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WO2025003305A1 - Résistance du poivre à meloidogyne enterolobii - Google Patents

Résistance du poivre à meloidogyne enterolobii Download PDF

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Publication number
WO2025003305A1
WO2025003305A1 PCT/EP2024/068072 EP2024068072W WO2025003305A1 WO 2025003305 A1 WO2025003305 A1 WO 2025003305A1 EP 2024068072 W EP2024068072 W EP 2024068072W WO 2025003305 A1 WO2025003305 A1 WO 2025003305A1
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Prior art keywords
plant
capsicum
qtl
enterolobii
resistance
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Isabelle JUSTAFRE
Sylvie Manin
Benoît GORGUET
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Vilmorin SA
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Vilmorin SA
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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
    • 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
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/822—Capsicum sp. [pepper]
    • 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
    • C12N15/8285—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 nematode resistance
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to Capsicum plants, seeds and parts thereof and related methods and uses.
  • Pepper Capsicum sp.
  • Solanaceae is one of the most important vegetable crops. Whilst its species are native to the Americas, it is now widely cultivated throughout the warm, temperate, tropical and subtropical countries, in field or greenhouse production. Pepper production is affected by a variety of diseases and parasites, including root-knot nematodes (Meloidogyne spp.) which are one of the three most economically damaging genera of plant-parasitic nematodes on horticultural and field crops. Root-knot nematodes are distributed worldwide, and are obligate parasites of the roots of thousands of plant species
  • M. Enterolobii is a polyphagous nematode, affecting in particular pepper (C. annuum), watermelon (C. lanatus), coffee (C. arabica), soybean (G. max), sweet potato (/. batatas), tomato (L. esculentum), tobacco (N. tabacum), bean (P. vulgaris), guava (P. guajava), eggplant (E. melongena) and ornamental plants and wild species.
  • Root-knot nematodes begin their lives as eggs that rapidly develop into J1 (first-stage juvenile) nematodes.
  • the J1 stage resides entirely inside the translucent egg case, where it molts into a J2 nematode.
  • J2s attack growing root tips and enter roots intercellu la rly , behind the root cap. They move to the area of cell elongation where they initiate a feeding site by injecting esophageal gland secretions into root cells.
  • These nematode secretions cause dramatic physiological changes in the parasitized cells, transforming them into giant-cells.
  • Root cells neighboring the giant-cells also enlarge and divide rapidly, presumably as a result of plant growth regulator diffusion, resulting in galls formation. Heavily galled roots provide minimal resources for the rest of the plant and severe infections result in reduced yields on numerous crops.
  • Root-knot nematodes are very difficult to manage because they are soilborne pathogens with a wide host range. Because root-knot nematodes live in the soil, chemical control requires applications of large amounts of chemicals with specialized equipment.
  • Resistance genes have been identified against certain root-knot nematode species, e.g. Meloidogyne incognita, M. javanica, M. arenaria, M. hapla.
  • the known genes of resistance to nematodes in pepper (Me1, Me2, Me3, Me4, Me5, Me6, Me7, N) are reported susceptible to M. enterolobii (Pinheiro et al, Nematropica 45.2 (2015): 184-188).
  • the present invention first provides a Capsicum plant comprising a quantitative trait locus (QTL) in its genome on chromosome 9, wherein said QTL confers to the plant resistance, in particular intermediate resistance, to Meloidogyne enterolobii.
  • QTL quantitative trait locus
  • the invention provides a Capsicum plant comprising in its genome a quantitative trait locus (QTL) on chromosome 9, wherein said QTL confers to the plant resistance to Meloidogyne enterolobii, and wherein said QTL is located within the chromosomal region delimited by single nucleotide polymorphism (SNP) PE-0057076 and SNP PE-0006730.
  • QTL quantitative trait locus
  • the invention provides a Capsicum plant comprising in its genome a quantitative trait locus (QTL) on chromosome 9, wherein said QTL confers to the plant resistance to Meloidogyne enterolobii, and wherein said QTL is located between positions 5905642 and 5921103 of the physical genome Zunla-1 v2.0.
  • QTL quantitative trait locus
  • said Capsicum plant comprises said QTL introgressed in its genome.
  • said QTL on chromosome 9 conferring resistance to Meloidogyne enterolobii is present in the genome of the seeds of Capsicum annuum PB21 SG7-1294, representative seeds of which have been deposited under accession number NCIMB 44105.
  • said QTL on chromosome 9 conferring resistance to Meloidogyne enterolobii can be identified by detecting one or more SNP markers selected from SNPs PE-0057076, PE-0057077, PE-0057080, PE-0057081 and PE-0006730.
  • said plant is a progeny of a Capsicum annuum plant PB21 SG7-1294, representative seeds of which have been deposited under accession number NCIMB 44105.
  • a further aspect of the invention relates to a cell of the plant according to the invention, wherein said cell comprises in its genome a quantitative trait locus (QTL) on chromosome 9 (QTL9), wherein said QTL confers to the plant resistance to Meloidogyne enterolobii, and wherein said QTL is located within the chromosomal region delimited by SNP PE-0057076 and SNP PE-0006730.
  • QTL quantitative trait locus
  • a further aspect of the invention relates to a plant part of a Capsicum plant according to the invention, wherein said plant part comprises cells according to the invention.
  • a further aspect of the invention relates to a Capsicum seed, which can be grown into a Capsicum plant according to the invention.
  • a further aspect of the invention relates to an in vitro cell or tissue culture of regenerable cells of a Capsicum plant according to the invention, wherein the regenerable cells are derived from a seed, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole or flower of a plant according to the invention.
  • a further aspect of the invention relates to a method of producing a Capsicum plant resistant to Meloidogyne enterolobii, comprising:
  • a further aspect of the invention relates to the use of a Capsicum plant or seed according to the invention, as a breeding partner in a breeding program for conferring M. enterolobii resistance to progeny Capsicum plants.
  • a further aspect of the invention relates to a method for producing a Capsicum plant resistant to M. enterolobii, comprising the steps of:
  • step b) optionally self-pollinating and/or backcrossing one or several times the plant selected at step b) and selecting in the progeny thus obtained a plant comprising the QTL on chromosome 9 conferring the resistance to M. enterolobii.
  • a further aspect of the invention relates to a method for producing a Capsicum plant resistant to M. enterolobii, comprising the steps of:
  • said marker is selected from SNPs PE-0057076, PE-0057077, PE-0057080, PE- 0057081 and PE-0006730.
  • said allele is selected from allele T of SNP PE-0057076, allele T of SNP PE-0057077, allele A of SNP PE-0057080, allele T of SNP PE-0057081 and allele G of SNP PE-0006730.
  • At least 2, preferably at least 3, still preferably at least 4, even more preferably at least 5 markers are detected at step (b).
  • a further aspect of the invention relates to a method for producing a Capsicum plant resistant to M. enterolobii, comprising the step of introgressing into a plant a QTL on chromosome 9 conferring resistance to M. enterolobii, wherein said QTL is located within the chromosomal region delimited by SNP PE-0057076 and PE-0006730.
  • a further aspect of the invention relates to a method for detecting and/or selecting plant resistant to M. enterolobii, wherein said method comprises the detection of at least one genetic marker linked to a QTL on chromosome 9 conferring the resistance to M. enterolobii.
  • said method comprises the detection of at least one genetic marker located within the chromosomal region delimited by SNP PE-0057076 and PE-00006730, or a genetic marker linked with said chromosomal region.
  • the method comprises the detection of a genetic marker within 40 cM, 20 cM, 10 cM, 5 cM, 2 cM or 1 cM of said chromosomal region.
  • the method comprises the detection of a genetic marker within 10 Mb, 5 Mb, 2 Mb, 1 Mb, 500 kb, 200 kb, 100 kb, 90 kb, 50 kb, 20 kb or 10 kb of said chromosomal region.
  • said method comprises the detection of a genetic marker selected from SNPs PE- 0057076, PE-0057077, PE-0057080, PE-0057081 and PE-0006730, or a genetic marker linked with one or more of said SNPs.
  • the method comprises the detection of a genetic marker within 40 cM, 20 cM, 10 cM, 5 cM, 2 cM or 1 cM of one or more of said SNPs.
  • the method comprises the detection of a genetic marker within Mb, 5 Mb, 2 Mb, 1 Mb, 500 kb, 200 kb, 100 kb, 90 kb, 50 kb, 20 kb or 10 kb of one or more of said SNPs.
  • a further aspect of the invention relates to the use of a marker for identifying a plant resistant to M. enterolobii, wherein said marker is linked to a QTL on chromosome 9 conferring the resistance to M. enterolobii.
  • said marker is located within the chromosomal region delimited by SNP PE- 0057076 and PE-00006730.
  • said marker is associated with at least one allele selected from: allele T of SNP PE-0057076, allele T of SNP PE-0057077, allele A of SNP PE-0057080, allele T of SNP PE-0057081 , and allele G of SNP PE-0006730, with a p-value of 0.05 or less.
  • said marker located within the chromosomal region delimited by SNP PE-0057076 and PE-00006730 and associated with at least one allele selected from: allele T of SNP PE-0057076, allele T of SNP PE-0057077, allele A of SNP PE-0057080, allele T of SNP PE-0057081 , and allele G of SNP PE-0006730, with a p-value of 0.05 or less.
