaagaacct gaatcaatgc catgcaataa tattgcaagt ttggaagatc 960 tttcattctc aacattaaga tctctatcag gtgagttttc agtcgaccct ccagaagaag 1020 agaacatatc atctgcakgt acgcagt 1047
SEQ ID NO 6: Sequence of the ToLCNDV resistant donor plant comprising SNP 06.
SEQ ID NO: 6
gcgtacmtgc aggcaaagaa tggcacagta cagtaatggt agctcatctg ttcatcaagg 60 agaatcaagc tgcctcagcc attcagtatc ggttccaccc ctgatcatct cctacaatga 120 tcgcattcgt cctctccttg atgctgttga caagcttcgt cacctcatga tcatgagaga 180 aggcatccaa ctgcctacca tagttgttgt tggtgatcag tcawccggta agtcaagtgt 240 cctcgagtcg ttggctggga tcagcctacc tcgaggtcag ggcatctgca ccagggtccc 300 tctgataatg aggctccaaa accatcctga tcccgaaccc gagcttgttt tggagtacaa 360 tgggaaaaag atccacaccg acgaatcctt cattgctgaa gacatctgta cagctacaga 420 ggagattgct ggcagtggca aaggaatatc gaaagcgcca ttgactttga ttgtgaagaa 480 aaatggtgtt cctgatctta caatggttga tctccctgga attacragag tgcctgttaa 540 agatcagcct gaagacattt atgaccaaat aaaagatata atcatggaac atatcaagcc 600 agaagagagc atcatcttga atgtcttgtc tgcgacggtt gattttccaa cttgtgaatc 660 gatacggatg tctcaaagtg tcgacaagac gggaatgaga acgttggcag ttgtgactaa 720 gtctgacaag gcaccagaag gcctacacga gaaggtcacc rcggatgatg tcagtatcgg 780 ccttggttat gtttgcgtta ggaaccgaat tggcaatgag acatatgagg aagctcgggt 840 tgcagaagcc aaattgtttt caactcatcc tcttctctcc aaaattgaca aatctgttgt 900 gggcattcca gtcttggctc agaagttggt gcaaattcaa gcaggtaccc aaactaattc 960 ctgactcaaa agctaggttc cgttagataa ccattttgtt ttagaaaatc aagtttattt 1020 tctctaaatm gtgtaccatg attttcatct ttcttaaata aaaaagttgm attcttwact 1080 aaattttaaa agcaaaaaca agttttaata cttttttt 1118
SEQ ID NO 7: KASP- -assay primer for FAM allele of SNP _01.
SEQ ID NO 8: KASP- -assay primer for VIC allele of SNP 01.
SEQ ID NO 9: KASP- -assay common primer for SNP 01.
SEQ ID NO 10 :KASP- -assay primer for FAM allele of SNP _02.
SEQ ID NO 11 :KASP- -assay primer for VIC allele of SNP 02.
SEQ ID NO 12: :KASP- -assay common primer for SNP 02.
SEQ ID NO 13 :KASP- -assay primer for FAM allele of SNP _03.
SEQ ID NO 14 :KASP- -assay primer for VIC allele of SNP 03.
SEQ ID NO 15: :KASP- -assay common primer for SNP 03.
SEQ ID NO 16 :KASP- -assay primer for FAM allele of SNP _04.
SEQ ID NO 17: :KASP- -assay primer for VIC allele of SNP 04.
SEQ ID NO 18: :KASP- -assay common primer for SNP 04.
SEQ ID NO 19 :KASP- -assay primer for FAM allele of SNP _05.
SEQ ID NO 20: :KASP- -assay primer for VIC allele of SNP 05.
SEQ ID NO 21 :KASP- -assay common primer for SNP 05.
SEQ ID NO 22: :KASP- -assay primer for FAM allele of SNP _06.