  • a further aspect of the invention relates to a method for identifying a molecular marker linked with a QTL conferring the resistance to M. enterolobii, said QTL being as present in representative seeds deposited at the NCIMB under accession number NCIMB 44105, comprising: a. identifying a molecular marker in the Capsicum genome, in the chromosomal region delimited on chromosome 9 by markers PE-0057076 and PE-0006730; and b. determining whether an allele or state of said molecular marker is associated with resistance to M. enterolobii in a segregating population comprising Capsicum plants exhibiting said resistance.
  • a further aspect of the invention relates to a method for improving the yield of pepper production in an environment infected by M. enterolobii, comprising growing a Capsicum plant according to the invention.
  • a further aspect of the invention relates to a method for protecting a field, tunnel, greenhouse or glasshouse of pepper from an infection by M. enterolobii, comprising growing a Capsicum plant according to the invention.
  • a further aspect of the invention relates to the use of a Capsicum plant according to the invention for limiting or controlling an infection by M. enterolobii.
  • a further aspect of the invention relates to a method of producing pepper fruit comprising: a) growing a Capsicum plant according to the invention; b) allowing said plant to set fruit; and c) harvesting fruit of said plant, preferably at maturity and/or over-maturity; and optionally d) processing said pepper fruit into a pepper processed food.
  • the term “pepper”, “pepper plant” or “Capsicum plant” relates to any species, variety, cultivar, or population of the Capsicum spp genus.
  • the Capsicum genus is known to comprise around 20-27 species, five of which are domesticated: Capsicum annuum, Capsicum baccatum, Capsicum frutescens, Capsicum chinense, and Capsicum pubescens.
  • the vast majority of commercial varieties of peppers belong to the species Capsicum annuum, which comprises sweet peppers (peppers with a no pungency) and hot peppers (peppers with pungency from low level to high level).
  • Sweet peppers include bell peppers, which are fruit of plants in the Grossum cultivar group of the species Capsicum annuum. Sweet pepper plants can be produced in different colors, from immature green color to mature color of red, yellow, orange, white or purple.
  • the Capsicum plant may be of one of the following types: Dulce Italiano, Lamuyo, Blocky Florida, Open field and blocky protected, Ancho, Anaheim, Marconi, Jalapeno, Cayenne, Charleston or Sivri.
  • the Capsicum plant is preferably not a plant of one of the reference lines CM334, Maor, Dempsey, UCD10X or Zunla-1.
  • plant part refers to any part of a plant including but not limited to the shoot, root, stem, seeds, fruits, leaves, petals, flowers, ovules, branches, petioles, internodes, pollen, stamen, rootstock, scion and the like.
  • allele refers to any of several alternative or variant forms of a genetic unit, such as a gene, which are alternative in inheritance because they are positioned at the same locus in homologous chromosomes.
  • Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused, for example, by chemical or structural modification, transcription regulation or post-translational modification/regulation.
  • the two alleles of a given gene or genetic element typically occupy corresponding loci on a pair of homologous chromosomes.
  • the term "genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism (e.g., a plant), or group of organisms.
  • locus refers to any site that has been defined genetically, this can be a single position (nucleotide) or a chromosomal region.
  • a locus may be a gene, a genetic determinant, or part of a gene, or a DNA sequence, and may be occupied by different sequences.
  • a locus may also be defined by a marker, such as a SNP (Single Nucleotide Polymorphism), by several markers (e.g. SNPs), or by two flanking markers (e.g. SNPs).
  • QTL Quality of Life
  • a QTL may for instance comprise one or more genes of which products confer genetic resistance or tolerance.
  • a QTL may for instance comprise regulatory genes or sequences of which products influence the expression of genes on other loci in the genome of the plant thereby conferring the resistance or tolerance.
  • the QTL of the present invention may be defined by indicating its genetic location in the genome of the respective pathogen-resistant accession using one or more molecular genomic markers. One or more markers, in turn, indicate a specific locus.
  • centimorgan 1 % recombination between loci (marker).
  • regulatory sequence refers to a promoter, enhancer, repressor, insulator or any DNA segment that regulates the expression of a gene, generally by the binding of regulatory proteins, e.g. transcription factors, to the regulatory sequence.
  • resistance is as defined by the ISF (International Seed Federation) Vegetable and Ornamental Crops Section for describing the reaction of plants to pests or pathogens, and abiotic stresses for the Vegetable Seed Industry. Specifically, by resistance, it is meant the ability of a plant variety to restrict the growth and development of a specified pest or pathogen and/or the damage they cause when compared to susceptible plant varieties under similar environmental conditions and pest or pathogen pressure. Resistant varieties may exhibit some disease symptoms or damage under heavy pest or pathogen pressure.
  • High resistance refers to plant varieties that highly restrict the growth and/or development of the specified pest and/or the damage it causes under normal pest pressure when compared to susceptible varieties. These plant varieties may, however, exhibit some symptoms or damage under heavy pest pressure.
  • Intermediate resistance refers to plant varieties that restrict the growth and/or development of the specified pest and/or the damage it causes but may exhibit a greater range of symptoms or damage compared to high resistant varieties. Intermediate resistant plant varieties will still show less severe symptoms or damage than susceptible plant varieties when grown under similar environmental conditions and/or pest pressure.
  • tolerance is meant the ability of a plant variety to endure biotic and abiotic stress without serious consequences for growth, appearance and yield.
  • the term “susceptible” refers to a plant that is unable to restrict the growth and development of a specified pest or pathogen.
  • an offspring plant refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof.
  • an offspring plant may be obtained by cloning or selfing of a parent plant or by crossing two parent plants and include selfings as well as the F1 or F2 or still further generations.
  • An F1 is a first-generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of a second generation (F2) or subsequent generations (F3, F4, etc.) are specimens produced from selfing of F1 ’s, F2s, etc.
  • An F1 may thus be (and usually is) a hybrid resulting from a cross between two true breeding parents (true-breeding is homozygous for a trait), while an F2 may be (and usually is) an offspring resulting from self-pollination of said F1 hybrids.
  • cross refers to the process by which the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.
  • heterozygote refers to a diploid or polyploidy cell or plant having different alleles (forms of a given gene or sequences) present at at least one locus.
  • heterozygous refers to the presence of different alleles (forms of a given gene or sequences) at a particular locus.
  • homozygote refers to an individual cell or plant having the same alleles at one or more loci on all homologous chromosomes.
  • homozygous refers to the presence of identical alleles at one or more loci in homologous chromosomal segments.
  • hybrid refers to any individual cell, tissue or plant resulting from a cross between parents that differ in one or more genes.
  • inbred or “line” refers to a relatively true-breeding strain.
  • phenotype refers to the observable characters of an individual cell, cell culture, organism (e.g. a plant), or group of organisms which results from the interaction between that individual genetic makeup (i.e. genotype) and the environment.
  • introgression refers to the process whereby genes of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species.
  • the crossing may be natural or artificial.
  • the process may be optionally be completed by backcrossing to the recurrent parent, in which case introgression refers to infiltration of the genes of one species into the gene pool of another through repeated backcrossing of an interspecific hybrid with one of its parents.
  • An introgression may be also described as a heterologous genetic material stably integrated in the genome of a recipient plant.
  • molecular marker refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences.
  • indicators are restriction fragment length polymorphism (RFLP) markers, amplification fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location.
  • RFLP restriction fragment length polymorphism
  • AFLP amplification fragment length polymorphism
  • SNPs single nucleotide polymorphisms
  • SSRs single nucleotide polymorphisms
  • SCARs sequence-characterized amplified regions
  • CAS cleaved amplified polymorphic sequence
  • marker-based selection or “marker-assisted selection (MAS)” or “marker- assisted breeding (MAB)” or “marker assisted selection program” refers to the use of genetic markers to detect one or more nucleic acids from a plant, wherein the nucleic acid is associated with a desired trait to identity plants that carry genes for desirable (or undesirable) traits, so that those plants can be used (or avoided) in a selective breeding program.
  • MAS marker-assisted selection
  • MAB marker- assisted breeding
  • the term “primer” refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primers extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • a pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.
  • a single nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C, or G — in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes in an individual.
  • SNP single nucleotide polymorphism
  • DNA strand and allele designation and orientation for the markers (i.e. SNPs) disclosed in the present application are mentioned according to the TOP/BOT method developed by Illumina (https://www.illumina.com/documents/products/technotes/technote_topbot.pdf).
  • a chromosomal region delimited by two markers X and Y refers to the section of the chromosome lying between the positions of these two markers and comprising said markers, therefore the nucleotide sequence of this chromosomal region begins with the nucleotide corresponding to marker X and ends with the nucleotide corresponding to marker Y, i.e. the markers are comprised within the region they delimit.
  • association or genetic association, and more specifically linkage or genetic linkage, it is to be understood that a polymorphism of a genetic marker (e.g. a specific allele of the SNP marker) and the phenotype of interest occur simultaneously, i.e. are inherited together, more often than would be expected by chance occurrence, i.e. there is a non-random association of the allele and of the genetic sequences responsible for the phenotype, as a result of their genomic proximity.
  • the association or linkage is with a p-value of preferably less than 0.05, and most preferably less than 0.01 or even less.
  • lite or “commercial” variety or line means a variety or line that has resulted from breeding and selection for superior horticultural performance for use in agriculture.
  • a number of commercial pepper types have been developed, which are agronomically elite and appropriate for commercial cultivation.
  • SEQ ID NO: 1 shows a sequence surrounding SNP marker PE-0057076.
  • SEQ ID NO: 2 shows the sequence of a forward primer for detecting the resistant allele of SNP marker PE-0057076.
  • SEQ ID NO: 3 shows the sequence of a forward primer for detecting the susceptible allele of SNP marker PE-0057076.
  • SEQ ID NO: 4 shows the sequence of a common reverse primer for SNP marker PE-0057076.
  • SEQ ID NO: 5 shows a sequence surrounding SNP marker PE-0057077.
  • SEQ ID NO: 6 shows the sequence of a forward primer for detecting the resistant allele of SNP marker PE-0057077.
  • SEQ ID NO: 7 shows the sequence of a forward primer for detecting the susceptible allele of SNP marker PE-0057077.
  • SEQ ID NO: 8 shows the sequence of a common reverse primer for SNP marker PE-0057077.
  • SEQ ID NO: 9 shows a sequence surrounding SNP marker PE-0057080.
  • SEQ ID NO: 10 shows the sequence of a forward primer for detecting the resistant allele of SNP marker PE-0057080.
  • SEQ ID NO: 11 shows the sequence of a forward primer for detecting the susceptible allele of SNP marker PE-0057080.
  • SEQ ID NO: 12 shows the sequence of a common reverse primer for SNP marker PE-0057080.
  • SEQ ID NO: 13 shows a sequence surrounding SNP marker PE-0057081 .
  • SEQ ID NO: 14 shows the sequence of a forward primer for detecting the resistant allele of SNP marker PE-0057081 .
  • SEQ ID NO: 15 shows the sequence of a forward primer for detecting the susceptible allele of SNP marker PE-0057081 .
  • SEQ ID NO: 16 shows the sequence of a common reverse primer for SNP marker PE-0057081 .
  • SEQ ID NO: 17 shows a sequence surrounding SNP marker PE-0006730.
  • SEQ ID NO: 18 shows the sequence of a forward primer for detecting the resistant allele of SNP marker PE-0006730.
  • SEQ ID NO: 19 shows the sequence of a forward primer for detecting the susceptible allele of SNP marker PE-0006730.
  • SEQ ID NO: 20 shows the sequence of a common reverse primer for SNP marker PE-0006730.
  • SEQ ID NO:21 shows the sequence of SNP Marker A
  • SEQ ID NO:22 shows the sequence of SNP Marker B
  • SEQ ID NO:23 shows the sequence of SNP Marker C
  • SEQ ID NO:24 shows the cDNA sequence of the Long-chain-fatty-acid-CoA ligase family protein gene
  • SEQ ID NO: 25 shows the protein sequence of the cDNA sequence of the gene annotated WD repeated-containing protein VIP3
  • SEQ ID NO:26 shows the sequence of the CA09g16830, the Me1 candidate gene published by Wang et al., 2018
  • Figure 1 Box-plot distribution of the main known nematodes resistance genes described in Pepper compared to the claimed resistance (named R parent).
  • Figure 2 Dunnett’s test using the resistant parent ME-231-02 as control.
  • Figure 3 Box-plot distribution of the F6 and F7 RILs, with the S parent, line ME-231-02 and several tested lines.
  • Figure 4 Distribution of 173 F6 and F7 individuals on a 1 to 9 scoring scale. The % indicate the amount of individuals per score.
  • Figure 5 Penetration assays of M. Enterolobii.
  • the present invention is directed to a Capsicum plant comprising a quantitative trait locus (QTL) in its genome on chromosome 9, wherein said QTL confers to the plant resistance to Meloidogyne enterolobii.
  • QTL quantitative trait locus
  • the inventors have indeed identified a QTL on chromosome 9 which confers resistance to Meloidogyne enterolobii. Said QTL is unrelated to and located at a different position on chromosome 9 than the known loci of resistance to root knot nematodes.
  • the QTL of the present invention can be introgressed into various genetic background, including elite/commercial plants, thereby confers a new type of resistance to these plants.
  • said QTL is introgressed in the Capsicum plant genome.
  • said QTL is present on chromosome 9 in the genome of a seed of Capsicum annuum PB21 SG7-1294.
  • Representative seeds of Capsicum annuum PB21 SG7-1294 have been deposited at NCIMB accession number NCIMB 44105.
  • said QTL is located on chromosome 9 within the chromosomal region delimited by markers PE-0057076 and PE-0006730.
  • said QTL is located on chromosome 9 within the chromosomal region delimited by positions 5905642 and 5921103 on the public genome Capsicum annuum Zunla-1 v2.0.
  • all references to positions on the public genome Zunla-1 orZunla, in the present disclosure referto the public genome Capsicum annuum Zunla-1 v2.0 accessible on http://www.solgenomics.net (Qin et al., Proc Natl Acad Sci USA. 2014 Apr 8;1 11 (14):5135-40).
  • said QTL is located on chromosome within the chromosomal region delimited by markers PE-0057077 and PE-0006730.
  • said QTL is located within the chromosomal region delimited by any two of single nucleotide polymorphisms (SNP) PE-0057076, PE-0057077, PE-0057080, PE-0057081 and PE-0006730.
  • SNP single nucleotide polymorphisms
  • the QTL position is markedly distinct from the position of CA09g16830, the Me1 candidate gene (SEQ ID NO:26) as identified in Wang, Xueying, et al. Molecular breeding 38 (2016): 1-10, and which is localized at position 6486175 on Zunla-1 public genome.
  • the plant preferably comprises the QTL homozygously in its genome.
  • the present inventors have indeed found that the resistance to Meloidogyne enterolobii was not expressed in hybrid plants obtained by crossing resistant and susceptible plants.
  • the QTL on chromosome 9 was therefore identified as a recessive QTL, i.e. which requires homozygosity to confer Meloidogyne enterolobii resistance to the plant.
  • Capsicum plants comprising the QTL homozygously in their genome express the resistance to Meloidogyne enterolobii.
  • said QTL on chromosome 9 conferring resistance to M. enterolobii can be identified by detecting one or more genetic markers associated with the resistance phenotype.
  • said QTL on chromosome 9 conferring resistance to M. enterolobii can be identified by detecting one or more SNP markers selected from SNPs PE-0057076; PE-0057077, PE-0057080, PE-0057081 and PE-0006730.
  • the QTL can be identified by detecting any combination of said SNP markers, in particular at least 2, more particularly at least 3, even more particularly at least 4 said markers, or all 5 markers.
  • said QTL on chromosome 9 conferring resistance to M. enterolobii can be identified by detecting one or more genetic markers selected from PE-0057076, PE-0057077, and PE-0057081 or PE-0057077, PE-0057080, PE-0057081 and PE-0006730, more particularly PE-0057080, PE- 0057081 and PE-0006730.
  • said QTL can be identified by detecting one or more genetic markers selected from PE-0057077, PE-0057080, PE-0057081 and PE-0006730, in particular selected from PE-0057077, PE-0057080 and PE-0057081 or PE-0057077, PE-0057080, PE-0057081 and PE-0006730, more particularly PE-0057080, PE-0057081 and PE-0006730.
  • the QTL can be identified by detecting any combination of said SNP markers, in particular at least 2, more particularly at least 3 or even more particularly at least 4 or all of said markers.
  • the QTL can be identified by detecting a resistant allele of the genetic markers disclosed herein. Said resistant allele is different from the corresponding allele of a reference susceptible line, in particular the CM334 line. More particularly, at least 2, more particularly at least 3 or at least 4 or all 5 of said markers in said QTL have an allele which is different from the alleles of the corresponding markers in the reference susceptible line.
  • said QTL on chromosome 9 conferring resistance to M. enterolobii can be identified by detecting one or more alleles selected from allele T of SNP PE-0057076, allele T of SNP PE-0057077, allele A of SNP PE-0057080, allele T of SNP PE-0057081 and allele G of SNP PE-0006730.
  • the QTL can be identified by detecting any combination of these alleles, in particular at least 2, more particularly at least 3 or even more particularly at least 4 or all of said markers.
  • said QTL on chromosome 9 conferring resistance to M. enterolobii can be identified by detecting one or more alleles selected from allele T of PE-0057076, allele T of PE-0057077 and allele T of PE-0057081 .
  • said QTL can be identified by detecting one or more alleles selected from allele T of SNP PE-0057077, allele A of SNP PE-0057080, allele T of SNP PE- 0057081 and allele G of SNP PE-0006730, in particular one or more alleles selected from allele T of SNP PE-0057077, allele A of SNP PE-0057080 and allele T of SNP PE-0057081 .
  • the QTL can be identified by detecting any combination of said alleles, in particular at least 2, more particularly at least 3 of said alleles or even more particularly at least 4 or all of said alleles.
  • said detection of a marker on chromosome 9 is performed using two forward primers, one being specific for the resistant allele and one being specific for the susceptible allele, and one common reverse primer, for instance as shown in Table 2.
  • Said primers may be selected so as to enable amplifying a nucleic acid comprising or consisting respectively of SEQ ID NO:1 , 5, 9, 13, 17, or a fragment thereof including the SNP shown in said sequences.
  • the forward primer for detecting the resistant allele of the PE-0057076, PE-0057077, PE-0057080, PE-0057081 and PE- 0006730, markers may consist in the sequence set forth in Table 2, i.e. the sequence of SEQ ID NO: 2, 6, 10, 14 and 18, respectively.