SEQ ID NO 23: :KASP- -assay primer for VIC allele of SNP 06. SEQ ID NO 24:KASP-assay common primer for SNP 06. Description of the Figures
Fig. 1 : Shown is a ToLCNDV sensitive recurrent plant (upper picture) and a recurrent plant into which ToLCNDV resistance was integrated (lower picture) by introgression of a fragment comprising the sequence of the donor in-between SNP 01 and SNP 06. The picture was taken 25 days post infection (dpi) with ToLCNDV by whitefly transmission.
Fig. 2: Shown are the symptom levels 35 days post infection (dpi) with ToLCNDV by whitefly transmission of a donor plant being ToLCNDV resistant (Wild Donor), a recurrent plant (Recurrent) and a plant obtained after introgression of the ToLCNDV resistance from the donor plant into the recurrent plant (Introgression). The symptom levels were determined as described herein under "General Methods".
General Methods
1. Determination of symptom level on ToLCNDV infected plants 1.1 Plants and pathogens (virus)
A melon plant (Cucumis melo) infecting strain of ToLCNDV is used for infection of melon plants. In the present invention a ToLCNDV strain isolated in Murcia, Spain was used as inoculum.
1.2 ToLCNDV propagation
The ToLCNDV inoculum source is maintained on living infected melon plants. It must be ensured, that pure virus isolates are used and that neither the virus source, nor the whiteflies are contaminated with other diseases, in particular with other viruses (e.g. CGMMV, CYSDV, CYVY SqMV). For pre- multiplication of the ToLCNDV inoculum whiteflies (Bemisia tabaci) are fed on ToLCNDV sensitive (susceptible), infected melon plants in an insect proven cage. Before infection of test plants, put ToLCNDV infected plants into an insect proven cage, release whiteflies to the same cage and allow the whiteflies to feed for approximately 3 days on the ToLCNDV infected plants.
1.3 Inocculation of plants to be tested
For each genotype of melon plants to be analysed 14 plants were grown until the first true leaf is expanded (normally 12 - 15 days after sowing), 12 of which were infected and 2 were mock infected. Also 12 plants of susceptible varieties were included, in this experiment variety Gandalf Fl (Hild Samen) and variety Vedantrais. The 12 plants per genotype to be tested on ToLCNDV resistance were put into an insect proof cage, infected whiteflies obtained as described under 1.2 above, were released into the cage to infect the plants. It has to be ensured that at least 5 -10 whiteflies are available for each test plant in the cage. Whiteflies and test plants are kept in the cage for approximately 48 hours, before the whiteflies are eliminated with an appropriate insecticide. Also two plants per genotype were mock infected, i.e. they were treated in the same manner as the test plants apart from that the whiteflies used for infection were free of ToLCNDV.
1.4 Growing infected test plants
Infected test plants obtained as described under 1.3 were transplanted to bigger pots, transferred into a greenhouse with cooling equipment. The plants were grown at approximately 18°C night temperature and approximately 25°C day temperature in a timeframe of 14 to 16 hours daylight. The infected plants for each infected genotype were grown in two replicates in two different plots, each of which comprises 6 ToLCNDV infected plants and 1 mock infected plant. The plots are randomized in respect to the growing area.
1.5. Scoring the symptom level of ToLCNDV infection The scoring of the symptom level may already be done approximately 15 days post infection (dpi) with ToLCNDV but is preferably done approximately 30 days post infection (dpi) with ToLCNDV, or later. In case plants are present which show recovery from the virus infection, a further scoring of the symptom is done approximately 45 days post infection (dpi) with ToLCNDV.