  • the forward primer for detecting the susceptible allele of the PE- 0057076, PE-0057077, PE-0057080, PE-0057081 and PE-0006730 markers may consist in the sequence set forth in Table 2, i.e. the sequences of SEQ ID NO: 3, 7, 11 , 15 and 19, respectively.
  • the common reverse primer for detecting the resistant allele of the PE-0057076, PE-0057077, PE- 0057080, PE-0057081 and PE-0006730 markers may consist in the sequence set forth in Table 2, i.e. the sequence of SEQ ID NO: 4, 8, 12, 16 and 20, respectively. Using these 3 primers for each marker, detection of the resistant allele rather than the susceptible allele, as indicated in Table 2, indicates the presence of the QTL on chromosome 9 conferring resistance to M. enterolobii.
  • said Capsicum plant is characterized by the presence on chromosome 9 in its genome of one or more of allele T of the sequence set forth in SEQ ID NO:21 , allele A of the sequence set forth in SEQ ID NO:22 and allele T of the sequence set forth in SEQ ID NO: 23.
  • any 2 of said alleles or any 3 of said alleles may be present.
  • said QTL on chromosome 9 is linked with the gene Capana09g000172.
  • Capana09g000172 encodes a long-chain-acyl-CoA ligase, or synthetase, is also named LACSs and is involved in activation of long-chain fatty acids for both synthesis of cellular lipids, and degradation via beta-oxydation.
  • LACs are reported to be involved in plant tolerance to abiotic stress (Wei et al., Int J Mol Sci. 2022 Jul 29;23(15):8401) and in plant biotic stress response (Lai and Chye, Cells. 2021 Apr 30;10(5):1064.)
  • said resistance to M. enterolobii is caused by at least one mutation within the Capana09g000172 gene or a regulatory sequence thereof, in comparison with the corresponding gene or sequence of a susceptible Capsicum plant and/or line.
  • a reference coding sequence of the Capana09g000172 gene of a susceptible plant is set forth in SEQ ID NO: 24.
  • the mutation may consist in at least one nucleotide substitution, insertion or deletion in the sequence of the Capana09g000172 gene or a regulatory sequence thereof, in particular the coding sequence of Capana09g000172 as set forth in SEQ ID NO: 24, including the deletion of the full gene or a fragment thereof.
  • the mutation is a loss-of-function mutation.
  • the mutation may induce one or more amino acid substitutions in the sequence of the protein encoded by the Capana09g000172 gene, and impair the function of the protein.
  • the loss-of-function mutation is a null mutation.
  • a null mutation prevents expression of an active protein, for instance by causing a premature stop in the translation of the mRNA into a protein, resulting into the expression of a truncated form of the protein.
  • Capana09g000173 is a candidate gene responsible for the trait of resistance to M. enterolobii.
  • Capana09g000173 gene is annotated as a WD repeatcontaining protein VIP3 on Zunla-1 reference genome. It is a component of the PAF1 complex (PAF1 C) which is involved in histone modifications such as methylation on histone H3 'Lys-4' (H3K4me3).
  • Capana09g000173 gene comprises allele G of SNP PE-0006730 (SEQ ID NO: 5).
  • CSN5 is a subunit of the COP9 signalosome that has been identified as an interactor with a cell-penetrating protein of Meloidogyne incognita (Bournaud et al. (2016), Frontiers in plant science, 9, 904).
  • said resistance to M. enterolobii is caused by at least one mutation within the Capana09g000173 gene or a regulatory sequence thereof, in comparison with the corresponding gene or sequence of a susceptible Capsicum plant and/or line.
  • a reference coding sequence of the Capana09g000173 gene of a susceptible plant is set forth in SEQ ID NO: 25.
  • the mutation may consist in at least one nucleotide substitution, insertion or deletion in the sequence of the Capana09g000173 gene or a regulatory sequence thereof, in particular the coding sequence of Capana09g000173 as set forth in SEQ ID NO: 25, including the deletion of the full gene or a fragment thereof.
  • the mutation is a loss-of-function mutation.
  • the mutation may induce one or more amino acid substitutions in the sequence of the protein encoded by the Capana09g000173 gene, and impair the function of the protein.
  • the loss-of-function mutation is a null mutation.
  • a null mutation prevents expression of an active protein, for instance by causing a premature stop in the translation of the mRNA into a protein, resulting into the expression of a truncated form of the protein.
  • the Capana09g000173 gene of a Capsicum plant according to the invention comprises allele G of SNP PE-0006730.
  • the resistance to Meloidogyne enterolobii is preferably determined by comparison to a susceptible line, preferably a susceptible reference line, for example the CM334 line or any other reference susceptible line.
  • a susceptible line preferably a susceptible reference line, for example the CM334 line or any other reference susceptible line.
  • the resistance is preferably determined as detailed in the Examples, on the basis of an inoculation test of the plant.
  • the resistance to M. enterolobii is an intermediate resistance.
  • the Capsicum plant according to the invention has a M. enterolobii reproduction index of 30% or less, preferably 20% or less, more preferably 10% or less, still preferably 5% or less, even more preferably 3% or less, in comparison to the reproduction index of susceptible Capsicum plants, particularly in comparison to plants from the CM334 line, in the same environmental and infestation conditions.
  • the reproduction index may be measured according to Soares et al. 2018 Bioscience Journal, 34(2), 912-925.
  • the reproduction index is measured at 7 weeks after inoculation of at least 400 eggs and/or second-stage juvenile (J2) root-knot nematodes, in particular at least 3000 eggs and/or second-stage juvenile (J2) root-knot nematodes, more particularly about 4500 eggs and/or second-stage juvenile (J2) root-knot nematodes.
  • Inoculation is preferably carried out on plants at 4 expanded leaves.
  • the percentage of attacked root system is reduced by a factor 1 .5 or more, preferably 2 or more, in comparison to susceptible Capsicum plants in the same environmental and infestation conditions, particularly in comparisons to plants from the CM334 line.
  • the plants are under conditions of heavy infestation by Meloidogyne enterolobii, for instance by injecting at least 400 second-stage juvenile (J2) root-knot nematodes per Capsicum plant or by injecting at least 4000 eggs of root-knot nematodes per Capsicum plant.
  • J2 second-stage juvenile
  • the Capsicum plant according to the invention may advantageously comprise one or more genes responsible for a trait of agronomic interest such as, but not limited to, genes that confer resistance to pests or disease, genes that confer resistance or tolerance to an herbicide, genes that control male sterility, genes that affect abiotic stress resistance (e.g., against salt, heavy metal, flooding), and other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth or plant architecture, fruit growth, shape or taste or resistance to a pest or a disease.
  • genes responsible for a trait of agronomic interest such as, but not limited to, genes that confer resistance to pests or disease, genes that confer resistance or tolerance to an herbicide, genes that control male sterility, genes that affect abiotic stress resistance (e.g., against salt, heavy metal, flooding), and other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth or plant architecture, fruit growth, shape or taste or resistance to a pest or
  • it comprises at least one resistance to a pathogen selected from Colletotrichum spp., Ralstonia solanacearum, Rhizoctonia solani, Pythium spp, Fusarium oxysporum, Phytophthora capsici, Sclerotium rolfsii, Verticillium albo-atrum, Verticillium dahliae, Leveillula taurica, Xanthomonas campestris, viruses such as PMMV, TMV, TSWV, PVY, Geminivirus or CMV, or insects such as thrips (e.g. Frankliniella occidentalis and Thrips parvispinus).
  • a pathogen selected from Colletotrichum spp., Ralstonia solanacearum, Rhizoctonia solani, Pythium spp, Fusarium oxysporum, Phytophthora capsici, Sclerotium rolfsii, Verticillium albo-atrum, Verticill
  • said plant also contains in its genome one or more loci conferring resistance to root knot nematodes, in particular to Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica and/or Meloidogyne hapla.
  • said locus conferring resistance to root knot nematodes is selected from Me1, Meeh 2, Me2, Me3, Me4, Me5, Me7, Meehl and N, preferably N, Me1, Me3 and Me7.
  • loci of resistance to root knot nematodes is useful to obtain plants resistant to a broad spectrum of nematode species and isolates.
  • the loci N, Me1 or Me3 can provide a resistance to M. incognita, M. javanica and M. arenaria, in addition to the M. enterolobii resistance provided by the QTL of the invention.
  • the Capsicum plant according to the invention can be from any species within the Capsicum genus. In particular, it may be a Capsicum annuum, Capsicum baccatum, Capsicum frutescens, Capsicum chinense, Capsicum pubescens or Capsicum chacoense plant.
  • the plant according to the invention is a Capsicum annuum plant, more preferably a sweet bell pepper, in particular a blocky or lamuyo pepper.
  • the plant can also be a hot pepper, such as a jalapeno or habanero pepper.
  • the Capsicum plant can also be from any type.
  • Capsicum plant may be of one of the following types: Dulce Italiano, Lamuyo, Blocky, Blocky Florida, Ancho, Anaheim, Marconi, Jalapeno, Habanero, Cayenne, Charleston or Sivri.
  • the invention also relates to a population of Capsicum plants according to the invention, wherein said population comprises at least 5 plants, in particular at least 10 plants, more particularly at least 20 plants, even more particularly at least 50 or 100, or more particularly at least 1000 plants.
  • the present invention is directed to one or more parts of a plant according to the invention.
  • said QTL is present on chromosome 9 in the genome of a seed of Capsicum annuum PB21 SG7-1294, representative seeds of which have been deposited at NCIMB accession number NCIMB 44105.
  • the plant part according to the invention comprises a QTL conferring resistance to M. enterolobii located on chromosome 9 within the chromosomal region delimited by PE-0057076 and PE-0006730.
  • a plant part according to the invention comprises one or more alleles conferring resistance to M. enterolobii, preferably selected from allele T of SNP PE-0057076, allele T of SNP PE-0057077, allele A of SNP PE-0057080, allele T of SNP PE-0057081 , allele G for PE- 0006730
  • the QTL can be identified by detecting any combination of said alleles, in particular at least 2, more particularly at least 3 or even more particularly at least 4, or all of said alleles.
  • one or more of the QTL and alleles as described here above is as found in the genome of a plant corresponding to the deposited material PB21 SG7-1294, representative seeds of which have been deposited under NCIMB accession number NCIMB 44105.
  • the plant part is a seed, explant, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole or flower.
  • a particularly preferred plant part according to the invention is a seed produced by the plant of the invention.
  • the seed homozygously comprises the QTL on chromosome 9 conferring resistance to M. enterolobii.
  • the plant part is a plant cell.
  • one aspect of the invention relates to a cell of a Capsicum plant according to the invention, i.e. a plant cell comprising in its genome a quantitative trait locus (QTL) on chromosome 9, conferring resistance to M. enterolobii. More particularly, said plant cell comprises said QTL introgressed in its genome.
  • QTL quantitative trait locus
  • a plant cell according to the invention preferably comprises a QTL conferring resistance to M. enterolobii on chromosome 9 within the chromosomal region delimited by PE-0057076 and PE- 0006730.
  • said QTL is present homozygously in the genome of the cell.
  • a plant cell of the invention may have the capacity to be regenerated into a whole plant.
  • the invention is also directed to plant cells which are not regenerable, and thus are not capable of giving rise to a whole plant.
  • a plant cell according to the invention is derived from a seed, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole or flower.
  • the invention further relates to a seed of a Capsicum plant, giving rise when grown up to a Capsicum plant resistant to M. enterolobii as defined above.
  • the invention further relates to a seed from a plant of the invention, i.e. produced by such a plant after selfing or crossing.
  • said seed is capable of germinating into a plant resistant to M. enterolobii, wherein said resistance is conferred by as defined here above conferring said resistance.
  • the invention also provides Capsicum plants grown from the seeds of the invention.
  • the invention also relates to a population of Capsicum seeds according to the invention, wherein said population comprises at least 2 seeds, especially at least 10 seeds, particularly at least 100 seeds, even more particularly at least 10 5 or 10 6 seeds.
  • the present invention is also directed to an in vitro cell or tissue culture of regenerable cells of the plant as defined above according to the present invention.
  • the regenerable cells are derived from a seed, reproductive material, scion, cutting, fruit, root, root tip, rootstock, pollen, ovule, embryo, meristem, callus, cotyledon, hypocotyl, protoplast, leaf, anther, stem, petiole or flower of a plant of the invention, and comprise in their genome the QTL on chromosome 9 conferring resistance to M. enterolobii as described here above.
  • the tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing Capsicum plant, and of regenerating plants having substantially the same genotype as the foregoing Capsicum plant.
  • the present invention also provides Capsicum plants regenerated from the tissue cultures of the invention.
  • the invention also provides a protoplast of the plant defined above, or from the tissue culture defined above, said protoplast comprising in its genome the QTL on chromosome 9 conferring resistance to M. enterolobii as described here above.
  • the present invention also encompasses asexual processes of propagation. Accordingly, one aspect of the invention relates to a method of producing a Capsicum plant resistant to Meloidogyne enterolobii, comprising:
  • the present invention is also directed to the use of a Capsicum plant as detailed according to the first aspect of the invention, resistant to M. enterolobii, as a breeding partner in a breeding program for obtaining Capsicum plants resistant to M. enterolobii.
  • a Capsicum plant according to the first aspect harbors in its genome the QTL on chromosome 9, as defined here above conferring said resistance.
  • a plant having a different genotype for instance a susceptible or less resistant plant, it is possible to transfer this QTL, conferring the desired phenotype, to the progeny.
  • a plant according to the invention can thus be used as a breeding partner for introgressing the QTL conferring the desired phenotype into a Capsicum plant or germplasm.
  • the invention is also directed to the same use with plants or seeds of PB21 SG7-1294, representative seeds of which have been deposited at NCIMB under accession number NCIMB 44105, or with progeny of such plants or seeds.
  • the selection of the progeny displaying the desired phenotype, or bearing sequences linked to the desired phenotype can advantageously be carried out on the basis of the allele of the markers disclosed in the present specification, and/or any marker linked to the markers disclosed in the present specification.
  • Selecting the progeny may for instance comprise the detection of a polymorphism at one or more SNP markers selected from PE-0057076, PE-0057077, PE-0057080, PE-0057081 and PE-0006730.
  • Selecting the progeny may in particular comprise detecting one or more alleles selected from allele T of SNP PE-0057076, allele T of SNP PE- 0057077, allele A of SNP PE-0057080, allele T of SNP PE-0057081 , and allele G of SNP PE- 0006730.
  • the selection of the progeny having the desired phenotype can also be made by assessing the resistance phenotype of the progeny plants in conditions of a M. enterolobii infestation. Such assessment is disclosed inter alia in the Examples or with other tests well-known to the skilled reader.
  • a plant according to the invention in particular a plant, representative seeds of which have been deposited at NCIMB under accession number NCIMB 44105, is thus particularly valuable in a marker assisted selection program for obtaining commercial Capsicum lines and varieties resistant to M. enterolobii.
  • the invention is also directed to the use of said plants in a program aiming at identifying, sequencing and/or cloning the genetic sequences conferring the desired phenotype.
  • the invention also concerns methods for the production of Capsicum plants resistant to M. enterolobii, especially commercial/elite plants.
  • a method for the production resistant to M. enterolobii comprises the following steps:
  • step b) optionally self-pollinating and/or backcrossing one or several times the plant selected at step b) and selecting in the progeny thus obtained a plant comprising said QTL on chromosome 9 conferring the resistance to M. enterolobii.
  • the plant produced by the method is a hot pepper such as a Jalapeno or Habanero pepper, or a sweet bell pepper, such as a blocky pepper.
  • the selected plant is one of the following types: Dulce Italiano, Lamuyo, Blocky, Blocky Florida, Ancho, Anaheim, Marconi, Jalapeno, Habanero, Cayenne, Charleston or Sivri.
  • the first plant is a Capsicum annuum plant of PB21 SG7-1294, representative seeds of which have been deposited under accession number NCIMB 44105, or a progeny of said plant.
  • the self-pollination and backcrossing steps may be carried out in any order and can be intercalated, for example a backcross can be carried out before and after one or several self-pollinations, and self- pollinations can be performed before and after one or several backcrosses.
  • such a method is advantageously carried out by detecting the markers linked to the QTL on chromosome 9 as described in the present specification, for one or more of the selections carried out at step b) and/or c) for selecting plants comprising the QTL on chromosome 9.
  • the selection at step (b) and/or (c) is carried out by detecting a single nucleotide polymorphism at one or more SNP markers selected from PE-0057076, PE-0057077, PE- 0057080, PE-0057081 and PE-0006730.
  • the selection at step (b) and/or (c) comprise detecting comprise detecting one or more of , allele T of SNP PE-0057076, allele T of SNP PE-0057077, allele A of SNP PE-0057080, allele T of SNP PE-0057081 , and allele G of SNP PE-0006730.
  • any marker linked to the markers disclosed in the present specification as associated with the QTL and/or present within the boundaries defined for said QTL can also be used for marker-assisted selection, e.g. at step (b) and/or (c) of the method.
  • a marker within 40 cM, 20 cM, 10 cM, 5 cM, 2 cM or 1 cM, or within 10 Mb, 5 Mb, 2 Mb, 1 Mb, 500 kb, 200 kb, 100 kb, 90 kb, 50 kb, 20 kb or 10 kb of a marker associated with the QTL of M. enterolobii resistance on chromosome 9 as disclosed in the present specification can be used for marker-assisted selection.
  • the selection carried out at steps b) and/or (c) can also be made using any type of genetic marker linked to the QTL according to the invention on chromosome 9, in particular restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), simple sequence length polymorphisms (SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (Indels), variable number tandem repeats (VNTRs), random amplified polymorphic DNA (RAPD), isozymes, and other markers known to those skilled in the art.
  • RFLPs restriction fragment length polymorphisms
  • AFLPs amplified fragment length polymorphisms
  • SSRs simple sequence repeats
  • SSLPs simple sequence length polymorphisms
  • SNPs single nucleotide polymorphisms
  • Indels variable number tandem repeats
  • RAPD random amplified polymorph
  • the selection of the progeny having the desired phenotype can also be made on conditions of disease infection, as disclosed inter alia in the Examples or with other tests well-known to the skilled reader.
  • the method used for allele detection can be based on any technique allowing the distinction between two different alleles of a marker, on a specific chromosome.