The following symptom levels are to be used according to the phenotypes indicated in the following:
Symptom level Observed phenotype
1 Dead plant
2 Severe mosaic and curling, chlorosis and growth reduction. No recovery
3 Strong mosaic and curling, chlorosis and growth reduction. No recovery
4 Curling and mosaic, chlorosis, no or mild growth reduction. No recovery
5 Curling and mosaic, chlorosis, no growth reduction. Slight recovery of the upper plant zone
6 Mild curling, mosaic and chlorosis, no growth reduction. Recovery of the upper middle plant
7 Mild curling, mosaic and chlorosis, no growth reduction. Symptoms appear only in the lower plant zone
8 Faint mosaic
Figure imgf000047_0001
1.6 Optional additional tests
It is recommended to use at least one genotype highly resistant to ToLCNDV (symptom level 8 - 9) and one genotype highly sensitive to ToLCNDV (symptom level 1) in each experimental setup. It is further recommended to also include a genotype being intermediate resistant to ToLCNDV infection in each test setup. Best results are obtained when the just mentioned genotypes are included and the symptom levels of each genotype is scored relative to the results obtained for the highly resistant, highly sensitive and intermediate resistant genotypes. These genotypes also give a clear indication on the amount of infection of melon plants with ToLCNDV by the whiteflies. Furthermore, it is advisable to check infection and spreading of ToLCNDV in infected plants and control plants. This can be done by checking for the presence and amount of virus DNA in upper parts of the plants. A suitable way to check for the presence and amount of ToLCNDV DNA in upper plant parts is hybridization of plant material with a probe hybridizing with the DNA of the ToLCNDV strain used. Various hybridization techniques are well known in the art. A simple so called Dot Blot analysis is sufficient for obtaining valuable results. Likewise PCR or quantitative PCR techniques can be used.
Examples
1. Selection of ToLCNDV resistant donor plants
The symptom level of wild accessions of melon plants were tested for ToLCNDV resistance according to the test described under "General Methods". A wild donor plant was identified which has a high resistance to ToLCNDV infection, having a resistance level of about 7 (as seen further below the average disease score was 7.4, while the susceptible plant had an average score of 2.0).
2. Identification of genomic location of ToLCNDV resistance
Three mapping populations were developed including the use of the donor plant obtained in Example 1 to map the position of the ToLCNDV resistance conferring fragment (QTL) in the genome of donor melon plant.
Analysis in these mapping populations revealed one major QTL associated with resistance, located on chromosome 5 and showing dominant inheritance patterns.
The magnitude of the detected fragment QTL and observed inheritance patterns suggested a single locus dominant gene. From resistant material the inventors developed BC (back cross) lines to fine map and further investigate resistance from the donor. Genotypic results in 10 advanced BC families developed through phenotypic selection showed >93 % agreement with individual phenotypes.
The markers identified during fine mapping and their respective positions according to publicly known data from Diaz et al. (2015, Mol Breeding 35, 188) is shown in the following Table:
Pseudo-
SNP molecule
SNP Marker ID position coordinates
AI 13-H12 CM3.5 scaffold00003 7996720 27276249
SNP_ .01 mME11320_k CM3.5 scaffold00003 7824960
SNP_ 02 mME43070_k CM3.5 scaffold00003 6950286
SNP_ _03 mME10621_k CM3.5 scaffold00003 6553350
SNP_ 04 mME50729_k CM3.5 scaffold00003 5785550
SNP_ _05 mME32395_k CM3.5 scaffold00003 5202092
CMGAAN144 CM3.5 scaffold00003 5123876 24403405
CMPSNP682 CM3.5 scaffold00009 4703882 15854444
SNP_ _06 mME49184_k CM3.5 scaffold00009 2607437
CMPSNP460 CM3.5 scaffold00009 1816109 12966671
3. Development of KASP-assay
A KASP-assay was developed for identifying the SNPs flanking the QTL. The SNPs associated with the QTL can be determined by use of the following primers in a KASP-assay:
SNP FAM allele VIC allele Common Primer
SNP_ .01 SEQ ID NO 7 SEQ ID NO 8 SEQ ID NO 9
SNP_ 02 SEQ ID NO 10 SEQ ID NO 11 SEQ ID NO 12
SNP_ _03 SEQ ID NO 13 SEQ ID NO 14 SEQ ID NO 15
SNP_ 04 SEQ ID NO 16 SEQ ID NO 17 SEQ ID NO 18
SNP_ _05 SEQ ID NO 19 SEQ ID NO 20 SEQ ID NO 21
SNP_ _06 SEQ ID NO 22 SEQ ID NO 23 SEQ ID NO 24
4. Introgression of ToLCNDV into a cultivated melon plant
Backcrossing has been performed with cultivated melon plants and recurrent melon plants having an increased resistance to ToLCNDV have been obtained. The presence of the QTL in these lines was established by use of the KASP-assay sequences shown in Example 3. A representative result for the ToLCNDV symptom level achieved in those plants is given in the following Table and in Figure 2. The symptom levels have been determined 35 days post infection (dpi) according to the method described herein under "General Methods".