  • Detection of a polymorphism can be made by electrophoretic techniques including a single strand conformational polymorphism (Orita, et al. (1989) Genomics, 8(2), 271-278), denaturing gradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavage fragment length polymorphisms (Life Technologies, Inc., Gaithersburg, Md.), but the widespread availability of DNA sequencing often makes it easier to simply sequence amplified products directly.
  • PCR detection and quantification is carried out using two labeled fluorogenic oligonucleotide forward primers and an unlabeled common reverse primer, for example, KASParTM (KBiosciences).
  • Detection of a polymorphism can also be made by electrophoretic techniques including a single strand conformational polymorphism (Orita, et al. (1989) Genomics, 8(2), 271-278), denaturing gradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavage fragment length polymorphisms (Life Technologies, Inc., Gaithersburg, Md.).
  • electrophoretic techniques including a single strand conformational polymorphism (Orita, et al. (1989) Genomics, 8(2), 271-278), denaturing gradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavage fragment length polymorphisms (Life Technologies, Inc., Gaithersburg, Md.).
  • the widespread availability of DNA sequencing often also enables to sequence amplified products directly.
  • the present invention also concerns a Capsicum plant obtained or obtainable by the methods described herein.
  • a Capsicum plant obtained or obtainable by the methods described herein.
  • Such a plant is a Capsicum plant that comprises in its genome a QTL on chromosome 9 conferring resistance to M. enterolobii.
  • the present invention is also directed to a hybrid Capsicum plant obtainable by crossing a first Capsicum plant according to the first aspect of the invention with a second Capsicum plant having a different genotype.
  • the second plant can also be a plant according to the invention, comprising the QTL on chromosome 9 in its genome.
  • the first plant and/or the second plant can also be a plant obtainable by the methods of the present invention.
  • the hybrid Capsicum plant obtainable is a progeny of PB21 SG7-1294, representative seeds of which have been deposited under accession number NCIMB 44105.
  • the second Capsicum plant can also be a plant susceptible to M. enterolobii, or a plant with a different level of resistance to M. enterolobii than the first Capsicum plant, e.g. a lower level of resistance.
  • a particularly preferred hybrid Capsicum plant is a plant which displays any trait or phenotype of agronomical interest, in addition to the resistance to M. enterolobii.
  • the methods comprise crossing a first Capsicum plant according to the invention with itself or with a second Capsicum plant of a different genotype, and harvesting the resultant seeds.
  • the second Capsicum plant also comprises the QTL on chromosome 9 conferring resistance to M. enterolobii.
  • Capsicum plants of the invention are not exclusively obtained by means of an essentially biological process.
  • the invention relates to a Capsicum plant or seed, preferably a non- naturally occurring Capsicum plant or seed, which may comprise one or more mutations in its genome, which provides the mutant plant a resistance to M. enterolobii, in particular intermediate resistance to M. enterolobii.
  • said mutation is located within a gene selected from Capana09g000172 and Capana09g000173.
  • said mutation is as present, in the genome of plants of which a representative sample was deposited with the NCIMB under deposit number NCIMB 44105.
  • the mutations can have a natural cause (spontaneous mutations) or can be induced via methods such as mutagenesis.
  • Mutagenesis methods are known in the art and include chemical mutagenesis using ethyl methanesulfonate (EMS).
  • EMS ethyl methanesulfonate
  • Other chemical mutagenic agents include but are not limited to, diethyl sufate (des), ethyleneimine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N- nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea(enu), and sodium azide.
  • the mutations can be induced by means of irradiation, which is for example selected from x-rays, fast neutrons, UV radiation.
  • TILLING Targeting Induced Local Lesions IN Genomes
  • TILLING is a general reverse genetics technique that uses traditional chemical mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high throughput screens for the discovery of mutations.
  • TILLING combines chemical mutagenesis with mutation screens of pooled PCR products, resulting in the isolation of missense and non-sense mutant alleles of the targeted genes.
  • TILLING uses traditional chemical mutagenesis (e.g. EMS or MNU mutagenesis) or other mutagenesis methods (e.g.
  • S1 nucleases such as CEL1 or ENDO1
  • electrophoresis such as a LI-COR gel analyzer system, see e.g. Henikoff et al. Plant Physiology 2004, 135: 630-636.
  • TILLING has been applied in many plant species, including pepper (Kang, H.S., Kim, S.H., Lee, S.W. et al. Hortic. Environ. Biotechnol.
  • the mutation(s) is(are) the integration of one QTL conferring resistance to M. enterolobii, wherein said at least one QTL is present on chromosome 9, in replacement of the homologous sequences of a Capsicum plant.
  • the mutation is the substitution of the sequence delimited by SNP marker PE-0057076 and PE-0006730 on chromosome 9 of the genome of a target Capsicum plant, or a fragment of said sequence, by the homologous sequence on chromosome 9 present in the genome of a plant of which a representative sample was deposited with the NCIMB under deposit number NCIMB 44105, wherein the introduced sequence or fragment confers resistance to M. enterolobii, in particular intermediate resistance, when present in the genome of the target Capsicum plant.
  • the invention relates to a method for obtaining a Capsicum plant or seed carrying one or more mutations in its genome, which provides the plant with a resistance to M. enterolobii, in particular intermediate resistance.
  • a method for obtaining a Capsicum plant or seed carrying one or more mutations in its genome which provides the plant with a resistance to M. enterolobii, in particular intermediate resistance.
  • Such a method is illustrated in the Examples and may comprise: a) treating M0 seeds of a Capsicum plant to be modified with a mutagenic agent to obtain M1 seeds; b) growing plants from the thus obtained M1 seeds to obtain M1 plants; c) producing M2 seeds by self-fertilisation of M1 plants; and d) optionally repeating step b) and c) n times to obtain M2+n seeds.
  • the M2+n seeds are grown into plants and submitted to M. enterolobii infestation.
  • the surviving plants, or those with the milder symptoms of M. enterolobii infestation, are multiplied one or more further generations while continuing to be selected for their resistance to M. enterolobii.
  • the M1 seeds of step a) can be obtained via chemical mutagenesis such as EMS mutagenesis.
  • Other chemical mutagenic agents include but are not limited to, diethyl sufate (des), ethyleneimine (ei), propane sultone, N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU), N-ethyl-N-nitrosourea(enu), and sodium azide.
  • the mutations are induced by means of irradiation, which is for example selected from x-rays, fast neutrons, UV radiation.
  • the mutation(s) is(are) induced by means of genetic engineering.
  • Such mutations also include the integration of sequences conferring the resistance to M. enterolobii, as well as the substitution of residing sequences by alternative sequences conferring the resistance to M. enterolobii.
  • the genetic engineering means which can be used include the use of all such techniques called New Breeding Techniques which are various new technologies developed and/or used to create new characteristics in plants through genetic variation, the aim being targeted mutagenesis, targeted introduction of new genes or gene silencing (RdDM).
  • New Breeding Techniques which are various new technologies developed and/or used to create new characteristics in plants through genetic variation, the aim being targeted mutagenesis, targeted introduction of new genes or gene silencing (RdDM).
  • Example of such new breeding techniques are targeted sequence changes facilitated through the use of Zinc finger nuclease (ZFN) technology (ZFN-1 , ZFN-2 and ZFN-3, see U.S. Pat. No.
  • Oligonucleotide directed mutagenesis ODM
  • Cisgenesis RNA-dependent DNA methylation
  • RdDM RNA-dependent DNA methylation
  • Grafting on GM rootstock
  • Transcription Activator-Like Effector Nucleases TALENs, see U.S. Pat. Nos. 8,586,363 and 9,181 ,535, incorporated by reference in their entireties
  • the CRISPR/Cas system see U.S. Pat. Nos.
  • Such applications can be utilized to generate mutations (e.g., targeted mutations or precise native gene editing) as well as precise insertion of genes (e.g., cisgenes, intragenes, or transgenes).
  • the applications leading to mutations are often identified as site-directed nuclease (SDN) technology, such as SDN1 , SDN2 and SDN3.
  • SDN site-directed nuclease
  • the outcome is a targeted, non-specific genetic deletion mutation: the position of the DNA DSB is precisely selected, but the DNA repair by the host cell is random and results in small nucleotide deletions, additions or substitutions.
  • a SDN is used to generate a targeted DSB and a DNA repair template (a short DNA sequence identical to the targeted DSB DNA sequence except for one or a few nucleotide changes) is used to repair the DSB: this results in a targeted and predetermined point mutation in the desired gene of interest.
  • the SDN3 is used along with a DNA repair template that contains new DNA sequence (e.g. gene). The outcome of the technology would be the integration of that DNA sequence into the plant genome.
  • the present invention also provides methods for detecting and/or selecting a Capsicum plant that is resistant to M. enterolobii, in particular intermediate resistant to M. enterolobii, wherein said method comprises the step of detecting the presence of at least one QTL conferring resistance to M. enterolobii, wherein said at least one QTL is present on chromosome 9.
  • said QTL conferring resistance to M. enterolobii has the features as described in the present specification.
  • said QTL present on chromosome 9 can be identified by amplifying one or more SNP markers selected from PE-0057076, PE-0057077, PE-0057080, PE- 0057081 and PE-0006730; or any other markers within the chromosomal region delimited by SNP markers PE-0057076 and PE-0006730, in particularthe SNP markers present in the seeds deposited at NCIMB under accession number NCIMB 44105 within the chromosomal region delimited by SNP markers PE-0057076 and PE-0006730, e.g. the markers comprising a nucleic acid sequence having a SNP with respect to the corresponding nucleic acid sequence in a reference susceptible plant, such as CM334.
  • detection of the markers described in the application is performed by amplification, e.g. by PCR, using, for each marker, one forward primer which can be used for amplifying the resistant allele, one forward primer which can be used for amplifying the susceptible allele and one common reverse primer, for example using the KASPar (KBiosciences) technology.
  • the primers for amplifying each of said markers may have the sequences as described in the first aspect of the invention.
  • the amplification is as described in the examples.
  • the amplification is performed using a two-step touchdown method in which the elongation and annealing steps are incorporated into a single step.
  • the temperature used for the annealing stage determines the specificity of the reaction and hence the ability of the primers to anneal to the DNA template.
  • a touchdown PCR involves a first step of Taq polymerase activation, followed by a second step called the touchdown step that involves a high annealing temperature and incrementally decreasing the annealing temperature in each PCR cycle, and a third step of DNA amplification.
  • the higher annealing temperatures in the early cycles of a touchdown ensure that only very specific base pairing will occur between the DNA and the primer, hence the first sequence to be amplified is most likely to be the sequence of interest.
  • the annealing temperature is gradually decreased to increase the efficiency of the reaction.
  • the regions that were originally amplified during the highly specific early touchdown cycles will be further amplified and outcompete any non-specific amplification that may occur at the lower temperatures.
  • the amplification of SNP markers is performed as recommended in the KASPar assay and illustrated in the examples, namely by PCR cycles, comprising a first denaturation step at 94°C during around 15 minutes, at least 10 cycles of around 20 seconds at 94°C followed by around 60 second at a decreasing temperature from 65°C for the 1 st cycle to 57°C for the last cycle, and around 35 cycles of around 20 seconds at 94°C followed by around 60 seconds at 57°C.
  • This protocol can easily be adapted by a skilled person, depending on the type of primers used.
  • the present invention also provides one or more molecular markers that are linked to the QTL on chromosome 9 as defined here above conferring the resistance to M. enterolobii.
  • said molecular marker linked to the QTL conferring the resistance to M. enterolobii on chromosome 9 is linked to or selected from the SNP markers PE-0057076, PE- 0057077, PE-0057080, PE-0057081 , and PE-0006730 or any of their combinations.
  • molecular markers PE-0057076, PE-0057077, PE- 0057080, PE-0057081 and PE-0006730 or any of their combinations, or any other markers linked to one or more of said markers, or any markers within the chromosomal region delimited by marker PE- 0057076 and PE-0006730, for detecting a Capsicum plant that is resistant to M. enterolobii.
  • the invention is also directed to the use of one or more of the SNP markers PE-0057076, PE- 0057077, PE-0057080, PE-0057081 and PE-0006730 associated with a QTL on chromosome 9 conferring the resistance to M. enterolobii according to the invention, for identifying one or more alternative molecular markers associated with said QTLs.
  • said alternative molecular markers are in the chromosomal region delimited on chromosome 9 by marker PE-0057076 and PE- 0006730.
  • the alternative marker is preferably within 40 cM, 20 cM, 10 cM, 5 cM, 2 cM or 1 cM of one or more of the SNP markers PE-0057076, PE-0057077, PE-0057080, PE-0057081 and PE-0006730.
  • the 1 alternative molecular marker is preferably associated with said QTL with a p-value of 0.05 or less, preferably less than 0.01. The QTL is to be found in the deposited seeds NCIMB 44105.
  • the alternative marker may be within within 10 Mb, 5 Mb, 2 Mb, 1 Mb, 500 kb, 200 kb, 100 kb, 90 kb, 50 kb, 20 kb or 10 kb of one or more of the SNP markers PE-0057076, PE-0057077, PE-0057080, PE- 0057081 and PE-0006730.
  • the invention is also directed to a method for identifying a molecular marker associated with a QTL conferring resistance to M. enterolobii, comprising: a. identifying a molecular marker in the Capsicum genome, wherein said marker :
  • - is in the chromosomal region delimited on chromosome 9 by markers PE-0057076 (SEQ ID NO: 1) and PE-0006730 (SEQ ID NO: 17); and/or
  • - is linked with one or more of SNPs PE-0057076, PE-0057077, PE-0057080, PE-0057081 and PE-0006730, in particular within 40 cM, 20 cM, 10 cM, 5 cM, 2 cM or 1 cM of one or more of said SNPs, or within 10 Mb, 5 Mb, 2 Mb, 1 Mb, 500 kb, 200 kb, 100 kb, 90 kb, 50 kb, 20 kb or 10 kb of one or more of said SNPs.
  • b. determining whether an allele or state of said molecular marker is associated with resistance to M. enterolobii in a segregating population comprising Capsicum plants exhibiting said resistance.
  • said QTL is as present in representative seeds deposited at the NCIMB under accession number NCIMB 44105.
  • the population is preferably derived from a Capsicum plant exhibiting said resistance, in particular a plant of PB21 SG7-1294, representative seeds of which have been deposited under accession number NCIMB 44105, exhibiting the resistance to M. enterolobii as described in the invention.
  • association or linkage is as defined above.
  • association or linkage is with a p- value of preferably less than 0.05, and most preferably less than 0.01 or even less.
  • a molecular marker and the resistance phenotype are inherited together in preferably more than 90% of the meiosis, preferably more than 95%, even more preferably 98% or 99%.
  • the invention relates to method for the production of Capsicum plantlets or plants resistant to M. enterolobii, which method comprises: i. culturing in vitro an isolated cell or tissue of the Capsicum plant according to the invention to produce Capsicum micro-plantlets resistant to M. enterolobii, and ii. optionally further subjecting the Capsicum micro-plantlets to an in vivo culture phase to develop into Capsicum plants resistant to M. enterolobii.
  • the isolated cell or tissue used to produce a micro-plantlet is an explant obtained under sterile conditions from a Capsicum parent plant of the invention to be propagated.
  • the explant comprises or consists, for instance, of a cotyledon, hypocotyl, stem tissue, leaf, embryo, meristem, node bud, shoot apice, or protoplast.
  • the explant can be surface sterilized before being placed on a culture medium for micropropagation.
  • Micropropagation typically involves: i. axillary shoot production: axillary shoot proliferation is induced by adding cytokinin to the shoot culture medium, to produce shoots preferably with minimum callus formation; ii. adventitious shoot production: addition of auxin to the medium induces root formation, in order to produce plantlets that are able to be transferred into the soil. Alternatively, root formation can be induced directly into the soil.
  • Plantlets can be further subjected to an in vivo culture phase, by culture into the soil under lab conditions, and then progressive adaptation to natural climate, to develop into Capsicum plant resistant to M. enterolobii.
  • the resistant plants of the invention are advantageously grown in an environment infested or likely to be infested or infected by M. enterolobii; in these conditions, the resistant plants of the invention produce more marketable peppers than susceptible plants. Heavily galled roots of susceptible plants under pest pressure, provide minimal resources for the rest of the plant, resulting in reduced yield or non- harvestable or unmarketable fruits.
  • the invention is thus also directed to a method for improving the yield of Capsicum plants and/or fruits or for increasing the number of harvestable Capsicum fruits, in an environment infested by M. enterolobii comprising growing in said environment Capsicum plants resistant to M. enterolobii as defined, comprising on chromosome 9 the QTLs or sequences according to the invention and conferring to said plants resistance to M. enterolobii.
  • the invention is also directed to the use of the Capsicum plants of the invention for improving the yield of Capsicum plants and/or fruits, and/or for increasing the number of harvestable Capsicum fruits, in an environment infested by M. enterolobii.
  • the method comprises a first step of choosing or selecting a Capsicum plant comprising said sequences of interest conferring to said plants resistance to M. enterolobii.
  • the method can also be defined as a method of increasing the productivity of a Capsicum field, tunnel or glasshouse, or as a method of reducing the intensity or number of chemical or fungicide applications in the production of peppers.
  • the invention is also directed to a method for reducing the loss on Capsicum production in condition of M. enterolobii infestation, comprising growing a Capsicum plant as defined above.
  • the resistant plants of the invention are also able to restrict the growth of the pathogens responsible for M. enterolobii, thus limiting the infection of further plants and the propagation of the pathogens.
  • the invention is also directed to a method for protecting a field, tunnel or glasshouse, or any other type of plantation, from M. enterolobii infection, or of at least limiting the level of infection or limiting the spread of M. enterolobii.
  • Such a method preferably comprises the step of growing a resistant or tolerant plant of the invention, i.e. a plant comprising on chromosome 9 the sequences conferring resistance to M. enterolobii.
  • resistant plants according to the invention have the capacity to disrupt the lifecycle and reproduction of Meloidogyne enterolobii; making possible to use the plant as a pest management tool.
  • the invention thus concerns the use of a Capsicum plant resistant to M. enterolobii, according to the invention, in a field, tunnel or glasshouse, or other plantation.
  • the invention concerns the use of a Capsicum plant resistant to M. enterolobii according to the invention for controlling infestation in a field, tunnel, glasshouse or any other plantation, by M. enterolobii.
  • the invention relates to a use of the Capsicum plant according to the invention, for reducing nematode pressure in an environment infected by M. enterolobii, preferably a field, tunnel, greenhouse or glasshouse.