Figure imgf000049_0001
AVG: average value of tested genotype, STDV: standard deviation
Additional backcrossing is done to introgress the resistance in elite Piel de Sapo, Galia, Cantaloup and Charentais backgrounds. Genotypic results in 10 advanced BC families developed through phenotypic selection show >93 % agreement with individual phenotypes.

Claims

Claims
1. A melon plant cell comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 03 at nucleotide 68 of SEQ ID NO: 3 and SNP 04 at nucleotide 227 of SEQ ID NO: 4.
2. A melon plant cell according to claim 1, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between SNP 01 at nucleotide 101 of SEQ ID NO: 1 and SNP 06, at nucleotide 445 of SEQ ID NO: 6, or in-between SNP 02 at nucleotide 945 of SEQ ID NO: 2 and SNP 05 at nucleotide 839 of SEQ ID NO: 5, or in-between SNP 02 at nucleotide 945 of SEQ ID NO: 2 and SNP 04 at nucleotide 227 of SEQ ID NO: 4, or in-between SNP 03 at nucleotide 68 of SEQ ID NO: 3 and SNP 05 at nucleotide 839 of SEQ ID NO: 5.
3. A melon plant comprising melon plant cells according to claim 1 or 2.
4. A melon seed comprising plant cells according to claim 1 or 2.
5. A melon fruit comprising plant cells according to claim 1 or 2.
6. Melon plant propagation material comprising plant cells according to claim 1 or 2.
7. A method for producing a ToLCNDV resistant melon plant comprising the following steps
a) Selecting a ToLCNDV resistant donor plant,
b) Crossing the donor plant selected in step a) with a plant sensitive to ToLCNDV,
c) Obtaining seeds from the plants crossed in step b), and optionally
d) Verifying if the plants grown from the seeds obtained in step c) are resistant to ToLCNDV and/or comprise one or more of the SNP nucleotides from the donor plant selected from the group of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.
8. A method for producing melon seeds comprising the following steps: a) growing a melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, b) harvesting the fruits of the melon plants grown in step a), and optionally
c) collecting the seeds from the fruits obtained in step b).
9. A method for producing hybrid melon seeds comprising the following steps
a) providing a first inbred melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP_04,
b) providing a second inbred melon plant with or without a chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP_04,
c) crossing the plant provided in step a) with the plant provided in step b), and optionally d) selecting seeds obtained from the cross of step c).
10. A method for producing a melon fruit comprising the following steps:
a) growing a plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, the introgression fragment comprising the sequence of the donor plant in-between SNP 03 and SNP 04, b) harvesting the fruits produced by the plants grown in step a).
11. Use of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and/or SNP 06 for identification of a ToLCNDV resistant melon plant.
12. Use of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06 for introgression of ToLCNDV resistance into a ToLCNDV susceptible melon plant.
13. Use of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and/or SNP_06 in breeding ToLCNDV resistant melon plants.
PCT/EP2017/067078 2016-07-12 2017-07-07 Tolcndv resistant melon plants WO2018011075A1 (en)

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