  • the invention relates to a method for reducing nematode pressure in an environment infected by M. enterolobii, preferably a field, tunnel, greenhouse or glasshouse, wherein said method comprises growing a Capsicum plant according to the invention in said environment.
  • the invention relates to a method of growing a Capsicum plant , comprising :
  • the plant lacking said QTL on chromosome 9 is a isogenic plant.
  • the invention relates to a use of a Capsicum plant according to the invention to disrupt the lifecycle and/or reproduction cycle of M. enterolobii.
  • All the preferred features of the QTL are as defined in connection with the other aspects of the invention, in particular it is preferably present in the seeds of Capsicum annuum PB21 SG7-1294, representative seeds of which have been deposited under accession number NCIMB 44105, and it is identifiable by the markers as defined according to the present invention.
  • the present invention is also directed to a method for improving the yield of Capsicum plants in an environment infested by M. enterolobii comprising:
  • the yield of the Capsicum plants is increased, inter alia more marketable peppers can be harvested, or more seeds are obtained.
  • the invention relates to a method of producing pepper fruits comprising:
  • the method may advantageously comprise a further step of processing said peppers into a processed food.
  • the present invention also relates to a method of producing a food product, comprising mixing a pepper fruit of the invention, or part thereof, with one or more food ingredients.
  • the method comprises cooking and/or processing the pepper fruit of the invention, alone or in mixture with the one or more food ingredients.
  • food products that comprise pepper in raw, cooked or otherwise processed form include powders, soups, sauces, salsas, pastas, condiments, pastries, sweets and salads.
  • the present invention also relates to a food product made of a pepper fruit of the invention or parts thereof, optionally in processed form.
  • the invention relates to the use of a Capsicum plant according to the invention or a fruit thereof in the fresh cut market or for food processing.
  • Techniques for using pepper in food processing are well known from the skilled person, e.g. as an ingredient in a food product such as powders, soups, sauces, salsas, pastas, condiments, pastries, sweets and salads, and described, for instance, in Handbook of Food Science, Technology and Engineering, vol. 4, Y. H. Hui, Frank Sherkat. CRC Press.
  • the term “comprising” is to be interpreted as encompassing all specifically mentioned features as well optional, additional, unspecified ones.
  • the use of the term “comprising” also discloses the embodiment wherein no features other than the specifically mentioned features are present (i.e. “consisting of’).
  • a representative sample of seeds from the Capsicum plant according to the invention i.e. seeds from Capsicum annuum PB21SG7-1294 plant
  • NCIMB National Collection of Industrial, Food and Marine Bacteria
  • a deposit of the PB21SG7-1294 seeds is maintained by Vilmorin & Cie, 4 quai de la Megisserie, 75001 Paris, France.
  • Example 1 Evaluation of the resistance to Meloidogyne enterolobii.
  • the resistance of line ME-231-02 was evaluated in comparison to several other lines.
  • J2 second-stage juveniles
  • J2 Meloidogyne enterolobii
  • Water was collected every 2-3 days and filtered at 6pm to separate J2 from other nematodes stage and stored in a tube at 4°C. 1 .5 mL inoculum was then injected on plants at 3 or expanded leaf stage (about 33 days after sowing).
  • the dosage of the inoculum injection was about 450 J2/plants. 70 days after sowing, the resistance was evaluated using a scoring scale (Table 1) to collect comparative dataset from 1 (highly susceptible) to 9 (highly resistant).
  • Table 1 scoring scale of the M. enterolobii resistance evaluation on Pepper.
  • Line ME-231-02 produced few galls with egg-masses and exhibited a higher level of resistance to M. enterolobii than all other tested lines, including lines with known genes of resistance to nematodes.
  • ME-231-02 is the only line which can be classified as resistant.
  • the lines with known genes of resistance to other species of root knot nematodes (A/, Me1, Me2, Me3, Me4, Me5, Me7) are susceptible to M.enterolobii.
  • Figure 1 shows the box-plot distribution of the main known nematodes resistance genes described in Pepper compared to the claimed resistance (named R parent). The same letters on top of each bar indicates significant similar groups (Tukey’s, p ⁇ 5%). Line ME-231-02 was significantly more resistant than all other lines tested.
  • RILs F6 and F7 recombinant inbred lines
  • the 173 F6 and F7 RILs were phenotyped together with lines carrying known genes of resistance to nematodes. Additional lines were also phenotyped. The parental lines were also phenotyped together with the F1 (ME-231-02 x S. parent) hybrid.
  • the test was designed according to a Rep-Check mode based on the evaluation of 4 replications of 3 plants (total 12 plants) per F6 family.
  • the inoculation protocol was as described in Example 1 .
  • the scoring was semi-quantitative and done by evaluating the percentage of roots affected by galls and egg-masses.
  • the scoring scale is shown in Table 1 above.
  • the phenotyping scores are shown in Figure 3. They confirm that ME-231-02 is significantly more resistant than the other candidate sources, including known genes of resistance to nematodes (Me1, Me3, Me7, N).
  • the F1 (ME-231-02 x S. parent) was phenotyped as susceptible, suggesting a recessive inheritance of the resistance.
  • Phenotyping data set was analyzed using JMP ⁇ software and adjusted values were used for QTL mapping (see Example 3 below). The data distribution ( Figure 4) seems to indicate a single genetic control.
  • Example 3 QTL Mapping
  • F6 or F7 RILs from the population described in Example 2 were genotyped with 2652 SNPs, together with plants from the parent susceptible line and the ME-231-02 line. 6 plants were genotyped per F6 or F7 RIL family or line. The SNPs were filtered based on a Sample HetRate ⁇ 10% and a Marker Callrate >90%.
  • QTLs were identified with a QTL detection module and the CIM (Composite Interval Mapping) method was used with a QTL detection every 1 cM between two adjacent Markers. After a new filtering process, 9 unmapped markers and 19 markers with low allele frequency were rejected. 2640 SNPs were finally retained for the QTL detection
  • variable used for QTL detection was the adjusted means from the M. enterolobii resistance phenotype test done on F6 or F7 RILs (see Example 2).
  • the M. enterolobii QTL was mapped between markers PE-0057076 and PE-0006730.
  • 5 markers were selected as particularly relevant, i.e. linked to the intermediate resistant phenotype and unique to the resistant source. Those 5 markers are: PE-0057076, PE- 0057077, PE-0057080, PE-0057081 , and PE-0006730 (see Table 2 below).
  • the markers were tested using the KASPar technology, as described in the present specification.
  • the resistant allele was not detected in ME-231-02. It was also not detected in the susceptible parent and in the F1 (ME-231-02 x S. parent).
  • the markers were tested on a control hybrid plant known to exhibit the root knot nematode resistance linked to Markers A, B and C: the test was validated, as the control hybrid was shown to contain the alternate alleles (resistant) on those markers.
  • the markers were also tested on the F5 RIL population derived from ME-231-02 and the susceptible parent: the resistant allele was not detected in the F5 RIL plants. This confirms that the QTL of the present invention differs from the previously reported resistances.
  • Example 6 the Capsicum annuum PB21SG7-1294 deposited under accession number NCIMB 44105 confers partial resistance to M.Enterolobii by significantly decreasing the amount of egg-masses development and affecting the nematode fecundity.
  • Capsicum annuum plants PB21 SG7-1294 were evaluated for resistance to M.Enterolobii.
  • nematodes were extracted with a 1 % NaOCI solution according Hussey and Barker, 1972) and counted under a haemocymeter under a microscope (x40).
  • Table 4 summary table of the reproduction factor calculated from each genotype according to Soares et al., 2018. Highest index indicates a susceptible reaction. N indicates the number of tested plants. SD is the standard deviation.
  • Example 7 the Capsicum annuum PB21SG7-1294 deposited under accession number NCIMB 44105 confers partial resistance to M.Enterolobii by significantly decreasing the Meloidogyne enterolobii infection by penetration assays
  • roots were stained with 1 ml of 3.5% acid fuchsin stain, heated to the boiling level and then cooled at room temperature and washed in running water. The roots were kept in acidified glycerin. After staining, roots segments were observed under a binocular magnifier.
  • NCIMB44105 confers resistance to Meloidogyne enterolobii with different mechanisms: i) reduces the amount of infectious J2 penetration in the roots; ii) delays the lifecycle of Meloidogyne enterolobii within the root to accomplish the complete life cycle, iii) reduces the development of symptoms on the roots such as galls and egg-masses and iv) affects the nematode reproduction cycle.
  • Seeds of Capsicum plants are to be treated with EMS by submergence of approximately 2000 seeds into an aerated solution of either 0.5% (w/v) or 0.7% EMS for 24 hours at room temperature.
  • M2 seeds are harvested and bulked in one pool per variety per treatment.
  • the resulting pools of M2 seeds are used as starting material to identify the individual M2 seeds and the plants resistant to M. enterolobii.

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Abstract

La présente invention concerne une plante Capsicum comprenant un locus de caractère quantitatif (QTL) dans son génome sur le chromosome 9, ledit QTL conférant à la plante une résistance à Meloidogyne enterolobii. L'invention concerne également des procédés associés de production de plantes et des procédés d'identification de plantes résistantes à Meloidogyne enterolobii.
PCT/EP2024/068072 2023-06-28 2024-06-27 Résistance du poivre à meloidogyne enterolobii Pending WO2025003305A1 (fr)

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